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At least 595 records · Page 33Linked to original sources
PubMed2003

Efficient switching of mycobacteriophage L5-based integrating plasmids in Mycobacterium tuberculosis.

We previously used a mycobacteriophage L5-derived integrating vector to demonstrate that glnE and aroK are essential genes in Mycobacterium tuberculosis by showing that we were unable to excise the integrated vector when it carried the only functional copy of these genes. We tested three systems to replace the integrated copy with alternative alleles. The most efficient method was to transform the strain with a second copy of the integrating vector. Excision of the resident vector and integration of the incoming vector occurred at an extremely high efficiency. This technique will allow us to study the role and functionality of essential genes in this important human pathogen.

Alleles↗
PubMed2005

Under the skin: On the impartial treatment of genetic and environmental hypotheses of racial differences.

Environmental and genetic explanations have been given for Black-White racial differences in intelligence and other traits. In science, viable, alternative hypotheses are ideally given equal Bayesian prior weights; but this has not been true in the study of racial differences. This article advocates testing environmental and genetic hypotheses of racial differences as competing hypotheses. Two methods are described: (a) fitting means within structural equation models and (b) predicting means of interracial children. These methods have limitations that call for improved research designs of racial differences. One improvement capitalizes on biotechnology. Genetic admixture estimates--the percentage of genes of European origin that a Black individual possesses (independent of genes related to skin coloration)--can represent genetic influences. The study of interracial children can be improved by increasing sample size and by choosing family members who are most informative for a research question. Eventually, individual-admixture estimates will be replaced by molecular genetic tests of alleles of those genes that influence traits.

Environment↗
PubMed2003

Two novel mutations in the gene encoding thyroxine-binding globulin (TBG) as a cause of complete TBG deficiency in Taiwan.

OBJECTIVE: Thyroxine-binding globulin (TBG) encoded by the TBG gene on chromosome Xq22 is the major transport protein, carrying approximately 75% of circulating T4. Inherited defects in TBG are associated with three phenotypes based on the level of TBG in serum of affected hemizygous males: complete TBG deficiency (TBG-CD), partial TBG deficiency (TBG-PD) and TBG excess (TBG-E). In this study, we report two unrelated Han Chinese males with complete TBG deficiency who carry different mutations in the TBG gene. PATIENTS: Two index cases of Han males who were diagnosed as having TBG deficiency on the basis of undetectable serum TBG and an additional 75 (50 males and 25 females) normal Han Chinese. MEASUREMENT: Serum thyroid hormones were measured by chemiluminescent immunoassay, thyroid autoantibodies by an agglutination test, and TSH receptor antibody and TBG by radioimmunoassay. Genomic DNA extraction, polymerase chain reaction (PCR) and DNA sequence analysis of the TBG gene were performed with standard methods. RESULTS: One index case had one missense mutation in his copy of the gene, a G --> A transition in codon 52 that results in the replacement of serine by asparagine, and a known polymorphism in codon 283 (TTG --> TTt) that results in the replacement of leucine by phenylalanine. The allelic frequency of TBG-Poly allele in 75 normal Han Chinese (100 chromosomes) was 31%. A second index case was hemizygous for a nonsense mutation in codon 280 of exon 3 (TGG --> TGa). This mutation, located in the C-terminal of TBG, predicts a markedly truncated protein. CONCLUSIONS: This is the first report of complete thyroxine-binding globulin deficiency (TBG-CDT1 and TBG-CDT2) due to TBG gene mutations in Taiwan.

Case-Control Studies↗
PubMed2004

Generation and characterization of functional mutants in the translation initiation factor IF1 of Escherichia coli.

Three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. No clear function has been assigned to the smallest of these three factors, IF1. Therefore, to investigate the role of this protein in the initiation process in Escherichia coli we have mutated the corresponding gene infA. Because IF1 is essential for cell viability and no mutant selection has so far been described, the infA gene in a plasmid was mutated by site-directed mutagenesis in a strain with a chromosomal infA+ gene, followed by deletion of this infA+ gene. Using this approach, the six arginine residues of IF1 were altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. All mutants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Differences in growth phenotypes of the mutants were observed in both minimal and rich media. Some of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type infA+ allele. Several of these recombinants showed reduced growth rate and a partial cold-sensitive phenotype. This paper presents a collection of IF1 mutants designed for in vivo and in vitro studies on the function of IF1.

Base Sequence↗
PubMed2001

Role of the N-terminal forkhead-associated domain in the cell cycle checkpoint function of the Rad53 kinase.

