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THE y-TÜBÜLIN GENE FAMILY IN HOMO SAPIENS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State UniversiQr

By

Dawnne O'neal Wise, B.S.

*****

The Ohio State University 1999

Dissertation Committee: Dr. Berl R. Oakley, Adviser Dr. Arthur H. M. Burghes Approved! Dr. Thomas J. Byers Dr. Paul A. Fuerst Advisor Department of Molecular/Genetics UMI Number 9951743

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Bell & Howell Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Art)or, Ml 48106-1346 ABSTRACT

Y is an essential component of the organizing centers of eukaryotes. It is required for the nucléation of microtubule assembly from microtubule organizing centers in animal and fungal cells, and it is, consequently, essential for mitosis and for the organization of the microtubule in these organisms. Some of the most useful cancer chemotherapy agents target , and it has been proposed that

Y-tubulin may be the site of action for some experimental cancer chemotherapy agents (Bai e t al., 1993). In spite of the importance of y tubulin, little is known of the number and expression patterns o f y-tubulin genes in humans.

Zheng et al. (1991b) identified a human y-tubulin cDNA (Hyl) from a HeLa library. To determine if other y-tubulin genes are present in the human genome, I have carried out Southern hybridization analysis of HeLa DNA. The results indicate that there are probably no more than three y-tubulin sequences in the human genome. I have screened a HeLa cDNA library and isolated cDNA clones that contain partial sequences of a novel y-tubulin gene that I have named H 72 . hi order to obtain a full-length copy of H 72 ,1 have screened the human expressed sequence tag (EST) database (dbEST) and have obtained a clone I have designated H^^EST. I have also identified a processed pseudogene ii of Hyl. This is the first y-tubulin pseudogene identified in any organism.

Hyl has been mapped to chromosome 17 by several labs involved in the search for the BRCAl breast cancer susceptibility gene (Rommens e t al., 1995; Friedman e t a l.,

1995). I present data localizing Hy2 to the same 700 Kb Yeast Artificial Chromosome that carries Hyl.

Finally, I have analyzed the expression patterns of Hyl and H ^ in several human tissues by reverse transcriptase-polymerase chain reaction analyses. Both Hyl and Hy2 are expressed in all tissues examined.

ui Dedicated to my mother, Gwendolyn O’neal, who loved me first and loves me most; my grandparents, Laura and Isaiah Sneed, who showed me how to love unconditionally; my sister, Elisha O’neal, who loves me in spite of myself; my dad, Carlton O’neal, who learned to love me; and to my husband, Raymond Wise, who chose to love me and taught me the meaning of trae love.

IV ACKNOWLEDGEMENTS

In order for any person to accomplish any lofty or worthy goal he or she needs the advice, help, encouragement and assistance of many people. Although my graduate diploma will only have my name on it, 1 have matriculated through graduate school on the shoulders of many wonderful people and my simple thanks can never express how important you all have been to me. First, let me thank the triune God for giving me the strength to stand and allowing me to accomplish this goal in His time.

Professors: 1 wish to thank my advisor. Dr. Berl R. Oakley, for giving me the opportunity to start my scientific career in his laboratory. Dr Oakley, you have taught me the importance of carefully studying, planning and executing scientific experiments as well as the importance of maintaining the highest standards at all times even in the most minute details. Thanks Dr. Oakley. 1 also want to thank all of my committee members. Dr. Arthur

Burghes, Dr. Thomas Byers and Dr. Paul Fuerst for their support and scientific guidance during my graduate career. Dr. Fuerst, you gave me the fellowship that allowed me to enter graduate school. Dr. Byers, you allowed me to start my graduate training in your lab.

Dr. Burghes, thanks for all your advice about my project and thank you in advance for allowing me to start my postdoctoral training in your lab. I thank Dr. Roy Tassava for being my undergraduate advisor and inspiring me to go to graduate school. You all have contributed to the excellent training I have received in the Dept, of Molecular Genetics.

Collaborators and contributors: I would also like to thank Dr. Ralph Krahe and the members of his lab that helped us to do the chromosomal mapping work presented in

Chapter 2 of this thesis. Your help and willingness to lend your expertise to my project is greatly appreciated. I thank Dr. Joaima Rommens for sending me the HSD2 YAC strain to use in the chromosomal mapping work shown in Chapter 2. Your help and advice has been quite helpful. I thank Dr. Helmut Shraudolf for giving us the H^F genomic clone.

Many thanks to John Lucas, PhJ), Dan Luo, PhD and Tom Knobloch, for giving me

HeLa cells and assisting me in tissue culture experiments over the past few years.

The Oakley Lab Past and Present: When I entered the Oakley lab I completed my rotation under the guidance of Yixian Zheng. Thanks Yixian for helping me get started— I knew you were going to be a great scientist. Liz, what can I say! Chocoholics forever!

You are a great teacher! Your attention to detail at the bench and your editing skills are an asset to us all. I thank Yisang Yoon, Tetsuya Horio, and Tomohiro Akashi for all the help and friendship in the past. Mary Ann Martin and Yassmine Akkari, words cannot express how much you meant to me during the years we shared in the lab (Gary and Jeff I thank you too). I miss you both so much. Kathy Jung, you have only been gone a few weeks but it has seemed like an eternity. Thanks for all the advice, kind words and help over the years. You have been a great mentor to me; thanks. Rajinish, Yulia and Natalie— you all

v i have beea a pleasure to work with and I know our Mendships will continue in the future.

Friends: There have been many other graduate students that have helped me and made my graduate experience a pleasurable one. Thank you all: Eric Yang, ScotQr Wallœr, John

Carpten, Jennifer and Keith Thornton, Audrey Napier, Dolena LeDee, Gary Grumbling, and all the past members of the Burghes lab.

I thank all the "First Family" and the members of Faith Ministries Church that have encouraged me and prayed for me over the years. “Sisters of Faith" you all have been a blessing to me and a constant encouragement to me and I thank you for your wimess.

Bren'a and Andrea thanks for your prayers; they have availed. I thank my sister, Karen

(Dr. Alsbrooks) for sharing in my life, praying for me and for giving me a beautiful God- daughter-glory. Regina thanks for your fiiendship and words from on high; continue to let Him use you. Love ya sis. My sincere thanks and love go to my big sister, Priscilla.

Your friendship, encouragement, prayers, listening ears, gifts etc.... and so much more that I could never express in words have kept my spirit lifted. God has made me a millionaire because I have seen Him and experienced His love through all of you.

Family: To "the Family"- Bonnie and Frank, all I can say is thank you; I love you; 1 bless God for you; 1 could not have done it without you; your love and sacrifice will be rewarded and hopefully I will be able to bless your life in the years to come like you've blessed mine! Mom Lane, thanks for loving me and letting me be me—thanks for the com, combread and ho-cake too! And Sassy you are my favorite niece! Mom Wise, you are a jewel and I thank you for you love and encouragement over the years. I would also like to vii thank the entire Wise Family for loving me and accepting me with open arms. I would be remiss if I did not thank my grandparents, Laura and Isaiah Sneed, and the Sneed Clan h)r loving me unconditionally; you all are the greatest. Elisha Laurell thanks for making me smile like only you can. You have grown up to be a beautiful young women. I love ya sis. Daddy, we have come a long way over the years and I thank God for you and all the love you have shown me. You are a special man and I thank God for blessing me to have you as a father. Mom, I know no one is as happy for me as you are. I miss you so much and thank God for your support and prayers over the years—you were always the somebody praying for me. Raymond, I can only say 1 LOVE YOU. Thank you for the words of encouragement that kept me going. You have put up with so much during these years. You have let me pursue my dreams even when you had to sacrifice your own. We have only just begun. It does not yet appear what we shall be! Just think, we can look forward to doing this again next year when you graduate.

YUl VITA

November 26, 1967 ...... Bom, Bridgeport, CT, USA

1990...... B.S. Molecular Genetics, Ohio State University

1990-present...... Graduate Research and Teaching Associate, Dept, of Molecular Genetics, The Ohio State University, Columbus, Ohio

PUBLICATIONS

1. D.Wise and B.R. Oakley. Identification of a Second Gamma-Tubulin Gene in Homo Sapiens. Molecular Biology of the 6s:39a. 1995.

2. D. Wise and B.R.Oakley. Expression Patterns of Two Gamma-Tubulin G enes in Homo Sapiens. Molecular biology of the Cell 8s:45a. 1997.

FIELD OF STUDY

Major Field: Molecular Genetics

IX TABLE OF CONTENTS

EAGE

ABSTRACT ...... ü

DEDICATION ...... iii

ACKNOWLEDGEMENTS...... iv

VITA ...... vüi

L IS T O F...... TABLES...... xiii

LIST OF FIGURES...... xiv

CHAPTERS:

I. INTRODUCTION ...... 1

Microtubules ...... I General structure and function ...... 1 Microtubule dynamics ...... 6 T u b u lin ...... 11 General characteristics of tubulin ...... 11 Multigene families of a and P ...... 12 Functional significance of multiple tubulin isotypes 15 Microtubule organizing centers (MTOCs) ...... 18 ...... 19 Spindle pole bodies ...... 24 Plant MTOCs...... 26

y T u b u lin ...... 27 Discovery of y tubulin ...... 27 U b iq u ity ...... 28 Cellular localization and function ...... 29 Biochemical characterization of y tubulin and the yTuRC... 33 Multigene families of y tubulins ...... 39 Rationale for my dissertation research ...... 41

IDENTinCATION, CLONING AND ANALYSIS OF ADDITIONAL y- TUBULIN SEQUENCES IN HOMO SAPIENS...... 43

Introduction ...... 43 Materials and Methods ...... 49 Isolation of HeLa genomic DNA ...... 49 Southern analysis ...... 49 Sequence analysis of a genomic human y-tubulin clone (H‘)F) __ 51 Library screening ...... 51 EST database search, subcloning and sequencing of a full-length H y 2 c lo n e ...... 52 Construction of plasmid pASHy2 ...... 53 Chromosomal mapping ...... 55 Preparation of YAC DNA for PCR analysis ...... 57 PCR analysis and DNA sequencing of Hyp ...... 59 Blast search for HyF ...... 60 R e su lts...... 61 Estimation of the number of y-tubulin sequences in Homo sa p ie n...... s 61 Identification and characterization of a novel human y-tubulin cD N A , H y2 ...... 62 Somatic cell and radiation hybrid mapping of Hy2 ...... 80 is a fragment from a y-tubulin pseudogene ...... 92 D isc u ssio n ...... 104

3. ANALYSIS OF THE EXPRESSION PATTERNS OF y^TUBUUN GENES IN HOMO SAPIENS...... 107

XI Introduction ...... 107 Materials and methods ...... 109 Strains and media...... 109 Plasmid pASHy2F construction ...... 109 S. pom be transformation ...... I l l Isolation of total RNA firom 5. pombe strains...... 112 Isolation of total RNA firom HeLa cells and human tissues 112 RT-PCR analysis ...... 113 R e s u lts...... 114 Rationale for primer design and controls for RT-PCR ...... 114 Expression patterns of y-tubulin genes in human tissues 120 D isc u ssio n ...... 123

Bibliography ...... 127

XU LIST OF TABLES

Table Page

1. Organisms firom which y-tubulin genes or cDNAs have been isolated 30

2. Components of yTuRCs in different organisms ...... 38

3. Human a- and P-tubulin gene chart ...... 45

xm UST OF FIGURES

Figure

1. Microtubule lattice arrangements ...... 5

2. Low stringency Southern analysis of human genomic DNA using a 5'-HyI probe ...... 64

3. Low stringency Southern analysis of human genomic DNA using a B'-Hyl probe ...... 66

4. The nucleotide sequence of and its amino acid sequence 68

5. A comparison of the predicted amino acid sequences of and H y l ...... 71

6. The nucleotide and predicted amino acid sequence of clones pDOW? and pDOW21 ...... 73

7. The nucleotide and predicted amino acid sequence of clones pDOWla and pDOW12 ...... 76

8. A comparison of the predicted amino acid sequences of Hy2- clone pDOW7 and Hyl ...... 78

9. The nucleotide sequence of H’^EST and its predicted amino acid sequence ...... 82

10. The construction of plasmid pASHy2 ...... 85

11. A comparison of the predicted amino acid sequences of H]^EST

XIV and H y l ...... 87

12. A comparison of the nucleotide sequences in the 3UTR regions of Hyl and Hy2 ...... 89

13 Schematic diagram of human chromosome 17 showing Hyl, BRCAl, and Hy2 ...... 91

14. PCR analysis of HSD2 YAC DNA with Hy2 specific primers 94

15. A comparison of the predicted amino acid sequences ofH )f and Hyl with the H ]f specific primers underlined ...... 96

16. PCR amplification of H ]f from HeLa cell and human genomic DNA using HyF specific primers ...... 99

17. A comparison of the nucleotide sequence of human genomic clone "DJ1165K" with Hyl and HyF ...... 101

18. A comparison of the nucleotide sequences found in the 3XJTRs of Hyl and Hy2 ...... 117

19. The construction of plasmid pASH^ and S.pombe strain H M H G 2...... 119

20. PCR analysis of total RNA isolated firom three S. pombe strains used as controls for RT-PCR ...... 122

21 RT-PCR analysis of the expression patterns of Hyl and Hy2 in several human tissues...... 125

XV CHAPTER 1

INTRODUCTION

Microtubules

General structure and function of mlcrotubules

The eukaryotic cytoskeletal network is composed of three types of protein filaments— microtubules, filaments, and intermediate filaments— that provide the cell with strength, shape and the ability to coordinate movement Microtubules are cylindrical, dynamic cellular . They are involved in a varied of cellular fimctions including the maintenance of cellular morphology and polarity, chromosome movement during mitosis and meiosis, intracellular transport of cytoplasmic organelles and vesicles, and ciliary and flagellar motiliQr. In cilia, flagella, and most sperm tails, microtubules are arranged in a 9+2 structure in which outer doublet microtubules surround two central pair microtubules. Centrioles and basal bodies are composed of nine microtubule triplets surrounding an amorphous core.

Microtubules vary in length and are approximately 25 nm in diameter. They are composed of two globular subunits called a and P tubulin that form a heterodimer (Bryan

1 and Wilson, 1971). Both monomers aie ^iproximately 50 kdin size, 450 amino acids in length and they share 36-42% amino acid identic (reviewed in Little and Seehaus, 1988).

Tubulin heterodimers are arranged in a head-to-tail fashion forming longitudinal protofilaments which have a main 4 nmpeiiodici^ corresponding to the monomers and an

8 nm periodici^ corresponding to the heterodimers (Amos and Klug, 1974). Adjacent protofilaments are staggered by 0.9 nm forming a 3-start left-handed helix (Amos and

Klug, 1974; Linck and Amos, 1974; Mandelkow e t al., 1986).

The actual nature of the lateral interactions between monomers of adjacent protofilaments was a long-standing controversy in microtubule research. Originally, Amos and Klug (1974) analyzed diffiaction patterns of electron micrographs of flagella and suggested that the “A” microtubules of flagellar doublets formed what they called the “A” lattice and B-tubules formed a "B " lattice (see Figure la and lb). In the A-lattice (Figure la), a-tubulin monomers of one protofilament and P-tubulin monomers of adjacent protofilaments interact The A-lattice was favored as the general structure of microtubules for many years because 13-protofilament microtubules are helically symmetrical in this type of lattice. Song and Mandelkow (1993, 1995), however, used -decorated microtubules to show that the A and B tubules of flagellar outer doublets and in vitro assembled brain tubulin form a B-lattice. fii the B-lattice (see Figure lb), a-tubulin monomers of one protofilament interact with a monomers of adjacent protofilaments and p-tubulin monomers of adjacent protofilaments interact. These findings have been corroborated in vivo by Kikkawa e t al. (1994) and by the high resolution model of the 2 microtubule proposed by Nogales e t a l (1999).

In the majority of eukaryotes, microtubules are composed of 13 protofilaments

(Ledbetter and Porter, 1964; Porter, 1966; Tilney e t al., 1973). Exceptions to this rule

have been observed in specific cell types of crayfish, Caenorabditis elegans, guinea pigs.

Drosophila melanogasterand Homo sapiens (Burton et al., 1975; Savage e t al., 1989; Raff

et al., 1997; Kikuchi e t al., 1991). Protofilament number varies greatly when microtubules

are assembled in vitro, ranging from 11 to 16 protofilament microtubules, depending upon

the assembly conditions used in the experiments (McEwenand Edelstein, 1977; Scheeleer

al., 1982; Evans era/., 1985; Wade era/., 1990). Evans era/. (1985) demonstrated that the

protofilament number appears to be established by microtubule organizing centers

(M TOCs) in v itr o . Interestingly, genetic studies conducted in C. elegans and D. melanogaster suggest that protofilament number may not only be established by MTOCs but can also be controlled by specific P-tubulin isotypes (Savage e t al., 1989; Raff et al.,

1997).

Microtubules are intrinsically polar, dynamic polymers. The plus end of the microtubule, which, in cells, is distal to the MTOC, is fast growing, while the minus end of the microtubule, which is proximal to the MTOC, is slow growing (Bergen & Borisy,

1980). The polarity of microtubules is important because it facilitates the directional movement of cargos by motor proteins. For example, conventional are plus end- directed motors, while are minus end-directed motors. These motors transport essential cargo (e.g. organelles, other proteins, chromosomes) along microtubules in a 3 Figure 1: Microtubule lattice arrangements.

In the A-lattice, a-tubulin monomers of one protofîlament interact with P-tubulin monomers of adjacent protofilaments. A 13-protofilament microtubule with an A- lattice will form a helically symmetrical lattice. In the B-lattice a-tubulin monomers contact a- tubulin monomers in adjacent protofilaments and P-tubulin monomers contact P-tubulin monomers. The lateral interactions between protofilaments causes a 13-protofilament microtubule to have a seam, where a tubulin and P tubulin are in lateral contact, because one turn of a 3-start left-handed helix creates a rise of three tubulin monomers. Seam

a. A-Lattice b. B-Lattice

Figure 1. Courtesy of Tetsuya Horio directional manner (reviewed in Hirokawa e ta l., 1998; Barton and Goldstein, 1996).

Previous biochemical and sequence analysis of tubulin (Weisenberg e ta l., 1968;

Geahlan and Haley, 1977) revealed that tubulin is a GTP-binding protein. The GTP on P

tubulin is hydrolyzable, while the GTP on a tubulin is nonhydrolyzable ^acN eal and

Purich, 1978; Spiegelman et al., 1977). Mote recently, Mitchison (1993) found that GTP-

coated fluorescent beads bind specifically to the plus ends of microtubules indicating that

the exchangeable site must be exposed at the plus end. Nogales e t al. (1998a) used electron

crystallography to show that the GTP binding site on a tubulin is located at the intradimer

interface between the tubulin subunits, while the hydrolyzable site on P tubulin is partially

exposed in the dimer but buried after hydrolysis and addition of another dimer during

elongation of the microtubule (Nogales e t al., 1998a, 1999). Therefore, the orientation of

the tubulin dimer relative to the polariQr of the microtubule lattice has been established with

the P-tubulin subunit being terminal at the plus end and the a-tubulin subunit being

terminal at the minus end (Nogales et al., 1999).

Microtubule dynamics

Early in this century E 3 .Wilson (1925), along with other cytologists, realized that

the linear fibers that make up the mitotic spindle change rapidly with time. Later Inoué and

colleagues (1951) used a sensitive polarized light microscope to analyze the dynamic

formation and disappearance of these mitotic fibers. They demonstrated that the protein

subunits in the spindle polymer were in dynamic equilibrium with the subunits in the cell’s . It then became clear that a better understanding of the mechanism of 6 microtubule polymerization needed to be determined. Weisenberg (1972) demonstrated

that microtubules can be assembled in vitro from rat brain homogenates by warming the

homogenates in the presence of GTP, Mg+, and EGTA, a calcium chelator. Subsequently,

Gaskin et al. (1974) used turbidity measurements to study microtubule assembly m vitro

and found that assembly only occiurs above a specific concentration, called the critical

concentration for assembly.

Over the past twen^-five years three main theories have been advanced to explain

microtubule dynamics (reviewed in Desai and Mitchison, 1997). Initially the steady-state

theory of subunit polymerization postulated that when microtubules are in equilibrium with

the tubulin in the cytoplasm there is constant association and dissociation of tubulin dimers

at microtubule ends which allows the length of the microtubule to remain constant

(Oosawa, 1962). While studying the dynamic exchange of subunits on microtubule ends,

Margolis and Wilson (1978) proposed the theory of treadmilling in which a net gain of

tubulin at the plus end of the microtubule and a net loss of tubulin at the minus end of the

microtubule keeps the microtubule length constant at equilibrium.

Subsequently, Mitchison and Kirschner (1984a, b), inferred from length

measurements of microtubules in vitro, that within a population of microtubules some were growing, while others were shrinking— a phenomenon they called “dynamic instability”.

Later, Horio and Hotani (1986) demonstrated directly, by observing individual microtubules in vitro using dark field microscopy and real-time video recording, that growing and shrinking microtubules do coexist within a population, with the individual 7 microtubules alternating between these two phases. Later, Walker e ta l. (1988) coined the term "catastrophe” to refer to the microtubules* transition firom elongation to rapid shortening; whereas the reverse transition, from shortening to elongation, was called

"rescue". Dynamic instability of microtubules also occurs in vivo as has been shown by

Salm on et al. (1984), Saxton e t al. (1984), Koshland e t al. (1988) and Cassimeris e ta l.

(1988). When compared to the dynamic instability of pure tubulin assembled in vitro, microtubules assembled in vivo polymerize at a rate that is five to ten times faster than that of pure mbulin (at a similar concentration), and they exhibit a high fiequency of catastrophe

(reviewed in Cassimeris, 1993; McNally, 1996).

The exact mechanism by which dynamic instability occurs is not known. Originally biochemical analysis of tubulin (Weisenberg et a l., 1968) revealed that tubulin is a GTP binding protein. The GTP on P tubulin is hydrolyzable, while the GTP on a tubulin is nonhydrolyzable (MacNeal and Purich, 1978; Spiegelman et a/., 1977). Mitchison &

Kirschner (1984a) proposed the GTP cap model of dynamic instabiliDr, which postulates that a cap of GTP-tubulin at the end of microtubules stabilizes the microtubule and promotes assembly (reviewed in Erickson and O’Brien, 1992; Desai and Mitchison, 1997).

This model was based on the work of Carlier and Pantaloni (1981) which suggested that

GTP hydrolysis is not coupled to polymerization. As microtubules assemble, GTP-tubulin would be added to the ends of the microtubules forming GTP-tubulin caps. The GTP would be hydrolyzed at a rate independent of the microtubule assembly rate. At equilibrium GTP hydrolysis would overtake assembly in a probablistic fashion causing the 8 loss of the GTP cap at an end of some microtubules and rapid disassembly of those

microtubules. Other microtubules with GTP caps intact would continue to assemble.

Subsequent research has cast doubt on this theory because the ends of microtubules do not

disassemble totally after catastrophe as would be predicted by this model (Horio and

Hotani, 1986; Walker et al., 1988). Also measurements of the GTP cap indicates that it is

very small and that the original data of Carlier and Pantaloni (1981) were incorrect. Studies

of tubulin polymerization in the presence of nonhydrolyzable GTP analogs (GMPPNP and

GMPPCP) suggest that the primary role of GTP hydrolysis is to destabilize the microtubule lattice by creating GDP-bound subunits that have weaker interdimer contacts (Mandelkow

et al., 1991; Vale e t al., 1994). More recently Tran e t al. (1997a, b) have proposed an updated three-state conformational cap model of microtubule dynamic instabiliQr in vitro in which a metastable kinetic intermediate state exists between elongation and shortening states of dynamic instability.

Recently Rodionov and Borisy (1997) have revisited the theory of treadmilling by showing that cytoplasmic microtubules of fish melanophores detach from their nucléation site and depolymerize from their minus ends. These fi%e microtubules are released from centrosomes and move toward the periphery of the cell by treadmilling. Margolis and

Wilson (1998) suggest that while microtubules obviously undergo dynamic instabiliQr

(especially during the interphase to mitotic transition), treadmilling and microtubule motor proteins could account for a poleward migration of microtubules in the mitotic spindle known as flux and for microtubule dynamics of some interphase cells. Interestingly both 9 dynamic instability^ and treadmilling may occur in cells, with treadmilling occurring in

stable interphase microtubules and in the mitotic spindle causing a "flux" of subunits in the

kinetochore to pole direction, while dynamic instability is required for fast rearrangements

of microtubules needed for mitosis to occur (reviewed in Anderson, 1998; Margolis and

WUson, 1998).

