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Biomedical subjects

A G Clark

Publications and source records attributed to A G Clark.

At least 19 recordsLinked to original sources
PubMed1992

Sequencing errors and molecular evolutionary analysis.

Heuristic approaches were used to quantify the influence that sequencing errors have on estimates of nucleotide diversity, substitution rate, and the construction of genealogies. Error rates of less than 1 nucleotide/kb probably have little affect on conclusions about the evolutionary history of highly polymorphic organisms such as Drosophila and Escherichia coli, but organisms with very low nucleotide diversity, such as humans, require greater sequencing accuracy. A scan of GenBank for corrections of previous errors reveals that sequencing errors are highly nonrandom.

Animals↗
PubMed1992

Decrease in HIV provirus in peripheral blood mononuclear cells during zidovudine and human rIL-2 administration.

Quantification of human immunodeficiency virus (HIV) proviral DNA in peripheral blood mononuclear cells (PBMC) was performed in 13 HIV-seropositive asymptomatic individuals during 10-24 months by polymerase chain reaction amplification of multiple half-log dilutions of cellular DNA. At enrollment, subjects had a geometric mean titer of 100 copies of HIV provirus per 10(6) PBMC (mean +/- SD, 2 +/- 0.9 log10). In four untreated individuals there was no significant change in provirus levels during a mean period of 13.3 months. In eight patients treated with zidovudine (ZDV) and human recombinant interleukin 2 (rIL-2), HIV provirus copies declined to 13 per 10(6) cells (1.1 +/- 0.8 log10) at the end of the first course of ZDV and rIL-2 at week 20 (p less than 0.01), and to 40 per 10(6) cells (1.6 +/- 0.9 log10) after 12 months of treatment (p less than 0.04). Subsequent courses, which included 12 weeks of ZDV alone or 4 weeks of IL-2 alone, did not significantly change the already depressed provirus copy numbers. Proviral copy number also remained depressed during drug-free "washout periods" between courses. Finally, we observed a return to a geometric mean of 400 copies per 10(6) cells (2.6 +/- 0.3 log10) a mean of 7.9 months after discontinuation of therapy. Measurement of changes in HIV provirus should provide a direct marker for defining antiviral activity of drugs, biologics, and combination therapy.

DNA, Viral↗
PubMed1991

Excess nonsynonymous substitution of shared polymorphic sites among self-incompatibility alleles of Solanaceae.

The function of the self-incompatibility locus (S locus) of many plant species dictates that natural selection will favor high levels of protein diversity. Pairwise sequence comparisons between S alleles from four species of Solanaceae reveal remarkably high sequence diversity and evidence for shared polymorphism. The level of amino acid constraint was found to be significantly heterogeneous among different regions of the gene, with some regions being highly constrained and others appearing to be virtually unconstrained. In some regions of the protein, there was an excess of nonsynonymous over synonymous substitution, consistent with the strong diversifying selection that must operate on this locus. These hypervariable regions are candidates for the sites that determine functional allelic identity. Simple contingency table tests show that sites that have polymorphism shared between species have more nonsynonymous substitution than polymorphic sites that do not exhibit shared polymorphism. This is consistent with the idea that adaptive evolution favoring amino acid replacement is occurring at sites with shared polymorphism. Tests of clustered polymorphism reveal that an unusually low rate of recombination must be occurring in this locus, allowing very ancient alleles to preserve their identity.

Alleles↗
PubMed1991

Inhibition by inorganic anions of glutathione S-transferases from insect and mammalian sources.

Glutathione S-transferases 1-1, 3-3, 3-4 and 4-4 from rat liver and the major glutathione S-transferase from the wax moth (Galleria mellonella) are all inhibited by several simple inorganic anions. For each of 3-3, 3-4 and the insect enzyme, the order of inhibitory potency was ClO4- greater than or equal to SCN- greater than I- greater than NO3- greater than Br-. A more limited range of anions was tested on the isoenzymes 1-1 and 4-4, but the same trend was apparent. Values for Ki ranged from about 200 mM for Cl- to 6 mM for SCN- in the case of the insect enzyme and from 50 mM for Br- to 0.3 mM for SCN- for the rat isoform 3-3. Acetate, F-, SO4(2-) and PO4(3-) were not found to have significant inhibitory properties. The mode of inhibition was characterized as non-competitive in the case of the insect enzyme and rat transferase 1-1, whereas the mode of inhibition was partially non-competitive in the case of the rat isoforms 3-3, 3-4 and 4-4.

Animals↗
PubMed1991

The structure of human mitochondrial DNA variation.

