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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In avian leukosis virus, processing by the viral protease (PR) appears to activate reverse transcriptase (RT), since PR-defective virions have extremely feeble reverse transcriptase activity. We showed previously that when such detergent-treated virions are digested in vitro with PR, the Gag precursor is completely and properly matured, but the Gag-Pol precursor is not. In particular, the junction between Gag and Pol, i.e., between the PR and RT domains in Gag-Pol, remains refractory to cleavage, and reverse transcriptase is hardly activated. We have now investigated processing between Gag and Pol in greater detail, both in vitro and in vivo. In vivo, three mutations designed to destroy or alter the cleavage site at the N-terminus of RT failed to abrogate processing, suggesting that nearby cryptic cleavage sites can be used by PR, and thus that in virions this portion of Gag-Pol is in an extended conformation. By contrast, resistance to cleavage was observed in vitro in a series of N- and C-terminally truncated Gag-Pol substrates, produced by in vitro translation or in the baculovirus-insect cell system. This resistance was maintained even in short polypeptides, implying that the inability to be processed in vitro is a consequence of local conformation. In the previously described Gag mutant cs22, which is unable to undergo full activation of PR, we found that in vivo in quail cells the only cleavages made in the Gag-Pol polypeptide are at the NC-PR and the PR-RT junctions, suggesting that in wild-type avian leukosis virus, processing of Gag-Pol begins by cleavage immediately upstream and downstream of the PR domain. Taken together, these results suggest a model in which in immature virions the segment of polypeptide between PR and RT is held in an extended but inherently unstable conformation, and that in vivo the first cleavage in Gag-Pol must occur in this region. In the absence of virion structure this segment of polypeptide collapses into its most stable conformation, preventing cleavage. Based on amino acid sequence, we predict that this portion of Gag-Pol adopts a coiled coil conformation reminiscent of a leucine zipper.
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PMID:Proteolytic cleavage at the Gag-Pol junction in avian leukosis virus: differences in vitro and in vivo. 752 75

Among the 10(5) LINE-1 sequences (L1Hs) in the human genome are one or more 6-kb segments that are active retrotransposons. Expression of these retrotransposons appears to be favored in cells of germ line origin, as well as in some other tumor cells of epithelial origin. In such cells, the product of the first L1Hs open reading frame (ORF), a protein called p40, is detectable; p40 has no apparent similarity to gag proteins, but contains a leucine zipper region which may be responsible for the occurrence of p40 multimers. Transcription of L1Hs initiates at residue 1 although the transcriptional regulatory regions are downstream in the first 670 bp of the 5' untranslated region; deletion of a YY1-binding site in the first 20 bp reduces transcription by fivefold. Translation of the second ORF, which encodes reverse transcriptase, is independent of the translation of the frame encoding p40.
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PMID:LINE-1: a human transposable element. 827 57

We have identified a new member of the helix-loop-helix (H-L-H) and leucine zipper gene families via a reverse transcriptase-polymerase chain reaction based strategy. This new gene, CHUK (conserved helix-loop-helix ubiquitous kinase), may represent the founding member of a new class of interacting chimeric proteins. The nucleotide sequence of a near full-length murine CHUK cDNA clone revealed an encoded polypeptide specifying: a carboxyl-terminal H-L-H domain, an amino terminal serine-threonine kinase catalytic domain, and a leucine zipper-like amphipathic alpha-helix juxtaposed in between the H-L-H and kinase domains. CHUK is highly conserved in evolution and ubiquitously expressed in diverse types of established cell lines, whereas it is differentially expressed in normal murine tissues. The structural features of the CHUK polypeptide suggest that its putative kinase activity may be targetted to H-L-H and/or leucine zipper transcription factors. Alternatively, the dual amphipathic a helices may serve to control its intrinsic kinase activity by interactions with other cellular factors. CHUK may provide new insights into the regulated transmission of cytoplasmic signals to specific nuclear factors manifesting rapid alterations in patterns of cellular gene expression.
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PMID:CHUK, a new member of the helix-loop-helix and leucine zipper families of interacting proteins, contains a serine-threonine kinase catalytic domain. 877 33