Microtubule dynamics is also regulated in some cells by proteins called MAPs

(microtubule associated proteins). Originally MAPs such as MAPI, MAP2, MAP4 and

Tau were identified as proteins that were purified with tubulin when microtubules were purified firom mammalian brain tissue by repeated rounds of polymerization and depolymerization (Sloboda et a l., 1975; Murphy and Borisy, 1975; Weingarten cr af.,

1975). MAPs have been shown to stabilize microtubules and promote the assembly of microtubules by binding to the C-terminal region of tubulin (discussed in Nogales et at.,

1999) and lowering the critical concentration of tubulin assembly. MAPs also abolish microtubule dynamic instability. They are less prevalent in other tissues than in neuronal tissues, however. So their roles in microtubule dynamics in non-neuronal cells is open to question. For a detailed review of MAPs see Maccioni and Cambiazo (1995).

Microtubules also interact with motor proteins that exist in two main families known as kinesins and dyneins. These motors play an integral role in cellular mechanisms like transport and mitosis (reviewed in Hirokawa e t al., 1998; Vallee and Gee

(1998).

10 Tubulin

General characteristics of tubulin

Since the first complete sequences of a - and P-tubulin were published (Krauhs et aL, 1981; Ponstingl et aL, 1981), analysis of a- and p-tubulin sequences hrom phylogenetically diverse organisms has shown that all a tubulins are highly homologous to each other and all P tubulins are highly homologous to each other, except for their extreme

C-termini. All a tubulins share > 62.3% amino acid identic and all P tubulins share >

63.3% amino acid identity (reviewed in Little and Seehaus, 1988; Bums, 1991; 1994).

Because a and P tubulins firom diverse species share high amino acid homology.

Bums (1991) postulated that each tubulin monomer has a very similar tertiary stmcture.

Bums’ hypothesis has recently been confirmed by the publication of a 3.7Â atomic model of the a~P tubulin dimer, obtained by electron crystallography of tubulin sheets polymerized in the presence of zinc (Nogales et aL, 1998a). These data reveal that the stmcture of the two tubulin monomers is nearly identical. Each monomer has been divided into three functional domains based on its stmcture: 1) the N-terminal domain containing the GTP nucleotide binding region, 2) an intermediate domain, and 3) the C-terminal domain which has been implicated in the binding of MAPs and motor proteins (Nogales et al., 1998a). This model confirms the idea that regions of high homology between a and P tubulin have been conserved because of their functional significance. In addition to its binding GTP and MAPs, the tubulin dimer also binds to medicinally and agriculturally important dmgs such as taxol, nocodazole, benomyl, colchicine, and (reviewed 11 in Wilson and Jordan, 1994). Now that the structure of the dimer has been revealed, the sites of action of these drugs may be analyzed in further detail.

a and P tubulin share similar structures with each other and show overall structural similarity to the bacterial cell-division protein FtsZ (£ilamenting ten^rature-gensitive mutant Z), that is the leading candidate for a prokaryotic homolog of tubulin. Even though

FtsZ, a, and ^ tubulin only share 7% sequence homology at the amino acid level their tertiary structures are very similar and the regions of high sequence homology are those involved in GTP binding (Lowe and Amos, 1998; Nogales et aL, 1998b). FtsZ also polymerizes into tubular polymers, like tubulin, that form a ring of filaments or tubules needed for septation and proper cell division (Erickson, 1996; Nogales et aL, 1998b;

Bramhill etal., 1994; Erickson et aL, 1995).

Multigene families of a and P tubulins

In 1980, Cleveland and colleagues found that a and P tubulins are encoded by small multigene families using Southern blot analyses of chicken, sea urchin, angler fish, mouse, rat and human genomic DNA. Multiple a- and ^-tubulin genes have now been isolated and sequenced firom a variety of organisms ranging firom ciliated protozoans to humans (reviewed in Little and Seehaus, 1988; Bums et aL, 1991). The size of multigene families, within a species, ranges firom small families of one or two a- or P-tubulin genes

(as in fungi), to large gene families like those found in plants (nine expressed P-tubulin genes in Arabidopsis thaliana) (Raff et aL, 1994; Little and Seehaus, 1988). Southern blot analysis suggests that as many as 15-20 a - and P-tubulin sequences may exist in the

1 2 human genome (Cleveland et aL, 1980). Only four functional a-tubulin genes and four functional P-tubulin genes have been identified in humans while at least nine P-tubulin pseudogenes have been identified (Lee et aL, 1983; Wilde et aL, 1982; reviewed in

Sullivan, 1988; Luduena, 1998).

The multigene families that encode a - and P-tubulin sequences produce different

'isotypes' of each protein. In this thesis the term 'isotype' will be used to describe different genes and the different polypeptides encoded by these genes. The term 'isoform' will be used to describe an a- or P-tubulin protein that has been post-translationally modified, and they will be discussed later in this chapter. Multiple a - and P-tubulin isotypes have been identified in protists, fungi, plants and animals.

The IS C-terminal amino acids of a and P tubulins are highly variable. When sequences of avian and mammalian p-tubulins were compared however, it was found that isotype classes of tubulins exist. The 15 C-terminal amino acids of tubulins within an isotype class are conserved among organisms but they differ as much as 100% with tubulins of other isotype classes, even within the same organism. Vertebrate P-tubulin genes fall into six major isotype classes (Sullivan and Cleveland, 1986; Little and Seehaus,

1988; Luduena, 1988)

Within a given isoQrpe class, the 3' noncoding regions of a and P tubulins are often conserved across species (reviewed in Little and Seehaus, 1988; Cleveland and Sullivan,

1986). The same is true for the S' untranslated region of tubulins but to a lesser degree

(Cowan and Dudley, 1983; Little and Seehaus, 1988). The conservation of vertebrate 13 isotype classes suggests that different isotypes may have functional significance. Unlike the sequences of a and P tubulins in higher eukaryotes, a - and p-tubulin sequences in lower non-animal eukaryotes (/.e. fungi and protists) are postulated to have diverged fiom other tubulin sequences because of fewer structural constraints on tubulins in these organisms (Raff et aL, 1994; Luduena, 1998). a-Tubulin genes also fall into six major iso^rpe classes. These isotype classes are conserved in mammals but are not conserved in birds.

Another level of tubulin heterogenei^ is due to post-translational modification of tubulin. Post-translational modifications may be the only sources of tubulin diversiQ r in species that lack multiple tubulin isoQrpes (e.g. Chlamydomonas reinhardtii, Youngblom et aL, 1984). These modifications occur on either monomer or on both, as well as the microtubule polymer itself and they are enzymatically driven, a-tubulin modifications include acétylation and the detyrosination and ^osination of the C-terminus (reviewed in

MacRae, 1997; Luduena, 1998). A special form of a-tubulin called A2-tubulin found in cells involved in neuronal processes exists as a result of the removal of the C-terminal tyrosine and glutamine (Mary et aL, 1996; MacRae,1997). P-tubulins are modified by phosphorylation of residues like Qrrosine and serine (MacRae, 1997). Both a and P tubulin undergo polyglutamylation or polyglycylation (Edde etal., 1990; Redicker et aL,

1994). All these modifications seem to be associated with tubulin in stable microtubule populations like those found in axonemes, and brains and with subpopulations of stable microtubules found in the cytoplasm o f mammalian cells.

14 Functional significance of multiple tubulin isotypes

Although it has been known for almost twen^ years that tubulins are encoded by

multigene families, the functional significance of multiple tubulin isotypes is still being

debated today. Prior to the identification of tubulin as a heterodimer and prior to the

cloning of multiple tubulin genes, Behnke and Forer (1967) were the first to propose that

different tubulins are found in different microtubular organelles of cranefly spermatids

based on the different biochemical properties of the microtubules. Subsequently, Fulton

and Simpson (1976) proposed the "multi-tubulin hypothesis" which states that an organism

has multiple tubulin genes that encode different polypeptides which support different

microtubule functions. To date the only evidence that substantiates this theory has been

found with a D. melanogaster testis-specific p2 gene (Hoyle and Raff, 1990, Fackenthal et

aL, 1995; Raff et aL, 1997) and very specialized a- and P-tubulin isotypes found in

Caenorhabditis elegans (Savsige et aL, 1989; Fukushigeera/., 1999). Consequently, Raff

(1994) proposed that multiple tubulin genes may provide an organism with a mechanism to

regulate gene expression both temporally and spatially, while the expression of multiple

tubulin isotypes in a single cell may ensure that a sufficient pool of tubulin is expressed in the cell.

When considering the significance of the expression or existence of multiple tubulin isotypes Luduena (1998) suggested that there are three basic models to consider: (1) isotypes have no functional significance; ( 2 ) isotypes are adaptive but do not perform specific functions; and (3) isotypes perform specific functions. Examples that support each 15 of these models exist m nature.

The first model postulates that isotypes have no inherent functional significance.

For example, although mammalian p tubulins exist in six isotype classes that clearly are differentially expressed, all the cytoplasmic and mitotic microtubules in mammalian cells are mixed copolymers of all of the expressed P-tubulin isotypes (Lewis et nA, 1987; Lopata and Cleveland, 1987; Lewis and Cowan, 1988). This suggests that these isotypes are interchangeable and do not affect microtubule assembly or other characteristics. Many other examples of interchangeability of isotypes exists (Schatz et aL, 1986; Gu et aL, 1988;

May, 1989; Kirk and Morris, 1993).

According to the second model, having a large pool of tubulin isotypes allows an organism to adapt to environmental challenges but the isotypes are not preadapted to perform specific functions. For instance, in A. thaliana, transcription of difierent P- tubulin genes may be temperature dependent Transcription of p-tubulin isotypes TUB2,

TUBS, TUB6 , and TUB8 have decreases in transcription levels at low temperatures while

TUB9's transcription level increases. Luduena (1998) suggests that these plants are using their isotypes to adapt to environmental factors. There are many examples of isotype adaptability to drugs and extreme temperatures in plants and fungi (Modig et aL, 1999; Yan and Dickman, 1996; Chu etal., 1993; Ranganathan et aL, 1998; Sangrajrang et aL, 1998).

Interestingly, however, fungi and some other lower eukaryotes are able to adapt to a variety of enviroiunental factors although they only have one or two a- or P-tubulin genes.

The third model postulates that different isotypes are required for different 16 microtubule functions. The strongest argument supporting this model is the high conservation of isoQrpe di^iences over evolutionary time and the conservation of isotype classes across species. It has been suggested that constraints on isotype divergence resulted because of the essential functions microtubules provide to cells (Raff et aL, 1994).

Savage et al. (1989) were some of the first researchers to show that a specific P-tubulin isotype (mec-7) in C. elegans is required for the production of 15 promfilament microtubules in of touch receptor . More recently, Fukushige et al. (1999) have isolated another gene, Mec-12, an a-tubulin iso^pe in C. elegans, that is required along with Mec-7 to assemble the proper 15-protofilament microtubules needed in the axons of touch receptor neurons. Therefore, in this case, the assembly of a specific type of microtubule requires very specific a - and P-tubulin isotypes to be functional.

Experiments done over the past 18 years in the Raff lab have also advanced the notion that some tubulin isotypes have specific functions. Hoyle and Raff (1990) showed that a divergent, developmentally regulated D. melanogaster P3 isotype caimot support the specific axonemal functions of the testis-specific PZ isotype in PZ null flies. More recently.

R aff et al. (1997) wanted to determine if an orthologous PZ isotype fi-om a moth is functionally equivalent to the D. melanogaster pZ isotype. The moth pZ isotype was unable to support any microtubule assembly in a Z>. melanogaster pZ null background.

Coexpression of the moth pz at greater than 6% of the total tubulin pool results in accessory axonemal microtubules composed of 16 protofilaments (like those normally found in moths) instead of the normal 13 protofilament microtubules found in D. 17 melanogaster (R aff et aL, 1997). This finding along with the data on Mec-12 and Mec-7

in C. elegans suggests that protofilament number and microtubule architecture may be

regulated by specific tubulin isotypes. Consequently, Wilson and Borisy (1997) have

suggested that tubulins provide isoQfpe-specific functions in specialized cells.

Microtubule organizing centers

In the late 18(X)'s cytologist Theodar Boveri observed a cellular structure at the center of the mitotic in Ascaris eggs and he named this structure the

"" which means "central body" (Wilson, 1925). Later, using electron microscopy. Porter (1966) was able to show that microtubules are organized around centrosomes. Pickett-Heaps (1969) coined a more general name “microtubule organizing center” (MTOC) to refer to the morphologically diverse centers that share the ability to nucleate and organize microtubules. Although it has been shown that microtubules have the ability to self-assemble, microtubule assembly is often nucleated by MTOCs in vivo

(Mitchison and Kirschner, 1984b; Desai and Mitchison, 1997). The structure of MTOCs varies widely firom cell to cell and organism to organism. They include the centrosomes of higher eukaryotes, the spindle pole bodies (spb) of fungi, and blepharoplasts of lower plants. Interestingly, in higher plants there is no morphologically defined structure that acts as an MTOC.

While the primary function of MTOCs is to organize microtubules by nucleating microtubule assembly at a concentration of tubulin below the critical concentration of 18 assembly, MTOCs also appear to establish the protofilament number of nucleated

microtubules (Evans et ed. 1985). MTOCs also establish the orientation of the microtubules within cells by nucleating microtubules with their minus ends proximal to and plus ends distal fiom the MTOCs (McIntosh and Eutenuer, 1984). MTOCs organize microtubules into polar arrays that are essential for the directed movement of organelles along microtubules powered by members of the kinesin and families of motor proteins

(Ault and Rieder, 1994; Goldstein and Vale, 1991; Vallee, 1991). Kinesins are mostly plus-end directed motors that transport proteins (such as proteins needed in synaptic membranes at the end of axons) and organelles such as vesicles away fiom the MTOC

(reviewed in Hirokawa et a/., 1998). Conversely, dynein is a minus-end directed motor that transports certain proteins and vessicles toward the MTOC which is usually located near the nucleus (reviewed in Vallee and Gee, 1998). Both families of proteins may be involved in movement of chromosomes along kinetochore fibers during mitosis.

Centrosomes

As stated previously, the centrosomes of animals have been studied for more than

100 years. Centrosomes are small organelles, approximately 1 micron in diameter, which are composed of a pair of centrioles that lie perpendicular to each other and are surrounded by an amorphous electron-dense matrix called the pericentriolar material (PCM) ( reviewed in Doxsey, 1998; Steams and Winey, 1997; Balczon, 1996). Centrioles are cylindrical structures composed of nine short microtubule triplets (reviewed by Rose et aL, 1993;

Kuriyama and Kanatani, 1981). The centrosome is anchored to the surface of the nuclear 19 envelope in inteiphase cells (fkllogg et aL, 1994). Its location in the cell is incitant

because the centrosome s function in the cell depends on its location during interphase (at

the cell's center) and during mitosis (at each pole). Centrosomes have three basic

functions: ( 1) they nucleate the polymerization of microtubules, ( 2 ) they organize

microtubules into functional arrays, and (3) they duplicate once every cell cycle (Urbani

and Steams, 1999; Steams and Winey, 1997).

Centrosome duplication occurs once every cell cycle starting at the G 1/S transition

and ending at the start of mitosis (Kellogg et aL, 1994). During duplication a daughter'

centriole forms adjacent to, and perpendicular to, each pre-existing centriole (reviewed in

Marshall and Rosenbaum, 1999). The mother' or older centriole in the pair has spoke-like

appendages near its distal end and the two centrioles are connected by fibers at their

proximal ends (reviewed in Urbani and Steams, 1999). Cenexin, a 96 kd protein identified by monoclonal antibodies (mAbs) to mammalian centrosomal immunogens, is a marker of centriole maturation acquired at the G2/M transition by the immature centriole (Lange and

Gull, 1995). Until recently the molecular controls of duplication remained an enigma; however, recently the cdk 2 -cyclin£ complex has been identified as the regulator or trigger for centrosome duplication (Hinchcliffe et aL, 1999; Lacey et aL, 1999; reviewed in

Pennisi, 1999). A new gene, UNI3, has been cloned and shown to be required for the addition of the third microtubule of the triplet to the centriole. This gene encodes a new member of the tubulin superfamily of proteins, S tubulin (Dutcher et aL, 1998).

While the centrosome has been viewed as the preferred site of microtubule 20 assembly in vivo for a long time (Brinkley, 1985), it was Gould and Borisy (1977) who

found that the PCM is the actual site of microtubule nucléation at centrosomes. This

discovery led cell biologists to ask two questions that they have continued to investigate

until the present. What is the primary function of centrioles since they do not actually

nucleate microtubule assembly, and what specific components of the PCM actually nucleate

microtubule assembly? Recently Marshall and Rosenbaum (1999) have suggested that

centrioles may act as organizing centers that provide a scaffold around which microtubule

nucleating factors located in the PCM are recruited into a single focus, forming the

centrosome. Bobinnec et al. (1998) have reported that centrioles are needed to organize

pericentriolar components into a structurally stable organelle. Centrioles also seem to be

needed to assemble a functional centrosome that can nucleate astral microtubules which

provide proper nuclear spacing and migration during syncytial divisions in Sciara (de Saint

Phalleefa/., 1998).

Because the centrosome is a complex organelle that is contiguous with the

cytoplasm of the cell (unlike membrane-bound organelles) it has been difficult to isolate and

identify the protein components of the PCM. Biochemical and genetic analysis of

centrosomes has shown that over ICX) proteins localize to the centrosome during various

stages in the cell cycle (reviewed in Kalt and Schliwa, 1993; Rose et a/., 1993; Kellogg et aL, 1994; Balczon, 1996). Caution must be taken before assuming these proteins are functional components of the centrosome that are involved in its nucleating abiliQr. Some proteins may localize to the PCM because they are the cargo that is attached to minus-end 21 directed motors that travel toward the PCM along microtubules; others may be deposited at

the centrosome prior to mitosis to ensure that they are partitioned to both cells when

cytokinesis occurs. In essence, it is likely that the centrosome is composed of functional

proteins involved in nucléation and regulatory proteins that are involved in several cellular

processes that are under cell cycle control.

Initially, putative components of the centrosome were identified using polyclonal or

monoclonal antibodies, while some researchers characterized the centrosome using standard biochemical purification procedures (reviewed in Kimble and Kuriyama, 1992;

Kalt and Schliwa, 1993). Many centrosomal components have been categorized based on their localization patterns during the cell cycle. Some components are detectable at the centrosome throughout the cell cycle, while others are only detectable during mitosis.

There are also proteins that localize to the centrosome during mitosis but are found in other areas of the cell during the remainder of the cell cycle.

Cytocentrin and centrosomin are two proteins that localize to the centrosome during mitosis but are found in the cytosol during interphase. Cytocentrin is a Ral-GTPase binding protein that seems to be involved in the separation of centrosomes during mitosis in rat cell lines (Quaroni and Paul, 1999; Paul and Quaroni, 1993). Centrosomin was identified as a regulatory target of homeotic genes. It has been shown to be essential for the assembly and function of centrosomes during syncytial divisions in D. melanogaster

(Megraw et aL, 1999; Li and Kaufinan, 1996). Since homologs to neither protein have been characterized in other organisms, their overall functional significance is as yet 22 unknow n.

Several proteins are located at the centrosome during mitosis and in other

noncytosolic cellular locations during interphase. In D. melanogaster. R aff et at. (1993)

identified DMAP60 and DMAP190, two proteins that localize to the centrosome during

mitosis and have a microtubule binding capaci^. During interphase these two proteins

localize to the nucleus (Raff et aL, 1993). The function of these proteins remains

unknown, but neither seems to play a direct role in nucléation of microtubules (Oegema et aL, 1999).

One of the proteins most recently found to localize to the centrosome during mitosis and the nucleus during interphase is BRCAl, a suppressor of tumorigenesis in breast and ovarian cells (Hsu and White, 1998). BRCAl coimmunoprecipitates with y-tubulin and its function at the centrosome will be discussed in more detail in the y-tubulin section of this introduction.

Centrin, pericentrin, and y-tubulin are some of the proteins that reside at the centrosome throughout the cell cycle. Centrin, a member of the EF-hand superfamly of

Ca^ binding proteins, has been localized to the MTCXZs of plants, the SPBs of yeast and to animal centrosomes. Although two isotypes of centrin have been cloned in humans

(Errabolu et aL, 1994; Lee and Huang, 1993), its functional significance is still unknown.

Centrin is involved in SPB duplication in yeast and flagellar severing in Chlamydomonas reinhardtii indicating it has different functions in different organisms. A common molecular mechanism, such as the contraction of EF-hand proteins upon binding to C a\ 23 may underlie its functions (Schiebel and Boraens, 1995; Balczon, 1996). Pericentrin, a

220 kd coiled-coil protein that localizes to the PCM of X. laevis embryos, is required for proper formation of microtubule asters around centrioles and may provide scaffolding upon which Y tubulin and its associated proteins nucleate microtubule assembly (Doxsey etal.,

1994; Dictenburg etal., 1998).

Y tubulin, the third member of the tubulin superfamily (Oakley and Oakley, 1989), is required for the nucléation of microtubules at centrosomes and is located at the MTOC throughout the cell cycle. The details of its function will be discussed in the last section of this introduction.

Spindle Pole Bodies

The spindle pole body (SPB) serves as the MTOC in fungi and is the functional homolog of the animal centrosome. The SPB of Saccharomyces cerevisiae is the most extensively analyzed model of MTOC function because researchers have been able to use the power of yeast genetics and molecular genetics to analyze SPB components. The SPB has been shown to nucleate microtubules in vivo and in vitro, verifying that it is the MTOC of yeast (Byers etal., 1978; Hyams and Borisy, 1978). Byers and Goetsch (1975) used electron microscopy to analyze the substructure of SPBs and found that the SPB is embedded in the nuclear envelope throughout the cell cycle. SPBs have a trilaminar morphology consisting of a central plaque that is coplanar with the nuclear envelope, and an inner plaque and an outer plaque. More recently, Bullitt et a/.(1997) and OToole et al.

(1999) have characterized the fine structure of firozen SPBs using electron tomography and 24 have determined that the trilaminar SPB is actually composed of six distinct layers.

Over the past ten years several components of the yeast SPB have been identified via genetic screens for mutations in SPB components or biochemical analysis of isolated

SPBs. The Kilmartin lab was the first lab to biochemically characterize SPB con^nents biochemically (Rout and Kilmartin, 1990) and recently has used high-mass accuracy matrix-assisted laser desorption/ionization (MALDI) mass spectrometry mapping in combination with sequence database searching to identify 1 1 new gene products that localize to the SPB and 12 previously identified SPB components (Wigge et aL, 1998).

For an in-depth review of SPB components see Wigge et al. (1999), and Elliot et al.

(1999).

SpcllOp was one of the first proteins identified by Rout and Kilmartin (1990;

Kilmartin et al., 1993). It has been studied extensively and found to localize to the nuclear side of the SPB where it links or anchors the central plaque to the inner plaque by way of its central coiled-coil protein domain. More recently the N-terminal region of SpcllOp has been shown to mediate the binding of Spc98p and Spc97p, two other SPB components, to the inner plaque of the SPB via a direct interaction with Spc98p (Knop and Schiebel,

1997b; Sunberg and Davis, 1997; Nguyen et al., 1998). Spc98p and Spc97p bind to

Tub4p, the yeast y tubulin, forming the Tub4p complex. Another SPB component,

Spc72p, has been shown to tether the Tub4p complex to the cytoplasmic side of the SPB by interacting directly with Spc98p via its N-terminus (Knop and Schiebel, 1998; reviewed in Saunders, 1999) in a maimer analogous to SpcllOp.

25 Homologs of SpcI lOp, Spc98p, Spc97p have been found in various animals further substantiating the theory that some SPB components are general components of

MTOCs.