Restriction analysis of mitochondrial DNA (mtDNA) of 3065 humans from 62 geographic samples identified 149 haplotypes and 81 polymorphic sites. These data were used to test several aspects of the evolutionary past of the human species. A dendrogram depicting the genetic relatedness of all haplotypes shows that the native African populations have the greatest diversity and, consistent with evidence from a variety of sources, suggests an African origin for our species. The data also indicate that two individuals drawn at random from the entire sample will differ at approximately 0.4% of their mtDNA nucleotide sites, which is somewhat higher than previous estimates. Human mtDNA also exhibits more interpopulation heterogeneity (GST = 0.351 +/- 0.025) than does nuclear DNA (GST = 0.12). Moreover, the virtual absence of intermediate levels of linkage disequilibrium between pairs of sites is consistent with the absence of genetic recombination and places constraints on the rate of mutation. Tests of the selective neutrality of mtDNA variation, including the Ewens-Watterson and Tajima tests, indicate a departure in the direction consistent with purifying selection, but this departure is more likely due to the rapid growth of the human population and the geographic heterogeneity of the variation. The lack of a good fit to neutrality poses problems for the estimation of times of coalescence from human mtDNA data.

Alleles↗
PubMed1991

Population genetics of the Y chromosome of Drosophila melanogaster: rDNA variation and phenotypic correlates.

One means of examining the evolutionary significance of molecular variation on the Y chromosome is to identify phenotypes specifically affected by Y-linked genes, and to quantify the phenotypic variation and its correlation to the molecular variation. The functional importance of the Y-linked array of rRNA genes is demonstrated by the ability of Y chromosome to rescue X-linked bobbed lethal alleles, whose lethality is seen in homozygous females. Because low numbers of X-linked rDNA gene copies result in increased developmental time and shortened bristles, and because there is considerable natural variation in Y-linked copy number, a careful examination of Y-linked variation in these two traits may uncover a mode of selection acting on the multigene family. In this study, 36 Y-chromosome replacement lines were tested to detect subtle variation in bristle phenotypes and developmental rates. Correlations among these traits, rDNA gene copy number, and intergenic sequence length were quantified. The absence of significant correlations between phenotypic characters and rDNA copy number of intergenic sequence length suggests that the extant molecular variation in Y-linked rDNA can have at most very small selective effects.

Animals↗
PubMed1991

Mutation-selection balance and metabolic control theory.

The evolution of metabolic control is examined with models that unify approaches of classical quantitative genetics and metabolic control theory. The quantitative traits considered are the activities of enzymes embedded within metabolic pathways. In the models, polygenic mutation alters the enzyme activities (Vmax/Km) according to prescribed distributions, and the population evolves following classical haploid viability selection. Stabilizing selection operates on global properties of the metabolic pathway, including either flux or metabolite pool concentration. Analytical results and numerical simulations demonstrate several important properties of these characters, including skewed, non-Gaussian equilibrium distributions, and an expected positive correlation between activities of enzymes flanking a substrate pool undergoing stabilizing selection. The house-of-cards approximation proved to be accurate in predicting the equilibrium distribution of allelic effects for a biologically reasonable segment of the parameter space. Further experimental and theoretical work is needed before a clear assessment can be made whether the observed variance in enzyme activities is explicable by a mutation-selection balance, and this system provides an excellent opportunity for such a test.

Alleles↗
PubMed1991

Subfamily relationships and clustering of rabbit C repeats.

C repeats constitute the predominant family of short interspersed repeats (SINEs) in the rabbit genome. Determination of the nucleotide sequence 5' to rabbit zeta-globin genes reveals clusters of C repeats, and analysis of these and other sequenced regions of rabbit chromosomes shows that the C repeats have a strong tendency to insert within or in close proximity to other C repeats. An alignment of 44 members of the C repeat family shows that they are composites of different sequences, including a tRNA-like sequence, a conserved central core, a stretch of repeating CT dinucleotides, and an A-rich tract. Cladograms generated by both parsimony and cluster analysis subdivide the C repeats into at least three distinct subfamilies. Nucleotides at sites diagnostic for subfamilies appear to have changed in a punctuated and progressive manner during evolution, indicating that a limited number of progenitors have given rise to new repeats in waves of dispersion. C repeats that insert into preexisting C repeats belong to subfamilies that are proposed to have been propagated more recently; hence, these data support the model of dispersion in successive waves. The divergence among the oldest group of C repeats is greater than that observed for the analogous Alu repeats in humans, indicating that rabbit C repeats have been propagating longer than human Alu repeats. The improved consensus sequence for these repeats is similar to that of the predominant artiodactyl SINE in both the tRNA-like region and a central region. Because members of different subfamilies cross-hybridize very poorly, hybridization data with representatives of each subfamily provide a new minimal estimate, 234,000, for the copy number of C repeats in the rabbit haploid genome, although it is likely that the actual value is closer to 1 million.

