doi: 10.1093/nar/19.18.4819pmid: 16617493
Article PDF first page preview Close This content is only available as a PDF. © 1991 Oxford University Press
Tuddenham,, E.G.D.;Cooper,, D.N.;Gitschier,, J.;Higuchi,, M.;Hoyer,, L.W.;Yoshioka,, A.;Peake,, I.R.;Schwaab,, R.;Olek,, K.;Kazazian,, H.H.;Lavergne,, J.M.;Giannelli,, F.;Antonarakis,, S.E.
doi: 10.1093/nar/19.18.4821pmid: 1923751
Abstract Mutations at the factor VIII gene locus causing Haemophilia A have now been identified In many patients from many ethnic groups. Earlier studies used biased methods which detected repetitive mutations at a few CG dinucleotides. More recently rapid gene scanning methods have uncovered an extreme diversity of mutations. Over 80 different point mutations, 6 insertions, 7 small deletions, and 60 large deletions have been characterised. Repetitive mutation has been proved for at least 16 CpG sites. All nonsense mutations cause severe disease. Most missense mutations appear to cause instability of the protein, but some are associated with production of dysfunctional factor VIII molecules, thereby localising functionally critical regions of the cofactor. Variable phenotype has been observed in association with three of the latter class of genotype. This catalogue of gene lesions in Haemophilia A will be updated annually. This content is only available as a PDF. © 1991 Oxford University Press
Lloyd,, S.L.;Sargent,, C.A.;Chalmers,, J.;Lime,, E.;Habeebu,, S.S.M.;Affara,, N.A.
doi: 10.1093/nar/19.18.4835pmid: 1923752
Abstract We describe a new zinc finger gene sequence (CMPX1 or HGM symbol ZNF6; isolated by cross-hybridization of ZFY to clones in a testis cDNA library) which possesses a zinc finger domain closely related to the transcriptional activator gene ZFX. The putative acidic activation domain is only 11.5% homologous with ZFX, whereas the putative DNA binding domain shares 75% homology and shows the same organisation composed of a basic two fingered repeat unit. ZNF6 has an unusually large 5′ untranslated region (UTR) of 1.2 Kb which contains 26 potential ATG Initiation codons, only one of which is associated with a long open reading frame. Southern and Northern blot analysis has shown that this 5′ UTR is shared with many other sequences in the genome and transcribed associated with a large range of mRNA species. In situ hybridisation, analysis of somatic cell hybrids and male Individuals carrying deleted X chromosomes have mapped the gene to Xq21.1 – q21.3. The gene is highly conserved amongst the primates, in the mouse and can be detected weakly in the genome of a metatherian mammal (possum). Dosage In male and female mice indicates that it is also X-linked in this species. Possible origins of ZFX, ZFY and CMPX1 from a common ancestral gene are discussed. This content is only available as a PDF. © 1991 Oxford University Press
Pósfai,, György;Kim, Sun, C.;Szilák,, László;Kovács,, Attila;Venetianer,, Pál
doi: 10.1093/nar/19.18.4843pmid: 1923753
Abstract Individually inactive N- and C-terminal fragments of the m5C-methyltransferase M.flspRI can complement each other resulting in specific, in vivo methylatlon of the DNA. This was shown by cloning the coding regions for N- and C-terminal parts of the enzyme in compatible plasmids and co-transforming them into E.coli cells. The enzyme could be detached at several different sites, producing either non-overlapping or partially overlapping fragments capable of complementation. Reconstitution of the active methyltransferase from inactive fragments was demonstrated in vitro, as well. Another GGCC-specific methyltransferase, M.BsuRI, showed a similar complementation phenomenon. Moreover, interspecles complementation was observed between appropriate fragments of the two closely related enzymes M.BspRI and M.BsuRI. Fragments of structurally and functionally more different methyltransferases were unable to complement each other. This content is only available as a PDF. © 1991 Oxford University Press
Khanna, Navin, C.;Lakhani,, Sujata;Tewari,, K.K.
