Inhibition of mammalian polyadenylate polymerase by 2-aza-1,N6-etheno-adenosine triphosphate.

2-Aza-1,N6-etheno-adenosine triphosphate (aza-epsilonATP), a fluorescent analog of adenosine triphosphate, significantly inhibits polyadenylate [poly(A)] polymerase of bovine lymphosarcoma and calf thymus, with 50% inhibition at 200 muM (in the presence of an equal concentration of adenosine triphosphate). Calf thymus RNA polymerases II and III are inhibited 32 and 20%, respectively, by a 3.8-fold excess of aza-epsilonATP; DNA polymerase alpha is not inhibited. The inhibition of poly(A) polymerase by aza-epsilonATP appears to be competitive with adenosine triphosphate; incorporation of aza-epsilonATP is not observed. Polymers of 2-aza 1,N6-etheno-adenosine monophosphate are used as primers, but pootly. 1,N-Etheno-adenosine triphosphate and 9-beta-D-arabinofuranosyladenine triphosphate are poor inhibitors of poly(A) polymerase; adenosine diphosphate is ineffective. Deoxyadenosine triphosphate inhibits to the same extent as aza-epsilonATP, while other naturally occurring nucleotides inhibit poly(A) polymerase to varying degrees, with deoxynucleoside triphosphates more potent than ribonucleoside triphosphates. Inhibition of poly(A) polymerase by naturally occurring nucleoside triphosphates suggests that nucleotides may regulate the enzyme in vivo; inhibition by the fluorescent analog aza-epsilonATP suggests that this compound may be useful in elucidating poly(A) metabolism in both normal and neoplastic cells.

Poly (A) polymerase of bovine lymphosarcoma.

Poly(A) polymerase has been extensively purified from low-salt extracts of bovine lymphosarcoma. The enzyme is Mn2+ dependent, requires an oligonucleotide or RNA primer, incorporates only adenosine triphosphate, and is inhibited by other ribonucleotides or deoxynucleotides. Oligoadenylate and ribosomal RNA are good primers for the enzyme; transfer RNA and poly(A) are poor. RNA transcribed in vitro by homologous RNA polymerase is an efficient primer. The properties of the enzyme are similar to the properties of the Mn2+ -activated poly(A) polymerase of calf thymus. Approximately the same amount of enzyme appears to be present in lymphosarcoma and calf thymus.

RNA polymerase isolated from bovine lymphosarcoma by sequential low- and high-salt extraction.

Bovine lymphosarcoma tissue has been extracted with low- and high-salt buffers [0.05 M Tris-C1 plus or minus 0.3 M (NH4) 2S04]. Diethylaminoethyl-Sephadex chromatography of both the high-salt and low-salt extracts yields RNA polymerases I and II, although low-salt extraction releases only one-third as much activity. Extraction by high salt of the residue from the low-salt extract, followed by diethylaminoethyl-Sephadex chromatography, yields additional enzyme activity with properties of Form II. Purification of the low-salt extract by protamine precipitation, elution with sodium succinate, and phosphocellulose chromatography yields a preparation of RNA polymerase (RNAP) with hybrid properties, combining the salt optimum of Form I, diethylaminoethyl-Sephadex elution pattern of form II, and alpha-amanitin sensitivity of Form III. RNAP. transcribes native D,A and chromatin efficiently. More RNAPL is recovered from lymphosarcoma tissue than from calf thymus.

Abundance of alpha 1(I) and alpha 1(III) procollagen and p21 mRNAs in fibroblasts cultured from fetal and postnatal dermis.

The steady-state abundance of alpha 1(I) and alpha 1(III) procollagen mRNAs, p21Sdi1 mRNA, and beta-actin mRNA was determined in 29 skin fibroblast lines established from fetal, young and old donors. Donor ages ranged from 12 gestational weeks to nonagenarian. Adult donors were members of the Baltimore Longitudinal Study of Aging. The abundance of alpha 1(I) procollagen mRNA was decreased in cell lines from both young and old donors compared with fetal lines. Additionally, one alpha 1(I) transcript observed in the fetal lines was not detected in postnatal lines. The abundance of alpha 1(III) procollagen mRNA was decreased in postnatal lines from old donors compared with fetal lines. The abundance of beta-actin mRNA was lower in postnatal lines from both young and old donors compared to fetal lines. These results suggest that cultures of fetal skin fibroblasts exhibit a greater capacity for synthesis of procollagens and beta-actin than postnatal lines. In contrast, the abundance of p21Sdi1 mRNA was elevated in lines established from postnatal donors. These results are consistent with developmental changes in amounts of procollagen, beta-actin and p21.

The steady-state levels of type I collagen mRNA are reduced in senescent fibroblasts.

The decreased collagen content in aging skin could be a consequence of decreased synthesis or increased degradation. The possibility that decreased synthesis of collagen results from decreased synthesis of mRNAs for Type I collagen, the major collagen in skin, was investigated by assessing the steady-state levels of alpha 1(I) and alpha 2(I) collagen mRNAs in actively proliferating and senescent WI-38 fibroblasts. The levels of both alpha 1(I) mRNA and alpha 2(I) mRNA were significantly lower in senescent fibroblasts, suggesting that one factor contributing to the decreased collagen content of aging skin may be decreased synthesis of these collagen mRNAs by senescent fibroblasts. Both mRNAs were reduced to the same extent, suggesting that coordinate regulation of the two Type I collagen genes is maintained in senescent fibroblasts.

Transcription of repeat DNA in vitro by RNA polymerase III in cytosol extracts is inhibited when the extract is of the same species as the DNA.

