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PubMed1992

The human serum amyloid A locus SAA4 is a pseudogene.

We have isolated the human genomic DNA clone GSAA4 from a size-selected Bgl II library by hybridization to a probe derived from the human serum amyloid A gene GSAA1. Sequencing the 5' end of this clone revealed a region similar to the first exon of gene GSAA1 but with significant nucleotide differences and mutation of the 3' splice site. The restriction map of the GSAA4 clone corresponds to that for the locus "SAA4" recently reported by others. Sequence and hybridization details indicate that the locus in clone GSAA4 is a member of the human serum amyloid A gene family and contains a pseudogene. Isolating GSAA4 completes the collection of clones needed to account for all bands found in blot hybridizations of human DNA using serum amyloid A gene probes.

Base Sequence↗
PubMed1988

Identification and cloning of a plasmid-encoded erythromycin resistance determinant from Lactobacillus reuteri.

Plasmid analysis, plasmid curing, cloning, and hybridization experiments were used to study four Lactobacillus reuteri strains showing high resistance to erythromycin. Plasmid curing with acriflavine resulted in a loss of erythromycin resistance in a frequency of 1-10%. For three of the strains this was accompanied by a loss of a 6.9-MDa plasmid, which was shown to be identical for the different strains and designated pLUL631. The erythromycin (erm) gene was located on a 5.5-MDa plasmid in the fourth strain. A restriction map of pLUL631 was constructed and the location of the erm gene on the plasmid was identified by cloning in Escherichia coli. By using a Streptococcus lactis-E. coli shuttle vector, the erm gene was also transformed to S. lactis and expressed. The erm gene from L. reuteri was shown to be related to the erm gene from pIP501 (Streptococcus agalactiae) by DNA-DNA hybridization.

Cloning, Molecular↗
PubMed1999

Characterization of a novel bacteriocin-encoding plasmid found in clinical isolates of Staphylococcus aureus.

Plasmids specifying bacteriocin production and immunity to its action were found in three clinical isolates of Staphylococcus aureus obtained in different hospitals located in Rio de Janeiro. These plasmids (pRJ28, pRJ29 and pRJ30) of 8.0 kb were found to generate identical restriction fragment patterns upon digestion with several enzymes, although the range of strains susceptible to the respective bacteriocin varied among the producer strains, when different Gram-positive bacteria were used as indicators, pRJ29 was then chosen for further characterization in order to compare it with pRJ6 and pRJ9, two small bacteriocin-encoding plasmids previously described in strains isolated from food. pRJ29 was found to code for a bacteriocin with chemical properties (sensitivity to proteases, heat resistance, activity under anaerobiosis, and estimated molecular weight) similar to those of pRJ6-encoded bacteriocin, conferring cross-immunity to it. However, its restriction map differed from those of pRJ6 and pRJ9. These studies together with hybridization, incompatibility, and mobilization analyses using a derivative of pRJ29 tagged with Tn917-lac suggest that pRJ29 is a mosaic composed of genetic determinants found on pRJ6 and pRJ9, and that IS257 was not involved in the recombination events which gave rise to pRJ29.

Bacteriocins↗
PubMed1989

Shuttle plasmids constructed by the transformation of an Escherichia coli cloning vector into two Deinococcus radiodurans plasmids.

An Escherichia coli plasmid that confers kanamycin resistance (Kmr) was inserted into the large Deinococcus radiodurans cryptic plasmids pUE10 and pUE11, yielding pS28 and pS19. The method of insertion involved both in vitro splicing and the natural transformation of D. radiodurans and yielded full-length clones in E. coli of pUE10 and pUE11. Both pS28 and pS19 replicated and expressed Kmr in E. coli and D. radiodurans. In both pS28 and pS19, D. radiodurans plasmid sequences were immediately upstream from the Kmr determinant. Transformation experiments suggested that Kmr expression in D. radiodurans was initiated in upstream D. radiodurans sequences. Restriction maps of pS28 and pS19 showed that each plasmid contained three MraI sites. Both pS28 and pS19 transformed the MraI-producing D. radiodurans strain R1 at low frequencies. D. radiodurans strain Sark, which naturally contains pUE10 and pUE11, was transformed by pS28 and pS19 at much higher frequencies. A Sark derivative that was cured for pUE10 was isolated by screening Sark/pS28 subisolates for loss of kanamycin resistance.

Cloning, Molecular↗
PubMed1990

Long insertions within telomeres contribute to chromosome size polymorphism in Plasmodium berghei.

