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PubMed1992

Population genetics of polymorphism and divergence.

Frequencies of mutant sites are modeled as a Poisson random field in two species that share a sufficiently recent common ancestor. The selective effect of the new alleles can be favorable, neutral, or detrimental. The model is applied to the sample configurations of nucleotides in the alcohol dehydrogenase gene (Adh) in Drosophila simulans and Drosophila yakuba. Assuming a synonymous mutation rate of 1.5 x 10(-8) per site per year and 10 generations per year, we obtain estimates for the effective population size (N(e) = 6.5 x 10(6)), the species divergence time (tdiv = 3.74 million years), and an average selection coefficient (sigma = 1.53 x 10(-6) per generation for advantageous or mildly detrimental replacements), although it is conceivable that only two of the amino acid replacements were selected and the rest neutral. The analysis, which includes a sampling theory for the independent infinite sites model with selection, also suggests the estimate that the number of amino acids in the enzyme that are susceptible to favorable mutation is in the range 2-23 at any one time. The approach provides a theoretical basis for the use of a 2 x 2 contingency table to compare fixed differences and polymorphic sites with silent sites and amino acid replacements.

Alcohol Dehydrogenase↗
PubMed2001

The Q allele variant (GLN121) of membrane glycoprotein PC-1 interacts with the insulin receptor and inhibits insulin signaling more effectively than the common K allele variant (LYS121).

When overexpressed, the membrane glycoprotein PC-1 may play a role in human insulin resistance through the inhibition of insulin receptor (IR) autophosphorylation. A PC-1 variant (K121Q, with lysine 121 replaced by glutamine) is also associated with whole-body insulin resistance when not overexpressed. To better understand the effects of the Q allele on IR function and downstream signaling, we transfected cultured cells with cDNAs for either the Q or the K alleles. In human MCF-7 cells, the Q allele was severalfold more effective (P < 0.05-0.01) than the K allele in reducing insulin stimulation of IR autophosphorylation, insulin receptor substrate-1 phosphorylation, phosphatidylinositol 3-kinase activity, glycogen synthesis, and cell proliferation. Similar data on IR autophosphorylation inhibition were also obtained in mouse R-/hIR and human HEK 293 cell lines. In transfected MCF-7 cells, 125I-labeled insulin binding and IR content were unchanged, and PC-1 overexpression did not influence IGF-1 stimulation of IGF-1 receptor autophosphorylation. Both the Q and K alleles directly interacted with the IR, as documented by coimmunoprecipitation assays. This interaction was greater for the Q allele than for the K allele (P < 0.01), suggesting that direct PC-1-IR interactions are important for the PC-1 inhibitory effect on insulin signaling. In conclusion, the Q allele has stronger inhibitory activity on IR function and insulin action than the more common K allele, and this is likely a consequence of the intrinsic characteristics of the molecule, which more strongly interacts with the IR.

Alleles↗
PubMed1993

Allelic variations of human keratins K4 and K5 provide polymorphic markers within the type II keratin gene cluster on chromosome 12.

To appreciate point mutations in keratin genes as causes for hereditary epithelial diseases, the normal variation of these gene sequences in the population must be known. Because genetic polymorphism of keratins at the protein level due to allelic variation has been described for the type II keratins 4 and 5, we have analyzed their corresponding genes using single-strand conformation polymorphism gel electrophoresis and sequence analysis of polymerase chain reaction amplified genomic DNA. Although no sequence variations were found in the carboxyl-terminal and rod domains we were able to map the molecular differences among the alleles to their amino-terminal domains. In particular, we have identified three alleles of keratin 4. Two alleles differed by a nucleotide transition causing a neutral amino acid substitution (alanine to valine) and one allele had a 42-bp in-frame deletion corresponding to 14 amino acids within the V1 subdomain. Three alleles were also recognized for the keratin 5 locus, all being elicited by single nucleotide substitutions. Of these, only one altered the amino acid sequence, replacing an uncharged (glycine) with a charged (glutamic acid) amino acid in the H1 subdomain. Pedigree analyses in three families showed the alleles to be inherited as autosomal Mendelian traits. Thus, these normal alleles of keratins 4 and 5 will provide favorable polymorphic markers for linkage analysis directly within the cluster of type II keratin genes located on chromosome 12q to elucidate the potential involvement of these and other keratin genes in disorders of squamous cell differentiation.

Alleles↗
PubMed2006

Estrogen receptor alpha and risk for cognitive impairment in postmenopausal women.

