Nucleic Acids Research

Welsh,, John;McClelland,, Michael

doi: 10.1093/nar/18.24.7213pmid: 2259619

Abstract Simple and reproducible fingerprints of complex genomes can be generated using single arbitrarily chosen primers and the polymerase chain reaction (PCR). No prior sequence information is required. The method, arbitrarily primed PCR (AP-PCR), involves two cycles of low stringency amplification followed by PCR at higher stringency. We show that strains can be distinguished by comparing polymorphisms in genomic fingerprints. The generality of the method Is demonstrated by application to twenty four strains from five species of Staphylococcus , eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa (rice). This content is only available as a PDF. Author notes * Publication of this paper was delayed by the authors to allow simultaneous publication with a paper submitted later by another group. Nucleic Acids Research regrets that due to administrative errors the other paper, by Williams et al. , was published on pages 6531–6535 of issue 22. Both sets of authors agree that the two papers should be considered as published simultaneously and should be referred to together. © 1990 Oxford University Press

Matthews, Keith, R.;Shiels, Paul, G.;Graham, Sheila, V.;Cowan,, Catriona;Barry,, J.David

doi: 10.1093/nar/18.24.7219pmid: 2175429

Abstract In the mammalian bloodstream, African trypanosomes express variant surface glycoprotein (VSG) genes from a family of long and complex telomeric expression sites. VSG switching generally occurs by the duplication of different VSG genes into these sites by gene conversion involving a series of 70 base pair (70bp) repeats in the 5′ flank. In contrast, when VSG is first synthesised by trypanosomes in the tsetse fly at the metacyclic stage, a separate set of telomeric expression sites is activated. These latter telomeres appear not to act as recipients in gene conversion. We have found that the structure of two such expression sites is simple, with very short 70bp repeat regions and very little other sequence in common with bloodstream expression sites. However, the two telomeres readily act as donors in VSG gene conversion in the bloodstream and we show for one a consistent association of the conversion 5′ end point with the short 70bp repeat region. These findings help explain why a very predictable set of VSGs is expressed in the tsetse fly and have implications for VSG gene conversion mechanisms. This content is only available as a PDF. Author notes Present address: + Department of Internal Medicine, Yale MacArthur Center for Molecular Parasitology, Yale University School of Medicine, New Haven, CN, USA Present address: § Division of Molecular Biology, The Netherlands Cancer Institute (Antoni van Leeuwenhoek Huis) Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands © 1990 Oxford University Press

Ueda,, Hitoshi;Hirose,, Susumu

doi: 10.1093/nar/18.24.7229pmid: 2124348

Abstract Extracts from embryos and from posterior and middle silk glands of the silkworm, Bombyx mori contain a sequence specific DNA binding factor termed BmFTZ-F1. The factor binds to the recognition site of FTZ-F1, a positive regulator of the fushi tarazu gene in Drosophlla melanogaster . BmFTZ-F1 and FTZ-F1 share the same methylation interference patterns, the same chromatographic behaviors and similar protease digestion profiles. Anti-FTZ-F1 cross reacts with BmFTZ-F1. These results indicate that BmFTZ-F1 is a B.mori homologue of FTZ-F1. The mobility of the factor-DNA complex formed in the silk gland extract changes depending on the developmental stages. Purification of BmFTZF1 to an almost homogeneous state reveals that the factor is a 73 kd protein. This content is only available as a PDF. © 1990 Oxford University Press

