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PubMed1983

Restriction endonuclease mapping of the frog virus 3 genome.

A physical map for the frog virus 3 (FV 3) genome was constructed after digestion with the following restriction endonucleases: EcoRI, HindIII, KpnI, and XbaI. Mapping of the DNA was accomplished by partial digestion and recutting, double-digestion, and Southern blot hybridization with deduction of overlaps. Although the virion DNA is physically linear, the restriction map was circular, supporting the data that the FV 3 genome is circularly permuted (Goorha and Murti, Proc. Nat. Acad. Sci USA 79, 248-252, 1982), a unique feature among eukaryotic viruses.

DNA Restriction Enzymes↗
PubMed2005

Novel approach to mapping of resistance mutations in whole genomes by using restriction enzyme modulation of transformation efficiency.

Restriction enzyme modulation of transformation efficiencies (REMOTE) is a method that makes use of genome restriction maps and experimentally observed differences in transformation efficiencies of genomic DNA restriction digests to discover the location of mutations in genomes. The frequency with which digested genomic DNA from a resistant strain transforms a susceptible strain to resistance is primarily determined by the size of the fragment containing the resistance mutation and the distance of the mutation to the end of the fragment. The positions of restriction enzyme cleavage sites immediately flanking the resistance mutation define these parameters. The mapping procedure involves a process of elimination in which digests that transform with high frequency indicate that the restriction enzyme cleavage sites are relatively far away from the mutation, while digests that transform with low frequency indicate that the sites are close to the mutation. The transformation data are compared computationally to the genome restriction map to identify the regions that best fit the data. Transformations with PCR amplicons encompassing candidate regions identify the resistance locus and enable identification of the mutation. REMOTE was developed using Haemophilus influenzae strains with mutations in gyrA, gyrB, and rpsE that confer resistance to ciprofloxacin, novobiocin, and spectinomycin, respectively. We applied REMOTE to identify mutations that confer resistance to two novel antibacterial compounds. The resistance mutations were found in genes that can decrease the intracellular concentration of compounds: acrB, which encodes a subunit of the AcrAB-TolC efflux pump; and fadL, which encodes a long-chain fatty acid transporter.

Anti-Bacterial Agents↗
PubMed1991

Conservation of the gene for outer membrane protein OprF in the family Pseudomonadaceae: sequence of the Pseudomonas syringae oprF gene.

The conservation of the oprF gene for the major outer membrane protein OprF was determined by restriction mapping and Southern blot hybridization with the Pseudomonas aeruginosa oprF gene as a probe. The restriction map was highly conserved among 16 of the 17 serotype strains and 42 clinical isolates of P. aeruginosa. Only the serotype 12 isolate and one clinical isolate showed small differences in restriction pattern. Southern probing of PstI chromosomal digests of 14 species from the family Pseudomonadaceae revealed that only the nine members of rRNA homology group I hybridized with the oprF gene. To reveal the actual extent of homology, the oprF gene and its product were characterized in Pseudomonas syringae. Nine strains of P. syringae from seven different pathovars hybridized with the P. aeruginosa gene to produce five different but related restriction maps. All produced an OprF protein in their outer membranes with the same apparent molecular weight as that of P.aeruginosa OprF. In each case the protein reacted with monoclonal antibody MA4-10 and was similarly heat and 2-mercaptoethanol modifiable. The purified OprF protein of the type strain P. syringae pv. syringae ATCC 19310 reconstituted small channels in lipid bilayer membranes. The oprF gene from this latter strain was cloned and sequenced. Despite the low level of DNA hybridization between P. aeruginosa and P. syringae DNA, the OprF gene was highly conserved between the species with 72% DNA sequence identity and 68% amino acid sequence identity overall. The carboxy terminus-encoding region of P. syringae oprF showed 85 and 33% identity, respectively, with the same regions of the P. aeruginosa oprF and Escherichia coli ompA genes.

Amino Acid Sequence↗
PubMed1991

Cloning and restriction enzyme mapping of ribosomal DNA of Giardia duodenalis, Giardia ardeae and Giardia muris.

