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A new role for the tumour suppressor LIN-35 during meiotic recombination in Caenorhabditis elegans Lohn, Zoe Roy 2010

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A NEW ROLE FOR THE TUMOUR SUPPRESSOR LIN-35 DURING MEIOTIC RECOMBINATION IN CAENORHABDITIS ELEGANS by Zoe Roy Lohn B.Sc., Queen’s University, 2008  THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Medical Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2010  © Zoe Roy Lohn, 2010  
  ABSTRACT Meiosis is a fundamental biological process used by sexually reproducing species to ensure the faithful transmission of genetic material and to generate genetic diversity. In humans, failure to recombine properly during meiosis causes genetic conditions in the human conceptus such as aneuploidy and spontaneous abortion. An excellent model organism for the investigation of meiotic recombination is the nematode, Caenorhabditis elegans, which has many conserved meiotic processes. In this thesis, I have investigated the role of lin-35 in meiotic crossing over. LIN-35 is the C. elegans ortholog of the retinoblastoma (Rb) protein, well characterized with respect to its role in gene transcription and cell proliferation. My results show that mutation in the lin-35 gene alters recombination frequency differentially for several regions of the chromosome, causing increases in recombinationally suppressed regions and decreases in highly recombinogenic regions. In combination with Rec-1, a mutant known to alter crossover distribution, crossovers across the length of the entire chromosome, were decreased. In addition, other severely detrimental phenotypes were observed. For example, gametic viability was reduced dramatically in the double mutant, compared to either mutant alone. Thus, the Lin-35 and Rec-1 phenotypes were synergistic, indicating non-redundancy. In summary, lin-35 function plays a role in achieving normal levels of meiotic recombination, a role that may be related to its function in chromatin modification and gene transcription.  
  ii  TABLE OF CONTENTS ABSTRACT ...........................................................................................................................ii
 TABLE OF CONTENTS ........................................................................................................ iii
 LIST OF TABLES ..................................................................................................................v
 LIST OF FIGURES ................................................................................................................vi
 LIST OF ABBREVIATIONS ..................................................................................................vii
 ACKNOWLEDGEMENTS ................................................................................................... viii
 CHAPTER 1: INTRODUCTION ..............................................................................................1
 1.1 The role of meiotic recombination ...........................................................................1
 1.2 Meiotic prophase and recombination in Caenorhabditis elegans ............................1
 1.3 Rec-1 randomizes crossover distribution..................................................................6
 1.4 The role of chromatin structure and chromatin remodeling during meiotic recombination .................................................................................................................8
 1.5 The tumour suppressor and chromatin modifier LIN-35........................................10
 1.6 lin-35 (n745) ...........................................................................................................12
 1.7 Thesis objectives.....................................................................................................12
 CHAPTER 2: MATERIALS AND METHODS.........................................................................13
 2.1 General methods .....................................................................................................13
 2.2 Strain construction ..................................................................................................14
 2.3 Crosses ....................................................................................................................16
 2.4 Calculating recombination frequency.....................................................................16
 2.5 Brood analysis ........................................................................................................17
 2.6 RNAi.......................................................................................................................17
 
  iii  CHAPTER 3: RESULTS .......................................................................................................18
 3.1 Mutant LIN-35 affects recombination frequency in a chromosome cluster and a chromosome arm ..........................................................................................................18
 3.2 Like Rec-1, Lin-35 increases chances of CO in the central cluster of Chromosome III ......................................................................................................................................20
 3.3 Lin-35 and Rec-1 have similar meiotic phenotypes, which complement in trans..22
 3.4 Crossing over analysis in the double mutant, Lin-35 Rec-1, indicates that LIN-35 and REC-1 function non redundantly..................................................................................24
 3.5 Mutation in lin-35 reduces recombination across a whole chromosome ...............27
 3.6 The double mutant, Lin-35 Rec-1, has a more severe morphological phenotype than the single mutants ...............................................................................................................32
 3.7 Mutation in the histone deacetylase, had-4, does not influence crossing over.......34
 CHAPTER 4: DISCUSSION ..................................................................................................36
 4.1 Lin-35 affects meiotic crossing over in C. elegans.................................................36
 4.2 The Lin-35 mutant alters both the distribution and frequency of crossing over.....36
 4.3 Lin-35 in a Rec-1 background exhibits even fewer crossover events ....................38
 4.4 The phenotype of the Lin-35 Rec-1 double mutant is more severe than the single mutants or wild-type .....................................................................................................39
 4.5 Conclusion ..............................................................................................................41
 BIBLIOGRAPHY..................................................................................................................42
 APPENDIX 1 .......................................................................................................................48
  
  iv  LIST OF TABLES Table 1. Primers used to amplify mutant strains. ..............................................................15 Table 2. Crossing over in a Lin-35 mutant strain in the cluster region of Chromosome V and flanking arm region of Chromosome III. ..................................................................19 Table 3. Crossing over in a Rec-1 mutant strain in the cluster region of Chromosome I and Chromosome V and flanking arm region of Chromosome III. .................................21 Table 4. Complementation test data for Lin-35 and Rec-1 mutant strains in the cluster of Chromosome I. ..........................................................................................................23 Table 5. Crossing over in Lin-35 and Rec-1 mutant strains in the cluster of Chromosome V and the arm of Chromosome III. ...............................................................................25 Table 6. Crossing over in Lin-35 and Rec-1 mutant strains along Chromosome III. .......30 Table 7. Mutant Lin-35 and Rec-1 progeny analysis. .......................................................33 Table 8. Crossing over in a Hda-4 mutant strain in the dpy-5 unc-13 region of Chromosome Ι, the dpy-11 unc-42 region of Chromosome V, and the dpy-18 unc-64 region of Chromosome III.........................................................................................................35 Table A1. Strains that can be found at the C. elegans Genetics Center ............................48 Table A2. Strains constructed by Z. Lohn.........................................................................49 Table A3. Crossing over in Lin-35 and Rec-1 mutant strains along Chromosome III. Data includes multiple isolate labeled with strain numbers...............................................50  
  v  LIST OF FIGURES Figure 1. Schematic representation of the C. elegans adult hermaphrodite gonad and germ line. ..............................................................................................................................4 Figure 2. An overview of recombination pathways where DSB can resolve to either a crossover product or a non-crossover product.............................................................5 Figure 3. Comparison of the genetic map and the physical map in the Rec-1 mutant background. .................................................................................................................7 Figure 4. Genetic maps and physical maps for the cluster region of Chromosome V and the arm region of Chromosome III..................................................................................28 Figure 5. The standard genetic map of Chromosome III highlighting the five intervals examined in this study. ..............................................................................................31 Figure 6. The genetic map of Chromosome III in Rec-1 and Lin-35 mutant backgrounds.31  
  vi  LIST OF ABBREVIATIONS CO (crossover) COI (crossover interference) Dpy (dumpy) DSB (double stranded break) GFP (green fluorescence protein) Hda (histone deacetylase locus) Him (high incidence of males) LG (linkage group) Lin (cell lineage abnormal) m.u. (map units) p (recombination frequency) Rec (abnormal recombination) Rb (retinoblastoma) SNP (single nucleotide polymorphism) Unc (uncoordinated)  
  vii  ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor, Dr. Ann Rose, for providing me with an invaluable opportunity to learn in the medical genetics field. Your guidance and support provides me with a solid foundation to extend my career in the medical genetics realm. I would like to thank the entire Rose Lab for their assistance and encouragement including George Chung, Sanja Tarailo, Martin Jones, Jessica McLellan, Jim Huang and Shir Hazir. In particular, I would like to thank Nigel O’Neil for patient training and thought-provoking discussions. To my committee members, Dr. Don Riddle and Dr. Hugh Brock, thank you for graciously participating in the development and execution of my project. To Kevin, thank you for your technical knowledge and open ear.  
