Analysis of Actinobacillus pleuropneumoniae and related organisms by DNA-DNA hybridization and restriction endonuclease fingerprinting.

The objective of this study was to determine the degree of genetic relatedness of Actinobacillus pleuropneumoniae to selected members of the family Pasteurellaceae, with particular emphasis on species commonly associated with swine. Free-solution DNA-DNA hybridization studies revealed that representative strains of all 12 serotypes of A. pleuropneumoniae formed a homogeneous group, sharing 74 to 90% sequence homology with A. pleuropneumoniae serotype 1. All serotypes of A. pleuropneumoniae tested demonstrated a high degree of genetic relatedness (66 to 79%) to the type species of the genus Actinobacillus, A. lignieresii. Little homology (less than 20%) was detected between A. pleuropneumoniae strains and selected Haemophilus spp. and Pasteurella spp. Since free-solution hybridization methods are technically demanding and require large amounts of highly purified DNA, restriction endonuclease fingerprinting (REF) was examined to determine whether it could be a useful taxonomic tool for classification of members of the family Pasteurellaceae. REF profiles were compared, and the degree of similarity between organisms was quantitated by calculating Jaccard similarity coefficients. There was a significant positive relationship between the REF Jaccard coefficients and the DNA homology values determined from free-solution hybridization experiments.     

Analysis of production brewing strains of yeast by DNA fingerprinting

P. WIGHTMAN, D.E. QUAIN AND P. G. MEADEN. 1996. Production brewing strains of the yeast Saccharomyces cerevisiae were analysed by DNA fingerprinting, using a Southern blotting and hybridization procedure and employing the Tyl-15 transposon as a probe. The ability to differentiate readily between strains was very dependent on the restriction enzyme used to digest the DNA prior to Southern blotting and hybridization; the enzymes Eco RI, Pst I and Sal I were found to be particularly useful in this respect. The method was applicable to the differentiation of both ale and lager yeasts, and was sufficiently sensitive to distinguish between very closely related strains. DNA fingerprinting by this approach confirmed, for example, that a flocculent strain isolated during a production-scale fermentation with a lager yeast was genotypically different from the parent.     

Sequence-specific protection of plasmid DNA from restriction endonuclease hydrolysis by pyrrole-imidazole-cyclopropapyrroloindole conjugates

The pyrrole-imidazole (Py-Im) triamide-cyclopropa pyrroloindole (CPI) conjugates ImPyImLDu86 (7) and ImImPyLDu86 (14) were synthesized and their alkylating activities and inhibitory effects on DNA hydrolysis by restriction endonucleases were examined. Sequencing gel analysis demonstrated that conjugates 7 and 14 specifically alkylated DNA at 5'-CGCGCG-3' and 5'-PyGGCCPu-3', respectively. Agarose gel electrophoresis indicated that incubation of a supercoiled plasmid, pSPORT I (4109 bp), with conjugate 7 effectively inhibited its hydrolysis by BssHII (5'-G_CGCGC-3'), whereas conjugate 14 had no effect on this hydrolysis. These results suggest that conjugate 7 sequence-specifically inhibits the hydrolysis of DNA by BssHII. Sequence-specific alkylation by the Py-Im triamide-CPI conjugates was further confirmed by inhibition of the Eco52I (5'-C_GGCCG-3') hydrolysis of conjugate 14-treated pQBI PGK (5387 bp). In clear contrast, hydrolysis of pQB1 PGK by DraI (3'-TTT_AAA-3') was not inhibited by 5 micro M conjugate 14. That ImImPy did not inhibit the hydrolysis of pQB1 PGK indicates that covalent bond formation is necessary for inhibition. A similar experiment, using linear pQBI PGK, achieved the same extent of protection of the DNA with approximately half the concentration of conjugate 14 as was required to protect supercoiled DNA from hydrolysis.     

