A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.

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Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.

Yeast Sir2 is a heterochromatin component that silences transcription at silent mating loci, telomeres and the ribosomal DNA, and that also suppresses recombination in the rDNA and extends replicative life span. Mutational studies indicate that lysine 16 in the amino-terminal tail of histone H4 and lysines 9, 14 and 18 in H3 are critically important in silencing, whereas lysines 5, 8 and 12 of H4 have more redundant functions. Lysines 9 and 14 of histone H3 and lysines 5, 8 and 16 of H4 are acetylated in active chromatin and hypoacetylated in silenced chromatin, and overexpression of Sir2 promotes global deacetylation of histones, indicating that Sir2 may be a histone deacetylase. Deacetylation of lysine 16 of H4 is necessary for binding the silencing protein, Sir3. Here we show that yeast and mouse Sir2 proteins are nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases, which deacetylate lysines 9 and 14 of H3 and specifically lysine 16 of H4. Our analysis of two SIR2 mutations supports the idea that this deacetylase activity accounts for silencing, recombination suppression and extension of life span in vivo. These findings provide a molecular framework of NAD-dependent histone deacetylation that connects metabolism, genomic silencing and ageing in yeast and, perhaps, in higher eukaryotes.

Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase

The DNA of telomeres--the terminal DNA-protein complexes of chromosomes--differs notably from other DNA sequences in both structure and function. Recent work has highlighted its remarkable mode of synthesis by the ribonucleoprotein reverse transcriptase, telomerase, as well as its ability to form unusual structures in vitro. Moreover, telomere synthesis by telomerase has been shown to be essential for telomere maintenance and long-term viability.

Structure and function of telomeres

'The amino termini of histones extend from the nucleosomal core and are modified by acetyltransferases and deacetylases during the cell cycle These acetylation patterns may direct histone assembly and help regulate the unfolding and activity of genes

Histone acetylation in chromatin structure and transcription

Genetically identical cells exposed to the same environmental conditions can show significant variation in molecular content and marked differences in phenotypic characteristics. This variability is linked to stochasticity in gene expression, which is generally viewed as having detrimental effects on cellular function with potential implications for disease. However, stochasticity in gene expression can also be advantageous. It can provide the flexibility needed by cells to adapt to fluctuating environments or respond to sudden stresses, and a mechanism by which population heterogeneity can be established during cellular differentiation and development.

/pdf/stochasticity-in-gene-expression-from-theories-to-phenotypes-32urfuz0k3.pdf

Stochasticity in gene expression: from theories to phenotypes

https://www.cell.com/cell/pdf/0092-8674(90)90141-Z.pdf

Position Effect at S. cerevisiae Telomeres: Reversible Repression of Pol II Transcription

https://www.cell.com/cell/pdf/0092-8674(91)90049-5.pdf

Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae.

In Saccharomyces cerevisiae, telomeres repress transcription of genes located nearby. This region-specific gene inactivation is thought to involve the packaging of telomeric domains into silent chromatin. To gain insight into the mechanism of telomeric silencing, a genetic assay to examine the spread of silencing along the distal right arm of chromosome V was developed. The frequency of silencing a telomere-adjacent URA3 gene decreased with increasing distance of the gene's promoter from the telomere, irrespective of transcriptional orientation. The distance over which telomeric silencing of URA3 was observed was extended by weakening the gene's promoter--specifically, by deleting PPR1, the trans-activator of URA3. The silent telomeric domain was extended even farther by increasing the gene dosage of SIR3. These results suggest that a gene's promoter is a key determinant in controlling silencing on that gene and that SIR3 is a crucial component of the silent chromatin domain that initiates at the telomere and is assembled inwardly along the yeast chromosome. Finally, silencing is not observed on the centromeric side of an actively transcribed telomeric gene, demonstrating that the repressed telomeric domain is propagated continuously along the DNA. Taken together, these data reflect the complex and dynamic organization of eukaryotic genomes into functionally distinct regions.

/pdf/silent-domains-are-assembled-continuously-from-the-telomere-3ayj7kcxii.pdf

Barbara L. Billington

Papers

Position Effect at S. cerevisiae Telomeres: Reversible Repression of Pol II Transcription

Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae.

Silent domains are assembled continuously from the telomere and are defined by promoter distance and strength, and by SIR3 dosage.