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PubMed1988

Molecular genotyping of the HLA-DQ alpha gene region.

Restriction fragment analysis has been applied to genomic DNA extracted from human tumor cell lines. Polymorphic restriction fragments encompassing the HLA-DQ alpha gene were observed upon digestion with Bgl II, Eco RI, and Hind III. Analysis of these polymorphic fragments (or allogenotopes) showed that for each restriction enzyme a series of three differently sized allogenotopes existed. Clusters of cosegregating allogenotopes belonging to the different allelic series defined three different allogenotypes. Each allogenotype exhibited a distinctive restriction map generated by digestion with five restriction enzymes. Comparison of these restriction maps showed that generation of the polymorphisms observed at the HLA-DQ alpha region in these sets of cell lines is not caused by a single event. In some B- and T-lymphoma cell lines a fourth allogenotype was found. The restriction site map of genomic DNA from these cell lines suggested that the latter distribution of restriction enzyme sites was most probably generated by recombination between two of the previously observed allogenotypes at a crossover site(s) adjacent to the HLA-DQ alpha gene.

Alleles↗
PubMed1987

Progress towards construction of a total restriction fragment map of a human chromosome.

We present an approach to the construction of an overlapping restriction fragment map of a single human chromosome. A genomic cosmid library genome was constructed from a mouse-human hybrid cell line containing chromosome 17 as its only human genetic component. Cosmids containing human inserts were isolated by hybridisation to a human Alu sequence. DNAs from ninety-six randomly chosen cosmids were digested with either EcoRI or HindIII, end-labelled with 35S-dATP and analysed using agarose gel electrophoresis. Comparison of the restriction fragment patterns revealed two pairs of overlapping clones, that were confirmed by cross-hybridization of the overlapping fragments. The two pairs of cosmids both mapped to human chromosome 17, as shown by hybridization to a panel of somatic cell hybrids. These data demonstrate that the generation of an overlapping cosmid map along a human chromosome is feasible, representing an intermediate step towards the complete sequencing of a human chromosome.

Animals↗
PubMed1989

Retroviral insertions in the CB-1/Fim-3 common site of integration activate expression of the Evi-1 gene.

A common retroviral integration site (CB-1) was identified in two IL-3-dependent myeloid leukemia cell lines (DA-3, DA-34). The CB-1 locus was mapped to murine chromosome 3 and was shown to be closely linked to another myeloid common site of viral integrations in myeloid leukemias, Evi-1. A comparison of the CB-1 restriction map with published restriction maps for other common integration sites demonstrated that it was nearly identical to the restriction map of a common site of Friend MuLV integration in myeloid tumors termed Fim-3 (Bordereaux et al. 1987). Genomic clones representing approximately 110 kb of the CB-1 locus and 80 kb of the 5' region of the Evi-1 locus demonstrate no physical overlap of these viral integration sites. Previous studies (Morishita et al. 1988) have shown that retroviral insertions in the Evi-1 locus activate the expression of a gene potentially encoding a 120 kd zinc finger protein. Evi-1 expression is also activated in cell lines with viral integrations in the CB-1 locus. These results demonstrate that the CB-1/Fim-3 and Evi-1 loci constitute a large genomic region in which viral integrations activate the transcription of a new potential myeloid transforming gene.

Animals↗
PubMed1998

Familial hyperproinsulinaemia due to a mutation substituting histidine for arginine at position 65 in proinsulin: identification of the mutation by restriction enzyme mapping.

UNLABELLED: Familial hyperproinsulinaemia is a rare genetic disorder characterized by point mutations in the insulin gene which impair the conversion of proinsulin to insulin. We report here three members of a two-generation Caucasian family in whom this syndrome was identified by unexplained hyperinsulinism associated with normal glucose tolerance and normal insulin sensitivity. Plasma insulin immunoreactivity showed a reduced affinity for the insulin receptor and eluted mainly, on Biogel chromatography, at the position of proinsulin. Analysis of the PCR-amplified insulin gene by restriction enzyme mapping revealed a new recognition site for the enzyme Nla III, indicating a Arg65 to His mutation. Sequence analysis of exon 3 confirmed this mutation in one allele of the gene. CONCLUSION: This study reports a two-generation European-Caucasian family with hyperproinsulinaemia due to a substitution of His for Arg at position 65 in proinsulin, the seventh now identified worldwide and the second from Europe. The mutation generated a new restriction site on the insulin gene suggesting the usefulness of restriction enzyme mapping as a screening procedure.

Adult↗
PubMed1993

High resolution visual mapping of stretched DNA by fluorescent hybridization.

We describe a method for stretching DNA, which, when combined with fluorescent hybridization procedures, forms a new mapping technology that produces a high resolution, vivid, multi-colour image and map. Restriction fragments and cosmid probes were successfully mapped by this procedure with validation by standard restriction mapping. A long range map of a > 200 kilobase region containing five copies of the amplified dihydrofolate reductase gene was easily generated within two days. This DNA mapping procedure offers a significant and rapid alternative to a variety of standard mapping procedures.