Plant MTOCs

Lower land plants, like bryophytes and pteridophytes have structurally defined

MTOCs in some stages of their development (Marc, 1997; Vaughn and Harper, 1998), especially in those plants which have flagellated cells. Some of the discrete MTOCs of lower plants include bicentrioles (two centrioles joined end to end), polar organizers (an extension of the nuclear envelope), blepharoplasts (large spherical templates for centrioles), and a multilayered structure (a trilaminar strip of reorganized PCM) (reviewed in Vaughn and Harper, 1998).

In higher plants five different microtubule arrays form during the cell cycle: The interpbase radial cytoplasmic array; the cortical array (which forms during interphase); the preprophase band (seen in G 2 and early mitosis); the barrel-like acentriolar bipolar mitotic spindle; and the phragmoplast (Balczon, 1996; Vaughn and Harper, 1998).

Since plants do not have morphologically discrete MTOCs, the existence of homologs of common SPB or centrosomal nucleating elements in plants may indicate that plants have flexible MTOCs like those proposed by Mazia (1987). In recent years several centrosomal components have been localized to plants in areas where microtubule arrays are nucleated. These proteins include centrin, pericentrin and y-tubulin.

For many researchers the cloning of y-tubulin cDNAs from several plant genomes 2 6 (e.g. Allium cepia. Anemia phylitidis, Arabidopsis thaUand) has confinned that plants, like animals and fungi, have MTOCs that nucleate microtubule assembly, although they are not morphologically discrete QdcDonald etal., 1993; Liu etal., 1994; Marc, 1997).

y T ubulin

Discovery of y tu b u lin

In a search for genes whose protein products interact with microtubules, Weil et al.

(1986) identified two new loci, designated m ipA and mipB (microtubule-interacting proteins A and B), in Aspergillus nidulans. The mip loci were found in a genetic screen for revertants o f the A. nidulans P-tubulin mutant allele benA33. The benA33 allele confers heat sensitivity and seems to hyperstabilize microtubules at restrictive temperatures (Oakley and Morris, 1981). Revertants of benA33 suppressed its heat-sensMve phenoQrpe and in some cases conferred cold sensitivity. Three alleles at the mipA locus conferred allele- specific synthetic phenoQrpes in combination with various benA alleles.

The mipA gene product was cloned and sequenced by Oakley and Oakley (1989) and it encoded a novel member of the tubulin superfamily that they named y tubulin.

Disruption of the mipA gene blocks nuclear division and disrupts assembly of the mitotic apparatus, indicating that y tubulin is an essential gene that is necessary for the nucléation of microtubule assembly (Oakley et al., 1990). Further analysis of y tubulin using polyclonal antibodies revealed that y tubulin is a component of the SPB of interphase and mitotic cells in A. nidulans and of the centrosomes of HeLa and mouse (3T6) cell lines 27 (O akley etaL, 1990; 23ieng etal., 1991a, b). These findings led Oakley and colleagues

(1990; Zheng et aL, 1991b) to propose that y tubulin nucleates microtubule assembly at

MTOCs, attaches microtubules to MTOCs and establishes microtubule polarity in vivo.

Ubiquity ofy tubulin

Subsequent to the initial identification of y tubulin in A. nidulans, Zheng et aL

(1991b) cloned y-tubulin cDNAs firom D. melanogaster andHela cellcDNA libraries, thus demonstrating that y tubulin is probably a ubiquitous protein in eukaryotes. These two y- tubuUn cDNAs share 66.7% and 66.4% amino acid identity with the A. nidulans y-tubulin gene respectively. To date y-tubulin genes or cDNAs have been identified in 36 species

(see Table 1) ranging from plants to fungi to humans (reviewed in Bums, 1995a). All of these y-tubulin sequences are highly conserved sharing at least 66% amino acid identic except for C. elegans, S. cerevisiae (TUB4) and Candida albicans y tubulin genes which share approximately 44%, 36%, and 40% amino acid identiQr respectively with the other species identified to date (Bums, 1995b; Keeling and Doolittle, 1996; Akashi, 1999).

Although the 5. cerevisiae y tubulin is quite divergent it is essential to the microtubule nucleating capaciQr of the SPB (Spang et aL, 1996; Marschall et aL, 1996). Neither the human y-tubulin (Hyl) cDNA nor the Xenopus y-tubulin cDNA complements a mutant

TUB4 allele indicating that these proteins are not interchangeable (Spang et aL, 1996;

M arschall et aL, 1996). Marschall et al. (1996) also reported that TUB4 does not complement a S. pombe ytubulin null mutation. The y tubulin homologs in C. elegans and

S. cerevisiae may be highly divergent from other y-tubulin genes because they have 28 specialized functions in these organisms (reviewed in Oakley, 1999).

Sequence analysis of a diverse range of organisms reveals that y and P tubulins

share 35-36% amino acid identity, while y and a tubulins share 33-35% amino acid identity

(reviewed in Bums, 1995a). The N-terminal region of y tubulin (5' to amino acid 276) is

more homologous to the amino acids of a and P tubulin than is the C-terminal region.

Because the sequences of a, P, and y tubulins are similar they are likely to have similar

secondary and tertiary structures (Bums, 1994; 1995a). Many of the most highly

conserved regions shared between a, P, and y tubulins correspond to regions of the tubulin

dimer that have been shown to bind GTP (Bums, 1995a; Nogales et aL, 1998a; 1999).

Therefore, y tubulin was hypothesized to be a GTP binding protein like a and p tubulin.

Melki et al. (1993) showed that y tubulin preferentially binds to columns of immobilized

GTP ra±er than ATP and Oegema era/. (1999) showed that the y tubulin found in a small y tubulin complex of Drosophila preferentially binds to GDP.

Cellular localization and function

y Tubulin has now been localized to various Qrpes of MTOCs in several plants and animals, and fungi (reviewed in Bums, 1995a; Pereira and Schiebel, 1997). y Tubulin has also been localized to the basal bodies of specialized mammalian cells, ciliated protozoans, and lower plants (Mutesan et aL, 1993; Fuchs et aL, 1993; Liang et aL, 1996; Ruiz et aL,

1999; Silflow et aL, 1999). Interestingly, acentriolar MTOCs of certain animal and plant cell types also contain y tubulin McDonald etal., 1993; Julian et aL, 1993; Gueth-hallonet

29 Organism Citation

A spergillus n id u la n s O ak l^ and Oakley (1989) Drosophila melanogaster (2) Zheng, Jung & O ^ ey (1991) Homo sapiens Zheng, Jung & Oakley (1991) Schizosaccharomyces pombe Horio eta l. (1991) Xenopus laevis Steams et al. (1991) Yarrowia lypolytica Steams et al. (1991) Mus muscalis Joshietal. (1992) Euplotes octocarinates (2) Liang and Heckmann (1993) Ustilago violacea Luo and Perlman (1993) Anemia phyllitides L. Sw. Fuchs et al. (1993) Plasmodium falciparum Maessen et al. (1993) A rabidopsis thaliana (2) Liu et al. (1994) Cyanidium caldarium Akkari et al. (1994) Chlamydomonas reinhardtii SUflow & LaVioe (-1995) Zea m ays(3) Lopez et al. (1995) Physcomitrella patens Wagner et al. (1995) Leishmania mexicana Oakley & Fong (unpub.) Euplotes crassus (2) Weiligmann (Swissprot) Physarum polycephalum Lajoie-Mazenc et al (1996) Caenorhabditis elegans W ilson et al. (1996) E uplotes aediculatus Tan etal. (Genbank) Neurospora crassus Heckman et al. (Genbank) Reticulomyxa filosa Schliwa et al. (Genbank) Saccharomyces cerevisiae Johnston et al. (1996) Schizosaccharomyces japonicus Horio et al. (Genbank Trypanosoma brucei Scott et. ai (1997) Entamoeba histolytica Lohia & Sam uelson (1997) Dictyostelium discoideum Euteneuer et al. (1998) Tetrahymena pyriformes Li et al. (Genbank) Tetrahymena thermophila Li et al. (Genbank) Paramecium tetraurelia (2) Ruiz etal. (Genbank) Cochliobalus heterostrophus Parkinson et. al (Genbank) Oryza sativa Han et al. (Genbank) Microbutryum violaceum Perlman et al. (Genbank) Candida albicans Akashi et al. (Genbank) Rattus norvégiens Nakadaietal.(1999

Table 1: Organisms from which y-tubulin genes have been isolated.

3 0 et al., 1993; Paiocios and Joshi, 1993; Gard, 1994a; Debecera/., 1995; Endow and

Komma, 1998).

Y tubulin localizes along the length of kinetochore microtubules o f several plant cells

during mitosis, but it is not found at the kinetochore (Liu et aL, 1993; McDonald et aL,

1993). Lajoie-Mazenc and colleagues (1994) observed ytubulin staining at centrosomes

and in the mitotic spindle of several mammalian tissue culture cell lines, hnmunoelectron

microscopy data using these same cell lines suggests that y tubulin may associate with the

walls of spindle microtubules (Lajoie-Mazenc et aL, 1994). This localization pattern might

also be due to the presence of the staggered minus ends of microtubules found throughout

the spindle length (Liu et aL, 1994). Endow and Komma (1998) have shown that y-tubulin

(yrUB37CD) also localizes to a central spindle pole foimd in meiosis H D. melanogaster

oocytes. Localization of y tubulin to this central pole is dependent on the NCD minus-end

directed . Wilson and Borisy (1998), however, did not observe YTUB37CD

at the meiosis II central spindle pole. The cause for the discrepancies in these two papers is

not clear.

It is evident that y tubulin's localization to various Qrpes of MTOCs is linked to its

function as a nucleator of microtubule assembly, but y tubulin may also function in other

capacities as well. Several labs have independently localized y tubulin to the barrel of centrioles of mammalian cells and the basal bodies of Chlamydomonas reinhardtii and

Paramecium tetraurelia (DHtbayawaaet aL, 1995; Fuller ef a/., 1995; Moudjou era/., 1996;

Ruiz etal., 1999; reviewed in Marschall and Rosenbaum, 1999). Ruiz etal. (1999) have 31 shown that inactivatioa of y-tubulin genes in P. tetraurelia inhibits basal body duplication, indicating that y tubulin is requited for basal body duplication. These findings support the work of Fuller et aL (1995) who proposed that y tubulin is located on the outer walls of parental centrioles and acts as a template for daughter centriole formation.

Oakley's original hypothesis that y tubulin nucleates microtubule assembly in vivo at MTOCs (Oakley et al,, 1990) has been substantiated by several studies. Horio et aL

(1991) and Steams et aL (1991) independently disrupted the y-tubulin gene in

Schizosaccharomyces pombe and found that y tubulin is essential for cell viability and nuclear division. Plasmid loss experiments, in which a multicopy plasmid containing the

S. pombe y-tubulin gene is lost fiom into a haploid strain containing a disrupted y-tubulin gene, suggest that y tubulin is necessary for proper spindle structure and fimction (Horio et al., 1991). Later, Joshi (1992) demonstrated that anti-y-tubulin antibodies injected into mammalian cells inhibited cytoplasmic microtubule reassembly and the formation of functional mitotic spindles, y-tubulin function is also conserved in phylogenetically distant organisms as was shown when the human y-tubulin cDNA (Hyl) restored viability to S. pombe cells that lacked endogenous y tubulin (Horio and Oakley, 1994). Characterization of a mutant allele of D. melanogaster y tubulin (yTUB23CD) revealed that y tubulin is not only required for proper spindle function but it is also apparently required for proper

MTOC stmcture because the mutation results in the formation of abnormal MTCXZs although microtubule assembly still occurs in these mutants (Sunkel et a/.,1995).

Recently, Khodjakov and Rieder (1999) have reported the use of a y-tubulin green 32 fluorescent protein (yiG FP) in immunofluorescence studies to determine how y tubulin behaves during the cell cycle in mammalian cell lines. They show that yTGFPexists at low levels at the centrosome during interphase, but the amount of y tubulin increases by four fold at the onset of mitosis. yTGFPmigrates into the spindle during mitosis, corroborating earlier findings by Lajoie-Mazenc et al. (1994) and others. Two populations of yTGFP exist at the centrosome; one population rapidly exchanges with soluble y tubulin, while another population seems to be more stably bound to the centrosome and exchanges slowly with soluble y tubulin. The centrosome appears to become activated at the onset of mitosis to bind increased amounts of yTGFP.

Martin etal. (1997) analyzed the mipA (y-tubulin) gene disruption phenotype in A. nidulans conidia that were blocked in G2 and released. Results of this analysis indicated that y tubulin is absolutely required for nucléation of the mitotic spindle and nuclear division. The absence of functional y tubulin causes a transient mitotic block, y Tubulin is not required, however, for cell cycle progression from G1 to M nor for SPB duplication in

A. nidulans.

Biochemical characterization of y tubulin and the yXuRC

y tubulin is a minor cellular protein (in amount). It comprises only 0 .0 1 % of the cell’s total protein in unfertilized Xenopus eggs (Steams et al., 1991). This makes it difficult to purify y tubulin from cells. Since it is a minor protein, researchers tried to produce y tubulin in vitro as a fusion protein in E. coli, but like a and p tubulin, y tubulin forms insoluble inclusion bodies (Melki et al., 1993). y tubulin, like a and P tubulin, is 33 folded by TCPl chapercnin complexes prior to its release into the cytoplasm in a soluble form (Melki et aL, 1993). Unfolded or improperly folded y tubulin binds to prefoldin, a chaperone that forms a complex with TCPl where y is folded into its native form (Vainberg et aL, 1998). The absence of a TCPl complex in E. coli may preclude correct folding.

Subsequently, biochemical analysis of y tubulin in Xenopus laevis oocyte cell-fiee extracts revealed that y tubulin is recruited firom the oocyte cytoplasm in orcfer to activate the sperm centriole and form an active centrosome in vitro (Félix et al., 1994; Steams and

Kirschner, 1994). Steams and Kirschner (1994) found that cytoplasmic y tubulin sediments as a 25S complex they named the y-some. Zheng et al. (1995) developed a procedure using antibody-afGni^ chromatography to isolate this 25S y-some complex from

Xenopus egg extracts. Analysis of this complex showed that it contains y tubulin and at least six other proteins including a and P tubulin (Zheng et al., 1995). Electron microscopy analysis revealed that the complex is a 25 nm ring stmcture that was re-named the y-TuRC (y mbulin ring complex). The complex nucleates microtubule assembly and binds to the minus ends of microtubules in vitro (Zheng et al., 1995). hnmuno-electron microscopic tomogr^hy was used by Moritz et al. (1995) to localize y tubulin to 25-30 nm ring structures in the PCM of puriried D. melanogaster centrosomes. Therefore yTuRCs probably reside in the PCM where they nucleate microtubules at their minus ends thereby establishing the polarity of the microtubules. Subsequently, Vogel et al. (1997) reported that the PCM of Spisula solidissima oocyte centrosomes contain ring-shaped structures that are approximately 25 nm in diameter, like those seen by Moritz et al. (1995). Mcxe 34 recently, Moritz et aL (1998) have shown that D. melanogaster salt-stripped centromosomal scaffolds lack the abili^ to nucleate microtubule assembly. When D. melanogaster emhtyo extracts are added to the salt-stripped centrosomes, they regain the ability to nucleate microtubule assembly. If y-TuRCS are immunodepleted from the extracts, microtubules nucléation is not restored. If purified y-TuRCs and an as yet unidentified 220 kd protein are added to salt-stripped centrosomes microtubule nucléation is restored. Similarly, Schnackenberg e t al. (1998) were able to reconstitute salt-stripped, inactivated centrosomes isolated from S. solidissima using S. solidissima oocyte extracts which contain y tubulin and 25 nm ring structures. All these data support the original hypothesis of Oakley et al. (1990) that y tubulin nucleates microtubule assembly at

MTOCs. Li and Joshi (1995) also reported that y-tubulin binds to the minus ends of microtubules of axonemes in vitro further supporting another portion of Oakley’s hypothesis (Oakley etal., 1990).

As a consequence of the identification of y-TuR(Zs in XI laevis, other researchers have searched for cytoplasmic y-tubulin complexes in various organisms and have begun to characterize the y-TuRC subunits (reviewed in Wiese and Zheng, 1999). Components of y-TuRCs, called gamma tubulin ring proteins (grips), have been identified in D. melanogaster, mouse 3T3 cells, humans, sheep, A. nidulans and S. cerevisiae ^ o r itz et aL, 1998; Oegema et al., 1999; Murphy et aL, 1998; Tassin et aL, 1998; Detraves et aL,

1997; Akashi et aL, 1997; Knop and Schiebel, 1997a, b). These components are listed in

Table 2 (adapted from Wiese and Zheng, 1999) with homologous proteins located on the 35 same row. The S. cerevisiae Y-tubulin protein, Tub4p, is found in a 6 S complex that contains two molecules of Tub4p and one molecule each of Spc98p and Spc97p (Knop and

Schiebel, 1997b). This complex is required for the nucléation of microtubule assembly from both the cytoplasmic and nuclear sides of the SPB and it is tethered to the nuclear side of the SPB by SPClIOp (Geissler era/., 1996; Knob and Schiebel, 1997 a,b). Oegema er aL (1999) have isolated a conq>lex hom D. melanogaster, the Ytubulin small complex ( 7 ^

TuSQ that appears to be analogous to the Tub4p complex found in 5. cerevisiae. y-TuSC contains homologs of Spc97p and Spc98p that they have named Dgrip84 and Dgrip91

(Oegema et aL, 1999). Spc97p and Spc98p homologs have been identified in D. melanogaster, X. laevis, and H. sapiens (M artin et aL, 1998; Murphy et aL, 1998;

Oegema et aL, 1999; Tassin et aL, 1998). Spc97p and Spc98p are related and may be members of a protein superfamily.

Dictenberg and colleagues (1998) reported that pericentrin, a 220 kd protein that localizes to the PCM, and y tubulin are a part of a large complex that forms a centrosomal lattice that is conserved from mammals to amphibians. Since pericentrin has been predicted to be a coiled-coil protein it may form the base of a centrosomal lattice that organizes yiuRCs at the centrosome where they nucleate microtubules (Doxsey et aL, 1994;

Dictenberg et aL, 1998). As mentioned previously, Moritz and colleagues (1998) have shown that yTuRC and an unknown 220 kd protein are required to make salt-stripped centrosomal scaffolds competent to nucleate microtubule assembly in D. melanogaster.

Recently the Asp protein, a 220 kd product of the Asp (abnormal spindle) gene in D. 36 melanogaster, has been implicated as the protein that restores microtubule nucleating activiQr to these salt-stripped centrosome preparations in the presence of YTuRC (de Carmo

Avides and Glover, 1999). Asp seems to be required to focus the yTuRC within the PCM so it can nucleate asters of microtubules (de Carmo Avides and Glover, 1999). Since Asp does not copurify with YTuRC, Asp is probably not directly involved in microtubule nucléation; purified YTuRCs can nucleate microtubules but microtubules are not organized in discrete centers or asters unless Asp is present The functions of pericentrin and Asp seem to overlap and they may be functional homologs.

An unexpected recent finding is the localization of the BRCAl protein to centrosomes during mitosis and its coimmunoprecipitation with y-tubulin (Hsu and White,

1998). The BRCAl gene encodes a 1863 amino acid (aa) protein that is a suppressor of tumorigenesis in breast and ovary tissues (O'Brien et at., 1994; Wooster et aL, 1994).

BRCAl localizes to the nucleus during interphase and it is phosphorylated during late G1 and S phase (X u et aL, 1999). A hypophosphorylated form of BRCAl has been immunoprecipitated with y tubulin. Hsu and White (1998) have proposed that BRCAl may be a regulator of mitotic spindle assembly and of the G2/M transition. Xu et aL

(1999) reported that a mutant BRCAl gene expressed in mouse embryonic fibroblasts causes the G2/M checkpoint control to be defective and 25% of the mutant cells have amplified, functional centrosomes that form multiple spindle poles in each cell. Taken together these results link the centrosome s microtubule nucleating capacity and the control of cell cycle progression. 37 Organisms:

X. Laevis D. Melanogaster Mouse Human Sheep A. Nidulans S. Cerevisiae

250 Xgripl95 Dgripl63 211 195 Xgripl33 Dgripl28 128 HsSpcllO 130 Xgripl09 Dgrip91 101/GCP3 HsSpc98/hGCP3 105 -105 Spc98 Xgripl 10 Dgrip84 100/GCP2 hGCP2 -95 Spc97 Xgrip75s Dgrip75s 76 75 -80 OJ 00 Xgrip72 Dgrip72 71 y tubulin y tubulin y tubulin y tubulin y tubulin y tubulin Tub4p a/p-tubulin a/p-tubulin

♦Homologous yTuRC proteins are in the same row. Numbers refer to the molecular weight of the proteins in kilodaltons,

Table 2: Components of yTuRCs in different organisms. Multigene familles of y tubulins

Like a and P tubulin, y tubulins are encoded by multigene fa m ilies in some organisms. Initially, Zheng e t al. (1991 a,b) identified, cloned, and sequenced two y- tubulin cDNAs from D. melanogaster. Subsequently at least two y-tubulin genes have been isolated firom A. thaliana ^ u e t al., 1994), Zea mays (Lopez e t a l., 1995; Genbank),

Euplotes crassus (Tan and Heckman,1998), Euplotes octocarinatus (Liang and Heckman,

1993), and Paramecium tetraurelia (Ruiz e t al., 1999). The identity shared between the two isotypes ranges firom 81.6% (D. melanogaster) to 99.5% {Euplotes octocarinatus).

Lajoie-Mazenc and colleagues (1996) cloned a y-tubulin gene firom Physarum polycephalum that is transcribed into a single RNA but the polypeptide seems to be posttranslationally modified into two isoforms of y tubulin (referred to as a slow and fast form) that have different electrophoretic mobilities, apparent molecular masses (52 kd and

50 kd), and sedimentation properties.

Analysis of the expression patterns o f the two divergent D. melanogaster y-tubulin genes (YTub23C and YTub37CD) reveals that they are differentially expressed. yTub23C is zygotically expressed in males and females during all stages of development. It localizes to centrosomes of mitotic cells and germ cells and the basal bodies of post-meiotic spermatids

(Wilson e t al., 1997). Mutational analysis of YTub23C reveals that it is required for correct mitotic spindle assembly as well as proper maintenance of the structiue o f the centrosomes in larvae (Sunkel e t a l., 1995). The second Drosophila y-tubulin isotype, yTub37CD, is maternally expressed and localizes to ovaries, and developing egg chambers during 39 oogenesis, and is utilized by precellular and cellular embryos (Wilson e t aL, 1997;

Tavosanis e t aL, 1997; Wilson and Borisy, 1998). YTub37CD is required for the formation of bipolar mitotic spindles during oogenesis subsequent to germ cell proliferation and during early embryogenesis (Wilson and Borisy, 1998; Llamazares e t aL, 1999).

Wilson and Borisy (1998) have suggested that yrUB37CD is required for the proper activation of oocytes in meiosis I but is not required for proper meiosis I spindle formation although contradictory data has been presented (Tavosanis e t aL, 1997; Llamazares et aL,

1999).

The two Y-tubulin genes o f A. thaliana share 98% aa identity and they are co­ expressed in several tissues during different stages of development CLiu et aL, 1994). An analysis of the expression patterns of the two y-tubulin genes of Euplotes crassus has not been conducted. However, sequence analysis of the genes reveals that ytubl and 7 tub2 share 86.5% aa identiQr and differ in codon usage, transcriptional initiation sites and poly-

A-sites (Tan and Heckman, 1998). Euplotes octocarinatis has two isotypes that differ by only two amino acids. Apparently a third Euplotes species, Euplotes aediculatus, also has two y-tubulin isoQrpes that have not been deposited in Genbank as does another ciliate

Blepharisma japonicum (Tan and Heckman, 1998) indicating that multiple y-tubulin genes are the norm for ciliates. Since ciliates have multiple types of MTOCs (Adoutte and Fluery,

1996) they may require different isoQrpes for different MTOCs or the y-tubulin iso^pes may simply fulGU the spatial or temporal regulation of microtubule nucléation required by these organisms. Z m ays also has two highly homologous y-tubulin isotypes; however, 40 neither their expression patterns nor function has been analyzed (Lopez e t aL, 1995).

