Computer-aided construction of nucleic acid restriction maps using defined vectors

A new algorithm is described that will rapidly produce restrictionmaps of cloned DNA fragments. Information concerning the vectoris stored as a data file and used in constructing probable maps.As the program is based upon a permutation analysis it has twoprimary uses. First, preliminary restriction maps can be createdfrom fragment length data as a starting point for further analysis.Second, existing maps can be confirmed as being highly probable,and other probable maps examined to ensure certain combinationshave not been overlooked. Although primarily designed for linearvectors, the program can be used to calculate circular maps. Received on June 5, 1985; accepted on September 27, 1985     

Construction of restriction maps

A computer program is described, which constructs maps of restrictionendonuclease cleavage sites in linear or circular DNA molecules,given the fragment lengths in single and double digestions withtwo enzymes. The algorithm is based upon a partition methodand a very simple rule to chain fragments. The program is writtenin Prolog II. Received on July 28, 1987; accepted on December 31, 1987     

Estimation of restriction maps with known site order using a generalized linear model

A generalized linear model with Gamma errors is used to estimatethe coordinates of a restriction map when the site order isknown. This can be conveniently programmed in a wide range ofstatistical packages (e.g. Genstat 5, Minitab, SAS), and givesmaximum likelihood estimates with their associated optimal properties.Regression diagnostics allow the checking of assumptions andhelp to identify mis–specified, influential or discordantfragment lengths. A specific diagnostic for identifying fragmentlengths causing reversal of restriction site order is derived.Exact ‘fragment’ lengths from DNA sequencing canbe conveniently included in an approximate manner by givingthem a larger weight than observed restriction fragment lengths.Two examples and the Genstat 5 codes used in their analysisare presented.     

DNA sequence variation and phylogenetic relationships among strains of Pseudomonas syringae pv. syringae inferred from restriction site maps and restriction fragment length polymorphism.

We evaluated the restriction fragment length polymorphism of genomic DNA among 53 strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. Twenty-nine strains were isolated from beans, and the rest were isolated from 11 other hosts. Southern blots of DNA digested with EcoRI or HindIII were hybridized to two random probes from a cosmid library of P. syringae pv. syringae and a hrp (hypersensitive reaction and pathogenicity) cluster cloned from P. syringae pv. syringae. The size of hybridizing fragments was determined, and a similarity matrix was constructed by comparing strains on a pairwise basis for the presence or absence of fragments. The proportion of shared fragments was then used to estimate sequence divergence. Dendrograms were produced by using the unweighted pair group method with averages and the neighbor-joining method. For the hrp region, BamHI, EcoRI, EcoRV, and HindIII restriction sites were mapped for six representative bean strains and used to construct EcoRI and HindIII restriction maps for all 30 strains pathogenic on beans. Restriction mapping revealed the presence of a 3-kb insertion in nine bean strains and a probable second insertion or deletion event on the left-hand side of the hrp cluster that biased estimates of nucleotide sequence divergence from fragment comparisons. This demonstrated that the determination of phylogenetic relationships among bacteria by using restriction fragment length polymorphism data requires mapping restriction sites to remove the effect of insertion or deletion events on the analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

A procedure for the construction of linear restriction fragment maps of a 50- to 100-kb DNA.

A simple and effective procedure for the construction of linear restriction fragment maps was developed. Using a two-enzyme digestion, two-dimensional (2-D) electrophoresis procedure, all the restriction fragments in a 50- to 100-kb DNA can be individually resolved and displayed on a 2-D plane. This 2-D gel pattern, with appropriate markers, provides a fixed set of x, y coordinates for each fragment obtained from the single and double digestion as well as the relationship between the two steps. A matrix is constructed from the 2-D pattern. The vertical column shows all the singly digested individual fragments and their sizes obtained from each restriction enzyme treatment, and the dividing horizontal row shows all the doubly digested DNA fragments and their sizes after treatment with two enzymes. The order of arrangement is always from the smallest to the largest fragments. Using this matrix, two linear DNA restriction maps for these two enzymes can be simultaneously constructed in a self-reconfirming manner. As examples for this procedure, we describe the construction of two linear restriction fragment maps, a combination of EcoRI and BamHI digestion as well as a combination of EcoRI and HindIII digestion of lambda-phage DNA.     

