Biomedical subjects
A G Clark
Publications and source records attributed to A G Clark.
Dinucleotide repeat polymorphisms within the Flt-1 gene in minimal change nephropathy.
Dysregulation of Flt-1, a major receptor for vascular permeability factor (VPF), may provide a mechanism for the development of proteinuria in minimal change nephropathy (MCN). The gene for Flt-1 has a polymorphic dinucleotide repeat. We have demonstrated an 88% predominance of one allele and an 80% rate of homozygosity for this polymorphism with no association with MCN.
The size distribution of homozygous segments in the human genome.
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Molecular components of large conductance calcium-activated potassium (BK) channels in mouse pituitary corticotropes.
Large-conductance calcium- and voltage- activated potassium (BK) channels play a fundamental role in the signaling pathways regulating mouse anterior pituitary corticotrope function. Here we describe the cloning and functional characterization of the components of mouse corticotrope BK channels. RT-PCR cloning and splice variant analysis of mouse AtT20 D16:16 corticotropes revealed robust expression of mslo transcripts encoding pore-forming alpha-subunits containing the mouse homolog of the 59-amino acid STREX-1 exon at splice site 2. RT-PCR and functional analysis, using the triterpenoid glycoside, DHS-1, revealed that native corticotrope BK channels are not functionally coupled to beta-subunits in vivo. Functional expression of the STREX-1 containing alpha-subunit in HEK 293 cells resulted in BK channels with calcium sensitivity, single-channel conductance, and inhibition by protein kinase A identical to that of native mouse corticotrope BK channels. This report represents the first corticotrope ion channel to be characterized at the molecular level and demonstrates that mouse corticotrope BK channels are composed of alpha-subunits expressing the mouse STREX-1 exon.
Interleukin-4 and interleukin-4 receptor polymorphisms in minimal change nephropathy.
Minimal change nephropathy (MCN) is an important cause of nephrotic syndrome, especially in children, that is strongly associated with atopy and IgE production. The immunogenetics of MCN are poorly understood. Interleukin-4 (IL-4) is the critical cytokine involved in the development of atopy. Polymorphic regions in the genes encoding IL-4 itself and the IL-4 receptor have been demonstrated that may predispose to increased activity. We have analysed these polymorphisms in 149 patients with MCN and 73 controls to test the hypothesis that these loci are involved in genetic predisposition to MCN. In our populations there were no polymorphisms in the IL-4 promoter. We did confirm allelic variation in a dinucleotide repeat in the second intron of the IL-4 gene, but there was no significant difference between allele distributions in MCN and controls. Similarly, allele frequencies for the IL-4 receptor alpha chain polymorphism were similar in patients and controls. Genetic loci which are believed to influence IL-4 responsiveness and to predispose to atopy do not appear to be associated with susceptibility to MCN.
Chromatographic separation of fluorescent thiol adducts of 4-chloro-7-sulphobenzofurazan. Use as substrates for enzymes of the mercapturic acid xenobiotic pathway.
Fluorescent adducts of 4-chloro-7-sulphobenzofurazan with cysteine, cysteinylglycine, reduced glutathione and N-acetylcysteine were prepared. Adducts were separated by HPLC on a 3-mm Nova-Pak C18 reversed-phase column using isocratic elution with a solvent of acetonitrile-0.15 M phosphoric acid (5:95) buffered at pH 2.5. The adducts were detected using a fluorescence detector set at an excitation wavelength of 365 nm and an emission wavelength of 510 nm and an ultraviolet detector at 254 nm. The adduct of reduced glutathione was also formed by the action of the enzyme glutathione-S-transferase. This adduct acted as a substrate for the enzyme gamma-glutamyltranspeptidase and the product of this reaction, the 4-chloro-7-sulphobenzofurazanyl derivative of cysteinylglycine, acted as a substrate for either dipeptidase or aminopeptidase M. The sequential enzymic effects could be detected by changes in the relative fluorescence intensity of the solutions to which the respective enzymes had been added but were more appropriately followed by changes in the HPLC elution profiles after enzymic treatment of solutions.
Anaesthesia for Caesarean section in Takayasu's disease.
