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At least 919 records · Page 51Linked to original sources
PubMed2000

Rapid and sensitive genotyping of dopamine D4 receptor tandem repeats by automated ultrathin-layer gel electrophoresis.

Prior studies have revealed possible association between the presence of a seven repeat of the 48 bp variable number tandem repeat polymorphism of the human dopamine D4 receptor gene (DRD4) and some normal and pathological human traits, such as novelty seeking, hyperactivity disorders, and substance abuse. Some reports supported this finding whereas others did not. Incorrect genotyping could be one of the reasons for these controversial results, and might originate from preferential amplification of shorter polymerase chain reaction (PCR) products, resulting in the so-called allele dropout. In this paper we optimized the conditions for simultaneous amplification of shorter and longer amplicons of the 48 bp repeat region of the DRD4 gene in order to avoid the loss of the longer allele and consequent incorrect genotyping, using very low DNA template concentrations and partial replacement of 2'-deoxyguanosine-5'-triphosphate (dGTP) by 2'-deoxyinosine-5'-triphosphate (dITP). The optimized PCR method in combination with high throughput automated ultrathin-layer gel electrophoresis was suitable for rapid genotyping from less than a nanogram DNA using noninvasive sampling (buccal epithelial cells). All detected genotypes are presented, including such rear heterozygotes as the 2 x and 8 x 48 bp repeats in the same sample, showing the reliability of our novel detection method of longer alleles in the presence of shorter alleles.

Automation↗
PubMed2003

Angiotensin-converting enzyme polymorphisms and their potential impact on left ventricular myocardial geometry after aortic valve surgery.

BACKGROUND AND AIM OF THE STUDY: Genetic variants of the angiotensin-converting enzyme (ACE) cascade may influence left ventricular myocardial mass (LVMM) regression after aortic valve surgery. Postoperative long-term changes in LV indices were investigated in patients with asymptomatic aortic regurgitation (AR) and symptomatic aortic stenosis (AS) and related to alleles of ACE polymorphisms. METHODS: A total of 96 patients was included in the study, 21 with class IIa AR (22%) and 75 with class I AS (78%) recommendations for surgery. Patients were evaluated for demographic risk factors and underwent a thorough clinical examination including 3-D cardiac imaging by ultrafast-computed tomography. Genomic DNA was isolated for genotyping. RESULTS: AR patients were younger (55.8 +/- 8.9 versus 64 +/- 9.1 years, p = 0.0014), had a larger body surface area (1.92 +/- 0.21 versus 1.82 +/- 0.19 m2, p = 0.039), and were more likely to be asymptomatic (myocardial infarction, p = 0.04; syncope, p = 0.0099; thromboembolism, p = 0.03; NYHA class IV, p = 0.04). Postoperatively, the reduction in absolute LVMM (from 297.1 +/- 52.6 to 190.1 +/- 57.1 g versus 214.4 +/- 55.7 to 143.8 +/- 40.0 g; pT = 0.0000001) and indexed LVMM (from 156.0 +/- 31.7 to 99.3 +/- 28.4 g/m2 versus 118.7 +/- 28.3 to 79.3 +/- 20.6 g/m; pT = 0.0000001) over time was more significant in AR patients, but never reached normal values. Enforced ACE inhibitor medication resulted in significantly higher postoperative indexed LVMM differences in homozygote DD patients compared to AR patients with II/ID alleles of ACE 16 ins/del polymorphism. CONCLUSION: AR patients showed a statistically significant decrease in absolute/indexed LVMM during follow up, but never achieved LV mass recovery compared to standard values or to values in patients undergoing aortic valve replacement for AS. The benefits of ACE inhibitors were observed among AR patients with homozygote DD alleles of ACE 16 ins/del polymorphism.

Aged↗
PubMedDate not supplied

The influence of the p53 codon 72 polymorphism on ovarian carcinogenesis and prognosis.

