Parameters in gene conversion: An algebraic analysis of the hybrid DNA model at the gray locus of Sordaria fimicola

We have extended previous algebraic analyses of aberrant segregation at the gray locus of Sordaria fimicola (Whitehouse, 1965; Emerson, 1966; Fincham, Hill & Reeve, 1980) to the more complex situation where aberrant segregations are detected in three factor crosses involving two flanking markers. This algebra has been applied to seven gray alleles which have been extensively characterized for their pattern of gene conversion and postmeiotic segregation by Kitani & Olive (1967). It is based on seven major types of aberrant segregation which can be distinguished in the presence of flanking markers spanning the converting site, and allows us to use up to six parameters to describe hDNA formation and mismatch repair. We present solutions which predict a spectrum of aberrant segregation fitting the experimental data at the P > 0·05 level for six of the seven alleles tested. They are consistent with the following properties of hDNA at the gray locus: (1) the single stranded DNA transferred during hDNA formation has always the same chemical polarity. (2) hDNA is mostly, if not entirely, symmetric, and its probability of formation is constant over the whole gene. (3) Disparity in aberrant segregation is mostly, if not entirely due to disparity in mismatch repai

Ancient origin, functional conservation and fast evolution of DNA-dependent RNA polymerase III

RNA polymerase III contains seventeen subunits in yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and in human cells. Twelve of them are akin to the core RNA polymerase I or II. The five other are RNA polymerase III-specific and form the functionally distinct groups Rpc31-Rpc34-Rpc82 and Rpc37-Rpc53. Currently sequenced eukaryotic genomes revealed significant homology to these seventeen subunits in Fungi, Animals, Plants and Amoebozoans. Except for subunit Rpc31, this also extended to the much more distantly related genomes of Alveolates and Excavates, indicating that the complex subunit organization of RNA polymerase III emerged at a very early stage of eukaryotic evolution. The Sch.pombe subunits were expressed in S.cerevisiae null mutants and tested for growth. Ten core subunits showed heterospecific complementation, but the two largest catalytic subunits (Rpc1 and Rpc2) and all five RNA polymerase III-specific subunits (Rpc82, Rpc53, Rpc37, Rpc34 and Rpc31) were non-functional. Three highly conserved RNA polymerase III-specific domains were found in the twelve-subunit core structure. They correspond to the Rpc17-Rpc25 dimer, involved in transcription initiation, to an N-terminal domain of the largest subunit Rpc1 important to anchor Rpc31, Rpc34 and Rpc82, and to a C-terminal domain of Rpc1 that presumably holds Rpc37, Rpc53 and their Rpc11 partner

Isolation of Sets of a, α, a/α, a/a and α/α isogenic strains in Saccharomyces cerevisiae

A simple, quick technique for isolating sets of a, α, a/α, a/a and α/α isogenic strains of the yeast, Saccharomyces cerevisiae is described. Isogenic a/α diploids arise in haploid populations by a rare heterothallic switch of mating type followed by mating of the switched cell with one of the other cells in the population. Sucrose density gradient centrifugation was used to select for large elliptical diploid cells in a population of smaller haploid cells, since diploid cells are larger and more oval than haploid cells. From an a/α diploid strain obtained in this manner, ala and α/α cells were isolated by selecting for mating ability using a procedure similar to marker recovery. Finally isogenic a and α haploids were simply obtained by sporulation and dissection of an a/α isogenic diploid strain.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46978/1/294_2004_Article_BF00397636.pd

Non-canonical DNA transcription enzymes and the conservation of two-barrel RNA polymerases

DNA transcription depends on multimeric RNA polymerases that are exceptionally conserved in all cellular organisms, with an active site region of >500 amino acids mainly harboured by their Rpb1 and Rpb2 subunits. Together with the distantly related eukaryotic RNA-dependent polymerases involved in gene silencing, they form a monophyletic family of ribonucleotide polymerases with a similarly organized active site region based on two double-Ψ barrels. Recent viral and phage genome sequencing have added a surprising variety of putative nucleotide polymerases to this protein family. These proteins have highly divergent subunit composition and amino acid sequences, but always contain eight invariant amino acids forming a universally conserved catalytic site shared by all members of the two-barrel protein family. Moreover, the highly conserved ‘funnel’ and ‘switch 2’ components of the active site region are shared by all putative DNA-dependent RNA polymerases and may thus determine their capacity to transcribe double-stranded DNA templates

