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At least 433 records · Page 24Linked to original sources
PubMed1996

Rapid restriction mapping of cosmids by sequence-specific triple-helix-mediated affinity capture.

A simple and rapid strategy for restriction mapping based on sequence-specific triple-helix affinity capture (TAC) was developed. The strategy was applied to the analysis of cosmid clones by the construction of a new cosmid vector, ScosTriplex-II, containing two different triple-helix-forming sequences flanking the cloning site of the original SuperCos-1 cosmid vector. For restriction mapping, the recombinant cosmid DNA is digested with NotI restriction enzyme or with one of four intron-encoded endonucleases for excision of intact inserts followed by controlled partial digestion with a mapping enzyme used in conjunction with the corresponding methyltransferase. The partial digestion products are combined with biotinylated triple-helix-forming oligonucleotides to form a triple-helical complex. The triple-helix complexes are immobilized on streptavidin-coated magnetic beads, washed, and eluted with pH 9 buffer solution. The fragments are separated and directly sized by agarose gel electrophoresis. Bidirectional maps are obtained simultaneously by binding to the two different triple-helix-forming oligonucleotides. No probe labeling, gel drying, blotting to membranes, hybridization, or autoradiography is necessary. Also, TAC conditions that permit gel-free isolation of the terminal restriction fragments from cosmid inserts were found. These advantages afforded by ScosTriplex-II should facilitate the automation of cosmid restriction site fingerprinting needed for large-scale mapping and sequencing projects.

Base Sequence↗
PubMed1983

Analysis of tetracycline resistance encoded by transposon Tn10: deletion mapping of tetracycline-sensitive point mutations and identification of two structural genes.

Deletions in the tet genes derived from Tn10 were formed from different tet::Tn5 insertion mutations by removing DNA sequences located between a HindIII site in Tn5 and a HindIII site adjacent to the tet genes. Tetracycline-sensitive point mutations were mapped in recombination tests with the deletions and were thus aligned with the genetic and physical map of the tet region. Plasmids carrying point mutations were tested for complementation with derivatives of pDU938, a plasmid carrying cloned tet genes derived from Tn10 which had been inactivated by Tn5 insertions. Complementation occurred between promoter-proximal tet point mutations and distal tet::Tn5 insertions, suggesting the existence of two structural genes, tetA and tetB. These results, together with the analysis of polypeptides in minicells harboring pDU938tet::Tn5 mutants, suggested that tetA and tetB are expressed coordinately in an operon. The tetB gene encodes the previously characterized 36,000-dalton cytoplasmic membrane TET protein, but the product of tetA was not identified. Point mutations in either tetA or tetB led to the defective expression of the resistance mechanism involving tetracycline efflux. It is suggested that the tetA and tetB products interact cooperatively in the membrane to express resistance.

Biological Transport↗
PubMed1982

Tn7 and Tn501 Insertions into Pseudomonas aeruginosa plasmid R91-5: mapping of two transfer regions.

We constructed a restriction endonuclease map of the Pseudomonas aeruginosa narrow-host-range plasmid R91-5. Insertions of transposons Tn7 and Tn501 into the plasmid DNA were characterized physically and genetically. The distribution of sites of insertion showed some regional specificity for the insertion of these transposons, especially TN501. The insertion of Tn7 was unusual in that all 42 of 43 insertions were in the same orientation. By relating phenotypic changes to the site of insertion, the Tn1 transposon that was already present on R91-5 and coded for carbenicillin resistance was mapped, and its orientation was determined. Two major transfer regions were identified. We believe that Tra1 is involved in conjugal DNA metabolism, whereas Tra2 is involved mainly in production of the sex pili.

Adsorption↗
PubMed2001

The rice retrotransposon Tos17 prefers low-copy-number sequences as integration targets.

