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Results for “Restriction Mapping”

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PubMed1988

Restriction mapping and hybridization studies of a beta-lactamase-encoding fragment from Streptococcus (Enterococcus) faecalis.

A restriction map of a 5.1-kilobase EcoRI fragment encoding an enterococcal beta-lactamase was prepared and compared with the restriction map of a cloned staphylococcal beta-lactamase gene and with the published maps of several other staphylococcal beta-lactamase genes. Comparison and hybridization studies showed that there were identical restriction sites in the region of the bla structural gene but not in the region surrounding this gene.

DNA Restriction Enzymes↗

PubMed1992

An O (N2 log N) restriction map comparison and search algorithm.

We present an O (R log P) time, O (M+P2) space algorithm for searching a restriction map with M sites for the best matches to a shorter map with P sites, where R, the number of matching site pairs, is bounded by MP. As first proposed by Waterman et al. (1984, Nucl. Acids Res. 12, 237-242) the objective function used to score matches is additive in the number of unaligned sites and the discrepancies in the distances between adjacent aligned sites. Our algorithm is basically a sparse dynamic programming computation in which "candidate lists" are used to model the future contribution of all previously computed entries to those yet to be computed. A simple modification to the algorithm computes the distance between two restriction maps with M and N sites, respectively, in O (MN (log M+log N)) time.

Algorithms↗

PubMed1979

Restriction mapping of DNA of temperate Rhizobium meliloti phage 16-3: comparison of genetic and physical maps indicates a long, genetically silent chromosomal arm.

The complete restriction map of DNA (61.57 Kb) of temperate Rhizobium meliloti phage 16-3 has been constructed for enzymes BglII, HindIII, HpaI, KpnI, and a partial map for EcoRI. The strategy employed for mapping included the analysis of double, triple and partial digests; comparison of wild type and deletion mutants; and detailed analysis of subfragments, exploiting the presence of cohesive ends of the phage. Comparison of the genetic and physical maps indicates that one arm of the chromosome is genetically silent and/or contains nonessential genes.

Bacteriophages↗

PubMed1986

A simple nonisotopic method for restriction mapping in single-stranded DNA cloning vectors based on taking timepoints during primed Klenow synthesis.

A fast, simple, and nonisotopic method for restriction mapping inserts in single-stranded cloning vectors (such as M13 or single-stranded plasmids) is presented. The procedure uses a commercially available oligonucleotide sequencing primer to initiate Klenow-mediated, unidirectional DNA synthesis along the single-stranded insert DNA. Aliquots taken at very short timepoints from this reaction are quick-frozen, heat-inactivated, and restriction-digested with the restriction enzyme or enzymes of interest. When the samples are run on an agarose gel and stained with ethidium bromide, the restriction bands appear in the order of their proximity to the priming site. The method's advantages are that it is fast, unidirectional and thus relatively unambiguous, requires neither isotope nor elaborate DNA handling or extraction procedures, and resolves the ambiguities due to "near doublets" that often plaque double-digest mapping and partial-digest mapping. Tetranucleotide restriction maps extending up to 5 kb can be determined from a single priming experiment; more infrequent hexanucleotide restriction sites can be mapped over longer distances. Also, a single aliquot taken at an early timepoint can be restriction-digested to establish the orientation of cloned inserts.

Cloning, Molecular↗

PubMed1990

Construction of NotI restriction map of the Streptococcus mutans genome.

Streptococcus mutans and Streptococcus sobrinus are the major causative organisms of human dental caries. Pulsed-field gel electrophoresis (PFG) showed that the restriction enzyme NotI produced ten and six DNA fragments from the genomes of S. mutans strain MT8148 and S. sobrinus strain 6715, respectively. The sizes of the chromosomes of S. mutans and S. sobrinus were each estimated to be about 2200 kb. The NotI restriction map of S. mutans MT8148 genome was constructed by Southern blot analysis with probes that overlapped two adjacent NotI fragments. Several virulence-associated genes of S. mutans were placed on the NotI restriction map. In addition, unique 'fingerprints' of S. mutans chromosomal DNA digested with NotI were produced by PFG, and these may be useful for epidemiological studies.

