COMPARING DNA DAMAGE CAUSED BY FORMALDEHYDE [PDF]

COMPARING DNA DAMAGE CAUSED BY FORMALDEHYDE, GLUTARALDEHYE, CARNOY’S AND METHACARN IN CANCER TISSUE FIXATIONS

Chia-Jui Tsai

A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE December 2006 Committee: Scott O. Rogers, Advisor Carmen Fioravanti Mike Geusz

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ABSTRACT Scott O. Rogers, Advisor

Many molecular biotechniques are useful in detecting macromolecular changes. Disease stages may be analyzed accurately based on these molecular changes. Therefore, molecular methods have the potential to be applied as routine and highly informative clinical diagnostic tools. Fixation methods become an essential consideration while preserving tissues. This study investigates the frequency of DNA change caused by two aldehyde-based fixatives and two alcohol-chloroform-acetic acid fixatives. Aldehyde-based fixatives, especially 10% neutral buffered formalin (NBF) and 1% glutaraldehyde, are widely used in histological studies. Among these two fixatives, 1% glutaraldehyde preserves cellular structure better than formalin, and recent studies indicate that it preserves DNA better than formalin. In addition to the aldehyde-based fixatives, Carnoy’s, which is made with ethanol (60%), chloroform (30%) and acetic acid (10%), and Methacarn, which shares the same formula but uses methanol instead of ethanol were tested DNA changes for each fixation were compared this study. Human tissues (normal white blood cells, sarcomas, leukemias, and carcinomas) were fixed, separately using 10% NBF, 1% glutaraldehyde, Carnoy’s and Methacarn. DNA from the fixed tissues was extracted and segments (418 bp and 597 bp) of the nuclear small subunit ribosomal DNA (SSU rDNA) were amplified by polymerase chain reaction (PCR) and sequenced. The DNA sequences were analyzed by comparing the DNA changes between four

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fixative-treated and unfixed tissues. According to the results of the DNA statistical analyses of sequence alignments, we report that 10% NBF causes more DNA change than the other fixatives. Alcohol-chloroform-acetic acid-based fixatives, in general, caused less DNA changes and maintained the DNA integrity better than aldehyde-based fixatives.

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ACKNOWLEDGEMENTS Many people have contributed the completion of this study. The foremost gratitude goes to my major professor Dr. Scott R. Rogers for his guidance, kindness, and continued encouragement, patience through my study. It was my honor to work with him for these past two years. I also wish to express my deep appreciation to my committee members, Dr. Mike Geusz and Dr. Carmen Fioravanti, for their valuable suggestions and comments.

I would like to express my gratitude to my lab mates, Tom D'Elia, Lorena Harris, Zeynep Kocer, Seung-Geuk Shin, Farida Sidiq, and Ram Satish Veerapaneni, for their friendship and support. Special thanks go to Armeria Vicol for her critical reading and suggestions for improving the manuscripts. Gang Zhang is acknowledged for his helpful support and advice throughout the past two years.

Finally, my special thanks and deepest love are extended to my parents, Yung-Ta Tsai and Chang-Chang Chen, my brother, Chang-Yu Tsai, and my grand parents, Jiang-Hai Tsai and Chao-Chi Tsai-Hsu, for their continuous support and encouragement.

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TABLE OF CONTENTS Page CHAPTER 1. LITERATURE REVIEW…..……………………….….………..….

1

FIXATION………………………….………………………….…..…...

2

Introduction………………………………………..….….…...

2

Fixation methods…….……………………………….…….....

3

Ideal

fixation……….…………………………….…………

3

Heat…………………………………………………...............

4

Freezing……………………………………………….………

5

Chemical methods of fixation………………………………...

6

Formaldehyde…………………………………………............

7

Glutaraldehyde………………………………………………..

14

Factors involved in aldehyde-based fixation………………….

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Carnoy’s……………………………………………………….

24

Methacarn……………………………………………………..

25

CANCER…………………………………………………………….…

26

The causes of cancer……………………………………..…..

27

The development of cancer…………………………...………

36

Types of cancer…………..………………………….………..

43

Summary……………………………………………………...

46

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CHAPTER 2. DNA DAMAGE CAUSED BY FIXATIVES……………………….

47

INTRODUCTION…………………………………………………........

48

MATERIALS AND METHODS………………………………….....….

53

Specimens……………………………………………………..

53

Tissue preservation………………………………….…...........

54

DNA extraction………………………………………….…….

55

PCR amplification…………………………………………….

56

TOPO TA cloning and DNA sequencing……………………...

57

DNA sequence analysis……………………………………….

59

RESULTS…………………………………………………..…………....

61

DISCUSSION………………………………………………………....…

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LITERATURE CITED…………………………………………………………….….

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APPENDIX A: Carcinoma Sequence Data…………………………………………...

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APPENDIX B: Leukemia Sequence Data…………………………………………….

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APPENDIX C: Sarcoma Sequence Data……………………………………………...

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APPENDIX :D White Blood Cells Sequence Data……………………………….…..

141

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LIST OF FIGURES Figure

Page

CHAPTER 1. LITERATURE REVIEW 1.

Monomeric (a) and polymeric (b) formaldehyde reacts with water to form methylene hydrate……………………………………………...

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2.

Cannizzaro reaction of formalin……………………………………...

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3.

Methylene glycol adducts of protein functional groups……………...

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4.

Methylene bridge between proteins………………………………….

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5.

Exocyclic and endocyclic methylol adducts of DNA bases………….

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6.

Two DNA bases were cross-linked by methylene bridge…………….

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7.

DNA with apurine and apyrimidine (AP) sites………………………

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8.

Phosphodiester scission………………………………………………

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9.

Difunctional aldehyde groups of glutaraldehyde…………………….

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10.

The polymeric form of glutaraldehyde……………………………….

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11.

The reaction between the terminal aldehyde group and the amino

12.

groups of proteins….…………………………………………………

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Fixation of proteins by polymeric glutaraldehyde……………...……

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CHAPTER 2. DNA DAMAGE CAUSED BY FIXATIVES 13.

Agarose gel of PCR products from aldehyde-fixed and non-fixed tissues………………………………........…………………………...

14.

Agarose gel of PCR products from alcohol-chloroform-acetic acid-fixed and non-fixed tissues……………………………………...

15.

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A T-test comparing DNA difference rates of 10% NBF fixed tissue and non-fixed tissues……………………….………………………...

16.

68

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One-way ANOVA analysis comparing DNA change rates occurring in each fixative treatment…………………………………….………

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LIST OF TABLES

Table

Page

CHAPTER 1. LITERATURE REVIEW 1.

Viruses associated with human cancers……………………………...

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2.

Oncogenes and their functions……………………………………….

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3.

Tumor suppressor genes and their functions…………………………

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CHAPTER 2. DNA DAMAGE CAUSED BY FIXATIVES 4.

Results of PCR amplification………………………………………...

5.

Absolute numbers and percentages of A/C/G/T in the SSU rDNA that were amplified by PCR…………………...…………………….

6.

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The percentage of differences in DNA after fixation in each type of tissue by treatment……………………………………………………

7.

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Three types of the DNA sequence differences in each specimen by treatments…………………………………………………………….

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8.

The frequencies of DNA changes in each specimen by treatment…...

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9.

Standard deviations for DNA change percentages from each specimen type by fixative…………………………………………….

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CHAPTER 1

LITERATURE REVIEW

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FIXATION

Introduction From the moment tissues die, their cells are invaded by various bacteria from the extracellular environment, while the cells undergo autolysis to destroy themselves. The appearance and conformation of cells change, and these changes are called postmortem modifications. Fixation is a procedure that can preserve a specimen’s histologic structure and chemical composition and prevent postmortem decay. In order to achieve this goal, the specimen needs to be rapidly killed and degradative processes must be halted. Tissue fixation is an important procedure in histological methodology. With proper fixation, histologists can examine specimens that have as few alterations as possible compared to their living states. In addition, for some fixation methodologies, macromolecules, including proteins, nucleic acids, and carbohydrates can be studied. The solutions used during fixation are called fixatives or fixing solutions. In general, fixatives preserve tissues through two processes: fixatives first prevent autolysis caused by lysosomal enzymes and kill the microorganisms surrounding the specimen. Then they precipitate, coagulate, dehydrate, and/or cross-link the cellular macromolecules.

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Fixation methods Fixation methods can be divided into two categories: physical fixation and chemical fixation. Physical methods fix tissues by placing the specimen in extreme conditions. Mummies found by archeologists are preserved by physical methods in extremely dry environments from hot to very cold temperatures. In nature, fixation usually is by physical methods. Chemical methods, which have been used widely after the nineteenth century, a relatively short f./ixing time is required compared to physical methods. The duration of the fixation process for chemical methods depends on fixative penetration time and tissue size, and the effects of chemical fixation are mainly achieved through specific chemical molecules that link or coagulate components of the cells.

Ideal fixation Ideal fixatives should have the following characteristics (Presnell and Schreibman, 1997): (1) Be able to penetrate and fix tissues before postmortem conditions occur; (2) Convert cells into an insoluble state; (3) Strengthen the cell structure and prevent morphological changes in future processing; (4) Permit the specimen to be observable; (5) Ready the cell for staining to obtain adequate optical contrast; and (6) Augment the attachment of future treatment, such as dyes and

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proteins. Every fixative has some advantages and disadvantages. No single chemical can satisfy all the criteria of an ideal fixative. Therefore, a combination of several fixatives or fixing procedures is commonly used.

Heat Application of heat to a specimen is one of the treatments tested previously. It is widely used in microbiology because this method maintains the appearance and staining properties of microorganisms well enough that they are able to be distinguished. For eukaryotic cells, Bernhard (1974) reported that the stained sections were disfigured, the nuclei were shrunken, and the cytoplasm was clotted roughly when the specimen was heated alone at 80℃ in a microwave oven. The resemblance of the fixed specimen and its living state is poor. Therefore, heating is commonly applied along with chemical fixation. Ruijgrok et al. (1993 and 1994), and Hopwood et al. (1988), reported that high temperature used with formalin was effective, since microwave radiation can promote the diffusion of formaldehyde and accelerate the cross-linking of proteins. Microwave technology has reduced the times required for sample processing compared to routine processing protocols (Kahveci et al., 1997; and Giberson et al., 1997).

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Freezing Fixation by freezing is the best choice for the sections that contain lipids, enzymes, radioisotopes, and soluble components that may be lost during treatments with alcohol or heat. Liquid nitrogen (-196℃), solid carbon dioxide (dry ice and acetone mixture, -75℃), liquid nitrogen (-268℃), and isopentane (-170℃) are frequently used as freezing media (Bald et al., 1983; and Lemke et al., 1994). When freezing fixation is applied to a specimen, the formation of ice crystals needs to be avoided. Ice crystals can destroy and replace the tissue structure with meaningless artifacts. Ice crystal damage can be reduced with the aid of physical or chemical methods. Rapid freezing causes the formation of smaller ice crystals, compared to slow freezing. Thus, rapid freezing is preferable to slow freezing. Some cryoprotection agents can efficiently prevent the formation of ice crystals. The most common agents are: dimethylsulphoxide (DMSO) (Ashwood-Smith, 1971), glycerol (Holt, 1960), propylene glycol (Campbell et al., 1997), and sucrose (Terracio and Schwabe, 1981). In practice, a specimen is being dehydrated along with fixation by freezing. This can be completed by leaving the specimen in an evacuated chamber while it is freezing (termed freeze-drying) or using phosphorus pentoxide to absorb the water vapor (lyophilization). The proteins of specimens fixed by this method retain their activity

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(Orr, et al., 1962; Veltkamp, et al., 1993), but the process can cause structural deterioration of the cells (Goodwin and Grizzle, 1994). If ethanol or acetone is used as a dehydrating agent, the organic solution dissolves the ice but does not coagulate the proteins when the temperature is below -49℃ (Hippe-Sanwald, 1993).

Chemical methods of fixation Chemical fixation is the most widely used method for histological and histochemical purposes because proteins, nucleic acids, and macromolecular carbohydrates can be preserved easily in a short time. However, physical and chemical properties of tissues may be altered while fixing with chemical fixatives. Tissue size will be changed because they shrink or swell after being immersed in fixatives. The fixed tissue is about 60-70% of its original living volume when alcohols are used (Baker, 1958). Most fixatives also harden tissues which can facilitate sectioning although ecessive hardening makes sectioning difficult or impossible. The physical changes caused by chemical fixatives can be alleviated by mixing fixatives to balance undesirable side-effects. For example, acetic acid, which causes tissue swelling, often is combined with ethanol, which causes tissue shrinkage, in various fixative formulations. The fixative penetration rates dictate the duration of fixation and the maximum sizes of tissues that are suitable for fixation. Otherwise, incomplete

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or inconsistent fixation will occur. Chemical fixatives can be subdivided into two categories: additives and coagulants, according to the interaction between the fixatives and the specimen. Additives are fixatives that combine with biomolecules while coagulants are fixatives that precipitate biomolecules.

Formaldehyde Formaldehyde is the simplest aldehyde containing a single carbon. The melting point of this molecule is -117℃, and the boiling point -19.3℃. Formaldehyde is a gas at room temperature, but is readily soluble in water. Formalin is a solution of 37-40% (w/v) of formaldehyde gas in water, and paraformaldehyde is a solid polymer (HO(CH2O)nH), where n can be 6 to 100). In formalin, very few formaldehyde molecules are in monomeric form because formaldehyde reacts with water and forms methylene hydrate (methylene glycol) (Figure 1). Formaldehyde polymerizes in formalin and forms soluble polymers with the formula HO(CH2O)nH, where n = 2-8), which is also called polyoxymethylene glycol. Formalin, containing 10% methanol (v/v), can limit the polymerization because methanol and formaldehyde form a hemiacetal (methylal), which is more stable than methylene hydrate. Formaldehyde shows all the characteristics of an aldehyde. Formaldehyde undergoes Cannizzaro reactions (Figure 2) to produce formic acid and methanol in alkaline solution (Fox et

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a. Formaldehdye

b. Paraformaldehyde

water

Methylene hydrate

water

methylene hydrates

Figure 1: Monomeric (a) and polymeric (b) formaldehyde reacts with water to form methylene hydrate (Kiernan, 1990).

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Formaldehdye

methanol

formic acid

water

Figure 2: Cannizzaro reaction pf fpr,a;om. Cannizzaro reactions of formalin to form methanol, formic acid, and water, when pH is high (Kiernan, 1990).

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al., 1985). Formaldehyde is oxidized slowly by oxygen in the environment to generate formic acid. Formaldehyde can undergo addition reactions to lipids, proteins, and nucleic acids. The fixation is caused by adding methyl groups to the amino groups of phosphatidyl ethanolamine (Jones, 1972). Most lipids can be preserved by formaldehyde, especially insoluble phospholipids. Formaldehyde forms methyl hemiacetal-like adducts and methylene bridges to stabilize proteins. The methylene glycol molecule formed by formaldehyde reacts with the primary amines (N-terminal amino acid and lysine sidechains), the guanidyl group of arginine side chains, the sulphydryl group of cysteine, the aliphatic hydroxyl groups, and the amide nitrogen (Walker, 1964; Hopwood, 1969) (Figure 3). The free hydroxymethyl groups of hemiacetal-like adducts will further react with other proteins and form methylene bridges (Figure 4) (Mason and O’Leary, 1991). Gustavson (1956) reported that the majority of the cross-links between proteins occur between ε-amino groups of lysine and the amine nitrogen atoms of the peptide bond (Figure 4). The chemical reactions between formaldehyde and nucleic acids are similar to the reactions between formaldehyde and protein. Formaldehyde reacts with the bases of nucleic acids, and adds a hydroxymethyl (methylol) group to the amino and imino groups. Thus, the reaction can be either an exocyclic or an endocyclic addition (Auerbach et al., 1977; Douglas, 1997; Douglas and Rogers, 1998 ) (Figure 5). The free hydroxymethyl

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a. Methylene glycol molecule adds to the N-terminal amino acids:

b. Methylene glycol molecule adds to guanidyl groups:

c. Methylene glycol molecule adds to sulphydryl groups:

d. Methylene glycol molecule adds to aliphatic hydroxyl groups:

e. Methylene glycol molecule adds to amide nitrogen:

Figure 3: Methylene glycol adducts of protein functional groups. Methylene glycol adds to the functional groups of proteins to form hemiacetal adducts (Kiernan, 1990).

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Figure4. Methylene bridge between proteins. Hemiacetal groups form methlyene bridge between proteins (Kiernan, 1990).

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Figure 5: Exocyclic and endocyclic methylol adducts of DNA bases (Douglas, 1997; Douglas and Rogers, 1998).

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group is unstable and tends to be reactive. The amino nucleic bases undergo electrophilic attack by the active N-methylol, and form a stable methylene bridge between these two bases (Figure 6). Formaldehyde reacts with the N-glycosidic bond of the ribose and causes the detachment of purines and pyrimidines from nucleic acids by hydrolysis (Figure 7). The hydrolysis reaction triggered by formaldehyde also can change phosphodiester bonds to polydeoxyribose, and break the DNA backbone (Figure 8).

Glutaraldehyde Glutaraldehyde, also called 1,5-pentanedial, has a molecular weight of 100.12. It is a five-carbon molecule with bifunctional aldehyde groups at each end (Figure 9) and is one of the most widely used fixatives for electron microscopy. Glutaraldehyde, like formaldehyde, is easily oxidized and polymerizes in aqueous solution. The polymers are formed by aldol condensations: an olefinic double bond (C=C) is conjugated with the carbonyl (C=O) double bond of the aldehyde group (Kirnan, 1990; Monsan, Puzo, and Marzarguil, 1975, Figure 10). Most polymers are dimers (n=0) or trimers (n=1), and the number of polymers increases with increasing pH and temperature. Sabatini, Bensch, and Barrett (1963) report the importance of using high purity glutaraldehyde in fixation. Glutaraldehyde is commercially available as a

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Figure 6: Two DNA bases are cross-linked by methylene bridge (Douglas, 1997; Douglas and Rogers, 1998).

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Figure 7: DNA with apurine and apyrimidine (AP) sites. Apurinic and apyrimidinic (AP) sites with open ribose rings (Douglas, 1997; Douglas and Rogers, 1998).

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Figure 8: Phosphodiester scission. The broken phosphodiester backbone of DNA (MacMillan, 2001; Papavassiliou, 1995).

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Figure 9: Difunctional aldehyde groups of glutaraldehyde (Kiernan, 1990).

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Figure 10: The polymeric form of glutaraldehyde. Most polymers are dimers (n=0) or trimers (n=1) (Kiernan, 1990; Monsan, Puzo, and Marzarguil, 1975).

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concentrated (usually 25%, 50%, and 70%) aqueous solution at pH 3 to 5.5, and must be stored at 4℃. The condition of the glutaraldehyde solution can be checked spectrophotometrically: the maximum absorbance of pure glutaraldehyde is at 280 nm, but the aldehyde aldol condensation dimer absorbs at 235 nm. Prento (1995) reported that glutaraldehyde-based fixatives are still usable for up to eight hours after being diluted in neutral buffer. Hayat (1981) stated that storing glutaraldehyde in cacodylate buffer (pH 7.4) will induce less polymerization compared to storing it in phosphate buffer at the same pH. Glutaraldehyde is a stronger cross-linker compared to formaldehyde (Douglas and Rogers, 1998). It reacts with the amino groups, sulfhydryl groups, and possibly with the aromatic rings of proteins (Leong et al., 1991). Imines (compounds with C=N bonds, also called Schiff bases) are formed by the reaction of amino groups of proteins and either terminal aldehyde functional group of the glutaraldehyde monomer (Figure 11). Glutaraldehyde polymers can form imines in the same manner, but aliphatic imines are not stable. Therefore, the majority of imines are not formed at the ends of the polymer, but in the mid-chain of the molecule. The mid-chain aldehyde groups of polymeric glutaraldehyde have the best efficiencies of protein fixation (Figure 12) (Monsan, Puzo, and Marzarguil, 1975). Peter and Richards (1977) suggest that alpha-beta unsaturated aldehydes also can cross-link with proteins.

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Figure 11: The reaction between the terminal aldehyde group and the amino groups of proteins. The terminal aldehyde group reacts with the amino group of proteins to form imines (Kiernan, 1990).

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Figure 12: Fixation of proteins by polymeric glutaraldehyde. Polymeric glutaraldehyde fixes proteins in the middle of the chain and at the end (Kiernan, 1990).

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Glutaraldehyde forms stronger cross-linkages than formaldehyde, so the fixed specimen is harder, which increases the difficulty of sectioning. The penetration rate of glutaraldehyde is much lower than that of formaldehyde, so perfusing glutaraldehyde using a vacuum system is suggested.

Factors involved in aldehyde-based fixation Several aspects should be considered when choosing an aldehyde-based fixative: concentration, pH, temperature, rate of fixative penetration, and potential for chemical damage. The polymerization increases with increasing pH and temperature, but the length of DNA decreases with increasing fixation temperature.The concentration of formaldehyde affects DNA quality when fixing specimens at -5℃ (McGhee et al., 1975; Solomon and Varshavsky, 1985). The denaturation rate of DNA increased with increasing the concentration of formaldehyde. Neutral buffered 10% formalin (3.74% of formaldehyde, pH 7.2-7.4) is the most common by used fixative for histological and histochemical uses. The lowest DNA damage rates and DNA degradation were shown in tissues that were fixed at 4℃ (Nocguchi et al., 1997; Tokuda et al., 1990; and Yagi et al., 1996). Douglas and Rogers, (1998) reported that high DNA damage rates and low molecular weight DNA resulted in tissues fixed in formalin at pH 3.0 compared to the tissues fixed in neutral formalin. Formaldehyde

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penetrates into tissues faster than glutaraldehyde. The penetration rate of a fixative can be determined by the laws of diffusion: d = K√ t, where d is the depth of penetration, t is time; and K is a constant coefficient of diffusibility, which represents the distance in millimeters the fixative has diffused into the tissue in 1 hour. The K value of 4% formaldehyde is 0.78 (Leong et al., 1996). The K value of 4% glutaraldehyde is approximately half that of 4% formaldehyde at 4℃ (Bancroft et al., 1996).

