Nuclear DNA synthesis in yeast and the effect of irradiation.

The synthesis of DNA can be measured in yeast by following the uptake of 5-bromodeoxy-uridine-5'-triphosphate in a mutant that utilizes deoxythymidine-5'-monophosphate; approximately 60 per cent of the DNA is synthesized semi-conservatively before replication stops. Neither ultraviolet light (U.V.), nor ionizing radiation stimulates repair-type synthesis. Based on the ability to detect small amounts of synthesis, it appears that fewer than ten bases are synthesized per pyrimidine dimer removed.     

Dependence of yeast sporulation on mitochondrial protein synthesis

Sporulation in diploidSaccharomyces cerevisiae is not dependent on continued protein synthesis in the mitochondria. Using chloramphenicol, it is shown that proteins essential for respiration and sporulation are synthesized in mitochondria early during growth in a presporulation medium.     

Ribonucleoprotein particle appearing during sporulation in yeast.

During sporulation of Saccharomyces cerevisiae, most strains accumulate an unmethylated 20S RNA. Contrary to previous reports, this sporulation 20S RNA is distinct from the short-lived methylated 20S RNA precursor of 18S rRNA. This RNA species was found in a cytoplasmic 32S ribonucleoprotein particle consisting of one single-stranded 20S RNA molecule and 18 to 20 identical protein subunits of molecular weight 23,000. The ribonucleoprotein particle was resistant to ribonuclease digestion, although purified 20S RNA was ribonuclease sensitive. Both the RNA and the protein of the 32S ribonucleoprotein particle were only synthesized under conditions that induce sporulation. The accumulation of 20S RNA depended on continued protein synthesis but was actinomycin D insensitive, despite a high guanine-plus-cytosine content. Synthesis of 20S RNA stopped when cells were removed from sporulation conditions and placed in growth medium.     

New cytoplasmic genetic element that controls 20S RNA synthesis during sporulation in yeast.

Under conditions that induce meiosis and sporulation in Saccharomyces cerevisiae, most strains accumulate a 20S RNA, amounting to as much as 15% of the newly synthesized RNA. The ability of cells to accumulate this new RNA species depends on a dominant genetic element that is cytoplasmically inherited, but is distinct from the other cytoplasmic elements that have been previously identified. The ability to synthesize 20S RNA does not depend on mitochondrial DNA, 2-micron DNA, the translational suppressor psi, the genetic element carrying URE3, or double-stranded killer RNA. However, all 20S- strains examined were also nonkillers, although many nonkiller strains were 20S+. This work also shows that 20S RNA accumulating is not essential for sporulation even though it is induced only by conditions that initiate sporulation. Furthermore, strains that are unable to complete meiosis are still capable of producing 20S RNA when placed under the nitrogen starvation conditions that promote sporulation.     

DNA synthesis across an abasic lesion by yeast REV1 DNA polymerase

Abasic (apurinic/apyrimidinic) sites are among the most abundant DNA lesions in humans, and they present a strong block to replication. They are also highly mutagenic because when replicative DNA polymerases manage to insert a nucleotide opposite the lesion, they prefer to insert an A. Rev1, a member of Y-family DNA polymerases, does not obey the A-rule. This enzyme inserts a C opposite an abasic lesion with much greater catalytic efficiency than an A, G, or T. We present here the structure of yeast Rev1 in ternary complex with DNA containing an abasic lesion and with dCTP as the incoming nucleotide. The structure reveals a mechanism of synthesis across an abasic lesion that differs from that in other polymerases. The lesion is driven to an extrahelical position, and the incorporation of a C is mediated by an arginine (Arg324) that is conserved in all known orthologs of Rev1, including humans. The hydrophobic cavity that normally accommodates the unmodified G is instead filled with water molecules. Since Gs are especially prone to depurination through a spontaneous hydrolysis of the glycosidic bond, the ability of Rev1 to stabilize an abasic lesion in its active site and employ a surrogate arginine to incorporate a C provides a unique means for the “error-free” bypass of this noninstructional lesion.     

Protein degradation during yeast sporulation. Enzyme and cytochrome patterns.

The levels of several enzymes have been studied during sporulation of Saccharomyces cerevisia. The specific activities of ribonuclease and aminopeptidase I raised several-fold after transfer of the cells to sporulation medium, whereas the specific activities of phosphofructokinase, glucose-6-phosphate dehydrogenase, tryptophan synthase and pyruvate decarboxylase were not significantly altered. The specific activities of NAD-dependent glutamate dehydrogenase, isocitrate lyase, malate dehydrogenase and fructose bisphosphatase all decreased from the onset of sporulation. The inactivation of these latter enzymes was inhibited by cycloheximide and by inhibitors of energy metabolism. Hexokinase, alcohol dehydrogenase and glutamate oxaloacetate transaminase were partially lost from the cells during the period of ascus maturation. None of the enzyme changes observed proved to be 'sporulation-specific' in that it occurred exclusively in sporulating diploid yeast cells. Therefore it is postulated that the meiotic events and the metabolic changes required for ascospore formation are under separate genetic control in this organism. During sporulation, the cellular content of cytochromes b, c, and aa3 was reduced to 20% or less of that present in vegetative derepressed cells. Since the relative percentage of total to cycloheximide-insensitive mitochondrial protein synthesis was not significantly altered throughout sporulation, and the pattern of mitochondrially synthesized polypeptides was rather similar both in vegetative and in sporulating cells, it appeared that not only degradation but also synthesis and therefore turnover of the mitochondrially coded polypeptides of cytochromes b and aa3 took place during sporulation. The activity ratio of cytochrome c oxidase to F1-ATPase in submitochondrial particles isolated from vegetative cells and from purified asci was almost identical. This indicates that the loss of membrane-bound mitochondrial cytochromes during sporulation is probably due to a nonselective degradation of inner mitochondrial membrane proteins.     

