Search PubMed⌕ Search

Biomedical subjects

N Sarkar

Publications and source records attributed to N Sarkar.

At least 73 records · Page 4Linked to original sources
PubMed1983

Synthesis of polyadenylate-containing RNA in vitro in permeable cells of Escherichia coli B.

As a starting point for the study of the biosynthesis of polyadenylated RNA in bacteria, the characteristics of RNA synthesis by cells of Escherichia coli B made permeable to small molecules by treatment with toluene were examined. Such cells mediated the incorporation of radiolabeled ribonucleoside triphosphates into RNA in a reaction that was sensitive to inhibitors of RNA polymerase and required the simultaneous presence of the four ribonucleoside triphosphates. Between 10 to 15% of the RNA synthesized under these conditions was polyadenylated as shown by affinity chromatography on oligo(dT)-cellulose. The presence of orthophosphate or dADP, inhibitors of polynucleotide phosphorylase, had no effect on the reaction and the rate of RNA synthesis was indistinguishable in the polynucleotide phosphorylase-deficient strain PR-7 and in its otherwise isogenic parent strain PR-100. The poly(A) tracts associated with the newly synthesized RNA could be isolated after exhaustive digestion with pancreatic and T1 ribonucleases and accounted for 14% of the poly(A)-RNA. At least 74% of the poly(A) sequences were located at the 3' ends of RNA molecules and their weight-average length was 48 nucleotide residues. The size distribution of total RNA and poly(A)-RNA synthesized in the toluenized cell system was similar to that of the corresponding pulse-labeled fractions derived from growing cultures. The sequence complexity of poly(A)-RNA and unadenylated RNA synthesized in toluenized cells with [alpha-32P]CTP as the labeled substrate was analyzed by hybridization to fragments of Escherichia coli B DNA generated by digestion with EcoRI restriction endonuclease and immobilized on nitrocellulose sheets. Both RNA fractions hybridized with many DNA fractions, the hybridization patterns being similar with poly(A)-RNA and unadenylated RNA. This indicated that many different types of RNA transcripts synthesized in toluenized cells were subject to polyadenylation, but that polyadenylation was incomplete so that each transcript was present in both an adenylated and an unadenylated state.

Base Sequence↗
PubMed1983

Analysis of the 5'-termini of nascent DNA chains synthesized in permeable cells of Bacillus subtilis.

The nascent DNA synthesized by permeable cells of Bacillus subtilis in the presence of 5'-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP has been isolated and characterized. The newly synthesized DNA was isolated free from other cellular nucleic acids by affinity chromatography on thiol-substituted agarose. The number average chain length of the nascent DNA synthesized in one minute at 25 degrees C was 33 nucleotide residues, due to the chain-terminating action of 2',3'-dideoxyATP. Several lines of evidence indicated that at least 90% of the DNA thus isolated carried a terminally phosphorylated RNA moiety at its 5'-end: (1) the nascent DNA was resistant to exonucleolytic degradation by spleen phosphodiesterase unless first hydrolyzed by strong alkali or ribonuclease; (2) the 5'-termini of nascent DNA could not be phosphorylated by polynucleotide kinase unless first treated with alkaline phosphatase or subjected to hydrolysis by strong alkali or ribonuclease; (3) alkaline hydrolysis of nascent DNA labeled with 32P at the 5'-end released unlabeled DNA with a free 5'-terminus and 32P-labeled ribonucleoside 3',5'-bisphosphates; (4) ribonuclease degradation of similarly labeled material produced an unlabeled DNA-containing polynucleotide fraction and 32P-labeled ribo-oligonucleotides; (5) chromatography on dihydroxyboryl cellulose showed that the RNA moiety lacked a 3'-terminal cis-diol grouping (even after treatment with alkaline phosphatase) unless first subjected to the 3'-exonucleolytic action of bacteriophage T4 DNA polymerase. The sequence of the ribonucleotide chains was elucidated by end-group labeling with polynucleotide kinase and digestion with various ribonucleases. The ribonucleotide moiety was primarily three and four residues in length with the predominant sequence (pp)pApG(pC)1-2pDNA. The possibility that it represents a primer for discontinuous DNA synthesis is discussed.

