John J. Furth

Comparison of transcription of chromatin by claf thymus and E. coli RNA polymerases

Abstract Kinetic parameters of transcription of DNA and chromatin by calf thymus and E. coli RNA polymerases were studied. Using calf thymus RNA polymerase, the theoretical maximum velocity (Vmax) for chromatin is similar to that for DNA. The concentration of chromatin required to reach one-half the maximum velocity (Km) is greater than the Km for DNA. Using E. coli RNA polymerase, the Km for chromatin is similar to the Km for DNA, whereas the Vmax for chromatin is much less than the Vmax for DNA. These differences suggest that calf thymus RNA polymerase binds to specific, selected sites on chromatin and transcribes at all of these sites. E. coli RNA polymerase binds to many non-selected sites and transcribes at some or all of them.

DNA dependent RNA polymerase of KB cells I. Isolation of the enzymes and transcription of viral DNA, mammalian DNA and chromatin

Abstract 1. Two forms (nucleolar and nucleoplasmic) of RNA polymerase (ribonucleoside triphosphate: RNA nucleotidyl transferase, EC 2.7.7.6) have been isolated from KB cells, a neoplastic human cell line. The transcriptional properties of these enzymes have been studied using a variety of natural templates. 2. The properties of RNA polymerase obtained from adenovirus-infected cells are similar to those of polymerase from uninfected cells. 3. Adenovirus DNA is a relatively ineffective template for both forms of KB RNA polymerase. Optimal salt and cation conditions for transcription of adenovirus DNA differ from the conditions which are optimal for transcription of mammalian DNA. 4. Both nucleolar and nucleoplasmic forms of the enzyme transcribe chromatin. Optimal salt conditions for the transcription of chromatin differ from the conditions which are optimal for the transcription of DNA.

Deoxyribonucleic acid-dependent ribonucleic acid synthesis in Escherichia coli infected with bacteriophage T2

Following infection of Escherichia coli by the bacteriophage T2, ribonucleic acid polymerase activity in unfractionated extracts is reduced to approximately twenty per cent of the activity in unfractionated extracts of uninfected cells. The residual enzyme activity is sensitive to deoxyribonuclease, to ribonuclease and to actinomycin D. RNA polymerase can be purified from extracts of T2-infected cells with a yield of more than 200%. The purified enzyme has properties similar to the enzyme purified from uninfected cells. Deoxyribonucleic acid isolated from T2-infected cells is a mixture of high molecular weight bacterial and viral DNA. Both components of this mixture are active as templates for RNA polymerase in vitro, although only the viral DNA is a template in vivo. As reported by Skold & Buchanan (1964) , material which inhibits purified RNA polymerase in vitro appears in cell extracts following T2 infection. The inhibitory material is destroyed by acid treatment and reduced in amount by digestion with alkali. Incubation overnight at 37°C and heating at 100°C increase the amount of inhibitory material. In control experiments with uninfected E. coli, inhibitory material is not present in the initial extract or after treatment with acid. Treatment with alkali, incubation overnight at 37°C or heating at 100°C result in levels of inhibitory material similar to that in extracts of infected cells subjected to the same treatment.

The enzymatic synthesis of ribonucleic acid in animal tissue: III. Further purification of soluble RNA polymerase from lymphoid tissue and some general properties of the enzyme

Abstract 1. A procedure has been developed for the purification of RNA polymerase (ribonucleoside triphosphate: RNA nucleotidyltransferase EC 2.7.7.6) from lymphoid tissue, calf thymus and bovine lymphosarcoma, without first isolating nuclei. The preparation was soluble, free of DNA and dependent on DNA for activity, and RNA synthesized by the enzyme specifically hybridized with DNA used as template. 2. Although stimulated by KCl and (NH 4 ) 2 SO 4 , the reaction still ceased after approx. 2 h. 3. Actinomycin inhibited the mammalian enzyme at the same concentration as that which inhibited Escherichia coli enzyme. In contrast, rifamycin failed to inhibit the mammalian enzyme.

