Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Restriction Mapping”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,477 records · Page 82Linked to original sources
PubMed1990

Genetic analysis of a tetracycline resistance element from Clostridium difficile and its conjugal transfer to and from Bacillus subtilis.

A tetracycline resistance (Tcr) determinant from Clostridium difficile strain 630 was cloned into the Escherichia coli plasmid vector pUC13. The resulting plasmid pPPM20, containing an insert of 3.4 kbp, was mapped and a 1.1 kbp SacI-HindIII fragment wholly within the Tcr gene was identified. Dot-blot hybridization studies with the 1.1 kbp fragment showed that the Tcr gene belonged to hybridization class M. Tcr could be transferred between C. difficile strains and to Bacillus subtilis at a frequency of 10(-7) per donor cell. The element could be returned from B. subtilis to C. difficile at a frequency of 10(-8) per donor cell. This is the first demonstration of C. difficile acting as a recipient in intergeneric crosses. DNA from C. difficile transconjugants digested with EcoRV always has two hybridizing fragments of 9.5 and 11.0 kbp when probed with pPPM20. DNA from B. subtilis transconjugants digested with EcoRV produced one hybridizing band of variable size when probed with pPPM20. The behaviour of the element was reminiscent of the conjugative transposons. Therefore we compared the element to the conjugative transposon Tn916. The HincII restriction maps of the two elements differed and no hybridization was detected to oligonucleotides directed to the ends of Tn916. However, the elements do have some sequence homology, detected by hybridization analysis.

Bacillus subtilis↗
PubMed1989

Cloning and characterization of a histone gene family in Tilapia fish.

Genomic organization and expression of a histone gene family in the Tilapia (Cichlidae) fish was determined. A genomic library was prepared; four clones containing the complete set of core histones were analyzed. These clones differed significantly in their restriction maps, although three of them revealed a uniform internal gene arrangement of the core histone genes. Differential expression between two of the cloned clusters was observed when the hybridization pattern of those clusters to various RNA preparations was compared. The copy number of histone genes was estimated to be 100 to 110 for haploid Tilapia genome.

Animals↗
PubMed1994

Cloning and sequencing of the Leishmania major actin-encoding gene.

We report the cloning and characterization of the genomic sequence of the actin (Act)-encoding gene (act) from Leishmania major. Restriction maps of two genomic clones, as well as genomic Southern analysis strongly suggest that the act of L. major is a single-copy gene. A single 1.6-kb transcript is detected in Northern blots. The deduced amino-acid sequence shows 68-89% identity with Act sequences from other eukaryotes.

Actins↗
PubMed1995

Genomic organization of the human dystrophin gene across the major deletion hot spot and the 3' region.

The genomic organization of most of the human dystrophin gene has not been defined at single-exon level, owing to its enormous size (2300 kb). By taking advantage of a YAC-based restriction map of the gene previously constructed, we have localized individual dystrophin exons from 42 to 79 along the central and 3' regions of the gene. These data elucidate the general organization of this large portion of the gene (1250 kb) and, in particular, characterize the genomic region most frequently involved in deletion mutations responsible for Duchenne and Becker muscular dystrophies.

Dystrophin↗
PubMed1994

Structure and organization of a stable extrachromosomal element in human cells.

We have determined the structure and organization of a 630-kb extrachromosomal element (amplisome) containing the dihydrofolate reductase-encoding gene (DHFR) in a methotrexate (MTX)-resistant human cell line, HeLa-Bu25-10B3. The size and copy number of amplisomes have previously been found to remain remarkably stable with or without selection. Both linear and open circular 630-kb amplisomes are present in these cells. We have been able to isolate the linear amplisomes after pulsed-field gel electrophoresis (PFGE), and transfect the amplisomes into MTX-sensitive recipient cells by electroporation, thus demonstrating that DNA as large as 630 kb can be transfected into mammalian cells. The NotI restriction site immediately upstream from DHFR on the circular amplisome is devoid of methylation, suggesting that it is transcriptionally active. Restriction mapping by PFGE reveals that there is only one copy of DHFR per amplisome and no repetitive structure is observed. The small size of the amplisomes, their stability and our ability to transfect large DNA molecules provide the necessary ingredients for the development of mammalian cloning vectors for large DNA fragments.

Azacitidine↗
PubMed1991

Deletion of the entire cytochrome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism.