Forkhead-associated (FHA) domains are multifunctional phosphopeptide-binding modules and are the hallmark of the conserved family of Rad53-like checkpoint protein kinases. Rad53-like kinases, including the human tumor suppressor protein Chk2, play crucial roles in cell cycle arrest and activation of repair processes following DNA damage and replication blocks. Here we show that ectopic expression of the N-terminal FHA domain (FHA1) of the yeast Rad53 kinase causes a growth defect by arresting the cell cycle in G(1). This phenotype was highly specific for the Rad53-FHA1 domain and not observed with the similar Rad53-FHA2, Dun1-FHA, and Chk2-FHA domains, and it was abrogated by mutations that abolished binding to a phosphothreonine-containing peptide in vitro. Furthermore, replacement of the RAD53 gene with alleles containing amino acid substitutions in the FHA1 domain resulted in an increased DNA damage sensitivity in vivo. Taken together, these data demonstrate that the FHA1 domain contributes to the checkpoint function of Rad53, possibly by associating with a phosphorylated target protein in response to DNA damage in G(1).

Alleles↗
PubMed1999

Selective sweep at the Drosophila melanogaster Suppressor of Hairless locus and its association with the In(2L)t inversion polymorphism.

The hitchhiking model of population genetics predicts that an allele favored by Darwinian selection can replace haplotypes from the same locus previously established at a neutral mutation-drift equilibrium. This process, known as "selective sweep," was studied by comparing molecular variation between the polymorphic In(2L)t inversion and the standard chromosome. Sequence variation was recorded at the Suppressor of Hairless (Su[H]) gene in an African population of Drosophila melanogaster. We found 47 nucleotide polymorphisms among 20 sequences of 1.2 kb. Neutrality tests were nonsignificant at the nucleotide level. However, these sites were strongly associated, because 290 out of 741 observed pairwise combinations between them were in significant linkage disequilibrium. We found only seven haplotypes, two occurring in the 9 In(2L)t chromosomes, and five in the 11 standard chromosomes, with no shared haplotype. Two haplotypes, one in each chromosome arrangement, made up two-thirds of the sample. This low haplotype diversity departed from neutrality in a haplotype test. This pattern supports a selective sweep hypothesis for the Su(H) chromosome region.

Animals↗
PubMed1980

Duplicated genes producing transposable controlling elements for the mating-type differentiation in Saccharomyces cerevisiae.

Mutation of the two homothallic genes, HML alpha/HMLa and HMRa/HMR alpha, in homothallic strains of Saccharomyces cerevisiae was studied. Of 11 mutants of the HML alpha gene, eight were due to a phenotypic mutation from HML alpha to HMLa, i.e., a mutation causing a change in function of the original HML allele to that of the other HML allele (functional mutation), and three were due to a defective mutation at the HML alpha gene, i.e., a mutation causing a nonfunctional allele (nonfunctional mutation). All 14 mutants of the HMRa gene, on the other hand, were due to a phenotypic mutation from HMRa to HMR alpha i.e., a functional mutation. Phenotypic reverse mutations, i.e., HMLa to HML alpha and HMR alpha to HMRa, were also observed in the cultivation of EMS (ethyl methanesulfonate) treated spores having the HO HMR alpha HMLa genotype. Mutation from heterothallic cells to homothallism was observed in a nonfunctional mutant of the HML alpha gene, by mutagenesis with EMS, but not in the functional mutants of the HML alpha and HMRa genes or in the authentic strains having the alpha HO HMR alpha HML alpha (alpha Hp) and a HO HMRa HMLa (a Hq) genotypes. These observations suggest that the functional mutation is not caused by the direct mutation from a homothallic allele to the opposite, but by replacement of a transposable genic element produced from a homothallic locus with a region of a different homothallic locus. These observations also support the controlling-element model and the cassette model, which have been proposed to explain the mating-type differentiation by the homothallic genes.

DNA Transposable Elements↗
PubMed2002

Controlled elimination of clathrin heavy-chain expression in DT40 lymphocytes.

We exploited the high rate of homologous recombination shown by the chicken B cell line DT40 to inactivate the endogenous alleles for clathrin heavy chain and replace them with human clathrin complementary DNA under the control of a tetracycline-regulatable promoter. Clathrin repression perturbed the activities of Akt-mediated and mitogen-activated protein kinase-mediated signaling pathways and induced apoptosis; this finding suggests that in DT40 cells clathrin helps to maintain the integrity of antiapoptotic survival pathways. We also describe a variant cell line in which these signaling pathways were unaffected by clathrin down-regulation. This variant cell line did not undergo apoptosis in the absence of clathrin and was used to examine the effects of clathrin depletion on membrane-trafficking pathways. Receptor-mediated and fluid-phase endocytosis were both substantially inhibited, and transferrin-receptor recycling was modestly inhibited. Surprisingly, clathrin removal did not affect the morphology or biochemical composition of lysosomes.

