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At least 1,783 records · Page 99Linked to original sources
PubMed1999

The nucleotide sequence of the high-leukemogenic murine retrovirus SL3-3 reveals a patch of mink cell focus forming-like sequences upstream of the ecotropic envelope gene. Brief report.

We report the complete nucleotide sequence of the potent T-lymphomagenic murine retrovirus SL3-3. The non-LTR regions of the virus show 98% sequence identity to the endogenous ecotropic Akv murine leukemia virus. While the region encoding the surface envelope protein is completely identical to that of Akv, a approximately 200 nucleotide stretch in the integrase encoding region upstream of env is similar to the sequence of mink cell focus forming (MCF) viruses and shows a complete match with the mouse retrovirus 10A1. The history of SL3-3 may therefore include recombination involving an Akv-like virus and a virus with MCF-like sequences.

Animals↗
PubMed2005

A very large diversity space of synthetically accessible compounds for use with drug design programs.

We have constructed a very large virtual diversity space containing more than 10(13) chemical compounds. The diversity space is built from about 400 combinatorial libraries, which have been expanded by choosing sizeable collections of suitable R-groups that can be attached to each link point of their scaffolds. These R-group collections have been created by selecting reagents that have drug-like properties from catalogs of available chemicals. As members of known combinatorial libraries, the compounds in the diversity space are in general synthetically accessible and useful as potential drug leads. Hence, the diversity space can be used as a vast source of compounds by a de novo drug design program. For example, we have used such a program to generate inhibitors of HIV integrase enzyme that exhibited activity in the micromolar range.

Algorithms↗
PubMed2006

Analysis of human immunodeficiency virus type 1 integration by using a specific, sensitive and quantitative assay based on real-time polymerase chain reaction.

A novel real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type-1 (HIV-1) DNA with high specificity and sensitivity. This assay reproducibly allowed the detection of three copies of integrated HIV DNA in a background of 100,000 cell equivalents of human chromosomal DNA. The non-specific amplification of unintegrated HIV-1 DNA was significantly inhibited in this assay and the specificity of this assay was much higher than the previously reported method. This assay showed that kinetics in viral DNA synthesis was cell-type dependent and that the kinetics of HIV-1 DNA integration was very rapid in Jurkat T cell line. This method may provide new insights into the integration processes and be useful in evaluating future integrase inhibitors.

Base Sequence↗
PubMed2026

Site-specific gene integration by recombinase-mediated cassette exchange in anhydrobiotic Pv11 cells.

Pv11 cells, derived from Polypedilum vanderplanki, uniquely tolerate complete desiccation (anhydrobiosis). Although a CRISPR/Cas9-based precise integration method (CRIS-PITCh) has been developed for Pv11 cells, a CRISPR-free strategy that fixes both the genomic locus and transgene copy number has not yet been established. Here, we implement recombinase-mediated cassette exchange (RMCE) in Pv11 cells and generate master cell lines that retain anhydrobiosis following genetic engineering. We first evaluated the activity of multiple site-specific recombinases in Pv11 cells using a transient two-plasmid reporter assay. Flp, Cre, and Bxb1 recombinases all excised a test cassette and activated a green fluorescent protein (GFP) reporter, whereas phiC31 integrase mediated recombination at the DNA sequence level but did not induce reporter expression under our construct configuration. To enable genomic RMCE, we inserted an FRT/FRT3-landing pad (LP) into a previously identified genomic safe-harbor locus using CRIS-PITCh and isolated clonal master cell lines by single-cell sorting. Using the established master line, Flp-based RMCE achieved site-specific cassette exchange at the LP, producing HaloTag fluorescence and drug resistance upon successful exchange. In addition, the expected post-exchange sequence was confirmed by sequencing. We further established an all-in-one RMCE vector combining the Flp recombinase and donor cassette on a single plasmid. Together, these results demonstrate locus-defined, single-copy transgene integration in anhydrobiotic Pv11 cells via RMCE and provide a standardized, CRISPR-free workflow for routine genetic manipulation in this unique cell line. This workflow facilitates both fundamental research and applied biotechnological applications using desiccation-tolerant cells.

