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PubMed1995

Long, nearly identical untranslated sequences at the 3' terminal regions of the genomic RNAs of cherry leafroll virus (walnut strain).

Hybridization analyses of cDNA clones derived from the two genomic RNAs, RNA1 and RNA2, of the walnut strain of the nepovirus cherry leafroll nepovirus (wCLRV) demonstrated a long region of high homology between the two viral RNAs. Subsequent mapping and nucleotide sequencing revealed a long, noncoding, presumably untranslated, region (3' UTR) immediately 5' of the terminal polyadenylate, a region that is almost identical in the two RNAs. This 3' UTR is 1567 nucleotide residues long in RNA1. Homologies of about 80% were found with corresponding regions of genomic RNAs from other strains of CLRV, but not with the corresponding regions of other nepovirus genomic RNAs.

Base Sequence↗
PubMed1987

[The nature of virus strains isolated from patients with poliomyelitis in the Byelorussian SSR 1967-1985].

Oligonucleotide mapping of the RNA genome was applied for analysis of poliovirus clinical isolates in Byelorussia in 1967-1985. All the maps have been clearly shown to resemble the reference maps of the corresponding vaccine strains. Examination of the oligonucleotide maps of poliovirus isolates provides a measure of distribution of some of the mutations over the viral genome.

Antigens, Viral↗
PubMed1988

Haemolytic activity and characteristics of plasmid and chromosomally borne hly genes isolated from E. coli of different origin.

20 hly plasmids of different origin and source and 8 recombinant plasmids containing either chromosomally or plasmid encoded hly determinants were characterized with emphasis on their haemolytic activity. Remarkable differences among the hly genes tested were found in their haemolytic activity and in the regulation of the haemolytic activity by the extracellular iron concentration. Under iron-limiting growth conditions the haemolysin secretion encoded by 6 hly plasmids, but not by the remaining 14 hly plasmids and the chromosomal hly genes, was significantly induced. The 6 plasmids all derived from human isolates, belonged to the incompatibility groups FIV and FVI and showed similar restriction patterns. Two hly determinants, encoding an inducible haemolysin secretion, cloned from plasmids isolated in Berne and Paris, respectively, showed identical restriction maps, indicating a common origin. Compared to the hly determinant of a plasmid encoding a noninducible haemolysin secretion, distinct differences, mainly in the flanking regions, were observed.

Animals↗
PubMed1986

Structure of genes in the cytochrome P-450PBc subfamily: conservation of intron locations in the phenobarbital-inducible family.

The structures of P-450 genes in the rabbit phenobarbital-inducible subfamily of P-450 genes, defined by the cytochrome P-450PBc cDNAs, have been determined by restriction mapping and nucleotide sequencing. A genomic clone corresponding to the 5'-flanking region and the first six exons of the cytochrome P-450PBc2 gene has been examined in detail, and two other clones corresponding to exons 7-9 and the 3'-flanking region of the cytochrome P-450PBc3 and cytochrome P-450(form 1) genes have been characterized less extensively. The intron sequences adjacent to the exons are typical of vertebrate nuclear genes and contain GT and AG at their 5' and 3' ends, respectively. The intron locations in these genes are identical to those in the genes for rat cytochrome P-450b/e, members of the other phenobarbital-inducible subfamily, but completely different from those in the 3-methylcholanthrene-inducible cytochrome P-450 genes. Repetitive sequences are present in at least three introns of the cytochrome P-450PBc2 gene, but not in exons or the 5'-flanking region. The site of transcription initiation for the cytochrome P-450PBc2 gene was determined by primed reverse transcription and S1 mapping. Although the mRNA appeared to be heterogenous at the 5' terminus, the most probable start site is 18 bp downstream from a canonical TATA sequence and 23 bp upstream from the initiator ATG codon. The nucleotide sequence of 800 bp of the 5'-flanking region of the cytochrome P-450PBc2 gene has been determined. This region shares little homology with the 5'-flanking region of rat cytochrome P-450b/e genes, which are 55-60% homologous in the protein-coding region with the members of the P-450PBc subfamily. The amino-terminal sequence of cytochrome P-450PBc2, which could not be determined from the cDNA clones, was derived from the gene sequence. Comparison of the amino-terminal amino acid sequence of P-450PBc2 with those of other mammalian P-450s indicates that the rat isozymes f, g, h, i, and PB1 are in the same subfamily as the rabbit isozymes form 1, form 3b or P-450PBc3, P-450PBc1, and P-450PBc2.