PURPOSE: We present a case of Takayasu's Disease (Occlusive Thromboaortopathy-OTAP) in which general anaesthesia was used for Caesarean section with processed encephalographic monitoring to detect cerebral ischaemia. CLINICAL FEATURES: The patient was a 33-year-old woman in whom OTAP had been the cause of cerebrovascular events. She had the typical pulseless upper body of OTAP and had documented severe bilateral carotid artery stenoses. Regional anaesthesia had failed in the past and she demanded general anaesthesia for Caesarean section. Processed electroencephalography was used to assist in monitoring for signs of cerebral ischaemia and the surgery was uncomplicated. CONCLUSION: The use of processed electroencephalographic monitoring allows some assessment of cerebral haemodynamics during general anaesthesia in patients who refuse regional techniques.
DNA sequence diversity in a 9.7-kb region of the human lipoprotein lipase gene.
Lipoprotein lipase plays a central role in lipid metabolism and the gene that encodes this enzyme (LPL) is a candidate susceptibility gene for cardiovascular disease. Here we report the complete sequence of a fraction of the LPL gene for 71 individuals (142 chromosomes) from three populations that may have different histories affecting the organization of the sequence variation. Eighty-eight sites in this 9.7 kb vary among individuals from these three populations. Of these, 79 were single nucleotide substitutions and 9 sites involved insertion-deletion variations. The average nucleotide diversity across the region was 0.2% (or on average 1 variable site every 500 bp). At 34 of these sites, the variation was found in only one of the populations, reflecting the differing population and mutational histories. If LPL is a typical human gene, the pattern of sequence variation that exists in introns as well as exons, even for the small number of samples considered here, will present challenges for the identification of sites, or combinations of sites, that influence variation in risk of disease in the population at large.
Stress tolerance and metabolic response to stress in Drosophila melanogaster.
A potentially important physiological response to stress may be alteration in the gross regulation of energy metabolism. Different genotypes may respond differently to environmental stress, and the variation in these norms of reaction may be of key importance to the maintenance of genetic variation in metabolic traits. In the study reported here, a set of genetically defined lines of Drosophila melanogaster were exposed to four stresses (acetic acid, ethanol, starvation and thermal stress) in order to assess the magnitude of environmental effects and genotype x environment interactions. In addition to scoring metabolic traits, distributions of survival times under each stress were also quantified. Although both metabolic traits and survival times exhibited strong differences among genotypes, the correlations between enzyme traits and survival were generally weak. Many of the genetic correlations exhibit significant heterogeneity across environments. The results suggest that transient environmental stress may play an important role in the evolution of this highly intercorrelated set of metabolic traits.
Haplotype structure and population genetic inferences from nucleotide-sequence variation in human lipoprotein lipase.
Allelic variation in 9.7 kb of genomic DNA sequence from the human lipoprotein lipase gene (LPL) was scored in 71 healthy individuals (142 chromosomes) from three populations: African Americans (24) from Jackson, MS; Finns (24) from North Karelia, Finland; and non-Hispanic Whites (23) from Rochester, MN. The sequences had a total of 88 variable sites, with a nucleotide diversity (site-specific heterozygosity) of .002+/-.001 across this 9.7-kb region. The frequency spectrum of nucleotide variation exhibited a slight excess of heterozygosity, but, in general, the data fit expectations of the infinite-sites model of mutation and genetic drift. Allele-specific PCR helped resolve linkage phases, and a total of 88 distinct haplotypes were identified. For 1,410 (64%) of the 2,211 site pairs, all four possible gametes were present in these haplotypes, reflecting a rich history of past recombination. Despite the strong evidence for recombination, extensive linkage disequilibrium was observed. The number of haplotypes generally is much greater than the number expected under the infinite-sites model, but there was sufficient multisite linkage disequilibrium to reveal two major clades, which appear to be very old. Variation in this region of LPL may depart from the variation expected under a simple, neutral model, owing to complex historical patterns of population founding, drift, selection, and recombination. These data suggest that the design and interpretation of disease-association studies may not be as straightforward as often is assumed.
Genetic conflicts, multiple paternity and the evolution of genomic imprinting.
