Philip S. Perlman - US grants

The proposal is concerned with the mechanism underlying two important phenomena characteristic of mitochondrial genetics in yeast (Saccharomyces cerevisiae). These phenomena, polarity and suppressiveness, are characterized by the net loss of mitochondrial gene markers contributed by one parent in zygotes heterozygous for the mitochondrial omega locus or for petite mutations. We will test a new, specific hypothesis of the mechanism for this loss. Our hypothesis states that such cells are heterozygous for deletions or duplications of mitochondrial genes; during recombination, heteroduplexes, are formed in the mitochondrial DNA regions containing the deletions or duplications and a single stranded loop is formed. The preferential excision and repair of this loop leads to the loss of the gene markers contained in the longer genome and the conversion of closely linked markers. Experimental approaches are: (1) genetic experiments designed to test the postulate that recombination is required for both polarity and suppressiveness; (2) the genetic characterization of classes of petite mutants; followed by (3) the use of restriction endonuclease analysis of mtDNA to detect and map the hypothetical duplications or deletions; (4) the isolation and mapping of new mutations in the polarity region of mtDNA; (5) the analysis of mutant alleles of the omega locus. We propose that yeast mitochondria are a good system for investigating the general phenomenon of recombination involving duplications, deletions and frameshift mutations. Additionally these studies will contribute new insights to the fine structure mapping of yeast mtDNA.

This project will employ genetic and molecular approaches to analyze the structure and function of introns and the mechanism of RNA splicing using yeast mitochondria as the experimental system. Sequence analysis of existing and new cis-acting splicing defective mutations in introns 4 and 5 of the cytochrome b (cob) gene will be employed to define those intron sequences that serve as portions of the splicing substrate. The role of those sequences in splicing reactions will be assessed by characterizing partial revertants in experiments especially designed to learn the role of secondary and higher order structures in the splicing of those introns. The positions of splice junctions of those introns (and others) will be determined by cDNA sequencing and such sequences will be compared to learn whether one or more consensus sequences exist in this system. It already appears that such common splice junction sequences found in nuclear systems are different from those in this system. We will refine our current model of the expression of trans-acting splicing factors encoded by cob intron 4 by testing several submodels explicitly. We will also test the generality of the concept that introns which contain open reading frames encode intron specific splicing factors (maturases) by analyzing the four translatable introns of the oxi3 gene. Nuclear genes whose products participate in mitochondrial RNA splicing will be identified by the isolation of nuclear suppressors of mitochondrial splicing defective mutants. Those suppressors will be used to identify null alleles at the same loci; that is, we will use a novel genetic screening method to isolate nuclear mutants defective in splicing mitochondrial introns. Such mutants should be very useful in future attempts to study the RNA splicing apparatus in detail. These studies should lay a solid foundation for detailed molecular studies of the mechanism of RNA splicing in yeast mitochondria.

This is an application for partial funding of a Conference on Yeast RNA: Transcription, Splicing, Translation, Replication and Transposition, being held under the auspices of the Federation of Americal Societies for Experimental Biology (FASEB), from June 12-17, 1988 at the Vermont Academy, Saxton's River, Vermont. Participation will be limited to 155 scientist applicants who will be selected on the basis of their expertise and interests as being the most likely to help create a stimulating and informative meeting. The topic of the meeting emphasizes research on the roles of RNA molecules in biological processes using yeast as the experimental organism. While most participants will use baker's yeast, Saccharomyces cerevisiae, as an experimental system, scientists using other yeasts or filamentous fungi will be encouraged to participate. The conference will consist of nine scientific sessions, each with four speakers and a discussion leader; in addition, one or two afternoons will be devoted to poster sessions and/or informal group discussions. Most speakers will be invited in advance but about one speaker per session will be selected from among the enrolled participants. One session will be devoted to discussions of recent developments in RNA transposition, RNA replication and translation; two will be devoted to RNA processing and three to transcription. The ninth session will be determined later; it will either expand the depth of treatment of one of the above topics or deal with a new topic not presently included.

Splicing and genetic properties of group II introns will be investigated in mitochondria of bakers yeast, Saccharomyces cerevisiae. Introns I and 2 of the COXI gene of mtDNA code for a protein with reverse transcriptase (RT), endonuclease and maturase (splicing) functions essential for the splicing and homing/transposition by the introns. Both introns are highly efficient, site-specific retroelements clearly related to non-LTR- retrotransposons, including the LINE elements of the human genome. Major and minor intron homing pathways have been identified, all of which depend on a remarkable reaction in which a complex containing the intron-encoded RT and excised intron RNA lariat inserts the intron RNA into the sense strand of the intron-less DNA target site by a reverse splicing reaction. Subsequent steps vary widely among various RT- independent pathways. Bakers yeast has numerous attributes that make it a powerful and facile system for research on mechanisms of group II intron homing, and for the mechanisms of site-specific homing by group II introns to obtain a clear view of how the various pathways differ and, or importantly, to show how a common homing intermediate is partitioned among processes, transposition was likely a mechanism by which introns spread during evolution. A system will be developed to detect retrotransposition of a tagged intron from mitochondria to the nucleus. The third aim is to characterize aspects of group II intron molecular biology. The main emphasis is to identify nuclear will analyze genes that process the precursor to the intron-encoded RT, control the stability and degradation of excised group II intron RNAs, and assist the self-splicing of the introns. Finally, the RNA binding and splicing functions of the naturase domain of the intron-encoded protein will be studied.