Carnoy’s Carnoy’s fixative, which was introduced by J. B Carnoy in 1887, is made by mixing ethanol, chloroform, and acetic acid (6: 3:1). It was derived from Clark’s fixative, which contains ethanol and acetic acid (3: 1) and was published in 1851(Baker, 1958). Ethanol (CH2H5OH) is a coagulating fixative that denatures the non-water-soluble proteins at room temperature and above. However, many proteins will be precipitated without denaturing them, when the specimen is fixed at -5℃. Ethanol can extract lipids, but does not have any effect on carbohydrates. The acetic acid (CH3COOH) in the fixative mixture is used to preserve chromosomes by coagulating nucleic acids. It penetrates in tissues to a 1.2 mm depth in one hour at 4℃ (Backer, J R. 1958 ), which is faster than most fixatives. Acetic acid can break the

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cross-linkages between protein molecules and release lyophilic radicals that associate with water molecules (Presnell and Schreibman, 1997). Thus, tissues swell after being treated with acetic acid. Often, this property of acetic acid is used to prevent the tissue shrinkage and hardening caused by ethanol. The chloroform in the fixative is to speed the slow tissue penetration rate of alcohol. The extreme hydrophobicity of chloroform results in rapid tissue dehydration and penetration. James and Tas (1984) modified the proportion of ethanol: chloroform: acetic acid to 60:30:5. The RNA was better preserved with less acetic acid since acids cause nicks in RNA and DNA. The nucleic acids of tissues fixed by Carnoy’s were better preserved and easier to extract. Foss (1994) compared the effects of fixatives on tissues fixed in neutral buffered formalin and Carnoy’s, and reported that high-molecular weight RNA was extracted from Carnoy’s-fixed tissues. Giannella (1997) confirmed the result and suggested that the tissues, on which DNA/RNA analysis will be performed in the future, need to be preserved in ethanol-based fixatives.

Methacarn Puchtler et al. (1970) first reported the fixative known as “methacarn”, which is based on the formula of Carnoy’s fixative, except methanol is used instead of ethanol. Methacarn is made by mixing methanol, chloroform and acetic acid (6:3:1). The

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shrinkage temperature of collagen in methanol is 86℃ compared to 70℃ in Carnoy’s fixative. For tissues that are to be embedded in paraffin, a high shrinkage temperature is desirable. Methacarn precipitates nuclear proteins and coagulates nucleic acids. The precipitated proteins are inactive, but can be extracted with detergents (Shibutani et al., 2000). Thus, DNA remains intact. Uneyama et al. (2002) were able to extract high yields of rat liver DNA that was fixed in methacarn and to amplify a 522 bp fragment by PCR. The quantity of RNA extracted from unfixed and methacarn-fixed rat liver tissue was equivalent (Takagi et al., 2004). Shibutani et al. (2000) obtained a similar result with cultured cell lines. Methacarn fixed tissues can be stained and still retain antigen immunoreactivities without further treatment. Therefore, it is suitable for the fixation of tissues that need to undergo further protein, or nucleic acid, analysis (Beckstead et al., 1994; Borrebaeck, 1998; Takagi et al., 2004).

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Cancer

The Causes of Cancer In 1914, Theodor Boveri (1862-1915) first proposed that cancer was a disease caused by a chromosomal disruption (Macdonald, Ford, and Casson, 2004). Based on Boveri’s theory, cancer has been known for many years to be a disease caused by mutations ino DNA. The mechanisms of how carcinogens and microorganisms alter genes and induce cancer have been studied for many years. The substances that promote cancers are called carcinogens, and agents that have the ability to change DNA in ways that are inherited by daughter cells are called mutagens. Tabin et al., (1982) and Weinberg et al., (1988) first identified and characterized oncogenes and tumor suppression genes. These genes are categorized according to their functions. The modified genes that promote malignancy are called oncogenes, and the genes that restrict tumor growth are tumor suppression genes. The accumulation of multiple factors, carcinogens and altered genes, transform a normal cell into a cancerous one. Carcinogens that have the ability to promote cancer have various sources. Most people come into contact with carcinogens by consuming food. Plants, such as Aristolochia and Bracken, contain carcinogens, which will induce cancer by eating them over a long period of time. Carcinogens also are synthesized during food

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processing and storage. Over-cooked meats contain a potential carcinogen with a structure similar to benzo[a]pyrene, which is a carcinogen found in cigarette smoke. Aspergillus flavus and A. parasiticus grow on the grains and nuts stored under high temperature and high humidity conditions. Aflatoxin, a toxin produced by these microorganisms, can work alone as a carcinogen, but also has a synergistic carcinogenetic effect in case of concurrent infection with the hepatitis B virus (HBV). Williams et al. (2004) reported that HBV infections increase the time of aflatoxin persistence in the liver and the chance of DNA damage because HBV decreases the efficiency of aflatoxin metabolism in hepatocytes. Viruses induce cancer mainly in two ways: retroviral transduction and insertional mutagenesis. Rous sarcoma virus (RSV) is a retrovirus first isolated from chicken sarcoma by Peyton Rous (Rous, 1979). Sixty years afterwards, Stehelin et al. (1976) found that RSV was able to carry and express the SRC oncogene that was originally thought to belong to the host. Viruses cannot replicate by themselves, so they use the host to synthesize the proteins and nucleic acids needed for assembling the viruses. In order to reach this goal, viruses integrate their genome within the DNA of the host and use viral promoters to control cellular gene expression. The protooncogenes where the retrovirus inserts their genome become retroviral oncogenes by retroviral transduction. In addition to transforming retroviruses, some retroviruses, such as various animal

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leukemia viruses do not carry viral oncogenes, but induce cancer by insertional mutagenesis (Boland, 1998). The DNA of the provirus is integrated into the host genome close to a protooncogene. The transcriptional regulatory elements contained in the provirus that transform the host cell cause the synthesis of proteins with abnormal activity and at high levels (Hayward et al., 1981; Neel et al., 1981). Then the protooncogene is activated. A list of viruses and their corresponding tumors is found in Table 1. Chemicals also can cause DNA damage: there are more than 50 chemical compounds proven to cause carcinogenesis. Benzopyrene, nitrosonornicotine, and formaldehyde are the most common carcinogens found in either industrial or cigarette smoke. Other chemicals, like benzene, ketones, vinyl chloride, ethylene bromide (EBM), and dichloro-diphenyl-trichloroethane (DDT), are known carcinogens. All ionizing radiation can induce cancer in most tissues at all ages, so it has been categorized as a universal carcinogen. The magnitude of the contact with a chemical and radiation carcinogen, including the exposure time and the concentration, determine the potential risk of mutagenesis. Malignant cells grow without being controlled, because of a number of seriously mutated genes. In general, cancer patients have their oncogenes over-expressed and tumor suppression genes under-expressed. Both oncogenes and tumor suppression

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Table 1: Viruses associated with human cancers Virus

Associated tumors

DNA virus Epsein-Barr

Burkitt’s lymphoma, Nasopharyngeal cancer

Hepatitis B

Liver cancer

Papilloma virus

Benign warts, cervical cancer.

RNA virus Human immunodeficiency virus (HIV01)1

Kaposi’s sarcoma

Human T-cell leukemia virus Type 1 (HTVL-1)

Adult T-cell leukemia

HTLV-2

Hairy cell leukemia

HTLV-5

Cutaneous T-cell leukemia

This table was derived from Macdonald, Ford, and Casson. (2004).

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genes obtain their abnormality by mutation. In addition, one mutation in one gene usually is not sufficient to induce a cancer. Multiple mutations in several key genes have been shown to occur during tumorigenesis. Proto-oncogenes, under normal conditions, control cell growth, division, and signal transmission. Tumor suppression genes monitor cell behavior and signal apoptosis if the function of the cell is abnormal and not repairable. Even though they have different functions, the cooperation of oncogenes and tumor suppression genes keeps cells and groups of cells working properly. For tumorigenesis, a cell needs to have both oncogenes and tumor suppression genes dysfunctional in order to achieve uncontrolled mitoses. Therefore, at least two genetic changes are necessary for a cell to obtain tumorigenic competence (Hahn et al., 1999). Renan (1993) proposed that many types of cancer are age-dependent because four to seven random genetic alterations had accumulated during tumorigenesis, which normally requires time and replication cycles. An oncogene is a defective gene that can promote malignancy, and the normal homologues of the oncogenes are denoted proto-oncogenes. An oncogene that is carried by a virus is called a viral oncogene, and has its origin in the human genome, in a cellular oncogene. The products of proto-oncogenes play an important role in regulating cell growth, differentiation, signal transduction, and mitosis. When a proto-oncogene is mutated or over-expressed, these modifications turn the

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proto-oncogene into an oncogene. Thus, oncogenes which increase the chance of cellular malignancy play an important role in the early stages of tumorgenesis. The structure, activity, and concentration of the protein encoded by the oncogene may be altered. The modified protein structure may increase protein stability, which extends its influence on the cell, leading to misregulation of cell behavior. Some lose stability, some are expressed constitutively. More than one hundred oncogenes have been identified since the first oncogene was found through the study of retroviruses (Varmus, 1988). Oncogenes can be categorized into growth factors and protein kinases, according to their functions. The most common oncogenes are listed in Table 2 (MacDonal, Ford, and Casson, 2004). There is another group of genes which have opposite function to oncogenes are called tumor suppression genes (Table 3). The first human tumor suppressor gene, RB, was known by Friend et al. (1986). Currently, more than twenty tumor suppression genes are known (Fearon et al., 1997). Restriction fragment length polymorphism (RFLP) analysis has shown that various types of human cancers are characterized by a loss of heterozygosity (LOH) at multiple chromosomal loci with high frequency. LOH is one of the results of gene inactivation; therefore the gene that is inactivated by genetic alterations in cancer cells is denoted as tumor supressor gene. The mechanisms of gene alteration are not clear, but people have proposed that alterations

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in tumor supressor genes include point mutations, intragenic insertions/deletions, homozygous whole-gene deletions (Yokota, 2000), and methylational inactivation (Herman et al., 1995). During cell malignancy, tumor supressor genes are inactivated, while oncogenes are activated. Tumor supressor genes, like oncogenes, control cell growth and division. The former have effects opposite to those of oncogenes and prevent cells from progressing toward malignancy. Functional tumor supressor genes do not allow a cell with damaged DNA to divide. They either repair damaged DNA or trigger cell apoptosis when the DNA damage in the cells is irreversible. Tumor supressor genes inhibit cell proliferation by repressing the genes that promote cell division. Alfred Knudson (1971) proposed a hypothesis that a cancer cell has been through at least two mutational events, including LOH, by studying the relation between a tumor supressor gene and retinoblastoma. According to Knudson, children born with both defective copies of a gene contracted the disease before they were born, but the children who developed retinoblastoma at a later age were born with one defective copy of gene and one good copy, which subsequently mutated. Knudson’s hypothesis was confirmed (Friend et al., 1986), and the gene responsible for retinoblastoma is denoted as the RB tumor suppressor gene.

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Table 2. Oncogenes and their functions.

♦

This table was derived from Macdonald, Ford, and Casson (2004)..

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Table 3. Tumor suppressor genes and their functions.

♦

This table was derived from Macdonald, Ford, and Casson (2004).

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The Development of Cancer Normal cells respond to the signals that control the actions of cells. Cells that grow and divide indiscriminately are considered tumor cells. Tumors can be divided into two sub-types: benign and malignant tumors. The benign tumors are encapsulated by a fibrous capsule which limits their opportunity to invade surrounding tissues or to move to other areas, and the functions of benign tumor cells closely resemble those of the normal cells surrounding them. Therefore, benign tumors stay localized and remain the same size. The fibrous capsule makes them easy to distinguish from the surrounding tissue and easy to remove surgically. Malignant tumor cells grow and divide more rapidly than normal cells and are less differentiated than normal cells. Most malignant tumors do not remain in the places of their original appearances. They invade the surrounding normal tissue, spread out through circulatory or lymphatic system, and establish cell colonies in other areas of the body. This process is called metastasis. The malignant tumors, which have the ability of uncontrolled proliferation and metastasis, are considered to be cancer and are resistant to localized treatment. From the cellular biology point of view, it takes six mutational events during progression towards malignancy (Hanahan and Weinberg, 2000).These six events cells needed include: (1) secrete sufficient growth signals by themselves; (2) ignore anti-growth signals; (3) have limitless replicative potential; (4) evade apoptosis (5)

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tissue invasion and metastasis; (6) sustained angiogenesis is needed for increasing the size of the tumor and transferring to a secondary location. Mitogenic growth signals from the surrounding environment of the cells are the main factors that make normal cells enter a proliferative state from a quiescent state. Malignant cells have the ability of acquiring autocrine growth signal stimulation, and maximize the growth signals. To proliferate indefinitely, malignant cells need to have oncogenes that either can produce proteins that mimic the function of normal growth factors or overexpress their growth factor receptors to become hyperresponsive to growth signals. There are three types of signals: (1) diffusible growth factors, (2) extracellular matrix components, and (3) cell-to-cell adhesion/interaction molecules. All need transmembrane receptors to transmit the signal into the cells. Fedi et al. (1997) reported that glioblastomas and sarcomas obtain growth signal autonomy by producing platelet-derived growth factor and tumor growth factor α. On the other hand, malignant cells in the stomach, brain, and breast tumors have an upregulated epidermal growth factor receptor (EGF-R/erbB), while stomach and mammary carcinomas have overexpressed HER2/neu receptors (Fedi et al., 1997). Integrin is a plasma membrane protein that attaches the cell to the extracellular matrix and is able to transmit signals from the extracellular matrix to the intracellular end of the integrin through protein kinases. The signals that are transmitted via integrin provide the

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surrounding information to the cell, and cooperate with growth factors to control the cell’s behavior. Therefore, based on these signals, cells grow, divide, differentiate, remain quiescent or die. One of the most well studied mechanisms for obtaining growth signal autonomy is the SOS-Ras-Raf-MAP kinases pathway, which is triggered by ligand-activated growth factor receptors and integrins (Gianocotti and Ruoslahti, 1999). For many malignant cells, which maintain heterotypic signaling, they obtain growth signals that are produced by a different type of cell present in the same tumor (Skobe and Fusening, 1998). In addition to cooperating with surrounding normal cells, some tumors can induce the normal cells to malignancy and promote their transformation into cancer cells (Olumi et al., 1999 and Coussens et al., 1999). Acting in an opposite manner to growth signals, antiproliferative signals keep cells quiescent and maintain tissue homeostasis. Cells proliferate, remain quiescent, or enter a postmitotic state, based on the signals provided from the environment. Antiproliferative signals block cell proliferation, thus the cells either enter a quiescence phase (G0) or a postmitotic phase with specific differentiation. The antiproliferative signals are transmitted by soluble growth inhibitors and immobilized inhibitors embedded in the extracellular matrix or on the surfaces of surrounding cells. These inhibitors are carried to the intracellular space by a transmembrane protein, and one of the most well studied membrane protein is retinoblastoma protein (pRb).

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Retinoblastoma protein is the funnel for tumor growth factor β and can alter the function of E2F transcription factor that regulates the genes encoding proteins involved in DNA replication and it is essential for cells moving from the G1 to the S phase (Fynan and Reiss, 1993; Weinberg, 1995). Markowitz et al. (1995) discovered that in colon cancer, the insensitivity of antigrowth signals can be achieved due to a mutated or dysfunctional TGF-β receptor. Tumor cells are less differentiated than their surrounding normal cells. Normal cells need to have a Mad-Max transcription factor complex to express differentiation signals. The overexpression of oncogene c-myc will associate with Max and form Myc-Max complexes. This complex inhibits cell differentiation and promotes cell growth (Foley and Eisenman, 1999). Kerr et al. (1972) reported massive hormone-dependent tumor cell death due to hormone withdrawal. He concluded that apoptosis is one of the most essential barriers towards carcinogenesis. The malignant cell population is determined by the rate of cell proliferation and the rate of cell apoptosis. Therefore, the malignant cells need to develop a strategy to resist apoptosis. Sensors and effectors are two components in the apoptosis mechanism. Sensors can monitor the signals from both the extracellular and intracellular environments of a cell to decide whether the cell should live or die. The signals that extracellular sensors can detect are mainly from cell-cell adherence and cell-matrix interactions. Apoptosis will be triggered when the aberrant signals are

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detected (Ishizaki et al., 1995). Another manner to provoke cell death is when intracellular sensors recognize the loss of the integrity and normal functions of the cell, such as DNA damage and signaling abnormalities (Evan and Littlewood, 1998). There is a mechanism, which is not controlled by the cell-cell communication described above, that exists in all types of mammalian cells with a limited number of replication cycles (Hayflick, 1997). Telomeres can protect chromosomes from damage or fusing into rings by having a six basepair sequence that is repeated at the end of chromosome. There is a loss of 50-100 bp telomeric DNA at the 3’ end during every replication, so the telomere eventually will become shorter until it is gone. Without the protection of telomeres, chromosomes are more accessible to damage that will result in cell death. Malignant cells maintain the length of their telomere by upregulating the expression of the telomerase enzyme. Bryan and Cech (1999) proposed that in 80%-90% of malignant cells, hexanucleotide repeats were added at the end of chromosomes by the telomerase. This mechanism is inhibited forcefully in normal human cells to prevent unlimited duplication. However, every 1 in 107 cells acquire unlimited replication ability through this mechanism (Wright et al., 1989). The details of how telomerase makes a cell immortalize still needs further study. The dysfunctional ras, pRb and p53 genes were found to play important roles in malignant cells (Serrano et al., 1997; Hanahan and Weinberg, 2000).

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The supply of oxygen and nutrients and release of wastes are crucial for the growth and survival of tissues, and these molecules are delivered or sent away via the circulatory system. Thus, the growth of new blood vessels (angiogenesis) is essential for an increase in the tissue size. However, most mature cells do not have the ability to encourage angiogenesis, which limits tissue expansion. Tumors must develop strategies to gain the ability of angiogenesis during carcinogenesis (Hanahan and Folkman, 1996). The switch between positive and negative angiogenesis signals regulates the growth of blood vessels. Veikkola and Altitalo (1999) reported that one pathway to angiogenesis was triggered by vascular endothelial growth factor (VEGF) and fibroblast growth factors (FGF1 and 2). These factors are positive soluble inducers that bind to the transmembrane tyrosine kinase receptors on the endothelial cells. Integrin, cell adhesion molecules, also induce angiogenesis. Giancotti and Ruoslahti (1999) reported that different classes of integrins are expressed in different cell phases. The complex formed by extracellular proteases and pro-angiogenic integrins enhances the angiogenesis ability of endothelial cells (Stetler-Stevenson, 1999). Folkman (1997) reported that the growth of tumor cells was suppressed in mice inoculated by an antiangiogenic substance subcutaneously. Thrombospondin-1, an angiogenesis inhibitor, binds to a CD36 transmembrane receptor on endothelial cells activating Src-like tyrosine kinases, thus inhibiting angiogenesis (Bull et al.,

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1994). Dameron et al. (1994) stated that the level of thrombospondin-1 falls due to a loss of function of p53. As a result, the endothelial cells were not influenced by inhibitory signals. Malignant tumors activate angiogenesis by increasing the expression of angiogenic inducers and suppressing the inhibitors (Volpert et al., 1997). Gately et al. (1997) reported that various proteases can cleave the fibroblast growth factor, and turn it from an angiogenesis inducer to an inhibitor. The balance between angiogenesis inducers and inhibitors, and the regulation by proteolysis determines how angiogenesis occurs. Angiogenesis is a necessary step in the process of tumor enlargement. Ninety percent of human cancer deaths are caused by cancer invasion and metastasis (Sporn, 1996). Malignant tumors eventually invade the surrounding tissues and settle other colonies during their metastatic states. While malignant cells colonize new areas of the body, they need to develop new surviving strategies because of the change in their environment. Although invasion and metastasis are very complicated steps and the detailed mechanisms are not fully understood, cell adhesion molecules and extracellular proteases play an important part in the process. Christotori and Semb (1999) found that the expression of E-cadherin weakened the ability of cancer invasion and metastasis in a transgenic mouse. E-cadherin, a cell-to-cell interaction molecule that is expressed on epithelial cells, induces antigrowth signals connected to

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intracellular signaling circuits by interacting with β-cadherin. The E-cadherin loses its function in many carcinomas due to gene mutation, transcriptional repression, or proteolysis cleavage (Christotori and Semb, 1999). Integrin, which links the cell to the extracellular matrix, also has the ability to modulate invasion and metastasis. Verner and Cheresh (1996) stated that expression of integrin in cultured cells changes cell behavior during invasion and metastasis. In carcinoma cells, the expression of integrins was changed from integrins favoring cell binding to the extracellular matrix, to other integrins that prefer binding to stromal components produced by extracellular proteases (Lukashev and Werb, 1998). The proteases can facilitate malignant cell invasion into nearby stroma, across blood vessel walls and through normal epithelial cell layers (Hanahan and Weinber, 2000). The upregulated protease genes with downregulated protease inhibitor genes generate active proteases that are able to degrade the extracellular matrix (Coussens and Werb, 1996). Many matrix-degrading proteases are produced by stromal and inflammatory cells, and can be utilized by carcinomas (Werb, 1997). The proteases have transmembrane domains that can bind to protease receptors or integrins on the cell surface (Werb, 1997). The cells hooked with matrix-degraded proteases acquire the ability of invasion and metastasis. Types of Cancer Genetic alterations resulting in an unregulated proliferation of cells is typical of

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all cancers. Any of the different kinds of cells containing damaged DNA have the potential to become cancer cells. Customarily, the cancers are named by tumor location and the type of cell involved, such as breast carcinoma. Most tumors can be categorized into three groups: carcinomas, sarcomas, and leukemias; according to the type of cell from which they arise. Approximately ninety percent of human cancers are carcinomas. These malignant tumors derive from the endoderm or ectoderm, which will eventually differentiate to epithelial cells. Carcinomas can be sub-categorized into adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell undifferentiated carcinoma, according to their histological appearance. Adenocarcinoma originates from the epithelial cells of glandular tissues. It is not necessary for malignant cells to be located in any part of a gland. As long as they have secretory ability, they are classified as adenocarcinoma. Thirty to forty percent of lung cancers are adenocarcinomas. Squamous cell carcinoma is found mostly in skin, lung, esophagus, and cervix cancers and cause 90% of all head and neck cancers. Squamous cells are usually developed from epidermal and mucous membranes. Red, scaly skin that becomes open sores is characteristic of squamous carcinoma. Smoking is the main factor of causing small cell carcinoma, which usually is associated with lung cancer and cervical cancer. The characteristics of small cell carcinoma include ectopic

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production of the hormones antidiuretic hormone (ADH), and adrenocorticotropic hormone (ACTH) and early metastasis. Large cell carcinomas are aggressive and represent 10-20% of bronchogenic tumors. Large cell carcinoma does not show any squamous or glandular maturation. Therefore, it has no diagnostic features and is diagnosed by excluding other possible carcinomas. Sarcoma is a cancer of cells derived from the mesoderm, which differentiate into connective or supportive tissues. They are divided into two categories: bone sarcomas and soft tissues sarcomas. Of all cancers, 0.2% are sarcomas, and they can easily metastasize. According to the location of origin, bone sarcoma can be sub-grouped into three types. Osteosarcoma is a malignant tumor originating at the ends of long bones, where new bone tissue forms. This is one of the most common childhood cancers. Ewing’s sarcoma is a tumor developed from the middle of large bones, such as thigh, upper arms, ribs, and pelvis. Chondrosarcoma is a tumor of the cartilage between joints. Soft tissue sarcoma can be found in fat, muscles, nerves, fibrous tissues surrounding joints, blood vessel, or deep skin tissues, but only accounts for one percent of all sarcomas and the most common sites of origin are in the extremities. There are approximately seventy varieties of soft tissue sarcomas in humans. Leukemia, a hematological neoplasm, is one class of sarcoma. The abnormally proliferating cells can be either white or red blood cells, bone marrow or blood

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platelets, but mostly white blood cells. Acute leukemia is characterized by immature blood cells that cannot perform their normal functions. The blood cells in chronic leukemia are relatively mature but still abnormal. When the abnormal cells affected are lymphoid cells, it is called lymphocytic leukemia. Myelogenous leukemia is when myeloid cells are involved. The most common form of this hematological neoplasm is acute lymphocytic leukemia, which is a very common cancer in young children.

