Nucleic Acids Research

Shaw, Gray, D.;Boll,, Werner;Taira,, Hideharu;Mantei,, Ned;Lengye,, Peter;weissmann,, Charles

doi: 10.1093/nar/11.3.555pmid: 6188104

Abstract The mouse has an interferon-a (MuIFN-a) gene family containing at least four, and likely more than ten members. A segment of mouse chromosomal DNA and cDNAs encoding murine alpha IFNs have been cloned, and the sequence of two MuIFN-a DNAs determined. No intron was found in the chromosomal gene. The two coding sequences produced biologically active IFN when expressed in monkey cells under the control of an SV40 promoter, and in E.coli under the control of the ampicillinase promoter. MuIFN-al had no detectable activity on human cells, while MuIFN-a2 was 201 as active on human as on mouse cells. This content is only available as a PDF. Author notes *Present address:Biogen Inc., 241 Binney Street, Cambridge, MA 02142, USA +Department of Pure and Applied Sciences, University of Tokyo, 3-8-1 Komaba Meguro-ku Tokyo 153 Japan © IRL Press Limited

Bertram,, S.;Göringer,, U.;Wagner,, R.

doi: 10.1093/nar/11.3.575pmid: 6340062

Abstract SUMMARY Kethoxal modification of guanosines within phe-tRNAphe from E.Coli was studied for tRNA in the free state and specifically bound to the ribosomal A-site. Complex formation with the ribosome results in a protection from chemical modification of two distant sites in the tRNA molecule. The guanosines affected are G-18 and G-19, located in the D-loop, and G-34 in the anticodon loop. Modification of phe-tRNAphe in the absence of ribosomes leads to a destabilisation of the tRNA structure. Our data are consistent with the conclusion that mocification of G-34 at the anticodon loop triggers a conformational instability in distant parts of the tRNA molecule. This content is only available as a PDF. © IRL Press Limited

Pieler,, T.;Digweed,, M.;Bartsch,, M.;Erdmann, V., A.

doi: 10.1093/nar/11.3.591pmid: 6340063

Abstract 5S rRNAs from Splnaceaoleracea cytoplasmic and chloroplastic ribosomes have been subjected to digestion with the single strand specific nuclease S1 and to chemical modification of cytidines by sodium bisulphite in order to probe the RNA structure. According to these data, cytoplasmic 5S rRNA can be folded as proposed in the general eukaryotic 5S rRNA structure (1) and 5S rRNA from chloroplastides is shown to be more related to the general eubac-terial structure (2). This content is only available as a PDF. Author notes *On the occasion of his 60th birthday, we would like to dedicate this paper to professor Friedrich carner © IRL Press Limited

Silberklang,, M.;RajBhandary, U., L.;Luck,, A.;Erdmann, V., A.

doi: 10.1093/nar/11.3.605pmid: 6340064

Abstract E.coli 50S ribosomal subunits were reacted with monoper-phthalic acid under conditions in which non-base paired adenines are modified to their 1-N-oxides. 5S RNA was isolated from such chemically reacted subunits and the two modified adenines were identified as A73 and A99. The modified 5S RNA, when used in re-constitution of 50S subunits, yielded particles with reduced biological activity (50%). The results are discussed with respect to a recently proposed three-dimensional structure for 5S RNA, the interaction of the RNA with proteins E-L5, E-L18 and E-L25 and previously proposed interactions of 5S RNA with tRNA, 16S and 23S ribosomal RNAs. This content is only available as a PDF. Author notes *Current address: Merck Institute for Therapeutic Research, Rahway NJ 07065,USA +Institut for biochemic, FB chermie, Freie Universitat.Berlin Thielalle 69-73, D-1000 Berlin 33 (Dahlem), FRG §On the occasion of his 60th birthday, we would like to dedicate this paper to professor Friedrich Cramer © IRL Press Limited

Krieg, Paul, A.;Robins, Allan, J.;Andrea, Richard, D';Wells, Julian R., E.

doi: 10.1093/nar/11.3.619pmid: 6300759

Abstract An H5 cDNA clone was used to select H5 genomal recombinants from a chicken Charon 4A library. DNA sequence analysis shows that the H5 gene contains no introns. Putative 5' promoter elements and a 3' polyadenylation site are present within the 1.8 kb of DNA examined. Analysis of 41 kb of DNA surrounding the H5 gene shows that it is not closely linked to either HI or core hlstone genes. This content is only available as a PDF. © IRL Press Limited

Stewart, Monica, A.;Hall, Lucinda M., C.;Edward,, B.;Maden,, H.