Animals↗
PubMed1991

Evolution of ribosomal RNA gene copy number on the sex chromosomes of Drosophila melanogaster.

A diverse array of cellular and evolutionary forces--including unequal crossing-over, magnification, compensation, and natural selection--is at play modulating the number of copies of ribosomal RNA (rRNA) genes on the X and Y chromosomes of Drosophila. Accurate estimates of naturally occurring distributions of copy numbers on both the X and Y chromosomes are needed in order to explore the evolutionary end result of these forces. Estimates of relative copy numbers of the ribosomal DNA repeat, as well as of the type I and type II inserts, were obtained for a series of 96 X chromosomes and 144 Y chromosomes by using densitometric measurements of slot blots of genomic DNA from adult D. melanogaster bearing appropriate deficiencies that reveal chromosome-specific copy numbers. Estimates of copy number were put on an absolute scale with slot blots having serial dilutions both of the repeat and of genomic DNA from nonpolytene larval brain and imaginal discs. The distributions of rRNA copy number are decidedly skewed, with a long tail toward higher copy numbers. These distributions were fitted by a population genetic model that posits three different types of exchange events--sister-chromatid exchange, intrachromatid exchange, and interchromosomal crossing-over. In addition, the model incorporates natural selection, because experimental evidence shows that there is a minimum number of functional elements necessary for survival. Adequate fits of the model were found, indicating that either natural selection also eliminates chromosomes with high copy number or that the rate of intrachromatid exchange exceeds the rate of interchromosomal exchange.

Animals↗
PubMed1990

Synthesis and use of an isoform-specific affinity matrix in the purification of glutathione S-transferases from the housefly, Musca domestica (L.).

Glutathione may be linked to an agarose matrix which has been activated by treatment with epichlorhydrin. The resulting resin displayed group selectivity for the glutathione S-transferases of the housefly Musca domestica (L). The isoenzymes of low isoelectric point, which have little activity with substrates other than 1-chloro-2,4-dinitrobenzene, bound strongly to this matrix and were eluted with 10 mM glutathione at pH 7.4. On the other hand, the group of isoenzymes of higher isoelectric point, showing activity with other substrates such as 3,4-dichloronitrobenzene, did not bind. These isoenzymes did bind to a sulfobromophthalein-glutathione conjugate immobilized on agarose and could be eluted with 5 mM sulfobromophthalein at pH 7.4. The immobilized glutathione resin bound rat liver glutathione S-transferase subunits from all three molecular weight classes.

Animals↗
PubMed1990

Direct and correlated responses to artificial selection on lipid and glycogen contents in Drosophila melanogaster.

A large outbred population of Drosophila melanogaster was subjected to artificial selection on lipid and glycogen storage. In three separate experiments, two replicates underwent sib selection for both increased and decreased storage. In the first study, flies were selected on the basis of total triacylglycerol for ten generations. This experiment resulted in no significant direct response, but there was a significant change in total body weight, underscoring the importance of concern for the allometric relationship between body weight and lipid content. In the second study, selection was performed for 15 generations on the percentage of body composition that was triacylglycerol. A significant direct response was obtained, and the two replicates revealed heritability estimates of 0.40 and 0.43. The third study selected glycogen content for 15 generations, and produced a significant response with heritabilities of 0.25 and 0.31. A series of 12 biochemical and enzyme kinetic traits was examined at five generation intervals in all experiments, and a number of correlated responses were detected. The results are interpreted with respect to the evolutionary constraints on energy storage evolution and the genetic basis of the allometric relationship between body weight and fat content.

Animals↗
PubMed1990

Genes encoding the beta-chains of HLA-DR7 and HLA-DQw2 define major susceptibility determinants for idiopathic nephrotic syndrome.

1. We have investigated the frequencies of the major histocompatibility complex class II alleles by restriction fragment length polymorphism analysis, using DR, DQ and DP complementary DNA probes, in 40 Caucasoid steroid-sensitive nephrotic children. 2. A significant association with HLA-DR7 was demonstrated (P = 2 x 10(-5); aetiological fraction 0.6). The DQB1 gene of HLA-DQw2 was present in 83% of our patients (P = 2 x 10(-4); aetiological fraction 0.7). We present evidence that it contributes a second susceptibility allele. 3. A weak association between the uncommon HLA-DP-Cp63 allele and the disease was also observed. 4. Our data suggest that the immune events in steroid-sensitive nephrotic syndrome are defined by a particular immunogenetic background involving the beta-chain genes of HLA-DR7 and HLA-DQw2.

