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The alleles cse1-1 and cse2-l were cold-sensitive yeast mutants that display increased mitotic chromosome nondisjunction phenotypes. Two high dosage suppressors, SCM1 and SCM2, were isolated which suppress the cold-sensitive phenotypes of cse1-1 and cse2-1 cells, respectively. DNA sequence analysis revealed that SCM1 is identical to SRP1, which was previously isolated as an extragenic suppressor of an RNA polymerase I mutant. SRP1 encodes a 67 kD protein containing an arm (armadillo) motif that localizes to the periphery of the nucleus. Mutational analysis indicates that SRP1 is essential for viability and that the C-terminus is important for function. Genetic analysis indicates that SRP1 and CSE1 functionally interact in chromosome transmission. In addition, a putative mouse homolog of SRP1 called MSG1 was isolated, which also contains an arm motif in the central domain. Overexpression of MSG1 suppresses the cold-sensitive phenotype of cse1-1 cells suggesting a possible functional similarity between SRP1 and MSG1. In addition to the cold-sensitive and chromosome missegregation phenotypes, cse2-1 also causes temperature sensitivity. Overexpression of SCM2 only suppresses the cold-sensitive phenotype. SCM2 encodes a 65 kD protein that is highly homologous to known amino acid permeases. Disruption of SCM2 causes slow growth. The scm2$\Delta$ trp1-$\Delta$101 double mutant cells exhibit a synthetic cold-sensitive phenotype and grow much more slowly at the permissive temperature than cells with either a scm2$\Delta$ or trp1-$\Delta$101 single mutation. Amino acid uptake assays revealed a decreased accumulation of tryptophan in the scm$\Delta$ cells indicating that SCM2 encodes a tryptophan permease. A temperature-sensitive allele of phosphoinositide-specific phospholipase C gene in yeast, plc1-1, causes a 9-fold increase in chromosome missegregation. The temperature sensitivity and the chromosome missegregation phenotypes of plc1-1 cells were both partially suppressed by calcium. The mutant plc1-1 allele was cloned and found to encode a single amino acid substitution in the protein. The yeast PLC1 protein contains a putative EF-hand calcium binding domain and the enzymatic activity of PLC1 is dependent upon calcium. Five EF-hand mutants have been generated. Three of these mutants exhibit a temperature-sensitive phenotype that can be suppressed by calcium. The effects of the plc1 mutations on chromosome segregation and cell lysis were also investigated.

Protein-bound and free lysine contributed to the total lysine content of maize endosperm, and both of these can be significantly increased by the opaque-2 (o2) mutation. Elongation factor 1A is one of the lysine-rich proteins increased in o2 mutants, and its concentration is highly correlated with the protein-bound lysine content of the endosperm. However, the biological basis of this correlation is unknown. The mechanism by which the free amino acid content, including free lysine, is increased by the o2 mutation is also poorly understood. Understanding the biological basis of these traits could provide new insights for improving maize nutritional quality. A maize genetic linkage map consisting of 83 DNA simple sequence repeat markers was created for two inbred lines (Oh51Ao2 and Oh545o2) that differ in elongation factor 1A and free amino acid content. Quantitative trait locus analysis was performed and identified two significant loci that accounted for 25% of the variance for elongation factor 1A content. One of them was linked with a cluster of 22-kD α-zein genes on the short arm of chromosome 4; the other locus was on the long arm of chromosome 7 and may be linked to the 27-kD γ-zein genes. Quantification of protein and mRNA levels of the major storage proteins suggested that a higher level of α-zein gene expression co-segregates with higher elongation factor 1A content. Furthermore, measurement of protein body size and density predicted a greater protein body surface area (80% higher) in Oh51Ao2 than Oh545o2, and this may partially explain the higher level of elongation factor 1A in Oh51 Ao2 by creating a more extensive cytoskeletal network. Quantitative trait locus analysis of free amino acid content identified four loci accounting for about 46% of the variation for this trait. One locus on the long arm of chromosome 2 is tightly linked to monofunctional aspartate kinase and a bifunctional aspartate kinase-homoserine dehydrogenase gene. Biochemical characterization of these enzymes indicated the aspartate kinase in Oh545o2 is less sensitive to lysine than that in Oh51 Ao2. Consequently, aspartate kinase 2 is the more promising gene involved in this quantitative locus.

As animals develop, cell differentiation is controlled through the actions of numerous signaling pathways. Typically, differentiation signals lead to the regulation of various transcription factors, which in turn regulate expression of sets of genes. In certain cells incapable of realizing new gene product expression, alternate strategies must be used to control development. The transition of a C. elegans spermatid (which has no ribosomes) to a spermatozoon is one such example of an atypical developmental event. How does the signaling pathway that controls this cell differentiation differ from those whose endpoint is gene regulation? Are novel signaling mechanisms, used, or are ubiquitous signaling strategies merely adjusted to compensate for the inability of these cells to make new gene products? Here we describe progress towards understanding how the spermiogenesis initiation pathway is organized by using two distinct approaches. We have utilized a classical genetics approach to identify and characterize mutants with defects in sperm activation. The phenotypic and molecular characterization of two genes, the previously described spe-12 and the newly identified spe-29, are described. Based on the phenotypes of these mutants, we present a model for sperm activation in which a weak hermaphrodite-derived or strong male-derived signal can stimulate sperm activation. Additionally, we describe a microarray-based large-scale screen for genes expressed during spermatogenesis. Over 450 genes expressed only during spermatogenesis in the adult were identified. Surprisingly, almost none of these genes are present on the X chromosome, suggesting a difference in transcription between autosomes and the sex-chromosome during spermatogenesis. Characterization of select sperm genes with predicted molecular functions will further our understanding of sperm development and activation.