Removal of DNA from RNA by chromatography on acetylated N-[N′-(m-dihydroxylborylphenyl)succinamyl]aminoethyl cellulose

Abstract RNA synthesized in vitro can be separated from contaminating DNA by chromatography on acetylated N-[N′-(m -dihydroxyborylphenyl)succinamyl]aminoethyl cellulose. The recovery of RNA is greater than 95%, and essentially all the DNA is removed. This procedure is extremely useful in studies in which DNA contamination of RNA interferes with subsequent analysis.

THERMAL INACTIVATION OF THE PRIMER IN DNA-DEPENDENT SYNTHESIS OF RNA IN ANIMAL TISSUE.

An enzyme preparation obtained from bovine lymphosarcoma tissue catalyzes the DNA-dependent synthesis of RNA. Native DNA is a more efficient primer in the reaction than heat-denatured DNA.

Transcription of chromatin by an RNA polymerase of calf thymus which is sensitive to high concentrations of α-amanitin

Abstract RNA polymerase which is sensitive to high concentrations of α-amanitin has been obtained by low-salt extraction of whole calf thymus tissue. Some characteristics of this Form III-type polymerase have been compared with Form II. Both enzymes require a higher concentration of purine nucleoside triphosphates compared to pyrimidine nucleoside triphosphates. The Form III-type polymerase is larger than Form II. It transcribes native DNA better than denatured DNA and transcribes chromatin efficiently. Under various salt and metal conditions Form II transcribes native DNA and chromatin poorly relative to denatured DNA. These results suggest that Form III RNA polymerase is a useful enzyme with which to study the transcription of chromatin in vitro and may possess some factor (which Form II lacks) which is necessary for transcription of double-stranded templates.

Genes for collagen types I, IV, and V are transcribed in Hela cells but a postinitiation block prevents the accumulation of type I mRNA☆

Collagen mRNA synthesis in HeLa cells was evaluated by in vitro transcription of type I collagen DNA, nuclear run-on studies, and steady-state mRNA analysis. Type I collagen mRNA was accurately initiated by HeLa cell RNA polymerase II in nuclear extracts, and run-on analysis indicted that mRNAs for collagen types alpha 1(I), alpha 2(I), alpha 1(III), alpha 1(IV), and alpha 2(V) were synthesized in HeLa cells. However, on assessing the steady-state levels of mRNAs of collagen types alpha 1(I), alpha 2(I), alpha 1(IV), and alpha 2(V), no type I mRNA was found in HeLa cells while types alpha 1(IV) and alpha 2(V) collagen mRNAs were observed. These results suggest that a postinitiation process prevents the accumulation of type I collagen mRNAs in HeLa cells. Persistence of types IV and V collagen mRNAs is consistent with the involvement of types IV and V collagen in adhesion of HeLa cells to glass or plastic.

Duplication of single stranded DNA catalyzed by calf thymus DNA polymerase alpha.

Purified calf thymus DNA polymerase alpha is inactive with native DNA as template and shows little activity with denatured DNA. DNA synthesis with denatured DNA as template is greatly stimulated by the addition of a nuclease which initially copurifies with DNA polymerase but is separated from the polymerase on DEAE-cellulose chromatography. A limit digest of nuclease treated native DNA which is then denatured is replicated 80-95%; extensive replication is also obtained with native DNA partially degraded by pancreatic DNase and then denatured. The product of the reaction with calf thymus nuclease-treated DNA as template is double-stranded DNA with a hairpin (looped back) structure.

An endonuclease from calf thymus which introduces a limited number of single strand breaks into DNA

Summary A nuclease that initially co-purifies with calf thymus DNA polymerase has been separated from the polymerase by DEAE-cellulose chromatography. The nuclease introduces single-strand nicks into calf thymus DNA and degrades the DNA to a discrete size. Native DNA incubated with the nuclease averages 7S in alkaline sucrose gradients; denatured DNA incubated with the nuclease is largely 3S.