  viii  CHAPTER 1: INTRODUCTION 1.1 The role of meiotic recombination Fundamental to the reproductive success of most diploid organisms, meiosis is a cell division process essential for the maintenance of proper ploidy between generations. During meiosis, homologous recombination generates genomic variation and ensures genome integrity through proper chromosome segregation (Resnick 1976, HowardFlanders and Theriot 1996, Petronezki et al. 2003, reviewed by Zetka 2009). Failure to recombine properly during meiosis causes genetic conditions in the human conceptus such as aneuploidy and spontaneous abortion (Reiter et al. 1996, Lopes et al. 1998, reviewed by Handel and Schiment 2010). Reduced recombination is associated with meiosis I maternal segregation errors. Errors such as nondisjunction can occur when chromosomes do not pair and exchange genetic material properly. In humans, the direct result is aneuploid oocytes, which cause conditions such as Down Syndrome. Most pregnancies are not at a great risk of aneuploidy; however, the prevalence increases considerably with advanced maternal age (reviewed by Hassold and Hunt 2001).  1.2 Meiotic prophase and recombination in Caenorhabditis elegans The hermaphrodite nematode Caenorhabditis elegans has several features that make it an excellent model organism to study meiosis including easy maintenance, a relatively compact genome, the generation of haploid gametes of both sexes, and highly conserved meiotic processes (reviewed by Muse and Boulton 2007, Zetka 2009). In C. elegans, meiosis begins in the germline where the distal tip cell gives rise to nuclei, which undergo pre-meiotic replication followed by prophase of meiosis I (reviewed by 
  1  Zetka and Rose 1990 and more recently Muse and Boulton 2007) (Figure 1). The spatiotemporal organization of the transparent germline facilitates visualization of meiotic prophase (Albertson et al. 1997). The homologs pair and align, first at the homolog recognition region (Rosenbluth and Baillie 1981, McKim et al. 1993) and then along the whole chromosome (Jones et al. 2009). The synaptonemal complex joins homologous chromosomes to ensure that a close proximity is maintained for meiotic recombination (Zetka 2009). The axial elements promote chromosome condensation and pairing while inhibiting recombination between sister chromatids. The central element is important for synapsis and likely involved with maintaining distance between crossover events. A highly conserved topoisomerase, SPO-11, catalyzes the double-strand break (DSB) which results in the obligate crossing over event (Figure 2). In C. elegans, Spo-11 mutants exhibit extensive embryonic lethality and a severe Him (high incidence of males) phenotype as a result of nondisjunction due to the absence of CO (Dernburg et al. 1998). The RAD-51 family of recombinases catalyzes the strand-invasion and strand-exchange reactions, resulting in products that have either exchanged flanking DNA arms (crossovers) or have not undergone exchange (non-crossovers). The presence of one cross over event reduces the probability of a second event occurring nearby, termed crossover interference (COI) (reviewed in Zetka 2009). One advantage of studying cross over events in C. elegans is that there is only one cross over event between homologous chromosomes per meiosis, demonstrating complete COI (Brenner 1974, Hillers and Villeneuve 2003). The absence of double crossovers facilitates the study of the frequency and distribution of exchange events. While the  
  2  mechanism underlying COI is unclear, recent analysis by Youds et al. (2010) has shown that the anti-recombinase RTEL-1 promotes non-crossover events, and in the Rtel-1 mutant DSB become crossover events due to a lack of COI (Barber et al. 2008, Youds et al. 2010). Another striking aspect of meiotic recombination in C. elegans is the meiotic pattern. The meiotic pattern is the cumulative effect of crossing over along the chromosome. The nematode exhibits a clear meiotic pattern as the central clusters of autosomes have fewer crossovers per unit DNA than the flanking arm regions which are highly recombinogenic (Brenner 1974). Thus, the probability of a recombination event is more likely in some genomic regions (genetic hotspots) than in others (genetic coldspots), a phenomena conserved across most species. Many of the molecular mechanisms underlying meiotic recombination are well known, while the factors determining the distribution of CO are less clear. In C. elegans, a mutant known to alter CO distribution exists, Rec-1 (Rose and Baillie 1979a Zetka and Rose 1995).  
  3  Figure 1. Schematic representation of the C. elegans adult hermaphrodite gonad and germ line. The C. elegans gonad consists of two mirrored arms. A signal from the somatic gonadal distal tip cell keeps most distal germ line nuclei in mitosis and nuclei that migrate to the proximal region initiate meiosis, cellularlize and differentiate into oocytes. (Adapted from Minasaki et al. 2009)  
  4  Figure 2. An overview of recombination pathways where DSB can either form a crossover product or a non-crossover product. Two homologous unpaired chromosomes are shown at the top and the remainder of the figure focuses on segments of sister chromatids. Proteins that are known to contribute to double strand breaks (DSB) formation and DSB resolution are indicated. (Adapted from Handel and Schimenti 2010)  
  5  1.3 Rec-1 randomizes crossover distribution First identified as a recessive mutation in a group of ethylmethane sulfonate treated strains, Rec-1 (abnormal recombination) disrupts the distribution of crossovers along the chromosome. Interestingly, Rec-1 alters the location of crossover events without affecting the total number, causing no other observable phenotypic effect (Rose and Baillie 1979a, Zetka and Rose 1995, Rose et al. 2010) (Figure 3). The genetic map in Rec-1 is more similar to the physical map than the wild type genetic map. While the molecular function of rec-1 is unknown, the mutant provides a valuable opportunity to study aspects of crossover distribution.  
  6  Figure 3. Comparison of the genetic map and the physical map in the rec-1 mutant background.  The top line depicts the wild-type (N2) genetic map of autosome I as measured by Zetka and Rose (1995). The second line shows the position of the gene markers on the physical map as annotated in WormBase (http://www.wormbase.org). The bottom line is the position of gene markers in the Rec-1 mutant. The Rec-1 genetic map is more similar to the physical map than the wild-type genetic map. This indicates that crossover events are not randomly distributed along the chromosome as there are more crossover events in the chromosomal arms compared to the cluster. (Adapted from Rose et al. 2010)  
  7  1.4 The role of chromatin structure and chromatin remodeling during meiotic recombination  Gross chromosome structure has been linked to DSB formation and meiotic recombination. In Drosophila melanogaster, there are structure differences in the synaptonemal complex in euchromatin compared to heterochromatin, which lead to the absence of crossing over events in heterochromatin (Carpenter 1975). While Drosophila represents an extreme case, Murakami et al. (2003) have shown that in yeast, prior to DSB formation, chromosomes become more sensitive to micrococcal nuclease, an indicator of open chromatin. However, nuclease sensitivity is not required for recombination because deoxyribonuclease I hypersensitive sites were identified in one mouse hotspot, Eβ1, but not in another, Psmb9 (Mizuno et al. 1996, Shenkar et al. 1991). Recently, Mets and Meyer (2009) showed that partial loss of the C. elegans dosage compensation condensin, DPY-28, results in extended chromosome axes and far more or far less RAD-51 foci than wild type worms, implicating gross chromosome structure in the formation of DSB. These pieces of data demonstrate that large-scale chromosome structure promotes recombination events in certain regions of the chromosome and suppresses these events in others; however, it is clear that additional factors are also required to facilitate recombination. In addition to gross chromosome structure, chromatin structure influences recombination at a local scale. Chromatin structure has been documented to play a role in transcriptional control, DNA replication, repair and more recently, meiotic recombination (reviewed by Hirota et al. 2009, Cayrou et al. 2010, Szekvolgyi and  
  8  Nicolas 2010). In yeast, histone acetyltransferases open chromatin to activate transcription, and loss of this activation reduces both transcription and recombination activity at ade-M26 and HIS4 recombination hotspots (Yamada et al. 2004, Merker et al. 2008). In addition to acetylation, open chromatin is marked by H3K4me3, which has been associated with DSB in yeast (Kniewel and Keeney 2009). Loss of function of SET1, the only histone H3K4 methyltransferase in yeast, severely reduces DSB formation in 84% of recombination hotspots (Sollier et al. 2004, Borde et al. 2009, Kolasinska-Zwierz et al. 2009). In the mouse, PRDM9 was recently identified as encoding a histone methyltransferase which methylates H3K4 at recombination hotspots (Baudat et al. 2009, Myers et al. 2009). Interestingly, in spermatocytes lacking functional PRDM9, gametogenesis is disrupted at the pachytene stage (Hayashi et al. 2005). Taken together, this data shows that DSB formation and recombination events are influenced by open chromatin, presumably through increased access to DNA for SPO-11. To illustrate how closed chromatin rather than open chromatin has been associated with meiotic recombination, Reddy and Villeneuve (2004) describe the case of him-17. In C. elegans, the Him-17 mutant showed reduced histone three lysine nine monomethylation (H3K9me), less of the DNA strand exchange protein, RAD-51, and fewer DSB. H3K9me marks heterochromatin and transcriptionally inactive DNA. The authors propose that a certain degree of chromosomal compaction in one area of the chromatin may result in compensatory loosening in another area, suggesting that a variable chromatin environment facilitates meiotic recombination. In any case, it is clear that proper meiotic recombination requires both a certain chromosomal and chromatin configuration.  