The debrisoquine/sparteine polymorphism is associated with a clinically important genetic deficiency of oxidative drug metabolism. From 5% to 10% of Caucasians designated as poor metabolizers (PMs) of the debrisoquine/sparteine polymorphism have a severely impaired capacity to metabolize more than 25 therapeutically used drugs. The impaired drug metabolism in PMs is due to the absence of cytochrome P450IID6 protein. The gene controlling the P450IID6 protein, CYP2D6, is located on the long arm of chromosome 22. A pseudogene CYP2D8P and a related gene CYP2D7 are located upstream from CYP2D6. This gene locus is highly polymorphic. After digestion of genomic DNA with XbaI endonuclease, restriction fragments of 11.5 kb and 44 kb represent mutant alleles of the cytochrome CYP2D6 gene locus associated with the PM phenotype. In order to elucidate the molecular mechanism of the mutant allele reflected by the XbaI 11.5-kb fragment, a genomic library was constructed from leukocyte DNA of one individual homozygous for this fragment and screened with the human IID6 cDNA. The CYP2D genes were isolated and characterized by restriction mapping and partial sequencing. We demonstrate that the mutant 11.5-kb allele results from a deletion involving the entire functional CYP2D6 gene. This result provides an explanation for the total absence of P450IID6 protein in the liver of these PMs.

Base Sequence↗
PubMed1989

Demonstration of clonal variation amongst O-antigen serotype variants of Escherichia coli O2 and O18 using DNA probes to the rfb region of the E. coli strain B41 (O101:K99/F41).

The confirmation at the DNA level of the existence of clonal variants within Escherichia coli O2 and O18 serotypes has been shown by Southern hybridization analysis of restriction endonuclease digested genomic DNA and subsequent probing with contiguous subclones of the E. coli O101 rfb region. The O101 rfb subclones are believed to represent a conserved region of DNA (Heuzenroeder et al. Molec. Microbiol, in press) and identify serotype variants by means of restriction fragment length polymorphisms (RFLP) within homologous DNA of O2 and O18 E. coli. A number of different restriction enzymes have been used singly and in combination to digest the genomic DNA, thereby allowing construction of restriction maps of the region displaying homology to the O101 rfb region subclones. This analysis further substantiates previously defined evolutionary relationships between O2 and O18 E. coli. These simple probes appear to be able to provide the same clonal information as a battery of isoenzyme, outer membrane protein (OMP) and lipopolysaccharide (LPS) analyses.

Antigens, Bacterial↗
PubMed1989

Molecular cloning and in vitro expression of a silent phenoxazinone synthase gene from Streptomyces lividans.

Phenoxazinone synthase (PHS) catalyzes a step in actinomycin D biosynthesis in Streptomyces antibioticus. Two sequences from Streptomyces lividans that hybridize to the phs gene of S. antibioticus have been cloned in Escherichia coli K-12 using the plasmid pBR322. Although there was some similarity in the restriction maps of the two cloned fragments, neither insert appeared to be a direct subset of the other nor of the S. antibioticus phs gene. In vitro expression studies, in a streptomycete coupled transcription-translation system, showed that a 3.98-kb SphI fragment encoded a PHS-related protein. These observations provide additional support for the existence of silent genes for antibiotic production in streptomycetes.

Blotting, Southern↗
PubMed1990

Characterization of the genetic determinants of SsoII-restriction endonuclease and modification methyltransferase.

The genes encoding SsoI and SsoII restriction endonuclease (ENase) and methyltransferase (MTase) are located on the small plasmids P6 and P4, respectively, of Shigella sonnei strain 47. Functions provided by plasmids P5, P7 and P9, which include colicinogenicity and immunity to colicin E1, resistance to streptomycin (Sm), and conjugative DNA transfer, respectively, have also been identified. The genes of the SsoII restriction-modification (R-M) system have been cloned into Escherichia coli expressing the 35-kDa (ENase) and 43-kDa (MTase) products. A restriction map of the P4 plasmid DNA was determined, and the approximate location of the genes encoding SsoII ENase and MTase (ssoIIR and ssoIIM) on that have been established. SsoI is an isoschisomer of EcoRI and SsoII cleaves the 5'-/CCNGG/recognition sequence producing 5'-protruding 5-nt long cohesive ends.

Cloning, Molecular↗
PubMed1991

Streptococcus pneumoniae produces at least two distinct enzymes with neuraminidase activity: cloning and expression of a second neuraminidase gene in Escherichia coli.

A gene from Streptococcus pneumoniae was cloned in lambda EMBL301 and then expressed in Escherichia coli, which cleaved the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid. The cloned gene therefore encodes an enzyme with neuraminidase activity. On the basis of restriction mapping and DNA hybridization studies, this gene could be distinguished from another pneumococcal neuraminidase gene cloned previously (A. M. Berry, J. C. Paton, E. M. Glare, D. Hansman, and D. E. A. Catcheside, Gene 71:299-305, 1988). Both neuraminidase genes were found in each of five isolates, covering at least three serotypes, of pneumococci tested.