Animals↗
PubMed1983

Detailed restriction enzyme map of crown gall-suppressive IncW plasmid pSa, showing ends of deletion causing chloramphenicol sensitivity.

Detailed restriction enzyme maps of the antibiotic resistance plasmid pSa and of a chloramphenicol-sensitive spontaneous deletion mutant of pSa were constructed. The smaller plasmid contained one set of three restriction enzyme sites that appeared at both ends of the deleted region in the intact plasmid. The frequency of loss of chloramphenicol resistance was 1% after eight logs of growth and was dependent on the recA gene function of Escherichia coli. A previously published map lacks an 11-kilobase-pair SstII fragment present on this map.

Chloramphenicol↗
PubMedDate not supplied

Restriction site mapping of adenovirus type 8 genome types.

The DNA of 60 adenovirus type 8 (AV8) isolates (collected during the period 1961 to 1982, mostly in Western Germany) was analysed by 6 endonucleases and revealed 6 different genome types, thus implying that the variability of AV8 is relatively low. It was found that 45 isolates belonged to the genome type D1. Restriction site maps of a prototype D1 and of all deviating restriction variants were elaborated for enzymes BamHI, BglII, Hind-III and SalI.

Adenoviridae↗
PubMed1989

Molecular epidemiology and restriction site mapping of adenovirus 7 genome types.

From the United States, the Federal Republic of Germany, and other regions, 168 strains of AV7, isolated between 1961 and 1985, were analyzed by six restriction endonucleases and nine genome types were identified. The enzymes BamHI and HindIII were most discriminative. The genome type D5 (or 7b) predominated with 120 isolates since 1970 in both countries. Strains of D2 (7a) and D4 (7c) were isolated for a limited time, D3 for an extended time period. Several clusters of infections with the same genome type were found. Differences in pathogenicity could not be derived from our data. On the basis of restriction site mapping, most other genome types were similar to D5, one to D2 and one to the prototype (D1). The genomic relation between AV7 and AV3 is discussed and shown by a dendrogram.

Adenoviridae↗
PubMed1979

Restriction cleavage maps of coliphages 186 and P2.

The restriction enzymes BamHI, Bg/II, EcoRI, HindIII, PstI, XbaI and XhoI have been used to cleave DNA isolated from the related coliphages P2 and 186 for analysis on 1% agarose gels. Three approaches were used to map the sites of cleavage: a) analysis dependent upon the existence of cohesive termini and availability of viable P2-186 hybrids; b) analysis of double digests and redigests of isolated fragments with a second enzyme and c) analysis of partial digests by transfer to nitrocellulose and hybridization with a single fragment. This last approach and the results obtained from it are detailed in a separate paper (Saint and Egan, 1979). The number of sites of each enzyme are as follows: a) 186, BamHI-7, Bg/II-1, EcoRI-3, HindIII-2, PstI-22, XbaI-0 and Xho-I-1; b) P2, BamHI-3, Bg/II-2, EcoRI-3, HindIII-0, PstI-O, XbaI-1 and XhoI-O. All of these sites have been mapped with the exception of PstI for 186, where only the five sites in the right 35% (the control region) have been mapped.

Coliphages↗
PubMed1986

Restriction endonuclease mapping of ribosomal RNA genes: sequence divergence and the origin of the tetraploid treefrog Hyla versicolor.

Hyla chrysoscelis (2n = 24) and H. versicolor (2n = 48) are a diploid-tetraploid species pair of treefrogs. Restriction endonuclease mapping of ribosomal RNA (rRNA) gene repeat units of diploids collected from eastern and western populations reveals no differences within rRNA gene coding regions but distinctive differences within the nontranscribed spacers. A minimum of two physical maps is required to construct an rRNA gene map for the tetraploid, whose repeat units appear to be a composite, with about 50% of the elements resembling the "western" diploid population and about 50% resembling the "eastern" population. These results imply that this population of the tetraploid species may have arisen from a genetically hybrid diploid. Alternatively, the dual level of sequence heterogeneity in H. versicolor may reflect some type of gene flow between the two species. The coding region of the rRNA genes in the tetraploid differs from that in either diploid in about 20% of all repeat units, as exemplified by a BamHI site located near the 5' terminus of the 28 S rRNA gene. If the 20% variant class of 28 S rRNA gene coding sequences is expressed, then there must be two structural classes of ribosomes; if only the 80% sequence class is expressed, then a genetic control mechanism must be capable of distinguishing between the two different sequence variants. It is postulated that the 20% variant sequence class may be correlated with a partial functional diploidization of rRNA genes in the tetraploid species.

Animals↗
PubMed1980

Nucleotide sequence of the mitochondrial genes coding for tRNAglyGGR and tRNAvalGUR.

