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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The FLP recombinase of the 2 microns plasmid of Saccharomyces cerevisiae is a member of the integrase family of site-specific recombinases. Recombination catalyzed by members of this family proceeds via the ordered cleavage and religation of four strands of DNA. Although the amino acid sequences of integrase family members are quite different, each recombinase maintains an absolutely conserved tetrad of amino acids (R-191, H-305, R-308, Y-343; numbers are those of the FLP protein). This tetrad is presumed to reflect a common chemical mechanism for cleavage and ligation that has evolved among all family members. The tyrosine is the nucleophile that causes phosphodiester bond cleavage and covalently attaches to the 3'-PO4 terminus, whereas the other three residues have been implicated in ligation of strands. It has recently been shown that cleavage by FLP takes place in trans; that is, a FLP molecule binds adjacent to the site of cleavage but receives the nucleophilic tyrosine from a molecule of FLP that is bound to another FLP-binding element (J.-W. Chen, J. Lee, and M. Jayaram, Cell 69:647-658, 1992). These studies led us to examine whether the ligation step of the FLP reaction is performed by the FLP molecule bound adjacent to the cleavage site (ligation in cis). We have found that FLP promotes ligation in cis. Furthermore, using in vitro complementation analysis, we have classified several mutant FLP proteins into one of two groups: those proteins that are cleavage competent but ligation deficient (group I) and those that are ligation competent but cleavage defective (group II). This observation suggests that the active site of FLP is composed of several amino acid residues from each of two FLP molecules.
Mol Cell Biol 1993 Jun
PMID:Mechanism of cleavage and ligation by FLP recombinase: classification of mutations in FLP protein by in vitro complementation analysis. 849 47

The retrotransposon Tf1, isolated from Schizosaccharomyces pombe, contains a single open reading frame with sequences encoding Gag, protease, reverse transcriptase, and integrase (IN). Tf1 has previously been shown to possess significant transposition activity. Although Tf1 proteins do assemble into virus-like particles, the assembly does not require readthrough of a translational reading frame shift or stop codon, common mechanisms used by retroelements to express Gag in molar excess of the polymerase proteins. This study was designed to determine if Tf1 particles contain equal amounts of Gag and polymerase proteins or whether they contain the typical molar excess of Gag. After using two separate methods to calibrate the strength of our antibodies, we found that both S. pombe extracts and partially purified Tf1 particles contained a 26-fold molar excess of Gag relative to IN. Knowing that Gag and IN are derived from the same Tf1 primary translation product, we concluded that the excess Gag most likely resulted from specific degradation of IN. We obtained evidence of regulated IN degradation in comparisons of Tf1 protein extracted from log-phase cells and that extracted from stationary-phase cells. The log-phase cells contained equal molar amounts of Gag and IN, whereas cells approaching stationary phase rapidly degraded IN, leaving an excess of Gag. Analysis of the reverse transcripts indicated that the bulk of reverse transcription occurred within the particles that possess a molar excess of Gag.
Mol Cell Biol 1996 Jan
PMID:The retrotransposon Tf1 assembles virus-like particles that contain excess Gag relative to integrase because of a regulated degradation process. 852 13

A mammalian expression vector designed for production of HIV-1 integrase was found to enhance the stability of a linear reporter plasmid in COS-7 cells. The effect is strictly dependent on coexpression of the HIV-1 rev gene and on the inclusion of U3 and U5 portions of the HIV-1 LTR in the reporter plasmid. Integrase point mutations P109S and D116N drastically reduced stabilization whereas T115A and D64A had little or no effect. Immunoblot analysis revealed the presence of a 32-34kDa integrase protein in extracts of transfected COS-7 cells and of wild type and mutant integrase proteins at comparable levels. We conclude that integrase acts in trans in COS-7 cells, possibly by binding to the HIV-1 LTR in the plasmid. This transfection system may be useful for studying factors that stabilize the HIV-1 DNA genome prior to its integration into the host cell chromosome.
Biochem Mol Biol Int 1995 Jul
PMID:Human immunodeficiency virus type 1 integrase stabilizes a linearized HIV-1 LTR plasmid in vivo. 852 37

