Nucleic Acids Research

Joshi,, C.P.

doi: 10.1093/nar/15.23.9627pmid: 3697078

Abstract In animal and viral pre-mRNAS, the process of polyadenylation is mediated through several cis-acting poly (A) signals present upstream and downstream from poly (A) sites. The situation regarding polyadenylation of higher plant pre-mRNAS, however, has remained obscure so far. In this paper, a search for putative poly (A) signals is made by considering the published data from 46 plant genomic DNA sequences. Certain domains in the 3′ untranslated regions from nuclear genes of higher plants were compiled and occurrence of sequence motifs such as AATAAA, CAYTG, YGTGTTYY and YAYTG was scored in relation to poly (A) sites. Moreover, consensus sequences for important regions in the 3′ untranslated sequences and poly (A) signals were also deduced from the data. It was inferred that sequence motifs similar to poly (A) signals exist around poly (A) sites but some of them are in entirely different spatial relationship than observed in other eukaryotes. This indicates their probable non-involvement in the process of polyadenylation in higher plants necessitating a functional analysis approach to define the plant specific poly (A) signals. This content is only available as a PDF. © IRL Press Limited

Murchie, Alastair, I.H.;Lilley, David, M.J.

doi: 10.1093/nar/15.23.9641pmid: 3697079

Abstract There are two alternative pathways by which inverted repeat sequences in supercoiled DNA molecules may extrude cruciform structures, called C-type and S-type. S-type cruciforms, which form the great majority, are characterised by absolute requirement for cations to promote extrusion, which then proceeds at higher temperatures and with lower activation parameters than for C-type cruciforms. The mechanism proposed for S-type extrusion involves an initial opening of basepairs limited to the centre of the inverted repeat, formation of intra-strand basepairing and a four-way junction, and finally branch migration to the fully extruded cruciform. The model predicts that central sequence changes will be more kinetically significant than those removed from the centre. We have studied the kinetics of cruciform extrusion by a series of inverted repeats related to that of plRbke8 by either one or two mutations in the symmetric unit. We find that mutations in the central 8 to 10 nucleotides may profoundly affect extrusion rates - the fastest being 2000-fold faster than the slowest, whereas mutations further from the centre affect rates to a much smaller extent, typically up to ten-fold. These data support the proposed mechanism for extrusion via central opening. This content is only available as a PDF. © IRL Press Limited

Klobeck,, H.-Gustav;Zimmer,, Franz-Josef;Combriato,, Gabriele;Zachau, Hans, G.

doi: 10.1093/nar/15.23.9655pmid: 3122178

Abstract The linking of the human V K and J K C K gene regions (abbreviations in ref. 1) by chromosomal walking is reported. Hybridization experiments with the DNA of a somatic cell hybrid containing the region between J K C K and the telomer show that none of the major V K gene clusters is located downstream of C K . The distance between the V K and J K genes was found to be 23 kb. The J K proximal V K gene is the B3 gene which is the only representative of subgroup IV in the genome. This gene and the neighbouring B2 gene (accompanying paper) are arranged in opposite orientation to J K C K and can therefore rearrange only by an inversion mechanism. Tnis finding is used, together with previous data, to delineate the rearrangement processes in the Burkitt lymphoma derived cell line BL21 as comprising an inversion in the first and a deletion in the second step. This content is only available as a PDF. © IRL Press Limited

Lorenz,, Wulfing;Straubinger,, Bernhard;Zachau, Hans, G.

doi: 10.1093/nar/15.23.9667pmid: 3122179

Abstract Genomic regions containing numerous cloned V K genes (abbreviations in ref. 2) were investigated by pulsed-field gel electrophoresis. 31 and 32 genes were linked within 1.0 and 1.3 Mb NotI fragments, respectively; the latter fragment includes also the J K C K gene segment. A 0.25 Mb NotI fragment comprises further 10 V K genes. Since the transcriptional polarities of the VK genes within the genomic regions are known the linking of the regions allows us now to answer unequivocally some longstanding questions concerning the mechanism of V K J K rearrangement. The V K genes of the 1.3 Mb NotI fragment except for the two J K proximal ones (accompanying paper) are arranged in the same transcriptional polarity as J K C K , and therefore must rearrange by a deletion mechanism. The V K genes of the 1.0 Mb NotI fragment which has not yet been linked to V K J K have identical polarity within the fragment. They should be arranged in opposite polarity to J K C K since reciprocal recombination products derived from them are known to exist; such recombination products must have been formed by inversion of oppositely oriented gene segments. This content is only available as a PDF. © IRL Press Limited

