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PubMed1991

Restriction endonuclease mapping of the genome of feline herpesvirus type 1.

Restriction endonuclease mapping was used to determine the molecular structure of feline herpesvirus type 1 (FHV-1) strain B 927. FHV-1 DNA purified from infected cells by pulsed field gel electrophoresis was partially restricted with Sau 3A to generate a series of overlapping fragments of 10-20 kb. EMBL 4 DNA was digested with Bam HI and Sal I and viral DNA was inserted into the "stuffer" region. Recombinant phage were selected on E. coli strain Q359. Single and double digestions with the enzymes Eco RI, Bam HI, Sal I, Sst I and Xho I enabled construction of physical maps of the genome. The FHV-1 genome is approximately 126 kbp in length and is composed of a long unique region of 99 kbp and a short region of 27 kbp comprising an unique sequence of 8-9 kbp flanked by inverted repeats of 7-8.5 kbp.

Animals↗
PubMed1989

Restriction enzyme mapping of ribosomal DNA can distinguish between fasciolid (liver fluke) species.

Recognition sites for nine different restriction endonucleases were mapped on rDNA genes of fasciolid species. Southern blots of digested DNA from individual worms were probed sequentially with three different probes derived from rDNA of Schistosoma mansoni and known to span between them the entire rDNA repeat unit in that species. Eighteen recognition sites were mapped for Fasciola hepatica, and seventeen for Fasciola gigantica and Fascioloides magna. Each fasciolid species had no more than two unique recognition sites, the remainder being common to one or both of the other two species. No intraspecific variation in restriction sites was noted in F. hepatica (individuals from 11 samples studied; hosts were sheep, cattle and laboratory animals; geographical origins. Australia, New Zealand, Mexico, U.K., Hungary and Spain), or in F. gigantica (two samples; Indonesia and Malaysia). Only one sample of F. magna was available. One specimen of Fasciola sp. from Japan (specific identity regarded in the literature as uncertain) yielded a restriction map identical to that of F. gigantica. Almost all recognition sites occurred in or near the putative rRNA coding regions. The non-transcribed spacer region had few or no cut sites despite the fact that this region is up to about one half of the entire repeat unit in length. Length heterogeneity was noted in the non-transcribed spacer, even within individual worms.

Animals↗
PubMed1991

Restriction site mapping of subgenus D adenoviruses, prototypes 42-47, intermediate and atypical strains.

Restriction site maps with the endonucleases BamHI, BgIII, and HindIII were elaborated for 19 adenovirus strains of subgenus D, namely candidates AV42 to 47, 6 intermediate and 7 atypical strains. On the basis of several types mapped by biochemical methods, the physical maps for the 19 strains were constructed by adapting restriction fragments of defined molecular weight to the known maps, as the homology among strains within this subgenus is considerable. Other prototypes mapped by adaptation were also used for reference. The localization of the restriction sites on the genome was analyzed for all mapped types or strains of subgenus D (except AV8). The sites appeared to be randomly distributed; only one site was common to all strains.

Adenoviruses, Human↗
PubMed2002

Apolipoprotein E genotyping: a comparative study between restriction endonuclease mapping and allelic discrimination with the LightCycler.

BACKGROUND: The apolipoprotein E (apo E) polymorphism is associated with the risk of developing cardiovascular disease and the risk and the time of onset of Alzheimer's disease. Therefore, the interest in apo E genotyping is high, both for epidemiological research and for the purpose of diagnosing dyslipidemia or dementia. The aim of our study was to compare and evaluate two different methods for apo E genotyping, both on the basis of polymerase chain reaction (PCR). METHODS: Genomic DNA of 197 subjects was extracted from whole blood. The first method involved DNA amplification performing a PCR using specific primers and endonuclease restriction mapping. The second one was a DNA assay that used real-time PCR on the LightCycler instrument (Roche). RESULTS: We obtained a 100% concordance between the two methods and we found a relative allelic frequency distribution typical for an Italian population. CONCLUSIONS: The LightCycler (LC) allelic discrimination method for apo E genotyping seems to be rapid, simple and accurate, suggesting a possible successful use of this method for diagnostic purposes.

Alleles↗
PubMed1990

Long-range restriction site mapping of a syntenic segment conserved between human chromosome 1 and mouse chromosome 3.