The frequency of dysfunction of the p53 tumor suppressor gene in cancer has made the concept of gene replacement therapy with wild-type p53 an attractive strategy. Codon 72 of the p53 gene is highly polymorphic with a reported arginine/proline allelotype frequency of 0.65/0.35 for Caucasians and a reversal of this ratio in African-Americans. Ovarian cancer is more common and less aggressive in Caucasians. The arginine and proline alleles have different biochemical properties. Thus, we have hypothesized that these alleles may also have different biologic properties that could make one superior to the other for gene replacement therapy. To test this hypothesis in vivo, we investigated the prevalence of each allelotype in a population of 190 Midwestern American women with ovarian cancer and 52 healthy controls without a family history of cancer. We have found that: (1) the heterozygous arginine/proline allelotype is more common in probands with borderline cancers than in probands with invasive cancers (P = .0001) or healthy controls (P = .005); (2) despite a survival advantage (P = .006), probands homozygous for the arginine allele developed ovarian cancer at an earlier age (P = .01); (3) the frequency of tumor p53 mutations was independent of the germline p53 allelotype, but (4) when a loss of heterozygosity occurred in probands with invasive disease, the proline allele was lost preferentially (P = .002), and (5) any tumor which retained a proline allele was more prone to mutation (P = .04) than a tumor without a proline allele. Our results suggest that variation in the p53 codon 72 allelotype is an example of an intermediate risk polymorphism which interacts with epigenetic factors to play a role in ovarian carcinogenesis and may differentially influence cellular DNA repair and apoptotic pathways. These findings may have important ramifications in the choice of wild-type p53 genotype for gene replacement therapy of ovarian cancer.

Adult↗
PubMed1995

Polymorphisms in angiotensin-converting-enzyme gene and progression of IgA nephropathy.

We have investigated the influence of the functional insertion (I) and deletion (D) polymorphism in intron 16 of the gene for angiotensin-converting enzyme (ACE) in a retrospective study of 100 patients with IgA nephropathy. There was no difference in genotype frequency compared with normal subjects. However, patients homozygous for the D allele tended to present at an earlier age (medians: DD, 33; ID, 34; II, 42 years) and to require renal replacement therapy at a younger age (medians 37, 42, and 48 years, respectively). The rate of progression was significantly worse in patients homozygous for the D allele. The DD genotype is associated with increased severity of disease in patients with IgA nephropathy.

Adult↗
PubMed2002

Genetic determinants of mammographic density.

BACKGROUND: Changes in breast density are highly correlated with steroid hormone exposure. MATERIALS AND METHODS: In a cross-sectional study of 396 Caucasian and African-American women, we evaluated whether polymorphisms in genes involved in steroid hormone biosynthesis and metabolism, CYP17 (T27C), COMT (Val158Met), 17HSDB1 (Ser312Gly) and 3HSDB1 (Asn367Thr), predict mammographic density. We also evaluated whether associations vary by menopausal and hormone replacement therapy status. RESULTS: We found no strong consistent relationships between polymorphisms in these genes and breast density. African-American women homozygous for the Thr allele of 3HSDB1 had increased density (the absolute difference versus the Asn/Asn genotype was +19.7%; P trend = 0.02), while Caucasian homozygous women had decreased density (-5.1%; P trend = 0.04). Among premenopausal women, carriers of the Ser allele had (not significantly) greater density (versus Gly/Gly genotype: +7.1%; P trend = 0.07). In addition, among current users of hormone replacement therapy, we observed that women with the low-activity Met/Met genotype of COMT had greater breast density (versus the Val/Val genotype: +11.7%; P trend = 0.01). CONCLUSION: Additional large studies evaluating these and other candidate breast cancer genes will be required to determine what proportion, if any, of the interindividual differences in breast density are due to underlying genetic variation in genes involved in steroid hormone biosynthesis or metabolism.

17-Hydroxysteroid Dehydrogenases↗
PubMed1997

Molecular basis of the apolipoprotein H (beta 2-glycoprotein I) protein polymorphism.