During prolonged in vivo mitotic multiplication of a Plasmodium berghei ANKA clone (8417HP), parasites that contained an enlarged version of chromosome 4 were observed. Restriction mapping and hybridization results demonstrated that the extra DNA present in the enlarged chromosome consists of 2.3-kb tandem repeats, known to be normally located in subtelomeric position at several chromosomal ends but absent in the original chromosome. The inserted 2.3-kb units appeared to interrupt one of the original telomeres and to create an internal (approximately 1-kb-long) telomeric sequence.

Animals↗
PubMed1993

Characterization of a prolate-headed bacteriophage of Lactobacillus delbrueckii subsp. lactis, and its DNA homology with isometric-headed phages.

A new Lactobacillus delbrueckii subsp. lactis bacteriophage, JCL 1032, was characterized. JCL 1032 had a small, elongated prolate head, and a long non-contractile tail with cross-bars. The restriction map of JCL 1032 genome was constructed with five endonucleases. The genome was 45.8 kb in size, and it had cohesive ends (cos). Molecular masses of the phage structural proteins were also determined. JCL 1032 showed DNA homology with morphologically dissimilar, isometric-headed phages of Lb. delbrueckii (subsp. lactis and subsp. bulgaricus) when analyzed by Southern hybridization. Although in general JCL 1032 was only distantly related to isometric-headed phages, there were also a few short highly homologous (minimal homology 84%) DNA regions.

Bacteriophages↗
PubMed1995

The immunoglobulin kappa locus of primates.

The immunoglobulin kappa genes of nonhuman primates were studied by using sequence information and hybridization probes derived from the human kappa gene regions. The following results were obtained: (1) V kappa gene probes of the three major human kappa subgroups hybridized to restriction nuclease digests of DNA from the chimpanzees Pan troglodytes (PTR) and Pan paniscus (PPA), the gorilla Gorilla gorilla (GGO), the orangutan Pongo pygmaeus (PPY), the macaque Macaca mulatta (MMU), the marmoset Callithrix geoffrei (CGE), and the bushbaby Galago demidovii (GDE), yielding patterns of decreasing similarity to the patterns of the human V kappa multigene family. (2) The C kappa gene segments of PTR, GGO, and PPY were 99.6, 97, and 93%, respectively, identical in sequence to the human C kappa gene. A V kappa gene in PTR, GGO, PPY, and MMU was 98, 96, 96, and 95%, respectively, identical to the most C kappa proximal V kappa gene, called B3. The other two J kappa-C kappa proximal V kappa genes in human, B1 and B2, hybridize to restriction fragments of sizes identical to that of DNA from humans and great apes. (3) The long-range restriction maps of the human (HSA), PTR, and GGO kappa loci as established by pulsed-field gel electrophoresis (PFGE) are quite homologous. According to the maps, however, and to hybridization studies with 11 duplication-differentiating probes, there is only one copy of the locus in PTR and GGO. This means that the duplication of large parts of the kappa locus as found in humans occurred after the branch-point of human and great ape evolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Tandem repeat of the 23S and 5S ribosomal RNA genes in Borrelia burgdorferi, the etiological agent of Lyme disease.

The DNA fragments containing the rrl and rrf genes were subcloned from a EMBL3 recombinant phage of Borrelia burgdorferi strain B31 into pUC18 and were characterized by restriction analysis and Southern hybridization. A fine restriction map of the fragments was constructed and the organization of the genes was determined. The genomic hybridization using the gene probes from B. burgdorferi showed that there are two sets of rrl/rrf genes in that genome. The results also revealed the important fact that the gene sets are repeated directly by 3.2-kb long. This is the first report of this remarkable feature in the organization of the eubacterial rRNA genes.

Borrelia burgdorferi Group↗
PubMed1992

The 23S/5S ribosomal RNA genes (rrl/rrf) are separate from the 16S ribosomal RNA gene (rrs) in Borrelia burgdorferi, the aetiological agent of Lyme disease.

DNA fragments containing the rRNA genes for Borrelia burgdorferi strain B31 were cloned in bacteriophage lambda EMBL3. A restriction map of the fragments was constructed and the organization of the rRNA genes was determined by Southern hybridization. One genomic DNA fragment contained a single copy of the rrs sequence and another cloned fragment contained both rrl and rrf sequences. The results revealed that the rrs gene is located separately from the set of rrl/rrf genes, suggesting that these rRNA genes are expressed independently in B. burgdorferi.

