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At least 1,621 records · Page 90Linked to original sources
PubMed1993

Target sequence specificity of transposon Tn5 in the absence of major hotspots in the plasmid pBR322: identification of a new hotspot.

The plasmid pLB11-1 is a pBR322 derivative in which part of the tetracycline resistance (tet) gene (basepair coordinates 23 to 375), containing five hotspots for Tn5 insertion (D. E. Berg et al., Genetics 105, 813-828, 1983), has been replaced with a 5.1-kb fragment of Escherichia coli genomic DNA encoding an osmoregulatory function. Restriction mapping of 40 pLB11-1::Tn5 derivatives, chosen at random from 240 Tn5 insertion derivatives with an unaltered osmotolerant phenotype, placed Tn5 in the vector portion of 20 clones. The majority of these insertions (16/20) were located in a 0.09-kb region immediately downstream of the beta-lactamase (bla) gene. Nucleotide (nt) sequence analysis of seven insertions from this group revealed an identical site of insertion within pBR322, representing a hitherto unidentified hotspot for Tn5 insertion. The target sequence, 5'GTCTGACGC, was found to be duplicated in these cases.

Base Sequence↗
PubMed1997

Gene structure of the human MET proto-oncogene.

By direct sequencing of cosmids using primers designed from the known cDNA sequence, we identified 19 exons in the human MET proto-oncogene, and sequenced the corresponding 5' and 3' exon-intron junctions. By homology search in the database of the Washington University Genome Sequence Center (GSC), we identified one additional exon. These 20 exons, together with a previously reported exon, bring the total exon number of MET to 21. Oligonucleotide primers were designed to amplify each exon and adjacent intronic sequences to permit examination of each exon for mutations. By restriction mapping, we assembled a 110 kb genomic contig that covered almost the entire MET proto-oncogene. This information is relevant for the screening of recently reported mutations of the MET gene which cause hereditary papillary renal carcinomas and for the search for additional mutations of the same gene which may play a role in the pathogenesis of common human carcinomas including carcinomas of the breast, ovary and pancreas.

Base Sequence↗
PubMed1990

Genomic termini of equine herpesvirus 1.

After cell infection with the equine herpesvirus 1 (EHV-1), the termini of the linear double-stranded DNA genome fuse to form circular forms. To investigate the mechanisms in the generation and cleavage of such replicative-form DNAs, the genomic termini, the fusion of termini from replicative-form molecules, and the junction between the short and long genome segments have been analyzed by restriction mapping, blot hybridizations, cloning, and sequencing. The data suggest that the genome ends are not redundant and that the genomic termini are fused in replicative intermediates via 3' single-base extensions at the termini of the unique long segment (UL) and terminal repeat (TR). Adjacent to the EHV-1 termini are AT and gamma sequence elements highly conserved among different herpesviruses. We propose that both of these sequence elements are important for the cleavage of EHV-1 replicative forms.

Animals↗
PubMed1989

Absence of alpha 2(1) procollagen synthesis in a clone of SV40-transformed WI-38 human fibroblasts.

Normal diploid human embryonic lung fibroblasts (WI-38) produce type I collagen of chain composition [alpha 1(1)]2.alpha 2(1) together with small amounts of type III collagen. We have examined the synthesis and secretion of type I collagen in a clone of SV40-transformed WI-38 fibroblasts (SVWI-38). These cells produced only 20-25% of the total collagen synthesized by their normal counterparts, with no detectable synthesis of alpha 2(1) chains and deposited a type I trimer consisting of overmodified alpha 1(1) chains. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cyanogen bromide peptides of collagens isolated from cells cultured either in the presence or absence of alpha,alpha-dipyridyl revealed that this overmodification occurred along the entire length of the alpha 1(1) chains. Analysis of poly(A+) RNA by Northern blot analysis and total RNA by slot blot analysis using cloned type I procollagen cDNA probes revealed that no alpha 2(1) mRNA was present in the SVWI-38 cells. Primer extension of the RNA confirmed this finding. The SVWI-38 cells had a normal chromosome number, but contained 28 normal and 18 abnormal and marker chromosomes. Restriction mapping of the entire alpha 2(1) procollagen gene did not reveal any gross deletions or insertions within this gene, nor was the gene hypermethylated in the transformed cells, when compared with their normal counterparts. One interesting feature, however, was the fact that certain CpG dinucleotides in the alpha 2(1) gene were methylated in the normal as well as the transformed cells. These SV40-transformed WI-38 fibroblasts therefore do not produce any alpha 2(1) collagen chains due to transcriptional inactivation of their genes.

Cell Line, Transformed↗
PubMed1990

Class II genes of miniature swine. II. Molecular identification and characterization of B (beta) genes from the SLAc haplotype.

