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PubMed1990

Developmental regulation of topoisomerase II sites and DNase I-hypersensitive sites in the chicken beta-globin locus.

We have mapped DNase I-hypersensitive sites and topoisomerase II (topo II) sites in the chicken beta-globin locus, which contains four globin genes (5'-rho-beta H-beta A-epsilon-3'). In the 65 kilobases (kb) mapped, 12 strong hypersensitive sites were found clustered within the 25-kb region from 10 kb upstream of rho to just downstream of epsilon. The strong sites were grouped into several classes based on their tissue distribution, developmental pattern, and location. (i) One site was present in all cells examined, both erythroid and nonerythroid. (ii) Three sites, located upstream of the rho-globin gene, were present at every stage of erythroid development, but were absent from nonerythroid cells. (iii) Four sites at the 5' ends of each of the four globin genes were hypersensitive only in the subset of erythroid cells that were transcribing or had recently transcribed the associated gene. (iv) Another three sites, whose pattern of hypersensitivity also correlated with expression of the associated gene, were found 3' of rho, beta H, and epsilon. (v) A site 3' of beta A and 5' of epsilon was erythroid cell specific and present at all developmental stages, presumably reflecting the activity of this enhancer throughout erythroid development. We also mapped the topo II sites in this locus, as determined by teniposide-induced DNA cleavage. All strong teniposide-induced cleavages occurred at DNase I-hypersensitive sites, while lesser amounts of cleavage were observed in transcribed regions of DNA. Most but not all of the DNase I-hypersensitive sites were topo II sites. These data are consistent with the hypothesis that, in vivo, topo II preferentially acts on nucleosome-free regions of DNA but suggest that additional topo II regulatory mechanisms must exist.

Animals↗
PubMed1991

Two regions of the mouse mammary tumor virus long terminal repeat regulate the activity of its promoter in mammary cell lines.

In vivo expression of the mouse mammary tumor virus (MMTV) is restricted to a few organs, with the highest rate of transcription found in the mammary gland. Using a series of mammary and nonmammary murine cell lines, we have identified two regulatory elements, located upstream of the hormone responsive element, that specifically regulate the MMTV promoter. The first element displays an enhancerlike activity and is coincident with the binding of a nuclear factor (designated MP4; position -1078 to -1052 in the long terminal repeat) whose presence is apparently restricted to mammary cell lines. The second regulatory region mediates a repressive activity and is mapped to the long terminal repeat segment from -415 to -483. This repression is specific for a particular subtype of mammary cells (RAC cells) able to grow under two differentiation states (A. Sonnenberg, H. Daams, J. Calafat, and J. Hilgers, Cancer Res. 46:5913-5922, 1986). The MMTV promoter in mammary cell lines thus appears to be modulated by two cis-acting elements that are likely to be involved in tissue-specific expression in vivo.

Animals↗
PubMed1991

The herald waves of influenza virus infections detected in Sendai and Yamagata cities in 1985-1990.

The community surveillance of respiratory virus infections performed during 1985-1987 in Sendai and 1988-1990 in Yamagata has identified a total of five herald waves of influenza virus infections: A/H3N2 virus infections in 1985 and 1989, A/H1N1 virus infections in 1986 and 1988, and type B virus infections in 1989. To investigate the antigenic and genetic relationships between the herald wave and epidemic strains, influenza A/H1N1 viruses isolated during the 1986 and 1988 herald waves were compared with those isolated during the 1986-1987 and 1988-1989 epidemic seasons, respectively, utilizing hemagglutination inhibition tests with anti-hemagglutinin monoclonal antibodies and oligonucleotide mapping of total viral RNAs. The results showed that multiple variants differing in antigenic and genetic properties were cocirculating during the 1986 herald wave as well as during each of the two epidemics (only one strain was isolated in the 1988 herald wave). It was also observed that viruses which had the antigenic and/or genetic characteristics closely similar to those of the viruses circulating in the 1986 and 1988 herald waves, were isolated during the winters of 1986-1987 and 1988-1989, respectively.

