Detection of induced mitotic chromosome loss in Saccharomyces cerevisiae--an interlaboratory study

The diploid yeast strain D61.M was used to study induction of mitotic chromosome loss. The test relies upon the uncovering and expression of multiple recessive markers reflecting the presumptive loss of the chromosome VII homologue carrying the corresponding wild-type alleles. The underlying 'loss event' is probably complex since the predicted centromere-linked lethal tetrad segregations for chromosome VII are not recovered. Instead, the homologue bearing the multiple recessive markers is patently homozygous. An interlaboratory study was performed in which 16 chemicals were tested under code in 2 laboratories. The results generated by the Berkeley and Darmstadt laboratories were in close agreement. Acetonitrile, ethyl acetate, 4-acetylpyridine, propionitrile and nocodazole were identified as potent inducers of mitotic chromosome loss. Acetone, dimethyl sulfoxide and 2-methoxyethyl acetate either elicited weak responses or yielded ambiguous results. Water, carbon tetrachloride, 4-fluoro-D,L-phenylalanine, amphotericin B, griseofulvin, cadmium chloride, ethyl methanesulfonate and methylmercury(II) chloride failed to induce chromosome loss. These data suggest that the system described herein represents a reliable assay for chemically induced chromosome loss in yeast.     

Meiotic Gene Conversion Mutants in SACCHAROMYCES CEREVISIAE . I. Isolation and Characterization of pms1-1 and pms1-2

The pms1 mutants, isolated on the basis of sharply elevated meiotic prototroph frequencies for two closely linked his4 alleles, display pleiotropic phenotypes in meiotic and mitotic cells. Two isolates carrying recessive mutations in PMS1 were characterized. They identify a function required to maintain low postmeiotic segregation (PMS) frequencies at many heterozygous sites. In addition, they are mitotic mutators. In mutant diploids, spore viability is reduced, and among survivors, gene conversion and postmeiotic segregation frequencies are increased, but reciprocal exchange frequencies are not affected. The conversion event pattern is also dramatically changed in multiply marked regions in pms1 homozygotes. The PMS1 locus maps near MET4 on chromosome XIV. The PMS1 gene may identify an excision-resynthesis long patch mismatch correction function or a function that facilitates correction tract elongation. The PMS1 gene product may also play an important role in spontaneous mitotic mutation avoidance and correction of mismatches in heteroduplex DNA formed during spontaneous and UV-induced mitotic recombination. Based on meiotic recombination models emphasizing mismatch correction in heteroduplex DNA intermediates, this interpretation is favored, but alternative interpretations involving longer recombination intermediates in the mutants are also considered.     

Chemical Detection of Microbial Prey by Bacterial Predators

A motile, predacious bacterium which degraded Pythium debaryanum was strongly attracted to substances released into the medium by the fungus. A nonpredacious bacterium was not attracted to these substances. The predator bacterium was specifically attracted to cellulose and its oligomers which are known to be components of the cell wall of Pythium. Ethanol inhibited chemotaxis of the bacterium without affecting either its motility or its ability to degrade cellulose. A second predacious bacterium was isolated for the alga, Skeletonema costatum. The role of chemoreception in the detection of microbial prey by bacterial predators in natural habitats is discussed.     

The Action of Homothallism Genes in Saccharomyces Diploids during Vegetative Growth and the Equivalence of hma and HMalpha Loci Functions

The action of homothallism genes in vegetatively growing diploid cells was examined. The results demonstrate that homothallism genes function during regular vegetative growth cycles as well as during the first few divisions after spore germination. A procedure based on ultraviolet-induced reciprocal mitotic recombination monitored by homozygosity for cryptopleurine resistance (a recessive marker closely linked to the mating-type locus) allowed us to identify and recover Saccharomyces cerevisiae colonies sectored for the mating-type locus i.e., a/a and alpha/alpha. Homothallism genes can switch a/a or alpha/alpha vegetative diploid cells, generated from a strain with genotype a/alpha HO/ho HMalpha/HMalpha HMa/HMa, to a/alpha diploids or a/a/alpha/alpha tetraploids during a given mitotic division cycle. We found that both a/a and alpha/alpha sectors generated from a strain with genotype a/alpha HO/HO hmalpha/hmalpha hma/HMa switch to a/alpha diploids or a/a/alpha/alpha tetraploids. This finding supports Naumov and Tolstorukov's suggestion (1973) that the hm a allele provides for the same functions as the HMalpha allele, namely, a switch at the mating-type locus from alpha to a. The HO allele is dominant to ho but hma and HMa alleles are codominant. A loose linkage between the mating-type and the HMalpha loci ( approximately 55cM), confirming Harashima, Nogi and Oshima (1974) data, was observed.     

Effects of low-chloride solutions on action potentials of sheep cadiac purkinje fibers

The rapid repolarization during phase 1 of the action potential of sheep cardiac purkinje fibers has been attributed to a time- and voltage-dependent chloride current. In part, this conclusion was based on experiments that showed a substantial slowing of phase 1 when larger, presumably impermeant, anions were substituted for chloride in tyrode’s solution. We have re- examined the electrical effects of low-chloride solutions. We recorded action potentials of sheep cardiac purkinje fibers in normal tyrode’s solution and in low-chloride solutions made by substituting sodium propionate, acetylglycinate, methylsulfate, or methanesulfonate for the NaCl of Tyrode’s solution. Total calcium was adjusted to keep calcium ion activity of test solutions equal to that of control solutions. Propionate gave qualitatively variable results in preliminary experiments; it was not tested further. Low-chloride solutions made with the other anions gave much more consistent results: phase 1 and the notch that often occurs between phases 1 and 2 were usually unaffected, and the action potential duration usually increased. The only apparent change in the resting potential was a transient 3-6 mV depolarization when low-chloride solution was first admitted to the chamber, and a symmetrical transient hyperpolarization when chloride was returned to normal. If a time- and voltage-dependent chloride current exists in sheep cardiac purkinje fibers, our results suggest that it plays little role in generating phase 1 of the action potential.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.