  9  Leger (2007) examined chromatin modifier mutants, including Him-17, and their effect on meiotic recombination in C. elegans. Mutation in him-17 caused an increase in recombination in a chromosomal hybrid region while rec-1 mutation caused a decrease. Thus, mutation in Him-17 failed to recapitulate the Rec-1 pattern. Another gene analyzed by Leger (2007) encodes the tumour suppressor LIN-35 (abnormal cell lineage). Mutation in lin-35 caused an increase in recombination in a chromosome cluster and a decrease in an arm, a similar effect to rec-1 mutation. Since HIM-17 shares structural properties with LIN-35, Reddy and Villeneuve (2004) constructed the double mutant and analyzed RAD-51 foci staining along the germline axis. Although the role of lin-35 in meiotic recombination was not investigated, the Him17 Lin-35 double mutant showed a reduction in foci compared to wild-type and the Him17 single mutant, suggesting fewer crossing over events in the double mutant. These are the first pieces of evidence suggesting that lin-35 plays a role during meiotic recombination.  1.5 The tumour suppressor and chromatin modifier lin-35 Mutation in the Rb gene was identified in malignant tumours of the retina. Rb mutation is now recognized as one of the most common events preceding the onset of tumourigenesis in humans (Dunn et al. 1988, reviewed by Sherr and McCormick 2002, Giacinti and Giordano 2006). In C. elegans, LIN-35 is an ortholog of the Rb tumour suppressor family. LIN-35 has been well-studied with respect to several cellular processes including gene transcription, mRNA stability, cellular proliferation, cell cycle regulation, soma germline transformation and apoptosis (reviewed by Kirienko et al. 2010). 
  10  pRb family members not only prevent tumorigenesis but also regulate the general organization of chromosomes. Loss of pRb in mouse adult fibroblasts increased mobility of heterochromatin protein 1, and the authors suggested that pRb plays a role in formation of compact chromatin (Siddiqui et al. 2007). In Drosophila neuroblasts, loss of the pRB homolog exhibits fused and broken chromosomes. These pieces of evidence suggest a role for Rb in maintaining gross chromosome structure (Longworth and Dyson 2010). While pRb family members have been shown to influence chromosome structure, LIN-35 has been shown to be a chromatin modifier in C. elegans. As a member of the DRM (Db, Rb and Muv genes) complex, LIN-35 binds to the transactivation domain of E2F transcription factors that bind to gene promoters to regulate gene transcription (Harrison et al. 2006). The DRM complex recruits other complexes to gene promoters such as SWI/SNF (switch/sucrose nonfermentable) and NuRD (nucleosome remodeling and deacetylase complex), which are involved with chromatin remodeling and histone deacetylation respectively (Sawa 2000, Cui et al. 2004). Affecting chromatin structure causes an indirect effect on gene transcription. When lin-35 is non-functional, 535 genes are up-regulated and 175 are down-regulated, including several meiotic genes (such as syp-1, htp-1, him-3, rad-51) (Grishock et al. 2008). Altered transcription of indirect targets may provide a means for lin-35 mutation to influence recombination. Thus, lin-35 may influence recombination in two ways: directly through chromatin structure or indirectly through gene transcription.  
  11  1.6 lin-35 (n745) Since first identified in a screen for genes involved with vulval development, the n745 allele of lin-35 has been used in numerous experiments (Ferguson and Horvitz 1989). The n745 allele causes a single nucleotide change, “TGG” in wild type to “TGA” in the mutant, causing an opal stop in the fourth codon of the gene (Lu and Horvitz 1998). For this reason, n745 is considered a null allele. However, Ouellet and Roy (2007) showed mRNA levels were similar to wild type in the allele, rr33, which introduces an amber stop codon in the same position as n745. The authors hypothesized that readthrough resulted in this mRNA stabilization. Previous investigators have interpreted the n745 allele as a null allele, although definitive proof has not been obtained.  1.7 Thesis objectives Chromatin structure has been long proposed to play a role during meiotic recombination. However, the role of chromatin structure in establishment of the meiotic pattern is unclear in C. elegans. To initiate this investigation, this thesis aims to determine whether or not the chromatin modifier, Lin-35, affects meiotic recombination. Using pairs of linked markers (Dpy and Unc mutants), the frequency and distribution of crossing over will be measured along the entire length of a chromosome. In this way, information will be gained regarding whether or not mutation in the lin-35 influences recombination, providing a starting point for understanding its functional role in the recombination process.  
  12  CHAPTER 2: MATERIALS AND METHODS 2.1 General methods Strains were obtained from the C. elegans Genetics Center at the University of Minnesota. The following alleles were used (shown left to right): LGI, rec-1(s180), dpy5(e61), lin-35(n745), unc-13(e51); LGIII, unc-45(e286), dpy-1(e1), dpy-17(e164), unc32(e189), dpy-18(e364), unc-64(e246); LGV, dpy-11(e224), unc-42(e270); LGX, hda4(ok518). The chromosomal rearrangement, hT2 [bli-4], was employed in this study as a genetic balancer. hT2 is a reciprocal translocation that balances left LGI through unc-101 and right LG III through unc-59 (McKim et al. 1993). Refer to Appendix I for a complete listing of the strains used (Table 10, Table 11). All strains were maintained and mated at 20 Degrees Celsius (°C) on petri dishes containing nematode growth medium (NGM) streaked with Escherichia coli strain OP50 (Brenner 1974). The CB286 strain, unc-45 (e286), is temperature sensitive and was maintained at 15 °C. Worms were visualized on a M38 dissecting microscope. Males were generated by incubating L4 hermaphrodites at 30°C for 5 to 6 hours. At this temperature, hermaphrodites produce male progeny more frequently than at 20°C because there is an increase in X-chromosome nondisjunction with an increase in temperature. Hda-4 and Lin-35 Rec-1 animals produced males at 20°C so the aforementioned step was unnecessary for these strains.  
