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At least 469 records · Page 26Linked to original sources
PubMed1995

Abasic sites stimulate double-stranded DNA cleavage mediated by topoisomerase II. DNA lesions as endogenous topoisomerase II poisons.

Several clinically relevant anticancer drugs induce genomic mutations and cell death by increasing topoisomerase II-mediated DNA breakage. To determine whether endogenous DNA damage also affects this cleavage event, the effects of abasic sites (the most commonly formed spontaneous DNA lesion) on topoisomerase II activity were investigated. The presence of 3 abasic sites/plasmid stimulated enzyme-mediated DNA breakage > 6-fold, primarily by enhancing the forward rate of cleavage. This corresponds to a potency that is > 2000-fold higher than that of the anticancer drug, etoposide. These findings suggest that abasic sites represent endogenous topoisomerase II poisons and imply that anticancer drugs mimic the cleavage-enhancing actions of naturally occurring DNA lesions.

Animals↗
PubMed1996

The plastid-encoded ccsA gene is required for heme attachment to chloroplast c-type cytochromes.

A chloroplast gene, ycf5, which displays limited sequence identity to bacterial genes (ccl1/cycK) required for the biogenesis of c-type cytochromes, was tested for its function in chloroplast cytochrome biogenesis in Chlamydomonas reinhardtii. Targeted inactivation of the ycf5 gene results in a non-photosynthetic phenotype attributable to the absence of c-type cytochromes. The cloned ycf5 gene also complements the phototrophic growth deficiency in strain B6 of C. reinhardtii. B6 is unable to synthesize functional forms of cytochromes f and c6 owing to a chloroplast genome mutation that prevents heme attachment. The selected (phototrophic growth) as well as the unselected (holocytochrome c6 accumulation) phenotypes were restored in complemented strains. The complementing gene, renamed ccsA (for c-type cytochrome synthesis), is expressed in wild-type and B6 cells but is non-functional in B6 owing to an early frameshift mutation. Antibodies raised against the ccsA gene product recognize a 29-kDa protein in C. reinhardtii. The 29-kDa protein is absent in strain B6 but is restored in a spontaneous revertant (B6R) isolated from a culture of B6. Sequence analysis of the ccsA gene in strain B6R indicates that it is a true revertant. We conclude that the ccsA gene is expressed and that it encodes a protein required for heme attachment to c-type cytochromes.

Amino Acid Sequence↗
PubMedDate not supplied

Prediction of drug resistance in M. tuberculosis: molecular mechanisms, tools, and applications.

Management of Tuberculosis is complicated by the emergence of drug resistant strains of Mycobacterium tuberculosis and this poses a threat to the success of Tuberculosis control programmes. Drug susceptibility testing by culture is time-consuming and technically difficult. It is known that resistance to drugs is due to a number of genomic mutations in specific genes of M. tuberculosis. These mutations in combination with molecular techniques can be used as markers for drug resistance, since drug susceptible isolates lack the corresponding gene mutations. This review focuses on molecular mechanisms, methods and applications as a possible new diagnostic tool for the early molecular detection of drug resistance in M. tuberculosis.

Anti-Bacterial Agents↗
PubMed2003

Monophyletic clade of HIV-1 subtype B in Korea: evolutionary pressure or single introduction?

We have previously shown that many of the nef sequences from Korean HIV-1 subtype B carriers were grouped together in phylogenetic tree analyses. To determine whether this pattern was originated from either a single HIV-1-infected person or some biological pressure which directs the HIV-1 genomic mutation by the Korean specific immunological characters, we analyzed nef sequences from HIV-1-infected individuals with different time intervals. Thirty-two out of 46 analyzed patients formed a Korean monophyletic (KM) clade with 93% bootstrapping value. Eighteen patients' nef sequences were analyzed 1-9 years after first analysis. None of the patients shifted their clade from the first clustered clade (KM or non-KM), and all of the re-analyzed isolates were clustered close to the first analyzed clade. Isolates of the KM dade and non-KM clade in nef analysis showed the same pattern as in env analysis. Phylogenetic clustering evidences from both nef and C2/V3 trees strongly support the idea that introduction of the KM clade in subtype B strains originated from a common source. Thus, treatment and development of an AIDS vaccine may be somewhat easier than in other countries with multiple strains of HIV-1.

