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At least 631 records · Page 35Linked to original sources
PubMed2005

Mutation of a single conserved nucleotide between the cloverleaf and internal ribosome entry site attenuates poliovirus neurovirulence.

The chemical synthesis of poliovirus (PV) cDNA combined with the cell-free synthesis of infectious particles yielded virus whose mouse neurovirulence was highly attenuated (J. Cello, A. V. Paul, and E. Wimmer, Science 297:1016-1018, 2002). Compared to the wild-type PV1 (Mahoney) [PV1(M)] sequence, the synthetic virus genome harbored 27 nucleotide (nt) changes deliberately introduced as genetic markers. Of the 27 nucleotide substitutions, the UA-to-GG exchanges at nucleotides 102/103, mapping to a region between the cloverleaf and the internal ribosome entry site (IRES) in the 5'-nontranslated region, were found to be involved in the observed attenuation phenotype in mice. The UA/GG mutation at nt 102/103 in the synthetic PV1(M) [sPV1(M)] background conferred also a ts phenotype of replication to the virus in human neuroblastoma cells. Conversely, the exchange of GG to wild-type (wt) UA at 102/103 in an sPV1(M) background restored wt neurovirulence in CD155 transgenic (tg) mice and suppressed the ts phenotype in SK-N-MC cells. All poliovirus variants replicated well in HeLa cells at the two temperatures, regardless of the sequence at the 102/103 locus. Analyses of variants isolated from sPV(M)-infected CD155 tg mice revealed that the G(102)G(103)-to-G(102)A(103) reversion alone reestablished the neurovirulent phenotype. This suggests that a single mutation is responsible for the observed change of the neurovirulence phenotype. sPV1(M) RNA is translated in cell extracts of SK-N-MC cells with significantly lower efficiency than PV1(M) RNA or sPV1(M) RNA with a G(102)-to-A(102) reversion. These studies suggest a function for the conserved nucleotide (A(103)) located between the cloverleaf and the IRES which is important for replication of PV in the central nervous system of CD155 tg mice and in human cells of neuronal origin.

Animals↗
PubMed1989

Mapping of antibody specificities to VH gene families.

VH gene segments represent the products of the repeated duplication and subsequent diversification of a primordial V gene element. It is widely assumed that natural selection, operating via pathogens, has played the dominant role in this process. Here, we screen some 3.7 x 10(4) C mu+ colonies of mitogen-activated B cells for the production of antibodies specific for phosphorylcholine or hen egg lysozyme and expression of the VH X-24, S107, Q52, or J558 gene families. These gene families were expressed at frequencies proportional to their genomic complexity among both unselected and antigen-specific C mu+ colonies. Thus, the capacity to encode equivalent antibody-combining sites is dispersed uniformly among VH families. This result suggests that individual VH genes have not evolved to address specific antigens.

Animals↗
PubMed1987

Unicircular structure of the Brassica hirta mitochondrial genome.

Restriction mapping studies reveal that the mitochondrial genome of white mustard (Brassica hirta) exists in the form of a single circular 208 kb chromosome. The B. hirta genome has only one copy of the two sequences which, in several related Brassica species, are duplicated and undergo intramolecular recombination. This first report of a plant mitochondrial DNA that does not exist in a multipartite structure indicates that high frequency intramolecular recombination is not an obligatory feature of plant mitochondrial genomes. Heterologous filter hybridizations reveal that the mitochondrial genomes of B. hirta and B. campestris have diverged radically in sequence arrangement, as the result of approximately 10 large inversions. At the same time, however, the two genomes are similar in size, sequence content, and primary sequence.

Brassica↗
PubMed1989

An Escherichia coli chemoreceptor gene is temporally controlled in Caulobacter.

Flagellar and chemotaxis genes are transcribed at a discrete time in the Caulobacter cell cycle. We demonstrate here that the expression of the Escherichia coli chemoreceptor gene tsr, with 2.6 kilobases of its upstream sequence, is temporally controlled in Caulobacter crescentus. The tsr gene was placed on the chromosome in single copy or on a low-copy-number plasmid. It was found that the Tsr protein appeared at the same point in the cell cycle as an endogenous C. crescentus methyl-accepting chemotaxis protein. Nuclease S1 mapping experiments showed that the tsr transcript was also controlled by the cell cycle, suggesting that the E. coli tsr gene is regulated by C. crescentus factors that mediate the timing of transcription initiation. The apparent transcription start site of the E. coli tsr gene was determined in both E. coli and C. crescentus, and we found that in both backgrounds the promoter used conforms to the consensus sequence for the promoters of the flagellar and chemosensory genes of Bacillus subtilis and E. coli. The use of this promoter suggests that C. crescentus has a cognate sigma factor and predicts that other C. crescentus genes are expressed from this consensus promoter.

