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Cloning, characterization and functional analysis of UBC-2, a gene encoding a ubiguitin-conjugating enzyme… Zhen, Mei 1995

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Cloning, characterization and functional analysis of ubc-2, a gene encoding a ubiquitin-conjugating enzyme in the nematode Caenorhabditis elegans by Mei Zhen B. Sc., Wuhan University, 1990 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy in THE F A C U L T Y OF G R A D U A T E STUDIES Department of Biochemistry and Molecular Biology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A December 1995 © Mei Zhen, 1995 In presenting this thesis in partial fulfillment of the requirements for an advanced degree at The University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. '"Department of Biochemistry and Molecular Biology The University of British Columbia 2146 Health Sciences Mall Vancouver, Canada V6T 1Z3 Date: December 27,1995 Abstract ABSTRACT ubc-2, a gene encoding a ubiquitin conjugating enzyme (E2) in Caenorhabditis elegans was cloned, and the expression and functions of this gene during the development of the nematode were investigated using molecular biological and genetic approaches. The ubc-2 gene was cloned by virtue of its high amino acid similarity to the yeast and Drosophila genes UBC4/5 and UbcDl, respectively. UBC4 and UBC5 encode ubiquitin conjugating enzymes which mediate selective degradation of short-lived and abnormal proteins in yeast, and are essential for viability under stressed conditions, ubc-2 encodes a protein sharing 85% amino acid identity with UBC4/5, and 95% with its Drosophila homolog U b c D l . Yeast complementation tests showed that UBC-2 rescues the growth deficiency of a yeast ubc4ubc5 mutant strain under normal and stressed conditions. These results suggest that this type of E2 is conserved among eukaryotes, and may play fundamental roles within cells. Northern blot analysis showed that ubc-2 is abundantly expressed in all developmental stages throughout the life cycle of C. elegans. Unlike yeast, the expression of this gene is not induced by heat shock or cadmium. ubc-2-lacZ fusions were constructed and transformed into the nematode to study the tissue specificity of ubc-2 expression in different life stages. The expression of the transgenes was observed from early embryogenesis (gastrulation) onward. Transgene expression was largely tissue general in embryonic and early-mid larval stages, but restricted to the nervous system in adults. UBC-2 protein was overexpressed in E. coli, and polyclonal antibodies were raised against the purified protein. In situ immunofluorescent staining showed that the endogenous UBC-2 protein is indeed specifically expressed in the adult nervous system. UBC-2 is the first ubiquitin-conjugating enzyme found to be tissue-specific in animals. Transformation of the nematode with constructs expressing antisense R N A against ubc-2 was lethal in early-mid larvae, suggesting that this gene is essential for early stage development. EMS-generated lethal mutations which map near ubc-2 were tested for rescue by transformation with a ubc-2 clone. One of the lethal mutations, let-70, was rescued. Both existing mutant i i Abstract alleles of let-70, SI 132 and S689, contain point mutations in ubc-2. SI 132 has a C to T substitution which leads to a His to Tyr change at residue 75. S689 contains a G to A transition at the splice donor site of the last intron of the gene. Both alleles exhibit developmental arrest in larvae, but the blockage in si 132 seems to be earlier (L1-L2 larval stage) than that in s689 (L3 stage), suggesting that the mutation in sll32 results in a more severe defect in the protein's function. These results clearly show that His75 and the C-terminal (amino acids 134-147) of UBC-2 are important for its functions. Since yeast UBC4 and UBC5 encode proteins with redundant functions, and knocking out either gene alone reveals no apparent cellular defect, it has been commonly assumed that multiple genes encoding functionally similar E2s would exist in multicellular organisms and that single gene mutations would therefore have little or no effect on organism viability. However the studies presented in this thesis suggest that in multicellular organisms, related E2 proteins may have become specialized for different life stages, different tissues or different environmental conditions, and thus essential for development and survival. i i i Table of Contents T A B L E O F C O N T E N T S A B S T R A C T i i T A B L E OF CONTENTS iv LIST OF T A B L E S xi LIST OF FIGURES xii LIST OF ABBREVIATIONS xiv A C K N O W L E D G M E N T S xvii I. INTRODUCTION 1 1. Protein degradation in eukaryotic cells - overview 1 2. Discovery of the ubiquitin-proteolytic system - historical review 2 3. Mechanism and components of the ubiquitin-mediated proteolysis system 3 3.1. Ubiquitin 3 3.2. Ubiquitin conjugation 4 3.3. Breakdown of conjugated proteins by the proteasome 5 3.4. Ubiquitin recycling 6 4. Enzymatic systems for the ubiquitin conjugation reactions 7 4.1. Ubiquitin-activating enzyme (El) 7 4.2. Ubiquitin-conjugating enzyme (E2) 8 4.3. Ubiquitin ligase (E3) 10 5. Functions of ubiquitin conjugation mediated by various E2s 11 5.1. Bulk protein degradation 11 5.2. Stress response 12 5.3. Transcriptional regulation 13 5.4. Cell cycle control 15 5.5. Neuronal activity and nervous system development 17 5.6. D N A repair 17 5.7. Mammalian cell transformation 19 iv Table of Contents 5.8. Protein translocation 20 5.9. Peroxisome biogenesis and assembly 21 5.10. Viral infection 22 5.11. Antigen processing 22 5.12. Phytochrome degradation 23 5.13. Ubiquitination of membrane proteins 23 5.14. Spermatogenesis 24 6. Substrate recognition by ubiquitin conjugating enzymes 24 7. C. elegans as a model system 25 8. trans splicing of C. elegans gene transcripts 26 9. The present study 26 II. M A T E R I A L S A N D METHODS 27 1. General techniques for handling C. elegans 27 1.1. Culturing of C. elegans 27 1.2. Maintenance of the strains 27 1.3. Heat-shock and cadmium treatment of C. elegans 27 2. General D N A and R N A techniques 28 2.1. Bacterial transformation 28 2.2. Standard polymerase chain reaction (PCR) 28 2.3. Worm PCR 28 2.3.1. Single worm PCR 28 2.3.2. PCR with a pool of worms 29 2.4. Purification of plasmid D N A from E.coli 29 2.5. Generation of nested deletion D N A clones with Exonuclease UI 30 2.6. Packaging and preparation of ssDNA from pBluescript vector 31 2.7. Large scale preparation of X phage D N A 31 2.8. Double-stranded and single-stranded D N A sequencing 32 Table of Contents 2.9. Preparation of radioactive D N A probes 32 2.9.1. Nick translation 32 2.9.2. Random hexamer extension labeling 33 2.9.3. Primer extension M13 probes 33 2.10. Screening of recombinant D N A libraries 34 2.10.1. Screening of C. elegans bacteriophage XZAP c D N A library 34 2.10.2. Screening of C. elegans bacteriophage A,Charon4 genomic library 35 2.11. Large scale isolation of C. elegans genomic D N A 35 2.12. Genomic Southern blot analysis 36 2.13. Isolation of C. elegans total cellular R N A 36 2.13.1. Large scale preparation 36 2.13.2. Mini-scale R N A preparation 37 2.14. Northern blot analysis 37 2.15. SI nuclease protection analysis 38 3 . Methods related to transgenic assays in C. elegans 38 3.1. Construction of ubc-2/lacZ fusions 38 3.2. Construction of ubc-2 sense and antisense plasmids 39 3.3. Establishment of transgenic C. elegans strains 40 3.4. Establishment of integrated transgenic strains 40 3.5. in situ histochemical X-gal staining of transgenic worms 40 3.6. Identification of X-gal staining cells in transgenic C. elegans 41 3.7. ONPG assay 41 4. Protein and immunological techniques 41 4.1. Overexpression of UBC-2 fusion protein in E.coli 41 4.2. Large scale purification of UBC-2 fusion protein from E.coli 43 vi Table of Contents 4.3. Ubiquitin thiol ester assay 44 4.4. Raising polyclonal antibodies against UBC-2 protein 45 4.5. Affinity purification of antibodies against UBC-2 protein 45 4.6. Preparation of C. elegans protein extracts 46 4.7. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 46 4.8. Western blot analysis 46 4.9. In situ immunofluorescent staining of C. elegans 47 IH. RESULTS Section A . Cloning and analysis of ubc-2, a gene encoding a ubiquitin-conjugating enzyme in C. elegans 48 1. Isolation of ubc-2 cDNA and genomic clones 48 2. Comparison of the predicted protein sequence encoded by C. elegans ubc-2, Drosophila UbcDl and yeast UBC4 49 3. Functional substitution of UBC4 by ubc-2 in yeast cells (yeast complementation test) 49 4. Analysis of the ubc-2 genomic sequence 51 5. Genomic Southern blot analysis 57 6. The 5' flanking region of ubc-2 and the ubc-2 trans-spliced sequence 57 7. Localization of the ubc-2 gene 61 8. SI nuclease protection analysis 61 Section B . Studies on the temporal and spatial expression patterns of ubc-2 in C. elegans 63 1. Northern blot analysis 63 1.1. Developmental analysis of ubc-2 expression 63 1.2. ubc-2 expression after heat-shock 66 2. Temporal and spatial expression patterns of ubc-2/lacZ transgenes 66 2.1. Construction of ubc-2/lacZ translational fusions 66 vii Table of Contents 2.2. Establishment of transgenic lines and integrated lines carrying ubc-2/lacZ fusions 69 2.3. Constitutive expression of pZMI.l and pZMII.l throughout the C. elegans life cycle 69 2.4. Spatial expression patterns of pZMI.l and pZMII.l 69 2.5. Expression of pZMI.l and pZMII.l in the nervous system 73 2.6. Important regulatory elements for transgene expression 73 3. In situ immunofluorescent staining of C. elegans 75 3.1. Over-expression of UBC-2 in E. coli and purification of the protein 75 3.2. In vitro ubiquitin conjugating activity of UBC-2 fusion protein 75 3.3. Raising antibodies against UBC-2 fusion protein and affinity purification of the antiserum 77 3.4. In situ immunofluorescent staining of C. elegans with antibody against UBC-2 protein 77 4. Expression of UBC-2 protein after heat-shock and cadmium treatment 81 4.1. Western blot analysis on extracts from heat-shocked worms 81 4.2. Expression of ubc-2/lacZ transgene after heat-shock and cadmium treatment 81 Section C. Studies on the functions of ubc-2 in C. elegans development 83 1. Inhibition of ubc-2 function by expression of antisense R N A 83 1.1. Construction of ubc-2 antisense constructs ANTI-I, ANTI-II and sense constructs hsp-ubc-2 and ubc-2(SK) 83 1.2. ANTI-II induced early embryonic lethality in C. elegans 83 1.3. Establishment of ANTI-I integrated lines and hsp-ubc-2 extrachromosomal lines 83 1.4. Effects of ANTI-I and hsp-ubc-2 expression on ubc-2 transcript and protein levels 85 viii Table of Contents 1.5. Developmental effects of ANTI-I expression 85 2. Attempts to isolate ubc-2 null mutants using Tel transposoition 88 3. let-70, a C. elegans lethal mutant was rescued by ubc-2 gene 92 4. Both alleles of let-70 mutant have single point mutations in the ubc-2 locus. 92 5. C. elegans let-70(ubc-2) mutants display mid-larval lethal phenotype 93 6. Mutant UBC-2 proteins encoded by let-70(ubc-2) alleles are highly unstable .93 IV. DISCUSSION 97 1. ubc-2 encodes a ubiquitin-conjugating enzyme highly conserved among eukaryotes 97 2. Hypothetical structure of UBC-2 protein, a Class I ubiquitin-conjugating enzyme 98 3. Both trans and cis splicing are involved in the maturation of ubc-2 transcript 98 4. ubc-2 is constitutively expressed in all life stages 100 5. ubc-2/lacZ transgene assays suggest that ubc-2 expression has developmental tissue specificity 101 6. UBC-2 protein is prominently expressed in the nervous system in all stages, especially adults 101 7. Analysis of the ubc-2 promoter region 103 8. ubc-2 expression is neither heat nor cadmium inducible 104 9. Expression of ubc-2 antisense R N A causes early embryonic and larval stage lethality and retarded development 105 10. Ubc-2 corresponds to a gene essential for early development, let-70, in C. elegans 106 11. His75 and C-terminal (amino acid 134-147) of UBC-2 protein are important for the protein functions 107 ix Table of Contents 12. ubc-2-type E2s in multicellular organisms may be indispensable and mediate specialized functions in different developmental stages or tissues 108 13. Other ubiquitin-conjugating enzymes in C. elegans 109 14. Conclusions 109 15. Future prospects 110 V . REFERENCES 113 VI. APPENDIX 127 x List of tables LIST O F T A B L E S Table 1. Growth rates of yeast ubc mutants and Mbc-2-expressing ubc mutants 54 Table 2. The effect of ubc-2 antisense R N A expression on the development of C. elegans 89 xi List offigu, LIST O F FIGURES Fig. 1. Protein sequence comparison 50 Fig. 2. Strategy for ORF replacement of UBC4 by ubc-2 in yeast 52 Fig. 3. Growth of yeast strains at different temperatures 55 Fig. 4. Immunodetection of C. elegans UBC-2 expressed in a ubc4::ubc-2ubc5 yeast strain 56 Fig. 5. Nucleotide and deduced amino acid sequences of ubc-2 58 Fig. 6. Genomic Southern blot analysis 60 Fig. 7. Mapping of the ubc-2 gene 62 Fig. 8. SI nuclease protection analysis 64 Fig. 9. Northern blot analysis of ubc-2 during development 65 Fig. 10. Northern blot analysis: the expression of ubc-2 after heat-shock 67 Fig. 11. Construction of ubc-2/lacZ transgenes 68 Fig. 12. lacZ expression from the pZMI.l and pZMI.2 transgenes in different stages of C. elegans , 70 Fig. 13. lacZ expression of pZMI.l and pZMI.2 in different cell types 71 Fig. 14. lacZ expression of pZMI.l and pZMI.2 in the nervous system 74 Fig. 15. Expression and purification of the 6xHis-UBC-2 fusion protein in E. coli 76 Fig. 16. In vitro ubiquitin thiol ester assay 78 Fig. 17. E C L Western blotting analysis of UBC-2 79 Fig. 18. In situ immunofluorescent staining of adults with polyclonal antibodies against UBC-2 80 Fig. 19. ONPG assays of (3-galactosidase activity inpZMI.llnl andpZMI.lIn2 animals exposed to various concentrations of CdCi2 and different temperatures 82 Fig.20. Construction of the sense constructs hsp-UBC-2, UBC-2(SK), and antisense ANTI-I and ANTI-II 84 xii List of figures Fig.21. Northern blot analysis of strains carrying ANTI-I or hsp-ubc-2 before and after heat-shock treatment 86 Fig.22. Western blot analysis of strains carrying ANTI-I or hsp-ubc-2 constructs before and after heat-shock treatment 87 Fig.23. Strain YK10 carries a Te l insertion in ubc-2 90 Fig.24. Point mutations in ubc-2 genes of the let- 70 mutants 94 Fig.25. Western blot analysis of UBC-2 proteins in wild-type and heterozygous let-70 C. elegans 96 Fig.26. Tertiary structures of a Class I ubiquitin-conjugating enzyme, yeast UBC4 protein 99 xiii List of abbreviations LIST O F A B B R E V I A T I O N S A absorbance Amp ampicillin ASFV African swine fever virus ATP adenosine-5'-triphosphate bp(s) basepair(s) B S A bovine serum albumin BVDV bovine viral diarrhea virus cDNA complementary deoxyribonucleic acid C. elegans Caenorhabditis elegans C i Curie cpm count per minute DAPI 4,6-diamidino-2-phenylindole dATP deoxyadenosine-5 '-triphosphate dCTP deoxycy tidine- 5 '-triphosphate DEPC diethyl pyrocarbonate dGTP deoxy guanosine- 5 '-triphosphate DMF dimethyl formamide DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dTTP deoxythymidine-5'-triphosphate ECL enhanced chemiluminescence ER endoplasmic reticulum dNTPs deoxyribonucleoside triphosphates DTT dithiothreitol E. Coli Escherichia coli EDTA ethylenediaminetetraacetic acid xiv List of abbreviations EMS ethylmethanesulfonic acid EtBr ethidium bromide FITC fluorescein isothiocyanate HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid HSE heat shock element I inosine IPTG isopropyl-p-D-thiogalactopyranoside kd kiloDalton M H C major histocompatibihty complex MOPS 3-N-morpholino-propanesulfonic acid mRNA messenger ribonucleic acid NLS nuclear localization signal CD optical density ONPG o-nitrophenyl-p-D-galactosidase ORF open reading frame P A G E polyacrylamide gel electrophoresis PBS phosphate buffer saline PCR polymerase chain reaction PIPES piperazine-N,N'-bis (2-ethansulfonic acid) Pfu plaque forming units PMSF phenylmethylsulfonyl fluoride PVDF polyvinylidene difluoride R N A ribonucleic acid RNase ribonuclease rpm revolutions per minute S. cerevisiae Saccharomyces cerevisiae SDS sodium dodecyl sulphate xv List of abbreviations SSPE 180mM NaCl, ImM EDTA, lOmM N a H 2 P 0 4 , pH 7.4 SV40 simian virus 40 Tris tris(hydroxymethyl)aminomethane tPvNA transfer ribonucleic acid ts temperature sensitive U V ultra-violet X-gal 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside Y A C yeast artificial chromosome xvi Acknowledgement A C K N O W L E D G M E N T S First and foremost I would like to thank my supervisor Peter Candido for providing me the chance to explore not only the science world but also a whole new life in Canada. His support and valuable advice has been making this journey a very pleasant and memorable time. I am grateful to my committee members Caroline Astell and Ross MacGillivray for their guidance, support, and critical reading of my thesis in such short time. For people who have collaborated with me in the last five years, Don Jones, Dave Leggett, Jacqueline Schein and Ruth Heinlein, I hope you all have enjoyed the experience as much as I did. My sincere thanks goes to Don Moerman, Dave Baillie and Ann Rose's labs. Not even once was my request turned down by the 'Vancouver worm community': advice, methods, strains, reagents, even worm plates (not too often of course). You give me so much confidence in the worm breeders. To the past and present Candido lab members, Don, Eve, Esther, Robert, Mike, Dave, Michel, Sandi, and Tracy, thanks for being my colleagues, friends and free English tutors (even though you are more interested in teaching me the uncivilized slangs). You have always been my inspiration in science and life, especially after we all had enough drinks. To my friends in and not in the Biochemistry department, I thank you for being an important part of my life. Life is always easier when there are people there to share the experience with you. A hearty thank-you to my parents and brother in China, who fill my heart with love and care even though I am so far away. Thanks for believing in my choice and allowing me to live my life. To my husband, Yip, all I can say is that I can never thank God enough for this blessing to me. xvii Introduction I. I N T R O D U C T I O N 1. PROTEIN DEGRADATION IN EUKARYOTIC CELLS - OVERVIEW Two distinct proteolytic processes exist in eukaryotic cells, lysosomal and non-lysosomal degradation pathways. While proteins that enter the cells from the extracellular milieu are degraded in lysosomes, the turnover of cellular proteins is largely controlled by selective, nonlysosomal proteolytic systems. Under conditions of nutritional or hormonal deprivation, however, lysosomal autophagy of intracellular proteins is greatly accelerated (Dice, 1987). Lysosomal protein breakdown appears to be largely energy-independent and nonselective (Hershko and Ciechanover, 1982), although it has been suggested that certain cytosolic proteins are targeted to the lysosome by specific amino acid sequences (such as the KFERQ peptide motif) in the presence of a heat shock protein hsc73, ATP and M g 2 + (Olson et al, 1991; Terlecky et al, 1991; Dice, 1992). The selective degradation of cytosolic proteins in eukaryotic cells involves several nonlysosomal, energy-requiring proteolytic pathways. Relatively well studied ones include the ubiquitin-mediated and antizyme-mediated proteolytic systems. In both systems, substrate proteins are fully or partially degraded by a 26S protease complex following conjugation to ubiquitin or antizyme. To date a few substrates, such as ornithine decarboxylase (ODC) have been clearly shown to undergo antizyme-dependent proteolysis (Murakami et al, 1992; Tokunaga et al, 1994). Other unique pathways responsible for some cellular protein degradation may also exist (Duckworth et al, 1994). However, much evidence suggests that ubiquitin-dependent proteolysis is the major pathway for the breakdown of abnormal, short-lived, and regulatory proteins in cells (Rechsteiner, 1987; Hershko, 1991; Jentsch, 1992). Recent studies suggest that this ubiquitin-mediated proteolysis system is also involved in the partial breakdown and post-translational processing of some precursor proteins to generate biologically active units (Driscoll and Finley, 1992; Ciechanover, 1994). 1 Introduction 2. DISCOVERY OF THE UBIQUITIN PROTEOLYTIC PATHWAY - HISTORICAL REVIEW Ubiquitin-mediated selective protein degradation was first discovered in two unrelated fields of research. In vitro studies on eukaryotic protein degradation showed that proteolysis was stimulated by A T P (Etlinger and Goldberg, 1977) and required a proteinaceous co-factor (Ciechanover et al, 1978), which was later found to be ubiquitin (Wilkinson et al., 1980). ATP was shown to be necessary for the covalent attachment of ubiquitin to its proteolyic substrates (Hershko et al., 1980). Parallel studies on chromatin proteins led to the discovery that histone H2A is ubiquitinated (West and Bonner, 1980). Structural analysis of the first identified ubiquitin-protein conjugate showed that the C-terminus of ubiquitin is attached to a specific lysyl residue (lysine 119) of the histone via an isopeptide bond (Busch and Goldknopf, 1981). Since histones are rich in lysyl residues, these observations indicated that ubiquitination sites are structurally defined and probably recognized by enzymatic components of a ubiquitin-conjugation system. The conjugation of ubiquitin to protein substrates was then found to be a multistep process catalyzed by a series of enzymes (Hershko et al., 1983), including a ubiquitin-activating enzyme (El), one of the ubiquitin-conjugating enzymes (E2) and, in some cases, a ubiquitin-ligase (E3) for substrate recognition (Hass and Rose, 1982; Pickart and Rose, 1985; Ciechanover and Schwartz, 1988; Reiss and Hershko, 1989). While the monoubiquitinated conjugates, including ubiquitinated histone, ubiquitinated actin (Ball et al., 1987) and certain cell surface proteins (Leung et al., 1987; Siegelman et al., 1986) seemed to be metabolically stable, multiubiquitination of the protein substrates was demonstrated to promote their degradation (Chau et al., 1989). A large multicatalytic protease complex, the proteasome, was identified as responsible for the degradation of ubiquitin-conjugated proteins (Hough and Rechsteiner, 1987; Waxman, et al, 1985). It was suggested that after thorough or partial degradation of multiubiquitinated proteins, ubiquitin is released and re-used. The identification of ubiquitin C-terminal hydrolases (UCH) which are capable of removing ubiquitin from proteins and peptides by Matsui et al. (1982) and Hadari et al. (1992), supports this theory. 2 Introduction 3. MECHANISM AND COMPONENTS OF THE UBIQUITIN PATHWAY 3.1. Ubiquitin Ubiquitin is a small (76 amino acid) and stable protein present in all eukaryotic cells. It is one of the most highly conserved proteins among eukaryotes. The sequence of the protein is identical in organisms as diverse as cattle (Schlesinger et al, 1975), humans (Schlesinger and Goldstein, 1975), trout (Watson et al., 1978), toad (Dworkin-Rastl et al., 1984) and insects (Gavilanes et al., 1982). Yeast ubiquitin differs from the human protein at only three positions (Ozkaynak et al., 1984). The extraordinary conservation of ubiquitin in evolution indicates its basic and essential cellular functions. The crystal structure of ubiquitin has been elucidated at 2.8 A resolution (Vijay-Kumar et al, 1985). It is a compact, globular protein with hydrophobic core. The amino-terminal residue of the protein is buried, while the carboxy-terminal Gly-Gly protrudes from the core structure and has considerable freedom of motion. The free Gly-Gly structure is required for the covalent attachment of the C-terminal Gly of ubiquitin to other proteins. Ubiquitin is encoded by two types of genes, ubiquitin-ribosomal fusion genes and polyubiquitin genes (Finley and Chau, 1991, Ozkaynak et al, 1987, Ozakaynak et al, 1984). The ubiquitin-ribosomal genes encode a protein in which ubiquitin is fused to the N-terminal of a 40S ^ or 60S subunit of ribosomal protein (Finley et al, 1989, Redman and Rechsteiner, 1989). The polyubiquitin gene encodes polyubiquitin proteins in which single ubiquitin moieties are joined head to tail (Finley and Chau, 1991; Ozkaynak et al, 1987; Ozakaynak et al, 1984). The translation products of both types of genes are rapidly processed by specific hydrolases called U C H (ubiquitin C-terminal hydrolase) or Ubp (ubiquitin specific protease), which cleave the C-terminal of the ubiquitin moieties (Ozakaynak et al, 1984; Finley et al, 1987). As a result of this processing free ubiquitin molecules are released in the cell. The free ubiquitin is then attached to substrate proteins via the ubiquitin-conjugation system. 3 Introduction Ubiquitin can be found either free or covalently attached to other proteins in cells. Free ubiquitin may also exist in the form of multiubiquitin chains (van Nocker and Viestra, 1993). Ubiquitin conjugation to other proteins forms an isopeptide bond between the C-terminal glycyl residue and an internal lysyl residue of the target proteins. This reaction is catalyzed by enzymes of the ubiquitin conjugation system. 3.2. Ubiquitin conjugation A multistep enzymatic process is required for conjugation of ubiquitin to substrate proteins. The first step is catalyzed by a ubiquitin-activating enzyme (El). A ubiquitin adenylate is formed in the presence of ATP, and the C-terminus of ubiquitin (Glycine) is then linked to a thiol group of an internal cysteinyl residue on the E l enzyme (Haas and Rose, 1982). This activated ubiquitin is then transferred from the E l enzyme to a specific cysteinyl residue of one of the ubiquitin-conjugating enzymes (E2s) (Pickart and Rose, 1985). These E2 enzymes then donate the ubiquitin to protein substrates, forming ubiquitin-protein conjugates. An isopeptide bond is formed between the carboxyl group of the C-terminal glycyl residue of ubiquitin and the e-amino group of a specific internal lysyl residue of the target proteins. Some conjugation reactions require the help of accessory proteins, known as ubiquitin ligases (E3) for substrate recognition (Ciechanover and Schwartz, 1988; Hershko, 1991; Reiss et al., 1989). After the first ubiquitin is attached to the substrate molecule, ubiquitin itself can then become a substrate for ubiquitination, which leads to the formation of a multiubiquitin chain (Chau et al., 1989; Gregori et al., 1990). Among the seven lysyl residues in ubiquitin, at least three are known to be involved in multiubiquitin formation: lysines 48, 63 and 29 (Chau et al., 1989; van Nocker and Vierstra, 1993; Arnason and Ellison, 1994). When a single lysyl residue of ubiquitin is used, a linear polyubiquitin chain is attached to the substrate protein. The utilization of more than one lysyl residue leads to the formation of a branched multiubiquitin 'tree'. However no evidence suggests that this kind of structure actually exists in cells. Recent studies suggest that ubiquitin chains linked through different lysyl residues may be used for different cellular functions. Arnason and Ellison (1994) showed that 4 Introduction polyubiquitin chains formed through lysine 63 may play a critical role in stress resistance in yeast. The identification of specific E2s in animals and plants which are capable of creating free multiubiquitin chains in vitro (Chen and Pickart, 1990; Chen et al. 1991; van Nocker and Vierstra, 1991) has raised another possible mechanism for ubiquitin conjugation to substrate proteins. It was suggested that a multiubiquitin chain can first be preassembled from ubiquitin monomers, then transferred en masse to the target protein (Duckworth et al, 1994). 