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R S Ranu

Publications and source records attributed to R S Ranu.

At least 19 recordsLinked to original sources
PubMed2003

Cloning and expression of 1-aminocyclopropane-1-carboxylate synthase cDNA from rosa (Rosa x hybrida).

The role of 1-aminocyclopropane-1-carboxylate (ACC) synthase in rose flower petal senescence was investigated. A cDNA library from senescing petals of rose ( Rosa x hybrid cv. Kardinal) prepared in lambdacDNA ZAP Express Vector was probed with a rose-specific 400-bp probe, and seven putative positive ACC synthase clones were isolated. Except for differences in length, the sequences of these clones were identical. A full-length clone, RKacc7, 1,750 bp long, coded for an open reading frame of 480 amino acids that contained the 11 conserved amino acid residues, the substrate and pyridoxal 5'-phosphate binding sites, all of which are characteristic of all ACC synthases. The transcripts prepared in vitro from the full-length clone when translated in rabbit reticulocyte lysates exhibited a 55-KDa polypeptide that comigrated with a polypeptide synthesized from a mRNA fraction isolated from senescing petals, and both were immunoselected by anti-ACC synthase antibodies. Reverse transcriptase-PCR-based studies showed that in planta RKacc7 is specifically expressed in rose petals, ovary and sepals. The expression of ACC synthase increased dramatically as the flower matured to senescence and also correlated positively with ethylene levels. The results of genomic Southern blots probed with RKacc7 are consistent with a pattern expected from a multigene family.

Amino Acid Sequence↗
PubMed2000

Purification and characterization of an extracellular alkaline serine protease from Aspergillus terreus (IJIRA 6.2).

An extracellular alkaline serine protease has been purified from Aspergillus terreus (IJIRA 6.2). The purification procedure involved chromatography on DEAE-Sephadex A25, phosphocellulose, hydroxyapatite, casein-Sepharose, gel filtration on Sephacryl-S-300 and by glycerol density gradient centrifugation. The enzyme was further purified to apparent homogeneity through a combination of electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) with or without protease substrate (gelatin) and subsequent regeneration of its activity in situ by removal of SDS. The active enzyme was visualized in a zymogram or on the basis of protease activity exhibited on an X-ray film. The protein in the unstained segment of the gel was electroeluted. The eluted protein with protease activity exhibited a molecular mass of 37,000-daltons on electrophoresis in SDS-polyacrylamide gel. A sedimentation coefficient of 3.2S was obtained by glycerol density gradient contrifugation. Maximum activity of protease was observed at pH 8.5 and at 37 degrees C. Purified protease was active between pH 5.5 and 9.5 and was found to be stable up to 60 degrees C. With Na-caseinate, the K(m) of the purified protease was found to be 0.055 mM. Antipain, phenylmethane sulfonyl fluoride, and chymostatin served as non-competitive inhibitors. Substrate specificity was determined by using a synthetic chromogenic peptide containing N-P-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results showed that the protease cleaved the peptide on the -COOH end of arginine residue.

Aspergillus↗
PubMed1997

Direct cycle sequencing with delta Taq DNA polymerase.

The delta Taq DNA polymerase is a new, genetically modified version of standard Taq DNA polymerase which lacks the 5'-->3'-exonuclease activity. The present study was designed to investigate the use of delta Taq DNA polymerase for direct cycle sequencing. Results show that delta Taq DNA polymerase can be used for direct cycle sequencing of the PCR amplified DNA, either from an asymmetrically amplified template (by PCR), double stranded DNA template, PCR amplified DNA cloned into a plasmid vector or from a single stranded template. The primer to template ratio and number of cycles necessary for best sequence data have been determined. From these results we conclude that delta Taq DNA polymerase is a highly versatile enzyme which can be used for DNA sequence determinations by direct cycle sequencing.

DNA Primers↗
PubMed1996

DNA sequencing with [alpha-33P]-labeled ddNTP terminators: a new approach to DNA sequencing with Thermo Sequenase DNA polymerase.

A new approach to DNA sequencing is described. The method is based on the use of [alpha-33P]-labeled dideoxyribonucleoside triphosphate terminators and Thermo Sequenase DNA polymerase in cycle sequencing. Thermo Sequenase DNA polymerase incorporates ddNTPs as efficiently as dNTPs, allowing the use of low concentrations of these nucleotides in DNA sequencing. Because only the properly terminated chains are labeled and visualized on autoradiography of the sequencing gels, the sequence results are free of background. The intensity of DNA bands generated are remarkably uniform, which makes reading of DNA sequences easy. By staggered loading of the sequencing gel (at 2-3 hour intervals), it is possible to sequence DNA at least 450 to 500 nucleotides. Exposure time for autoradiography with [alpha-33P] labels is much shorter than with [35S] and does not substantially compromise autoradiographic resolution. Data can be obtained after only 12 hours of exposure of an X-ray film. Moreover, cycle sequencing requires very small amounts of single- or double-stranded template. Consequently, it is even possible to generate sequence data from a single bacterial colony. The details of the protocol are presented in a stepwise manner, and some important parameters to be considered for sequencing with this method are discussed.