Rationale for my dissertation research

After having previously identified a y-tubulin gene in humans (which I will call

Hyl) and finding two y-tubulin genes in D. melanogaster, Zheng (1992) conducted a preliminary search for additional y-tubulin genes by reprobing a HeLacDNA library. No additional y tubulins were found, but subsequent antisense RNA and oligodeoxynucleotide analyses failed to inhibit y tubulin function in HeLa cells. This failure of inhibition could have been due to the expression o f more than one functionally redundant y tubulin gene in

HeLa cells. Since multiple y-tubulin genes were identified in D. melanogaster, another metazoan, and because a and P tubulins exist in multigene families, it is plausible that multiple y tubulin genes exist in Homo sapiens.

In recent years the extensive research on the causes and effects of tumorigenesis has revealed that many tumor cells display multiple (supernumerary) centrosomes that nucleate multiple spindles and cause aberrant chromosome segregation leading to genetic instabiliQr in these cells. The recent localization of BRCAl, a tumor suppressor gene, to the mitotic centrosome and mutational analysis of this gene has shown that it is involved in the regulation of the G2/M checkpoint and regulation of centrosome duplication (Hsu and

White, 1998; Xu et aL, 1999). Since y tubulin has been shown to interact with BRCAl during mitosis and y tubulin localizes to supernumerary centrosomes in tumor cells it may be exploited as a target for chemother^y agents. Bai et aL (1993) also proposed that the cytotoxic antimitotic compound spongistatin 1 may be toxic because of its high affinity for

4 1 Y tubulin. Antimitotic agents could also be used to inhibit y tubulin function and thereby prevent tumorigenesis.

A prerequisite for understanding y tubulin function and y tubulin as a target for therapeutic agents in humans is understanding the number and expression patterns of y-tubulin genes. Since multiple a and P tubulin pseudogenes have been identified in humans. I, like Zheng (1992), chose to probe a HelacDNA library to search for additional y-tubulin genes. In chapter 2 of this dissertation I will describe how I used Southern analysis to estimate the total number of y-tubulin genes found in the human genome. Data is also presented on the identification of a Hyl processed pseudogene. Finally, I will also present data on the identification, cloning and sequencing o f a second human y tubulin gene we have named Hy2. The methods used, results obtained, and a discussion o f these results will be presented in Chapter 2.

Since the two y-tubulin genes are differentially expressed in Drosophila, and I have identified two y-tubulin genes in Homo sapiens, I have analyzed the expression patterns of these two y-tubulin genes in various human tissues using RT-PCR analysis. In Chapter 3,

1 will present a more detailed rationale behind the methodology used and results obtained from the RT-PCR analysis. Since Hyl has been mapped to human chromosome 17,1 will also describe how H"y 2 was mapped to a specific locus in the human genome.

42 CHAPTER 2

IDENTIFICATION, CLONING AND ANALYSIS OF ADDITIONAL y- TUBULIN SEQUENCES IN HOMO SAPIENS

Introduction

After y-tubulin genes were identified \n Drosophila melanogaster aad Homo sapiens

(Zheng e t aL, 1991a), as well as Xenopus laevis and Schizosaccharomyces pombe (Steams e t aL, 1991; Horio e t aL, 1991), it became obvious that y tubulin is found not only in

Aspergillus nidulans but it is a ubiquitous protein. To date, y tubulin has been identified in

36 species and at least two y-tubulin genes have been identified in six species. Therefore, y tubulins, like a and P tubulins, are encoded by multigene families in some organisms.

Three different y-tubulin cDNAs have been identified in Z. m ays (Lopez et aL, 1995;

Genbank) and Southern blot analysis suggests that as many as five y-tubulin genes may exist in A. thaliana, however only two have been isolated (Liu e t aL, 1994). Although

Zheng (1992) searched for additional y-tubulin genes in H. sapiens, none were identified.

Initially Cleveland et aL (1980) used Southern blots of human genomic DNA, probed with chicken a - or P-tubulin cDNAs, to estimate that 12-15 a - and P-tubulin genes each exist in the human genome. Only five a-tubulin sequences and ten P-tubulin

43 sequences have been identified and fully sequenced (see Table 3). Of these fifteen sequences, eight (four a and four p) encode functional genes, while seven (one a and six

P) encode pseudogenes (Cowanet a/., 1981; Wildeef a/., 1982a, b; Lee eta/., 1983; Hail and Cowan, 1985). Interestingly, five of the six P-tubulin pseudogenes are processed pseudogenes and only one is a "classical" pseudogene (see Table 3). Processed pseudogenes are inactive genomic sequences that resemble the mRNA transcript of a functional gene in that they lack introns and end with a poly(A)tail. Processed pseudogenes are often flanked by short direct repeats believed to have originated firom the reverse transcription of the mRNA of a functional gene followed by a transposition event in which the transcript is inserted at some random site in the genome (reviewed in Lewin,

1997). In order for processed pseudogenes to be propagated in the human genome, these sequences must be integrated in germline cells (reviewed in Lewis and Cowan, 1986).

Classical pseudogenes have the same structure as functional genes, but they have been inactivated by mutations that prevent the proper transcription or translation of the gene

(reviewed in Lewin, 1997). These pseudogenes are usually found adjacent to a functional gene since many of them appear to have resulted firom duplications of genomic sequences.

To date no y-tubulin pseudogenes have been identified in higher eukaryotes.

Several of the human ot- and P-tubulin genes have been mapped to chromosomes

(see Table 3). The human P-tubulin gene TUBE (Tubulin B, also designated M40 protein) has been localized to the short arm of chromosome 6 at 6p21.3 using

44 Gene Name Clone Name Description* Chromosome Reference**

TUBE M40 Expressed p 6p21 Floyd-Smith, 1986 TUBB2 P2 Expressed p -- Lewis, 1985 TUBB4 P4 Expressed p 16 Ranganathan, 1988 TUBB5 5P Expressed p 19 G wo-Shu Lee, 1984 TUBBPl 21p Processed-M40 8q21 Floyd-Smith, 1986 TUBBP2 Processed-M40 13 Floyd-Smith, 1986 — — — — 14P Processed-M40 Gwo-Shu Lee, 1983 ———— 7p Processed-M40 - — Gwo-Shu Lee, 1983 ----- IP Pseudo-5p -- Wilde, 1982 lip Processed Unique p -- Wilde, 1982 K-ALPHA-1 K al Expressed a -- Cowan, 1983 TUBA I H2a Expressed a 2q Gerhard, 1985 TUBA2 ------Expressed a ISqll Dode, 1998 TUBAS Bal Expressed a 2q Hall, 1985 ------Pseudo-Bal Hall,1985

*Expressed-functional gene; Pseudo-classical pseudogene; Processed-processed pseudogene

♦♦Only first authors are listed in the reference column.

Table 3: Human a- and p-tubulin gene chart. human/chinese hamster ovary somatic cell panels (Hoyd-Smith, 1986). Two processed pseudogenes of TUBE, designated TUBBPl (previously identified as 21P) and TUBBP2, localize to chromosome 8 q region 21, and chromosome 13, respectively (Floyd-Smith,

1987). The fact that TUBB and its two pseudogenes are dispersed in the human genome is characteristic of processed pseudogenes. Other examples of dispersed processed pseudogenes have been documented in several multigene families such as the globin gene family (Fritsch e t al., 1980; Leder et at., 1981). Two of the other three functional P~ tubulin genes TUBB4 and TUBBS (SP) have been localized to human chromosomes 16 and 19 respectively by various labs involved in the human genome project

(www.ncbi.nlm.nih.gov-select Human genome resources). TUBB2 (HP2) as well as the other P-tubulin pseudogenes listed in Table 3 have not been mapped to chromosomes.

The first a-tubulln gene identified in humans, K al, (K-alpha-1; Cowan e t at.,

1983) has not been localized to a chromosome. In an attempt to identify candidate genes involved in two types of human nonsyndromic deafness, Dode e t at. (1996) localized

TUBA2 (Tubulin alpha-2), another a-tubulin gene, to region q ll of chromosome 13.

However, mutations in TUBA2 are now known not to be the cause of nonsyndromic deafness. TUBAl (Ha2) and TUB A3 (bal) have been localized to the q arm of chromosome 2 by labs involved in the human genome project (www.ncbi.nlm.gov).

Although both of these genes localize to 2q, their position on chromosome 2 in relationship to each other has not been established.

In most of the organisms in which multiple tubulin genes have been identified, they 46 ate dispersed throughout their respective genomes. However, a - and P-tubulin genes are

arranged in clusters or tandem repeats in some unicellular parasites, sea urchins, antarctic

fish, and red algae (Alexandraki and Ruderman, 1983; Parker and Dietrich, 1997; Akkari,

1997). For example, in the parasitic protozoan, Leishmania, a - and P-tubulin genes are

arranged in separate tandem repeats, whereas a - and P-tubulin genes in Trypanosoma

brucei are arranged in alternating tandem repeats (Landfear et al., 1983; Thomashow e t al.,

1983). It has been suggested that these genes are arranged in tandem as a means to regulate

gene expression through common DNA regulatory elements (reviewed in Cleveland and

Sullivan, 1985). Of the six species in which multiple y-tubulin genes have been identified, only the two y-tubulin genes isolated from D. melanogaster (yrUB23C and yTUB37CD) have been mapped to chromosomes and, as is denoted by the numbers in the names of the genes, they are not arranged in tandem (Zheng, 1992; Wilson e ta l., 1997).

Since multiple y-tubulin genes have been identified in D. melanogaster, a metazoan, as well as five other species of plants and animals and because a and P tubulins are encoded by multigene families, it is plausible that multiple y-tubulin genes exist in Homo sapiens. I have, therefore, attempted to estimate the number of y-tubulin genes in the human genome using Southern analysis of human genomic DNA. From this analysis I have estimated that no more than three y-tubulin sequences exist in the human genome.

I have chosen to screen a HeLa cDNA library to search for additional y-tubulin genes. I have isolated cDNAs that are encoded by a novel y-tubulin gene which I have named Hy2. I have sequenced these cDNAs as well as a full-length cDNA product of this 47 gene identified initially as an Expressed Sequence lag (EST) clone. Hy2 is highly

homologous to Hyl and encodes a functional (expressed) gene. I have also sequenced a

genomic y-tubulin clone we obtained from Dr. Helmut Shraudolfs lab (Universitat Ulm,

Ulm, Germany) that I named H ^f for Human gamma fragment. Characterization of

using a BLASTN search has revealed that this fragment originated from a processed

pseudogene, located on human chromosome 7, that probably is derived from Hyl.

Although the Hyl gene has been identified in ± e form of a cDNA clone, a genomic

Hyl clone has not been identified. In the early 1990's while several labs were trying to

identify a locus which contains the gene that causes familial breast cancer, the Hyl genomic

locus was mapped to within -150 kb centromeric to the BRCAl (breast and ovarian cancer

I) susceptibility gene on region q21 of chromosome 17 (Miki e ta l., 1994; Friedman eta l.,

1995; Rommens et al., 1995). AU the labs involved in the search for BRCAl have used

YAC clones that contain the 1 Mb BRCAl region of chromosome 17 to probe cDNA libraries in order to identify any functional genes, such as Hyl, in the region (Brody et al.,

1995; Couch et al., 1995; Friedman et aL, 1995; Rommens e t al., 1995).

In coUaboration with Dr. Ralf Krahe's Lab (Dept, of Human Cancer Genetics,

OSU) we have determined the localization of Hy2 in the human genome. This mapping analysis has revealed that Hy2, like Hyl, is located on chromosome 17, between 17ql2 and 17q21. Further investigation of the location of Hyl in relation to Hy2 using PCR analysis has revealed that the Hy2 gene is located on the same 700 kb YAC as the Hyl gene. To date we have not determined if these genes are arranged in tandem from the 48 experiments we have performed.

Materials and Methods

Isolation of HeLa genomic DNA

HeLa cells were kindly provided by Dan Luo (Dept, of Molecular Genetics, OSU).

Genomic DNA was isolated from HeLa cells as described by the "Isolation of DNA firom

Mammaban Cells: Protocol I" in Sambrook e ta l. (1989). The absorbance of the DNA was measured at 260 nm and 280 nm in order to determine the concentration and puri^ of the

DNA and then it was visually analyzed on 0.7% agarose/TBE gels [AgaroseLE,

Boebringer Mannheim Biocbemicals (BMB); TBE is 5.4 g/1 of Tris

[bydroxymetbyl]amino-metbane, 2.7 g/1 boric acid and 0.465 g/1 EDTA).

Southern Analysis

Ten micrograms of DNA were digested with the appropriate restriction endonuclease as directed by the manufacturer’s protocol (New England Biolabs). Digested

DNA was concentrated in Microcon 30 filter cups (Amicon, Inc.) and run on 0.7% agarose/TBE gels (AgaroseLE, BMB). The gels were stained with 1 p,g/ml of etbidium bromide (Sigma), photographed, and denatured in 50 mM NaOH for 45 min at room temperature. They were rinsed three times in dB,0 and soaked for 30 min in (IH 2O. They were then dried onto Whatman 3M M paper (10 m in heat off, 15 m in at 65®C, 15 min heat off). The dried gels were floated off 3MM paper in dHjO and prehybridized in BLOTTO 49 (Bovine Lacto Transfer Technique Optimizer, Johnson e t al., 1984) for I hour at 65^C or

60^C. After prehybridization, probes were added to 1x10^ cpm/ml of BLOTTO and hybridizations were carried out at either 65^C or 60°C for 20-24 hours. Gels probed at

65®C were washed twice in 2x SSC (0.6 M NaCI, 34 mM sodium citrate, pH= 8 ), 0.1%

SDS , for 20 min/wash and twice in 0.2x SSC, 0.1% SDS, also 20 min/wash. All washes were at 65°C. Gels hybridized at 60°C were washed three times at 60°C for 10 min with

2x SSC, 0.1% SDS. A Molecular Dynamics phosphor screen or Kodak X-OMAT AR film was used for autoradiography.

Probes were made from 5' and 3' regions of a Hyl cDNA. Two 3' probes were produced as follows. Plasmid pH3 {a pBluescript sk^(Stratagene) based plasmid containing the Hyl cDNA in an EcoRI site} was digested with PstI and Clal to produce a

399 bp fragment corresponding to aa298 to 432 or with BamHI to produce a 528 bp fragment corresponding to aa339 to 451 plus 191 bp of the 3'UTR. These fragments were recovered from agarose gels by the Freeze-Squeeze method (Tautz et aL, 1983) and were radiolabeled by the method of Feinberg and Vogelstein (1983) using random nine-mers

(Stratagene). A 515 bp 5’ probe, that begins 24 bp 5’ to the start codon and ends at aal79, was generated by radioactive PCR. Thirty cycles of a first round PCR reaction of 100 pi were carried out with 10 ng of plasmid pH3 DNA, 0.1 pmoles each o f M13S-20 universal primer and HGTR2 (a Hyl reverse primer: cactgagtatgtct made by Operon Technologies),

0.2 mM each of dATP, dCTP, dGTP, dTTP (BMB), and 2.5 units of ID-Pol DNA polymerase (ID Labs, Inc.) in Ix NH^+ ID-Pol PCR buffer {16 mM (N H ^ 2S0 4 67 mM 50 Tris-HCl (ph= 8 .8 at25®C), 0.1% Tweea-20} with MgCl 2 added to a Gnal concentration of

0.75 mM. Five percent (5 ^i) of the first round product was used as a template for a radioactive, 25 cycle, second round PCR amplification using 0.6 pM [a^^P]-dATP (ICN) and 0.2 mM each of cold dCTP, dGTP, and dTTP (BMB). Amplification conditions for both rounds of PCR were I min at 94°C and 4 min at 50®C following an initial 2 min,

94°C hold. Excess primers and nucleotides were separated fiom all probes using

Microcon-30 microconcentrators (Amicon, Inc.).

Sequence analysis of a genomic human y-tubulin clone (HyF)

A 930 bp human genomic DNA fragment of a putative second y-tubulin gene, in plasmid pIBI (IBI-Eastman Kodak) was received from Dr. Helmut Schraudolfs lab

(Universitat Ulm, Ulm, Germany). The 930 bp insert was subcloned into an

EcoRI/BamHI site of M13mpl8 and M13mpl9. I named this new firagment HyF for human gamma firagment. Following single-stranded DNA preparations (Sambrook et aL,

1989) of M13mpl8 and M13mpl9 containing the insert DNA, I sequenced the clones using the Sequenase 2.0 (USB-Amersham Life Sciences) standard protocol.

Library screening

A HeLa XZapH cDNA library (Stratagene) was screened using the plaque lift and purification protocol described by Carlock (1988) with the following modification. The filters were neutralized by laying them plaque side of the filter up, on 3MM paper soaked in 51 3M NaCl, 0.5 M Tris-HCl, pH=7.0 for 10 min. After the filters were air-dried the plaque

DNA was cross-linked to the filters using a UV Stratalinker 1800 (Stratagene). Filters were prehybridized in BLOTTO for four hours and hybridized to an [a^^P] dATP random- labeled (Feinberg and Vogelstein, 1983) 930 bp, HyF probe, for 12-18 hours at 65°C.

Filters were washed twice in 2x SSC, 0.1% SDS at 65®C followed by two washes in 0.2x

SSC, 0.1% SDS for 25 minutes/wash. From a total of 2.5 xlO^ plaques screened, 21 positive clones were isolated.

XZapQ phage have been designed to allow the isolation of the Bluescript plasmid containing an insert from the phage using helper phage R408 (Stratagene). Clones were excised from positive plaques and recircularized using helper phage by following the

XZapn protocol provided by Stratagene. Rescued pBSsk" plasmids containing the inserts of interest were prepared using the standard large scale plasmid preparation protocol found in Sambrook et aL (1989), except that the volume of the prep and components used in the prep were reduced to 1/5. Plasmids were purified by two rounds of CsCl-ethidium bromide equilibrium densi^ centrifugation.

All 21 clones were sequenced at both ends using Sequenase 2.0 (USB-Amersham

Life Sciences) to determine if their sequences matched the sequences of Hyl or H ^ , or were novel. Four novel clones (pDOWla, pDOW7, pDOW12, and pDOW21) were obtained and were sequenced on both strands by primer walking using the Sequenase 2.0

(USB-Amersham Life Sciences) protocol.

52 EST database search, subcloning and sequencing of a full-length Ry2 clone

In order to obtain a full-length Hy2 clone, the human expressed sequence tags

(EST) database (www.ncbi.nlm.nih.gov/dbEST) was searched and 159 EST sequences

were analyzed. A full-length HyZ clone, that 1 named H 72 EST, was found. This clone

was purchased from Research Genetics, Inc. and was sequenced fully on both strands.

Most sequencing was carried out using the double-stranded sequencing protocol for

Sequenase 2.0 (USB-Amersham Life Sciences). A portion of one strand was sequenced

using the ABl Prism Dye Terminator Cycle sequencing protocol (Perkin Elmer). Cycle sequencing reactions were run on a Gene Amp PCR 2400 thermal cycler (Perkin Elmer) and sequences were read with an ABl 373A DNA Sequencer (Perkin Elmer). Basic sequence analysis was done using DNA Strider 1.2 (Marck, 1988). Alignments of the y-tubulin genes were carried out with the QustalW algorithm (Thompson et aL, 1994) and SeqVu software (Garvan Institute, Sydney, Australia).

Construction of plasmid pASHy2

Five nanograms of 2x CsCl purifred-plasmid pHy2EST [the HyZEST clone in plasmid Lafinid BA (ATCQ] DNA was used as a template to amplify a 1589 bp region of the plasmid that includes the entire coding region and 205 bp of the 3' UTR. Thirty cycles of amplification using 2.5 units of Taq DNA polymerase (Gibco BRL) were carried out using the components and amoimts recommended in the Gibco BRL protocol. Hy2EST specific primers (HGEST3F1: gaagatctgagcgatgccccgggaga; and HGEST3R1: 53 gaagatctctgacaaccaagctttat) with BglTT ends were used to amplify pH^lEST using the following thermocycling parameters: 94®C for 2 min; 30 cycles of 94°C for 1 min and

60°C for 4 min. Taq DNA polymerase was removed from the PCR reaction using

Strataclean Resin (Stratagene) and the sample was concentrated in a Microcon-30 microconcentrator (Amicon, Inc.). This sample was digested with BglU as directed by the manufacturer (New England Biolabs) and then purified from a 0.7% SeaplaqueGTG low melting point agarose gel (EMC) using Wizard PCR preparations (Promega Life Sciences).

Plasmid pAS248 (Toda e t aL, 1991) was digested with BamHI as directed by the manufacturer (New England Biolabs). The BamHI en ^m e was removed from the sample using Strataclean Resin (Stratagene) and the sample was concentrated in Microcon-30 microconcentrators (Amicon, Inc.). The digested sample was treated with 0.01 units of

Thermosensitive Alkaline Phosphatase (TsAP) (Gibco BRL) in TsAP buffer supplemented with MgCl 2 ^ frnal concentration of 3mM at 65®C for 20 min and then inactivated following the manufacturer’s protocol.

The 1589 bp, BgUI-digested HyZ clone was ligated [with T4 ligase (BMB)] into the

BamHI site of pAS248, which is located in a small polycloning site immediately downstream of the 5. pom be alcohol dehydrogenase promoter (Russell and Hall, 1983).

The ligation mix was used to transform CaC^ competent JM109 (E. coli) cells (Sambrook e t a t., 1989) and Wizard DNA minipreps were performed using the manufacturer's protocol (Promega Life Sciences). Test digests were performed on the miniprep DNAs to determine if the ligation was successful. A correct plasmid was identified and named 54 PASHt2. a standard large scale plasmid preparation (Sambrook e t a l., 1989) was performed and the plasmid was purified by two rounds of equilibrium centrifugation in

CsCl-EtBr gradients. Purified pASHy2 was sent to Dr. Tetsuya Horio for transformation into S. pombe strains.

Chromosomal mapping

Primer combinations were designed to give specific amplification of Hy2 and were used in PCR reactions to map Hy2. A forward primer, HUGAM1-3F1: aggacaactttgatgagatggaca, with a sequence common to the coding regions of Hyl and Hy 2 was manufactured by Operon Technologies. Two reverse primers corresponding to sequences in the 3'UTR of Hy2 and specific for Hy2, [HUGAM3R1: ctggagatgaaccaagaagggttg, and HUGAM3R2: atctagaaggagaaggagtagtg] were manufactured by Operon Technologies. HUGAM1-3F1 and HUGAM3R1 should amplify a band of 252 bp specifically firom Hy2, while HUGAM1-3F1 and HUGAM3E12 should amplify a 151 bp band specifically firom Hy2. Because the hybrid panels used in chromosomal mapping are composed of human genomic DNA and Chinese hamster ovary (CHO) DNA, it was important to determine if these primer combinations amplified bands firom CHO genomic

DNA. Therefore, CHO genomic DNA and human genomic DNA were tested for specificity of the primers for human genomic DNA. Thirty cycles of first round PCR reactions of 100 p.1 each were carried out with 100-200 ng of CHO genomic DNA or human genomic DNA, 0.2 pmoles each of HUGAM1-3F1 and HUGAM3R1 or 55 HUGAM3R2, dNTPs at 0.2 mM each, and 2.5 units of ID-Pol DNA polymerase (ID

Labs, Inc.) in IX NH^+ID-POL PCR buffer [16mM 67mM Tris-HCl

(pH= 8.8 at 25°C), 0.1% Tween-20] supplemented with 0.50 mM MgC^ (final concentration). The specificity of each of the aforementioned primer combinations for amplification ofHy2 versus Hyl was also tested prior to chromosomal mapping as stated in the Materials and Methods of Chapter 3.

