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 1,531 records · Page 85Linked to original sources
PubMed1995

Confirmation that DNA encoding the major fimbrial subunit of Av24 fimbriae is homologous to DNA encoding the major fimbrial subunit of F107 fimbriae.

Plasmid DNA from porcine enterotoxigenic Escherichia coli strain Av24 (O141:K85ab) was cloned into recipient E. coli strain JM105 using the plasmid vector pUC18. Clones were obtained that produced fimbriae which reacted with antisera specific to the fimbriae produced by strain Av24. Restriction mapping of cloned DNA, PCR with fedA primers and DNA sequencing showed a portion of the cloned DNA to be homologous to that encoding the major fimbrial subunit of F107 fimbriae. This confirms that the fimbriae possessed by strains of E. coli causing both edema disease and post-weaning diarrhoea in piglets are variants of the same fimbriae.

Animals↗
PubMed1995

Identification of chromosomal mobile element conferring high-level vancomycin resistance in Enterococcus faecium.

A clinical isolate of Enterococcus faecium that contains a chromosomally encoded vanA gene cluster, Tn1546::IS1251, transferred vancomycin resistance to the plasmid-free strain Enterococcus faecalis JH2-2 during filter matings. Hybridization of a vanHAXY probe to SmaI restriction-digested genomic DNA separated by pulsed-field gel electrophoresis showed that the vanA gene cluster was located on a 40-kb fragment in the original donor strain and on fragments of different sizes (150 to 450 kb) in the transconjugants. No hybridization to vanA gene cluster probes was obtained with plasmid DNA preparations from the donor or transconjugants. These results suggested that in each case, the van genes had integrated into the recipient chromosome. The transconjugants in turn could act as donors of vancomycin resistance, and resistance was transferable to a Rec- recipient. The results of restriction analyses and DNA hybridizations of genomic DNA from the donor and transconjugants were consistent with the transfer of a mobile element that includes the 12.3-kb Tn1546::IS1251 gene cluster and at least 13 kb of additional DNA. This element has been tentatively designated Tn5482. DNA sequence analysis of a fragment predicted to contain the left end of Tn5482 revealed two insertion sequence-like elements: IS1216V and an apparently truncated IS3-like element. Restriction mapping and DNA hybridization patterns of the van gene clusters of three additional clinical isolates from New York City showed an element similar to Tn5482. Transfer of Tn5482 and related elements may be involved in dissemination of vancomycin resistance.

Amino Acid Sequence↗
PubMedDate not supplied

[Preparation of T-lymphoblastoid cell lines from patients with T-cell leukemia and asymptomatic carriers of HTLV-I: analysis of the viral genome status].

Three HTLV-I-infected partially interleukin-2-dependent lymphoid cell lines were derived from a patient with T-cellular leukemia (ATL) from Georgia and a carrier of HTLV-I from Sakhalin. The strains cultured in the presence of 3-5% interleukin-2 were designated as NBK-1, NBK-2, and YE-1, respectively. Immunoblotting analysis showed typical HTLV-I proteins in them except NBK-2 which expressed nontypical proteins p40K and p28-29K. Unexpectedly, leukemic cell fraction ATL/NBK from a patient contained gag proteins p19 (CA), p24 (MA), Pr53, and unidentified protein p29. Southern blot analysis of primary leukemic cells NBK showed one full-length non-defective provirus with restriction sites Sacl in both LTRs. Limited restriction map of the provirus virtually did not differ from previously described HTLV-I prototypes. Although the mechanism of abnormal protein expression remains to be determined, this event can be explained by defective provirus formation in NBK-2 cell line during coculturing of leukemic cells with human umbilical cord blood lymphocytes.

Blotting, Southern↗
PubMed1991

[Marburg virus: the first determined nucleotide sequence of two genes].

The preparations of purified Marburg virus were isolated from blood plasma of infected guinea pigs and characterized. Viral RNA was extracted from the virions. The cDNA was synthesized on the isolated RNA matrix by the reverse transcriptase with the use of dissipated priming. The obtained cDNA was inserted into the plasmid pBR322 by the connector technique and the resulting recombinant plasmids were cloned in Escherichia coli cells. The specific clones selected by molecular hybridization method were analyzed by the restriction mapping and cross-hybridization. Four overlapping cDNA clones were found and the virus specific 5012 bp fragment of the viral genome was sequenced. Three open reading frames were found and the preliminary analysis of the coded amino acid sequence and corresponding genes was fulfilled.