Plasmid profiles and genomic DNA restriction endonuclease patterns of 30 independent Staphylococcus lugdunensis strains

Extrachromosomal DNA analysis and restriction endonuclease analysis of whole cellular DNA were used to characterize 30 Staphylococcus lugdunensis strains isolated from 13 different hospitals from 1977 to 1988. All the strains were susceptible to most of the antibiotics tested, including penicillin G. A single 3.2 kilobase plasmid was detected in 13 strains and one or two plasmids, ranging from 2.3 to 6.6 kilobases, were found in 7 strains. EcoRI, PstI and PvuII restriction patterns of total cellular DNA were identical for 23 isolates, indicating strong conservation of endonuclease sites in this species. One or two additional DNA bands occurred in seven isolates. Molecular markers show rather little variations between different S. lugdunensis isolates suggesting that they are closely related.     

Selective inhibition of restriction endonuclease cleavage by DNA intercalators

The preferred dye binding sites and the microenvironment of known nucleotide sequences within mitochondrial and plasmid pBR322 DNA was probed in a gross fashion with restriction endonucleases. The intercalating dyes, ethidium bromide and propidium iodide, do not inhibit a given restriction endonuclease equally at all of the restriction sites within a DNA molecule. The selective inhibition may be explained, in part, by the potential B to Z conformation transition of DNA flanking the restriction site and by preferred dye binding sites. Propidium iodide was found to be a more potent inhibitor than ethidium bromide and the inhibition is independent of the type of cut made by the enzyme.     

Modes of DNA cleavage by the EcoRV restriction endonuclease

The mechanism of action of the EcoRV restriction endonuclease at its single recognition site on the plasmid pAT153 was analyzed by kinetic methods. In reactions at pH 7.5, close to the optimum for this enzyme, both strands of the DNA were cut in a single concerted reaction: DNA cut in only one strand of the duplex was neither liberated from the enzyme during the catalytic turnover nor accumulated as a steady-state intermediate. In contrast, reactions at pH 6.0 involved the sequential cutting of the two strands of the DNA. Under these conditions, DNA cut in a single strand was an obligatory intermediate in the reaction pathway and a fraction of the nicked DNA dissociated from the enzyme during the turnover. The different reaction profiles are shown to be consistent with a single mechanism in which the kinetic activity of each subunit of the dimeric protein is governed by its affinity for Mg2+ ions. At pH 7.5, Mg2+ is bound to both subunits of the dimer for virtually the complete period of the catalytic turnover, while at pH 6.0 Mg2+ is bound transiently to one subunit at a time. The kinetics of the EcoRV nuclease were unaffected by DNA supercoiling.     

DNA binding and recognition by the IIs restriction endonuclease MboII

The type IIs restriction endonuclease MboII recognizes nonsymmetrical GAAGA sites, cutting 8 (top strand) and 7 (bottom strand) bases to the right. Gel retardation showed that MboII bound specifically to GAAGA sequences, producing two distinct complexes each containing one MboII and one DNA molecule. Interference analysis indicated that the initial species formed, named complex 1, comprised an interaction between the enzyme and the GAAGA target. Complex 2 involved interaction of the protein with both the GAAGA and the cutting sites. Only in the presence of divalent metal ions such as Ca(2+) is the conversion of complex 1 to 2 rapid. Additionally, a very retarded complex was seen with Ca(2+), possibly a (MboII)(2)-(DNA)(2) complex. Plasmids containing a single GAAGA site were hydrolyzed slowly by MboII. Plasmids containing two sites were cut far more rapidly, suggesting that the enzyme requires two recognition sites in the same DNA molecule for efficient hydrolysis. MboII appears to have a mechanism similar to the best characterized type IIs enzyme, FokI. Both enzymes initially bind DNA as monomers, followed by dimerization to give an (enzyme)(2)-(DNA)(2) complex. Dimerization is efficient only when the two target sites are located in the same DNA molecule and requires divalent metal ions.     

Protein assembly and DNA looping by the FokI restriction endonuclease

The FokI restriction endonuclease recognizes an asymmetric DNA sequence and cuts both strands at fixed positions upstream of the site. The sequence is contacted by a single monomer of the protein, but the monomer has only one catalytic centre and forms a dimer to cut both strands. FokI is also known to cleave DNA with two copies of its site more rapidly than DNA with one copy. To discover how FokI acts at a single site and how it acts at two sites, its reactions were examined on a series of plasmids with either one recognition site or with two sites separated by varied distances, sometimes in the presence of a DNA-binding defective mutant of FokI. These experiments showed that, to cleave DNA with one site, the monomer bound to that site associates via a weak protein–protein interaction with a second monomer that remains detached from the recognition sequence. Nevertheless, the second monomer catalyses phosphodiester bond hydrolysis at the same rate as the DNA-bound monomer. On DNA with two sites, two monomers of FokI interact strongly, as a result of being tethered to the same molecule of DNA, and sequester the intervening DNA in a loop.     

