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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used the conservation of reverse transcriptase and integrase domains among retroelements to PCR-amplify three well-known types of these mobile genetic elements. Reverse transcriptase sequences from Ty1-copia were identified in spruce in this way, as well as integrase sequences from the Ty3-gypsy group. Using these sequences as probes against a Picea glauca genomic bank, individual members from the LTR (long terminal direct repeat) groups were obtained. A partial Ty1-copia-type element named Spcl was isolated along with a Ty3-gypsy-type element named Spdl. Genomic Southern hybridizations revealed the complexity and high copy number of LTR retrotransposons in black and white spruce.
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PMID:Different classes of retrotransposons in coniferous spruce species. 1119 42

A novel Ty3/Gypsy retrotransposon, named Pyret, was identified in the plant pathogenic fungus Magnaporthe grisea (anamorph Pyricularia oryzae). Pyret-related elements were distributed in a wide range of Pyricularia isolates from various gramineous plants. The Pyret element is 7250 bp in length with a 475 bp LTR and one conceptual ORF. The ORF contains seven nonsense mutations in the reading frame, indicating that the Pyret clone is lightly degenerate. Comparative domain analysis among retroelements revealed that Pyret exhibits an extra domain (WCCH domain) beyond the basic components of LTR retrotransposons. The WCCH domain consists of approximately 300 amino acids and is located downstream of the nucleocapsid domain. The WCCH domain is so named because it contains two repeats of a characteristic amino acid sequence, W-X(2)-C-X(4)-C-X(2)-H-X(3)-K. A WCCH motif-like sequence is found in the precoat protein of some geminiviruses, viral RNA-dependent RNA polymerase and also in an Arabidopsis protein of unknown function. Interestingly, detailed sequence analysis of the gag protein revealed that Pyret, as well as some other chromodomain-containing LTR retrotransposons, displays significant sequence homology with members of the gammaretroviruses (MLV-related retroviruses) in the capsid and nucleocapsid domains. This suggests that chromodomain-containing LTR retrotransposons and gammaretroviruses may share a common ancestor with the gag protein.
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PMID:Pyret, a Ty3/Gypsy retrotransposon in Magnaporthe grisea contains an extra domain between the nucleocapsid and protease domains. 1160 Jun 99

The hepatitis C virus nonstructural 5B protein (NS5B) protein has been shown to require either magnesium or manganese for its RNA-dependent RNA polymerase activity. As a first step toward elucidating the nature and the role(s) of the metal ions in the reaction chemistry, we have utilized endogenous tryptophan fluorescence to quantitate the interactions of magnesium and manganese ions with this protein. The association of either Mg(2+) or Mn(2+) ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was used to determine the apparent dissociation constants for both ions. The apparent K(d) values for the binding of Mg(2+) and Mn(2+) ions to the free enzyme were 3.1 and 0.3 mm, respectively. Dual ligand titration experiments demonstrated that both ions bind to a single common site, for which they compete. The kinetics of real time metal ion binding to the NS5B protein were also investigated. Based on the results of our fluorescence and near-UV circular dichroism experiments, we show that NS5B undergoes conformational changes upon the binding of metal ions. However, this process does not significantly stimulate the binding to the RNA or NTP substrates. We envisage that the ion-induced conformational change is a prerequisite for catalytic activity by both correctly positioning the side chains of the residues located in the active site of the enzyme and also contributing to the stabilization of the intermediate transition state.
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PMID:Characterization of the metal ion binding properties of the hepatitis C virus RNA polymerase. 1245 24

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan catabolic enzyme that is widely distributed in various tissues. In peripheral blood mononuclear cells (PBMCs), production of IDO by macrophages or dendritic cells has been reported to inhibit T-cell activation and proliferation. In the present study, we have determined that other phenotypes of PBMCs also express IDO. In cultures of PBMCs, IDO was found predominantly in monocyte by immunohistochemistry. Reverse transcriptase polymerase chain reaction analysis showed that IDO mRNA was expressed in T lymphocytes, B lymphocytes and natural killer (NK) cells and that expression was increased upon activation with interferon-gamma. The cytotoxicity of NK cells against K562 and HepG2 cells was reduced by IDO inhibitor. These results suggest that IDO in NK cells is essential for NK cells to generate killing activity against cancer cells.
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PMID:Indoleamine 2,3-dioxygenase is necessary for cytolytic activity of natural killer cells. 1487 Dec 94