  13  2.2 Strain construction In order to measure the recombination frequency between various markers in mutant backgrounds, several strains were constructed (Table 11). Dpy and Unc mutants were followed with their visible phenotypes while Lin-35, Rec-1 and Hda-4 required further analysis. lin-35 has been mapped to +0.46 cM on LGI (WormBase). The n745 lin-35 allele causes a base pair change that does not introduce a restriction enzyme digestion site, so it cannot be followed with PCR. Rather, quantitative brood size analysis and hT2::GFP were used to follow the non-green homozygous Lin-35 mutant. Since hT2 balances the genomic region on Chromosome I where lin-35 has been mapped, it served as an effective means to assure a homozygous Lin-35 mutant. In some cases when hT2 could not be used, brood size analysis distinguished strains because homozygous Lin-35 mutant animals have significantly fewer progeny than wild-type or heterozygous Lin-35 mutants. A molecular marker was used to follow Rec-1. PCR products were tested by running 5 µl of PCR product on a 1% agarose gel stained with ethidium bromide to distinguish between wild-type, Rec-1 homozygous and heterozygous mutants. Scoring recombination confirmed the Rec-1 phenotype. hda-4 has been mapped to +24.06 on the X Chromosome (WormBase). The hda4 (ok518) allele is a 1090 bp deletion mutant, which was tracked through PCR products (Table 1). The same PCR method was applied to hda-4 mutants as the rec-1 mutants.  
  14  Table 1. Primers used to amplify mutant strains. Primer Name  Annealing Temperature  Primer Sequence (5’ to 3’)  ZL7  60°C  cgt tag cat ggg atc tca cc  ZL8  60°C  tgc taa ggg atc agc aaa cc  ZL9  60°C  ttg att tag gtt gcc gaa gg  hda-4(ok518)  
  15  2.3 Crosses Hermaphrodites have not yet produced oocytes at the L4 larval stage and the vulva has not yet developed (Schedl 1997, Greenwald 1997). Thus, L4 hermaphrodites are selected at this stage because they have neither mated nor self-fertilized. Matings were set up with 8-10 males and 2-3 hermaphrodites. To note, crosses homozygous for the double mutant Lin-35 Rec-1 included 8-10 males and 8-10 hermaphrodites due to the reduced viability of this double mutant. Large numbers of males in the first filial generation (F1) indicates a successful mating. F1 hermaphrodites were picked to individual plates and allowed to self-fertilize to produce the F2 generation. The F2 generation was scored for most crosses.  2.4 Calculating recombination frequency Recombination frequency in the hermaphrodite was measured at 20°C by scoring the number of recombinant progeny from hermaphrodites that were cis-heterozygous for selected visible markers (Rose and Baillie 1979b). Recombination frequency (p) was calculated according to the following formula where p = 1 – (1 – 2R)1/2 (Brenner 1974). R is the total number of recombinant progeny divided by the total number of progeny. Recombinants were segregated on individual plates to ensure that the phenotype had been correctly identified. Both classes of recombinants were used in all calculations. For strains constructed during this thesis, multiple isolates were scored in some cases to further validate the data. Refer to appendix I for the scoring results of individual isolate strains (Table 12). Data represented in results is pooled from the isolates.  
  16  Confidence intervals (95% CI) were calculated using the statistics of Crow and Gardner (1959) where fewer than 300 recombinants were observed. When more than 300 recombinants were scored, the CI was calculated with the following formula where n ± 1.96 (nxy) 1/2 where n is the number of recombinants, x is the number of recombinants divided by the number of recombinants plus wild-type progeny, and y is 1-x.  2.5 Brood analysis To characterize distinguishing features, brood analysis was performed. Hermaphrodites were picked at the L4 stage to individual plates and allowed to selffertilize. The F1 progeny were counted to determine brood size and examined for distinguishing features such as the protruding vulva phenotype (Pvl). Data represents mean values from ten broods of each strain and error represents one standard deviation from the mean.  2.6 RNAi To phenocopy the results of the brood analysis, RNA-mediated interference (RNAi) was employed. RNAi constructs were obtained by the RNAi feeding library of Kamath et al. (2003). Bacteria expressing double stranded RNA from lin-35 was administered by feeding as described by Fraser et al. (2000). Worms were transferred to fresh RNAi plates each day for three consecutive days and scored for features after five days. Ten wild-type hermaphrodites and ten Rec-1 mutants were subjected to lin-35 (RNAi) by feeding. Data includes mean values from ten broods of the strain.  
  17  CHAPTER 3: RESULTS 3.1 Mutant LIN-35 affects recombination frequency in a chromosome cluster and a chromosome arm The first question addressed was whether or not mutation in lin-35 would alter the frequency of crossing over in recombinantly suppressed or enhanced regions. To do this, the recombination frequency (p) within two genomic intervals was analyzed: dpy-11 unc42 in the cluster region of Chromosome V and dpy-18 unc-64 in the arm region of Chromosome III (Table 2). The strain mT10430 lin-35(n745) was used and compared to the VC2010 N2 wild-type strain. The genetic distance between dpy-11 and unc-42 (LGV) in wild type was 1.7±0.5 map units (m.u.) and 5.4±1.1 m.u. in Lin-35. The genetic distance between dpy-18 and unc-64 (LGIII) in wild type was 9.9±0.3 m.u. and 6.5±1.4 m.u. in Lin-35. Lin-35 caused an increase in crossing over in the cluster of one chromosome and a decrease in the arm of another.  
  18  Table 2. Crossing over in a Lin-35 mutant strain in the cluster region of Chromosome V and flanking arm region of Chromosome III. Recombinants wild-type progenya  Dpy  Unc  Total  p x 100  95% C.I.b  dpy-11 unc-42/++  2320  26  26  52  1.69  1.24-2.18  dpy-11 unc-42/++; lin-35/lin-35  1554  65  44  109  5.38  4.51-6.67  dpy-18 unc-64/+ +  2972  212  194  406  9.91  9.56-10.10  dpy-18 unc-64/+ +; lin-35/lin-35  1079  32  61  93  6.50  5.42-8.23  Genotype  a b  
  Male progeny included. C.I. = 95% confidence interval; See Materials and Methods.  19  3.2 Like Rec-1, Lin-35 increases chances of CO in the central cluster of  Chromosome III The initial result for Lin-35 is similar to previous analysis of the Rec-1 phenotype. To investigate this further, crossing over within three genomic intervals was analyzed in the Rec-1 mutant: dpy-5 unc-13 in the cluster region of Chromosome I, dpy-11 unc-42 in the cluster region of Chromosome V, and dpy-18 unc-64 in the arm region of Chromosome III (Table 3). In both cluster intervals, Rec-1 increased crossing over more than three fold in; dpy-5 unc-13: 1.8±0.4 m.u. (wild type), 6.6±1.0 m.u. (Rec-1) and dpy11 unc-42: 1.7±0.5 m.u. (wild type), 7.1±1.1 m.u. (Rec-1). Within the arm, recombination was reduced in the Rec-1 mutant compared to wild type; dpy-18 unc-64: 9.9±0.3 m.u. (wild type), 6.5±0.8 m.u. (Rec-1). I observed the published result for the LGV cluster (7.6 m.u. in Rec-1 compared to 2.7 m.u. in wild type) (Zetka and Rose 1995) and observed the expected decrease in the arm of LGIII, a region not previously investigated.  