The rice retrotransposon Tos17 is highly activated by tissue culture. To evaluate the impact of transposition of Tos17 on the rice genome and examine its utility for insertional mutagenesis, more than 100 sequences flanking newly transposed Tos17 copies were characterised. The 5-bp target-site duplications flanking Tos17 did not show any consensus sequence, and preferred nucleotides, A/T and G/C, were only found at the second and third nucleotides from both ends of the target site duplications, respectively, indicating that Tos17 has relatively low target-site specificity at the nucleotide sequence level. Integration targets were widely distributed over the chromosomes; however, preferential integration into the sucrose synthase 2 gene and into Tos17 itself was demonstrated by PCR screening using pooled DNA prepared from the mutant population. Hybridisation studies indicated that Tos17 preferentially integrates into low-copy-number regions of the genome. In agreement with this result, about 30% of flanking sequences examined showed significant homology to known genes. Taken together, these results show that Tos17 can have a significant impact on the rice genome and can be used as a tool for efficient insertional mutagenesis.

Binding Sites↗
PubMed1996

Gene transfer and transposition mutagenesis in Streptomyces roseosporus: mapping of insertions that influence daptomycin or pigment production.

Streptomyces reseosporus, the producer of the cyclic lipopeptide antibiotic daptomycin, was shown to be a suitable host for molecular genetic manipulation. S. roseosporus does not appear to express significant restriction barriers based upon bacteriophage plaque formation studies. Plasmid DNA can be introduced into S. roseosporus by bacteriophage-FP43-mediated transduction and by conjugation from Escherichia coli. The streptomycete transposons Tn5096 and Tn5099, derived from IS493, transpose in S. roseosporus, and Tn5099-induced transposition mutants altered in the production of daptomycin, red pigment or black pigment were identified, and mapped to Dral and Asnl fragments. Three auxotrophic mutations (argB1, ade-1 and metB1) were identified among 100 individual Tn5096 insertions. Alignment and physical mapping of several Tn5099 insertions in Dral-E and Asnl-B fragments was facilitated by the presence of Dral and Asnl cleavage sites in Tn5099.

Bacteriophages↗
PubMed1981

Use of a cloned double stranded cDNA coding for a major androgen dependent protein in rat seminal vesicle secretion: the effect of testosterone in gene expression.

The abundant class of poly(A+)RNA [poly(A+)RNA11S] from rat seminal vesicle was used to synthesize ds-cDNA11S. The ds-cDNA11S was inserted and cloned into the Pst I site of pBR-322 using E. coli RR1 as host. Colony filter hybridization and restriction mapping was used to demonstrate that a 620 NTP long insert in a plasmid clone (pSV2) represents the almost full length structural gene coding for a precursor to the seminal vesicle secretion protein IV (SVS IV). The entire insert was sequenced and the coding region was matched with the known amino acid sequence. Most of the signal peptide sequence was derived from the DNA sequence. The insert in pSV2 was labelled and used to study the effect of testosterone on the accumulation of mRNA SVS IV. Administration of testosterone to castrated rats resulted in the induction of mRNA SVS IV from a few molecules per cell to levels of over 100,000 after 96 h of hormone treatment.

Animals↗
PubMed1989

Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii.

Determination of a 28,793-base-pair DNA sequence of a region from the Azotobacter vinelandii genome that includes and flanks the nitrogenase structural gene region was completed. This information was used to revise the previously proposed organization of the major nif cluster. The major nif cluster from A. vinelandii encodes 15 nif-specific genes whose products bear significant structural identity to the corresponding nif-specific gene products from Klebsiella pneumoniae. These genes include nifH, nifD, nifK, nifT, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifW, nifZ, nifM, and nifF. Although there are significant spatial differences, the identified A. vinelandii nif-specific genes have the same sequential arrangement as the corresponding nif-specific genes from K. pneumoniae. Twelve other potential genes whose expression could be subject to nif-specific regulation were also found interspersed among the identified nif-specific genes. These potential genes do not encode products that are structurally related to the identified nif-specific gene products. Eleven potential nif-specific promoters were identified within the major nif cluster, and nine of these are preceded by an appropriate upstream activator sequence. A + T-rich regions were identified between 8 of the 11 proposed nif promoter sequences and their upstream activator sequences. Site-directed deletion-and-insertion mutagenesis was used to establish a genetic map of the major nif cluster.