The estrogen receptor alpha (ESR1) gene has been implicated in the process of cognitive impairment in elderly women. In a paired case-control study, we tested whether two ESR1 gene polymorphisms (the XbaI and PvuII sites) are risk factors for cognitive impairment as measured by the six-item Orientation-Memory-Concentration test in postmenopausal Danish women. Hormone replacement therapy, age and executive cognitive ability were examined as covariates for ESR1 gene effects on cognitive impairment. The XbaI polymorphism showed a marginal effect on cognitive abilities (P=0.054) when adjusted for executive cognitive ability. Using a dominant genetic model for the X allele, we found an elevated risk (executive cognitive ability adjusted P=0.033) for cognitive impairment. Hormone replacement therapy also had a borderline effect on cognitive ability (P=0.049) and this effect was reflected in executive cognitive ability. These data support that the ESR1 gene variants affect cognitive functioning in postmenopausal women.

Aged↗
PubMed1999

Molecular standardization of mating type terminology in the Gibberella fujikuroi species complex.

Mating type in the Gibberella fujikuroi species complex is controlled by a single locus with two alleles and is usually identified following sexual crosses with standard, female-fertile tester isolates. The mating type alleles have been arbitrarily designated "+" and "-" within each biological species, and the nomenclature is tied to the standard tester strains. We developed a pair of PCR primers that can be used to amplify a unique fragment of one of the mating type alleles (MAT-2) from at least seven of the biological species in this species complex. Based on the amplification pattern, we propose a replacement for the existing, arbitrary +/- terminology that is presently in use. The new terminology is based on DNA sequence similarities between the mating type allele fragments from the biological species of the G. fujikuroi species complex and the corresponding fragments from other filamentous ascomycetes.

Crosses, Genetic↗
PubMed2001

Identification of a new HLA-DRB1 allele (DRB1*0318) in three members of a Caucasian Spanish family.

This communication reports the identification of a new allele HLA-DRB1*03 in three members of a Caucasian Spanish family. The new allele has been officially named HLA-DRB1*0318 by the World Health Organization Nomenclature Committee. The exon 2 sequence of this new allele is identical to that of DRB1*03011 except for the first nucleotide of codon 45. The nucleotide change (C replacing G) leads to the amino acid substitution of glycine to arginine (GGG-->CGG) at position 45. This position of the beta1 domain shows very little polymorphism among DRB1* alleles (nucleotide changes at this position have only been reported for DRB1*1436 and DRB1*0105) and locates in the vicinity of the highly polymorphic position 47, which is a constituent of the groove's pocket interacting with the amino acid position 7 of the antigen peptide. The familial study showed that the new allele was maternally transmitted into the HLA-A*3002, -B*1801, -Cw*0501, -DRB1*0318, -DRB3*0202, -DQB1*0201 haplotype. Interestingly, the two siblings of the family, which were HLA identical and suffered of insulin-dependent diabetes mellitus (IDDM), were carriers of the two HLA haplotypes (DRB1*03/DQB1*0201 and DRB1*04/DQB1*0302) reported as susceptibility markers to IDDM in Caucasians.

Alleles↗
PubMed1989

Characterization and expression of the human rhoH12 gene product.

The rho genes constitute an evolutionarily conserved family having significant homology to the ras oncogene family. These genes have been found in Saccharomyces cerevisiae, Drosophila melanogaster, rat, and human; their 21,000-dalton products show strong conservation of structure. In humans, three classes of rho cDNA clones have been identified which differ by virtue of the presence of variable C-terminal domains: rhoH12, rhoH6, and rhoH9. The predicted 193 amino acids of human rhoH12 protein show 88% similarity with those of the human rhoH6 clone, 96.8% similarity with those of the Aplysia rho product, and 81.8% similarity with those of the yeast RHO1 protein. Rat-1 and NIH 3T3 mouse fibroblasts were transfected with clones containing the normal human rhoH12 allele as well as the variants encoding valine in place of the glycine and leucine in place of the glutamine normally found at residues 14 and 64, respectively. These replacements mirror the changes responsible for oncogenic activation of the related ras-encoded p21 proteins. These mutant rhoH12 clone alleles did not cause focus formation in monolayers or growth in soft agar. However, amplification of normal rhoH12 via cotransfection with a dihydrofolate reductase gene resulted in colonies that displayed reduced dependence on serum for growth, grew to higher saturation densities, and were tumorigenic when inoculated into nude mice. Normal p21rho protein was detected in the transfected cell lines as well as in normal cell lines by Western immunoblot and immunoprecipitation analysis with rabbit antibodies raised against the peptide corresponding to amino acids 122 to 135.