3.3. Breakdown of conjugated proteins by the proteasome Certain proteins have been shown to be monoubiquitinated in vivo, such as histone H2A, actin and a few cell surface proteins. These ubiquitin-protein conjugates are metabolically stable. Multiubiquitination of substrates appears to be necessary for protein degradation in vivo. Specific degradation of these multiubiquitinated proteins is mediated by the proteasome, a large multicatalytic protease complex. In initial studies, two types of protease complex were purified from eukaryotic cells, 20S and 26S in size. In vitro both the 20S and 26S proteasomes exhibit multiple catalytic sites with trypsin-like, chymotrypsin-like and peptidyl-glutamyl-hydrolyzing activities. However, unlike the 26S complex, the 20S proteasome is unable to degrade ubiquitin-protein conjugates in vitro (Hough and Rechsteiner, 1987; Waxman et al, 1985). Later studies suggested that the 20S proteasome is part of the 26S complex. It was reported that the incubation of 20S proteasomes with two other protein fractions and ATP forms a 26S complex, which can degrade ubiquitin-conjugated proteins (Driscoll and Goldberg, 1990; Driscoll et al, 1992; L i and Etlinger, 1992). Depending on the organism, the 20S proteasome is composed of 9-15 polypeptide subunits of similar sizes (21 kd-31 kd), but different charges (isoelectric points range from 3-10). The 20S proteasome is a large hollow cylindrical particle composed of four stacked rings each containing six peptides. The two inner rings are composed of P type subunits, and the two outer ones are of a type subunits (Tanaka and Ichihara, 1990; Orlowski, 1990; Goldberg, 1992; Lowe et al, 1995). The subunit proteins characterized so far are related in sequence, but show no homology to any known proteases. 5 Introduction Genes encoding some proteasome subunits have been cloned from various organisms. In Saccharomyces cerevisiae, all of the genes cloned so far except that encoding subunit y l3 , are essential for viability (Fujiwara et al, 1990; Emori et al, 1991; Heinemeyer et al, 1991; Georgatsou et al, 1992; Hilt et al, 1993). Some missense mutations of the genes cause a slow growth phenotype. Analysis of the strains indicates that they also show defects in protein turnover and degradation (Hilt et al, 1993; Seufert and Jentsch, 1992). This strongly supports the premise that the proteasome mediates the selective degradation of cellular proteins. Recent studies suggest that some ubiquitin-conjugated proteins may be selectively processed by the proteasome, rather than completely degraded as in the usual case. This type of action has been implicated in processing of the p50 subunit precursor of the transcription factor NF-KB(Thanos and Maniatis, 1995), in antigen processing (Brown etal, 1991; Ortiz-Navarrete et al, 1991; Driscoll and Finley, 1992) and in the turnover of viral proteins (Andersson et al, 1985; Scheffner ei al, 1990; Guarino, 1990; Driscoll and Finley, 1992). 3.4. Ubiquitin recycling The stability of ubiquitin suggests that it can be used repeatedly in the degradation pathway. The removal of ubiquitin from proteins and peptides is carried out by ubiquitin C-terminal hydrolases (UCHs) or ubiquitin-specific proteases (Ubps). A large family of UCHs (or Ubps) exists in eukaryotes (Mayer et al, 1991; Wilkinson et al, 1989; Woo et al, 1995). In yeast four Ubps (Ubpl-4) have been cloned using functional screens (Agell et al, 1991; Baker, et al, 1992; Papa and Hochstrasser, 1993; Tobia and Varshavsky, 1991), while the yeast genome sequencing project identified genes encoding another 10 Ubps based on the homology of the conserved sequence domains (R. Baker, personal communication). Ubps are thiol proteases with little sequence homology except two conserved functional domains, a Cys box and a His box (Baker, et al, 1992). These enzymes seem to have distinct substrate specificities. Some are associated with 26S proteasome and used to cleave ubiquitin from ubiquitin-protein conjugates, and some are required for the processing of polyubiquitin precursors. Genetic analysis shows that they all play 6 , Introduction distinct cellular roles. For example, Ubp2 is found to generate free ubiquitin during stress conditions, and Ubp6 is involved in the cleavage of ubiquitin from both N-end rule and non-Nrend rule substrates. The studies of Ubps in multicellular organisms, still in the early stages, already suggest interesting aspects of their cellular functions. The yeast DOA4 (Ubp4) gene encodes a protein related to the product of the human tre-2 oncogene (Papa and Hochstrasser, 1993). The Drosophila gene fat facets (faf), which is required only for cell interactions leading to specific cell fate decisions during eye development was demonstrated to encode a Ubp (Fisher-Vize et al., accepted by Science). These studies suggest that Ubps may be highly cell type and substrate specific in multicellular animals. .4. ENZYMATIC SYSTEM FOR THE UBIQUITIN CONJUGATION REACTIONS 4.1. Ubiquitin-activating enzyme (El) E l catalyzes the activation of ubiquitin in the presence of ATP by a two-step reaction. First a ubiquitin-adenylate is formed with the displacement of PPi from ATP, and then the ubiquitin is transferred to a thiol ester site on the enzyme with the release of A M P (Haas and Rose, 1982). Genes encoding E l s have been cloned from various organisms including yeast, mammals, and plants either through genetic screening or by ubiquitin affinity-chromatography followed by protein sequencing and hybridization screening (Handley et al., 1991; Hatfield et al., 1990; Kay etal, 1991; McGrath et al, 1991). A l l E l genes encode proteins with molecular weights slightly higher than 100 kd and containing a nucleotide-binding consensus sequence G l y - X - G l y - X - X - G l y (McGrath et al, 1991). Abundant E l proteins are detected in both the cytosol and the nucleus (Cook and Chock, 1991; McGrath et al, 1991; Schwartz et al, 1992). They seem to act as homodimers (Ciechanover et al, 1982) and can form complexes with ubiquitin-conjugating enzymes (E2s) (S. Jentsch; M . Ellison, personal communication). Unlike other members of the ubiquitin pathway such as E2, E3 and Ubps, the E l family seems to consist of a small number of Introduction members. A single E l gene, UBA1, has been isolated from yeast so far (Hoefer and Cook, 1991; McGrath et al., 1991). In wheat, three isoforms of E l , UBA1, UBA2 and UBA3 have been identified (Hatfield and Vierstra, 1989). Two human E l proteins have been described (El-1 and El-2) (Cook and Chock, 1992; Handley et al., 1991). It is not clear thus far whether the different E l s interact with different E2s, or are involved in different cellular functions. 4.2. Ubiquitin-conjugating enzymes (E2) From E l , the activated ubiquitin is transferred to the thiol site of a ubiquitin conjugating enzyme, E2. Multiple E2s have been identified in various organisms using genetic or biochemical approaches. So far ten different E2s have been cloned in yeast, which makes E2 the second largest family in the ubiquitin pathway (Jentsch, 1992). Genes encoding multiple E2s have also been isolated in rabbit (Pickart and Rose, 1985), human (Kaiser et al, 1994, 1995; Scheffner et al, 1994), Drosophila (Koken et al, 1991; Trier and Seufert, 1992), C. elegans (Zhen et al, 1993; Leggett et al, 1995), wheat (Sullivan and Vierstra, 1989), and Arabidopsis (Bartling etal, 1993; Genschik et al, 1994; Watts et al, 1994). Sequence comparisons reveal that all E2s contain a conserved 16 kd U B C (Ubiquitin conjugating) domain, which shares approximately 35% identity among all known E2s. In the center of the UBC domain is a highly conserved cysteine residue which is required for thiol ester bond formation between E2 and ubiquitin. The sequence bracketing this cysteine residue (GXICLDIL) is almost identical among all E2s. At the N-terminal of the U B C domain is a highly conserved basic motif. Deletion of this motif results in reduced activity for thio-ester formation suggesting that this domain is involved in El-binding (Sullivan and Vierstra, 1991). Ubiquitin-conjugating enzymes are divided into four groups according to their structure (Jentsch et al, 1990). Class IE2 enzymes consist of only a U B C domain, examples being wheat 15k-E2 protein, yeast UBC4, UBC5, UBC7 and UBC9, Drosophila U b c D l , and C. elegans UBC-2. This type of E2 is able to accept the activated ubiquitin from E l in vitro. However, 8 Introduction without the addition of supplementary cell fractions containing potential E3 proteins, it is unable to transfer the ubiquitin to substrates (Girod and Vierstra, 1993). Class II E2 enzymes contain various C-terminal extensions of the U B C domain. Yeast UBC1, UBC2(RAD6), UBC3(CDC34), and UBC8 all belong to this type. It has been proposed that the C-terminal extension contributes the substrate specificity of E2 proteins. The C-terminal extensions of UBC2 and UBC3 proteins are both highly acidic and promote interactions with basic substrates such as histone. Interestingly, replacing the C-terminal tail of UBC2 with that of UBC3 generates a chimeric protein which can rescue the cell cycle arrest of a ubc3 null strain (Kolman et al., 1992; Silver et al., 1992). This suggests that the C-terminal extension of UBC3 protein plays an essential role in substrate recognition directly or indirectly (such as through E3 protein). However, when the C-terminal extension was deleted from UBC2, the truncated ubc2 protein retained all cellular functions except for sporulation (Silver et al., 1992). These results indicate that the C-terminal extension may not be the only site for substrate recognition by the Class II E2s. In addition to a role in providing substrate specificity, the C-terminal extension also functions in the cellular localization of some E2 proteins. Yeast UBC6 has a hydrophobic signal-anchor sequence at the C-terminal end of the extension, which localizes the protein to the endoplasmic reticulum (ER) (Sommer and Jentsch, 1993). Class III E2 enzymes have N-terminal extensions in addition to the U B C domain. So far only two E2s belong to this group, one from mouse and one from Drosophila (Hauser, unpublished; M . Treier and S. Jentsch, personal communication). While the U B C domains of the mouse and Drosophila enzymes share 98% amino acid identity, the N-terminal extension sequences are totally unrelated to each other. The functional significance of the N-terminal extensions is unknown. Class IV E2 enzymes are considered 'novel' E2 proteins. They contain both N-terminal and C-terminal extensions in addition to the U B C domains. Only two examples of this type of enzyme have so far been found. One is encoded by a murine gene, UbcMl, identified by Jentsch et al. (personal communication). U b c M l protein is a 500 kd membrane protein. It has a small C-9 Introduction terminal extension, and a long N-terminal extension with several putative transmembrane domains (Hauser and S. Jentsch, personal communication). Another member of this E2 class has been isolated and cloned from rabbit reticulocytes. It is a 230 kd soluble protein that is highly induced during reticulocyte differentiation suggesting it plays a role in this process. The E2 cysteine catalytic core is located in the center of the protein. Both in vivo and in vitro assays indicate that another cysteinyl residue located downstream of the E2 cysteine core sequence is essential for transferring ubiquitin to its substrate, but is not required for accepting ubiquitin from E l . This suggests that the C-terminal extension contains an E3-related domain. It has been proposed that this E2 protein is actually an E2/E3 hybrid enzyme (C. Pickart, personal communication). 4.3. Ubiquitin ligase (E3) E3 proteins are required as accessory factors by some E2 enzymes for the recognition of protein substrates. The first two E3 enzymes E3a and E3b were isolated from rabbit reticulocytes. E3a recognizes substrates with basic or bulky hydrophobic N-terminal amino acid residues, while E3b binds to substrates with small uncharged N-terminal amino acids (Reiss et al, 1989; Reiss and Hershko, 1990). The yeast homolog of E3a, UBR1, has been purified and its gene cloned (Bartel et al, 1990). E3 proteins seem to form a complex with specific E2 enzymes. For instance, UBR1 was found to form a tight complex with UBC2 (RAD6) (Dohmen et al, 1991). It is proposed that specific E2s and E3s can form an active complex, which possesses substrate specificity and the ability to ubiquitinate the substrates. Two different hypotheses have been proposed to explain how E3 facilitates the transfer of a ubiquitin chain from E2 to substrates. One model proposes that the ubiquitin chain is first transferred to the thiol site of an E3, then passed to the substrate that interacts with the E3 protein (Scheffner et al, 1995). The other model suggests that E3 enzyme functions only as a 'docking protein' to bring the E2 and substrate together; in this model the E2 enzyme can pass the ubiquitin chain to the substrates directly (Varshavsky, 1992; Watkins etal, 1993). 10 Introduction E3a, E3b and UBR1 all recognize substrates through their N-terminal residues. That the identity of its N-terminal amino acid affects the half-life of a protein is commonly known as the N -end rule. However, studies suggest that the N-terminus is not the only recognition signal for the ubiquitin pathway, and not even the major one (Dohmen et al, 1991; Varshavsky, 1992). Other E3 enzymes that are capable of recognizing internally localized signals of target proteins exist in cells. These E3 proteins may or may not interact directly with E2. A good example of this type of E3 is the complex formed by the E6 oncoprotein encoded by papillomavirus, and E6-AP, a host protein. In mammalian cells, the interaction of the tumor suppressor protein p53 with E6 and E6-A P triggers the ubiquitination and degradation of p53 (Scheffner, et al, 1993). Certain heat shock proteins, such as chaperonins, might also be classified as E3 proteins due to their requirement in the ubiquitination and turn-over of some cellular proteins (Burel et al, 1992). It is suggested that these heat-shock proteins bind to misfolded proteins, and could either help them to refold correctly, or trigger their ubiquitination and degradation. 5. FUNCTIONS OF UBIQUITIN CONJUGATION MEDIATED BY VARIOUS Els 5.1. Bulk protein degradation Studies on a mouse mutant cell line ts85A first revealed that a major part of cellular bulk protein degradation is mediated through the ubiquitin system (Ciechanover et al, 1984). This cell line expresses a temperature sensitive ubiquitin-activating enzyme. At high temperatures, ts85 exhibits severe defects in the degradation of short-lived and abnormal proteins. Since E l catalyzes the first step of the ubiquitin pathway, this defect suggests that the ubiquitin system plays a major role in bulk protein degradation. Several ubiquitin-conjugating enzymes (E2s) which are involved in this cellular function have been identified in yeast (Seufert et al, 1990; Seufert and Jentsch, 1990). Three genes encoding these E2 proteins, UBC1, UBC4 and UBC5, together play essential roles in cell growth and viability (Seufert et al, 1990). UBC4 and UBC5 are closely related in their amino acid 11 Introduction sequences, and functionally complement each other. ubc4 and ubc5 single null mutant strains grow as well as the wild type strain under normal conditions, while a ubc4ubc5 double mutant strain shows severe growth defects. Pulse chase experiments show that the turnover of short-lived and abnormal proteins is markedly reduced in this mutant (Seufert and Jentsch, 1990). UBC1 is also involved in the degradation of abnormal and short-lived proteins. Overexpression of UBC1 can partially complement the growth defect of ubc4ubc5 mutants. However, UBC1 seems to be specifically required for cell growth after germination of ascospores, suggesting that UBC1 has a specific function during this transition period in the life cycle of yeast (Seufert et al., 1990). Homologs of yeast UBC4 and UBC5 genes have been cloned from wheat (Girod and Vierstra, 1993), Drosophila (Treier, et al, 1992), C. elegans (Zhen et al, 1993, the present work), rat (S. Wing, personal communication), rabbit (Chen et al, 1991) and human (Scheffner et al, 1994). Proteins encoded by these genes share 80-85% amino acid identity with yeast UBC4 and UBC5, and over 95% identity among themselves. The extremely high conservation of the amino acid sequence of UBCs among different species suggests that they play essential and similar functions in eukaryotic cells. This speculation is supported by the fact that the expression of Drosophila and C. elegans homologous genes, UbcDl and ubc-2, respectively rescue the growth and protein degradation defects of yeast ubc4ubc5 mutants (Treier, et al, 1992; Zhen et al, 1993). 5.2. Stress response Ubiquitin-dependent proteolytic pathways play essential roles in the eukaryotic stress response. In yeast (Finley etal, 1987) and Dictyostelium (Miiller-Taubenberger et al, 1988) the. expression of the polyubiquitin gene is stress-inducible. Similarly the transcription of yeast UBC4 and UBC5 is also induced by heat-shock (Seufert and Jentsch, 1990), and the transcription of another yeast E2 gene, UBC7, is cadmium-inducible (Jungmann et al., 1993). A ubc4ubc5 null mutant is not viable at elevated temperature, and expresses heat-shock proteins at normal temperatures. A ubc7 null mutant shows no growth defect until the cells are treated with 12 Introduction cadmium. These results suggest that UBC4/UBC5 and t7BC7-mediated proteolysis is probably involved in the stress response induced by heat-shock or cadmium treatment. Th& Drosophila gene UbcDl and the C. elegans gene ubc-2, which have high sequence homology to-yeast UBC4/5, rescue the viability of yeast ubc4ubc5 null mutants (Trier et al, 1992; Zhen et al., 1993). Genes homologous to yeast UBC7 have also been cloned from various organisms including C. elegans (Zhen et al., in preparation) and wheat (Van Nocker and Vierstra, 1991). Interestingly, the wheat UBC7 homolog catalyzes the formation of multiubiquitin chains in vitro. The functions of these homologous UBC7 genes in multicellular organisms are unknown. It has recently been discovered that yeast UBC4 and UBC5 proteins have the ability to form multiubiquitin chains with different inter-ubiquitin-linkages when involved in different cellular functions (Arnason and Ellison, 1994): While the bulk degradation of short-lived and abnormal proteins involves lysine-48 linked multiubiquitinations of the substrates, the heat-shock , response involves the formation of multiubiquitin chains through lysine 63 (Arnason arid Ellison, 1994). ,.>•••/" 5.3. Transcriptional regulation Higher organisms have a chromatin structure high in ubiquitinated histones. Up to 15% of histone H2A and about 5% of H2B is ubiquitinated, mostly monoubiquitinated in vertebrates (Rechsteiner, 1987). The monoubiquitinated forms of these histones seem to be metabolically stable. Some studies have suggested that the.ubiquitination of histone leads to a looser chromatin structure, which might correspond to regions being actively transcribed. However, direct evidence for eukaryotic transcriptional regulation by. histone ubiquitination is still lacking. This hypothesis is also questioned by the fact that some organisms including yeast, seem to lack ubiquitinated histones (Swerdlow era/., 1990). Some transcriptional regulators are subject to ubiquitin-dependent proteolysis. Two relatively-well studied examples are the yeast transcriptional repressor MAToc2 (Chen et al., 1993; Hochstrasser et al., 1991) and the mammalian transcriptional activator NF-KB (Palombella et al., 13 Introduction 1994; Thanos and Maniatis, 1995). M A T a 2 functions in yeast mating type control, and has a short half-life of about 4 minutes. Deletion analysis has shown that there are at least two distinct degradation signals, Degl and Deg2 sequences in this protein. Surprisingly, different sets of ubiquitin conjugating enzymes, including UBC4, UBC5, UBC6 and UBC7 are involved in the degradation of M A T a 2 repressor by two distinct ubiquitination pathways. UBC4 and UBC5 define one of the pathways, which involves a degradation signal distinct from both Degl and Degl. UBC6 and UBC7, which may form a complex in vivo, define the other pathway and target the Degl degradation signal. When E2s in one of the pathways were disrupted, the half-life of M A T a 2 only increased 2-3 fold. When E2s involved in both pathways were disrupted, the half-life of the protein increased 12-15 fold. Since UBC6 was shown to be localized to the ER membrane and possibly also the nuclear envelope, it is possible that a UBC6/UBC7 complex controls the half-life of MAToc2 before it passes through the nuclear pore. This suggests that the short half-life of the M A T a 2 transcriptional repressor is determined by ubiquitin-mediated proteolytic pathways catalyzed by particular E2 enzymes at specific cellular locations (Chen et al., 1993). N F - K B regulates a variety of genes involved in the immune and inflammatory responses. It is a heterodimeric transcriptional activator consisting of p50 ( N F - K B I ) and p65 (RelA) subunits. The precursor of the p50 subunit, pl05, was shown to be processed by ubiquitin-mediated proteolysis. Unlike all the examples described previously, the breakdown of pl05 precursor by the proteasome is only a partial one. Leaving the N-terminal p50 domain intact, only the C-terminal of the protein is degraded (Palombella et al., 1994). Ubiquitin-mediated proteolysis is also involved in another aspect of the activation of N F - K B . In most cells N F - K B stays in the cytoplasm forming a complex with an inhibitor protein IKBOC . Translocation of N F - K B into the nucleus to function requires the removal of IKBCC , which masks the nuclear localization sequence of p50 and p65. Recent studies show that after the phosphorylation of I K B O C , which can be induced by extracellular signals, this inhibitor is quickly ubiquitinated and degraded by the 14 Introduction proteasome (Thanos and Maniatis, 1995). It is not clear which E2(s) and E3(s) are involved in the ubiquitination of pl05 and iKBa . 5.4. Cel l cycle control Several lines of evidence first indicated that the ubiquitin-mediated proteolytic pathway is involved in cell cycle control. 1). Some mammalian mutant cell lines with thermolabile ubiquitin-activating enzymes, such as ts85 and tsAlS9, exhibit cell cycle arrest in different stages (Eki et al, 1990; Zacksenhaus and Sheinin, 1990). 2). One of the yeast cdc mutants (cell division cycle mutation), cdc34, was found to correspond to a mutant allele of UBC2, an E2 gene (Goebl et al, 1988). This mutant is arrested at the G l /S border. 3). Cyclins, a family of important regulatory proteins during cell cycle progression are ubiquitinated, and tightly regulated by proteolysis during the cell cycle (reviews: Glotzer etal, 1991; Murray, 1995). The eukaryotic cell cycle is composed of G l , S, G2 and M (mitosis) phases. The progression of the cell cycle is regulated by the activity of a protein kinase, the p34 c dc2_ C y C i m complex. Depending on which subfamily of cyclin binds to p34C Q'c2> m j s complex allows the transition from G l to S phase and G2 to M phase. The cyclin is then degraded through the ubiquitin-proteolytic pathway, thus triggering the cells to exit from S and M phases. In G l , yeast G l cyclins (Clnl , Cln2 and Cln3) activate p34°dc2 through the formation of a p34 cdc2_Gl cyclin complex, which promotes the cell's entry into S phase. Three events including bud formation, spindle pole body duplication and D N A replication are initiated almost simultaneously. After activating p34C Q ,c2 5 Q\ cyclins Cln2 and Cln3 are extensively phosphorylated, conjugated with multiubiquitin chains and degraded by the proteasome. The ubiquitination and instability of Cln2 are partially facilitated by UBC2 (Cdc34) (Deshaies et al, 1995). It is reported that, in yeast Saccharomyces cerevisiae, D N A replication also requires the activation of Cdc28, a yeast homolog of Cdc2, by B-type cyclins (Clb5 and Clb6). A potent inhibitor of the Clbs, p 4 0 ^ ^ , is normally degraded by the UBC2 (Cdc34)-mediated ubiquitin pathway in order to permit the activation of Cdc28 (Schwob et al, 1994). Thus probably one of the reasons that UBC2(cdc34) mutants 15 Introduction cannot enter the S phase is that they fail to destroy the p40 G l inhibitor. Another E2 protein, UBC9, may also be involved in the G l / S transition, since the Clb5 protein is stabilized throughout the cell cycle in ubc9 mutants (Seufert et al., 1995). The mitotic transition involves another class of cyclin proteins, cyclin B (Clbl , Clb2, Clb3 and Clb4). The p34 c dc2_ C y C i m B complex, normally known as the mitosis-promoting factor (MPF), is necessary for the G 2 - M transition. After cells enter prophase, M P F activates the ubiquitin-dependent proteolytic system, causing both cyclin destruction and the initiation of anaphase. In the last step, the destruction machinery for cyclinB is turned off and the cell cycle is reset (Murray et al, 1995). The ubiquitination and degradation of cyclinB requires the involvement of multiple E2s. In Xenopus, UbcH5, a yeast UBC4 homolog, has been identified through a biochemical approach as one of the E2s supporting cyclinB ubiquitination (Mahaffery et al., 1993; King et al., 1995). UBC9, another E2 cloned from yeast through genetic screening, may also be involved in the degradation of cyclinB. Deletion of UBC9 leads to G2-M phase arrest and the stabilization of Clb2. In Xenopus, the ubiquitination of cyclinB by UBC4 requires a 20S complex containing Cdc27 and Cdcl6 (King et al., 1995). It is possible that this 20S protein complex functions as an E3 in the ubiquitination pathway. A conserved peptide sequence among cyclins, named the 'destruction box', has been identified as the signal for ubiquitination and degradation (Glotzer etal, 1991). Among the E2s so far known to be involved in cell cycle control, UbcH5 is a Class I enzyme sharing high homology with yeast UBC4, which has been described in detail in Sections 4.1 and 4.2 above. UBC3(Cdc34) is a 34 kd class II enzyme with C-terminal extension (Goebel et ah, 1988). This C-terminal tail is highly acidic and required for E3-independent histone ubiquitination in vitro. In vivo the C-terminal tail is required for cell cycle regulation. It was shown by Kolman et al. (1992) that only 39 residues adjacent to the U B C domain are necessary and sufficient for full cell cycle functions. UBC9 is an 18 kd class I protein which shares about 35% identity with other E2s (Seufert et al., 1995). 