A linkage map determined from segregation analysis of 338 meiotic events in an interspecific mouse cross was utilized to help investigate genomic organization of a linkage group conserved between human chromosome 1p and mouse chromosome 3. Using pulsed-field gel electrophoresis, the genes encoding the lymphocyte adhesion molecule human CD2/murine Ly-37, the alpha 1-subunit of Na, K-ATPase, the beta-subunit of thyrotropin, the beta-subunit of nerve growth factor, and muscle adenylate deaminase were similarly positioned on long-range restriction maps in both species. These studies indicate that the development of detailed genetic maps using interspecific Mus crosses facilitates rapid analysis of murine genomic organization and may enable physical mapping of syntenic regions within the human genome. Moreover, the data suggest profound conservation of genomic organization during mammalian evolution.

AMP Deaminase↗
PubMed1984

Structure of Marek's disease virus DNA: detailed restriction enzyme map.

Purified virion DNA (120 X 10(6) molecular weight [MW]) of Marek's disease virus strain GA was cleaved with BamHI restriction endonuclease, and 27 out of the 29 fragments were cloned into bacterial plasmids. Restriction maps for BamHI, BglI, and SmaI endonucleases were constructed. The genomic structure of Marek's disease virus DNA was found to be similar to that of herpes simplex virus types 1 and 2. A long unique region (75 X 10(6) MW, located at 10 X 10(6) to 85 X 10(6) MW [10-85] from the left end of the genome), which was subdivided into segment 1 (22 X 10(6) MW, located at 10-32) and segment 2 (51 X 10(6) MW, located at 34-85) by direct repeats (32-34), was flanked by a long terminal region (10 X 10(6) MW, located at 0-10) and a long inverted region (10 X 10(6) MW, located at 85-95). A short unique region (8 X 10(6) MW, located at 103-111) was flanked by a short terminal region (8 X 10(6) MW, located at 111-119) and a short inverted region (8 X 10(6) MW, located at 95-103). The direct repeat fragments (0.9 X 10(6) could be isolated by cleavage with SmaI. The right terminal end was found to be heterogenous .

Animals↗
PubMed1982

Restriction enzyme mapping of the DNA of Streptomyces bacteriophage B alpha and its deletion derivatives.

Cleavage analysis of actinophage B alpha DNA was done with several restriction enzymes, and a restriction map of the DNA was determined. The DNA appeared to carry cohesive ends. Deletion mutants of actinophage B alpha were isolated by five cycles of treatment with 15 mM PPi. Both mutants had deletions of 2.5 of 1.8 megadaltons near one end of the genome, and one of them lost the single EcoRI cleavage site.

Bacteriophages↗
PubMed1990

Bovine herpesvirus 4 genome: cloning, mapping and strain variation analysis.

The restriction map of the bovine herpesvirus 4 (BHV-4) genome (V. Test strain) was established for the restriction enzymes EcoRI, BamHI and HindIII by analysis of clones from a lambda library (Sau3AI partial digestion) and from a plasmid library (EcoRI fragments). One genome unit was defined as the length of the unique central part, flanked at both ends by one of the terminal tandem repeats called polyrepetitive DNA (prDNA) and was estimated to be 113 +/- 2 kbp. A restriction map of the prDNA of the V. Test strain showed internal 200 bp tandem repeats of different sequences. This region in the prDNA was highly polymorphic between BHV-4 strains, even in a viral DNA preparation from a plaque-purified strain. The right junction between the repeated and the unique sequence of the genome occurred at an almost constant site, but the left junction contained a modified prDNA and was variable between BHV-4 strains. The unique central part of the genome was very similar in the four strains under consideration, with a few variations due to the presence or absence of a restriction site and four length variations were observed, located at positions 0.006 to 0.034 (left end), 0.211 to 0.225, 0.864 to 0.881 and 0.962 to 0.984 (right end). The total length variation of 1 genome unit does not exceed 1 kbp.

Animals↗
PubMed1988

Restriction site mapping is in separation theory.

A computer algorithm for restriction-site mapping consists of a generator of partial maps and a consistency checker. This paper examines consistency checking and argues that a method based on separation theory extracts the maximum amount of information from fragment lengths in digest data. It results in the minimum number of false maps being generated.

Algorithms↗
PubMed1981

Restriction enzyme mapping and heteroduplex analysis of the rat milk protein cDNA clones.

Detailed restriction enzyme maps have been determined for the three major rat casein and the fourth principal milk protein, alpha-lactalbumin, cDNA clones. Each of the milk protein genes exhibited unique and characteristic restriction enzyme sites. A comparison of the restriction enzyme maps of the three rat caseins revealed no apparent sequence homology among their gene sequences. The orientation of each cDNA gene sequence within the parent plasmid, pBR322, was determined by hybridization with a 3' specific cDNA probe synthesized from a partially hydrolyzed total poly(A) mRNA preparation following isolation by chromatography on oligo(dT)-cellulose. This technique provided a rapid procedure for determining the 5'-3' orientation of the cloned DNA sequences. Three casein clones were selected, which were in the same orientation, and were employed for a heteroduplex analysis to determine whether minor regions of homology existed within the alpha-, beta-, and gamma-casein genes. No heteroduplex formation was observed among these genes even under the low stringency conditions of hybridization employed, suggesting that considerable sequence divergence has occurred within the rat casein gene family.