Amino Acid Sequence↗
PubMed1993

A yeast artificial chromosome contig linking the steroid sulfatase and Kallmann syndrome loci on the human X chromosome short arm.

In this report we describe the construction of a yeast artificial chromosome (YAC) contig linking the steroid sulfatase (STS) and Kallmann syndrome (KAL) loci on Xp22.3. Four human YAC libraries were screened initially with sequences from DXS237 (GMGX9), DXS278 (S232B), and KAL and later with primers from exon 10 of the STS gene and the end fragment of a YAC clone YGX3 to fill the gaps. Fifteen clones were isolated and the sizes of their human inserts were determined by pulsed-field gel electrophoresis followed by Southern hybridization with labeled total human DNA. Overlaps between the YAC clones were evaluated using more than 20 DNA markers, including the screening probes, the end fragments, and the Alu-PCR products of the YAC clones. The extent of overlapping between the clones was further determined by long-range restriction mapping. In combination with our previously reported YAC contigs around STS and KAL, a total of 2 Mb of Xp22.3 have been isolated in YAC clones. These clones will facilitate the isolation of new genes and the characterization of deletions and translocations which occur at very high frequency in this region of the human X chromosome.

Arylsulfatases↗
PubMed1996

YAC clone contigs covering 5 Mb of a repeat sequence island on the mouse X chromosome.

We have initiated work towards the construction of YAC clone contigs across a repeat sequence island region on the mouse X Chromosome (Chr). The repeat sequence island region-the 141 island-located at band A3 contains 50 copies of a localized long complex repeat unit (LCRU). We have isolated 87 YAC clones from the 141 island and have used a dual faceted approach towards the construction of contigs across the repeat sequence island. First, we have identified YAC clones originating from the same region of the island by the identification of commonly held LCRU restriction site variants. Second, we have constructed rare cutter restriction maps of each YAC clone. Taken together, we have been able to assemble one large contig of 2.8 Mb and a number of smaller contigs. In total, contigs covering 5Mb of the island region have been identified. The island region would appear to represent a major component of the A3 Giemsa dark band on the mouse X Chr.

Animals↗
PubMed1990

Detection of a novel chloramphenicol resistance plasmid from "equine" Staphylococcus sciuri.

A small chloramphenicol resistance (Cm) plasmid of 4.65 kB could be detected in an "equine" Staphylococcus sciuri-culture. This plasmid, designated as pSC3, was identified by interspecific protoplast transformation. On the basis of restriction endonuclease analyses a detailed restriction map of pSC3 could be constructed. This allowed structural comparisons of pSC3 with Cm-plasmids of other staphylococcal species from infections of humans and animals and identification of pSC3 as a member of the pC 221-family of staphylococcal Cm-plasmids. The pSC3-plasmid encoded an inducible chloramphenicol acetyltransferase as confirmed by enzymatic assays. This enzyme could be demonstrated in cell-free lysates of Cm-induced pSC3-transformants.

Animals↗
PubMed1998

PCR amplification and characterization of the intergenic spacer region of the ribosomal DNA in Pyrenophora graminea.

Successful amplification of the whole intergenic spacer region of the nuclear ribosomal repeat (IGS) in Pyrenophora graminea was obtained with a PCR-based assay. Single amplification products showed length differences. Depending on the length of the IGS-PCR product, ca. 3.8 or 4.4 kb, two groups of isolates could be identified. The RFLP patterns of isolates obtained with the 6-base cutting enzymes Apal, BglII, DraI, EcoRV, HindIII and SacI were similar within each group and different between the two groups. Restriction patterns of IGS-PCR products digested with the 4-base cutting enzyme AluI were polymorphic among isolates in spite of their IGS-PCR product length. In order to characterize the long and short IGS-PCR products the restriction map is shown. The long product shows an additional HindIII site and a BglII site that is lacking in the short product. However, the latter shows a SacI site that is not present in the long IGS-PCR product. Therefore, the described PCR-RFLP analysis of the IGS appears to be a useful tool to resolve genetic variation between P. graminea isolates.

Ascomycota↗
PubMed1997

The genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 reside on a large plasmid whose loss leads to degeneration of the strain.