Codon, Terminator↗
PubMed1996

In vitro translation of the full-length RNA transcript of figwort mosaic virus (Caulimovirus).

The circular DNA genome of FMV consists of seven tandemly arranged genes placed successively on a full-length RNA transcript that spans the entire circular viral genome. This transcript is a tentative mRNA for at least five of the six major conserved genes of this virus (genes I-V) that are positioned on this transcript. The sixth major gene (gene VI) is expressed as a separate monocistronic transcript. A long 5'-nontranslated leader (598 nucleotides), a small nonconserved gene (VII), and a short intergenic region (57 nucleotides) precede the five major conserved genes (I through V) on the full-length transcript. A reporter gene (CAT), as a separate cistron or fused in-frame, to viral cistrons in various downstream positions in cloned versions of the viral genome was used in a transcription vector to generate artificial full-length transcripts of FMV. When these mRNAs were translated in vitro (rabbit reticulocyte lysate system), the reporter gene was translated efficiently in all positions. Translation of internal native viral gene positioned on the full-length transcript of FMV was also determined (the gene VI product). These observations suggest that the full-length FMV transcript functions as a polycistronic mRNA in plants. Results are best explained on the basis of translational coupling/relay race model.

Animals↗
PubMed1987

Characterization of wheat germ initiation factor eIF-2.

The initiation factor eIF-2 that specifically binds Met-tRNAf and GTP in ternary complex (eIF-2. GTP. Met-tRNAf) has been purified to apparent homogeneity from wheat germ ribosomal salt wash. The purified factor exhibits a sedimentation coefficient of 5 X 5S and an aggregate molecular weight of 122,000-daltons for the native protein.

Centrifugation, Density Gradient↗
PubMed1987

Purification of 2',5'-oligoadenylate synthetase from rabbit reticulocytes.

The 2',5'-oligoadenylate synthetase (2-5A synthetase) from rabbit reticulocytes has been purified to apparent homogeneity. The purification procedure consists of (NH4)2SO4 fractionation (30-50% cut), specific binding of the 2-5A synthetase to and elution from the affinity matrix of polyinosinic-polycytidylic-cellulose, another (NH4)2SO4 precipitation step, and finally chromatography on DEAE-cellulose. Upon electrophoresis in sodium dodecyl sulfate polyacrylamide gel (10%), the purified enzyme migrates as a single polypeptide with an apparent molecular weight of 110,000 daltons. A sedimentation coefficient of 5.8S is obtained by glycerol density gradient centrifugation. The synthesis of 2',5'-oligoadenylate by the purified enzyme is dependent on the presence of double-stranded (ds) RNA, in the absence of which the enzyme is highly unstable. Biochemical characteristics of the purified enzyme have been defined.

2',5'-Oligoadenylate Synthetase↗
PubMed1987

Characterization of Bdellovibrio bacteriovorus bacteriophage MAC-1.

The bacteriophage MAC-1, which specifically infects Bdellovibrio bacteriovorus, was plaque purified and raised to high titre. The phage was purified by NaCl/polyethylene glycol precipitation, followed by two cycles of isopycnic density gradient centrifugation in CsCl. The purified phage exhibited a density of 1.363 g cm-3 and a sedimentation coefficient of 94S. Nucleic acid isolated from purified phage was resistant to hydrolysis under alkaline conditions and to digestion with RNAase, but it was hydrolysed by DNAase, providing evidence that the phage genome is made up of DNA. The lack of hyperchromic effect upon denaturation, hydrolysis of phage DNA by S1 nuclease, characteristic fluorescent staining with acridine orange, and resistance to digestion with a variety of restriction endonucleases are consistent with the DNA being single-stranded. A buoyant density of 1.722 g cm-3 and a sedimentation coefficient of 17.9S were obtained for the phage DNA. The molecular mass of phage DNA was determined as 1.58 MDa by agarose gel electrophoresis with single-stranded DNA as standards. Electron microscopy of the DNA showed that the genome is circular in nature. In addition, using Southern blots, the two replicative forms, RF1 (supercoiled) and RF2 (circular) have been identified and isolated from infected cell extracts.

Bacteriophages↗
PubMed1986

Regulation of protein synthesis in rabbit reticulocyte lysates. Requirement of initiation factor eIF-2 holoprotein for substrate specificity of heme-regulated protein kinase.