Antibodies, Monoclonal↗
PubMed1989

A transcriptional map of visna virus: definition of the second intron structure suggests a rev-like gene product.

Visna virus is the prototype lentivirus, with a genome structure similar to that of the human immunodeficiency viruses HIV-1 and HIV-2. We have analysed in vitro the transcription pattern of this virus in lytic infections of choroid plexus cells. Northern blot analysis shows the presence of spliced subgenomic mRNA species of 4.9, 4.3, 4.0, 1.7 and 1.4 kb. Use of appropriate subgenomic probes shows that the first three of these species encode envelope protein (but also potentially the small open reading frames Q and S). The 1.7 kb RNA could contain S. In order to elucidate the translational coding potential of the smallest RNA, and to characterize further all the transcripts, S1 mapping was performed across those parts of the genome which were close to exon/intron boundaries. This allowed the definition of acceptor splice sites following both introns 1 and 2 as well as donor sites preceding intron 2. The data suggest that the 1.4 kb RNA encodes a protein derived from the F reading frame that may form part of a precursor protein and also contains sequences with some degree of homology to the rev (trs/art) protein of HIV, as well as a typical protease cleavage site between these sequences and the F protein.

Amino Acid Sequence↗
PubMed1990

Two lectin genes differentially expressed in Dolichos biflorus differ primarily by a 116-base pair sequence in their 5' flanking regions.

Previous studies in our laboratory have shown that the Dolichos biflorus plant contains two similar lectins, a seed lectin and a stem and leaf lectin called DB58, that are present at different stages in the plant's life cycle. We have now established that each of these lectins is encoded by a separate gene by isolating these lectin genes from a library of D. biflorus nuclear DNA. Restriction mapping and nucleotide sequencing analyses show that the seed lectin and DB58 genes are located in the same transcriptional orientation within 3-kilobase pairs of one another. The lectin genes contain no introns and show greater than 90% nucleotide sequence identity in their protein coding and untranslated regions. This sequence similarity extends to both the 5' and 3' flanking regions of the genes; the major exception is that a 116-base pair segment located at position -215 to -100 from the transcription start site of the seed lectin gene is missing in the 5' flanking region of the DB58 gene. The possible significance of this segment with respect to the differential expression of these genes is discussed.

Base Composition↗
PubMedDate not supplied

[Genetic variability of epidemic strains of influenza virus serotypes H1N1 and H3N2 during antigenic drift].

Data are presented on structural variability of individual genes of selected variants of epidemic influenza viruses H1N1 (1977-1979) and H3N2 (1968-1979) in the course of antigenic drift obtained by oligonucleotide mapping. Six out of 8 genes of H1N1 viruses were found to be more variable than the corresponding genes of H3N2 viruses. Only HA and NS genes of H3N2 viruses underwent greater structural changes as compared with the analogous genes of H1N1 viruses. In viruses of both serotypes, most variable were the genes coding for hemagglutinin and matrix protein. Possible causes of greater structural variability of the matrix protein gene in the course of antigenic drift are discussed.

Antigenic Variation↗
PubMed1988

p55 IL-2 receptor mRNA precursors in murine T lymphocyte nuclei.

An unusual family of cDNA clones homologous to human p55 IL-2R sequences was isolated from the murine HT-2 Th cell line. These clones were mapped, partially sequenced, and compared with previously published human and mouse IL-2R sequences. They appear to consist of various combinations of exons and introns, suggesting that they are derived from p55 IL-2R mRNA precursors. The configuration of exons in the splicing intermediates indicates that the murine and human gene organizations are similar and that the 3' end of intron 3 is well conserved between the two species. RNA mapping experiments using nuclear, cytoplasmic, and total RNA and probes derived from various parts of the p55 IL-2R gene support and extend the sequence data. They indicate that detectable amounts of immature p55 IL-2R mRNA are found specifically in the cell nucleus of the HT-2 cell line. Similar data were obtained for the Th cell clone 52.3 and the cytotoxic T cell line CTLL. All these results indicate that the T cell nucleus contains significant amounts of immature p55 IL-2R mRNA.