Amino Acid Sequence↗
PubMed2001

G649, an allelic variant of the human H2 receptor with low basal activity, is resistant to upregulation upon antagonist exposure.

Orange et al reported an allelic variant of the human histamine H2 receptor, in which adenine 649 was replaced with guanine, to be more frequent in the schizophrenic population than controls in British Caucasians. The A649 to G change causes an Asn to Asp transition at amino acid position 217 in the third intracellular region, which is postulated to be important for receptor function. Herein, we analyzed the functional significance of this variant using wild-type and variant receptors expressed in Chinese hamster ovary cells. The variant receptor was associated with markedly lower basal cAMP productions than the wild-type receptor. Histamine-dependent cAMP productions via the variant receptor were lower as well. Treatment of cells expressing variant receptors with 10(-5) M ranitidine for 24 h resulted in a reduced degree of receptor upregulation as compared with the wild-type receptor. Thus, this is the first report of an allelic variant of the human H2 receptor which confers altered receptor function. To analyze gastric acid secretion in individuals with this variant, we examined 100 Japanese control subjects. However, neither heterozygotes nor homozygotes were found, suggesting that this variant, if present, is uncommon in the Japanese population.

Alleles↗
PubMed1990

Frequency of three Hex A mutant alleles among Jewish and non-Jewish carriers identified in a Tay-Sachs screening program.

Mutations in the HEX A gene, encoding the alpha-subunit of beta-hexosaminidase A (Hex A), are the cause of Tay-Sachs disease as well as of juvenile, chronic, and adult GM2 gangliosidoses. We have examined the distribution of three mutations--a 4-nucleotide insertion in exon 11, a G----C transversion at a 5' splice site in intron 12, and a 269Gly----Ser amino acid substitution in exon 7--among individuals enzymatically diagnosed as carriers of Hex A deficiency. Mutation analysis included polymerase chain reaction (PCR) amplification of the relevant regions of genomic DNA, followed by allele-specific oligonucleotide hybridization; another test for heterozygosity of the exon 11 insertion was based on the formation of heteroduplex PCR fragments of low electrophoretic mobility. The percentage distribution of the exon 11, intron 12, exon 7, and unidentified mutant alleles was 73:15:4:8 among 156 Jewish carriers of Hex A deficiency and 16:0:3:81 among 51 non-Jewish carriers. Regardless of the mutation, the ancestral origin of the Jewish carriers was primarily eastern and (somewhat less often) central Europe, whereas for the non-Jewish carriers it was western Europe. Because a twelfth of the Jewish carriers and four-fifths of the non-Jewish carriers of Hex A deficiency had mutant alleles other than the three common ones tested, enzyme-based tests cannot be replaced by DNA-based tests at the present time. However, DNA-based tests for two-carrier couples could identify those at risk for the chronic/adult GM2 gangliosidoses rather than for infantile Tay-Sachs disease.

Alleles↗
PubMedDate not supplied

A common genetic polymorphism associated with lower coagulation factor VII levels in healthy individuals.

We have identified a genetic polymorphism of factor VII that is strongly associated with plasma factor VII coagulant activity (factor VIIc) in healthy individuals from the United Kingdom. This polymorphism was detected after Msp I digestion of polymerase chain reaction-amplified genomic DNA. In a sample of 284 men, the frequency of the M2 allele (loss of cutting site) is 0.1, and individuals with the M1M2 genotype have factor VIIc levels 22% below the sample mean (p less than 0.0001). Msp I genotype was found to be the strongest predictor of factor VIIc, accounting for 20.2% of the variance, with cholesterol accounting for an additional 3.5%. The base change that gives rise to the Msp I polymorphism is a G-to-A substitution in the codon for amino acid 353, leading to replacement of arginine (Arg) with glutamine (Gln) in the protein product of the M2 allele (designated Gln 353). Three individuals homozygous for the M2 allele have both low factor VIIc and low factor VII protein concentrations. The conformation of the Gln 353 molecule may be different from that of the Arg 353 protein, affecting its intracellular processing, secretion, turnover in plasma, or activity. In view of its association with lower factor VIIc levels, possession of the M2 allele may confer protection against thrombosis and myocardial infarction.

Aged↗
PubMed2003

Molecular markers for rapidly identifying candidate genes in Chlamydomonas reinhardtii. Ery1 and ery2 encode chloroplast ribosomal proteins.