16 Introduction 5.5. Neuronal activity and nervous system development The involvement of ubiquitin and the ubiquitin-conjugating system in neuronal tissue in mammals has been recognized for many years. Several degenerative diseases of the nervous system including Alzheimer's disease, Pick disease, Parkinson's disease, and PSP (progressive supranuclear palsy) are characterized by the presence of neuronal inclusions made of straight filaments (Gallo and Anderton, 1989). Ubiquitin is present in the filaments in all of these inclusions (Perry et al, 1987; Mantto et al, 1988). The cerebral soluble ubiquitin content in patients with Alzheimer's disease is significantly higher than that in normal individuals (Tadder et al, 1993). In cultured rat D R G neurons, stress results in increased formation of ubiquitin conjugates (Morandi etal, 1989). In the moth Manduca sexta, ubiquitin levels increase markedly in neurons undergoing programmed cell death (Fahrbach et al, 1994). A l l of these observations suggest that the ubiquitin system may play roles in neuronal activity or development, probably by regulating the activities and half-lives of proteins in the nervous system. More direct evidence of this theory has come from the Drosophila bendless mutation (Charles et al, 1994). The bendless mutation affects the connectivity between the giant fiber and the tergotrochanter motor neuron, and results in the failure of the flies to exhibit the escape response consisting of a jump and subsequent flight. This gene has been recently cloned and found to encode a protein (ben) with highest similarity to the yeast E2 proteins UBC5 and UBC4. The ben protein also contains a perfect match to the E2 active site, which strongly suggests that it is a ubiquitin-conjugating enzyme. 5.6. DNA repair Yeast UBC2 corresponds to the yeast D N A repair gene, RAD6 (Jentsch et al, 1987). ubc2(rad6) mutants show a variety of phenotypical defects in D N A repair: moderately slow growth (Kupiec and Simchen, 1984), hypersensitivity to various D N A damaging reagents, defects in induced mutagenesis and post-replication repair and meiotic recombination, and increased spontaneous and induced mitotic recombination (Madura etal, 1990). In addition, ubc2(rad6) strains show enhanced base-pair transitions and G C - T A transversions (Kang et al, 1992). 17 Introduction Retrotransposons (Ty elements) are transposed in ubc2(rad6) mutant strains at a rate 100 fold higher than that in the wild-type strain (Picologlou. ef al, 1990). These data suggest that ubc2(rad6) mutants have a mutator phenotype. Moreover, yeast ubc2(rad6) homozygous diploids are sporulation deficient (Jentsch et al, 1987). The UBC2(RAD6) gene is transcriptionally activated by DNA-damaging agents (Madura et al, 1990), which underscores the role of the ubiquitin system in D N A repair pathways. This gene encodes a 20 kd class IIE2 protein which contains a striking C-terminal extension of 14 negatively charged residues (Jentsch et al, 1987; Reynolds et al, 1985). This C-terminal domain is required for mono- and multi-ubiquitination of histone in vitro (Sung et al, 1988). It may also be required for the ubiquitination of other positively charged protein substrates in vivo. Studies suggest that in vivo, the C-terminal acidic extension is required only for sporulation. The U B C domain of the UBC2 protein is sufficient for its D N A repair functions (Silver et al, 1992). UBC2 ubiquitinates histone in vitro without the additional requirement for E3 proteins (Jentsch et al, 1987). However, it can also form a complex with the auxiliary E3 protein UBR1 for other functions. In vitro, together with UBR1, UBC2 ubiquitinates proteins according to the identity of their N-terminal amino acid residues for protein degradation by the N-end rule pathway (Dohmen et al, 1991; Varshavsky, 1992). The cellular importance of the UBC2-mediated N-end rule pathway is not yet very clear. A yeast ubrl deletion mutant has no obvious phenotype, which suggests that this pathway does not play a significant role in total protein degradation (Bartel et al, 1990). Thus far the only in vivo protein substrate found to be degraded through this pathway in yeast is G a protein (Madura and Varshavsky, 1994). It is possible that UBC2 interacts with E3 proteins other than UBR1 for the ubiquitination of other protein substrates (Sharon et al, 1991). Functional homologs of yeast UBC2 have been cloned from S. pombe (Reynolds etal, 1990), Drosophila (Koken et al, 1991), C. elegans (Leggett, et al, 1995) and human (Schneider et al, 1990). Except for the homolog from C. elegans, all of these proteins are class I enzymes without the C-terminal extension. When expressed in ubc2 mutants, these homologs can restore the D N A repair and induced mutagenesis functions, but cannot rescue the sporulation deficiency of 18 Introduction the mutant cells (Reynolds et al, 1990; Koken et al, 1991; Koken et al, 1991). Even the C. elegans homolog ubc-1, which also contains a negatively-charged C-terminal extension, could not rescue the sporulation deficiency (Leggett et al, 1995). Thus the C-terminal extension of UBC1 provides a strong specificity for certain substrates in the yeast spbmlatiori pathway. The surprising diversity of functions shown by UBC2 suggests that it mediates selective degradation of regulatory proteins for D N A repair, sporulation, and retrotransposition. Whether UBC2 ubiquitinates histone in vivo, and thus affects chromatin structure, is questionable. Studies show that ubiquitination of histone is hot required for normal growth in yeast (Swerdlow et al, 1990). 5.7. Mammalian cell transformation Recent discoveries show that defective or misregulated ubiquitin-conjugation events can lead to mammalian cell transformation. Two examples involvethe degradation of p53 and c-Jun. The mammalian tumor-suppressor protein p53 is believed to activate transcription for cell proliferation. Theabsence of p53 predisposes animals to neoplastic disease (Donehower, et al, 1992). In the presence of the virus-encoded oncoproteins, including E6 by-papillomavirus and E1A by adenovirus, p53 is targeted by the ubiquitin system, and degraded by the proteasome (Scheffner et al, 1993). A human E2 protein', UbcH5, which is very similar to the yeast E2 enzyme UBC4/5, has been shown to be able to ubiquitinate p53 with the aid of E6 (Rolfe et al, 1995). Another example of misregulated ubiquitin-conjugation leading to cell transformation involves c-Jun. It has been shown that c-Jun can be efficiently multiubiquitinated and turned over by the proteasome; however, its transforming counterpart; v-Jun, manages to escape ubiquitin-mediated proteolysis due to its lack of a degradation signal, the 8-domain of the protein (Trier et al, 1994). The accumulation of Jim, which is believed to positively regulate cell proliferation, leads to oncogenesis (Bos etal, 1990; Morgan et al, 1993). 19 Introduction 5.8. Protein translocation Yeast UBC6 encodes a 28.4 kd Class II ubiquitin-conjugating enzyme with a C-terminal signal anchor sequence (Sommer and Jentsch, 1993). UBC6 is an integral membrane protein with its enzymatic domain facing the cytosol. In situ immunofluorescent staining shows that it localizes to the endoplasmic reticulum (ER), and possibly to the nuclear envelope. A ubc6 null mutant exhibits no apparent phenotype (Sommer and Jentsch, 1993). However UBC6 (identical to D0A4) contributes to a complex degradation pathway of the yeast MAToc2 transcription repressor (Chen et al, 1993) ( for a detailed discussion see Section 4.3.). Genetic evidence suggests that UBC6 is also involved in protein translocation, probably through its association with yeast SEC61 (Sommer and Jentsch, 1993). Associated with other SEC proteins, SEC61 is thought to be part of a multisubunit translocation apparatus of the ER which is essential for the translocation of proteins across the ER membrane. At the restrictive temperature of a conditional lethal sec61 mutant, translocation of proteins across the ER is strongly reduced and precursor proteins accumulate. Interestingly, this deficiency is restored nearly to wild type levels by a ubc6 null mutation. However the lethality of a sec61 deletion mutant is not suppressed in a ubc6 null mutant background. This suggests that the suppression is specific for defects caused by mutant sec61 proteins. It was noticed that Sec61 mutant protein is stable in cells, suggesting that itself is not a target for UBC6-mediated degradation. Strong overexpression of UBC6 also leads to weak suppression of the ts sec61 phenotype and partial restoration of the translocation of some ER proteins. Since the same extent of suppression can be obtained by overexpression of the ubc6^er87 mutant protein, this suppression is probably mediated through a titration effect (such as specific E3 proteins associated with UBC6). A model for UBC6 function in secdl cells was proposed by Sommer and Jentsch. In sec61 cells at the non-permissive temperature, the protein translocation apparatus is structurally distorted, leading to UBC6-catalyzed ubiquitination of one of its subunits and its subsequent degradation. Proteolytic elimination of a subunit in the apparatus subsequently results in the translocation defect. The absence of UBC6-mediated degradation, 20 Introduction either in a ubc6 null background, or through depletion of UBC6-specific E3 proteins, restores translocation to a certain extent (Sommer and Jentsch, 1993). Ubiquitin conjugation may also be involved in protein translocation into mitochondria. The in vitro uptake of monoamine oxidase B into mitochondria is inhibited by anti-ubiquitin antibodies (Zhaung and McCauley, 1989). Elucidation of the biological function and mechanism of the ubiquitin system in this protein translocation process awaits further study. 5.9. Peroxisome biogenesis and assembly Peroxisomes are almost universal components of eukaryotic cells which are involved in a wide range of metabolic processes, including the (5-oxidation of fatty acids, elimination of hydrogen peroxide via catalase, synthesis of plasmalogens and cholesterol, etc. (Lazarow and Fujiki, 1985). Some genes required for peroxisome biogenesis and proliferation have been shown to encode ubiquitin conjugating enzymes. The Saccharomyces cerevisiae PAS2 gene was cloned from a mutant lacking functional peroxisomes due to impairment of the peroxisome import machinery. D N A sequence analysis showed that it encodes a 21.1 kd E2 enzyme (UBC10). The proposed function of the Pas2 protein as a ubiquitin conjugating enzyme is supported by the fact that mutant Pas2 proteins containing substitutions in the conserved cysteine residue of the U B C domain fails to complement Pas mutant strains. Localization studies suggest that Pas2(UBC10) is a peroxisomal protein (Wiebel and Kunau, 1992). In the yeast Pichia pastoris, the PAS4 gene, which is required for peroxisome assembly, also encodes a ubiquitin-conjugating enzyme. PAS4 is a 24 kd protein located on the cytoplasmic surface of peroxisomes, and is induced during peroxisome proliferation. 5. cerevisiae PAS2(UBC10) shares 48% amino acid similarity with Pichia pastoris PAS4 (Crane et al, 1994) although both encode Class III E2s with N-terminal extensions. PAS2(UBC10) does not complement Pichia pastoris pas4 mutations (Crane et al., 1994). The functions of PAS2- and PAS4-mediated ubiquitination in peroxisome assembly and biogenesis remain to be determined. 21 Introduction 5.10. Viral infection Several observations suggest that the ubiquitin system may play roles in viral infection. Ubiquitin is encoded by two isolates of togavirus B V D V , CP1 and Osloss (Meyers et al, 1991). The insertion of ubiquitin sequences into the genome of B V D V seems functionally significant since it is not found in the genome of their noncytopathogenic counterpart. Baculovirus, a D N A virus that infects insect cells, encodes a ubiquitin-like protein which is distinct from the insect ubiquitin. It has been suggested that the viral ubiquitin-like protein could function as an inhibitor of some conjugation reactions by binding irreversibly to specific E2s (Guarino, 1990). The animal virus A S F V (African swine fever virus) encodes a Class II E2 protein with an acidic C-terminal extension (Hingamp et al., 1992), which is expressed early and continuously during virus infection. The significance of this enzyme for viral propagation is still unclear. 5.11. Antigen processing During the vertebrate immune response,- antigenic peptides are generated from endogenously synthesized cellular proteins or viral proteins. Binding to the newly synthesized class I M H C (major histocompatibility complex) in the ER, these peptides are transported to the cell surface for presentation to cytotoxic T cells (Monaco, 1995). Several observations suggest that the ubiquitin-dependent proteolytic pathway plays essential roles in the production of those antigenic peptides through the ubiquitination and subsequent partial degradation of target proteins. In the mutant ts20 cells carrying a temperature sensitive allele of E l , M H C class I-restricted antigen presentation is inhibited at the nonpermissive temperature (Michalek et al., 1993). Proteasome inhibitors have been found to block antigen presentation (Rock et al., 1994). Furthermore, two proteins whose genes map to the M H C region, LMP2 and LMP7, can be assembled into the proteasome as its J3-subunits (Gaczynska et al, 1993; Fruh et al, 1994). A current model for the function of the LMP2 and LMP7 is that they alter the cleavage specificity of the proteasome so that targeted proteins undergo controlled breakdown instead of complete degradation. They also 22 Introduction change the cleavage specificity to give a higher proportion of peptides with C-terminal hydrophobic residues, which are favored for class I M H C binding. 5.12. Phytochrome degradation Phytochrome is the photoreceptor which controls red light-mediated morphogenesis in plants. After a brief light pulse, it is photoconverted from its stable red light-absorbing form Pr to a far-red light-absorbing form Pfr. Pfr is then quickly multiubiquitinated and degraded through the ubiquitin-proteolytic system (Shanklin et al., 1987). While the half-life of Pr is more than 100 hours, Pfr has a half-life less than 1 hour. It is not clear which E2 enzyme is involved in the multiubiquitination, or what type of structural change provides the degradation signal. 5.13. Ubiquitination of membrane proteins Several membrane proteins have been found to be ubiquitinated, yet metabolically stable. The lymphocyte homing;receptor gp90^EL" 14 is ubiquitinated oh its extracellular domain (Siegelman et al, 1986). Two other cell surface proteins, the platelet derived growth factor (PDGF) receptor and the growth hormone (GH) receptor are ubiquitinated on their cytoplasmic domains (Yarden et al, 1986). The ubiquitination of gp90 seems to be functionally significant. Blocking the ubiquitin branch of the protein with a monoclonal antibody against ubiquitin prevents lymphocyte binding (Siegelman et al, 1986). An intriguing question is how E2 enzymes gain access to these membrane proteins, especially in the case of gp90. It has been proposed that these receptors are ubiquitinated in the ER prior to membrane insertion (Siegelman et al, 1986). Two ER membrane-located E2 enzymes, UBC6 in yeast (Sommer and Jentsch, 1993) and U b c M l in mouse (Hauser and Jentsch, in preparation), are considered to be candidate ubiquitin-conjugating enzymes for membrane ubiquitination. 23 ' • • : \ Introduction 5.14. Spermatogenesis Three loci encoding proteins with strong sequence similarity to ubiquitin-activating enzymes (El) have been identified in the mouse genome. Two reports suggest that one of these, the Als9Y-l gene, is a candidate spermatogenic gene (Mitchell et al, 1991). Als9Y-I is located in the Sxr (sex-reserved) region on the short arm of the mouse Y chromosome, and maps close to the Spy locus, which is required for the survival and proliferation of spermatogonia during spermatogenesis. The expression of the AIs9Y-I gene is largely restricted to testis, and this supports the hypothesis that Als9Y-l locus is identical to Spy (Kay et al, 1991). Recently, Wing, S. et al. cloned a rat UBC4/5 homolog which is specifically expressed in testis (personal communication). A l l of this indirect evidence suggests that the ubiquitin system may have functions in spermatogenesis. 6. SUBSTRATE RECOGNITION BY UBIQUITIN CONJUGATION ENZYMES As discussed above,- the ubiquitin systenphas striking functional diversity and individual ubiquitin-conjugation enzymes are functionally specialized. (Jentsch, 1992). This specialization suggests that the enzymes possess narrow substrate specificities, and recognize specific signals on substrates. These signals can be short amino acid sequences, or specific protein conformations. Some proteins carry several distinctdegradation signals which are recognized by several ubiquitin-conjugating enzymes, such as MATcc2 and cyclins. In addition to degradation signals; the target proteins also must carry a lysyl residue in close proximity as an acceptor for ubiquitination (Bachmair and Varshavsky, 1986). -Evidence suggests that the substrate specificity of distinct E2s is accomplished with the assistance of.E3 proteins. Thus far two types of E2 enzymes, UBC2 and UBG4 are known to require an additional factor to ubiquitinate substrates (Bartel et al, 1990; Girod and Vierstra, 1993). Among E3s, UBR1, the yeast E3 specific to UBC2, is the best characterized. Substrates of the UBR1-UBC2 pathway are recognized and degraded by the identity of their N-terminal residues (N-end rule). The N-terminus of the substrate is bound by UBR1 and ubiquitinated by 24 Introduction UBC2, which forms a ternary complex with UBR1. However, this N-end rule pathway is not the major pathway for ubiquitination in cells (Bartel et al., 1990). The basis for the recognition of other degradation signals and the mechanisms for other E3 functions, such as E6 for UBC4-related degradation of p53, are at present unclear. 7. C. ELEGANS AS A MODEL SYSTEM Caenorhabditis elegans is a small, free living soil nematode which possesses many unique advantages for developmental, genetic and molecular studies. C. elegans has a short life cycle of three days at 25°C, and it can be easily maintained on plates containing nutrient growth media spread with a lawn of bacteria. Large quantities of the worms can be obtained through culturing in liquid in large bottles (Sulston and Brenner, 1974). The transparency and relatively simple anatomy of this nematode make it a very attractive system for developmental studies. The complete cell lineage, including cell migration, programmed cell death* and neurocircuitry is known (Sulston, 1976; Sulston and Horvitz, 1977; Sulston etal, 1983; Chalfie et al, 1985). Genetic tools are available for C. elegans. While most of the worms in a population are self-fertilizing hermaphrodites, males arise spontaneously by non-disjunction at a frequency of 1 in 500 at 20°C, allowing for crosses. To uncover genes playing essential functions in nematode development and behavior, numerous mutants have been isolated using various mutagens and mapped. C. elegans D N A transformation, both integrative and extrachromosomal, has been developed and improved into an efficient process which allows reverse genetics to be employed to study gene expression and cell biology (Mello et al, 1991). Even though techniques for gene disruption through homologous recombination are still lacking, several laboratories are now developing systems to knock out gene functions using transposon insertion and excision (Plasterk and Groenen, 1992). The C. elegans genome is very compact, with a size about 5 times that of yeast, and two thirds that of Drosophila . Most of the genome has now been cloned and ordered as a series of 25 Introduction cosmid and Y A C (yeast artificial chromosome) clones (Coulson et al, 1988). Sequencing of the genome and of cDNAs is in progress. 8. T R A N S SPLICING OF C. E L E G A N S GENE TRANSCRIPTS Many C. elegans gene transcripts undergo trans splicing, a process in which a short leader from a distinct species of small nuclear R N A is attached to the 5' end of the mRNA precursor, usually at the position corresponding to a 3' consensus splice site (Becktesh and Hirsh, 1988). The splice site for trans splicing is the same as that for cis splicing, except that it lacks the 5' donor consensus site. Two spliced leaders, SL1 and SL2, have been identified in C. elegans. Although both R N A leaders are 22 bases in length, they differ in their sequences (Huang and Hirsh, 1989; Krause and Hirsh, 1987). SL1 is used near the 5' ends of pre-mRNAs while SL2 is used at internal trans-splicing sites of polycistronic pre-mRNAs (Blumenthal, 1995). It has been estimated that about 70% of C. elegans gene products are trans spliced, and about one quarter of all genes are contained in polycistronic clusters (Blumenthal, 1995): 9. THE PRESENT STUDY Only recently has it been appreciated that ubiquitin-conjugating enzymes (E2s) exist as a large multi-enzyme family in eukaryotic cells. The amazing diversity of cellular functions for the ubiquitin system leads to the hypothesis that different ubiquitin conjugating enzymes (E2), perhaps with the help of other components of the ubiquitin pathway, target specific substrate proteins important for different aspects of cell regulation. At the beginning of these studies, most of the work on E2 genes and their functions had been carried out in yeast. Studies of E2s in C. elegans were initiated in order to elucidate the functions of the ubiquitin system in a multicellular animal. A gene encoding an E2 enzyme was cloned and regulation of its expression in different tissues and life stages was studied. The functions of the E2 enzyme in the development of C. elegans was extensively investigated. The work presented in this thesis suggests that the regulation of E2 functions may be very different in the nematode compared to yeast. 26 Materials and methods II. M A T E R I A L S A N D M E T H O D S 1. GENERAL TECHNIQUES FOR HANDLING C. ELEGANS 1.1. Culturing of C.elegans C. elegans Bristol (N2) was maintained on N G plates (0.3% NaCl, 0.25% tryptone, 5mg/ml cholesterol, ImM CaCl2, 1 mM MgS04, 25 mM K H 2 P O 4 pH 6.0 and 1.7% agarose) spread with a lawn of Escherichia coli OP50 as described by Brenner (1974). Normally the nematode was grown at room temperature (20-22°C). 1.2. Maintenance of the strains C. elegans strains were maintained on NG plates by transferring adult worms to fresh plates after each generation. For short term storage, L2 stage larvae were allowed to starve on the plates and develop into dauer larvae. Dauer larvae can survive on plates for up to three months. For long term storage, strains were frozen in liquid nitrogen (Wood, 1988). To obtain maximum recovery from the freezing procedure, worms were washed off the plates with M9 buffer, and mixed with an equal volume of the freezing buffer (Sterile Basal S containing 30% glycerol). Aliquots of 1 ml were frozen at -70°C overnight before transfer to liquid nitrogen. Typically, survival rates of the larval stages range from 60-80%, while some adults also survive freezing but become sterile by the procedure. Embryos do not survive freezing. 1.3. Heat-shock and cadmium treatment of C. elegans C. elegans can be heat-shocked either on NG plates or in liquid medium. To heat-shock worms on N G plates, plates containing nematodes in various stages were placed in a 33°C incubator for 1-2 hours. A 15 minute recovery at room temperature was allowed after the heat-shock. Another 1-2 hour incubation at 33°C followed the recovery when a double heat-shock 27 Materials and methods treatment was intended. For heat-shocks in liquid, sterile air was bubbled through the medium during the treatment. 2. GENERAL DNA AND RNA TECHNIQUES 2.1. Bacterial transformation Plasmid ligation products were transformed into competent E.coli DH5oc (GIBCO-BRL) cells following the procedures provided with the product. For transformation of purified plasmids, 50 ixl of the competent cell suspension was mixed with 10 ng of the D N A , the mixture was incubated on ice for 15 minutes and plated. YT-Ampicilin plates were used for colony selection. 2.2. Standard Polymerase Chain Reaction (PCR) Standard polymerase chain reactions were carried out in a 50p:l reaction mix, containing lf i l template D N A , 50 pmol of each oligonucleotide primer, 50fiM each deoxynucleoside triphosphate, and 1 unit of Taq D N A polymerase (Promega) in the PCR buffer as described by the manufacturer (Promega). The amplification consisted of 35 cycles of 94°C for 30 seconds, 45-60°C (depending on the length and G C content of the oligonucleotide) for 90 seconds, and 72°C for 90 seconds. An additional 10 minute incubation at 72°C followed the final cycle. A l l PCR reactions were carried out in the TwinBlock™ system (ERICOMP Inc.). 2.3. Worm P C R 2.3.1. Single worm PCR For single worm PCR reactions, a nematode was picked and placed in 2.5|_tl PCR-Lysis solution (50mM KC1, lOmM Tris pH 8.3, 2.5mM MgCi2, 0.45% Tween 20, 0.01% gelatin, and 2.5mg/ml Proteinase K). The solution was then overlayed with two drops of mineral oil and incubated at 60°C until the worm dissolved, usually 30-45 minutes. The solution was then heated 28 Materials and methods at 95°C for 15 minutes to inactivate Proteinase K. To the same tube was then added 22.5p:l PCR reaction mix comprised of n.5p:l dH20, 2.5LL1 10X concentrated buffer (Pharmacia), l.OjLtl dNTPs (5mM of each), 0.5p:l of each oligonucleotide (50pmol/p:l) and 1 unit of Taq polymerase (Pharmacia). The amplification consisted of 30 cycles of 94°C for 30 seconds, 55°C for 1 minute, and 72°C for 2 minutes. An additional 10 minute incubation at 72°C followed the final cycle. 