Stability and synthesis of the penicillin-binding proteins during sporulation

The penicillin-binding proteins (PBPs) of Bacillus subtilis were examined after incubation of vegetative and sporulating cultures with chloramphenicol, an inhibitor of protein synthesis. The results indicate that the sporulation-specific increases in vegetative PBPs 2B and 3 and the appearance of two new PBPs, 4* and 5*, depend on concurrent protein synthesis, which is most likely to be de novo synthesis of the PBPs rather than synthesis of an activator or processing enzyme. It was also learned that in vivo the PBPs differ in their individual stabilities, which helps to explain some of the quantitative changes that occur in the PBP profile during sporulation. All the membrane-bound PBPs, except possibly PBP 1, were found to be stable in the presence of crude extracts of sporulating cells that contained proteolytic activity.     

Variation of the lipid content of yeast cells during sporulation

Changes in the lipid content of yeast cells during sporulation were studied. The lipid content increases during sporulation. The unsaponifiable fraction represents the major part of lipids extracted at the moment sporulation sets in.     

Protein-template-directed synthesis across an acrolein-derived DNA adduct by yeast Rev1 DNA polymerase

Acrolein is generated as the end product of lipid peroxidation and is also a ubiquitous environmental pollutant. Its reaction with the N2 of guanine leads to a cyclic gamma-HOPdG adduct that presents a block to normal replication. We show here that yeast Rev1 incorporates the correct nucleotide C opposite a permanently ring-closed form of gamma-HOPdG (PdG) with nearly the same efficiency as opposite an undamaged G. The structural basis of this action lies in the eviction of the PdG adduct from the Rev1 active site, and the pairing of incoming dCTP with a "surrogate" arginine residue. We also show that yeast Polzeta can carry out the subsequent extension reaction. Together, our studies reveal how the exocyclic PdG adduct is accommodated in a DNA polymerase active site, and they show that the combined action of Rev1 and Polzeta provides for accurate and efficient synthesis through this potentially carcinogenic DNA lesion.     

Cell signaling in yeast sporulation

Gametogenesis is essential for the propagation of all sexually reproducing organisms and consists of halving the chromosome number through meiosis, and the subsequent packaging of the haploid products into gametes. Meiosis and gamete formation must be tightly coupled to ensure the formation of viable progeny; perturbations result in infertility, inviability, and birth defects. In the yeast Saccharomyces cerevisiae, sexual reproduction occurs via sporulation and is similar in many respects to gametogenesis in mammals. An increasing number of conserved signaling molecules have been shown to be essential for yeast sporulation; recent studies reveal molecular insights into how these molecules regulate this intricate differentiation program.     

Nuclear accumulation of HMG1 protein is correlated to DNA synthesis

The subcellular localization of HMG1 protein was studied by immunoelectron microscopy during growth of CV1 cells in culture and in confluent CV1 cells subsequently lytically infected with SV40. HMG1 was always detected in the cytoplasm of both non-infected and infected cells. On the other hand, this protein displayed a nuclear localization only in those cells active in cellular and/or viral DNA replication, that is, in actively dividing non-infected cells and in confluent cells following SV40 infection. The combination of electron microscope immunocytochemistry and autoradiography revealed that during SV40 lytic infection, HMG1 accumulates at sites of active viral DNA replication. Since HMG1 is a single-stranded DNA binding protein and acts in vitro as a physiological nucleosome assembly factor, we suggest that its presence in the nucleus is related to its requirement in the DNA replication process.     

Regulation of yeast mitochondrial DNA synthesis

Summary The expression and stability of Escherichia coli F-primes in Proteus mirabilis is examined. It is possible to consecutively introduce, and stably maintain, the DNA of several E. coli F-primes in P. mirabilis in the absence of selective pressure for all or some of the plasmids. Additionally, we can recover more than one F-prime from certain P. mirabilis recipient strains which carry DNA derived from several independent matings with E. coli F-prime donors.     

Regulation of yeast mitochondrial DNA synthesis

Summary A single recessive nuclear gene mutation has been isolated from strain 123.1 C of Saccharomyces cerevisiae which is conditionally deficient in mitochondrial DNA metabolism and has been termed tpi. Growth of this mutant strain in media containing galactose at 36°C causes a reduction of mitochondrial DNA synthesis as analyzed by incorporation of radioactive adenine into the mitochondrial DNA. These cells continue to grow and divide producing petite cells which are neutral and have been found to lack mitochondrial DNA as measured by radioactive incorporation of ³H-adenine into the mitochondrial DNA in the presence of cycloheximide at the permissive temperature. The rate of mitochondrial DNA synthesis of the mutant strain grown at the restrictive temperature in dextrose or glycerol containing media was found to be greatly reduced following two hours of exposure to the restrictive temperature. In addition, the action of this mutant gene has been found to be independent of the respiratory capacity of the mutant strain.