Bacillus subtilis↗
PubMed1982

The synthesis of DNA complementary to polyadenylate-containing RNA from Bacillus subtilis.

We had shown earlier (Gopalakrishna, Y., Langley, D., Sarkar, N. (1981) Nucleic Acid Res. 9, 3545-3554) that a substantial fraction of mRNA of various bacterial species carries 3'-terminal polyadenylate sequences. In this paper, we show that poly(A)-containing RNA from Bacillus subtilis can serve as template for the synthesis of complementary DNA by avian myeloblastosis virus reverse transcriptase, provided that oligodeoxythymidylate is added as primer. Poly(A)-RNA purified by affinity chromatography on oligo(dT)-cellulose was 20 times more effective as template for cDNA formation than total bacterial RNA, whereas rRNA was inactive. The average chain length of the cDNA was 400 nucleotides (range = 230-800 nucleotides), and 95% of the cDNA could be degraded by the single-strand specific S1 nuclease after denaturation. The small fraction (5%) that was resistant to S1 nuclease may represent duplex hairpin structures. Annealing with poly(A)-RNA protected cDNA from degradation by S1 nuclease, indicating that cDNA indeed contains nucleotide sequences complementary to poly(A)-RNA. These results constitute independent evidence that a large fraction (about 40%) of B. subtilis mRNA is polyadenylated. Moreover, the synthesis of cDNA to bacterial mRNA provides an important new tool for the study of bacterial mRNA structure.

Bacillus subtilis↗
PubMed1982

Transforming activity of mercury-substituted DNA synthesized in vitro by permeable cells of Bacillus subtilis.

Mercurated DNA was synthesized in permeable cells of Bacillus subtilis, using 5-mercurideoxycytidine triphosphate as one of the substrates, and was separated from parental unsubstituted DNA by isopycnic centrifugation in CsCl gradients. The ability of mercurated DNA to transform auxotrophic strains of B. subtilis to prototrophy was compared with that of normal DNA and was 10-20% of the latter. Mercurated and normal DNA bound with similar affinities to identical surface receptors of the recipient cells, but the efficiency of the bound mercurated DNA in promoting transformation was one-tenth to one-fifth of that of normal DNA. The transforming activity of mercury-substituted DNA synthesized in an in vitro system opens the way for the use of mercury as a probe to study the mechanism of bacterial transformation and various other kinds of genetic exchange.

Bacillus subtilis↗
PubMed1981

Detection of high levels of polyadenylate-containing RNA in bacteria by the use of a single-step RNA isolation procedure.

A new one-step procedure for the isolation of bacterial RNA, involving lysis by proteinase K in the presence of sodium dodecyl sulfate, is described. Pulse-labeled RNA isolated by this procedure for Bacillus brevis, Bacillus subtilis, and Escherichia coli B has been found to contain a substantial fraction (15-40%) of polyadenylated RNA as determined by adsorption to oligo(dT)-cellulose. This contrasts with RNA isolated by procedures involving phenol extraction, a process which appears to lead to the selective loss of polyadenylated RNA. The presence of polyadenylated RNA in E. coli was confirmed by an independent method which involved hybridization with [3H]polyuridylic acid. Using the proteinase K method for RNA isolation, it was possible to demonstrate the in vitro synthesis of polyadenylated RNA by toluene-treated cells of B. brevis, B. subtilis, and E. coli.

Bacillus↗
PubMed1981

ATP requirement for in vitro DNA synthesis in permeable cells of Bacillus brevis.