Summary Cancer is a genetic disease, which is caused by mutations. Gene alterations can be induced by chemical carginogens, radiation, viruses, and cell mutations. The abnormal genes found in tumorigenesis can be divided into two groups: oncogenes and tumor-supressor genes. During development to tumor malignancy, cells need to obtain six changes: autocrine growth signaling, anti-growth signal insensitivity, unlimited replication, apoptosis evasion, tissue invasion and metastasis, and angiogenesis. Cancers are named according to tumor location and type of cell involved. The research techniques used to conduct cancer research are mainly targeted on gene coding DNA, and gene products (RNA or proteins). With the cooperation of the traditional molecular methods and advanced microarray techniques, cancer profiling now can add a further layer of understanding tumorigenesis mechanisms in a productive way.

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CHAPTER 2

DNA DAMAGE CAUSED BY FIXATIVES

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INTRODUCTION

Many genetic diseases can be diagnosed by screening genes and gene products before the symptoms appear. Advanced molecular techniques, based on information about macromolecules, can provide information relating to the disease status of all organisms. Generally, the macromolecules that are used are DNA, RNA, and proteins. According to the expression level of these macromolecules, disease conditions can be determined accurately and rapidly (Florell et al., 2001). Polymerase chain reaction (PCR), DNA sequencing, and DNA microarrays are standard DNA analysis methods widely used in research laboratories, but not in hospital laboratories. Some researchers and clinicians have been trying to integrate these molecular profiling techniques into daily diagnosis protocols to facilitate disease tracking. In addition to screening the DNA sequences directly, gene products, RNA and proteins, can be used to track the disease stages. However, the relationships between these gene products and disease are more complex, such that further basic studies are necessary before applying the knowledge to medical diagnosis. Traditionally, for many diseases, especially cancer, diagnosis and determination of the disease stage are performed by tissue morphology analysis. Tissues need to be fixed before analysis, in order to maintain their form and

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ultrastructure so that they can be examined and/or archived. Huge collections of fixed and preserved tissues of many rare or unknown diseases have accumulated. Shibata et al. (1988) extracted human DNA from tissues fixed 40 years previously in 10% buffered formalin and embedded in paraffin. Even though the DNA extracted was highly degraded, people started to be interested in studying archived tissues using molecular methods. Previously, maintaining the tissue structure was the main purpose of specimen fixation. Currently, preservation of fragile macromolecules that can be used for molecular analysis is also an important consideration in specimen fixation. Improper preservation will result in degraded or denatured macromolecules, and the results, which are based on these defective molecules, may be misleading. Therefore, investigations of the effects of fixatives on macromolecules and optimization of fixation conditions should increase the efficiency and accuracy of molecular profiling. This thesis research has focused on the DNA damage caused by fixatives. Normal white blood cells served as control tissue for three malignant tissue types: carcinomas, leukemias, and sarcomas. Four common fixatives were tested: (1) Neutral buffered 10% formalin (NBF); (2) glutaraldehyde (1%); (3) Carnoy’s (60% ethanol, 30% chloroform, and 10% acetic acid); and (4) methacarn (60% methanol, 30% chloroform, and 10% acetic acid) Formalin (3.7~4% formaldehyde) and glutaraldehyde are aldehyde fixatives

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because their aldehyde functional groups play an important role in the fixation process. Formaldehyde has only one carbon with a single reactive aldehyde, but there are five carbons and di-aldehyde groups at the each end of the molecule for glutaraldehyde. When formaldehyde molecules dissolve in water, they become methylene hydrate (methylene glycol), and may continue to polymerize forming polyoxymethylene glycol. The methylene hydrate from formaldehyde adds a hydroxymethyl (methylol) group to the amino and imino groups of DNA bases. The methylol group addition can be exocyclic or endocyclic (Auerbach et al., 1977, and Feldman, 1973). Other free hydroxymethyl groups can form methylene bridges between DNA bases through electrophilic attack (Chaw et al., 1980). Aldehyde fixatives cause purine/pyrimidine detachments (called apurine/apyrimidine [AP] sites) from the DNA and hydrolysis reactions occur at the N-glycosidic bond of the ribose in nucleic acids, yielding 2-deoxy-D-ribose (Loeb and Preston, 1986). In addition to the AP sites, the hydrolysis reaction also modifies the phosphodiester, which severs the backbone of DNA, eventually breaking it into small fragments. Carnoy’s and methacarn are fixatives made from several organic components. Carnoy’s fixative was developed by J. B Carnoy (Backer, 1958). The components of Carnoy’s are ethanol (60%), chloroform (30%) and acetic acid (10%). Methacarn was first introduced by Puchtler et al. (1970), and shared the same formula but used

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methanol instead of ethanol. The ingredients in the alcohol-chloroform-acetic acid fixatives each serve a particular purpose in fixation. Ethanol (CH3CH2OH) is a coagulating fixative by itself. It denatures the non-water-soluble proteins at room temperature and above, but precipitates most of proteins at -5℃ without denaturing them. The drawbacks of ethanol fixation include tissue shrinkage, hardening, and slow tissue penetration. Therefore, other organic solutions are used to balance the side effects. According to Puchtler et al. (1970), methanol has a higher shrinkage temperature than ethanol, thus methanol fixed tissues are less likely to shrink than those that are ethanol fixed. Acetic acid can be used to counteract the shrinkage effects caused by alcohol fixation. Acetic acid makes tissues swell because it can break the cross-linkages between protein molecules and release lyophilic radicals that associate with water molecules (Presnell and Schreibman, 1997). The acetic acid in the fixative mixture is used to preserve chromosomes by coagulating nucleic acids. It penetrates in tissues to a 1.2 mm depth in one hour at 4℃ (Leong, 1996), which is faster than most fixatives. Chloroform is highly hydrophobic, and can penetrate and dehydrate tissues in a short period of time. Therefore, chloroform is used to accelerate the fixation process. Carcinomas, sarcomas, leukemias and normal white blood cells were used as specimens. Each was separately treated with each fixative to study the effects of the

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fixative on DNA. The DNA samples extracted from the fixed tissues were subjected to PCR amplification of parts of their small subunit (SSU) ribosomal DNA (rDNA) gene. The DNA changes caused by fixatives were compared by multiple sequence alignments. Statistical analyses were applied to determine the extent of DNA damage caused by fixatives in these four kinds of human tissues.

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MATERIALS AND METHODS

Specimens Human cancerous tissues used in this study were obtained from The Cooperative Human Tissue Network (CHTN: http://www-chtn.ims.nci.nih.gov). Three different kinds of malignant tissues (carcinomas, leukemias, and sarcomas) were used as experimental material and normal human white blood cells were used as a control in this project. All of the tissues were frozen in a -80℃ freezer before being subjected to any further treatment. Solid tissues were resized to 5mm x 5mm x 2mm by cutting with a sterile razor blade on a pre-frozen metal block. White blood cells were obtained by spinning down 1 ml of normal blood in 1.5 ml microcentrifuge tubes at 10,000 rpm for 3 min. After discarding the blood serum, the red blood cell pellet and blood serum remaining in the tubes were washed by adding 1 ml of pre-chilled phosphate buffered saline (PBS, 0.02 M sodium phosphate buffer with 0.15 M sodium chloride, pH adjusted to 7.4) into the microcentrifuge tubes and centrifuged at the same speed. The washing process was repeated two additional times to make sure only white blood cells were left in the tubes. The cells were kept on ice during the washing process, in order to minimize DNA degradation.

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Tissue Preservation Tissues were fixed in the following fixatives: neutral buffered 10% formalin (NBF), 1% glutaraldehyde, Carnoy’s, and Methacarn. NBF contained 3.7% w/v formaldehyde dissolving in PBS, and 1% glutaraldehyde fixative contained 1% glutaraldehyde in PBS. The pH of NBF and 1% glutaraldehyde were adjusted to 7.2 to 7.4 using 100 mM NaOH. Carnoy’s fixative was made by mixing absolute ethanol, chloroform, and glacial acetic acid in a 6:3:1 ratio. Methacarn was made by the same protocol of making Carnoy’s but using methanol instead of ethanol. The pH of Carnoy’s and methacarn was between 2.2 to 2.4. The fixatives were freshly made prior to the fixation treatment, and were kept in 250 ml glass bottles with caps tightly sealed. The fixatives were cooled to 4℃ in a refrigerator for 1 hour before being used. For solid tissue fixation, two pieces of tissue of the same origin were fixed in 50 ml of the cooled fixatives. Fixation was for 12 hours at 4℃ in the dark for the aldehyde fixatives (10% NBF, and 1% glutaraldehyde) and 6 hours at 4℃ in the dark for the alcohol-chloroform-acetic acid fixatives (Carnoy’s and methacarn). The tissues fixed in aldehyde fixatives were washed with an excess of distilled water for one hour twice and then soaked in distilled water for 12 hours. Carnoy’s fixed tissues were washed twice in absolute ethanol, and methacarn fixed tissues were washed twice in

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methanol for one hour, then left in the alcohol for 6 hours. All processes were carried out at 4℃. For the fixation of white blood cell pellets, 1 ml of fixative was added into the 1.5 ml microcentrifuge tubes, which contained the pelleted cells. The fixation time for aldehyde-based fixatives was 10 min, and that of alcohol-based fixatives was 5 min. During the fixation process, the microcentrifuge tubes were kept at 4℃. Following fixation, the cells were centrifuged at 10,000 rpm for 3 min and the fixatives were discarded. For aldehyde-based fixed cells, 1 ml distilled water was added into the microcentrifuge tubes to wash the remaining fixative out of the tube. For Carnoy’s fixed cells, 1 ml of absolute ethanol was added, and 1 ml of methanol was added into the tubes that contained methacarn-fixed cells. The tubes were centrifuged at 10,000 rpm for 1 min. Then, the washing was repeated for each.

DNA Extraction Fixed tissues were incubated with an equal volume of proteinase K (200 ug/ml) for 10 hours at 55℃ followed by 10 min of protein denaturation at 99℃ in a Thermal Controller (MJ Research, Watertown, MA). The samples were kept at 4℃ prior to DNA extraction. White blood cell pellets, both non-fixed and fixed, were treated with proteinase K (as above), but the DNA was not extracted. DNA was

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extracted from fresh and fixed tissues using a modified CTAB (hexadecyltrimethylammonium bromide) method (Rogers and Bendich, 1985).

PCR Amplification The SSU rDNA was amplified by polymerase chain reaction (PCR) using 2 sets of primers. For the DNA extracted from solid tissue, primers human 18S1F (5’-GGTTGATCCTGCCAGTAGCATAT-3’) and human 18S3R (5’-TGACGGGGAATCAGGGTT-3’), were used to amplify a 418 bp fragment of DNA. For the white blood cell DNA, primers18S1F (5’-GGTTGATCCTGCCAGTAGCATAT-3’) and human 18S4R (5’-TTTAACGAGGATCCATTGGAGGG-3’) were used to amplify a 597 bp fragment. PCR was performed using a Takara Ex TaqTM Hot Start Version kit (Takara Bio Inc.). The volume of each reaction was 25 μl, which included less than 10 ng of genomic DNA, 25 pmoles of each primer, 5 pmoles of each dNTP, 1 Unit Taq DNA polymerase, 10 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 0.5% Tween® 20, 0.5% Nonidet P-40 ® and sterilized distilled water. The PCR reaction was overlaid with 50 μl light mineral oil (Sigma, St. Louis, MO). The thermal cycling program was 1 min. at 95℃ for template denaturation, followed by 40 cycles of 95 ℃ (1 min.), 55℃ (1 min.), and 72℃ (2 min.) using a PTC-100 Thermal Controller

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from MJ Research (Watertown, MA). After the cycles were completed, the samples were incubated for 10 min at 72℃. The quantity of the PCR products were determined by UV fluorescence following gel electrophoresis at 5 v/cm for 90 min. on 1.0% agarose gels in TBE (89 mM Tris-base, 90 mM boric acid, 2 mM EDTA, pH8.0) containing 0.5 ug/ml ethidium bromide. Then, the PCR products were separated on 1.0% low-melting point agarose gels (NuSieve GTG, FMC, Rockland, ME) in TBE containing 0.5 ug/ml ethidium bromide, at 5 V/cm for 120 min. The DNA bands were visualized by UV, and were cut with sterile razor blades and purified using a QIAquick Gel extraction kit (QIAGEN Inc. Valencia, CA), as directed by the manufacturer. The final concentrations of PCR products were 20~30 ng/μl after purification.

TOPO TA Cloning and DNA Sequencing PCR products were cloned using a TOPO TA Cloning® kit (Invitrogen, Catalog no. K4500-01) as directed by the manufacturer. Ten recombinant (white) colonies were selected and added into 1.5 ml microcentrifuge tubes containing 20 μl 0.1X TE buffer (1mM Tris [pH 8.0] and 0.1mM EDTA [pH 8.0]). The DNA of each colony was amplified by PCR using an M13 forward primer (GTAAAACGACGGCCAG) and M13 reverse primer (CAGGAAACAGCTATGAC). The DNA quantity and size were

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determined by UV fluorescence on 1.0% agarose gels in TBE containing 0.5 ug/ml ethidium bromide, following gel electrophoresis at 5 V/cm for 90 min. The PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN Inc. Valencia, CA) according to the directions provided by the manufacturer. For sequencing, the final concentration of the PCR products was adjusted to 30~40 ng/μl for blood samples, and 20~25 ng/μl for solid tissues. The purified PCR products were sequenced both in our lab and at GeneGateway, LLC (Hayward, CA). For the purified PCR products that were sequenced in our lab, the 20 μl of sequencing reaction components consisted of 8.0 μl of ABI PRISM® BigDye™ Terminator v3.0 Ready Reaction Cycle Sequencing reagent (the dye terminators [ddNTP], dNTP, Ampli Taq DNA polymerase, FS, magnesium chloride, and buffer) (Applied Biosystems, Columbia, MD), 60-100 ng of purified PCR product, 5 pmoles of primer (M13 forward or M13 backward), in a total volume of 20μl. The cycle sequencing program was: 94℃ for 1 min, followed by 30 cycles each of 94℃ for 10 s, 50℃ for 30 s, and 60℃ for 4 min. The products were purified by ethanol precipitation: Ethanol was added into the microcentrifuge tubes that contained the cycle sequencing products to a final concentration of 60%~65%, and the tubes were incubated at room temperature for 30 min. Then the tubes were centrifuge at 13,200 rpm for 30 min and the supernatants were removed. Ethanol (80%) was added into the

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microcentrifuge tube and centrifuged at 13,200 rpm for 10 min. The supernatants were removed and dried in vacuum centrifuge for 1 hour. Template suppression reagent (12.5 μl) was added to the cycle sequencing products that had been dried, and the products were heated at 95℃ for 3 min. Then, the products were placed on ice immediately for 2 min before being transfered into 200 ul ABI sequencing tubes. The tubes were placed in the ABI PRISM® 310 Genetic Analyzer (Applied Biosystems, Columbia, MD) and the sequences were determined.

DNA Sequence Analysis Alignments were performed using Clustal X 1.8 (Thompson et al 1997; .Jeanmougin et al., 1998; Chenna et al., 2003). After sequence alignment, the gaps were adjusted within each DNA sequence in order to complete the sequence alignments. DNA sequences were adjusted manually by careful examination of the chromatographs. The number of base pair changes and types of change were recorded. The DNA damage rates were obtained by dividing the total number of changed bases in each sequence by the total number of bases in each sequence. The decision-making process for whether the DNA sequences were significantly damaged by fixative treatment was based on the probability value (p-value) in statistical analyses. Both T-test and ANOVA analyses were performed using MINITAB 14.1 statistical software

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(Minitab Inc., State College PA). The percentage of DNA damage after each fixative treatment was compared with the DNA damage rates of unfixed tissues by performing T-test analysis. Boxplots, which indicated the DNA damage rate variance and outliers were generated with p-values during T-test analysis.. The DNA damage rates of tissues that were treated by four kinds of fixative and unfixed tissues were compared together by ANOVA analysis. The comparisons were performed according to fixatives, thus one-way ANOVA with Tukey’s method was applied. For the statistical analyses purpose, the standard deviations and p-values, were generated during ANOVA analysis of variance based on the damage rates. For graphical analysis, residuals versus the fitted values graphs, which displayed nonconstant variance and the pattern of spreading residuals across fitted values.

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RESULTS

Each fixation treatment was repeated 6 times, (12 pieces of tissue in total), for each kind of tissue, and two attempts to amplify rDNA from each DNA sample were made. Therefore, a total of 240 fixations (Table 4) and 490 PCR amplifications were performed in this study. For these fixations and amplifications, the DNA from the fresh non-fixed tissue was the most easily extracted and amplified by PCR. The percentages of attempts to successfully generate PCR amplifications are: 73% (35/48) for NBF fixed tissues, 58% (34/48) for 1% glutaraldehyde fixed tissues, 40% (19/48) for Carnoy’s fixed tissues, 38% (18/48) for methacarn fixed tissue, and 98% (47/48) for non-fixed fresh tissues (Table 4). Therefore, all fixation methods resulted in lower yields of useful DNA. According to the data obtained from this study, more than half of the tissues fixed by aldehyde fixatives yielded DNA that could be amplified, only about one-third of tissues fixed by alcohol-chloroform-acetic acid fixatives yielded DNA that could easily be amplified. Therefore, the DNA of the tissues treated by aldehyde fixatives was either more accessible than that from alcohol-chloroform-acetic acid fixed tissues. Within the 480 PCR amplification attempts, the successful PCR percentages are: 45% for 10% NBF fixed tissues, 49% for 1% glutaraldehyde fixed tissues, 65% for

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Carnoy’s fixed tissues, 60% for methacarn fixed tissues, and 94% for non-fixed fresh tissues. It was shown that it is more difficult to amplify DNA from the fixed tissues than from those untreated. In addition, the PCR products generated from the tissues fixed by alcohol-chloroform-acetic acid were of higher molecular weight than aldehyde fixed tissues (Figures 13 and 14). Around half of the DNA from the aldehyde fixed tissues could be amplified by PCR. At the same time, more than 60% of the DNA from the alcohol-chloroform-acetic acid fixed tissues could be amplified. Therefore, the DNA can be amplified by PCR more effectively from the alcohol-chloroform-acetic acid fixed tissues than from aldehyde fixed tissues. In addition to the effect on PCR, there was a difference in the concentrations of PCR products of the tissues fixed in different fixative. The average mass of PCR product from the fixed tissues is 4ng/μl, and 20ng/μl for unfixed tissues (Figures 13 and 14). Thus, the fixation in general, reduces the efficiency of PCR. The rDNA sequences were subjected to further analysis. Two sets of primers were used to amplify fragments that were 418 bp and 597 bp. The 418 bp fragment contains 19.4% A, 29.43% C, 28.5% G, and 22.7% T (Table 5). The 597 bp fragment contains 22.6% A, 28.0% C, 28.1 % G, and 21.3% T (Table 5). The average DNA change rates were 3.36% for NBF fixed tissue, 1.63% for 1% glutaraldehyde fixed tissues, 1.14% for Carnoy’s fixed tissues, 1.04% for methecarn fixed tissues, and

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1.38% for non-fixed fresh tissues. The percentage of DNA damage caused by each treatment is listed in Table 6. The lowest DNA damage rate was found in carcinoma was for non-fixed tissues (0.58%). Among the four kinds of fixatives, the highest DNA damage rate was for 10% NBF fixed tissues for all kinds of the tissues: 1.92% in carcinoma, 2.82% for leukemia, 1.97% for sarcoma, and 6.73% for the non-malignant tissue (Table 6). In general, methacarn caused less DNA damage compared with the other fixatives. Methacarn-fixed sarcomas and white blood cells had the least DNA damage in each tissue category (0.58% in sarcomas, and 0.87% in non-malignant white blood cells). Carnoy’s fixative caused the second lowest DNA damage in sarcomas (0.87%) and white blood cells (1.43%), and ranked the second best for DNA preservation. Analysis of the frequency of changed DNA (Table 7) showed that about one third of DNA errors were transitions (34.50% for 10% NBF, and 33.62% for 1% glutaraldehyde), and a little more than 10% of the errors were transversions (11.24% for 10% NBF, and 10.34% for 1% glutaraldehyde). Base indels (insertions and deletions) were the most common (43.06%) changes in Carnoy’s-fixed tissues. Other changes were ambiguous bases (34.73%) and transitions (20.83%). Most DNA changes in methacarn-fixed tissues were base transitions (36.13%), ambiguous bases (27.73%), and base indels (26.89%). For the DNA errors from the non-fixed tissues,

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more than 60% of errors were composed by ambiguous bases (32.38%) or base indels (32.38%). Further base change analysis (Table 8) showed that a cytosine to non-specific base change was the most frequent change (12.2%) followed by a guanine to adenine transition (9.88%) in the 10% NBF-fixed tissues. For 1% glutaraldehyde-fixed tissues, cytosine to thymine (8.98%) changes appeared the most often, and guanine to adenine transitions (8.29%) ranked second. Cytosine to non-specific base changes (19.5%) were most frequent within the base damage followed by cytosine addition (15.3%) in Carnoy’s fixed tissues. Methacarn-fixed tissues had adenine to guanine transitions (12.7%) and guanine to adenine transitions (11.5%) appearing most often when the base damage occurred. Cytosine to non-specific base changes (12.4%) and guanine to adenine changes (10.2%) were the DNA changes frequently observed in the DNA of the non-treated tissues. These findings were based on the results of multiple sequence alignments of fixed/unfixed tissues. Since DNA is double stranded, a GÆA change on one strand would result a CÆT change on the other strand. Therefore, it was uncertain that the base changes found from this study were caused by fixatives damage or were the complementary bases generated during PCR. Several statistical analyses were used to determine the significance of the DNA changes caused by the four fixatives. To compare the base damage percentages

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between the treated and non-treated specimens, a T-test was employed. The DNA damage rate with 10% NBF-fixed tissue was the only one that yielded a significant difference, as indicated by T-test comparisons between the DNA damage percentages caused by fixatives compared to the untreated tissues. The P-value for the T-test done by comparing the 10% NBF treated tissues and non-treated frozen tissues is 0.069 (Figure 15). The DNA damage percentages generated from the tissues fixed by other fixatives were not significantly different (P-value > 0.1) than the DNA differences among non-treated tissues. Also one way ANOVA analysis of variance was used to compare DNA changes from the four fixatives and one untreated tissue. The result of this statistical analysis resulted in a P-value of 0.029, which indicated that a significant difference exists in this comparison (Figure 16). Further analysis showed that there was a significant difference between the DNA changes in methacarn-fixed tissues, which had the lowest DNA base damage overall, and those in 10% NBF-fixed tissues, which has the worst DNA base damage. To study the distribution of the DNA base damage, standard deviation (Table 9) and the graph of residuals versus the fitted values (Figure 16) was generated. The standard deviation for the 10% NBF-fixed tissues was the largest regardless of what type of tissues were treated (0.025 for carcinomas, 0.05 for leukemias, 0.01 for sarcomas, and 0.07 for white blood cells). Methacarn-fixed carcinomas and white

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blood cells had the lowest standard deviations (0.005 for carcinomas, and 0.00 for white blood cells). The smallest standard deviations for leukemias and non-treated controls were in the Carnoy’s-fixed leukemia (0.012) and non-treated control sarcomas (0.002). The graph of residuals versus the fitted values indicated that the DNA base changes from 10% NBF treated tissues were distributed in a wide range on the right side of the graph. The base damage percentages generated from the tissues fixed by other fixatives were more condensed and located in the left side of the graph.