Degeneration is the process whereby Clostridium acetobutylicum ATCC 824 loses the capacity to produce acetone and butanol after repeated vegetative transfers or in continuous culture. Two degenerate mutants (M5 and DG1) of C. acetobutylicum ATCC 824 do not contain the four genes (ctfA, ctfB, adc, and aad) for acetone and butanol formation. Strain ATCC 824 contains a 210-kb plasmid (pSOL1) which is absent in M5 and DG1. pSOL1 carries the four acetone and butanol formation genes. A restriction map of pSOL1 was constructed by using ApaI, SmaI, SstII, and NarI digestions. M5 and DG1 could be complemented for acetone and butanol formation by expressing the corresponding genes (ctfA, ctfB, and adc for acetone; aad for butanol) on the plasmid. Degeneration of this strain thus appears to be the result of pSOL1 loss.

Acetone↗
PubMed1991

Scarcity of ars sequences isolated in a morphogenesis mutant of the yeast Yarrowia lipolytica.

Previous attempts is isolate autonomously replicating sequences (ars) from the dimorphic yeast Yarrowia lipolytica have been unsuccessful. We isolated a Fil- mutant unable to produce hyphae and growing only in a yeast form to facilitate ars isolation. This mutant was transformed with a Y. lipolytica DNA bank and several unstable clones were obtained. Extrachromosomal plasmids were evidenced in yeast, recovered in Escherichia coli and characterized by restriction mapping. They were able to retransform Fil- and Fil+ yeast strains at high frequency and transformants displayed a slightly unstable phenotype. The detailed analysis of the plasmids showed that only two different ars sequences had been isolated, each of them corresponding to a unique sequence in the Y. lipolytica genome. We concluded that functional ars sequences that can be cloned on plasmids are rare in this yeast.

Base Sequence↗
PubMed1990

Characterization of a repetitive DNA probe for Babesia bigemina.

A plasmid (p16) containing a Babesia bigemina DNA insert was selected and labeled with 32P. This probe was evaluated for specificity and sensitivity by dot blot hybridization. The probe was specific and hybridized with only Babesia bigemina DNA, and not DNA from Babesia bovis, bovine leukocyte, Trypanosoma brucei or Anaplasma marginale. The DNA probe detected as little as 10 pg of Babesia bigemina DNA. The probe hybridized with Babesia bigemina isolates from Mexico, the Caribbean region and Kenya. Genomic Babesia bigemina DNA of a Kenyan isolate was digested with restriction endonucleases, and the fragments were separated by gel electrophoresis and Southern blotted. The filter was hybridized with labeled p16 and each endonuclease digestion produced at least 16 resolvable DNA fragments. The inserted Babesia bigemina DNA was approximately 6.3 kb in size. A partial restriction map was constructed. A simple whole blood dot blot procedure was utilized to evaluate the sensitivity of the DNA probe. This probe would detect as few as 150 Babesia bigemina infected erythrocytes contained in a 1-microliter sample. The DNA probe has the potential to be a very sensitive and specific diagnostic tool.

Animals↗
PubMed1991

Virulent human strains of group G streptococci express a C5a peptidase enzyme similar to that produced by group A streptococci.

Specific proteolytic destruction of the human chemotaxin, C5a, is a property of group A and B streptococcal pathogens. Here we show that virulent group G streptococci from human sources also express C5a peptidase activity. The enzyme responsible for this activity is approximately the same size as and is antigenically similar to that produced by group A streptococci. On the basis of Southern hybridization analysis with an internal fragment of the group A C5a peptidase gene (scpA) as a probe, a copy of this gene was found in the genome of all group G human isolates tested. Comparison of partial restriction maps of scpA and scpG revealed significant similarity between the two genes. Group G strains isolated from dogs and cows were found to lack C5a peptidase activity and did not hybridize to the scpA-specific probe. The association of this activity with three streptococcal species suggests that elimination of phagocyte chemotactic attractants is a more universal virulence mechanism than originally anticipated.

Adhesins, Bacterial↗
PubMed1990

Cloning of midecamycin(MLS)-resistance genes from Streptomyces mycarofaciens, Streptomyces lividans and Streptomyces coelicolor A3(2).

