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)

Previous in vitro data on type I collagen self-assembly into fibrils suggested that the amino acid 776-796 region of the alpha1(I) chain is crucial for fibril formation because it serves as the recognition site for the telopeptide of a docking collagen monomer. We used a natural collagen mutation with a deletion of amino acids 766-801 to confirm the importance of this region for collagen fibril formation. The proband has type III osteogenesis imperfecta and is heterozygous for a COL1A1 IVS 41 A(+4) --> C substitution. The intronic mutation causes splicing of exon 41, confirmed by sequencing of normal and shorter reverse transcriptase-PCR products. Reverse transcriptase-PCR using RNA from proband dermal fibroblasts and clonal cell lines showed the mutant cDNA was about 15% of total alpha1(I) cDNA. The mutant transcript is translated; structurally abnormal alpha chains are demonstrated in the cell layer of proband fibroblasts by SDS-urea-PAGE. The proportion of mutant chains in the secreted procollagen was determined to be 10% by resistance to digestion with MMP-1, since chains lacking exon 41 are missing the vertebral collagenase cleavage site. Secreted proband collagen was used for analysis of kinetics of binding of alpha1(I) C-telopeptide using an optical biosensor. Telopeptide had slower association and faster dissociation from proband than from normal collagen. Purified proband pC-collagen was used to study fibril formation. The presence of the mutant molecules decreases the rate of fibril formation. The fibrils formed in the presence of 10-15% mutant molecules have strikingly increased length compared with normal collagen, but are well organized, as demonstrated by D-periodicity. These results suggest that some collagen molecules containing the mutant chain are incorporated into fibrils and that the absence of the telopeptide binding region from even a small portion of the monomers interferes with fibril growth. Both abnormal fibrils and slower remodeling may contribute to the severe phenotype.in
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PMID:Procollagen with skipping of alpha 1(I) exon 41 has lower binding affinity for alpha 1(I) C-telopeptide, impaired in vitro fibrillogenesis, and altered fibril morphology. 1170 4

Human infection with Trypanosoma brucei may result in meningo-encephalitis, neuronal demyelination, blood-brain-barrier dysfunction, peri-vascular infiltration, astrocytosis and neuronal apoptosis. Prevention of the short- or long-term, parasite-induced, neuronal assault requires a better understanding of the host's responses to the infection at the molecular level. Northern analysis, cDNA micro-arrays, reverse-transcriptase-PCR (RT-PCR), SDS-PAGE and immunohistology were therefore used to investigate global gene and protein expression in the brains of mice infected with T. brucei. Temporal and spatial expression of neuroleukin (NLK), a predominant neurotrophin which is associated with neuronal protection and regeneration during neuronal assault in the brain, was then assessed. Expression of 20 of the 588 genes investigated (representing pro- and anti-inflammatory immuno-modulators, growth factors, neurotransmitters, and pro- and anti-apoptosis factors) was significantly altered (P < 0.05). TUNEL analysis revealed extensive apoptosis at peak parasitaemia, mainly in the cerebellum. RT-PCR analysis of two regulators of apoptosis, Bcl-x(L) (anti-apoptotic) and Bax (pro-apoptotic), revealed equivalent increases in levels of expression. NLK expression was up-regulated in punctated fashion in brain and was mainly localized to abnormal (stellate) catecholamine neurons (CN) in the locus coeruleus (LC) of infected [and, to a lesser degree, the normal (polygonal) cells of uninfected] brainstem. Expression of NLK receptor (NLK-R) was inversely correlated with that of NLK. At peak parasitaemia, trypanosome infection apparently induces cerebellar apoptosis and a corresponding increase in NLK expression. NLK may be modulating inflammation and is probably involved in protecting CN and the cerebellum against apoptosis.
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PMID:Trypanosoma brucei infection induces apoptosis and up-regulates neuroleukin expression in the cerebellum. 1178 34