Genomic clones corresponding to class II beta genes of the SLAc haplotype of miniature swine have been isolated and characterized. These genes have been grouped into seven non-overlapping clusters on the basis of restriction mapping. Ordering of exons within each cluster was accomplished by hybridization of Southern blots of restriction fragments with exon-specific probes. The two clusters (clusters 2 and 3) encoding the DRB and DQB genes were identified on the basis of hybridization with locus-specific 3' untranslated cDNA probes. Cluster 4 contained exons of both DOB and DQB genes, the basis for which remains to be determined. The remaining four clusters (1, 5, 6, 7) were identified as containing DP, DR, and DO coding sequences, respectively, on the basis of sequence analysis. The porcine class II region appears very similar to that of man in number and nature of the class II genes identified and in the intron/exon organization of corresponding genes.

Animals↗
PubMed1993

Natural populations of Escherichia coli and Salmonella typhimurium harbor the same classes of insertion sequences.

Despite their close phylogenetic relationship, Escherichia coli and Salmonella typhimurium were long considered as having distinct classes of transposable elements maintained by either host-related factors or very restricted gene exchange. In this study, genetically diverse collections of E. coli and S. typhimurium (subgroup I) were surveyed for the presence of several classes of insertion sequences by Southern blot analysis and the polymerase chain reaction. A majority of salmonellae contained IS1 or IS3, elements originally recovered from E. coli, while IS200, a Salmonella-specific element, was present in about 20% of the tested strains of E. coli. Based on restriction mapping, the extent of sequence divergence between copies of IS200 from E. coli and S. typhimurium is on the order of that observed in comparisons of chromosomally encoded genes from these taxa. This suggests that copies of IS200 have not been recently transferred between E. coli and S. typhimurium and that the element was present in the common ancestor to both species. IS200 is polymorphic within E. coli but homogeneous among isolates of S. typhimurium, providing evidence that these species might differ in their rates of transfer and turnover of insertion sequences.

Base Sequence↗
PubMed1993

Rapid divergence of Agrobacterium vitis octopine-cucumopine Ti plasmids from a recent common ancestor.

The octopine/cucumopine (o/c) Ti plasmids of the grapevine-associated Agrobacterium vitis strains constitute a family of related DNA molecules. Restriction maps were established of two limited-host-range o/c Ti plasmids, pTiAg57 and pTiAB3, and of the wide-host-range o/c Ti plasmid pTiHm1. Together with the previously obtained map of the wide-host-range o/c Ti plasmid pTiTm4, about 1000 kb were mapped with a resolution of 0.2 kb, allowing a detailed comparison of the various structures. One region of the o/c Ti plasmids is highly conserved and differs mainly by the presence or absence of relatively small DNA fragments (0.9-2.7 kb); the other region has been modified more extensively and carries large sequences specific for each Ti plasmid type. The sequence similarity within large conserved regions shows that these plasmids have diverged recently and that their evolution was driven by large-scale genetic events rather than single nucleotide changes. These results have important implications for studies on bacterial evolution.

Arginine↗
PubMed1989

Cloning and sequence analysis of the genomic DNA fragment encoding oryzacystatin.

A genomic DNA clone encoding oryzacystatin (Oc), a cysteine proteinase inhibitor (cystatin) of rice, was isolated from a lambda EMBL3 phage library constructed with Sau3AI partial digests of rice chromosomal DNA, by screening with an oc cDNA as a probe. The restriction map of the isolated DNA fragment was consistent with the pattern of the genomic Southern-blot analysis using a cDNA probe, and consequently, the gene is considered to be a single-copy gene. The oc gene is about 1.4 kb long and composed of three exons and two introns. The first intron (336 bp) intervenes between Ala-38 and Asp-39. The second intron (372 bp) exists in the 3'-noncoding region at the G residue next to the stop codon. S1 nuclease mapping showed the major transcription start point (tsp) at A, 104 bp upstream from the start codon (ATG). Typical CAT and TATA box sequences were found in the 5'-upstream region of the tsp. The nucleotide sequences around the TATA box, the tsp, the start codon, and the stop codon essentially matched the consensus sequences of other higher plant genes. The intron boundaries of the oc gene were quite different from those of the human kininogen-encoding gene and the human salivary cystatin (cystatin S)-encoding gene.

Amino Acid Sequence↗
PubMed1988

Cloning of mtr, an amino acid transport gene of Neurospora crassa.

Translocation of neutral aliphatic and aromatic amino acids across the plasma membrane of the ascomycete Neurospora crassa requires a functional gene product of the mtr locus. Mutations at this locus are defective in transport of those amino acids. We have cloned the mtr+ gene of Neurospora crassa from an ordered cosmid library of genomic DNA and produced a preliminary restriction map of 2.9 kilobases of genomic DNA that encompasses the mtr coding region. We have confirmed that the cloned DNA regions contain the mtr gene sequence by restriction fragment length polymorphism mapping and have determined that the cloned sequence codes for a messenger RNA transcript of approximately 1200 nucleotides in length.