Characterization of strains of Leuconostoc mesenteroides by analysis of soluble whole-cell protein pattern, DNA fingerprinting and restriction of ribosomal DNA

Of 215 leuconostocs isolated from field grass, natural whey cultures and water-buffalo milk, 178 were identified as Leuconostoc mesenteroides ssp. mesenteroides while 37 strains could not be identified Biochemical characterization allowed seven groups to be defined. Representative strains of each group and different habitat and nine reference strains were selected for further analyses. Protein profiles appeared suitable for species discrimination, but did not differentiate between the three subspecies of Leuc. mesenteroides. The technique also showed some differences among equivocal strains. DNA fingerprinting for most strains of Leuc. mesenteroides ssp. mesenteroides examined showed a different restriction pattern from that of the type strain. Ribotyping was not useful for discriminating species and subspecies of the genus Leuconostoc: Leuc. mesenteroides ssp. mesenteroides and ssp. dextranicum showed the same ribopattern as Leuc. lactis while Leuc. mesenteroides ssp. cremoris exhibited a pattern distinct from all the other species examined. On the basis of ARDRA-PCR, two main groups could be distinguished: the larger group included Leuc. mesenteroides, Leuc. lactis, Leuc. pseudomesenteroides and some unidentifiable strains; the second one included Leuc. citreum, Leuc. fallax, Weissella paramesenteroides and some unidentified strains.     

The DNA sequence recognised by the HinfIII restriction endonuclease

HinfIII is a type III restriction enzyme (Kauc &; Piekarowicz, 1978) isolated from Haemophilus influenzae Rf. Like other type III restriction endonucleases, the enzyme also catalyses the modification of susceptible DNA. It requires ATP for DNA cleavage and S-adenosyl methionine for DNA methylation. We have determined the DNA sequence recognised by HinfIII to be:

$\text{5′-C-G-A-A-T-3′}\text{·····3′-G-C-T-T-A-5′}$

In restriction, the enzyme cleaves the DNA about 25 base-pairs to the right of this sequence. In the modification reaction only one of the strands is methylated, that containing the 5′-C-G-A-A-T-3′ sequence.     

Discrimination between DNA sequences by the EcoRV restriction endonuclease

The EcoRV restriction endonuclease cleaves not only its recognition sequence on DNA, GATATC, but also, at vastly reduced rates, a number of alternative DNA sequences. The plasmid pAT153 contains 12 alternative sites, each of which differs from the recognition sequence by one base pair. The EcoRV nuclease showed a marked preference for one particular site from among these alternatives. This noncognate site was located at the sequence GTTATC, and the mechanism of action of EcoRV at this site was analyzed. The mechanism differed from that at the cognate site in three respects. First, the affinity of the enzyme for the noncognate site was lower than that for the cognate site, but, by itself, this cannot account for the specificity of EcoRV as measured from the values of kcat/Km. Second, the enzyme had a lower affinity for Mg2+ when it was bound to the noncognate site than when it was bound to its cognate site: this appears to be a key factor in limiting the rates of DNA cleavage at alternative sites. Third, the reaction pathway at the noncognate site differed from that at the cognate site. At the former, the EcoRV enzyme cleaved first one strand of the DNA and then the other while at the latter, both strands were cut in one concerted reaction. The difference in reaction pathway allows DNA ligase to proofread the activity of EcoRV by selective repair of single-strand breaks at noncognate sites, as opposed to double-strand breaks at the cognate site. The addition of DNA ligase to reactions with EcoRV made no difference to product formation at the cognate site, but products from reactions at noncognate sites were no longer detected.     