2.3.2. PCR with a pool of worms Worms were washed off N G plates when the E: coli was almost depleted and rinsed several times with dH20. For each 30ul of the compact worm suspension, 25 uT of PCR-Lysis solution was added and the mixture was incubated at 60°C for 4 hours. Alternatively, the worm suspensions could also be frozen at -70°C and lyophilized before addition of the PCR-lysis solution. After heat-inactivatiori of Proteinase K at 90°C for 20 minutes, the worm lysate was centrifuged at 14,000g for 10 minutes, and the supernatant was collected for PCR reactions. PCR amplifications were carried out as described for single worm PCR, using 1/50-1/10 of the supernatant as the template DNA. 2.4. Purification of plasmid DNA from E.coli A standard alkaline-SDS lysis protocol for rapidsmall scale plasmid preparation (Maniatis et al, 1982) was slightly modified. To prepare plasmid D N A for double stranded D N A sequencing, 3ml of the E.coli culture was briefly centrifuged (14,000g) and the cell pellet was resuspended in 200jil GTE buffer (Maniatis et al, 1982). Cells were lysed by the addition of 400uT alkaline-SDS solution (Maniatis), and the mixture was neutralized with 300|il 7.5M NH4OAC. The supernatant was centrifuged at 14,000g for 5 minutes, and only the top 700jil of the supernatant was collected and precipitated with 420pil isopropanol. The D N A pellet was rinsed with 70% ethanol, and resuspened in 80p:l dH^O. Typical yield from this procedure was 15-20|ig of plasmid DNA. 29 Materials and methods To prepare plasmids for microinjection, the standard Maniatis procedures were followed. After dissolving the crude D N A pellet in d f^O, the D N A solution was mixed with an equal volume of 4.4mM L i C l . The tube was incubated on ice for 2 hours, arid R N A was collected by 14,000g centrifugation for 10 minutes. The D N A in the supernatant was precipitated with ethanol as before, and resuspended in H2O to give a final plasmid concentration of 200ng/|il. The Magic Plasmid Preparation Kit (Promega) was also used to purify plasmid from E.coli cultures as described by the manufacturer. Plasmid D N A prepared using this kit is free of protein and RNA, and was suitable for all experiments described in this thesis. 2.5. Generation of nested deletion DNA clones with Exonuclease III A series of nested deletion clones of large D N A fragment inserts in plasmids were generated according to the method described by Henikoff (1984) with small modifications. Purified plasmid D N A (5 pig) was completely digested with two restriction enzymes, so as to leave a 4 base 3'- extension protecting the vector and a 5'- extension or blunt end adjacent to the insert. Digested D N A was then precipitated with ethanol, redissolved in 30 | i l Exo buffer (66 mM Tris pH 8.0, 0.66 mM MgCl2), and pre-warmed at 37°C for 5 minutes. To the D N A was then added 250 units of exonuclease III (Promega) and the solution was quickly mixed. During the incubation at 37°C, 2.5 p;l aliquots were removed at 30 second intervals and.added to cold centrifuge tubes containing 7.5 u\l SI mix (30 mM KOAc pH 4.6,;250 mM NaCl, 5% Glycerol, 0.1 m M ZnSC>4, 2 units of SI nuclease). The tubes containing the aliquots were incubated at room temperature for 30 minutes, then 1 p:l of the SI stop solution (0.3 M Tris, 0.05M EDTA) was added to each, and the incubation was continued at 70°C for 10 minutes to inactivate SI nuclease. The tubes were then transferred to 37°C, and incubated with 2 i l l of Klenow mix (20 m M Tris pH 7.5, 100 m M MgCl2, and 0.2 units of Klenow D N A polymerase) for 5 minutes. Another 5 minute incubation at 37°C followed the. addition of 2 u l dNTPs (0.125. mM each). In variation from Henikoff s protocol, the deleted D N A fragments were purified by agarose gel electrophoresis at this stage. The purified D N A fragment was then ligated in a mixture containing 1 X One-for-all buffer 30 Materials and methods (Pharmacia), 1 mM ATP, and 0.5 unit of T4 D N A ligase at room temperature overnight, before transformation into DH5oc competent cells. 2.6. Packaging and preparation of ssDNA from pBluescript vector The + strand of pBluescript plasmid can be packaged into a phage since this plasmid contains a phage f l replication sequence. X L 1-Blue cells containing the plasmid were grown to an ODgQO °f 0.3-0.5, and helper phage R408 (Stratagene) was added to the culture at a multiplicity of infection of 20:1 (phage to cells). The infected culture was left growing at 37°C overnight. An aliquot of this culture (1.5 ml) was centrifuged at 14,000g for 10 minutes, and 1.3 ml of the supernatant was transferred to a tube containing 0.3 ml of 2.5 M NaCl -20% PEG 8000. The sample was left for 15 minutes at room temperature and centrifuged as before. The resultant phage pellet was resuspended in 200ul low Tris buffer (20 mM Tris-HCl pH 7.5, 20 m M NaCl, 1 mM EDTA), extracted twice with an equal volume of phenolxhloroform (1:1), and the phage D N A was precipitated from 0.3 M NaOAc and 2 volumes of 95% ethanol and centrifuged at 14,000g for 10 minutes. The D N A pellet was dried and resuspended in 25 pJ of low Tris buffer. 2.7. Large scale preparation of X phage DNA Large scale A, phage D N A preparation was carried out according to Sambrook et al. (1989) with some modifications. An overnight LE392 bacterial culture (200 ml) was first pre-incubated with 200 [i\ A, phage stock (10 6 pfu/|il) in X dilution buffer at 37 °C for 15 minutes. Then 20 ml of N Z Y C medium was added, and the culture was incubated at 37°C overnight with vigorous shaking. Three ml of chloroform was then added to the culture, and the incubation was continued for another 15 minutes to lyse the bacterial cells. The culture was decanted from the chloroform and centrifuged twice (14,000g for 10 minutes) to remove the bacterial debris. Three ml of 5 M NaCl and 3g of PEG 8000 were added to the supernatant, and the solution was incubated at 4°C for two hours. The precipitated phage particles were collected by centrifugation at 14,000g for 10 minutes. The phage pellet was resuspended in 500 p:l of DNase buffer (50mM HEPES pH 7.5, 5 31 Materials and methods m M MgCl2, 0.5 mM CaCl2) containing 100 |ig RNaseA and 5p:g DNase I, and incubated at 37°C for 1 hour. To this mixture 150 \ig of proteinase K and 50 i l l of 10 X SET (100 m M Tris-HCl pH 7.5, 200 mM EDTA, 5% SDS) were added, and the solution was incubated at 65°C for 2 hours. After extraction twice with an equal volume of phenolxhloroform (1:1), the A, phage D N A in the liquid phase was precipitated with 2 volumes of 95% ethanol, and centrifuged at 14,000g for 10 minutes. The phage D N A pellet was resuspended in 50 (J,l dK^O. 2.8. Double-stranded and single-stranded DNA sequencing Double-stranded and single-stranded D N A were sequenced by the dideoxy chain termination method using a Sequenase kit (U.S. Biochemical). Single-stranded D N A sequencing was performed following the protocols described by the manufacturer of the Sequenase kit (U.S. Biochemical). For double-stranded D N A sequencing, 2-5 \ig of purified plasmid D N A in 18 ixl of distilled water was denatured by mixing with 2 p.1 of freshly prepared 2N NaOH solution and incubation at room temperature for 5 minutes. Two p:l of 3M NaOAc (pH 5.5) was then added, and the D N A was precipitated with 2 volumes of 95% ethanol. The denatured D N A was resuspended in 7 [i\ of d t ^ O , and used for the sequencing reactions. Annealing to the oligonucleotide sequencing primer, primer extension reactions, and termination of the extension reactions were carried out as described by the manufacturer for single-stranded D N A sequencing. 2.9. Preparation of radioactive DNA probes Several different protocols were used to make radioactive D N A probes in the present work. 2.9.1. Nick translation Nick-translated radioactive D N A probes were prepared by standard procedures described by Sambrook et al. (1989) with [a-32p] dATP and [a- 3 2 P] dGTP. 32 Materials and methods 2.9.2. Random hexamer extension labeling The dsDNA fragment (0.5 |!g-1.0 |ig in 6.6 pi) was mixed with 1.4 | i l of random hexamer oligonucleotide (90 A26O units/ml in 1 mM Tris, 1 mM EDTA, pH 7.5), boiled for 5 minutes, and quickly chilled on ice. The 17 ui reaction mix including 5 pi of 5 X buffer (1M HEPES, pH 6.6), 1 ui of B S A (10 mg/ml), 5 | i l of dNTP mix QOOpiM each dGTP, dTTP, dCTP in 250 m M Tris pH 8, 25 m M MgCl2 and 50 mM fj-mercaptoethanol), 5 ul of [oc-3 2P] dATP, and 1 unit of Klenow D N A polymerase fragment was added to the tube and incubated at 37°C for two hours. The reaction was terminated with 75 pi of 1 X T A E buffer, and the unincorporated nucleotides and short D N A fragments were separated from the D N A probes on a 1 ml Sephadex G-50 spun column as described by Maniatis, et al. (1987). 2.9.3. Primer extension Ml3 probes This method was used to generate single-stranded radioactive D N A probes for SI nuclease protection analysis. Five p i of the M13 template ssDNA (500 ng) was mixed with 1 | i l of universal M13 primer (1 pmol/pi) (or primers complementary to the insert D N A sequences) and 2 III of 10 X annealing buffer (100 mM Tris pH 7.5, 600 mM NaCl, 70 m M MgCl 2 ) . The solution was incubated at 65°C for 10 minutes, and gradually cooled to 30°C in 30 minutes. One | i l each of 1 m M dCTP and dTTP, 1.5 ul each of [a - 3 2 P]dATP and [a- 3 2 P]dGTP, and 1 unit of Klenow D N A polymerase fragment were added to the solution, and the mixture was incubated at 37 °C for 10 minutes. The chase reaction was carried out by adding 1 ui of 1 mM dATP and dGTP to the reaction, followed by another 37°C incubation for 10 minutes. The reaction was terminated by heat-inactivation at 70°C for 10 minutes. The primer extended D N A was digested with the appropriate restriction enzymes for 1 hour. Free nucleotides and small D N A fragments were separated from the labeled D N A probe by Sephadex G-50 spin column dialysis as outlined by Maniatis etal. (1982). 33 Materials and methods 2.10. Screening of recombinant DNA libraries 2.10.1 Screening ofC. elegans bacteriophage XZAP cDNA library A C. elegans bacteriophage AZAP cDNA library constructed by Barstead and Waterston, Washington University, St. Louis, was used to screen for c D N A clones encoding UBC-2. The X phage was propagated in E.coli BB4 and plated out on N Z Y C plates. For this purpose, the BB4 strain was grown overnight in TB broth supplemented with 0.2% maltose and 10 m M MgS04-The cells were collected by centrifugation, and resuspended in 1/2 volume 10 m M MgSC>4. Approximately 30,000 phage in up to 100 pi ADIL (10 mM Tris, pH 7.5, 8 m M M g S 0 4 ) were incubated with 350 (ii of the BB4 cell stock at 37°C for 15 minutes, then quickly mixed with 7 ml of N Z Y C top agarose solution pre-heated at 55°C, and poured onto 9 cm N Z Y C media plates. Two such plates were poured, and left at 37°C for 6-8 hours to allow phage propagation. When plaques had grown to a diameter of approximately 1.0-1.5 mm and were just beginning to make contact with each other, the phage particles were transferred to nitrocellulose or nylon membranes (Amersham) by plaque lift. To prevent the agarose from sticking to the membranes during plaque lifts, plates could be chilled in the cold room for 30 minutes before the transfer. The phage D N A was then released, denatured and fixed on the membranes by soaking the filters successively in the denaturing solution (0.5M NaOH, 1.0M NaCl), renaturing solution (0.5M Tris pH 8, 1.0M NaCl), and washing solution (2XSSPE) for 3 minutes each. The membranes were baked at 70°C for 30 minutes. After this treatment, the membranes could be stored at 4°C for up to several months. The library was screened with the nick translated, or random hexamer oligonucleotide-labeled 312bp PCR fragment (Zhen et al, 1993) as described by Sambrook et al, 1989. Eight positive clones containing the entire coding region were isolated out of 60,000 plaques screened, and 5 of these contained the complete coding sequence of ubc-2. A l l positive clones were purified by secondary screens. The purified positive phage clones were converted into pBluescript plasmids by superinfection with the helper phage R408 (Stratagene). Medium-sized, purified 34 Materials and methods positive plaques were picked individually into 1 ml XDIL. After vigorous vortexing for 3 minutes, 200 pi of the phage stock was mixed with 200.pl BB4 cell stock, 10 pi R408 helper phage and 500 ul XDIL, and incubated at 37°C for 15 minutes. Five ml of TB++ media (Sambrook, et al., 1989) was added and the incubation at 37°C with vigorous shaking was continued for 4 to 8 hours. The culture was heated at 70°C for 20 minutes, and centrifuged at 1,000 g for 5 minutes. The supernatant contained the pBluescript plasmid packaged in the M13 or f 1 phage particle. To plate the pBluescript plasmids, the supernatant was diluted 500 fold with TB++ medium, and 25 uT of the diluted plasmid stock, 300 pi BB4 cell stock, and 225 pi TB++ media were combined and incubated at 37°C for 15 minutes, before plating out on YT-Amp plates spread with 10 ul of 100 mM IPTG and 50 pi of 20% X-gal in DMF. 2.10.2. Screening of bacteriophage XCharon4 genomic library A ACharon4 library was previously derived from a partial Saw3A digestion of C. elegans genomic D N A and cloned into the £coRI sites ofthe X phage vector by Terry Snutch of Simon Fraser University. The phage library was propagated in E. coli LE392 in N Z Y C medium. For screening, 8,000 phage were mixed with 250 pi of the LE392 stock (an overnight LE392 cell culture resuspended in 1/2 volume of 10 mM MgS04), heated at 37°C for 10 minutes, and plated on N Z Y C plates together with 3 ml of N Z Y C top agarose. The phage D N A was transferred to nylon membranes and screened for clones encoding ubc-2 as described in 2.10.1. The complete ubc-2 cDNA fragment was labeled and used as the probe. Of 70,000 phage screened, 4 positive recombinant clones were obtained, of which three identical ones encoded ubc-2, and one contained a sequence homologous to ubc-2 . All positive clones were purified by rescreening. 2.11. Large scale isolation of C. elegans genomic DNA High molecular weight genomic DNA was isolated from nematodes by the method of Emmons et al, (1979), modified by Jones et al, (1986). Three grams of frozen nematodes were suspended in 20 ml of proteinase K buffer (100 mM Tris-HCl, pH 8.5; 50 mM E D T A ; 200 mM 35 Materials and methods NaCl; 1% SDS) with 3 mg proteinase K. After a 2-hour digestion at 65°C, this solution was extracted three times with an equal volume of phenol:ehloroform (1:1). in a separation funnel and once with chloroform in polypropylene tubes. The aqueous phase was transferred to a glass tube, chilled on ice, and overlaid with 2 volumes of -20°C 95% ethanol. The glass tube was slowly rotated at an angle while the genomic DNA was precipitated as shown by the white pellet at the interface. When only the white DNA pellet was left in the lower phase, the ethanol was discarded, and the D N A pellet was washed with 95% ethanol followed by 75% ethanol. The DNA pellet was air-dried for 10 minutes, and redissolved in 1 ml T E buffer while still moist. Typical yield was about 3 mg genomic DNA per gram of frozen nematodes. 2.12. Genomic Southern blot analysis : Three |ig of C. elegans genomic DNA was digested with various restriction enzymes, and the digested DNA fragments were separated on 0.7% agarose gels following standard protocols (Maniatis et al, 1982). The DNA bands were depurinated, denatured and blotted onto nylon or nitrocellulose membranes in 20 X SSPE as described (Southern, 1975). 32p_iat>eiecj DNA probes were prepared by nick translation or. random hexamer extension labeling. Pre-hybridization and hybridization were performed according to standard procedures (Maniatis etal., 1982) in Seal-a-Meal bags. Background nonspecific hybridization was eliminated, by the inclusion of 5 mg of heparin (sodium salt; Sigma) per ml in the hybridization solutions. 2.13. Isolation of C. elegans total cellular RNA 2.13.1. Large scale preparation Large scale total cellular RNA was isolated by the method of Antonucci (1985). Three to five mg of frozen nematodes were powdered in a chilled mortar and pestle, and dissolved in 3 ml of homogenization buffer (7.5 M guanidinium hydrochloride, 25 mM sodium citrate, pH 7.0, 0.1 M fi-mercaptoethanol). The homogenate was passed ten times through a 21g needle to reduce the 36 Materials and methods viscosity by shearing genomic D N A , then overlaid onto a 1 ml cesium chloride solution (5.7 M CsCl, 25 m M sodium citrate, pH 5.0 which had been treated with 0.07% DEPC for 15 minutes and autoclaved), and centrifuged (220,000 g) for 16 hours at 22°C. The R N A pellet was dissolved in 0.3 ml of sterile, DEPC-treated water. The aqueous RNA was precipitated with 1/10 volume of DEPC-treated 3M NaOAc (pH 5.2) and 2 volumes of 95% ethanol at -70°C for 15 minutes. The R N A pellet was washed with 75% ethanol, and redissolved in DEPC-treated distilled water. Typical yields were 3 mg of total cellular RNA per gram of frozen nematodes. 2.13.2. Mini-scale RNA preparation Approximately 200 C. elegans were washed off N G plates, rinsed in distilled water, and collected in a final volume of 200 p.1. Equal volumes of lysis buffer (4 M Guanidine isothiocyanate, 0.13% Sarkosyl, 33 m M Tris pH 8.0, 0.5% p-mercaptoethanol, 6.7 m M EDTA) and phenol: chloroform (2:1:1) were added to the worm slurry. In the presence of fine glass beads, the mixture was vigorously shaken (Vortex) for 4 minutes. After a brief centrifugation, the aqueous phase was transferred to a clean Eppendorf tube, and the R N A was precipitated by the addition of 1/40 volume of 1M HOAc and 3/4 volume of 95% ethanol followed by storage at -20°C for 1 hour. The R N A was collected by centrifugation at 14,000 g for 20 minutes. The R N A pellet was re-dissolved in distilled water, and re-precipitated with NaOAc and ethanol as described above. 2.14. Northern blot analysis Northern blot analysis was performed following standard procedures (Maniatis et al, 1982). R N A ( 5-20 pg in 4.5 pi) samples were dissolved in a denaturing loading buffer (3.5 pi of formaldehyde, 10 p i of formamide, 2 pi of 10 X R N A loading buffer which contained 50% glycerol, 1 m M EDTA, 0.4% bromophenol blue, and 0.4% xylene cyanol), heated at 55°C for 15 minutes, and separated on 1% formaldehyde-agarose gels in 1 X MOPS buffer as described (Sambrook et al., 1989). After blotting of the RNA to nylon membranes, the membranes were 37 Materials and methods hybridized with 32p_i a h e led D N A probes overnight, and washed according to standard procedures. Five mg of heparin (sodium salt; Sigma) per ml was included in the the pre-hybridization and hybridization solutions to eliminate nonspecific hybridization. 2.15. SI nuclease protection analysis Nuclease SI protection analysis was performed as described by Berk and Sharp (1977). Single-stranded D N A probes were prepared by primer extension. Oligonucleotide OZM7 which is complementary to ubc-2 sequence (from nucleotides -28 to -45) was used as the primer. The ssDNA probe, which complements the ubc-2 sequence from nucleotides -28 to -1,300 was separated on 4% denaturing polyacrylamide-urea gels, and recovered from the gel slice by electroelution in 0.5 X T B E buffer containing 0.05% SDS. 1 X 10^ cpm of purified probe was mixed with 10-20 (ig of total C. elegans cellular R N A in 30 | i l of hybridization buffer (60% formamide, 0.4 M NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 0.025% acrylamide, 1 unit of RNasin). The solution was heated to 70°C for 15 minutes, immediately transferred to 50°C, and incubated overnight. Hybridization was terminated by the addition of 170 (il of ice-cold nuclease SI buffer (280 m M NaCl, 50 mM NaOAc pH 4.6, 4.5 mM ZnS04) containing 150 units of nuclease SI (Pharmacia). The mixture was incubated at 37°C for one hour and 40 \il of 4 M NH4OAC, 0.1 M E D T A solution was added to terminate the digestion. Nuclease SI protected D N A fragments were then precipitated with an equal volume of isopropanol using 100 mg/ml of E. coli tRNA as carrier. Samples were directly dissolved in 10 \i\ formamide dye mix, and 5 | i l was analyzed on a thin 6% 8 M urea-polyacrylamide gel. 3. METHODS RELATED TO TRANSGENIC ASSAYS IN C. ELEGANS 3.1. Construction of ubc-2/lacZ fusions Five ubc-2/lacZ fusion constructs, namely CI, pZMI.l, pZMI.2, pZMII.l and pZMII.2, were prepared for transformation into C. elegans. CI is an in-frame translational fusion of the lacZ 38 Materials and methods coding region with the second exon of ubc-2. A 1.4 kb HindYR-BgUl D N A fragment from a ubc-2 genomic clone was inserted into the Hindlll-Bglll region of the pPD16.43 vector (Fire et al., 1989) as the promoter fragment. A polyadenylation signal was provided by a 0.6 kb Xhol fragment downstream of the stop codon of ubc-2. To make constructs pZMI.l, pZMI.2, pZMII.l and pZMII.2, the lacZ coding region, a 3.3 kb BamHl-Apal fragment from pPD16.43, was inserted in-frame into the second exon (Bglll-Apal sites) of the ubc-2 genomic clone. A l l four constructs contained a complete ubc-2 coding sequence including five exons and four introns. In pZMI.l, a 6 kb BgUl fragment preceding the initiation methionine codon and 2.6 kb of sequence downstream of the T A G stop codon were included in the construct to provide regulatory elements for expression. pZMI.2 included the same 5' non-coding sequence but less 3' non-coding sequence (the 0.6 kb Xhol fragment). pZMII.l contained the 1.4 kb Hindlll-Bglll upstream sequence and the 2.6 kb 3' non-coding sequence. pZMII.2 is identical to pZMII.l except that the 0.6 kb Xhol fragment was ligated to the 3' end of the fusion. A nuclear localization signal (NLS) from SV40 was included in all constructs at the beginning of the lacZ coding sequence. 3.2. Construction of ubc-2 sense and antisense constructs Two sense constructs ubc-2(SK) and hspl6-ubc-2, and two antisense constructs ANTI-I and ANTI-II were constructed to study the functions of ubc-2 during C. elegans development. For ANTI-I, a 1.0 kb Kpnl-Sacl fragment recovered from a ubc-2 cDNA clone, ubc-P2, was inserted between the Kpnl-Sacl sites of the pPD49.78 vector (provided by A . Fire). This fragment contained the complete cDNA sequence except the polyA track. hspl6-ubc-2 is the same as ANTI-I except that the ubc-2 cDNA fragment was cloned in a reversed orientation in pPD49.78, so that this vector will express the sense ubc-2 transcript after heat-shock. ANTI-II was made directly from the ubc-2 genomic clone (the sense construct, ubc-2(SK)) digested with Apal and Xhol. This resulted in two D N A fragments, one containing the ubc-2 coding sequence and the other containing the vector and ubc-2 upstream and downstream sequences. The two fragments were 39 Materials and methods blunt-ended and re-ligated together. Restriction digestion and sequencing were used to select the clones containing the inverted ubc-2 coding region sequence. 3.3. Establishment of transgenic C. elegans strains Plasmid D N A for transforming C. elegans was prepared by methods described in section 2.3. The D N A was suspended in distilled water to give a final concentration of 200 ng/ul The constructs of interest were injected into the gonad of a C. elegans rol-6 null strain with a marker plasmid pRF4 as described by Mello et al, 1991. Transformed progeny were identified by their right-rolling phenotype caused by the mutant collagen encoded by the pRF4 plasmid. Each selected transgenic animal was placed on a separate plate for propagation. Lines were established after the selectable phenotype had been passed on successfully for three generations. 3.4. Establishment of integrated transgenic strains Twenty-five transgenic animals (in late L3 or early L4 stage) carrying mixed extrachromosomal arrays of the constructs of interest and the pRF4 marker plasmid were mutagenized with 3,800 rads of y rays emanating from a ^ C o source. Healthy F l rollers (125-150) were picked onto separate plates and allowed to propagate. Five F2 rollers were picked from each plate on which over 50% of the progeny were rollers, and were individually screened for those producing only rolling progeny. The homozygous rolling F3s were outcrossed to the N2 (Bristol) strain for seven generations to eliminate other potential mutations in the genome. 3.5. in situ histochemical X-gal staining of transgenic worms C. elegans carrying ubc-2/lacZ constructs were stained as described by Fire et al. (1992). Transgenic animals were washed in distilled water, placed on glass slides, and vacuum-dried. A few drops of cold acetone were added to the dried worms, and the slides were left for a few minutes to allow the acetone to evaporate. Meanwhile 500 pil of the staining solution consisting of 125 p:l of 0.8 M Na3P04, 50 pi of the oxidation buffer (19.6 mg/ml potassium ferricyanide, and 40 Materials and methods 21.1mg/ml potassium ferrocyanide), 2 pi of 1% SDS, 0.5 pi of 1M MgCl2, 7.5 pi of Kanamycin (5mg/ml), and 6 pi of X-gal (2% in DMF) was prepared, and 50-100 pi of the solution was applied to each slide. All slides were incubated at 37°C for two hours to overnight in a moist environment. 3.6. Identification of (5-galactosidase staining cells in transgenic C. elegans Transgenic animals stained with X-gal were permanently mounted in 80% glycerol, 20 mM Tris pH 8.0, 200 mM sodium azide prior to examination by Nomarski microscopy. The identities of stained cells were determined by the size and shape of their nucleus and its position in the animal relative to other nuclei as defined by Sulston (1976), Sulston and Horvitz (1977), Albertson and Thomson (1976), and Sulston et al. (1983). 3.7. O N P G assay Total P-galactosidase activity expressed by transgenic C. elegans was quantified using the colorimetric substrate ONPG. For ONPG assays, worms were washed in PBS buffer several times, and fixed in 5 ml cold acetone for 2 minutes. The worms were then air-dried, and 200-800 pi of the ONPG assay mix containing 1 X Z buffer (60 mM Na2HPO>4, 40 mM NaH2P04, 10 mM KC1 and 1 mM MgS04, pH 7.0), 38 pM P-Mercaptoethanol, and 1 mg/ml ONPG was added tothe worm pellet. After incubation for several hours at 37°C, the reaction was stopped by the addition of 1/2 volume 1 M Na2C03, and the absorbance of the supernatant was measured at 460nm. 4. PROTEIN AND IMMUNOLOGICAL TECHNIQUES 4.1. Overexpression of UBC-2 fusion protein in E. coli The open reading frame of ubc-2 was amplified by PCR with two oligonucleotides O Z M l and OZM2 using the ubc-2 cDNA as a template. O Z M l and OZM2 contain convenient restriction 41 > Materials and methods sites, //mdlJJ and PstI, respectively, at their 5'-end for cloning purposes. The 456 bp amplified PCR fragment was digested with PstI and Hindlll, and cloned into the HindlW-Pstl sites of the expression vector pRSETC (Invitrogen). After confirming the nucleotide sequence of the PCR fragment by DNA sequencing, this pRSETC-UBC-2 construct was transformed into the host E.coli strain BL21. A rapid screen for small scale expression of the fusion protein was performed as described in the information booklet, 'The QIAexpressionist'.