Animals↗
PubMed1997

Multiple-complete-digest restriction fragment mapping: generating sequence-ready maps for large-scale DNA sequencing.

Multiple-complete-digest mapping is a DNA mapping technique based on complete-restriction-digest fingerprints of a set of clones that provides highly redundant coverage of the mapping target. The maps assembled from these fingerprints order both the clones and the restriction fragments. Maps are coordinated across three enzymes in the examples presented. Starting with yeast artificial chromosome contigs from the 7q31.3 and 7p14 regions of the human genome, we have produced cosmid-based maps spanning more than one million base pairs. Each yeast artificial chromosome is first subcloned into cosmids at a redundancy of x15-30. Complete-digest fragments are electrophoresed on agarose gels, poststained, and imaged on a fluorescent scanner. Aberrant clones that are not representative of the underlying genome are rejected in the map construction process. Almost every restriction fragment is ordered, allowing selection of minimal tiling paths with clone-to-clone overlaps of only a few thousand base pairs. These maps demonstrate the practicality of applying the experimental and software-based steps in multiple-complete-digest mapping to a target of significant size and complexity. We present evidence that the maps are sufficiently accurate to validate both the clones selected for sequencing and the sequence assemblies obtained once these clones have been sequenced by a "shotgun" method.

Base Composition↗
PubMed1983

Restriction site maps of the human adenovirus type 8 DNA.

Restriction site maps of human adenovirus 8 (Ad h 8) were constructed with BamHI, HindIII, PstI, SalI and KpnI endonucleases. The genome size was found to be 22.1 to 22.3 X 10(6) Mr. Comparison of the results with the data available on h Ad subgenera A, B, C showed that the SalI enzyme revealed subgenus-specific differences in the genomes. Similar patterns of the SalI fragments in both type 8 and 10 suggest that the differences were specific for the subgenus D of h Ad.

Adenoviridae↗
PubMed1995

Systematics of cetaceans using restriction site mapping of mitochondrial DNA.

Phylogenetic analysis of 14 cetacean species, including members from two baleen whale families and three toothed whale families, was undertaken using restriction site mapping of mitochondrial DNA and using cladistic and distance measures to infer phylogenies. The amount of between-taxa sequence divergence inferred from the data was lower than expected from the standard interpretation of the fossil record, but more consistent with some recent estimates of sequence divergence in cetacean mitochondrial DNA or nuclear DNA. This implies either that the rate of molecular evolution of cetacean DNA is much lower than that of other mammalian orders or that the fossil record of cetaceans requires reinterpretation. The incompleteness of the cetacean fossil record precludes resolution of the paradox at the present time. However, this discrepancy could in part be attributed to the sampling error inherent in the restriction site mapping technique, as comparative studies using the complete mtDNA genome and restriction site data of the blue and fin whales (genus Balaenoptera) indicate that the restriction site maps underestimate sequence divergence by about 40%. In contrast to a recent study suggesting that toothed whales were paraphyletic, with the sperm whales being more closely related to the rorquals than to the other toothed whales, the restriction data tend to support the monophyly of the baleen and the toothed whales, a finding which is consistent with a recent molecular-based study and with morphological and paleontological data. Topologies of the subfamily and generic levels are generally consistent with morphologically based schemes.

Animals↗
PubMed1982

2-micrometers DNA-like plasmids in the osmophilic haploid yeast Saccharomyces rouxii.

