Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mutations were introduced by recombinant DNA techniques into 9 genes of an infectious molecular clone of human immunodeficiency virus type 1. The 24 mutants generated were characterized biochemically and biologically by transfection and infection experiments. None of the mutants which have mutations in gag (p17, p24, and p15 regions), pol (protease, reverse transcriptase, and endonuclease domains), env (gp120 region), tat, or rev were infectious, whereas vif, vpr, vpu, some of env (gp41) and nef mutants could grow in human CD4+ cells to various degrees. Of the non-infectious mutants, only endonuclease (pol) and gp41 mutants exhibited normal phenotypes with respect to the production of functional reverse transcriptase, the expression of gag, pol, and env proteins, and the generation of progeny virions, when examined in transient assays. All infectious mutants killed the CD4+ cells with the exception of a mutant carrying a defect in the vif gene.
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PMID:Generation and characterization of the human immunodeficiency virus type 1 mutants. 170 90

Human endogenous retroviral element S71 had previously been shown to contain gag- and pol-related regions and a 3' LTR-like sequence. The nucleotide sequence of S71 was determined and compared with the corresponding regions of SSV and its helper virus SSAV. The 1.48-kb S71 gag region consists of matrix protein p15 (MA)-, capsid protein p30 (CA)-, and nucleocapsid protein p10 (NC)-related sections and the 1.82-kb pol region of tether, RNase H (RH), and endonuclease/integrase (IN) sections. The S71 nucleotide sequence contains a 167 amino acid open reading frame encompassing MA. The boundaries of the S71 element are delimited by direct repeats and the entire element is 5.4 kb long. Similarity between S71 and the v-sis-bearing, defective SSV provirus also covers overall structural organization, including the presence of presumably nonretroviral sequences. Both the gag and the pol regions of S71 contain sequences highly conserved in numerous retroviruses. Phylogenetic analysis with conserved CA, RH, and IN sequences showed that of all other (C-type) human retroviral elements available for comparison, S71 is most closely related to infectious primate and murine retroviruses. This suggests that S71 represents a phylogenetic subgroup of its own. In addition we identified short ranges of conserved amino acid sequences within C-type retroviral gag and pol genes sufficient for phylogenetic analysis. Use of these may facilitate large-scale phylogenetic evaluation of C-type retroviral elements and allow rapid classification of new elements.
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PMID:S71 is a phylogenetically distinct human endogenous retroviral element with structural and sequence homology to simian sarcoma virus (SSV). 215 93

Three pol gene products have been identified in avian retroviral particles: the full-length 95-kilodalton (kDa) beta chain of reverse transcriptase and two proteolytic cleavage products of beta, a 63-kDa reverse transcriptase alpha chain derived from the amino terminus of beta and a 32-kDa (pp32) endonuclease from its carboxy terminus. By using molecularly cloned retroviral DNA and synthetic oligonucleotides to introduce initiator ATGs and codons corresponding to the authentic N termini, we constructed two bacterial-expression clones; one clone contains the entire pol gene, and the other contains the region encoding the pp32 domain. A 99-kDa protein was synthesized in Escherichia coli by the full-length clone, and a 36-kDa protein was synthesized by the endonuclease domain clone. The recombinant proteins exceeded the size of both the mature viral beta chain and the pp32, respectively, by approximately 4 kDa. These larger sizes, however, are consistent with predictions from the DNA sequence of the pol gene. Processing of the recombinant pol proteins was examined by using p15 protease purified from virus particles and antisera directed against synthetic peptides corresponding to three domains in pol. Proteolytic digestion of the 99-kDa product with p15 produced a 63-kDa protein that comigrated on polyacrylamide gels with the alpha chain of reverse transciptase and a 36-kDa fragment that comigrated with the endonuclease domain product. Further digestion of the 36-kDa protein yielded a 32-kDa protein that comigrated with viral pp32 endonuclease. Thus, we concluded that two p15-sensitive sites exist in pol. Cleavage at the previously identified site produces alpha, and cleavage at the newly discovered site removes approximately 4 kDa from the C terminus of the primary protein product. Since the 36-kDa protein was also detected in protein isolated from virus particles, it seems probable that processing at the C-terminal site is a normal step in the production of mature beta and pp32 endonuclease products.
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PMID:Proteolytic processing of avian sarcoma and leukosis viruses pol-endo recombinant proteins reveals another pol gene domain. 243 65

