Molecular Determinants of Faithful Chromosome Transmission and Cell Cycle Checkpoint Regulation. A fundamental requirement of the cell division cycle is the maintenance, replication, and segregation of chromosomal DNA. Failure of complex mechanisms involved in maintaining genome integrity has been implicated in cancer, aging, and congenital birth defects. Research in our laboratory focuses on the molecular mechanisms of high fidelity chromosome transmission, the organization of chromatin structure, and the checkpoint regulatory mechanisms that ensure the proper execution of the cell cycle in Saccharomyces cerevisiae and the study of its human homologs. We are also interested in the application of genome technologies such as serial analysis of gene expression (SAGE) and DNA microarrays for analysis of transcription profiles and the identification of small non-annotated open reading frames (NORFs).Molecular Determinants of Faithful Chromosome Transmission. CEN DNA sequences and the trans-acting kinetochore components (centromere-specific DNA binding proteins) are required for high fidelity chromosome transmission. Additionally, a higher order chromatin structure provides a framework for interactions of histones, CEN DNA, and the kinetochore. We have used genetic analysis to characterize genes that are required for the structure/function of the kinetochore and studied the corresponding human homolog. We analyzed the phenotype of the ctf (chromosome transmission fidelity) mutants in secondary genetic screens for kinetochore integrity and identified five putative kinetochore mutants. We have studied two of these mutants, s138 and s141. The gene complementing the s138 mutation was shown to be the S. cerevisiae SPT4 gene. We showed that the spt4 mutants exhibit genetic interactions with mutations in cis-acting CEN DNA sequences and trans-acting kinetochore proteins. We have recently established that Spt4p is a component of centromeric and heterochromatic chromatin with roles in kinetochore function and gene silencing. A human homolog of SPT4, HsSPT4, is able to functionally complement the spt, chromosome missegregation and silencing phenotypes of S. cerevisiae spt4mutants. These studies represent one of the first examples of the functional complementation of silencing defects of a yeast mutant (spt4) by a human gene (HsSPT4) and the in vivo, association of a human protein to the kinetochores of budding yeast. Our results highlight the evolutionary conservation of pathways required for genome stability in yeast and humans and demonstrate that the yeast model system can be used to study the fundamental process of chromosome segregation. Further studies of the second putative kinetochore mutant s141 showed that the mutation is allelic to a nucleoporin mutation nup170. The S. cerevisiae NUP170 and NUP157 genes are highly homologous to each other and have a mammalian counterpart, NUP155. In collaborative efforts, we have established important physical and functional link between members of the Nup170p protein complex and mitotic spindle checkpoint proteins Mad1p and Mad2p. We have also defined the minimal domain of Mad1p that is required for association with the nuclear pore complex and checkpoint and chromosome segregation functions. These studies as well other results from our laboratory have led to a comprehensive study on the localization of the spindle checkpoint proteins, Mad1p, Mad2p and Bub3p and demonstrated for the first time that a spindle checkpoint protein can associate in vivo with a single defective kinetochore. Future studies will help us elucidate the domains of checkpoint proteins required for chromosome segregation, checkpoint and nucleopore functions.Cell cycle checkpoint responses to DNA damage and replication arrest. We have used SAGE (Serial Analysis of Gene Expression) to identify, quantitate, and compare global gene expression patterns from hydroxyurea-arrested (S-phase), nocodozole-arrested (G2/M phase), and logarithmically growing cells of S. cerevisiae. SAGE analysis was done in collaborative efforts with Dr. Hieter, Dr. Kinzler, Dr. Velculescu and Dr. Vogelstein. SAGE has permitted the identification of at least 302 previously unidentified transcripts from NORFs corresponding to proteins with <100 amino acids, some of which are expressed in a cell cycle-regulated manner. The genome sequencing efforts have not annotated any ORF with <100 amino acids in length. Several of the NORF genes are evolutionarily conserved and have homologs in either human, mouse, or C. elegans. Further studies have shown that transcription of one of these, NORF5/HUG1 (hydroxyurea, ultraviolet, gamma induced), is induced by DNA damage and this induction requires MEC1, a homolog of the ataxia telangiectasia-mutated (ATM) gene and genes in the MEC1 pathway. Overexpression of HUG1 is lethal in the presence of DNA damage or replication arrest. A deletion of HUG1 rescues the lethality due to a mec1 null allele. Future studies will shed light on the role of HUG1 in the DNA damage and replication arrest-induced pathways regulated by cell cycle checkpoint genes. Identification of a human homolog of HUG1 may further our understanding of similar pathways in humans.We have also recently established a novel and functional relationship between the oxidative stress genes SOD1 and its copper chaperone LYS7 and the MEC1 pathway. Our results establish that signaling through the MEC1 pathway is sensitive to the redox balance in the cell. DNA microarrays for study of chromosome structure and function. The S. cerevisiae microarray project initiated in FY2000 as an institutional collaboration between our laboratory and four other NCI laboratories (PIs M. Lichten, D. Garfinkel, J Strathern and C. Wu). The primary aim of this project was to construct microarrays containing all the open reading frames (ORFs) and intergenic regions (IGRs) from the budding yeast genome. We have completed the construction of these arrays by PCR amplification of the genome. These arrays are being used for study of chromosome structure, function and the identification of NORFS (non-annotated ORFs) and non-coding RNAs. Small is Beautiful and Meaningful: Identification and Characterization of Non-Annotated ORFs (NORFs) in S. cerevisiae. Annotation of the S. cerevisiae genome revealed 6275 ORFs which includes genes that were previously characterized and those that encode for proteins of at least 100 amino acids. Computational identification of small ORFs (smORFs) (<100 amino acids) based on sequence analysis alone is severely limited by high false positive rates. Also, smORFs have been missed in traditional genetic screens due to their small target size. Hence, it is a challenge to identify small genes which encode for biologically important class of molecules and are """"""""buried"""""""" in an enormous pile of meaningless short ORFs. Evidence for the presence of hundreds of smORFs has been accumulating using independent approaches. These data are derived primarily from two approaches: (a) RNA and protein based expression analysis and (b) comparative genomics. In the first of such studies, Serial Analysis of Gene Expression (SAGE) revealed over 300 small NORFs that were expressed at more than one copy per cell. Microarrays and global analysis of proteins confirmed and extended the SAGE study to validate the presence of NORFs. NORFs were also identified based on expression of a gene containing a transposon bearing a lacZ reporter. In a second approach, comparitive genomics identified new NORFs and showed that a subset of NORFs identified through expression analyses are evolutionarily conserved between different fungal species including Ashbyii gossypii