HUGAMI-3F1, HUGAM3R1, and HUGAM3R2 were given to Dr. Ralph Krahe's lab in the Dept, of Human Cancer Genetics at the Ohio State University. They used two independent human-CHO cell hybrid clone panels to map Hy2 to a chromosome. Panel 1 was a multi-chromosomal 17-member human-CHO cell hybrid clone panel (Stallings e t aL,

1988). At least two independent PCRs were carried out on Panel I using each set of primers. The PCR parameters for HUGAM1-3F1 and HUGAM3RI were 94®C for 5 min, followed by 35 cycles at 94®C for 30 sec, 67®C for 30 sec, and 72®C for 30 sec, and a final extension for 10 min at 72® C. The parameters were the same for the HUGAM1-3F1 and HUGAM3R2 combination except that the annealing temperature during the 35 cycles of amplification was at 55®C. PCR products were analyzed by standard 1.5% agarose gel electrophoresis. Human and CHO genomic DNAs were used as positive and negative controls for amplification with each PCR reaction. Discordancy analysis was calculated for each chromosome using Lotus 123.

The second panel (Panel 2) was a mono-chromosomal human-CHO cell hybrid clone panel and was used to verify the mapping results obtained using the multi- 56 chromosomal panel. The same PCR reaction parameters as those used on the multi-

chromosomal panel were used to amplify H 7 2 DNA 6 0 m this panel.

In order to localize H 72 to a more specific region of a human chromosome, the 93- member Whitehead bstitute Genebridge 4 radiation hybrid (GB4RH) panel was used.

PCR analysis was carried out using the same parameters used on Panel 1 and Panel 2.

PCR reactions were resolved on 6% denaturing polyacrylamide gel electrophoresis followed by silver staining. They were then scored to generate a linear vector of numbers that reflect the presence or absence of positive PCR amplification (reviewed in Miano et al.,

1999) and the results were submitted to the Whitehead Institute/MIT center for Genome

Research Radiation Hybrid mapping server (http://www.genome.wi.mit.edu). The map positions and distances of STS markers are expressed in centiRays (cR) where 1 cR equals

~ 300 kb for the GB4RH panel. The map position of Hy2 was determined relative to an

STS marker and its distance was estimated in cR.

Preparation of YAC DNA for PCR analysis

We received a yeast strain from Dr. Johanna Rommen’s lab (University of Toronto) containing yeast artificial chromosome (YAC) HSD2 subclone 'C (Rommens e t al., 1995) which maps to the HSD17B locus on the long arm of chromosome 17 (17q21) and carries the Hyl gene. This strain was grown on YPD (2% w/v peptone, 1% w/v yeast extract, 2% glucose, 1.8% w/v agar) plates at 30®C. Low molecular weight YAC DNA was prepared by modifications of the protocols of Carpten (1994) and Kaiser et al. (1994). Five milliliter 57 cultures inoculated with single colonies from YPD plates were grown in 25 ml erlenmeyer

flasks to saturation, (30°C for 36 hours) in YPD medium with shaking at -200 rpm. Cells

were pelleted at lOOOXg and rinsed with LOG ^tl of 50 mM EDTA, pH=8.0. Pellets were

resuspended in 500 jil of SPEM buffer (IM sorbitol, 100 mM sodium phosphate, pH=7.4,

60 mM EDTA, pH=8.0, and 90 mM p-mercaptoethanol). Zymolyase lOOT (Seikagaku

KOGYO, Co., LTD., Tokyo, Japan) was added to a concentration of 0.5 mg/ml and cells

were incubated at 37®C for 1-2 hours to give 90-100% spheroplasting. Cells were

examined for spheroplasting using phase contrast light microscopy after treating 2.5 |il of

cells with 10 )xl of 5% SDS. Spheroplasts were harvested by centrifugation at 600Xg and

resuspended in 500 ^il of yeast lysis buffer (0.5 M NaCl, 50 mM Tris-Cl, pH=8.0, 25 mM

EDTA, pH=8.0, 30 mM P-mercaptoethanol, 1% w/v SDS, 0.1% v/v NP-40). Cells were

lysed by incubation at 68 °C for 15 minutes and transferred to 2 ml microcentrifuge tubes.

200 jil of 5M potassium acetate was added to the samples and they were incubated on ice

for 1 hour. After a 5 minute centrifugation in a microcentrifuge the supernatant was

transferred to a fresh microcentrifuge tube and precipitated with 1 volume of 100%

isopropanol at room temperature. The DNA was pelleted by centrifugation and the pellet

was air-dried and resuspended in 100 |il of TE, pH=7.4. RnaseA (Dnase-free) (Sigma)

was added to a final concentration of 1 mg/ml and the sample was incubated at 37^C for 30

minutes. One-tenth volume of 3M sodium acetate, pH=5.2, was added to the sample and it was precipitated with 200 pi of 100% isopropanol. The pellet was air-dried then resuspended in 100 pi of TE, pH=7.4. The concentration of the YAC DNA was 58 determined by measuring its absorbance at 260 nm.

YAC DNA from HSD2 subclone C was given to Dr. Ralf Krahe's lab (Dept, of

Human Cancer Genetics, OSU. They used the same primer combinations (HUGAMI-3FI in combination with HUGAM3RI or HUGAM3R2) and PCR conditions that they used in the chromosomal mapping experiments in the previous section in order to determine if the

H72 gene maps to YAC-HSD2. PCR products were run on a 1.5% agarose gel and visualized using EtBr.

PCR analysis and DNA sequencing of HyF

Two specific primers, HGENOIB: gcggatccgccccaagggcacca and HGEN02E: ggaattcggtccatagtcagct, were designed from the sequence of the 930 bp clone, to amplify a

761 bp fragment from human genomic DNA. These primers were manufactured by

Operon Technologies. HeLa cells were obtained from Dr. James Lang's lab (Dept, of

Internal Medicine, OSU) and genomic DNA was isolated as is described in Sambrootc et al.

(1989). Human DNA, obtained from a patient at the OSU Hospital, was given to us by

Dr. Arthur Burghes' Lab (Dept, of Neurology, OSU). HGENOIB and HGEN02E were used to amplify the insert from this HeLa genomic DNA and human patient genomic DNA.

A second specific reverse primer, HGENOR2: gtgcaggatctggtc, was designed and used with HGENOIB to amplify a smaller 576 bp region of H ^ from human genomic DNA.

Platinum Taq DNA polymerase (2.5 units/100 |il reaction) (Gibco BRL) was used to amplify 500 ng of human genomic DNA using the manufacturer’s suggested PCR 59 solutions. The thennocyling parameters for both sets of primers were 94°C for 2 min, followed by 30 cycles at 94®C for I min and 55®C for 4 min. Ten percent (10 pi) of the first round PCR product was used as a template for 25 cycles of second round amplification. Second round PCR products were run on a 1% SeaplaqueGTG low melting point agarose gel (FMQ and were purified using the Wizard PCR prep system (Promega

Life Sciences). Purified samples of the 576 bp fragment of human genomic DNA were sent to the University of Georgia’s Molecular Instrumentation Facüity (Athens, Ga; www.mgif.uga.edu) for sequence analysis. Basic DNA analyses were carried out using

DNA Strider 1.2 software (Marck, 1988). Alignments of the sequence were done with the

ClustalW Algorithm (Thompson et al., 1994) and SeqVu software (Garvan Institute,

Sydney, Australia).

Blast search for RyF

In order to find a full-length clone containing HyF, the Hyl sequence and the 930 bp HyF sequence were used to search the Washington University (St. Louis) human genomic DNA database (http://genome.wustl.edu/gsc) using the default parameters and selecting the GSS— genomic sequence survey database of the BLASTN program (Altschul e t a/., 1990). Sequence alignments were carried out using ClustalW (Thompson et al.,

1994) and SeqVu (Garvan Institute, Sydney, Australia) to compare the clones to Hyl and

HyF.

60 R esults

Estimation of the number ofy-tubulin sequences inHomo sapiens

In order to estimate the number of y-tubulin sequences in Homo sapiens, I

performed Southern hybridizations on HeLa genomic DMA digested with four different restriction enzymes (EcoRL HindUL BamHI, and SacI). The hybridizations were carried out at 60°C or 65^C under the salt conditions given in the Materials and Methods. In 60^C hybridizations, the 5' probe revealed two or three bands of hybridization in each restriction enzyme digest (Figure 2). This probe also hybridized faintly to a 4.8 kb band in the A. nidulans lane, that is the expected size of an EcoRI fragment that contains the A. nidulans y-tubulin gene. This probe shares 54.2% nucleotide identity with the corresponding A. nidulans sequence. Since this probe detected an A. nidulans y-tubulin sequence at 60°C, we can conclude that any human y-tubulin not detected under our conditions would have to be extremely divergent from Hyl. The 3' BamHI probe also showed two or three bands of hybridization (Figure 3) in each digest at 60®C but did not show a band of hybridization in the A. nidulans DNA. The 3' probe shares only 32.1% with the corresponding A. nidulans y-tubulin sequence and, therefore, may be too divergent to hybridize with A. nidulans DNA under the conditions used. Hybridizations carried out at 65°C using either the 5' probe or the 3' (Pstl/Clal) probe showed similar results except that neither probe showed hybridization to A. nidulans DNA (data not shown). From these data we can conclude that no more than three y-tubulin sequences are detected under these conditions 61 and therefore there are probably no more than three y-tubulin sequences in the human genome.

Identification and characterization of a novel human y-tuhuiin cDNA, Hy2

A 930 bp human y-tubulin fiagment was obtained by PCR amplification of human genomic DNA and was provided to us by Dr. Helmut Schraudolf (Universitat Ulm). I subcloned this fiagment into M13mpl8 and M13mpl9 to facilitate single-stranded dideoxy sequencing. Sequencing analysis of this fiagment which I named H ^ (Figure 4) revealed that it shares 91.6% nucleotide identity and 86.1% amino acid identity with the corresponding region of Hyl (Figure 5). These results indicated that HyF is a novel sequence and it had not been amplified firom Hyl. The 930 bp/310 aa H*^ clone spans more than two-thirds of the Hyl cDNA sequence (see Figure 5) and, interestingly, it does not contain any intron sequences.

In an effort to clone a cDNA corresponding to H ^ , I used the H ^ fiagment as a probe to screen a HeLa A, ZapII cDNA library at high stringency. 1 screened 2.5 x 10® plaques over four rounds of purification and isolated 21 positive clones. Partial sequencing of the clones revealed that none of them corresponded to HyF. Fifteen of the clones corresponded to Hyl, one clone contained a human a-tubulin sequence, and four of the clones, (pDOWla, pDOW12, pDOW7, pDOW21) represented a novel human y-tubulin

62 Figure 2: Low stringency Southern analysis of human genomic DNA using a. 5 -Hyl probe.

Hela genomic DNA samples (10 |ig each) were digested with EcoRI, HindlU, BamHI, or SacI. A. nidulans genomic DNA (labeled AspE) was digested with EcoRI in order to serve as a control for hybridization stringency. Digests were run on 0.7% agarose gels, processed for Southern analysis, and probed with a 515 bp 5 -Hyl probe at 60®C. The positions of the XHindHI molecular weight marker bands are shown to the right of the gel.

63 m indm

. 23.1 kb

. 9.4 kb

_ & 7kb

_ 4.4 kb

2.3 kb 2.0 kb

Figure 2.

64 Figure 3: Low stringency Southern analysis of human genomic DNA using a 3'-Hyl probe.

HeLa genomic DNA samples (10 jig each) were digested with EcoRI, HindUI, BamHI, or SacI. Digests were run on 0.7% agarose gels, processed for Southern analysis, and probed with a 528 bp 3 -Hyl sequence at 600C.

65 5 1

u>

EcoRl g; I 11 I

g Figure 4: The nucleotide sequence of and its predicted amino acid sequence.

The genomic clone we received from Dr. Helmut Shraudoif and sequenced on both strands. This clone is 930 base pairs long, encodes a 310 amino acid protein, and lacks introns.

67 1 /1 g g g t t t t g g a a a e a g e t g t g e a c t gag c a t g g t ate age eee aag gge a e e a t g g a g g ag g i y p h e t r p l y s g i n l e u e y s t h r g l u h i s g l y ile ser pro lys g l y t h r m a t g lu g l u 6 1 /2 1 t t c g e t a c t g a g g g e a c t g a e e a e a a g g ae a t e t t t t t e t a e eag gca gae gat g a g e a e p h e a l e t h r g l u g l y t h r a s p h i s l y s a s p i l e p h e phe t y r g i n a l a a s p a s p g l u h i s 1 2 1 /4 1 t a c a t e eee egg get gtg e t g e t g g a e e t g g a g eee egg gtg ate eae tee ate ete aae tyr ile pro arg a l a v a l l e u l e u asp leu glu pro arg val ile h i s ser ile leu asn 1 8 1 /6 1 t e e c e e t g t g e e a a g e t c t a e a a e e e a g ag a a c ate tae etg teg g a g c a t ggg g g a a g a s e r p ro e y s a l a l y s l e u t y r a s n p ro g lu asn lie tyr leu ser g l u h i s g l y g l y a r g 2 4 1 /8 1 g e t g g e a a e a a e t g g g e e a g e a g n t t e t e e e a g agg gag aag ate eat gag g a c a t t t t t a l a g l y a s n a s n t r p a l a s e r a r g p h e s e r g i n a r g g lu ly s i l e h i s g lu a s p i l e p h e 3 0 1 /1 0 1 aac ate aca gae eag g a g g c a g a t g g t a g t g a e a a t e t a g a t g g e t t t g tg e t g t g t c a g asn ile thr asp g i n g l u a l a a s p g l y s e r a s p a s n le u a s p g l y p h e v a l l e u e y s g i n 3 6 1 /1 2 1 t t e a t t g e t gg g ggg a e a g g e t e t g g e e t g gge tee tae ete t t a g a a tg g e t g a a t g a e p h e i l e a l a g l y g l y t h r g l y s e r g l y l e u g l y s e r t y r le u l e u g l u t r p l e u a s n a s p 4 2 1 /1 4 1 agg tat eet aag aag etg gtg e a g a e a t a e t e a g t g ttt cee aac eag gae a a g a t g a g e a r g t y r p ro l y s l y s l e u v a l g i n t h r t y r s e r v a l phe p ro a s n g i n a s p l y s m e t s e r 4 8 1 /1 6 1 aae gtg gtg gte eag ett tat a a t t e a e t c e t e a e a e t c a a g a g g e t g a t g e a g a a e g a a asn val val val g i n l e u t y r a s n s e r le u leu thr leu lys arg leu met gin asn glu 5 4 1 /1 8 1 g a c t g t g t g g t g g t g e t g e a e a a e a e a gee etg aae eag att gee aea gae eag ate etg a s p c y s v a l v a l v a l l e u h i s a s n t h r a l a l e u a s n g in i l e a l a t h r a s p g i n i l e le u 6 0 1 /2 0 1 e a e a t e e a g a a e e e a t e e t t e t e t e a g a e e a a e e a g e t g g tg tee aee ate ate t e g g e e h i s i l e g i n a s n p ro s e r p h e s e r g i n t h r a s n g i n le u v a l s e r t h r ile ile ser ala 6 6 1 /2 2 1 age aec ate aee etg ege t a e c e e a g e t a e a t g g a e a a t g ae e t c a t a gge e t e a t e g e e s e r t h r i l e t h r l e u a r g t y r p ro s e r t y r a s p a s n a s p l e u i l e g ly le u i l e a l a 7 2 1 /2 4 1 t e a e t e a t t e e e a e e e e a tg g e t e e a e t t t e t e a t g a c t gge tac aee eag etg a c t a t g s e r l e u i l e p ro t h r p ro t r p l e u h i s phe l e u SM t t h r g ly t y r t h r g in l e u t h r m et 7 8 1 /2 6 1 g a e c a a t e a g t g g e e a g e g t g a g g a a g a e e a t g g t e e t g g a t g t e a t g ag g tg g e t g e t g a s p g i n ser val ala ser v a l a r g l y s t h r n e t v a l le u a s p v a l m e t a r g t r p l e u l e u 8 4 1 /2 8 1 c a g c e e a a g a a e g t g a t g g t a t e e a e a gge e g a g a e ege c a g a e c a a e e a e t e e t a e a t e gin pro lys asn val val s e r t h r g ly a r g a s p a r g g in thr asn his ser tyr ile 9 0 1 /3 0 1 a e e a t e e t e a a e a t e a t t e a a g g e g a a g tg t h r i l e l e u a s n i l e i l e g i n g l y g l u v a l

Hgure 4-

68 cDNA that I have named Ky2 (Human gam m a 2). None of these four clones, however, encoded a full-length Ht^ sequence. Two clones ^DOW7 and pDOW21) (Figure 6) contained 1465 bp inserts with identical sequences and two clones (pDOWla and pDOW12) (Figure 7) contained 809 bp inserts that are identical to each other and to the corresponding regions within the larger inserts. The two larger clones lacked the region corresponding to the N-terminal 48 amino acids of Hyl (see Figure 8 ) but extended into the

3' UTR region which contains a poly(A) signal (AATAAA) and ended in a poly (A) tail.

In order to identify a full-length Hy2 clone, I searched the human Expressed

Sequence Tag database (dbEST) maintained by the National Center for Biotechnology

Information (NCBI). Thirty-nine of 159 EST sequences analyzed encoded partial Hy2 sequences (the other ESTs encoded Hyl). A full-length cDNA clone (Accession No

H10668 and HI0669) obtained from a human infant (73 days post natal) brain cDNA library was identified as corresponding to Hy2 based on the EST sequence which covered approximately 450 bp at each end of the clone. This cDNA clone (which I named

HylEST) was obtained from Research Genetics and sequenced on both strands. The cDNA is 1857 bp in length and contains the entire Hy2 coding region as well as 244 bp of the 5'UTR and 232 bp of the 3'UTR (Figure 9). The sequence of Hy2EST was identical to the corresponding regions of the cDNA in pDOW7 and pDOW21, except that Hy2EST contained a 27 bp in-frame insert within the coding region that was not present in the corresponding region of pDOW7 and pDOW21, nor the same region in pDOWla and

69 Figure 5: A comparison of the predicted amino acid sequences of and Hyl.

Abbreviations; H.gl-predicted amino acid sequence of Hyl; H.gF-predicted amino acid sequence of HyF.

The H ^ clone spans more than two-thirds of the Hyl cDNA sequence and shares 86.1% aa identity with the corresponding region of the Hyl cDNA.

70 H.gl MPREIITLQLGQC6NQI6FEFNKQLCAEH6ISPBAIVEEFATB6TDRKDVFFYQADDEHX 60 H #gF G——————T——————K6TM————————"E"" I" """"""""""" 41

H.gl IPRAVLLDLEPRVZHSILNSPZAKLXNPENIXLSEHGGGAGNNIfASGFSQGEKIBEDZFD 120 H . g F ------C------R------R-- R N 101

H.gl ZZDREADGSDSLEGFVLCHSZAGGTGSGLGSZLLERLNDRZPKKZ.VQTZSVFPNQDEMSD 180 H.gF -T-Q----- M-D-----QF------K------K— M 161

H.gl VWQPZNSLLTLKRLZQNADCVWLDNTALNRZATDRLRZQNPSFSQZNQLVSTZIfSAST 240 H . g F L------— M— E----- H---- Q--- Q------T------Z-----221

H.gl TTLRYPGYMNNDLZGLZASLZPTPRLHFLMTGYTPLTTDQHVAHVRRTTVLDVNRRLLQP 300 H.gF Z--- -S—D------W------Q— M— ------M------W,---- 281

H.gl KNVMVSTGRDRQTNHCYZAZLNZZQGEVDPTQVHKSLQRZRERKZJiNFZPIfGPASZQVAL 360 H . g F ------S—T------310

H.gl SRKSPYLPSAHRVSGZJ0IANHTSZSSLFERTCRQYDRLRKREAF1.EQFRKEDNFRDHFDE 420

H.gl MDTSREZVQQIiZDEYHAATRPDYZSirGTQEQ 451

Figure 5. Figure 6: The nucleotide and predicted amino acid sequence of clones pDOW7 and pDOW2L

The sequence shown in this figure corresponds to the 1465 bp sequence of two identical clones, pDOW7 and pDOW21, that were identified by screening a HeLa cDNA library with Hyp. The sequence o f these clones spans 89.4% of the coding region of a novel y-tubulin gene, Hy2. The predicted amino acid sequence of the novel y-tubulin gene contains 404 amino acids in the coding region of the gene. The stop codon is underlined in the figure. This sequence contains a polyadenylation signal sequence (underlined) and it ends at the poly(A)tail.

72 1/1 aat tcc age aag gac gte ttt ttc tac cag gca gac gat gag cac tac ate ccc egg gcc asn ser ser lys asp val phe phe tyr gin ala asp asp gin his tyr ile pro arg ala 6 1 /2 1 gtg etg etg gac ttg gaa ccc egg gtg ate cac tcc ate etc aac tcc ccc ta t gcc aag val lea leu asp leu glu pro arg val ile his ser ile leu asn ser pro tyr ala lys 1 2 1 /4 1 etc tac aac cca gag aac ate tac etg teg gaa cat gga gga gga get gge aac aac tgg leu tyr asn pro glu asn ile tyr leu ser glu his gly gly gly ala gly asn asn trp 1 8 1 /6 1 gcc age gga ttc tcc cag ggt gag aaa a tt cat gaa gac ate ttt gac ate ata gac cga ala ser gly phe ser gin gly glu lys ile his glu asp ile phe asp ile ile asp arg 2 4 1 /8 1 gaa gca gat gga agt gac agt ttg gag gge ttc gtg etg tg t cac tcc ate get ggg ggt glu ala asp gly ser asp ser leu glu gly phe val leu eys his ser ile ala gly gly 3 0 1 /1 0 1 acg ggt te t gge etg gge tcc tac etc etg gag cga etg aat gac agg tac ccc aag aag thr gly ser gly leu gly ser tyr leu leu glu arg leu asn asp arg tyr pro lys lys 3 6 1 /1 2 1 eta gtg cag act tat tea gtg ttt ccc tac cag gac gag atg age gac gta gtg gtt cag leu val gin thr tyr ser val phe pro tyr gin asp glu met ser asp val val val gin 4 2 1 /1 4 1 ccc tac aat tea etc etg aca etc aag agg etg acg cag aac gca gat tg t gtg gtg gtg pro tyr asn ser leu leu thr leu lys arg leu thr gin asn ala asp cys val val val 4 8 1 /1 6 1 etg gac aac aca gcc etg aac egg a tt gcc aca gac cgc etg cac ate cag aac ccg tcc leu asp asn thr ala leu asn arg ile ala thr asp arg leu his ile gin asn pro ser 541/181 572/191 ttc tcc cag ate aac cag etg gtg tcc acc ate atg teg gcc age acc acc acc etg cgc phe ser gin ile asn gin leu val ser thr ile met ser ala ser thr thr thr leu arg 6 0 1 /2 0 1 tac ccc gge tac atg aac aat gac etc ate gge etc ate gcc teg etc att ccc acc cca tyr pro gly tyr met asn asn asp leu ile gly leu ile ala ser leu ile pro thr pro 6 6 1 /2 2 1 egg etc cac ttt etc atg acc gge tac acc ccg etc act aca gac cag tea gtg gcc age arg leu his phe leu met thr gly tyr thr pro leu thr thr asp gin ser val ala ser 7 2 1 /2 4 1 gtg agg aag acc acg gte etg gat gte atg agg egg etg etg cag ccc aag aac gtg atg val arg lys thr thr val leu asp val met arg arg leu leu gin pro lys asn val met 7 8 1 /2 6 1 gtg tcc aca gge cga gac cgc cag acc aac cac tgc tac ate gcc ate etc aac ate ate val ser thr gly arg asp arg gin thr asn his cys tyr ile ala ile leu asn ile ile 8 4 1 /2 8 1 cag gga gag gtg gac ccc acc cag gte cac aag age etg cag agg ate egg gaa egg aag gin gly glu val asp pro thr gin val his lys ser leu gin arg ile arg glu arg lys 9 0 1 /3 0 1 ttg gcc aac ttc ate ccg tgg gge ccc gcc age ate cag gtg gcc etg teg agg aag te t leu ala asn phe ile pro trp gly pro ala ser ile gin val ala leu ser arg lys ser 9 6 1 /3 2 1 ccc tac etg ccc teg gcc cac egg gte age ggg etc atg atg gcc aac cac acc age ate pro tyr leu pro ser ala his arg val ser gly leu met met ala asn his thr ser ile (To be continued)

Figure 6 . (Hgure 6 continued)

1 0 21 /341 tcc teg etc ttt gaa agt tcc tgc cag cag ttt gac aag etg egg aag egg gat gcc ttc ser ser lea phe glu ser sec cys gin gin phe asp lys lea arg lys arg asp ala phe 1 0 81 /361 etc gag cag ttc cgt aag gag gac atg ttc aag gac aac ttt gat gag atg gac agg te t lea gla gin phe acg lys gla asp swt phe lys asp asn phe asp gla met asp arg ser 1 1 41/381 agg gag g tt g tt cag gag etc a tt gat gag tac cat gcg gcc acc cag cca gac tac a tt arg gla val val gin gla lea lie asp gla tyr his ala ala thr gin pro asp tyr lie 1 2 01/401 tcc tgg gge acc cag gag cag tea tttccctccccactactccttctccttctagatggtaaccacagcctc ser trp gly thr gin gla gin OPA 1275 gaccatgcctgctccctctgacccagcttcacctcatggacaacccttcttggttcatctccagcccgtgagctggtcct gcttcctcccttccatQ ccctaacttttaatatacttattcaactctaataaaataataaaacttacrttatcagtaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

74 Figure 7: The nucleotide and predicted amino acid sequence of clones pDOWla and pDOWI2.

The nucleotide sequence shown on this figure corresponds to the 809 bp sequences of two identical cDNA clones, pDOWla and pDOW12, that were identified by screening a HeLa cDNA library with HyF. The predicted am ino acid sequence contains 269 amino acids. These clones encode a portion (59%) of a novel y-tubulin gene, Hy2. This sequence is contained within the sequence of clones pDOWT and pDOW21.