We describe a method for correlating polymerase activity with a particular polypeptide band in an SDS-polyacrylamide gel which does not require renaturation of the SDS-denatured enzyme. The method involves the following steps: (i) transfer of proteins from an SDS-polyacrylamide gel onto nitrocellulose; (ii) incubation with excess antiserum raised against a partially purified polymerase preparation to link one Fab site of an antibody molecule to the denatured enzyme on the nitrocellulose; (iii) binding of native polymerase to the other Fab site of the antibody molecule in the immune complex to generate a specific polymerase 'sandwich'; (iv) assaying of the nitrocellulose filter for antibody-linked native polymerase activity using an appropriate template and a radioactive substrate followed by treatment with trichloroacetic acid to precipitate in situ the radioactive product. The essential feature of this method is that the use of both non-specific anti-polymerase serum and a partially purified enzyme preparation is sufficient to allow identification of a specific protein following SDS-polyacrylamide gel electrophoresis. This antibody-linked polymerase assay has been developed to identify a 130,000-dalton RNA-dependent RNA polymerase from cowpea leaves. Possible applications of this type of assay as a tool for identifying a wide variety of proteins are discussed.
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PMID:Antibody-linked polymerase assay on protein blots: a novel method for identifying polymerases following SDS-polyacrylamide gel electrophoresis. 1189 32

Mammalian reovirus is an enteric virus that contains a double-stranded RNA genome. The genome consists of ten RNA segments that encode eight structural and three non-structural proteins. The structural proteins form a double-layered structure. The innermost layer, called the core, consists of five proteins (lambda1, lambda2, lambda3, micro 2, and sigma2). Protein lambda3 is the RNA-dependent RNA polymerase (RdRp) and micro 2 is thought to be an RdRp cofactor. Translation of most reovirus proteins is known to commence at the first start codon. However, the translation initiation site of the viral core protein micro 2, encoded by the M1 RNA segment, has been in dispute. Although the theoretical molecular weight of micro 2 is 83 267 Da the actual molecular weight is unknown because micro 2 runs aberrantly in SDS-PAGE and has resisted characterization by Edman degradation, indicating that the amino terminus is post-translationally modified. In this study, we used proteolysis coupled with MALDI-Qq-TOFMS to determine that translation of micro 2 initiates at the first AUG codon, that its actual molecular weight approximates the theoretical value of 83 kDa, that the amino terminal methionine residue is removed, and that the next amino acid (alanine) is post-translationally acetylated.
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PMID:Mammalian reovirus core protein micro 2 initiates at the first start codon and is acetylated. 1247 77

Nodamura virus (NoV) was the first isolated member of the Nodaviridae, and is the type species of the alphanodavirus genus. The alphanodaviruses infect insects; NoV is unique in that it can also lethally infect mammals. Nodaviruses have bipartite positive-sense RNA genomes in which RNA1 encodes the RNA-dependent RNA polymerase and the smaller genome segment, RNA2, encodes the capsid protein precursor. To facilitate the study of NoV, we generated infectious cDNA clones of its two genomic RNAs. Transcription of these NoV1 and NoV2 cDNAs in mammalian cells led to viral RNA replication, protein synthesis, and production of infectious virus. Subgenomic RNA3 was produced during RNA replication and encodes nonstructural proteins B1 and B2 in overlapping ORFs. Site-directed mutagenesis of these ORFs, followed by SDS-PAGE and MALDI-TOF mass spectrometry analyses, showed synthesis of B1 and two forms of B2 (B2-134 and B2-137) during viral replication. We also characterized a point mutation in RNA1 far upstream of the RNA3 region that resulted in decreased RNA3 synthesis and RNA2 replication, and a reduced yield of infectious particles. The ability to reproduce the entire life cycle of this unusual nodavirus from cDNA clones will facilitate further analysis of NoV RNA replication and pathogenesis.
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PMID:Recovery of infectivity from cDNA clones of nodamura virus and identification of small nonstructural proteins. 1257 89

We have employed a pET-ubiquitin expression system to produce two his-tagged forms of hepatitis C virus (HCV) non-structural protein 5A (NS5A) in Escherichia coli. One derivative contains the full-length protein extended to include a carboxy-terminal hexahistidine tag; the other derivative contains an amino-terminal hexahistidine tag in place of the 32 amino acid amphipathic helix that mediates membrane association. At least 1 mg of each derivative at a purity of 90% could be produced from a 1-L culture. The purified derivatives produced high titer antibody that recognized both p56 and p58 forms of NS5A in Huh-7.5 cells expressing an HCV subgenomic replicon. The NS5A derivatives were efficiently phosphorylated by casein kinase II, leading to at least 5 mol of phosphate incorporated per mole of protein. Interestingly, this level of phosphorylation did not alter the migration of the protein in an SDS-polyacrylamide gel, suggesting that hyperphosphorylation alone is not sufficient to generate the p58 form of NS5A observed in Huh-7 cells. Neither NS5A derivative was capable of inhibiting the eIF2alpha-phosphorylation activity of the activated form of the double-stranded RNA-activated protein kinase, PKR, suggesting that NS5A phosphorylation may be required for this function of NS5A. However, both unphosphorylated derivatives were shown to interact with NS5B, the HCV RNA-dependent RNA polymerase, in solution by using a novel kinase-protection assay. The availability of purified HCV NS5A will permit rigorous biochemical and biophysical characterization of this protein, ultimately providing insight into the function of this protein during HCV genome replication.
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PMID:Purification and characterization of hepatitis C virus non-structural protein 5A expressed in Escherichia coli. 1529 92