Amino Acid Sequence↗
PubMed1979

Characterization of the major altered leader sequence of late mRNA induced by SV40 deletion mutant d1-1811.

d1-1811 is a viable SV40 mutant with a 40 base pair deletion that includes the major wild-type capping site of late mRNA at map position 0.72. The late viral mRNAs induced by d1-1811 have now been further characterized by inversed S1-mapping analysis. The S1-resistant, 32P-labeled RNA fragments derived from the leader region were examined by fingerprinting and by analysis of RNase T2-generated 5'-terminal cap structures. The results show that most if not all of the mutant leader fragments analyzed have their 5' terminus to the left of the d1-1811 deletion site, i.e., closer to the origin of DNA replication. The major altered leader fragment is a continuous transcript from the DNA in the region 0.716 to 0.761 map unit and its 5' terminus has been precisely mapped at nucleotide L290. The observation that the cap structure of the major altered leader is only a minor cap species in wild-type late RNA suggests regulation in the use of different capping sites in SV40.

Base Sequence↗
PubMed1989

RFLP-based genetic map of Phanerochaete chrysosporium ME446: lignin peroxidase genes occur in clusters.

We describe the construction of an RFLP-based genetic map of the white rot fungus Phanerochaete chrysosporium ME446. The map is deduced from the allele distributions of 38 RFLP markers in a test set of 53 meiotically derived haploid recombinants of this strain. The map includes a cellulase gene, a matingtype locus and a family of lignin peroxidase (and related) genes that are arranged in two unlinked clusters.

Alleles↗
PubMed1985

Polymorphism of mitochondrial DNA in pigs based on restriction endonuclease cleavage patterns.

Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) of pigs and Japanese wild boars were analyzed using 17 enzymes which recognize six nucleotides. The map of cleavage sites was made by double-digestion methods. Polymophism of mtDNA was detected in the digestion by BglII, EcoRV, ScaI, and StuI. The restriction cleavage patterns were identical among the breeds of Landrace, Hampshire, Duroc I, and Large White I (A type). The patterns of Large White II were the same as those of Japanese wild boars (B type). A difference between the A type and the B type of mtDNA was found in the case of three restriction enzymes, BglII, ScaI, and StuI, and the nucleotide alterations between them were estimated as more than six. On the other hand, a difference between mtDNA from almost all pigs and mtDNA from Duroc II was detected using EcoRV. We suggest that the difference of mtDNA between the A type and the B type of mtDNA could result from the different origin of boars, that is, whether they were of European or Asian origin.

Animals↗
PubMed1991

The first isolation of swine H1N1 influenza viruses from pigs in Thailand.

Two influenza A viruses were isolated from pigs in Thailand in January 1988 during the early febrile stage of an influenza-like illness. The isolates contained hemagglutinin and neuraminidase antigens related to those of swine H1N1 influenza virus. This result based on the virus isolation is compatible with the epizootiological evidence that, unlike the human influenza with peak activity in summer (May-July), swine influenza virus is prevalent in the winter season (November-January) in Thailand. The proportion of sera with hemagglutination-inhibiting antibody was higher to A/NJ/8/76 than to A/sw/Iowa/15/30. Likewise, hemagglutination-inhibition tests with monoclonal antibodies indicated that hemagglutinin antigen of the isolates was very similar to that of A/NJ/8/76 virus. In agreement with the serological survey and antigenic characteristic, genetic relatedness between the isolates from Thailand and A/NJ/8/76 virus was also demonstrated by the oligonucleotide mapping of RNA, suggesting that they may be of the same origin.

