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At least 865 records · Page 48Linked to original sources
PubMed2005

Molecular footprint of drug-selective pressure in a human immunodeficiency virus transmission chain.

Known human immunodeficiency virus (HIV) transmission histories are invaluable models for investigating the evolutionary and transmission dynamics of the virus and to assess the accuracy of phylogenetic reconstructions. Here we have characterized an HIV-1 transmission chain consisting of nine infected patients, almost all of whom were treated with antiviral drugs at later stages of infection. Partial pol and env gp41 regions of the HIV genome were directly sequenced from plasma viral RNA for at least one sample from each patient. Phylogenetic analyses in pol using likelihood methods inferred an evolutionary history not fully compatible with the known transmission history. This could be attributed to parallel evolution of drug resistance mutations resulting in the incorrect clustering of multidrug-resistant virus. On the other hand, a fully compatible phylogenetic tree was reconstructed from the env sequences. We were able to identify and quantify the molecular footprint of drug-selective pressure in pol using maximum likelihood inference under different codon substitution models. An increased fixation rate of mutations in the HIV population of the multidrug-resistant patient was demonstrated using molecular clock modeling. We show that molecular evolutionary analyses, guided by a known transmission history, can reveal the presence of confounding factors like natural selection and caution should be taken when accurate descriptions of HIV evolution are required.

Anti-HIV Agents↗
PubMed2006

Dynamics of hepatitis B virus resistance to lamivudine.

Lamivudine was the first approved inhibitor of hepatitis B virus (HBV) reverse transcriptase (RT). Lamivudine resistance develops in 53% to 76% of patients after 3 years of treatment. We extensively characterized the dynamics of HBV quasispecies variant populations in four HBV-infected patients who developed lamivudine resistance. Virological breakthrough was preceded by 2 to 4 months by the emergence of quasispecies variants bearing amino acid substitutions at RT position 204, i.e., within the YMDD catalytic motif (rtM204V/I). Three patients had a gradual switch from a YMDD variant population at baseline to a 100% lamivudine-resistant variant population, whereas the remaining patient had a fluctuating pattern of resistance variant dynamics. Careful analysis of amino acid substitutions located outside domain C of HBV RT, including those known to partially restore replication capacities in vitro, showed that the in vivo replication of HBV variants is driven by multiple forces, including intrinsic replicative advantages conferred by mutations accumulating outside domain C and the changing environment in which these variants replicate. Our findings also suggest that individual treatment optimization will require sensitive methods capable of detecting the emergence of viral resistance before the relevant variants acquire optimal replicative capacities.

Adult↗
PubMed2005

Reverse transcriptase and integrase of the Saccharomyces cerevisiae Ty1 element.

Integrase (IN) and reverse transcriptase (RT) play a central role in transposition of retroelements. The mechanism of integration by IN and the steps of the replication process mediated by RT are briefly described here. Recently, active recombinant forms of Ty1 IN and RT have been obtained. This has allowed a more detailed understanding of their biochemical and structural properties and has made possible combined in vitro and in vivo analyses of their functions. A focus of this review is to discuss some of the results obtained thus far with these two recombinant proteins and to propose future directions.

Amino Acid Sequence↗
PubMed2003

The non-LTR retrotransposons in Ciona intestinalis: new insights into the evolution of chordate genomes.

BACKGROUND: Non-long terminal repeat (non-LTR) retrotransposons have contributed to shaping the structure and function of genomes. In silico and experimental approaches have been used to identify the non-LTR elements of the urochordate Ciona intestinalis. Knowledge of the types and abundance of non-LTR elements in urochordates is a key step in understanding their contribution to the structure and function of vertebrate genomes. RESULTS: Consensus elements phylogenetically related to the I, LINE1, LINE2, LOA and R2 elements of the 14 eukaryotic non-LTR clades are described from C. intestinalis. The ascidian elements showed conservation of both the reverse transcriptase coding sequence and the overall structural organization seen in each clade. The apurinic/apyrimidinic endonuclease and nucleic-acid-binding domains encoded upstream of the reverse transcriptase, and the RNase H and the restriction enzyme-like endonuclease motifs encoded downstream of the reverse transcriptase were identified in the corresponding Ciona families. CONCLUSIONS: The genome of C. intestinalis harbors representatives of at least five clades of non-LTR retrotransposons. The copy number per haploid genome of each element is low, less than 100, far below the values reported for vertebrate counterparts but within the range for protostomes. Genomic and sequence analysis shows that the ascidian non-LTR elements are unmethylated and flanked by genomic segments with a gene density lower than average for the genome. The analysis provides valuable data for understanding the evolution of early chordate genomes and enlarges the view on the distribution of the non-LTR retrotransposons in eukaryotes.

