Nucleic Acids Research

Sharp,, Stephen;Garcia,, Alonzo;Cooley,, Lynn;Söll,, Dieter

doi: 10.1093/nar/12.20.7617pmid: 6093044

Abstract Transcription of isolated repeat units of D. molanogaster 5S DNA in Drosophila KcO cell extract revealed three types of template activities. 5S1 DNA encodes the known 5S rRNA of D. molanogaster and has a relatively high transcription efficiency. 58II DNA is identical to 5SI DNA except for a two-nucleotide deletion at 5S rRNA positions 28 and 29; the efficiency of transcription is approximately 40% that of 5SI DNA and because of the deletion, the primary transcript is two nucleotides shorter. 5SIII DNA does not support in vitro tranacription (≪2% 5SI DNA), but has the same sequence as 5SI DNA except for a single G to A transition at position 86. This is the first reported point-mutation in a 5S RNA gene reaulting in loss of transcription function. Of approximately 23 5S rRNA gene copies in a cloned 5S DNA sub-cluster (p12D1) 19 appear to be of the transcriptionally inactive 5SIII DNA type. This content is only available as a PDF. © IRL Press Limited

Strittmauer,, G.;Kössel,, H.

doi: 10.1093/nar/12.20.7633pmid: 6093045

Abstract The termini of rRNA processing intermediates and of mature rRNA species encoded by the 3' terminal region of 23S rDNA, by 4.5S rDNA, by the 5' terminal region of 5S rDNA and by the 23S/4.5S/5S intergenic regions from Zea maya chloroplast DNA were determined by using total RNA isolated from maize chloroplasts and 32 P-labelled rDNA restriction fragments of these regions for nuclease S 1 and primer extension mapping. Several processing sites detect able by both 3' and 5' terminally labelled probes could be identified and correlated to the secondary structure for the 23S%4.5S intergenic region. The complete 4.5S%SS intergenic region can be reverse transcribed and a common processing site for maturation of 4.5S and SS rRNA close to the 3' end of 4.5S rRNA was detected. It is therefore concluded that 23S, 4.5S and 5S rRNA are co-transcribed. This content is only available as a PDF. © IRL Press Limited

Rigolet,, Muriel;Galibert,, Francis

doi: 10.1093/nar/12.20.7649pmid: 6093046

Abstract The E4 region of Adenovirus 2 is a leftward transcribed part of the viral genome. Its nucleotide sequence has already been analysed. From this sequence several open reading frames were defined, which could be used in the coding of the E4 proteins. Using S1 digestion of mRNA-DNA hybrids a precise mapping of donor and acceptor sites was done. Their use in various combinations allows the synthesis of mRNAs, able to direct the synthesis of at least 7 polypeptides, ranging in size from 9K to 34K. Comparison of the sequences of the different acceptor sites indicates that they all conform to the consensus sequence. Analysis of the ATG surrounding sequence shows that initiator ATG may be positively selected according to Kozak's rule. This content is only available as a PDF. © IRL Press Limited

Tessier,, Luc-Henri;Sondermeyer,, Paul;Faure,, Thérèse;Dreyer,, Dominique;Benavente,, Annie;Villeval,, Dominique;Courtney,, Michael;Lecocq,, Jean-Pierre

doi: 10.1093/nar/12.20.7663pmid: 6093047

Abstract Parameters influencing the efficiency of expression of the human ininune interferon (IFW-γ) gene in E.coli were studied by comparing a series of eight in vitro -derived gene variants. These contained all possible combinations of silent mutations in the first three codons of the mature IFN-γ polypeptide coding sequence. Expression levels varied up to 50-fold among the different constructions. Comparison of messenger RNA secondary structure models for each variant suggested that the presence of stem-loop structures blocking the translation initiation signals could drastically decrease the efficiency of IFN-γ synthesis. With variants displaying no stable n secondary structure in the region, a C+U transition at position +11 after the AUG resulted in a 5-fold increase in expression indicating that RNA primary structure also plays an important role in expression. In addition we demonstrate that, in this system, a spacing of 8 nucleotides between the Shine-Dalgarno region and AUG was optimal for gene expression and that the steady-state production level of IFN-γ rose exponentially with increasing rate of synthesis. This content is only available as a PDF. © IRL Press Limited