In an attempt to study Giardia at the DNA sequence level, the rRNA genes of three species, Giardia duodenalis, Giardia ardeae and Giardia muris were cloned and restriction enzyme maps were constructed. The rDNA repeats of these Giardia show completely different restriction enzyme recognition patterns. The size of the rDNA repeat ranges from approximately 5.6 kb in G. duodenalis to 7.6 kb in both G. muris and G. ardeae. These size differences are mainly attributable to the variation in length of the spacer. Minor differences exist among these Giardia in the sizes of their small subunit rRNA and the internal transcribed spacer between small and large subunit rRNA. The genetic maps were constructed by sequence analysis of the DNA around the 5' and 3' ends of the mature rRNA genes and between the rRNA covering the 5.8S rRNA gene and internal transcribed spacer. Comparison of the 5.8S rDNA and 3' end of large subunit rDNA from these three Giardia species showed considerable sequence variation, but the rDNA sequences of G. duodenalis and G. ardeae appear more closely related to each other than to G. muris.

Amino Acid Sequence↗
PubMed1994

A partial digest approach to restriction site mapping.

We present a new, practical algorithm to resolve the experimental data in restriction site analysis, which is a common technique for mapping DNA. Specifically, we assert that multiple digestions with a single restriction enzyme can provide sufficient information to identify the positions of the restriction sites with high probability. The motivation for the new approach comes from combinatorial results on the number of mutually homeometric sets in one dimension, where two sets of n points are homeometric if the multiset of n(n-1)/2 distances they determine are the same. Since experimental data contain errors, we propose algorithms for reconstructing sets from noisy interpoint distances, including the possibility of missing fragments. We analyse the performance of these algorithms under a reasonable probability distribution, establishing a relative error limit of r = theta(1/n2) beyond which our technique becomes infeasible. Through simulations, we establish that our technique is robust enough to reconstruct data with relative errors of up to 7.0% in the measured fragment lengths for typical problems, which appears sufficient for certain biological applications.

Algorithms↗
PubMed1993

A partial digest approach to restriction site mapping.

We present a new practical algorithm to resolve the experimental data of restriction site analysis, which is a common technique for mapping DNA. Specifically, we assert that multiple digests with a single restriction enzyme can provide sufficient information to identify the positions of the restriction sites with high probability. The motivation for the new approach comes from combinatorial results on the number of mutually homeometric sets in one dimension, where two sets of n points are homeometric if the multiset of (n2) distances they determine are the same. Since experimental data contains error, we propose algorithms for reconstructing sets from noisy interpoint distances, including the possibility of missing fragments. We analyze the performance of these algorithms under a reasonable probability distribution, establishing a relative error limit of r = theta (1/n2) beyond which our technique becomes infeasible. Through simulations, we establish that our technique is robust enough to reconstruct data with relative errors of up to 7.0% in the measured fragment lengths for typical problems, which appears sufficient for certain biological applications.

Algorithms↗
PubMed1982

Restriction endonuclease maps of the beta-like globin gene cluster in the British and Greek forms of HPFH, and for one example of G gamma beta + HPFH.

We report here restriction endonuclease maps of the beta-like globin gene cluster for the British form of HPFH and for a case of G gamma beta + HPFH, and also confirm and extend previous reports of the map for the Greek form of HPFH. These results show that all these conditions belong to a group lacking any substantial deletion or rearrangement of DNA sequence in this gene cluster. The absence of any gross disruption of the structure of this region of the genome, together with evidence that the HPFH genotype is either allelic with, or closely linked to, the beta-like globin gene cluster, suggests that the responsible lesions are nearer to being purely regulatory in nature than in forms of HPFH due to substantial deletions. Thus these conditions promise to provide less equivocal evidence about the regulation of beta-like globin gene expression than has so far been available.

Chromosome Mapping↗
PubMed1990

Genomic maps of some strains within the Mycoplasma mycoides cluster.