Animals↗
PubMed1997

Expression of an antisense hla fragment in Staphylococcus aureus reduces alpha-toxin production in vitro and attenuates lethal activity in a murine model.

Isogeneic bacterial strains that differ only in the production of a single microbial factor have been invaluable in studying the pathogenesis of bacterial infections. The targeted, intentional inactivation of a gene encoding a potential virulence determinant generally requires homologous recombination to replace the gene with an inactivated allele. To determine whether the insertion and expression of a fragment of a bacterial gene in an antisense orientation could be used as a rapid alternative to allelic inactivation for producing paired isogeneic isolates, we inverted a 600-bp fragment of the Staphylococcus aureus gene encoding alpha-toxin, hla, behind its native promoter on an Escherichia coli-S. aureus shuttle vector. A transformant of an S. aureus strain carrying the antisense hla fragment produced antisense hla RNA and made 16-fold less alpha-toxin than either its parent or an isogeneic transformant containing vector DNA without hla. Also, intraperitoneal injection of 1.5 x 10(9) CFU of the antisense hla-containing transformant was significantly less lethal in a murine model than that of the parent (1 of 10 versus 7 of 10 mice expired [P < 0.02]) or the transformant without hla (1 of 10 versus 7 of 7 mice expired [P < 0.001]). We conclude that the expression of a fragment of hla in an antisense orientation in S. aureus on a plasmid vector reduces alpha-toxin production and the lethal activity of the strain in a murine model. The antisense strategy for creating isogeneic strains of bacteria may facilitate molecular investigations into the pathogenesis of infection. It also may be useful in creating novel live-attenuated strains of bacteria for use as vaccine candidates.

Animals↗
PubMed1999

Citrobacter rodentium espB is necessary for signal transduction and for infection of laboratory mice.

Citrobacter rodentium is the causative agent of transmissible murine colonic hyperplasia and contains a locus of enterocyte effacement (LEE) similar to that found in enteropathogenic Escherichia coli (EPEC). EPEC espB is necessary for intimate attachment and signal transduction between EPEC and cultured cell monolayers. Mice challenged with wild-type C. rodentium develop a mucosal immunoglobulin A response to EspB. In this study, C. rodentium espB has been cloned and its nucleotide sequence has been determined. C. rodentium espB was found to have 90% identity to EPEC espB. A nonpolar insertion mutation in C. rodentium espB was constructed and used to replace the chromosomal wild-type allele. The C. rodentium espB mutant exhibited reduced cell association and had no detectable fluorescent actin staining activity on cultured cell monolayers. The C. rodentium espB mutant also failed to colonize laboratory mice following experimental inoculation. The espB mutation could be complemented with a plasmid-encoded copy of the gene, which restored both cell association and fluorescent actin staining activity, as well as the ability to colonize laboratory mice. These studies indicate that espB is necessary for signal transduction and for colonization of laboratory mice by C. rodentium.

Actins↗
PubMed1976

Enhanced transformability with heterospecific deoxyribonucleic acid in a Streptococcus sanguis mutant impaired in ribonucleic acid polymerase activity.

We have induced with nitrosoguanidine in Streptococcus sanguis a mutation conferring inability to grow and synthesize ribonucleic acid (RNA) at 42 C, the optimal temperature for growth and RNA synthesis in the parental strain. The mutation (ts) is transferable via transforming deoxyribonucleic acid (DNA) and is replaceable by its wild-type allele with fairly high efficiency in transformation reactions. The ts mutation is unlinked to the sites of mutation conferring resistance of rifampin (rifr) and streptolydigin (stgr), known to affect the beta subunit of DNA-dependent RNA polymerase. Extracts from strains carrying the ts mutation are more sensitive to elevated temperatures than are parental extracts when assayed for DNA-dependent RNA polymerase. The conclusion that the mutation causes a temperature-sensitive defect in some component of this enzyme (other than beta) is supported by the finding that the polymerase activity of a heat-inactivated ts stgr extract cannot be increased by addition of an unheated ts stgs extract, which is itself inactivated by streptolydigin. S. sanguis recipients carrying the ts mutation are highly transformable with heterospecific DNA, especially at the restrictive temperature.