We have purified from Moloney murine leukemia virus (Mo-MuLV) a protease that has the capacity of accurately cleaving the polyprotein precursor Pr65gag into the mature viral structural proteins. Both the NH2- and COOH-terminal amino acid sequences have been determined and aligned with the amino acid sequence deduced from the DNA sequence of Mo-MuLV by other workers. The results show that: (i) the protease is located at the 5' end of the pol gene, and the first four amino acids are overlapped with the 3' end of the gag gene; (ii) the fifth amino acid residue is glutamine, which is inserted by suppression of the UAG termination codon at the gag-pol junction; and (iii) the protease is composed of 125 amino acids with calculated Mr = 13,315, and the COOH terminus of the protease is adjacent to the NH2 terminus of reverse transcriptase. The map order of the gag-pol gene is proposed to be 5'-p15-p12-p30-p10-protease-reverse transcriptase-endonuclease-3'.
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PMID:Murine leukemia virus protease is encoded by the gag-pol gene and is synthesized through suppression of an amber termination codon. 388 15

The primary structure analysis of the gag gene products of human T-cell leukemia virus (HTLV)-ICR has been nearly completed. A comparison of the amino acid sequences with the published nucleotide sequence of HTLV-IATK established that i) p19 which is known to share antigenic determinants with a protein present in normal thymic epithelium, is nevertheless virally coded. ii) The gene order and complete primary structure of the gag precursor (Pr55) which has been shown to be myristylated (My) at its N-terminus is My-p19-p24-p15-OH; and iii) the Pr55gag amino acid sequences of HTLV-ICR and HTLV-IATK are nearly identical showing only a single residue difference in the C-terminal region of p15. Antibodies to synthetic peptides inferred from the nucleotide sequence of the env gene of HTLV-IATK were also raised and used to identify and purify env precursor gPr62-68, surface glycoprotein gp46-51 and transmembrane protein p21. While most of the peptide sera were shown to be subgroup specific some of them detected antigenic determinants shared between protein homologs of viruses of subgroups I and II. Partial or complete amino acid sequences of both the gag and env gene coded proteins of bovine leukemia virus (BLV) structural proteins have also been determined. These extensive protein data together with nucleotide sequences confirm and extend our initial finding that HTLV and BLV are structurally and antigenically related and may have originated from common ancestor. The structural and immunological studies revealed also relationships between HTLV and a number of type C and type D retroviruses studied. One of the highly conserved sequences is shared by the transmembrane proteins of these retroviruses which have been implicated in immunosuppression. It is conceivable that these common regions have common biological function. Two previously unidentified proteins of BLV have also been purified and structurally characterized. Nucleotide sequences capable of coding for related products are present in HTLV. The nature and possible biological functions of these new BLV proteins and the putative HTLV gene products will be discussed. The size and complexity of the genome of the replication competent retroviruses are similar but not identical. The 35S RNA of all replication competent helper viruses is divided into three genes encoding the viral structural proteins: the gag (group-specific antigen) gene codes for the internal structural proteins, the pol (polymerase) gene codes for the enzymes protease, reverse transcriptase and endonuclease and the env (envelope) gene codes for the proteins of the viral envelope.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structural and antigenic characterization of the proteins of human T-cell leukemia viruses and their relationships to the gene products of other retroviruses. 610 Jun 35

Four cloned rat cell lines (NX-1 to -4) infected with Moloney murine leukemia virus and defective in virus replication were found to be all different by viral protein and nucleic acid analyses. All four clones produced noninfectious particles and, except for NX-2, at about the same level as wild type. Compared with wild-type virions these defective particles contained larger amounts of gag precursor proteins and very little or no p30 or p15. Analysis of intracellular precursor proteins revealed that NX-2 to -4 synthesized normal Pr65gag, whereas NX-1 produced a slightly smaller precursor. Both NX-1 and NX-4 synthesized an intracellular polyprotein with a size similar to that of wild-type Pr180 gag-pol. Restriction endonuclease analysis of NX-1 to -4 cellular DNA showed that each clone contained a single integrated provirus which possessed large terminal repeat sequences at both the 5' and 3' ends. The proviruses of NX-1 to -3 appeared normal by restriction endonuclease analysis, but NX-4 provirus had a deletion of 1,700 base pairs comprising part of the polymerase region. The noninfectious particles produced by all four clones packaged Moloney viral RNAs and rat RNAs of two different sizes.
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PMID:Four Moloney murine leukemia virus-infected rat cell clones producing replication-defective particles: protein and nucleic acid analyses. 616 41