Yeast mitochondrial DNA-pBR322 recombinant DNA molecules known to contain tRNA genes from a tRNA rich region of the yeast genome were used as a source of DNA for restriction mapping and tRNA gene sequence analysis. We report here restriction maps of two segments of yeast mitochondrial DNA and the sequence of mitochondrial genes coding for tRNAglyGGR and tRNAvalGUR. Both genes are flanked by A + T rich DNA and neither has an intervening sequence nor codes for a 3' CCA end. The tRNA structures deduced from the genes have the usual cloverleaf structures and invariant nucleotides. This combination of DNA sequencing and restriction mapping has enabled us to determine that the tRNAvalGUR and a previously sequenced tRNA, the tRNApheUUY are transcribed from the same strand of DNA.

Base Sequence↗
PubMed1987

Comparison of porcine parvovirus to other parvoviruses by restriction site mapping and hybridization analysis of Southern Blots.

The genomic relationship between porcine parvovirus (PPV) and several other autonomous parvoviruses was examined by restriction site and hybridization analysis. Restriction site maps of the PPV genome were prepared by digesting the double-stranded replicative form of the viral DNA with each of eight restriction enzymes. Subsequent comparison of such maps with those previously reported for PPV, canine parvovirus (CPV), feline panleukopenia virus (FPV), minute virus of mice (MVM), H-1 virus (H-1) and bovine parvovirus (BPV) revealed that while the maps of CPV, FPV, MVM and H-1 had a number of features in common, those of PPV and BPV were substantially different. For hybridization analysis radioactive probes prepared by nick translation of PPV, CPV and BPV genomes were tested under conditions of both low and high stringency for homologous hybridization and for heterologous hybridization with each of the other two viruses and with FPV. The results of these tests indicated homology among the genomes of PPV, CPV and FPV, but little or no homology between the genome of BPV and those of any of the other viruses tested. Additional tests with restriction fragments of PPV and a CPV probe indicated that heterologous hybridization was confined primarily to a segment of the genome between 1.85 and 2.7 kb from the 3' end. Based on transcriptional maps previously determined for several of the rodent parvoviruses, this interval is likely to include part of the coding sequences for both non-structural and structural proteins and may be the genetic basis for the replicative as well as the antigenic similarities between PPV and both CPV and FPV.

Animals↗
PubMed1983

Restriction endonuclease mapping of gamma-delta-beta-globin region in G gamma (beta)+ HPFH and a Chinese A gamma HPFH variant.

Restriction endonuclease mapping of the beta-globin genomic region was used for studying the molecular basis of two variants of hereditary persistence of fetal hemoglobin (HPFH): an African G gamma (beta)+ HPFH and a Chinese HPFH variant with predominant synthesis of A gamma chains. HPFH and control DNA samples were digested with a battery of restriction enzymes, and the fragments were identified by hybridization to a family of discrete probes. DNA fragments from the A gamma HPFH (Chinese) and the G gamma (beta)+ HPFH individuals were identical with those of the normal controls. These findings suggest that the two mutants are the result of small structural anomalies of DNA sequences that play a role in the regulation of the expression of gamma-globin genes.

Chromosome Mapping↗
PubMed2001

Genetic and physical mapping of two centromere-proximal regions of chromosome IV in Aspergillus nidulans.

Chromosome IV is the smallest chromosome of Aspergillus nidulans. The centromere-proximal portion of the chromosome was mapped physically using overlapping clones of a cosmid genomic library. Two contiguous segments of a physical map, based on restriction mapping of cosmid clones, were generated, together covering more than 0.4 Mb DNA. A reverse genetic mapping approach was used to establish a correlation between physical and genetic maps; i.e., marker genes were integrated into physically mapped segments and subsequently mapped by mitotic and meiotic recombination. The resulting data, together with additional classical genetic mapping, lead to a substantial revision of the genetic map of the chromosome, including the position of the centromere. Comparison of physical and genetic maps indicates that meiotic recombination is low in subcentromeric DNA, its frequency being reduced from 1 crossover per 0.8 Mb to approximately 1 crossover per 5 Mb per meiosis. The portion of the chromosome containing the functional centromere was not mapped because repeat-rich regions hindered further chromosome walking. The size of the missing segment was estimated to be between 70 and 400 kb.

Aspergillus nidulans↗
PubMed1986

Restriction enzyme map of cryptic plasmid accompanying pR711b and pR409; identity of pR409 and pR388.

A reference strain containing the IncW plasmid, pR409, was found to contain also a cryptic, conjugative plasmid, here named pJR15. After the separation of the two plasmids into different strains of bacteria, pR409 was found to have an identical restriction enzyme map to the IncW plasmid, pR388. Both pR409 and pR388 were isolated from the same hospital in London and confer sulphonamide and trimethoprim resistance. Originally pR409 was reported to confer resistance to tetracycline, but this was not confirmed by the Plasmid Reference Center. pR409 appears to represent another isolate of pR388 in a different host background. The restriction enzyme map of the cryptic plasmid, pJR15 (39 kb), has been determined. pJR15 was found compatible with plasmids from 15 different incompatibility groups and has been found also in the reference strain of the IncD plasmid, pR711b. The HindIII and BglII digests of pR711b are shown. The possible presence of the conjugative plasmid, pJR15, should be examined for studies on the sex pili or chromosomal mobilization properties of pR711b.

DNA Restriction Enzymes↗