The specificity of the heme-regulated protein kinase (HRI) was investigated further by utilizing the isolated 38,000 Da subunit (alpha subunit) polypeptide of eIF-2 as the substrate. For this purpose, the three subunit polypeptides of eIF-2 (38,000 Da, alpha; 50,000 Da, beta; and 52,000 Da, gamma) were resolved by reversed-phase high performance liquid chromatography (HPLC). Results show that HRI is incapable of phosphorylating the 38,000 Da subunit separated from the other two eIF-2 polypeptides. Data suggest that the substrate specificity of HRI is determined by the quaternary structure assumed by the alpha subunit in association with the other two subunits in the eIF-2 holoprotein.

Animals↗
PubMed1986

Regulation of protein synthesis in rabbit reticulocyte lysates. Thiophosphorylation of initiation factor eIF-2 by heme-regulated protein kinase.

The heme-regulated protein kinase, which specifically phosphorylates the 38-kDa subunit of initiation factor eIF-2, can utilize adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma S]) as a substrate. The rate of thiophosphorylation is 5-6-times slower than that observed with ATP. It is of special interest that thiophosphorylated derivatives of eIF-2 are resistant to dephosphorylation catalyzed by eIF-2 phosphoprotein phosphatase. The thiophosphorylated eIF-2 is less effective in promoting protein synthesis in hemin-deficient lysates under physiological conditions. In addition, ATP[gamma S] could also be utilized by the self-phosphorylation activity intrinsically associated with HRI.

Animals↗
PubMed1983

Regulation of protein synthesis in rabbit reticulocyte lysates: separation of heme regulated protein kinase into a high and a low molecular weight species.

Protein synthesis in rabbit reticulocyte lysates is regulated by heme. In heme deficiency, a heme regulated protein kinase (HRI) is activated that phosphorylates initiation factor eIF-2. Consequently, eIF-2 is inactivated. Results described in this report show that HRI exists in crude and highly purified preparations in two forms; a high molecular weight component which sediments at a sedimentation co-efficient of 14-15S and a previously described 5.8S component (Ranu, R. S. and London, I. M. (1976) Proc. Natl. Acad. Sci. USA 73, 4349-4353). The 14-15S HRI selfphosphorylates poorly and undergoes dissociation into the 5.8S component via an intermediate of 8.5-9S. The 5.8S HRI, on weight basis, is about 5-10 times more active than the 14-15S HRI. In addition, a phosphoprotein phosphatase has been detected in lysates that dephosphorylates selfphosphorylated HRI. This observation suggests that phosphate on HRI turns over. These findings may be relevant to the mechanism of activation and inactivation of HRI in the absence and presence of heme in situ.

Animals↗
PubMed1983

Inhibition of eukaryotic protein chain initiation by vanadate.

Vanadate inhibits protein chain initiation in rabbit reticulocyte lysates. The evidence that supports this conclusion is as follows: (i) the biphasic kinetics of inhibition in which protein synthesis is maintained at the control rate for 1-2 min is followed by an abrupt decline in the rate of synthesis; (ii) inhibition is associated with a marked disaggregation of polyribosomes and a concomitant increase in 80S ribosomes; and (iii) vanadate concentrations that inhibit protein chain initiation do not inhibit polypeptide chain elongation or the aminoacylation of tRNA. In partial reactions of protein chain initiation, vanadate concentrations that inhibit protein synthesis have no detectable effect on the formation of eukaryotic initiation factor eIF-2-promoted ternary complex with Met-tRNAf and GTP and on the assembly of 40S ribosomal subunit-Met-tRNAf complexes. On the addition of mRNA, the 40S ribosomal subunit-Met-tRNAf complexes also are transformed into 80S ribosome-mRNA-Met-tRNAf complexes, termed 80S initiation complexes. In vanadate-treated samples, however, these 80S initiation complexes are defective and unable to proceed beyond this step.

Animals↗
PubMed1982

Regulation of protein synthesis in rabbit reticulocyte lysates: effect of of Ca2+, Mg2+, and Mn2+ on self-phosphorylation and heterophosphorylation catalyzed by the heme-regulated and double-stranded-RNA-activated protein kinases.

The influence of divalent cations Mg2+, Mn2+, and Ca2+ on the cyclic-AMP-independent protein kinases of the heme-regulated and double-stranded-RNA-activated translational inhibitory protein kinases on self-phosphorylation and heterophosphorylation of the substrate (the 38 000-dalton subunit of initiation factor eIF-2) has been examined. Results show that Mg2+, Mn2+, and Ca2+ affect the activities of these enzymes in the following fashion. Mg2+ supports both self-phosphorylation and heterophosphorylation efficiently. Mn2+ on the other hand supports self-phosphorylation but to a lesser degree the heterophosphorylation. Ca2+ promotes neither self-phosphorylation nor heterophosphorylation.

Animals↗