Biomedical subjects
C Wills
Publications and source records attributed to C Wills.
Forensic DNA typing.
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The role of molecular biology in human evolutionary studies: current status and future prospects.
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Maintenance of multiallelic polymorphism at the MHC region.
Models that purport to explain the maintenance of MHC polymorphism must be able to explain a variety of phenomena. (1) The range of MHC allele frequencies at some of the loci is very large, with some alleles quite common and many others rare, while at others the range of allele frequencies is far narrower. (2) MHC alleles and their frequencies often have long persistence times, in some cases tens of millions of years. (3) Random-mating populations appear to be in Hardy-Weinberg equilibrium for MHC. (4) There is no obvious, strong and consistent selection pressure yet detected that acts differentially on different MHC genotypes. (5) Because the allelic composition of the MHC polymorphism does change over evolutionary time, the MHC system must be capable of accommodating new alleles with similar properties without destruction of the equilibria that permit the maintenance of the older alleles. In this review I examined the degree to which a large number of models that have been proposed fit these criteria. These include heterosis, marginal overdominance, conditional heterosis, assortative mating, maternal-fetal incompatibility, molecular mimicry, minority advantage, pathogen adaptation, and optimum allele frequency models. Most of the models do poorly at accounting for a number of the above phenomena. The last class, optimum allele frequency models, have the most satisfactory set of properties. However, optimum allele frequency models require mechanisms that somehow "feed back" from the frequency of an allele in the population to the fitness of an organism carrying that allele. Thus, these models require that MHC polymorphisms be maintained by some type of group selection. Evidence for an against optimum allele frequency selection, and ways in which this type of selection might be detected experimentally, are presented.
Population size bottleneck.
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Regulation of sugar and ethanol metabolism in Saccharomyces cerevisiae.
This review briefly surveys the literature on the nature, regulation, genetics, and molecular biology of the major energy-yielding pathways in yeasts, with emphasis on Saccharomyces cerevisiae. While sugar metabolism has received the lion's share of attention from workers in this field because of its bearing on the production of ethanol and other metabolites, more attention is now being paid to ethanol metabolism and the regulation of aerobic metabolism by fermentable and nonfermentable substrates. The utility of yeast as a highly manipulable organism and the discovery that yeast metabolic pathways are subject to the same types of control as those of higher cells open up many opportunities in such diverse areas as molecular evolution and cancer research.
An efficient selection producing structural gene mutants of yeast alcohol dehydrogenase resistant to pyrazole.
Selection for resistance to allyl alcohol in respiration-incompetent Saccharomyces cerevisiae produces a high proportion of mutants that can be localized within the ADH2 structural gene and that still, because of the type of selection employed, retain enzyme activity. We show here that a similar type of selection produces a similarly high proportion of mutants resistant to the competitive inhibitor pyrazole. The first four mutants examined, picked at random from a collection of spontaneous pyrazole-resistant mutants, show altered--usually increased--KM values for ethanol and NAD+, and markedly increased K1 values for pyrazole, compared with the wild type. When these kinetic measures and their electrophoretic mobilities were compared, all the mutants could be clearly distinguished from each other as well as from wild type. Genetic analysis shows these mutants to be close to and probably resident in the structural gene. For a variety of reasons, these mutants are even more favorable subjects for population genetic analysis and the dissection of molecular microevolution than are allyl alcohol-resistant mutants.
Macro- and trace-mineral intakes of exclusively breast-fed infants.
Intakes of calcium, phosphorus, magnesium, zinc, sodium, potassium, iron, and copper of 45 exclusively breast-fed infants were determined during the first 4 mo of life. Direct 24-h measurements of milk intake and mineral contents of human milk were used to estimate mineral intakes. Daily intakes of Ca, P, Zn, K, Na, Fe, and Cu decreased significantly over the study period while the intake of Mg increased. With the exception of Mg, mineral intakes on a weight basis displayed significant quadratic trends over the 4 mo. In spite of seemingly low-mineral intakes, growth progressed satisfactorily.
Functional alcohol dehydrogenase mutants of Saccharomyces cerevisiae conferring temperature-conditional allyl alcohol resistance.