To take advantage of available expressed sequence tags and genomic sequence, we have developed 64 PCR-based molecular markers in Chlamydomonas reinhardtii that map to the 17 linkage groups. These markers will allow the rapid association of a candidate gene sequence with previously identified mutations. As proof of principle, we have identified the genes encoded by the ERY1 and ERY2 loci. Mendelian mutations that confer resistance to erythromycin define three unlinked nuclear loci in C. reinhardtii. Candidate genes ribosomal protein L4 (RPL4) and L22 (RPL22) are tightly linked to the ERY1 locus and ERY2 locus, respectively. Genomic DNA for RPL4 from wild type and five mutant ery1 alleles was amplified and sequenced and three different point mutations were found. Two different glycine residues (G(102) and G(112)) are replaced by aspartic acid and both are in the unstructured region of RPL4 that lines the peptide exit tunnel of the chloroplast ribosome. The other two alleles change a splice site acceptor site. Genomic DNA for RPL22 from wild type and three mutant ery2 alleles was amplified and sequenced and revealed three different point mutations. Two alleles have premature stop codons and one allele changes a splice site acceptor site.

Alleles↗
PubMed1993

Phenotypic revertant mutations of a new OmpR2 mutant (V203Q) of Escherichia coli lie in the envZ gene, which encodes the OmpR kinase.

The Escherichia coli ompR2 allele ompR472 contains a valine-to-methionine point mutation at position 203, resulting in an OmpF-constitutive OmpC- outer membrane phenotype. In the present study, OmpR residue V-203 was replaced with glutamine (V203Q mutation), resulting in the same outer membrane phenotype. However, unlike the OmpFc OmpC- phenotype conferred by the OmpR(V203M) mutant protein, the OmpFc OmpC- phenotype produced by the OmpR(V203Q) mutation was suppressed by the envZ11(T247R) allele. Additional suppressors of OmpR(V203Q) were isolated by random mutagenesis. All suppressor mutations were found in the envZ gene and conferred an OmpC+ OmpF- phenotype in the presence of the wild-type ompR. These envZ11-like mutations mapped to a region different from those previously reported and were incapable of suppressing the ompR(V203M) allele. Our results indicate that while methionine or glutamine replacements could cause similar effects on OmpF and OmpC expression, they conferred different abilities on the mutant proteins to be suppressed by envZ.

Bacterial Outer Membrane Proteins↗
PubMed2005

Investigating the aspartic acid (D) repeat of asporin as a risk factor for osteoarthritis in a UK Caucasian population.

OBJECTIVE: A compelling genetic association with osteoarthritis (OA) of 2 functional alleles in the aspartic acid (D) repeat of the asporin gene was recently reported in a Japanese population. Allele D13 of the repeat encoded OA protection, whereas allele D14 encoded OA susceptibility. The 2 alleles mediate differences in the capacity of asporin to inhibit the cartilage growth factor transforming growth factor beta, with the D14 allele being a particularly potent inhibitor. Our objective was to assess whether the D repeat is associated with OA in UK Caucasians. METHODS: The repeat was genotyped in 1,247 patients who had undergone elective joint replacement of the hip or the knee due to end-stage primary OA and in 748 age-matched controls. RESULTS: The D13 allele was more common in controls, and the D14 allele was more common in patients. However, this trend was significant only for men who had undergone hip replacement (P = 0.016, odds ratio 1.48, 95% confidence interval 1.09-2.01). CONCLUSION: Our data suggest that the asporin polymorphism is not a major influence on OA etiology in Caucasians. The results of our study do not question the veracity of the Japanese report. Instead, our study highlights the complex, heterogeneous nature of OA genetic susceptibility.

Aged↗
PubMed1995

A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori.

A homologous gene transfer system for Aspergillus awamori for site-specific integration is described, based on two components. First, a defined A. awamori pyrG mutant strain constructed by a selection strategy for gene-replacement in fungi. Second, a vector with a homologous pyrG selection marker containing a defined mutation at a site different from that of the mutations in the pyrG gene of the defined mutant strain. Defined mutation in the A. awamori pyrG gene, isolated from a genomic library by heterologous hybridisation with the A. niger pyrG gene as a probe, were introduced by specifically altering sequences at restriction sites in the coding region of the gene. After transformation of the A. awamori wild-type strain with vectors containing these mutated pyrG genes, and selection for 5-fluoro-orotic acid resistance (5-FOAR), on the average 60% of the 5-FOAR colonies originated from replacement of the wild-type pyrG gene by the mutated pyrG allele. After transformation of a mutant strain, carrying a mutation near the 5' end of the pyrG gene with vectors containing a mutation near the 3' end of the pyrG gene, 35% of the resulting transformants contained one copy of the vector at the pyrG locus.