Biological Evolution↗
PubMed2006

Silencing of HIV-1 subtype C primary isolates by expressed small hairpin RNAs targeted to gag.

Discovery of sequence-specific silencing by activating the RNA interference (RNAi) pathway has led to exciting new strategies for treating infection with human immunodeficiency virus type 1 (HIV-1). Of the HIV-1 subtypes, C is especially common in areas of the world that are worst affected. Although prone to mutation, genome plasticity of this subtype is limited in functionally important regions. We identified conserved sequences within the HIV-1 subtype C gag open reading frame and assessed whether they are suitable targets for inhibition of viral replication by RNA Pol III-driven small hairpin RNAs (shRNAs). Initially, the efficacy of each of a panel of 10 shRNAs against HIV-1 was determined using a reporter assay. shRNAs A and B, which targeted the 5 end of gag, were most effective and were used to assess inhibition of replication in cultured cells of two R5 isolates (Du151 and Du422) and one X4 virus (SW7). These shRNAs diminished intracellular HIV-1 gag RNA and HIV-1 protein concentrations as well as p24 secretion by up to 80% without inducing an interferon response. However, shRNA-mediated knockdown efficacy against each of these viral isolates varied slightly. These data show successful activation of RNAi to inhibit the replication of biologically distinct HIV-1 subtype C isolates. The effector shRNAs described here are potential candidates for gene therapy applications against the most common global subtype of HIV-1.

Cell Line↗
PubMed1997

Disequilibria in the distribution of rpoB alleles in rifampicin-resistant M. tuberculosis isolates from Germany and Sierra Leone.

The effectiveness of culture-independent resistance predictions by molecular techniques is dependent on the number and the frequency of accessible resistance-associated genomic mutations. We have characterized an rpoB gene region involved in rifampicin resistance in 49 Mycobacterium tuberculosis isolates resistant to rifampicin from Germany and Sierra Leone. The determined frequencies of mutations differed between both countries of origins as well as with respect to previously reported distributions of resistance mutations. It is concluded that at least for some isolates the acquisition of mutations leading to rifampicin resistance in clinical samples of M. tuberculosis is a non-random process which may lead to a geographical and temporal dependence of the sensitivities of molecular typing techniques for rifampicin resistance predictions.

Alleles↗
PubMed2000

An inbreeding model of associative overdominance during a population bottleneck.

Associative overdominance, the fitness difference between heterozygotes and homozygotes at a neutral locus, is classically described using two categories of models: linkage disequilibrium in small populations or identity disequilibrium in infinite, partially selfing populations. In both cases, only equilibrium situations have been considered. In the present study, associative overdominance is related to the distribution of individual inbreeding levels (i.e., genomic autozygosity). Our model integrates the effects of physical linkage and variation in inbreeding history among individual pedigrees. Hence, linkage and identity disequilibrium, traditionally presented as alternatives, are summarized within a single framework. This allows studying nonequilibrium situations in which both occur simultaneously. The model is applied to the case of an infinite population undergoing a sustained population bottleneck. The effects of bottleneck size, mating system, marker gene diversity, deleterious genomic mutation parameters, and physical linkage are evaluated. Bottlenecks transiently generate much larger associative overdominance than observed in equilibrium finite populations and represent a plausible explanation of empirical results obtained, for instance, in marine species. Moreover, the main origin of associative overdominance is random variation in individual inbreeding whereas physical linkage has little effect.

Animals↗
PubMed1990

Clastogenic effects of known and suspect spindle poisons studied by chromosome analysis in mouse bone marrow cells.