Amino Acid Sequence↗
PubMed1984

Overlapping promoters transcribed by bacillus subtilis sigma 55 and sigma 37 RNA polymerase holoenzymes during growth and stationary phases.

A 471-base pair HindIII DNA fragment of Bacillus subtilis contains two overlapping promoters which are recognized in vitro by sigma 55- and sigma 37-containing RNA polymerase holoenzymes from B. subtilis. In vitro transcript analyses and S1 nuclease mapping experiments with in vivo RNA indicate that both enzymes initiate transcription from the same putative +1 site. Physiological studies with the promoter-containing DNA fragment inserted into the expression probe plasmid pCED6 and quantitative S1 nuclease mapping experiments with RNA isolated from various stages of growth indicate that expression from these overlapping promoters is greater during the early stationary phase than during growth. We propose that the cryptic gene controlled by these promoters is transcribed by the sigma 55 enzyme during growth and by the sigma 37 enzyme during early stationary phase.

Bacillus subtilis↗
PubMed1977

Atrial pacing at multiple sites in the Wolff-Parkinson-White syndrome.

Atrial pacing at multiple sites was used in an attempt to predict the site of pre-excitation in 5 patients with Wolff-Parkinson-White syndrome with 5 different anomalous pathway locations (right anterior, right posterior, septal, left posterior, and left lateral). At least 3 atrial pacing sites were tested in each patient. Pacing sites tested included high right atrium, low lateral right atrium, low septal right atrium, proximal coronary sinus, and distal coronary sinus. Atrial stimulation sites with shortest and longest stimulus-delta intervals could be identified in each patient, the shortest stimulus-delta interval in each case ranging from 60 to 80 ms. The difference between the shortest and longest stimulus-delta interval in each case ranged from 60 to 110 ms. It was suggested that the site with the shortest stimulus-delta interval corresponded to a site close to the atrial insertion of the anomalous pathway. This hypothesis was confirmed in all cases (3 with epicardial mapping and 2 with retrograde atrial activation data). In conclusion, atrial pacing at multiple sites is helpful in predicting the site of anterogradely conducting anomalous pathways, and appears particularly useful for differentiation of right posterior, left posterior, and septal pre-excitation.

Adolescent↗
PubMed2002

Long-range mapping of the Streptococcus agalactiae phylogenetic lineage restriction digest pattern type III-3 reveals clustering of virulence genes.

Human isolates of serotype III Streptococcus agalactiae (group B streptococcus [GBS]) can be divided into three separate phylogenetic lineages based on analysis of the restriction digest patterns (RDPs) of chromosomal DNA. Nine DNA sequences that are present in all isolates of the RDP III-3 phylogenetic lineage, but not in the other lineages, were identified by genomic subtractive hybridization. A complete physical map of a III-3 chromosome was constructed. Six of the nine III-3-specific sequences mapped to a 340-kb Sse8387I fragment which contains or is located close to known GBS virulence genes. One of the III-3-specific probes, AW-10, encodes part of GBSi1, a group II intron that is inserted at two sites within the GBS genome. The second chromosomal site for GBSi1 was isolated, sequenced, and mapped to a location near the locus responsible for hemolysin production. These findings suggest that the genetic variation that distinguishes the RDP type III-3 strains from other serotype III strains occurs largely within localized areas of the genome containing known or putative virulence genes.

Base Sequence↗
PubMed1993

Retroviral insertions into a herpesvirus are clustered at the junctions of the short repeat and short unique sequences.