Bacteria↗
PubMed1991

Promoter region of the human platelet-derived growth factor A-chain gene.

The platelet-derived growth factor (PDGF) A- and B-chain genes are widely expressed in mammalian tissues and their homodimeric gene products appear to regulate the autocrine growth of both normal and transformed cells. In this study, we analyzed the 5' flanking sequences of the human PDGF A-chain gene to seek elements important to regulating its transcription. The promoter region was exceptionally G + C-rich and contained a "TATA box" but no "CAAT box." The transcription start site was identified 845 base pairs 5' to the translation initiation site by S1 nuclease mapping and by primer extension. Both in vitro transcription and transient expression of the chloramphenicol acetyltransferase gene linked to the PDGF A-chain 5' flanking sequences established that the putative promoter region was active, and RNase H mapping established that the three characteristic mRNAs (1.9, 2.3, and 2.8 kilobases) used the same transcription start site, which was used in normal endothelial cells and in two human tumor cell lines that express high levels of A-chain transcripts. The results established an exceptionally G + C-rich promoter region and a single transcription start site active for each of the three mRNAs of the PDGF A-chain gene. DNA sites of potential importance in mediating the activation of the PDGF A-chain gene in normal cells and in transformed cell lines expressing high levels of PDGF A chain were identified.

Base Sequence↗
PubMed1990

High-resolution TLC mapping and characterization of 32P-postlabeled monophosphate 7,12-dimethylbenz[a]anthracene-DNA adducts.

Previous work has shown that 7,12-dimethylbenz[a]anthracene (DMBA)-DNA adducts can be converted by 32P-postlabeling to different types of radiolabeled derivatives (nucleoside 3',5'-bisphosphates, nucleoside 5'-monophosphates and dinucleotides), and that the 32P-labeled 3',5'-bisphosphate derivatives can be further characterized by cross-referencing with 3H-labeled nucleoside DMBA adducts, for which structural information is available. This work has now been extended by TLC comparisons of 5'-monophosphate and 3',5'-bisphosphate DMBA adducts. To this end, DMBA-modified DNA was enzymatically hydrolyzed to 3'-monophosphates (Xp + Np) or to dinucleotides (XpN), and these digestion products were 32P-postlabeled by published procedures to yield 3',5'-bisphosphate (*pXp) or 5'-monophosphate (*pX) adducts. Individual *pXp and *pX fractions were isolated from polyethyleneimine (PEI)-cellulose TLC maps and chromatographically compared after enzymatic 3'-dephosphorylations of the 3',5'-bisphosphate (*pXp) derivatives (*pXp----*pX + Pi). Four reactions were standardized and employed for this purpose: (i) 3'-dephosphorylation by extensive digestion with nuclease P1; (ii) 3'-dephosphorylation catalyzed by polynucleotide kinase; (iii) partial dephosphorylation by bacterial alkaline phosphatase; and (iv) partial dephosphorylation by prostatic acid phosphatase. Individual DMBA adducts displayed marked differences with regard to their susceptibility to enzymatic dephosphorylation. The three major and most minor postlabeled 5'-monophosphate DMBA adducts were cross-referenced this way with 3',5'-bisphosphate and nucleoside adducts, so that specific dihydrodiol epoxide-nucleoside 5'-monophosphate adducts can now be identified and measured by 32P-postlabeling.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1987

Sequence homologies among the three yolk polypeptide (Yp) genes in Drosophila melanogaster.

To identify candidates for cis-acting sequences that regulate the sex-, stage-, and cell-specific expression of three coordinately regulated yolk polypeptide genes (Yp) in Drosophila melanogaster, we have mapped the Yp3 transcript, sequenced a 4278 bp DNA fragment containing the Yp3 gene, compared Yp3 region sequences to corresponding parts of Yp1 and Yp2, and compared the predicted amino acid sequence of YP3 to YP1 and YP2. The results showed that the Yps are largely homologous in translated regions, especially in the 3' half of the genes. Untranscribed flanking regions had little homology. A conserved inverted repeat (the H-box) has homology both to vertebrate steroid hormone receptor binding sites and to the ecdysone control region of Drosophila's hsp23. These results identify sequences to mutate in order to define elements that regulate Yp gene expression and govern YP polypeptide function.