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Table 4: Results of PCR amplification. 10% NBF

1% glutaraldehyde

Carcinoma

8/12

6/12

5/12

4/12

12/12

Leukemia

7/12

6/12

3/12

5/12

12/12

Sarcoma

9/12

5/12

5/12

4/12

11/12

Normal tissue

10/12

9/12

6/12

5/12

12/12

Total

34/48

28/48

19/48

18/48

47/48

Percentage

73%

58%

40%

38%

98%

Carnoy’s

Methacarn

None-fixed

Results are shown with two numbers separated by a slash. In all cases, 12 pieces of tissues (the number on the right) were subjected to PCR amplification testing. The numbers on the left show the number of tissues from which PCR products could be obtained.

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A

B L

1

2

3

4

5

6

400 bp (30 ng)

Figure 13: Agarose gel of PCR products from aldehyde-fixed and non-fixed tissues. The PCR products resulting from DNA amplification of aldehdye fixed tissues separated on 1% agarose gel. (A) DNA ladder (Hyperladder II from Bioline, Randolph, MA) indicating the size and mass of DNA. (B) Amplification products from fixed tissue. The DNA ladder (L) is in the left most lane. Duplicate amplifications are shown for 10% NBF fixed tissues (lanes 1 and 2), 1% glutaraldehdye fixed tissues (lanes 3 and 4), and non-fixed frozen tissue (lanes 5 and 6).

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A

B 1

2

3

4

5

6

L

600bp 30 ng

Figure 14: Agarose gel of PCR products from alcohol0chloroform-acetic acid-fixed and non-fixed tissues. The PCR products resulting from DNA amplification of alcohol-chloroform-acetic acid fixed tissues separated on 1% agarose gel. (A). Amplification products from fixed tissues. The DNA ladder (L) is in the right most lane. Duplicate amplifications are shown for Carnoy’s fixed tissues (lanes 1 and 2), methacarn fixed tissues (lanes 3 and 4), and non-fixed frozen tissue (lanes 5 and 6). (B) DNA ladder (hyperladder II from Bioline, Randolph, MA) indicating the size and mass of DNA

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Table 5: Absolute numbers and the percentages of A/C/G/T in the SSU rDNA fragments (418 bp and 597 bp) that are used in this study before fixation. base

A

C

G

T

418 bp

81/418 (19.4%)

123/418 (29.4%)

119/418 (28.5%)

95/418 (22.7)

597 bp

135/597 (22.6%)

167/597 (28.0%)

168/597 (28.1%)

127/597 (21.3%)

size

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Table 6: The percentage of differences in DNA after fixation in each type of tissue, by treatment. Tissues

Carcinoma

Leukemia

Sarcoma

White blood cells

Mean (by treatment)

10% NBF

1.92%

2.82%

1.97%

6.73%

3.36%

1% Glutaraldehyde

0.60%

2.19%

1.36%

2.36%

1.63%

Carnoy’s

0.79%

1.48%

0.87%

1.43%

1.14%

Methacarn

1.03%

1.68%

0.58%

0.87%

1.38%

Unfixed

0.58%

1.49%

1.84%

1.61%

1.38%

Mean (by tissue)

0.98%

1.93%

1.32%

2.60%

Fixatives

The differences are out of a total of 418 bp (white blood cells samples) or 597 bp (carcinomas, leukemias, and sarcomas). The first column on the left of the table represents the mean of the DNA difference percentages for each fixative. The last row at the bottom of the table represents the mean of DNA difference percentage for each type of tissues.

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Table 7: Three types of the DNA sequence differences in each specimen by treatments. The differences are out of a total of 418 bp (white blood cells sample) or 597 bp (carcinomas, leukemias, and sarcomas).

a b

Transitions: Pyrimidine bases are changed to another type of pyrimidine bases (A ÅÆ G; CÅÆT). Transversions: Pyrimidine bases are changed to purine bases or purine bases are changed to pyrimidine bases (A ÅÆ C/T; CÅÆG/A; GÅÆC/T; TÅÆG/A).

c

Indels: Can be either insertion or deletion.

d

Ambiguos bases: Base that cannot be determined by sequencer.

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Table 8: The frequencies of DNA changes in each specimen, by treatment. The changes are out of a total of 418 bp (white blood cells sample) or 597 bp (carcinomas, leukemias, and sarcomas).

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Table 8 (cont.): The frequencies of DNA changes in each specimen, by treatment. The changes are out of a total of 418 bp (white blood cells sample) or 597 bp (carcinomas, leukemias, and sarcomas).

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Table 8 (cont.): The frequencies of DNA changes in each specimen, by treatment. The changes are out of a total of 418 bp (white blood cells sample) or 597 bp (carcinomas, leukemias, and sarcomas).

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Table 8 (cont.): The frequencies of DNA changes in each specimen, by treatment. The changes are out of a total of 418 bp (white blood cells sample) or 597 bp (carcinomas, leukemias, and sarcomas).

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Table 8 (cont.): The frequencies of DNA changes in each specimen, by treatment. The changes are out of a total of 418 bp (white blood cells sample) or 597 bp (carcinomas, leukemias, and sarcomas).

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Boxplot of Treatment, Control 0.20

Data

0.15

0.10

0.05

0.00 Treatment

Control

Figure 15: A T-test comparing DNA difference rates of 10% NBF fixed tissue (treatment) and non-fixed tissues(control). The y-axis shows the DNA difference percentage. The means of DNA difference rate for treatment are shown as open circles (3.36% for the treatment and 1.40% for the control). The gray bars represent the areas where the most DNA difference rates are located. The asterisks represent the DNA difference rates that are far away from the majority.

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R e s id u a ls V e r s u s th e F itte d V a lu e s ( r e s p o n s e is R e s p o n s e )

0 .1 5

Residual

0 .1 0

0 .0 5

0 .0 0

- 0 .0 5 0 .0 1 0

0 .0 1 5

1% glutaraldehyde

Methacarn Carnoy’s

0 .0 2 0 Fit t e d V a lu e

0 .0 2 5

0 .0 3 0

10% NBF

Non-Fixed

Figure 16: One-way ANOVA analysis comparing DNA change percentages occurring in each fixative treatment. The fitted value (x-axis) is the mean of DNA change rate and the residual (y-axis) is the distance from the mean. Each dot represents the DNA change rate in each specimen by treatment. The DNA change rates are 1.04%, 1.14%, 1.38%, 1.63% and 3.36% for methacarn, Carnoy’s 1%, non-fixed, 1% glutaraldehyde, and 10% NBF fixed tissue respectively.

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Table 9: Standard deviations for DNA change percentages from each specimen type by fixative. Fixatives Tissues

10% NBF

1% glutaraldehyde

Carnoy’s

Methacarn

Unfixed

Carcinoma

2.49%

0.63%

0.53%

0.52%

0.58%

Leukemia

5.99%

1.48%

1.21%

2.17%

1.23%

Sarcoma

1.44%

1.45%

0.44%

0.47%

0.22%

White blood cells

7.62%

0.85%

0.10%

0.09%

0.11%

The tissues fixed in 10% NBF show higher standard deviations in all tissue types, which means that the DNA change rates from human tissues are more variable after being treated by 10% NBF.

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DISCUSSION From biological sciences to disease studies, archived specimens have become a valuable resource for many areas of research (Srinivasan, Sedmak, and Jewell., 2002; Vachot and Monnert, 1996) because of advanced molecular profiling techniques. The advantage of these techniques is that a small size of sample is needed for providing a huge amount of detailed data and they are readily available in abundance. Many researchers have shifted their focus to macromolecules to study the mechanisms and the fundamental principles of tissue development and gene regulation. Many diseases are due to dysfunctional macromolecules therefore, they can be diagnosed and potentially cured by manipulating the molecules. Molecular methods have been applied in cancer research because the disease not only is one of the major causes of death in the United States but also is caused by gene mutations. Archived tissues can be used for a variety of cancer research projects. Human cancer can be identified by screening for genetic markers from archived tissues (Borrebaeck, 1998; Fields, Kohara , and Lockhart, 1999). Both fixed human malignant tissues (carcinomas, leukemias, and sarcomas) and normal tissues (white blood cells) were used as the specimens in this project to study the potential of using archived tissues in DNA sequence analysis. The molecular techniques are so sensitive that the integrity of the examined molecules can be inferred from the data.

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In addition, DNA extraction and PCR amplification are fundamental to most DNA assays, and the accessibility and fidelity of the DNA is important for further analyses. Fixatives, used for preserving archived tissues, not only can be used for maintaining the tissue structures, but some protect macromolecules from degradative processes. Therefore, obtaining the optimal quality of macromolecules from fixed tissues (Vachot and Monnert, 1996; Schander and Halanych, 2003) and understanding how fixatives affect the molecular quality and interfere with the results of molecular assays is important for prospective researchers who intend to use archived tissues (Srinivasan, Sedmak, and Jewell, 2002). Recent studies have been focused on the effects of fixatives on preserving the quantity of macromolecules (Shibutani et al., 2000; Uneyama et al., 2002; Williams et al., 1999). This study used human malignant tissues treated by two aldehyde-based fixatives (10% NBF, 1% glutaraldehyde) and two alcohol-chloroform-acetic acid-based fixatives (Carnoy’s, methacarn) DNA sequencing analysis was applied and study the effects of fixatives on DNA integrity. For formalin-fixed tissues, Ortiz-Pallardo et al. (2000) and Poncin et al. (1999) stated that DNA fragments sized around 200 bp could be amplified by PCR regularly, and the largest fragment size that has been ever published was 959 bp (Akalu and Reichardt, 1999). Two sets of primers were used in this study to

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amplify the SSU rDNA. The amplicons in this case were 418 bp and 597 bp. For fixed tissues, the longer amplicon was rarely observed. This situation was especially consistent for the solid tissues. Therefore, most of the DNA extracted from fixed tissues was subjected to amplification of 418 bp fragments, to facilitate the comparison of DNA damage caused by different fixatives. In addition to the limit of DNA size that can be amplified from fixed tissues, DNA extracted from 10% NBF-fixed tissues was difficult to amplify compared to the DNA from the tissues fixed by other fixatives. This confirms previous findings which stated low efficiencies in PCR amplification (Bramwell and Burns, 1988; Burmer, et al., 1989; Douglas and Rogers, 1998; Hamazaki, et al., 1993; Shibata, et al., 1988) and mentioned the limit of the DNA length (around 450 bp) that can be amplified (Vacho and Monnerot, 1996). Burner, et al. (1989) used the c-Ki-ras-2 gene of human colon carcinomas as their target. They found that NBF reduced the accessibility of DNA, but it was still usable as the template for producing 126 bp PCR products. Vachot and Monnerot (1996) used 10% formalin-fixed amphibian tissues to amplify the 16S rRNA gene using PCR. They found that amplification of a 450 bp fragment was successful regularly, but amplification of a 600 bp product was difficult, and concluded that the DNA in the fixed tissues was degraded by the fixative. However, Dubeau et al.

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(1986), Greer et al. (1990), and Shedlock et al. (1997) concluded the opposite. Dubeau et al. (1986) obtained good quality DNA from the tissues that were fixed by formalin, but the DNA from the tissues fixed by the fixatives containing picric acid or mercuric chloride were not intact. Dubeau stated that the DNA from formalin-fixed tissues was suitable for quantitative and qualitative analysis by Southern or dot blotting analysis. Greer et al. (1990) compared the effects of fixatives on tissues and the ability of DNA from the tissues to serve as PCR templates. They found that tissues treated by acetone and 10% NBF were the most suited for performing PCR. Other fixatives that were found suitable for amplification by PCR were Zamboni's (4% paraformaldehyde, 0.3% picric acid, and 0.1 M phosphate buffer), Clarke's (3:1 of a solution of absolute ethanol: glacial acetic acid), paraformaldehyde (polymer form of formaldehyde, HO(CH2O)nH [n being 6-100]), formalin-alcohol-acetic acid, methacarn, and acetone. On the other hand, DNA from Carnoy's-, Zenker's-, or Bouin's- treated tissues were amplified with difficulty. The finding regarding formalin and mercury containing fixatives were conflicting with Douglas and Rogers (1998), and the results found in this experiment: Dubeau et al. (1986) and Geer et al. (1990) mentioned the importance of fixation times, since the DNA was less likely to act as a suitable PCR template as the fixation processing times lengthened. However, what they found about the DNA

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integrity damage caused by mercury containing fixatives were conflict with the results from Douglas and Rogers (1998), who stated that few DNA changes resulted from fixation with Zenker’s (a mercuric chloride-based fixative). Shedlock, et al. (1997) found that during the DNA extraction process from formalin-fixed samples, the step which caused major DNA loss was during phenol/chloroform protein extraction. However, this situation did not occur with alcohol-fixed samples. They concluded that the DNA from formalin-fixed specimens was associated with protein complexes, which favored recovery of low molecular weight of DNA, and proposed a proportion of phenol/chloroform that helped to improve the yield of DNA extraction. The revised phenol/chloroform extraction protocol provided by Shedlock (1997) was as follows: (1) Tissues were digested by extraction buffer (1% sodium dodecyl sulfate [SDS], 25 mM Tris-HCL, pH 7.5, 100 mM EDTA, 10 mg/ml proteinase K, and 10 mg/ml DNase-free RNase ). (2)The digestions were extracted in the equilibrated phenol for 3 times followed by 25:24:1 solution of phenol: chloroform: isoamyl alcohol, and 24:1 solution of chloroform: isoamyl alcohol. Coombs et al. (1999) and Serth et al. (2000) also confirmed that poor DNA extraction efficiency for formaldehyde-based fixatives was due to the cross-linkage of the polypeptides, which surround the genomic DNA. Yagi et al. (1996) reported that a formaldehyde solution containing DNase-neutralizing EDTA could efficiently

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inhibit DNase degradation of DNA, compared to a formaldehyde solution alone. In addition to fixation time and the DNA extraction method, Koshiba et al. (1993) and Douglas and Rogers (1998) proposed two other factors that influence the use of fixed tissues in PCR amplification. Koshiba et al. (1993) stated that the fixation temperature and DNA extraction method played important roles in extracting high molecular DNA from aldehyde fixed tissues. They found that the tissues fixed by buffered formalin at 4 ℃, and treated with cell lysing buffer (155 mM NH4Cl; 10 mM KHCO3; 0.1 mM Na2 EDTA; pH 7.4) that contained urea would yield high molecular DNA. Douglas and Rogers (1998) reported that the fixation conditions that caused less DNA damage were low temperature (4 ℃) and neutral pH, when assayed using direct sequencing of fungal specimens. In addition, Zsikla et al. (2004) extracted and analyzed the quantity of β actin DNA with real-time PCR from buffered and unbuffered formalin-fixed upper gastrointestinal biopsies. Their results also confirmed the importance of using buffered formalin. In order to obtain the maximum yield of PCR products, minimal DNA damage, and maximum utility for future clinical applications, the fixation temperature, pH, and duration must be carefully controlled during fixation. All the fixations in this project, including aldehyde-based fixatives and alcohol-chloroform-acetic acid-based fixatives were done at 4 ℃. The pH of the

87

aldehyde-based fixatives was buffered to pH 7.2, but alcohol-chloroform-acetic acid-based remained around pH 2.2 to 2.4. The fixation times that were used in this project were based on previously published research (Douglas and Rogers, 1998; Dubeau et al., 1986; Greer et al., 1990; Kieman, 1990) and were tested for DNA amplification feasability. The fixation times for aldehyde-based and alcohol-chloroform-acetic acid-based fixatives were 12 hours and 6 hours, respectively. With equal amounts of tissue for each fixative, the numbers of tissues that yielded usable DNA for PCR amplification reactions were higher after an aldehyde-based fixation than after an the alcohol-chloroform-acetic acid-based. However, the DNA accessibility for PCR and the molecular weights of DNA extracted from the aldehyde fixative-treated tissues were lower compared to the tissues treated by alcohol-chloroform-acetic acid based fixatives and to untreated frozen tissues. Even though the PCR yield was low, the DNA from the aldehyde fixed tissues still was able to serve as PCR template. Comparing the performance of aldehyde-based, and alcohol-chloroform-acetic acid-based fixation, methacarn fixation is close to the results obtained from unfixed specimens. Thus, methacarn fixation is better than that the other fixations in providing high yield DNA for PCR amplification (Uneyama et al., 2002; Takagi et al., 2004). Puchtler et al. (1970) who first proposed the formula of methacarn, found

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that methacarn fixation was superior to Carnoy’s for fibrous protein, glycogen, and erythrocytes by producing more intensive staining. Mitchell et al. (1985) stated that methacarn and Carnoy’s treated tissues had a better performance in immunohistochemical localization of tissues antigens than those treated by formalin, because the immunoreactivity of some antigens was reduced by formaldehyde and generated false negative results. Urieli-Shoval et al. (1992) stated that Carnoy’s fixed tissues contained high molecular weight RNA with slight degradation, while only a small percentage of RNA was extractable from paraformaldehdye-fixed tissues compared to the effects of other fixatives (Carnoy’s, 85% ethanol, 5% paraformaldehyde and 0.1% glutaraldehyde) for tissues to be used for in situ hybridization and immunohistochemistry. Shibutani et al. (2000) stated that the RNA and protein extracted from methacarn-fixed tissues were suitable for RT-PCR, Western blot, and immunochemistry analyses. Uneyama et al. (2000) were able to extract DNA with high yield from methacarn-fixed rat liver and amplified a 4-kb fragment by PCR. Additionally, the methacarn protocols did not require proteinase K treatments, which are required for DNA extraction of formalin-fixed tissues (Dietmaier et al., 1999; Murase et al., 2000; Hirose et al., 2001). Thus, it was easier to extract DNA from methacarn fixed tissues than from those fixed using formalin. Uneyama et al. (2002) performed PCR on methacarn-fixed immunostained

89

microdissected cerebral cortex (1mm x 1mm x 10-µm) tissues. Amplification up to 2.8 kb was obtained by nested PCR, and a fragment up to 552 bp was obtained by single-step PCR. Uneyama et al. (2002) concluded that the results were superior to the same DNA analysis performed on microdissected tissues fixed by formalin. Takagi et al. (2004) used real-time RT-PCR to quantify the expression from mRNA (cytochrome P450 2B1) from methacarn-fixed and unfixed rat liver. They found that RNA yield of methacarn-fixed specimens was equivalent to that of unfixed specimens. Unlike cross-linkage caused by formaldehyde, methacarn coagulates the nuclear proteins and as well as cytoplasmic and nuclear DNases. The proteins treated in this manner are inactivated, but still can be extracted by detergents (Shibutani et al., 2000). Therefore, the DNA preserved using methacarn is protected from degradation and is suitable for use in PCR amplification. These DNA change rates were subjected to T-tests and ANOVA to determine whether the changes were statistically significant. In the T-test, the DNA damage rates for fixed tissues were compared to those for unfixed tissues. The only significant value (p-value = 0.069) appeared when applying a T-test to the damage rates for 10% NBF fixed tissues and unfixed tissues. On the other hand, the number of DNA errors caused by other fixatives was not statistically different from untreated samples. This showed that the number of DNA errors caused by 10%

90

NBF is significantly higher than the tissues fixed by other fixatives. These results confirm a previous finding, which stated that the DNA damage was more severe in 10% NBF-fixed tissues than in glutaraldehyde-fixed tissues (Douglas and Rogers, 1998) and methacarn-fixed tissues (Uneyama et al., 2002). The results of ANOVA showed that there was a significant difference (p-value = 0.029) between methacarn-fixed tissues and 10% NBF-fixed tissues based on the DNA change rates sorted by fixative. This result corresponded to the report of Takagi et al. (2004), who proposed that the performance of methacarn fixation is superior to that of formalin in maintaining the RNA integrity. In addition, the results of this project showed that the DNA damage rates for alcohol-fixed tissues were not only relatively low compared to aldehyde fixation, but also close to that of the unfixed tissue. However, there was not a significant difference in DNA damage rates when comparing the four fixative treatments on a single type of tissue. The significant differences appeared when the analysis was based on all fixed tissues, regardless of tissues types, that have been treated by the different fixatives because the number of single tissue type samples was too limited to achieve statistical support. This means larger scale of the fixation causing DNA damage is needed before reaching a conclusion about the effects of fixatives on each type of tissue. According to the statistical analyses, the DNA damage rates generated by the