The TGA genes encode a family of basic domain-leucine zipper (bZIP) transcription factors that are conserved in higher plants. We have continued to unravel the complexity of this gene family by using a polymerase chain reaction (PCR)-based approach. Taking advantage of the conserved amino acid sequence in the bZIP domain found in all members of this gene family, two degenerate oligonucleotides were synthesized based on the sequence of this region in order to amplify by PCR the analogous genomic fragments from the various TGA loci in Arabidopsis. This approach has led us to the finding of a new member of the TGA gene family, and subsequently the isolation of a gene designated as TGA6. Further characterization of the TGA6 locus confirmed our prediction that the gene structure of this family is remarkably conserved. Genomic Southern blot analysis revealed that TGA6 is a single-copy gene in Arabidopsis. Based on the genomic sequence information, gene-specific primers were synthesized for isolating the cDNA that corresponds to the coding region. Subsequently, the cDNA for TGA6 was cloned and sequenced. Gel mobility shift assays with in vitro translated TGA6 protein showed that TGA6 is more like TGA5 in terms of its in vitro DNA-binding properties. The expression of TGA6 in different tissues was estimated by using reverse transcriptase (RT)-PCR and further analyzed in transgenic Arabidopsis lines expressing a TGA6 promoter-GUS fusion. TGA6 promoter activity is found primarily in roots of young seedlings. As seedlings develop, TGA6 is expressed in aging cotyledons, mesophyll cells of hydathodes on leaf margins, vascular tissue and trichomes of senescing rosette leaves, but is very low in primary roots of mature plants. High levels of expression persist in young lateral roots and in regions of the primary root where lateral roots are emerging. In flowers, the activity is localized predominantly to mature pollen grains. The expression pattern of TGA6 reported here is strikingly similar to that of an Arabidopsis acidic chitinase gene. Possible biological significance of TGA6 in cellular defense against pathogens and abiotic stress is discussed.
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PMID:DNA-binding properties, genomic organization and expression pattern of TGA6, a new member of the TGA family of bZIP transcription factors in Arabidopsis thaliana. 922 52

To identify light-regulated genes in Arabidopsis thaliana (L.) Heynh. a clone was isolated which contains a cDNA fragment with sequence similarity to receptor-like protein kinases (RLKs). Sequence analysis of the corresponding genomic DNA as well as determination of transcribed regions revealed that the gene comprises 12 exons. Sections of the deduced polypeptide exhibit homologies with kinase domains and the entire protein possesses structural features indicating that it is a novel member of the RLK family. The protein consists of a signal peptide, a putative receptor site including a leucine zipper region with a new motif, a transmembrane helix and 11 subdomains characteristic of serine/threonine kinases. The gene is designated light-repressible receptor protein kinase (lrrpk), as the specific mRNA is predominantly expressed in the absence of light. The lrrpk mRNA steady-state levels were assessed by competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and found to be very low after light pulses, irrespective of the wavelength applied. Blue light was least effective in this respect, and the repression was not reversible by far-red light. Employment of in-situ RT-PCR revealed elevated lrrpk mRNA levels in the cotyledons of etiolated seedlings. The mRNA was also increased in the outer regions of the roots of greenhouse-grown A. thaliana, but was not detectable in any other part of the plants. An explanation of the relatively low lrrpk mRNA levels and the photophobic expression of the gene could be the finding that in the 5' upstream region of the lrrpk gene sequence elements are present that are similar to those identified in promoters of phytochrome A genes.
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PMID:Light-repressible receptor protein kinase: a novel photo-regulated gene from Arabidopsis thaliana. 926 89

