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At least 1,711 records · Page 95Linked to original sources
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↗
PubMed1989

Factors that bind to adeno-associated virus terminal repeats.

We have identified and characterized a DNA-protein complex that forms with the adeno-associated virus (AAV) terminal repeats. The complex formed only if the terminal palindrome was in the covalently closed or hairpin configuration; little if any binding was detected with the open duplex form of the terminal repeat. This fact suggested that both secondary structure and primary sequence are essential elements of recognition. DNase I protection studies indicated that virtually all of the A-A' palindrome and significant portions of the B-B' and C-C' palindromes are protected. The postulated terminal resolution site of AAV also is protected. Restriction mapping of the sequences necessary for binding indicated that almost all of the terminal palindrome must be present for binding to occur. Hairpins which are similar in size and shape to the AAV termini did not exhibit competition for binding, and the complex formed only if AAV-infected extracts were used. Thus, the binding reaction is specific for AAV sequences. The viral-coded nonstructural proteins Rep78 and Rep68 comigrated with the DNA-protein complex on neutral acrylamide gels, suggesting that one or both of these proteins are components of the complex. The characteristics of the complex suggested that it has a role in AAV DNA replication.

Base Sequence↗
PubMed1994

[Use of a plasmid with integrative function of phage phiC31 for transfer of cloned genes into Streptomyces strains].

Opportunities for application of integrative vectors carrying the attP site and the int gene of the temperate actinophage phi C31 for cloning genes in Streptomyces strains were demonstrated. The behavior of the integrative vectors pZAT22 and pTO1 in the model strain S. lividans TK64 and in the bialaphos-producing strain S. hygroscopicus, respectively, was characterized. Restriction maps of the S. lividans and S. hygroscopicus chromosomal regions containing attB sites were constructed. The bar gene of resistance to bialaphos was incorporated into the chromosome of the model strain S. lividans via the integrative pZAT22 vector, the level of expression of this gene within the chromosome of a heterologous host was determined. The possibility of amplification of the bar gene in the chromosome of the strain S. lividans was shown. The conjugative integrative vector pTO1, which carried the cloned bar gene, was introduced into the bialaphos-producing strain by intergeneric matings of Escherichia coli and S. hygroscopicus. The effect of an additional copy of the gene in the chromosome of S. hygroscopicus on strain resistance and productivity was studied.

Bacteriophages↗
PubMed1993

Complex organization of the soybean mitochondrial genome: recombination repeats and multiple transcripts at the atpA loci.

Identification of the soybean mitochondrial atpA open reading frame (atpA ORF) was based on sequence similarity with atpA genes in other plant mitochondria and partial protein sequencing. The atpA reading frame ends with four tandem UGA codons which overlap four tandem AUG codons initiating an unidentified reading frame, orf214. The atpA-orf214 region is found in multiple sequence contexts in soybean mitochondrial DNA (mtDNA), which can be attributed to the presence of two recombination repeats. A 1-kb repeat spans 600 nucleotides (nt) of atpA N-terminal coding region and 400 nt of upstream sequence. Its four configurations correspond to two full-length atpA-orf214 genes and two truncated pseudogenes. A 2-kb repeat lies 3 kb downstream from the 1-kb repeat. Restriction maps of cosmid clones suggest that a 10-kb segment containing both repeats is itself duplicated in the mt genome. With two recombination repeats present in a total of three copies per genome, soybean mtDNA is expected to consist of a complex population of subgenomic molecules. Transcription of the atpA loci was analysed by Northern blotting and S1 nuclease protection. The atpA genes express multiple transcripts with one major 3' end and heterogeneous 5' sequences extending several kb upstream of the atpA coding region. The atpA gene and orf214 are co-transcribed on all major transcripts. The pseudogenes do not express stable RNAs.

Amino Acid Sequence↗
PubMed1989

Isolation and characterization of the promoter and flanking regions of the gene encoding the human protein-synthesis-initiation factor 2 alpha.