Animals↗
PubMed1991

Monitoring bovine viral diarrhea virus vaccines for adventitious virus, using T1 ribonuclease viral RNA oligonucleotide fingerprinting.

Viral RNA oligonucleotide fingerprinting was used to discriminate 3 cytopathic vaccine bovine viral diarrhea viruses (BVDV) grown in medium supplemented with serum contaminated with noncytopathic BVDV from the same 3 viruses grown in cell culture free of BVDV. Oligonucleotide fingerprinting also effectively discriminated between reference Singer BVDV, NADL BVDV, and New York-1 BVDV grown in BVDV-free noncontaminated or BVDV-contaminated cell cultures. Oligonucleotide fingerprint mapping of viral RNA maybe used to determine the purity of virus stocks, as well as that of BVDV vaccines.

Animals↗
PubMed1987

Mapping of DNA markers close to the fragile site on the human X chromosome at Xq27.3.

We report the identification of a new RFLP detected by the DNA probe MN12, which is linked to both the fragile site on the X chromosome at Xq27.3 and the highly polymorphic locus detected by St14 (DXS52). In situ mapping confirms the localisation of MN12 distal to the fragile site. A detailed physical analysis of this region of the X chromosome using pulsed-field gel electrophoresis has shown that MN12, St14 and DX13 (DXS15) are physically linked within a region of 470kb. A long range restriction map around the MN12 locus reveals at least two candidate HTF islands, suggesting the existence of expressed sequences in this region.

Base Sequence↗
PubMed1994

Solution structure of the 3'-end of brome mosaic virus genomic RNAs. Conformational mimicry with canonical tRNAs.

The conformation of the last 201 nucleotides located at the 3'-end of brome mosaic virus (BMV) RNAs was investigated in solution using different chemical and enzymatic probes. Bases were probed with dimethylsulfate (which methylates N-1 positions of A, N-3 positions of C and N-7 positions of G), a carbodiimide (which modifies N-1 positions of G and N-3 positions of U) and diethylpyrocarbonate (which modifies N-7 positions of A). Ribonucleases T1, U2 and S1 were used to map unpaired nucleotides and ribonuclease V1 to monitor paired bases or stacked nucleotides. Cleavage or modification sites were detected by gel electrophoresis either indirectly by analyzing DNA sequence patterns generated by primer extension with reverse transcriptase of the modified RNAs or by direct identification within the statistical cleavage patterns of the RNA. On the basis of these biochemical results, an atomic model was built by computer modeling and its stereochemistry refined. The deduced secondary structure of the RNA confirms data previously proposed by others but contains additional base-pairs (A27-U32, A28-G31, G41-A134, G64-C68, U80-A99, G81-A98, G88-U91, G100-U126, U104-U125, G162-G166 and A172-A191), one new tertiary long-range interaction (U103-U164) and a small triple helical conformation with (G41-A134)-A18 and (C42-G133)-A17 interactions. The new secondary structure also indicates the existence of a second pseudoknot involving pairing between residues A181 to A184 and residues U197 to U194, outside the domain conferring tyrosylation ability to BMV RNA. The main outcome from the model stems from its intricate folding, which allows a new assignment for the domains mimicking the anticodon- and D-loop regions of tRNA. Interestingly, the stem and loop region found structurally to be analogous to the anticodon arm of tRNA(Tyr) does not contain the tyrosine anticodon involved in the aminoacylation process. The structural analogies with canonical tRNA(Tyr) illustrate the functional mimicry existing between the BMV RNA structure and canonical tRNA(Tyr) that allows for their efficient aminoacylation by tyrosyl-tRNA synthetase. This structural model rationalizes mutagenic and footprinting data that have established the importance of specific regions of the viral RNA for recognition by its replicase, (ATP,CTP):tRNA nucleotidyl-transferase and yeast tyrosyl-tRNA synthetase. The new fold has biological implications that can be used as a predictive tool for elaborating new experiments.