  20  Table 3. Crossing over in a Rec-1 mutant strain in the cluster region of Chromosome I and Chromosome V and flanking arm region of Chromosome III. Recombinants wild-type progenya  Dpy  Unc  Total  p x 100  95% C.I.b  dpy-5 unc-13/++  4142  51  48  99  1.79  1.46-2.20  dpy-5 unc-13/++; rec-1/rec-1  2086  84  95  179  6.64  5.65-7.66  dpy-11 unc-42/++  2320  26  26  52  1.69  1.24-2.18  dpy-11 unc-42/++; rec-1/rec1  2085  97  100  197  7.11  6.08-8.19  dpy-18 unc-64/+ +  2972  212  194  406  9.91  9.56-10.10  dpy-18 unc-64/+ +; rec-1/rec-1  3215  119  170  289  6.53  5.98-7.58  Genotype  a b  
  Male progeny included. C.I. = 95% confidence interval; See Materials and Methods.  21  3.3 Lin-35 and Rec-1 have similar meiotic phenotypes, which complement in trans Results thus far have shown that mutations in Lin-35 and Rec-1 display similar meiotic phenotypes compared to wild type, that is, there is an increase in crossing over in the chromosome cluster and a decrease in crossing over in the chromosome arm. Both lin-35 and rec-1 map to LG I. Lu and Horvitz (1998) mapped lin-35 to the cluster of LGI between dpy-5 and unc-13. Although previous strain constructions eliminated the dpy-5 unc-13 interval as a position for the rec-1 gene, I constructed the trans-heterozygote and examined the phenotype (Baillie and Rose 1979, Zetka and Rose 1995). lin-35(n745) complements rec-1(sI80) (Table 4), indicating that lin-35 and rec-1 are not allelic and that there is no dominant interaction.  
  22  Table 4. Complementation test data for Lin-35 and Rec-1 mutant strains in the cluster of Chromosome I. Recombinants wild-type progenya  Dpy  Unc  Total  p x 100  95% C.I.b  dpy-5 unc-13/++  4142  51  48  99  1.79  1.46-2.20  dpy-5 unc-13 rec-1/++rec-1  1792  74  85  159  7.01  5.89-8.15  dpy-5 unc-13 rec-1+/+++lin-35  1918  20  26  46  1.86  1.37-2.43  Genotype  a b  
  Male progeny included. C.I. = 95% confidence interval; See Materials and Methods.  23  3.4 Crossing over analysis in the double mutant, Lin-35 Rec-1, indicates that LIN-35 and REC-1 function non redundantly To analyze the relationship between lin-35 and rec-1, I constructed a Lin-35 Rec1 double mutant. Between dpy-11 and unc-42 of LGV, the genetic map distance in the double mutant was 2.1±0.4 m.u. compared to 1.7±0.5 m.u. in wild type, 7.1±1.0 m.u. in the Rec1 mutant, and 5.4±1.1 m.u. in the Lin-35 mutant (Table 5, Figure 5). The double mutant recombination frequency was less than the single mutants and similar to wild type. To observe an arm interval, the dpy-18 unc-64 arm interval on Chromosome III was scored. The recombination frequency of the double mutant was lower than both wild type and the single mutants (wild type: 9.9±0.3 m.u., Lin-35: 6.5±0.9 m.u., Rec-1: 6.5±0.8 m.u., Lin-35 Rec-1: 4.6±1.5 m.u.) (Table 5, Figure 5). Clearly, in this case the wild-type distance was not restored. Nor was the distance the same as the single mutants, as it was reduced. Within both the cluster and arm intervals, the recombination frequency of the double mutant was less than the single mutants.  
  24  Table 5. Crossing over in Lin-35 and Rec-1 mutant strains in the cluster of Chromosome V and the arm of Chromosome III. Recombinants wild-type progenya  Dpy  Unc  Total  p x 100  95% C.I.b  dpy-11 unc-42/++  2320  26  26  52  1.69  1.24-2.18  dpy-11 unc-42/++; rec-1/rec1  1867  82  89  171  6.86  5.78-7.85  dpy-11 unc-42/++; lin-35/lin-35  1554  65  44  109  5.38  4.51-6.67  dpy-11 unc-42/++; lin-35 rec-1/lin-35 rec-1  3321  44  47  91  2.07  1.67-2.54  dpy-18 unc-64/+ +  2972  212  194  406  9.91  9.56-10.10  dpy-18 unc-64/+ +; rec-1/rec-1  3215  119  170  289  6.53  5.98-7.58  dpy-18 unc-64/+ +; lin-35/lin-35  1079  32  61  93  6.50  5.42-8.23  dpy-18 unc-64/+ +; lin-35 rec-1/lin-35 rec-1  634  19  23  42  4.58  3.33-6.24  Genotype  a b  
  Male progeny included. C.I. = 95% confidence interval; See Materials and Methods.  25  Figure 4. Genetic maps and physical maps for the cluster region of Chromosome V and the arm region of Chromosome III. A. The top portion of the figure depicts the cluster or Chromosome V between the markers dpy-11 and unc-42 B. The bottom portion depicts the right arm of Chromosome 3 between dpy-18 and unc-64. The position of the gene markers on the physical map is shown for each interval for wildtype (N2) worms as annotated by WormBase (http://www.wormbase.org). The genetic map of Lin-35 and Rec-1 mutants is more similar to the physical map than the genetic map.  
  26  3.5 Mutation in lin-35 reduces recombination across a whole chromosome To further analyze the relationship between Lin-35 mutation and meiotic recombination, the entire length of Chromosome III (LGIII) was analyzed. The chromosome was divided into five sections comprising of the chromosome arms (dpy-1 unc-45, dpy-18 unc-64), cluster (dpy-17 unc-32) and two halves (dpy-17 unc-45, dpy-17 unc-64) (Figure 6, Table 6, Figure 7). Determining the genetic length of these segments serves to test previous findings, in addition to determining effects along an entire chromosome. The genetic distance within the cluster and arm intervals in the Lin-35 mutant was similar to intervals previously analyzed. Within the cluster of LGIII between dpy-17 and unc-32, the genetic distance in Lin-35 was 3.4±0.8 m.u., higher than wild type worms (1.9±0.6 m.u.). Rec-1 exhibited a more striking effect in this interval (5.1±0.94 m.u.). Within the left arm of LGIII between dpy-1 and unc-45, wild type worms displayed a genetic distance of 11.1±0.4 m.u. while in Lin-35 mutants the distance was 6.4±1.2 m.u. and in Rec-1 mutants the distance was 5.6±0.9 m.u.. The right arm interval, dpy-18 unc64, was described in a previous section (Table 5). These results confirmed previous observations that Lin-35 increased crossing over in chromosomal clusters and reduced it in the flanking arms. To determine the genetic length of the whole chromosome, the two halves of Chromosome III were added together. In wild type, left half of LG III between dpy-17 and unc-45 is 22.3±0.9 m.u.. In Rec-1, this distance was unchanged (22.9±0.9 m.u.); since this interval included both a cluster which increases and an arm with reduces, this  
  27  result was expected (Zetka and Rose 1995). The Lin-35 mutant measured gave 21.0±1.0 m.u.. For the right half of Chromosome III, between dpy-17 and unc-64, wild type and Rec-1 worms showed similar genetic distances (p = 26.3±1.0 m.u. and 24.8±0.9 m.u. respectively) while the Lin-35 mutant was 21.3±3.3 m.u.. Adding together the two halves gives a genetic length for the whole chromosome of 48.6 m.u. in wild type, 47.7 m.u. in Rec-1 and 42.3 m.u. in Lin-35. Thus, wild type and Rec-1 worms were close to the theoretical value of 50 m.u., whereas the Lin-35 mutant chromosome was shorter at 42.3 m.u. The left and right halves of LGIII was investigated in the Lin-35 Rec-1 double mutant. Between dpy-17 and unc-64, the double mutant displayed a genetic distance that was less (14.7±0.7 m.u.) than wild type (26.3±1.0 m.u.) and the single mutants (Rec-1: 24.8±0.95 m.u., Lin-35: 21.3±3.3 m.u.) (Table 6, Figure 7). Similarly, between dpy-17 and unc-45, the double mutant displayed a genetic distance that was less (13.3±1.7 m.u.) than wild type (22.3±0.9 m.u.) and the single mutants (Rec-1: 22.9±0.9 m.u., Lin-35: 21.0±1.0 m.u.). Thus, the length of Chromosome III in the Lin-35 Rec-1 double mutant is severely truncated at 28.0 m.u.. These results show that the genetic length of Chromosome III is shorter than wild type in both Lin-35 single mutants and Lin-35 Rec-1 double mutants.  