Zacharias,, Wolfgang;Larson, Jacquelynn, E.;Kilpatrick, Michael, W.;Wells, Robert, D.

doi: 10.1093/nar/12.20.7677pmid: 6093048

Abstract The capacity of the modification methylase (M Hha I) and restriction endonuclease ( Hha I) from Haemophilue haemolyticus to methylate and cleave, respectively, recognition sites which are in right-handed B or left-handed Z structures was determined in vitro Plasmids containing tracts of (dCdG) S8 well as numerous individual d(GCGC) sites distributed around the vector were studied. Negative aupercoiling was used to convert the (dC-dc) tracts (˜30bp in length) from a right-handed to a left-handed conformation. (Methyl-was used to localize and quarititate modified d(GCGC) recognition sites, whereas cleavage by Hhat was used to detect unmethylated sites. In the left handed Z-form, the (dc-dc) blocks were not methylated by MHha I and not cleaved by Hha I. A two-dimensional gel analysis of a family of 33 topoisomers treated with M Hha I revealed that the lack of methylation in the (dc-dc) blocks was directly correlated to the supercoil-induced B to Z transition in these segments. These results are significant with respect to enzyme-DNA interactions in general and provide the basis for using Hha I and M Hha I as probes for different DNA structures and conformational transitions under physiological conditions. This content is only available as a PDF. © IRL Press Limited

Unger,, Bernhard;Klock,, Gerd;Hillen,, Wolfgang;Wells, Robert, D.

doi: 10.1093/nar/12.20.7693pmid: 6093049

Abstract The tetracycline resistance determinant of RA1 was cloned. It consists of at least two genes oriented with opposite polarity, tetA for resistance and tetR for regulation. The transcriptional control sequence was identified and analyzed. It consists of overlapping promotors with divergent orientation and a tandem arrangement of operators. Nucleotide sequencing revealed two open reading frames. One codes for a protein which was identified as a Tet repressor by comparing its primary structure with those of other let repressors. The RA1 tetR gene codes for 218 amino acids with a calculated molecular weight of 24.4kDa. In the primary sequence of the RA1-, p5C101-, Tn10-, and RP1/Tn1721- encoded let repressors, 36% of the amino acids are identical. This homology is clustered within the first 150 amino acids, 49% of which are identical among all four proteins. These results are discussed with respect to their structure and function In comparison to other DNA binding proteins. This content is only available as a PDF. © IRL Press Limited

Meyerhof,, Wolfgang;Klinger-Mitropoulos,, Stella;Stalder,, Jürg;Weber,, Rudolf;Knöchel,, Walter

doi: 10.1093/nar/12.20.7705pmid: 6093050

Abstract We present the complete nucleotide sequence of the larval β I -globin gene of Xenopus laevis including 240 nucleotides of the 5' flanking region and 594 nucleotides beyond the polyadenylation site. The site of transcription initiation was mapped by S 1 nuclease, and the site of polyadenylation was determined by comparison with corresponding cDNA clones. The larval Xenopus β I -globin gene shows the same internal structure as the β-globin genes of higher vertebrates, viz. 3 exons interrupted by 2 intervening sequences. The first intervening sequence, which is of exceptional length, spans over 564 nucleotides and interrupts the coding sequence at amino acid 30, whereas the second one comprises 968 nucleotides and is located between the amino acids 104 and 105. The second intervening sequence contains a long inverted repeat of almost perfect homology. The 5' flanking region contains a TATA- and a CAAT-box at positions -33 and -58, respectively. An additional TATA-box is located at -197 and two more CAAT-boxes occur at positions -105 and -237. This content is only available as a PDF. © IRL Press Limited

