The budding yeast (S. cerevisiae) centromere component, Cse4p is an evolutionarily conserved histone H3-like protein, with homologues identified in fission yeast, worm, fly and human. All histone H3-like proteins have C-terminal histone fold domains (HFD) that are highly similar to the HFD of H3, but carry very different N-termini with unknown functions. The Cse4p N-terminus contains 135 residues, with a large portion of charged amino acids and a high concentration of serines within the first 22 residues. Based on the current model that suggests that Cse4p replaces H3 in a specialized centromeric nucleosome, the Cse4p N-terminus would extend out from the putative Cse4p-nucleosome and may play a variety of roles in centromere function. To elucidate the function of the Cse4p N-terminus, we conducted two comprehensive and systematic mutagenesis studies involving alanine scanning and sequence deletions, and we defined a 33-amino acid domain that is essential for cell viability and chromosome segregation. This essential N-terminal domain (END) has functions distinct from that of the HFD as demonstrated by interallelic complementation between cse4 END and HFD mutant alleles and heterodimer formation of END-HFD mutant proteins. Mutating all the potential posttranslational sites in the END indicates that the END function does not require posttranslational phosphorylation or acetylation. Genetic studies involving dosage suppression, synthetic lethality and two-hybrid analysis reveal that the END interacts with the Ctf19p/Mcm21p/Okp1p kinetochore complex. These results are consistent with the current Cse4p-nucleosome model. Although Cse4p has an HFD resembling that of H3, unlike H3, Cse4p exclusively localizes at the centromere. An important question is whether the N-terminus of Cse4p is responsible for the specific centromere targeting of the protein. Lethal Cse4p proteins lacking regions of the N-terminus can localize to the centromere in the presence or absence of wildtype Cse4p as determined by chromatin immunoprecipitation. In contrast, some lethal Cse4p HFD mutant proteins as well as chimeric proteins consisting of the Cse4p N-terminus fused to the HFD of either H3 or the Cse4p human homologue, CENP-A, fail to localize to the centromere. We conclude that the N-terminus of Cse4p is not required for centromere targeting of the protein and that the Cse4p HFD is necessary and sufficient to confer centromere localization.

An investigation of the mechanism responsible for genetic hypervariability in Halobacterium halobium gas vesicle synthesis was conducted. Four partially vacuolated mutants (Vac$\sp{\delta-})$ H. halobium mutants were analyzed by Southern hybridization, cloning, and DNA sequence analysis. In each mutant a different halobacterial insertion element was responsible for the observed phenotype. The insertions mapped upstream of the H. halobium gvpA gene. DNA sequence analysis of the 5$\sp\prime$ and 3$\sp\prime$ regions of gvpA revealed 10 open reading frames; gvpD, E, F, G, H, I, J, K, L, and M; 5$\sp\prime$ to the gvpA gene in the opposite strand and two open reading frames, gvpC and N, in the region 3$\sp\prime$ to gvpA with the same transcriptional orientation as gvpA. A study was conducted to determine if the products of the gvpA, gvpC, gvpD, gvpE, gvpF, gvpJ and gvpM genes could be detected in purified H. halobium gas vesicles or whole cell lysates using immunological techniques. To do so, LacZ-Gvp fusion proteins were produced in E. coli and used to immunize rabbits. The antisera and protein-A column purified antibodies were used in immunoblot analysis of purified gas vesicles and cell lysates. The antiserum directed against the LacZ-GvpC fusion protein was successful in identifying a protein present in both purified gas vesicles and whole cell lysates, and this indicates that the gvpC gene product is a structural gas vesicle protein. Techniques were developed to allow for genetic analysis of gas vesicle synthesis in H. halobium. An H. halobium/E. coli shuttle vector, pJHGV3, which contains the gvpA gene cluster was constructed. Transformation of Vac$\sp-$ H. halobium strains, in which the gvpA gene cluster is deleted, with pJHGV3 resulted in complementation of gas vesicle synthesis. Methods were developed to allow non-polar mutations to be introduced into gvp genes present on pJHGV3. A plasmid containing a disruption of the region 3$\sp\prime$ to the gvpN gene, pJHGV33$\sp\prime$::$\kappa,$ was constructed and used to transform Vac$\sp-$ deletion mutants. Resulting transformants were Vac$\sp+$ indicating that there are not additional contiguous gvp genes downstream from gvpN. Together these techniques will provide useful tools in further analysis of the gas vesicle structure and its assembly.