Selection for allyl alcohol resistance in respiratory incompetent yeast is a highly specific method for isolating functional mutations at ADH1, the gene coding for the cytoplasmic alcohol dehydrogenase, ADHI. Because of the nature of this selection scheme, the ADHI activity of such mutants is retained, but the kinetic characteristics of the enzymes are altered. The high specificity for targeting functional mutations at this locus suggested that selection for enzyme variants with more subtle phenotypic effects might be possible. Here, we describe functional ADHI mutants that are temperature-conditional in their allyl alcohol resistance. Haploid cells of one of these mutants grow well on plates at 10 mM allyl alcohol at 19 degrees, but not at 37 degrees, the restrictive temperature. A second mutant grows well at 10 mM at 37 degrees, but its growth is restricted at 19 degrees. What distinguishes these mutants from other temperature-sensitive mutants is that the temperature-conditional growth phenotypes described here must be due to interactions between allyl alcohol levels and ADHI functional properties and cannot be due to lability of the enzyme at the restrictive temperature. This system shows promise for the investigation of functional enzyme variants that differ by only one or two amino acid residues but have significant temperature- and substrate-conditional effects on growth phenotypes in both the haploids and the diploids.
Conditional overdominance at an alcohol dehydrogenase locus in yeast.
Documented examples of heterosis attributable to overdominance at specific protein-encoding gene loci have rarely been reported, the association of sickle cell hemoglobin with malarial resistance being the best documented example of this phenomenon. Here we report an example of overdominance that is temperature- and allyl alcohol-dependent and due to heterozygosity at the ADH1 locus, involving two ADHI functional mutants. Overdominance appears to be due in part to an intermediate level of ADHI activity in the heterozygote. Unlike previous work with this this system using haploid strains, the NAD+/NADH ratios show no negative correlation with allyl alcohol resistance. This system is formally equivalent to that of sickle cell hemoglobin and shows promise as a tool for investigating the physiological basis for overdominance.
Null and electrophoretic mobility mutants in the structural gene for L-lactate dehydrogenase of Saccharomyces cerevisiae.
A mutant lacking L-lactate dehydrogenase (EC 1.1.2.3) of Saccharomyces cerevisiae was isolated by its inability to grow on minimal medium with L-lactate as a carbon source. A simple activity gel assay for visualization of this enzyme and the two D-lactate dehydrogenases in this organism (EC 1.1.2.4 and 1.1.1.28) was developed. This enabled us to screen spontaneous and ethylmethanesulfonate-induced back mutants for electrophoretic mobility. Two mutants with a mobility faster than that of the wild type were isolated, and proved to be allelic to the L-lactate dehydrogenase negative mutant.
Effect on gluconeogenesis of mutants blocking two mitochondrial transport systems in the yeast Saccharomyces cerevisiae.
Two mutants of Saccharomyces cerevisiae, ccr1 and tpy1, have been found to interfere with the transport of small molecules across the inner mitochondrial membrane. Both also have the effect of interfering with the synthesis of a number of cytoplasmically located enzymes involved in gluconeogenesis, even when the cells are released from glucose repression. The ccr1 mutant, defective in the transport of dicarboxylic acids across the inner membrane, represses the synthesis of gluconeogenic enzymes almost totally, but synthesis can be induced on complete medium without a carbon source. This mutant has low levels of intracellular malate under all growth conditions tested. The tpy1 mutant, defective in the transport of pyruvate across the inner membrane, shows repression of gluconeogenesis enzymes under some growth conditions, particularly high levels of ethanol in the medium. These conditions also lead to low levels of malate in the cells. Intracellular levels of malate in these mutants, and in the wild type, are correlated with the levels of gluconeogenic enzymes present. The ability of isolated mutant mitochondria to phosphorylate ADP is shown to be consistent with the interpretation that they are defective in inner membrane transport, although as yet no evidence is available that these defects are the primary lesions in the two mutants. The data are consistent with two general models. In one, the exhaustion of an extramitochondrial corepressor or introduction of a coinducer by mitochondrial activity triggers the induction of gluconeogenic enzyme synthesis. In the second, the mitochondria themselves trigger this induction, but only when the tricarboxylic acid cycle is able to operate at a high level.
Alcohol dehydrogenase II and fructose-1,6-bisphosphatase appear to be co-regulated in wild-type yeast.
An activity gel assay for fructose-1,6-bisphosphatase (FBP), the enzyme catalyzing the final step in gluconeogenesis in yeast, has been developed which can be used in conjunction with spectrophotometric assays to show that it is tightly co-regulated with the inducible alcohol dehydrogenase, ADHII. Both enzymes are repressed coordinately in aerobically grown yeast by the addition of high levels of glucose or ethanol, and induced on minimal medium by the addition of yeast extract. A mutant deficient in FBP segregates independently of the ADHII structural gene locus. This phenomenon is of interest because of the discovery of Ciriacy [(1979) Mol. Gen. Genet. 176, 427-431] of mutants (ccr, or carbon catabolite repression) which repress both FBP and ADHII simultaneously, along with several other enzymes.