Genomic restriction maps for the small colony (SC) strains (PG1, KH3J, Gladysdale, and V5) of Mycoplasma mycoides subsp. mycoides (the agent of contagious bovine pleuropneumonia) and for Mycoplasma strain PG50 (classified as bovine serogroup 7), with respective sizes of 1,280, 1,280, 1,260, 1,230, and 1,040 kbp, were compared with the map (1,200 kbp) for a large colony strain (Y goat) of M. mycoides subsp. mycoides. The number and order of all mapped restriction sites were fully conserved in the SC genomes, as were the approximate positions of mapped loci. A number of these restriction sites in the Y genome and some, but fewer, in the PG50 genome appeared to be conserved. The SC and large colony strains shared conservation in the relative positions of the mapped loci, except for rpoC.

DNA Probes↗
PubMed1989

Restriction fragment mapping of nontypable haemophilus influenzae: a new tool to study this middle ear pathogen.

Isolates of nontypable haemophilus influenzae (NTHI) recovered from paired nasopharyngeal (NP) and middle ear (ME) fluid cultures performed simultaneously on seven children with otitis media with effusion (OME) were studied by analysis of the restriction fragment patterns produced from total genomic DNA. This method provides a sensitive measure of the genetic similarities between strains. In the seven pairs examined, the NP and ME strains were indistinguishable from one another, whereas each pair was distinct from the other, except for two siblings in whom all four strains were identical. This information provides evidence that the pathogenesis of OME caused by NTHI involves spread of the bacteria from NP to the ME. Analysis of paired NP and ME isolates from three children with recurrent OME caused by NTHI indicated that the second episode was caused by the reinfection with a different strain rather than persistence and reemergence of the first strain. Finally, DNA analysis of strains from two siblings with concurrent OME suggested that person-to-person transmission of NTHI can occur among children. These findings suggest that at any one time the NTHI population in the NP is relatively homogenous, at least at the time of acute upper respiratory disease. Our observation that the strains present at the second episode of otitis media were different than the strains present in the first episode is consistent with strain replacement in the NP. Restriction fragment mapping analysis of genomic DNA of NTHI could provide a powerful tool for investigating the rate of turnover of the NTHI population in the NP.

Child, Preschool↗
PubMed1988

A cladistic analysis of phenotype associations with haplotypes inferred from restriction endonuclease mapping. II. The analysis of natural populations.

Genes that code for products involved in the physiology of a phenotype are logical candidates for explaining interindividual variation in that phenotype. We present a methodology for discovering associations between genetic variation at such candidate loci (assayed through restriction endonuclease mapping) with phenotypic variation at the population level. We confine our analyses to DNA regions in which recombination is very rare. In this case, the genetic variation at the candidate locus can be organized into a cladogram that represents the evolutionary relationships between the observed haplotypes. Any mutation causing a significant phenotypic effect should be imbedded within the same historical structure defined by the cladogram. We showed, in the first paper of this series, how to use the cladogram to define a nested analysis of variance (NANOVA) that was very efficient at detecting and localizing phenotypically important mutations. However, the NANOVA of haplotype effects could only be applied to populations of homozygous genotypes. In this paper, we apply the quantitative genetic concept of average excess to evaluate the phenotypic effect of a haplotype or group of haplotypes stratified and contrasted according to the nested design defined by the cladogram. We also show how a permutational procedure can be used to make statistical inferences about the nested average excess values in populations containing heterozygous as well as homozygous genotypes. We provide two worked examples that investigate associations between genetic variation at or near the Alcohol dehydrogenase (Adh) locus and Adh activity in Drosophila melanogaster, and associations between genetic variation at or near some apolipoprotein loci and various lipid phenotypes in a human population.

Alcohol Dehydrogenase↗
PubMed1994

Molecular characterization of the porcine MHC class I region.

A porcine cosmid library was screened with a human MHC class I cDNA. Four positive clones were isolated and mapped with different restriction endonucleases. Altogether nine SLA class I genes were identified and their positions located within restriction maps. Sizes of class I homologous DNA sequences varied between 3600 and 5800 bp. The distances between these regions ranged from 11,900 to 22,200 bp.

Animals↗
PubMed2004

Single-molecule approach to bacterial genomic comparisons via optical mapping.