Base Sequence↗

PubMed1992

Patterns of naturally occurring restriction map variation, dopa decarboxylase activity variation and linkage disequilibrium in the Ddc gene region of Drosophila melanogaster.

Forty-six second-chromosome lines of Drosophila melanogaster isolated from five natural populations were surveyed for restriction map variation in a 65-kb region surrounding the gene (Ddc) encoding dopa decarboxylase (DDC). Sixty-nine restriction sites were scored, 13 of which were polymorphic. Average heterozygosity per nucleotide was estimated to be 0.005. Eight large (0.7-5.0 kb) inserts, two small inserts (100 and 200 bp) and three small deletions (100-300 bp) were also observed across the 65-kb region. We see no evidence for a reduction in either nucleotide heterozygosity or insertion/deletion variation in the central 26-kb segment containing Ddc and a dense cluster of lethal complementation groups and transcripts (greater than or equal to 9 genes) compared to that seen in the adjacent regions (totaling 39 kb) in which only a single gene and transcript has been detected, or to that observed for other gene regions in D. melanogaster. The distribution of restriction site variation shows no significant departure from that expected under an equilibrium neutral model. However insertions and deletions show a significant departure from neutrality in that they are too rare in frequency, consistent with them being deleterious on average. Significant linkage disequilibrium among variants exists across much of the 65-kb region. Lower regional rates of recombination combined with the influence of polymorphic chromosomal inversions, rather than epistatic selection among genes in the dense cluster, probably are sufficient explanations for the creation and/or maintenance of the linkage disequilibrium observed in the Ddc region. We have also assayed adult DDC enzyme activity in these same lines. Twofold variation in activity among lines is observed within our sample. Significant associations are observed between level of DDC enzyme activity and restriction map variants. Surprisingly, one line with a 5.0-kb insert within an intron and one line with a 1.5-kb insert near the 5' end of Ddc each show normal adult DDC activities.

Animals↗

PubMed1985

Demonstration of the colinearity of human cytomegalovirus genomes and construction of restriction maps of unknown isolates using cloned subgenomic fragments.

In this study, we have established the colinearity of human cytomegalovirus (HCMV) genomes using stringent conditions of DNA-DNA filter hybridization of HCMV HindIII fragments and cosmid-cloned AD169 strain HCMV DNA fragments. Large cosmid-cloned fragments of AD169 DNA were used for the preparation of radioactive probes by nick translation. These probes were hybridized to HindIII digests of DNA from three fresh isolates of HCMV and to that of the Davis strain. Using published HindIII restriction maps for the AD169 strain as a reference, the results obtained by hybridization allowed us to construct HindIII restriction maps for the genomes of the three fresh isolates. Confirmation of our methodology was found in the correspondence between the HindIII map we constructed for the Davis strain and that published previously. Furthermore, it is shown that variation in the restriction profiles of the unique regions of the genome are due to the absence or gain of restriction sites, and not to a rearrangement of fragments. This technique allows rapid construction of physical maps of the DNA of any fresh isolate for a given restriction enzyme provided the corresponding restriction map of a strain to be used as reference is available.

Base Sequence↗

PubMed1979

Restriction map of the region surrounding the EcoRI site in the pCR1 plasmid and analysis of an inserted ovalbumin gene.

We have determined a restriction map of a 1650 base pair region surrounding the EcoRI site of the bacterial plasmid, pCR1. We have used pCR1 as a vector in cloning synthetic ovalbumin double-stranded cDNA. Using the pCR1 restriction map, we have characterized the ovalbumin sequences inserted in one recombinant plasmid, pOvE12. POvE12 appears to contain all, or nearly all, of the sequences found in full length, double-stranded cDNA synthesized in vitro.

Base Sequence↗

PubMed1986

[Restriction map of pesticinogenicity plasmid pYP1 of Yersinia pestis].

