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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↗
PubMed1999

Endonuclease-mediated long PCR and its application to restriction mapping.

The polymerase chain reaction (PCR) is the most widely used technique for the study of DNA. Applications for PCR have been extended significantly by the development of "long" PCR, a technique that makes it possible to amplify DNA fragments up to 40 kb in length. This article describes two novel applications of the long PCR technique, one which simplifies restriction mapping and another which enhances amplification specificity and yield. The same primers used to perform the long PCR amplification can be used as probes to perform restriction mapping of the DNA fragment amplified. Restriction digestion performed prior to long PCR amplification can be used to selectively suppress the amplification of members of families of closely related DNA sequences, thereby making it possible to selectively amplify one of a group of highly homologous sequences. These two complimentary techniques, both involving use of the long PCR paired with restriction digestion, have potential application in any laboratory in which PCR is performed.

DNA Restriction Enzymes↗
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↗
PubMed1995

A 405-kb cosmid contig and HindIII restriction map of the progressive myoclonus epilepsy type 1 (EPM1) candidate region in 21q22.3.

As a step toward identifying the molecular defect in patients afflicted with progressive myoclonus epilepsy type 1 (EPM1), we have assembled a cosmid contig of the candidate EPM1 region in 21q22.3. The contig constitutes a collection of 87 different cosmids spanning 405 kb based on a derived HindIII restriction map. Potential CpG-rich islands have been identified based on the restriction map generated from eight different rare-cutting enzymes. This contig contains the genetic material required for the isolation of expressed sequences and the identification of the gene defective in EPM1 and possibly other disorders mapping to this region.

Chromosome Mapping↗
PubMed1993

A long-range restriction map of human chromosome 5q21-q23.

A long-range restriction map encompassing the APC (adenomatous polyposis coli) gene has been constructed. The map includes 35 DNA markers and consists of two segments of 10 and 2.5 Mb. Published genetic markers have been connected using additional, nonpolymorphic DNA probes. The map clarifies marker order and allows comparison of physical and genetic data.

Adenomatous Polyposis Coli↗
PubMed1994

Restriction map polymorphism in the forked and vermilion regions of Drosophila melanogaster.

Restriction map polymorphism at two X linked foci, forked and vermilion of Drosophila melanogaster was studied in three natural populations. The estimates of nucleotide variation were theta = 0.003 and pi = 0.002 for the forked region and theta = 0.004 and pi = 0.002 for the vermilion region. Three insertions (> 500 bp) were observed at each locus. Typical of other regions of this species each of these large insertions was unique in the sample. Non-random association among polymorphisms was common at the vermilion locus, while the forked locus was not polymorphic enough to test linkage disequilibrium. The amounts of restriction site and size variation in the vermilion and forked were within the range observed for other loci of D. melanogaster.

Animals↗
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↗
PubMed1998

A long-range restriction map of deletion interval 6 of the human Y chromosome: a region frequently deleted in azoospermic males.

Deletion interval 6 (DI6) of the human Y chromosome, located at the distal end of the long arm euchromatic region, is required for normal spermatogenesis. About 10% of males with idiopathic azoospermia or oligospermia have microdeletions in this region. Six gene families, including RBMY (RNA binding motif, Y chromosome), DAZ (deleted in azoospermia), and four recently isolated genes, have been mapped to this interval. Genes from all of these families show testis-specific expression and are thus candidates for azoospermic factor (AZF). DI6 is also rich in Y-specific repetitive sequences, which may be responsible for its frequent deletion. To understand the sequence organization of this region, a 5-Mb restriction map was constructed based on YAC clones and was partially verified on genomic DNA. The locations of five gene family members, as well as numerous STSs, were determined. The map shows several inverted and direct repeats several hundred kilobases in size. The restriction map of DI6 will facilitate future mapping of deletion breakpoints in infertile males and elucidation of mechanisms behind frequent deletions.

Blotting, Southern↗
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↗
PubMed1997

Dissection of the 5.5 Mbp region directly telomeric of HLA-B including a long range restriction map, YAC and PAC contigs.

A large number of diseases are associated with the human major histocompatibility (HLA) complex located in 6p21.3. The underlying defect of most of these has not yet been determined even after detailed analysis of the HLA region. Due to the extended haplotypes found in this area, several of the HLA-linked disease genes may be located also telomeric of the class I region. In order to analyse the area covering the 4 megabases directly telomeric of HLA-F in close detail, we have generated 50 new markers. These and other markers have been used to establish a SalI restriction map from 46 YACs. A subset of 42 markers was applied to construct a genomic long range restriction map from an HLA-A2/B13 haplotype. Both maps have been compared revealing the presence of additional 150 kb in the HLA-A2 haplotype close to the RFP locus. Additionally, 47 PACs have been selected mapping to this region and grouped into 7 contigs. Sequencing of these PAC contigs has already been initiated.

Chromosomes, Artificial, Yeast↗
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↗
PubMed1980

A restriction map of the bacteriophage T4 genome.

We report a detailed restriction map of the bacteriophage T4 genome and the alignment of this map with the genetic map. The sites cut by the enzymes Bg/II, XhoI, KpnI, SalI, PstI, EcoRI and HindIII have been localized. Several novel approaches including two-dimensional (double restriction) electrophoretic separations were used.

Chromosome Mapping↗
PubMed1993

A cosmid contig and high resolution restriction map of the 2 megabase region containing the Huntington's disease gene.

The quest for the mutation responsible for Huntington's disease (HD) has required an exceptionally detailed analysis of a large part of 4p16.3 by molecular genetic techniques, making this stretch of 2.2 megabases one of the best characterized regions of the human genome. Here we describe the construction of a cosmid and P1 clone contig spanning the region containing the HD gene, and the establishment of a detailed, high resolution restriction map. This ordered clone library has allowed the identification of several genes from the region, and has played a vital role in the recent identification of the Huntington's disease gene. The restriction map provides the framework for the detailed analysis of a region extremely rich in coding sequences. This study also exemplifies many of the strategies to be used in the analysis of larger regions of the human genome.

Base Sequence↗
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↗