75 1 /1 g a c a t c t t t g a c a t c a t a g a c c g a g a a g c a g a t g g a a g t g a c a g t t t g gag gge t t c g t g a s p i l e p h e a s p i l e i l e a s p arg glu ala asp g ly s e r a s p s e r l e u g lu g l y p h e v a l 6 1 /2 1 e t g t g t c a c t c c a t c g e t ggg g g t a c g g g t t e t g g e e t g g g e t c c t a c e t c e t g g a g c g a l e u c y s h i s s e r i l e a l a glyg ly t h r g l y s e r g ly l e u g l y s e r t y r l e u l e u g l u a r g 1 2 1 /4 1 e t g a a t g a c ag g t a c c c c a a g a a g c t a g t g c a g a c t t a t t e a g t g t t t c c c t a c c a g g a c leu asu asp arg t y r p r o l y s lys leu val gin t h r t y r s e r v a l p h e p ro t y r g i n a s p 1 8 1 /6 1 g a g a t g a g c g ac g t a g t g g t t c a g c c c t a c a a t t e a e t c e t g a c a e t c a a g a g g e t g a c g glu met ser asp v a l v a l v a l g in p ro t y r a s n s e r l e u l e u t h r l e u l y s a r g l e u t h r 2 4 1 /8 1 c a g a a c g c a g a t t g t g t g g t g g tg e t g g a c a a c a c a g c c e t g a a c c g g a t t g c c a c a g a c g i n a s n a l a a s p c y s val val val leu asp asn t h r a l a l e u a s n a r g i l e a l a t h r a s p 3 0 1 /1 0 1 c g c e t g c a c a t c c a g a a c c c g t c c t t c t c c c a g a t c a a c c a g e t g g tg t c c a c c a t c a t g a r g l e u h i s i l e g i n a s n p ro s e r p h e s e r g i n i l e a s n g i n l e u v a l s e r t h r i l e s w t 3 6 1 /1 2 1 t c g g c c a g c a c c a c c a c c e t g c g c t a c c c c g g e t a c a t g a a c a a t g a c e t c a t c g g e e t c s e r a l a s e r t h r t h r t h r l e u a r g t y r p r o g l y t y r m e t a s n a s n a s p le u i l e g l y l e u 4 2 1 /1 4 1 a t c g c c t c g e t c a t t c c c a c c c c a c g g e t c c a c t t t e t c a t g a c c gge t a c a c c c c g e t c i l e a l a s e r l e u i l e p r o t h r p ro a r g l e u h i s phe l e u n w t t h r g l y t y r t h r p r o l e u 4 8 1 /1 6 1 a c t a c a g a c c a g t e a g t g g c c a g c g tg a g g a a g a c c a c g g t e e t g g a t g te a t g a g g c g g t h r t h r a s p g in s e r v a l a l a s e r v a l a r g l y s t h r t h r v a l l e u asp val met arg a r g 5 4 1 /1 8 1 e t g e t g c a g c c c aa g a a c g t g a t g g tg t c c a c a gge c g a g a c c g c c a g a c c a a c c a c tg c l e u l e u g in p ro ly s a s n v a l sm t v a l s e r t h r g ly a r g a s p a r g g in t h r a s n h i s c y s 6 0 1 /2 0 1 t a c a t c g cc a t c e t c a a c a t c a t c c a g g g a g ag g tg g a c c c c a c c c a g g te c a c a a g ag c t y r i l e a l a i l e le u a s n i l e i l e g in g l y g l u v a l a s p p r o t h r g in v a l h is l y s s e r 6 6 1 /2 2 1 e t g c a g a g g a t c c g g g a a c g g aa g t t g g c c a a c t t c a t c c c g tg g g g e c c c g c c a g c a t c l e u g i n a r g i l e a r g g l u a r g ly s l e u a l a a s n phe i l e p ro t r p g l y p ro a l a s e r i l e 7 2 1 /2 4 1 c a g g t g g c c e t g tc g a g g a a g t e t c c c t a c e t g cc c t c g g c c c a c c g g g te ag c g g g e t c g i n v a l a l a le u s e r a r g l y s s e r p ro t y r l e u p ro s e r ala his arg val s e r g l y l e u 7 8 1 /2 6 1 a t g a t g g c c a a c c a c a c c a g c a t c t c c m e t m e t a l a a s n h is t h r s e r i l e s e r

Figure 7.

76 Figure 8 : A comparison o f the predicted amino acid sequences of H ^-cione pDOW7 and

H yl.

Abbreviations; Hgaml predicted amino acid sequence of Hyl; Hy2/pD7-predicted amino acid sequence of the Hy2 insert in pDOW7.

The predicted amino acid sequence o f the pDOW7, partial Hy2 insert is highly homologous to Hyl. The clone lacks the region corresponding to the N-terminal 48 amino acids of Hyl. Amino acid differences are indicated by asterisks.

77 h g a m l MPREIITLQI, GQCCaiQIGEE FWKQLCAEH6 XSPEAIVEEF ATEGTDSKDV H g 2 /p D 7 ...... KDV

h g a m l FFYQADDBHY IPRAVLLDLB PRVIHSILNS PYAKLYMSEN lYLSEHGGGA H g 2 /p D 7 FFYQADDEHY IPRAVLLDLE PRVIHSILNS PYAKLYMPEN lYLSEHGGGA

h g a m l GNNWASGFSQ GBKIHEDIPD IIDREADGSD SLEŒVLCHS lAGGTGSGLG H g 2 /p D 7 GNMWASGFSQ GEKIHEDIFD IIDREADGSD SLEGFVLCHS lAGGTGSGLG

h g a m l SYLLERLNDR YPKKLVQTYS VFPNQDEMSD WVQPYNSLL TLKRLTQNAD H g 2 /p D 7 SYLLERLNDR YPKKLVQTYS VFPYQDEMSD WVQPYNSLL TLKRLTQNAD

h g a m l CLWLDNTAL NRIATDRLHI QNPSFSQINQ LVSTIMSAST TTLRYPGYMN H g 2 /p D 7 CVWLDNTAL NRIATDRLHI QNPSFSQINQ LVSTIMSAST TTLRYPGYMN

h g a m l NDLIGLIASL IPTPRLHFLM TGYTPLTTDQ SVASVRKTTV LDVMRRLLQP H g 2 /p D 7 NDLIGLIASL IPTPRLHFLM TGYTPLTTDQ SVASVRKTTV LDVMRRLLQP

h g a m l KNVMVSTGRD RQTNHCYIAI LNIIQGEVDP TQVHKSLQRI RERKLANFIP H g 2 /p D 7 KNVMVSTGRD RQTNHCYIAI LNIIQGEVDP TQVHKSLQRI RERKLANFIP

h g a m l WGPASIQVAL SRKSPYLPSA HRVSGLMMAN HTSISSLFER TCRQYDKLRK H g 2 /p D 7 WGPASIQVAL SRKSPYLPSA HRVSGLMMAN HTSISSLFES SCQQFDKLRK

h g a m l REAFLEQFRK EDMFKDNFDE MDTSREIVQQ LIDEYHAATR PDYISWGTQE H g 2 /p D 7 RDAFLEQFRK EDMFKDNFDE MDRSREWQE LIDEYHAATQ PDYISWGTQE

h g a m l QZVPQDRGPS SALLVGPSPA ZLTTPSEHRS GTSRISFS ...... YTWTLCW H g 2 /p D 7 Q ZFPS...PL LLLLLDGNHS LDHACSLZPS FTSWTTLLGS SPARELVLLP

h g a m l p a n t f t s . p l MRLFIFNKAL H g 2 /p D 7 PFHALTFNML VQLZZSNKAW

Figure 8 .

78 pDOW12. Although it is possible that the insert resulted firom tissue-specific alternative

splicing, it occurs in a highly conserved region of the y-tubulin sequence.

Interestingly, this insert appears to render the Hy2 gene toxic and lethal to S. pombe. Horio and Oakley (1994) found that expression of a Hyl cDNA supported the

growth of S. pombe cells that lacked endogenous y tubulin. To determine if this was the case for Hy2EST, I cloned Hy2EST into plasmid pAS24S such that its expression was driven by the S. pombe alcohol dehydrogenase (ADH) promoter. This promoter is constitutive and moderately strong. The resulting plasmid, pASHy2, was sent to Dr.

Tetsuya Horio. Dr. Horio transformed pASHyZ into 5. pombe strain NC377, selecting for the leucine marker on pASHy2. NC377 is a diploid strain which carries one fimctional y- tubulin gene and one y tubulin disrupted by a URA4+ gene. Only a few transformants were obtained compared to hundreds of transformants with control DNA. Dr. Horio suggested that the small number of transformants is significant and it seems likely that expression of pASHy2 is toxic to S. pombe.

But for the 27 bp insert in Hy2EST, the coding regions of Hyl and Hy2 share

94.6% nucleotide identic and 97.3% amino acid identity (Figure 11). Many of the base pair differences that exist between the two genes are in the third position of the codon and cause no amino acid difierences. Interestingly, eight of the twelve amino acid differences observed between the two sequences are in the C-terminal 61 amino acids of the proteins.

The 3' UTRs of the two genes share 54% nucleotide identity (Figure 12). The poly(A)tail

79 of Hyl is 191 bp 3* to the stop codoa and the poly(A) tail of Hy2 is 204 bp 3' to the stop

codon.

Somatic Cell and Radiation Hybrid Mapping of Hy2

I designed two combinations of primers that specifically amplify 224 bp and 145 bp

regions of Hy2 (see Figure 18 in Chapter 3). These primers were given to Dr. Ralph

Krahe's lab (Dept of Human Cancer Genetics, OSU) who used them to amplify genomic

DNA from three different chromosomal panels; (1) a human-CHO multi-chromosomal

hybrid mapping panel, (2) a human-CHO mono-chromosomal panel, and (3) a GB4RH

radiation hybrid panel. Analysis of the multi-chromosomal panel revealed that Hy2, like

Hyl, maps to Chromosome 17. PCR analysis of the mono-chromosomal mapping panel

unambiguously confirmed that Hy2 maps to chromosome 17.

Further refinement of Hy2's chromosomal location using the radiation hybrid panel

indicated that Hy2 maps to 17ql2-21 a region that includes the Hyl gene (see Figure 13)

(Friedman et al., 1995; Rommens et al., 1995; Couch et al., 1995; Brody et al., 1995).

Because of these results we wanted to map the genes more precisely relative to each other.

Since Dr. Joanna Rommen’s lab had mapped a Hyl cDNA to a 7CX) kb YAC, HSD2

(Rommens et al., 1995), I obtained a strain containing this YAC from Dr. Rommens and isolated DNA from the strain. Dr. Krahe's lab used the Hy2 specific primers to determine if Hy2 is also located on this 7(X) kb YAC. As is shown in Figure 14, the primer combinations amplified Hy2 fragments. These results indicate that Hyl and HyZ are within

80 Figure 9; The nucleotide sequence of H 72 EST and its predicted amino acid sequence.

The Hy2EST cDNA is 1857 base pairs in length and contains the entire H 72 coding region as well as a 27 base pair in-frame insert within the coding region of the gene. The cDNA contains 244 base pairs of the 5' UTR and 232 base pairs of the 3'UTR. The clone ends at the poly(A)tail which is preceded by a polyadenylation signal (AATAAA) that is underlined in the tigure. The start and stop codons are in bold face type.

81 1 a a g c t t acgaggaattc'ttggatgacatccctcageatgtctgagcgactgctgagggagaaaatgatgcccagg^tggtccccgg cccaccgcagagaggcctgcgctgcacacgcgcagaccgagcatccgcgtcaagaggcgaagagagcgcgeccccacgte ctgegetcctggetgccgggcattcgtetcagccgtgactctcgccaggccggggctggcgcgcccacgtctgaagagcg 2 5 0 /1 atg ccc cgg gag ate ate acc etg cag etg gge cag tge gge aae eag a tt ggg ttc gag Met pro arg gla ile ile thr lea gla lea gly gin cys gly asn gin ile gly phe gla 3 1 0 /2 1 ttc tgg aaa cag etg tgc gcc gag cat ggt atc agc ccc gag gge atc gtg gag gag ttc phe trp lys gin lea eys ala gla his gly ile ser pro gla gly ile val gla gla phe 3 7 0 /4 1 gcc acc gag gge act gac cgc aag gae gte ttt ttc tae cag gca gac gat gag cac tac ala thr gla gly thr asp arg lys asp val phe phe ty r gin ala asp asp gin his tyr 4 3 0 /6 1 atc ccc cgg gcc gtg etg etg gac ttg gaa ccc cgg gtg atc cac tcc atc etc aac tcc ile pro arg ala val lea lea asp lea gla pro arg val ile his ser ile lea asn ser 4 9 0 /8 1 ccc ta t gcc aag etc tac aae cca gag aac atc tac etg tcg gaa cat gga gga gga get pro tyr ala lys lea ty r asn pro gla asn ile tyr lea ser gla his gly gly gly ala 5 5 0 /1 0 1 gge aac aac tgg gcc age gga ttc tcc cag ggt gag aaa a tt cat gaa gae atc ttt gac gly asn asn trp ala ser gly phe ser gin gly gla lys ile his gla asp ile phe asp 6 1 0 /1 2 1 atc ata gac cga gaa gca gat gga agt gac agt ttg gag etc cag aac agc tcc aga gtg ile ile asp arg gla ala asp gly ser asp ser lea gla lea gin asn ser ser arg val 6 7 0 /1 4 1 ata aag gge ttc gtg etg tgt cac tcc atc get ggg ggt acg ggt tet gge etg gge tcc ile lys gly phe val lea cys his ser ile ala gly gly thr gly ser gly lea gly ser 7 3 0 /1 6 1 tac etc etg gag cga etg aat gac agg tac ccc aag aag cta gtg cag act ta t tea gtg tyr lea lea gla arg lea asn asp arg tyr pro lys lys lea val gin thr tyr ser val 7 9 0 /1 8 1 ttt ccc tac cag gac gag atg agc gac gta gtg g tt cag ccc tac aat tea etc etg aca phe pro tyr gin asp gla met ser asp val val val gin pro tyr asn ser lea leu thr 8 5 0 /2 0 1 etc aag agg etg acg cag aac gca gat tg t gtg gtg gtg etg gac aac aca gcc etg aac lea lys arg lea thr gin asn ala asp cys val val val lea asp asn thr ala lea asn 9 1 0 /2 2 1 cgg a tt gcc aca gac cgc etg cac atc cag aac ccg tcc ttc tcc cag atc aac cag etg arg ile ala thr asp arg leu his ile gin asn pro ser phe ser gin ile asn gin lea 9 7 0 /2 4 1 gtg tcc acc atc atg tcg gcc agc acc acc acc etg cgc tac ccc gge tac atg aac aat val ser thr ile met ser ala ser thr thr thr lea arg tyr pro gly tyr a»t asn asn 1 0 3 0 /2 6 1 gac etc atc gge etc atc gcc tcg etc att ccc acc cca cgg etc cac ttt etc atg acc asp lea ile gly lea ile ala ser lea ile pro thr pro arg lea his phe lea met thr 1 0 9 0 /2 8 1 gge tac acc ccg etc act aca gac cag tea gtg gcc agc gtg agg aag acc acg gte etg gly tyr thr pro lea thr thr asp gin ser val ala ser val arg lys thr thr val lea

(To be continued) Figure 9.

82 (Figure 9 continued)

1 1 5 0 /3 0 1 gat gtc atg agg egg ctg ctg cag ccc aag aac gtg atg gtg tec aca ggc cga gac cgc asp val net azg arg lea leu gin pro lye asn val net eel ser thr gly arg asp arg 1 2 1 0 /3 2 1 cag ace aac cac tgc tac ate gee ate etc aae ate ate cag gga gag gtg gac cce aec gin thr asn his eys tyr lie ala lie leu asn ile lie gin gly gin val asp pro thr 1 2 7 0 /3 4 1 cag gtc eae aag age ctg eag agg ate egg gaa egg aag ttg gee aae ttc ate ecg tgg gin val his lys ser leu gin arg ile arg glu arg lys leu ala asn phe ile pro trp 1 3 3 0 /3 6 1 gge eee gee age ate eag gtg gee etg teg agg aag te t cee tae etg cee teg gee eae gly pro ala ser ile gin val ala leu ser arg lys ser pro tyr leu pro ser ala his 1 3 9 0 /3 8 1 egg gte age ggg etc atg atg gee aae eac aee age ate tee teg etc ttt gaa agt tee arg val ser gly leu net met ala asn his thr ser ile ser ser leu phe glu ser ser 1 4 5 0 /4 0 1 tge eag eag ttt gae aag etg egg aag egg gat gee ttc etc gag eag tte cgt aag gag eys gin gin phe asp lys leu arg lys arg asp ala phe leu glu gin phe arg lys glu 1 5 1 0 /4 2 1 gae atg tte aag gae aae ttt gat gag atg gae agg te t agg gag gtt gtt cag gag etc asp net phe lys asp asn phe asp glu net asp arg ser arg glu val val gin glu leu 1 5 7 0 /4 4 1 a tt gat gag tae eat geg gee aee eag eea gae tae a tt tee tgg gge aee cag gag eag ile asp glu tyr his ala ala thr gin pro asp tyr ile ser trp gly thr gin glu gin 1 6 3 0 /4 6 1 tgattteeeteeceaetaetectteteettetagatggtaaeeaeageetegaeeatgeetgeteeetctgaeeeaget 0P& teaeeteatggacaaeeettettggtteateteeageeegtgagetggteetgctteeteeetteeatgeeetaaettt taatataettatteaaetetaataaaataataaaQettaattuteaatataaaaaaaaaaaaaaaaaaaa

83 Figure 10: The construction o f plasmid pASHy2.

Abbreviations: ADH-alcohol dehydrogenase promoter; LEU- leucine selectable marker; Ps- PstI site; Bm-BamHI site; Sm-Smal site; Sc-SacI site; Bgl-BglQ ends of Hy2 specific primers. The coding region plus 205 bp of the 3'UTR of Hy2EST was amplified from plasmid pHyZEST with primers containing Bgin ends. Following amplification the Hy2EST PCR product was digested with BgUI. In parallel, plasmid pAS248 was digested with BamHI; the BamHI site is in the polylinker of pAS248. The BgUI digested Hy2EST PCR product was ligated into the BamHI-digested pAS248 downstream of the ADH promoter. The resulting plasmid was named pASHy2.

84 L«u

Amplify the coding region plus 205 bp of the 3’ ÜTR of HylEST located In pHg2EST with Hy2 specific primers containing Bgin ends.

AOH % B g l"

■B9I I SC Bffll iBgX Digest pAS248 %vith BamHI.

Digest Hy2EST PCR product with BgUI.

Ligate Hy2EST into pAS248. The new plasmid construct is called pASHy2.

L o u

AO

Figure 10. 85 Figure 11: A compansou of the predicted amino acid sequences of H 72 EST and HyL

Abbreviations: Hgaml-predicted amino acid sequence of Hyl; Hg2EST-predicted amino acid sequence of H 72 EST.

The coding regions of Hyl and Ht2£ST (minus the 27 bp insert that is underlined in this figure) share 97.3% amino acid identity. Only twelve amino acid differences exist between Hyl and Hy2 and eight of the differences are clustered in the C-terminal 61 amino acids of the proteins. Most of the nucleotide differences that exist between the two genes occur in the third position of the codon thus enabling the amino acid sequences of the two genes to be highly conserved. Amino acid differences are marked by asterisks.

86 hgaml MPSEIXTLQI. GQCQIQXGFE FWKQLCAEH 6 XSPEAXVEEF ATEGTDRKDV 50 agZ est MPKEXXTLQL GQCGNQXGFE FWKQLCAEH 6 XSPEGXVEEF ATEGTDRKDV 50

hgam l FFYQADDEHY XPRAVLLDLE FRVXHSXLNS PYAKI.YNFBN XYX.SEHGGGA 100 H g2est FFYQADDEHY XPRAVDLDXg FRVXHSXLNS PYAKLYHFEN XYLSEHGGGA 100

hgaml (RINWASCTSQ GEKXHEDXFD XXDREADGSD SLE .CTVLCHSX 141 H g2est GNNWASGFSQ GEKXHEDXFD XXDREADGSD 1 5 0

hgam l AGGTGSGLGS YLLERLNDRY PKKLVQTYSV FPNQDEMSDV WQPYNSLLT 191 H g2est AGGTGSGLGS YLLERLNDRY PKKLVQTYSV FPYQDEMSDV WQPYNSLLT 200

hgam l LKRLTQNADC LWLDNTALN RXATDRLHXQ NPSFSQXNQL VSTXMSASTT 241 H g2est LKRLTQNADC VWLDNTALN RXATDRLHXQ NPSFSQXNQL VSTXMSASTT 250

hgam l TLRYPGYMNN DLXGLXASLX PTPRLHFLMT GYTPLTTDQS VASVRKTTVL 291 H g2est TLRYPGYMNN DLXGLXASLX PTPRLHFLMT GYTPLTTDQS VASVRKTTVL 300

hgam l DVMRRLLQPK NVMVSTGRDR QTNHCYXAXL NXXQGEVDPT QVHKSLQRXR 341 H g2est DVMRRLLQPK NVMVSTGRDR QTNHCYXAXL NXXQGEVDPT QVHKSLQRXR 350

hgaml ERKLANFXPW GPASXQVALS RKSPYLPSAH RVSGLMMANH TSXSSLFERT 391 H g2est ERKLANFXPW GPASXQVALS RKSPYLPSAH RVSGLMMANH TSXSSLFESS 400

hgam l CRQYDKLRKR EAFLEQFRKE DMFKDNFDEM DTSREXVQQL XDEYHAATRP 441 H g2est CQQFDKLRKR DAFLEQFRKE DMFKDNFDEM DRSREWQEL XDBYHAATQP 450

hgaml DYXSWGTQEQ . 4 5 1 . . Hg2est DYXSWGTQEQ 450

Figure 11.

87 Figure 12: A comparison of the nucleotide sequences in the 3' UTR regions of Hyl and

Hy2.

Abbreviations: H gam l-Hyl 3' UTR nucleotide sequence; Hgam2-Hy2 3'UTR nucleotide sequence. The 3' UTR’s of Hyl andHy2 share 54% nucleotide identity. The polyadenylation signal (AATAAA) of both sequences is underlined. The poly(A)tail ofHy2 is 204 base pairs 3' to the stop codon.