Cytosol extracts containing RNA Polymerase III were obtained from cultured human (HeLa) and bovine (MDBK) cells. Human extract efficiently transcribed genomic bovine DNA and cloned bovine alu-type repeat DNA while genomic human DNA and cloned human alu repeat DNA were inefficient templates. Conversely, genomic bovine DNA and cloned bovine alu-type repeat DNA were less effective templates for bovine extract than were genomic human DNA and alu repeat DNA. These results suggest that species specific factor(s) present in mammalian cells are responsible for the observed in vivo inhibition of transcription of most alu-type repeat DNA.

Transcription of genomic bovine and Xenopus laevis DNA species by RNA polymerase III in HeLa-cell cytosol extracts.

RNA transcribed from genomic Xenopus laevis DNA by RNA polymerase III in HeLa-cell extract was found in discrete size classes and was transcribed from at least two different Xenopus repeat DNA species. Very little 5S ribosomal RNA was transcribed, contrasting with results obtained on transcription of genomic Xenopus DNA by Xenopus extract [Bogenhagen et al. (1982) Cell 28, 413-421]. The low transcription was not due to an inability to use 5S rDNA templates, since the cloned Xenopus 5S ribosomal gene and pseudogene were effective templates for RNA polymerase III in HeLa extract. RNA transcribed from genomic bovine DNA by RNA polymerase III in HeLa-cell cytosol extract consisted of 120-nucleotide RNA and a larger amount of heterogeneously sized RNA (180-650 nucleotides). Only a small portion of the 120-nucleotide RNA was 5S rRNA. Most of the 120-nucleotide RNA and the larger RNA species were transcribed from one bovine repeat DNA. Genes for 5S rRNA and bovine repeat DNA were transcribed roughly in proportion to their frequency in Bos, contrasting with results in a homologous system in which transcription of repeat genes is repressed [Furth (1985) Biochem. Biophys. Res. Commun. 131, 551-556]. Bovine 5S rRNA genes appear to be concentrated on one DNA fragment obtained by restriction-enzyme-HindIII digestion of genomic bovine DNA.

RNA synthesized in vitro by calf thymus RNA polymerase III (C), as well as by E. coli RNA polymerase, is restricted to a subset of calf thymus DNA.

RNA synthesized in vitro from chromatin and DNA by calf thymus RNA polymerase III was evaluated by hybridization in vast DNA excess. The RNA contains RNA complementary to both moderately repeated and unique DNA sequences. Very highly repeated DNA is not transcribed. A greater portion of RNA transcribed from DNA by RNA polymerase III hybridizes to moderately repeated DNA than RNA transcribed by Escherichia coli RNA polymerase. In studies utilizing DNA absorbed to filters, RNA transcribed from chromatin in short incubations hybridized to a greater extent than RNA transcribed for longer times. Similar results were obtained with RNA transcribed from DNA by E. coli RNA polymerase. These results suggest: 1) RNA polymerase III may be responsible for the synthesis of RNA species in addition to tRNA and 5 S ribosomal RNA and a portion of this RNA is transcribed from unique DNA; and 2) in vitro there may be selectivity in the initiation of transcription by both E. coli RNA polymerase and calf thymus RNA polymerase III.

Mammalian endonuclease, DNase V. Purification and properties of enzyme of calf thymus.

An endonuclease present in partially purified preparations of calf thymus DNA polymerase has been purified to homogeneity. It has a molecular weight of 53,000 +/- 2,500 as determined by sucrose gradient sedimentation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates the protein is composed of four subunits, each polypeptide possessing a molecular weight of 13,000. Its isoelectric point is 10.3 +/- 0.2. The endonuclease has a pH optimum at 6.6, requires Mg2+ or Mn2+ for activity, and does not attack RNA. The enzyme appears to be present in tissues other than calf thymus. The enzyme catalyzes the endonucleolytic cleavage of both denatured and native eukaryotic DNA. The enzyme introduces a limited number of single strand nicks into native DNA; hydrolysis of denatured DNA produces acid-soluble oligonucleotides. The average size of the limit product, sedimented in an alkaline sucrose gradient, is 1200 nucleotides for native DNA. The product contains 5'-phosphoryl and 3'-hydroxyl termini. While all four deoxynucleotides are found at the 5' termini, pyrimidine residues predominate. Calf thymus DNase V degrades closed circular duplex SV40 DNA and glucosylated T4DNA but not poly(dA-dT). The rate of hydrolysis of homopolymers is: poly(dT) greater than poly(dA) greater than poly(dC) greater than poly(dG) in the presence of Mg2+, and poly(dT) greater than poly(dC) greater than poly(dA) = poly(dG) in the presence of Mn2+.

De novo synthesis of a polymer of deoxyadenylate and deoxythymidylate by calf thymus DNA polymerase alpha.

In a reaction mixture containing calf thymus DNA polymerase alpha (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7), calf thymus DNA unwinding protein, DNA, deoxyadenosine 5'-triphosphate and deoxythymidine 5'-triphosphate, a copolymer of deoxyadenylate and deoxythymidylate is synthesized after a lag period of 1-2 hr. In the presence of the four deoxyribonucleoside triphosphates only deoxyadenylate and deoxythymidylate are incorporated into the polymer and the rate of synthesis is decreased. The reaction variably occurs in the absence of DNA or DNA unwinding protein but with a greatly entended lag period. The optimal Mg2+ concentration for synthesis of the polymer of deoxyadenylate and deoxythymidylate is 1 mM, in contrast to an optimal Mg2+ concentration of 8 mM for DNA synthesis with activated DNA as template. Characterization of the product of de novo synthesis indicates that it is the alternating copolymer, poly(dA-dT).