DNA plasmids were detected in two independent strains of Saccharomyces rouxii among 100 yeast strains other than Saccharomyces cerevisiae tested. The plasmids, pSR1 and pSR2, had almost the same mass (approximately 4 X 10(6) daltons) as 2-micrometers DNA of S. cerevisiae. pSR1 and pSR2 gave identical restriction maps with restriction endonucleases BamHI, EcoRI, HincII, HindIII, and XhoI, and both lacked restriction sites for PstI, SalI, and SmaI. These maps, however, differed significantly from that of S. cerevisiae 2-micrometers DNA. Restriction analysis also revealed two isomeric forms of each plasmid and suggested the presence of a pair of inverted repeat sequences in the molecules where intramolecular recombination took place. DNA-DNA hybridization between the pSR1 and pSR2 DNAs indicated significant homology between their base sequences, whereas no homology was detected between pSR1 and pJDB219, a chimeric plasmid constructed from a whole molecule of 2-micrometers DNA, plasmid pMB9, and a 1.2-kilobase DNA fragment of S. cerevisiae bearing the LEU2 gene. A chimeric plasmid constructed with pSR1 and YIp1, the larger EcoRI-SalI fragment of pBR322 ligated with a 6.1-kilobase DNA fragment of S. cerevisiae bearing the HIS3 gene, could replicate autonomously in an S. cerevisiae host and produced isomers, presumably by intramolecular recombination at the inverted repeats.

Base Sequence↗
PubMed1996

A neurotransmitter transporter encoded by the Drosophila inebriated gene.

Behavioral and electrophysiological studies on mutants defective in the Drosophila inebriated (ine) gene demonstrated increased excitability of the motor neuron. In this paper, we describe the cloning and sequence analysis of ine. Mutations in ine were localized on cloned DNA by restriction mapping and restriction fragment length polymorphism (RFLP) mapping of ine mutants. DNA from the ine region was then used to isolate an ine cDNA. In situ hybridization of ine transcripts to developing embryos revealed expression of this gene in several cell types, including the posterior hindgut, Malpighian tubules, anal plate, garland cells, and a subset of cells in the central nervous system. The ine cDNA contains an open reading frame of 658 amino acids with a high degree of sequence similarity to members of the Na+/Cl(-)-dependent neurotransmitter transporter family. Members of this family catalyze the rapid reuptake of neurotransmitters released into the synapse and thereby play key roles in controlling neuronal function. We conclude that ine mutations cause increased excitability of the Drosophila motor neuron by causing the defective reuptake of the substrate neurotransmitter of the ine transporter and thus overstimulation of the motor neuron by this neurotransmitter. From this observation comes a unique opportunity to perform a genetic dissection of the regulation of excitability of the Drosophila motor neuron.

Amino Acid Sequence↗
PubMed1985

A cosmid vector that facilitates restriction enzyme mapping.

We describe the construction and use of a cosmid vector, loric, which is derived from the phage lambda origin of replication and appears to be more stable than ColE1-derived cosmids. Loric recombinants can be efficiently packaged in vivo to yield 100-300 micrograms of DNA per liter that is linear and has single-stranded cos ends. We call such molecules "phosmids." Phosmid restriction maps can be rapidly generated by labeling either the left or right cos site by annealing on a 32P-labeled oligonucleotide complementary to either cos-L or cos-R. Partial restriction enzyme digestion, agarose gel electrophoresis, and autoradiography are used to size restriction fragments of increasing length, all of which terminate at the labeled cos site. The procedures have been tested by isolating and mapping a region of the H-2 locus of mouse chromosome 17.

Bacteriophage lambda↗
PubMed1983

Restriction endonuclease mapping of globin genomic regions of HEL (human erythroleukemia) line.

The restriction endonuclease map of the alpha and beta globin genomic region of the new human erythroleukemia line, HEL, was compared with that of normal human DNA. The HEL line, which produces mainly fetal (G gamma and A gamma) but no adult (delta and beta) globin chains, was shown to have the same pattern of DNA fragments as that of normal human DNA. This suggests that the selective expression of the gamma globin genes observed in HEL cells is not due to a major deletion or rearrangement in the epsilon-G gamma-A gamma-delta-beta gene complex. Thus, the HEL line provides a model for studying the control of globin developmental switching in cells with structurally intact globin gene regions.

Cell Line↗
PubMed1989

Physical mapping of the Myxococcus xanthus genome by random cloning in yeast artificial chromosomes.

Random segments of Myxococcus xanthus DNA were cloned in yeast artificial chromosomes (YACs) to construct a physical map of the genome. EcoRI restriction maps of 409 YAC clones with inserts averaging 111 kilobase pairs (kb) were determined. Comparison to the map of a 300-kb region of M. xanthus obtained from clones in Escherichia coli indicates that segments of DNA cloned in YACs are stably maintained in yeast and that their sequences accurately reflect the structure of the Myxococcus genome. The 409 YAC inserts were ordered within 60 map segments (contigs) by aligning their EcoRI restriction maps and by hybridization with 18 gene-specific DNA probes. These 60 map segments may represent the entire Myxococcus genome and could be used to organize its genetic information. This study illustrates the utility of YACs for cloning large segments of DNA and for reliable long-range genomic mapping.

Chromosome Mapping↗