Aminoglycosides↗
PubMed1987

Construction and characterization of Pseudomonas aeruginosa algB mutants: role of algB in high-level production of alginate.

The algB gene, which is involved in the production of alginate in Pseudomonas aeruginosa, was localized to approximately 2.2 kilobases of DNA from strain FRD by using transposon Tn501 insertion mutagenesis, subcloning, and complementation techniques. The previously reported alg-50(Ts) mutation, which confers the phenotype of temperature-sensitive alginate production, was here designated as an algB allele. A transduction-mediated gene replacement technique was used for site-directed mutagenesis to isolate and characterize algB::Tn501 mutants of P. aeruginosa FRD. Although algB::Tn501 mutants had a nonmucoid phenotype (indicating an alginate deficiency), they still produced about 1 to 5% of wild-type levels of alginate in most growth media and up to 16% in very rich media. The algB::Tn501 mutations had no apparent effect on growth rate or growth requirements. Using another gene replacement technique called excision marker rescue, we constructed a chromosomal algB deletion (delta algB) mutant of P. aeruginosa FRD. The delta algB mutant also produced low levels of alginate as did the algB::Tn501 mutants. The alginate produced by algB::Tn501 mutants resembled wild-type alginate by all criteria studied: molecular weight, acetylation, and proportion of mannuronic and guluronic acids. Thus, the algB gene product is apparently involved in the high-level production of alginate by P. aeruginosa and is not directly involved in the pathway leading to its biosynthesis. Chromosomal mapping of an algB::Tn501 insertion showed linkage to the trp-2 marker on the FRD chromosome as does the algB50(Ts) mutation. The excision marker rescue technique was also used to place the algB::Tn501 marker on the chromosome of characterized strains of P. aeruginosa PAO. The algB::Tn501 mutation mapped near 21 min on the PAO chromosome.

Alginates↗
PubMed1992

Use of electroporation to construct isogenic mutants of Haemophilus ducreyi.

Little is known about the genetics of Haemophilus ducreyi, the etiologic agent of chancroid. To develop a method for constructing isogenic mutants of this organism that could be utilized in pathogenesis-related studies, electroporation techniques were evaluated as a means of introducing DNA into this organism. Electroporation of the plasmid shuttle vector pLS88 into H. ducreyi yielded approximately 10(6) antibiotic-resistant transformants per microgram of plasmid DNA. Studies of the feasibility of moving mutated genes into H. ducreyi were initiated by using NotI linker insertion and mini-Tn10kan mutagenesis techniques to introduce insertion mutations into cloned H. ducreyi genes encoding cell envelope antigens. In the former case, a gene encoding chloramphenicol acetyltransferase was then inserted into the NotI linker site created in the cloned H. ducreyi gene. The recombinant Escherichia coli strains containing these mutated plasmids no longer expressed the homologous H. ducreyi cell envelope antigens, as evidenced by their lack of reactivity with monoclonal antibody probes for these H. ducreyi proteins. Subsequent electroporation of both circular and linearized forms of plasmids carrying these mutated H. ducreyi genes into the homologous wild-type strain of H. ducreyi yielded antibiotic-resistant transformants which also lacked reactivity with the cell envelope antigen-specific monoclonal antibodies. Southern blot analysis confirmed that homologous recombination had occurred in these monoclonal antibody-unreactive transformants, resulting in the replacement of the wild-type allele with the mutated allele. Allelic exchange was most efficient when linear DNA molecules were used for electroporation. These results indicate that electroporation methods can be utilized to construct isogenic mutants of H. ducreyi.

Blotting, Southern↗
PubMed1999

A novel mutation of CHRNA4 responsible for autosomal dominant nocturnal frontal lobe epilepsy.

OBJECTIVE: To identify the mutation responsible for autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) in a nonwhite family. BACKGROUND: ADNFLE is newly recognized as an entity of idiopathic partial epilepsy. Recently, two different mutations of the neuronal nicotinic acetylcholine receptor alpha4 subunit (CHRNA4) gene were identified in a white family as a cause of ADNFLE. METHODS: Four affected and three unaffected individuals in three generations of a Japanese family with ADNFLE, and 100 unrelated healthy Japanese volunteers were studied. Clinical features and EEG findings in affected individuals were consistent with those of ADNFLE reported in white families with ADNFLE. Mutations within the CHRNA4 gene were screened for using single-strand conformation polymorphism analysis (SSCA) and were determined by direct sequencing. The mutation identified was sought in volunteers by the amplification refractory mutation system. RESULTS: A C-to-T exchange (C755T) was found in exon 5 of the CHRNA4 gene on one allele of affected individuals. C755T segregated in affected individuals and was not found in 200 alleles obtained from the volunteers. C755T replaced serine 252 (Ser252) in the second membrane-spanning domain (M2) of CHRNA4 with a leucine. Ser252 is conserved characteristically in the alpha-subunit of acetylcholine receptor and is considered to play an important role in channel function. CONCLUSION: C755T is a novel missense mutation of the CHRNA4 gene causing autosomal dominant nocturnal frontal lobe epilepsy in this Japanese family.