The blade and sheath of a maize leaf are separated by a linear epidermal fringe, the ligule, and two wedge-like structures, the auricles. In plants homozygous for the null mutation, liguleless2-reference (lg2-R), the ligule and auricles are often absent or positioned incorrectly and the blade-sheath boundary is diffuse. This phenotype is in contrast to that of liguleless1-reference (lgl-R) mutant plants, which have a more defined boundary even in the absence of the ligule and auricles. Additionally, mosaic analysis indicates the lg2-R phenotype is cell-nonautonomous and the lg1-R phenotype is cell-autonomous. Using scanning electron microscopy we show that lg2-R mutant plants are affected before the first visible sign of ligule and auricle formation. We have cloned the Lg2+ gene through a Mutator-8 transposon insertion allele, and verified it with five independently derived alleles. The comparison of genomic DNA and cDNA sequences reveals an open reading frame encoding a protein of 531 amino acids with partial homology to a subclass of plant basic leucine zipper (bZIP) transcription factors. Although a large body of molecular and biochemical characterization exists on this subclass of bZIP proteins, our work represents the first report of a mutant phenotype within this group. A specific reverse transcriptase (RT)-PCR assay shows LG2 mRNA expression in meristem/developing ligule regions. RT-PCR also shows that LG2 mRNA accumulation precedes that of LG1 mRNA. The mutant phenotype and expression analysis of lg2 suggest an early role in initiating an exact blade-sheath boundary within the young leaf primordia.
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PMID:The maize gene liguleless2 encodes a basic leucine zipper protein involved in the establishment of the leaf blade-sheath boundary. 949 Feb 65

The molecular cloning of the t(5;10)(q33;q22) associated with atypical chronic myeloid leukemia (CML) is reported. Fluorescence in situ hybridization (FISH), Southern blot, and reverse transcriptase- polymerase chain reaction analysis demonstrated that the translocation resulted in an H4/platelet-derived growth factor receptor betaR (PDGFbetaR) fusion transcript that incorporated 5' sequences from H4 fused in frame to 3' PDGFbetaR sequences encoding the transmembrane, WW-like, and tyrosine kinase domains. FISH combined with immunophenotype analysis showed that t(5;10)(q33;q22) was present in CD13(+) and CD14(+) cells but was not observed in CD3(+) or CD19(+) cells. H4 has previously been implicated in pathogenesis of papillary thyroid carcinoma as a fusion partner of RET. The H4/RET fusion incorporates 101 amino acids of H4, predicted to encode a leucine zipper dimerization domain, whereas the H4/PDGFbetaR fusion incorporated an additional 267 amino acids of H4. Retroviral transduction of H4/PDGFbetaR, but not a kinase-inactive mutant, conferred factor-independent growth to Ba/F3 cells and caused a T-cell lymphoblastic lymphoma in a murine bone marrow transplantation assay of transformation. Mutational analysis showed that the amino-terminal H4 leucine zipper domain (amino acids 55-93), as well as H4 amino acids 101 to 386, was required for efficient induction of factor-independent growth of Ba/F3 cells. Tryptophan-to-alanine substitutions in the PDGFbetaR WW-like domain at positions 566/593, or tyrosine-to-phenylalanine substitutions at PDGFbetaR positions 579/581 impaired factor-independent growth of Ba/F3 cells. H4/PDGFbetaR is an oncoprotein expressed in t(5;10)(q33;q22) atypical CML and requires dimerization motifs in the H4 moiety, as well as residues implicated in signal transduction by PDGFbetaR, for efficient induction of factor-independent growth of Ba/F3 cells. (Blood. 2001;97:3910-3918)
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PMID:H4(D10S170), a gene frequently rearranged in papillary thyroid carcinoma, is fused to the platelet-derived growth factor receptor beta gene in atypical chronic myeloid leukemia with t(5;10)(q33;q22). 1138 34

The t(10;11)(p13;q14-21) is a non-random translocation described in acute lymphoblastic and myeloid leukaemias. It results in the fusion of the gene CALM, which encodes a clathrin assembly protein, on 11q14 to the gene AF10, a putative transcription factor on 10p13. Here we describe for the first time, the occurrence of a CALM-AF10 fusion in a case of acute megakaryoblastic leukaemia. Fluorescence in situ hybridisation and reverse transcriptase polymerase chain reaction were used to confirm the presence of a CALM-AF10 fusion. A novel splice variant of CALM missing nt 1927-2091 was also detected. Though CALM is a cytoplasmic protein, the chimaeric fusion product is able to localise to both the nucleus and cytoplasm. Analysis of the fusion variants suggests, however, that the critical fusion product is likely to be cytoplasmic and contain the interactive leucine zipper of AF10.
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PMID:Identification and molecular characterisation of a CALM-AF10 fusion in acute megakaryoblastic leukaemia. 1141 76