Animals↗
PubMed1988

Organization of the ribosomal RNA genes of Schizophyllum commune.

The 18, 5.8, 25 and 5S ribosomal RNA (rRNA) cistrons have been mapped on the ribosomal DNA (rDNA) unit repeat of Schizophyllum commune strain 4-40. These genes are spatially ordered in the sequence given. The presence of a large primary precursor rRNA which is processed to form the mature 18, 5.8 and 25S rRNAs has been demonstrated. We have mapped the site of transcriptional initiation for this rRNA primary precursor. The sequence surrounding this site has been determined and shown to be highly conserved, with considerable identity to those in Neurospora crassa and Dictyostelium discoideum. The direction of transcription of the rRNA genes has been determined. The 5S rRNA cistron is transcribed in the same direction as the other rRNAs, however it is not transcribed as a part of the large primary precursor. The previously identified rDNA strain-specific length polymorphisms (Specht et al. 1984) are shown to be located within the transcribed region of the rDNA unit repeat.

Basidiomycota↗
PubMed1992

Stable heteroplasmy for a large-scale deletion in the coding region of Drosophila subobscura mitochondrial DNA.

Due to the extremely economic organization of the animal mitochondrial genome, large-scale deletions are rarely found in animal mtDNA. We report the occurrence of a massive deletion in the coding region of mtDNA in Drosophila subobscura. Restriction mapping and nucleotide sequence analysis revealed that the deletion encompasses six protein genes and four tRNAs. All individuals of an isofemale strain proved to be heteroplasmic for normal and deficient mtDNA molecules. This type of heteroplasmy resembles one observed in patients with mitochondrial myopathies but differs in that the fitness of heteroplasmic flies is not significantly reduced even though the mutant mtDNA constitutes 50-80% of total mtDNA in most of the individuals studied. The heteroplasmic strain is genetically stable: despite extensive screening not a single homoplasmic fly was observed since the foundation of the line.

Amino Acid Sequence↗
PubMed1997

Fast-atom bombardment mass spectrometry for mapping of endogenous methylated purine bases in urine extracts.

Fast-atom bombardment (FAB) mass spectrometry, linked with tandem mass spectrometry (MS/MS), was employed for the identification of methylated purine bases in four urinary extracts of healthy subjects and fourteen urinary extracts of patients bearing colorectal tumors. In order to obtain an easy structural identification of the species present in urinary extracts, the MS/MS spectra of MH+ species of twenty nine diagnostically relevant purine bases were studied. Even if definitive quantitative data cannot be obtained by this approach, FAB mass spectra of urine extracts lead to a readily reproducible mapping of endogenous purine bases, allowing a distinction between healthy and sick subjects. Bases such as 9-ethyladenine, N6-2-isopentenyladenine and N6-benzyladenine were detected only in urine samples of colorectal tumor bearing patients. The detection in urine of compounds such as 7-methylguanine and 1-methylguanine, and their increase in the urine of colorectal tumor bearing patients, has been justified either by a more rapid turnover of nucleic acids in tumor tissue or by an increase in the extent of their methylation. The obtained results indicate that the method can be employed for diagnostic purposes.

Colorectal Neoplasms↗
PubMed1987

Structure of the cloned Locusta migratoria mitochondrial genome: restriction mapping and sequence of its ND-1 (URF-1) gene.

We have cloned the entire mitochondrial genome of Locusta migratoria in four fragments and characterised by restriction mapping. In addition, we have sequenced a 1,095 kb region containing the ND-1 (URF-1) gene. The inferred primary structure of the protein is highly homologous to its Drosophila counterpart (68%). The gene is flanked at the 5' end by the tRNA(CUNleu) gene, interrupted by the sequence TTG. The 3' end is flanked by the tRNA(UCNser) gene, followed by a sequence homologous to the 3' end of D. yakuba cytochrome b. The relative position of the genes is conserved between Locusta and Drosophila, thus indicating conservation of mitochondrial gene order in insects.