Mutations of RNA polymerase II activate key genes of the nucleoside triphosphate biosynthetic pathways

The yeast URA2 gene, encoding the rate-limiting enzyme of UTP biosynthesis, is transcriptionally activated by UTP shortage. In contrast to other genes of the UTP pathway, this activation is not governed by the Ppr1 activator. Moreover, it is not due to an increased recruitment of RNA polymerase II at the URA2 promoter, but to its much more effective progression beyond the URA2 mRNA start site(s). Regulatory mutants constitutively expressing URA2 resulted from cis-acting deletions upstream of the transcription initiator region, or from amino-acid replacements altering the RNA polymerase II Switch 1 loop domain, such as rpb1-L1397S. These two mutation classes allowed RNA polymerase to progress downstream of the URA2 mRNA start site(s). rpb1-L1397S had similar effects on IMD2 (IMP dehydrogenase) and URA8 (CTP synthase), and thus specifically activated the rate-limiting steps of UTP, GTP and CTP biosynthesis. These data suggest that the Switch 1 loop of RNA polymerase II, located at the downstream end of the transcription bubble, may operate as a specific sensor of the nucleoside triphosphates available for transcription

Maize haplotype with a helitron-amplified cytidine deaminase gene copy

BACKGROUND: Genetic maps are based on recombination of orthologous gene sequences between different strains of the same species. Therefore, it was unexpected to find extensive non-collinearity of genes between different inbred strains of maize. Interestingly, disruption of gene collinearity can be caused among others by a rolling circle-type copy and paste mechanism facilitated by Helitrons. However, understanding the role of this type of gene amplification has been hampered by the lack of finding intact gene sequences within Helitrons. RESULTS: By aligning two haplotypes of the z1C1 locus of maize we found a Helitron that contains two genes, one encoding a putative cytidine deaminase and one a hypothetical protein with part of a 40S ribosomal protein. The cytidine deaminase gene, called ZmCDA3, has been copied from the ZmCDA1 gene on maize chromosome 7 about 4.5 million years ago (mya) after maize was formed by whole-genome duplication from two progenitors. Inbred lines contain gene copies of both progenitors, the ZmCDA1 and ZmCDA2 genes. Both genes diverged when the progenitors of maize split and are derived from the same progenitor as the rice OsCDA1 gene. The ZmCDA1 and ZmCDA2 genes are both transcribed in leaf and seed tissue, but transcripts of the paralogous ZmCDA3 gene have not been found yet. Based on their protein structure the maize CDA genes encode a nucleoside deaminase that is found in bacterial systems and is distinct from the mammalian RNA and/or DNA modifying enzymes. CONCLUSION: The conservation of a paralogous gene sequence encoding a cytidine deaminase gene over 4.5 million years suggests that Helitrons could add functional gene sequences to new chromosomal positions and thereby create new haplotypes. However, the function of such paralogous gene copies cannot be essential because they are not present in all maize strains. However, it is interesting to note that maize hybrids can outperform their inbred parents. Therefore, certain haplotypes may function only in combination with other haplotypes or under specialized environmental conditions

Parameters in gene conversion: An algebraic analysis of the hybrid DNA model at the gray locus of Sordaria fimicola.

We have extended previous algebraic analyses of aberrant segregation at the gray locus of Sordaria fimicola (Whitehouse, 1965; Emerson, 1966; Fincham, Hill & Reeve, 1980) to the more complex situation where aberrant segregations are detected in three factor crosses involving two flanking markers. This algebra has been applied to seven gray alleles which have been extensively characterized for their pattern of gene conversion and postmeiotic segregation by Kitani & Olive (1967). It is based on seven major types of aberrant segregation which can be distinguished in the presence of flanking markers spanning the converting site, and allows us to use up to six parameters to describe hDNA formation and mismatch repair. We present solutions which predict a spectrum of aberrant segregation fitting the experimental data at the P > 0·05 level for six of the seven alleles tested. They are consistent with the following properties of hDNA at the gray locus: (1) the single stranded DNA transferred during hDNA formation has always the same chemical polarity. (2) hDNA is mostly, if not entirely, symmetric, and its probability of formation is constant over the whole gene. (3) Disparity in aberrant segregation is mostly, if not entirely due to disparity in mismatch repair