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At least 1,441 records · Page 80Linked to original sources
PubMed1992

Characterization of an 8.7-kilobase thiostrepton resistance-encoding plasmid (pGIF3) of Streptomyces incarnatus.

The low-copy-number 8.7-kb plasmid pGIF3 of Streptomyces incarnatus was studied after cloning in the Escherichia coli vector pBR322; a restriction map was constructed. Southern blot analysis showed that pGIF3 in S. incarnatus occurs predominantly as integrated in a larger replicon. The plasmid carries a gene for thiostrepton resistance having no homology with the known thiostrepton resistance gene from Streptomyces azureus.

Blotting, Southern↗
PubMed2005

Mycobacterium marinum infections in fish and humans in Israel.

Israeli Mycobacterium marinum isolates from humans and fish were compared by direct sequencing of the 16S rRNA and hsp65 genes, restriction mapping, and amplified fragment length polymorphism analysis. Significant molecular differences separated all clinical isolates from the piscine isolates, ruling out the local aquaculture industry as the source of human infections.

Animals↗
PubMed1990

Cloning and expression of the dermonecrotic toxin gene of Pasteurella multocida ssp. multocida in Escherichia coli.

A DNA library of Pasteurella multocida ssp. multocida strain CVI 47459 was constructed in the Lambda GEM-11 vector. Recombinant clones that encoded dermonecrotic toxin (DNT) were identified immunologically with antiserum raised against purified DNT. By comparing the DNA restriction maps of the immunoreactive recombinants, we located the DNT gene. Hybridization studies with 10 strains of P. multocida ssp. multocida suggested that strains that do not produce the DNT do not contain sequences homologous to the DNT gene.

Bacterial Toxins↗
PubMed1991

Direct and general selection for lysogens of Escherichia coli by phage lambda recombinant clones.

We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda. This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.

Bacteriophage lambda↗
PubMed1990

[Structural organization of a segment of chromosome, containing the vir gene of Bordetella pertussis].

To study the structural arrangement of the chromosomal region containing vir genes of Bordetella pertussis the corresponding 15 kb fragment of Bordetella pertussis chromosomal DNA has been cloned. The sequence homology to an earlier characterized Bordetella pertussis genetical element RSBP1 and flanked by two 400 bp inverted repeats has been shown to be located at an end of a BamHI fragment. The restriction map of Bordetella pertussis 475 coincides with the previously published maps of Bordetella pertussis Tohama and 18323 permitting one to conclude the definite conservatism of the cloned sequence. The preliminary data obtained make possible mapping of the RSBP1 homologous sequence adjacent to adenylate cyclase, agglutinin 2 and pertussis toxin genes. The possible role of RSBP1 elements in the regulation of Bordetella virulence is suggested.

Bordetella pertussis↗
PubMed1992

Genomic amplification and expression of delta-endotoxin fragment of Bacillus thuringiensis.

delta-Endotoxin gene of Bacillus thuringiensis HD-1 var kurstaki codes for the insecticidal crystal protein (ICP) specific for lepidopteran insects. Since the N-terminal half of the toxin is sufficient both for insect specificity and toxicity, the coding sequence of this part of the gene CryIA(b) was amplified by PCR and cloned in pUC19. As there was no expression of immunologically detectable delta-endotoxin in this clone in E. coli, the amplified ICP gene was transferred to an expression vector pGEx2T. Restriction mapping and immunoblotting confirmed the presence and expression of the CryIA(b) gene. This insert should be suitable for expression in plant system if it is mobilized into a plant binary vector.

Bacillus thuringiensis↗
PubMed1992

Small antibiotic resistance plasmids in Staphylococcus intermedius.

Antibiograms and plasmid profiles were evaluated for 116 Staphylococcus intermedius isolates collected from dogs in Germany and in the USA. Of the 26 S. intermedius isolates from Germany, 9 (34.6%) carried plasmids, while 20 (22.2%) of the 90 S. intermedius isolates from the USA were found to be plasmid-positive. Eight small resistance plasmids were identified and characterized using protoplast transformations and restriction endonuclease analyses. Five plasmids (3.8 and 3.9 kb) encoded for chloramphenicol resistance, 2 plasmids (each 2.5 kb) carried determinants for macrolide-lincosamide resistance, and one plasmid (4.5 kb) conferred resistance to tetracycline. Detailed restriction maps of these plasmids were constructed and served for structural comparisons with other small resistance plasmids found in staphylococci. These comparisons implied marked structural homologies with those prototype plasmids initially characterized in S. aureus of human origin.

Ampicillin Resistance↗
PubMed2001

Diagnostic methods for detecting forms and strains of Helicobacter pylori and evaluation of its eradication.