doi: 10.1093/nar/11.3.629pmid: 6300760

Abstract Ribosomal DNA (rDNA) from Xenopus laevis contains several heterogeneities in all three transcribed spacers, as revealed by analysis of cloned and uncloned amplified rDNA from oocytes and cloned chromosomal rDNA from erythrocytes. Heterogeneities include single base changes and length variants of one to several nucleotides. Sites of variation are widely but non-uniformly distributed, some occurring only a short distance outside the boundaries of the rRNA coding regions. No two transcription units that we have yet examined are identical throughout their transcribed spacer regions. This content is only available as a PDF. © IRL Press Limited

Cooper, David, N.;Taggart, Mary, H.;Bird, Adrian, P.

doi: 10.1093/nar/11.3.647pmid: 6188105

Abstract We have detected a fraction that is rich in unmethylated HpaII and Hhal sites by end-abelling HpaII fragments of chicken DNA. The fraction is not obvious when DNA fragments are stained with ethidium bromide as it amounts to less than 2% of the genome. The average frequency of sites for HpaII is over thirteen times greater in the unmethylated fraction than in total DNA. Partial digests indicate that the unmethylated sites are clustered in the genome. Similar unmethylated fractions were detected in six other vertebrates in both somatic and germ line DNA. This content is only available as a PDF. © IRL Press Limited

Cheng,, Sheue-Yann;Merlino, Glenn, T.;Pastan,, H.

doi: 10.1093/nar/11.3.659pmid: 6188106

Abstract We describe a simple, general method to link proteins covalently to ONA. The method uses two reagents, N-acetyl-N'-(p-glyoxylylbenzoyl)cystam1ne and 2-1m1noth1olane. The former reacts specifically with nonpaired quanine residues and upon reduction generates a free sulfhydryl group. The latter reacts with a protein to provide another sulfhydryl group which 1s subsequently conjugated to DNA by an intermolecular disulfide Interchange reaction. Using this method α2- macroglobulin was conjugated to plasmid DNA encoding the Herpes simplex virus-1 thymidine kinase gene or a DNA fragment containing the E.coli chlor-amphenicol acetyltransferase gene. Up to 20% of the total DNA was conjugated to α2-macroglobul1n and the α2-macroglobulin-DNA conjugate had a prote1n/DNA molar ratio of approximately two. The whole reaction takes place under very mild conditions 1n aqueous solution. The structure of DNA appears not to be significantly affected by the chemical modification. This method may prove useful In ligand directed gene transfer studies. This content is only available as a PDF. © IRL Press Limited

Kajitani,, Masayuki;Ishihama,, Akira

doi: 10.1093/nar/11.3.671pmid: 6300761

Abstract Invitrotranscription was performed in a single reaction mixture, which contained three species of truncated E.coliDNA template, each carrying one specific promoter, lacP(UV5), trpP orrpljp, and the transcripts of distinct sizes were analyzed by electrophoresis on the same gel. Using this “mixed transcription” system, the order of the promoter strength, i.e., the capacity to form stable open complex, was determined in the single-round transcription under the standard conditions (50mMNaCl and 37°C) to be lacP > trpP > rplJp the latter two promoters being 30˜40 and 5˜10% the strength of lacP, respectively. After the multiple-round transcription, however, the level of trp transcription was the lowest due to low cyclic-reaction rate but became the highest when another trp fragment containing the natural terminator was used as the template. The order of the transcription level also varied depending on the ionic strength and the reaction temperature and, as a result, lacP was no more the strongest under high salt concentration and at high temperature. This content is only available as a PDF. © IRL Press Limited

Haynes,, Joel;Weissman,, Charles

doi: 10.1093/nar/11.3.687pmid: 6188107

Abstract Hybrid plasmids containing the mouse dihydrofolate re-ductase (dhfr) and a human interferon (either IFN-α5 or IFN-γJO coding sequence under the control of viral promoters were trans-fected into dhfr– Chinese hamster ovary (CHO) cells. Dhfr+ colonies produced IFN at 10-1000 units-ml–1-day–1. Clones selected in methotrexate had a 20-50-fold increase in the IFN-a5 and dhfr DNA and mRNA content and secreted IFN at 20,000-100,000 units.ml–1.day–1”1. SDS-polyacrylamide gel electrophoresis of partially purified 35s-HuIFN-γ from CHO cells showed a multiplet of labeled bands with a mobility corresponding to 22,400 to 23,400 daltons which was absent in the supernatants of non-transformed CHO cells. The higher apparent molecular weight of human IFN-Jγ from CHO cells as compared to that of human IFN-γ from E.coli (about 18,800) suggests that the former was glycosylated. This content is only available as a PDF. Author notes *Present address: Connaught Research Institute, 1755 Steeles Avenue West, Willowdale, Ontario M2R 3T4, Canada © IRL Press Limited