Preparations of purified Rauscher murine leukemia virus were found to contain an endodeoxyribonuclease after disruption of the virus with nonionic detergents. The enzyme makes single-strand breaks in linear or covalently closed circular phage double-stranded DNA molecules. The enzyme was partially purified by ion-exchange chromatography on DEAE- and carboxymethyl-Sepharose columns followed by electrophoresis in DNA-containing polyacrylamide gels. The enzyme was separated from reverse transcriptase (p80pol), and the final endonuclease preparation contained no detectable reverse transcriptase activity. The DEAE-Sepharose column-purified endonuclease activity contained a polypeptide of about 40,000 Mr that we term p40. Peptide mapping experiments demonstrated that p40 shares methionine-labeled tryptic peptides with Pr200gag-pol and Pr135pol. Six major methionine-labeled tryptic peptides derived from p40 were found in Pr200gag-pol, but only five of these were detected in Pr135pol. The four core proteins (p30, p15, pp12, and p10) and p80pol plus p40 account for most, but not all, of the peptide sequences of Pr200gag-pol. The endonuclease-associated p40 is similar in size and precursor origin to the avian retrovirus-coded endonuclease (p32). In view of these similarities to the avian p32 endonuclease and its association with partially purified Rauscher murine leukemia virus-associated endonuclease preparations, we propose that p40 is the Rauscher murine leukemia virus-coded endonuclease.
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PMID:Endodeoxyribonuclease activity associated with Rauscher murine leukemia virus. 626 Sep 82

Two commercially available expression vectors were modified to generate plasmids pGEXcPk and pQ9cPk. Proteins expressed from pGEXcPk and pQ9cPk had a short oligopeptide tag termed Pk at their carboxy termini and either glutathione S-transferase (GST) or a small histidine (His) tag, respectively, at their N termini. GST fusion proteins can be purified on immobilized glutathione and proteins coupled to the His tag selectively bind to Ni(2+)-NTA columns. The Pk tag is recognized by monoclonal antibody (MAb) SV5-P-k, previously produced in our laboratory. Thus proteins expressed from the pGEXcPk and pQ9cPk vectors can be purified in a two-step procedure, first via the N-terminal tag and second via the C-terminal tag. The combination of two affinity purification steps significantly improves the antigen purity and selects for full-size proteins. Moreover, by using the MAbSV5-P-k in the second purification step, Pk-linked antigens can be assembled directly into solid matrix-antibody-antigen (SMAA) complexes for use as vaccines. The genes for nef, endonuclease, p15, p17, p27, protease, Rev, reverse transcriptase (rt), tat, vif, vpr, and vpx of simian immunodeficiency virus (SIV mac 251) were cloned and expressed as both GST-SIV-Pk and His-SIV-Pk proteins. Multivalent SMAA complexes were made that contained His-p17-Pk, His-p27-Pk, His-rt-Pk, His-vpx-Pk, and His-vpr-Pk. Following two immunizations of mice with this mixture, antibodies could be detected to all five SIV antigens. When compared to single-protein immunizations, the immunogenicity of some of the proteins in this cocktail was either enhanced or decreased. Mice were also immunized with His-p17-Pk or His-p17-Pk-antibody complexes in the presence or absence of alum. The antibody-antigen complexes induced two- to four-fold higher antibody levels than antigen alone but did not appear to be more immunogenic in inducing lymphoproliferative responses. Sera from SIV-infected macaques were tested for the presence of antibodies reacting with the recombinant proteins by Western blot analysis. Antibodies to endonuclease, p15, p17, p27, rt, and vif were readily detected, antibodies against protease and vpx were present at much lower levels, but no antibodies were detected to nef, rev, tat, or vpr. Thus, we have developed a comprehensive range of reagents (available on request) that can be used to examine immune responses to SIV in both mice and monkeys.
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PMID:Expression and purification of nonglycosylated SIV proteins, and their use in induction and detection of SIV-specific immune responses. 807 30