Amino Acid Sequence↗
PubMed2003

Gene replacement method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability.

We describe a method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability. This method utilizes a chloramphenicol-resistant plasmid containing a temperature-sensitive (ts) replication origin. In this method, the target gene is first cloned into the ts vector and the recombinant plasmid is used to transform wild-type B. subtilis. The second step involves transformation of the resulting strain with a linear DNA fragment containing a second antibiotic resistance marker (tet) that disrupts the gene of interest. Selection for tetracycline resistance forces a double crossover between the chromosomal and fragment-borne copies of the gene, thereby replacing the wild-type gene in the chromosome with the disrupted allele. Cells survive even if the gene is essential by virtue of the complementing plasmid. Transformants are then grown at the non-permissive temperature for plasmid replication under various growth conditions. Isolation of chloramphenicol-sensitive colonies indicates that the gene is dispensable, whereas the inability to isolate chloramphenicol-sensitive colonies indicates that the gene is essential. The general utility of this method is demonstrated by allowing disruption of mtrA and trpE under conditions that render each gene non-essential, but not under growth conditions in which each gene is essential.

ATP-Binding Cassette Transporters↗
PubMed1998

Construction of a recA mutant of Burkholderia (formerly Pseudomonas), cepacia.

A recA mutant was constructed of a soil isolate of Burkholderia cepacia, strain ATCC 17616. Prior to mutagenesis, the recA gene was cloned from this strain by its ability to complement the methyl methanesulfonate sensitivity of an Escherichia coli recA mutant. Sequence analysis of the strain showed high sequence similarity (94% nucleic acid and 99% amino acid identity) with the recA gene previously cloned from a clinical isolate of B. cepacia, strain JN25. The subcloned recA gene from B. cepacia ATCC 17616 restored UV resistance and recombination proficiency to recA mutants of E. coli and Pseudomonas aeruginosa, as well as restoring the ability of D3 prophages to be induced to lytic growth from a RecA- strain of P. aeruginosa. The recA mutant of B. cepacia ATCC 17616 was constructed by lambda-mediated Tn5 mutagenesis of the cloned recA gene in E. coli, followed by replacement of the Tn5-interrupted gene for the wild-type allele in the chromosome of B. cepacia by marker exchange. The RecA- phenotype of the mutant was demonstrated by the loss of UV resistance as compared to the parental strain. Southern hybridization analysis of chromosomal DNA from the mutant indicated the presence of Tn5 in the recA gene, and the location of the Tn5 insertion in the recA allele was identified by nucleotide sequence analysis. A test using the recA mutant to see if acquired resistance to D-serine toxicity in B. cepacia might be a result of RecA-mediated activities proved negative; nevertheless, RecA activity potentially contributes to the overall genomic plasticity of B. cepacia and a recA mutant will be useful in bioengineering of this species.

Amino Acid Sequence↗
PubMed1999

Use of sulfite resistance in Saccharomyces cerevisiae as a dominant selectable marker.

Two S. cerevisiae genes were found to exhibit dominant phenotypes useful for selecting transformants of industrial and laboratory strains of S. cerevisiae. FZF1-4, which confers sulfite resistance, was originally isolated and identified as RSU1-4, but the two genes are shown here to be allelic. Cysteine 57 in wild-type Fzf1p was found to be replaced by tyrosine in Fzf1-4p. Multicopy SSU1, which also confers sulfite resistance, was found to be somewhat less efficient. In both cases, a period of outgrowth in non-selective medium following transformation was found to be necessary. The number of transformants obtained was found to be strain-dependent, and also to depend on the sulfite concentration used during selection. Undesirable background growth of non-transformants was not observed at cell densities as high as 2.5 x 10(7)/plate. In two ura3 laboratory strains where selection for URA3 was applied independently of that for sulfite, the transformation efficiency for sulfite resistance was about 50% that for uracil prototrophy.

Anion Transport Proteins↗
PubMed1989

Fixation probabilities for advantageous mutants: a note on multiplication and sampling.

If the average number of gametes produced by the individual is small, as may be the case for haploid organisms, then sampling with and without replacement can lead to considerable differences in the fixation probabilities of mutant alleles. As a function of the population size N, these probabilities converge quickly to the survival probabilities given by branching processes with Poisson or Bernoulli offspring distributions.

Biological Evolution↗