We previously described the integration of a nonacute retrovirus, reticuloendotheliosis virus (REV), into the genome of a herpesvirus, Marek disease virus (MDV), following both long-term and short-term coinfection in cultured fibroblasts. The long-term coinfection occurred in the course of attenuating the oncogenicity of the JM strain of MDV and was sustained for > 100 passages. The short-term coinfection, which spanned only 16 passages, was designed to recreate the insertion phenomenon under controlled conditions. We found that REV integrations into MDV were common and could occur within the first passage following coinfection. Now we have mapped the integration sites. After 5-16 passages in vitro, 17 out of 19 REV junction sites are clustered in two 1-kilobase regions at the junctions of the short unique and short repeat region of MDV. In the long-term cocultivation experiment, 6 out of 10 insertions also mapped in this region. In both cases, integrated proviruses are unstable and undergo subsequent recombinative deletion, often leaving a solitary long terminal repeat. The long terminal repeat sequences are, however, stably maintained for many rounds of passaging in vitro. This clustering of insertions presumably is influenced by selection for viable and fast-growing viruses, and occurs in a region of the MDV genome which shows significant size heterogeneity in several strains.

Animals↗
PubMed1999

Chromosome map of Xanthomonas campestris pv. campestris 17 with locations of genes involved in xanthan gum synthesis and yellow pigmentation.

No plasmid was detected in Xanthomonas campestris pv. campestris 17, a strain of the causative agent of black rot in cruciferous plants isolated in Taiwan. Its chromosome was cut by PacI, PmeI, and SwaI into five, two, and six fragments, respectively, and a size of 4.8 Mb was estimated by summing the fragment lengths in these digests. Based on the data obtained from partial digestion and Southern hybridization using probes common to pairs of the overlapping fragments or prepared from linking fragments, a circular physical map bearing the PacI, PmeI, and SwaI sites was constructed for the X. campestris pv. campestris 17 chromosome. Locations of eight eps loci involved in exopolysaccharide (xanthan gum) synthesis, two rrn operons each possessing an unique I-CeuI site, one pig cluster required for yellow pigmentation, and nine auxotrophic markers were determined, using mutants isolated by mutagenesis with Tn5(pfm)CmKm. This transposon contains a polylinker with sites for several rare-cutting restriction endonucleases located between the chloramphenicol resistance and kanamycin resistance (Kmr) genes, which upon insertion introduced additional sites into the chromosome. The recA and tdh genes, with known sequences, were mapped by tagging with the polylinker-Kmr segment from Tn5(pfm)CmKm. This is the first map for X. campestris and would be useful for genetic studies of this and related Xanthomonas species.

Base Sequence↗
PubMed1987

Molecular cloning and physical mapping of the genome of insect iridescent virus type 6: further evidence for circular permutation of the viral genome.

A defined and complete gene library of the Chilo iridescent virus (CIV) genome was established. The CIV DNA was cleaved with restriction endonucleases EcoRI, NcoI, SphI, and BamHI or double digested with BamHI/SalI and the resulting DNA fragments were inserted into the corresponding sites of the bacterial vectors pACYC184, pKm2, pL-ES-C3, and pAT153 using T4 DNA ligase. All cloned fragments were identified by digestion of the recombinant plasmids with different restriction enzymes and checked by hybridization of recombinant plasmid to viral DNA. This analysis revealed that sequences representing 100% of the viral genome were cloned into the EcoRI site of pACYC184. Although the CIV genome is linear, all 32 EcoRI fragments have been cloned directly. This suggests that the CIV genome is circularly permuted. In addition, NcoI(72%), SphI(40.7%), BamHI (11.6%), and BamHI/SalI(39.7%) DNA fragments of the viral genome were inserted into the corresponding sites of pKm2, pL-ES-C3, and pAT153, respectively. The physical map of the viral genome was constructed using the established gene library for restriction enzymes ApaI, BamHI, EcoRI, NcoI, SalI, and SmaI. Although the CIV genome is linear, this analysis revealed that the restriction maps of the viral genome are circular. This finding supports the hypothesis that the CIV genome is circularly permuted.