(QIAGEN). Briefly, single colonies were picked and grown overnight at 37°C in 1.5 ml LB/Kanamycin/Ampicillin. This dense culture was diluted 1:10 into 10 ml of the same medium the next day, and the incubation at 37°C continued until the Agpo of the culture reached 0.7-0.9. After the addition of IPTG to a final concentration of 2 mM, the culture was grown for another 5 hours. One ml samples of the induced culture were taken at 1 hour intervals, and the cells were collected by centrifugation. Cell pellets were dissolved in 200 p.1 Buffer B (8 M urea, 0.1 M Na-phosphate, 0.01 M Tris/HCl pH 8.0) by gentle mixing, and the lysates were collected after a 14,000 g centrifugation for 20 minutes. Thirty |i l of a 50% slurry of Ni-NTA resin (QIAGEN) was added to each lysate, and mixed gently at room temperature for 30 minutes. The resins were collected after brief centrifugation at 14,000 g, and washed three times with 1 ml of Buffer C (8 M urea, 6.1 M Na-phosphate, 0.01 M Tris/HCl pH 6.3). The protein specifically bound to the resin was eluted with 20 (ll of Buffer C/100 mM . E D T A , and analyzed on 12.5% SDS-polyacrylamide gels. This rapid screen showed that the UBC-2 fusion protein was successfully expressed in the E.coli cells, and 3 hours of IPTG induction gave me highest yield of the protein. The solubility and localization of the expressed UBC-2 protein were also examined following the protocol described by QIAGEN. A 100 ml of the culture was grown and induced with IPTG for 3 hours, as previously described. The culture was then harvested and divided into two aliquots. For one aliquot, the cell pellet was resuspended in 5 ml of sonication buffer (50, mM Na-phosphate pH 7.8, 300 mM NaCl). After three freeze-thaw.cycles, the cell suspension was sonicated for 2 minutes at setting 2 using the sonicator by Mandel Inc.. The supernatant containing the soluble cytoplasmic protein was collected after a 10,000 g centrifugation for 20 minutes. The 42 Materials and methods pellet was resuspended in 5 ml of sonication buffer, and taken as a suspension of the insoluble cytoplasmic proteins. The cell pellet of the second aliquot was resuspended in 10 ml Tris pH 8.0, 20% sucrose. E D T A was added to a final concentration of 1 mM, and the cells were shaken at room temperature for 10 minutes. After the supernatant was removed, the cell pellet was resuspended in 10 ml ice-cold 5 mM MgSC>4, and incubated for 10 minutes at 0°C with occasional shaking. The cell suspension was centrifuged again, and the resulting supernatant consisted of the osmotic shock fluid containing the proteins of the periplasmic space. Ten u\l of each sample i.e., the soluble fraction, the insoluble fraction and the periplasmic proteins was analyzed on 12.5% SDS-polyacrylamide gels. The expressed UBC-2 fusion proteins were found to exist in both the soluble and insoluble fractions of the cytoplasmic proteins. 4.2. Large scale purification of UBC-2 fusion protein from E. coli A large-scale expression culture was grown, and the expressed UBC-2-6xHis fusion protein was purified on Ni-NTA resin following the protocols described in 'The QIAexpressionist' (QIAGEN) with small modifications. For each purification attempt, pRSETC-UBC-2, the expression plasmid, was transformed into BL21 cells, and the fresh transformants were inoculated into 10-20 ml of LB/ampicillin/kanamycin medium and grown overnight. One liter of the same medium was inoculated at 1:100 with the overnight culture, and the incubation at 37°C continued until the A<500 reached 0.7- 0.9. After a three hour induction with 2 m M IPTG, the cells were harvested by centrifugation at 4,000 g for 10 minutes. The cell pellet was either processed immediately or stored at -70°C. Since some of the expressed UBC-2-6xHis fusion protein was soluble in the cell, native proteins were purified under non-denaturing conditions at 0°C. PMSF was added to all the solutions used in the purification process to a final concentration of 0.2mM. The cell pellet was first resuspended in 5 volumes of sonication buffer (as in Section 4.1) per gram of E.coli (wet weight). The samples were frozen in a dry ice/ethanol bath, and thawed in cold water. The cells were then broken by sonication on ice (1 minute bursts/1 minute cooling for a total of 5 minutes). 43 Materials and methods After passing the lysate through a 21 gauge syringe needle several times to shear the D N A and R N A , it was centrifuged at 10,000 g for 20 minutes, and the supernatant was collected. Four ml of 50% slurry of Ni -NTA resin was mixed with the supernatant, followed by gentle stirring at 0°C for 1 hour. The resin was then loaded into a column, washed with sonication buffer and wash buffer (50 mM Na-phosphate, 300 mM NaCl, 10% Glycerol, pH 6.0) until the A280 of the flow through was less than 0.01. The bound proteins were finally eluted with a 30 ml pH 6.0 to pH 4.0 gradient in the wash buffer. The UBC-2-6xHis fusion protein was found to elute at pH 4.0. The pH of the recovered protein solution was adjusted to 6.5-7.0 with Tris buffer before storage at -70°C. Typically 6-10 mg of the native UBC-2-6xHis fusion protein was obtained from 1 liter of BL21 culture. The insoluble fusion protein was purified using denaturing solutions. The cell pellet was resuspended in Buffer A (6 M GuHCl, 0.1 M Na-phosphate, 0.01 M Tris, pH 8.0) at 5 ml per gram wet weight, and stirred for an hour at room temperature. The supernatant of the cell lysate was collected after centrifugation, followed by mixing with 8 ml of a 50% slurry of the Ni -NTA resin for 45 minutes. The resin was then loaded into a column, and washed successively with Buffer A , Buffer B, and Buffer C (same as in 4.1), respectively, till the A28O of the flow through was less then 0.01. The recombinant protein was eluted with 10 ml of Buffer D (8 M urea, 0.1 M Na-phosphate, 0.01 M Tris, pH 4.5). Eluted protein was dialyzed against 4 L PBS solution overnight at 0°C. 4.3. Ubiquitin thiol ester assay The 6xHis-UBC-2 fusion protein was assayed for its ability to form a thiol ester bond with El-activated ubiquitin. The assay was carried out as described by Hatfield and Vierstra, 1992 with minor modifications. Reactions contained a mixture of 30 ng of E l (prepared from E. coli overexpressing the wheat E l protein, UBA1), 500 ng of 1 2 5I-Ubiquitin, 1-3 pg of UBC-2 protein, and 1 unit of inorganic pyrophosphatase in 15 pi of 50 mM Tris (pH 7.6), 10 m M M g C l 2 , 1 mM ATP and 0.1 m M DTT. Reactions were incubated at 30°C for 10 minutes, and terminated by 44 Materials and methods boiling for 10 minutes in SDS-PAGE sample buffer, or in the same sample buffer without DTT or P-mercaptoethanol. Samples were subjected to 12.5% SDS-PAGE, and the 1 2 5I-ubiquitin thiol ester adducts were detected by autoradiography. 4.4. Raising polyclonal antibodies against UBC-2 protein Three New Zealand White rabbits were immunized with 0.5 mg of UBC-2-6xHis fusion protein (denatured by boiling in 0.1% SDS for 10 minutes) as an emulsion with an equal volume of Freund's complete adjuvant. The animals were boosted with the antigen in Freund's incomplete adjuvant for seven months, at two or three week intervals. Blood samples were taken and allowed to clot at 37°C for four hours. Serum, with NaN3 added to a final concentration of 0.1%, was then collected and stored at 0°C. 4.5. Affinity purification of antibodies against UBC-2 protein IgG was first precipitated from the serum with 45% ammonium sulfate, followed by 10,000 x g centrifugation for 10 minutes. The precipitate was dissolved in 1/2 volume of 10 mM Tris pH 8.0, and dialyzed twice against 2 liters of 10 mM Tris pH 8.0. IgG was further affinity-purified on a UBC-2-6xHis fusion protein column. To prepare the column, 5 mg of purified UBC-2 protein was first dialyzed against 20 m M PIPES pH 6.7 overnight, and mixed with 0.5 ml of Affi-gel 10 (Bio-Rad) at 4°C for 4 hours. The resin was loaded into a column at room temperature and washed with several bed volumes of PBS. The column was further washed with 0.1 M glycine pH 2.5 and 10 mM Tris pH 8.0, respectively, until the A28O was 0. This column could be stored in the cold room in PBS-0.1% NaN3. To purify antibodies against the fusion protein, ammonium sulfate precipitated serum was agitated with the Affi-gel resin at room temperature for 1 hour. The slurry was again packed in the column, washed extensively with PBS and eluted with 0.1 M glycine pH 2.5. Fractions were neutralized with 1M Tris base (15 pl/ml eluant). The protein content in each fraction was measured by the absorbance at 280 nm. The IgG containing fractions were pooled, dialyzed against 2 liters of PBS, and stored 45 Materials and methods in aliquots at -70°C. If the resulting antibody still cross-reacted with unrelated proteins, the column was washed with 1 M urea, 1 M NaCl or 0.5% Triton-X before elution. 4.6. Preparation of C. elegans protein extract Worms were collected from N G plates or liquid culture and washed with distilled water. For each 200 ul of packed worm slurry , 160 ul of distilled water and 80 ul of 5 X SDS-PAGE loading buffer without dye (250 mM Tris pH 6.8, 500 mM DTT, 10% SDS, 50% glycerol) was added. The mixture was heated at 90°C for 15 minutes, and the supernatant was collected after brief centrifugation at 14,000 g. If necessary, the solution viscosity was reduced by passage through a 21 gauge needle to shear the D N A . The protein content in the crude extract was determined by microbradford assays according to the protocol by Bio-Rad. 4.7. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) Discontinuous SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) was used to separate various protein samples dissolved in 1 X SDS sample buffer (50 m M Tris pH 6.8, 100 m M DTT, 2% SDS, 10% glycerol, 0.1% bromophenol blue). A 'mini-gel' apparatus (Mini-PROTEANII cell, Bio-Rad) was used to pour and run the gels (0.75 mm X 7.5 cm X 8.0 cm) according to the protocols described by the manufacturer. 4.8. Western blot analysis Proteins separated on SDS-polyacrylamide gels were electro-blotted to polyvinylidene difluoride (Immobilon-PVDF, Millipore) membranes. The gels were agitated in transfer buffer (25 m M Tris, 192 mM Glycine, 20% methanol, pH 8.2) for 10 minutes. The Immobilon membranes were moistened in methanol, then soaked in transfer buffer before assembly into the filter paper-gel-membrane-filter paper 'sandwich'. The transfer was carried out for 1 hour at 250 raA in an electro-blotting apparatus (Trans-blot cell, Bio-Rad) filled with transfer buffer. After transfer the blots were briefly rinsed in distilled water and dried at room temperature. 46 Materials and methods Immunodetection of proteins on the blots was performed as described by Millipore. By taking advantage of the hydrophobicity of PVDF, this method eliminates the blocking step and reduces the washing time dramatically. The dry blots were directly immersed in primary antibody (1:2000 dilution in TBS-0.05% Tween for UBC-2 antibody) for 1 hour, and washed twice with TBS-0.05% Tween for 10 seconds. The blots were then incubated in secondary antibody (1:10,000 dilution of peroxidase labeled anti-rabbit antibody, Amersham) for 30 minutes, followed by two 10 second washes. The protein-antibody complexes on the blots were detected using the E C L (Enhanced chemiluminescence) western blotting kit (Amersham). 4.9. in situ immunofluororescent staining of C. elegans The protocol was based on that of Mclntire etal. (1992). Worms were harvested from N G plates and fixed with freshly prepared 4% paraformaldehyde in 100 m M potassium phosphate buffer (pH 7.4) for 4-6 hours. The animals were then incubated in 1% Triton X-100, 100 m M Tris (pH 7.0), 1% P-mercaptoethanol overnight to reduce the disulfide bonds in the cuticle. After several washes with PBS, the worms were permeabilized by incubation in 1000 U/ml collagenase IV (Sigma) at 37°C for 3 hours. The fixed and permeabilized worms could be stored in A b A buffer (PBS, 0.1% Triton X-100, 1% BSA, 0.05% NaN 3 ) at 4°C for up to one week. The permeabilized worms were incubated with horse serum for 6 hours at room temperature in AbA buffer followed by overnight incubation with antibodies against UBC-2. Both serum and antibody were used at 1:500 dilution. After three washes in AbB buffer (PBS, 0.1% Triton X-100, 0.1% BSA, 0.05% NaN 3 ) , worms were incubated with 1:1,000 dilution FITC-conjugated goat anti-rabbit IgG (Sigma) for 6 hours. After three washes in AbB buffer, worms were mounted on slides in 70% glycerol, 100 m M potassium phosphate (pH 7.4), 5% n-propylgallate to minimize photobleaching, and 1 mg/ml 4,6-diamidirio-2-phenylindole (DAPI). The latter is a fluorescent DNA-binding agent which permits visualization of cell nuclei. 47 , , Results 111. RESULTS SECTION A. CLONING AND SEQUENCING OF UBC-2, A GENE ENCODING A UBIQUITIN-CONJUGATING ENZYME IN C. ELEGANS 1. Isolation of ubc-2 cDNA and genomic clones In yeast, UBG4 and UBC5 are the two most important E2s for bulk protein degradation, and the degradation of short-lived and damaged proteins. Their Drosophila homolog, UbcDl , shares 85% amino acid identity with'UBC4/5. To clone the homologous genes of UBC4/5 and UbcDl in C. elegans, two degenerate oligonucleotides, OPC9 and DJ2, were designed based on conserved protein sequences. These oligonucleotide sequences are as follows: OPC9 5' GAGGATCCTA(T,C)GA(A,G)GG(T,A)GG(T,A)GT(T,C)TT(T,C)TT 3' DJ2 5" CTGTCGACAC(A,G,C,T)GC(G,A)TA(C,T)TT(C,T)TT(A,G,C,T)GTCCA (C,T)TC 3' These two primers were used to amplify cDNA synthesized from C elegans RNA using . the polymerase chain reaction (PCR). A 312 bp DNA fragment was amplified, and shown to encode part of an E2 protein by dsDNA sequencing. Using this fragment as a probe to screen a C. elegans cDNA library prepared in XZAP, several positive clones containing a complete cDNA sequence encoding an E2 were isolated and the positive X.ZAP clones were converted into pBluescript plasmids. by superinfection with the helper phage R408 (See Materials and Methods). The plasmids were sequenced and the corresponding gene was named ubc-2. To isolate ubc-2 genomic recombinant clones, a C. elegans ACharon4 genomic library was screened with the complete ubc-2 cDNA as a probe. Several positive clones were obtained from which phage, DNAs were prepared. By restriction digestion of the phage DNA and hybridization with the entire cDNA probe, these positive clones were divided into two types, PC2#1 and PC2#2. From PC2#1, a 5.0 kb EcoRI fragment was subclohed into pBluescript (referred to as pSK5.0k). A series of nested deletion clones were generated from pSK5.0k and subjected to sequencing 48 Results analysis. D N A sequencing of these clones revealed that the 5.0 kb EcoRI fragment contained the complete coding region for ubc-2, 1.5 kb upstream of the coding region and 2.6 kb of 3' downstream sequence. From PC2#2 a 1.8 kb EcoRI fragment which reacted with the ubc-2 cDNA probe was subcloned. Sequencing of nested deletions generated from this clone showed that it contained regions identical to ubc-2, separated by small stretches of apparendy random sequence. This strongly suggested that this phage clone contain a pseudogene copy of ubc-2. 2. Comparison of the protein sequences encoded by C. elegans ubc-2, Drosophila UbcDl and yeast UBC4. Sequencing of the ubc-2 cDNA indicated that it encodes a 16.7 kDa protein. The cysteine at position 85 corresponds to the active site residue required for conjugation of ubiquitin. Comparison of the predicted protein sequences encoded by ubc-2, ubcDl and UBC4 (Figure 1) shows that UBC-2 shares striking amino acid identity to its yeast and Drosophila homologs. The amino acid similarity between UBC-2 and UBC4/5 and UbcDl are 90% and 98%, respectively. The core region of E2 proteins, an eight amino acid peptide flanking the cysteinyl residue at position 85, is identical in all four proteins. This strikingly high degree of conservation suggests that this type of E2 plays essential and fundamental roles in eukaryotic cells. 3. Functional substitution of UBC4 by ubc-2 in yeast cells (yeast complementation test) To determine whether ubc-2 encodes a protein that is functionally equivalent to yeast UBC4, C. elegans ubc-2 was introduced into yeast cells and tested for complementation of the growth deficiencies of a ubc4ubc5 mutant. A precise open reading frame (ORF) replacement of UBC4 by ubc-2 was performed as outlined in Figure 2. Two restriction sites, Spel and Ncol, had been introduced at the beginning and end of the ORF of a UBC4 genomic D N A clone by Trier et al. (1992). Spel and Ncol sites were added to the 5' and 3' ends of the ubc-2 ORF by PCR amplification of the ubc-2 cDNA with oligonucleotides OZM3 and OZM4 (See appendix for the 49 Results i 1 20 40 60 j ubc-2 C. elegans RLFLL KR IQKELQDLGREPPRQCSRGPUGDDLFHUQRT IHGPPESPVQGGUFFLT IHFPTDVPFKPPKURFTTR I I UbcDl Drosophila N D ; UBC4 Veast - S S S — f l — S S V S RD — f l S S —-K-; 80 1 00 1 20 HO ubc-2 C, elegans YHPMINSNGSICLDILRSQUSPflLTI SKULLS ICSLLCDPNPDDPLUPE I R R IVKTDRERVNQLRREUTQKVfln UbcDl Drosophila K K - - E R UBC4. Veast fl — N KD T-fl 0 PK-ERT K U > Figure 1. Protein sequence comparison A comparison of the amino acid sequences of the proteins encoded by C. elegans ubc-2, Drosophila UbcDl and yeast UBC4 . The arrowhead marks the cysteine residue which is essential for enzyme activity. Identical residues are indicated by dashes. C. elegans UBC-2 shares 85% amino acid identity with yeast UBC4, and 95% identity with Drosophila UbcDl . 50 Results oligonucleotide sequences). The chimeric gene carrying the UBC4 regulatory region and ubc-2 ORF was generated and transformed into a yeast ubc4ubc5 double mutant, in which UBC4 was disrupted by a HIS3 marker (by Ruth Heinlein). If ubc-2 can functionally replace UBC4 in yeast, the recombinant should be phenotypically similar to a ubc5 single mutant, which grows at 37°C. The recombinant was therefore selected for viability at 37°C and histidine auxotrophy. Integration of the C. elegans sequence in the genome of the recombinant was confirmed by Southern analysis (data not shown). As shown in Table 1 and Figure 3, yeast ubc4ubc5 double mutants do not grow at 37°C and display impaired growth at 30°C, as indicated by the 4 fold increase in the doubling time compared with that of the wild-type strain. A ubc5 single mutant shows normal growth at both temperatures. Driven by UBC4 promoter, the expression of C. elegans ubc-2 in the recombinant strain rescues the growth defect at 30° and 37°C. The ubc4::ubc-2ubc5 strain is phenotypically similar to the ubc5 single mutant, its growth rate being very close to that of the wild type. This indicates that ubc-2 encodes an E2 enzyme which can functionally substitute for UBC4 in yeast. The expression of C. elegans UBC-2 protein in the recombinant strain was detected by Western blot analysis (Figure 4, by Ruth Heinlein). Protein extracts from wild-type, ubc4ubc5, ubc4::ubc-2ubc5 and ubc4::ubcDlubc5 cells were prepared and reacted with a polyclonal antibody raised against Drosophila ubcDl protein (Zhen et al., 1993). This antiserum recognized not only the Drosophila ubcDl and C. elegans UBC-2 protein, but also UBC4 in the wild-type strain. In the ubc4::ubc-2ubc5 extract, a protein band of the same mobility as that of UbcDl reacts with the antibody. This indicates that this yeast strain does express UBC-2. 4. Analysis of the ubc-2 genomic sequence Subfragments from ubc-2 genomic clone pSK5.0k, the 1.4 kb EcoRI-Bglll and 1.5 Bglll fragments, were subcloned into pBluescript. Nested deletion clones were generated from each of the subclones and sequenced. The D N A sequence of part of the ubc-2 genomic clone is shown in Figure 5. The coding region of the gene consists of five exons divided by four introns. These are 51 Results C H R O M OSOM E HIS3 52 Results Figure 2. Strategy for O R F replacement of UBC4 by ubc-2 in yeast A, B: Ncol (N) and Spel (S) sites were introduced at the beginning and end, respectively, of the ORFs of the ubc-2 cDNA and UBC4 genomic DNA by site-directed mutagenesis or PCR. C, D: The chimeric gene carrying a UBC-4 control region and the ubc-2 ORF was transformed into a yeast ubc4ubc5 double mutant, in which UBC4 was disrupted by a HIS3 marker. Homologous recombinants were selected by their viability at 37°C. 53 Results Table 1. Growth fates of yeast ubc mutants and ubc-2-expressing ubc mutants Mutant Doubling time (h) Wild type... : 1.5 ubc4 2:0 ubc4ubc5 ..... 6.0 ubc4::VbcDlubc5......... ;."..•.,;.:'..„ T . . . . . . . . , 1.5 ubc4: :ubc-2ubc5 1.8 (Table provided by Ruth Heinlein and Stefan Jentsch) 54 Results Figure 3. Growth of yeast strains at different temperatures Growth on plates of yeast ubc5, a ubc4ubc5 double mutant and a UBC-2-expressing double mutant (ubc4 \\ubc-2ubc5) at normal growth temperature (30°C) and heat shock temperature (37°C). The ubc4ubc5 double mutant displays impaired growth at 30°C, and does not grow at 37°C. The expression of C. elegans UBC-2 protein in the ubc4::ubc-2ubc5 strain rescues the growth defect at both 30°C and 37 °C (figure provided by Ruth Heinlein and Stefan Jentsch). 55 Results lSkDa • ^^^^^^^^^^^^^ .a**- j^llBBl^fc-^ 14kDa Figure 4. Immunodetection of C. elegans U B C - 2 expressed in a ubc4::ubc-2ubc5 yeast strain Protein extracts were prepared from the ubc4ubc5 double mutant strain Y W 0 2 2 (lane 1), wild-type Y W O l (lane 2), ubc4::ubc-2ubc5 expressing UBC-2 (Ycuc217, lane 3) and ubc4::ubcDlubc5 expressing UbcDl (YW056, lane 4). Identical amounts of protein (10 pg/lane) were separated on an 18% polyacrylamide-SDS gel, blotted onto a PVDF membrane (Millipore) and reacted with an antiserum raised against UbcDl . The blot was developed with [125I]-protein A followed by autoradiography (figure provided by Ruth Heinlein and Stefan Jentsch). 56 Results typical short C. elegans introns, disrupting the ORF after positions 24, 246, 398, and 537. Thus as-splicing is involved in the maturation of the ubc-2 transcript. Stretches of 1300bp upstream and 800 bp downstream of the gene, respectively, were also sequenced and are shown in Figure 5. The trans-splicing signal (TTTTCAG) and the putative polyadenylation signal ( A A T A A A ) are underlined in Figure 5. In ubc-2 cDNA clones, the polyA tail was found 16 nucleotides downstream of the polyadenylation signal. 5. Genomic Southern blot analysis Genomic D N A was prepared from C. elegans embryos, and 2-3 p.g was digested with various restriction enzymes, and probed with the nick-translated 312 bp PCR fragment encoding amino acids 45 to 145 of UBC-2. The result of the Southern blot analysis is shown in Figure 6. For each restriction enzyme digest, only one or two D N A fragments hybridized with the probe. In comparing these results with a Southern blot analysis of phage clones PC2#1 and PC2#2, it was concluded that one of the two bands shown in the EcoRI digestion lane in the genomic Southern must arise from a different gene locus. The higher 5.0 kb band represents the functional ubc-2 gene, while the 1.8 kb band originates from a ubc-2 pseudogene (PC2#2, see Results, Section A . l ) . This pseudogene copy is not detected in the other lanes due to the restriction enzymes chosen to digest the genomic D N A . These results strongly suggest that ubc-2 is a single copy gene. On the other hand, when the stringency of hybridization was decreased, other bands appeared (data not shown), suggesting that there are multiple genes encoding other less closely related ubiquitin-conjugating enzymes in C. elegans, as expected. 