Amino Acid Sequence↗
PubMed1995

Promoter region of the transcriptional unit for human alpha 1-chimaerin, a neuron-specific GTPase-activating protein for p21rac.

alpha 1-chimaerin is a neuron-specific GTPase-activating protein for p21rac, a protein involved in morphological events. The mRNA is highly expressed in certain brain regions. It is also detected in cultured neuronal, but not in non-neuronal cells. As a first step towards understanding the mechanisms underlying this regulation, genomic clones containing the 5'-flanking region of the human alpha 1-chimaerin transcriptional unit were isolated and characterised. A cluster of multiple transcription start sites of alpha 1-chimaerin mRNAs was detected by primer-extension and S1-mapping analyses. The cluster was mapped to nucleotides -464 to -434 (relative to nucleotide A in the initiation codon) in genomic DNA. The 5'-proximal region contained no TATA box, initiator motif and Sp1-binding site. A 210-bp fragment with approximately 110 bp 5'-flanking sequence could function as a minimal promoter upon analysis using hybrid chloramphenicol acetyltransferase reporter constructs and transient transfection. Internal deletion and point-mutation experiments revealed that a GGCCAATC sequence located at nucleotides -519 to -512 was essential for alpha 1-chimaerin promoter activity. Mobility-shift assay showed the specific binding of nuclear factor(s) to this region, which was competed by the oligonucleotides corresponding to wild-type but not mutant forms. The data also suggest the existence of possible novel CCAAT-binding factor(s) interacting with the alpha 1-chimaerin CCAAT box binding site. A cell-type-preferred suppressor located in the 5'-distal region was found which may play a role in controlling neuron-specific expression of alpha 1-chimaerin mRNA. These findings of a specific promoter for alpha 1-chimaerin transcription will facilitate further studies on its neuronal-specific expression and function.

Amino Acid Sequence↗
PubMed1988

Physicochemical properties of Marburg virus: evidence for three distinct virus strains and their relationship to Ebola virus.

The physicochemical and antigenic properties of three groups of Marburg (MBG) virus isolates, separated temporally and geographically, were compared to each other and to another member of the same family, Ebola (EBO) virus. Each MBG isolate contained seven virion proteins, one of which was a glycosylated surface protein. Peptide mapping of glycoproteins, nucleoproteins (NP) and viral structural protein (VP40) demonstrated extensive sequence conservation in the proteins of viruses isolated over a 13-year period, but homology was not evident in VP24. Some homology between the NPs of MBG and EBO was observed. A close antigenic relationship between MBG strains was found by radioimmunoassay but no evidence was found of antigenic cross-reactivity with EBO viruses. MBG virion proteins are produced from virus-specific monocistronic mRNA species. Five of the seven viral proteins were produced by in vitro translation of these RNAs. MBG virions contained one RNA species with an Mr of 4.2 x 10(6) and virions had a density of 1.14 g/ml in potassium tartrate. Virus isolates from different outbreaks had distinct T1 oligonucleotide maps, but had approximately 95% homology in base sequence. No two geographically distinct virus pairs were more closely related to each other than to a third virus isolate. MBG viruses are thus similar to EBO viruses in morphology and other physicochemical properties and are very similar to each other in RNA and protein composition. Each of the three geographically and temporally distinct MBG virus outbreaks appears to have been due to a genetically distinguishable, but antigenically closely related virus strain. In addition, these studies confirm the belief that MBG and EBO viruses are members of the new virus family, the Filoviridae.

Animals↗
PubMed2003

Mapping of viral RNA sequences required for assembly of peanut clump virus particles.

RNA sequences required for assembly into rod-shaped virions of RNA-1 and RNA-2 of Peanut clump virus (PCV) were mapped by testing the ability of different RNA-1 and -2 deletion mutants to be encapsidated in vivo in an RNase-resistant form. Encapsidation of RNA-1 was found to require a sequence domain in the 5'-proximal part of the P15 gene, the 3'-proximal gene of RNA-1. On the other hand, the subgenomic RNA which encodes P15 was not encapsidated, suggesting that other features of RNA-1 are important as well. Two sequences which could drive encapsidation of RNA-2 deletion mutants were located. One was in the 5'-proximal coat protein gene and the other in the P14 gene near the RNA 3' terminus. There were no obvious sequence homologies between the different assembly initiation sequences.