Blotting, Southern↗
PubMed1992

Physical and functional maps of the luminescence gene cluster in an autoinducer-deficient Vibrio fischeri strain isolated from a squid light organ.

Vibrio fischeri ES114 is an isolate representing the specific bacterial light organ symbiont of the squid Euprymna scolopes. An interesting feature of this strain of V. fischeri is that it is visibly luminous within the light organ of the squid host but is nonluminous when grown under standard laboratory conditions. Luminescence can be restored in laboratory culture, however, by the addition of autoinducer, a species-specific inducer of the V. fischeri luminescence (lux) genes. Most other isolates of V. fischeri produce autoinducer in sufficient quantities to induce luminescence in laboratory culture. We have cloned an 8.8-kb DNA fragment from V. fischeri ES114 that encodes all of the functions necessary for luminescence in Escherichia coli in the absence of exogenous autoinducer. This DNA contains both of the recognized V. fischeri lux regulatory genes, one of which (luxI) directs E. coli to synthesize autoinducer. The organization of the individual lux genes within this DNA fragment appears to be the same as that in the other strains of V. fischeri studied; the restriction map of the V. fischeri ES114 lux DNA has diverged substantially, however, from the largely conserved maps of V. fischeri MJ1 and ATCC 7744. Although E. coli containing the V. fischeri ES114 lux DNA synthesizes considerable amounts of autoinducer, V. fischeri ES114 synthesizes autoinducer only in small amounts, even when transcription of the lux genes, including luxI, is activated by the addition of exogenous autoinducer. Nonetheless, transconjugants of V. fischeri ES114 that contain multicopy plasmids bearing the ES114 lux genes synthesize sufficient autoinducer to induce luminescence. These results suggest that V. fischeri ES11r does not lack a functional luxl, nor is it deficient in the ability to synthesize metabolic precursors for autoinducer synthesis.

4-Butyrolactone↗
PubMed1991

Typing and molecular characterization of human papillomaviruses in genital warts from South African women.

Condyloma acuminata from 12 women were examined for the presence of human papillomavirus DNA. Six of the women had HPV 6a, two had HPV 11a, one had a new HPV 6 subtype, and one a new HPV 11 subtype. The new HPV 6 subtype could be distinguished from other HPV 6 subtypes on the basis of Pst I fragments as well as Hind II fragments. The new HPV 11 subtype had a different Hpa II restriction endonuclease pattern. Restriction maps of both new subtypes were constructed. Two of the biopsies did not contain detectable HPV DNA when probed with HPV 6, HPV 11, and HPV 16. Biopsies were taken from normal tissue 1 cm away from the condyloma in 11 of the patients. Only one of these normal tissue biopsies was positive for HPV by Southern blot hybridization.

Condylomata Acuminata↗
PubMed1990

Isolation and characterization of a distantly related member of the beta-gamma crystallin super gene family from Xenopus.

In an attempt to isolate gamma crystallin genes from Xenopus genomic library, we have obtained seventeen clones which strongly hybridized to the gamma 1 cDNA probe from Rana temporaria. Of these four clones are characterized. A restriction map was derived by using single and multiple digests with a variety of enzymes. Hybridization selection of lens mRNAs and in vitro translation produced a protein of 26,000 daltons. These data suggest that the above clones may contain at least part of the gamma or beta crystallin sequences.

Animals↗
PubMed1992

Molecular epidemiology of respiratory syncytial virus: rapid identification of subgroup A lineages.

Methods for the rapid analysis of samples of respiratory syncytial (RS) virus are described using the polymerase chain reaction (PCR) followed by restriction mapping. Isolates (either clinical samples or tissue culture grown virus) can readily be divided into subgroups and then further classified into lineages. These methods enable examination of large numbers of isolates by molecular techniques, thereby facilitating research into the molecular epidemiology of the virus.

Base Sequence↗
PubMed1993

Molecular cloning and expression of murine thromboxane synthase.

The complementary DNA (cDNA) for murine thromboxane synthase (TS) was isolated from a lung cDNA library. The full-length cDNA (1,910 bp) encodes a 533 amino acid protein (Mr 58,220) sharing 78% identity with human TS. Sequence comparison indicated that one of the two N-glycosylation sites, eight of the eleven cysteine residues, and a heme-binding domain are conserved in both murine and human TS sequences. The authenticity of the cDNA was confirmed by transient expression of a catalytically active TS in cos-1 cells. Northern analysis indicated that murine TS gene was expressed primarily in lung, kidney, and spleen. Interestingly, the size of mRNA in the kidney was approximately 100 to 150 bp shorter than that in the lung or spleen (2.2 Kb). RT-PCR and restriction mapping indicated that neither the coding sequence nor the 3' untranslated region could account for the observed size difference, suggesting a shorter 5' untranslated region and/or poly (A) tail in the kidney transcript.