The promoter region of the gene (eIF-2 alpha) for eukaryotic initiation factor 2 alpha (eIF-2 alpha) was isolated from a human genomic library and its structure was determined by restriction mapping and nucleotide (nt) sequence analysis. The promoter region and twelve in vivo transcriptional start points (tsp) have been identified by endonuclease S1 mapping and their location confirmed by primer-extension analysis, using RNA isolated from human cells. The untranslated leader is 102 to 140 nt long depending upon the tsp, and the 5' region of the mRNA has the potential for forming stable stem-loop structures. The nt sequence of the regions upstream and downstream from the tsp contains neither a 'TATA box' nor a 'CAAT box', but does contain several direct and inverted repeats, as well as palindromic sequences near the tsp. In addition, multiple consensus binding sites for a wide variety of regulatory proteins are present throughout upstream and downstream tsp-flanking regions.

Base Sequence↗
PubMed1991

Structures and characterization of sex-specific mouse cytochrome P-450 genes as members within a large family. Duplication boundary and evolution.

We characterized two female-specific testosterone 16 alpha-hydroxylase mouse cytochrome P-450 genes, 16 alpha oh-a and 16 alpha oh-b. Gene 16 alpha oh-a, consisting of nine exons, is approximately 38 kbp in size. The exon sequence of this P-450 gene is identical to cDNA pf26 nucleotide sequence [Noshiro, M., Lakso, M., Kawajiri, K. & Negishi, M. (1988) Biochemistry 27, 6434-6443], which encodes female-specific testosterone 16 alpha-hydroxylase regulated by the murine Rip locus. Gene 16 alpha oh-b, containing nine exons with the same junctions as the 16 alpha oh-a, spans at least 20 kbp, and encodes a cytochrome P-450 whose deduced amino acid sequence is 90% similar to the hydroxylase. Nucleotide sequences revealed that duplication of the two genes occurred 4-22 million years ago, and that the 5' duplication boundary is located 1336 bp upstream from the putative transcription-start site. In the flanking regions of both genes, there is a long stretch (100 bp) of CA repeats in addition to other motifs, including TATA box, glucocorticoid-response-element-core and Simian-virus-40-enhancer sequences and IgG light-chain gene promoter. We isolated many genomic DNA clones which contain exon 1 sequences, and compared their restriction maps, cross-hybridization and nucleotide sequences. The results indicate that these genomic clones represent closely related genes in the 16 alpha oh family with a minimum of 16 members, which is further divided into classes a, b and c. 16 alpha oh-a and 16 alpha oh-b belong to the first and second classes, respectively. Moreover, extensive segmental gene conversion and nonreciprocal recombination were noted among the genes, particularly among those in class b. All genes in that class contain the long ATTT repeat sequences in intron 1, which may have triggered a rapid gene conversion and/or stabilize the duplicated genes.

Animals↗
PubMed1999

Persistent infection of bovine herpesvirus type 4 in bovine endothelial cell cultures.

Herpesviruses can establish a persistent infection in the cells and tissues of their natural hosts and thus may produce diseases due to cytolytic infections. We have isolated a herpesvirus from a bovine vascular endothelial cell culture after continuous subculturing. Typical cytopathic changes were observed in bovine endothelial cell cultures 2 days after inoculation of the virus. The virus had an icosahedral nucleocapsid of 100-150 nm in diameter and an envelope. The sequences of some DNA fragments of the virus were highly homologous to those of the bovine herpesvirus type 4 (BHV-4) strains. The DNA restriction maps of the virus and the reference strains of BHV-4, DN 599 and Movar 33/63 were very similar but not identical. Therefore, the newly isolated virus has been designated Taiwan strain. The presence of BHV-4 DNA in apparently normal bovine endothelial cell cultures was shown by Southern blot hybridization with the BamHI fragment of the newly isolated BHV-4 and was further confirmed by digestion of the DNA with BamHI plus AccI. In conclusion, we have demonstrated that BHV-4 persisted in the bovine endothelial cell cultures and continuous subcultures could lead to the production of infectious viral particles.