doi: 10.1093/nar/19.18.4849pmid: 1717936
Abstract We have used photoaffinity labelling to examine the chloroplast RNA polymerase components which come into contact with nascent transcripts during the in vitro transcription of plastld DNA. The transcripts were synthesized in the presence of a photoactive analogue (4-thio UTP) and α-32P-ATP, using enriched pea chloroplast RNA polymerase preparation and a recombinant plasmid containing the plastid 16S rRNA promoter. Brief Irradiation of the transcriptional complex crosslinked the photoactive nascent RNA to proximal proteins. Labelling of the transcriptional complex was dependent on 4-thlo UTP and template DNA. Two polypeptides of 51 and 54 kDa were consistently crosslinked to the nascent transcripts; about 60% of the total radioactivity of the crosslinked RNA was associated with these polypeptides. In some experiments, two additional polypeptides of 38 and 75 kDa were also found to be associated with about 13% and 17% of the total crosslinked RNA radioactivity, respectively. The UV-crosslinked transcriptional complexes were stable to either DNase or S1 nuclease hydrolysis but partially sensitive to RNase T1. Insensltivity of the complex to hydrolysis with RNase H suggested that the nascent transcripts were not crosslinked to the template. The complexes could also be hydrolysed by proteinase K and thermolysin. No crosslinkage was observed when labelled RNA molecules containing 4-thio UMP residues were added after synthesis to the polymerase preparation. This suggested that the method Identified only those polypeptides which came into close contact with the transcript during its synthesis. Antibodies raised against the RNA-proteln complex confirmed the presence of the polypeptides in the chloroplast RNA polymerase preparation on Western blots. Preincubatlon of these antibodies with the chloroplast RNA polymerase inhibited plastid DNA transcription. These data showed that the transcript-binding polypeptides were functional components of the chloroplast transcriptional complex. This content is only available as a PDF. © 1991 Oxford University Press
doi: 10.1093/nar/19.18.4857pmid: 1656379
Abstract The size and physical structure of the Leptospira Interrogans genome was characterized using contourclamped homogenous electric field (CHEF) gel electrophoresis. The L. Interrogans genome is approximately 4750 kb in size and is composed of two molecular species of DNA: a 4400 kb chromosome; and a 350 kb plasmid, pLIN1. A physical map of the chromosome was constructed with the restriction enzymes Nott and Sfll. A physical map of pLIN1 was constructed with Apal, Notl, Sse8387l, SgrAl, and Smal. Both the L. Interrogans chromosome and pLIN1 are circular. This content is only available as a PDF. © 1991 Oxford University Press
Belt, Peter, B.G.M.;Jongmans,, Wim;, de Wit, Jan;Hoeijmakers, Jan, H.J.;, van de Putte, Pieter;Backendorf,, Claude
doi: 10.1093/nar/19.18.4861pmid: 1656380
Abstract Human cells are, in general, poor recipients of foreign DNA, which has severely hampered the cloning of genes by direct phenotypic correction of deficient human cell lines after DNA mediated gene transfer. In this communication a methodology is presented which largely circumvents this problems. The method relies on the use of a recently developed episomal Epstein - Barr-virus-derived cDNA expression vector (Belt et al. (1989) Gene 84, 407–417). The cloning of hypoxanthine phosphoribosyltransferase (HPRT) cDNA, corresponding to a low abundant mRNA in wild type cells is used as a model system. Size fractionated poly (A)+ RNA from wild type cells, which resulted in an approximately 10 fold enrichment In HPRT mRNA, was used to construct a cDNA library of 25,000 independent clones in the pECV25 vector. An HPRT deficient human cell line was transfected and subsequently selected with hygromycin B for DNA uptake. In a small scale experiment only 7000 hygromycin BR transfectants were sufficient to isolate 2 independent HATR clones which were shown to replicate eplsomes harbouring HPRT cDNA. The first insert had a 5′ untranslated region (UTR) and a 3′ UTR perfectly in agreement with published data. The second cDNA clone harboured an unusually long 5′ UTR and a shorter 3′ UTR due to alternative polyadenylation of the HPRT transcript which has not been previously recognized. This content is only available as a PDF. Author notes +Present address: Department of Molecular Biology (EDH), Central Veterinary Institute, PO Box 65, 8200 AB Lelystad, The Netherlands © 1991 Oxford University Press