  28  Figure 5. The standard genetic map of Chromosome III highlighting the five intervals examined in this study. The genetic distance between various markers is shown (adapted from WormBase). The regions indicated are genetic intervals that were analyzed in this study: unc-45 dpy-1 (left arm), dpy-17 unc-32 (cluster), dpy-18 unc-64 (right arm), dpy-17 unc-45 (left half), dpy17 unc-64 (right half).  
  29  Table 6. Crossing over in Lin-35 and Rec-1 mutant strains along Chromosome III. Recombinants wild-type progenya  Dpy  Unc  Total  px 100  95% C.I.b  dpy-18 unc-64/+ +  2972  212  194  406  9.9  9.56-10.1  dpy-18 unc-64/+ +; rec-1/rec-1  3215  119  170  289  6.5  5.98-7.58  dpy-18 unc-64/+ +; lin-35/lin-35  2112  71  90  161  5.93  5.01-5.92  dpy-18 unc-64/+ +; lin-35 rec-1/lin-35 rec-1  634  19  23  42  4.58  3.33-6.24  dpy-17 unc-32/+ +  2008  21  27  48  1.88  1.36-2.47  dpy-17 unc-32/+ +; rec-1/rec-1  2033  64  74  138  5.11  4.35-6.22  dpy-17 unc-32/+ +; lin-35/lin-35  2011  40  45  85  3.35  2.62-4.12  dpy-1 unc-45/+ +  2021  146  157  303  11.1  10.7-11.52  dpy-1 unc-45/+ +; rec-1/rec-1  2021  79  80  159  5.79  4.88-6.74  dpy-1 unc-45/+ +; lin-35/lin-35  1414  63  57  120  6.38  5.25-7.65  dpy-17 unc-45/+ +  2052  276  320  596  22.3  21.4-23.1  dpy-17 unc-45/+ +; rec-1/rec-1  2037  325  268  593  22.9  22.0-23.8  dpy-17 unc-45/+ +; lin-35/lin-35  1450  197  162  359  21.0  20.0-22.0  dpy-17 unc-45/+ +; lin-35 rec-1/lin-35 rec-1  1688  137  133  270  13.3  11.6-15.0  dpy-17 unc-64/+ +  2035  379  338  717  26.3  25.3-27.3  dpy-17 unc-64/+ +; rec-1/rec-1  2004  290  373  663  24.8  23.9-25.8  dpy-17 unc-64/+ +; lin-35/lin-35  702  109  101  210  21.3  18.5-25.1  dpy-17 unc-64/+ +; lin-35 rec-1/lin-35 rec-1  2083  186  192  378  14.7  14.0-15.3  Genotype  a b  
  Male progeny included. C.I. = 95% confidence interval; See Materials and Methods.  30  Figure 6. The genetic map of Chromosome III in Rec-1 and Lin-35 mutant backgrounds. The five markers (unc-45, dpy-1, dpy-17, unc-32, dpy-18, unc-64) are labeled on the wild-type chromosome in both the physical and genetic map (top). Genetic distances between markers (map units, m.u.) are shown where measured in the genetic maps. The total genetic length of the chromosome was determined by adding the two halves together.  
  31  3.6 The double mutant, Lin-35 Rec-1, has a more severe morphological phenotype than the single mutants In constructing the double mutant, Lin-35 Rec-1, somatic and meiotic characteristics were scored. The progeny of ten hermaphrodite worms were counted and analyzed for morphological changes, viability and fertility. The double mutant progeny displayed protruding vulvae (Pvl), everted vulvae (Evl), reduced fertility and arrested embryonic progeny (Table 7). These features were phenocopied when RNAi was directed against lin-35 (Table 7). To note, the standard deviation included for each piece of data is higher for some values compared to others, indicating phenotypic variation. Embryonic viability was reduced in the double mutant. Clearly, the Lin-35 Rec-1 double mutant exhibits a more severe phenotype than the single mutants.  
  32  Table 7. Mutant Lin-35 and Rec-1 progeny analysis. Genotype  Brood Size1  %Unhatched Eggs  % Vulval Defects (Pvl, Evl)2  % Male  +3  253±25  0.1±0.2%  0  0.2±0.4%  rec-13  234±48  0.2±0.2%  0  0.1±0.2%  lin-353  115±29  0.8±3.3%  0  0.4±0.5%  lin-35 rec-13  101±28  3.4±9%  14.3±1.5%  1.5±0.6%  + ; lin-35 (RNAi)4  194±39  0  0  0  rec-1; lin-35 (RNAi)4  184±42  0  7.6±3.8%  0  1  Brood size excludes unfertilized oocytes. Pvl, protruding vulva phenotype; Evl, everted vulva phenotype. 3 Mutant alleles were employed to observe rec-1 and lin-35 mutants. Data includes mean values from ten broods of each strain. 4 Lin-35 and Rec-1 mutants were observed through interference RNA (RNAi). Ten wildtype hermaphrodites and ten Rec-1 mutants were subjected to lin-35(RNAi) by feeding. Data includes mean values from ten broods of the strain. Error represents one standard deviation from the mean. 2  
  33  3.7 Mutation in the histone deacetylase, Had-4, does not influence crossing over Histone acetyltransferases (HAT) modify chromatin to activate transcription (Struhl 1998,Yamada et al. 2004). In yeast, histones near recombination hotspots are often hyperacetylated by HAT, and suppression of HAT reduces recombination activity (Krebs et al. 1999, Vogeleuer et al. 2000). Recently, Petes et al. (2007) showed that mutation in the histone deacetylase, SIR-2, affected the distribution of Spo-11 induced DSB in yeast. In C. elegans, there are at least four genes encoding histone deaceytlases. One of these, hda-4, results in a viable null mutant, facilitating the scoring of recombination. HDA-4 regulates chemoreceptor gene expression and deacetylates histones. It is proposed to function independently of the LIN-35 pathway (Choi et al. 2002). The recombination frequency resulting from mutation in hda-4(ok518) was analyzed for three genomic intervals: dpy-5 unc-13 in the cluster of Chromosome Ι, dpy11 unc-42 in the cluster of Chromosome V, and dpy-18 unc-64 in the arm of Chromosome III (Table 9). Within all three intervals examined, the recombination frequency of Hda-4 mutants did not deviate significantly from wild type; dpy-5 unc-13: 1.8±0.4 (wild type) and 1.5±0.3 (hda-4); dpy-11 unc-42: 1.7±0.5 (wild type) and 2.0±0.4 (hda-4); dpy-18 unc-64: 9.9±0.3 (wild type) and 9.9±1.3 (hda-4). These results indicate that hda-4 does not play a role in meiotic recombination.  