Modern comparative genomics has been established, in part, by the sequencing and annotation of a broad range of microbial species. To gain further insights, new sequencing efforts are now dealing with the variety of strains or isolates that gives a species definition and range; however, this number vastly outstrips our ability to sequence them. Given the availability of a large number of microbial species, new whole genome approaches must be developed to fully leverage this information at the level of strain diversity that maximize discovery. Here, we describe how optical mapping, a single-molecule system, was used to identify and annotate chromosomal alterations between bacterial strains represented by several species. Since whole-genome optical maps are ordered restriction maps, sequenced strains of Shigella flexneri serotype 2a (2457T and 301), Yersinia pestis (CO 92 and KIM), and Escherichia coli were aligned as maps to identify regions of homology and to further characterize them as possible insertions, deletions, inversions, or translocations. Importantly, an unsequenced Shigella flexneri strain (serotype Y strain AMC[328Y]) was optically mapped and aligned with two sequenced ones to reveal one novel locus implicated in serotype conversion and several other loci containing insertion sequence elements or phage-related gene insertions. Our results suggest that genomic rearrangements and chromosomal breakpoints are readily identified and annotated against a prototypic sequenced strain by using the tools of optical mapping.

Escherichia coli K12↗
PubMed1992

SfiI genomic cleavage map of Escherichia coli K-12 strain MG1655.

An SfiI restriction map of Escherichia coli K-12 strain MG1655 is presented. The map contains thirty-one cleavage sites separating fragments ranging in size from 407 kb to 3.7 kb. Several techniques were used in the construction of this map, including CHEF pulsed field gel electrophoresis; physical analysis of a set of twenty-six auxotrophic transposon insertions; correlation with the restriction map of Kohara and coworkers using the commercially available E. coli Gene Mapping Membranes; analysis of publicly available sequence information; and correlation of the above data with the combined genetic and physical map developed by Rudd, et al. The combination of these techniques has yielded a map in which all but one site can be localized within a range of +/- 2 kb, and over half the sites can be localized precisely by sequence data. Two sites present in the EcoSeq5 sequence database are not cleaved in MG1655 and four sites are noted to be sensitive to methylation by the dcm methylase. This map, combined with the NotI physical map of MG1655, can aid in the rapid, precise mapping of several different types of genetic alterations, including transposon mediated mutations and other insertions, inversions, deletions and duplications.

Base Sequence↗
PubMed1991

[5-Variable genome sequence of the LIVP vaccinia virus. A possible role for short direct repeats in the formation of deletions].

HindIII-O/N DNA fragments of vaccinia virus (VV) of the LIVP strain were mapped using thirteen restriction endonucleases. Nucleotide sequences of the HindIII-O fragment (1530 bp) as well as of a site of the HindIII-N genome fragment 353 bp in size were determined. Comparison of restriction maps and nucleotide sequences of VV strains (WR and LIVP) demonstrated that DNA of VV LIVP contained % deletions and 2 insertions. "Reliable" short direct repeats were localized and their possible role in formation of DNA deletions was shown. It was suggested that VV endonuclease and DNA-ligase participate in replication and repair processes. Mechanism of formation of variable sequences of viral genomes is discussed.

Amino Acid Sequence↗
PubMed1989

The human alpha-amylase multigene family consists of haplotypes with variable numbers of genes.

Polymorphic amylase protein patterns have suggested the presence in the human genome of various haplotypes encoding these allozymes. To investigate the genomic organization of the human alpha-amylase genes, we isolated the pertinent genes from a cosmid library constructed of DNA from an individual expressing three different salivary amylase allozymes. From the restriction maps of the overlapping cosmids and a comparison of these maps with the restriction enzyme patterns of DNA from the donor and family members, we were able to identify two haplotypes consisting of very different numbers of salivary amylase genes. The short haplotype contains two pancreatic genes (AMY2A and AMY2B) and one salivary amylase gene (AMY1C), arranged in the order 2B-2A-1C, encompassing a total length of approximately 100 kb. The long haplotype spans about 300 kb and contains six additional genes arranged in two repeats, each one consisting of two salivary amylase genes (AMY1A and AMY1B) and a pseudogene lacking the first three exons (AMYP1). The order of the amylase genes within the repeat is 1A-1B-P1. All genes are in a head-to-tail orientation except AMY1B, which has the reverse orientation with respect to the other genes. Analysis of somatic cell hybrids confirmed the presence of these short and long haplotypes. Furthermore, we present evidence for the existence of additional haplotypes in the human population and propose a general model for the evolution of the human alpha-amylase multigene family. A general designation 2B-2A-(1A-1B-P)n-1C can describe these haplotypes, n being 0 and 2 for the short and the long haplotypes presented in this paper, respectively.

