No description available.

No description available.

No description available.

No description available.

No description available.

During female meiotic prophase in many organisms, a specialized chromatin structure is formed in the oocyte nucleus. This structure is known as the karyosome, and has been proposed to be important for the formation of the female meiotic bipolar spindle. However, how the karyosome is formed and maintained is not very well understood. To identify proteins involved in the formation and maintenance of the karyosome, I carried out a cytological screen on a collection of 220 mutant fly lines for mutants that were defective in karyosome morphology. The screen identified 46 mutants on the X and 2nd chromosome with abnormal karyosomes. Genetic analysis of these 46 mutants, followed by molecular analysis of one mutant, identified SRPK (SR Protein Kinase) as a protein that is important for the proper formation of the karyosome. NHK-1 (Nucleosomal Histone Kinase 1) was previously identified as a protein that is essential for the formation of the karyosome via its phosphorylation of BAF (Barrier-to-Autointegration Factor). NHK-1 phosphorylation of BAF leads to the release of chromatin from the nuclear membrane, an essential step for the formation of the karyosome, however, the regulation of this process is unclear. In order to identify genes that interact with NHK-1, I carried out a genetic modifier screen using a semi-lethal allele of NHK-1, NHK-1trip. After screening a collection of 44 deficiencies located on the 2nd chromosome, I identified a genetic region (44B8-44D1) containing a gene that interacts with NHK-1 and, when gene dosage is halved, enhanced the semi-lethal phenotype of NHK-1trip.

There is a vast diversity of karyotypes in nature, yet mechanisms that have facilitated such diversity are unclear. Alterations to an organism’s karyotype can have major negative fitness consequences in meiosis through non-disjunction and aneuploidy. Here, I investigated the role of biased segregation in female meiosis, i.e., meiotic drive, as a force that contributes to the evolution of karyotype form. The closely related species pair, Drosophila americana and Drosophila novamexicana, is an exemplar for understanding mechanisms of karyotype evolution. Since their recent divergence nearly half a million years ago, D. americana has evolved two different centromeric fusions: one fusion between the 2nd and 3rd chromosomes (Muller elements C and D), and the other fusion between the X and 4th chromosomes (Muller elements A and B). The 2-3 fusion is fixed in D. americana. However, the X-4 centromeric fusion remains polymorphic within the species. I uncovered biased transmissions for both fused chromosomes in D. americana such that the X-4 fused chromosome was inherited by 57% of the offspring from heterozygous females and the 2-3 chromosome was inherited by 62% of the offspring. Introgression experiments shoed the fused X-4 and the unfused X and 4th chromosomes are segregating at a 50/50 ratio in D. novamexicana. I have isolated the fused X-4 centromeric region as a possible player in the observed meiotic drive. However, the centromere is not sufficient to cause meiotic drive without a secondary factor. I also measured heterochromatin content between the fused and unfused X and 4th homologs. No obvious size differences were uncovered, but possible compositional differences were revealed. This suggests that if the centromere itself is involved in meiotic drive, either differences in the number of centromeres or compositional differences between the centromeres are influencing meiotic drive. Overall, I have identified and characterized meiotic drive as a force driving karyotype evolution in D. americana but appears to be absent in D. novamexicana, and I have begun to dissect the mechanisms of meiotic drive.

p53 promotes maintenance of genetic information either by causing apoptosis of damaged cells, or by altering the cell cycle and repair pathways such that damage can be accurately repaired. The nematode Caenorhabditis elegans possesses only one p53 family member, CEP-1, that controls apoptosis and the cell cycle in response to genotoxic stress. Mutation in the meiotic gene him-5 increases nondisjunction of the X chromosome, resulting in increased frequencies of XO male and XXX Dpy progeny, and it affects the frequency of meiotic recombination on X. him-5 is allelic to the ORF D1086.4, which encodes a putative basic protein with no clear homologues or domain structure. The modest embryonic lethality (Emb) of him-5 mutants is dramatically increased by mutation of cep-1 but no change is seen in the proportion of XO male or XXX Dpy progeny. The synergistic effects of cep-1 and him-5 mutation are independent of CEP-1's DNA damage regulators and other meiotic mutants, and they do not involve deregulated apoptosis. cep-1; him-5 double mutants have abnormal chromatin morphology in diakinesis-arrested oocytes reminiscent of that seen in double strand break (DSB) repair mutants. This phenotype depends on the presence of SPO-11-induced meiotic DSBs, suggesting CEP-1 and HIM-5 function together to promote accurate recombination during meiosis. In support of this hypothesis, cep-1; him-5 show a significant reduction in crossover frequency between autosomal markers compared to wild-type or either single mutant alone, suggesting they function together to promote meiotic crossing over. The X chromosome nondisjunction in both him-5 and cep-1; him-5 is a result of failure of DSB formation and subsequent chiasma formation on the X. However, the embryonic lethality phenotype of him-5 and cep-1; him-5 is caused by a defect either downstream or in parallel to meiotic DSB formation. The diakinesis chromatin phenotype of cep-1; him-5 suggests this defect may be in meiotic DSB repair. This is confirmed by the fact that cep-1; him-5 animals show more persistent meiotic DSB-associated RAD-51 foci staining compared to wild-type, suggesting CEP-1 and HIM-5 may function in efficient resolution of SPO-11-induced DSBs during meiosis. A role for CEP-1 in promoting accurate repair of DSBs during meiosis may be related to p53's function in promoting faithful meiotic recombination in mammalian cells. HIM-5's role in DSB formation and repair suggests another mechanistic link between these recombination steps. Meiotic recombination is vital for genome stability, and characterization of the role of CEP-1 and HIM-5 will increase our understanding of the p53 family and genetic redundancy at multiple steps in this process.