Diagnostic methods for detecting forms and strains of Helicobacter pylori isolated from biopsy specimens of gastric mucosa in 28 patients with duodenal ulcers and evaluation of its eradication were compared. Biopsy specimens from all patients were tested for the presence of H. pylori by the urease test, histological method, and PCR with species-specific primers before and after treatment. H. pylori infection was detected in all patients before treatment, the mean titer of serum IgG being 36.7+/-16.6 U/ml. Biopsy specimens positive for H. pylori in PCR were subjected to restriction analysis of specific PCR-amplified genes or their fragments. The fingerprint analysis gave electrophoregrams of restriction products amplified fragment of flaA gene of H. pylori in 7 patients. Differences in restriction maps indicate the presence of 5 H. pylori strains in the studied samples.

Adolescent↗
PubMed1990

Evolution of the T1 retroposon family in the Anopheles gambiae complex.

The T1 family of retrotransposable elements is interspersed and moderately repeated in five member species of the Anopheles gambiae sibling-species complex and has diverged little since the radiation of the complex. T1 includes two closely related but independent subfamilies, defined by the presence or absence of linked sets of restriction sites, in all but one species, although the relative abundance of the subfamilies differs within each. Sequence analysis of a 349-bp region from 21 clones isolated from A. gambiae confirmed the bipartite organization by revealing 19 coordinated nucleotide differences between the two subfamilies--T1 alpha and T1 beta. Sequence divergence is not only greater between than within subfamilies, but divergence within T1 beta is less than that within T1 alpha. Between-species comparisons of genomic consensus restriction maps revealed that T1 alpha is fixed for species-diagnostic differences in all species. With one exception, these subfamilies account for approximately 70% of detectable T1 copies in the genome. The results support retroposition as the dominant mechanism underlying the evolution of the T1 family.

Animals↗
PubMed2001

[The cloning and verification of the Sox9 gene of the rice field eel].

The Sox 9 gene plays important roles in sex determination, which is conserved in a variety of species including mammals and chicken. The positive lambda clone of the Sox 9 gene of the rice field eel was analyzed by restriction enzymes and the restriction map was constructed. This clone was further verified to be the Sox9 gene by PCR amplification and sequencing of the HMG box region. The cloning of the Sox9 gene of the rice field eel further suggests the evolutionary conservation of this gene.

Animals↗
PubMed2003

Molecular basis of the TaqI p49a,f polymorphism in the DYS1 locus containing DAZ genes.

The previously described TaqI p49a,f polymorphism at the DYS1 locus in the non-recombinant part of the Y chromosome is widely exploited to investigate many facets of human population genetics. It has been shown that the DYS1 locus corresponds to the four DAZ genes in the AZF-c region of the Y chromosome. As the DNA sequence of the DAZ genes is known in its entirety, it is now possible to establish correspondences between the main Southern polymorphic TaqI bands (A, C, D, F and I) at the DYS1 locus and TaqI fragments deduced from the sequence, by way of comparing band sizes and sequence homologies between hybridized fragments. Transitions between polymorphic forms for each variable TaqI fragment can be explained regarding the restriction maps, by postulating a parsimonious number of TaqI site losses during human evolution. Most of the codon changes caused by TaqI site losses located in the exons of the four DAZ genes have potentially high selective values.

Chromosomes, Human, Y↗
PubMed1989

Isolation and partial characterization of plasmids found in three Halobacterium volcanii isolates.

Three new isolates of Halobacterium volcanii were screened for the presence of plasmids. Each of the different isolates was found to contain one plasmid. These plasmids do not show any homology to each other, nor to the previously isolated plasmid pHV2. Partial restriction maps of these plasmids were determined. One of the plasmids contains chromosomal repetitive sequences as judged by the existence of homologous sequences in the chromosomal DNA of the three isolates. Using the protoplast fusion technique, we showed that at least one of the newly isolated plasmids is compatible with pHV2.

Cloning, Molecular↗
PubMed2005

Identification of an EcoRI restriction site for a rapid and precise determination of beta-asarone-free Acorus calamus cytotypes.

Calamus (Acorus calamus L., Araceae) is an aromatic herb, indigenous to Central Asia and Eastern Europe. The fragrant oils obtained by alcoholic extraction of the rhizome are mainly used in the pharmaceutical and oenological industries. Nevertheless, the occurrence of beta-asarone [(Z)-1,2,4-trimethoxy-5-prop-1-enyl-benzene] limits the possibility of its use due to the carcinogenic properties of this compound. The aim of this work was to identify a diploid beta-asarone-free A. calamus by using chemical and molecular approaches. For these purposes alcoholic extracts of both diploid and triploid A. calamus were analyzed by gas chromatography-mass spectrometry (GC-MS) and comparison of the 700 bp sequence of the non-transcribed spacer (NTS) in the 5S-rRNA gene was also performed. Alcoholic extracts of the triploid A. calamus were characterized by a higher percentage of beta-asarone (11%), which was the main compound, followed by higher percentages of camphene (2.27%), E-beta-ocimene (3.28%), camphor (1.54%), calarene (1.42%), alpha-selinene (5.02%) and tau-cadinol (2.00%), when compared to the diploid A. calamus. The latter had higher percentages of iso-shyobunone (8.62%), beta-sesquiphellandrene (3.28%), preiso calamendiol (22.81%) and acorone (26.33%), and completely lacked of beta-asarone. The 5S-rRNA spacer region of both diploid and triploid A. calamus were amplified by PCR using a pair of primers located at the 3' and 5' ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 700 bp) were gel purified, subcloned into pGEM-T Easy vector and sequenced. By aligning the isolated nucleotide sequences of the two varieties and the sequences from different A. calamus chemotypes present in Genbank, sequence diversities were found in the spacer region. Furthermore, the PCR products were digested by using EcoRI. The restriction profile of the spacer domain resulted different for the two cytotypes. Along with chemical analysis of alcoholic extracts, sequence analysis coupled to restriction mapping was demonstrated to represent a powerful tool to distinguish the A. calamus diploid cytotype from the others. The security and effective usage of the diploid beta-asarone-free A. calamus was also discussed.