88 STOP hgam2 GACTACATTT CCTGGGGCAC CCAGGAGCAG TGATTTCCCT CC,.CCACTA hgaml 6ACTACATCT CCTGGGGCAC CCAGGAGCAG TGAGTCCCCC AGGACAGGGG hgam2 CTCCTTCTCC TTCTAGATGG TAACCACAGC CTCGACCATG CCTGCTCCCT hgaml ACCCTCATCT GCCTTACTGG TTGGCCCAAG CCCTGCC.TG ACTGACCACC hgam2 CTGACCCAGC TTCACCTCAT GGACAACCCT TCTTGGTTCA TCTCCAGCCC hgaml CCCTCAGAGC A.CAGATCAG GGACCTCACG CATCTCTTTC TCATATACAT hgam2 GTGAGCTGGT CCTGCTTCCT CCCTTCCATG CCCTAACTTT TAATATGCTT hgaml GGACTCTCTG TTGGCCTGCA AACACATTTA CTTCTCCTCT TA.TGAGACT hgam2 GTTCAGCTCT AATAAAGTAA TAAAGCTTGG TTGTCAGTAT AAAAAAAAAA hgaml ATTTATCTTT AATAAAGCAC TGGG...... hgam2 AAAAAAAAAA

Figure 12. Figure 13: Schematic diagram of human chromosome 17 showing Hyl, BRCAI, and Hy2.

This diagram of human chromosome 17 shows the position of Hyl in relation to the BRCAI, breast cancer susceptibiliQr gene at region q21. Hy2 maps within the 700 kb region that is magnified and shown to the left of the bracket.

90 p arm

700 kb ? Hy 2 HSD2

Hyl q arm BRCAI

17

F igure 13.

91 a 700 kb region of DNA that is located at region q21 on chromosome 17. Currently, Dr.

Krahe's lab is using a high resolution (50-100 kb resolved) TNG radiation hybrid map to refine the mapping of Hyl and Hy2.

HyF is a fragment from a y-tubulin pseudogene

No cDNAs were found in our probing of a HeLa cDNA library. This could mean that the sequence is fiom agene expressed at very low levels in HeLa cells, that the H ^ sequence is from a pseudogene, or that H ^ is a PCR amplification artifact (Le. not of human origin). To determine if HyF is of human origin I designed primers,

HGENOIB, HGEN02E, and HGENOR2 (underlined in Figure 15) that are specific for

HyF (specificity was tested using plasmids containing Hyl and HyF) to use in PCR analyses of HeLa genomic DNA and human patient genomic DNA. PCR analysis was carried out as described in the Materials and Methods and results are shown in Figure 16.

Fragments of the sizes expected for HyF (a 761 bp HGEN01B/2E band and a 576 bp

HGEN01B/R2 band. Figure 16) were amplified from human patient genomic DNA and

HeLa genomic DNA. These results verified that Dr. Shraudolf s original clone was really of human origin. I decided to sequence the 576 bp human genomic DNA PCR product to verify that the primers used were amplifying a subset of the HyF clone. Sequence analysis of both strands of the clone revealed that this clone is identical to the analogous region in the 930 bp-H"^ clone.

92 Figure 14: PCR analysis of HSD2 YAC DNA with Hy2 specific primers.

DNA was isolated firom YAC clone HSD2 and amplified with two Hy2 specific primer combinations. The 224 bp Hy2 firagment in lanes 2, 3, and 5 was amplified with HUGAM1-3F1 and HUGAM3R1, while HUGAM1-3F1 and HUGAM3R2 were used to amplify a 145 bp firagment of Hy2 shown in lanes 8, 9, and 11. A X/Haein molecular weight marker is shown in lane 1 and a 100 bp ladder is shown in lane 12. The positive amplification of Hy2 fi'om HSD2 YAC DNA, using both combinations of primers, is shown in lanes 5 and 11 respectively. Lanes 2, 3, 8, and 9 show positive amplification of human genomic DNA used as a positive control for amplification conditions.

93 224 bp 145 b p

Figure 14.

94 Figure 15: A comparison of the predicted amino acid sequences of H 7 F and Hyl with the

H7 F specific primers underlined.

Abbreviations: Hgaml-predicted amino acid sequence of Hyl; HgamF-predicted amino acid sequence of H^F.

One common forward primer, HGENOIB, and two reverse primers, HGEN02E and HGENOR2 were designed to amplify firagments of H^^. The primers were tested for specificity of amplification and shown to specifically amplify H ^ DNA and not Hyl DNA (data not shown). Both of these primer combinations were used to amplify H ^ form HeLa genomic DNA and human genomic DNA. Dashes indicate that the amino acids in the HyF sequence are conserved. The asterisk represents a gap in the amino acid sequence of Hyl to compensate for an insertion in the H ^ sequence.

95 HgenolB Hgaml MPREIITLQLGQCGNQIGFBFIfKQLCABHGISPEAIVEBFATBGTDRKDVFFYQADDBHY 60 Mgamf G——————T————— KGTM—————————H——Z—————————— 41

Hgaml IPRAVXJ.DLBPRVXHSXLNSPYAKLYNPBNXYLSBHGGGAGNll1fASGFSQGBKIHBDIFD 120 H g a m f ------C------R------R-- R N 101

Hgaml XXDRBADGSDSLBGFVXÆHSXAGGTGSGLGSYLLBRLNDRYPKKLVQTYSVFPNQDBNSD 180 Hgamf -T-Q----- M-D---- QF------W ------K— M 161 BggaoR2 Hgaml WVQPYNSLLTLKRLTQNADCVWLDNTALIIRXATDR*LHXQNP8F8QXNQLVSTXNSAST 240 H g a m f L------M—B------H-----Q-- -OX— ------T------X-----222 Mggno28 Hgaml TTLRYPGYMNNDLXGI.XASLXPTPRLHFLIITGYTPLTTDQSVASVRKTTVXJ>VNRRLLQP 300 ^ Hgamf X -^S— D------If------0--M^--- H----- K--- 282

Hgaml KNVNVSTGRDRQTHHCYXAXLHXXQGBVDPTQVHKSLQRIRBRKLANFXPIIGPASXQVAL 360 H g a m f ------S—T------310

Hgaml SRKSPYLPSAHRVSGLNNANHTSXSSLFBRTCRQYDKLRKRBAFLBQFRKBDMFKDHFDB 420

Hgaml MDTSRBXVQQLXDBYHAATRPDYXSHGTQBQ 451

Figure 15. To shed light on we used the HyF sequence in a BLASTN search of the

Washington University (St. Louis) Human Genomic Sequencing Center database.

Sequences corresponding to H yF were found on two PAG clones

(H_DJI165K10.contig74 and H_DJ1022I14.contig59)(see Figure 17), both from chromosome 7. The two H^^-Iike sequences differ by only 2 bp (a GC in one sequence is

CG in the other) in the region of interest. I surmised that these two sequences are of the same genomic region and the 2 bp differences are simply a sequencing error. Analysis of the two genomic clones reveals that they contain a y-tubulin-like sequence that lacks introns. A functional y-tubulin cDNA can not be expressed from these sequences for two reasons. First, there is no initiation codon in either genomic sequence. The codon corresponding to the initiation codon has been mutated from ATG to ATA. Second, the genomic fragment contains a one bp deletion and a 5 bp insert that disrupts the y-tubulin reading frame. Consequently, I have concluded that the genomic sequence from which

originated is a y-tubulin processed pseudogene.

Further inspection of the sequences suggests that although the sequences have diverged, this pseudogene was probably derived from H yl. The Hyl cDNA and the pseudogene share 92.4% nucleotide identity in the region corresponding to the coding region of the Hyl cDNA, 91.4% identity in the 187 bp region corresponding to the Hyl

3'UTR and 92.0% identity in the 25 bp of the S' UTR available for Hyl (Figure 17). In comparison, the HyZ cDNA and the pseudogene share 41.6% nucleotide identity in the S'

97 Figure 16: PCR amplification of H 7F from HeLa cell and human genomic DNA using specific primers.

(16a) specific primers HGENOIB and HGEN02E were used to amplify a 761 bp fragment from HeLa genomic DNA (Lane 1) using the parameters given in the materials and methods. A 123 bp ladder (BRL) molecular weight marker is shown in lane 2. (16b) A 576 bp and a 761 bp fragment is shown in lanes 1 and 2 respectively. A plasmid named pIBI-yF, that contains the 930 bp HyF sequence, was used with both primer combinations as a positive control of amplification and the results are shown in lanes 3 and 4. Lane 5 contains the no DNA control for PCR contamination.

98 N m î O' ON I

» 123bp

« I "4 « 3 2 O' O' u lOObp myw Eyw pIBl-yr plBX-yP lOObp N o DNA Figure 17: A comparison of the nucleotide sequence of human genomic clone "DJl 165k" with Hyl, and H ^

Abbreviations: Hgaml Hyl sequence; Hgam2-Hy2sequence; Hgamf-HyF sequence; DJ116K-DJ1165k genomic sequence.

The nucleotide sequence of human genomic clone DJ1165K is compared to the cDNA sequence of Hyl, and the nucleotide sequence of HyF. The genomic clone lacks a proper initiation codon (underlined in the figure), and does not contain any introns within the 1351 base pairs corresponding to the Hyl coding region. The Hyl cDNA and DJ1165K share 92.4% nucleotide identity in the region corresponding to the coding region of Hyl and 91.4% nucleotide identity in the region corresponding to the Hyl 3'UTR.

100 h g a m l CGCAACGCC6 GTGCCTGAGG AGCGATOCCG AGG6AAATCA. TCACCCTACA h g a m f D J l 1 6 5 .GCAACTCT6 GTGCCTGAGG AGCGATACCA AGAGAAATCA TCACCC.ACA h g a m l GTTGGGCCAG TGCGGCAATC AGATTGGGTT CGAGTTCTG6 AAACAGCTGT h g a m f ...... GGGTTTTGG AAACAGCTGT D J 1 1 6 5 ATTGGGCCAG TGCAGCAATC AGATTGGGTT CGAGTTCTG6 AAACAGCTGT h g a m l GCGCCGAGCA TGGTATCAGC CCCGAGGCGA TCGTGGAGGA GTTCGCCACC h g a m f GCACTGAGCA TGGTATCAGC CCCAAGGGCA CCATGGAGGA GTTCGCTACT D J 1 1 6 5 GCACTGAGCA TGGTATCAGC CCCAAGGGCA CCATGGAGGA GTTCGCTACT h g a m l GAGGGCACTG ACCGCAAGGA CGTCTTTTTC TACCAGGCAG ACGATGAGCA h g a m f GAGGGCACTG ACCACAAGGA CATCTTTTTC TACCAGGCAG ACGATGAGCA D J 1 1 6 5 GAGGGCACTG ACCACAAGGA CATCTTTTTC TACCAGGCAG ACGATGAGCA h g a m l CTACATCCCC CGGGCCGTGC TGCTGGACTT GGAACCCCGG GTGATCCACT h g a m f CTACATCCCC CGGGCTGTGC TGCTGGACCT GGAGCCCCGG GTGATCCACT D J 1 1 6 5 CTACATCCCC CGGGCTGTGC TGCTGGACCT GGAGCCCCAG GTGATCCACT h g a m l CCATCCTCAA CTCCCCCTAT GCCAAGCTCT ACAACCCAGA GAACATCTAC h g a m f CCATCCTCAA CTCCCCCTGT GCCAAGCTCT ACAACCCAGA GAACATCTAC D J 1 1 6 5 CCATCCTCAA CTCCCCCTGT GCCAAGCTCT ACAACCCAGA GAACATCTAC h g a m l CTGTCGGAAC ATGGAGGAGG AGCTGGCAAC AACTGGGCCA GCGGATTCTC h g a m f CTGTCGGAGC ATGGGGGAAG AGCTGGCAAC AACTGGGCCA GCAGATTCTC D J l 1 6 5 CTGTCGGAGC ATGGGGGAAG AGCTGGCAAC AACTGGGCCA GCAGATTCTC h g a m l CCAGGGAGAA AAGATCCATG AGGACATTTT TGACATCATA GACCGGGAGG h g a m f CCAGAGGGAG AAGATCCATG AGGACATTTT TAACATCACA GACCAGGAGG D J 1 1 6 5 CCAGAGGGAG AAGATCCATG AGGACATTTT TAACATCACA GACCAGGAGG h g a m l CAGATGGTAG TGACAGTCTA GAGGGCTTTG TGCTGTGTCA CTCCATTGCT h g a m f CAGATGGTAG TGACAATCTA GATGGCTTTG TGCTGTGTCA GTTCATTGCT D J 1 1 6 5 CAGATGGTAG TGACAATCTA GATGGCTTTG TGCTGTGTCA GTTCATTGCT h g a m l GGGGGGACAG GCTCTGGACT GGGTTCCTAC CTCTTAGAAC GGCTGAATGA h g a m f GGGGGGACAG GCTCTGGCCT GGGCTCCTAC CTCTTAGAAT GGCTGAATGA D J 1 1 6 5 GGGGGGACAG GCTCTGGCCT GGGCTCCTAC CTCTTAGAAT GGCTGAATGA h g a m l CAGGTATCCT AAGAAGCTGG TGCAGACATA CTCAGTGTTT CCCAACCAGG h g a m f CAGGTATCCT AAGAAGCTGG TGCAGACATA CTCAGTGTTT CCCAACCAGG D J 1 1 6 5 CAGGTATCCT AAGAAGCTGG TGCAGACATA CTCAGTGTTT CCCAACCAGG

(continued)

Figure 17.

101 (H gure 17 continuecO

h g a m l A CG AGATGA6 CGATGTGGTG GTCCAGCCTT ACAATTCACT CCTCACACTC h g a m f ACAAG&TGAG C A A C G T G G T 6 GTCCAGCTTT ATAATTCACT CCTCACACTC D J 1 I 6 5 ACAAGATGAGCAACGTGGTG GTCCAGCTTT ATAATTCACTCCTCACACTC

h g a m l AAGAGGCTGA CGCAGAATGC AGACTGTCTG GTGGTGCTGGACAACACAGC h g a m f AAGAGGCTGATGCAGAACGAAGACTGTGTG GTGGTGCTGCACAACACAGC D J l 1 6 5 AAGAGGCTGATGCAGAACGC AGACTGTGTGGTGGTGCTGCACAACACAGC

h g a m l CCTGAACCGG ATTGCCACAG ACCG ------CCT GCACATCCA6 AACCCATCCT h g a m f CCTGAACCAG ATTGCCACAG ACCAGATCCT GCACATCCAGAACCCATCCT D J 1 1 6 5 CCTGAACCAG ATTGCCACAGACCAGATCCT GCACATCCAGAACCCATCCT

h g a m l TCTCCCAGATCAACCAGCTGGTGTCTACCA TCATGTCAGCCAGCACCACC h g a m f TCTCTCAGAC CAACCAGCTG GTGTCCACCATCATCTCGGC CAGCACCATC D J 1 1 6 5 TCTCTCAGAC CAACCAGCTG GTGTCCACCATCATCTCGGC CAGCACCATC h g a m l ACCCTGCGCT ACCCTGGCTA CATGAACAATGACCTCATCG GCCTCATCGC h g a m f ACCCTGCGCT ACCCCAGCTA CATGGACAATGACCTCATAGGCCTCATCGC D J 1 1 6 5 ACCCTGCGCT ACCCCAGCTACATGGACAAT GACCTCATAGGCCTCATCGC h g a m l CTCGCTCATT CCCACCCCACGGCTCCACTT CCTCATGACCGGCTACACCC h g e u n f CTCACTCATT CCCACCCCATGGCTCCACTT TCTCATGACT GGCTACACCC D J 1 1 6 5 CTCACTCATT CCCACCCCATGGCTCCACTT TCTCATGACTGGCTACACCC h g e u n l CTCTCACTAC GGACCAGTCAGTGGCCAGCG TGAGGAAGACCACGGTCCTG h g a m f AGCTGACTAT GGACCAATCAGTGGCCAGCG TGAGGAAGAC CATGGTCCTG D J 1 1 6 5 AGCTGACTAT GGACCAATCAGTGGCCAGCG TGAGGAAGAC CATGGTCCTG h g a m l GATGTCATGA GGCGGCTGCTGCAGCCCAAG AACGTGATGG TGTCCACAGG h g a m f GATGTCATGA GGTGGCTGCTGCAGCCCAAGAACGTGATGGTATCCACAGG D J 1 1 6 5 GATGTCATGA GGTGGCTGCTGCAGCCCAAGAACGTGATGGTATCCACAGG h g a m l CCGAGACCGC CAGACCAACCACTGCTACATCGCCATCCTC AACATCATCC h g a m f CCGAGACCGC CAGACCAACCACTCCTACAT CACCATCCTC AACATCATTC D J 1 1 6 5 CCGAGACCGC CAGACCAACCACTCCTACATCACCATCCTC AACACCATCC h g a m l AGGGAGAGGT GGACCCCACCCAGGTCCACA AGAGCTTGCA GAGGATCCGG h g a m f AAGGCGAAGT G ...... D J l 1 6 5 AGGGAGAGGT GGACCCCACC CAGGTCCACA AGAGCCTGCA GAG GA TCCG6 h g a m l GAACGCAAGT TGGCCAACTTCATCCCGTGG GGCCCCGCCA GCATCCAGGT h g a m f D J l 1 6 5 GAATGGAAGT TGGCCAACTTCATCCTGTTG GGCCCCACCA GCATCCAGGT

(continued)

102 (Hgure 17 continued)

hgaml GGCCCTGTCG AGGAAGTCTC CCTACCTGCC CTCGGCCCAC CGGGTCAGCG h g a m f ...... DJ1165 GGCCCTGTCG AGGAAGTCTC CTTACCTGCC CTTGGCCCAC CGGGTCAGTG hgaml GGCTCATGAT GGCCAACCAC ACCAGCATCT CCTCGCTCTT CGAGAGAACC h g a m f ...... DJ1165 GGCGCATGAT GGCCAACCAC ACCAGCATCT CCTCACTCTT CTAGAGAACC hgaml TGTCGCCAGT ATGACAAGCT GCGTAAGCGG GAGGCCTTCC TGGAGCAGTT h g a m f ...... DJ11&5 TGTCGCCAAT ATGACAAGCT GCGGAAGTG6 GAGGCCTTCC TGGAGCCGTT hgaml CCGCAAGGAG GACATGTTCA AGGACAACTT TGATGAGATG GACACA. . . . h g a m f ...... DJ1165 CCGCAAGGAG GACATGTTCA AGGACAACTT TGATGAGATG GACACAACAC hgaml .TCCAGGGA6 ATTGTGCAGC AGCTCATCGA TGAGTACCAT GCGGCCACAC h g a m f ...... DJ1165 ATCTAGGGAG ATTGTGCAGC AGCTCATGGA TGAGTACCAC ATGGCCACAT hgaml GGCCAGACTA CATCTCCTGG GGCACCCAGG AGCAGXGAGT CCCCCAGGAC h g a m f ...... D Jl 165 GGCCAGACTA CATCTCCTGG GGCACCCAGG AGCAGXGAGT CCCCTAGGAC hgaml AGGGGACCCT CATCTGCCTT ACTGGTTGGC CCAAGCCCTG CCTGACTGAC h g a m f ...... D Jl 165 AGGGAGCC.T CATCTGCCTT ACTGGTTGAC CAAGGCCCTG CCTGACTGAC hgaml CACCCCCTCA GAGCACAGAT CAGGGACCTC ACGCATCTCT TTCTCATATA h g a m f ...... D Jl165 CACTCACTTA GAGCACAGAT CAGGGACCTC ACACATCTCT TTCTCATATA hgaml CATGGACTCT CTGTTGGCCT GCAAACACAT TTACTTCTCC TCTTATGAGA h g a m f ...... D Jl165 CATGCATTTT CTGTTAGTCT GCGAACACAT T.ACTTCTCC TCTTATGAGA

hgaml CTATTTATCT TTAATAAAGC ACTGGG ...... h g a m f ...... DJ1165 CTATTTATCT TTAATAAAGC AGTGGATATAAATAAATTTTTTTTAAAAA

103 UTR, 89.6% identity in the coding region and 45.1% identiQr in the 3TJTR.

Discussion

Southern analyses indicate that there are probably no more than three y-tubulin sequences in the human genome and three y-tubulin sequences have now been identified:

(1) Hyl, the first human y-tubulin gene isolated by Zheng e t al.y (1991a,b); (2) Hy2, the second human y-tubulin gene which I report in this study; and (3) a Hyl processed pseudogene corresponding to sequences in HyF and two PAC clones. Consequently, I conclude that all the y-tubulin sequences in Homo sapiens have now probably been identified.

The predicted products of Hyl and Hy2 share 97.3% amino acid identity. The high amino acid identity shared between these two y-tubulin cDNAs also exists between the pairs of y-tubulin genes that have been isolated from Arabidopsis thaliana (Liu e t aL,

1994), Zea m ays (Lopez e t aL, 1995), Paramecium tetraurelia (Ruiz eta L , 1999), and

Euplotes octocarinatus (Genbank, 1999), each of which share greater than 98% amino acid identity.

In order to obtain a full-length Hy2 clone, I have searched the EST database and obtained a Hy2EST clone that contains the entire coding region of Hy2 as well as the

5'UTR and 3'UTR sequences to the poly(A)tail of the cDNA. Comparison of Hy2EST to our four Hy2 cDNAs has revealed that Hy2EST is identical to pDOW7 andpDOW21

104 except for a 27 bp in-frame insert that is located in a highly conserved region of the y-

tubulin sequence. Interestingly, this insert begins at the same position as a 5' intron splice

site that has been conserved in the genomic y-tubulin clones identified in A. nidulans, S. pom be and Ustilago violacea (Oakley and Oakley, 1989; Horio et aL, 1991; Luo and

Perlman, 1993). Therefore, this insert may actually be part of an intron that was

misspliced. Unfortunately no y-tubulin genomic clones have been sequenced fix)m higher

eukaryotes, therefore there is no information on intron position in these organisms.

Rommens etal. (1995) have reported that Northern blot analysis shows that a Hyl cDNA probe hybridizes to a doublet (1.8 kb and 1.85 kb) of y-tubulin transcripts from total

RNA isolated from the frontal cortex of adult brain. The y-tubulin probe hybridized to a

single 1.8 kb transcript when Caco-2 intestinal cell line, lymphoblast, HL60 and breast carcinoma cell line total RNAs were probed. Rommens and colleagues (1995) suggest that brain tissue may contain two y-tubulin mRNAs produced by alternative splicing or difrerential polyadenylation. Since the Hy2EST clone originated from a 73 day post-natal infant brain cDNA library, the 27 bp insert may have resulted from brain-specific alternative splicing. However, it is important to note that the 27 bp insert would cause a nine amino acid insert in a highly conserved region of y tubulin. In addition Dr. Tetsuya

Horio has found that the transformation of a S. pom be strain, lacking endogenous y tubulin, with plasmid pASHy2 is toxic. The 27 bp insert in pASHyZ may alter the Hy2 protein in a way that causes it to create a dominant negative effect when expressed in S.

105 pom be cells. Since HyZEST's function has only been tested in S. pom be the possibility exists that this form of y tubulin is only found in specific human tissues such as brain tissue.

We (our lab in collaboration with Dr. Krahe's lab) have used PCR amplification with H^Z-specific primers to map Hy 2 to the same chromosomal interval and, eventually to the same 700 kb YAC as Hyl. It is interesting that the Hy2 was not detected on the YAC carrying Hyl since this YAC has been analyzed by several labs involved in the search for the breast cancer susceptibility gene. The entire YAC was extensively analyzed and other novel and previously identified genes were found. Since these investigators used YAC and cosmid clones to screen cDNA libraries to find expressed genes that map to the YACs, the

H72 cDNA may have simply been overlooked or Hy2 may not be expressed at detectable levels in the tissue or cell lines fiom which the cDNA was derived {i.e. human mammary gland, human breast, human breast cancer cell lines a human ovary Xgtl 1 cDNA libraries,

Rommens et aL, 1995). Note that Hy2 is expressed in ovaries (see Chapter 3). It is also possible, given the similarities of Hyl and Hy2, that Hy2 cDNAs were mistakenly identified as Hyl cDNAs.

Finally analysis of the HyF y-tubulin clone has shown that it is a processed pseudogene of Hyl. This is the first identification of a y-tubulin pseudogene in any organism and likely completes the family of y-tubulin sequences in humans.