Blotting, Southern↗
PubMed1993

Analysis of the genome of the five Bifidobacterium breve strains: plasmid content, pulsed-field gel electrophoresis genome size estimation and rrn loci number.

The genomes of the five Bifidobacterium breve strains available from culture collections were compared by restriction endonuclease analysis. Electrophoretic migration of undigested DNA allowed us to detect a 5.6-kb circular plasmid in two of these strains. A restriction map of this plasmid was constructed using 10 enzymes. With DraI endonuclease, pulsed-field gel electrophoresis has allowed the determination of the five B. breve genome sizes to 2.1 Mb. This estimation was further confirmed for CIP 6469 (type strain) and ATCC 15698 using XbaI and SpeI enzymes. In addition, rRNA gene regions were used as probes for strain characterization and suggest that there are at least three rrn loci in B. breve.

Bifidobacterium↗
PubMed1989

Presence of a small plasmid in clinical isolates of Streptococcus pneumoniae.

We have detected the presence of a small (2.95 kb) plasmid in a clinical isolate of Streptococcus pneumoniae. A restriction map was constructed for this plasmid and for pDP1 (the only previously reported pneumococcal plasmid); no apparent differences were observed and the two plasmids hybridized strongly to each other. Portions of pDP1 were then cloned into Escherichia coli K-12, using the vector pUC19, and the pneumococcal DNA insert was used as a probe to screen 500 clinical isolates of S. pneumoniae for pDP1 sequences. The plasmid was detected in a total of 8 isolates. These were of various serotypes and no correlation could be found between the presence of the plasmid and the geographical location from which it came, the type of infection, or with resistance to antibacterial drugs. Although no function has yet been assigned to pDP1, it may form the basis of a useful vector for cloning in S. pneumoniae, as it contains at least seven unique restriction sites.

Blotting, Southern↗
PubMed1983

Multiplicity of constant kappa light chain genes in the rabbit genome: a b4b4 homozygous rabbit contains a kappa-bas gene.

We have constructed a genomic library of homozygous b4b4 rabbit DNA in the pJB8 cosmid vector. Clones containing Ckappa-like sequences were screened with a b4 cDNA probe and were characterized by restriction mapping. One of the clones contained a Ckappa sequence different from the b4 allotype normally expressed by the animal. We report here the nucleotide sequence of this gene and show that it probably corresponds to a kappa-bas form of the Basilea allotype. It appears to be a structurally complete gene without any stop codons within the coding region and containing the dinucleotide AG as a splice site acceptor for the J-C junction, just 5' of the coding block. Comparison with the b4 cDNA nucleotide sequence shows a separate evolution of the Ckappa-coding and 3'-untranslated sequences, since the 3'-untranslated regions are more conserved than the coding regions. Genomic blot analysis would suggest that the kappa-bas gene is isotypic in the domestic rabbit population, since it lies within a genomic EcoRI or PstI restriction fragment, which was shown to be common to all homozygous b4, b5, b6 and b9 rabbit DNAs.

Amino Acid Sequence↗
PubMed1992

Isolation of two distinct type I angiotensin II receptor genes.

A rat genomic Southern blot, probed with a type I angiotensin II receptor probe, demonstrated that two highly homologous type I angiotensin II receptors were present. A rat genomic library was subsequently screened and four clones were isolated. From restriction mapping, differential hybridization, polymerase chain reaction amplification and sequence analyses we have determined that there are two unique type I angiotensin II receptor genes. The first of these genes corresponds to the published rat vascular complementary DNA sequence; the second, corresponds to a novel receptor not previously described.

Amino Acid Sequence↗
PubMed1994

Production of a plasmid mediated AmpC-like beta-lactamase by a Klebsiella pneumoniae septicaemia isolate.