Amino Acid Sequence↗
PubMed1992

Recent advances and prospective researches on molecular epidemiology of dengue viruses.

The determination of amino acid changes in the envelope protein by direct sequencing of either genomic RNA or PCR-amplified cDNA fragments provides useful informations for assessing the genetic variability and the geographic distribution of the actually most widespread dengue-2 serotype. The possible link of variations in the envelope protein-gene and virus virulence is discussed.

Dengue↗
PubMed1999

Novel patterns of amino acid mutations in the hepatitis B virus polymerase in association with resistance to lamivudine therapy in japanese patients with chronic hepatitis B.

Lamivudine is effective in suppressing replication of hepatitis B virus (HBV). However, the emergence of HBV variants resistant to lamivudine is a concern. Lamivudine resistance has been attributed mainly to a substitution of isoleucine or valine for methionine at residue 550 (M550I or M550V) in the catalytic site of the virus polymerase. A substitution of methionine for leucine at residue 526 (L526M) has also been identified. To examine such virus genotypic mutations in Japanese patients, we studied five patients with chronic hepatitis B, who showed HBV breakthrough while on a 1-year lamivudine treatment. The entire nucleotide and deduced amino acid sequences of the proposed reverse transcriptase domain of the polymerase gene were determined on HBV DNA amplified by polymerase chain reaction from patient sera collected at the start and at the end of therapy. The HBV sequences from all five patients were of genotype C. In four patients, a substitution of valine or isoleucine for leucine at residue 426, which has not been reported previously, emerged in combination with M550I. One also harbored L526M. In the remaining patient, an alteration of leucine to methionine at residue 428 co-occurred with M550V. Longitudinal study of the mutations showed that the two or three mutations in each patient emerged almost simultaneously 4 weeks before or at the time of breakthrough and were replaced by wild-type virus after completing the therapy. Our results indicate that occurrence of HBV polymerase mutations at residue 426 in combination with M550I is frequent in Japanese or genotype C virus-in- fected patients who develop resistance to lamivudine.

Adult↗
PubMed1999

Comparative genomics and evolutionary dynamics of Saccharomyces cerevisiae Ty elements.

The availability of the complete genome sequence of Saccharomyces cerevisiae provides the unique opportunity to study an entire genomic complement of retrotransposons from an evolutionary perspective. There are five families of yeast retrotransposons, Ty1-Ty5. We have conducted a series of comparative sequence analyses within and among S. cerevisiae Ty families in an effort to document the evolutionary forces that have shaped element variation. Our results indicate that within families Ty elements vary little in terms of both size and sequence. Furthermore, intra-element 5'-3' long terminal repeat (LTR) sequence comparisons indicate that almost all Ty elements in the genome have recently transposed. For each family, solo LTR sequences generated by intra-element recombination far outnumber full length insertions. Taken together, these results suggest a rapid genomic turnover of S. cerevisiae Ty elements. The closely related Ty1 and Ty2 are the most numerous elements in the genome. Phylogenetic analysis of full length insertions reveals that reverse transcriptase mediated recombination between Ty1 and Ty2 elements has generated a number of hybrid Ty1/2 elements. These hybrid Ty1/2 elements have similar genomic structures with chimeric LTRs and chimeric TYB (pol) genes. Analysis of the levels of nonsynonymous (Ka) and synonymous (Ks) nucleotide variation indicates that Ty1 and Ty2 coding regions have been subject to strong negative (purifying) selection. Distribution of Ka and Ks on Ty1, Ty2 and Ty1/2 phylogenies reveals evidence of negative selection on both internal and external branches. This pattern of variation suggests that the majority of full length Ty1, Ty2 and Ty1/2 insertions represent active or recently active element lineages and is consistent with a high level of genomic turnover. The evolutionary dynamics of S. cerevisae Ty elements uncovered by our analyses are discussed with respect to selection among elements and the interaction between the elements and their host genome.

Amino Acid Sequence↗
PubMed2000

BCL-1 rearrangements and p53 mutations in atypical chronic lymphocytic leukemia with t(11;14)(q13;q32).