<p> This is a dissertation on the neurodegenerative tauopathy corticobasal degeneration (CBD). Because CBD is a rare disorder and has &lt;50% antemortem diagnostic accuracy, we sought to learn as much as possible from our invaluable resource, having the largest, single neuropathologically-confirmed CBD cohort in the world. This is a culmination of several different projects, all focused on neuropathologic, genetic, and clinical features of CBD, with the overall intent being to help patients who will suffer from this devastating disorder. Each study/chapter had a specific hypothesis, aimed to elucidate various unanswered questions about CBD. Essentially, the only possible approach to further our understanding of CBD is what we have done here: use patient tissue and DNA samples who have donated their brains to research. </p><p> <u>Richardson syndrome is one of the most common clinical presentations of CBD:</u> Clinicopathologic assessment of CBD patients presenting with Richardson syndrome (CBD-RS) (i.e. patients misdiagnosed as progressive supranuclear palsy), identified neuropathologic and clinical signs and symptoms that were able to differentiate CBD-RS from PSP patients. Digital microscopy and image analysis were used to quantify tau pathology in multiple brain regions and found CBD-CBS (corticobasal syndrome) cases had greater peri-Rolandic tau burden and CBD-RS had greater hindbrain and limbic tau pathology. CBD-RS patients exhibited a frontal/dysexecutive syndrome and urinary incontinence more often than PSP patients. We also describe an unusual variant of CBD with olivopontocerebellar atrophy, of which two of the patients presented with Richardson syndrome and had greater hindbrain tau pathology than CBD-CBS, and interestingly had significant TDP-43 (protein inclusions found in FTLD and ALS) pathology in affected regions. Regarding TDP-43 pathology in CBD, we found that >25% of cases have TDP-43 immunoreactive neuronal and glial cytoplasmic inclusions and threads, and the greatest burden was in the basal ganglia. With these studies we were able to show that Richardson syndrome is one of the most common clinical presentations of CBD.</p><p> Identification of a novel MAPT mutation (p.N410H) in a CBD case that is neuropathologically indistinguishable from sporadic CBD: We screened our autopsy-confirmed CBD cohort for MAPT mutations and identified a novel mutation in MAPT exon 13 (p.N410H) in a case that is neuropathologically indistinguishable from sporadic CBD. This is not only the first MAPT mutation identified to cause CBD, but it is also the first mutation to cause CBD. Functional studies with this mutation showed that there was decreased ability for mutant tau to promote microtubule assembly and exhibited a marked increase in filament formation in vitro. In this study we also identified two rare variants in the 3' untranslated regulatory region of MAPT that associate with CBD, and one of these variants also associated with PSP. CBD and PSP have shared and unique genetic risk factors: Our last study as part of my dissertation, is the first ever CBD genome-wide association study. We performed a discovery stage, replication stage, and meta-analysis in 152 CBD cases versus 3,111 control individuals (discovery), with 67 CBD cases versus 457 controls (replication). This identified two genome genome-wide significant associations with CBD at MAPT and KIF13B/DUSP4. Using a candidate gene approach, we tested for CBD association with the top progressive supranuclear palsy GWAS SNPs. This identified strong associations at MOBP and MAPT H1c haplotype. Another novel genetic association for CBD patients was identified at SOS1. Expression/SNP associations were tested from ~400 brain samples of cerebellum and temporal cortex and found significant associations at MAPT, MOBP, and SOS1. The genome-wide significant association at KIF13B/DUSP4 with CBD will require additional studies to determine which gene is responsible for the association signal. In summary, these findings show for the first time, that CBD and PSP share common genetic variation, other than MAPT, at MOBP which confers disease risk. Together, warranting future studies to understand the mechanism by which MOBP contributes to the CBD and PSP disease processes.</p>

The molecular mechanisms underlying plant reproductive development are poorly understood. Segregation distortion has recently been employed as a screening criterion to identify genes required for the development and function of the gametophyte generation. This dissertation presents analyses of the genetic, developmental, and molecular basis of the segregation distortion used to identify one such Arabidopsis thaliana mutant, gametophytic factor 1 (gfa1). The genetic basis of segregation distortion was examined by quantifying transmission of two gfa1 alleles in reciprocal crossed to wildtype. Both alleles displayed a complete lack of transmission of the gfa1 allele through the female parent, and a significant reduction of transmission through the male parent. In addition, self-pollinated siliques had a reduced seed set phenotype characteristic of a female gametophytic mutation. Both alleles co-segregated with the seed set phenotype, indicating that mutations in the GFA1 gene were responsible for the seed set phenotype. The developmental basis of gfa1 segregation distortion was identified by first examining gfa1-1 male (MG) and female (FG) gametophytes using confocal microscopy. Approximately half of the FGs scored in gfa1-1/+ siliques showed abnormalities in the number and position of the nuclei within the FG, as well as defects in cellularization. Although there were no obvious abnormalities in gfa1-1 pollen grains, analysis of the position of kanamycin-resistant seeds in gfa1-1/+ siliques showed that fewer gfa1-1 pollen fertilized ovules at the basal (receptacle) end of the silique. Taken together, these results suggest that gfa1 mutations affect primarily FG development and MG function. The GFA1 gene was cloned and found to belong to the U5-116 kD small nuclear ribonucleoprotein family of RNA splicing factors first identified in mammals and yeast. In addition, a second gene-family member, GFA1-like (GFL), was found in the Arabidopsis genome. GFA1 expression was found throughout plant development, and evidence of GFL expression was also found. The paternal GFA1 allele did not appear to be silenced during early seed development. Therefore, we conclude that a mutation in an essential splicing factor is responsible for the reduced transmission and seed set phenotypes observed in gfa1 mutants.