Acorus↗
PubMed1997

Structural analysis of rDNA in the genus Nicotiana.

The nucleotide sequence of the intergenic spacer (IGS) region between the 25S and the 18S rRNA coding regions has been determined for tobacco (Nicotiana tabacum). The IGS (5140 bp in length) can be subdivided into several regions (I-VII) two of which, upstream and downstream of the putative transcription initiation site (TIS), contain prominent subrepeats (A and C). The unique sequence in the central part of the IGS (region IV) preceding the TIS is extremely AT-rich. The distance from the putative TIS to the 5' end of the 18S rRNA gene is 3005 bp. The IGS sequences are compared with potato (Solanum tuberosum) and tomato (Lycopersicon esculentum) IGS. Restriction mapping of 13 Nicotiana species shows that considerable rDNA repeat length heterogeneity in this genus is probably due to different numbers of A and C subrepeats.

Base Composition↗
PubMed1988

Molecular and phenotypic variation of the white locus region in Drosophila melanogaster.

Restriction site and insertion/deletion polymorphism in a 45-kb region of the white locus on the X chromosome in Drosophila melanogaster was investigated for 64 X chromosome lines with six 6-cutter and ten 4-cutter restriction enzymes. A total of 109 polymorphisms were detected (54 restriction sites and 55 insertions/deletions). Estimated heterozygosity per nucleotide for this region (0.004-0.008) was similar to those of the Adh and 87A heat-shock locus regions located on the autosomes in D. melanogaster. This is contrary to a simple prediction based on the theory of mutation selection-balance of partially recessive deleterious mutants which predicts less variation on X chromosomes. Large linkage disequilibria between pairs of polymorphisms (including insertions and deletions) within the transcriptional unit (especially the 3' end of the 1st intron) were observed. As expected from population genetics theory, linkage disequilibria between these polymorphisms were greater for those pairs that are physically closer on the restriction map. Linkage equilibrium was typically observed when the pairs of sites were separated by 2 kb or more. Although significant between-line variation in eye pigment was observed (P less than 0.05), there is little evidence for strong associations between this phenotype and the polymorphisms at the DNA level.

Animals↗
PubMed1990

Structural changes in the ribosomal genes of immortalized and transformed mouse embryo C3H/10T1/2 cell lines.

Comparative studies of DNA isolated from adult C3H mouse liver, immortalized mouse embryo cells (C3H/10T1/2 Cl 8) and the tumorigenic methylcholanthrene transformed C3H/10T1/2 Cl 16 cell line have been carried out in order to analyze possible structural changes in the ribosomal genes associated with the immortalization and tumorigenic transformation of mouse cells. Southern blot hybridization experiments revealed a mutation hotspot within repetitive sequences 13 kb upstream from the 18S rRNA genes in the non-transcribed spacer (NTS). Other DNA changes were localized near the initiation and termination regions of rRNA transcription. The differences found in the restriction maps of the 5'-region resided 5 to 6 kd upstream from the 18S 5'-end and the changes located in the 3'-end mapped approximately 5 kb downstream from the 28S 3'-end. Thus, oncogenic transformation of the C3H/10T1/2 Cl 8 cells by methylcholanthrene treatment was associated with stable genetic changes in the 18S rRNA gene. There was no evidence for rRNA gene amplification.

Animals↗
PubMed1993

von Hippel-Lindau disease: identification of deletion mutations by pulsed-field gel electrophoresis.

Von Hippel-Lindau disease (VHL) is an inherited multisystem neoplastic disorder. We prepared a 2.5-megabase (Mb) restriction map of the region surrounding the VHL gene and identified and characterized overlapping deletions in three unrelated patients affected with VHL. The smallest nested deletion (100 kb) was located within a 510-kb NruI fragment detected by 19-63'. The rearrangements detected will be useful in isolating and evaluating candidate cDNAs for the VHL gene. The detailed physical map will be useful in studying the organization and structure of genes in the VHL region.

Adult↗