Animals↗
PubMed1985

Cloning and physical mapping of Yaba monkey tumor virus DNA.

The physical map positions for the BamHI, EcoRI, and SalI restriction fragments of Yaba monkey tumor pox virus DNA were determined using cloned virus DNA fragments as probes for hybridization as well as analyzing the secondary digests of larger DNA restriction fragments. Digests of EcoRI A and B fragments and SalI A and B fragments with BamHI allowed for the orientation of most of the BamHI restriction map. These secondary digest products were confirmed and the map positions for the EcoRI fragments were established using cloned BamHI fragments. Yaba monkey tumor virus DNA was cloned using the plasmid vector pBR322.

Animals↗
PubMedDate not supplied

Identification, cloning, and sequence analysis of the nitrogen regulation gene ntrC of Agrobacterium tumefaciens C58.

We describe the cloning of an ntrC gene of Agrobacterium tumefaciens C58 by interspecific complementation of an Escherichia coli ntrC mutant. Restriction mapping and Southern blot analysis of the complementing clone identified a 1.7-kb EcoRI-PvuII DNA fragment whose sequence was determined. Analysis of this sequence revealed coding regions corresponding to a complete ntrC gene and the C-terminal region of an ntrB gene. Amino acid sequence comparisons of A. tumefaciens NTRC protein with NTRC sequences from Rhizobium meliloti, Bradyrhizobium sp. (Parasponia), Klebsiella pneumoniae, E. coli, and Salmonella typhimurium show strong sequence conservation supporting DNA hybridization data, demonstrating strong evolutionary homology among ntrC genes of Rhizobiaceae. The C58 NTRC protein has been identified, by 35S-labeling, in a T7 RNA polymerase (pT7-7) expression vector system.

Agrobacterium tumefaciens↗
PubMed1990

Transcriptional control plays an important role for the production of heat-labile enterotoxin in enterotoxigenic Escherichia coli of human origin.

The production of heat-labile enterotoxin (LT) in 76 strains of human enterotoxigenic Escherichia coli (ETEC) varied by a factor of 100. Three ETEC strains that differ in the levels of LT production were chosen for the cloning of LT genes (toxAB) into plasmid pBR322, and the gene structure and expression were compared in E. coli HB101. The recombinant of the low LT-producing strain produced LT at the same level as that of the moderate LT-producing strain, but that of the high-level producer continued to produce at a level 14-21 times higher than the others. The restriction maps of the coding regions of the cloned LT genes (toxAB) were identical, but the flanking regions were dissimilar. The content of LT mRNA per cell, examined by Northern blot analysis, was higher in the high producer than the others by 6 times. The promoter strengths of the recombinants were all alike. LT mRNA of the high producer was more stable than that of the moderate one by 1.3 times, but the difference was not large enough to explain the difference of the content of LT mRNA. It was shown that LT production can be controlled at a transcriptional step, and DNA structure of the flanking regions may be involved in the control of the LT gene expression.

Bacterial Toxins↗
PubMed1990

Polymorphism in Brucella spp. due to highly repeated DNA.

The species of Brucella are very closely related, but Brucella ovis does not express detectable amounts of a protein, designated BCSP31, that is common to the other species. We studied the lack of expression of BCSP31 by Southern analysis. DNAs from the B. ovis culture collection strains and field isolates were probed with a 1.3-kb HindIII fragment encoding BCSP31 of Brucella abortus. The probe hybridized to a 1.6-kb HindIII fragment of all B. ovis strains tested, showing that the gene is present in B. ovis but occurs on a larger restriction fragment. DNA linkage studies and restriction mapping of the cloned polymorphic region of B. ovis showed that the polymorphism was due to a DNA insertion of approximately 0.9 kb at a site downstream of the BCSP31-coding region. When the 1.6-kb polymorphic B. ovis fragment was used to probe a HindIII Southern blot of cellular DNA of strains of B. ovis and of B. abortus, at least 24 fragments of B. ovis and 6 fragments of B. abortus hybridized to the inserted DNA. Specimens of B. ovis collected over a 30-year period on two continents had similar hybridization patterns. The large difference between B. ovis and B. abortus in the number of copies of the repeated DNA is interesting in the context of the closeness of the Brucella species.