The restriction map of Yersinia pestis pesticinogenicity plasmid pYP1 has been constructed with the use of 18 restriction endonucleases. Plasmid dimensions (6.3 Md) have been specified, the genes for pesticin synthesis, for pesticin immunity protein, fibrinolysin and plasmocoagulase have been localized by molecular cloning of single plasmid DNA fragments in vector plasmid pBR322.

Bacteriocins↗

PubMed1991

Detection of the Hb Quong Sze mutation in a Chinese family by selective amplification of the alpha 2-globin gene and restriction map analysis with Msp I.

The polymerase chain reaction technique combined with restriction map analysis with Msp I and hybridization with synthetic oligonucleotide probes has been used to identify Hb Quong Sze [alpha 125(H8)Leu----Pro] in a Chinese family in Guangxi (Quong Sze), P. R. China. Our data and those described in an earlier publication (1) indicate that the Hb Quong Sze carriers originate from the same province of the People's Republic of China, namely Guangxi.

Adult↗

PubMed1989

Completion of the detailed restriction map of the E. coli genome by the isolation of overlapping cosmid clones.

Ordered sets of cosmids derived from E. coli K-12 803 overlap the 6 remaining gaps left in the physical map of strain W3110. We present detailed restriction maps of the gaps and surrounding regions, thus providing a comparison of about 30% of the genome of the two E. coli strains. Our analysis shows that there is a high degree of homology between the strains, with only occasional restriction fragment differences. However, the large inversion occurring between rrnD (72.1') and rrnE (90.4') in strain W3110 is absent in strain 803. Instead, a new inversion and adjacent deletion near argF is present in strain 803. The distribution of cosmid clones at, and adjacent to, the gaps shows that all gaps except one were difficult to clone in both lambda and cosmid clones. A low copy number cosmid vector, pOU61cos, developed previously, was essential for cloning 3 of the 8 gaps.

Chromosome Mapping↗

PubMed1988

Long-range restriction maps of Plasmodium falciparum chromosomes: crossingover and size variation among geographically distant isolates.

Homologous chromosomes from the human malaria parasite Plasmodium falciparum exhibit striking size polymorphism from isolate to isolate. To examine the structural basis for these variations, we have determined full-length restriction maps of chromosome 4 from three P. falciparum clones. Two clones, HB3 and 3D7, are derived from geographically distant strains, while the third, XP5, is the product of an HB3/3D7 cross. The restriction maps show that, while the overall structure and organization of chromosome 4 from each clone are similar, large-scale variations occur within a few hundred kilobase pairs of the chromosome ends. An apparent crossover between the 3D7 and the HB3 parent chromosomes accounts for a chromosome of intermediate size in clone XP5. Similar restriction studies extended to other parasite chromosomes will ultimately yield a long-range physical map of the P. falciparum genome.

Animals↗

PubMed1988

Enhanced recovery and restriction mapping of DNA fragments cloned in a new lambda vector.

In this paper we describe a modification to the lambda vector EMBL3 which greatly expedites the construction of restriction maps of cloned DNA sequences. In the modified vector, EMBL3cos, all the phage coding sequences are placed to the right of the cloning sites so that the left cohesive end is separated by only 200bp, rather than 20kb (as in conventional lambda vectors), from the inserted DNA fragment. We show that reliable restriction maps can be rapidly constructed from partial digests of clones made in this vector by labelling the left cohesive end with a complementary 32P-labelled oligonucleotide. In addition, we quantify the restriction of clones containing human DNA by the McrA and McrB systems of E. coli and show that the use of Mcr- plating strains can increase the yield of recombinant phage up to tenfold, to give cloning efficiencies of greater than or equal to 10(7) pfu/microgram of human DNA.

Bacteriophage lambda↗

PubMed1989

A 12 megabase restriction map at the cystic fibrosis locus.

We have constructed a physical map of the chromosomal region containing the cystic fibrosis locus using seven DNA markers and pulsed-field gel electrophoresis methods. The map includes cleavage sites for 8 rare-cutting restriction enzymes and spans over 12 megabases (Mb) of DNA, with one unlinked probe covering an additional 5 Mb. To our knowledge, this is the largest segment of human DNA which has been restriction-mapped to date. We can identify thirteen putative HTF islands spaced at intervals of 0.3-3.2 Mb. The region between loci D7S8 and MET, where the CF gene lies, includes 1.4-1.9 Mb of DNA.

