Transport of 6-deoxyglucose in Saccharomyces cerevisiae.

The uptake of 6-deoxyglucose was measured in wild-type Saccharomyces cerevisiae, in a double mutant strain lacking activity for hexokinases A and B (hxkl hxk2), in a triple mutant strain lacking activity for both hexokinases and glucokinase (hxkl hxk2 glk), and in the triple mutant with high levels of activity of single kinases restored by introduction of the cloned genes. In the wild-type strain, uptake of the glucose analog showed two components, with Km values of ca. 20 mM ("high affinity") and 250 mM ("low affinity"), respectively. The double mutant also had high- and low-affinity components, but the triple mutant showed only low-affinity uptake. Reintroduction of the single kinases to the triple mutant restored high-affinity uptake. (Other experiments on 6-deoxyglucose uptake are also presented, including the apparent use of the galactose transport system when induced.) These results show that the recent implication of the kinases in transport of glucose (L.F. Bisson and D.G. Fraenkel, Proc. Natl. Acad. Sci. U.S.A. 80:1730-1734, 1983) applies equally to the nonmetabolized analog 6-deoxyglucose and suggests that the role of the kinases in transport is not merely a consequence of metabolism of the transported compound.     

Inhibition of DNA replication by transformation in a Haemophilus influenzae mutant carrying an altered Rec-1 protein.

In the HM5 mutant of Haemophilus influenzae, which carries a mutation in the rec-1 gene region and in which the replication of donor-recipient DNA complexes formed in transformation is inhibited, the transformation frequency could be greatly enhanced by inhibition of protein synthesis during transformation, indicating that transformation in the HM5 mutant induces the synthesis of a protein that inhibits the replication of the donor-recipient DNA complexes. This induction occurred in an early step of the recombination. Synthesis of the wild-type Rec-1 protein after transformation of the HM5 mutant with wild-type DNA could diminish the inhibiting effect on DNA replication. The HM5 mutant synthesized an altered Rec-1 protein (molecular weight, 38,000) whose pI differed from that of the wild type. As a result of the mutation in the rec-1 gene, two other proteins (molecular weights, 37,500 and 43,000) are lacking in the HM5 mutant.     

A Genetic Analysis of CANDIDA ALBICANS: Isolation of a Wide Variety of Auxotrophs and Demonstration of Linkage and Complementation

Naturally occurring strains of Candida albicans appear to be diploid and heterozygous for a limited number of nutritional markers. Additional heterozygosity can be induced by treatment with mutagens; nitrous acid alone or in combination with UV is a potent mutagen in terms of both efficacy and efficiency in the production of a wide variety of mutations. Spheroplast fusion followed by regeneration on selective media revealed complementation among four histidine-requiring mutants analyzed. Some of the fusion products appeared to be stable prototrophs, whereas in others several kinds of segregants resulted, apparently due to chromosomal or nuclear elimination. The results are suggestive of both heterokaryosis as well as nuclear fusion. The procedures described can be successfully used for generating new mutants and studying allelism. Three sets of linkage relationships have been derived from evidence provided by concomitant appearance or cosegragation of several auxotrophic markers.     

Genetic Analysis of Murine Arylsulfatase C and Steroid Sulfatase

SWR/J mice possess two- to threefold higher 4-methylumbelliferyl sulfate (4MUS), dehydroepiandrosterone sulfate (DHEAS) and estrone sulfate (E1S) sulfatase activities in liver and kidney extracts than do A/J mice. These interstrain activity differences are maintained throughout the 6- to 45-day postnatal period. Characteristics of the hepatic activities of SWR/J mice suggest that all three activities reside in the same enzyme. Biochemical properties of the SWR/J and A/J enzyme were not significantly different. Expression of hepatic enzyme activity is subject to regulation by an autosomal locus possessing two alleles with additive effects. Postnuclear E1S- and DHEAS-sulfatase activities are primarily microsomal. Although postnuclear hepatic 4MUS-sulfatase activity is predominantly microsomal, renal activity is primarily nonmicrosomal. Only that portion of 4MUS-sulfatase occurring in cell membranes appears capable of hydrolyzing E1S and DHEAS. The hepatic- and renal-specific subcellular distributions of 4MUS-sulfatase activity may reflect tissue differences in enzyme processing. Renal 4MUS-sulfatase activity is also controlled by an autosomal gene with two alleles having additive effects. Positive correlation between hepatic and renal 4MUS-sulfatase activities indicates that both activities are most likely influenced by the same gene.     