The Haloferax mediterranei nar operon has been sequenced and its regulation has been characterized at transcriptional level. The nar operon encodes seven open reading frames(ORFs) (ORF1 narB, narC, ORF4, narG, narH, ORF7 and narJ). ORF1, ORF4 and ORF7 are open reading frames with no assigned function, however the rest of them encoded different proteins. narB codes for a 219-amino-acid-residue iron Rieske protein. narC encodes a protein of 486 amino acid residues identified by databases searches as cytochrome-b (narC). The narG gene encodes a protein with 983 amino acid residues and is identified as a respiratory nitrate reductase catalytic subunit (narG). NarH protein has been identified as an electron transfer respiratory nitrate reductase subunit (narH). The last ORF encodes a chaperonin-like protein (narJ) of 242 amino acid residues. The respiratory nitrate reductase was purified 21-fold from H. mediterranei membranes. Based on SDS-PAGE and gel-filtration chromatography under native conditions, the enzyme complex consists of two subunits of 112 and 61 kDa. The optimum temperature for activity was 70 degrees C at 3.4 M NaCl and the stability did not show a direct dependence on salt concentration. Respiratory nitrate reductase showed maximum activity at pH 7.9 and pH 8.2 when assays were carried out at 40 and 60 degrees C, respectively. The absorption spectrum indicated that Nar contains Fe-S clusters. Reverse transcriptase (RT-PCR) shows that regulation of nar genes occurs at transcriptional level induced by oxygen-limiting conditions and the presence of nitrate.
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PMID:Respiratory nitrate reductase from haloarchaeon Haloferax mediterranei: biochemical and genetic analysis. 1534 13

PSP94 (prostate secretory protein of 94 amino acids), an abundant protein within semen, has reported local functions within the reproductive tract and reported systemic functions. Mechanisms of action remain poorly understood, but binding to undefined molecules within the prostate, pituitary, testis and blood may initiate some of these actions. PSP94 serum measurements, especially of bound and free forms, have potential clinical utility in prostate cancer management. Identification of the binding molecules will help in the understanding of PSP94's action, and enable further development of PSP94 serum assays. PSPBP (PSP94-binding protein) was purified from human serum by ammonium sulphate fractionation, ion-exchange and affinity chromatography. The glycosylated protein ran as two bands on SDS/PAGE (70 and 95 kDa). N-terminal sequencing yielded a 30-amino-acid sequence, identical with the translated N-terminal region of a previously published cDNA (GenBank accession number AX136261). Reverse transcriptase PCR and plaque hybridization demonstrated PSPBP mRNA in peripheral blood leucocytes and in a prostate cDNA library. Northern blotting showed 2 kb mRNA species in prostate, testis, ovary and intestine. Immunohistochemistry demonstrated PSPBP in tissues, including pituitary and Leydig cells, supporting a role for PSP94 in hormonal control at the pituitary gonadal axis. ELISA demonstrated that PSPBP levels were significantly lower (P=0.0014) in the serum of a prostate cancer population (n=65) compared with a control population (n=70). PSPBP identification will help the understanding of PSP94's functions and facilitate ELISA development to address the clinical value of PSP94 serum assays.
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PMID:Identification, purification and characterization of a novel human blood protein with binding affinity for prostate secretory protein of 94 amino acids. 1534 9