Improved methods of bone regeneration are needed in the craniofacial rehabilitation of patients with significant bone deficits secondary to tumor resection, congenital deformities, and prior to prosthetic dental reconstruction. In this study, a gene-enhanced tissue-engineering approach was used to assess bone regenerative capacity of Sonic hedgehog (Shh)-transduced gingival fibroblasts, mesenchymal stem cells, and fat-derived cells delivered to rabbit cranial bone defects in an alginate/collagen matrix. Human Shh cDNA isolated from fetal lung tissue was cloned into the replication-incompetent retroviral expression vector LNCX, in which the murine leukemia virus retroviral LTR drives expression of the neomycin-resistance gene. The rat beta-actin enhancer/promoter complex was engineered to drive expression of Shh. Reverse transcriptase-polymerase chain reaction analysis demonstrated that the transduced primary rabbit cell populations expressed Shh RNA. Shh protein secretion was confirmed by enzyme-linked immunosorbent assay (ELISA). Alginate/ type I collagen constructs containing 2 x 10(6) Shh-transduced cells were introduced into male New Zealand White rabbit calvarial defects (8 mm). A total of eight groups (N=6) were examined: unrestored empty defects, matrix alone, matrix plus the three cell populations transduced with both control and Shh expression vectors. The bone regenerative capacity of Shh gene enhanced cells was assessed grossly, radiographically and histologically at 6 and 12 weeks postimplantation. After 6 weeks, new full thickness bone was seen emanating directly from the alginate/collagen matrix in the Shh-transduced groups. Quantitative two-dimensional digital analysis of histological sections confirmed statistically significant (P<0.05) amounts of bone regeneration in all three Shh-enhanced groups compared to controls. Necropsy failed to demonstrate any evidence of treatment-related side effects. This is the first study to demonstrate that Shh delivery to bone defects, in this case through a novel gene-enhanced tissue-engineering approach, results in significant bone regeneration. This encourages further development of the Shh gene-enhanced tissue-engineering approach for bone regeneration.
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PMID:Sonic hedgehog gene-enhanced tissue engineering for bone regeneration. 1551 Jan 77

A double-stranded (ds) RNA (2411 bp) from a strain V18 of the violet root rot basidiomycetous fungus, Helicobasidium mompa was sequenced. Using the fungal mitochondrial genetic code in which UGA codes for tryptophan, the positive strand of V18 dsRNA was found to contain a long open-reading frame with the potential to encode a protein of 700 amino acids (molecular mass 79,805 Da), including conserved motifs characteristic of RNA-dependent RNA polymerase (RDRP). This putative RDRP was shown to be related to putative RDRPs of several fungal mitochondrial viruses. It is proposed that V18 dsRNA is assigned to the genus Mitovirus in the family Narnaviridae and designated as H. mompa mitovirus 1-18 (HmMV1-18). Like other mitoviruses, HmMV1-18 RNA can be folded into potentially stable stem-loop structures at both the 5'- and 3'-termini, and both terminal sequences have inverted complementarity with the potential to form panhandle structure. BLAST analysis indicates that the RDRP encoded by HmMV1-18 is more closely related to those encoded by mitochondrial viruses of some ascomycetes than to that of the unassigned RsM2-1A1 dsRNA in the basidiomycetous Rhizoctonia solani. HmMV1-18 is the first member of the genus Mitovirus from basidiomycete fungi.
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PMID:Nucleotide sequence of a mitochondrial RNA virus from the plant pathogenic fungus, Helicobasidium mompa Tanaka. 1556 32

Analysis of the genome sequence of the small hyperthermophilic archaeal parasite Nanoarchaeum equitans has not revealed genes encoding the glutamate, histidine, tryptophan and initiator methionine transfer RNA species. Here we develop a computational approach to genome analysis that searches for widely separated genes encoding tRNA halves that, on the basis of structural prediction, could form intact tRNA molecules. A search of the N. equitans genome reveals nine genes that encode tRNA halves; together they account for the missing tRNA genes. The tRNA sequences are split after the anticodon-adjacent position 37, the normal location of tRNA introns. The terminal sequences can be accommodated in an intervening sequence that includes a 12-14-nucleotide GC-rich RNA duplex between the end of the 5' tRNA half and the beginning of the 3' tRNA half. Reverse transcriptase polymerase chain reaction and aminoacylation experiments of N. equitans tRNA demonstrated maturation to full-size tRNA and acceptor activity of the tRNA(His) and tRNA(Glu) species predicted in silico. As the joining mechanism possibly involves tRNA trans-splicing, the presence of an intron might have been required for early tRNA synthesis.
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PMID:Nanoarchaeum equitans creates functional tRNAs from separate genes for their 5'- and 3'-halves. 1569 44

The presence of albumin in the human epidermis has been reported more than a decade ago, but until now, it was assumed that this protein is synthesized in the liver and transported to the avascular skin. To our knowledge, transcription of albumin in the human epidermis was never considered. In this report, we present for the first time evidence for autocrine synthesis of albumin in the human epidermis in keratinocytes in situ and in vitro. Using double immunofluorescence labelling, we identified that albumin colocalized together with its transcription factor PCD/DCoH/HNF-1alpha in suprabasal keratinocytes in human full-thickness skin sections and in keratinocytes cultured in serum-free medium. Moreover, albumin and HNF-1alpha protein expression was confirmed by Western blotting in undifferentiated and differentiated keratinocytes as well as in human epidermal suction blister roof extracts. Reverse-transcriptase polymerase chain reaction analysis from human epidermal keratinocytes and epidermal suction blister roofs revealed the transcription of albumin. Using in vivo fluorescence excitation spectroscopy at the surface of human skin, we confirmed albumin as a major constituent yielding a lambda(max) at 295 nm, which was assigned to the single tryptophan 214 fluorophore in this protein. This in vivo result is in agreement with albumin concentrations of 10(-3) M, underlining the importance of this protein in epidermal homeostasis.
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PMID:In vivo and in vitro evidence for autocrine DCoH/HNF-1alpha transcription of albumin in the human epidermis. 1574 May 90