BACKGROUND AND OBJECTIVES: The translocation t(11;14) (q13;q32), typically described in mantle cell lymphomas (MCL), has also been found in some cases of non-MCL lymphoproliferative disorders, such as splenic lymphoma with villous lymphocytes (SLVL), multiple myeloma (MM), prolymphocytic leukemia (PLL), typical and atypical chronic lymphocytic leukemia (CLL and aCLL). In order to define better the genetic features of aCLL with t(11;14), which could represent a distinct disease subset, we looked for genetic lesions in the BCL-1 locus and in BCL-2, BCL-6, c-myc and p53 genes. DESIGN AND METHODS: We investigated a panel of B-lymphoproliferative disorders with translocation t(11;14)(q13;q32) including nine aCLL, six MCL and one MM. Southern and Northern blot analysis was used to investigate DNA structure and RNA expression; SSCP and direct sequencing were used to detect and characterize p53 point mutations; cytofluorimetric analysis was used to quantify p53 protein. RESULTS: Alterations of BCL-2, BCL-6 and c-myc were not detected. Conversely, BCL-1 rearrangements were present in 4 out of 7 aCLL and in 2 out of 4 MCL. A high incidence of p53 gene alterations was found, almost equivalent in aCLL and MCL. INTERPRETATION AND CONCLUSIONS: Our results indicate that the occurrence of BCL-1 locus lesions in aCLL selected for t(11;14) is as high as in MCL. Interestingly, rearrangements in the mTC1 (minor translocation cluster 1) were only found in aCLL. Therefore, the two B-cell chronic lymphoproliferative disorders share similar molecular rearrangements and the t(11;14) identifies a subset of B-CLL sharing molecular features with MCL and characterized by aggressive clinical evolution.

Adult↗
PubMed2002

[Copia-like retrotransposon in Amaranthus].

This study analyzed the characteristics of reverse transcriptase sequences of copia-like retrotransposons in Amaranthus via PCR amplification and sequence approaches. The results are as follows: (1) The reverse transcriptase sequences of copia-like retrotransposons were detected in all 30 Amaranthus accessions studied, indicating that this class of retrotransposons is ubiquitous in Amaranthus; (2) Twenty-eight reverse transcriptases in A. quitensis were cloned and sequenced, showing extensive heterogeneity in base deletion mutation and stop codon mutation; (3) Cluster and alignment analyses of the 28 sequences and other published sequences of the reverse transcriptase from other species (such as rice, tomato and Drosophila) showed that the copia-like retrotransposons in A. quitensis have a close relationship with those in dicot and monocot plants. The evolution of copia-like retrotransposons in A. tricolor, which might be of the same origin as the copia and 1731 in Drosophila, was discussed.

Amaranthus↗
PubMed2004

[Screening of cDNA clone for putative RNA polymerase subunit of Cysticercus cellulosae].

OBJECTIVE: To obtain related genes of Cysticercus cellulosae from spliced leader (SL) cDNA library. METHODS: Spliced leader library of Cysticercus cellulosae was constructed using SL specific primer and oligo (dT) 15 with M13M4 primer, and positive clones were then screened randomly, identified with enzyme restriction, followed by sequencing and homologous analysis. RESULTS: The amino acid sequence, encoded by the positive clone with a poly (A)22 tail and a complete open reading frame (ORF), was with homology of RNA polymerase subunit genes of human, B. napus, fission yeast, A. thaliana, C. elegans and fruit fly up to 71.6%. CONCLUSION: The protein, RNA polymerase subunit encoded putatively by the clone, is high conservative in different species.

Amino Acid Sequence↗
PubMed1994

Study of the roles of proline 391 and a highly conserved sequence in the carboxyl-terminal region of members of the serpin family in the secretion of alpha 1-proteinase inhibitor.

Truncation of alpha 1-proteinase inhibitor prior to Pro391 prevents its secretion. This residue is the carboxyl terminus of a highly conserved sequence in serpins, suggesting that either Pro391 or the conserved sequence may serve as at least a part of a signal recognized by components of the secretory pathway. To evaluate these possibilities, we have determined the effects of replacement of Pro391 on the secretion of alpha 1-proteinase inhibitor and have examined the ability of the 9-residue conserved sequence to mediate secretion. We find that replacement of Pro391 with hydrophobic residues yields variants that are well secreted, but replacement with other classes of amino acids severely restricts secretion. These results show that while alpha 1-proteinase inhibitor is secreted most efficiently when proline occupies position 391, Pro391 is not an absolute requirement for its secretion. Our results show that the 9-amino acid conserved sequence found near the carboxyl termini of proteins of the serpin family is not sufficient to direct the secretion of alpha 1-proteinase inhibitor. We conclude that mutations affecting residue 391 and other positions in the conserved region lead to structural changes, possibly very minor, that influence the secretion of alpha 1-proteinase inhibitor.