  34  Table 8. Crossing over in a Hda-4 mutant strain in the dpy-5 unc-13 region of Chromosome Ι, the dpy-11 unc-42 region of Chromosome V, and the dpy-18 unc-64 region of Chromosome III. Recombinants wild-type progenya  Dpy  Unc  Total  p x 100  95% C.I.b  dpy-5 unc-13/+ +  4142  51  48  99  1.79  1.46-2.20  dpy-5 unc-13/+ +; hda-4/hda-4  5949  60  54  114  1.50  1.24-1.79  dpy-11 unc-42/+ +  2320  26  26  52  1.69  1.24-2.18  dpy-11 unc-42/+ +; hda-4/hda-4  4712  61  64  125  2.01  1.67-2.38  dpy-18 unc-64/+ +  2972  212  194  406  9.91  9.56-10.1  dpy-18 unc-64/+ +; hda-4/hda-4  2086  125  140  265  9.88  8.69-11.2  Genotype  a b  
  Male progeny included. C.I. = 95% confidence interval; See Materials and Methods.  35  CHAPTER 4: DISCUSSION In this thesis, I have shown a meiotic phenotype for Lin-35, and a synthetic interaction with Rec-1, a well-characterized meiotic mutant in C. elegans.  4.1 Lin-35 affects meiotic crossing over in C. elegans A chromosome cluster (dpy-11 unc-42) and a chromosome arm (dpy-18 unc-64) were first analyzed because these intervals have been well studied previously and represent both recombinantly suppressed and enhanced regions. Within the cluster interval, Lin-35 mutation caused a three-fold increase in recombination (wild type: 1.7±0.5 m.u., lin-35: 5.4±1.1 m.u.) while the arm demonstrated an approximate 30% decrease (wild type: 9.9±0.3 m.u., Lin-35: 6.5±1.4 m.u.) (Table 2). Analysis of Rec-1 mutation yielded similar results to Lin-35 mutation (dpy-11 unc-42: 7.1±1.1 m.u., dpy-18 unc-64: 6.5±0.8 m.u.) (Table 3). This initial data indicated that LIN-35 functions to alter the distribution of the crossover events along the chromosome. Similar findings were observed previously in the Rose Lab by M. Leger (Leger 2007). I have furthered this analysis by investigating the effect of Lin-35 on the frequency of crossing over for an entire chromosome.  4.2 The Lin-35 mutant alters both the distribution and frequency of crossing over To determine the genetic length of a whole chromosome, recombination was scored across five intervals on Chromosome III: the two halves, the two arms, and the  
  36  cluster (Figure 6). Since crossing over interference is high in C. elegans autosomes, only one crossover per chromosome pair is expected, producing a genetic map of 50 m.u. per linkage group. The genetic length of Chromosome III in Lin-35 was 42.3 m.u., shorter than wild-type worms and Rec-1 (48.8 m.u., 47.8 m.u. respectively) (Table 6, Figure 7). This is the first piece of evidence showing that the phenotype of Lin-35 differs from that of Rec-1. Zetka and Rose (1995) scored recombination across LGI and found that the genetic length of Chromosome I was similar in Rec-1 mutants and wild-type worms (45.3 m.u., 43.5 m.u. respectively), indicating that Rec-1 mutation alters the distribution of crossing over but not the frequency; moreover, Rec-1 affects the location of crossovers while maintaining the normal number. Here, it can be concluded that Lin-35 reduced the frequency of crossing over and consequently the distribution, as crossovers were located in different regions compared to wild-type. To determine whether or not there are in fact fewer crossover events in Lin-35 and Lin-35 Rec-1 mutants, RAD-51 foci staining could be employed. In C. elegans, RAD-51 is a strand exchange protein that can be used to quantify the number of DSB (Alpi et al. 2003, Martin et al. 2005). This technique has been applied to Him-17 Lin-35 double mutants where a reduction in DSB was observed compared to wild type and Him17 single mutants (Reddy and Villeneuve 2004). Since RAD-51 foci staining was not shown for Lin-35 single mutants, this would be a valuable avenue for additional research. In addition to lending insight into the number of crossover events, the Chromosome III genetic map can be compared to the physical map. Zetka and Rose (1995) determined that the Rec-1 genetic map for Chromosome I is more similar to the  
  37  physical map than the wild type genetic map in terms of the spacing of genes (Zetka and Rose 1995 Figure 3). Similarly, results here show that the Rec-1 and Lin-35 genetic maps for Chromosome III are more similar to the physical map than the wild-type genetic map, reflecting the effects these mutants have on the frequency and distribution of crossing over. These results indicate that mutation in lin-35 reduces the ability to crossover in C. elegans.  4.3 Lin-35 in a Rec-1 background exhibits even fewer crossover events The four intervals analyzed in the Lin-35 Rec-1 double mutant shed light on the relationship between Lin-35 and Rec-1; however, when examined independently, these intervals point towards different types of genetic relationships. Within the cluster of chromosome V (dpy-11 unc-42), the recombination frequency of the double mutant was similar to wild type and less than the single mutants (Table 5). The phenotype of Lin-35 was suppressed in a Rec-1 mutant background (or vice versa). In contrast, the arm of chromosome III (dpy-18 unc-64) exhibited a partially additive relationship between Lin35 and Rec-1 because the recombination frequency of the double mutant was less than wild type and each single mutant (Table 5). Finally, the left and right halves of chromosome III demonstrated a synergistic relationship as the recombination frequency of the double mutant was even less than the reductions of each single mutant (Table 6). While these relationships appear to be contradictory, it is more informative to draw conclusions from the whole chromosome as chromosome arms and clusters behave differently in terms of meiotic recombination. The genetic length of the chromosome in the Lin-35 Rec-1 mutant was 28.0 m.u., considerably shorter than wild-type worms (48.8 m.u.), Rec-1 mutants (47.8 m.u.) or Lin
  38  35 mutants alone (42.3 m.u.) (Table 6, Figure 7). This data represents a synergistic relationship between lin-35 and rec-1 because the recombination frequency of the double mutant is less than the single mutants across the chromosome. This non-redundancy may result from lin-35 and rec-1 functioning in distinct pathways. Thus, not only do Lin-35 and Rec-1 have different phenotypes, but genetic interaction analysis indicates independence of function. The severe reduction in crossing over in the Lin-35 Rec-1 double mutant is supported by the percentage of males observed in the Lin-35 Rec-1 double mutant brood analysis. There were more than seven times more males in Lin-35 Rec-1 double mutants compared to wild-type worms (0.2% in wild-type and 1.5% in Lin-35 Rec-1) (Table 7). Wild-type hermaphrodites (5A XX) are self-fertilizing by producing sperm, and males (5A XO) arise spontaneously through X-chromosome loss or nondisjuction at a rate of 1.07 per 1000 wild-type progeny at 20°C (Rose and Baillie 1979). A reduction in crossing over, as observed in the double mutant, is associated with increased chromosomal nondisjunction, manifesting as an increase in male worms in C. elegans (Lamb et al. 2005).  4.4 The phenotype of the Lin-35 Rec-1 double mutant is more severe than the single mutants or wild-type The brood analysis of the Lin-35 Rec-1 double mutant showed morphological changes, and a reduction in viability and fertility compared to the single mutants and wild type (Table 7). Discussion here will draw links between my data, additional Lin-35 phenotypes, and meiotic recombination where possible to substantiate results and raise further questions. 