The present study was performed within the project 'Genomic Mutations' (sponsored by the Commission of the European Communities) in order to gather all possible experimental information on 10 chemicals selected on the basis of their possible capacity to induce aneuploidy. An analysis of chromosomal aberrations was carried out in bone marrow cells of mice with the first five chemicals: colchicine (COL), econazole (EZ), chloralhydrate (CH), hydroquinone (HQ) and diazepam (DIAZ). The experiments were performed in parallel to micronucleus tests with the objective to distinguish a positive micronucleus response due to chromosomal breakage from that obtained by lagging chromosomes. The results of the micronucleus tests will be reported elsewhere. COL, CH, EZ and DIAZ showed no clastogenic effects in mouse bone marrow cells after single intraperitoneal injection. Polyploid cells were significantly more frequent after COL treatment. HQ showed a dose-dependent induction of chromosomal aberrations at 6 and 24 h after treatment. After 24 h, cells with multiple aberrations up to complete chromosome fragmentation were frequently observed. They indicate that a small fraction of the cell population, probably related to a specific stage of the cell cycle, was particularly sensitive to HQ. A sex difference in clastogenic response to HQ was not observed. It is concluded that of the five chemicals tested only HQ was clastogenic in mouse bone marrow cells under the present experimental conditions.

Aneuploidy↗
PubMed1991

In vitro micronucleus test with kinetochore staining: evaluation of test performance.

In the framework of the coordinated programme 'Genomic Mutations' sponsored by the Commission of European Communities, eight known or suspected spindle poisons (cadmium chloride, chloral hydrate, colchicine, diazepam, econazole, hydroquinone, pyrimethamine, thiabendazole, thimerosal and vinblastine) were tested in a modified in vitro micronucleus test in Chinese hamster cells. Micronuclei (MN) with or without kinetochore were analyzed by staining of the kinetochore with an antikinetochore antibody (CREST staining). Mitotic index and ana-telophase:mitosis ratio were also recorded to evaluate cytotoxicity and c-mitotic effects of tested chemicals. CREST-positive MN were induced by diazepam, thiabendazole and vinblastine. Hydroquinone, pyrimethamine, econazole and cadmium chloride induced MN that contained both entire chromosomes and acentric fragments. Negative results were obtained with thimerosal. The results obtained indicate that the detection of CREST-positive MN is a powerful assay to identify spindle poisons. Furthermore, this method provides a useful tool to ascertain the origin(s) of induced MN.

Aneuploidy↗
PubMed1996

Effects of RNA editing and gene processing on phylogenetic reconstruction.

RNA editing is a ubiquitous phenomenon affecting most mitochondrial and chloroplast, and some nuclear genomes, where mutations in genomic DNA are "corrected" in the mRNA during transcriptional processing. Most editing in plants and animals corrects T-to-C substitutions at nonsynonymous first or second base positions, and the overall effect is an mRNA and protein sequence that differs from that predicted by the DNA. It has been suggested that genomic sequences that undergo editing should not be used in phylogenetics. We contend that editing will have little or no effect on DNA-based phylogenetic reconstruction because it is an intrinsic transcriptional process that does not affect the historical information in the DNA sequence. The only effect of editing on protein-coding DNA should be an increase in the rate of T-to-C transitions. Here we test the effects of RNA editing on phylogenetic reconstruction, using two data sets with high levels of editing, plant coxII and coxIII. Even with high levels of editing, phylogenies based on DNA and edited mRNA are virtually identical. The two types of sequences should not be used in the same analysis, however, because the particular forms of the gene will tend to group together. We also examine the effects of processed paralogs--a term proposed for mRNA sequences that are reverse transcribed and reinserted into the genome as intact gene sequences. Processed paralogs result in a distinct and under-appreciated source of conflict among gene trees because of RNA editing. Analyses with unidentified processed paralogs may yield incorrect phylogenies, and the sequences may evolve at different rates if the gene has been transferred from one genetic compartment (nuclear, mitochondrial, chloroplast) to another. Although RNA editing itself is not a problem in phylogenetic reconstruction, analyses should not combine mRNAs with DNAs, and processed paralogs should be either excluded or analyzed with caution.

Cyclooxygenase 2↗
PubMed1997

Accelerated evolution of sites undergoing mRNA editing in plant mitochondria and chloroplasts.