Cloning, Molecular↗
PubMed1980

Genetic mapping of the ecotropic virus-inducing locus Akv-2 of the AKR mouse.

A combination of somatic cell hybridization and standard mendelian breeding techniques was used to map the AKR ecotropic virus inducibility locus Akv-2 to the centromeric end of chromosome 16. This assignment of Akv-2 further emphasizes the endogenous ecotropic retroviruses are inserted at multiple sites in mouse chromosomes.

Animals↗
PubMed1999

Identification of a promoter sequence from the BETL1 gene cluster able to confer transfer-cell-specific expression in transgenic maize

The maize (Zea mays L.) betl1 locus, encoding a basal endosperm transfer layer-specific protein, has been mapped and molecularly cloned in its entirety. The locus is shown to consist of three gene copies in the maize inbred line A69Y. To distinguish the three transcription units from the locus name, we have termed them BETL1a, BETL1b, and BETL1c. Two of the copies are expressed, whereas one is inactive and contains retrotransposon-like insertions in both promoter and intron regions. Based on this information, and a restriction site map covering 17 kb around the BETL1 locus, a DNA fragment putatively containing an active promoter sequence was identified. This fragment was tested for its ability to confer transfer-cell-specific expression in transient and stably transformed maize tissues. The transgenic maize plants obtained showed the predicted cell-type specificity of expression restricted to the basal endosperm transfer cells, although there were minor deviations in promoter strength and timing and accumulation of the transgene product from the corresponding BETL-1 endogene expression pattern.

Journal Article↗
PubMed1992

Lambdoid phages as elements of bacterial genomes (integrase/phage21/Escherichia coli K-12/icd gene).

The lambdoid phages are a group of related temperate bacteriophages that lysogenize by site-specific recombination with the bacterial chromosome. Various members of the group have different specific chromosomal insertion sites, despite the fact that the enzymes catalyzing the insertion (integrases) appear to be all descended from a common ancestor. Insertion sites are not located randomly on the E. coli chromosome but are restricted to one segment of the map; also, most prophages are oriented in the same direction along the chromosome. Lambdoid phage 21 inserts within the isocitrate dehydrogenase gene and introduces an alternative 165 bp 3' end for that gene. A defective element (e14) inserts at the same position. We suggest that this mode of insertion arose from insertion of an ancestral phage to the right of icd which then picked up part of the icd gene by abnormal excision and speculate that, at an earlier time, phages may have arrived at their present locations by a process of chromosomal walking.

Bacterial Proteins↗
PubMed1997

Epitope mapping and tagging by recombination PCR mutagenesis.

We describe a rapid PCR method that directly inserts an epitope tag into an open reading frame (ORF) to facilitate protein detection. This project was performed within a varicella-zoster virus (VZV) system. In earlier work, we produced a monoclonal antibody (MAb 3B3) to one VZV ORF called gE. MAb 3B3 bound to its epitope under extreme denaturing conditions. To further characterize the epitope, we devised a technique that identified the epitope by its insertion into another protein of interest. The 3B3 epitope was mapped to 11 residues (residues 151-161; QRQYGDVFKGD) in the gE ectodomain by using the technique of recombination PCR. At the same time, the 3B3 epitope was inserted in-frame into another VZV protein for which no MAb was available. The end result, VZV gL3B3.11, was a unique construct possessing a 33-bp insertion that expresses gL-3B3 protein recognized by the MAb 3B3. The 3B3 epitope was verified to be both highly functional and stable. An important advantage of this recombination PCR method of epitope mapping and tagging is that the epitope sequence can be inserted anywhere along the nucleotide sequence of an ORF, regardless of existing restriction sites.

Amino Acid Sequence↗