Apolipoprotein H (apoH, protein; APOH, gene) is considered to be an essential cofactor for the binding of certain antiphospholipid autoantibodies to anionic phospholipids. APOH exhibits a genetically determined structural polymorphism due to the presence of three common alleles (APOH*1, APOH*2 and APOH*3) detectable by isoelectric focusing (IEF) and immunoblotting. The APOH*3 allele can be further characterized into two subtypes, APOH*3w and APOH*3B, based upon its reactivity with monoclonal antibody 3D11. In this study we have determined the molecular basis of the APOH protein polymorphism and its distribution in three large U.S. population samples comprising 661 non-Hispanic whites, 444 Hispanics and 422 blacks. By direct DNA sequencing of PCR amplified fragments corresponding to the eight APOH exons, we identified two missense mutations that correspond to the APOH*1 and APOH*3w alleles. A missense mutation (G-->A) in exon 3, which alters amino acid Ser to Asn at codon 88 and creates a restriction site for TSP509 I, was present in all APOH*1 allele carriers. A second missense mutation (G-->C) at codon 316 in exon 8, which replaces amino acid Trp with Ser and creates a restriction site for BSTBI, was present in all APOH*3w carriers. The distribution of the Ser 88 Asn and Trp 316 Ser mutations was significantly different between the three racial groups. The frequency of the Asn-88 allele was 0.011, 0.043, and 0.056 in blacks. Hispanics and non-Hispanic whites, respectively. While the Ser-316 allele was observed sporadically in blacks (0.008), it was present at a polymorphic frequency in Hispanics (0.027) and non-Hispanic whites (0.059). The identification of the molecular basis of the APOH protein polymorphism will help to elucidate the structural-functional relationship of apoH in the production of antiphospholipid autoantibodies.

Alleles↗
PubMed1990

Structure of the gene of tum- transplantation antigen P35B: presence of a point mutation in the antigenic allele.

Mutagen treatment of P815 tumour cells produces tum- variants that are rejected by syngeneic mice because they express new transplantation antigens. These 'tum-' antigens elicit a cytolytic T lymphocyte (CTL) response but no detectable antibody response. The DNA of tum- variant P35 was transfected into P815 cell line P1.HTR. Transfectants expressing tum- antigen P35B were identified on the basis of their ability to stimulate anti-P35B CTL. This was repeated with a cosmid library and a cosmid carrying the sequence encoding antigen P35B was recovered from a transfectant expressing the antigen. Gene P35B is 6 kb long and contains 11 exons. The sequence shows no homology with the previously identified tum- gene P91A nor with any gene presently recorded in the data banks. The antigenic allele of gene P35B differs from the normal allele by a point mutation located in exon 5. This mutation, which replaces a Ser by an Asn residue, was shown by site-directed mutagenesis to be responsible for the expression of the antigen. A synthetic decapeptide covering the sequence surrounding the tum- mutation rendered P815 cells sensitive to lysis by anti-P35B CTL. Surprisingly, the homologous peptide corresponding to the normal sequence of the gene had the same effect, indicating that this tum- mutation does not exert its effect by generating the aggretope or the epitope of the antigenic peptide. As observed previously with gene P91A, we found that fragments of gene P35B containing only exons 4 and 5, which were cloned in non-expression vectors, transferred efficiently the expression of the antigen.

Alleles↗
PubMed1994

The STR system in the human phenylalanine hydroxylase gene: true fragment length obtained with fluorescent labelled PCR primers.

We present a simple, fast, non-radioactive method for the analysis of the polymorphic short tandem repeat (STR) system in the human phenylalanine hydroxylase gene. Previously, sizing of the STR marker involved radiolabelling of PCR amplified fragments and resolution on denaturing polyacrylamide gels using M13 sequencing ladder as a standard. However, this method consistently gave sizes 2 bp longer than the known sequence. The fluorescent method presented here employs internal lane standards and enables accurate sizing of the fragments. To avoid confusion, we suggest that the true fragment lengths are used as reference values in the future. The analysis of STR alleles is valuable for population genetic studies and for targeted mutation screening in phenylketonuria (PKU). It can replace RFLP-based haplotype analysis for carrier detection, and we report its use for prenatal diagnosis in a Northern Irish family with PKU. The analysis of 250 Northern Irish chromosomes, including 128 PKU alleles, showed no significant difference between normal and PKU alleles, with fragment lengths of 238 and 242 bp most common in both groups.

Genetic Testing↗
PubMed1994

Distribution of the molossinus allele of Sry, the testis-determining gene, in wild mice.