The selective constraints influencing mRNA editing in plant organelles are largely unknown. To investigate these, we compared patterns of editing between monocot and dicot mitochondrial mRNA. On average, 24% of sites that are edited form C to U in one species have been substituted during evolution by a genomic T in the other: this is four times the rate of evolution seen at nonedited synonymously variable C residues. A similar, but weaker trend (not statistically significant) is seen at sites edited in chloroplast mRNA. The elevated substitution rate does not appear to be a consequence of a higher mutability of the trinucleotide motif (T-C-purine) associated with editing. nor to be a result of reverse transcription from mature mRNA. Selection to replace the genomic C with a T may account for the accelerated evolution, either due to elimination of inefficient transcripts and protein products or as a consequence of the prior loss of components outside the edit site which are necessary for editing; the latter hypothesis is supported by the frequent loss of editing without genomic mutation at third codon positions. Whatever the cause, the rapid rate of evolution indicates that editing confers little selective advantage at most sites.

Base Sequence↗
PubMed2003

A new subclass of the luteinizing hormone/chorionic gonadotropin receptor lacking exon 10 messenger RNA in the New World monkey (Platyrrhini) lineage.

The luteinizing hormone receptor (LHR) plays an essential role as a mediator of LH and CG action during embryonic sexual differentiation and in gametogenesis. In a hypogonadal male patient, we recently demonstrated that a genomic deletion of exon 10, located in the hinge region of the extracellular domain, results in discrimination of LH and hCG action. In the common marmoset (Calltithrix jacchus), exon 10 of the LHR is naturally missing at the mRNA level. In order to investigate whether this is an isolated species-specific phenomenon, we performed a phylogenetic screening, searching for the presence of LHR exon 10 mRNA in a number of primate species representative for the major lineages of primate evolution. The expressed LHR region encompassing exon 10 was amplified from testicular tissue by RT-PCR, cloned, and sequenced. In addition, we performed Southern blot analysis of the LHR of selected New World and Old World primates. The results revealed that exon 10 mRNA is lacking in the complete New World monkey (Platyrrhini) lineage but is present in both more primitive and more advanced primates. However, exon 10 seems to be present at the genomic level, arguing for a splicing failure possibly due to a genomic mutation or the lack of appropriate splicing factors. Considering that, in the human, LH is far less active than hCG on the LHR lacking exon 10, we addressed the question whether the existence of such a receptor has any consequences on the dual hormone LH/CG system present in Platyrrhini. Using primers specific for the known marmoset CG beta cDNA, we amplified the CG beta subunit cDNA from male common marmoset pituitaries by RT-PCR, while LH beta could not be amplified, suggesting a possible physiological role of pituitary CG in this species. In conclusion, we demonstrated for the first time that the LH mRNA without exon10 is the natural wild-type LHR in the Platyrrhini lineage. We propose that this LHR represents a new subclass of receptors that should be named LHR type II. In addition, the high expression of CG beta in the marmoset pituitary suggests a physiological role of CG in the reproductive function of these primates beyond pregnancy.

Amino Acid Sequence↗
PubMed1998

Heteroduplex analysis: a useful screening method for glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSDIa), also known as von Gierke disease, is the most common and severe disease of glycogenoses and is caused by a deficiency of glucose-6-phosphatase (G6Pase) and transmitted by an autosomal recessive trait. The encoding gene of G6Pase is composed of only five exons and each exon is short. With heteroduplex analysis (HDA) method, we analyzed the genomic DNA from a patient diagnosed with GSDIa and from her parents. Exons II and IV of the patient showed heteroduplex bands. The mother had a heteroduplex band of exon II, and the father had a heteroduplex band of exon IV. In a mini-slab electrophoresis, exons II and IV of the patient did not show clear heteroduplex bands, but they appeared broader than the others, which made us suspect that they were heteroduplex bands. HDA is an easy and simple method and can verify mutant homozygous DNA fragments by adding wild-type DNA. We think that HDA may be a very useful screening method for the detection of novel genomic mutation in GSDIa in large-scale and mini-slab electrophoresis.

Adult↗
PubMed1983

Analysis of Theiler's virus isolates from persistently infected mouse nervous tissue.

The DA strain of Theiler's virus causes a chronic progressive demyelination in mice following intracerebral inoculation. Virus was isolated from chronically infected mice, and then grown in cell culture, and the isolates were compared with the parent virus used for inoculation. No defective interfering particles or temperature-sensitive virus were recovered, and capsid proteins appeared identical by SDS-PAGE. One of three isolates had evidence of genomic mutation by Tl ribonuclease oligonucleotide fingerprinting. The significance of these findings with regard to the generation and maintenance of persistence and to adaptation to cell culture is discussed. Also of interest was the marked difference between the DA fingerprint and that of GD VII, a serologically related strain with different biological activity.