Amino Acid Sequence↗
PubMed1987

Tumorigenic poxviruses: transcriptional mapping of the terminal inverted repeats of Shope fibroma virus.

A composite transcriptional map for the entire 12.4-kb terminal inverted repeat (TIR) region of the Shope fibroma virus (SFV) genome has been determined. Northern blotting and S1-nuclease mapping were used to determine the regions which are transcribed, their temporal relationships, as well as the transcriptional initiation sites. Sequences representing the entire TIR are transcribed into poly(A)+ mRNA at both early and late times in the infection. Fifteen transcriptional initiation sites were mapped, 12 within the TIRs and 3 within the unique sequences close to the junction between the right TIR and the unique internal sequences. Ten of the 12 transcriptional initiation sites within the TIR and 2 of the 3 sites outside the right TIR correspond to the 5'-ends of the major open reading frames (ORFs) T1 to T9 plus the SFV growth factor gene. The 3 other initiation sites map within ORFs but near potential start codons for shorter polypeptides. All the expressed ORFs are tandemly arranged and transcribed toward the hairpin terminus. At early times during SFV infection of cultured rabbit cells, transcription of each ORF gives rise to a transcript of distinct size, while at late times termination of transcription is imprecise and substantial read-through into downstream sequences occurs. These results are discussed in light of recent observations on the related recombinant leporipoxvirus, malignant rabbit fibroma virus, which suggest that one or more gene products from this region of the SFV genome are implicated in viral tumorigenicity.

Base Sequence↗
PubMed1987

RNA 3' processing regulates histone mRNA levels in a mammalian cell cycle mutant. A processing factor becomes limiting in G1-arrested cells.

Post-transcriptional regulation of histone gene expression in a mouse mastocytoma cell cycle mutant (21-Tb) depends largely on conserved DNA sequences that are essential for RNA 3' processing. We have analyzed whether this regulation occurs at the level of RNA 3' processing. We show, by RNase mapping, that nuclear H4 mRNA precursors, which are hardly detectable in total RNA from exponentially dividing cells, accumulate in G1-arrested cells, i.e. when mature mRNAs are drastically reduced. Furthermore, we show that a heat-labile component of the processing apparatus, recently identified in HeLa cell nuclear extracts, is limiting in extracts from G1-arrested 21-Tb cells. In contrast, this activity is in excess in extracts from exponentially dividing cells, whereas both extracts contain similar amounts of snRNPs of the Sm serotype. These fluctuations in the heat-labile activity may generally contribute to proliferation or cell cycle dependent histone gene regulation.

Animals↗
PubMed1992

Isolation of new temperature-sensitive mutants of Saccharomyces cerevisiae deficient in mannose outer chain elongation.

We have isolated two temperature-sensitive Saccharomyces cerevisiae mutants which exhibit a deficiency in mannose outer chain elongation of asparagine-linked oligosaccharide. The size of yeast glycoprotein, secretory form of invertase, of one mutant (och1) was slightly larger than that of the sec18 mutant at the non-permissive temperature, while that of the other mutant (och2) was almost the same as that of the sec18 mutant. Unlike sec mutants, the och mutants were not deficient in secretion of invertase. The och1 mutant showed a 2+:2- cosegregation with regard to the temperature sensitivity and mannose outer chain deficiency, suggesting that a single gene designated as OCH1 is responsible for these two phenotypes. The och1 mutant stopped its growth at the early stage of bud formation and rapidly lost its viability at the non-permissive temperature. The och1 mutation was mapped near the ole1 on the left arm of chromosome VII. The och1 mutant cells accumulated the external invertase containing a large amount of core-like oligosaccharides (Man9-10GlcNAc2) and a small amount of high mannose oligosaccharides (greater than Man50GlcNAc2) at the non-permissive temperature. Production of the active form of human tissue-type plasminogen activator was increased in the och1 mutant compared with the parental strain, suggesting the potential advantage of this mutant for the production of mammalian-type glycoproteins which lack mannose outer chains in yeast.