Amino Acids↗
PubMed1989

Identification and characterization of the potato leafroll virus putative coat protein gene.

Complementary DNA clones representing approximately 6100 nucleotides of potato leafroll virus (PLRV) were generated, restriction-mapped, and partially sequenced. Within one of the cDNA clones an open reading frame (ORF) encoding a 23K protein was identified and further characterized. Amino acid sequence comparison of this protein showed significant homology (47.1%) with the barley yellow dwarf virus (BYDV-PAV) coat protein. This and other observations suggested that this gene encodes the PLRV coat protein. Other similarities were observed between PLRV and BYDV sequences in this region of their genomes, including an ORF of 17K within the ORF encoding the 23K putative coat protein.

Amino Acid Sequence↗
PubMed1990

Cloning, sequencing and homologies of the cbh-1 (exoglucanase) gene of Humicola grisea var. thermoidea.

Studies on the enzymes of the cellulase complex of the thermophilic fungus Humicola grisea var. thermoidea are described. A genomic library was constructed in the phage vector EMBL 4, and from this library two clones were isolated using as a probe the cloned cbh-1 (exoglucanase, EC 3.2.1.91) gene of Phanerochaete chrysosporium, a cellulolytic basidiomycete fungus. These clones were analysed by restriction mapping and Southern blotting, and one of them (lambda 3) was sub-cloned into the M13 phage vectors mp18 and mp19. The gene sequence was determined by the dideoxy chain-termination method. Sequence comparison with the equivalent genes from P. chrysosporium and Trichoderma reesei was made: in terms of primary sequence there is about 60% homology between the three species. Secondary structure prediction of the H. grisea sequence was also computed.

Amino Acid Sequence↗
PubMed1990

Purification of acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824 and cloning of the acetoacetate decarboxylase gene in Escherichia coli.

In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of acetoacetate to acetone. We report here the purification of the enzyme from C. acetobutylicum ATCC 824 and the cloning and expression of the gene encoding the acetoacetate decarboxylase enzyme in Escherichia coli. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was screened by plaque hybridization, using oligodeoxynucleotide probes derived from the N-terminal amino acid sequence obtained from the purified protein. Phage DNA from positive plaques was analyzed by Southern hybridization. Restriction mapping and subsequent subcloning of DNA fragments hybridizing to the probes localized the gene within an approximately 2.1 kb EcoRI/Bg/II fragment. A polypeptide with a molecular weight of approximately 28,000 corresponding to that of the purified acetoacetate decarboxylase was observed in both Western blots (immunoblots) and maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. Although the expression of the gene is tightly regulated in C. acetobutylicum, it was well expressed in E. coli, although from a promoter sequence of clostridial origin.

Amino Acid Sequence↗
PubMed1990

Attaching effacement of the rabbit enterocyte brush border is encoded on a single 96.5-kilobase-pair plasmid in an enteropathogenic Escherichia coli O111 strain.

An enteropathogenic Escherichia coli (EPE) O111 serotype a,b,H- strain carried the following four plasmids: pLV501 (96.5 kilobase pairs [kbp]) specifying resistance to chloramphenicol, tetracycline, and kanamycin; pLV502 (8 kbp) specifying ampicillin resistance; pLV503 (1.9 kbp) specifying streptomycin resistance; and pLV504 (80 kbp) with no resistance markers. This EPEC attached to HEp-2 cells to produce localized clumps of bacteria (localized adhesion) and attached intimately to the enterocyte surface, leading to loss of the brush border (attaching effacement). Plasmid pLV501 was also found to specify the ability to produce localized adhesion on HEp-2 cells and attaching effacement in a rabbit ileal explant model system. Restriction maps showed considerable dissimilarities between pLV501 and pMAR-2, an EPEC plasmid carrying the EPEC adherence factor (EAF) genes. Furthermore, pLV501 did not hybridize with the EAF probe, whereas pLV504 did. There was sequence homology between pLV501 and large plasmids in all seven other well-characterized EPEC, only five of which hybridized with the EAF probe. These findings indicate that pLV501 carries at least one of the genes responsible for production of the brush border damage characteristic of EPEC.

Animals↗
PubMed1990

A physical map of the genome of Haemophilus influenzae type b.