6. The 5' flanking region of ubc-2 and the ubc-2 trans-spliced sequence Analysis of 1300 bp of ubc-2 upstream sequence reveals no consensus regulatory elements such as the T A T A box. Two putative heat shock element (HSE) sequences (-319 to -307, -249 to -236) were identified in this region. To test if they are functional regulatory elements, the 57 13 00 g a a t t c g t a t t t t a a a a a t a a c t t c a a c t c c a c a t g t g a g -1261 1260 t a g t g a t t a g g g a a t t t a g t g c t c g a t c a a t a a t t t g a a c a a c a t t t t t g c a a a a c t t a t -1201 1200 t t c t c a a a a t t a a a g t c a c t C c c g a a g t a t g a t t t g g c c t a t t t t a t a c c t c t t t g t t t t -1141 1140 c g t t g t c c c a c g t g a c t c a t a t a g c a c g a a a t a a g a a a a c a c a c t c a a g t t c a t t c g t c c -1081 1080 t a a a a t t t t a t a a a t c c g c g t t c a a a t t t c a g t c a t c t a g t a a c c a t c a g a a a a g t g c a a -1021 1020 g t t c a c t c a c g t c c a c a c t g a a t t g a a t c a g t g g a a t c a c a t g a t c a a c t g c t t c t c t c a -961 -960 c t c t c t c g c t t t g t c a t t a g a t a t c t c t a c c t c t c t g c g t t t c g t g t c t a a g t g c t t a t a -901 -900 g t t t c g c t t c t c t c c g c t t t c a a t c c c c a t t g c g a t g c g a c t g c a g c t a g t o t g c a g t g a -841 -840 g t g t g a c g g t c g t c t c a a g a t t t a t g g c g c t g c g t a t c g a t c g t t g c c t c g t a c c c t c t t - 7 81 -780 a c g t t t c c t g t g t t a t t t c t g t c t c t g t c g t t c t g a c a c t t c a g c g c t c a c c c c g t c g c t -721 -720 c a t t t t c a c t c a c a g a c a g c a c a c a t a c t c a c t c a c t c g c a g c c t a t g g c c a t t t c t t c c -661 -660 t t c c t c a e c a t c t c g t g a g g c g a c a a g t c c g g t g t c t t t a g t c g a t t t c a g a g t g a t t a t -601 -600 c t g g a a a t c c a t t t g t t t c a a t c a g a a a a t a a t a c g g t a c a t g c c g a a t a g t t t t t t a a a -541 -540 a a a c t a a c c a g n c a a t t t t g t g a t a c g t c t t g a a t t t t a a t t t t t g c c c a g t c t c g a t t c -481 -480 a t t g t g a a g a a a a t t t a c a a g a t t t g a a t c g c t t g c t t t t t g t g c g t a a t a t t c c a g t c c -421 -420 a t t t c t t t t a a g c g t t a a a a t t t t t t t c g a g t g t c a c t g g c t t g t a g c c g t a a a a a t g t g -3 61 -3 60 t c a t a t t t g t c a t c a t t t c t c a t c g t t a g t t a t t t t a t t a g g a a a t t t c c g a a a t t t a t g -3 01 -300 t t a t t t t g g t t t t c a a a t t t a t a t c t g t t c t a t t t t t c c t c a t t t t t c g c t t t c t a g a a t -241 -240 g t t c c a c t a g t t t c g a a a t g t g a c g t a a c a g t t c a c c a c g a a g g g c a a a c t g a g t a c t t t -181 -180 g t a c c g c c t c t a a a t a t c g a t t t t t c g c g c g c c g c g c c g t a c t c t t t a t g t t a t t t c a t g -121 -120 c g a a a a t c c t c a c t t t c a a t t t t t c c c t a t a g c a g a a g c t c a a t t t c a c c g t a t t t a t c g -61 - 60 a a a t t t a t E i t c ^ g a g t a c t c c g g g t g t a g c c g g g t c a a t c a g c a a a t c a g a a c a c c a a c -1 1 ATG GCT CTC AAA AGA ATC CAG AAG g t a a g g t t t c a a c a c t t a a c a a c a a t a a 52 1 M A L K R I Q K 8 53 a a t t a t t a t g t a c g t t t c a g GAA CTC CAA GAT CTC GGC CGT GAT CCA CCC 102 9 E L Q D L G R D P P 18 103 GCA CAA TGC TCC GCT GGA CCA GTT GGT GAT GAT TTG TTC CAT TGG 147 19 A Q C S A G P V G D D L F H W 33 148 CAA GCT ACG ATT ATG GGC CCA CCA GAG TCT C C C TAT CAG GGA GGT 192 3 4 Q A T I M G P P E S P Y Q G G 48 193 GTC TTC TTC CTC ACT ATC CAC TTC CCA ACA GAC TAT CCA TTC AAA 237 4 9 V F F L T I H F P T D Y P F K 63 238 CCA CCA AAG g t a t t g a t c g a a a t t g g a a a a a a a t a a a t t t a a t t t g t t t t c t t c a g 293 64 P P K 66 294 GTT GCC TTC ACC ACT CGA ATT TAT CAT CCG AAC ATC AAT TCA AAC 3 38 6 7 V A F T T R I Y H P N I N S N 81 3 3 9 GGA AGC ATC TGC C T T GAC ATT CTC C G T T C G CAG TGG T C G C C G G C T 3 8 3 82 G S I A L D I L R S Q W S P A 96 384 C T G A C C A T T T C G A A A G g t t g a t a t t a t c a t t a t t g t t c g c g t t c t a a c t t t t a a 4 3 7 97 L T I S K V 102 438 t t t t c a g T T CTG C T T TCG ATC TGC TCG CTG CTC TGT GAT CCA AAT 482 103 L L S I C S L L C D P N 114 483 CCG GAT GAT CCA CTT GTG CCA GAG ATT GCA CGC ATC TAC AAG ACG 527 115 P D D P L V P E I A R I Y K T 129 528 GAT CGT GAA AG g t g a a t t t a g a t t t t c t t t t a a c t a c t a a a a a t a a t c g a t t t t c a 583 130 D R E R 133 584 g G TAC AAT CAA TTG GCT AGA GAA TGG ACG CAA AAG TAC GCT ATG 627 134 Y N Q L A R E W T Q K Y A M 147 628 TGA g g a g g c t a a c a c c a t t c a t a t a a g a a c g c a g c c a a a c c a a t c a a t a a a c c a t g t g t 686 148 * 148 687 t t c t t t t c c c t c t c t g t g t c t c c c a t t c a t c t c c a a a c c c t t t c c a t c t t a t t c a a t c c a 746 7 47 c a c c a a a c c c c g g c g a t t c c t c t t t g t a c t a t a a a t t g t g t t c t c t t t t t a t c g a t t t t g 806 807 t t c t c c c c c a c a c a t t c t t c t c c t t c a a a a a a t a c c c a c t t t c a t a t t a t t t t c t g t c g t 866 867 c a g c a a a g c a t g t a a a a t a t a t a t g g t c t t t t a t a t t t c t t c c t t a t a a t t c a a a a g t a a 926 927 a t c g a a c g g t g t a g a t t g a t c c c c t c a a t t c t c t c t c a t c c c c t t t a a a c t a t t t t g t c t 986 987 c c a a a t c t c c c t c c t c c c t t c c t c t g a t t t t t c t c g a g a a a t t g a a a a a g g a t t t c t t c c 1046 1047 a a a c c a c c a c a c a t t c g t t c t t c g t t t a t t t c a t c t g g c g g t c t g a g a a t c c t g a a t a a a 1106 1107 t t a c t g a a g a g c g t c t t a t a t c t c g a c a t t g t t t t t c t t t c a g a a t c g a t g g c c a c g t t t g 1166 1167 t t c c c t t t g t t a c t t g t c t t g a t a c a g g t t t t t g g a a c g a a g t a g a c a a g a a a a a g c t c a 1226 1227 a t c g a t t g g a a a c t c g a t g a g a c g c c a a a g t g t a t c t c a a g t c a a t t a t t g c t t c g t a a g 1286 1287 a a t t g c c t a t a t c g t a t c a g a t g a t t t t a a t t t a a a t t c a g a t c a a a c c g a a g g t t t a a a 13 46 1347 t g t c a t c t o t c c t t a a g t t a c g a c a g t t t g a g c a g t t t g g a g a g c a c a a c t g g a t g t c a a 1406 1407 t g t c g t a c c c t t c t t c t g t a c a a t a c a a t t g a a a g t t t c a a g a t g g t t g a c a a a a g t g t t 1466 1467 t g a t a a g a a g t g a a g c a g a a a a g g t a g a a t a a c t t t c g a t a g t t a a t c a a c a t c g t c a g e 1526 1527 a a t t t c a g a t c t 1538 _ " " " " Results Figure 5. Nucleotide and deduced amino acid sequences of ubc-2 The 2837 bp ubc-2 genomic sequence is shown. Intron sequences are indicated in lower case, coding sequence in upper case. The consensus sequence of the trans-splicing signal is underlined. Two solid triangles denote the 5' end points of transcripts determined from SI nuclease protection experiments. The putative polyadenylation signal is underlined with dots, and the site of polyadenylation is shown by an arrow. The active site cysteine is starred. 59 Results 1 2 3 4 5 6 7 8 k b - 2 3 . 0 - 9 . 4 - 6 . 5 -4 .3 -2 .3 -2 .0 - 0 . 5 6 Figure 6. Genomic Southern blot analysis. Genomic D N A (2-3 (ig) prepared from C. elegans embryos was digested with Xbal, Xhol, Pstl, Apal or BamHl (lanes 1-5, respectively), Bglll plus EcoKI (lane 6), BglTl (lane 7) or EcoRl (lane 8), and separated by a 0.7% agarose gel. The hybridization probe was a nick-translated 312 bp PCR fragment encoding amino acids 45-145 of ubc-2. Only one or two D N A bands are visible in each lane. The positions of lambda Hindlll size standards are shown on the right in kb. 60 Results expression levels of ubc-2 transcript and protein before and after heat-shock treatment were compared (See Section 1.1.2 below). These studies suggest that these HSEs are not functional. Many C. elegans gene transcripts are known to undergo trans splicing, a process in which a short splice leader from a distinct small nuclear R N A is attached to the 5' end of the mRNA precursor. The position for attachment corresponds to a 3' consensus trans-splicing signal. Comparison of the ubc-2 genomic and cDNA sequences reveals that part of an SL1 splice leader sequence (5' A A G T T T G A G 3') is present at the 5' end of the cDNA. A consensus splicing signal (TTTTCAG) is found at the relevant position in the genomic D N A (Figure 5). This indicates that the ubc-2 transcript undergoes trans splicing. 7. Localization of the ubc-2 gene The genomic phage clone PC2#1 was mapped using a fingerprinting method (Coulson, et al, 1986) by Alan Coulson (MRC, Cambridge, England). PC2#1 phage D N A was digested with HindlTI, and the ends of the fragments were labeled with 3 5 S-dATP by reverse transcriptase. The fragments were then further digested with Sau3A, and separated on a polyacrylamide gel. The pattern revealed by autoradiographic exposure was compared with that of a series of cosmids covering the C. elegans genome. Overlapping cosmids containing ubc-2 were identified, and the gene was mapped to the right arm of chromosome IV between lin-3 and unc-22 (Figure 7). 8. SI nuclease protection analysis S1 nuclease protection experiments were performed in an attempt to localize the site of transcription initiation of ubc-2. The ssDNA probe complements D N A sequence from nucleotide -28 to -1,300. Total cellular R N A was prepared from embryos growing at normal temperature. Two main protected probe fragments were seen, corresponding to SI cleavage at nucleotides -49 and -47 of the gene, adjacent to the 3' end of the splicing sequence (Figure 8, lane 2). The two bands are likely due to the identity in sequence (with one mismatch) between the 3'-most 6 nucleotides of SL1 and the 3' splicing signal. Thus the cleavage at nucleotide -49 may represent 61 Results £ oo T oo g 3 C I • i—l T 3 CN i I 3 oo J3 S O CN CN CN Cli T3 c 3 CN "3 0 1 Map units CHROMOSOME IV (PART OF THE RIGHT ARM) Figure 7. Mapping of the ubc-2 gene The ubc-2 gene (PC2#1 clone) was mapped to the right arm of chromosome IV by fingerprinting, as part of the C. elegans physical mapping project (Coulson et al, 1986). The flanking markers are also shown in this figure. Each map unit covers about 150 kb D N A sequence. 62 Results protection of the 3' portion of the SL1 sequence joined to the ubc-2 3' splicing site. The start site of the transcript could not be determined, probably due to the extremely low levels of unspliced ubc-2 heterogeneous nuclear R N A present. Since previous studies have shown that elevated temperature may block the process of trans splicing (Graham et al., 1988), another SI nuclease protection analysis was performed using R N A prepared from embryos heat-shocked at 34°C for two hours. The results were the same as with non-heat-shocked R N A (Figure 8, lane 2 and lane 3) suggesting that trans splicing occurs very rapidly and the heat-shock treatment is insufficient to block the process, hence unspliced mRNA is not detectable. SECTION B. STUDIES ON THE TEMPORAL AND SPATIAL EXPRESSION PATTERNS OF UBC-2 IN C. ELEGANS 1. Northern blotting analysis 1.1. Developmental analysis of ubc-2 expression Total cellular R N A prepared from various life stages of C. elegans was separated on a 1% denaturing agarose gel. To avoid detecting other possible E2 transcripts, a fragment from the 3' untranslated region of ubc-2 was used as the probe. A transcript close to the predicted size, 1.2 kb, was detected at all life stages (Figure 9). As a control, the same blot was stripped and reprobed with the C. elegans actinl gene, which is constitutively expressed throughout the nematode life cycle (Krause and Hirsh, 1986). No significant differences in the levels of the ubc-2 transcripts were observed at different stages. This suggests that constitutive expression of ubc-2 occurs at all life stages of C. elegans. 63 Results 1 2 probe — (1,310 bp) " P i C (-49) G (-47) Figure 8. SI nuclease protection analysis Total cellular R N A prepared from C. elegans embryos was hybridized with a 3 2P-labeled ssDNA probe (complementary to nucleotides -1300 to +10, lane 1), digested by SI nuclease, and subjected to 8% polyacrylamide electrophoresis. Lane 2 shows the protected fragments using R N A prepared from embryos at normal temperature. In Lane 3, total cellular R N A was prepared from heat shocked embryos. The two main protected fragments represent SI cleavage sites at -49 (C) and -47 (G). 64 Results 1 2 3 4 5 6 7 k b 1 2 3 4 5 6 7 9.5 -7.5 -4 .4 -2 .4 -1.4 -- 0 . 2 4 -UbC-2 act in 1 Figure 9. Northern blot analysis of ubc-2 during development Total cellular R N A (5-20 pg) was prepared from the major stages of development: egg, adult, L I , L2, L3, L4 and dauer larva (lanes 1 to 7 respectively), separated by electrophoresis on a 1% formaldehyde-agarose gel, and probed with a 423 bp cDNA fragment specific to ubc-2 (left panel) or to the actin gene (right panel). The amount of RNA loaded in Lane 3 and 7 was less than that in the other lanes. 65 Results 1.2. ubc-2 expression after heat-shock. The expression of ubc-2 after heat-shock was investigated since the yeast homologs UBC4 and UBC5 are heat-inducible. Total cellular R N A was prepared from C. elegans mixed cultures and from cultures of selected stages, following either maintenance at normal temperature or 33°C for 2 h. After Northern blot analysis with'the same ubc-2 probe as used in 1.1, the blot was stripped and re-probed with.an actinl gene probe, as a control for equal R N A loading. No significant change in the level of the ubc-2 transcripts was seen after heat-shock (Figure 10). The >: same blot was stripped and re-hybridized with a probe from an hsp-16 gene, a strictly inducible heat shock gene. This showed the expected high levels of induction in the heat shock R N A sample, while no signal was seen with the RNA from the unshocked animals at any stage (data not shown). 2. Temporal and spatial expression patterns of ubc-2/lacZ transgenes 2.1. Construction of ubc-2/lacZ translational fusions .» To study the temporal and spatial expression patterns of ubc-2, in-frame translational fusions of the lacZ coding region with ubc-2 were constructed. A l l ubc-2/lacZ translational fusion constructs are illustrated in'Figure 11. CI is an in-frame translational fusion of the lacZ coding region with the second exon of ubc-2, and contains 1.4 kb of ubc-2 5' non-coding sequence, extending to codon 12 (Asp) of the coding region. A polyadenylation signal is contained within the 0.6 kb 3' non-coding region. In constructsi pZMI.l,pZMI.2, pZMII.l and pZMII.2, the lacZ coding region was inserted in the second exon of the ubc-2 genomic clone. These constructs all contain the complete ubc-2 coding sequence, including five exons and four introns, but differ in the extent of their 5' and 3' non-coding sequences, as described in Materials and Methods and Figure 11. A nuclear localization signal (NLS) from SV40 was included in a l l constructs at the beginning of the lacZ coding sequence. . 66 Results 1 2 3 4 5 6 7 8 k b - 7.5 -- 4 . 4 -- 2 . 4 -- 1 . 4 -0 . 2 4 -1 2 3 4 5 6 7 8 ubc-2 act in / Figure 10. Northern blot analysis: the expression of ubc-2 after heat-shock Total cellular RNA (5-20 ug) was prepared from control and heat-shocked C. elegans in different developmental stages: mixed culture (lanes 1 and 2), egg (lanes 3 and 4), L I (lanes 5 and 6) and L2 (lanes 7 and 8). Lanes 1, 3, 5 and 7 are from control cultures; lanes 2, 4, 6 and 8 are from heat-shocked cultures (33°C, 2 hours). Electrophoresis and hybridization probes were as in Figure 9. The blot was stripped and exposed by X-ray film for three days to ensure there was no residual activity left from the ubc-2 probe. It was then hybridized with a probe specific to hspl6-2 to confirm the effectiveness of the heat-shock for samples 2, 4, 6 and 8 (data not shown). 67 Results ubc-2 CI pZMI.l pZMI.2 1400bp Bgii i Apai coding region Bglll Apal — AATAAA-260Cbp 260Cbp pZMII.l pZMII.2 Bglll Apal Bglll Apal 600bp Figure 11. Construction of ubc-2/lacZ transgenes With the exception of CI, all constructs consist of the complete ubc-2 coding region with varying 5' and 3' noncoding sequences, and the E. coli lacZ gene inserted in-frame into the second exon of ubc-2. CI includes 5' and 3' noncoding sequences of ubc-2, but only the first 12 residues of the coding region, as well as the first intron. Shaded rectangles represent ubc-2 exons, while clear rectangles indicate introns. NLS is the SV40 nuclear localization signal. 68 Results 2.2. Establishment of transgenic lines and integrated lines carrying ubc-2/lacZ fusions Each construct illustrated in Figure 11 was injected as a circular plasmid, either alone or together with a rol-6(sul006) selection marker plasmid pRF4 into the C. elegans gonad. Transgenic lines carrying mixed extrachromosomal arrays of the rol-6 plasmid and each construct were identified by their clockwise rolling phenotype. Two integrated lines carrying pZMI.l and pRF4 were generated from the relevant extrachromosomal transgenic line by y radiation (See Materials and Methods). 2.3. Constitutive expression of pZMI.l and pZMII.l throughout the C. elegans life cycle Transgenic lines carrying pZMI.l or pZMII.l extrachromosomal arrays were fixed, permeabilized and stained with X-gal. With both constructs, (3-galactosidase was found to be expressed in embryos from gastrulation onward, and in larvae and adult stages (Figure 12). X-gal staining was very intense, suggesting that ubc-2 expression is driven by a relatively strong promoter. Staining of embryos and L I larvae was visible within 2 h, and was saturated in 5 h. Staining in L4 larvae and young adults was first visible after 3 h, while older adults began to show staining after 4-5 h. Blue precipitate was localized in nuclei as expected, due to the NLS included in the constructs. 2.4. Spatial expression patterns of pZMI.l and pZMII.l Expression of the ubc-2/lacZ fusion protein from pZMI.l and pZMII.l was consistently observed in embryos, larvae and adults, p-galactosidase was expressed in most cells in the embryos. In L I , L2 and L3 larvae, most somatic tissues were intensely stained, including neurons, pharynx, hypodermis and body muscle. In a small percentage of animals, intestinal cells were also stained. Examples of some cell types expressing P-galactosidase are shown in Figure 13. At the onset of the L4 stage, however, staining became more restricted to neurons, pharynx and hypodermis. In adults, P-galactosidase staining was seen only in the ventral nerve cord, pharyngeal ganglia, retrovesicular ganglia and a few hypodermal cells in the head and tail. These 69 Results Figure 12. lacZ expression from the p Z M I . l and pZMI.2 transgenes in different stages of C. elegans Transgenic nematodes were dried on glass slides, fixed by cold acetone, and stained with the X-gal staining solution as described in Materials and methods. Staining was usually carried out at 37°C for 4 hours, (a), staining in comma stage embryo. Note X-gal staining throughout the embryo. Magnification 500x. (b),(c),(d), staining in L I , L2 and L3 larvae respectively. Blue precipitate is seen in body muscle, hypodermal, neural and some intestinal nuclei. Magnification 500x. (e), adult animal showing specific expression in the nervous system and in a few hypodermal cells in the head and tail, and strong expression in embryos. Magnification lOOx. 70 Results Results Figure 13. lacZ expression of p Z M I . l and pZMI.2 in different cell types The expression of the pZMI. l and pZMI.2 transgenes was observed in many cell types, including body muscle (bm), hypodermis (hyp), nerve (nr), P-lineage cells (pi), vulval muscle (vu). Worms shown in A, B and D were in late larval stages (L3-L4), while the ones in C and D were adults. Magnificaton 100X for A and D, 500X for B , C and E. 72 . Results results suggest that the expression of ubc-2 is tissue general in embryos and early larvae but is largely restricted to the nervous system in adults. _ 2.5. \ Expression ofpZMI.l andpZMII.l in the nervous system Despite differing tissue specificities in larvae and adults, the expression of fi-galaetosidase from pZMI.l and II. 1 was seen constitutively in the nervous system at all postembryonic stages. From L I onward, intense staining was observed in the ventral nerve cord, including P-cells and neurons, pharyngeal ganglia and retrovesicular ganglia (Figure 14). The expression level of P-galactosidase in the nervous system was also the highest among all the tissues. This was evident by the fact that during X-gal staining, blue precipitate showed up first in the pharyngeal ganglia. The constitutive and high level expression from pZMI.l and pZMII.l transgenes in the nervous system suggests that ubc-2 plays important roles in neuronal function or nervous-system development, 2.6. Important regulatory elements for transgene expression The p-galactosidase expression levels of the five constructs described above were compared to localize potential transcriptional regulatory elements. CI failed to produce any P-galactosidase, pZMI.2 and II.2 were only weakly expressed in nerve ganglia around the pharynx (data not shown), while pZMI.l and pZMII.l showed similar, strong and most tissue-general expression. This suggests that 5' regulatory elements are included within the 1.4 kb.sequence. Both the coding region and distal 3' non-coding sequences (within 3 kb) of the gene are also necessary for maximal expression of the transgene. : 73 Results Figure 14. lacZ expression of p Z M I . l and pZMI.2 in the nervous system X-gal staining was carried out at 37°C for 4 h. Magnification 98x (a, b and d), 166X (c, e). (a) L4 larva carrying pZMI. l or pZMI.2. Staining is seen in the ventral nerve cord (vc), pharyngeal ganglia (shown as nerve ring (nr) in this figure), and retrovesicular ganglia (rg). A few hypodermal cells in the head and tail are also stained, (b)-(e) X-gal staining in adults carrying pZMI.l or pZMI.2. The same staining pattern as in (a) can be observed. 74 Results 3. In situ immunofluorescent staining with polyclonal antibodies against ubc-2 protein 3.1. Over-expression of UBC-2 in E. coli and purification of the protein To determine if the expression patterns of the ubc-2/lacZ transgenes resembled those of endogenous ubc-2, rabbit polyclonal antibodies were raised against UBC-2, and the localization of the protein was examined by in situ immunofluorescent staining. To over-express UBC-2 in E. coli, a 450 bp BamHI-HindUl fragment containing the ubc-2 open reading frame was inserted into pRSETC (Invitrogen). This generated the reading frame for a fusion protein consisting of a 4 kDa amino-terminal sequence containing six histidyl residues followed by the 16.7 kDa UBC-2 protein. This plasmid was transformed into E. coli BL21 (k DE3 pLysS) and the 21 kd fusion protein was expressed by induction with 1-2 m M IPTG. Since 40% of the expressed fusion protein in E. coli cells was soluble, it was purified in its native form on a N i -NTA resin column (Materials and Methods). After this one-step purification, the fusion protein migrated as a single band on SDS-polyacrylamide gels stained with Coomassie blue (Figure 15). Twenty mg of the protein was obtained from a 4 L £ coli culture. 3.2. In vitro ubiquitin conjugating activity of the UBC-2 fusion protein The recombinant UBC-2 fusion protein was assayed for its activity as a ubiquitin-conjugating enzyme (E2) in vitro. This assay detects thiol ester bond formation between an E2 and ubiquitin, in an El-dependent reaction. U B A 1 , an E l protein from wheat, was over-expressed in E.coli (provided by P. M . Hatfield, University of Wisconsin-Madison). The E.coli extract was used in the assays as a source of E l . As shown in Figure 16, UBC-2- 1 2 5 I-Ubiquitin conjugates were detected only when p-mercaptoethanol was not included in the SDS-PAGE sample loading buffer. This strongly suggests that UBC-2 forms a thiol-ester bond with ubiquitin. The formation of UBC-2-ubiquitin-thiol ester adducts was also E l and A T P dependent, 75 Results u b c - 2 f u s i o n p r o t e i n Figure 15. Expression and purification of the 6xHis-UBC-2 fusion protein in E. coli. E. Coli BL21 was grown to an A^oo of 0.7-0.9, and the expression of 6xHis-UBC-2 fusion protein was induced by 2mM IPTG. The fusion protein was purified by nickel resin affinity chromatography (Qiagen). Lane 1, protein molecular weight marker; lanes 2 to 4, 6xHis-UBC-2 fusion protein after nickel resin affinity chromatography. A band of the expected size was observed by Coomassive blue staining. The total E.coli protein extract before and after IPTG induction are shown in lane 5 and lane 6, respectively. 76 Results as expected. These results demonstrate that recombinant UBC-2 protein is active in at least the first step of the conjugation reaction, El-dependent ubiquitin thioester formation. 3.3. Raising antibodies against UBC-2 fusion protein and affinity-purification of the antiserum UBC-2 fusion protein was denatured with 0.1% SDS and used to immunize New Zealand White rabbits (0.4 mg/rabbit). After boosting at two week intervals with the same antigen for up to eight months, the rabbits began to show weak reaction to the fusion protein. This slow response is probably due to the extremely highly conserved nature of this E2 among animals. To increase the titer and purity of the antibody, rabbit antiserum was affinity purified using UBC-2 fusion protein affixed to an Affi-gel 10 column (Bio-Rad). E C L western blotting analysis using this purified antibody showed that it recognizes C. elegans UBC-2 (Figure 17). 3.4. In situ immunofluorescent staining of C. elegans with antibody against UBC-2 protein The localization of UBC-2 in C. elegans was examined by in situ immunofluorescent staining. As shown in Figure 18, UBC-2 antibody specifically reacts with neuronal cells, especially the ventral nerve cord in adults. This is consistent with the localization of lacZ activity seen with ubc-2/lacZ transgenes. Unlike the X-gal staining with transgenic strains, however, the antibody staining was seen not only in the ventral nerve cord itself (Figure 18a), but also in the neuronal processes connecting the ventral and dorsal cord (Figure 18b). This difference is likely due to the fact that a NLS, which is included in each ubc-2/lacZ fusion construct, restricts the protein expression to nuclei only. The ventral cord contains many nucleated cell bodies, but these are largely absent from the dorsal cord and neuronal processes. Therefore, both transgene expression analysis and in situ immunofluorescent staining indicate that ubc-2 is highly expressed in the adult nervous system of C. elegans. 11 Results Figure 16. In vitro ubiquitin thiol ester assay The activity of the 6xHis-UBC-2 fusion was assayed for its ability to form a thiol ester bond with ubiquitin in an El-dependent reaction. Reactions in Lanes 1 and 2 included a mixture of E l , 1 2 5I-ubiquitin, UBC-2 protein, and ATP in the appropriate buffer (see Materials and methods). Reactions were incubated at 30°C for 10 minutes, and terminated by boiling for 10 minutes in SDS-PAGE sample buffer (lane 1), or in the sample buffer without DTT or (3-mercaptoethanol (lane 2). In Lanes 3-5, individual components (E l , UBC-2 or ATP, respectively) were omitted from the reaction, and samples were boiled in SDS-PAGE sample buffer without (5-mercaptoethanol. Lane 6 was loaded with 1 2 5I-ubiquitin only. No ubiquitin-UBC-2 conjugates were formed in the latter reactions. 78 Results 1 2 43.3 k d -28.3 k d -18.1 kd -15.4 kd -Figure 17. E C L Western blotting analysis of U B C - 2 Extracts were prepared from asynchronous cultures and 20ug protein was loaded in each lane. Lane 1, extract from worms without heat-shock treatment. Lane 2, extract from worms after a 2 h heat-shock at 33°C. Polyclonal antibodies against UBC-2 were used at 1:1,500 dilution. The antibodies bound to the membrane were detected by the E C L Western blotting analysis system (Amersham). 79 Results Figure 18. In situ immunofluorescent staining of adults with polyclonal antibodies against UBC -2 N2 wild-type C. elegans was fixed using 4% paraformaldehyde, stained with anti-UBC-2 primary antibodies (1:500 dilution) and visualized with FITC-conjugated goat anti-rabbit secondary antibodies at 1:1,000 dilution (Sigma). Antibody staining is seen in the ventral nerve cord (vc), and also in neuronal processes (np) connecting the ventral and dorsal cords. Magnification 630x. 80 Results 4. Expression of UBC-2 after heat-shock and cadmium treatment 4.1. Western blot analysis on extracts from heat-shocked worms UBC4 and UBC5, the yeast homologs of ubc-2, play important roles in the stress response and their expression levels increase dramatically following a heat-shock or cadmium exposure. Although ubc-2 complements the growth defects of a yeast ubc4ubc5 null mutant, its transcript level was found to be unaltered by heat-shock in the nematode (Figure 10). With the availability of UBC-2 antibody, UBC-2 protein levels were examined in extracts prepared from worms before and after heat-shock, using Western blot analysis (Figure 17). After a 2 h heat-shock at 33°C, the level of UBC-2 protein detected was similar to that seen in the control worms. 4.2. Expression of ubc-2/lacZ transgene after heat-shock and cadmium treatment The amount of (3-galactosidase expressed from the ubc-2 promoter in transgenic animals before and after heat-shock or cadmium treatment was quantified by ONPG assays and compared (Figure 19). Two lines carrying the integrated construct pZMI.l, referred to as pZMI.llnl and pZMI.l!a2, were used for ONPG assays. Figure 19a clearly shows that after a heat-shock at 33°C for 2 h, the activity of f3-galactosidase remained similar to that of non heat-shocked animals. Similarly, following cadmium treatments at a range of 10-200 u M , the expression level of p1-galactosidase remained virtually unchanged (Figure 19b). 