Hong,, G.-F.;Burn,, J.E.;Johnston,, A.W.B.

doi: 10.1093/nar/15.23.9677pmid: 3320955

Abstract In Rhizobium leguminosarum biovar viciae , the regulatory nodulation nodD gene has at least two functions. It constitutively represses its own transcription and in the presence of inducer flavonoid molecules, it activates the expression of two other nod gene transcriptional units, nodABCIJ and nodFE . Upstream of nodA and nodF is a conserved sequence, the nod box, which has been implicated in nodD -mediated transcriptional activation of these genes. DNA fragments spanning the nod boxes that precede nodA and nodF were end-labelled and were exposed to cell-free extracts obtained from strains of Rhizobium . Using the gel retardation technique, it was shown that a complex between protein and these DNA fragments was formed, but only if the extract contained a functional nodD gene. Evidence that the protein that binds to the regulatory sequences is the nodD gene product came from the observation that a complex was formed between the nod box preceding nodA and protein from a cell-free extract isolated from Escherichia coli containing the cloned nodD gene. Extracts from Rhizobium strains containing mutant forms of nodD which were specifically affected in autoregulation or in flavonoid-dependent activation formed either no protein DNA complex or formed a complex with altered mobility compared to that obtained with extracts from wild-type strains. This content is only available as a PDF. Author notes * Present address: Shanghai Institute of Biochemistry, Academia Sinica, Shanghai, People's Republic of China © IRL Press Limited

Gut, Stephan, H.;Bischoff,, Men;Hobi,, Reinhard;Kuenzle, Clive, C.

doi: 10.1093/nar/15.23.9691pmid: 3697080

Abstract Three Z-DNA-binding Droteins of Mr 31, 33 and 58 kO were isolated from mature bull testis. They were obtained in a native state suitable for binding studies. These are the first examples of Z-DNA-binding proteins from a mammalian tissue. Purification involved tissue extraction with 0.35 M NaCl, cation exchange chromatograohy on CM-Trisacryl M and two consecutive anion exchange FPLC runs on Mono Q. The proteins appeared virtually homogeneous by anion exchange FPLC, SDS polvacrylamide gel electrophoresis and reverse phase HPLC (58 kD protein only). Yields from 50 g of testis tissue were: 31 kD protein, 40 μg; 33 kD protein, 100 μg; and 58 kD protein, 150 μg. Z-DNA binding was determined by Scatchard analysis of filter binding data using brominated poly(dG–dC).poly(dG–dC) as a conformation-specific ligand. Dissociation constants (K z , in mol nucleotide/liter) were: 31 kD protein, 7 × 10 −7 M; 33 kD protein, 8 × 10 − 7 M; 58 kD protein, 6 × 10 −8 M (primary binding site) and 6 × 10 −7 M (secondary binding site). B-DNA binding to Poly (dG–dC) · poly (dG–dG) was too low for reliable determination under the conditions of assay. This attested to a high degree of conformational specificity of the three proteins. The 58 kD protein bound Z-DNA at the primary site with an affinity almost equivalent to that of a polyclonal anti-Z-DNA antiserum raised in a rabbit (K Z , 4 × 10 −8 M). This content is only available as a PDF. © IRL Press Limited

Rupp, Ralph, A.W.;Sippel, Albrecht, E.