Pyruvate carboxylase deficiency in yeast: a mutant affecting the interaction between the glyoxylate and Krebs cycles.
A single-gene nuclear mutant has been isolated in Saccharomyces cerevisiae which cannot grow on minimal medium supplemented with ethanol, acetate, pyruvate, aspartate, or oxaloacetate as sole carbon sources. It will grow on complete medium with these carbon sources, and on minimal medium with dextrose as carbon source. The only supplement which will permit growth on minimal medium with ethanol or pyruvate is aspartate, so the mutant is an aspartate auxotroph when grown on these nonfermentable substrates. It exhibits enhanced levels of phosphoenolpyruvate carboxykinase (EC 4.1.1.49) when grown on dextrose. The mutant can survive as an alcohol dehydrogenase-negative, indicating that the defect is not in the Krebs Cycle or in electron transport. When grown on pyruvate, it produces two to three times as much free alanine and half as much aspartate plus asparagine as the wild type. Two different assays show that the mutant phenotype is due to a deficiency of pyruvate carboxylase (EC 6.4.1.1), an important anaplerotic enzyme. Inferences that can be drawn from the characteristics of this mutant include (a) the glyoxylate cycle is probably located entirely outside the mitochondria, (b) the inner mitochondrial membrane appears to be impermeable to oxaloacetate, and (c) a succinate-malate exchange across the inner mitochondrial membrane connects the glyoxylate and Krebs cycles when yeast is grown on minimal medium with ethanol as a sole carbon source.
Feeding patterns of exclusively breast-fed infants during the first four months of life.
Milk intake and feeding patterns of 45 exclusively breast-fed infants were documented longitudinally over the first four months of life. Two prevailing feeding patterns were identified: one in which feedings were distributed throughout the 24-h day and one in which feedings were excluded from the early 12:00 a.m. to 6:00 a.m. period. No significant differences in total milk intake (g/24 h) were detected the day irrespective of feeding pattern. Feeding frequency and duration declined in successive months of lactation. Total milk intake was not significantly correlated to feeding frequency or duration.
Prediction of body density from skinfold measurements in lactating women.
Regression equations predicting body density from skinfold measurements were derived for a group of lactating women. It was concluded that specific equations for lactation were not necessary, since the resultant equations were not significantly different from those published for non-lactating women. The ability to predict an individual's body density from skinfold thickness measurements was unsatisfactory for either clinical or research applications.
Effect of mutants and inhibitors on mitochondrial transport systems in vivo in yeast.
We have reported elsewhere (Wills, C. and Martin, T. (1984) Biochim. Biophys. Acta 782, 274-284) that one or more mitochondrial transport systems may be involved in the regulation of the inducible alcohol dehydrogenase of yeast, ADH-II. In order to investigate this phenomenon further, it was necessary to determine which of these systems operate in the cell in vivo. We give in this paper preliminary evidence that inhibitors of the malate-phosphate (n-butyl malonate), malate-citrate (hydroxycitrate) and malate-alpha-ketoglutarate (aminooxyacetate or cycloserine) transport systems all operate in vivo. While the demonstration of the in vivo inhibitory activity of n-butyl malonate and hydroxycitrate is entirely by physiological methods, that of the transaminase inhibitors aminooxyacetate and cycloserine depends in part on the isolation of mutants capable of growth on glycerol in minimal medium. On this medium these mutants depend on the malate-aspartate shuttle for growth, and as expected the transaminase inhibitors prevent their growth. Two of the mutants show an enhanced rate of mitochondrial glutamate uptake. A preliminary survey of the properties of the glycerol growth mutants is presented, showing that the probable mode of action of these mutants is an increase in the efficiency of the malate-aspartate shuttle.
Extracellular conditions affecting the induction of yeast alcohol dehydrogenase II.
The alcohol dehydrogenases in yeast form one of the best-understood eukaryotic regulatory systems at the genetic level, but very little is known about their regulation at the biochemical level. We report on a simple whole-cell assay system for the induction of the inducible isozyme, alcohol dehydrogenase II, which has been used to show that no single compound added to the medium is responsible for the induction. The compounds which show the greatest inducing activity--malate, glutamate and fumarate--are all directly or indirectly involved in mitochondrial transport systems. No single compound can be purified from extracts of yeast cells to give inducing activity at low concentrations, suggesting that the inducing activity is an endogenous function of the cell. Tentative models for regulation of this isozyme involving the mitochondrion are proposed, and suggestions are made for testing these models further.