81 Results I i i E3 pZMI.Hnl M pZMI.Hn2 CdCl2 (uM) A420 pZMI.Hnl pZMI.Hn2 l l l l l ^ 25°C 33°C 25°C 33°C Figure 19. (3-galactosidase activity in p Z M I . H n l and pZMI.Hn2 animals exposed to various concentrations of C d C h and different temperatures Approximately 1,000-1,500 worms of mixed stages were treated with acetone and whole animals were used for the assays. Left panel: Both nematode lines were exposed to 0-200uM CdCl2 for 24 h, and the {3-galactosidase activity was determined by ONPG assays. Right panel: pZMI. l In l and In2 animals were heat-shocked at 33°C for 2 h. The P-galactosidase activity was assayed and compared to the level in worms without heat-shock treatment (25°C). 82 Results SECTION C. STUDIES ON THE FUNCTIONS OF UBC-2 IN C. ELEGANS 1. Inhibition of ubc-2 function by expression of antisense R N A 1.1. Preparation of ubc-2 antisense constructs ANTI-I and ANTI-II and the sense constructs hsp-ubc-2 and ubc-2 (SK) Two antisense R N A constructs, ANTI-I and ANTI-II and two sense constructs, hsp-UBC-2 and UBC-2(SK) were created as described in Materials and Methods. The structures of these constructs are shown in Figure 20. ANTI-I contains an inversion of the entire ubc-2 cDNA under the control of the hspl6-2 promoter, which drives expression in most somatic tissues at temperatures higher than 29°C. On the other hand, hsp-ubc-2 should express sense ubc-2 transcript at temperatures higher than 29°C. ANTI-II contains the complete ubc-2 genomic sequence with a large part (nucleotides 162-1018) of the coding region inverted. Expression of antisense R N A with ANTI-II is driven by the ubc-2 promoter. 1.2. ANTI-II-induced C. elegans early developmental lethality When ANTI-II was injected into the C. elegans gonad at concentrations of 40 to 200 ng/pJ, along with pRF4 marker plasmid, no transformants were obtained. Injection with the sense construct, ubc-2 (SK), however, gave a normal transformation efficiency (20-30 rollers per successful injection). This strongly suggests that ANTI-II, which expresses ubc-2 antisense R N A under the control of its own promoter, causes early developmental lethality. 1.3. Establishment of ANTI-I integrated lines and hsp-ubc-2 extrachromosomal lines ANTI-I transformants were obtained at the expected frequency of about 20 to 30 rollers per successful injection, and several extrachromosomal lines were established at 25°C. From one of these lines, several integrated lines were derived as described in Materials and Methods. A l l 83 Results X ^-Transcription P (hsp l6 -2 hsp-ubc-2 ANTI-I Apal 1,400bp ^ Xhol | 2,600bp 3' ubc-2(SK) ANTI-II Figure 20. Construction of the sense constructs hsp-UBC-2 , UBC-2(SK), and antisense constructs A N T I - I and ANTI-II (a) A 1.0 kb Kpnl-Sacl fragment containing the complete ubc-2 c D N A sequence was inserted into pPD 16.49 in two orientations, resulting in hsp-UBC-2 and ANTI-I. The hspl6-2 promoter in these constructs drives the expression of sense and antisense R N A under heat-shock conditions, (b) ANTI-II was directly derived from a UBC-2(SK) genomic clone. The Apal-Xhol fragment was blunt-ended and inverted. Shaded regions represent exons, while clear regions indicate introns. 84 Results strains were maintained at 25°C. As a control, the sense construct, hsp-ubc-2, was also injected. Stable lines carrying extrachromosomal arrays of hsp-ubc-2 and rol-6 were established. 1.4. Effects, of hsp-ubc-2 and ANTI-I expression on ubc-2 transcript and protein levels To test if ANTI-I and hsp-ubc-2 constructs drove the expression of antisense ubc-2 R N A and sense ubc-2 RNA, respectively, and whether the expressed antisense R N A affected the UBC-2 protein level in C. elegans, Northern blot and Western blot analyses were performed. C. elegans lines carrying either an integrated ANTI-I construct or a non-integrated hsp-ubc-2 construct were subjected to a 30°C heat shock for 2 h. Two hundred worms from each line were picked and total cellular R N A was extracted as described in Materials and Methods. Northern blot analysis using a ssDNA probe specific to the sense ubc-2 transcript (Figure 21) showed that the line carrying hsp-ubc-2 has an increased level of sense ubc-2 transcript after heat shock, suggesting that the hspl6 promoter induced the expression of transcript upon heat shock. The result of Northern blot for the ANTI-I integrated line, however, was inconclusive. The UBC-2 protein level in the ANTI-I integrated line was therefore.tested before and after heat shock by Western blotting (Figure 22). It is clear that the expression of antisense ubc-2 R N A by ANTI-I leads to a decrease in UBC-2 protein levels. 1.5. Developmental effects of ANTI-I expression ANTI-I integrated lines were subjected to a 30°C heat shock for 2 h to induce antisense R N A expression at various life stages. The effect of the expressed antisense R N A on nematode development was investigated and the results are shown in Table 2. When ubc-2 expression was inhibited in the embryonic or early larval stages (LI, L2 and early L3), 85% of the animals died within one day and the remaining 15% developed into adults within 4 days at 25 °C. When worms of the parental strain were subjected to the same heat-shock treatment, nearly 100% of them developed into adults within 3 days. When antisense R N A expression was induced in later larval stages (late L3 and L4), almost all survived and developed 85 Results 1 2 1 2 Figure 21. Northern blot analysis of strains carrying hsp-ubc-2 before and after heat-shock treatment Total cellular R N A was prepared from 200 worms carrying hsp-ubc-2 both before and after heat shock treatment, separated on a 1% formaldehyde-agarose gel, and subjected to Northern blot analysis. Lane 1 (in both panels), R N A prepared from worms before heat shock; Lane 2 (both panels) , R N A prepared after heat shock, (a) a M13 ssDNA probe specific to ubc-2 sense transcripts was hybridized with the blot, (b) To check the R N A loading in each lane, the same blot was stripped and probed with an actinl specific probe. The results show that heat-shock induces the expression of sense ubc-2 transcript in the strain carrying the sense construct, hsp-ubc-2. The heat shock was for 2 h at 30°C. 86 Results Figure 22. Western blot analysis of strains carrying A N T I - I before and after heat-shock treatment Protein lysate was prepared from worms carrying the ANTI-I construct before and after heat-shock treatment (2 h, 30°C) and separated by electrophoresis on a 12.5% SDS-polyacrylamide gel. E C L Western blotting was carried out using UBC-2 antibodies. Lanel, extract from worms after heat-shock; lane 2, extract from worms before heat-shock. The total amount of protein loaded in both lanes was similar as determined by A28O reading and Coomassie blue staining. 87 Results into adults, although their developmental time was lengthened by two days relative to identically treated control worms. Expression of the antisense R N A in adults produced no apparent effect. It is worth noticing, however, that the progeny number of these adults was decreased to 20% of that of the control worms. 2. Attempts to isolate a ubc-2 null mutant using T e l transposition To examine the function of ubc-2 on C. elegans development, attempts were made to isolate a ubc-2 null mutant by the method of Te l transposon insertion and imprecise excision. Using PCR screening and sibling selection, Dr. Yoshiki Andachi of the National Institute of Genetics, Japan, isolated YK10, a strain which has a Tel transposon inserted in the second intron of ubc-2. This strain was obtained from Dr. Andachi, and the insertion of the Te l transposon was confirmed by single worm PCR (Figure 23). A D N A fragment of the correct size was amplified with oligonucleotides OZM11 (corresponding to part of the ubc-2 sequence) and O Z M l 2 (complementary to part of the Tel sequence) from YK10, but not from the wild-type strain. Since the transposon insertion is in an intron, ubc-2 gene function is not disrupted in YK10. To isolate a ubc-2 null mutant, PCR was used to screen for worms carrying deletions in ubc-2 induced by Tel imprecise excision, starting from a pool of YK10 animals. Since C. elegans is a diploid, it was expected that a mutation could be detected either in a homozygous or heterozygous state. In several attempts, putative positive signals from the primary screening were lost after one or two rounds of sibling selection, suggesting they were just somatic excision. So far no ubc-2 mutant strain has been isolated by this method. This may be due to the fact that Te l excision happens at a high frequency in somatic cells, but is very rare in the germ line, combined with a low frequency of Tel transposition at the ubc-2 locus. The latter is inferred by the difficulty in isolating the original Tel insertion in this gene (Y. Andachi, personal communication). 88 Results Table 2. The effect of ubc-2 antisense R N A expression on the development of C. elegans Stage of rol-6 null rol-6 null carrying integrated heat shock ANTI-I Embryo, L I and 100% survival and 85% showed growth arrest and L2 larva development into adults died after heat-shock, 15% within 2 days survived and developed into adults within 4 days L3 and L4 100% survival and 95% survived and larva development developed into adults in 3 into adults within 1 day days Adult No detectable effect No detectable effect rol-6 null and integrated ANTl-Vrol-6 null strains were subjected to a 2 h 30°C heat-shock at various life stages. The proportion of animals surviving and developing to adults was scored in each case. 89 Results coding region O Z M l l ubc-2 1400bp OZMl 2 YK10(mut-2(r459), dpy-19(n!347), ubc-2(mslO::Tcl)) 90 Results Figure 23. Strain YK10 carries a Tel insertion in ubc-2 (A) The position of the Tel insertion in the ubc-2 locus in YK10 strains. The regions covered by two oligonucleotides, OZM11 and OZM12 which were used to confirm the insertion are also shown. The shaded boxes indicate the exons of the open reading frame, while the clear ones represent the introns. (B) PCR amplification of DNA from YK10 and N2 (wild-type) strains with O Z M 11 and OZM12. Lane 1, PCR control reaction with dri^O as template; lanes 2 and 3, DNA from ^(wild-type) and YK10 worm pools, respectively; lane 4, the product of a single YK10 worm PCR reaction. Only the DNA from YK10 worms can be amplified with oligonucleotides OZM11 and O Z M 12. 91 Results 3. let-70, a C. elegans lethal mutant was rescued by ubc-2 Since the antisense R N A studies described here suggested that mutations in ubc-2 may be lethal in early developmental stages, another attempt to isolate ubc-2 mutant strains by rescuing lethal mutations located near the ubc-2 locus on the genetic map was carried out. Approximately ten EMS-generated lethal mutations have been mapped very close to the position of ubc-2 by Dr. David Baillie's laboratory at Simon Fraser University. To investigate the possibility that one of these is within ubc-2, two transgenic lines carrying a ubc-2 and pRF4 mixed extrachromosomal array were established as described by Mello (1991). The extrachromosomal array from these strains was crossed into the various lethal mutant strains by Jacquie Schein, Simon Fraser University. One of the mutants, let-70 (Rogalski et al, 1985; Clark et al, 1988; Clark and Baillie, 1992), was successfully rescued by the extrachromosomal array. This result strongly suggested that let-70 contains a mutation in the ubc-2 gene. 4. Both alleles of let-70 contain single point mutations in the ubc-2 locus To confirm that let-70 mutants are altered in ubc-2, genomic D N A was prepared from single worms homozygous for let-70 mutations and the ubc-2 coding region was PCR-amplified with oligonucleotides OZM3 and OZM4. Homozygous let-70 worms were identified by their twitching phenotype, caused by the genetic marker (unc-22) on the mutant chromosome. The amplified PCR product was digested with BaraHI and Spel restriction enzymes and cloned into the corresponding sites in pBluescriptSK. The insert D N A was sequenced with oligonucleotides T3, T7, OZM3, OZM4, Mei20-2 and Mei20-4. A single point mutation in the ubc-2 coding region was discovered in each let-70 mutant allele. Strain BC1903 which carries the let-70(sll32) allele contains a C to T transition at nucleotide 317 of ubc-2, resulting in substitution of histidine75 for a tyrosine (Fig.24a). His75 is conserved in all ubiquitin-conjugating enzymes sequenced to date, with the exception of yeast UBC6. The other strain, BC2020, which carries the let-70(s689) allele, contains a G to A change at the splice donor site of the last intron of ubc-2 (Fig.24b). The splice site mutation could result in a mutant UBC-2 protein either lacking the last exon, or 92 Results containing an altered and extended C-terminal sequence, depending upon whether or not the potential cryptic splice sites are utilized. To eliminate the possibility that the single base pair changes seen were due to PCR error, Pfu polymerase (NEB), which possesses 3' proof reading ability was used for PCR amplification instead of Taq polymerase. This should increase the amplification accuracy by 15 times. More than ten worms from each let-70 allele were individually tested by PCR and sequencing. The same mutations were found in the ubc-2 locus in each animal. 5. C. elegans let-70(ubc-2) mutants display an early to mid-larval lethal phenotype Both let-70(ubc-2) alleles cause developmental arrest in larval stages. The si 132 allele shows an earlier blockage (LI to L2 stage) than the s689 allele (late L2 to early L3). This suggests that the mutation in si 132 leads to a more severe defect in the normal protein functions. Microscopic examination showed that tissue development before the L3 stage in the homozygous let-70 worms (both alleles) was relatively normal until developmental arrest occurred. Detailed analysis of the phenotype of the mutant worms hasn't been carried out yet. 6. Mutant UBC-2 proteins encoded by let-70(ubc-2) alleles are highly unstable Protein extract was prepared from the heterozygous let-70(ubc-2) adults (both alleles), and immediately used for Western blot analysis with antibodies against UBC-2 protein (Figure 25). Proteins representing wild-type UBC-2 were detected with the antibody, along with several other lower molecular weight polypeptides. When an equal amount of the protein extract was prepared from wild-type (N2) nematodes using the same method and used for Western blotting, only the wild-type UBC-2 was detected. This suggests that the lower molecular weight protein bands seen in the heterozygous let-70 worms are the degradation products of the mutant UBC-2 proteins. This may also imply that the mutant UBC-2 proteins encoded by both let-70 alleles are highly unstable in cells. 93 Results Results 1 ATG GCT CTC AAA AGA ATC CAG AAG g t a a g g t t t c a a c a c t t a a c a a c a a t a a 52 1 M A L K R I Q K 8 53 a a t t a t t a t g t a c g t t t c a g GAA CTC CAA GAT CTC GGC CGT GAT CCA CCC 102 9 E L Q D L G R D P P 18 103 GCA CAA TGC TCC GCT GGA CCA GTT GGT GAT GAT TTG TTC CAT TGG 147 19 A Q • C S A G P V G D D L F H W 33 148 CAA GCT ACG ATT ATG GGC CCA CCA GAG TCT CCC TAT CAG GGA GGT 192 34 Q A T I M G P P E S P Y Q G G 48 193 GTC TTC TTC CTC ACT ATC CAC TTC CCA ACA GAC TAT CCA TTC AAA 237 49 V F F L T I H F P T D Y P F K 63 238 CCA CCA AAG g t a t t g a t c g a a a t t g g a a a a a a a t a a a t t t a a t t t g t t t t c t t c a g 293 64 P P K 66 sll32 T (Y) t 294 GTT GCC TTC ACC ACT CGA ATT TAT CAT CCG AAC ATC AAT TCA AAC 338 67 V A F T T R I Y H P N I N S N 81 339 GGA AGC ATC TGC CTT GAC ATT CTC CGT TCG CAG TGG TCG CCG GCT 383 82 G S I C L D I L R S Q W S P A 96 384 CTG ACC ATT TCG AAA G g t t g a t a t t a t c a t t a t t g t t c g c g t t c t a a c t t t t a a 437 97 L T I S K V 102 438 t t t t c a g TT CTG CTT TCG ATC TGC TCG CTG CTC TGT GAT CCA AAT 482 103 L L S I C S L L C D P N 114 483 CCG GAT GAT CCA CTT GTG CCA GAG ATT GCA CGC ATC TAC AAG ACG 527 115 P D D P L V P E I A R I Y K T 129 s689 a X 528 GAT CGT GAA AG g t g a a t t t a g a t t t t c t t t t a a c t a c t a a a a a t a a t c g a t t t t c a 583 130 D R E R 133 584 g G TAC AAT CAA TTG GCT AGA GAA TGG ACG CAA AAG TAC GCT ATG 627 134 Y N Q L A R E W T Q K Y A M 147 628 TGA g g a g g c t a a c a c c a t t c a t a t a a g a a c g c a g c c a a a c c a a t c a a t a a a c c a t g t g t 686 148 * 148 (C) Figure 24. Point mutations in ubc-2 genes of the let-70 mutants Single homozygous wild type, let-70(sll32) and let-70(s689) nematodes were lysed and the ubc-2 gene coding region was PCR-amplified with oligonucleotides OZM3 and OZM4. Sequencing of the PCR products show that there is a C to T shift (marked by arrows and starts) in si 132 allele (Panel a), and a G to A change (marked by arrows and starts) in s689 (Panel b). Panel (c) shows the positions of the mutations in ubc-2 gene and the amino acid change caused by the mutation in si 132. 95 Results N2 wild-type let-70(sll32) heteozygous let-70(s689) heterozygous 18 kd 14 kd _ubc-2 (wild-type protein) IS-* Degradation %/~*~ products Figure 25. Western blot analysis of UBC-2 proteins in wild-type and heterozygous let-70 C. elegans Protein extract was made from wild-type and heterozygous let-70 C. elegans cultures by boiling in 5xSDS-sample buffer. 20 pg protein from each strain was loaded on a 15% SDS-PAGE gel immediately after the extract was made. Protein transfer and Western blot analysis was carried out as described in Materials and Methods. 96 Discussion IV. DISCUSSION /. UBC-2 ENCODES A UBIQUITIN-CONJUGATING ENZYME WHICH IS CONSERVED AMONG EUKARYOTES , . ' Comparisons of the predicted protein sequence encoded by ubc-2 with its homologs in yeast, UBC4 and UBC5, and Drosophila ubcDl suggest that it encodes a ubiquitin-conjugating enzyme that is highly conserved among eukaryotes. It shares over 85% amino acid identity with UBC4 and UBC5, and more than 95% identity with UbcDl. If conservative substitutions are. included, the similarity with UBC4/5 and UbcDl increases to 90 and 98%, respectively. This strong evolutionary conservatism suggests that the enzymes encoded by these genes play similar and fundamental roles in eukaryotic cells. The above suggestion is supported by the ability of both C. elegans ubc-2 and Drosophila UbcDl to complement the growth defects of a yeast ubc4ubc5 double mutant. When expressed from the UBC4 promoter, UBC-2 protein rescues the growth defect of a ubc4ubc5 double mutant at normal temperature, as well as the viability of this mutant at elevated temperatures. The doubling time of a ubc4::ubc-2ubc5 strain is identical to that of a w£c5 single mutant, and very close to that of wild-type cells. UBC4 and UBC5 function in the ubiquitin-dependent degradation of both abnormal and short-lived proteins, by mediating the formation of high molecular weight ubiquitin-protein conjugates. Most such conjugates disappear from ubc4ubc5 mutants and are restored in cells expressing Drosophila UbcDl . Given the high degree of sequence conservation, it is very likely that UBC-2 also functions in the elimination of abnormal proteins and in the degradation of short-lived proteins in C. elegans. The UBC-2 protein was over-expressed in E.coli and tested for its ability to form a thiol ester bond with ubiquitin in vitro. The results of this assay demonstrated that the formation of UBC-2-ubiquitin-thiol ester adducts is ATP- and E l - dependent, which is characteristic of ubiquitin-conjugating enzymes. 97 Discussion 2. HYPOTHETICAL STRUCTURE OF UBC-2-A CLASS I UBIQUITIN-CONJUGATING ENZYME E2 enzymes are grouped into four distinct classes. Class I enzymes are the smallest E2s and contain only core domain sequences (about 120-150 amino acids) in which the ubiquitin-accepting cysteine is located. Class II and Class III enzymes contain extra extensions from the core domain, at the C-terminal and N-terminal, respectively. Class IV enzymes have both N -terminal and C-terminal extensions. UBC-2 belongs to Class I ubiquitin-conjugating enzymes, based on its amino acid sequence. Cook et al. (1993) reported that the tertiary structures of Class I ubiquitin-conjugating enzymes are highly conserved. The structures of two E2 proteins, yeast UBC4 and Arabidopsis thaliana Ubcl , are superimposable eventhough they only share 40% amino acid identity (Cook et al, 1992; Cook et al, 1993). This leads to a strong possibility that the overall folding of the UBC-2 molecule is very similar to that of UBC4. Generally speaking, Class I E2 is an ct/p protein with four a-helices and one four-stranded antiparallel P-sheet (Figure 26). The ubiquitin-accepting cysteine is located in a cleft between two loops in the long stretch between the fourth strand of the P-sheet and the second a-helix. Most of the conserved amino acids in ClassI E2 proteins are either buried inside the molecule or clustered on one surface that lies adjacent to the ubiquitin-accepting cysteine. It was predicted that this conserved surface region provides a binding site for E l or ubiquitin during the formation of the E2-ubiquitin adduct, while variable sequences may form the non-conserved surfaces which might be utilized in binding proteins targeted for ubiquitination. 3. BOTH TRANS AND CIS SPLICING ARE INVOLVED IN THE MATURATION OF THE UBC-2 TRANSCRIPT Sequence analysis of ubc-2 shows that this gene is made up of five exons divided by four introns. Thus cis splicing is involved in the maturation of the ubc-2 transcript. The presence of a consensus splicing signal (TTTTCAG) at the 5' end of the gene suggests that ubc-2 transcripts also 98 Figure 25. Tertiary structure of a Class I ubiuitin -conjugating enzyme, yeast UBC4 protein Ribbon drawing of the yeast UBC4 molecule cc-helices are shown in red, /?-strand are shown in blue, and the remainder of the molecule is in yellow. The ubiquitin accepting cysteine (Cys 86) is shown in green (Cook et al. 1993). 99 Discussion undergo trans splicing. Sequencing of ubc-2 cDNA confirmed the presence of SL1 at the 5' end of the transcript. Therefore both trans and cis splicing are involved in the maturation of ubc-2 mRNA. Trans splicing is a very rapid process, making it difficult to determine the start site of transcription for such genes. SI nuclease protection experiments detected only the probe fragments protected by trans spliced transcript. Graham et al. (1988) found that heat-shock treatment can slow the splicing process, and lead to an elevated level of unspliced heterogeneous nuclear RNA. S1 nuclease mapping was therefore performed with R N A prepared from C. elegans embryos after a 2 h heat-shock treatment at 33°C. The results were the same as those obtained using the non-heat-shocked R N A sample. Thus the heat-shock treatment did not result in the accumulation of sufficient un-processed ubc-2 transcript to allow mapping of the start site. The success of Graham et al. with this approach is likely due to the fact that the gene they were studying, the polyubiquitin gene, is transcribed at a much higher level than ubc-2. 4. UBC-2 IS CONSTITUTIVELY EXPRESSED IN ALL LIFE STAGES Both Northern blot analysis and transgene assays indicate that ubc-2 is expressed in all life stages. Since all E2 enzymes share 30% protein sequence similarity, it was important to use a probe which would be specific to the ubc-2 transcript in the Northern blot analysis. A 600 bp c D N A fragment from the 3' transcribed, but untranslated region was chosen as the probe for Northern analysis. A transcript of the expected size (around 1.1 kb) was detected in all stages. Compared to control actin transcript levels, it is apparent that ubc-2 is transcribed at all life stages, and that the abundance of ubc-2 transcripts is similar at all stages. Analysis of transgenic worms carrying the ubc-2/lacZ fusions pZMI.l and pZMII.l shows that fi-galactosidase is expressed in embryos from gastrulation onward, and in larvae and adults. Thus the ubc-2 promoter drives protein expression throughout the C. elegans life cycle. This 100 Discussion result and the fact that ubc-2 expression is turned on early in embryos suggests that this gene may encode important functions for growth and development. 5. UBC-2/LACZ TRANSGENE ASSAYS SUGGEST THAT UBC-2 EXPRESSION HAS DEVELOPMENTAL TISSUE SPECIFICITY Although ubc-2 is expressed in all life stages, the spatial pattern of expression undergoes alteration during development as seen in transgenic animals carrying ubc-2/lacZ fusions. While |3-galactosidase expression in these strains is quite tissue-general in larvae, it is restricted in later stages. In post-gastrulation embryos, almost every cell stains with X-gal. Most somatic tissues stain in early larval stages, including muscle, hypodermis, nerve and intestine. In L3 and L4 larvae (3-galactosidase expression was seldom seen in intestinal cells. In adults, (3-galactosidase expression could be found only in the nervous system, including the ventral nerve cord and nerve ganglia, and in a few hypodermal cells in the head and tail. The distribution of p-galactosidase activity in these transgenic animals seems to indicate a progressive restriction of expression with developmental stage. This phenomenon raises interesting questions regarding ubc-2 function. It seems likely that ubc-2 is essential and functions in a tissue general manner in early development. Since UBC-2 is highly conserved relative to its yeast homologues UBC4 and UBC5, it is possible that this enzyme is involved in the degradation of abnormal proteins and short-lived proteins, including key regulatory proteins for embryonic and early larval development in C. elegans. In later developmental stages, restricted distribution of the gene product suggests either that its functions are no longer required in some tissues or that they are taken over by other E2s. 6. UBC-2 IS PROMINENTLY EXPRESSED IN THE NERVOUS SYSTEM IN ALL STAGES, ESPECIALLY ADULTS A striking aspect of ubc-2 gene expression is its prominence in the nervous system. First indicated by ubc-2/lacZ transgene assays, the constitutive expression of ubc-2 in the nervous system in C. elegans was then confirmed by in situ localization of the endogenous UBC-2 protein. 