Lack of maturation of phagosomes containing pathogenic Mycobacterium tuberculosis within macrophages has been widely recognized as a crucial factor for the persistence of mycobacterial pathogen. Host molecule tryptophan-aspartate containing coat protein (TACO) has been shown to play a crucial role in the arrest of such a maturation process. The present study was addressed to understand whether or not polyphenols derived from green tea could down-regulate TACO gene transcription. And if yes, what impact TACO gene down-regulation has on the uptake/survival of M. tuberculosis within macrophages. The reverse-transcriptase polymerase chain reaction and reporter assay technology, employed in this study, revealed that the major component of green tea polyphenols, epigallocatechin-3-gallate had the inherent capacity to down-regulate TACO gene transcription within human macrophages through its ability to inhibit Sp1 transcription factor. We also found out that TACO gene promoter does contain Sp1 binding sequence using bioinformatics tools. The down-regulation of TACO gene expression by epigallocatechin-3-gallate was accompanied by inhibition of mycobacterium survival within macrophages as assessed through flow cytometry and colony counts. Based on these results, we propose that epigallocatechin-3-gallate may be of importance in the prevention of tuberculosis infection.
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PMID:Green tea polyphenol inhibits Mycobacterium tuberculosis survival within human macrophages. 1635 57

Arom gene, encoding a single polypeptide that catalyses steps two to six of the aromatic amino acid (phenylalanine, tyrosine and tryptophan) biosynthetic pathway, has been amplified from Scleortinia sclerotiorum genomic DNA by PCR and sequenced. In order to identify the fragment encoding AROM protein experimentally and search a method of obtaining the enzyme in a large amount, the open reading frame of arom gene of S. sclerotiorum was amplified by Pyrobest DNA Polymerase and inserted between Kpn I and Not I sites of the vector pYES2 to construct the expression vector pYES2-arom. The construct was transformed into Saccharomyces cerevisiae H158 by the method of LiAc/ SSDNA/PEG. The rate of transformation was 2 x 10(2)/microg DNA, which was enough for the selection of the positive transformants. PCR using the extracted plasmids as the templates and restriction enzyme analysis of the plasmids extracted from E. coli cells transformed by the above plasmids were performed respectively to screen the positive S. cerevisiae transformants since the copy number of the plasmid in S. cerevisiae was low. Subsequently, the transformant activated by the SC-U medium containing 2% raffinose was inoculated into the SC-U medium containing 2% galactose and the SC-U medium containing 2% glucose respectively to induce and depress the expression of the foreign arom gene. The results of RT-PCR analysis showed: there was not any DNA band in the negative control without the anti-transcriptase, which indicated there was no DNA contamination in the extracted total RNA; there was an expected DNA band in the positive control using the expression vector pYES2-arom as the template, which indicated the used amplification condition was proper; there was not any DNA band in the negative control using total RNA from the depressed transformant as the template, which indicated the DNA bands amplified from total RNA of the induced transformant were not false; there were the expected DNA bands in the samples using total RNA of the transformant induced for 48h, 60h, 72h or 84h as the templates, which indicated the heterogeneous arom gene was transcribed in S. cerevisiae H158 cells. The result of Northern hybridization was consistent with that of RT-PCR, and showed that arom gene of S. sclerotiorum had been transcribed in S. cerevisiae H158 cells when the cells were induced for 48h in the SC-U medium containing 2% galactose at 30 degrees C at 180r/min. 5-enolpyruvylshikimate-3-phosphate synthase activity of the transformant, which was one of AROM protein activities, was measured by estimating the rate of Pi release to check the expressed AROM protein was active or not. The results of enzyme assay in the different culture period indicated that the transformant had 5-enolpyruvylshikimate-3-phosphate synthase activity and the activity reached the peak when the transformant was induced for 72h in SC-U medium at 30 degrees C at 180r/min. The molecular weight of AROM protein is high, it exists in cytoplasm as a dimmer and its expression is controlled by the amounts of amino acids. Therefore, it is very difficult to purify the enzyme. A great lot protein can be obtained by heterogeneous expression. S. cerevisiae expression system has the merits of safe status, authentic posttranslational modification, fast cultivation etc. and usually is the first choice eukaryotic expression system. S. cerevisiae expression system of AROM protein from S. sclerotiorum was successfully constructed for the first time, which provided the basis for the research on the catalysis mechanism of the enzyme and an economical means of simultaneously synthesizing five aromatic amino acid biosynthetic pathway enzymes.
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PMID:[Cloning and expression of arom gene of Sclerotinia sclerotiorum]. 1657 63


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