Amino Acid Sequence↗
PubMed1994

[Expression of the gene coding for the D1-protein of barley photosystem II in Escherichia coli].

Previously characterized by us barley chloroplast psbA gene, which encodes one of the main Photosystem II components--D1 protein, has been inserted in a set of special plasmid vectors and its expression in vitro and in vivo has been investigated. Experiments on the in vitro expression in the rabbit reticulocyte lysate system revealed a major product with a molecular weight ca. 33.5 kD, which corresponds to the unprocessed D1 barley protein. A lower molecular weight protein (about 29 kD) was also observed. These results are in agreement with the existence of two potential translation start sites in the psbA gene in the same reading frame, the second one starting from Met37 residue. The results fully correlate with the earlier data on the in vitro expression of psbA genes of maize, pea, and tobacco. Experiments on the in vivo expression of psbA gene in E. coli cells with the above constructions also revealed proteins with m. w. about 33.5 and 29 kD. The yield of the target recombinant protein in some cases was about 25-30% of the total E. coli cellular protein. The correspondence of the bands to the desired products was proved by the immunoenzyme analysis with the use of polyclonal antibodies. The data obtained show for the first time the construction of E. coli strains producing recombinant D1 protein of cereals in a high level.

Amino Acid Sequence↗
PubMed1996

[Identification and characterization of new and unknown coronaviruses using RT-PCR and degenerate primers].

A modified method of the reverse transcription followed by polymerase chain reaction (RT-PCR) was developed in order to examine the genome of a recently discovered virus, the porcine epidemic diarrhoea virus (PEDV), which resembled morphologically the coronaviruses. The published sequences of the genomes of various coronaviruses were compared. On the level of the amino acid sequence, conserved regions, common to all coronaviruses, were found in the gene encoding the nonstructural protein 1b as well as in the genes coding for the major structural proteins (S, M, and N). Due to the degeneration of the genetic code, some amino acids may be encoded by different nucleotide triplets. In order to compensate for this degeneration, mixtures of primers were synthesized, containing a variety of nucleotide sequences which together represented all possible codons for the conserved amino acid sequences. This method allowed to amplify and clone approximately 4000 base pairs of the genome of PEDV. An analysis of the genomic sequences revealed that PEDV holds an interesting intermediate position between human coronavirus 229E and Transmissible Gastroenteritis virus. We postulate that the method presented in this contribution may be useful to study and characterize other unknown viruses, especially viruses for which no cell cultures for propagation are available.

Amino Acid Sequence↗
PubMedDate not supplied

Genetic divergence of Asiatic Bdellocephala (Turbellaria, Tricladida, Paludicola) as revealed by partial 18S rRNA gene sequence comparisons.

Polymerase chain reaction (PCR) and direct sequencing of small ribosomal RNA genes were used for analysis of genetic differences among Asiatic species of freshwater triclad genus Bdellocephala. Representatives of four species and four subspecies of this genus were used to establish homology between nucleotides in the 5'-end portion of small ribosomal RNA gene sequences. Within 552 nucleotide sites of aligned sequences compared, six variable base positions were discovered, dividing Bdellocephala into five different genotypes. Sequence data allow to distinguish two groups of these genotypes. One of them unites species from Kamchatka and Japan, another one unites Baikalian taxa. Agreement between available morphological, cytological and sequence data is discussed.

Animals↗
PubMed1998

D2 dopamine antisense RNA expression vector, unlike haloperidol, produces long-term inhibition of D2 dopamine-mediated behaviors without causing Up-regulation of D2 dopamine receptors.