Blotting, Southern↗
PubMed1990

Cloning of Clostridium difficile toxin B gene and demonstration of high N-terminal homology between toxin A and B.

High titered Clostridium sordellii lethal toxin antiserum, cross-reactive with C. difficile cytotoxin B (ToxB), was used to isolate toxB fragments from a C. difficile expression library. Recombinant clones containing toxB fragments of the 5' and 3' end were isolate. A 2.5-kb HincII fragment of chromosomal DNA overlaps both groups of clones. A partial restriction map of the total toxB gene is presented. The gene is positioned upstream of utxA and toxA, toxB has a size of 6.9 kb, corresponding to a 250-kDa polypeptide. A partial sequence of the 5' end of toxB was determined. The sequence contains 398 bp upstream of toxB with a putative Shine-Dalgarno box (AGGAGA) and 609 bp of the toxB open reading frame. The N-terminal 203 amino acids of ToxB were compared with the N-terminal amino acids of the enterotoxin A (ToxA). A homology of 64% of the residues was detected, which proves the relatedness of ToxA and ToxB of C. difficile.

Amino Acid Sequence↗
PubMedDate not supplied

Mitochondrial DNA polymorphism in subspecies of the Japanese Sika deer, Cervus nippon.

Variations in the mitochondrial DNA (mtDNA) of Sika deer (Cervus nippon) from seven localities in Japan were examined to assess the degree of genetic differentiation among subspecies. The entire mtDNA of C. n. centralis, which is about 16,600 base pairs in length, was cloned into plasmids. Using various clones as hybridization probes, we constructed restriction maps of mtDNA for 59 deer and identified six different mtDNA genotypes. The genotypes observed in the subspecies centralis from Honshu Island (the mainland) were distinct from that of the same subspecies from the Tsushima Islands. By contrast, the subspecies yesoensis, which is distributed on Hokkaido Island, shared at least two mtDNA genotypes with the mainland centralis. Analysis of genetic distances showed that Sika deer on the Japanese islands consist of two distinct clusters, namely, the Hokkaido-Honshu (yesoensis-mainland centralis) and the Yakushima-Tsushima (yakushimae-Tsushima centralis) populations. The molecular phylogenetic data obtained for these subspecies do not reflect the present taxonomic status of these subspecies that is based on comparative morphometry. Genetic differentiation of Sika deer in the Japanese islands is a possible example of vicariance.

Animals↗
PubMed1989

Stability of Escherichia coli strains harboring recombinant plasmids for L-threonine production.

Escherichia coli recombinant strains bearing the thr operon have been previously selected for threonine production and phenotypically classified according to antibiotic resistance properties (Nudel et al. 1987). Further analysis of those strains permitted the isolation and restriction mapping of two different plasmids of 13 kb and 18.6 kb. The smaller one, which expressed tetracycline resistance gave better results on threonine accumulation but it was rather unstable when grown without antibiotic pressure. Therefore, other hosts were transformed with those plasmids to improve stability. A threonine-auxotrophic strain was a better host for plasmid maintenance and expression of thr operon. Host influence in plasmid-mediated threonine production was studied in terms of specific yields (the ratios of threonine accumulated to biomass values) and of plasmid maintenance (percent of AprTcr clones after cultivation in non selective media). We also determined that semisynthetic media of defined composition were better than rich media for threonine expression, due to feed-back controls exerted by undesired catabolites accumulated in complex media.