Biological Evolution↗
PubMed2001

[Isolation and functional-structural characteristics of Bacillus polymyxa RP4::Mucts62 transcipients].

Conjugative-like transfer of hybrid plasmid RP4::Mucts62 from Escherichia coli to plasmid-free Bacillus polymyxa was carried out. Bacillus transcipients are detected by the markers of kanamycin and tetracyclin resistance RP4 and thermal sensitivity to growth at 40-42 degrees C, determined by prophage Mucts62 in the plasmid. The technique of transception using millipore filters on solid media has been improved. For comparison with experimental samples, restriction mapping of the native plasmid in donor E. coli GA570 strain was carried out with identification of the prophage location in point 30.5 n. p. of RP4 map with counter-clockwise orientation of the right terminal towards IS21 element. Two phenotypes of bacillus transcipients selected for restriction mapping were singled out. Phenotype KmRTcRTS retains all markers of donor strain plasmid, and by the sum of restricts electrophoretically either corresponds to intact hybrid plasmid or contains deletions in RP4 sites (1-8 n. p.) adjacent to the left arm of prophage or rarely on the right arm (up to 3 n. p.). KmRTcRTS transcipients lose kanamycin resistance and on restriction map show greater prolongation of deletions from the prophage right terminal to kan RP4 gene, shortening it to 13 n. p. without involving the prophage proper. Its left terminal is retained, but 2-7 n. p. sites are deleted in the RP4 area adjacent to it. The possibility of transception in nature and horizontal routes of drug resistance dissemination among genetically remote bacteria are discussed.

Bacillus↗
PubMed1984

Restriction site map of African swine fever virus DNA.

Treatment of African swine fever virus DNA (about 170 kbp) with the restriction endonucleases SalI, EcoRI, KpnI, PvuI, and SmaI yielded 14, 31, 17, 13, and 11 fragments, respectively. The order of the restriction fragments produced by each nuclease was established by identifying the crosslinked EcoRI and SalI terminal fragments and then finding overlapping fragments. The five restriction fragment maps were integrated into a single map by locating SalI, KpnI, PvuI, and SmaI sites in cloned EcoRI fragments, and orienting each fragment in the overall map.

African Swine Fever Virus↗
PubMed2005

A RT/PCR-partial restriction enzymatic mapping (PREM) method for the molecular characterisation of the large satellite RNAs of Arabis mosaic virus isolates.

The satellite RNA of the grapevine isolate NW of Arabis mosaic virus (ArMV) was cloned and sequenced, and showed 75% identity at the nucleotide level to the satellite RNA of the lilac isolate of ArMV. In order to survey ArMV isolates from various geographical origins and natural hosts for the presence of large satellite RNAs and analyse their degree of variability, a RT/PCR-partial restriction enzymatic mapping (PREM) method was developed. The method is based on the incorporation of 5-methyl-dCTP in the RT/PCR reaction, and the subsequent digestion of the RT/PCR products by methyl-sensitive restriction enzymes. Satellites RNAs were detected by RT/PCR in eight isolates out of 47, six of them originating from grapevine, one from hop and one from lilac. The partial restriction digestion patterns allowed to distinguish six different types of satellites. Cloning and sequencing of the different satellites confirmed these results, the PREM proving able to discriminate sequences with 96% identity. The sizes of the different satellites varied between 1092 and 1139 nucleotides, their encoded proteins between 338 and 360 amino acids. Conserved domains were found in the amino and carboxy-termini between the sequences of the proteins encoded by the satellites of the different isolates of ArMV.

Amino Acid Sequence↗