doi: 10.1093/nar/15.23.9707pmid: 3122180

Abstract The TGGCA protein, the chicken homologue of HeLa cell NF-I, was purified to homogeneity from liver tissue by a procedure which includes preparative mobility shift electrophoresis (PMSE) as the final step. PMSE was here adjusted for the isolation of the TGGCA protein, but can be used as a general method to characterize the protein moiety of specific DNA-binding proteins. The TGGCA protein is a family of 6 protein species, which show minor differences in molecular weight from 36.8kd to 29.8kd. This microheterogeneity differs from the size distribution reported for HeLa cell NF-I polypeptides. All species of the TGGCA protein bind identically to a synthetic DNA-binding site and appear to be highly related in primary structure. We discuss the possible functional importance of this microheterogeneity. This content is only available as a PDF. © IRL Press Limited

Uemura,, Tadashi;Morino,, Kazuhiko;Uzawa,, Satoru;Shiozaki,, Kazuhiro;Yanagida,, Mitsuhiro

doi: 10.1093/nar/15.23.9727pmid: 2827111

Abstract We cloned the structural gene top1+ for Schizosaccharomycespombe DNA topoisomerase I (topo I) by hybridization. An eight-fold increase of topo I relaxing activity was obtained in S . pombe cells transformed with multicopy plasmid with top1+ insert. Nucleotide sequence determination showed a hypothetical coding frame interrupted by two short introns, encoding a 812 residue polypeptide (M.W. 94,000), 43 residues longer than and 47% homologous to Saccharomyces cerevisiae topo I. We show that the top1 (null) strain made by gene disruption is viable, although its generation time is 20% longer than that of wild type. The top1 locus is mapped in the long arm of chromosome II, using the Leu + marker integrated with the cloned top1+ sequence. We constructed a double mutant top1 (null) top2 (ts) and found its defective phenotype similar to that of previously obtained top1 (heat sensitive) top2 (ts). The other double mutant top1 (null) top2 (cs), however, was lethal. Our results suggest that top1+ gene of S . pombe is dispensable only if topo II activity is abundant. This content is only available as a PDF. Author notes 1 Present address: The Howard Hughes Medical Institute, University of California School of Medicine, San Francisco, CA 94143, USA 2 Present address: Mitsubishi Gas Chemical Company Inc., Niigata Research Laboratory, Niigata 950-31, Japan © IRL Press Limited

Pollwein,, Peter;Wagner,, Susanne;Knippers,, Rolf

doi: 10.1093/nar/15.23.9741pmid: 3697081

Abstract Simian Virus 40 (SV40) large T antigen is a DNA binding protein with high affinity for segments of the viral genome. To find out whether T antigen also binds to sequences of genomic cellular DNA we mixed T antigen and SAU 3 A restricted mouse DNA under stringent DNA binding conditions. Resulting protein-DNA complexes were immunoprecipitated using T antigen specific monoclonal or polyclonal antibodies. The DNA fragments in the immunoprecipitates were cloned in plasmid vectors. Four plasmid clones were selected for a detailed investigation of the inserted mouse DNA fragments. Nucleotide sequencing and DNase I footprint experiments showed that T antigen binds to sites in these fragments consisting of two tandemly oriented G(A)AGGC pentamers separated by AT rich spacers of different lengths. The cellular binding sites are very similar in their architecture to the SV40-DNA binding site I. The isolated cellular DNA fragments with T antigen binding sites occur only once or a few times in the mouse genome. Our data help to further define the structure of T antigen's DNA binding sites. The genetic functions of the isolated cellular DNA elements are not known. This content is only available as a PDF. Author notes * Present address: Deutsches Krebsforschungszentrum, D-6900 Heidelberg, FRG © IRL Press Limited

Smith, Terry, J.;Forrest, Susan, M.;Cross, Gareth, S.;Davies, Kay, E.

doi: 10.1093/nar/15.23.9761pmid: 3697082

Abstract We have isolated overlapping human fetal muscle cDNAs encompassing 2.6kb which are localised very close to the 5′end of the Duchenne muscular dystrophy (DMD) gene. Using DNA from patients with deletions of previously reported genomic probes, we have mapped the exons across the region. Investigation of deletions in both DMD and Becker muscular dystrophy (BMD) patients shows the deletions to be present in 10% of cases and heterogeneous. This content is only available as a PDF. © IRL Press Limited