Faraonio,, Raffaella;Musy,, Margherita;Colantuoni,, Vittorio

doi: 10.1093/nar/18.24.7235pmid: 2259620

Abstract The Retinol-Binding Protein (RBP) is expressed primarily in the liver. The regulatory elements involved in its tissue-specific expression have been Identified and mapped to the 5′ flanking region of the RBP gene. In this paper heterokaryons and somatic cell-hybrids have been produced and analysed in order to demonstrate that the RBP gene is subject to extinction and to identify the target sequences of this phenomenon. We show here that the gene is extinguished in fusions of hepatoma with a variety of cells of different species and embryonic lineages. The repression is not due to loss of the gene and occurs also when chromosome 10, where the gene is located, is inherited from the expressing parental cell-type. Hybrid clones were transfected with constructs carrying DNA segments of different lengths from the 5′ flanking region of the RBP gene fused to a reporter gene. We demonstrate that extinction takes place also on an exogenous RBP-CAT gene, mimicking the phenomenon observed with the endogenous gene in its chromosomal location. Moreover, we identify and map the target sequences of the putative extinguishing function. Our data thus show that extinction of RBP is mediated through the DNA segment that is involved in its tissue-specific expression. This content is only available as a PDF. © 1990 Oxford University Press

Bell,, Andrew;Gaston,, Kevin;Williams,, Roy;Chapman,, Karen;Kolb,, Annie;Buc,, Henri;Minchin,, Stephen;Williams,, Jackie;Busby,, Stephen

doi: 10.1093/nar/18.24.7243pmid: 2259621

Abstract The effects of a number of mutations in the E.coli cyclic AMP receptor protein (CRP) have been determined by monitoring the in vivo expression and in vitro open complex formation at two semi-synthetic promoters that are totally CRP-dependent. At one promoter the CRP-blnding site is centred around 41.5 base pairs upstream from the transcription start whilst at the other promoter it is 61.5 base pairs upstream. The CRP mutation E171K reduces expression from both promoters whilst H159L renders CRP totally inactive: neither mutation stops CRP binding at either promoter. The mutations K52N and K52Q reverse the effect of H159L and ‘reeducate’ CRP to activate transcription. CRP carrying both H159L and K52N activates transcription from the promoter with the CRP site at −41.5 better than wild type CRP. In sharp contrast, this doubly changed CRP is totally inactive with respect to the activation of transcription from the promoter carrying the CRP site at −61.5. Our results suggest that CRP can use different contacts and/or conformations during transcription activation at promoters with different architectures. This content is only available as a PDF. © 1990 Oxford University Press

Kendall,, E.;Sargent,, C.A.;Campbell,, R.D.

doi: 10.1093/nar/18.24.7251pmid: 2259622

Abstract A new cluster of genes has been defined in the human Major Histocompatibility Complex class III region. The seven novel genes, G12 to G18, are localised in a 160 kb segment of DNA extending from the complement gene cluster towards HLA-DR. The genes were identified by isolation of cDNA clones using cosmid genomic inserts as hybridisation probes, and by the detection of the corresponding transcripts in Northern blot analysis. Characterisation of the cosmid genomic DNA inserts, in conjunction with pulsed field gel electrophoresis analysis of uncloned DNA, for the presence of clustered sites for infrequently cutting restriction endonucleases has revealed that at least 5 of the 7 genes are associated with HTF-islands. These unmethylated CpG-rich sequences are frequently found at the 5′ ends of ubiquitously expressed genes. Together with previously published data 36 genes have now been defined in a 680kb stretch of DNA within the MHC. With one gene approximately every 20kb of DNA this represents the most densely packed region of the human genome so far characterised, and is of major significance in relation to the mapping and sequence analysis of the rest of the genome. This content is only available as a PDF. © 1990 Oxford University Press