Araki,, Hiroyuki;Hamatake, Robert, K.;Morrison,, Alan;Johnson, Anthony, L.;Johnston, Leland, H.;Sugino,, Akio
doi: 10.1093/nar/19.18.4867pmid: 1923754
Abstract DNA polymerase II purified from Saccharomyces cerevisiae contains polypeptldes with apparent molecular masses of >200, 80, 34, 30 and 29 kDa, the two largest of which (subunlts A and B) are encoded by the essential genes POL2 and DPB2. By probing a λgt11 expression library of yeast DNA with antiserum against DNA polymerase II, we isolated a single gene, DPB3, that encodes both the 34- and 30-kDa polypeptides (subunit C and C′). The nucleotide sequence of DPB3 contained an open reading frame encoding a 23-kDa protein, significantly smaller than the observed molecular masses, 34- or 30-kDa, which might represent post-translationally modified forms of the DPB3 product. The predicted amlno acid sequence contained a possible NTP-binding motif and a glutamate-rich region. A dpb3 deletion mutant (dpb3δ) was viable and yielded a DNA polymerase II lacking the 34- and 30-kDa polypeptides. dpb3δ strains exhibited an increased spontaneous mutation rate, suggesting that the DPB3 product is required to maintain fidelity of chromosomal replication. Since a fifth, 29-kDa polypeptide was present in DNA polymerase II preparations from wild-type cell extracts throughout purification, the subunit composition appears to be A, B, C (or C and C′) and D. The 5′ nontranscribed region of DPB3 contained the Mlul-related sequence ACGCGA, while the 0.9-kb DPB3 transcript accumulated periodically during the cell cycle and peaked at the G1/S boundary. The level of DPB3 transcript thus appears to be under the same cell cycle control as those of POL2, DPB2 and other DNA replication genes. DPB3 was mapped to chromosome II, 30 cM distal to his7. This content is only available as a PDF. Author notes +Present address: Department of Virology, the Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08540, USA © 1991 Oxford University Press
doi: 10.1093/nar/19.18.4873pmid: 1923755
Abstract I have cloned a yeast gene, RGM1, which encodes a proline-rich zinc, finger protein. rgm1 mutants do not show any obvious phenotype but overexpression of RGM1 gene greatly impairs cell growth. The prolinerich region of RGM1 attached to a heterologous DNA binding domain is able to repress the expression of the target gene. RGM1 shares similar zinc finger motifs with the mammalian Egr (early growth response) proteins as well as proline-rich sequences with a high serine and threonlne content, suggesting that RGM1 and Egr proteins could have functional similarities. This content is only available as a PDF. © 1991 Oxford University Press
Jang,, Kyung-Lib;Pulverer,, Bemd;Woodgett, James, R.;Latchman, David, S.
doi: 10.1093/nar/19.18.4879pmid: 1656381
Abstract Lytic infection with herpes simplex virus (HSV) results in the repression of most host cell protein synthesis but produces an increased activity of the cellular AP-1 transcription factor. This increase Is paralleled by an increase In the transcription rate of the proto-oncogene encoding the AP-1 component, c-Jun resulting in an increase in c-Jun protein In infected cells. The increased AP-1 activity in infected cells is dependent upon the HSV immediate-early protein ICPO. Thus a mutant lacking the gene encoding this protein falls to increase AP-1 activity whilst an ICPO expression plasmid can specifically increase the activity of an AP-1 dependent promoter in co-transfection experiments. The Implications of these effects in the interaction of HSV with cultured cells are discussed. This content is only available as a PDF. © 1991 Oxford University Press
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