Cell Cycle Proteins↗
PubMedDate not supplied

[Genetic recombination between natural isolates of influenza virus serotypes H1N1 and H3N2].

Oligonucleotide mapping of individual genes was used for search of possible genetic recombinants between natural isolates of influenza H1N1 and H3N2 viruses isolated in the USSR in 1977-1979. No antigenic hybrids and recombinants with the antigenic structure H3N2 were found, however, it was shown that isolates of H1N1 viruses of 1979 (the A/USSR/61/79 strain) might represent genetic recombinants carrying genes P1 + P2 from H3N2 viruses, the M-gene of the USSR/61/79 virus being closest in its structure to the analogous gene of the earliest isolate of H3N2 viruses, namely A/Hong Kong/1/68. Possible selective advantages of virus recombinants having M-genes from viruses of a different serotype are discussed.

Genes, Viral↗
PubMed1990

Intrinsic bends and integration host factor binding at F plasmid oriT.

F plasmid oriT DNA extending from the F kilobase coordinate 66.7 (base pair [bp] 1 on the oriT sequence map) rightward to bp 527 was analyzed for intrinsic bends (by permutation assays) and for binding of integration host factor (IHF) (by gel retardation and DNase footprinting). Intrinsic bending of the 527-bp fragment (bend center approximately at bp 240) was represented as a composite of at least two components located near bp 170 and near bp 260. IHF bound primarily to a site extending from bp 165 to 195 and with lower affinity to a site extending from bp 287 to 319. The intrinsic curvature and sequences to which IHF binds (IHF is known to bend DNA) may play a structural role in oriT function.

Bacterial Proteins↗
PubMed1988

A long range restriction map of the pseudoautosomal region by partial digest PFGE analysis from the telomere.

The analysis of partial digestion products extending from the telomere of the human X and Y chromosomes, visualised by hybridisation to a probe located close to the telomere, was used to establish a restriction map of the pseudoautosomal region. In this highly polymorphic region with a 10-fold elevated recombination frequency in males we identified site or methylation differences between 7 different in male and female cell lines and tissues, and derived an estimate of the size of the pseudoautosomal region of approximately 3 Megabases by comparing X and Y chromosomes. This size correlates well with previous estimates based on genetic arguments and argues against a strongly enhanced rate of exchange near telomeres in general. We identified a CpG rich and hypomethylated region within 500 kbp from the telomere, which might reflect structural features of mammalian telomeres, and a small number of (additional) CpG islands, which might represent candidate genes for the Turner phenotype in XO females.

Animals↗
PubMed1989

A simple and precise aberrant translocation of the rat c-myc gene into the epsilon-heavy chain switch region of the IgE-producing immunocytoma, IR162.

We report a simple type of reciprocal chromosomal translocation in the LOU rat IgE-secreting immunocytoma cell line, IR162, involving the c-myc protooncogene and the switch region of the epsilon immunoglobulin heavy chain, c-myc/S epsilon. By cloning and sequencing the translocation-associated and the homologous normal c-myc and S epsilon DNAs, we have identified the position of the translocational junction in both the c-myc 5'-flanking region and the repetitive elements of the S epsilon region. The translocational recombination was precise, and no insertion or N-addition was found in the junctional region, leaving all the c-myc exons, together with two promoter sites, intact. RNase mapping confirmed that the same promoters were utilized in IR162 and normal LOU spleen cells. No point mutation was found in the 5'-flanking region and the 3'-portion of exon 1 of the translocated c-myc gene. However, the putative silencer region was lost with the translocation. It was also noticed that a strikingly AT-rich sequence associated with S epsilon region had translocated to the 5'-flanking region of c-myc gene. We discuss the possibility that a change of DNA topology, perhaps either due to the juxtaposition of an AT-rich sequence of the S epsilon region, or to the loss of the putative silencer element, may contribute to c-myc gene deregulation in IR162.

Animals↗
PubMed1992

Scheme for the generation of a truncated endogenous murine leukaemia virus, the Fv-4 resistance gene.