DNA containing genes for midecamycin(Mdm)-resistance was cloned from Streptomyces mycarofaciens ATCC 21454 (mdmA gene), Streptomyces lividans 66 (lrm gene) and Streptomyces coelicolor A3(2). The phenotype imparted to S. lividans and Streptomyces griseofuscus transformants by the cloned DNA segments indicates that they encode an MLS-type of resistance activity. The mdmA and lrm genes could be distinguished by the phenotype they conferred in S. lividans and S. griseofuscus, whereas the S. lividans lrm and S. coelicolor MLS genes appears to be identical on the basis of their restriction maps and behavior in S. lividans and S. griseofuscus. The DNA sequence of a 1.4-kb BamH I DNA fragment containing the mdmA gene indicates the presence of one complete orf whose deduced product exhibits a high similarity to the deduced product of the Streptomyces thermotolerans carB gene and several other bacterial MLS-resistance genes.

Amino Acid Sequence↗
PubMed1990

Molecular cloning of a DNA segment essential for replication from Streptomyces hygroscopicus strain 10-22 and localization of its minimal functional region.

The 13 kb DNA segment responsible for replication has been isolated from Streptomyces hygroscopicus subsp. yingchengensis strain 10-22 using a pBR322-derived Streptomyces replication-probe vector pIJ2703. The restriction map of the hybrid plasmid, pHZ54, has been constructed. Following a series of deletion and subcloning experiments, the minimal functional region for replication was located within a 2.86 kb DNA fragment. Subsequently, this region was further shortened into a 2 kb region by exonuclease III deletion. A few small bifunctional plasmids able to replicate in E. coli as well as in Streptomyces, were obtained during the above process. Some plasmids could be used as shuttle vectors.

Cloning, Molecular↗
PubMed1996

Analysis of the EHEC hly operon and its location in the physical map of the large plasmid of enterohaemorrhagic Escherichia coli O157:h7.

Almost all clinical enterohaemorrhagic Escherichia coli (EHEC) O157:H7 isolates harbour a large virulence plasmid designated pO157. In this study, pO157 of EHEC O157:H7 reference strain EDL 933 was characterized at the molecular level. A restriction map was constructed by using seven restriction enzymes, with appropriate gel electrophoretic and hybridization methods. The molecular size of pO157 was determined to be 93.6 kb. By sequencing the DNA region extending in the 3'-direction of the previously described EHEC hlyC and hlyA genes, two further genes were discovered and analysed; these were termed EHEC hlyB and EHEC hlyD. The newly discovered genes together with the EHEC hlyC and hlyA genes constitute a typical RTX (Repeats in ToXin) determinant (EHEC hly operon) with the gene order CABD. The map position of the operon was determined by hybridization experiments. Analysis of a DNA fragment carrying the downstream flanking region of the EHEC hly operon revealed an open reading frame which was highly homologous to orf1 of RepFIB, a basic replicon of IncF plasmids. It was located close to the EHEC hly operon.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of a novel human gene (D11S4896E) at chromosomal region 11p15.5.

A novel gene (GOK) has been cloned from human chromosome region 11p15.5 that is believed to contain a gene or genes associated with a number of pediatric malignancies, including Wilms tumor. A 4-kb cDNA has been cloned and it encodes a predicted protein of approximately 84 kDa that could be translated in vitro. Computer analysis predicted that the protein had a signal peptide and may contain a transmembrane helix. Restriction mapping by pulsed-field electrophoresis indicates that GOK is located 1.7 kb telomeric of RRM1, and both genes are transcribed in the same direction. GOK displays high evolutionary conservation: cloning and partial sequencing of a mouse genomic clone revealed 90% identity with the human gene at both the nucleotide and the predicted amino acid levels.

Amino Acid Sequence↗
PubMed2005

Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.).

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-to-tail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp b-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.

Animals↗
PubMed1992

Cloning of Candida boidinii DNA fragments promoting autonomous replication of plasmids in Saccharomyces cerevisiae.

Fragments of Candida boidinii chromosomal DNA were inserted into the integrative vector YIp-kanr and examined for the presence of sequences promoting autonomous replication of plasmids in Saccharomyces cerevisiae. Restriction maps of two plasmids, designated S6/4 and S6/5, originating from the same S. cerevisiae transformant, were constructed. Southern hybridization data confirmed that the plasmids carry sequences from the C. boidinii chromosome. Both plasmids transform S. cerevisiae strains at 4-5-fold higher frequency than cloning vectors based on the replication origin of the 2 microns plasmid. Mitotic stability of the constructed plasmids is similar to that of the 2 mu-based vector pNF2 in S. cerevisiae.

Blotting, Southern↗