When the Y chromosome of the laboratory inbred mouse strain C57BL/6 (B6) is replaced by the Y of certain strains of Mus musculus domesticus, testis determination fails and all XY fetuses develop either as hermaphrodites or XY females (XY sex reversal). This suggests the presence of at least two alleles of Sry, the male-determining gene on the Y:M. m. domesticus and B6. The B6 Y chromosome is derived from the Japanese house mouse, M. m. molossinus and therefore carries a molossinus Sry allele. As a first step to determine how the molossinus Sry allele evolved, its distribution pattern was determined in wild mice. The cumulative data of 96 M. musculus samples obtained from 58 geographical locations in Europe, North Africa, and Asia show the molossinus Sry allele is restricted to Japan and the neighboring Asian mainland and confirm that Japanese M. m. molossinus mice were derived in part from a race of M. m. musculus from Korea or Manchuria. Sry polymorphisms, as illustrated by the molossinus Sry allele, can serve as molecular markers for studies on the evolution of wild M. musculus populations and can help determine the role sex determination plays in speciation.

Alleles↗
PubMed1989

Allelic variation in HLA-B and HLA-C sequences and the evolution of the HLA-B alleles.

Several new HLA-B (B8, B51, Bw62)- and HLA-C (Cw6, Cw7)-specific genes were isolated either as genomic cosmid or cDNA clones to study the diversity of HLA antigens. The allele specificities were identified by sequence analysis in comparison with published HLA-B and -C sequences, by transfection experiments, and Southern and northern blot analysis using oligonucleotide probes. Comparison of the classical HLA-A, -B, and -C sequences reveals that allele-specific substitutions seem to be rare events. HLA-B51 codes only for one allele-specific residue: arginine at position 81 located on the alpha 1 helix, pointing toward the antigen binding site. HLA-B8 contains an acidic substitution in amino acid position 9 on the first central beta sheet which might affect antigen binding capacity, perhaps in combination with the rare replacement at position 67 (F) on the alpha 1 helix. HLA-B8 shows greatest homology to HLA-Bw42, -Bw41, -B7, and -Bw60 antigens, all of which lack the conserved restriction sites Pst I at position 180 and Sac I at position 131. Both sites associated with amino acid replacements seem to be genetic markers of an evolutionary split of the HLA-B alleles, which is also observed in the leader sequences. HLA-Cw7 shows 98% sequence identity to the JY328 gene. In general, the HLA-C alleles display lower levels of variability in the highly polymorphic regions of the alpha 1 and alpha 2 domains, and have more distinct patterns of locus-specific residues in the transmembrane and cytoplasmic domains. Thus we propose a more recent origin for the HLA-C locus.

Amino Acid Sequence↗
PubMed1980

A criterion for the establishment of a stable polymorphism of higher order with an application to the evolution of polymorphism.

This note gives some further useful properties of the constant fitness selection model for multiple alleles which pertain to the effects of adding a new allele to n preexisting alleles in stable equilibrium. In particular the conditions are derived for the establishment of a stable equilibrium involving all n + 1 alleles. For 3 alleles (i.e. n = 2) I give a complete qualitative solution, including the case of the replacement of one diallelic polymorphism by another. As an application I discuss a possible mechanism for the evolution of polymorphism using Monte Carlo methods similar to Lewontin, Ginzburg and Tuljapurkar (1978).

Alleles↗
PubMed1993

Mutations of the intronic IgH enhancer and its flanking sequences differentially affect accessibility of the JH locus.

To investigate the role of intronic immunoglobulin heavy chain (IgH) enhancer (E mu) in generating accessibility of the JH locus for VDJ recombination, we generated ES cells in which E mu or its flanking sequences were mutated by replacement with or insertion of an expressed neor gene. Heterozygous mutant ES cells were used to generate chimeric mice from which pre-B cell lines were derived by transformation of bone marrow cells with Abelson murine leukemia virus (A-MuLV). Comparison of the rearrangement status of the normal and mutated alleles in individual pre-B cell lines allowed us to assay for cis-acting effects of the mutations. Replacement of a 700 bp region immediately downstream from the core E mu [which includes part of the 3' matrix associated region (MAR) and the I mu exon] had no obvious effect on rearrangement of the targeted allele, indicating that insertion of a transcribed neor gene into the JH-C mu intron does not affect JH accessibility. In contrast, replacement of an overlapping 1 kb DNA fragment that contains the E mu resulted in a dramatic cis-acting inhibition of rearrangement, demethylation and germline transcription of the associated JH locus. Surprisingly, insertion of the neor gene into the 5' MAR sequence approximately 100 bp upstream of the core E mu also dramatically decreased recombination of the linked JH locus; but, in many lines, did not prevent demethylation of this locus. We conclude that integrity of the E mu and upstream flanking sequences is required for efficient rearrangement of the JH locus and that demethylation of this locus, per se, does not necessarily make it a good substrate for VDJ recombination.