Anhydrobiosis↗
PubMed2006

Clinical news from the XVI International AIDS Conference: The attempt of a summing up.

Topics highlighted at the XVI International AIDS Conference in Toronto included HIV/AIDS vaccine research, entry inhibitors, integrase inhibitors, new NNRTIs and PIs, single-agent therapies, pre-exposure prophylaxis and microbicides. Beside the large scientific part, policy and funding were of great concern. Within this article we are trying to focus on topics with direct clinical relevance. This includes new epidemiological and resistance data, results from current studies investigating established and novel antiretroviral drugs and drug classes as well as new findings in therapy management and strategies.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Cell protein cross-linking by erbstatin and related compounds.

The protein-tyrosine kinase inhibitor and stable erbstatin analogue methyl 2,5-dihydroxycinnamate (4) cross-links cell proteins by a non-physiological chemical mechanism (Stanwell et al., Cancer Res 55: 4950-4956, 1995). To determine the structural requirements for this effect, erbstatin (1) and fifteen related compounds, including caffeic acid phenylethyl ester (9) were synthesized and examined for their ability to induce cross-linking of cellular protein at concentrations ranging from low micromolar up to 1000 microM. Tests were conducted in NIH3T3 fibroblasts as well as mouse keratinocytes. Potent cross-linking of cellular protein was observed for a number of analogues, including erbstatin, at concentrations as low as 10-50 microM. The inactivity of methoxy and fluoro as compared with their corresponding dihydroxylated counterparts indicated that free aromatic hydroxyls were essential for cross-linking. Additionally, compounds containing phenyl rings with 1,4-dihydroxy substituents were more potent than those having 1,2-dihydroxylated patterns. As with the prototype compound 4, cross-linking was induced at both 37 degrees and 4 degrees, suggesting a chemical rather than physiological mechanism. Consistent with the data, a mechanism of action is proposed which involves initial oxidation to reactive quinone intermediates that subsequently cross-link protein nucleophiles via multiple 1,4-Michael-type additions. Similar alkylation of protein by protein-tyrosine kinase inhibitors, such as herbimycin A, has been invoked. While the latter benzoquinoid ansamycin antibiotics contain performed quinone moieties, results of the present study suggest that other hydroxylated kinase inhibitors can potentially participate in similar phenomena. A large number of potential therapeutics, including HIV integrase inhibitors, possess polyhydroxylated nuclei. The non-specific nature of the protein cross-linking reaction demonstrated for these erbstatin analogues, and the fact that cross-linking can occur at micromolar concentrations, may limit the therapeutic usefulness of such compounds to specific applications.

Animals↗
PubMed1991

Mutating P2 and P1 residues at cleavage junctions in the HIV-1 pol polyprotein. Effects on hydrolysis by HIV-1 proteinase.

Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (-Leu*Phe-) and (b) between the p51 and RNase H domain (-Phe*Tyr-) within p66 of RT in the HIV-1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a beta-branched (Ile) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a beta-branched (Val) residue in the P2 position flanking such -Hydrophobic*Hydrophobic- junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.

Base Sequence↗
PubMed1987

Description of the topological entanglement of DNA catenanes and knots by a powerful method involving strand passage and recombination.

We utilize a recently discovered, powerful method to classify the topological state of knots and catenanes. In this method, each such form is associated with a unique polynomial. These polynomials allow a rigorous determination of whether knotted or catenated DNA molecules that appear distinct actually are, and indicate the structure of related molecules. A tabulation is given of the polynomials for all possible stereoisomers of many of the knotted and catenated forms that are found in DNA. The polynomials for a substrate DNA molecule and the products obtained from it by either recombination or strand passage by a topoisomerase are related by a simple theorem. This theorem affords natural applications of the polynomial method to these processes. Examples are presented involving site-specific recombination by the transposon Tn3-encoded resolvase and the phage lambda integrase, in which product structure is predicted as a function of crossover mechanism.