Culture Media↗
PubMed1991

Rapid genotyping of the Chlamydia trachomatis major outer membrane protein by the polymerase chain reaction.

Twenty one Chlamydia trachomatis reference strains and 40 clinical isolates belonging to the lymphogranuloma venerum (LGV) and trachoma biovars were genotyped by differential restriction mapping of the major-outer-membrane-protein gene (MOMP) obtained by the polymerase-chain reaction (PCR). AluI digestion of the PCR product distinguishes eight MOMP-genotypes corresponding to 8 serovars. Six additional enzymes (NlaIII, CfoI, EcoRI, HinfI, DdeI and FokI) further permit the discrimination of 10 MOMP-genotypes corresponding to the 10 remaining serovars of the species. AluI alone allows direct typing of 78% of the clinical isolates. AluI digestion patterns of mouse C. trachomatis biovar, a C. pneumoniae and two C. psittaci strains, studied for comparison, were clearly distinguishable from one another and from the C. trachomatis LGV and trachoma strains. These results indicate that MOMP genotyping by PCR is a valuable molecular tool for studying C. trachomatis epidemiology.

Bacterial Outer Membrane Proteins↗
PubMed1996

Molecular cloning and expression of the glucose/xylose isomerase gene from Streptomyces sp. NCIM 2730 in Escherichia coli.

A partial genomic library of Streptomyces sp. NCIM 2730 was constructed in Escherichia coli using pUC8 vector and screened for the presence of the D-glucose/xylose isomerase (GXI) gene using an 18-mer mixed oligonucleotide probe complementary to a highly conserved six-amino acid sequence of GXI from actinomycetes. Eight clones which hybridized with the radiolabelled oligoprobe showed the ability to complement xylose isomerase-defective E. coli mutants. The restriction map of the insert from one (pMSG27) of the eight GXI-positive clones showing detectable GXI activity was constructed. GXI-deficient strains of E. coli were able to utilize xylose as the sole carbon source for their growth upon transformation with pMSG27. E. coli JM105 (pMSG27) and E. coli JC1553 (pMSG27) were inducible by IPTG suggesting that the expression of the cloned gene was under the control of the lacZ promoter. Western blot analysis revealed that the cloned gene is expressed as a fusion protein of M(r) 110. This is the first report of expression of a catalytically active GXI from Streptomyces in Escherichia coli.

Aldose-Ketose Isomerases↗
PubMed1991

Molecular cloning and analysis of forked locus in Drosophila ananassae.

Drosophila ananassae, in spite of its unique genetic characters including high mutability and high frequency of male recombination has been little studied at the molecular level. Since the species is very similar to D. melanogaster, it is natural to expect that the high spontaneous mutation rate and male recombination may be caused by inserted mobile DNA elements, as in D. melanogaster. The present study was designed to determine whether or not most spontaneous mutations of the forked locus of D. ananassae are caused by insertion sequences as is found in D. melanogaster. I cloned the forked locus of D. ananassae, using forked DNA from D. melanogaster as a probe and investigated the molecular structure and transcription of the gene by Southern and Northern analyses. The results suggest that the restriction map and transcriptional patterns of the forked locus of D. ananassae are similar to those of D. melanogaster, while the spontaneous mutations available in D. ananassae are induced quite differently from those that have been described in D. melanogaster; in four (f;cd, f10-14, f49 and f86) out of eight forked alleles, neither insertions nor deletions were detected around the forked coding region. Forked transcripts are expressed in a pattern which is very similar to that of D. melanogaster and were all of normal size in these mutants. The other four mutants (f10-3, f9-10, f83i and f79b) had insertion sequences upstream of the forked coding region, while transcripts of the forked gene were of normal size. Hence, none of the eight mutations studied appear to affect the structure of the forked transcripts.

Animals↗
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↗