p53 promotes maintenance of genetic information either by causing apoptosis of damaged cells, or by altering the cell cycle and repair pathways such that damage can be accurately repaired. The nematode Caenorhabditis elegans possesses only one p53 family member, CEP-1, that controls apoptosis and the cell cycle in response to genotoxic stress. Mutation in the meiotic gene him-5 increases nondisjunction of the X chromosome, resulting in increased frequencies of XO male and XXX Dpy progeny, and it affects the frequency of meiotic recombination on X. him-5 is allelic to the ORF D1086.4, which encodes a putative basic protein with no clear homologues or domain structure. The modest embryonic lethality (Emb) of him-5 mutants is dramatically increased by mutation of cep-1 but no change is seen in the proportion of XO male or XXX Dpy progeny. The synergistic effects of cep-1 and him-5 mutation are independent of CEP-1's DNA damage regulators and other meiotic mutants, and they do not involve deregulated apoptosis. cep-1; him-5 double mutants have abnormal chromatin morphology in diakinesis-arrested oocytes reminiscent of that seen in double strand break (DSB) repair mutants. This phenotype depends on the presence of SPO-11-induced meiotic DSBs, suggesting CEP-1 and HIM-5 function together to promote accurate recombination during meiosis. In support of this hypothesis, cep-1; him-5 show a significant reduction in crossover frequency between autosomal markers compared to wild-type or either single mutant alone, suggesting they function together to promote meiotic crossing over. The X chromosome nondisjunction in both him-5 and cep-1; him-5 is a result of failure of DSB formation and subsequent chiasma formation on the X. However, the embryonic lethality phenotype of him-5 and cep-1; him-5 is caused by a defect either downstream or in parallel to meiotic DSB formation. The diakinesis chromatin phenotype of cep-1; him-5 suggests this defect may be in meiotic DSB repair. This is confirmed by the fact that cep-1; him-5 animals show more persistent meiotic DSB-associated RAD-51 foci staining compared to wild-type, suggesting CEP-1 and HIM-5 may function in efficient resolution of SPO-11-induced DSBs during meiosis. A role for CEP-1 in promoting accurate repair of DSBs during meiosis may be related to p53's function in promoting faithful meiotic recombination in mammalian cells. HIM-5's role in DSB formation and repair suggests another mechanistic link between these recombination steps. Meiotic recombination is vital for genome stability, and characterization of the role of CEP-1 and HIM-5 will increase our understanding of the p53 family and genetic redundancy at multiple steps in this process.

The presence of an unpaired copy of a gene during meiosis triggers the silencing of every copy of that gene in the diploid ascus cell of Neurospora crassa, a phenomenon called Meiotic Silencing. This phenomenon has two stages: trans-sensing and meiotic silencing. If a DNA region is not detected on the opposite homologous chromosome early in meiosis (a trans-sensing failure), a signal corresponding to the unpaired region is produced that transiently silences expression of all homologous sequences. Meiotic silencing is related to RNA Silencing, a phenomenon that employs RNA-dependent RNA Polymerases (RdRPs), Argonautes, and Dicers. Dicers cleave double-stranded RNA (dsRNA) into 21-23 nucleotide RNAs. In the filamentous fungus Neurospora crassa, two RNA silencing pathways have been identified; one is active during mitosis, and the other is active during meiosis. The mitotic RNA silencing pathway, known as “quelling”, involves an RdRP (quelling-deficient-1--qde-1), an Argonaute-like protein (quelling-deficient-2--qde-2), and two Dicer-like proteins (dicer-like-1--dcl-1 and dicer-like-2--dcl-2). Previous studies in N. crassa also revealed the involvement of an RdRP (Suppressor of ascus dominance-1--Sad-1) and an Argonaute-like protein (Suppressor of meiotic silencing-2--Sms-2) in meiotic silencing, suggesting that meiotic silencing is RNA-dependent and raising the question of whether a Dicer is involved in meiotic silencing. In this work, we tested the participation in meiotic silencing of the dcl-1 gene of N. crassa, which codes for a Dicer-like protein we call Suppressor of meiotic silencing-3--Sms-3. Crosses homozygous for mutant alleles of Sms-3 are barren, indicating that the gene is also essential for sexual development. Due to this homozygous sterility, we could only test the involvement of Sms-3 in meiotic silencing in heterozygous crosses. Under these conditions, we observed suppression of the meiotic silencing which would have otherwise been induced by the presence of unpaired DNA of reporter genes. We conclude that the Dicerlike protein Sms-3 is required for both meiotic RNA silencing and sexual development.