Analysis of Meiosis-Defective Mutations in Yeast by Physical Monitoring of Recombination

We have developed a method by which the extent of physical exchange of DNA molecules can be determined throughout meiosis in the yeast Saccharomyces cerevisiae. We have used this technique to analyze the effect of five meiosis-defective mutations (rad6, rad50, rad52, rad57 and spo11) on the physical exchange of DNA molecules. In the same experiments, we have also measured other meiotic parameters, such as premeiotic DNA synthesis, commitment to intragenic recombination, haploidization, ascus formation, and viability. rad50 and spo11 diploids make an undetectable amount of physically recombined DNA and less than 1% of wild-type levels of viable intragenic recombinants. In contrast, diploids homozygous for rad52, rad6 or rad57 all yield significant amounts of novel restriction fragments which arise by recombination. rad57 diploids make nearly wild-type levels of the recombined restriction fragments, although they produce less than 10% of the wild-type levels of viable intragenic recombinants. rad52 strains are also capable of a significant (33%) amount of exchange of DNA molecules, but make less than 1% of wild-type levels of viable intragenic recombinants. rad6 diploids are also capable of undergoing a high level of exchange, as measured by the appearance of the recombined restriction fragment. In addition, rad6 diploids show an unusual allele- or locus-specific variability in the level of viable intragenic recombinants produced. Although rad6 diploids produce no viable spores, they are able to complete a significant amount of haploidization upon return to vegetative growth conditions.     

A Complete Set of Marked Telomeres in Saccharomyces Cerevisiae for Physical Mapping and Cloning

Each telomere in a single strain (S288C) of Saccharomyces cerevisiae was marked with a URA3 containing integrating vector having telomeric TG(1-3) sequences. Efficiency of integrative transformation was enhanced by creating single random double-strand breaks in the integrating vector using DNAseI in the presence of Mn(2+) ions. A total of 327 transformants were screened by CHEF gels of intact chromosomal DNA. Transformants with homology to the vector at particular chromosomal bands were then screened by Southern analysis with several restriction enzymes to confirm telomeric locations. CHEF gels of NotI and/or SfiI digests were also analyzed to determine left or right arm locations. In some cases allelism of marked telomeres was determined genetically. Transformation was performed by lithium acetate and electroporation with varying results. Electroporation resulted in 50% (75/150) of the integrants at the internal URA3 location rather than telomeres. There were also two rearrangements involving URA3 and the telomere of another chromosome. Lithium acetate transformation resulted in fewer integrants at the internal URA3 location (5/84) and no rearrangements. All telomeres were marked with approximately the same efficiency ranging from 0 to 11 hits in the first 240 transformants. These marked telomeres can be used to complete the physical maps of chromosomes in which the telomere regions are absent. The marked telomeres can be cloned with the appropriate restriction enzymes, thus completing the cloning of individual chromosomes for sequencing projects. The analysis of these clones will lead to a better understanding of telomere region biology. The methodology can also be applied to telomeres of other organisms once they are cloned as telomeric YACs.     

Passage of serum-destined proteins through the Golgi apparatus of rat liver. An examination of heavy and light Golgi fractions

The participation of hepatic Golgi apparatus in the intracellular transport of blood-destined proteins has been analyzed using Golgi fractions enriched in cis and trans components of the Golgi apparatus. SDS-polyacrylamide gel electrophoresis of the liver Golgi fractions showed several proteins corresponding in relative proportions and mobilities with serum proteins. After a pulse injection of labeled leucine, the secretory content of the cis Golgi fraction was labeled earlier than the trans Golgi fraction. Taken together, the results show the participation of the liver Golgi apparatus in the secretion of most of the serum proteins and provide documentation for a sequential progression of secretory protein through the cis and trans components of the Golgi apparatus.     

The alpha-glucosidases of Dictyostelium discoideum. II. Developmental regulation and cellular localization

Four isozymes of α-glucosidase in Dictyostelium discoideum have been identified and some of their enzymatic and physical properties characterized (R. H. Borts and R. L. Dimond, 1981, Develop. Biol.87, 176–184). In this report the cellular localization and developmental regulation of three of these isozymes are determined. α-Glucosidase-1 is the major isozyme of vegetative amoebae. It is lysosomally localized and secreted from the cell under certain conditions. It has an acidic pH optimum and carries the common antigenic determinant found on all lysosomal enzymes in this organism. The specific activity of this isozyme begins to decrease within a few hours after the initiation of development and is no longer detectable in the mature fruiting body. α-Glucosidase-2 has a neutral pH optimum and is neither lysosomal nor secreted. Rather it is membrane bound and is possibly located on the cisternal side of microsomal vesicles. This isozyme does not possess the common antigenic determinant. α-Glucosidase-2 comprises 20–40% of the total α-glucosidase activity of the vegetative cell. Its specific activity increases threefold during development. This isozyme appears to be developmentally controlled since it fails to accumulate in aggregation deficient mutants. Its accumulation is also dependent upon continued protein synthesis. α-Glucosidase-4, like α-glucosidase-1, has an acidic pH optimum. It does not appear to be lysosomally localized nor membrane bound. Approximately 30% of the activity is precipitable by antibody against the common antigenic determinant indicating that it is less highly modified or fewer molecules are modified. The isozyme is undetectable during vegetative growth and does not begin to accumulate until late aggregation. Activity peaks in mature fruiting bodies where it is the predominant acidic α-glucosidase activity. Accumulation of α-glucosidase-4 is blocked in morphologically deficient mutants and by inhibitors of protein synthesis.     