Base Sequence↗
PubMed2005

Characterization and expression analysis of the groESL operon of Bartonella bacilliformis.

The groESL operon of Bartonella bacilliformis, a facultative intracellular, Gram-negative bacterium and etiologic agent of Oroya Fever, was characterized. Sequence analysis revealed an operon containing two genes of 294 (groES) and 1632 nucleotides (groEL) separated by a 55-nt intergenic spacer. The operon is preceded by a 72-nt ORF (ORF1) that encodes a hypothetical protein with homology to a portion of the HrcA repressor for groESL. A divergent fumarate hydratase C (fumC) gene lies further upstream. Deduced amino acid sequences for B. bacilliformis GroEL and GroES revealed a high degree of identity with homologues from other Bartonella and alpha-Protebacteria. A single transcriptional start site (TSS) was mapped 79 nucleotides upstream of the groES start codon, regardless of incubation temperature. The TSS was located immediately 5' to a potential controlling inverted repeat of chaperonin expression (CIRCE) element and is preceded by a sigma70-like promoter. The operon is followed by a predicted rho-independent transcriptional terminator. Northern blot analysis indicated that groES and groEL are co-transcribed as a single mRNA of approximately 2.4 kb. A 6-h time course analysis by qRT-PCR showed that groEL expression increases 1.3-fold within 30 min of a temperature upshift from 30 to 37 degrees C, with maximum transcription reached after 60 min (approximately 4.3-fold), followed by a steady decrease to background (30 degrees C) transcription levels by 6 h. Western blot analysis revealed a 1.4- and 1.5-fold increase in GroEL synthesis following a temperature upshift or by inhibiting DNA supercoiling with coumermycin A1, respectively. Functional expression and complementation of temperature-sensitive Escherichia coli groES or groEL mutants with the cloned operon allowed them to grow at otherwise restrictive temperatures.

Amino Acid Sequence↗
PubMed1983

Microtubule assembly with the guanosine 5'-diphosphate analogue 2',3'-dideoxyguanosine 5'-diphosphate.

The GDP analogue 2',2'-dideoxyguanosine 5'-diphosphate (ddGDP) supports efficient tubulin polymerization. Microtubule-associated protein (MAP) dependent microtubule assembly occurs in 0.1 M 2-(N-morpholino)-ethanesulfonate, and sheets of parallel protofilaments are formed in 1.0 M glutamate without MAPs. The nucleotide is bound to tubulin in the course of polymerization, presumably in the exchangeable GTP site. The ddGDP is not hydrolyzed, however, and is completely stable in the reaction mixture. Nor was the nonexchangeable GTP bound to tubulin hydrolyzed in ddGDP-supported polymerization: equivalent amounts of GTP remained associated with polymerized tubulin after polymerization with either ddGDP or GTP. Higher concentrations of ddGDP than GTP were required under all conditions. Nevertheless, under optimum conditions for the ddGDP-supported reaction, polymerization began with a shorter lag period and both the rate and extent of polymerization were greater with ddGDP than with GTP. The MAP-dependent reaction with ddGDP is temperature dependent, cold reversible, and inhibited by calcium and antimitotic drugs. It differs from the GTP-supported reaction in being most vigorous at minimal Mg2+ concentrations and exquisitely sensitive to GDP inhibition.

Animals↗
PubMed1997

Identification and characterization of ssb and uup mutants with increased frequency of precise excision of transposon Tn10 derivatives: nucleotide sequence of uup in Escherichia coli.

