The proposed research represents an integrated genetic and biochemical approach to mtDNA recombination in Saccharomyces cervisiae. Particular emphasis will be given to the analysis of the molecular features of non-reciprocal recombination at the 21S rRNA locus. Evidence will be sought for the suggestion from preliminary data that an open reading frame within a 1.1 kb intron of one of the 21S rRNA alleles encodes a trans-acting factor required for non-reciprocal recombination. We posit that this factor, perhaps with other proteins that may be associated with it, introduces a site specific double-strand cut in the intron-less 21S allele and that this double-strand cut is an intermediate in the non-reciprocal exchange. This proposition will be examined by the analysis of mutants of both alleles of the 21S gene, in vitro biochemical studies to reproduce aspects of the in vivo phenomenon, immunological approaches to identify and characterize the putative ORF product, and by comparisons of the properties of non-reciprocal exchange at the 21S locus in S. cerevisiae with reciprocal recombination occurring at other loci in the mitochondrial genome. Additional studies are proposed to take advantage of methodology we have developed to introduce productively into S. cerevisiae cells, the mitochondrial genomes of various industrial species that harbor useful polymorphisms and genetic properties. These studies include measurements of the gradient of flanking sequence co-conversion associated with intron transfer and how sequence variations in the 21S rRNA gene in strains which lack the intron, but yield atypical outputs in heteropolar crosses, affect recombinational events there. Finally, studies are proposed to develop methods to obtain and characterize nuclear mutants with defects in general mtDNA recombination and to initiate studies on the molecular mechanisms of var1 recombination--the only other locus on the yeast mitochondrial genome that is known to undergo non-reciprocal exchange.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM035510-01
Application #
3288388
Study Section
Genetics Study Section (GEN)
Project Start
1985-09-05
Project End
1990-08-31
Budget Start
1985-09-05
Budget End
1986-08-31
Support Year
1
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Type
Overall Medical
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Henke, R M; Butow, R A; Perlman, P S (1995) Maturase and endonuclease functions depend on separate conserved domains of the bifunctional protein encoded by the group I intron aI4 alpha of yeast mitochondrial DNA. EMBO J 14:5094-9
Kennell, J C; Moran, J V; Perlman, P S et al. (1993) Reverse transcriptase activity associated with maturase-encoding group II introns in yeast mitochondria. Cell 73:133-46
Moran, J V; Wernette, C M; Mecklenburg, K L et al. (1992) Intron 5 alpha of the COXI gene of yeast mitochondrial DNA is a mobile group I intron. Nucleic Acids Res 20:4069-76
Wernette, C; Saldanha, R; Smith, D et al. (1992) Complex recognition site for the group I intron-encoded endonuclease I-SceII. Mol Cell Biol 12:716-23
Wenzlau, J M; Perlman, P S (1990) Mobility of two optional G + C-rich clusters of the var1 gene of yeast mitochondrial DNA. Genetics 126:53-62
Anziano, P Q; Moran, J V; Gerber, D et al. (1990) Novel hybrid maturases in unstable pseudorevertants of maturaseless mutants of yeast mitochondrial DNA. Nucleic Acids Res 18:3233-9
Wernette, C M; Saldahna, R; Perlman, P S et al. (1990) Purification of a site-specific endonuclease, I-Sce II, encoded by intron 4 alpha of the mitochondrial coxI gene of Saccharomyces cerevisiae. J Biol Chem 265:18976-82
Perlman, P S; Butow, R A (1989) Mobile introns and intron-encoded proteins. Science 246:1106-9
Wenzlau, J M; Saldanha, R J; Butow, R A et al. (1989) A latent intron-encoded maturase is also an endonuclease needed for intron mobility. Cell 56:421-30
Zinn, A R; Pohlman, J K; Perlman, P S et al. (1988) In vivo double-strand breaks occur at recombinogenic G + C-rich sequences in the yeast mitochondrial genome. Proc Natl Acad Sci U S A 85:2686-90

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