Blotting, Southern↗

PubMed1992

Long-distance restriction mapping of the proximal long arm of human chromosome 21 with Not I linking clones.

Human chromosome 21 is the smallest of the 22 autosomes and 2 sex chromosomes. Hybridization of the human repetitive sequence Alu to pulsed-field gel-fractionated Not I-digested genomic DNA from a human-mouse hybrid cell line containing chromosome 21 as the sole human component identified chromosome 21 Not I restriction fragments. A Not I restriction map of regions of the chromosome was constructed, by identifying neighboring Alu bands with Not I linking clones. This approach simplifies the task of physical mapping and avoids ambiguities in Not I fragment assignments that arise from gel-to-gel mobility variations. A contiguous map was constructed with six Not I linking clones that covers at least the proximal one-third of the long arm of chromosome 21 and spans 20 megabases. A more detailed restriction map revealed 11 likely CpG islands in this region and localized 11 additional DNA markers.

Chromosomes, Human, Pair 21↗

PubMed1979

Isolation and restriction mapping of plasmids containing ribosomal DNA sequences from the rrn B cistron of E. coli.

Recombinant plasmids containing the entire 16S RNA gene from the rrn B cistron of E. coli inserted in Col E1 and pBR322 plasmid vectors have been constructed. These plasmids have been mapped using several restriction endonucleases as well as by DNA-RNA hybridization. These maps reveal previously undetected restriction sites in the rrn B cistron and in Col E1 plasmid DNA.

DNA Restriction Enzymes↗

PubMed1991

Improved algorithms for searching restriction maps.

We present algorithms for searching a DNA restriction enzyme map for a region that best matches a shorter 'probe' map. Our algorithms utilize a new model of map alignments, and extensive experiments prove our model superior to earlier approaches for certain applications. Let M be the number of map sites and P be the number of probe sites. Our first algorithm, which optimizes only over a restricted class of alignments, requires O(MP log P) worst-case time and O(M + P) space. Our second algorithm, which optimizes over all alignments, runs in O(MP3) time and O(M + P2) space, under reasonable assumptions about the distribution of restriction enzyme cleavage sites. Combining the algorithms gives a map-searching method that optimizes over all alignments in O(MP log P) time in practice. The algorithms' effectiveness is illustrated by searches involving a genomic restriction map of Escherichia coli.

Algorithms↗

PubMed1989

An extended long-range restriction map of the human sex-determining region on Yp, including ZFY, finds marked homology on Xp and no detectable Y sequences in an XX male.

We have used pulsed-field gel electrophoresis to study the short arm of the Y chromosome by using a pseudoautosomal probe (MIC2Y) and adjacent Y-specific sequences 27a and 47z (DSXY5) in XX males and XY females, in order to detect chromosomal breakpoints which may have given rise to these individuals. The preliminary published long-range restriction map was used as a basis for this study. Our data confirm the reported fragment sizes and resolve some discrepancies. In addition, the recently cloned ZFY locus, pDP1007, the putative sex-determining locus, has been used to extend this long-range restriction map on Yp. Thus far, the X and Y copy of this sequence appear to have conserved GC islands around this locus, since it is found on a 280-kb fragment in males and females by using SacII, BssHII, NarI, and NotI. Only two Y-specific sequences of 50 and 70 kb have been detected at the pulsed-field level by using SfiI and NaeI, respectively. No translocation breakpoints have been detected in any of the patients studied. One XX male, GM1889, however, does not have any of the Y-specific fragments detected using conventional or pulsed-field gel electrophoresis. This is one of the few typical XX males who therefore does not have the ZFY copy of the TDF clone. Since all the other XX males hybridized to 47z, which is centromeric to ZFY, a series of DNA loci that are centromeric to 47z need to be studied in order to detect chromosomal breakpoints.

DNA Probes↗