An algorithm for searching restriction maps

This paper presents an algorithm thai searches a DNA restrictionenzyme map for regions that approximately match a shorter 'probe'map. Both the map and the probe consist of a sequence of address-enzymepairs denoting restriction sites, and the algorithm penalizesa potential match for undetected or missing sites and for discrepanciesin the distance between adjacent sites. The algorithm was designedspecifically for comparing relatively short DNA sequences witha long restriction map, a problem that will become increasingcommon as large physical maps are generated. The algorithm hasbeen used to extract information from a restriction map of theentire Escherichia coli genome. Received on October 28, 1989; accepted on February 2, 1990     

Revised restriction maps of Bacillus subtilis bacteriophage phi 105 DNA.

Physical mapping of Bacillus subtilis temperate phage phi 105 DNA was carried out by using restriction endonucleases EcoRI, SmaI, and KpnI, and a new revised EcoRI cleavage map is presented. In addition, the EcoRI cleavage maps of six specialized transducing phages carrying sporulation genes of B. subtilis were revised.     

Ligation independent cloning irrespective of restriction site compatibility.

Here we report the use of exonuclease to expose complementary DNA between an insert and vector such that annealing becomes independent of restriction site compatibility. We demonstrate that unusual and, in some cases, previously impossible cloning strategies can be readily and efficiently achieved as long as the flanking sequences of the linear vectors are highly related. Furthermore, we show that the bacterial repair system resolves the residual mismatches, overhangs or gaps in a predictable fashion to generate excisable inserts. This approach facilitates cloning regardless of restriction site compatibility and overcomes an important limitation in current cloning techniques.     

The cleavage site of the restriction endonuclease Ava II.

We have determined that the type II restriction enzyme Ava II, isolated from Anabaena variabilis, recognizes and cuts the sequence (formula: see article). The eight Ava II sites of pBR322 have been mapped, as well as a unique site for Ava I.     

A program package applicable to the detection of overlaps between restriction maps.

A computer program package for the storage, change, and comparison of restriction maps is described. The programs are intended to detect overlaps between relatively short (about 10-40 kb; abbreviations ref.2) maps and to merge the overlapping fragments into large restriction maps. They run on a 16-bit-microcomputer with limited memory and addressing capability. Due to the restricted reliability of restriction maps compared with DNA sequence data a particular storage method was used. The source code of the programs is freely available (+).     

Sa/I restriction endonuclease maps of FII incompatibility group R plasmids.

SalI restriction endonuclease maps of FII incompatibility group R plasmids NR1, NR84, and R6 have been determined by sequential digestion of plasmid DNA with EcoRI and SalI and subsequent analysis of the fragments by electrophoresis on agarose gels. In the composite R plasmid NR1 there are five SalI sites, one in the r-determinants component and four in the RTF-Tc component. SalI cleavage of transitioned NR1 DNA, which contains tandem sequences of the r-determinants in a head-to-tail orientation, produces the five original bands plus a single new amplified band whose mobility on agarose gels corresponds to the monomer r-determinants DNA. NR84 has a total of four SalI sites. It is missing one of the SalI sites near the repA locus. R6 has five SalI sites, four the same as those of NR84, and one additional site within the Km transposon Tn601.     

RsrII--a novel restriction endonuclease with a heptanucleotide recognition site.

A sequence-specific endonuclease present in extracts of Rhodopseudomonas sphaeroides 630 has been purified and characterized. The enzyme, Rsr II, recognises and cleaves the palindromic heptanucleotide sequence: (sequence; see test) By virtue of its unusual specificity, RsrII cuts most DNA molecules very infrequently which should facilitate the physical mapping of large genomes.     

Construction of DNA restriction maps based on a simplified experiment

MOTIVATION: A formulation of a new problem of the restriction map construction based on a simplified digestion experiment and a development of an algorithm for solving both ideal and noisy data cases of the introduced problem. RESULTS: A simplified partial digest problem and a branch and cut algorithm for finding the solution of the problem.     

Errors between sites in restriction site mapping

Restriction site mapping programs construct maps by generatingpermutations of fragments and checking for consistency. Unfortunatelymany consistent maps often are obtained within the experimentalerror bounds, even though there is only one actual map. A particularlyefficient algorithm is presented that aims to minimize errorbounds between restriction sites. The method is generalizedfor linear and circular maps. The time complexity is derivedand execution times are given for multiple enzymes and a rangeof error bounds. Received on July 17, 1987; accepted on November 3, 1987     

EcoRI restriction endonuclease cleavage site map of bacteriophage P22DNA.