91

alcohol-chloroform-acetic acid fixatives were lower (average of 0.01) than the aldehyde fixatives (average of 0.03). The same results were confirmed by the residuals versus the fitted values graph plotted by ANOVA. The standard deviations and graphic analysis conclude that the high average DNA damage rate from 10% NBF fixation is due to a wide range of inconsistent damage rates. To analyze which DNA bases were more often damaged and what kind of damage was caused by the fixative treatment, the percentage of DNA errors for each was obtained. In order to analyze the pattern of DNA changes for the fixed tissues, the percentages of 12 types of base changes were obtained (Table E). These numbers showed that cytosine and guanine were the bases that were most frequently altered. In addition, cytosine bases were changed to undeterminable bases in many cases, and guanine bases were changed to adenine bases. One-way ANOVA analysis was used to determine whether any of the bases was significantly more frequently damaged by the fixatives. However, no significant p-value was obtained. The possible reasons of the high damage rate on cytosine and guanine might be due to: (1) the sequence of SSU rDNA contains more cytosine (29.3%) and guanine (28.45%), which resulted in increased damage rates for cytosine and guanine; (2) Hengen (1996) found that G/C-rich regions were more difficult to sequence because this region generates more stable secondary structures which prohibit the

92

denaturation, and this inhibited primer annealing and DNA extension. Therefore, higher DNA sequence alteration shown in cytosine and guanine in this experiment is not due to fixation, and all of the fixatives are not changing the DNA sequence in a base-specific manner. DNA damage caused by aldehyde-based fixatives was analyzed by Crosby et al. (1988), De Giorgi et al. (1994), Douglas and Rogers (1998), Hamazaki et al. (1993), Shedlock et al. (1997), and Williams et al. (1999), but the damage pattern caused by alcohol-chloroform-acetic acid-base fixatives has not been studied. Of those who have performed DNA damage analysis, only Hamazaki et al. (1993), who used lambda phage DNA as their samples, and Shedlock et al. (1997), using parts of the 16S rDNA (which was 570 bp) and the cytochrome b gene (which was 470 bp), found that DNA obtained from fresh tissues and formalin fixed tissues were identical. Similar to what was found in this study, Douglas and Rogers (1998) obtained the DNA damage rates from several common cytological fixatives. They found that the 10% formalin caused more DNA errors than glutaraldehyde in fixed fungal tissues when the fixations were performed under the same conditions. However, there was no single pattern of DNA change. Guanine transitions occurring in the DNA of fixed tissues were slightly higher than other type of DNA changes, this result supporting the previous findings of Crosby et al. (1988) and De Giorgi et al. (1994). Crosby et

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al. (1988) used formalin-fixed lymphoblasts and E. coli to determine the DNA change caused by fixatives. They found that most DNA changes were G to A transitions in human lymphoblasts. In addition to human samples, analysis using GPT (green fluorescence protein) gene sequences from fixed E. coli cells indicated that A to G and G to A transitions were at the highest frequencies. The same study reported that G to C, G to T, and A to C transversions occurred very often. Similar results obtained by De Giorgi et al. (1994), who analyzed DNA pattern changes caused by formalin, stated that A to G transitions occurred at high frequency in tissues fixed using 2.5% formalin. Next to transitions, several severe multiple nucleotide insertions and deletions produced ambiguous shifts and increased the difficulty of sequence alignment. Shearman and Loeb (1979) found a direct proportional relationship between misincorporation of non-complementary nucleotides and the number of AP sites in the template. Furthermore, the results of Williams et al. (1999) showed that up to one mutation artifact per 500 bp was found in formalin-fixed archival specimens, but the artifacts were not reproducible. The most common DNA mutations found in their study were C-T or G-A changes. Thus, they proposed a possible reason why formaldehyde molecules may influence the accuracy of PCR. The cytosine is more accessible to be cross-linked by formaldehyde, and the altered cytosine bases may not be recognized by Taq DNA

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polymerase. The polymerase placed adenosines instead of guanosines during the incorporation. Williams et al. (1999) stated that the damage needed to be present at the beginning of the amplification process for the PCR product that contained detectable DNA damage. From the results obtained from this project, the DNA damage rates were significantly higher in 10% NBF-fixed tissues because the rates varied even if they were fixed under the same conditions at the same time. The four interactions between formaldehyde and DNA: (1) hydroxymethyl additions; (2) methylene cross-links; (3) apurinic and apyrimidinic (AP) sites; and (4) phosphodiester bond fractures, were fully understood and described by Douglas and Rogers (1998); and Srinivasan, Sedmak, and Jewell (2002). As one of the aldehyde fixatives, glutaraldehyde has a similar effect on DNA as formaldehyde (Srinivasan, Sedmak, and Jewell, 2002), and Monsan, Puzo, and Marzarguil (1975) state that only the mid-chain aldehyde groups of glutaraldehyde polymers are involved in fixation. This makes cross-linking a characteristic of glutaraldehyde fixation. The cross-link stabilizes DNA structures and reduces the possibilities for DNA damage. Thus, the DNA damage rate is lower in glutaraldehyde-fixed tissues than in those fixed by formaldehyde. Carnoy’s and methacarn are made of organic solvents and do not modify nucleotides or polypeptides in the way that aldehyde fixatives do (Takagi et al., 2004). The alcohols of these fixatives, ethanol and methanol, dehydrate

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coagulate, and precipitate nucleic acids and they preserve high molecular weight DNA and RNA. Chloroform prevents the activity of nucleases by denaturing them. However, the low pH caused by the acetic acid from the fixative can cause DNA damage. Storing DNA in an acidic environment can cause AP sites through two spontaneous hydrolysis reactions: (1) cleavage of N-glycosydic bond of the deoxyribose and the base; (2) depurination (and to a lesser extent depyrimidiction) of deoxyribose-phosphate backbone. Ke et al., (2001) found that acetic acid can damage DNA in its form and structure: the phosphodiester backbone can be disrupted, hydrogen bonds can be broken, and the DNA bases can be stacked. Thus, similar to aldehyde fixation, the fixatives containing acetic acid cause breakage of the DNA backbone and generate AP sites. In addition, Ke et al. (2001) found that the frequency of damage of DNA bases, from high to low, affected cytosine, thymine, guanine, and adenine. In this study, we found that the base damage degree, from high to low, was 10% NBF, 1% glutaraldehyde, Carnoy’s, and methacarn. In addition, the damage rates of 1% glutaraldehyde-, Carnoy’s-, methacarn-fixed and non-fixed tissues are closer together than to 10% NBF. This result confirms the previous studies (Takagi et al., 2004; Miething et al., 2006), which sated that methacarn, Carnoy’s and glutaraldehyde are more suitable for tissue fixation than buffered formalin, which produced variable results. The alcohol-based fixatives,

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including Carnoy’s and methacarn, are suggested to be an alternative for preserving the tissues that will subsequent be used for DNA sequencing or RNA hybridation analysis (Giannella et al., 1997). In conclusion, 10% NBF fixation can generate various nonreproducible sequence alterations with high frequency. The DNA damage rates for 1% glutaraldehyde-fixed tissues was lower than for 10% NBF, but methacarn and Carnoy’s caused the least damage and exhibited more consistency of sequences among the tissues that were treated by these fixatives. Even while the fixatives may not have damaged tissue DNA in a specific manner, methacarn and alcohol-chloform-acetic acid fixatives generated less DNA damage compared to aldehyde fixatives. In addition to the low damage rates, the DNA damage rates were more consistent for alcohol-chloroform-acetic acid fixed tissues. Carnoy’s and methacarn-fixed tissues yielded better PCR products in both the PCR amplification rate and the concentration of the products. On the other hand, the DNA extraction was successful more often with tissues fixed with aldehyde fixatives. Therefore, alcohol-chloroform-acetic acid fixatives are preferred over aldehyde-based fixatives for DNA analytical procedures. There may be methods to improve DNA yield from tissues fixed with these solutions.

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111

APPENDIX A: CARCINOMA SEQUENCE DATA

Note: • • • • • •

CF: Carcinoma fixed by 10% neutral buffered formalin. CG: Carcinoma fixed by 1% glutaraldehyde. CC: Carcinoma fixed by Carnoy’s. CM: Carcinoma fixed by methacarn. CPC: Carcinoma, unfixed. CJT(number): The number of PCR products (from the same specimen) that were sequenced by Chia-Jui Tsai.

•

Template 18S: Human 18S ribosomal RNA (accession number: X03205.1 GI:36162), published by McCallum, and Maden, 1985.

112 1

10

20

30

40

50

CF_CJT27_112005_ GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CPC_CJT36_112005 GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CG_CJT30_112005_ GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCCAAGATTA

A-GCCATGCA

CPC_CJT40_112005 GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

AAGCCATGCA

CPC_CJT38_112005 GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CC_CJT34_120105_ GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CM_CJT45_120105_ GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CF_CJT33_120105_ GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CM_CJT44_120105_ GGTTGATCNT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CM_CJT42_120105_ GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CM_CJT46_120105_ GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CC_CJT34_112005

GGTTGATCCT

GCCAGTAGCA

TATGCTTGTT

TCAAAGATTA

A-GCCATGCA

CC_CJT36_120105_ GGTTGATCCT

GCCAGTAGCA

TATGCTTGTT

TCAAAGATTA

A-GCCATGCA

CFCJT29_112005_

GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CG_CJT20_122205_ GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CM_CJT40_120105_ GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

Template_18S

GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CG_CJT18_122205_ GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CC_CJT33_112005_ GGTTGATCCT

GCCCGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CM_CJT41_120105_ GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CPC_CJT39_112005 GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CG_CJT19_122205_ GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CG_CJT17_122205_ GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CC_CJT32_112005

GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CM_CJT39_120105_ GGTTGATCCT

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

CF_CJT28_112005

GCCAGTAGCA

TATGCTTGTC

TCAAAGATTA

A-GCCATGCA

GGTTGATCCT

113 51

60

70

80

90

100

CF_CJT27_112005_ TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CPC_CJT36_112005 TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CG_CJT30_112005_ TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CPC_CJT40_112005 TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CPC_CJT38_112005 TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CC_CJT34_120105_ TGTNTAAGT ACGCACGGCC- GGTACAGTGA AACTGNGAAT GGCTCATTAA CM_CJT45_120105_ TGTNTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CF_CJT33_120105_ TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CM_CJT44_120105_ TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CM_CJT42_120105_ TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CM_CJT46_120105_ TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CC_CJT34_112005

TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA

CC_CJT36_120105_ TGTNTAANT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CFCJT29_112005_

TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA

CG_CJT20_122205_ TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CM_CJT40_120105_ TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA Template_18S

TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA

CG_CJT18_122205_ TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CC_CJT33_112005_ TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCAATAA CM_CJT41_120105_ TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CPC_CJT39_112005 TGTCTAAGT ACGCACGGCCC GGTACAGTGA AACTGCGAAT GGCTCATTAA CG_CJT19_122205_ TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CG_CJT17_122205_ TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CC_CJT32_112005

TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA

CM_CJT39_120105_ TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA CF_CJT28_112005

TGTCTAAGT ACGCACGGCC- GGTACAGTGA AACTGCGAAT GGCTCATTAA

114 101

110

120

130

140

150

CF_CJT27_112005_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG CPC_CJT36_112005 ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG CG_CJT30_112005_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG CPC_CJT40_112005 ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG CPC_CJT38_112005 ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG CC_CJT34_120105_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT NGGATAACTG CM_CJT45_120105_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT NGGATAACTG CF_CJT33_120105_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT NGGATAACTG CM_CJT44_120105_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCNTACT NGGATAACTG CM_CJT42_120105_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG CM_CJT46_120105_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG CC_CJT34_112005

ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG

CC_CJT36_120105_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTNTCCTACT TGGATAACTG CFCJT29_112005_

ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG

CG_CJT20_122205_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG CM_CJT40_120105_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG Template_18S

ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG

CG_CJT18_122205_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG CC_CJT33_112005_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG CM_CJT41_120105_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG CPC_CJT39_112005 ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG CG_CJT19_122205_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG CG_CJT17_122205_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG CC_CJT32_112005

ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG

CM_CJT39_120105_ ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG CF_CJT28_112005

ATCAGTTATG GTTCCTTTGG TCGCTCGCTC CTCTCCTACT TGGATAACTG

115 151

160

170

180

190

200

CF_CJT27_112005_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CPC_CJT36_112005 TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CG_CJT30_112005_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CPC_CJT40_112005 TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CPC_CJT38_112005 TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CC_CJT34_120105_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CM_CJT45_120105_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CF_CJT33_120105_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACT CCCTTTCGCG CM_CJT44_120105_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CM_CJT42_120105_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CM_CJT46_120105_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CC_CJT34_112005

TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG

CC_CJT36_120105_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CFCJT29_112005_

TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG

CG_CJT20_122205_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CM_CJT40_120105_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG Template_18S

TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG

CG_CJT18_122205_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CC_CJT33_112005_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CM_CJT41_120105_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CPC_CJT39_112005 TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CG_CJT19_122205_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CG_CJT17_122205_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CC_CJT32_112005

TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG

CM_CJT39_120105_ TGGTAATTCT AGAGCTAATA CATGCCGACG GGCGCTGACC CCCTT-CGCG CF_CJT28_112005

TGGTAATTCT AGAGCTAATG CATGCCGAAG GGCGCTGACC CCCTT-CGTG

116 201

210

220

230

240

250

CF_CJT27_112005_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT CPC_CJT36_112005 GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT CG_CJT30_112005_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-GGCCCGG T-CAGCCCCT CPC_CJT40_112005 GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AGCCCGG T-CAGCCCCT CPC_CJT38_112005 GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT CC_CJT34_120105_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT CM_CJT45_120105_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CCCAACCCGG T-CAGCCCCT CF_CJT33_120105_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT CM_CJT44_120105_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CCAAACCCGG T-CAGCCCCT CM_CJT42_120105_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CCAAACCCGG NTCAGCCCCT CM_CJT46_120105_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CCAACCCCGG GTCAGCCCCT CC_CJT34_112005

GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT

CC_CJT36_120105_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT CFCJT29_112005_

GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT

CG_CJT20_122205_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT CM_CJT40_120105_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CCAACCCTGG T-CAGCCCCT Template_18S

GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT

CG_CJT18_122205_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT CC_CJT33_112005_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT CM_CJT41_120105_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT CPC_CJT39_112005 GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT CG_CJT19_122205_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT CG_CJT17_122205_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT CC_CJT32_112005

GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT

CM_CJT39_120105_ GGGGGGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT CF_CJT28_112005

GGGAAGATGC GTGCATTTAT CAGATCAAAA CC-AACCCGG T-CAGCCCCT

117 251

260

270

280

290

300

CF_CJT27_112005_ CTCCGG—CC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG

GTGACTCTAG

CPC_CJT36_112005 CTCCGG—CC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG

GTGACTCTAG

CG_CJT30_112005_ CTCCGG—CC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG

GTGACTCTAG

CPC_CJT40_112005 CTCCGG—CC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG

GTGACTCTAG

CPC_CJT38_112005 CTCCGG—CC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG

GTGACTCTAG

CC_CJT34_120105_ CTCCGG—CC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG

GTGACTCTAG

CM_CJT45_120105_ CTCCCGGCCC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG GTGACTCTAG CF_CJT33_120105_ CTCCGG-CCC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG GTGACTCTAG CM_CJT44_120105_ CTCCGG-CCC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG GTGACTCTAG CM_CJT42_120105_ CTCCGG-CCC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG GTGACTCTAG CM_CJT46_120105_ CTCCGG-CCC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG GTGACTCTAG CC_CJT34_112005

CTCCGG-CCC CGGGCCGGGG GGCGGGCGCC GGCGGCTTTG GTGACTNTAG

CC_CJT36_120105_ CTTCGG—CC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG

GTGACTCTAG

CFCJT29_112005_

CTCCGG—CC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG

GTGACTCTAG

CG_CJT20_122205_ CTCCGG—CC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG

GTGACTCTAG

CM_CJT40_120105_ CTCCGG-CCC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG GTGACTCTAG Template_18S

CTCCGG—CC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG

GTGACTCTAG

CG_CJT18_122205_ CTCCGG—CC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG

GTGACTCTAG

CC_CJT33_112005_ CTCCGG-CCC C-GGCCGGGG GGCGGGCGCC GGCGGCTTTG GTGACTCTAG CM_CJT41_120105_ CTCCGG-CCC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG GTGACTCTAG CPC_CJT39_112005 CTCCGG-CCC C-GGCCGGGG GGCGGGCGCC GGCGGCTTTG GTGACTCTAG CG_CJT19_122205_ CTCCGG-CCC C-GGCCGGGG GGCGGGCGCC GGCGGCTTTG GTGACTCTAG CG_CJT17_122205_ CTCCGG-CCC CGGCCCGGGG GGCGGGCGCC GGCGGCTTTG GTGACTCTAG CC_CJT32_112005

CTCCGG-CCC CGGCC-GGGG GGCGGGCGCC GGCGGCTTTG GTGACTCTAG

CM_CJT39_120105_ CTCCGG—CC CCGGCCGGGG GGCGGGCGCC GGCGGCTTTG CF_CJT28_112005

GTGACTCTAG

CTCTGG-CCC CGGCCAGGGG GTCGGGTGCC ACCAGCTTTG GTGACTCTAG

118 301

310

320

330

340

350

CF_CJT27_112005_ ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGCGA CGACCCATTC CPC_CJT36_112005 ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGCGA CGACCCATTC CG_CJT30_112005_ ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGCGA CGACCCATTC CPC_CJT40_112005 ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGCGA CGACCCATTC CPC_CJT38_112005 ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGCGA CGACCCATTC CC_CJT34_120105_ ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGCGA CGACCCATTC CM_CJT45_120105_ ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGGGCGGCGA CGACCCATTC CF_CJT33_120105_ ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGGGCGGCGA CGACCCATTC CM_CJT44_120105_ ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGGGCGGCGA CGACCCATTC CM_CJT42_120105_ ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGGGCGGCGA CGACCCATTC CM_CJT46_120105_ ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGGGCGGCGA CGACCCATTC CC_CJT34_112005

ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGNGA CGACCCATTC

CC_CJT36_120105_ ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGGGCGGCGA CGACCCATTC CFCJT29_112005_

ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGCGA CGACCCATTC

CG_CJT20_122205_ ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGCGA CGACCCATTC CM_CJT40_120105_ ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGCGA CGACCCATTC Template_18S

ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGCGA CGACCCATTC

CG_CJT18_122205_ ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGCGA CGACCCATTC CC_CJT33_112005_ ATAACCTCGG GCCGATCGCA CGCCCCCCCG TGG-CGGCGA CGACCCATTC CM_CJT41_120105_ ATAACCTCGG GCCGATCGCA CGCCCCCCCG TGG-CGGCGA CGACCCATTC CPC_CJT39_112005 ATAACCTCGG GCCGATCGCA CGCCCCCCCG TGG-CGGCGA CGACCCATTC CG_CJT19_122205_ ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGCGA CGACCCATTC CG_CJT17_122205_ ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGCGA CGACCCATTC CC_CJT32_112005

ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGCGA CGACCCATTC

CM_CJT39_120105_ ATAACCTCGG GCCGATCGCA CGCCCCCC-G TGG-CGGCGA CGACCCATTC CF_CJT28_112005

ATAGCCTCGG GCCAATCGCA CACCCCCC-G TGG-CAGCGA CNACCCATTA

119 351

360

370

380

390

400

CF_CJT27_112005_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTATCCATGG CPC_CJT36_112005 GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTAGCCATGG CG_CJT30_112005_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CPC_CJT40_112005 GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CPC_CJT38_112005 GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CC_CJT34_120105_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CM_CJT45_120105_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CF_CJT33_120105_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CM_CJT44_120105_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CM_CJT42_120105_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CM_CJT46_120105_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CC_CJT34_112005

GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG

CC_CJT36_120105_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CFCJT29_112005_

GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG

CG_CJT20_122205_ GAACGTTTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CM_CJT40_120105_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG Template_18S

GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG

CG_CJT18_122205_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CC_CJT33_112005_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CM_CJT41_120105_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CPC_CJT39_112005 GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CG_CJT19_122205_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CG_CJT17_122205_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CC_CJT32_112005

GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG

CM_CJT39_120105_ GAACGTCTGC CCTATCAACT TTCGATGGTA GTCGCCGTGC CTA-CCATGG CF_CJT28_112005

GAACGTCTGC CCTATCAACT TTTGATGGTA GTCGCTGTGC CTA-CCANGG

120 401

410

420

430

CF_CJT27_112005_ TGA-CCACGG GTGACGGGGA AT-CAGAGGT T CPC_CJT36_112005 TGA-CCACGG GTGACGGGGA ATACAGAAGT T CG_CJT30_112005_ TGAGCCACGG GTGACGGGGA AT-CAGAGGT T CPC_CJT40_112005 TGA-CCACGG GTGACGGGGA AT-CAGGGTT CPC_CJT38_112005 TGA-CCACGG GTGACGGGGA AT-CAGGGTT CC_CJT34_120105_ TGA-CCACGG GTGACGGGGA AT-CAGGGTT CM_CJT45_120105_ TGA-CCACGG GTGACGGGGA AT-CAGGGTT CF_CJT33_120105_ TGA-CCACGG GTGACGGGGA AT-CAGGGTT CM_CJT44_120105_ TGA-CCACGG GTGACGGGGA AT-CAGGGTT CM_CJT42_120105_ TGA-CCACGG GTGACGGGGA AT-CAGGGTT CM_CJT46_120105_ TGA-CCACGG GTGACGGGGA AT-CAGGGTT CC_CJT34_112005

TGA-CCACGG GTGACGGGGA AT-CAGGGTT -

CC_CJT36_120105_ TGA-CCACGG GTGACGGGGA AT-CAGGGTT CFCJT29_112005_

TGA-CCACGG GTGACGGGGA AT-CAGGGTT -

CG_CJT20_122205_ TGA-CCACGG GTGACGGGGA AT-CAGGGTT CM_CJT40_120105_ TGA-CCACGG GTGACGGGGA AT-CAGGGTT Template_18S

TGA-CCACGG GTGACGGGGA AT-CAGGGTT -

CG_CJT18_122205_ TGA-CCACGG GTGACGGGGA AT-CANGGTT A CC_CJT33_112005_ TGA-CCACGG GTGACGGGGA AT-CAGGGTT CM_CJT41_120105_ TGA-CCACGG GTGACGGGGA AT-CAGGGTT CPC_CJT39_112005 TGA-CCACGG GTGACGGGGA AT-CAGGGTT CG_CJT19_122205_ TGA-CCACGG GTGACGGGGA AT-CAGGGTT CG_CJT17_122205_ TGA-CCACGG GTGACGGGGA AT-CAGGGTT CC_CJT32_112005

TGA-CCACGG GTGACGGGGA AT-CAGGGTT -

CM_CJT39_120105_ TGA-CCATGG GTGACGGGGA AT-CAGGGTT CF_CJT28_112005

TGA-CCACGG GTGACGGGGA AT-CAGGGTT -

121

APPENDIX B: Leukemia Sequence Data

Note: • • • • • •

LF: Leukemia fixed by 10% neutral buffered formalin. LG: Leukemia fixed by 1% glutaraldehyde. LC: Leukemia fixed by Carnoy’s. LM: Leukemia fixed by methacarn. LPC: Leukemia, unfixed. CJT(number): The number of PCR products (from the same specimen) that were sequenced by Chia-Jui Tsai.