Amino Acid Sequence↗
PubMed1995

Isolation of cryptic plasmids from moderately halophilic eubacteria of the genus Halomonas. Characterization of a small plasmid from H. elongata and its use for shuttle vector construction.

Three cryptic plasmids have been isolated from moderately halophilic eubacteria belonging to three species of the genus Halomonas. These three plasmids were designated pHE1 (4.2 kb, isolated from H. elongata ATCC 33174), pHI1 (48 kb, isolated from "H. israelensis" ATCC 43985), and pHS1 (ca. 70 kb, isolated from H. subglaciescola UQM 2927). Because of its small size, the plasmid pHE1 was selected for further characterization and construction of a shuttle vector for Halomonas strains. pHE1 was cloned into pBluescript KS and a detailed restriction map was constructed. Hybridization experiments excluded the existence of sequences homologous to pHE1 in total DNA from other strains of the genus Halomonas. Moreover, no DNA homology with pMH1, the only plasmid described so far from moderate halophiles, was found. Since pHE1 appeared to be unable to replicate in Escherichia coli cells, a number of mobilizable pHE1-derived hybrid plasmids were constructed that could be selected and maintained both in E. coli and in H. elongata. Finally, an improved shuttle vector, pHS15, was generated. The vector pHS15 contains an origin of replication from E. coli as well as one from H. elongata, a streptomycin resistance gene for positive selection in moderate halophiles, a number of unique restriction sites commonly used for cloning, and the mobilization functions of the broad host range IncP plasmid RK2. The vector pHS15 was readily mobilized by the RK2 derivative pRK2013 to all Halomonas strains tested so far. This is the first report on the development of a cloning vector useful for moderately halophilic eubacteria.

Genetic Vectors↗
PubMed2002

Fine analysis of the Pneumocystis carinii f. sp. carinii genome by two-dimensional pulsed-field gel electrophoresis.

Pneumocystis carinii is a general designation for a group of unusual unicellular fungal parasites responsible of pneumopathy in animal hosts. Divided into several subgroups termed the 'special forms', P. carinii is prone to an extensive karyotype variation. In previous studies, the nuclear genome of these organisms has been considered to be haploid and a set of 16 chromosomes has been assigned to P. carinii f. sp. carinii, a special form known to infect rats. We report the analysis of the genome of an isolate representative of the karyotype 1 of this special form, using two-dimensional pulsed-field gel electrophoresis procedures. The 'karyotype and restriction display' (KARD) fingerprints indicated the presence of 17 different chromosomes. The haploid genome size was estimated to be 8.4 Mbp. Some homologous chromosomes were distinguished on the basis of a single restriction fragment length polymorphism, which raises the possibility of a diploid nucleus. A restriction map of the chromosome 15, characterized by two homologues with a size difference of 7 kb, was constructed. Hybridization data indicated that insertion/deletion events may have occurred within subtelomeric regions which carry genes encoding the major surface glycoprotein (MSG) of Pneumocystis.

Bacterial Proteins↗
PubMed1992

Gene transfer in magnetic bacteria: transposon mutagenesis and cloning of genomic DNA fragments required for magnetosome synthesis.

Broad-host-range IncP and IncQ plasmids have been transferred to the aerobic magnetic bacterium Aquaspirillum sp. strain AMB-1. Conjugal matings with Escherichia coli S17-1 allowed high-frequency transfer of the RK2 derivative pRK415 (4.5 x 10(-3) transconjugant per recipient cell) and the RSF1010 derivative pKT230 (3.0 x 10(-3) transconjugant per recipient). These plasmids successfully formed autonomous replicons in transconjugants and could be isolated and transformed back into E. coli, illustrating their potential as shuttle vectors. A mobilizable plasmid containing transposon Tn5 was transferred to Aquaspirillum sp. strain AMB-1 and also to the obligately microaerophilic magnetic bacterium Aquaspirillum magnetotacticum MS-1. Five nonmagnetic kanamycin-resistant mutants of Aquaspirillum sp. strain AMB-1 in which Tn5 was shown to be integrated into the chromosome were obtained. Different genomic fragments containing the mutagenized regions were cloned into E. coli. Two genomic fragments were restriction mapped, and the site of Tn5 insertion was determined. They were shown to be identical, although derived from independent transposon insertions. One of these clones was found to hybridize strongly to regions of the A. magnetotacticum MS-1 chromosome. This is the first report of gene transfer in a magnetic bacterium.

Cloning, Molecular↗