  39  Lin-35 mutants are synthetic multi-vulva class B (synMuv B) mutants such that synthetic vulval defects arise when combined with a synMuv A or synMuv C mutant (Lu and Horvitz 1998). The C. elegans vulva is an excellent model system to study development because the vulva phenotype responds to inducers, which create visible changes (reviewed by Fay and Yochem 2007). Since the vulva is susceptible to these changes, it is not surprising to observe defective vulval phenotypes (Pvl and Evl) in the Lin-35 Rec-1 double mutant. However, a link between these phenotypes and recombination is not clear. Lin-35 also exhibits increased penetrance and strength for germline, embryonic, and post-embryonic RNAi phenotypes (Lehner et al. 2006). Thus, mutation in lin-35 has wide affects on both transcription and mRNA stability. This complicates analysis because several genes affected by lin-35 mutation may have caused the meiotic, viability and fertility phenotypes observed. To shed light on this issue, Grishok et al. (2008) examined how mutant LIN-35 influences gene transcription. Loss of lin-35 function results in the up-regulation of 535 genes and the down-regulation of 175 genes. Since several of these genes have a meiotic function, this provides an indirect means for meiotic control. The meiotic genes that are up-regulated in Lin-35 mutants include several synaptonemal complex genes (syp-1, syp3, htp-1, him-3) and the strand exchange protein, rad-51. While the loss of function of these genes results in aberrant recombination events, it is unclear how an enhancement of gene transcription would affect meiotic recombination. Analyzing the consequences of up-regulating these secondary transcripts would be an intriguing avenue of future study.  
  40  In addition to transcriptional effects, Lin-35 may affect meiosis by modifying chromatin. While it is clear that Lin-35 interacts with several chromatin modifying complexes as part of the DRM complex, the precise histone modifications influenced by lin-35 have not been determined (Sawa 2000, Cui et al. 2004, Harrison et al. 2006). Immuno-staining C. elegans germline with molecular antibodies for specific histone modifications may shed light on this question. It is unclear whether the effects lin-35 has on recombination are direct and local through chromatin structure, or indirect and global through gene transcription. In fact, it is likely a combination of these because a change in chromatin structure alters the transcription environment. Further experimental support is required to elucidate the relationship between chromatin modifications, transcriptional regulation and meiotic recombination. A unified model may include a mechanistic link between distinct DNA loci and chromatin structure to encompass the factors known to influences meiotic recombination (reviewed by Wahls and Davidson 2010).  4.5 Conclusion The findings presented in this thesis expand upon the knowledge surrounding meiotic recombination, establishing a new role for the Rb ortholog, LIN-35, during meiosis. LIN-35 maintains wild type levels of meiotic recombination, possibly through its role in modifying chromatin and regulating gene transcription.  
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  47  APPENDIX 1 Table A1. Strains that can be found at the C. elegans Genetics Center Strain Name N2  Genotype Wild-type, variety Bristol  BC313  rec-1(s180)  MT10430  lin-35(n745)  BC26  dpy-5(e61) unc-13(e51)  KR177  dpy-5(e61) unc-13(e51) rec-1(s180)  BC502  dpy-18(e364) unc-64(e246)  KR4479  rec-1(s180); dpy-18(e364) unc-64(e246)  BC251  dpy-11(e224) unc-42(e270)  CB164  dpy-17(e164)  KR1234  hT2/[bli-4(e937)]  BC503  unc-64(e246)  CB286  unc-45(e286)  CB1  dpy-1(e1)  RB758  hda-4(ok518)  C32F10.2  lin-35(RNAi)  dpy, dumpy; unc, uncoordinated; lin, abnormal cell lineage; rec, abnormal recombination; hda, histone deacetylase.  
  48  Table A2. Strains constructed by Z. Lohn Strain Name  Genotype  KR4841  lin-35(n745) rec-1(s180)  KR4918  lin-35(n745); dpy-18(e364) unc-64(e246)  KR4919  rec-1(s180) lin-35(n745); dpy-18(e364) unc-64(e246)  KR4830  lin-35(n745); dpy-11(e224) unc-42(e270)  KR4868  rec-1(s180) lin-35(n745); dpy-11(e224) unc-42(e270)  KR4821  dpy-17(e164) unc-64(e246)  KR4839  lin-35(n745); dpy-17(e164) unc-64(e246)  KR4829  rec-1(s180); dpy-17(e164) unc-64(e246)  KR4825  dpy-17(e164) unc-45(e286)  KR4870  lin-35(n745); dpy-17(e164) unc-45(e286)  KR4867  rec-1(s180); dpy-17(e164) unc-45(e286)  KR4917  rec-1(s180) lin-35(n745); dpy-17(e164) unc-45(e286)  KR4820  dpy-1(e1) unc-45(e286)  KR4823  rec-1 (s180); dpy-1(e1) unc-45(e286)  KR4869  lin-35 (n745); dpy-1(e1) unc-45(e286)  KR4912  lin-35 (n745); dpy-17(e164) unc-32 (e189)  KR4822  hda-4 (ok518); dpy-18 (e364) unc-64 (e246)  KR4819  hda-4 (ok518); dpy-11 (e224) unc-42 (e270)  KR4840  hda-4 (ok518); dpy-5 (e61) unc-13 (e51)  KR4824  dpy-17(e164) unc-32 (e189)  dpy, dumpy; unc, uncoordinated; lin, abnormal cell lineage; rec, abnormal recombination; hda, histone deacetylase.  
  49  Table A3. Crossing over in Lin-35 and Rec-1 mutant strains along Chromosome III. Data includes multiple isolate labeled with strain numbers. Recombinants wild-type progenya  Dpy  Unc  Total  px 100  95% C.I.b  dpy-17 unc-32/+ +  2008  21  27  48  1.88  1.36-2.47  dpy-17 unc-32/+ +; rec-1/rec-1  2033  64  74  138  5.11  4.35-6.22  dpy-17 unc-32/+ +; lin-35/lin-35 (Isolate 1)  1127  22  24  46  3.45  2.55-4.56  dpy-17 unc-32/+ +; lin-35/lin-35 (Isolate 2)  884  18  21  39  3.22  2.28-4.34  dpy-17 unc-32/+ +; lin-35/lin-35 (Combined)  2011  40  45  85  3.35  2.62-4.12  dpy-1 unc-45/+ +  1884  141  148  289  11.3  9.9-12.7  dpy-1 unc-45/+ +; rec-1/rec-1 (Isolate 1)  1269  55  51  106  6.12  4.92-7.33  dpy-1 unc-45/+ +; rec-1/rec-1 (Isolate 2)  752  24  29  53  5.23  3.92-6.81  dpy-1 unc-45/+ +; rec-1/rec-1 (Combined)  2021  79  80  159  5.79  4.88-6.74  dpy-1 unc-45/+ +; lin-35/lin-35  1414  63  57  120  6.38  5.25-7.65  dpy-17 unc-45/+ +  2052  276  320  596  22.3  21.4-23.1  dpy-17 unc-45/++; rec-1/rec-1 (Isolate 1)  1477  201  191  392  21.2  20.1-22.0  dpy-17 unc-45/+ +; rec-1/rec-1 (Isolate 2)  560  124  77  201  27.6  23.2-32.2  dpy-17 unc-45/+ +; rec-1/rec-1 (Combined)  2037  325  268  593  22.9  22.0-23.8  dpy-17 unc-45/+ +; lin-35/lin-35  1450  197  162  359  21.0  20.0-22.0  dpy-17 unc-64/+ +  1812  343  302  645  26.5  25.3-27.4  dpy-17 unc-64/+ +; rec-1/rec-1 (Isolate 1)  943  137  179  316  24.9  23.6-26.4  dpy-17 unc-64/+ +; rec-1/rec-1 (Isolate 2)  920  138  182  321  25.9  24.6-27.4  dpy-17 unc-64/+ +; rec-1/rec-1 (Combined)  2004  290  373  663  24.8  23.9-25.8  dpy-17 unc-64/+ +; lin-35/lin-35  702  109  101  210  21.3  18.5-25.1  dpy-17 unc-64/+ +; lin-35 rec-1/lin-35 rec-1  2083  186  192  378  14.7  14.0-15.3  Genotype  a b  
  Male progeny included. C.I. = 95% confidence interval; See Materials and Methods. 50  

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