We present nine diallelic models of genetic conflict in which one allele is imprintable and the other is not to examine how genomic imprinting may have evolved. Imprinting is presumed to be either maternal (i.e., the maternally derived gene is inactivated) or paternal. Females are assumed to be either completely monogamous or always bigamous, so that we may see any effect of multiple paternity. In contrast to previous verbal and quantitative genetic models, we find that genetic conflicts need not lead to paternal imprinting of growth inhibitors and maternal imprinting of growth enhancers. Indeed, in some of our models--those with strict monogamy--the dynamics of maternal and paternal imprinting are identical. Multiple paternity is not necessary for the evolution of imprinting, and in our models of maternal imprinting, multiple paternity has no effect at all. Nevertheless, multiple paternity favors the evolution of paternal imprinting of growth inhibitors and hinders that of growth enhancers. Hence, any degree of multiple paternity means that growth inhibitors are more likely to be paternally imprinted, and growth enhancers maternally so. In all of our models, stable polymorphism of imprinting status is possible and mean fitness can decrease over time. Neither of these behaviors have been predicted by previous models.
Female genotypes affect sperm displacement in Drosophila.
Differential success of sperm is likely to be an important component of fitness. Extensive variation among male genotypes in competitive success of sperm in multiply mated females has been documented for Drosophila melanogaster. However, virtually all previous studies considered the female to be a passive vessel. Nevertheless, under certain conditions female fitness could be determined by her role in mediating use of sperm from multiple males. Here we ask whether females differ among genotypes in their tendency to exhibit last-male precedence. Competition of sperm from two tester male genotypes (bwD and B3-09, a third-chromosome isogenic line from Beltsville, MD) was quantified by doubly mating female lines that had been rendered homozygous for X, second, or third chromosomes isolated from natural populations. The composite sperm displacement parameter, P2', was highly heterogeneous among lines, whether or not viability effects were compensated, implying the presence of polymorphic genes affecting access of sperm to eggs. Genetic variation of this type is completely neutral in the absence of pleiotropy or interaction between variation in the two sexes.
Negative covariance suggests mutation bias in a two-locus microsatellite system in the fish Sparus aurata.
Constraints on microsatellite length appear to vary in a species-specific manner. We know very little about the nature of these constraints and why they should vary among species. While surveying microsatellite variation in the Mediterranean gilthead sea bream, Sparus aurata, we discovered an unusual pattern of covariation between two closely linked microsatellite loci. One- and two-locus haplotypes were scored from PCR amplification products of each locus separately and both loci together. In a sample of 211 fish, there was a strong negative covariance in repeat number between the two loci, which suggests a mechanism that maintains the combined length below a constrained size. In addition, there were two clusters of the same combined haplotype length, one consisting of a long repeat array at one locus and a short array at the other and vice versa. We demonstrate that several models of biased mutation or natural selection, in theory, could generate this pattern of covariance. The common feature of all the models is the idea that tightly linked microsatellites do not evolve in complete independence, and that whatever size dependence there is to the process, it appears to "read" the combined size of the two loci.
HLA-DRB and -DQB1 alleles in Polish patients with hepatitis B associated membranous nephropathy.
We have studied the HLA-DRB and -DQB1 alleles of 42 paediatric patients who have suffered from membranous nephropathy associated with a hepatitis B infection (HBVMN). These patients were all from the Gdansk area of Northern Poland and the disease was diagnosed by light and electron microscopy. The control population consisted of 55 healthy children, approximately age matched, from schools in Gdansk. In addition we have also analysed 40 patients chronically infected with hepatitis B, without any renal involvement, as hepatitis B disease controls. The HLA alleles were defined using PCR/SSP. As idiopathic membranous nephropathy and low responsiveness to hepatitis B vaccine have been found to be associated with DR3 in Caucasoids, our hypothesis was that the HBVMN patients would show an increase in DR3. Our results indicate that, although there is a small increase in the frequency of DRBl*0301 in the HBVMN patients (16/42 38%) when compared to the healthy controls (15/55 31%), this does not approach significance. There is a significant increase in the frequency of DQB1*0303 in the HBVMN patients vs the healthy controls, after correction for the number of antigens detected (Pc)(13/42 vs 2/55, RR=11.6, P=0.0007, Pc=0.02). A similar increase in DQB1*0303 is seen in the HBVMN patients when compared to the hepatitis controls (13/42 vs 4/40) but this is only significant before correction (RR=4.3, P=0.04).