Contour-clamped homogeneous electric field pulsed-field gel electrophoresis (PFGE) was used in combination with Southern hybridization to construct a genomic restriction map for the pathogen Haemophilus influenzae serotype b, strain Eagan. Sites for four restriction endonucleases, EagI, NaeI, RsrII and SmaI, were located on the 2100 kbp circular chromosome. Twelve potential virulence loci have been placed on the map together with certain loci essential for growth of the bacteria (e.g. ribosomal RNA operons). PFGE also provided a valuable tool for characterizing ten capsulated, type b isolates (other than Eagan) known to be genetically heterogeneous and two laboratory-derived variants (transformants) derived through complex recombinational events involving random uptake of high-molecular-mass donor genomic DNA.

Base Sequence↗
PubMed1990

Isolation and characterization of the two distinct genes for human glutamate dehydrogenase.

Two genomic clones containing a part of the glutamate dehydrogenase gene were isolated from a human genomic library. The restriction map of both clones were distinctly different from one another, although the nucleotide sequences of the three exons that they contained were virtually the same in each clone. Southern blotting analysis of the genomic DNAs from several unrelated human individuals revealed that in every case the probe hybridized with at least two DNA fragments of different sizes, each characteristic to one of the two clones. These results strongly suggest that the two clones presently obtained do not result from polymorphism but are generated from two different gene loci for glutamate dehydrogenase on the human chromosome.

Amino Acid Sequence↗
PubMed1994

Isolation and characterization of the human genomic locus coding for the putative metastasis control gene nm23-H1.

Nm23-H1 gene expression is inversely correlated with tumor metastatic potential in certain tumors, including melanomas, breast carcinomas, and hepatocellular carcinomas. Using nm23-H1 c-DNA primer and genomic polymerase chain reaction (PCR) amplification, we purified three PCR fragments (one of 4kb and two of 2 kb) covering the whole human genomic locus of the gene (8.460bp). We recombined the PCR products into pUC18 and produced a restriction map to perform subcloning. Complete sequencing of genomic PCR fragments, including the whole coding region of nm23-H1, revealed that the gene consists of five exons and four introns spanning 8.5kb. A sequence homology analysis between human nm23-H1 and the homolog gene of the rat (NDP-K beta) shows that exon-intron boundaries are well conserved between these two species.

Amino Acid Sequence↗
PubMed1990

Characterization of a plasmid involved with cointegrate formation and lactose metabolism in Lactococcus lactis subsp. lactis OZS1.

A 55 kilobase (kb) plasmid (pOZS550) in the non-clumping Lactococcus lactis subsp. lactis strain OZS1 carrying genes for lactose metabolism was characterised. A mobilizable cointegrate plasmid which is formed between pOZS550 and pOZS448 carries the necessary information for conjugation and transfer. Cointegrate formation was found to involve an insertional element located on pOZS550. The insertion sequence was found to be identical to ISS1 located on pSK08 in the clumping L. lactis subsp. lactis strain ML3. Restriction maps of pOZS550 and pSK08 were similar suggesting a close ancestral relationship, although pSK08, in addition to the lactose metabolism genes, expressed genes for proteinase activity and cell clumping, which were not expressed by pOZS550, and carried two copies of ISS1 compared to one on pOZS550. Furthermore, hybridization of the 18 base pair inverted repeat, of the insertion sequence, with various L. lactis subsp. lactis strains and two L. lactis subsp. cremoris strains showed moderate to strong hybridization to one plasmid in each organism.

Base Sequence↗
PubMed1993

Cloning, characterization and sequencing of two haemagglutinin genes from Eikenella corrodens.

Eikenella corrodens is emerging as an important human pathogen, in both extra-oral and periodontal infections. From a clone bank of Eikenella corrodens chromosomal DNA produced in Escherichia coli JM109, twenty-two clones expressed Eikenella antigens and of these, two expressed functional haemagglutinins. By virtue of different restriction maps and a lack of homology by Southern hybridization, the two cloned fragments encoding the two haemagglutinins have been shown to be distinct. Maxicell analysis revealed that clone 1, carrying plasmid pVKR201, produces three Eikenella proteins, one of 31.5 kDa and two of approximately 14 kDa each. Expression of each of the proteins appears to be under the control of an Eikenella promoter(s). Clone 2, carrying plasmid pVKR301, produces two proteins, one of 93 kDa and the second of 17 kDa. Expression of both of these proteins in E. coli requires the lac promoter in the vector. By preparing a series of subclones and testing each by maxicell analysis and for haemagglutination activity, a functional map of the insert of clone 1 was deduced and the 31.5 kDa polypeptide identified as the haemagglutinin. Using similar methods, the 17 kDa protein was found to be the haemagglutinin of clone 2. The nucleotide sequences of both haemagglutinin genes were determined and are presented. Computer analysis revealed no homology between the two haemagglutinins, and no homology to any previously sequenced proteins. These are the first genes of this genus to be cloned and sequenced.

Amino Acid Sequence↗