Mullineaux, Philip, M.;Guerineau,, François;Accotto,, Gian-Paolo

doi: 10.1093/nar/18.24.7259pmid: 2175430

Abstract We have used a polymerase chain reaction (PCR) procedure to analyse low abundance complementary sense RNAs of Digitaria streak virus (DSV) from infected leaves of Digitaria setigera . This study has confirmed that both spliced and unspliced RNAs are synthesised by the same transcription unit. The position of the intron has been proven from sequencing cDNAs corresponding to the spliced RNA. Although the majority of cDNAS have 3′ ends at coordinate 1063, downstream from a consensus polyadenylation sequence, a minor population of RNAs with heterogeneous 3′ ends has also been identified. Two major RNA species with alternative splice sites or 3′ ends, previously identified by nuclease S1 protection assays, could not be detected, but a cDNA species was observed with an apparent 90bp insertion at the 5′ end of the intron. In transgenic tobacco containing integrated dlmers of DSV DNA, the major unspliced RNA could readily be detected, but no spliced RNA was present. This may be a reason why DSV DNA did not replicate in tobacco. In addition, neither the minor population of heterogeneous RNAs nor the cDNA species with the insertion could be detected. The failure of the intron to be spliced in tobacco and its low activity in Digitaria is discussed in relation to recent studies on RNA splicing in plants and has led us to the conclusion that the geminivlrus introns may be intrinsically inefficient. This content is only available as a PDF. © 1990 Oxford University Press

Dryden, Sylvia, C.;Kaplan,, Samuel

doi: 10.1093/nar/18.24.7267pmid: 1701878

Abstract We have identified, cloned and sequenced the three ribosomal RNA (rRNA) operons ( rrn ) present in the facultative photoheterotroph Rhodobacter sphaeroides . DNA sequence analysis has identified the 16S, 23S, and 5S rRNAs, two tRNAs (ile and ala) in the spacer region between the 16S and 23S rRNAs, and an f-met tRNA immediately following the 5S rRNA gene of all three operons. Physical mapping, genetic analysis, and Southern hybridization data indicate that rrnA is contained on a large chromosome and rrnB and rrnC are contained on a second smaller chromosome. These findings are discussed in relation to the origins of diploidy. This content is only available as a PDF. © 1990 Oxford University Press

Murata,, Tetsuya;Iwai,, Shigenori;Ohtsuka,, Eiko

doi: 10.1093/nar/18.24.7279pmid: 2259623

Abstract A dodecadeoxyribonucleotide containing a cis-syn thymine dimer with a phosphorodithioate linkage was synthesized on a solid support using a dinucleotide coupling unit prepared by UV-irradiation of dithymidine monophosphorodithioate followed by S - and 5′- O -protection and 3′-phosphitylation. A photodimer-containing dodecamer without phosphate modification was also synthesized. The dodecamers were hybridized to the complementary sequence, and the duplexes used as substrates for T4 endonuclease V. This enzyme cleaved the phosphate-modified substrate more slowly than the unmodified duplex with the same dissociation constant. This content is only available as a PDF. © 1990 Oxford University Press

Roy,, Christine;Tounekti,, Naceur;Mougel,, MarylÈne;Darlix,, Jean-Luc;Paoletti,, Claude;Ehresmann,, Chantal;Ehresmann,, Bernard;Paoletti,, Jacques

doi: 10.1093/nar/18.24.7287pmid: 2259624

Abstract The genome of Moloney murine leukemia vlrus(MoMuLV) is composed of two identical RNA molecules joined at their 5′ ends by the dimer linkage structure(DLS). Recently it was shown that in vitro generated MuLV RNA formed dimeric molecules and that dimerization sequences are located within the Psi encapsidation domain between positions 215 and 420. Conditions for the spontaneous dimerization of a MuLV RNA fragment encompassing the Psi domain have been investigated. The rate of spontaneous MuLV RNA dimer formation is dependent upon RNA, NaCl and MgCl 2 concentrations as well as temperature. Thermal denaturation of in vitro generated dimer RNA of 350 nt, from positions 215 to 565, gave a Tm of about 58°C in 100 mM NaCl. This Tm value is very close to that found for RNA corresponding to the 5′ 755 nt and to the genomic 70 S RNA isolated from virions. According to thrermodynamic parameters derived from denaturation curves of MuLV dimer RNA generated in vitro, the dimer linkage structure probably involves short sequences. This content is only available as a PDF. © 1990 Oxford University Press