Animals↗
PubMed1994

Targeted insertions of two exogenous collagen genes into both alleles of their endogenous loci in cultured human cells: the insertions are directed by relatively short fragments containing the promoters and the 5' ends of the genes.

Previous studies demonstrated that type II procollagen is synthesized by HT-1080 cells that are stably transfected with constructs of the human COL2A1 gene that contain the promoter and 5' end of either the COL2A1 gene or the human COL1A1 gene. Since the host HT-1080 cells were from a human tumor line that synthesizes type IV collagen but not type II or type I procollagen, the results suggested that the constructs were integrated near active enhancers or promoters. Here, however, we demonstrate that a 33-kb construct of the COL2A1 gene containing a 5' fragment from the same gene was inserted into both alleles of the endogenous COL2A1 gene on chromosome 12, apparently by homologous recombination by a nonconservative pathway. In contrast, a similar construct of the COL2A1 gene in which the 5' end was replaced with a 1.9-kb fragment from the 5' end of the COL1A1 gene was inserted into both alleles of the locus for the COL1A1 gene on chromosome 17. Therefore, targeted insertion of the gene construct was not directed by the degree of sequence homology. Instead, it was directed by the relatively short 5' fragment from the COL1A1 gene that contained the promoter and the initially transcribed sequences of the gene. After insertion, both gene constructs were expressed from previously inactive loci.

3T3 Cells↗
PubMed1997

Effect of 360His mutation in apolipoprotein A-IV on plasma HDL-cholesterol response to dietary fat.

In order to determine whether genetic variability of apolipoprotein (apo) A-IV is responsible for the improvement in lipid profile when dietary saturated fats are replaced by carbohydrates or monounsaturated fats, 41 healthy male subjects were studied: 33 were homozygous for the 360Gln allele and 8 were heterozygote carriers of the 360His allele. These were administered three consecutive 4-week diets. The first was a diet rich in saturated fat (SAT diet, with 38% fat, 20% saturated. This was followed by a low fat diet (NCEP-I, with < 30% fat, < 10% saturated). The final diet was rich in monounsaturated fat (MUFA diet, with 38% fat, 22% monounsaturated). There was no difference in plasma lipid and apolipoprotein levels of both groups of individuals after consuming the SAT diet. Switching from this diet to the NCEP-I diet, carriers of the 360His allele presented a greater decrease in high density lipoprotein-cholesterol (HDL-C) (-10 vs. -1 mg/dL, P < 0.004) and apoA-I levels (-19 vs. -8 mg/dL, P < 0.037). Similarly, replacement of carbohydrates by monounsaturated fats produced a greater increase in HDL-C (9 vs. 1 mg/dL, P < 0.003) and apoA-I levels (9 vs. 2 mg/dL, P < 0.036) in carriers of the 360His mutation. Lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) activities and apoA-IV levels were also measured. However, no genotype-related differences were observed for these parameters. Our results suggest that variability in HDL-C and apoA-I response to diet is, at least partially, determined by the 360His mutation of apoA-IV.

Adult↗
PubMed1988

Mitotic gene conversion, reciprocal recombination and gene replacement at the benA, beta-tubulin, locus of Aspergillus nidulans.

