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 regulatory mechanisms that ensure the proper execution of the cell cycle in Saccharomyces cerevisiae and study of its human homologs. We are also interested in the application of genome technologies such as SAGE and CHIPS 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, higher order chromatin structure provides a framework for interactions of histones, CEN DNA and the kinetochore. We have characterized genes that are important 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 genetic screens for kinetochore integrity. From a total of 29 ctf mutants, five ctf mutants tested positive in these screens. We have studied two of these mutants, s138 and s141 and will pursue studies on the other three putative kinetochore mutants in the future. 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. In collaborative efforts with Dr. Fred Winston's laboratory we have shown that a human homolog of SPT4, HsSPT4, is able to functionally complement the spt- as well as the chromosome missegregation phenotypes of S. cerevisiae spt4 mutants. We are studying the role of Spt4p in chromosome transmission and would like to determine if Spt4p is a component of the heterochromatin that may be present at the S. cerevisiae centromere. We will also use yeast cDNA microarrays to identify downstream targets of Spt4p. 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. We have determined that the chromosome missegregation phenotype of nup170 mutants may be due to defects in kinetochore integrity. Unlike several other nucleoporins, Nup170p is a unique nucleoporin required for chromosome segregation. We are currently pursuing studies to determine the molecular role of Nup170p in chromosome transmission and spindle checkpoint function. 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. Vogelstein and Dr. Kinzler (Johns Hopkins Univ.). SAGE has permitted the identification of at least 302 previously unidentified transcripts from NORFs (Non-Annotated Open Reading Frames) corresponding to proteins with <100 amino acids some of which are expressed in cell-cycle regulated manner. The genome sequencing efforts have not annotated any ORF <100 amino acids in length. Several of the NORF (Nonannotated ORF) 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 ataxia telangiectasia mutated (ATM) gene and genes in the MEC1 pathway. Overexpression of HUG1 is lethal in combination with a mec1 mutation in the presence of DNA damage or replication arrest, whereas a deletion of HUG1 rescues the lethality due to a mec1 null allele. We are currently pursuing approaches to determine the molecular role of HUG1 in the checkpoint mediated pathway to DNA damage and replication arrest. Identification of a human homolog of HUG1 may further our understanding on similar pathways in humans. DNA microarrays for study of chromosome structure and function The Saccharomyces cerevisiae microarray project was initiated in FY2000 as an intramural collaboration between our laboratory and four laboratories in the Division of Basic Science (PIs M. Lichten, D. Garfinkel, J. Strathern and C. Wu). The primary aim of this project is to manufacture microarrays containing all the open reading frames (ORFs) and intergenic regions (IGRs) in the Saccharomyces cerevisiae genome. Our group and the Lichten group are the lead laboratories on this project, with primary responsibility for microarray manufacture. We have successfully completed the PCR amplification of about 6,200 ORFs and IGRs from the yeast genome. The ORFs have be spotted on glass slides at the NCI Advanced Technology Center. The ORF microarrays are currently being tested for optimization and data analysis. The arrays will be used by the five collaborating laboratories for the analysis of yeast gene expression and chromosome structure as part of their research programs. In the project's initial phase, we amplified ORFs and IGRs covering all of chromosme III of S. cerevisiae (about 300 PCR products), which were then used to manufacture a chromosome III-only array. The main purpose of this initial phase was to trouble-shoot and optimize all aspects of the array manufacture process. The resulting chromosome III arrays have already been used by the Lichten laboratory to examine meiotic chromosome structure (Gerton JL, DeRisi J, Shroff R, Lichten M, Brown PO, Petes TD, Proc. Natl. Acad. Sci, USA 97:11383-90). The IGRs will be spotted along with the ORFs to create a whole-genome array. These arrays will be important for the study of yeast chromosome structure, and for the analysis of expression of non-annotated ORFs (NORFs) and of non-coding RNAs, neither of which are contained in ORF-only arrays.