Hepatitis E is an acute hepatitis casused by hepatitis E virus (HEV) in developing countries, where it occurs as cases sporadic and in epidemics form. The causative agent, hepatitis E virus, is transmitted primarily by the fecal-oral route. HEV is icosahedron non-enveloped virus, and its genome is a single-stranded, positive-sense, 3'-polyadenylated RNA about 7.5 kb in length. It contains three open reading frames (ORFs). Of which ORF1 codes for a polyprotein of 1693 amino acids and contain domains homologous to a viral methyltransferase, a papainlike cysteine protease, an RNA helicasre, and an RNA-dependent RNA polymerase, besides the most hypervariable region of the HEV genome. And ORF3 codes for a 123-amino-acide-long polypeptide with unknown function. While the major viral capsid protein (pORF2, ORF2 codes) of 660 amino acid was showed to contain the protective epitope. The bacterially expressed polypeptide disignated as NE2 has been proved to be a protective antige. And the anti-NE2 monoclonal antibodies (mAb) was screend, two of these mAbs 8C11 and 8H3 were showed to be against separate conformational neutralization epitope of hepatitis E virus (HEV). And these two mAb were used to screen for binding peptides from a 7-peptides phage display library. After four rounds of panning, tweenty-one positive monoclonal phages (11 for 8C11, and 10 for 8H3) were selected and the inserted fragments were sequenced. The DNA sequence coding for the obtained dominant peptide 8C11 (N'-His-Pro-Thr-Leu-Leu-Arg-Ile-C', named 8C11A) and 8H3 (N'-Ser-Ile-Leu-Pro-Tyr-Pro-Tyr-C', named 8H3A) were then synthesized and cloned to insert between amino acid 78 to 83 of hepatitis B core antigen (HBcAg), then expressed in E. coli. The recombinant proteins aggregate into homodimer or polymer on SDS-PAGE, and could bind with mAb 8C11 and 8H3 in Western blotting. Respectively, the recombinant protein C8C11A showed to be dimer mainly, which can bind with mAb 8C11. The monomer and dimer of C8H3A are in the same amount on SDS-PAGE, but only the dimer could bind with mAb 8H3 on Western blotting. The renatured recombinant proteins were all showed to aggregate into virus like particles which were similar as HBcAg on transmission electron micrograph. The dominant peptide 8H3A (N'-Ser-Ile-Leu-Pro-Tyr-ProTyr-C') that selected out by mAb 8H3 was further chemo-synthesized, and its binding activity was confirmed by BIAcore biosensor. The result showed that this 7-peptide can bind with mAb 8H3 in a big Ka and Kd form, which means the binding is not stable. These results implicated that conformational dependent neutralization epitope could be partially modeled by short peptide, which provided a feasible route for subunit vaccine development.
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PMID:[Selection of a peptide mimic the neutralization epitope of hepatitis E virus with phage peptide display technology]. 1597 79

In researches involving in vitro protein synthesis and self-replication system, Qbeta replicase is one of the key enzymes, which are demanded for the high availability. Qbeta replicase is a RNA-dependent RNA polymerase of Qbeta coliphage. It consists of four subunits (alpha, beta, gamma, and delta subunit), where the beta-subunit is encoded by the viral genome, while the other three subunits are host proteins normally involved in protein synthesis, namely, ribosomal protein S1 (alpha), elongation factors EF-Tu (gamma) and EF-Ts (delta). To increase the production of the Qbeta replicase holoenzyme, several types of expression vectors, including pKK, pET and others, were employed to produce Qbeta replicase. However, the beta-subunit was almost in the precipitate fraction. Considering that the four subunits of Qbeta replicase holoenzyme are in equivalent molar ratio and the amount of the subunits, ribosomal S1 and EF-Ts, being produced by the host cells is relatively low, co-expression of beta-subunit with the other three subunits was performed to know whether the availability of the host subunits is the contributing factor for the solubility of the Qbeta replicase. pBAD33-rep was constructed by cloning the beta-subunit gene into pBAD 33, a pACYC derivative, and pET21a(+) was employed as expression vector for the three other subunits. Among the different combinations of co-expression experiments, solubility was found to slightly increase by SDS-PAGE analysis when the beta-subunit was co-expressed with EF-Tu-Ts. And the replicase activity assay showed this soluble enzyme is in active form. The expression of beta-subunit was enhanced by decreasing the level of inducer IPTG in co-expression, and more soluble enzyme were obtained.
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PMID:[Co-expression of beta-subunit with other subunits of Qbeta replicase]. 1611 Sep 60


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