Mutation-selection balance with multiple alleles.
Human genetic disorders provide an extraordinary richness of data on the diversity of defective alleles. Well over 100 defective alleles for each of several human genetic disorders have been identified, including breast cancer (BRCA1), cystic fibrosis (CFTR), muscular dystrophy (DZM), and phenylketonuria (PAH). These observations raise the classical question of balance between the action of mutation generating new defective alleles and selection removing those alleles from the population. The problem of multiple-allele, mutation-selection balance was considered by Crow and Kimura, who obtained some approximate results showing that the level of dominance and degrees of interallelic complementation are important in determining the equilibrium allele frequencies. Here those deterministic results are reviewed and extended, showing that there are conditions yielding surprisingly high equilibrium frequencies of defective alleles. Just as the equilibrium mutation load is independent of the level of dominance, it is also independent of the number of defective alleles.
Neutral behavior of shared polymorphism.
Several cases have been described in the literature where genetic polymorphism appears to be shared between a pair of species. Here we examine the distribution of times to random loss of shared polymorphism in the context of the neutral Wright-Fisher model. Order statistics are used to obtain the distribution of times to loss of a shared polymorphism based on Kimura's solution to the diffusion approximation of the Wright-Fisher model. In a single species, the expected absorption time for a neutral allele having an initial allele frequency of 1/2 is 2.77 N generations. If two species initially share a polymorphism, that shared polymorphism is lost as soon as either of two species undergoes fixation. The loss of a shared polymorphism thus occurs sooner than loss of polymorphism in a single species and has an expected time of 1.7 N generations. Molecular sequences of genes with shared polymorphism may be characterized by the count of the number of sites that segregate in both species for the same nucleotides (or amino acids). The distribution of the expected numbers of these shared polymorphic sites also is obtained. Shared polymorphism appears to be more likely at genetic loci that have an unusually large number of segregating alleles, and the neutral coalescent proves to be very useful in determining the probability of shared allelic lineages expected by chance. These results are related to examples of shared polymorphism in the literature.
Stress and metabolic regulation in Drosophila.
Environmental changes that result in stress (defined here as decreased absolute viability and/or fecundity) result in extrinsic changes in metabolism that are to some extent compensated by altered gene expression. The fact that different genotypes may respond differently to environmental stress may be of key importance to the maintenance of genetic variation in metabolic traits. Here we quantify a set of metabolic characters in genetically defined lines of Drosophila melanogaster subjected to four stresses (3% acetic acid, 3% ethanol, starvation and thermal stress) in order to assess the magnitude of environmental effects and genotype x environment interactions. Genetic correlations were quantified, and many exhibit significant heterogeneity across environments. Pleiotropically related traits may exhibit the phenomenon of apparent selection, whose effects may be particularly strong in stressful environments. This transient apparent selection may have a large consequence on the maintenance of genetic variation.
Comparisons of the nucleotide substitution process among repetitive segments of the alpha- and beta-spectrin genes.
The actin-cross-linking protein spectrin is a prominent component of the membrane cytoskeleton. Spectrin is a tetramer of two antiparallel alphabeta-dimers which share a unique and ancient gene structure. The alpha-spectrin and beta-spectrin genes are composed primarily of tandemly repeated 106-amino-acid segments, each of which forms a triple alpha-helical coiled coil. Both the genes and the repeats themselves are homologous. The two genes are thought to be the result of a gene duplication event, and each gene is the product of duplications of the 106-amino-acid repeats. In this work we compare the process of molecular evolution across the repeated segments of the alpha- and beta-spectrin genes. We find that the alpha-spectrin segments have, for the most part, evolved in a homogeneous fashion, while considerable heterogeneity is found among beta-spectrin segments. Several segments with unique known functions are found to have evolved differently than the others. On the basis of heterogeneity of the evolutionary process, we suggest that at least one repeat has a unique function that has yet to be documented. We also present new statistical methods for comparing the evolutionary process between different regions of DNA sequences.