We have developed a procedure for determining the rates of mitotic recombination of an interrupted duplication created by integration of transforming plasmid sequences at the benA, beta-tubulin, locus of Aspergillus nidulans. Transformation of a strain carrying a benomyl-resistant benA allele with plasmid AIpGM4, which carries the wild-type benA allele and the pyr4 (orotidine-5'-phosphate decarboxylase) gene of Neurospora crassa, creates an interrupted duplication with plasmid sequences flanked by two benA alleles, one wild type and one benomyl resistant. Such transformants will not grow in the presence of high levels of benomyl. Mitotic recombination causes the loss of the wild-type benA allele or conversion of the wild-type to the mutant allele resulting in nuclei carrying only the benomyl-resistant allele. Conidia containing such nuclei can be selected on media with high benomyl allowing easy quantitation of mitotic recombination. We found that the rate of recombination giving rise to benomyl-resistant conidia was 4.6 x 10(-4). Reciprocal recombination leading to benomyl-resistant conidia lacking plasmid sequences occurred at a rate of 2.0 x 10(-4) and gene conversion leading to benomyl-resistant conidia occurred at a rate of 2.6 x 10(-4). We selected for reciprocal recombination leading to loss of pyr4 sequences on 5-fluoro-orotic acid and used this selection for two-step gene replacement of a mutant benA allele with the wild-type allele.

Alleles↗
PubMed1992

Polymorphism and divergence in the Mst26A male accessory gland gene region in Drosophila.

Drosophila males, like males of most other insects, transfer a group of specific proteins to the females during mating. These proteins are produced primarily in the accessory gland and are likely to influence the female's reproduction. The results of studies of DNA sequence polymorphism and divergence in two genes coding for male accessory gland proteins of Drosophila are reported here. The Mst26Aa and Mst26Ab transcription units are tandemly arranged in a approximately 1.6-kb segment in Drosophila sechellia, Drosophila mauritiana and Drosophila simulans as they were reported to be in Drosophila melanogaster. The DNA sequences of 10 alleles from D. melanogaster and one allele each from the three sibling species reveals a high degree of amino acid replacement variation. A substantial part of the variation is due to insertion/deletion differences. Possible functional significance of these amino acid sequence changes is discussed. Statistical analyses based on the neutral theory of molecular evolution show that the distribution of polymorphism over the 1.6-kb region is inconsistent with the pattern of divergence between the species. The amount of 4-cutter restriction map polymorphism in a larger sample of 75 alleles from the same D. melanogaster population is similar to that obtained from the DNA sequence of the 10 alleles (a pairwise average of 0.007 difference per site). The 6-cutter restriction map survey of a 18-kb region containing the Mst26A genes indicates that polymorphism in the region flanking these genes maybe higher. The failure of polymorphisms and divergence in the Mst26A region to conform to the expectations of a simple mutation-drift-equilibrium model indicates that selection in or near this region has played a role in the history of these genes.

Amino Acid Sequence↗
PubMed1998

Molecular population genetics of the Arabidopsis CAULIFLOWER regulatory gene: nonneutral evolution and naturally occurring variation in floral homeotic function.

The evolution of interspecies differences in morphology requires sufficient within-species variation in developmental regulatory systems on which evolutionary forces can act. Molecular analyses of naturally occurring alleles of the Arabidopsis thaliana CAULIFLOWER locus reveal considerable intraspecific diversity at this floral homeotic gene, and the McDonald-Kreitman test suggests that this gene is evolving in a nonneutral fashion, with an excess of intraspecific replacement polymorphisms. The naturally occurring molecular variation within this floral regulatory gene is associated with functionally different alleles, which can be distinguished phenotypically by their differential ability to direct floral meristem development.

Arabidopsis↗
PubMed1988

The joining of germ-line V alpha to J alpha genes replaces the preexisting V alpha-J alpha complexes in a T cell receptor alpha, beta positive T cell line.

To determine whether T cell receptor genes follow the same principle of allelic exclusion as B lymphocytes, we have analyzed the rearrangements and expression of TCR alpha and beta genes in the progeny of the CD3+, CD4-/CD8- M14T line. Here, we show that this line can undergo secondary rearrangements that replace the pre-existing V alpha-J alpha rearrangements by joining an upstream V alpha gene to a downstream J alpha segment. Both the productively and nonproductively rearranged alleles in the M14T line can undergo secondary rearrangements while its TCR beta genes are stable. These secondary recombinations are usually productive, and new forms of TCR alpha polypeptides are expressed in these cells in association with the original C beta chain. Developmental control of this V alpha-J alpha replacement phenomenon could play a pivotal role in the thymic selection of the T cell repertoire.

Alleles↗