We have previously isolated and characterized a novel human gene HUEL (C4orf1) that is ubiquitously expressed in a wide range of human fetal, adult tissues and cancer cell lines. HUEL maps to region 4p12-p13 within the short arm of chromosome 4 whose deletion is frequently associated with bladder and other carcinomas. Here we present the genomic organization, sizes and boundaries of exons and introns of HUEL. The GC-rich upstream genomic region and 5' untranslated region (UTR) together constitute a CpG island, a hallmark of housekeeping genes. The 3250 bp HUEL cDNA incorporates a 1704 bp ORF that translates into a hydrophilic protein of 568-amino acids (aa), detected as a band of approximately 70 kDa by Western blotting. We have isolated the murine homolog of HUEL which exhibits 89% nucleotide and 94% amino acid identity to its human counterpart. The HUEL protein shares significant homology with the minimal DNA-binding domain (DNA-BD) of the DNA repair protein encoded by the xeroderma pigmentosum group A (XPA) gene. Other notable features within HUEL include the putative nuclear receptor interaction motif, nuclear localization and export signals, zinc finger, leucine zipper and acidic domains. Mimosine-mediated cell cycle synchronization of PLC/PRF/5 liver cancer cells clearly portrayed translocation of HUEL into the nucleus specifically during the S phase of the cell cycle. Yeast two-hybrid experiments revealed interactions of HUEL with two partner proteins (designated HIPC and HIPB) bearing similarity to a mitotically phosphorylated protein and to reverse transcriptase. Co-immunoprecipitation assays validated the interaction between HUEL and HIPC proteins in mammalian cells. HUEL is likely to be an evolutionarily conserved, housekeeping gene that plays a role intimately linked with cellular replication, DNA synthesis and/or transcriptional regulation.
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PMID:The novel human HUEL (C4orf1) protein shares homology with the DNA-binding domain of the XPA DNA repair protein and displays nuclear translocation in a cell cycle-dependent manner. 1190 20

By using particle-associated reverse transcriptase (RT) activity as an assay for Pol incorporation into human immunodeficiency virus type 1 (HIV-1) Gag virus-like particles (VLPs), it has been found that truncated, protease-negative, Gag-Pol missing cis Gag sequences is still incorporated into Gag VLPs, albeit at significantly reduced levels (10 to 20% of the level of wild-type Gag-Pol). In this work, we have directly measured the incorporation of truncated Gag-Pol species into Gag VLPs and have found that truncated Gag-Pol that is missing all sequences upstream of RT is still incorporated into Gag VLPs at levels approximating 70% of that achieved by wild-type Gag-Pol. Neither protease nor integrase regions in Pol are required for its incorporation, implying an interaction between Gag and RT sequences in the Pol protein. While the incorporation of Gag-Pol into Gag VLPs is reduced 12-fold by the replacement of the nucleocapsid within Gag with a leucine zipper motif, this mutation does not affect Pol incorporation. However, the deletion of p6 in Gag reduces Pol incorporation into Gag VLPs four- to fivefold. Pol shows the same ability as Gag-Pol to selectively package tRNA(Lys) into Gag VLPs, and primer tRNA(3)(Lys) is found annealed to the viral genomic RNA. These data suggest that after the initial separation of Gag from Pol during cleavage of Gag-Pol by viral protease, the Pol species still retains the capacity to bind to both Gag and tRNA(3)(Lys), which may be required for Pol and tRNA(3)(Lys) to be retained in the assembling virion until budding is completed.
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PMID:Incorporation of pol into human immunodeficiency virus type 1 Gag virus-like particles occurs independently of the upstream Gag domain in Gag-pol. 1469 38


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