Asparagine↗
PubMed1987

Mapping the B and D gene promoters of bacteriophage S13 by footprinting and exonuclease III analysis.

The region of the bacteriophage S13 genome which contains the B, K, and C genes and most of the A, D, and E genes (map positions 627 to 2198) was analyzed for Escherichia coli RNA polymerase-binding sites by combined DNase I footprinting, exonuclease III analysis, and DNA sequencing. Two high-affinity binding sites that correspond to the B and D gene promoters were mapped at positions 936 to 995 and 1779 to 1848, respectively. Positions 936 to 995 are in gene A, preceding the B gene, and positions 1779 to 1848 are in gene C, just preceding the D gene. In addition, two lower-affinity binding sites were identified at positions 1527 to 1538 and 1705 to 1756 preceding the C and D genes, respectively. The footprinting studies described here, in combination with previous studies on transcription, provide definitive evidence on the position of the B and D gene promoters in S13 and the closely related phage phi X174.

Coliphages↗
PubMed1992

Antigenic and genetic analyses of eight influenza C strains isolated in various areas of Japan during 1985-9.

Eight strains of influenza C virus isolated in various areas of Japan between January 1985 and January 1989 were compared using monoclonal antibodies to the haemagglutinin-esterase (HE) glycoproteins and by oligonucleotide mapping of total vRNA. Five of six strains isolated during 1986-9 were closely related to one another and also resembled the virus, C/Aichi/1/81, isolated in 1981 in Aichi prefecture. This suggests that the C/Aichi/1/81-related viruses had an epidemiological advantage over any co-circulating viruses at least during that period. One of two 1985 isolates (C/Nara/1/85) was antigenically indistinguishable from the C/Mississippi/1/80 strain though their oligonucleotide patterns were markedly different from each other. This raises the possibility that C/Nara/1/85 may be a recombinant virus which receives its HE gene from the C/Mississippi/1/80-related parent.

Antigens, Viral↗
PubMed1990

Differential regulation of the hepatitis B virus surface gene promoters by a second viral enhancer.

The hepatitis B virus surface gene is transcribed from two promoters, and the resulting mRNA species code for three distinct forms of the surface antigen. We show here that the viral transcriptional trans-activator, X protein, has no effect on either promoter. However, a cis-acting element in the downstream half of the X gene, distinct from the previously mapped viral enhancer, selectively activates the major surface gene promoter. Nuclease protection and gel-shift assays reveal that multiple cellular factors bind to two sites within this DNA fragment, both of which are necessary for enhancer activity. Since this region of the viral genome is frequently deleted upon integration into the hsot chromosome in chronic hepatitis B, loss of this second enhancer can alter the relative amounts of the three forms of the surface antigen in infected hepatocytes and thus possibly contribute to cellular damage.

Base Sequence↗
PubMed1988

R-loop mapping of RNA transcripts from bovine papillomavirus type 4.

RNA isolated from bovine oesophageal warts was hybridised to BPV-4 genomic DNA cloned in a plasmid vector. In R-loop preparations, six major classes of transcripts were mapped. Class I, due to an unspliced transcript encompassing the E4/E5 ORF region, is most common. In Class II the E4/E5 region is spliced at its 5' end to the E6 ORF region, and the RNAs appear to have different transcription start points in the E6 ORF. Some Class I R-loops may represent shorter Class II-type transcripts not hybridised to the E6 region. Transcripts that form Class III R-loops have not been previously described for BPV-4 and contain the E4/E5 ORF exon spliced at its 3' end to the LI ORF. In Class IV, transcripts map to the 3' end of the EI exon, artificially truncated by the Bam HI site used for cloning the BPV-4 genome, and are spliced to the 5' end of the exon containing the E4/E5 ORF. Class V transcripts are ambiguously located in the cloned BPV-4 genome, and could be derived from the EI or LI ORF. The former case may represent the remainder of the transcript from Class IV R-loops. The rare Class VI R-loops are due to the L2 ORF spliced at its 3' end to the LI ORF.

Bovine papillomavirus 1↗