DNA synthesis in permeable cells of Bacillus brevis is almost completely dependent on the presence of ATP. Nevertheless, DNA synthesis in this system reflects primarily DNA repair rather than replication, as shown by the response to replication inhibitors, density shift experiments, and product size analysis. The apparent dependence of DNA synthesis on ATP was due to the stabilization of the deoxyribonucleoside triphosphate pools against degradation. These observations indicate that dependence of DNA synthesis on ATP is not a sufficient criterion for semiconservative DNA replication.

Adenosine Triphosphate↗
PubMed1981

Inhibition of deoxyribonucleic acid replication in Bacillus brevis by ribonucleic acid polymerase inhibitors.

The incorporation of [3H]thymidine into deoxyribonucleic acid by exponentially growing cells of Bacillus brevis was inhibited by streptolydigin and rifampin in the same concentration range in which these drugs inhibit ribonucleic acid synthesis. Complete inhibition occurred within one-third generation time after drug addition, suggesting an effect on deoxyribonucleic acid chain elongation.

Aminoglycosides↗
PubMed1979

Comparison of the effect of linear gramicidin analogues on bacterial sporulation, membrane permeability, and ribonucleic acid polymerase.

Various analogues of linear gramicidin were tested for their biological activity in restoring the normal spore phenotype of gramicidin-negative mutants of Bacillus brevis and for their ability to increase cation conductivity of black lipid membranes and to inhibit bacterial RNA polymerase. Whereas many biologically active gramicidin analogues had no effect on membrane permeability, all biologically active peptides were able to inhibit ribonucleic acid (RNA) polymerase. These observations make it unlikely that membranes are the site of action of gramicidin during bacterial sporulation, but they are consistent with the notion that gramicidin functions to control RNA synthesis during the transition from vegetative growth to sporulation (Sarkar & Paulus, 1972). The relationship between peptide structure and the ability to restore normal sporulation and inhibit RNA polymerase showed that the eight amino-terminal residues have little influence on the function of gramicidin, whereas the highly nonpolar repeating sequence D-leucyl-L-tryptophan is essential for biological activity and may represent the site of interaction with RNA polymerase.

Bacillus↗
PubMed1979

The leukocyte migration inhibition response to certain breast cancer-related antigens (MCF-7 and MuMTV): their potential as discriminants.

Certain oncogenic viruses have been implicated in human breast cancer, including the murine mammary tumor virus (MuMTV) and the Mason-Pfizer monkey virus (MPMV). We have used the leukocyte migration inhibition (LMI) response to assay the response to several potential breast cancer-related antigens, including MuMTV, MPMV, and a breast cancer cultured cell line, MCF-7, in 96 breast cancer patients, in 32 women with benign breast disease, and in 67 normal women. The lowest tenth percentile of control (LMI) responses was used as the cutoff point to designate responders. Breast cancer patients showed significant responses to MuMTV (49% and to MCF-7 (50%), but not to MPMV (29%). In a paired-antigen study using MuMTV and MCF-7, 75% of the breast cancer patients responded, versus 18% of the normal women (P less than 0.0050). The potential for this assay to distinguish "normal" from "breast cancer" was analyzed using a migration index derived from discriminant analysis. The ability of the assay to discriminate "normal" from "cancer" was significant (P less than 0.001) and showed a sensitivity of detecting "cancer" of 75%. The overall responses to MuMTV and MCF-7 were analyzed with reference to certain prognostic factors, but showed no relation to age, menstrual status, estrogen receptor status, or stage of disease. The above reactions suggest that a large proportion of breast cancer patients exhibit presensitization to antigenfs found in MuMTV and MCF-7, which may be cross-reactive with antigens in the primary cancer. These responses appear to be independent of major prognostic variables. Further refinement of this assay may yield one which is more highly discriminating for breast cancer.

Adult↗
PubMed1978

Isolation and characterization of polyadenylate-containing RNA from Bacillus brevis.