A septicaemia Klebsiella pneumoniae isolate from Greece (Ath1) was shown to be resistant to third generation cephalosporins, aztreonam and cefoxitin and this resistance was not decreased in the presence of clavulanic acid. The gene coding for the resistance phenotype, associated with a beta-lactamase showing cephalosporinase activity and a pI of 8.6, could be transferred into Escherichia coli K-12 by conjugation and transformation. DNA-hybridisation showed that this gene was located on two different plasmids, 7.8 and 80 kb respectively. The larger, conjugative plasmid also carried genes coding for another beta-lactamase (pI 6.6) and resistance to aminoglycosides, tetracycline, chloramphenicol and trimethoprim. Under highly stringent conditions the gene coding for the pI 8.6 beta-lactamase hybridised with chromosomal DNA from Citrobacter freundii OS60 but not from E. coli or Enterobacter cloacae. Furthermore, the restriction map of this beta-lactamase gene corresponded to that of ampC from C. freundii OS60. The regulatory ampR gene could not be detected in the plasmid DNA from the Ath1 K. pneumoniae by DNA hybridisation. The relationship between a plasmid-mediated extended spectrum beta-lactamase in a K. pneumoniae septicaemia isolate and chromosomal AmpC beta-lactamase from C. freundii was demonstrated.

Citrobacter freundii↗
PubMed1993

Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae.

Strains of Pseudomonas syringae pv. syringae resistant to copper, streptomycin, or both compounds were recovered from symptomless and diseased tissue of four woody hosts in three nurseries in Oklahoma. In strains resistant to copper and streptomycin (Cur Smr), resistance to both compounds was cotransferred with a single plasmid which was either 68, 190, or 220 kilobase pairs (kb). All Cus Smr strains contained a 68-kb conjugative plasmid. Cur Sms strains contained one plasmid which varied in size from 60 to 73 kb. All conjugative plasmids which transferred streptomycin resistance contained sequences homologous to the strA and strB Smr genes from the broad-host-range plasmid RSF1010. The Smr determinant was subsequently cloned from a 68-kb Cur Smr plasmid designated pPSR1. A restriction map detailing the organization of the homologous Smr genes from pPSR1 and RSF1010 and cloned Smr genes from P. syringae pv. papulans and Xanthomonas campestris pv. vesicatoria revealed the conservation of all sites studied. The Cur genes cloned from P. syringae pv. tomato PT23 and X. campestris pv. vesicatoria XV10 did not hybridize to the Cur plasmids identified in the present study, indicating that copper resistance in these P. syringae pv. syringae strains may be conferred by a distinct genetic determinant.

Copper↗
PubMed1991

Arrangement of nitrogenase structural genes in an aerobic filamentous nonheterocystous cyanobacterium.

Members of the marine filamentous, nonheterocystous cyanobacterial genus Trichodesmium not only are capable of fixing nitrogen aerobically in the light but when grown under a light-dark cycle will fix nitrogen only during the light phase. In this study, we constructed a restriction map of the structural nitrogen fixation genes (nifHDK) in Trichodesmium sp. strain NIBB 1067. We found that the organization of the nif genes in Trichodesmium sp. strain NIBB 1067 is contiguous, as found in other nonheterocystous cyanobacteria and in heterocysts. Furthermore, the nif gene arrangement was identical when the cultures were grown with combined nitrogen or under nitrogen-fixing conditions. Therefore, no gene rearrangements occur, such as those that occur during the development of heterocysts in heterocystous species.

Blotting, Southern↗
PubMedDate not supplied

An unusual rRNA gene organization in Mycoplasma fermentans (incognitus strain).

The macro-restriction map of Mycoplasma fermentans (incognitus strain) was constructed and its rRNA genes were located on the map. It was found that this organism contains two sets of rRNA genes. The 16S and 23S rRNA genes were closely linked as two clusters. However, both 5S rRNA genes were separated from the 16S and 23S genes. The two 16S-23S rRNA gene clusters were arranged in an unusual tail to tail orientation.

Base Sequence↗
PubMedDate not supplied

[Mutual location of the rRNA operon and tuf gene in the Mycoplasma gallisepticum strain S6 genome].

The genomic library of Mycoplasma gallisepticum was constructed and two clones, selected by hybridization on E. coli 16S rRNA, were analyzed. The restriction map of the clones indicate that both clones belong to the same region of the M. gallisepticum genome. The results of Southern hybridization with either E. coli 16S rRNA, or E. coli 23S rRNA, or oligonucleotide synthesized as a part of M. gallisepticum 5S rRNA, led to the conclusion that the unspliced rRNA operon was cloned. The order of genes in the operon is common for eubacteria: 16S-23S-5S. The tuf gene of M. gallisepticum was mapped inside the cloned region 5 kb upstream of 16S gene by hydridization on E. coli tufA gene and oligonucleotide synthesized on the basis of M. gallisepticum tuf gene sequence. The direction of transcription of the gene and the expected direction of transcription of the rRNA operon coincide.

Bacteriophages↗