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Biomedical subjects

A Dasgupta

Publications and source records attributed to A Dasgupta.

At least 235 records · Page 13Linked to original sources
PubMed1986

Mechanism of in vitro synthesis of covalently linked dimeric RNA molecules by the poliovirus replicase.

Four RNA fragments of approximately 1,000 to 1,200 nucleotides, representing both the 5' and 3' termini of poliovirus plus- and minus-strand RNAs, were generated by transcription of poliovirus cDNA by using bacteriophage SP6 RNA polymerase. The copying of these templates by the poliovirus replicase invariably produced RNA products approximately twice the size of the templates. In experiments with templates uniformly labeled with 32P it was shown that some of the apparently double-length products were generated by extension from an internal site of the template. Filter hybridization of the labeled in vitro-synthesized products with various unlabeled templates suggested a second mechanism by which double-length molecules could be synthesized; the results can be best explained by de novo synthesis of the first strand by copying of the template RNA, followed by snap-back of the newly synthesized RNA, generating a template-primer structure for the synthesis of the second strand. Highly purified poliovirus replicase was able to support the synthesis of double-length RNA products in response to these templates. These reactions did not require host factor. In contrast, synthesis of genome-length copies of poliovirion RNA by the same replicase was absolutely dependent on added host factor. The synthesis of double-length RNA products did not require either the 3'-terminal poly(A) of plus RNA or sequences within the 3' termini of both plus- and minus-strand RNAs.

Base Sequence↗
PubMed1985

The host protein required for in vitro replication of poliovirus is a protein kinase that phosphorylates eukaryotic initiation factor-2.

The HeLa cell protein (host factor) required for in vitro replication of poliovirus has been identified as a 67,000 dalton phosphoprotein. The purified protein displays three activities in vitro: stimulation of poliovirus RNA synthesis in the presence of poliovirus replicase, apparent self-phosphorylation, and phosphorylation of the alpha-subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2). All three activities can be removed or inhibited by an antibody to host factor. Partially purified preparations of reticulocyte eIF-2 contain a similar phosphoprotein and display host factor activity in the viral RNA synthesis assay in vitro. In vitro phosphorylation of the 67 kd protein can be stimulated by low concentrations of double-stranded RNA. Addition of phosphorylated host factor in an in vitro RNA synthesis assay significantly changes the kinetics of viral RNA synthesis, indicating that protein phosphorylation may play an important role in viral RNA replication.

Adenosine Triphosphate↗
PubMed1985

Primer-dependent eukaryotic RNA polymerase capable of accurate transcription from the adenovirus major late promoter in a reconstituted system.

A sensitive assay for detection of eukaryotic RNA polymerase II has been developed. This assay depends on the ability of polymerase II to elongate a small RNA primer, oligo(U), hybridized to a single-stranded homopolymeric DNA template, poly(dA). The poly(dA).oligo(U)-dependent RNA polymerase II from calf thymus has been purified approximately 10,000-fold using this assay. The purified enzyme contains four polypeptides of apparent Mr 180,000, 140,000, 24,000, and 16,000 and is fully active in accurate initiation of transcription from the adenovirus major late promoter in the presence of transcription factors from HeLa cells. The poly(dA).oligo(U)-dependent RNA polymerase activity can be detected in crude cell extracts from a variety of tissue culture cells and appears to be largely due to polymerase II, since 90-95% of this activity is inhibited by alpha-amanitin at a concentration of 1 microgram/ml.

Adenoviruses, Human↗
PubMed1985

In vitro synthesis of infectious poliovirus RNA.

Replication of the infectious RNA genome of poliovirus is accomplished in cells by the viral RNA polymerase through negative-strand RNA intermediates. Full-length negative-strand poliovirus RNA was synthesized in vitro by transcription of infectious poliovirus cDNA with bacteriophage SP6 DNA-dependent RNA polymerase. When provided with this negative-strand RNA as template, the poliovirus RNA-dependent RNA polymerase synthesized full-length positive-strand molecules. The positive-strand RNAs synthesized in vitro were infectious when transfected into HeLa cells. In contrast, positive-strand copies of poliovirus RNA synthesized in vitro by SP6 polymerase, using a poliovirus cDNA template, were not infectious. Production of infectious positive-strand RNA by the poliovirus polymerase was not observed when magnesium or negative-strand RNA template was omitted from the reaction mixture. Infectivity of the product RNA was not destroyed by DNase treatment. The specific infectivity in HeLa cells of in vitro-synthesized positive-strand RNA was 4 X 10(4) plaque-forming units/micrograms of RNA.

DNA↗
PubMed1985

Purification of a soluble template-dependent rhinovirus RNA polymerase and its dependence on a host cell protein for viral RNA synthesis.