A retrotransposon from the fungal plant pathogen Fusarium oxysporum f. sp. lycopersici has been isolated and characterized. The element, designated skippy (skp) is 7846 bp in length, flanked by identical long terminal repeats (LTR) of 429 bp showing structural features characteristic of retroviral and retrotransposon LTRs. Target-site duplications of 5 bp were found. Two long overlapping open reading frames (ORF) were identified. The first ORF, 2562 bp in length, shows homology to retroviral gag genes. The second ORF, 3888 bp in length, has homology to the protease, reverse transcriptase. RNase H and integrase domains of retroelement pol genes in that order. Sequence comparisons and the order of the predicted proteins from skippy indicate that the element is closely related to the gypsy family of LTR-retrotransposons. The element is present in similar copy numbers in the two races investigated, although RFLP analysis showed differences in banding patterns. The number of LTR sequences present in the genome is higher than the number of copies of complete elements, indicating excision by homologous recombination between LTR sequences.
Mol Gen Genet 1995 Dec 20
PMID:Skippy, a retrotransposon from the fungal plant pathogen Fusarium oxysporum. 854 29

SIVsm chronically infected cultures were obtained after infection of CEMX174 cells with either SIVsmH3 or SIVsmE660. These phenotypically CD4 cells, formed syncytia but only when cocultivated with CD4+ cells. Single cell clones were derived from these cultures and examined for the production of virus-specific proteins. The majority of the clones expressed SIV p27 antigen and low levels of virus reverse transcriptase activity. Western blot analysis, performed with either monoclonal or polyclonal sera, showed that a chronically infected clone (B7) produced particles which contained envelope (gp135 and gp43), gag precursors and gag proteins (p27, p16 and p8). However, these particles (SIVsmB7) lacked detectable levels of vpx and of integrase, and contained several fusion proteins which expressed viral protease antigens. This defective virus failed to infect established CD4+ cell lines, as well as primary cultures of macrophages and of peripheral blood lymphocytes, obtained both from humans and from rhesus macaques. Lack of infection correlated with lack of viral DNA detection by PCR amplification of genomic DNA extracted from these cell cultures. In addition, SIVsmB7 virus lacked infectivity in vivo. Rhesus macaques inoculated with high concentrations of SIVsmB7 showed no viremia and their PBMC were PCR negative. Thus, B7 cells produced stable, non-infectious virus mutants, which contained env and gag proteins, but lacked detectable amounts of vpx and of enzymes required for virus replication. Due to the high constitutive expression of this virus-like particle, we are now testing this preparation as a vaccine.
Cell Mol Biol (Noisy-le-grand) 1995
PMID:Properties of virus-like particles produced by SIV-chronically infected human cell clones. 857 47

We extended our previous study with 3'-azido-3'-deoxythymidine nucleotides [Proc. Natl. Acad. Sci. USA 91:5771-5775 (1994)] and examined the effects on human immunodeficiency virus type 1 (HIV-1) integrase of the nucleotides of three nucleoside analogues currently under evaluation in clinical trials: beta-D-2',3'-didehydro-3'-deoxythymidine, beta-D-2'-ara-fluoro-2', 3'-dideoxyadenosine, and beta-L-2',3'-dideoxy-3'-thiacytidine. Beta-D-2',3'-Didehydro-3'-deoxythymidine and beta-D-2'-ara-fluoro-2',3'-dideoxyadenosine nucleotides had IC50 values for strand transfer of 100 and 200 microM, respectively, whereas the corresponding 2',3'-dideoxynucleoside triphosphates, ddT triphosphate and ddA triphosphate, did not inhibit the integrase at 800 and 200 microM, respectively. Beta-L-2',3'-Dideoxy-3'-thiacytidine triphosphate had no effect up to 500 microM. The L-enantiomers of 5-fluoro-2',3'-dideoxycytidine monophosphate and triphosphate had IC50 values of approximately 40 microM, whereas their D-enantiomer isomers showed no inhibition at 200 microM. NAD, pyridoxal phosphate, and coumermycin A1, which exhibit no antiviral activity but are typically used to probe nucleotide binding sites, were also tested. NAD was inactive, and its etheno derivative exhibited activity at 1 mM. In contrast, pyridoxal phosphate (IC50 = 18 microM and coumermycin A1 (IC50 = 5 microM were potent inhibitors. None of the coumermycin monomeric derivatives were active integrase inhibitors. The physiological ribonucleotides ATP and GTP inhibited HIV-1 integrase at or near cellular concentrations, suggesting that they may regulate HIV-1 integrase activity in cells. In general, the active nucleotides tested inhibited binding of HIV-1 integrase to its substrate DNA an inhibited an integrase deletion mutant containing only amino acids 50-212, indicating that nucleotides bind to the enzyme catalytic core. Consisently, the choice of nucleophile in the 3'-processing reaction was blocked to the same extent regardless of the nucleotide used (water, glycerol, or the viral DNA hydroxyl) by the enzyme. These observations suggest new strategies for antiviral drug development that could be based on nucleotide analogues as inhibitors of HIV-1 integrase.
Mol Pharmacol 1996 Apr
PMID:Effects of nucleotide analogues on human immunodeficiency virus type 1 integrase. 860 89