Subunit assembly for DNA cleavage by restriction endonuclease SgrAI

The SgrAI endonuclease usually cleaves DNA with two recognition sites more rapidly than DNA with one site, often converting the former directly to the products cut at both sites. In this respect, SgrAI acts like the tetrameric restriction enzymes that bind two copies of their target sites before cleaving both sites concertedly. However, by analytical ultracentrifugation, SgrAI is a dimer in solution though it aggregates to high molecular mass species when bound to its specific DNA sequence. Its reaction kinetics indicate that it uses different mechanisms to cleave DNA with one and with two SgrAI sites. It cleaves the one-site DNA in the style of a dimeric restriction enzyme acting at an individual site, mediating neither interactions in trans, as seen with the tetrameric enzymes, nor subunit associations, as seen with the monomeric enzymes. In contrast, its optimal reaction on DNA with two sites involves an association of protein subunits: two dimers bound to sites in cis may associate to form a tetramer that has enhanced activity, which then cleaves both sites concurrently. The mode of action of SgrAI differs from all restriction enzymes characterised previously, so this study extends the range of mechanisms known for restriction endonucleases.     

Computer-assisted restriction endonuclease analysis of plasmid DNA in field strains of Salmonella enteritidis.

Computer-assisted restriction endonuclease analysis of plasmid DNA in field strains of Salmonella enterica serovar Enteritidis (S. enteritidis) is described. The procedure consists of plasmid DNA purification, its digestion with restriction endonuclease TaqI, electrophoresis, charge-coupled device camera scanning of the gels, and an analysis of the restriction patterns with the software Gel Manager. The system allowed us to analyse, in detail, results of plasmid profiling in more than 600 field strains of S. enteritidis. In addition to plasmid-free and virulence plasmid only containing strains, 15 additional plasmid types were detected. All the images and detailed protocols are available at the Web site http://www.clark.cz/vri/salmon.htm.     

HLA-DQ polymorphism analyzed by sequential restriction endonuclease DNA digestion

A direct, physical correspondence between certain Pst I-generated genomic DNA fragments and Taq I-generated fragments, revealed with HLA-DQor -DQ gene probes, has been demonstrated. As an immediate consequence, the nature of the DQ and DX hybridized genes contained in the fragments was established. Taq I-generated DQ allelic forms which associate with serologically defined DRl1 DR2, and DRw6 specificities were also proven to be sensu stricto splits of the Pst I-generated allelic form associated with all three DR specificities.     

Methylation of f1 DNA by a restriction endonuclease from Escherichia coli B

Bacteriophage f1 duplex DNA containing hybrid SB sites, the genetic sites which confer upon DNA sensitivity to Escherichia coli B-specific restriction and modification, were prepared in vitro. The hybrid SB sites (modified and mutant) were tested for their ability to be methylated in vitro by endonuclease R · EcoB, the enzyme responsible for both B-specific restriction and modification in vivo. DNA containing hybrid (modified) SB sites can be methylated. One methyl group is added to the DNA per hybrid (modified) SB site. On the other hand, DNA containing hybrid (mutant) SB sites is refractory to modification.The nature and the function of the SB site as well as the implications of these observations for f1 recombination are discussed.     

Characterization of mitochondrial DNA in various Candida species: isolation, restriction endonuclease analysis, size, and base composition.

A practical and effective method for the extraction of mitochondrial DNA from Candida species was developed. Zymolyase was used to induce yeast protoplasts, and mitochondrial DNA was extracted from DNase I-treated mitochondrial preparations. Restriction endonuclease analyses of mitochondrial DNAs from 19 isolates representing seven species of Candida (C. albicans, C. kefyr, C. lusitaniae, C. maltosa, C. parapsilosis, C. shehatae, and C. tropicalis) and Lodderomyces elongisporus revealed different cleavage patterns that appeared to be specific for the species. Few common restriction fragments were evident. The genome sizes of the mitochondrial DNAs ranged from 26.4 to 51.4 kilobase pairs, and the guanine-plus-cytosine contents ranged from 20.7 to 36.8 mol%. There was no correlation between the base compositions of nuclear and mitochondrial DNAs. Eight isolates of C. parapsilosis, including the type culture, and an ascosporogenous strain of L. elongisporus, which was once proposed as the teleomorph of C. parapsilosis, had similar mitochondrial DNA molecular sizes (30.2 and 28.8 kilobase pairs); however, restriction endonuclease patterns of these organisms were distinct. These data provide additional support for discrimination of these two species. The results of our experiments demonstrate that mitochondrial DNA analyses may provide useful criteria for the differentiation of yeast species.