•

Template 18S: Human 18S ribosomal RNA (accession number: X03205.1 GI:36162), published by McCallum, and Maden, 1985.

122 1

10

20

30

40

50

LG_CJT13_112005

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LG_CJT15_112005_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCCCATGC

LF_CJT27_120105_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LC_CJT50_120105_

TNGNTGATCC TGCCAGTAGC ATATGCTTNT TC-AAAGATT AAGCC-ATGC

LF_CJT04_122205_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LF_CJT07_122205_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

Template_18S_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LG_CJT10_112005

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LM_CJT15_122205_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LPC_CJT21_112005

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LM_CJT10_122205_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LPC_CJT22_112005

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LM_CJT14_122205_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LF_CJT29_120105_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LF_CJT31_120105_

-TGNTGATCC TGCCANTAGC ATATGCTN-T CTCNAAGATT AAGCC-ATGC

LM_CJT18_112005

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LF_CJT32_120105_

-GGTTGATCN TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LPC_CJT23_112005

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LG_CJT14_112005_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LM_CJT19_112005

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LPC_CJT24_112005

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LM_CJT08_122205_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT ATGCC-ATAC

LPC_CJT20._112005 -GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC LG_CJT16_112005_

-GGTNGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LPC_CJT26_112005_ -GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC LG_CJT11_112005

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LM_CJT17_112005

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LPC_CJT25_112005

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LF_CJT30_120105_

TGGTTGATNC TGCCAGTAGC ATATGCTTGT CTCAAAGAT- AAGCC-ATGC

LC_CJT47_120105_

-GGTTGATC- TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LC_CJT49_120105_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LF_CJT01_122205_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LF_CJT03_122205_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LC_CJT51_120105_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LF_CJT28_120105_

-GGTTGATCC TGCCAGTAGC ATATGCTTGT NTCAAAGATT AAGCC-ATGC

LG_CJT12_112005

-GGTTGATCC TGCCAGTANC ATATGCTTGT CTCAAAGATT AAGCC-ATGC

LF_CJT02_122205_

---------- ---------- -------TTT TTCAAAGATT NAGCC-NNGC

123 51

60

70

80

90

100

LG_CJT13_112005

ACGTCTAAGT ACGCA-CGGG CCCGTACAGT GAAACTGCGA ATGGCTCATT

LG_CJT15_112005_

ACGTCTAAGT ACGCAACGGG CCCGTACAGT GAAACTGCGA ATGGCTCATT

LF_CJT27_120105_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LC_CJT50_120105_

ATGT-TAAGT ACGCA-CGG- CCGGTACAGT GAAANTGCGA ATGGCTCATT

LF_CJT04_122205_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LF_CJT07_122205_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

Template_18S_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LG_CJT10_112005

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LM_CJT15_122205_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LPC_CJT21_112005

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LM_CJT10_122205_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LPC_CJT22_112005

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LM_CJT14_122205_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LF_CJT29_120105_

ATGTNTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LF_CJT31_120105_

ATGTNTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LM_CJT18_112005

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LF_CJT32_120105_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGTGA ATGGCTCATT

LPC_CJT23_112005

ATGTNTAAGT ACGCA-CGGG CCGGTACAGT GAAACTGCGA ATGGCTCATT

LG_CJT14_112005_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LM_CJT19_112005

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LPC_CJT24_112005

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LM_CJT08_122205_

ATGTCTAAGT ACACA-GGG- CCAGTACAGT GAAACTGCGA ATGGCTCATT

LPC_CJT20._112005 ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT LG_CJT16_112005_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LPC_CJT26_112005_ ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT LG_CJT11_112005

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LM_CJT17_112005

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LPC_CJT25_112005

ATGTCTAAGT ACGCA-CGG- CAGGTACAGT GAAACTGCGA ATGGCTCATT

LF_CJT30_120105_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LC_CJT47_120105_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LC_CJT49_120105_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGG ATGGCTCATT

LF_CJT01_122205_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LF_CJT03_122205_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LC_CJT51_120105_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LF_CJT28_120105_

ATGTCTAAGT ACGCA-CGG- CCGGTACAGT GAAACTGCGA ATGGCTCATT

LG_CJT12_112005

ATGTCTAAAT ACGCA-CGG- CCSGTACAGT GAAACTGCGA ATGGCTCACT

LF_CJT02_122205_

ANNTTNANNT NCCC--CNG- CCNGTNACGG GAAAATNNGA ANGGNCCATT

124 101

110

120

130

140

150

LG_CJT13_112005

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LG_CJT15_112005_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LF_CJT27_120105_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LC_CJT50_120105_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LF_CJT04_122205_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LF_CJT07_122205_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

Template_18S_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LG_CJT10_112005

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LM_CJT15_122205_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LPC_CJT21_112005

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LM_CJT10_122205_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LPC_CJT22_112005

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LM_CJT14_122205_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LF_CJT29_120105_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTNCTCCT ACTTGGATAA

LF_CJT31_120105_

AAATCAGTTA TGGT-CCNTT GGTCGCTCGC TCCTN-TCCT ACTNGGATAA

LM_CJT18_112005

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTN-TCCT ACTNGGATGA

LF_CJT32_120105_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTNGGATAA

LPC_CJT23_112005

GAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTNGGATAA

LG_CJT14_112005_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LM_CJT19_112005

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTNGGATAA

LPC_CJT24_112005

AAATCAGATA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LM_CJT08_122205_

AAATCAGTTA TGGTTCTTTT GATCGCTCGC TCCTC-TCCT ACTTTGAAAA

LPC_CJT20._112005 AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTNGGATAA LG_CJT16_112005_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LPC_CJT26_112005_ AAATCAGATA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA LG_CJT11_112005

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LM_CJT17_112005

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LPC_CJT25_112005

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LF_CJT30_120105_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LC_CJT47_120105_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LC_CJT49_120105_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LF_CJT01_122205_

AAGTCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LF_CJT03_122205_

AAGTCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LC_CJT51_120105_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LF_CJT28_120105_

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCT ACTTGGATAA

LG_CJT12_112005

AAATCAGTTA TGGTTCCTTT GGTCGCTCGC TCCTC-TCCC ACTTGGATAA

LF_CJT02_122205_

AAATCAATTA NGGTTCCTT- GGTCGNTCGN TCCTT-TCCT AANTGGATAA

125 151

160

170

180

190

200

LG_CJT13_112005

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LG_CJT15_112005_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LF_CJT27_120105_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTCC

LC_CJT50_120105_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LF_CJT04_122205_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LF_CJT07_122205_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

Template_18S_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LG_CJT10_112005

CTGTGGTAAT TCTAGAGCTA ATACCATGCC GACGGGCGCT GACCCCCTTC

LM_CJT15_122205_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LPC_CJT21_112005

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LM_CJT10_122205_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LPC_CJT22_112005

TTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LM_CJT14_122205_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LF_CJT29_120105_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LF_CJT31_120105_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LM_CJT18_112005

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LF_CJT32_120105_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LPC_CJT23_112005

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LG_CJT14_112005_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LM_CJT19_112005

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LPC_CJT24_112005

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LM_CJT08_122205_

CTGTGGTAAT TCTAGAGCTA ATGC-ATGCC GAAGGGCGCT GACCCCCTTC

LPC_CJT20._112005 CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC LG_CJT16_112005_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LPC_CJT26_112005_ CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC LG_CJT11_112005

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LM_CJT17_112005

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LPC_CJT25_112005

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LF_CJT30_120105_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LC_CJT47_120105_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LC_CJT49_120105_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LF_CJT01_122205_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LF_CJT03_122205_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LC_CJT51_120105_

CTGTGGTAAT TCTAGAGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LF_CJT28_120105_

CTGTGGTAAT TCTAGGGCTA ATAC-ATGCC GACGGGCGCT GACCCCCTTC

LG_CJT12_112005

CTGTGGTAAT TCTNGAGCTN ATAC-ATGCN GACGGGCNCT GACCCCCTTC

LF_CJT02_122205_

ACGGGGTAAT TNTAGAGNTA ATAC-ATGCC GACGGGCGCT GACCCCNTTN

126 201

210

220

230

240

250

LG_CJT13_112005

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LG_CJT15_112005_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LF_CJT27_120105_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LC_CJT50_120105_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LF_CJT04_122205_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LF_CJT07_122205_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

Template_18S_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LG_CJT10_112005

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LM_CJT15_122205_

GCGGGGGGGA TGCGCGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LPC_CJT21_112005

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LM_CJT10_122205_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LPC_CJT22_112005

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LM_CJT14_122205_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LF_CJT29_120105_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LF_CJT31_120105_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC CGGTCAGCCC

LM_CJT18_112005

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LF_CJT32_120105_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LPC_CJT23_112005

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LG_CJT14_112005_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LM_CJT19_112005

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LPC_CJT24_112005

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LM_CJT08_122205_

GTGGGGAAGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LPC_CJT20._112005 GTGGGGAAGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC LG_CJT16_112005_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LPC_CJT26_112005_ GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC LG_CJT11_112005

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GATCAGCCC

LM_CJT17_112005

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LPC_CJT25_112005

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LF_CJT30_120105_

GCCGGGGGAA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LC_CJT47_120105_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC CGGTCAGCCC

LC_CJT49_120105_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LF_CJT01_122205_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LF_CJT03_122205_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LC_CJT51_120105_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LF_CJT28_120105_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCAACCC -GGTCAGCCC

LG_CJT12_112005

NCGGGGGGGA TGCGTGCATT TATCACATCA AAACCAACCC -GGTCAGCCC

LF_CJT02_122205_

GCGGGGGGGA TGCGTGCATT TATCAGATCA AAACCNANCC CGGTCAGCCC

127 251

260

270

280

290

300

LG_CJT13_112005

CTNTCCGGCC CCGGCC-GGG GGGCGGGCGC NGGCGGCTTT GGTGACTNTA

LG_CJT15_112005_

CTNTCCGGCC CCGGCC-GGG NGGCGGNCGC NGGCGGCTTT GGTGACTCTA

LF_CJT27_120105_

CTCTCTGGCC CCGGCCAGGG GGTCGGGTGC CACCAGCTTT GGTGACTCTA

LC_CJT50_120105_

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LF_CJT04_122205_

CTCTCCGGCC CCGGCCCGGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LF_CJT07_122205_

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

Template_18S_

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LG_CJT10_112005

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LM_CJT15_122205_

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LPC_CJT21_112005

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LM_CJT10_122205_

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LPC_CJT22_112005

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LM_CJT14_122205_

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LF_CJT29_120105_

CTCTCCGGCC CCCGGCCGGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LF_CJT31_120105_

CTCTCCGGCC CCCGGCCGGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LM_CJT18_112005

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LF_CJT32_120105_

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LPC_CJT23_112005

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LG_CJT14_112005_

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LM_CJT19_112005

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGATTCTA

LPC_CJT24_112005

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LM_CJT08_122205_

CTCTCCGGCC CCGGCC-GGG AGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LPC_CJT20._112005 CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA LG_CJT16_112005_

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LPC_CJT26_112005_ CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA LG_CJT11_112005

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LM_CJT17_112005

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACGCTA

LPC_CJT25_112005

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LF_CJT30_120105_

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LC_CJT47_120105_

CTCTCCGGCC CCCGGCCGGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LC_CJT49_120105_

CTCTCCGGCC CCCGGCCGGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LF_CJT01_122205_

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LF_CJT03_122205_

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LC_CJT51_120105_

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LF_CJT28_120105_

CTCTCCGGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTA

LG_CJT12_112005

CTCTTCTGCC CCGGCC-GGG GGGCGGGCGC CGGCGGCTTT GGTGACTCTC

LF_CJT02_122205_

NTNTCCGGCC CC-GGCCGGG GGGCGGGCGC CGACGGCTTT GGTGACTCTA

128 301

310

320

330

340

350

LG_CJT13_112005

GATAA-CCTC GGGCCAATCG CACACCCCCC GTGGC—AGCG ACGACCCAT

LG_CJT15_112005_

GATAA-CCTC GGGCCAATCG CACACCCCCC GTGGC—AGCG ACGACCCAT

LF_CJT27_120105_

GATAG-CCTC GGGCCAATCG CACACCCCCC GTGGG-CAGCG ACGACCCAT

LC_CJT50_120105_

GATAA-CCTC GGGCCAATCG CACGCCCCC- GTGGC-AG-CG ATGACCCAT

LF_CJT04_122205_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LF_CJT07_122205_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

Template_18S_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LG_CJT10_112005

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LM_CJT15_122205_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LPC_CJT21_112005

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LM_CJT10_122205_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GAGGC-GG-CG ACGACCCAT

LPC_CJT22_112005

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GGGCG ACGACCCAT

LM_CJT14_122205_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LF_CJT29_120105_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGG-CGGCG ACGACCCAT

LF_CJT31_120105_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGG-CGGCG ACGACCCAT

LM_CJT18_112005

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LF_CJT32_120105_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LPC_CJT23_112005

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LG_CJT14_112005_

GATAAACCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LM_CJT19_112005

GATAACCCTN GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LPC_CJT24_112005

GATAA-CCTC GGGCCGATCG CACGCCCCTC GTGGGCGGGCG ACGACCCAT

LM_CJT08_122205_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LPC_CJT20._112005 GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCTAT LG_CJT16_112005_

GATAA-CCTC GGGCCGATCG CACGCCCCTC GTGGC-GG-CG ACGACCCAT

LPC_CJT26_112005_ GATAA-CCTC GGGCCGATCG CACGCCCCTC GTGGC-GG-CG ACGACCCAT LG_CJT11_112005

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LM_CJT17_112005

GATAA-CCTC GGGCCGATCG CACG-CCCCC GTGGC-GG-CG ACGACCCAT

LPC_CJT25_112005

GATAA-CCTC GGGCCGATCG CACGTCCCCC GTGGC-GG-CG ACGACCCAT

LF_CJT30_120105_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGGCGG-CG ACGACCCAT

LC_CJT47_120105_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGGCGG-CG ACGACCCAT

LC_CJT49_120105_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGGCGG-CG ACGACCCAT

LF_CJT01_122205_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LF_CJT03_122205_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LC_CJT51_120105_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LF_CJT28_120105_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

LG_CJT12_112005

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGNC-GG-CG ACGACCCAT

LF_CJT02_122205_

GATAA-CCTC GGGCCGATCG CACGCCCCCC GTGGC-GG-CG ACGACCCAT

129 351

360

370

380

390

400

LG_CJT13_112005

TAGAA-CGT- CTGCCCTATC AA-CTTTTGA TGG—TAGTC GCT-GT-GCC

LG_CJT15_112005_

TAGAA-CGT- CTGCCCTATC AA-CTTTTGA TGG—TAGTC GCT-GT-GCC

LF_CJT27_120105_

TAGAA-CGT- CTGCCCTATC AA-CTTTTGA TGG—TAGTC GCT-GT-GCC

LC_CJT50_120105_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LF_CJT04_122205_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LF_CJT07_122205_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

Template_18S_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LG_CJT10_112005

TCGAA-CGT- CTGCCCTATC AAACTTTCGA TGG—TAGTC GCC-GT-GCC

LM_CJT15_122205_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LPC_CJT21_112005

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LM_CJT10_122205_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LPC_CJT22_112005

TCGAAACGT- CTGCCCTATC AAACTTTCGA TGG—TAGTC GCC-GT-GCC

LM_CJT14_122205_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LF_CJT29_120105_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LF_CJT31_120105_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LM_CJT18_112005

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LF_CJT32_120105_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LPC_CJT23_112005

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LG_CJT14_112005_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LM_CJT19_112005

TCGAA-CGT- ATGCCCTATC AA-CTTTCGA GAAGTTCGTC CCCTGTAGCC

LPC_CJT24_112005

TCGAAACGTT CTGCCCTATC AAACTTTCGA TGG—TAGTC GCC-GT-GCC

LM_CJT08_122205_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LPC_CJT20._112005 TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC LG_CJT16_112005_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LPC_CJT26_112005_ TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC LG_CJT11_112005

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LM_CJT17_112005

TCGAA-CGT- CTGCCCTATC AG-CTTTCGA TGG—TAGTC GCC-GT-GCC

LPC_CJT25_112005

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC ACC-GT-GCC

LF_CJT30_120105_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LC_CJT47_120105_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LC_CJT49_120105_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LF_CJT01_122205_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LF_CJT03_122205_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LC_CJT51_120105_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LF_CJT28_120105_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LG_CJT12_112005

TCNAA-CGT- CGGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

LF_CJT02_122205_

TCGAA-CGT- CTGCCCTATC AA-CTTTCGA TGG—TAGTC GCC-GT-GCC

130 401

410

420

430

440

LG_CJT13_112005

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

LG_CJT15_112005_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

LF_CJT27_120105_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

LC_CJT50_120105_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

LF_CJT04_122205_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

LF_CJT07_122205_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

Template_18S_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

LG_CJT10_112005

TA-CCATGGT GA-CCACGGG TGA-CGGGGA AATCAGGGTT-

LM_CJT15_122205_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

LPC_CJT21_112005

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

LM_CJT10_122205_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

LPC_CJT22_112005

TA-CCATGGT GA-CCACGGG TGA-CGGGGA AATCAGGGTT-

LM_CJT14_122205_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

LF_CJT29_120105_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

LF_CJT31_120105_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

LM_CJT18_112005

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

LF_CJT32_120105_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

LPC_CJT23_112005

TA-CCATGGT GA-CCACGGG TGA-CGGGGA AATCAGGGTT-

LG_CJT14_112005_

TA-CCATGGT GA-CCGCGGG TGA-CGGGGA AT-CAGAAGTT

LM_CJT19_112005

TAGCCTTGGT GA-CCACGGG TGA-CGGGNA ATACACAAGTT

LPC_CJT24_112005

TA-CCATGGT GAACCACGGG TGNACGGGGN AATCAGAAGTT

LM_CJT08_122205_

TA-CCATGGC GA-CCACGGG TGA-CGGGGA AT-CAGGGTT-

LPC_CJT20._112005 TA-CCATGGT GA-CCACGGG TGA-CGGGGA AT-CAGAGGTT LG_CJT16_112005_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA AT-CAGGGTT-

LPC_CJT26_112005_ TA-CCATGGT GA-CCACGGG TGA-CGGGGA AT-CAGGGTTLG_CJT11_112005

TA-CCATGGT GA-CCACGGG TGA-CGGGGA AT-CAGGGTT-

LM_CJT17_112005

TA-CCATGGT GA-CCACGGG TGA-CGGGGA AT-CAGGGTT-

LPC_CJT25_112005

TA-CCATGGT GA-CCACGGG TGA-CGGGGA AT-CAGGGTT-

LF_CJT30_120105_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA AT-CAGGGTT-

LC_CJT47_120105_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA AT-CAGGGTT-

LC_CJT49_120105_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA AT-CAGGGTT-

LF_CJT01_122205_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA AT-CAGGGTT-

LF_CJT03_122205_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA AT-CAGGGTT-

LC_CJT51_120105_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA AT-CAGGGTT-

LF_CJT28_120105_

TA-CCATGGT GA-CTACGGG TGA-CGGGGA AT-CAGGGTT-

LG_CJT12_112005

TA-CCANGGT GA-CCACGGG TGA-CGGGGA AT-CAAGGTTA

LF_CJT02_122205_

TA-CCATGGT GA-CCACGGG TGA-CGGGGA A-TCAGGGTT-

131

APPENDIX C: SARCOMA SEQUENCE DATA

Note: • SF: Sarcoma fixed by 10% neutral buffered formalin. • SG: Sarcoma fixed by 1% glutaraldehyde. • SC: Sarcoma fixed by Carnoy’s. • SM: Sarcoma fixed by methacarn. • SPC: Sarcoma, unfixed. • CJT(number): The number of PCR products (from the same specimen) that were sequenced by Chia-Jui Tsai. •

Template 18S: Human 18S ribosomal RNA (accession number: X03205.1 GI:36162), published by McCallum, and Maden, , 1985.