Animals↗
PubMed1994

Mutagenesis of Legionella pneumophila using Tn903 dlllacZ: identification of a growth-phase-regulated pigmentation gene.

Study of the molecular basis for Legionella pneumophila pathogenicity would be facilitated with an efficient mutagen that can not only mark genomic mutations, but can also be used to reflect gene expression during macrophage infection. A derivative of Tn903, Tn903dlllacZ, is shown to transpose with high efficiency in L. pneumophila. Tn903dlllacZ encodes resistance to kanamycin (KmR) and carries a 5' truncated 'lacZ gene that can form translational fusions to L. pneumophila genes upon transposition. The cis-acting Tn903 transposase is supplied outside Tn903dlllacZ, and hence chromosomally integrated copies are stable. KmR LacZ+ insertion mutants of L. pneumophila were isolated and shown by DNA hybridization to carry a single Tn903dlllacZ inserted within their chromosomes at various locations. One particular KmR LacZ+ mutant, AB1156, does not produce the brown pigment (Pig-) characteristic of Legionella species. Tn903dlllacZ is responsible for this phenotype since reintroduction of the transposon-linked mutation into a wild-type background results in a Pig- phenotype. L. pneumophila pigment production is normally observed in stationary-phase growth of cells in culture, and beta-galactosidase activity measured from the pig::lacZ fusion increased during the logarithmic-phase growth and peaked at the onset of stationary phase. Interestingly, pig::lacZ expression also increased during macrophage infection. The pigment itself, however, does not appear to be required for L. pneumophila to grow within or kill host macrophages.

Alleles↗
PubMed1992

Hypothesis for the control of clotting factor VIII inhibitory antibodies by decreasing potency of helper T-cell-recognized epitopes in factor VIII.

The study of the immunobiology of FVIII inhibitors may lead to new therapies for this potentially severe complication of haemophilia A and to new principles for the use of therapeutic proteins. In order to characterize the idiotype-anti-idiotype networks regulating FVIII inhibitors, we developed rabbit anti-idiotypic sera to 7 murine inhibitors and found at least 12 independent FVIII loci to which inhibitors could be raised. Rabbit antisera to the FVIII peptide, Ser1687-Thr1695, characterized one functional site to which about 46% of patients' inhibitor sera reacted. The multiplicity of inhibitor-recognized epitopes in FVIII makes it impractical, at the present time, to develop clinically useful specific anti-idiotypic therapies for FVIII inhibitors. Alternatively, one might induce genomic mutations in recombinant FVIII molecules to decrease immunogenicity of epitopes recognized by T helper cells. Methods to design such altered therapeutic proteins are presented, based on changing the longitudinal hydrophobic strip-of-helix which is in or near many T-cell-presented epitopes.

Animals↗
PubMed2004

Impact of methoxymycolic acid production by Mycobacterium bovis BCG vaccines.

BCG vaccines are a family of closely related daughter strains of an attenuated isolate of Mycobacterium bovis derived by in vitro passage from 1908 to 1921. During subsequent laboratory propagation of the vaccine strain until its lyophilization in 1961, BCG Pasteur underwent at least seven further genomic mutations. The impact of these mutations on the properties of the vaccine is currently unknown. One mutation, a glycine-to-aspartic acid substitution in the mmaA3 gene, occurred between 1927 and 1931 and impairs methoxymycolic acid synthesis in BCG strains obtained from the Pasteur Institute after this period. Mycolic acids of the cell wall are classified into three functional groups (alpha-, methoxy-, and ketomycolic acids), and together these lipids form a highly specialized permeability barrier around the bacterium. To explore the impact of methoxymycolic acid production by BCG strains, we complemented the functional gene of mmaA3 into BCG Denmark and tested a number of in vitro and in vivo phenotypes. Surprisingly, restoration of methoxymycolic acids alone had no effect on cell wall permeability, resistance to antibiotics, or growth in cultured macrophages and C57BL/6 mice. Our results demonstrate that the loss of methoxymycolic acid production did not apparently affect the virulence of BCG strains.

Amino Acid Substitution↗