A substantial fraction (30--40%) of pulse-labeled RNA from exponentially growing cells of Bacillus brevis contains polyadenylate sequences, as measured by adsorption to oligo(dT)-cellulose. The weight-average length of poly(A) tracts obtained after digestion with pancreatic and T1 ribonucleases is 60 nucleotide residues. Susceptibility to degradation by snake venom phosphodiesterase after ribonuclease degradation indicates that the poly(A) sequences are located near the 3' ends of the RNA chains, but that in 40% of the material at least one internal pyrimidine nucleotide residue intervenes between the poly(A) tract and the 3'-hydroxyl terminus. These pyrimidine nucleotides consist of 65% cytidylate and 35% uridylate residues. In the remaining RNA chains, the poly(A) sequence is directly at the 3'-terminus, but the possibility cannot be excluded that a small fraction of this material may contain a 3'-hydroxyl terminal guanylate residue. The weight-average sedimentation coefficient of poly(A)-containing RNA is 12.5 S, corresponding to a polynucleotide chain length of 800--900 residues. This is in a size range expected for messenger RNA, a possibility which is also supported by the observation that pulse-labeled RNA has a considerably higher poly(A) content than long-term labeled RNA.

Bacillus↗
PubMed1977

Biological function of gramicidin: selective inhibition of RNA polymerase.

This paper describes a novel biochemical effect of gramicidin, a class of peptide antibiotics produced by Bacillus brevis during the transition from vegetative growth to sporulation. Gramicidin inhibits RNA synthesis by purified RNA polymerase (nucleosidetriphosphate:RNA nucleotidyl-transferase, EC 2.7.7.6) by interfering with the binding of RNA polymerase to DNA. This effect seems to involve the destabilization of the "open" RNA polymerase-DNA complex, a mode of action consistent with the control of promoter selection. Selectivity in the inhibition of RNA synthesis by gramicidin is observed when transcription is partially blocked by low levels of actinomycin D. Since the inhibition of RNA synthesis by gramicidin is obtained in a highly purified system devoid of membranes, it must be distinct from the ionophoretic activity of the antibiotic. It is possible that this new mode of action reflects the function of gramicidin during bacterial sporulation.

Bacillus↗
PubMed1975

A guanosine 3':5'-monophosphate-sensitive nuclease from Bacillus brevis.

In toluene-treated cells of Bacillus brevis, newly synthesized RNA is rapidly degraded in a reaction that is inhibited by cyclic guanosine 3':5'-monophosphate (cGMP) and by 1,10-phenanthroline. This appears to be due to a ribonuclease found in cell-free extracts of B. brevis which is inhibited by cGMP and related compounds as well as by 1,10-phenanthroline. The cGMP-sensitive nuclease hydrolyzes synthetic polynucleotides, yielding nucleoside 5'-monophosphates as the sole products, even during the early stages of hydrolysis. Synthetic polynucleotides terminated by a 3'-phosphate are resistant to hydrolysis. While with 3'-hydrolysis of the polymer. The enzyme is therefore an exonuclease that degrades polynucleotides from the 3' end to product 5'-mononucleotides. It also acts on denatured but not on native DNA. Activity is greatest in the presence of Mn2+ and is not affected by the presence of monovalent cations. 1,10-Phenanthroline, but not 1,7-phenanthroline, inhibits the nuclease even when Mn2+ is present in excess. The inhibition of the enzyme by cGMP is noncompetitive, and cGMP itself is not hydrolyzed. The sensitivity of the nuclease to inhibition depends strikingly on the nature of the substrate and is lost when the enzyme is assayed at high pH. These observations suggest that cGMP inhibits the nuclease by combining with an allosteric site on the enzyme. Although cGMP was found to be the most effective inhibitor, other nucleoside 3':5'-monophosphates and derivatives of 5'-GMP can also inhibit the nuclease. Since measurements of cGMP in B. brevis have not revealed detectable amounts (less than 5 times 10-8 M), the substance that modulates the activity of the nuclease under physiological conditions remains to be identified.

Bacillus↗