DNA↗
PubMed1990

Type I (R1) and type II (R2) ribosomal DNA insertions of Drosophila melanogaster are retrotransposable elements closely related to those of Bombyx mori.

Approximately 50% of the ribosomal DNA (rDNA) units of Drosophila melanogaster are inactivated by two different 28 S RNA ribosomal gene insertions (type I and type II). We present here the nucleotide sequence of complete type I and type II elements. Conceptual translation of these sequences revealed open reading frames (ORFs) encoding amino acid residues conserved in all retrotransposable elements. Full-length type I elements are 5.35 x 10(3) base-pairs in length and contain two overlapping ORFs. The smaller ORF (471 amino acid residues) has similarity to gag genes, while the larger ORF (1021 residues) has similarity to pol genes. Full-length type II elements are 3.6 x 10(3) base-pairs and contain one large ORF (1056 residues) that appears to represent a gag-pol fusion. Type I and type II elements are similar in structure, in the proteins they encode, and in insertion specificity to the R1Bm and R2Bm retrotransposable elements of Bombyx mori. We suggest that the D. melanogaster elements be called R1Dm and R2Dm, to reflect their structure as retrotransposons. Comparison of the R1 and R2 elements from these two widely different species revealed regions of the ORF that are likely to play an important role in the propagation of the elements. Four distinct regions of sequence conservation separated by regions of little or no sequence similarity were detected for both the R1 and R2 elements: (1) cysteine motifs of the gag gene, with three such motifs for R1 and one motif for R2; (2) a reverse transcriptase domain; (3) an integrase domain located carboxyl terminal to the reverse transcriptase region; and (4) a small region amino terminal to the reverse transcriptase domain, whose function is not known. The level of identity of the amino acid residues for these segments is 28 to 34% between the R1 elements, and 34 to 39% for the R2 elements. Finally, it may be predicted that the mechanism of unequal crossover might eventually eliminate R1 and R2 from the rDNA locus. The long history of selection at the protein level exhibited by these elements indicates that it is their active transposition that maintains them in the locus. The high level of sequence homogeneity between copies of each element within the same species is consistent with the high turnover rate expected to result from these processes.

Amino Acid Sequence↗
PubMed1991

Programmed translational frameshifting and initiation at an AUU codon in gene expression of bacterial insertion sequence IS911.

The proteins expressed by insertion sequence IS911, a member of the widespread IS3 family of elements, have been analyzed. The results indicate that three major species are produced from two consecutive reading frames. A protein of Mr 11,500, ORFA, is synthesized from an upstream reading frame. A larger protein, ORFAB, uses the same initiation codon and is produced by a -1 programmed translational frameshift between orfA and a downstream frame, orfB, whose amino acid sequence shows significant homology with retroviral integrase proteins. The orfB frame is also expressed independently in two alternative forms: the first uses a rare AUU initiation codon in the orfB phase whereas the second appears to initiate in the orfA phase and is produced by a -1 frameshift mechanism similar to that used in ORFAB expression. A specific IS911 integration reaction using a minimal active junction composed of 51 base-pairs of the right inverted repeat and a flanking phase lambda sequence resembling a second end in inverted orientation has been developed to analyze the functions of these proteins by transcomplementation in vivo. The orfA and orfB frames are shown to be essential and production of ORFAB is shown to stimulate integration in this system, suggesting that this fusion protein is the IS911 transposase.

Amino Acid Sequence↗
PubMed1992

Mutagenesis of a conserved region of the gene encoding the FLP recombinase of Saccharomyces cerevisiae. A role for arginine 191 in binding and ligation.