132 1

10

20

30

40

50

SG_CJT09_120105_

-GGTTGATCN TGCCAGTAGC ATTATGCTCG TCTC-AAAGA CTNAGCCTCG

SG_CJT10_120105_

-GGTTGATCN TGCCAGTAGC ATTATGCTCG TCTC-AAAGA CTMAGCCTCG

SF_CJT01_CJT01_120105_ -GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG SG_CJT12_120105_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTCCAAAGA TTAAGCCATG

SF_CJT07_120105_

---------- ---------- -----GNTTG TNTC-AAAGA TTAAGCCATG

SPC_CJT22_120105_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TNTC-AAAGA TTAAGCCATG

SF_CJT04_120105_

-GGTTGATC- TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SG_CJT13_120105_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TAAAGCCATG

SPC_CJT23_120105_

TGGTTGATCN TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TAA-GCCATG

SF_CJT02_120105_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAGGA TTAAGCCATG

SM_CJT09_112005

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SM_CJT06_112005

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SG_CJT14_120105_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SF_CJT03_120105_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SG_CJT04_112005

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

Template_18S

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SF_CJT08_120105_

-GGTTGATCC TGCCAGTAGC AT-ATACTTG TCTC-AAAGA TTAAGCCATG

SG_CJT03_112005

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SC_CJT16_120105_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SG_CJT02_112005_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SM_CJT08_112005_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SM_CJT05_112005_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SM_CJT07_112005_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SG_CJT01_112005_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TCAAGCCATG

SC_CJT15_120105_

-GGTTGATCN TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SC_CJT17_120105_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SC_CJT19_120105_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SC_CJT18_120105_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SF_CJT05_120105_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TNTC-AAAGA TTAAGCCATG

SG_CJT11_120105_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SF_CJT06_120105_

-GGTTGATCC TGCCAGTAGC AT-ATGCTTG TCTC-AAAGA TTAAGCCATG

SPC_CJT24_120105_

---------- --------GC AT-ATGCT-G TCTC-AAAGA ATAAGCCATG

SPC_CJT25_120105_

---------- --------GC AT-ATGCT-G TCTC-AAAGA TTAA-CCATG

SPC_CJT26_120105_

-GGTTGATCC TGCCACTAGC AT—TGCTTG TTTT-AAAGA TTAAGCCA-G

133 51

60

70

80

90

100

SG_CJT09_120105_

CACGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGNTCAT

SG_CJT10_120105_

CACGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGNTCAT

SF_CJT01_CJT01_120105_ CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT SG_CJT12_120105_

CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SF_CJT07_120105_

CATGTNTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SPC_CJT22_120105_

CATGTNTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SF_CJT04_120105_

CATGTNTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SG_CJT13_120105_

CATGTNTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SPC_CJT23_120105_

CATGTNTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SF_CJT02_120105_

CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SM_CJT09_112005

CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SM_CJT06_112005

CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SG_CJT14_120105_

CATGTCTAAG TACTCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SF_CJT03_120105_

CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SG_CJT04_112005

CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

Template_18S

CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SF_CJT08_120105_

CATGTCTAAG TACGTA-CGG CC-GGTACAG TGAAACTGCG GATGGCTCAT

SG_CJT03_112005

CATGCCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SC_CJT16_120105_

CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACCGCG AATGGCTCAT

SG_CJT02_112005_

CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SM_CJT08_112005_

CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SM_CJT05_112005_

CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SM_CJT07_112005_

CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AGTGGCTCAT

SG_CJT01_112005_

CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SC_CJT15_120105_

CATGTNTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SC_CJT17_120105_

CATGTNTGAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SC_CJT19_120105_

CATGTNTAAG TACGCA-CGG CC-GGTACAG TGAAACTGNG AATGGCTCAT

SC_CJT18_120105_

CATGTNTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SF_CJT05_120105_

CATGTCTAAG TACGNAACGG CCCNGTACAG TGAAACTGCG AATGGCTCAT

SG_CJT11_120105_

CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SF_CJT06_120105_

CATGTNTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG AATGGCTCAT

SPC_CJT24_120105_

CATGTCTAAG TACGCA-CGG CC-GGTACAG TGAAACTGCG ANTGGCTCAT

SPC_CJT25_120105_

CAT-TCTAAG TACGCA-CGG CC-GGTACAG G-AAACTGCG AATGGCTCAT

SPC_CJT26_120105_

CA-GTCTAAG TACGCA-CGG CC-GGTACA- TGAAACTGCG AATGGCTCAT

134 101

110

120

130

140

150

SG_CJT09_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTNT-CC TACTNGGATA

SG_CJT10_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTNT-CC TACTNGGATA

SF_CJT01_CJT01_120105_ TAAATCAGTT ATGGTTCCCT TGGTCGCTCG CTCCTNT-CC TACTNGGATA SG_CJT12_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTNT-CC TACTNGGATA

SF_CJT07_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTNT-CC TACTNGGATA

SPC_CJT22_120105_

TAAATCAGTT ATGGTTCCTT TGGTNGCTCG CTCCTNT-CN TACTNGGATA

SF_CJT04_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTNTTCN TACTNGGATA

SG_CJT13_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTNT-CC TACTNGGATA

SPC_CJT23_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTNT-CC TACTNGGATA

SF_CJT02_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

SM_CJT09_112005

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

SM_CJT06_112005

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

SG_CJT14_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

SF_CJT03_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

SG_CJT04_112005

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

Template_18S

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

SF_CJT08_120105_

TAAATCAGTT ATGGTTCCTT CGGTCGCTCG CTCCTCT-CC TACTTGGATA

SG_CJT03_112005

TAAATCAGTT ATGGTTCCTT CGGTCGCTCG CTCCTCT-CC TACTTGGATA

SC_CJT16_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

SG_CJT02_112005_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

SM_CJT08_112005_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

SM_CJT05_112005_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

SM_CJT07_112005_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CT TACTTGGATA

SG_CJT01_112005_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

SC_CJT15_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

SC_CJT17_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

SC_CJT19_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

SC_CJT18_120105_

TAAATCAGTT ACGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

SF_CJT05_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTNGGATA

SG_CJT11_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTNGGATA

SF_CJT06_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTNGGATA

SPC_CJT24_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-C- TACTTGGATA

SPC_CJT25_120105_

TAAATCAGTT ANGGTTCCTT TG-TCGCTCG CTCCTCT-CC TACTTGGATA

SPC_CJT26_120105_

TAAATCAGTT ATGGTTCCTT TGGTCGCTCG CTCCTCT-CC TACTTGGATA

135 151

160

170

180

190

200

SG_CJT09_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGNCGCT GACTCCC-TT

SG_CJT10_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACTCCC-TT

SF_CJT01_CJT01_120105- ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT SG_CJT12_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCTC-TT

SF_CJT07_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCC--TT

SPC_CJT22_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SF_CJT04_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCCTTN

SG_CJT13_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SPC_CJT23_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SF_CJT02_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SM_CJT09_112005

ACTGTGGTAA TTCTGGAGCT AATACATGCC GGCGGGCGCT GACCCCC-TT

SM_CJT06_112005

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SG_CJT14_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SF_CJT03_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SG_CJT04_112005

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

Template_18S

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SF_CJT08_120105_

ACTGTGGTAA TTCTAGAGCT AATGCATGCC GACGGGCGCT GACCCCC-TT

SG_CJT03_112005

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SC_CJT16_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SG_CJT02_112005_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SM_CJT08_112005_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SM_CJT05_112005_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCACC-TT

SM_CJT07_112005_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SG_CJT01_112005_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SC_CJT15_120105_

ACTGTGGTAA TTCTAGAGWT AATACATGCC GACGGGCGCT GACCCCC-TT

SC_CJT17_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SC_CJT19_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SC_CJT18_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SF_CJT05_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SG_CJT11_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SF_CJT06_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-TT

SPC_CJT24_120105_

ACTGTGGTAA TTNTAGAGWT AATACATGCC GACGGGCGCT GACCCCC-TT

SPC_CJT25_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC AACGGGCGCT GACCCCC-TT

SPC_CJT26_120105_

ACTGTGGTAA TTCTAGAGCT AATACATGCC GACGGGCGCT GACCCCC-AT

136 201

210

220

230

240

250

SG_CJT09_120105_

CGCGGGGGGG ATGTGTGCAT TTATCAGATC AAAACC—AC CCC-GGTCAG

SG_CJT10_120105_

CGCGGGGGGG ATGTGTGCAT TTATCAGATC AAAACC—AC CCC-GGTCAG

SF_CJT01_CJT01_120105_ CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG SG_CJT12_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SF_CJT07_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SPC_CJT22_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SF_CJT04_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAANCCAAC CCCGNKTCAG

SG_CJT13_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SPC_CJT23_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SF_CJT02_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SM_CJT09_112005

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-GAC CC--GGTCAG

SM_CJT06_112005

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SG_CJT14_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SF_CJT03_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SG_CJT04_112005

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

Template_18S

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SF_CJT08_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SG_CJT03_112005

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SC_CJT16_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SG_CJT02_112005_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SM_CJT08_112005_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SM_CJT05_112005_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SM_CJT07_112005_

CGCGGGGAAG ATGCCTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SG_CJT01_112005_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SC_CJT15_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SC_CJT17_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SC_CJT19_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CCC-GGTCAG

SC_CJT18_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CCC-GGTCAG

SF_CJT05_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AA- CCC-GGTCAG

SG_CJT11_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AA- CCC-GGTCAG

SF_CJT06_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAA CCC-GGTCAG

SPC_CJT24_120105_

NGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SPC_CJT25_120105_

CGCGGGGGGG ATGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

SPC_CJT26_120105_

CGCGGGGGGG GTGCGTGCAT TTATCAGATC AAAACC-AAC CC--GGTCAG

137 251

260

270

280

290

300

SG_CJT09_120105_

CCCCTNTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SG_CJT10_120105_

CCCCTNTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SF_CJT01_CJT01_120105_ CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT SG_CJT12_120105_

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SF_CJT07_120105_

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SPC_CJT22_120105_

CCCCTCTCCG GCCCCCGGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SF_CJT04_120105_

CCCTCTCCCG GCCCCCGGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SG_CJT13_120105_

CCCCTCTTCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SPC_CJT23_120105_

CCCCTCTCCG GCCCCCGGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SF_CJT02_120105_

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SM_CJT09_112005

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SM_CJT06_112005

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCAGCGGC TTGGGTGACT

SG_CJT14_120105_

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SF_CJT03_120105_

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SG_CJT04_112005

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

Template_18S

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SF_CJT08_120105_

CCCCTCTCCG ACCCCCGGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SG_CJT03_112005

CCCCTCTCCG GCCCC-GGCC GGGGGGCAGG CGCCGGCGGC TTTGGTGACA

SC_CJT16_120105_

CCCCTCTCCG GCCCCCGGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SG_CJT02_112005_

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SM_CJT08_112005_

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SM_CJT05_112005_

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SM_CJT07_112005_

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SG_CJT01_112005_

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SC_CJT15_120105_

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SC_CJT17_120105_

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SC_CJT19_120105_

CCCCTCTCCG GCCCCCGGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SC_CJT18_120105_

CCCCTCTCCG GCCCCCGGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SF_CJT05_120105_

CCCCTCTCCG GCCCCCGGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SG_CJT11_120105_

CCCCTCTCCG -CCCCCGGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SF_CJT06_120105_

CCCCTCTCCG GCCCCCGGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SPC_CJT24_120105_

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SPC_CJT25_120105_

CCCCTCTCCG GCCCC-GGCC GGGGGGCGGG CGCCGGCGGC TTTGGTGACT

SPC_CJT26_120105_

CCCCTCTCCG GCCCC-GGCC GGGGGGCAGG CGCCGGCGGC TTTGGTGACT

138 301

310

320

330

340

350

SG_CJT09_120105_

CTAGATAACC TCGGGCCGAT CGCACGTCCC CCGTGG-CGG CGACGACCCA

SG_CJT10_120105_

CTAGATAACC TCGGGCCGAT CGCACGTCCC CCGTGG-CGG CGACGACCCA

SF_CJT01_CJT01_120105_ CTAGATAACT TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA SG_CJT12_120105_

CTATATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SF_CJT07_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CAG CGACGACCCA

SPC_CJT22_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SF_CJT04_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGGGCGG CGACGACCCA

SG_CJT13_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SPC_CJT23_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGGGCGG CGACGACCCA

SF_CJT02_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SM_CJT09_112005

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SM_CJT06_112005

CTAGATAACC TCGGGCCGAC CGCACGCCCC CCGTGG-CGG CGACGACCCA

SG_CJT14_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SF_CJT03_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SG_CJT04_112005

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

Template_18S

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SF_CJT08_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC TCGTGG-CGG CGACGACCCA

SG_CJT03_112005

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SC_CJT16_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SG_CJT02_112005_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SM_CJT08_112005_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SM_CJT05_112005_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SM_CJT07_112005_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SG_CJT01_112005_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SC_CJT15_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGGGCGG CGACGACCCA

SC_CJT17_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGGGCGG CGACGACCCA

SC_CJT19_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGGGCGG CGACGACCCA

SC_CJT18_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGGGCGG CGACGACCCA

SF_CJT05_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGGGCGG CGACGACCCA

SG_CJT11_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SF_CJT06_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGGGCGG CGACGACCCA

SPC_CJT24_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SPC_CJT25_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

SPC_CJT26_120105_

CTAGATAACC TCGGGCCGAT CGCACGCCCC CCGTGG-CGG CGACGACCCA

139 351

360

370

380

390

400

SG_CJT09_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SG_CJT10_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SF_CJT01_CJT01_120105_ TTC-GAACGT CTGCCCTATC AACCTTCGAT GGTAGTCGCC GTGCCTACCA SG_CJT12_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SF_CJT07_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGCCGCC GTGCCTACCA

SPC_CJT22_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SF_CJT04_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SG_CJT13_120105_

TTC-GAACGT CTGCCCTATC AACTTCCGAT GGTAGTCGCC GTGCCTTCCA

SPC_CJT23_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SF_CJT02_120105_

TTNCGAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SM_CJT09_112005

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SM_CJT06_112005

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SG_CJT14_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SF_CJT03_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SG_CJT04_112005

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

Template_18S

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SF_CJT08_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SG_CJT03_112005

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SC_CJT16_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SG_CJT02_112005_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SM_CJT08_112005_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SM_CJT05_112005_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SM_CJT07_112005_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SG_CJT01_112005_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SC_CJT15_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SC_CJT17_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SC_CJT19_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SC_CJT18_120105_

TTC-GAACGT CTGCCCTATC AACTTCCGAT GGTAGTCGCC GTGCCTACCA

SF_CJT05_120105_

TTC-GAACAT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SG_CJT11_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SF_CJT06_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SPC_CJT24_120105_

TTC-GAACGT CTGCCCTATC AACTTTCAAT GGTAGTCGCC GTGCCTACCA

SPC_CJT25_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

SPC_CJT26_120105_

TTC-GAACGT CTGCCCTATC AACTTTCGAT GGTAGTCGCC GTGCCTACCA

140 401

410

420

430

SG_CJT09_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SG_CJT10_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SF_CJT01_CJT01_120105_ TGGTGACCAC GGGTGACGGG GAATCAGGGT T SG_CJT12_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SF_CJT07_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SPC_CJT22_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SF_CJT04_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SG_CJT13_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SPC_CJT23_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SF_CJT02_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SM_CJT09_112005

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SM_CJT06_112005

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SG_CJT14_120105_

TGGTGACCAC GTGTGACGGG GAATCAGGGT T

SF_CJT03_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SG_CJT04_112005

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

Template_18S

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SF_CJT08_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SG_CJT03_112005

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SC_CJT16_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SG_CJT02_112005_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SM_CJT08_112005_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SM_CJT05_112005_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SM_CJT07_112005_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SG_CJT01_112005_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SC_CJT15_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SC_CJT17_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SC_CJT19_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SC_CJT18_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SF_CJT05_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SG_CJT11_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SF_CJT06_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SPC_CJT24_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SPC_CJT25_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

SPC_CJT26_120105_

TGGTGACCAC GGGTGACGGG GAATCAGGGT T

141

APPENDIX :D WHITE BLOOD CELLS SEQUENCE DATA

Note: • FWBC: White blood cells fixed by 10% neutral buffered formalin. • GWBC: White blood cells fixed by 1% glutaraldehyde. • CWBC: White blood cells fixed by Carnoy’s. • MWBC: White blood cells fixed by methacarn. • PCWBC: White blood cells, unfixed. • CJT(number): The number of PCR products (from the same specimen) that were sequenced by Chia-Jui Tsai. •

Template 18S: Human 18S ribosomal RNA (accession number: X03205.1 GI:36162), published by McCallum, and Maden, , 1985.

142 1

10

20

30

40

50

PCWBC_CJT14_091505_ GGTTGATCCT GCC-AGTAGC ATATGC---- ---TTGTCTC AAAGATTAAG PCWBC_CJT15_091505_ GGTTGATCCT GCC-AGTAGC ATATGC---- ---TTGTCTC AAAGATTAAG GWBC_CJT08_091505_

GGTTGATCCT GCC-AGTAGC ATATGC---- ---TTGTCTC AAAGATTAAG

MWBC_CJT32_082305_

GGTTGATCCT GCC-AGTAGC ATATGC---- ---TTGTCTC AAAGATTAAG

CWBC_CJT29_082305_

GGTTGATCCT GCC-AGTAGC ATATGC---- ---TTGTCTC AAAGATTAAG

CWBC_CJT30_082305_

GGTTGATCCT GCC-AGTAGC ATATGC---- ---TTGTCTC AAAGATTAAG

CWBC_CJT28_082305_

GGTTGATCCT GCC-AGTAGC ATATGC---- ---TTGTCTC AAGGATTAAG

PCWBC_CJT35_082305_ GGTTGATCCT GCC-AGTAGC ATATGC---- ---TTGTCTC AAAGATTAAG Template_

GGTTGATCCT GCC-AGTAGC ATATGC---- ---TTGTCTC AAAGATTAAG

FWBC_CJT01_082305_

GGTTGATCNT GCC-AGTAGC ATATGC---- ---TTGTCTC AAAGATTAAG

GWBC_CJT06_091505_

GGTTGATCCT GCC-CGTAGC -CATATG--- ---TTTTCTC AAAGCTTTAG

GWBC_CJT07_091505_

GGTTGATCCT GCC-AGTAGC ATATGCT--- ---TGTCTNC AAAGANTNAG

MWBC_CJT10_091505_

GGTTGATCCT GCC-AGTAGC ATATGC---- ---TTGTCTC AAAGATTAAG

MWBC_CJT11_091505_

GGTTGATCCT GCC-AGTAGC ATATGC---- ---TTGTCTC AAAGATTAAG

FWBC_CJT04_091505_

GGTTGATCCT GCCCNGTAGC ATATGC---- ---TTGTCTC AAAGATTAAG

FWBC_CJT02_091505_

GGTTGATCCN GCC-NGTNGC ATATGC---- ---TTGTCTC AAAGATTNNG

PCWBC_CJT16_091505_ GGTTGATCCT GCC-AGTAGC ATATGC---- ---TTGTNTC AAAGATTAAG FWBC_CJT05_091505_

GGTTGATCCT GCC-ANTAGC ATATGN---- ---TTGTCTC NAAGNTTAAG

FWBC_CJT01_091505_

GGTNGNNCCC CCCCCNTTNN CCANCCTGCC CTTTCNTCCC CAGCNNTNNG

143 51

60

70

80

90

100

PCWBC_CJT14_091505_ CC-ATGCATG --TCTAAGTA CGC----ACG GCC-GGTACA GTG-AAACTG PCWBC_CJT15_091505_ CC-ATGCATG --TCTAAGTA CGC----ACG GCC-GGTACA GTG-AAACTG GWBC_CJT08_091505_

CC-ATGCATG --TCTAANTA CGC----ACG GCC-GGTACA GTG-AAACTG

MWBC_CJT32_082305_

CC-ATGCATG --TCTAAGTA CGC----ACG GCC-GGTACA GTG-AAACTG

CWBC_CJT29_082305_

CC-ATGCATG --TCTAAGTA CGC----ACG GCC-GGTACA GTG-AAACTG

CWBC_CJT30_082305_

CC-ATGCATG --TCTAAGTA CGC----ACG GCC-GGTACA GTG-AAACTG

CWBC_CJT28_082305_

CC-ATGCATG --TCTAAGTA CGC----ACG GCC-GGTACA GTG-AAACTG

PCWBC_CJT35_082305_ CC-ATGCATG --TCTAAGTA CGC----ACG GCCCGGTACA GTG-AAACTG Template_

CC-ATGCATG --TCTAAGTA CGC----ACG GCC-GGTACA GTG-AAACTG

FWBC_CJT01_082305_

CC-ATGCATG --TCTAAGTA CGC----ACG GCC-GGTACA GTG-AAACTG

GWBC_CJT06_091505_

CC-CTGCATG --TCTAAGTA CGCC---ACG GCC-GGTACA GTG-AAACTG

GWBC_CJT07_091505_

CC-NTGCATG --TCTAAGTA CGC----ACG GCC-GGTACA GTG-AAACTG

MWBC_CJT10_091505_

CC-ATGCATG --TCTAAGTA CGC----ACG GCC-GGTACA GTG-AAACTG

MWBC_CJT11_091505_

CC-ATGCATG --TCTAAGTA CGC----ACG GCC-GGTACA GTG-AAACTG

FWBC_CJT04_091505_

CCCATGCATG --TCTAAGTA CGC----ACG GCC-GGTACA GTG-AAACTG

FWBC_CJT02_091505_

CC-NTGCANG --TCTAAGTA NGC----ACG GCC-GGTNCA GTG-AAACTG

PCWBC_CJT16_091505_ CC-NTGCATG --TNTAAGTA CGC----ACG GNCCGGTACA GTNGAAACTG FWBC_CJT05_091505_

CC-CTGCANG --TCTAAGTN CGC----ACG GCC-GGTACA GTG-AAANTG

FWBC_CJT01_091505_

CNCNCCCNNC GTCCTANNTN CCNTCACTCN CCCCGGTNCA CNGAAACCNC

144 101

110

120

130

140

150

PCWBC_CJT14_091505_ C-GAATGGCT CATT—AAAT C—AGTTATG GTTCC-TTT- -GGTCGCTCPCWBC_CJT15_091505_ C-GAATGGCT CATT—AAAT C—AGTTATG GTTCC-TTT- -GGTCGCTCGWBC_CJT08_091505_

C-GAATGGCT CATT—AAAT C—AATTATG GTTCC-TTT- -GGTCGCTC-

MWBC_CJT32_082305_

C-GAATGGCT CATT—AAAT C—AGTTATG GTTCC-TTT- -GGTCGCTC-

CWBC_CJT29_082305_

C-GAATGGCT CATT—AAAT C—AGTTATG GTTCC-TTT- -GGTCGCTC-

CWBC_CJT30_082305_

C-GAATGGCT CATT—GAAT C—AGTTATG GTTCC-TTT- -GGTCGCTC-

CWBC_CJT28_082305_

C-GAATGGCT CATT—AAAT C—AGTTATG GTTCC-TTT- -GGTCGCTC-

PCWBC_CJT35_082305_ C-GAATGGCT CATT—AAAT CC-AGTTATG GTTCC-TTT- -GGTCGCTCTemplate_

C-GAATGGCT CATT—AAAT C—AGTTATG GTTCC-TTT- -GGTCGCTC-

FWBC_CJT01_082305_

C-GAATGGCT CATT—AAAT C—AGTTATG GTTCC-TTT- -GGTCGCTC-

GWBC_CJT06_091505_

CCGAATGGCT CATT—AAAT C—ACTTATG GTTCCCTTT- -GGTCGCTC-

GWBC_CJT07_091505_

C-GAATGGCT CATT—AAAT C—AGTTATG GTTCC-TTT- -GGTCGCTC-

MWBC_CJT10_091505_

C-GAATGGCT CATT—AAAT C—AGTTATG GTTCC-TTT- -GGTCGCTC-

MWBC_CJT11_091505_

C-GAATGGCT CATT—AAAT C—AGTTATG GTTCC-TTT- -GGTCGCTC-

FWBC_CJT04_091505_

C-GAATGGCT CNTT—AAAT C—AGTTATG GTTCC-TTT- -GGTCACTC-

FWBC_CJT02_091505_

C-GAATGGCT CCTT—AAAT C—AGTTANG GTTCC-TTT- -GGTCGCTC-

PCWBC_CJT16_091505_ C-GAATGGCT CATT—AAAT C—AGTTATG GTNCC-TTT- -GGTCGCTCFWBC_CJT05_091505_

C-GAATGGCT CCATCCAAAT C—AGTTNTG GTTCC-TTT- -GGTCGCTC-

FWBC_CJT01_091505_

CGAANTGGCT CCACTNAAAT CCCNNTNACG GTTCCCNTNC GGNCCGCTCC

145 151

160

170

180

190

200

PCWBC_CJT14_091505_ GCTCCTCTCC -TA-CTTGGA TAACTG-TGG TAATTC—TA GAGCTAATAC PCWBC_CJT15_091505_ GCTCCTCTCC -TA-CTTGGA TAACTG-TGG TAATTC—TA GAGCTAATAC GWBC_CJT08_091505_

