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A G Clark

Publications and source records attributed to A G Clark.

At least 145 records · Page 8Linked to original sources
PubMed1995

Spontaneous mutation rate of modifiers of metabolism in Drosophila.

A rigorous test of our understanding of evolutionary quantitative genetics would be to predict accurately the equilibrium distribution of a character from empirical estimates of the relevant parameters in a mutation-selection-drift balance model. An aspect of this problem that is amenable to experimental analysis is the distribution of the effects of new mutations. This study quantifies the divergence among 200 lines of Drosophila melanogaster as they accumulated mutations on the second chromosome and estimates the rate of increase of variation and covariation in metabolic characters. Amounts of stored triacylglycerol and glycogen and the activities of a series of 12 metabolic enzymes were assayed in a subset of lines at generations 0, 11, 22, 33 and 44. Analyses of the rate of increase in the among-line variance in each trait allowed estimation of Vm/Ve, the ratio of among-line variance added per generation to the environmental variance. Values of Vm/Ve for the second chromosome ranged from 0.0004 to 0.0289 per generation. Six of the 16 characters showed significant departure from a normal distribution, and several lines exhibited large changes in more than one character. The covariance of pairs of traits also was partitioned into a within-line component (environmental covariance, Cov(e)) and an among-line component (mutational covariance, Covm). Both variances and covariance among lines increased over time, as assessed by linear regression, whereas environmental covariance showed no such trend. Results indicate that the quantitative genetic parameters describing the variation in metabolic traits are similar to those of other continuous characters.

Analysis of Variance↗
PubMed1994

Heart fatty acid binding protein is a novel regulator of cardiac myocyte hypertrophy.

Heart fatty acid binding protein (H-FABP) is a 15kDa protein that mediates the passage of fatty acids from the plasma membrane to sites of lipid synthesis. We report here, for the first time, that H-FABP is a potent inducer of cardiac myocyte hypertrophy, stimulating an increase in cell surface area, protein synthesis and c-jun expression. A high affinity receptor for H-FABP has also been identified. Taken together, these data suggest that H-FABP may participate in the regulation of cardiac myocyte growth and differentiation.

Animals↗
PubMed1994

Invasion and maintenance of a gene duplication.

The ubiquity of multigene families is evidence for the frequent occurrence of gene duplication, but the origin of multigene families from a single gene remains a little-studied aspect of genome evolution. Although it is clear that a duplication can arise and become fixed in a population purely by random genetic drift and that the rate of fixation is accelerated if the duplication is directly advantageous, the nature of gene duplication suggests that other factors may influence the fate of a novel duplication. In the face of disadvantageous loss-of-function mutations, duplication of a functional gene may provide a buffer against such mutations. Here the conditions for invasion of a rare duplication starting from a mutation-selection balance are derived with formal population genetic models in both haploids and diploids. Recurrent duplication protects the duplicated chromosome from loss and can be very effective in increasing its frequency in a population. In the absence of recurrent duplication, one might suppose that a duplication would be favored by natural selection because it can mask the effects of deleterious mutations. However, the models show that a duplication can invade only if it provides a direct advantage to the organism. This result is closely related to recent theoretical work on the evolution of ploidy.

Diploidy↗
PubMed1994

Nature screen: an efficient method for screening natural populations of Drosophila for targeted P-element insertions.

The efficiency of molecular techniques is making it increasingly necessary to rely on reverse genetics to understand the function of genes. Tissue-specific libraries allow one to identify numerous genes that can be cloned, sequenced, and mapped and whose temporal and tissue-specific pattern of expression are well characterized but whose function remains unknown. In such cases, it is desirable to generate targeted mutations to examine the phenotype of loss-of-function lesions. Here we describe a method for identifying naturally occurring variants of Drosophila melanogaster with specific genes tagged by a nearby P element. Imprecise P-element excision can then be used to generate a series of small deletions in or near the gene. In the method described here, large numbers of wild-caught males were crossed to balancer females, and inserts were identified in pooled samples by the polymerase chain reaction with one primer from each target gene and one primer from the P-element terminal repeat. We present the calculations for the probability of successfully tagging a gene and show that it is greatly improved by simultaneously screening inserts into several genes. If a large natural population is available, a nature screen is faster and easier than inducing P-element transposition in the laboratory, but the resulting lines, being genetically heterogeneous, may require more subsequent work to isolate. Using this method to screen the genomes of approximately 10,400 males, we found P-element inserts in close proximity to 3 of 10 genes that were screened.

Animals↗
PubMed1993

Inhibition of glutathione S-transferases from rat liver by basic triphenylmethane dyes.

(1) Basic triphenylmethane dyes related to pararosanilin inhibit class alpha glutathione S-transferases (GSTs) from rat liver. The inhibitory potency of each dye correlates with its octanol-water partition coefficient. Values of Ki determined at pH 6.5 ranged from about 1 x 10(-7) M for Ethyl violet to 7 x 10(-5) M for Methyl green. GST 3-3, a class mu isoenzyme, was an order of magnitude less sensitive to inhibition by Ethyl violet. (2) All of the dyes tested were bleached to varying degrees by glutathione. The bleaching appears to result from the formation of an adduct between the dye and glutathione. At pH 6.5, adduct formation is significant only for Malachite green and Methyl green. There is kinetic evidence that for these dyes the adduct contributes significantly to the overall inhibition. It is probable that at physiological pH, all of the dyes would exist to a significant extent in the adduct form. (3) The dyes are excreted extensively in the bile, at least partly as the glutathione adduct. The free dye is regenerated on standing, it is assumed as a result of removal of glutathione by oxidation.

Animals↗
PubMed1993

Conservation of alternative splicing and genomic organization of the myosin alkali light-chain (Mlc1) gene among Drosophila species.

The Mlc1 gene of Drosophila melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlc1 pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlc1 genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%-99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC1 isoforms, suggests that exon 5 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis-regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlc1 gene to maintain proper expression and function of the two MLC1 polypeptides.

Alternative Splicing↗
PubMed1993

Prospects for estimating nucleotide divergence with RAPDs.

The technique of random amplification of polymorphic DNA (RAPD), which is simply polymerase chain reaction (PCR) amplification of genomic DNA by a single short oligonucleotide primer, produces complex patterns of anonymous polymorphic DNA fragments. The information provided by these banding patterns has proved to be of great utility for mapping and for verification of identity of bacterial strains. Here we consider whether the degree of similarity of the banding patterns can be used to estimate nucleotide diversity and nucleotide divergence. With haploid data, fragments generated by RAPD-PCR can be treated in a fashion very similar to that for restriction-fragment data. Amplification of diploid samples, on the other hand, requires consideration of the fact that presence of a band is dominant to absence of the band. After describing a method for estimating nucleotide divergence on the basis of diploid samples, we summarize the restrictions and criteria that must be met when RAPD data are used for estimating population genetic parameters.

Computer Simulation↗
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↗