The FLP recombinase from the 2 microns plasmid of Saccharomyces cerevisiae contains a region from amino acid 185 to 203 that is conserved among several FLP-like proteins from different yeasts. Using site-directed mutagenesis, we have made mutations in this region of the FLP gene. Five of twelve mutations in the region yielded proteins that were unable to bind to the FLP recombination target (FRT) site. A change of arginine at position 191 to lysine resulted in a protein (FLP-R191K) that could bind to the FRT site but could not catalyze recombination. This mutant protein accumulated as a stable protein-DNA complex in which one of the two bound FLP proteins was covalently attached to the DNA. FLP-R191K was defective in strand exchange and ligation and was unable to promote protein-protein interaction with half-FRT sites. The conservation of three residues in all members of the integrase family of site-specific recombinases (His305, Arg308, Tyr343 in FLP) implies a common mechanism of recombination. The conservation of arginine 191 and the properties of the FLP-R191K mutant protein suggest that this arginine also plays an important role in the mechanism of FLP-mediated site-specific recombination.

Amino Acid Sequence↗
PubMed1988

Close evolutionary relatedness of the hepatitis B virus and murine leukemia virus polymerase gene sequences.

Previous work indicates that hepatitis B virus (HBV) and retroviruses utilize a unique mechanism for genome replication by reverse transcription of RNA and share homology in biologically important nucleotide and protein sequences. The data presented here extend previous findings of sequence homology among the genomes of the members of these virus families. HBV was found to possess sequences homologous to the retrovirus protease and reverse transcriptase gene sequences. Homology was not found to the retrovirus integrase sequence consistent with the observation that hepadnaviruses do not integrate into cellular DNA as a necessary step in their replication cycle. Overall, the homology of the hepadnavirus polymerase gene was strongest with that of the murine leukemia viruses (MLVs). Also, the hepadnavirus polymerase shares organizational similarities to the MLV polymerase sequence. Analysis suggests that the ancestor of both hepadnaviruses and retroviruses possessed an overlapping long open reading frame in the polymerase gene sequence. In addition, low stringency blot hybridization using hepadnavirus DNA probes indicates that HBV is more closely related to MLV sequences than the sequences of MLV-related viruses and endogenous retrovirus-like genetic elements. Taken together, the data indicate that the polymerase gene sequence of the hepadnavirus and MLV genomes are organized in a similar fashion which suggests that these viruses evolved from a common ancestor.

Amino Acid Sequence↗
PubMed1989

An insertion mutation in the pol gene of Moloney murine leukemia virus results in temperature-sensitive pol maturation and viral replication.

An insertion mutation in the pol gene of Moloney murine leukemia virus (M-MuLV) was found to render the virus temperature-sensitive for replication. A provirus containing a 12-bp insertion at the boundary between the reverse transcriptase (RT) and integrase (IN) domains induced the formation of mutant virions containing a partially processed RT-IN fusion protein. Some proteolytic processing to form mature RT and IN was observed at 32 degrees, but only aberrantly processed proteins were detected at 39 degrees. The uncleaved precursor was found to exhibit DNA polymerase activity, even though it could not support replication of the virus in vivo at 39 degrees.

Animals↗
PubMed1992

Isolation and characterization of a highly divergent HIV-2[GH-2]: generation of an infectious molecular clone and functional analysis of its rev-responsive element in response to primate retrovirus transactivators (Rev and Rex).

A highly divergent HIV-2 designated as HIV-2[GH-2] was obtained from an AIDS-related complex (ARC) patient in Ghana. A full-length molecular clone of this isolate was obtained and a biologically active clone was constructed. Its restriction pattern differed from that of prototype HIV-2[GH-1] in 25 of 35 restriction sites, but was strikingly similar to a previously characterized HIV-2 isolate from a Ghanaian (HIV-2ALT). The conserved integrase region (288-bp fragment) previously displayed 95% identity with that of ALT but 17-20% divergence from the HIV-2 prototype member, and a new distinct subgroup (HIV-2b) of HIV-2 consisting of GH-2 and ALT was postulated (Miura et al. 1991.) These isolates, however, were biologically distinguishable from each other by its replication capacity in a monocyte line, U937, in which GH-2 could not grow but ALT grew well. In addition, the nucleotide sequence of the LTR of this new isolate displays 21% divergence from that of prototype HIV-2[GH-1], but the core enhancer, Sp1 binding sites and TATA box were conserved. Although the 3' half of the env gene sequence which is deleted in HIV-2ALT clone showed 27% diversity from the prototype, functional differences in the rev-responsive element were not observed.