GCTCCTCTCC -TA-CTTGGA TAACTG-TGG TAATTC—TA GAGCTAATAC

MWBC_CJT32_082305_

GCTCCTCTCC -TA-CTTGGA TAACTG-TGG TAATTC—TA GAGCTAATAC

CWBC_CJT29_082305_

GCTCCTCTCC -TA-CTTGGA TAACTG-TGG TAATTC—TA GAGCTAATAC

CWBC_CJT30_082305_

GCTCCTCTCC -TA-CTTGGA TAACTG-TGG TAATTC—TA GAGCTAATAC

CWBC_CJT28_082305_

GCTCCTCTCC -TA-CTTGGA TAACTG-TGG TAATTC—TA GAGCTAATAC

PCWBC_CJT35_082305_ GATCNTCTCC -TA-CTTGGA TAACTG-TGG TAATTC—TA GAGCTAATAC Template_

GCTCCTCTCC -TA-CTTGGA TAACTG-TGG TAATTC—TA GAGCTAATAC

FWBC_CJT01_082305_

GCTCCTTTCC -TA-CTTGGA TAACTG-TGG TAATTC—TA GAGCTAATAC

GWBC_CJT06_091505_

GCTCCTCTCC -TA-CTTGGA TAACTG-TGG TAATTC—TA GAGCTCATAC

GWBC_CJT07_091505_

GCTCCTCTCC -TA-CTTGGA TAACTG-TGG TAATTC—TA GAGCTAATAC

MWBC_CJT10_091505_

GCTCCTTTCC -TA-CTTGGA TAACTG-TGG TAATTC—TA GAGCTAATAC

MWBC_CJT11_091505_

GCTCCTCTCC -TA-CTTGGA TAACTG-TGG TAATTC—TA GAGCTAATAC

FWBC_CJT04_091505_

GCTCCTCTCC -TA-CTTGGA TAACTG-TGG TAATTC—TA GAGCTAATAC

FWBC_CJT02_091505_

GCTCCTCTCC -TA-CTNGGA TAACTG-TGG TAATTC—NA GAGCTNATAC

PCWBC_CJT16_091505_ GNTCCTCTCC -NA-CTNGGA TAACTG-TGG TAATTC—TA GAGCTNATAC FWBC_CJT05_091505_

GCTCCTCTCC CTA-CTTAGA TAACTG-TGG TAATTC—TA GAGCTAATAC

FWBC_CJT01_091505_

GCCCCTTTCC CTAACTTGGA TAACCCCTGG TGACTNCCTA CAGCTCATNC

146 201

210

220

230

240

250

PCWBC_CJT14_091505_ -ATGCCGACG GG-CGCTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT PCWBC_CJT15_091505_ -ATGCCGACG GG-CGCTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT GWBC_CJT08_091505_

-ATGCCGACG GG-CGCTGAC CCCCTT—AG CGGGGGGGAT GC-GTGCATT

MWBC_CJT32_082305_

-ATGCCGACG GG-CGCTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT

CWBC_CJT29_082305_

-ATGCCGACG GGGCGCTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT

CWBC_CJT30_082305_

-ATGCCGACG GG-CGCTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT

CWBC_CJT28_082305_

-ATGCCGACG GG-CGCTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT

PCWBC_CJT35_082305_ -ATGCCGACG GG-CGCTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT Template_

-ATGCCGACG GG-CGCTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT

FWBC_CJT01_082305_

-ATGCCGACG GG-CGCTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT

GWBC_CJT06_091505_

-CTGCCGACG GG-CGCTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT

GWBC_CJT07_091505_

-ATGCCGACG GG-CGCTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT

MWBC_CJT10_091505_

-ATGCCGACG GG-CGCTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT

MWBC_CJT11_091505_

-ATGCCGACG GG-CGCTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT

FWBC_CJT04_091505_

-ANGCCGACG GG-CGNTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT

FWBC_CJT02_091505_

-ATGCCGACT GG-CGCTGAC CCCCTT—NG CGGGGGGGAT GC-GTGCATT

PCWBC_CJT16_091505_ -ATGCCGACG GG-CGNTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT FWBC_CJT05_091505_

-ATGCCGACG GG-CGNTGAC CCCCTT—CG CGGGGGGGAT GC-GTGCATT

FWBC_CJT01_091505_

ANNNCCGACG GG-CGCTGAC CCCCTCTCNG CGGGGGGGAT GCCGNGCATT

147 251

260

270

280

290

300

PCWBC_CJT14_091505_ TATCANATCA AAACCAACCC GGTCAGCCCC TCTCTGGCCC CGGCC--GGG PCWBC_CJT15_091505_ TATCANATCA AAACCAACCC GGTCAGCCCC TCTCCGGCCC CGGCC--GGG GWBC_CJT08_091505_

TATCANATCA AAACCAACCC GGTCAGCCCC TCTCCGGCCC CGGCC--GGG

MWBC_CJT32_082305_

TATCAGATCA AAACCAACCC GGTCAGCCCC TCTCCGGCCC CGGCC--GGG

CWBC_CJT29_082305_

TATCAGATCA AAACCAACCC GGTCAGCCCC TCTCCGGCCC CGGCCCGGGG

CWBC_CJT30_082305_

TATCAGATCA AAACCAACCC GGTCAGCCCC TCTCCGGCCC CGGCC--GGG

CWBC_CJT28_082305_

TATCAGATCA AAACCAACCC GGTCAGCCCC TCTCCGGCCC CGGCC--GGG

PCWBC_CJT35_082305_ TATCAGATCA AAACCAACCC GGTCAGCCCC TCTCCGGCCC CGGCC--GGG Template_

TATCAGATCA AAACCAACCC GGTCAGCCCC TCTCCGGCCC CGGCC--GGG

FWBC_CJT01_082305_

TATCAGATCA AAACCAACCC GGTCAGCCCC TCTCCGGCCC CGGCC--GGG

GWBC_CJT06_091505_

TATCAGATCA AAACCAACCC GGTCAGCCCC TCTCCGGCCC CGGCC--GGG

GWBC_CJT07_091505_

TATCAGATCA AAACCAACCC GGTCAGCCCC TCTCCGGCCC CGGCC--GGG

MWBC_CJT10_091505_

TATCAGATCA AAACCAACCC GGTCAGCCCC TTTCCGGCCC CGGCC--GGG

MWBC_CJT11_091505_

TATCAGATCA AAACCAACCC GGTCAGCCCC TTTCCGGCCC CGGCC--GGG

FWBC_CJT04_091505_

TATCAGATCA AAACCAACCC GGTCAGCCCC TTTCCGGCCC CGGCC--GGG

FWBC_CJT02_091505_

TATCAGATCA AAACCAACCC GGTCAGCCCC ACTCCGGCCC CGGCC--GGG

PCWBC_CJT16_091505_ TATCAGATCA AAACCAACCC GGTCAGCCCC TNTCCGGCCC CGGCC--GGG FWBC_CJT05_091505_

TATCAGATCA AAACCAACCC GGTCAGCCCC TNTCCGGCCC CGGCC--GGG

FWBC_CJT01_091505_

TATCAGATCA AAACCAACCC GGTCAGCCCC TTTCCGGCCC CGGCC-GGGG

148 301

310

320

330

340

350

PCWBC_CJT14_091505_ GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTC GGGCCGATCG PCWBC_CJT15_091505_ GGGCGGGCGC CGGCGGCATT GGTGACTCTA G-ATAACCTC GGGCCGATCG GWBC_CJT08_091505_

GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTC GGGCCGATCG

MWBC_CJT32_082305_

GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTC GGGCCGATCG

CWBC_CJT29_082305_

GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTC GGGCCGATCG

CWBC_CJT30_082305_

GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTC GGGCCGATCG

CWBC_CJT28_082305_

GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTC GGGCCGATCG

PCWBC_CJT35_082305_ GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTC GGGCCGATCG Template_

GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTC GGGCCGATCG

FWBC_CJT01_082305_

GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTT GGGCCGATCG

GWBC_CJT06_091505_

GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTC GGGCCGATCG

GWBC_CJT07_091505_

GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTC GGGCCGATCG

MWBC_CJT10_091505_

GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTC GGGCCGATCG

MWBC_CJT11_091505_

GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTC GGGCCGATCG

FWBC_CJT04_091505_

GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTC GGGCCGATCG

FWBC_CJT02_091505_

GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTC GGGCCGATCG

PCWBC_CJT16_091505_ GGGCGGGCGC CGGCGGCTTT GGTGACTCTA G-ATAACCTC GGGCCGATCG FWBC_CJT05_091505_

GGGCGGGCGC CGGCGGCTTT GGTGACTCCN GGATAACCTC GGGCCGATCG

FWBC_CJT01_091505_

GGNNGGGCGC CGGCGGCTTT NGTGACTCTA G-ATAACCTC GGGCCGATCG

149 351

360

370

380

390

400

PCWBC_CJT14_091505_ CACGCCCCCC -GTGGCGGCG ACNACCCATT CGAACGTCTG CCCTATCAAC PCWBC_CJT15_091505_ CACGCCCCCC -GTGGCGGCG ACNACCCATT CGAACGTCTG CCCTATCAAC GWBC_CJT08_091505_

CACGCCCCCC -GTGGCGGCN ACGACCCATT CGAACGTCTG CCCTATCAAC

MWBC_CJT32_082305_

CACGCCCCCC -GTGGCGGCG ACGACCCATT CGAACGTCTG CCCTATCAAC

CWBC_CJT29_082305_

CACGCCCCCC -GTGGCGGCG ACGACCCATT CGAACGTCTG CCCTATCAAC

CWBC_CJT30_082305_

CACGCCCCCC -GTGGCGGCG ACGACCCATT CGAACGTCTG CCCTATCAAC

CWBC_CJT28_082305_

CACGCCCCCC -GTGGCGGCG ACGACCCATT CGAACGTCTG CCCTATCAAC

PCWBC_CJT35_082305_ CACGCCCCCC -GTGGCGGCG ACGACCCATT CGAACGTCTG CCCTATCAAC Template_

CACGCCCCCC -GTGGCGGCG ACGACCCATT CGAACGTCTG CCCTATCAAC

FWBC_CJT01_082305_

CACGCCCCCC -GTGGCGGCG ACGACCCATT CGAACGTCTG CCCTATCAAC

GWBC_CJT06_091505_

CACGCCCCCC -GTGGCGGCG ACGACCCATT CGAACGTTTG CCCTATCAAC

GWBC_CJT07_091505_

CACGCCCCCC -GTGGCGGCG ACGACCCATT CGAACGTCTG CCCTATCAAC

MWBC_CJT10_091505_

CACGCCCCCC -GTGGGGGCG ACGACCCATT CGAACGTCTG CCCTATCAAC

MWBC_CJT11_091505_

CACGCCCCCC CGTGGCGGCG ACGACCCATT CGAACGTCTG CTCTATCAAC

FWBC_CJT04_091505_

CACGCCCCCC -GTGGCGGNG ACGACCCATT CGAANGTTTG CCCTATCAAC

FWBC_CJT02_091505_

CACGCCCCCC -GTGGCGGNG ACGACCCNTT NGAANGTNTG CCCTATCAAC

PCWBC_CJT16_091505_ CACGCCCCCC -GTGGCGGCG ACGACCCATT NGAANGTNTG CCCTATCAAC FWBC_CJT05_091505_

CACGCCCCCC -GTGGCGGCG ACGACCCNTT CGAACGTNTG CCCTATCAAC

FWBC_CJT01_091505_

CANGCCCCCC -GTGGNGGGG ACGACCCNTT NGAANGTTTG CCCNATCAAC

150 401

410

420

430

440

450

PCWBC_CJT14_091505_ TTTCGANGGT AGTCGCCGTG CCTACCAGGG TG-ACCACGG GTGACGGGGPCWBC_CJT15_091505_ TTTCGATGGT AGTCGCCGTG CCTACCANGG TG-ACCACGG GTGACGGGGGWBC_CJT08_091505_

TTTCGATGGT AGTCGCCGNG CCTACCANGG TG-ACCACGG GTGACGGGG-

MWBC_CJT32_082305_

TTTCGATGGT AGTCGCCGTG CCTACCANGG TG-ACCACGG GTGACGGGG-

CWBC_CJT29_082305_

TTTCGATGGT AGTCGCCGTG CCTACCATGG TGGACCACGG GTGACGGGGG

CWBC_CJT30_082305_

TTTCGATGGT AGTCGCCGTG CCTACCATGG TG-ACCACGG GTGACGGGG-

CWBC_CJT28_082305_

TTTCGATGGT AGTCGCCGTA CCTACCATGG TG-ACCACGG GTGACGGGGN

PCWBC_CJT35_082305_ TTTCGATGGT AGTCGCCGTG CCTACCATGG TG-ACCACGG GTGACGGGGTemplate_

TTTCGATGGT AGTCGCCGTG CCTACCATGG TG-ACCACGG GTGACGGGG-

FWBC_CJT01_082305_

TTTCGATGGT AGTCGCCGTG CCTACCATGG TG-ACCACGG GTGACGGGG-

GWBC_CJT06_091505_

TTTCGATGGT AGTCGCCGTG CCTACCATGG TG-ACCACGG GTGACGGGG-

GWBC_CJT07_091505_

TTTCGATGGT AGTCGCCGTG CCTACCATGG TG-ACCACGG GTGACGGGG-

MWBC_CJT10_091505_

TTTCGATGGT TGTCGCCGTG CCTACCATGG TG-ACCACGG GTGGCGGGG-

MWBC_CJT11_091505_

TTTCGATGGT TGTCGCCGTG CCTACCATGG TG-ACCACGG GTGACGGGG-

FWBC_CJT04_091505_

TTTCGATGGT NGTNGCCGTG CCTACCNTGG TG-ACCACGG GTGACGGGG-

FWBC_CJT02_091505_

TTTCGATGGT NGTCGCCGTG CCTACCNTGG TG-ACCACGG GTGACGGGG-

PCWBC_CJT16_091505_ TTTCGATGGT TGTCGCCGTG CCTACCATGG TG-ACCACGG GTGACGGGGFWBC_CJT05_091505_

TTTCGATGGT AGTCGCCGTG CCTACCNTGG TG-ACCACGG GTGACGGGG-

FWBC_CJT01_091505_

NTTCGATGGT TGTNGCCGTG CCTACCNTGG TG-ACCACGG GTGACGGGG-

151 451

460

470

480

490

500

PCWBC_CJT14_091505_ AATCAGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC PCWBC_CJT15_091505_ AATCAGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC GWBC_CJT08_091505_

AATCAGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC

MWBC_CJT32_082305_

AATCAGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC

CWBC_CJT29_082305_

AATCAGGGTT CGATTCCCGG AGAGGGAGCC TGAGAAACGG CTACCACATC

CWBC_CJT30_082305_

AATCAGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC

CWBC_CJT28_082305_

AATCAGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC

PCWBC_CJT35_082305_ AATCAGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC Template_

AATCAGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC

FWBC_CJT01_082305_

AATCAGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC

GWBC_CJT06_091505_

AATCAGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC

GWBC_CJT07_091505_

AATCAGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC

MWBC_CJT10_091505_

AATCAGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC

MWBC_CJT11_091505_

AATCAGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC

FWBC_CJT04_091505_

AATCAGGGTT CGATTCC-GG AGAGGGAGCC NGAGAAACGG CTACCACATC

FWBC_CJT02_091505_

AATCAGGGTT NGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC

PCWBC_CJT16_091505_ AATCAGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC FWBC_CJT05_091505_

AATCAGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC

FWBC_CJT01_091505_

AATCNGGGTT CGATTCC-GG AGAGGGAGCC TGAGAAACGG CTACCACATC

152 501

510

520

530

540

550

PCWBC_CJT14_091505_ CNAGG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGAGGT PCWBC_CJT15_091505_ CAAGG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGAGGT GWBC_CJT08_091505_

CNAGG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGGAGG

MWBC_CJT32_082305_

CNAGG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGAGGT

CWBC_CJT29_082305_

CNAGGGAAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGGAGG

CWBC_CJT30_082305_

CNAGG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGAGGT

CWBC_CJT28_082305_

CNAGG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGAGGT

PCWBC_CJT35_082305_ CAAGG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGAGGT Template_

CAAGG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGAGGT

FWBC_CJT01_082305_

CAAGG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGAGGT

GWBC_CJT06_091505_

CAAGG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGAGGT

GWBC_CJT07_091505_

CAAGG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGAGGT

MWBC_CJT10_091505_

CAAGG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGAGGT

MWBC_CJT11_091505_

CAAGG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGAGGT

FWBC_CJT04_091505_

CNAGG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCNNC CCGGGGAGGT

FWBC_CJT02_091505_

CAAGG-AAGG CAGCAGGNGC GTAAATTACC CACTCCCGAC CCGGGGAGGT

PCWBC_CJT16_091505_ CAAGG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGAGGT FWBC_CJT05_091505_

CAAAG-AAGG CAGCAGGCGC GCAAATTACC CACTCCCGAC CCGGGGAGGT

FWBC_CJT01_091505_

CAAGG-AAGG CAGCAGGNGC GCAAATTACC CACTCCCGAC CCGGGGAGGT

153 551

560

570

580

590

600

PCWBC_CJT14_091505_ GGGTGGACGA AAAATAACAA TACAGG—AC TCTTTCGAGG --CCCT-GTA PCWBC_CJT15_091505_ AGT—GACGA AAAATAACAA TACAGG—AC TCTTTCGAGG --CCCT-GTA GWBC_CJT08_091505_

TAGT-GACGA AAAATAACAA TACNGGG-AC TCTTTCGAGG --CCCT-GTA

MWBC_CJT32_082305_

AGT—GACGA AAAATAACAA CACAGG—AC TCTTTCGAGG --CCCT-GTA

CWBC_CJT29_082305_

TAGT-GACGA AAAATAACAA TACAGG—AC TCTTTCGAGG --CCCT-GTA

CWBC_CJT30_082305_

AGT—GACGA AAAATAACAA TACAGG—AC TCTTTCGAGG --CCCT-GTA

CWBC_CJT28_082305_

AGT—GACGA AAAATAACAA TACAGG—AC TCTNTCGAGG --CCCT-GTA

PCWBC_CJT35_082305_ AGT—GACGA AAAATAACAA TACAGG—AC TCTTTCGAGG --CCCT-GTA Template_

AGT—GACGA AAAATAACAA TACAGG—AC TCTTTCGAGG --CCCT-GTA

FWBC_CJT01_082305_

AGT—GACGA AAAATAACAA TACAGG—AC TCTTTCGAGG --CCCT-GTA

GWBC_CJT06_091505_

AGT—GACGA AAAATAACAA TACAGG—AC TCTTTCGAGG --CCCTTGTA

GWBC_CJT07_091505_

AGT—GACGA AAAATAACAA TACAGG—AC TCTTTCGAGG --CCCT-GTA

MWBC_CJT10_091505_

AGT—GACGA AAAATAACAA TACAGG—AC TCTTTCGAGG --CCCT-GTA

MWBC_CJT11_091505_

AGC—GGCGA AAAATAACAA TACAGG—AC TCTTTCGAGG --CCCT-GTA

FWBC_CJT04_091505_

AGT—GANGA AAAATAACNA TACNCGCGAN TCTTTCGAGG ACTCCTAGTA

FWBC_CJT02_091505_

AGC—GACGA AAAATAACAA TACAGG—AC TCTTTCGAGG --CCCT-GTA

PCWBC_CJT16_091505_ AGT—GACGA AAAATAACAA TACAGG—AC TCTTTCGAGG --CCCT-GTA FWBC_CJT05_091505_

AGT—GACGA AAAATAACAA TACAGG—AC TCTTTCGAGG --CCCT-GTA

FWBC_CJT01_091505_

AGT—GACGA AAAATAACAA TACAGG—AC TCTTTNGAGG --CCCT-GTA

154 601

610

620

630

640

650

PCWBC_CJT14_091505_ ATTGN-AATG AGTCCACTTT AAATCCNTTA ACG-AGNATC CNTNGGAGGG PCWBC_CJT15_091505_ ATTGG-AATG AGTCCACTTT AAATCCTTTA ACG-AGGATC CNTTGGAGGG GWBC_CJT08_091505_

ATTGG-AATG AGTCCACTTT AAATCCTTTA ACG-AGNATC CNTTGGAGGG

MWBC_CJT32_082305_

ATTGG-AATG AGTCCACTTT AAATACTTTA ACNGAGGATC CATTGGAGGG

CWBC_CJT29_082305_

ATTGG-AATG AGTCCACTTT AAATCCTTTA ACG-AGGATC CATTGGAGGG

CWBC_CJT30_082305_

ATTGG-AATG AGTCCACTTT AAATCCTTTA ACG-AGGATC CATTGGAGGG

CWBC_CJT28_082305_

ATTGGGAATG AGTCCACTTT AAATCCTTTN ACG-AGGANC N-TTGGAGGG

PCWBC_CJT35_082305_ ATTGG-AATG AGTCCACTTT AAATCCTTTA ACG-AGGATC CATTGGAGGG Template_

ATTGG-AATG AGTCCACTTT AAATCCTTTA ACG-AGGATC CATTGGAGGG

FWBC_CJT01_082305_

ATTGG-AATG AGTCCACTTT AAATCCTTTA ACG-AGGATC CATTGGAGGG

GWBC_CJT06_091505_

ATTGG-AATG AGTCCACTTT AAATCCTTTA ACG-AGGATC CATTGGAGGG

GWBC_CJT07_091505_

ATTGG-AATG AGTCCACTTT AAATCCTTTA ACG-AGGATC CATTGGAGGG

MWBC_CJT10_091505_

ATTGG-AATG AGTCCACTTT AAATCCTTTA ACG-AGGATC CATTGGAGGG

MWBC_CJT11_091505_

ATTGG-AATG AGTCCACTTT AAATCCTTTA ACG-AGGATC CATTGGAGGG

FWBC_CJT04_091505_

ATTNG-AATG AGTCCACNTT AAATCCTTTA ATCGAGGATC CATTGGAGGG

FWBC_CJT02_091505_

ATTGG-AATG AGTCCACTTT AAATCCTTTA ACG-AGGATC CATTGGAGGG

PCWBC_CJT16_091505_ ATTGG-AATG AGTCCACTTT AAATCCTTTA ACG-AGGATC CATTGGAGGG FWBC_CJT05_091505_

ATTGG-AATG AGTCCACTTT AAATCCTTTA ACG-AGGATC CATTGGAGGG

FWBC_CJT01_091505_

ATTGG-AATG AGTCCACTTT AAATCCTTTA ACG-AGGATC CATTGGAGGG