We have identified a family of repetitive sequences in the genome of Nicotiana alata named Tna1 (Transposon of N. alata). The first element we characterised was a genomic clone for the N. alata s6-ribonuclease (S6-RNase), a gene required for self-incompatibility in this species. The DNA sequence of this element resembles the integrase domain of retrotransposons of the gypsy class and is most similar to a retrotransposon from Lilium henryi. A transcript present in N.alata styles (self-incompatibility genotype S6S6) hybridized to Tna1 and accumulated in the style following either pollination or touching. This transcript was cloned from a cDNA library and was encoded by second, partial Tna1 elements. Neither the transcribed sequence nor the original Tna1 element contain an open reading frame or is likely to be able to transpose. The second element was mapped using a population of N.alata plants segregating for alleles of the self-incompatibility locus and is closely linked to the S6-allele. The Tna1 element is present in a number of Nicotiana species and appears to have been active at least twice during the evolution of this genus.
Mol Gen Genet 1996 Feb 05
PMID:A retrotransposon-like sequence linked to the S-locus of Nicotiana alata is expressed in styles in response to touch. 862 17

The integrase domain of RNA-mediated elements (class I) and the transposase domain of DNA-mediated transposable elements (class II) were compared. A number of elements contain the DDE signature, which plays an important role in their integration. The possible relationships between mariner-Tc1 and IS elements, retrotransposons, and retroviruses were analyzed from an alignment of this region. The mariner-Tc1 superfamily, and LTR retrotransposons and retroviruses were found to be monophyletic groups. However, the IS elements of bacteria were found in several groups. These results were used to propose an evolutionary history that suggests a common ancestor for some integrases and transposases.
J Mol Evol 1996 Mar
PMID:Relationships between transposable elements based upon the integrase-transposase domains: is there a common ancestor? 866 97

The barley BARE-1 is a transcribed, copia-like retroelement with well-conserved functional domains, an active promoter, and a copy number of at least 3 x 10(4). We examined its chromosomal localization by in situ hybridization. The long terminal repeat (LTR) probe displayed a uniform hybridization pattern over the whole of all chromosomes, excepting paracentromeric regions, telomeres, and nucleolar organizer (NOR) regions. The integrase probe showed a similar pattern. The 5'-untranslated leader (UTL) probe, expected to be the most rapidly evolving component, labeled chromosomes in a dispersed and non-uniform manner, concentrated in the distal regions, possibly indicating a targe site preference.
Plant Mol Biol 1996 Mar
PMID:Retrotransposon BARE-1 is a major, dispersed component of the barley (Hordeum vulgare L.) genome. 870 40

A virulence-associated region in the genome of Dichelobacter nodosus has been shown to contain an integrase gene which is highly related to the integrases of Shigella flexneri phage Sf6 and coliphages P4 and phi R73, together with open reading frames (vapB, C and D) related to genes borne on plasmids in Neisseria gonorrhoeae, Escherichia coli, Actinobacillus actinomycetemcomitans and Treponema denticola. Similar to P4 and phi R73, the vap region is bracketed by putative bacteriophage att sites and is adjacent to a tRNA gene, which suggests that the vap region has been derived by the integration of a bacteriophage, or a plasmid carrying a bacteriophage-related integrase gene. Many similarities in genes and genes clusters encoding virulence determinants have been found in distantly related bacteria. These genes are often located on plasmids in one organism but on the chromosome in others, implying that transmission of the genes has been followed by integration. Thus, the events which have generated the vap regions of D. nodosus may represent a common mechanism for transfer of virulence determinants. A number of genes involved in the virulence of bacterial pathogens are found on integrated bacteriophages, and we suggest that others will prove to be associated with tRNA genes and/or integrase genes derived from bacteriophages. The use of tRNA genes as integration sites for many bacteriophages and plasmids may favour intergeneric transmission, as tRNA genes are highly conserved.
Mol Microbiol 1995 Oct
PMID:A role for bacteriophages in the evolution and transfer of bacterial virulence determinants. 870 40


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