The F plasmid is able to co-transfer (mobilize) the small, chimeric R plasmid pBR322 during conjugation only at a very low frequency (Bolivar et al., 1977). Mobilization has been found here to be invariably (> 99%) associated with a structural alteration of pBR322. The alteration was shown, by restriction endonuclease analysis and electron microscopy, to be an insertion of the F attachment sequence λδ (2.8 to 8.5F). λδ is, therefore, an insertion sequence.     

Determination of isocyanate biomarkers in construction site workers.

4,4'-Methylenediphenyl diisocyanate (MDI) is the most important isocyanate in the manufacture of polyurethanes, dyes, pigments and adhesives. High concentrations of isocyanates are a potent respiratory irritant. Therefore, it is important to develop methods to monitor exposure to such compounds. We monitored biological samples from 40 non-exposed and 45 exposed construction site workers. 4,4'-Methylenedianiline (MDA) and N-acetyl-4,4'-MDA (AcMDA) were determined from untreated urine (U-MDA, U-AcMDA) and MDA was analysed from acid-treated urine (U-MDA-tot). Haemoglobin (Hb) adducts of MDA (Hb-MDA) were determined in all workers. The levels of biomarkers decreased in the following order: U-MDA-tot>U-AcMDA>U-MDA>Hb-MDA. The same order was found for the percentage of samples, which were found positive in exposed workers: 100%, 91%, 91%, 27%. The urine levels U-MDA-tot correlate with U-MDA, U-AcMDA and Hb-MDA with r=0.79, 0.86 and 0.39, respectively (Spearman rank order, p<0.01). U-AcMDA correlates with U-MDA and Hb-MDA with r=0.77 and 0.47, respectively (p<0.01). U-MDA correlates with Hb-MDA (r=0.38, p<0.05). The levels in the controls were significantly lower than in the exposed workers for all compounds (Mann-Whitney test, p<0.01). The median isocyanate-specific IgE-level was higher in the exposed workers, but the difference was statistically not significant. The change of the biomarker levels was compared in a group of workers (n=20), which were analysed prior to isocyanate exposure and after the exposure for approximately 4-7 months. All urine MDA metabolites and the Hb-adduct levels increased significantly (Wilcoxon sign test, p<0.01). Total IgE increased significantly after the exposure with isocyanate activity (p<0.01). With the present work it could be shown that outdoor workers are exposed to a similar extent as workers from a MDI factory.     

A simple computer program for calculating, modifying and drawing circular restriction maps.

An HPL program is described which constructs and draws circular restriction maps given a set of cleavage sites, together with deletion of insertion data if required. This program is of great use in the routine interpretation of restriction patterns for the analysis of recombinant DNA molecules.     

Neuron-specific restriction of a herpes simplex virus recombinant maps to the UL5 gene.

We have previously shown that, when compared with either parent, a herpes simplex virus type 1/herpes simplex virus type 2 intertypic recombinant (R13-1) is attenuated by 10,000-fold with respect to neurovirulence in mice. Despite this, after intracranial inoculation, R13-1 replicated to titers of 10(5) PFU per brain. We present evidence that the restriction is specific for replication in neurons and have taken a three-step approach in determining the basis of the attenuation by (i) characterizing cellular tropism of the virus in both central and peripheral nervous systems, (ii) defining where in the viral replication cycle the restriction is manifest, and (iii) identifying the genetic basis of the restriction through marker rescue analysis. Following inoculation into the animal, R13-1 viral antigens predominate in nonneuronal cells, and the block to replication in neurons was found to be beyond the level of adsorption and penetration. Despite the restricted replication within neurons, the virus established a latent infection in spinal ganglia and could be reactivated by in vitro cocultivation of the ganglia. In studies carried out in cell culture, R13-1 was found to replicate normally in mouse embryo fibroblasts and primary mouse glial cells but was restricted by 1,000-fold in primary mouse neurons and PC12 cells. R13-1 appeared to produce normal levels of early RNA in these cells, but production of DNA and late RNA was less than that of the wild type. Marker rescue analysis localized the fragment responsible for restoring neurovirulence to UL5, a component of the origin-binding complex implicated in replication of the viral genome. Our results with this virus, with a cell-specific restriction, suggest that a neuron-specific component is involved in viral replication.