101 Discussion In transgenic nematodes carrying ubc-2/lacZ fusion constructs p%galactosidase was expressed in the pharyngeal ganglia, ventral nerve cord (including P-cells and neuronal cells), and retrovesicular ganglia at every developmental stage. Rabbit polyclonal antibodies raised against UBC-2 protein were found to react specifically with neuronal cells in adults, interestingly, the antibody staining was observed not only in the ventral cord, but also in nerve bundles connecting the ventral and dorsal cords. The reason that ubc-2 expression in neuronal processes could be detected with antibody, but not via ubc-2/lacZ transgene expression is likely due to the fact that the NLS from SV40 was included in all ubc-2/lacZ fusion constructs. This signal would restrict the expressed (3-galactosidase to nuclei only. The high expression level of ubc-2 in the nervous system suggests a role for this gene in neuronal function. The involvement of the ubiquitin conjugating system in neural tissues has been noted in several other systems (see Introduction). For example, in humans, the cerebral soluble ubiquitin content in patients with Alzheimer's disease is significantly higher than that of normal individuals (Tadder et al., 1993). In patients with Alzheimer's disease, Pick disease and Parkinson's disease, neurons contain inclusions composed of abnormal filaments together with ubiquitin (Gallo and Anderton, 1989). In cultured rat D R G neurons, stress results in increased formation of ubiquitin conjugates (Morandi etal., 1989). More recently, bendless, a Drosophila mutation affecting the connectivity between the giant fiber and the tergOtrochanter motor neuron, was shown to encode an E2 homolog (Charles et al., 1994). Although the biological functions of ubiquitin and E2s in the nervous system have not been elucidated in detail, a reasonable hypothesis would be that the ubiquitin system targets specific proteins in neuronal cells for degradation or protein modification, thus affecting the development of the nervous system and neuronal activity. The expression pattern of ubc-2 suggests that it may be involved in both the development of the nervous system and neuronal activity. In the embryonic and early larval stages, ubc-2 may be required for general tissue development, as indicated by its tissue-general expression pattern in embryos and early larvae. However in the later stages, when most somatic tissues, including the nervous system, have completed their development, ubc-2 expression is largely restricted to the 102 Discussion nervous system. This suggests that at this stage ubc-2 may be involved in the maintenance of neuronal activity. The prominent UBC-2 protein expression seen in neuronal processes may provide some clues to its function and potential substrates. A recent paper oh G a o , the a subunit of the heterotrimeric guanosine triphosphate-binding protein Go, shows that this protein has an almost identical expression pattern as that of UBC^2 in C. elegans adults, and that alteration of Goto activity affects nematode behavior. Reduction-of-function mutations in goa-1, the gene encoding G a o protein in C. elegans, caused hyperactive movement. The expression of the activated form of this protein, on the other hand, resulted in lethargic movment (Mendel, 1995). Considering that the yeast G a subunit was shown to be degraded (at least partially) by a RAD6-mediated,N-end rule pathway (Madura and Varshavsky, 1994), it would be very interesting to determine whether the level of Gao is controlled by the ubiquitin system, especially by aubc-2-mediated pathway, in C. elegans. 7. ANALYSIS OF THE UBC-2 PROMOTER REGION Recent studies reveal that the transcriptional regulation of C. elegans genes is more complex than expected. Many genes contain enhancers downstream of the coding region, an example being the myosin heavy chain gene unc-54 (Okkema et al, 1993). Enhancers can also be present within the introns and coding regions (Stone and Shaw, 1993). The discovery of polycistronic genes and genes whose transcripts undergo alternative trans-splicing demonstrates that some genes can be regulated by upstream DNA sequence many kilobases away (Blumenthal, 1995). To ensure that the staining patterns of ubc-2/lacZ fusions reflect the localization of endogenous UBC-2 protein, five constructs containing different D N A regions of ubc-2 were prepared, and their spatial and temporal expression patterns were compared. Among the five ubc-2/lacZ fusions created, pZMI.l and pZMII.l were found to have the most tissue-general and the highest level of P-galactosidase expression in C. elegans. Since the only difference between these two constructs is that pZMI.i includes 6 kb of upstream sequence, while pZMII.l contains only 1.4 kb, it seems that all the 5' regulatory elements required are 103 Discussion located in the 1.4 kb region. With construct CI, in which only the 1.4 kb upstream sequences and the polyadenylation signal were present, no expression was seen in transgenic animals. This indicates that other D N A regions required for maximum expression lie within the coding region and/or the 3 kb of downstream sequences. The addition of the complete coding region, including four introns and five exons in constructs pZMI.2 and pZMII.2, only partially restored the expression, as indicated by the low expression level and the tissue-restricted staining pattern. Even in larvae, staining was observed only in nerve ganglia around the pharynx. Only when additional downstream 3' non-coding sequences were included in the translational fusion was maximum expression observed. This suggests that important regulatory elements, perhaps strong enhancers or sequences conferring message stability, exist in the 3 kb 3' non-coding region. ubc-2 is not likely to be a member of a polycistronic gene grouping. This was first suggested by the fact that the ubc-2 transcript undergoes splicing to SL1 rather than to SL2, since in most cases, only SL2 isfrarcs-spliced to the 5'-end of the transcripts from a polycistronic gene. The lack of polyadenylation signals in the 5' upstream sequence of ubc-2 is also consistent with this conclusion. Comparison of the expression patterns of pZMI.l and pZMII.l corroborates the suggestion that ubc-2 expression is not under the regulation of remote 5' upstream D N A sequences. 8. UBC-2 IS NOT INDUCED BY HEAT OR CADMIUM Although ubc-2 is both structurally and functionally very similar to yeast homologs UBC4 and UBC5, it is regulated differently in response to heat-shock treatment or cadmium exposure. While UBC4 and UBC5 transcript levels show a more than 50% increase when yeast cells are treated at 38°C for 30 minutes, and UBC5 transcription is strongly induced after lOOuM cadmium treatment for 30 minutes, Northern blot analysis, Western blot analysis and ONPG assays (See Results) showed that ubc-2 transcript and protein levels were unaltered by either heat-shock or cadmium treatment. Along with the fact that the expression of ubiquitin is heat-inducible in yeast, but constitutive in C. elegans (Graham, 1992), these results indicate that at least some components 104 Discussion of the ubiquitin system are regulated in different ways in yeast and C. elegans. Since the heat and cadmium inducibility of ubiquitin and UBC4/5 type E2s has been reported so far only in yeast, this pattern of regulation may be the exception rather than the rule among species. It is well known that heat-shock and cadmium treatment cause protein damage in living cells. If C. elegans does not increase the level of UBC-2 in response to stress, there are several alternatives it might utilize to deal with the increased protein damage. The simplest explanation is, of course, that the basal level of ubc-2 expression is sufficient to tag the damaged proteins, even after heat-shock or cadmium treatment. This possibility is supported by the observed intense UBC-2 antibody staining and high level expression of (3-galactosidase driven by the ubc-2 promoter. Alternatively, there may exist other E2 genes in C. elegans that are either stress-inducible or which specifically target damaged proteins induced by stress. In this regard, ubc-7, a C. elegans homolog of yeast UBC7, a gene conferring cadmium resistance, has recently been discovered and is currently being characterized. A yeast ubc7 null mutant shows no growth defect under normal conditions, but dies when exposed to 30uM cadmium. This demonstrates that UBC7 is specifically involved in a cadmium resistance pathway. It is possible that in C. elegans, ubc-7 plays a role in cadmium resistance. 9. EXPRESSION OF UBC-2 ANTISENSE RNA CAUSES EARLY EMBRYONIC AND LARVAL STAGE LETHALITY AND RETARDED DEVELOPMENT To investigate the role of ubc-2 in C. elegans development, several attempts were made to isolate a C. elegans ubc-2 null mutant strain. This was based on Te l insertion mutagenesis followed by imprecise Te l excision, an approach developed by Plasterk et al. (Plasterk and Groenen, 1992). This method has been used successfully in several cases. However, a collaboration with Ronald Plasterk to isolate a Tcl-insertion mutation in ubc-2 was unsuccessful. Of the loci studied to date, ubc-2 proved to be one of the regions rare for Tel targeting. When a Te l insertion into ubc-2 was finally isolated, several attempts to derive a deletion from it were unsuccessful, suggesting that excision was also a very low frequency event at this locus. 105 Discussion Following these unsuccessful attempts, an alternative approach, expressing antisense R N A , was used to inactivate ubc-2 function. The results confirmed the hypothesis that this gene plays essential roles in early development. No transformants were obtained with ANTI-II, a construct which expresses ubc-2 antisense R N A under control of the ubc-2 promoter, suggesting that expression of ubc-2 antisense R N A during early development is lethal. ANTI-I, a construct which expresses antisense R N A only at higher temperature, was designed to allow investigation of the effect of the antisense transcript on worms of various stages. To eliminate non-specific interaction between the antisense R N A and transcripts of genes other than ubc-2, a ubc-2 cDNA without the polyA tract was inverted and fused to an hspl6 promoter. Introduction of ANTI-I was also lethal in embryos and early stage larvae. However, when expression was induced in L3 and L4, it caused only delayed development. Little effect was observed when antisense R N A was induced in adult animals. How might one explain the differences in the effect of antisense R N A at different stages? One hypothesis is that ubc-2 function is essential at all stages, but the protein is sufficiently stable that a transient block in synthesis at later stages has a less severe effect due to the presence of UBC-2 accumulated earlier. A second possibility is that in later stages, the function of the gene is restricted to certain cell types and tissues, and may not be as 'essential' as it was in the early stages. Finally, other functionally-redundant E2 enzymes might be expressed in later stages, thus reducing the effect of antisense RNA in these stages. 10. UBC-2 CORRESPONDS TO A GENE ESSENTIAL FOR EARLY DEVELOPMENT, LET-70, IN C. ELEGANS The let-70 mutation was first generated by EMS treatment and genetically mapped in Dr. David Baillie's laboratory at Simon Fraser University (Rogalski et al., 1985; Clark et al., 1988; Clark and Baillie, 1992). Only known for being essential for larval viability, the product of this gene was unidentified. The fact that both alleles of let-70 were rescued by the ubc-2 gene, and that point mutations in the ubc-2 gene were identified in both let-70 alleles proved that ubc-2 106 Discussion corresponds to let-70. The requirement of UBC-2 protein for early development in C. elegans corroborates the results of previous antisense studies. The fact that ubc-2 encodes a ubiquitin-conjugating enzyme suggests that the lethality of let-70(ubc-2) mutations is probably due to the failure of targeting and/or removing specific cellular proteins. Such targets may include regulatory proteins such as transcription factors and cell cycle regulators. In support of this hypothesis, a human E2 which mediates the partial degradation of the tumor suppressor protein p53 has recently been cloned and shown to be closely related to yeast UBC4/5 and C. elegans ubc-2 (Scheffner et al., 199 A). 11. HIS75 AND THE C-TERMINAL (AMINO ACIDS 134-147) OF UBC-2 PROTEIN ARE IMPORTANT FOR THE PROTEIN FUNCTIONS Lethality of the homozygous let-70(ubc-2) alleles suggests that both mutations abolish, at least partially, the functions of UBC-2 protein. This is the first time that His75 and the C-terminal sequence of a type I E2 have been implicated in the function of these enzymes. Conformational changes caused by these mutations could lead to a decrease or complete loss of the protein function. Based on the tertiary structure of the yeast UBC4 protein, which should be very similar to that of C. elegans UBC-2 (See Discussion 2, Figure 26), the imidazole nitrogen of His75 forms a hydrogen bond with the carbonyl oxygen of Leu 109 (2.48A between the two atoms). When this His is replaced by Tyr, the bulky aromatic side chain would reduce the distance to 0.7A, necessitating a conformational change to accomodate the large side chain. The negatively charged hydroxyl group also contributes to the repelling force. Since Leu 109 is located in the relatively rigid structure (the second a-helix) and Tyr75 is in a flexible loop, it is likely that the Tyr75 would be pushed away. This change will lead to a conformational alteration of the Cys85-containing active core surface, which is located on the same loop. Thus it is quite possible that the SI 132 mutation results in a defect in transfer of the activated ubiquitin from the E l protein. For the S689 protein, the structural changes will depend on whether other cryptic splice sites are utilized. If not, the mutated splice site would be read through and the fourth helix would be deleted. Judging from 107 Discussion the tertiary structure of UBC4, this helix seems to be a relatively independent structure from the rest of the molecule. Loss of the C-terminal E2 sequence should not have much effect on the transfer of ubiquitin from E l . However this C-terminal domain might be important for interactions with other proteins, such as another E2 molecule, an E3 or even substrates. So it is likely that the S689 mutant protein is defective in transferring the activated ubiquitin to substrates. If a cryptic splice site is utilized in S689, the reading frame of the C-terminal of the molecule wil l be altered and the secondary structure of the C-terminal of the protein will be changed. How much this change will affect the overall structure of the protein will depend on the amino acid sequence and is difficult to predict at this time. The attempt to detect potential truncated UBC-2 protein from s689 worms was unsuccessful since Western blot analysis suggests that both mutant proteins are unstable (Figure 26). This instability of the mutant UBC-2 proteins from both alleles may also contribute to the loss of their function in cells. 12. UBC-2-TYPE E2S IN MULTICELLULAR ORGANISMS MAY BE INDISPENSABLE AND MEDIATE SPECIALIZED FUNCTIONS IN DIFFERENT DEVELOPMENTAL STAGES OR TISSUES Since yeast has two functionally redundant ubc-2 type of E2s, UBC4 and UBC5, and deletion of either gene alone shows no apparent phenotype, it has always been commonly assumed that multicellular organisms would also possess several genes with a similar function, and that individual genes of this group would be dispensable. However the studies presented here show, for the first time, that in multicellular organisms this type of E2 may have developed different functions and be regulated differently. The fact that the expression of ubc-2 is not induced by heat-shock or cadmium suggests that it is specialized to target protein degradation under normal conditions. The tissue-specific expression pattern of ubc-2 in adults and the larval lethality caused by mutations in this gene strongly indicate that, this type of E2 may have become specialized in different developmental stages or tissues, and thereby indispensable. 108 Discussion 13. OTHER UBIQUITIN-CONJUGATING ENZYMES IN C. ELEGANS So far ten ubiquitin-conjugating enzymes have been found in yeast. Multiple E2s have also been reported in other eukaryotic systems such as Drosophila, Arabidopsis, wheat, rabbit, and human. It is reasonable to predict that C. elegans also has multiple E2 family members: A n E2 which is homologous to yeast UBC2/RAD6 has recently been cloned and characterized from this nematode (Leggett et al., 1995). Besides these two genes, the ongoing C. elegans genome sequencing project being carried out by the GenOme Sequencing Center at the Washington University School of Medicine and the Sanger Center in Cambridge, U K , and the c D N A sequencing project by the Gene Library Lab at the National Institute of Genetics, Japan have revealed 7 genomic and cDNA clones encoding putative E2 members. It seems likely that more genes encoding' E2s will be isolated. The diversity of E2 genes suggests that the ubiquitin system is involved in many aspects of cell regulation. It will be particularly interesting to look at the regulation of these E2s in a multicellular organism such as C. elegans. 14. CONCLUSIONS 1) The ubc-2 gene, which encodes a Class Iubiquitin-conjugating enzyme in C. elegans, has been cloned. The UBC-2 protein shares extremely high amino acid sequence similarity to its yeast and Drosophila homologs, and complements the growth defects of a yeast ubc4ubc5 null mutant. This suggests that this type of E2 is conserved among eukaryotes, and plays fundamental roles in cells. -2) The expression of ubc-2 is observed in somatic tissues from the gastrulation stage onwards, by transgene assays. It is constitutively expressed in all stages, but shows interesting developmental specificity in the post embryonic stages. While the expression is tissue general in embryos and early larvae, it becomes more restricted in later developmental stages, being expressed mainly m the nervous system in adults. 3) Unlike that in yeast, heat shock had no effect on the expression level of M £ C - 2 transcript and protein in C. elegans. Heat shock and cadmium treatment did not alter the intensity or 109 Discussion distribution of the p-galactosidase staining pattern in ubc-2AacZ transgenic animals. Thus ubc-2 is neither heat nor cadmium inducible. 4) ubc-2 is essential for early development. Blocking the expression of the gene with antisense R N A and the early to middle larval stage lethality of let-70(ubc-2) mutants clearly show that this gene is required for C. elegans early stage development. Conclusions 2 to 4 above suggest that in multicellular organisms the i75C4/5-type of E2s may have become specialized in their functions in different developmental stages, or different tissues under normal conditions. 5) The expression of ubc-2 is under the regulation of multiple elements, which are included in 1.5 kb of upstream sequence, the coding region, and 2.3 kb of downstream sequence. The existence of an enhancer in the downstream sequence is postulated, since removing this region abolished transgene expression in most somatic tissues, and greatly decreased the expression level • of the reporter gene. 15. FUTURE PROSPECTS This work has focused on analyzing the expression and function of ubc-2 in the development of C. elegans. Future studies of ubc-2 could follow several interesting and fruitful directions: 1) Identification of the mutations in the two mutant alleles of ubc-2 provides very powerful genetic 'handles' to carry out functional analysis of the gene in C. elegans. For example, suppressor screening will help to identify proteins interacting with UBC-2, which may include ubiquitin pathway components and substrate proteins. The larval stage lethality of the let-70 mutants suggests that, both alleles, especially si 132 may represent the null state of the gene. Thus temperature sensitive (ts) alleles of the gene could be constructed in vivo and introduced into the let-70(ubc-2) homozygous strain by microinjection. It has been found that point mutations (Pro71—»Ser or Gly58->Arg) in CDC34 will generate ts mutant enzymes (Ellison et al., 1991; Prendergast et al., 1995). If the same mutations in ubc-2 also make ts alleles, then these ubc-2 ts mutants could be used to screen for suppressors, which could lead to the identification of proteins 110 Discussion playing roles in the ubiquitin pathway and targeted substrates for degradation in different life stages. 2) Biochemical studies of the mutant UBC-2 proteins will test the effect of the mutations on the protein functions. Both mutant proteins could be overexpressed and purified from E.coli, and in vitro ubiquitination assays could be carried out to see if they still retain the ability to accept activated ubiquitin from E l enzyme and to transfer the ubiquitin to substrates. Ubiquitination assays supplemented with C. elegans extract using either wild type UBC-2 protein or the mutated versions of the protein could identify the proteins subjected to UBC-2-mediated proteolysis. A proteasome inhibitor, lactacystin, was recently reported to specifically block the ubiquitin pathway in cells (Fenteany et al, 1995). Including this inhibitor in ubiquitination assays using C. elegans extract could also help to identify substrates of the protein degradation pathway. 3) More alleles of ubc-2 gene could be isolated with the same method used to isolate let-70. This would help to identify important functional domains of the protein. If different alleles led to developmental arrest in different stages, this; could provide insight into the different functions this gene may have in different life stages. 4) Promoter analysis of ubc-2 has identified multiple elements required for proper expression of the gene. Further dissection of these regions would be desirable to identify these sequences. Comparisons with other promoters which drive expression in specific tissues, especially in the nervous system, might identify useful tissue-specific enhancers. 5) The expression pattern of ubc-2 has provided some information on potential substrates targeted by this enzyme in C. elegans. The specific expression of the protein in the adult nervous system strongly suggests that this protein plays roles in neuronal activity, perhaps by regulating key proteins in nerve transmission pathways. Of special interest in this regard is Gao, a subunit of the G proteins which functions in signal transduction for hormones, neurotransmitters, and other cellular regulators. The biological functions of the Gao protein remain to be elucidated. It has recently been reported that reduction-of-function mutations of goa-1, which encodes the Gao subunit in C. elegans, cause hyperactive movement, while overexpression of the subunit leads to 111 Discussion lethargic movement (Segalat et al, 1995; Mendel, et al, 1995). An interesting feature of this gene, is that like ubc-2, it is also abundantly expressed in most neurons in the nematode. Since a yeast Goto protein has been reported to be at least partially degraded by a ubiquitin-dependent pathway, it would be worthwhile investigating whether the ubiquitin pathway, especially a ubc-2-mediated ubiquitin pathway, is involved in the regulation of Gcto protein levels, thus regulating aspects of nematode behavior. Immunological tools such as Western blotting and immunoprecipitation could be applied to detect the ubiquitination of Gao and the interaction of the protein with components of the ubiquitin pathways. Biochemical and genetic experiments such as blocking proteasome function with specific inhibitors (Fenteany et al, 1995), generating strains that overexpress components of the ubiquitin pathway or carry mutant alleles of the genes encoding the components, and studying the development and behavior of the strains wil l also help elucidate the roles of the ubiquitin pathway. 6) Identifying the expression patterns of other E2 genes The C. elegans genome and cDNA sequencing projects have revealed at least five genomic and cDNA clones encoding proteins homologous to ubiquitin-conjugating enzymes. One of these shows highest homology to yeast UBC7, which plays roles in a cadmium-resistance pathway. C. elegans ubc-7/lacZ transgene assays suggest that this gene has a distinct expression pattern compared to ubc-2 (M. Zhen and D. Leggett, unpublished). It is abundantly expressed in muscle and hypodermis, and very limited expression was found in the nervous system. 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C,T)GTCCA(C,T)TC 3' MEI20-2 5' G G T T G C C T T C A C C A C T C G A A T 3' MEI20-4 5' G G T G A G A G C C G G C G A C C A C T G 3' OZMl 5' G A C C C T Q C A Q C T A T G G C T C T C A A A A G A A T C C A G 3' ' P s t l ' : OZM2, 5' G C C A A G ^ I T T T A G C C T C C T C A C A T A G C G T A 3' Hindlll OZM3 5' G A G C J A T C C A rGGCTCTC A A A A G A A T C C A G A A G 3' BamlU Ncol . - , OZM4 5' A G T A C T ^ G T l T A G C C T C C T C A C A T A G C G T A 3' Spel OZMl . 5' G G C T A C A C C C G G A G T A C T 3' OZM11 5' G A C C C T C C A G A T T G C A C G C A r C T A C A A G A C G 3' OZM12 5' G C C A A G C T T G C T G A T C G A C T C G A T G C C A C G T C G 3' //mdlll SUMMARY OF E.COLI, YEAST AND C. ELEGANS STRAINS AND THEIR GENOTYPES E.-coii. strains BB4 hsdR514 supE44 supF58 galK2 galT22 trpR55 metBl tonA MacU169 FlproAB + lacN lacZAMIS TnlO(tetr)] Appendix recAl endAl gyrA96 thi-1 hsdR.17 supE44 relAl AlacU169 (<p80 lacZAM15) hsdS gal (Xclts857 indl Sam7 nin5 lacUV5-T7 genel) supE44 supF58 hsdR514 galK2 galT22 metBl trpR55 lacYl supE44 hsdR17 recAl endAl gyrA46 thi relAl lac F' [proAB + lacN lacZAMIS TnlO(tef)\ wild type (haploid MA 7a strain congenic to DF5) ubc4::HIS3, ubc5::LEU2 (haploid MATa strain congenic to DF5) ubc4::ubc-2, ubc5::LEU2 (haploid MATa strain congenic to DF5) ubc4::UbcDl, ubc5::LEU2 (haploid MATa strain congenic to DF5) 2. C. elegans strains (obtained from other laboratories) YK10 mut-2(r459), dpy-19(nl347), ubc-2(mslO::Tcl) BC1903 let-70(s689), unc-22(s7)MTl(IV); +/nTl (V) BC2020 let-70(sll32), unc-22(s7), unc-31(el69)/nTl (IV); +MT1 (V) ro/-6~null rol-6(nl27(el87)) 3. Transgenic and integrated strains produced in the present work Strain Genotype Selection Construct PC81 ubEx75 pRF4 pZMI.l PC82 ubEx76 pRF4 pZMI.l PC83 ubEx77 pRF4 pZMI.l PC84 ubEx78 pRF4 pZMII.l PC85 ubEx79 pRF4 pZMI.l DH5ot BL21(DE3) LE392 X L 1-Blue 2. Yeast strains YWOl YW022 Ycuc217 YWQ56 128 Appendix PC86 ubEx80 pRF4 CI PC87 ubEx81 pRF4 pZMI.l PC88 ubEx82 pRF4 pZMII.l PC89 ubEx83 pRF4 pZMI.l PC90 ubEx84 pRF4 CI PC95 ubln7 pRF4 pZMI.l PC96 ubln8 pRF4 pZMI.l PC97 ubln9 pRF4 pZMI.l PC98 ublnlO pRF4 pZMI.l PC99 ubEx89 pRF4 ANTI-I PC 100 ubEx90 pRF4 ANTI-I PC101 ubEx91 pRF4 ANTI-I PC 102 ubEx92 pRF4 ANTI-I PCI 11 ublnl4 pRF4 ANTI-I PCI 12 ublnl5 pRF4 ANTI-I PC 115 ubExlOO pRF4 ubc-2(SK) PC116 ubExlOl pRF4 ubc-2(SK) PC 123 ubExl08 pRF4 hspl6-ubc-2 PC 125 ubExl09 pRF4 hspl6-ubc-2 PC, laboratory strain designation; ub, laboratory allele designation; Ex, extrachromosomal; In, integrated. 129 

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