The yeast Rad6 protein: a mediator of homologous recombination across the scaffold attached region at the replication origin ARS1.

Here we show that the ubiquitin-conjugating enzyme Rad6p plays a crucial role in locus-specific replacement recombination in the TRP1-ARS1 region. In rad6-1 strains, where this ubiquitination activity is modified, homologous recombination across a 150 bp continuous region is completely abolished. Our results unambiguously identified the ARS1 scaffold attached region (SAR) as being the region where this impediment for replacement recombination is located, since a merging of the location of the recombination impediment and binding properties in a scaffold exchange assay with deletion mutations was observed. Our observations strongly support the notion of torsionally separated chromosomal domains being organized by SARs and scaffold proteins, and being dynamically realigned as a consequence of ubiquitination and proteolysis.

Replication properties of ARS1 plasmids in Saccharomyces cerevisiae: dependence on the carbon source.

The replication behaviour of a number of ARS1-based plasmids was investigated on propagation in Saccharomyces cerevisiae grown with either glucose or galactose as carbon source. Growth on galactose results in reduced plasmid stability, as well as in reduced replication efficiency, when the entire 1.5-kb TRP1-ARS1 fragment is present on a plasmid. The galactose sensitivity is mediated by a 0.13-kb fragment harbouring part of the GAL3 promoter. This fragment exerts its effect when situated either 5' or 3' to the ARS core consensus at distances up to 0.9 kb. The endogenous 2 microns plasmid remained unaffected by the choice of carbon source.

A search for an essential function of the replication origin ARS1 in the life cycle of Saccharomyces cerevisiae.

We have investigated the significance of the chromosomal replication origin, ARS1, during the entire life cycle of yeast. This was done by substituting the chromosomal copy with a series of ars1 deletion mutants. It was shown that the ARS1 replication origin is not essential for mitotic or premeiotic DNA replication since no effect on growth, chromosomal loss rate and spore viability was observed in the ars1 mutant strains. We conclude that replication origins are abundantly, present in the yeast genome and that the removal of a single replication origin is compensated for by replication forks emanating from neighbouring origins.

Mutational analysis of a variant of ARS1 from Saccharomyces cerevisiae.

A naturally occurring single base-pair G to A transition, creating a 10/11 near-match close to the essential 11 base-pair core consensus of ARS1, was used to investigate the importance of near-match sequences. The 10/11 near-match can not substitute for the core consensus since an ARS- phenotype is observed when the core consensus is deleted. However, deletion mutations revealed that this near-match together with a short palindromic sequence, also situated in the B-flanking region, comprise a single element crucial for optimal ARS function. The palindrome has the potential of forming a stem-loop structure. Rather precise observations concerning the borders of the B-region were achieved. The four base pairs separating the near-match from the core consensus perform a spacing function where the identity of the bases are unimportant. However, this spacing is highly important since deletion of these four base pairs leads to an ARS- phenotype.

Maintenance and copy number control of ARS1 plasmids in Saccharomyces cerevisiae. Evidence of a mating type effect.

Following mating of a and alpha isogenic haploids we observe that the frequency of plasmid bearing cells, during selective growth, increases three fold. By examining the mitotic stability, the frequency of plasmid bearing cells during the cell cycle and the copy number of ARS1 plasmids in isogenic haploid and diploid cells, we show that the apparent stability of circular ARS1 plasmids in a/alpha cells is largely due to a diminished copy number in these cells. This observation is fully comprehensible with the model for plasmid segregation as presented by Murray and Szostak (1983). In order to account for the differences in copy numbers, alpha and alpha/alpha isogenic strains were compared. Likewise a number of mating type nonspecific sterile mutants were compared with the parental Ste+ strain. It seems that a diminished copy number is established when the MATa1/MAT alpha 2 regulatory system (Klar et al. 1981) is switched on, since the effect is observed in Sir- strains only.

Chromosomal proteins in Saccharomyces cerevisiae : II. Chromosome dosage effect on the cellular and nuclear content of nonhistone proteins.

The effect of varying the chromosomal dosage in aneuploids for chromosome I or VII on the synthesis of individual nonhistone proteins was revealed. The nuclear content of most nonhistone proteins seemed to be little impaired in a strain with tetrasomy for chromosome VII as compared with a strain which is disomic for chromosome VII. On the contrary, tetrasomy for chromosome I as compared with disomy for this chromosome seems to impair both the cellular and the nuclear content of a number of chromosomal proteins, including the tubulin subunits.

Chromosomal proteins in Saccharomyces cerevisiae : I. number and properties of individual proteins.

Proteins were isolated from purified yeast chromatin and subjected to two-dimensional electrophoresis. The cellular and the chromosomal content of the major nonhistone proteins was measured. Two polypeptides of molecular weights 55,000 and 53,000, identified as α and ß tubulin, and a polypeptide of molecular weight 63,000, associated with the nuclear DNA to a very high degree, account for nearly 50% of the nonhistone proteins present in chromatin. Only one tenth of the RNA polymerase subunit with the molecular weight of 23,000 was associated with nuclear DNA following chromatin purification in metrizamide gradients.

Peptide chain elongation rate and ribosomal activity in Saccharomyces cerevisiae as a function of the growth rate.

The peptide-chain elongation rate of Saccharomyces cerevisiae at two different growth rates was estimated by the kinetics of radioactive labelling of nascent and finished polypeptides as described by Gausing, 1972, and Young and Bremer, 1976. The elongation rates of a diploid strain cultured in yeast nitrogen base supplemented with glucose or acetate were 9.3 amino acids/s and 5.5 amino acids/s at 30 degrees C, respectively. These data together with published values on the "ribosomal efficency" as a function of growth rate (Waldron and Lacroute, (1975) enable us to estimate the rate of synthesis of ribosomal proteins as a function of the rate of total protein synthesis, alpha r, and the fraction of ribosomes that one active in protein synthesis. We conclude that in S. cerevisiae alpha r is largely independent of the growth rate while the fraction of active ribosomes decreases with decreasing growth rate.