Circadian Rhythm↗
PubMed2000

A novel mutation of KCNQ3 (c.925T-->C) in a Japanese family with benign familial neonatal convulsions.

At present, only one mutation of KCNQ3, a KCNQ potassium channel gene, has been identified as a cause of benign familial neonatal convulsions type 2 (BFNC2). We found a T to C substitution (c.925T-C) on one allele of affected individuals in a Japanese family with BFNC but not on 200 alleles from healthy subjects. c.925T-->C replaced Trp309, a conserved residue within the P-loop of the KCNQ potassium channel family that holds the channel pore open, with an Arg (W309R). We report c.925T-->C as the second mutation of KCNQ3 responsible for BFNC2.

Amino Acid Sequence↗
PubMed1992

Novel deletion and a new missense mutation (Glu 217 Lys) at the catalytic site in two adenosine deaminase alleles of a patient with neonatal onset adenosine deaminase- severe combined immunodeficiency.

Mutations at the adenosine deaminase (ADA) locus result in a spectrum of disorders, encompassing a fulminant neonatal onset severe combined immunodeficiency (SCID) and childhood onset immunodeficiency, as well as apparently normal immune function. The extent of accumulation of the toxic metabolite, deoxyATP, correlates directly with severity of disease. We have now determined the mutations on both alleles of a child with fulminant, neonatal onset ADA- SCID and accumulation of extremely high concentrations of deoxyATP. The genotype was consistent with the severely affected phenotype. One allele carried a large deletion that arose by non-homologous recombination and included the first five exons and promoter region. The second allele carried a missense mutation (G649A) resulting in replacement of Glu217, an amino acid involved in the catalytic site, by Lys and predicting a major alteration in charge. Expression of the mutant cDNA in Cos cells confirmed that the mutation abolished enzyme activity. We have previously reported that a missense mutation at the preceding codon is similarly associated with neonatal onset ADA- SCID and accumulation of extremely high deoxyATP. These findings suggest that genotype-phenotype correlations may be apparent for ADA- SCID, despite the role that random variation in exposure to environmental pathogens may play in the initial phenotype. Such genotype-phenotype correlations may be important to consider in evaluating results of ongoing trials of "gene" and enzyme replacement therapy.

Adenosine Deaminase↗
PubMedDate not supplied

[Evolution as resistance to entropy. II. A conservative role of the sexual reproduction].

It is claimed that biological meaning of the sex and meiotic genetic recombination is a creation of a barrier for evolution. The transition to sexuality is not merely a change in reproduction mode but a leap to a new known as cohesion. In a sexual population, the lineages of different individuals become tangled into multidimensional net, resulting in a creation of gene pool and new superindividual entity--biological species. A sexual individual can not reproduce its particular genocopy and its fitness is sacrificed to some extent for the fitness of the species. The competition between individuals is replaced with the competition between gene alleles given that the competitors have after all the common offspring. The genotype of the "outstanding" individuals with a highest fitness are not transmitted to next generation, being scattered and shuffled in new combination after unavoidable crossing with the "ordinary" partners. So, the sexual reproduction can evolve only as a whole. Genetic recombination in meiosis changes a character of mutations distribution among gametes enhancing the classes with mutation load both lower and higher than average. By this, an efficiency of the truncated selection (elimination of the individuals with multiple mutations) is enhanced and an ability to restore the initial genotype appears. Evolution within the species becomes reversible, which is equivalent to its virtual cessation. The species acquires an evolution resistance that can be overcome by rare concurrence of circumstances.

Animals↗
PubMedDate not supplied

[The genetic polymorphism of somatotropin].

This review deals with the growth hormone genetic polymorphism and presents information about somatotropin variants, which are synthesized in the pituitary gland and in the placenta. The detailed results of somatotropin DNA polymorphism study in different animal species are presented. It is supposed that some DNA alleles which find their source in replacements in non-coding part of the gene, express their phenotype by the deviations in the expression control. The polymorphous somatotropin genetic system may be used as the marker system in the QTL study.

Animals↗