Direct estimate of the mutation rate and the distribution of fitness effects in the yeast Saccharomyces cerevisiae

Estimates of the rate and frequency distribution of deleterious effects were obtained for the first time by direct scoring and characterization of individual mutations. This was achieved by applying tetrad analysis to a large number of yeast clones. The genomic rate of spontaneous mutation deleterious to a basic fitness-related trait, that of growth rate, was U = 1.1 x 10(-3) per diploid cell division. Extrapolated to the fruit fly and humans, the per generation rate would be 0.074 and 0.92, respectively. This is likely to be an underestimate because single mutations with selection coefficients s < 0.01 could not be detected. The distribution of s > or = 0.01 was studied both for spontaneous and induced mutations. The latter were induced by ethyl methanesulfonate (EMS) or resulted from defective mismatch repair. Lethal changes accounted for approximately 30-40% of the scored mutations. The mean s of nonlethal mutations was fairly high, but most frequently its value was between 0.01 and 0.05. Although the rate and distribution of very small effects could not be determined, the joint share of such mutations in decreasing average fitness was probably no larger than approximately 1%.     

A role for the mismatch repair system during incipient speciation in Saccharomyces

The cause of reproductive isolation between biological species is a major issue in the field of biology. Most explanations of hybrid sterility require either genetic incompatibilities between nascent species or gross physical imbalances between their chromosomes, such as rearrangements or ploidy changes. An alternative possibility is that genomes become incompatible at a molecular level, dependent on interactions between primary DNA sequences. The mismatch repair system has previously been shown to contribute to sterility in a hybrid between established yeast species by preventing successful meiotic crossing-over leading to aneuploidy. This system could also promote or reinforce the formation of new species in a similar manner, by making diverging genomes incompatible in meiosis. To test this possibility we crossed yeast strains of the same species but from diverse historical or geographic sources. We show that these crosses are partially sterile and present evidence that the mismatch repair system is largely responsible for this sterility.     

Interactions among oscillatory pathways in NF-kappa B signaling

Background  

Sustained stimulation with tumour necrosis factor alpha (TNF-alpha) induces substantial oscillations—observed at both the single cell and population levels—in the nuclear factor kappa B (NF-kappa B) system. Although the mechanism has not yet been elucidated fully, a core system has been identified consisting of a negative feedback loop involving NF-kappa B (RelA:p50 hetero-dimer) and its inhibitor I-kappa B-alpha. Many authors have suggested that this core oscillator should couple to other oscillatory pathways.     

Meiotic recombination: too much of a good thing?

Proper chromosome segregation in meiosis requires the right number and distribution of crossovers. Recent work in budding yeast has revealed a meiosis-specific role for RecQ helicase in limiting crossovers, distinct from its known somatic role in maintaining genome stability.     

Adsorption of ethylenediaminetetraacetic dianhydride modified oxalate decarboxylase on calcium oxalate

We used ethylenediaminetetraacetic acid dianhydride (EDTAD) to modify oxalate decarboxylase (OXDC) to improve its adsorption on calcium oxalate stones. The modified sites were identified by Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and the adsorption mechanism of the EDTAD-modified OXDC on calcium oxalate (CaOx) was investigated. We investigated adsorption time, initial enzyme concentration, temperature and solution pH on the adsorption process. Data were analyzed using kinetics, thermodynamics and isotherm adsorption models. UPLC-MS showed that EDTAD was attached to OXDC covalently and suggested that the chemical modification occurred at both the free amino of the side chain and the α-NH₂ of the peptide. The adsorption capacity of the EDTAD-OXDC on calcium oxalate was 53.37% greater than that of OXDC at the initial enzyme concentration of 5 mg/ml, pH = 7.0, at 37° C. The modified enzyme (EDTAD-OXDC) demonstrated improved oxalate degradation activity at pH 4.5?6.0. Kinetic data fitting analysis suggested a pseudo second order kinetic model. Estimates of the thermodynamic parameters including ΔG⁰, ΔH⁰ and ΔS⁰ of the adsorption process showed it to be feasible, spontaneous and endothermic. Isotherm data fitting analysis indicated that the adsorption process is reduced to monolayer adsorption at a low enzyme concentration and to multilayer adsorption at a high enzyme concentration. It may be possible to apply OXDC to degradation of calcium oxalate stones.