Linkage Modification with Mixed Random Mating and Selfing: A Numerical Study

Although recombination cannot increase under conditions of random mating or complete selfing in regimes of constant selection, with mixed random mating and selfing, selection for increased recombination can occur. For some fitness regimes there may be selection for reduced recombination with both low and high degrees of selfing but selection for increased recombination with moderate degrees of selfing. With some fitness regimes there is a historical effect: depending on which equilibrium a population starts from, there may be selection for either increased or decreased recombination. In other cases the direction of selection may be determined by the present state of individuals within the population. If recombination is already fairly limited, there may be selection for further reduction. If recombination is already fairly frequent, there may be selection for increased recombination. For certain symmetric viability systems there may be an intermediate value of the recombination fraction between 0 and 0.5 toward which the population will evolve. Although it is not yet possible to classify precisely those fitness matrices that can exhibit selection for increased recombination, it does appear that selection for increased recombination can occur only if at least two of the double homozygotes are less fit than would be expected on the basis of a comparison of the fitnesses of the single and double heterozygotes on an additive scale.     

Flies as a source of enteric pathogens in a rural village in Thailand.

The village of Ban Pong in northeastern Thailand was studied from January through December 1981 to determine the importance of flies as a source of enteric pathogens. The number of flies (predominantly Musca domestica) increased in kitchens and animal pens in the hot dry spring, when the incidence of diarrhea was highest in the village. Enterotoxigenic Escherichia coli, Shigella spp., non-O1 Vibrio cholerae, and Vibrio fluvalis were isolated from fly pools in yards (69%), animal pens (38%), bathrooms (35%), and kitchens (8%). Enterotoxigenic E. coli was isolated from one fly pool in May and from another in June, when the incidence of such infections was highest in the village. Flies often carry and presumably disseminate enteric pathogens in rural Thailand.     

Longevity of Salmonella typhimurium in Tilapia aurea and water from pools fertilized with swine waste.

Salmonella typhimurium declined rapidly when inoculated into Tilapia aurea culture pools fertilized with fresh swine waste. Within the water column, a 95% decline of viable cells occurred during the first 6 h. Isolation of viable salmonellae was possible at 16 days post-inoculation, but not at 32 days. Similarly, salmonellae could be detected in the viscera and epithelium of T. aurea at 16 days, although not at 32 days. Salmonellae were not isolated from the fish flesh, nor was there evidence of septicemic infection.     

Selection of Wine Yeasts for Growth and Fermentation in the Presence of Ethanol and Sucrose

To optimize the conversion of carbohydrates to ethanol, strains of several Saccharomyces species were examined for the ability to grow and ferment in a range of sucrose and ethanol concentrations. A total of 632 wine yeasts, most of them isolated from wineries in Andalusia and Extremadura, southwestern Spain, were subjected to screening and selection. Growth and fermentative capacity in different ethanol and sucrose concentrations varied from one strain to another. There was no correlation between growth and fermentative capacity. The best 35 strains grew in 15% ethanol and fermented in 18% ethanol. Ethanol accumulated, although at a reduced rate, after the cells stopped growing. Most yeast strains were highly fermentative in 50% sucrose. Some of them effectively utilized the carbohydrates of the culture, yielding final ethanol concentrations of > 14%. Of the 35 selected strains, 16 were promising for genetic analysis and breeding because of their capacity to sporulate. These strains were homothallic, and their spores were viable. The meiotic products analyzed so far were also homothallic.     

Inactivation of Naegleria gruberi cysts by chlorinated cyanurates.

The resistance of Naegleria gruberi cysts to chlorine in the presence of cyanuric acid was compared at pH 5 and 7. An amperometric membrane electrode was used to measure HOCl concentrations independently of the chlorinated cyanurate species, thus permitting an analysis of the role of free chlorine versus chlorinated cyanurates in cyst inactivation. In the presence of cyanuric acid, the products of the HOCl residual and the contact time required for 99% cyst inactivation were 8.5 mg . min/liter and 13.9 mg . min/liter at pH 5 and 7, respectively. The Watson's Law coefficients of dilution (n) were 1.3 and 1.6 at pH 5 and 7, respectively. The results strongly suggest that HOCl is the predominant cysticide with no measurable cysticidal effect of the chlorinated cyanurate species.