The soluble phase of the cytoplasm of human rhinovirus type 2-infected cells contains an enzymatic activity able to copy rhinovirion RNA without an added primer. This RNA-dependent RNA polymerase (replicase) makes a specific copy of the added rhinovirion RNA, as shown by hybridization of the product to its template RNA but not to other RNAs. The same replicase preparation also contains a virus-specific polyuridylic acid [poly(U)] polymerase activity which is dependent on added polyadenylic acid-oligouridylic acid template-primer. Both activities purify together until a step at which poly(U) polymerase but no replicase activity is recovered. Addition of a purified HeLa cell protein (host factor) to this poly(U) polymerase completely reconstitutes rhinovirus replicase activity. Host factor activity can be supplied by adding oligouridylic acid, suggesting that the host cell protein acts at the initiation step of rhinovirus RNA replication. A virus-specific 64,000-dalton protein purifies with both poly(U) polymerase and replicase activities.

Cell Transformation, Viral↗
PubMed1984

Phospholipid studies of marine organisms: new branched fatty acids from Strongylophora durissima.

The phospholipids of the sponge Strongylophora durissima were analyzed. The major phospholipids present were phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG) and phosphatidylinositol (PI). The major fatty acid components of the phospholipids consisted of short chain (C14-C19) and very long chain (C25 -C30) "Demospongic" acids. Three novel branched delta 5 monounsaturated acids, Z-19-methyl-5-pentacosenoic, Z-19-methyl-5-hexacosenoic and Z-19-methyl-5-heptacosenoic acids were encountered in the sponge. The 3-saturated counterparts of these compounds, 19-methylpentacosanoic, 19-methylhexacosanoic and 19-methylheptacosanoic acids, as well as 19-methyltetracosanoic and 20-methyloctacosanoic acids also are hitherto undescribed acids present in the sponge. Trace amounts of 2 very long chain acids also were detected and their structures tentatively assigned as 19,21-dimethylheptacosanoic and 20,22-dimethyloctacosanoic acids. The distribution of these fatty acids according to phospholipid head groups also was described.

Animals↗
PubMed1984

Antibody to poliovirus genome-linked protein (VPg) precipitates in vitro synthesized RNA attached to VPg-precursor polypeptide(s).

Antibody to poliovirus genome-linked protein VPg, specifically precipitated RNA synthesized in vitro by the poliovirus replicase and host factor in response to poliovirion RNA. A significant amount of the immunoprecipitated RNA was RNAase T1 resistant, sedimented at approximately 2-4 S and was shown to be largely polyuridylic acid. RNAase A digestion or alkali hydrolysis of the immunoprecipitated RNA left [32P]UMP-labeled material which comigrated on SDS-polyacrylamide gels with known VPg-precursor polypeptides. The results presented in this paper suggested that VPg was involved in the host factor-dependent, poliovirus replicase-catalyzed in vitro RNA synthesis, most probably in the form of a larger precursor protein.

Antibodies, Viral↗
PubMed1984

Lymphatic filariasis in man: demonstration of circulating antigens in Wuchereria bancrofti infection.

Five hundred and twenty-nine sera obtained from people dwelling in an area endemic for bancroftian filariasis were analysed for the presence of soluble circulating antigens (SCA) of filarial origin by counter immunoelectrophoresis and 303 were found positive. It, therefore, appears that 57.3% subjects could be diagnosed by the detection of SCA irrespective of their clinical status. Of the three groups investigated, microfilaraemic, amicrofilaraemic and clinical, SCA could be demonstrated in maximum number of sera obtained from clinical cases. The parasite specificity of the SCA was determined by enzyme linked immunosorbent assay (ELISA), immunofluorescent antibody test (IFAT), counter immunoelectrophoresis (CIEP) and gel diffusion (GD) using antisera raised against Litomosoides carinii in rabbits and antigens derived from Wuchereria bancrofti microfilariae or antigens present in sera of patients with bancroftian filariasis. This anti-carinii hyperimmune serum under study did not show positive reactions with parasites other than filaria, or with the sera obtained from pre-immunized rabbits and non-endemic controls. A demonstration of a distinct precipitin band in CIEP and GD indicates that either monospecific hyperimmune sera or monoclonal antibodies could be raised for the development of a simple method for immunodiagnosis of bancroftian filariasis.

Antigens↗
PubMed1984

ATP is required for initiation of poliovirus RNA synthesis in vitro: demonstration of tyrosine-phosphate linkage between in vitro-synthesized RNA and genome-linked protein.

Poliovirus replicase- and host factor-catalyzed copying of 3'-terminal polyadenylic acid [poly(A)] of poliovirion RNA was studied. Host factor-stimulated synthesis of polyuridylic acid [poly(U)] by the replicase required ATP in addition to UTP. ATP was not required for the oligouridylic acid-primed copying of 3'-terminal poly(A) of virion RNA. GTP, CTP, and AMP-PCP (5'-adenylyl beta-gamma methylenediphosphate, an ATP analog) could not replace ATP in host factor-stimulated synthesis of poly(U). Antibodies to poliovirus genome-linked protein (VPg) specifically precipitated in vitro-synthesized poly(U) from a host factor-stimulated reaction. The poly(U) synthesized in a host factor-stimulated reaction was shown to be attached to VPg precursor polypeptide(s) via a tyrosine-phosphate bond as found in poliovirion VPg-RNA.

Adenosine Triphosphate↗