106 CHAPTER 3

ANALYSIS OF THE EXPRESSION PATTERNS OF y-TUBULIN GENES IN HOM O SAPIENS

Introduction

After multiple y-tubulin genes were identified in D. melanogaster, Oakley (1994) suggested that one of the future directions of y-tubulin research would be to determine the number of y-tubulin genes expressed in animals and plants, their patterns of expression in these organisms and whether sequence differences between y tubulins within a species correlate with functional differences. While multiple y-tubulin isoQrpes exist in six species other than humans, the expression patterns of only two of these species (Drosophila melanogaster and Arabidopsis thaliana) have been studied. Analysis of the expression patterns of the two Z>. melanogaster y-tubulin proteins (YTUB23CD and YTUB37CD)

(using antibodies specific for each protein) reveals that they are differentially expressed. yrUB23CD is zygotically expressed in male and female flies during all stages of development, while yrUB37CD is expressed in ovaries and the developing egg chambers during oogenesis and in developing embryos (Wilson etal.\ 1997). Northern blot analysis

107 of the two y-tubulin genes in Arabidopsis reveals that both genes are expressed in whole seedlings, roots, flowers, and suspension cell cultures (Idu e t aL, 1994). In both of these cases it is difflcult to determine the relationship between the expression patterns of the two genes and their functions. Although multiple y-tubulin iso^pes exist, specific y-tubulin isotype classes have not been identified among the species in which multiple y-tubulin genes have been identified.

In Chapter 2,1 reported the identification of a second y-tubulin gene, Hy2, in Homo sapiens. In this chapter I have chosen to investigate the expression patterns of Hyl and

HyZ in ten different human cell types using multiplex reverse transcription PCR (RT-PCR).

Because Hyl and Hy2 cDNA share 94.6% base pair identity within their coding regions I was not able to find areas in their coding regions that were sufficiently different to allow synthesis of primers that could be used for specific amplification of the two sequences. To circumvent this problem 1 have designed specific reverse primers firom sequences that lie within the 3' UTR of each gene to use in multiplex reverse transcription (RT) reactions. A forward primer, with a sequence common to Hyl and Hy2, has been designed and used in combination with each reverse primer to give specific amplification of the two sequences.

Multiplex RT-PCR has revealed that both Hyl and Hy2 are expressed in all the tissues analyzed, although the two genes may not be expressed to the same extent within a given tissue or among different tissues.

108 Materials and Methods

Strains and Media

As positive controls for multiplex RT-PCR analysis, total RNA was isolated from

S. pombe stains expressing all or portions of Hyl and HyZ. A haploid strain, AH004, carrying a disrupted y-tubulin gene and a plasmid, pTH5, which expresses Hyl was constructed by Dr. Tetsuya Horio (Horio and Oakley, 1994). An 5. pom be haploid strain

HM123 (hr, leuI-32) was used as a wild-type strain and RNA from this strain was used as a negative control. Strain HMHG2 expresses a portion of Hy2 (see plasmid pASHyZF construction and 5. pombe transformation below).

Strain HM123 was streaked to single colony on YPD (1% yeast extract, 2% peptone, 2% dextrose and 1.7% agar for solid medium) plates. YPD medium was inoculated with single colonies of HM123 for transformation purposes and for total RNA preparations (see below). Strain AH004 was grown on YPD supplemented with 70 pg/ml of adenine for total RNA preparations. Strain HMHG2 was grown on EMM minimal medium (BIO 101, Inc.) to select for plasmid pASHyZF which contains the S. pombe

LEU2 gene.

Plasmid pASHy2F Construction

Ten nanograms of purified plasmid pDOW7 (a Hy2 partial clone in pBS sk-) DNA were used as a template to amplify a 1214 bp region of Hy2 (see Figure 19). Thirty cycles

109 of amplifîcatioii using 2.5 units of Taq DNA polymerase (Gibco BRL) were carried out

using the components and amounts recommended in the Gibco BRL protocol. Hy2-

specifîc primers with Bgin ends [Helagam3F: gaagatctcatgaagacatctttgac and HGEST3RI: gaagatctctgacaaccaagctttat (Operon Technologies)] were used to amplify a 1214 bp region

of the H 72 cDNA in pDOWT using the following thermocycling parameters: 94®C for 2 min; followed by 30 cycles of 94°C for 1 min and 55®C for 4 min. The Taq DNA polymerase was removed &om the PCR reaction using Strataclean Resin (Stratagene) and then the sample was concentrated in a Microcon-30 microconcentrator (Amicon, Inc.).

This sample was digested with BgUI as directed by the manufacturer (New England

Biolabs) and then purified from a 0.7% SeaplaqueGTG low melting point agarose gel

(EMC) using Wizard PCR preparations (Ptomega life sciences).

Plasmid pAS248 (Toda et aL, 1991) was digested with BamHI as directed by the manufacturer (New England Biolabs) (see Figure 19). The BamHI enzyme was removed from the sample using Strataclean Resin (Stratagene) and the DNA was concentrated in

Microcon-30 microconcentrator (Amicon, Inc.). The digested sample was treated with

0.01 units of Thermosensitive Alkaline Phosphatase (TsAP) (Gibco BRL) in a 10 pi mixture containing TsAP buffer diluted to Ix concentration and supplemented with MgCl^ to a final concentration of 3 mM at 65°C for 20 min and then inactivated by following the manufacturer’s protocol.

The 1214 bp Hy2, BgUI-digested firagment was ligated into the BamHI site in a

110 small polycloning site immediately downstream of the pom be alcohol dehydrogenase promoter (Russell and Hall, 1983). Ligation mixes were used to transform TOPIO £1 colt competent cells (Invitrogen) using the manufacturer’s protocol. Wizard DNA minipreps were performed using the manufacturer's protocol (Ehromega Life Sciences). Test digests were performed on the miniprep DNAs to determine if the ligation was successful. The correct plasmid was identified and named pASH^F (F=fi:agment). A standard large scale plasmid preparation (Sambrook e t aL, 1989) was performed and pASHy2F was purified twice using equilibrium centrifugation in CsQ-EtBr gradients. Purified pASHyZF was used to transform 5. p o m b e.

S. pom be Transformation

Haploid wild-type S. pom be strain HMI23 (hr, leul-32) was transformed with pASHylF as described in the Lithium Acetate Procedure H protocol found in the Fission

Yeast Handbook (www.bio.uva.nl/pombe) with the following modifications. A 20 ml culture of strain HM123 was grown at 32®C to an 00^^^= 0.5. After cells were harvested and washed they were resuspended in O.IM lithium acetate at a density of 4 x 10^ cells per milliliter. One microgram of plasmid DNA (pASH^ZF or pAS248) was added to the cells.

Cells were incubated in 50% PEG at 32®C for 1 hour and then they were washed in I ml of sterile ddH^O. Cells were resuspended in 2(X) pi of sterile ddH^O and l(X)-200 ul aliquots were spread onto EMM plates. Cells were allowed to grow 4-5 days at 30®C. A positive transformant was given the designation HMHG2 (Figure 19). HM123 was also

111 transformed with pAS248 as a positive control for transformation efGciency.

Plasmid pASHy2F DNA was isolated firom strain HMHG2 using the Plasmid

Recovery protocol (procedure A) found in the Fission Yeast Handbook

(www.bio.uva.nl/pombe). This DNA was amplified using the Hy2-specific primers

Helagam3F and HGESTRl and the conditions described previously (see Plasmid pASHy2F construction above), in order to verify that strain HMHG2 contains the correct

H72 -containing plasmid DNA.

Isolation of Total RNA from S.Pombe Strains

Total RNA was extracted firom S. pombe strains HM123, AH004, and HMHG2 using the "hot" phenol extraction method of Aves e t al. (1985) with the following modifications. Cells were harvested by centrifugation and then they were firozen in 50 ml

Falcon tubes in a dry-ice/ethanol bath. The firozen cell pellet was lysed using the liquid nitrogen cryo-impacting method of Smucker and Pfister (1975). All three RNA samples were treated with RNase-fiee DNase I (BMB) using a standard DNase I treatment protocol found in Sambrook e t at. (1989) to ensure that the samples were not contaminated with

DNA prior to reverse transcription.

Isolation of Total RNA from HeLa cells and Human tissues

We used standard methods to grow HeLa cells in culture (Freshney e t a/., 1992) for

RNA extraction. All human tissues were obtained through the Cooperative Human Tissue

Network (CHTN) firom the Tissue Procurement Division of the Dept, of Pathology of the

1 1 2 OSU Medical Center and tissues were homogenized following the TRIzol protocol using a

Tekmar SDTIOOEN Tissumizer (Tekmar Co.). Total RNA was extracted firom human

tissues (kidney, liver, stomach, ovary, colon, heart, lung and testicles) and HeLa cells

using TRIzol reagent (Gibco BRL) as described in the manufacturer’s protocol. The RNA

firom each tissue was treated with RNase-firee DNase I (BMB) using the standard DNase I treatment protocol found in Sambrook e t aL (1989).

RT-PCR Analysis

Total RNA (8 jig of each type of human tissue RNA or 1 p,g of HM123 RNA or 1 fxg of AH004 and HMHG2 RNA mixed together) was reverse-transcribed at 42°C for 1 hour using Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV-RT) (Gibco

BRL) in a final volume of 20 |xl. A multiplex reverse-transcription approach was used in which Hyl, Hy2, and human P-actin reverse primers (listed below) were combined in the same tube to facilitate simultaneous reverse transcription of these RNAs. Total RNA, lOX amplification buffer (Sambrook e t aL, 1989), reverse primers (1 ^iM final), and ddH^O were mixed and heated to 75°C for 5 min. Mixtures were placed on ice 5 min and then 10 units of rRNasin (Promega), dNTPS (each 1 mM final), and M-MLV-RT (200U) were added to the sample. After a one hour incubation at 42^C, the M-MLV-RT was inactivated at 95°C for 5 minutes.

Multiplex PCR was performed in the GeneAmp 2400 PCR System (Perkin Elmer) with 2.5 pi of each RT reaction in a 50 pi final volume containing 3 mM MgCl^. Each

113 primer was used at 0.4 except for HuGAMl-3Fl which was used at 0.8 ^M. Cold dNTPS were used at 0.4 mM each and 5 ftCi of dATP (3000Ci/mmoi, ION) was used for each 50 pi reaction along with 2.5 units of Platinum Taq DNA polymerase (Gibco

BRL). PCR parameters were 30 cycles of I min at 94°C and 4 min at 60^C, after an initial dénaturation of 2 min at 94^C. The following nucleotide primers were synthesized for use in RT and PCR analysis by Operon Technologies: HUGAM1-3F 1-common forward primer-aggacaactttgatgagatggaca; HUGAMIR2-Y1 specific reverse primer- gtgtttgcaggccaacagagagtc; HU GAM 3R2-y 2 specific reverse primer- atctagaaggagaaggagtagtg; HUACTFI p-actin forward primer-ttctacaaatgagctgcgtgtggct;

HUACTRl P-actin reverse primer-gcttctccttaatgtcacgcacga. The RT-PCR products were run on 3.5% agarose gels [Agarose LE, (BMB)] that were dried and placed in a Molecular

Dynamics phosphor cassette for 18-20 hours. Images were analyzed with ImageQuant version 4.1 on a Molecular Dynamics 445SI Phosphorimager.

Results

Rationale for Primer Design and Controls for RT-PCR

The existence of two functional y tubulin genes has raised the important question of whether these genes are differentially expressed in various human tissues. RNase protection assays and Northern blot analysis are often used to study the expression patterns of genes. Both approaches were problematic for examination of the expression of Hyl and

114 Hy 2, however, because of the great sequence similarities of the mRNAs encoded by the

two genes and because each of the two genes is likely to be expressed at relatively low

levels. I chose, instead, to use two-step multiplex reverse transcription PCR (RT-PCR)

(Chelly and Khan, 1994). In this technique multiple mRNAs from a tissue are first reverse

transcribed and then an aliquot of the reverse transcription mixture is used for PCR with

gene-specific primers.

In order to use RT-PCR analysis to analyze Hyl and Hy2 expression patterns, it was necessary to design primers specific for each gene. The similarity of Hyl and Hyl sequences within the coding region precluded designing specific primers. However, the 3'

UTR of the two genes were sufficiently divergent that specific primers were feasible. I chose to use a forward primer common to the coding region of Hyl and Hy2 and two reverse primers, each specific for the 3' UTR of Hyl or Hy2 (see Materials and Methods).

This combination of primers should amplify a 252 bp fragment specific to Hyl and a 145 bp fragment specific to Hy2 (see Figure 18). The specificity of the primers was first verified by PCR amplification of plasmids containing Hyl and Hy2 cDNAs (results not shown). Neither primer combination falsely amplified Hyl or Hy2 plasmid DNA. As a further and more important control, RT-PCR was carried out with the multiplex primer mixture on RNA isolated from three Schizosaccharomyces pombe strains. One strain

(AH004) produced an Hyl transcript, a second strain (HMHG2) (Figure 19) produced a

115 Figure 18: A comparison of the nucleotide sequences found in the 3' UTRs of Hyl and

Hy2.

Abbreviations: H gam l- Hyl 3'coding and 3'UTR nucleotide sequence; Hgam2 Hy2 clone pDCW7 3' coding and 3UTR nucleotide sequence.

A common forward primer, HUGAMI-3F1 (labeled HG1-3F1) and two specific reverse primers, HUGAM3R2 (labeled HG3R2) and HUGAM1R2 (labeled HG1R2) are underlined. Both reverse primers were used in multiplex reverse transcription reactions. Aliquots of the cDNAs were then used in PCR reactions in which HUGAM1-3F1 and HUGAM1R2 amplify a 252 bp Hyl firagment, while HÜGAM1-3F1 and HUGAM3R2 amplify a 145 bp H 72 firagment.

116 hgam2 GGCCCTGTCG AGGAAGTCTC CCTACCTGCC CTCGGCCCAC CGGGTCAGCG 985 hgam l GGCCCTGTCG AGGAAGTCTC CCTACCTGCC CTCGGCCCAC CGGGTCAGCG 1148

hgam2 GGCTCATGAT GGCCAACCAC ACCAGCATCT CCTCGCTCTT TGAAAGTTCC 1035 hgam l GGCTCATGAT GGCCAACCAC ACCAGCATCT CCTCGCTCTT CGAGAGAACC 1198

hgam2 TGCCAGCAGT TTGACAAGCT GCGGAAGCGG GATGCCTTCC TCGAGCAGTT 1085 hgam l TGTCGCCAGT ATGACAAGCT GCGTAAGCGG GAGGCCTTCC TGGAGCAGTT 1248

aoi-ari hgam2 CCGTAAGGAG GACATGTTCA AGGACAACTT TGATGAGATG GACAGGTCTA 1135 hgam l CCGCAAGGAG GACATGTTCA AGGACAACTT TGATGAGATG GACACATCCA 1298

hgam2 GGGAGGTTGT TCAGGAGCTC ATTGATGAGT ACCATGCGGC CACCCAGCCA 1185 hgam l GGGAGATTGT GCAGCAGCTC ATCGATGAGT ACCATGCGGC CACACGGCCA 1348

STOP H03lt2 hgam2 GACTACATTT CCTGGGGCAC CCAGGAGCAG %@&TTTCCCT CC. .CCACTA 1233 hgam l GACTACATCT CCTGGGGCAC CCAGGAGCAG TQAGTCCCCC AGGACAGGGG 1398

hgam2 CTCCTTCTCC TTCTAGATGG TAACCACAGC CTCGACCATG CCTGCTCCCT 1283 hgam l ACCCTCATCT GCCTTACTGG TTGGCCCAAG CCCTGCC.TG ACTGACCACC 1448

hgam2 CTGACCCAGC TTCACCTCAT GGACAACCCT TCTTGGTTCA TCTCCAGCCC 1333 hgam l CCCTCAGAGC A.CAGATCAG GGACCTCACG CATCTCTTTC TCATATACAT 1498

hgam2 GTGAGCTGGT CCTGCTTCCT CCCTTCCATG CCCTAACTTT TAATATGCTT 1383 hgam l GGACTCTCTG TTGGCCTGCA AACACATTTA CTTCTCCTCT TA.TGAGACT 1547 H01R2 hgam2 GTTCAGCTCT AATAAAGTAA TAAAGCTTGG TTGTCAGTAT AAAAAAAAAA 1433 hgaml ATTTATCTTT AATAAAGCAC TGGG ...... 1 5 7 1 h g S e s AAAAAAAAAA 1 4 4 2

Figure 18.

117 Figure 19; The construction of plasmid pASIfyF and S. pombe strain HMHG2.

A 1214 bp region of the H"y2 clone in pDOW7 was amplified using H 72 specific primers that contain BgUI ends. The H 72 PCR product was then digested with BgUI. In paraUel, plasmid pAS248 was digested with BamHI. The BgUI digested H 72 PCR product was Ugated into the BamHI site of pAS248 downstream of a constitutive alcohol dehydrogenase promoter. The new plasmid was named pASH72F. WUd-type S. pom be strain HM123 was transformed with pASHy2F to create strain HMHG2. Total RNA was isolated from this strain and used as a positive control for RT-PCR analysis of Hy2's expression in various human tissues.

118 A m pT

** pDOWTisapBSsk'lMMdplannidwitha HyZ partial done in the EcoRI site of the P 0 0 N 7 poiyiinker.

Am plify a 1214 bp région o f bp oK f il using primers with BglS ends. Bflrx

F a S c mgx

Digest Hy2 Bgin PCR product with BgllL \

Digest pAS 248 with BamHI. /

Ligate Hy2EST into pAS248. The imw plasmid construct is called pASHy2.

L ou

Transform strain HM123

I w/2 partial elom S.pombe strain HMHG2

Figure 19.

119 transcript that covers most of the sequence (including the 3' end and 3' UTR), and a third strain (HM123) only transcribed the endogenous S. p o m b e y-tubulin gene. As anticipated, only the 252 bp Augment specific to Hyl was amplified from AH004, only the

145 bp fragment specific to Hy2 was amplified from HMHG2 and neither fiagment was amplified from HM123 (Figure 20). When RT-PCR was carried out on a mixture of

AH004 and HMHG2 RNA, both fragments were amplified (Figure 20). These results indicated that multiplex RT-PCR with these primers under the conditions used is sufficiently sensitive and specific to determine if Hyl and Hy2 are expressed in various tissues.

Expression Patterns of y-tubulin Genes in Human Tissues

I isolated total RNA from the ten tissues shown in Figure 21 and carried out RT-

PCR with the Hyl and HyZ primer mixture. The mixture also included primers specific for human P-actin to serve as an internal control for the qualiQr of RNA in the samples and as an internal control for the RT-PCR reactions. Examination of the expression patterns of

Hyl and Hy2 (Figure 21) revealed that both genes are expressed in all the tissues examined, but there is some apparent variation in the levels of expression of the two genes.

Although RT-PCR is, at best, semiquantitative, the relative amounts of Hyl and Hy2 seem to be different in some tissues and the level of expression of each of them seems to vary among tissues. For example, Hyl and Hy2 are both expressed in testicular tissue (Figure

21), but if the testicular Hyl band is compared to the Hyl band from HeLa cells (Figure

1 2 0 Figure 20; PCR analysis of total RNA isolated from three S. pom be strains used as controls for RT-PCR.

The AH004/HMHG2 labelled lane contains the multiplex RT-PCR products of S. pombe strains AH004 and HMHG2 that express Hyl and Hy2 respectively. These RT- PCR products served as positive controls for the PCR parameters used in the analysis of y- tubulin expression patterns in various human tissues. The HM123 labelled lane is blank and served as a negative control because neither Hyl nor Hy2 specific primers amplify the endogenous S. pom be y-tubulin gene expressed by this strain. The Hyl primer combination amplifies a 252 bp band and the Hy2 specific primer combination amplifies a 145 bp band. A 100 bp ladder (Gibco BRL) is also shown.

1 2 1 51

o I g

Î v. t

100 Op ladder to B M iia 21, lane 1), the Hyl band seems more intense in HeLa cells. The HeLa cell y-tubulin bands are more intense than bands firom other tissues and may simply reflect the fact that

HeLa cells are aneuploid. Conversly increased y-tubulin expression in HeLa cells may be due to the presence of supernumerary centrosomes that are often found in tumor cells. The no-RT control reactions for each tissue (lanes 14-23), the HM123 negative y-tubulin control (lane 24) and the no -DNA PCR control were all negative. Therefore the amplification products we obtained were not caused by contamination or amplification of tissue DNA.

Discussion

I have used multiplex RT-PCR to analyze the expression patterns of Hyl and Hy2 in several human tissues. RT-PCR analysis has shown that both Hyl and Hy2 are expressed at varied levels in all of the tissues assayed. A survey of y-tubulin clones in the human EST database (www.ncbi.nlm.nih.gov/dbEST) has revealed that Hyl clones have been isolated firom 27 different tissue/cell cDNA libraries including nine tumor cell libraries and a multiple sclerosis cell line, while Hy2 clones have been isolated firom 11 diflerent tissue/cell cDNA libraries including two tumor cell libraries. This corroborates our findings that both Hyl and Hy2 are widely expressed in specific human cell Qrpes and tissues.

Although Hyl and H ^ are highly homologous, they encode y-tubulins with

123 Figure 21: RT-PCR analysis of the expression patterns of Hyl and Hy2 in several human tissues.

Radioactive multiplex RT-PCR was conducted as described in the Materials and Methods. RT-PCR products were run on 3.5% agarose gels. Molecular Dynamics phosphor screens were exposed to gels 18-20 hours. Lanes 1-10 contain RT-PCR products firom the Human tissues indicated above the lanes. Lane 11 contains the Hyl and Hy2 positive control transcripts that were expressed in 5. pom be strains AH004 and HMHG3. Lanes 14-23 contain negative control reactions for each of the tissues found in lanes 1-10. These reactions lacked reverse transcriptase and were run to make sure samples were firee from DNA contamination. S. pom be wild-type strain HM123 expresses endogenous y tubulin. Lane 24 which contains the HM123 negative control, confirms the specificiQr of the Hyl and Hy2 primers. A final PCR reaction (labelled No cDNA) was also run that lacked any cDNA in order to ensure that PCR solutions were not contaminated. The P-actin, Hyl, and Hy2 bands’ expected sizes are indicated.

124 SZl

3 ] X X *g *6 3 c K > t O' 3

w stom ach K id n e y L iver T e stic le

M u scle Ovary

C o lo n AH004/HMHy2

HM 123

No cDNA slightly different sequences. Vertebrate P tubulins and mammalian a tubulins are classified

into at least six "isotype classes", based on the conservation of their C-terminal 15 am ino

acids and conservation of their function, as is seen within the same and across different

species (Sullivan and Cleveland, 1986; Lewis et uL, 1985; Villisante et aL, 1986). To

date, no isotype defining motif has been identified in any of the species fiom which

multiple y-tubulin genes have been isolated. Thus while y tubulins encode different

isotypes, there is no evidence as yet for “isotype classes”.

As has been previously mentioned, Hyl and share 97.3% amino acid identi^.

Why would an organism need two y-tubulin isotypes, that encode nearly identical y-tubulin

proteins? These two very homologous genes may exist to encode proteins that perform

specific functions in cells. Recent studies by Ruiz et al. (1999) have revealed that y tubulin

is required for basal body duplication and therefore may also be involved in centriole

duplication. H only one y-tubulin isotype is involved in the basal body duplication then

there probably would be increased expression of this isotype in cells that are heavily ciliated

such as lung endothelium and intestines or cells that contain basal bodies like sperm.

At the present time, however, there is no evidence for this functional specificity and it seems unlikely that the functional specificity of y-tubulin is determined by such a small number of amino acid differences. In the case of Hyl and H 72 this functional specificity would have to be determined by the twelve amino acid differences that exist between the two genes.

126 The two genes could exist to facilitate regulation of the levels of Y-tubulin synthesis in different tissues. Our RT-PCR experiments suggest that this may be the case but RT-

PCR is semi-quantitative at best and we must be cautious when interpreting the variations in levels of Hyl and H 72 message we see.

Finally, it is possible that the two y-tubulin genes exist for simple functional redundancy, y tubulin is essential for viability in all organisms that have been studied and having two genes instead of one would greatly reduce the chance that a y-tubulin mutation would lead to loss of viabili^.

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