Adolescent↗
PubMed1990

Polymorphism at the self-incompatibility locus in Solanaceae predates speciation.

Sequences of 11 alleles of the gametophytic self-incompatibility locus (S locus) from three species of the Solanaceae family have recently been determined. Pairwise comparisons of these alleles reveal two unexpected observations: (i) amino acid sequence similarity can be as low as 40% within species and (ii) some interspecific similarities are higher than intraspecific similarities. The gene genealogy clearly illustrates this unusual pattern of relationships. The data suggest that some of the polymorphism at the S locus existed prior to the divergence of these species and has been maintained to the present. In support of this hypothesis, the number of shared polymorphic sites was found to exceed the number found in simulations with independent accumulation of mutations. Strictly neutral evolution is exceedingly unlikely to maintain the polymorphism for such a long time. The allele multiplicity and extreme age of the alleles is consistent with Wright's classic one-locus population genetic model of gametophytic self-incompatibility. Similarities between the plant S locus and the mammalian major histocompatibility complex are discussed.

Alleles↗
PubMed1990

Two tests of Y chromosomal variation in male fertility of Drosophila melanogaster.

Deficiency mapping with Y autosome translocations has shown that the Y chromosome of Drosophila melanogaster carries genes that are essential to male fertility. While the qualitative behavior of these lesions provides important insight into the physiological importance of the Y chromosome, quantitative variation in effects on male fertility among extant Y chromosomes in natural populations may have a significant effect on the evolution of the Y chromosome. Here a series of 36 Y chromosome replacement lines were tested in two ways designed to detect subtle variation in effects on male fertility and total male fitness. The first test involved crossing males from the 36 lines to an excess of females in an attempt to measure differences in male mating success (virility) and male fecundity. The second test challenged males bearing each of the 36 Y chromosomes to competition in populations with males bearing a standard, phenotypically marked (BsY) chromosome. These tests indicated that the Y chromosome lines did not differ significantly in either male fertility or total fitness, but that interactions with autosomes approached significance. A deterministic population genetic model was developed allowing Y autosome interaction in fertility, and it is shown that, consistent with the experimental observations, this model cannot protect Y-linked polymorphism.

Analysis of Variance↗
PubMed1990

Inference of haplotypes from PCR-amplified samples of diploid populations.

Direct sequencing of genomic DNA from diploid individuals leads to ambiguities on sequencing gels whenever there is more than one mismatching site in the sequences of the two orthologous copies of a gene. While these ambiguities cannot be resolved from a single sample without resorting to other experimental methods (such as cloning in the traditional way), population samples may be useful for inferring haplotypes. For each individual in the sample that is homozygous for the amplified sequence, there are no ambiguities in the identification of the allele's sequence. The sequences of other alleles can be inferred by taking the remaining sequence after "subtracting off" the sequencing ladder of each known site. Details of the algorithm for extracting allelic sequences from such data are presented here, along with some population-genetic considerations that influence the likelihood for success of the method. The algorithm also applies to the problem of inferring haplotype frequencies of closely linked restriction-site polymorphisms.

Algorithms↗
PubMed1989

Rapid enzyme kinetic assays of individual Drosophila and comparisons of field-caught D. melanogaster and D. simulans.

Techniques for performing numerous enzyme kinetic assays with minimum time and effort would be valuable to studies of the evolutionary genetics of metabolic control and the quantitative genetics of determinants of kinetic parameters. Microtiter plate readers have been used for a variety of repetitious analytical techniques, and instruments are available that can take repetitive readings with sufficient speed to perform kinetic assays. The ability of these instruments to assay rapidly the kinetic properties of small samples makes them potentially useful for a number of problems in population genetics. While the ability to handle large numbers of samples is very attractive, the small sample volumes and optical imprecision of microtiter plates result in some sacrifice in accuracy. This paper presents methods for performing kinetic assays on individual field-caught Drosophila, quantifies the precision of these methods, and characterizes differences among Drosophila melanogaster and D. simulans from samples caught in California and Pennsylvania. Comparisons between field-caught and laboratory reared D. melanogaster show that most of the characters are very similar, with the exception of alpha GPDH, which has a threefold higher mean activity among field-caught flies. The phenotypic correlations are presented with a brief discussion of their relevance to assessing the evolution of metabolic control of these enzymes.

Animals↗