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S T Jacob

Publications and source records attributed to S T Jacob.

At least 127 records · Page 7Linked to original sources
PubMed1975

DNA-dependent RNA polymerases I and II from kidney. Effect of polyamines on the in vitro transcription of DNA and chromatin.

DNA-dependent RNA polymerases I and II were purified from pig kidney nuclei by chromatography on DEAE-Sephadex and phosphocellulose. When nonlimiting amounts of double-stranded DNA were used as the template, the in vitro transcription was markedly stimulated by spermidine and spermine. Maximal stimulation of RNA polymerase I occurred at 2-5 mM spermidine and 0.5-2 mM spermine, whereas optimal polyamine concentrations for RNA polymerase II were 5-10 and 1-5 mM for spermidine and spermine, respectively. DNA transcription by polymerase II was stimulated to a greater extent than that of polymerase I. Higher spermine (5-10 mM) concentrations were strong inhibitors of both polymerases under these conditions. The apparent Km of RNA polymerases I and II for UTP was unchanged at optimal polyamine concentration; under the same conditions the maximal reaction velocity was increased two- to three-fold and was essentially due to an increase in the rate of chain elongation. Thus, in a typical experiment the average chain length as determined by the UMP/uridine ratio increased from 570 to 1330 and the chain elongation rate increased from 0.64 to 1.44 nucleotides times sec-1 in the presence of spermine. When limiting quantities of native DNA were employed as the template, both RNA polymerases I and II were inhibited by 1-2 mM spermine. Kidney chromatin could be transcribed by homologous RNA polymerases with an efficiency ranging from 2 to 10% of that with native DNA. When chromatin was used in nonlimiting amounts instead of DNA, RNA polymerase II activity was again stimulated about two-fold at 2 mM spermine. Under these conditions, RNA polymerase I activity was inhibited by spermine. The inhibition of RNA synthesis in vitro at limiting quantities of templates (DNA or chromatin) could be overcome by preincubation of the enzyme with templates before polyamines were added. This inhibition thus appears to be due to a block in the initiation of RNA chains. Similar inhibition of transcription by RNA polymerase II was also observed with limiting quantities of chromatin as the template.

Animals↗
PubMed1975

Mechanism of inhibition of RNA polymerase II and poly(adenylic acid) polymerase by the O-n-octyloxime of 3-formylrifamycin SV.

Factors affecting the inhibition of RNA polymerase II from rat liver by the O-n-octyloxime of 3-formylrifamycin SV (AF/013) were investigated. Using either native or denatured calf-thymus DNA as template, almost complete inhibition of RNA polymerase II was observed when AF/013 was added directly to the enzyme. Considerable resistance to AF/013 was observed when RNA polymerase II was preincubated with denatured DNA at either 0 or 37 degrees. However, under similar conditions, no resistance was observed when enzyme was preincubated with native DNA. Only when AF/013 was added to the ongoing reaction using native DNA did a resistance to AF/013 occur. The inhibition of RNA polymerase II by AF/013 was competitive with respect to all four nucleoside triphosphate substrates. The inhibition by AF/013 remaining after enzyme-DNA complex formation also appeared competitive with nucleoside triphosphate levels. The effect of exogenous protein (bovine serum albumin, BSA) on the inhibition of RNA polymerase II was also investigated. BSA reduced the extent of inhibition by AF/013, but did not alter the competitive nature of inhibition. Concurrently, the inhibition of highly purified nuclear poly(A) polymerase from rat liver, a template independent enzyme which incorporates AMP in a chain elongation reaction, was examined. As in the case of RNA polymerase, poly(A) polymerase was inhibited by AF/013 in a manner competitive with the nucleoside triphosphate substrate. The competitive nature of inhibition of RNA polymerase by AF/013 with respect to all four nucleoside triphosphate substrates, before and after enzyme-DNA complex formation, as well as the competitive nature of inhibition of poly(A) polymerase with respect to ATP tend to indicate that the major effect of AF/013 on RNA polymerase II is at the level of the substrate binding as opposed to a specific inhibition of initiation.

Animals↗
PubMed1975

Mitochondrial poly(A) polymerase from a poorly differentiated hepatoma: purification and characteristics.

Poly(A) polymerase (EC 2.7.7.19) solubilized from mitochondria of a poorly differentiated rat tumor, Morris hepatoma 3924A, was purified more than 1000-fold by successive column chromatography on phosphocellulose, DEAE-Sephadex, and hydroxylapatite. Purified enzyme catalyzed the incorporation of ATP into poly(A) only upon addition of an exogenous primer. Of several primers tested, synthetic poly(A) was the most effective. The enzyme utilized mitochondrial RNA as a primer at least five times as efficiently as nuclear RNA. The enzyme required Mn2+, and had a pH optimum of 7.8-8.2. The enzyme utilized ATP exclusively as a substrate; the calculated K-m for ATP was 28 muM. The polymerization reaction was not inhibited by RNase, ethidium bromide, distamycin, or alpha-amanitin. The reaction was sensitive to O-n-octyloxime of 3-formylrifamycin SV (AF/013). As estimated from glycerol gradient centrifugation and acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight of the enzyme was 60,000. The product was covalently linked to the polynucleotide primer and the average length of the poly(A) formed was 600 nucleotides.

Animals↗
PubMed1974

Role of RNA in induction of hepatic microsomal mixed function oxidases.

Induction of hepatic microsomal cytochrome P-450 and ethylmorphine N-demethylase activity by phenobarbital requires de novo synthesis of mRNA. Inhibition of RNA synthesis by alpha-amanitin given up to 8 hr after phenobarbital administration substantially inhibits this induction. However, beyond 8 hr after phenobarbital administration, RNA synthesis is not required for induction of these hepatic microsomal systems. Thus, mRNAs for cytochrome P-450 and ethylmorphine N-demethylase appear to be stable. Furthermore, these experiments reveal that the lag period for RNA synthesis approximates the length of the lag period for induction of the hepatic microsomal enzyme systems.

Animals↗
PubMed1974

Inhibition of poly(A) polymerase by rifamycin derivatives.

The effect of several rifamycin derivatives on poly(A) synthesis in vitro was tested using purified rat liver mitochondrial poly(A) polymerase assayed with an exogenous primer. When used at a concentration of 300 mug/ml, derivatives AF/013, PR/19, AF/AETP, M/88 and AF/ABDP completely inhibited activity corresponding to 50 mug of enzyme protein. Under similar conditions, derivatives DMAO and AF/MO failed to inhibit enzyme activity. Studies with PR/19 showed that the drug interacted directly with the enzyme molecule and did not affect the enzyme-primer complex formation. The inhibition by the drug could be reversed by increasing the substrate (ATP) concentration. It is concluded that some rifamycin derivatives can specifically inhibit template-independent nucleotide chain elongation reactions.

Adenosine Triphosphate↗