Walton Lawrence Fangman - US grants

Yeast chromosomal DNAs contain terminal interstrand cross-links. Specific fragments containing cross-links will be analyzed with enzyme probes to test the notion that a terminus has a hairpin structure. Terminal sequences will be cloned and the clones used to map this telomere DNA, to look for relatedness between different telomeres and to probe the nucleotprotein organization of the telomere. The timing and topology of telomere DNA replication will be examined using hybridization probes and electron microscopy. Control of the activation of replication origins in yeast will be studied. The 2 microns DNA plasmid will be examined for alterations associated with origin activation and its refractoriness to secondary activation. The replication of new yeast plasmids, formed from chromosomal DNA segments, will be examined to determine the extent to which their origins remain under normal replication controls. These plasmids will also be used as hybridization probes in cell synchrony and dense isotope transfer experiments to measure the temporal program of origin activation under different growth conditions. The quantitative transmission of different yeast multiple copy genetic elements through yeast meiosis and sporulation will be determined using radiolabel, dense isotope experiments. These experiments will include an assessment of the possibility that double strand RNA-containing virus-like particles are transmitted as intact structures. In addition, the general mechanism of double strand RNA replication will be determined.

Replication is central to the biological role of DNA as the molecule of inheritance. The control of DNA replication ensures that chromosomes are duplicated in a timely and precise way. All available evidence indicates that controls operate at the point of initiation of replication at individual origins scattered at high density over eukaryotic chromosomes. It is the control of replication origins that we propose to investigate. Our understanding of these controls is very poor, primarily because replication origins themselves have been elusive. Recently, two sensitive gel electrophesis techniques have been developed for identifying origins in chromosomal DNA. Our studies in the yeast Saccharomyces cerevisiae have revealed that the initiation activity of origins depends to a large extent on their chromosomal context. Three aspects of contextual control of origin use in yeast will be examined. (I) When origins are located in close tandem arrays--in plasmid multimers and in the rDNA locus--many potential origins are not used. What is responsible for the inactivity? Are there spacing constraints? And, how are the active origins chosen? (II) Replication initiation in the transcriptionally active rDNA repeats occurs in the non-transcribed spacer and replication is unidirectional with the active fork moving the transcriptional direction. Does the transcriptional activity of other chromosomal regions influence origin use? Has evolution favored the placement of origins that permits replication forks to follow transcription complexes through actively transcribed genes, rather than colliding head-on with them? (III) Chromosomal origins located near telomeres (the physical ends of chromosomes) are activated later in S phase than origins located at internal sites. What feature of telomeres influences origin activation times, and are some origins inactive because of their proximity to telomeres? Our approach to these questions involves making directed replacements and alterations in yeast plasmids and chromosomes and using our recently developed 2-D gel technique to identify active origins and to estimate the efficiency of their activation. The answers to these questions will lead to a greater understanding of the regulation of chromosome replication in normal cells and in those with altered growth properties, such as cancer cells.

The principal objective of our program is to train graduate students to function effectively as geneticists, enabling them to apply the rigor of genetic methods to pertinent issues in contemporary biology and medicine. The variety of approaches taken by the training faculty ensures a broad scope of training that encompasses microbial genetics, human and clinical genetics, yeast and plant geneticists, the developmental genetics of dipterans and nematodes, immunogenetics, aging, population genetics, as well as new directions in genomics. The predoctoral trainees supported by this program gain specific expertise from thesis research in one of these subdisciplines while gaining a broader intellectual outlook from formal coursework, first-year research rotations, and journal club. A well-established program of seminars and other forums for exchanges of research findings lends further perspective and provides valuable interaction with postdoctoral fellows, whose research training is also integrated into the program. Access to medical and clinical aspects of genetics derives both from formal coursework and from association with the Division of Medical Genetics in the Department of Medicine. A greater breadth of expertise is afforded the trainees by our affiliation with other units of the University, including the Departments of Botany and Zoology in the College of Arts and Sciences and the Departments of Biochemistry, Pathology, and Pediatrics in the School of Medicine, as well as with the Basic Sciences Division of the Fred Hutchinson Cancer Research Center.

The mechanism of replication initiation of the mitochondrial DNA (mtDNA) in the yeast Saccharomyces cerevisiae is poorly understood, in part because a high rate of recombination has made it difficult to use common physical approaches. However, the accumulated evidence suggests that two mechanisms of replication initiation may exist. One, analogous to initiation in mammalian mtDNA, may employ a promoter and transcript cleavage to generate a primer. The other mechanism that operates in the absence of transcription, may initiate replication forks through end/internal recombination events. We will examine these mechanisms using a combination of structural and genetic approaches. A new 2-D gel electrophoresis technique will be used to systematically search the mitochondrial genome for various possible initiation structures. Mutants with a temperature sensitive defect in the initiation of mtDNA replication will be isolated and characterized. Double mutants defective in transcription and in possible recombination pathways will be examined for failure to maintain mtDNA. A mutant that abolishes the biased inheritance of a class of mitochondrial deletion genomes, thought to have a replication advantage because of a high concentration of replication origins, will be studied.