The Fv-4 resistance (Fv-4r) gene is a truncated endogenous murine leukaemia virus (MuLV) containing a 3' portion of pol, the entire env gene and the 3' long terminal repeat. Env expression renders mice resistant to infection by ecotropic MuLVs, probably via receptor interference. Previous studies have suggested that the flanking cellular sequences are also important for Fv-4 env gene expression. To establish how the truncated retrovirus was generated and the nature of the cellular sequences involved, the Fv-4 susceptible (Fv-4s) allele DNA was cloned, and its restriction map and nucleotide sequence were compared with those of the Fv-4r allele. A likely mechanism for generation of the truncated endogenous MuLV is suggested by the results; integration of a prototype MuLV provirus at a site within the Fv-4s allele about 6 to 8 kb downstream of a non-retroviral promoter region, followed by deletion of the 5' half of the provirus, with an accompanying loss of only 7 or 10 bp of cellular flanking sequences. The deletion may have led to the expression of the Fv-4r env gene under control of the non-retroviral promoter.

3T3 Cells↗
PubMed1988

Localization and physical mapping of a plasmid-borne 23-kb nif gene cluster from Enterobacter agglomerans showing homology to the entire nif gene cluster of Klebsiella pneumoniae M5a1.

A physical and genetical map of the plasmid pEA3 indigenous to Enterobacter agglomerans is presented. pEA3 is a 111-kb large plasmid containing a 23-kb large cluster of nif genes which shows extensive homology (Southern hybridization and heteroduplex analysis) to the entire nif gene cluster of Klebsiella pneumoniae (Kp) M5a1. All the nif genes on pEA3 are organized in the same manner as in K. pneumoniae, except nifJ, which is located on the left end of pEA3 nif gene cluster (near nifQB). A BamHI restriction map of pEA3 and a detailed restriction map of the 23-kb nif region on pEA3 is also presented. The nif genes of pEA3 showed a low level of acetylene reduction in Escherichia coli, demonstrating that these genes are functional and contain the whole genetic information required to fix nitrogen. The origin of vegetative replication (OriV) of pEA3 was localized about 5.5 kb from the right end of the nif gene cluster. In addition to pEA3, large plasmids from four other strains of E. agglomerans showed homology to all the Kp nif genes tested, indicating that in diazotrophic strains of E. agglomerans nif genes are usually located on plasmids. In contrast, in most of the free-living, nitrogen-fixing bacteria the nif genes are on chromosome.

DNA Restriction Enzymes↗
PubMed1990

The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei.

The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site specific transcripts. The start of transcription was determined by hybridization and RNase protection analysis of nascent RNA. The 5' ends of the major transcripts coming from the initiation region map at nucleotide sequences that do not strongly resemble rRNA transcriptional starts even though the transcripts are synthesized by an RNA polymerase highly resistant to alpha-amanitin. The cloned VSG gene 221 ES transcription initiation region promotes high CAT gene expression, when reintroduced by electroporation into T. brucei. We show that the activity of this expression site is controlled at or near transcription initiation in bloodstream trypanosomes. The 221 ES is inactivated without any sequence alteration within 1.4 kb of the transcription start site. This excludes mechanisms of promoter inactivation involving DNA rearrangements in the vicinity of the transcription start site, e.g. promoter inversion or conversion.

Animals↗
PubMed1989

Transcriptional and posttranscriptional regulation contributes to the sex-regulated expression of two sequence-related genes at the janus locus of Drosophila melanogaster.

We investigated the structure and developmental pattern of expression of two genes clustered at the janus locus, located at 99D.3R. Data obtained from genomic and cDNA sequencing and from a combination of S1 mapping and primer extension experiments indicated a very unusual organization of this locus, which appeared to be composed of two partially overlapping genes, designated janA and janB. These two genes were found to be transcribed in the same direction. janA encoded one minor and two major transcripts. The 5' end of the janB mRNA mapped within the 3' untranslated region of the janA transcribed sequence. The overlapping region was 118 bases long. Similarities observed between these two genes with respect to both peptidic sequence and intron position strongly suggested that this locus originated from the duplication of an ancestral transcription unit. However, each of the resulting genes has acquired its own specificity of expression linked to sex determination. The janB transcript was detected only in males, and its expression at the adult stage was restricted to germ line cells. The janA gene displayed a much more complex expression; one of the major mRNAs was found in both sexes and at all stages, whereas the two other janA transcripts were expressed only in males.

Amino Acid Sequence↗