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

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

Les outils du génie génétique

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La génétique moléculaire des levures

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The genetics of RNA polymerases in yeasts.

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