A Lac+ papillation assay was used to identify mutants (tex) of Escherichia coli that exhibit an increased frequency of precise excision of a lacZ::Tn10dKan insertion. Three tex strains had suffered mutations in the gene (ssb) encoding the essential single-stranded DNA-binding protein SSB, which resulted in the following alterations in the 177-residue protein: G4D; L10F, P24S; and V102M. The phenotypes of these ssb mutants indicated that they were largely unaffected in other functions mediated by SSB, such as DNA replication, recombination, and repair. Strains with multicopy ssb+ exhibited a decreased frequency of Tn10dKan precise excision. Three other tex mutants had insertion mutations in the locus designated uup at 21.75 min on the linkage map. The nucleotide sequence of uup was determined, and the gene was inferred to encode a 625-amino-acid hydrophilic protein that belongs to the superfamily of ABC-domain proteins (with two pairs of the Walker A and B motifs), which are postulated to be involved in coupling ATP hydrolysis with other biological processes. The uup gene product shares extensive homology with the deduced sequences of two proteins of Haemophilus influenzae. The uup gene is also situated immediately upstream of (and is transcribed in the same direction as) the paraquat-inducible SoxRS-regulated pqi-5 gene, two reported promoters for which are situated within the uup coding sequence.

ATP-Binding Cassette Transporters↗
PubMed1988

The histone H3 and H4 mRNAs are polyadenylated in maize.

Northern blot analysis revealed that the histone H3 and H4 mRNAs are of unusual large size in germinating maize embryos. S1-mapping experiments show that the 3'-untranslated regions of the mRNAs transcribed from 3 H3 and 2 H4 maize genes previously described are much longer than in the non-polyadenylated histone mRNAs which represent a major class in animals. Moreover, oligo d(T) cellulose fractionation of RNAs isolated at different developmental stages indicates that more than 99% of the maize H3 and H4 mRNAs are polyadenylated. A putative polyadenylation signal is present in all five genes 17 to 27 nucleotides before the 3'-ends of the mRNAs.

Base Sequence↗
PubMed1992

Baboon apolipoprotein C-I: cDNA and gene structure and evolution.

We have isolated and characterized cDNA and genomic clones for apolipoprotein C-I (apoC-I) encoded by the APOC1 locus in baboons. Baboon apoC-I cDNA is only 410 bp in size, but the gene spans 4.5 kb including four small exons and three introns containing a large number of Alu repeats. The coding sequences of apoC-I cDNA and genomic clones are identical, indicating that this genomic clone contains the functional gene for apoC-I rather than a pseudogene like human APOC1'. We also detected a second gene in Southern blots of baboon genomic DNA that may correspond to the human APOC1' pseudogene. Two start sites for baboon APOC1 transcription were mapped to nucleotides that are 7 and 9 bp downstream from the predominant start site for human APOC1 transcription. Alignment of Alu repeats showed that the 5' region of the baboon APOC1 gene is more similar to that of the human pseudogene APOC1', and the 3' region and coding sequences are more similar to those of human APOC1. These regions are separated by an Alu repeat that is present only in the baboon gene, perhaps reflecting its role in gene rearrangement or conversion. Sequence comparisons from baboon, human, dog, and rat showed extensive differences in apoC-I amino acid sequences, which are less conserved than nucleotide sequences. However, comparisons of hydrophilicity profiles show significant conservation of protein domains that may be important for apoC-I function.

Amino Acid Sequence↗
PubMed1981

The nucleotide sequence of the gene for protein IVa2 and of the 5' leader segment of the major late mRNAs of adenovirus type 5.

We present here the primary structure of the region of human adenovirus 5 (Ad5) DNA from nucleotide 4001 through the HindIII site at nucleotide 6246 (map positions 0.11 to 0.17). The corresponding region in the closely related adenovirus type 2 (Ad2) encodes the spliced mRNA for viral protein IVa2 (Chow et al., 1979; Persson et al., 1979). Reverse transcription of the Ad5 pIVa2 mRNA localized the 5' terminus of the mRNA to approximately position 5840, and its splice coordinates to positions 5706 and 5427. From the data of Aleström et al. (1980) for Ad2, the 3' end of this mRNA was inferred to be specified by Ad5 nucleotide 4060. The nucleic acid data allow us to predict an Mr of 50873 for the IVa2 protein of Ad5, which is close to the experimentally determined value of 50000 (Persson et al., 1979). The DNA sequence described here also includes the information for the 5'-terminal leader segment of the major late mRNAs of Ad5.

Adenoviruses, Human↗