Cloning, Molecular↗
PubMed1995

V(D)J recombination of immunoglobulin genes.

We have constructed transgenic mice carrying an artificial substrate of V(D)J recombination. In this substrate, the only DNA fragments derived from Ig genes were short stretches of recombination signal sequences. This artificial substrate was rearranged at high frequency in lymphocytes, although in non-lymphoid cells no rearrangement was detected even by a sensitive PCR assay. This result indicates that the V(D)J recombination requires only the signal sequences and that a recombination similar to the V(D)J recombination does not occur in non-lymphoid tissues including the central nervous tissue. A protein binding to the V(D)J recombination signals was purified and its cDNA was cloned. This protein, termed RBP-J kappa, was initially considered to be involved in V(D)J recombination because of its DNA binding specificity and structural similarity to site-specific recombinases known as the integrase family. However, further study on the Drosophila homolog of RBP-J kappa indicated that RBP-J kappa probably functions as a transcription factor in the differentiation of the peripheral nervous tissues. The exact function of RBP-J kappa is still unknown. Analogous to the Drosophila gene, it is suggested that mouse RBP-J kappa participates in the regulation of differentiation of various tissues.

Amino Acid Sequence↗
PubMed1988

Functional expression of a sequence-specific endonuclease encoded by the retrotransposon R2Bm.

A fraction of the 28S ribosomal genes in certain insect species is interrupted by the insertion elements R1 and R2. These two elements from the silkworm Bombyx mori (R1Bm and R2Bm) are retrotransposons capable of transposing in a highly sequence-specific manner. We report here the functional expression in E. coli of the entire single open reading frame of R2Bm and show that it encodes a double-stranded endo-nuclease (integrase) that can specifically cleave the 28S gene at the R2 insertion site. The resulting cleavage is a 4 bp staggered 5' overhang. Deletion analysis of the 28S gene revealed that the DNA sequence required for specific cleavage is asymmetric with respect to the actual insertion (cleavage) site, with fewer than 10 bp required at one side and at least 24 bp at the other side of the site. A model is proposed based on these and previous data to account for the sequence-specific integration of the R2 retrotransposon.

Animals↗
PubMed1989

Site-specific recombination by Tn3 resolvase: topological changes in the forward and reverse reactions.

Site-specific recombination catalyzed by Tn3 resolvase proceeds with a linkage change, delta Lk, of +4 in the forward resolution reaction and -4 in the catenane fusion reverse reaction. The reverse reaction occurs only at low superhelical densities and gives unknotted circular products, consistent with plectonemic and not solenoidal wrapping of the two recombination sites. The strand exchange topologies are consistent with a mechanism in which resolvase cleaves all four DNA strands and religates them after a 180 degrees rotation of two duplex partners in a right-handed sense for the "forward" reaction, and in a left-handed sense for the "reverse" action. This could be achieved by a 180 degrees rotation of two resolvase subunits within a tetramer with D2 symmetry; we suggest that a different symmetry applies to phage lamda integrase catalysis.

Chloroquine↗
PubMed1989

Half-att site substrates reveal the homology independence and minimal protein requirements for productive synapsis in lambda excisive recombination.

The early events in site-specific excisive recombination were studied with phage lambda half-att sites that have no DNA to one side of the strand exchange region; they carry a single core-type integrase binding site and either P or P' arm flanking DNA. These half-attR and half-attL sites exhibit normal properties for the initial (covalent) top-strand transfer and form stable intermediates independent of later steps in the reaction. With these novel substrates we show that Xis specifically promotes the first strand exchange and that attL enhances Int cleavage at the top-strand site of attR. It is also shown that synapsis and initial strand transfers do not require DNA-DNA pairing but are mediated by protein-protein and protein-DNA interactions. These involve the two top-strand Int binding sites (required for the first strand exchange) and, in addition, one of the two bottom-strand sites (C') responsible for the second strand exchange.

Attachment Sites, Microbiological↗