Long-term inhibition of D2 dopamine receptors using classic D2 dopamine receptor antagonists such as haloperidol often causes a compensatory up-regulation of the D2 dopamine receptors. We investigated whether the long-term inhibition of D2 dopamine receptors using an eukaryotic expression vector housing a cDNA sequence encoding an antisense RNA directed to the D2 dopamine receptor transcript (D2 antisense vector) would also produce up-regulation of the D2 receptors. Single, bilateral injections of the D2 antisense vector into the corpora striata of mice inhibited the stereotypy induced by acute challenge injections with the D2/D3 dopamine receptor agonist quinpirole but did not inhibit the grooming induced by acute challenge injections with the D1 agonist SKF 38393. Similar treatment with the D2 antisense vector produced a long-term (>1 month) cataleptic response without producing tolerance to challenge injections with haloperidol. By contrast, catalepsy induced by a single injection of haloperidol lasted only approximately 2 days, and tolerance developed to its effects after long-term treatment. Repeated treatment of mice with haloperidol resulted in an inhibition of apomorphine-induced climbing behavior throughout the time of treatment with haloperidol, but the climbing behavior markedly increased to levels significantly higher than that of the control mice immediately after withdrawal from haloperidol treatment. This increased climbing was accompanied by increased levels of D2 dopamine receptors in the striatum. By contrast, single, bilateral intrastriatal injections of the D2 antisense vector significantly inhibited apomorphine-induced climbing for approximately 30 days but failed to increase the climbing behavior or the levels of D2 dopamine receptors in striatum over those of the control values. These results suggest that a single injection of a D2 antisense RNA expression vector into mouse striatum produces specific, long-term inhibition of D2 dopamine receptor behaviors without causing a compensatory increase in the levels or function of D2 dopamine receptors.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1998

The Bacillus subtilis regulator SinR inhibits spoIIG promoter transcription in vitro without displacing RNA polymerase.

Initiation of sporulation in Bacillus subtilis is controlled by several regulators which affect activation by phosphorylation of the key response regulator Spo0A or transcription of Spo0A-P-dependent genes. In vivo overexpression of one of these regulators, sinR , results in suppression of transcription from the Spo0A-P-dependent promoters of spo0A , spoIIA , spoIIE and spoIIG and in vitro SinR binds to the promoters of the spoIIA operon and the spo0A gene. In this study we have demonstrated that in vitro SinR directly repressed Spo0A- P-dependent transcription by B.subtilis RNA polymerase from the spoIIG operon promoter. SinR inhibited transcription prior to formation of heparin-resistant complexes but did not displace RNA polymerase from the spoIIG promoter. DNase I protection studies demonstrated that SinR protected a large region of the spoIIG promoter and induced DNase I hypersensitive sites, particularly around the 0A boxes, at the same positions as those induced by zinc. Since binding of zinc induces bends in the DNA, we concluded that SinR binding also altered the conformation of the spoIIG promoter. We propose that SinR-induced conformational changes in Spo0A-dependent promoters prevent activation of trans-cription by Spo0A-P.

Bacillus subtilis↗
PubMed1994

A role for the H4 subunit of vaccinia RNA polymerase in transcription initiation at a viral early promoter.

The vaccinia virus H4 gene encodes an essential subunit of the DNA-dependent RNA polymerase holoenzyme encapsidated within virus particles (Ahn, B., and Moss, B. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3536-3540; Kane, E. M., and Shuman, S. (1992) J. Virol. 66, 5752-5762). The role of this protein in transcription of viral early genes was revealed by the effects of affinity-purified anti-H4 antibody on discrete phases of the early transcription reaction in vitro. Anti-H4 specifically prevented the synthesis of a 21-nucleotide nascent RNA chain but had no impact on elongation of the 21-mer RNA by preassembled ternary complexes. Inhibition of initiation but not elongation was also observed with affinity-purified anti-D6 antibody directed against the 70-kDa subunit of the vaccinia early transcription initiation factor (ETF). Native gel mobility-shift assays showed that anti-H4 prevented the NTP-dependent recruitment of RNA polymerase to the preinitiation complex of ETF bound at the early promoter. Two species of ternary complexes could be resolved by native gel electrophoresis. Addition of anti-H4 to preformed complexes elicited a supershift of both ternary species but not of the preinitiation complex. Supeshift by anti-D6 revealed that the more rapidly migrating species of ternary complex did not contain immunoreactive ETF. Loss of ETF from the ternary complex was time-dependent. Thus, whereas the H4 protein was a stable constituent of the elongation complex, ETF was dissociable. We suggest that H4 functions as a molecular bridge to ETF and thereby allows specific recognition of early promoters by the core RNA polymerase. H4 is unlike bacterial sigma factor in that it remains bound to polymerase after the elongation complex is established.

Amino Acid Sequence↗