Although human immunodeficiency virus (HIV) infection is progressive, the rate of decline in CD4+ lymphocyte counts varies. The role of immune system components in limiting HIV infection has yet to be defined, but a previous report on the U.S. Navy HIV Seropositive Cohort reported that strong reactivity in the anti-p55 (core precursor), p24 (core) and p53 (reverse transcriptase) Western blot bands was associated with higher CD4+ lymphocyte counts at the first clinical evaluation for HIV. The previous report examined the cross-sectional association between Western blot banding patterns and initial CD4+ lymphocyte counts. This report examines the association between these banding patterns in individuals who progressed rapidly as compared with patterns of patients who did not, based on their trends in repeated CD4+ lymphocyte counts as a marker of progression. Rapid and slower progressors were identified from a cohort of 3414 Navy and Marine Corps personnel who had a first positive HIV Western blot during 1986-1991. For purposes of this study, rapid progressors were defined as individuals whose CD4+ lymphocyte counts declined to < 500 cells/mm3 within 1 year of seroconversion. A total of 325 individuals met these criteria. A comparison group of 63 slower progressors also was identified; this group consisted of those whose CD4+ lymphocyte counts remained at > or = 500 cells/mm3 for a minimum of 5 years of follow-up after their first positive Western blot. Rapid progressors were slightly younger than slower progressors and were more likely to be never married but did not differ significantly from slower progressors in race or sex. Rapid progressors had weaker reactivity in the anti-p55 core precursor (P < 0.0001), p15 core (P < 0.01), gp41 transmembrane (P < 0.01) and p31 endonuclease (P < 0.05) bands on the Western blot. The odds ratio for rapid progressor status associated with weak or absent reactivity was 7.8 in the anti-p55 band and ranged from 2.0 to 3.2 in the anti-p31, p15, and gp41 bands. These associations remained significant after adjustment for age, race, and sex. The p55 association persisted in repeated Western blots during routine clinical evaluation during a period of 5 years after the first positive Western blot. It was concluded that several possible explanations may account for the weaker reactivity of rapid progressors: (i) weak anti-p55 reactivity might have been a marker of early immune system damage; (ii) high concentrations of p55 or related proteins in the serum may have bound the available anti-p55 antibodies in rapid progressors, making them difficult to identify on the Western blot; or (iii) lack of anti-p55, p15, gp41, or p31 reactivity might have allowed more rapid progression.
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PMID:Western blot banding patterns of HIV rapid progressors in the U.S. Navy Seropositive Cohort: implications for vaccine development. Navy Retroviral Working Group. 887 45

To explore the possibility of a new therapeutic strategy for leukemia by intervening in the DNA methylation to re-express p15 suppressor gene, methylation inhibitors, 5-Aza-2'-deoxycytidine (5-Aza-CdR) and cell differentiation agent (CDAII) were used to treat myelogenous leukemia cell line KG1a in which p15 gene expression was suppressed due to DNA hypermethylation. The biological characteristics of KG1a cells untreated or treated with the agents were investigated and analyzed using morphology, methylation specific-PCR (MSP), (3)H-labeled microassay technique, restriction endonuclease reaction, flow cytometry and immunofluorescence methods. The results indicated that both agents showed concentration-dependent and time-dependent inhibition of cell proliferation. 5-Aza-CdR and CDAII induced apoptosis and cell differentiation with G(2) and G(0)/G(1) arrest respectively. Furthermore, DNA methyltransferase activity and level of methylation in genomic DNA were decreased and p15 protein was re-expressed partially. It is concluded that it is possible to treat leukemia by intervening in the DNA methylation using methyltransferase inhibitors and it is worth to make a thorough study on mechanism of the new strategy.
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PMID:[Inhibitory effect on proliferation of KG1a cell line by methyltransferase inhibitors]. 1251 59


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