Bajwa,, Wajeeh;Meyhack,, Bernd;Rudolph,, Hans;Schweingruber,, Anne-Marie;Hinnen,, Albert

doi: 10.1093/nar/12.20.7721pmid: 6093051

Abstract We have sequenced the genetically linked genes for repressible ( PH05 ) and and constitutive ( PH03 ) acid phosphatase from S. cerevisiae Both genes are located on a 3.91 Kb BamHI and HpaI fragment, in the order (5') PH05. PH03 (3'). The mRNA transcripts have been analysed by S1-nuclease mapping. They show heterogenous initiation sites. Each of the PH05 and PH03 genes codes for 467 amino acids as deduced from the DNA sequence. The coding regions of the two genes show homology both at the nucleotide (82%) and the amino acid (87%) level. In the coding sequences, long stretches of homologous regions are flanked by small non-homologous regions. The nucleotide homology (65%) extends to some length into the 5' and 3' non-coding flanking sequences. Further upstream sequences are unrelated. The comparison of the NH 2 -terminal amino acid sequence deduced from the nucleotide sequence, with that of purified repressible acid phosphatase revealed the presence of a putative signal peptide. This content is only available as a PDF. Author notes * Present address: Biotechnology Department, CIBA-GEIGY AG, CH-4002 Basel, Switzerland © IRL Press Limited

Bertrand-Burggraf,, E.;Schnarr,, M.;Lefevre,, J.F.;Daune,, M.

doi: 10.1093/nar/12.20.7741pmid: 6387626

Abstract Supercoiling of DNA is now known to have considerable effects on transcription in bacteria. By abortive initiation reaction (6) we have determined the binding constant K B and the forward rate of isomerization k 2 as a function of temperature, pH and buffer for the tet promoter in a supercoiled plasmid. If the activation energy of isomerization is very similar to that obtained previously under the same conditions on a linearized plasmid (6) (respectively 21±5 kcal/rnole and 13±5 kcal/mole) the supercoiling introduces very important and not well understood changes in the thermodynamic parameters of the association polymerase - promoter. Using the technique of superhelical DNA relaxation by eukaryotic topoisome rase I, we have determined the specific unwinding by RNA polymerase of the tet promoter of pBR322 (430°). This unwinding differs only slightly from the mean value (470°) obtained for all the promoters of pBR322. This content is only available as a PDF. © IRL Press Limited

Andrews, Deborah, L.;Millstein,, Larry;Hamkalo, Barbara, A.;Gottesfeld, Joel, M.

doi: 10.1093/nar/12.20.7753pmid: 6093052

Abstract We have constructed hybrid plasmids bearing both Xenopus 5S RNA genes and satellite I sequences in order to test the effect of satellite DNA on 5S gene transcription. Satellite sequences inactivate 5S transcription in both HeLa S100 and Xenopus oocyte microinjection transcription assays. Inactivation of 5S transcription by satellite DNA is observed both in cis and in trans Transcription of a tRNA gene is also precluded by satellit DNA. The xenopus satellite I repeat contains an RNA polymerase III transcription unit which is highly active in both assay systems. This promoter element is 10- to 25-fold more efficient than the 5S gene in transcription competition assays. This quantitative difference in affinity for transcription components may explain the inactivation of 5S transcription by satellite sequences. The satellite I promoter forms stable transcription complexes in vitro which do not dissociate for at least 30 rounds of transcription. Although stable complex formation on the promoter is largely temperature independent over the range of 0–20°, complex formation on both 5S and tRNA genes exhibits a steep temperature dependence characteristic of DNA helix unwinding. The DNA sequence requirements for stable complex formation on 5S genes have been determined using 5' deletion mutants. This content is only available as a PDF. © IRL Press Limited