Restriction endonuclease analysis for the identification of baculovirus pesticides.

Gel electrophoresis of deoxyribonucleic acid (DNA) fragments generated by digesting the DNA genomes of nuclear polyhedrosis viruses (NPV) with restriction endonucleases provides DNA fragment patterns that may be used to identify different viruses of this group. Characteristic fragment patterns were obtained for three NPVs, which are important as biological pesticides (Autographa californica NPV, Orgyia pseudotsugata NPV, and Heliothis zea NPV). The DNA fragment patterns of the A. californica NPV genoms did not change with passage through the alternate insect host, Trichoplusia ni. Heterogeneity in one preparation of O. pseudotsugata NPV was observed. The identification procedure is direct and precise. Applications of this procedure include quality control of commercial preparations of viral pesticides and screening for genetic alterations in the viruses.     

Restriction endonuclease analysis of four Borrelia burgdorferi strains

Abstract A restriction endonuclease analysis was performed on four strains of Borrelia burgdorferi : one isolated from man (SF), one from Ixodes dammini (B31) and two form I. ricinus (BITS in Italy and B45 in Germany). Digestion by Taq I and Hae III gave the best resolution of the DNA fragments. Three different restriction patterns were obtained: BITS and B45 showed only one band difference. These results correlate with the reactivity of the four strains with monoclonal antibodies.     

Restriction endonuclease analysis of four Borrelia burgdorferi strains

A restriction endonuclease analysis was performed on four strains of Borrelia burgdorferi: one isolated from man (SF), one from Ixodes dammini (B31) and two from I. ricinus (BITS in Italy and B45 in Germany). Digestion by Taq I and Hae III gave the best resolution of the DNA fragments. Three different restriction patterns were obtained: BITS and B45 showed only one band difference. These results correlate with the reactivity of the four strains with monoclonal antibodies.     

Restriction endonuclease analysis of the ilvGEDA operon of members of the family Enterobacteriaceae.

Four of the genes required for the biosynthesis of isoleucine and valine form the ilvGEDA operon in Escherichia coli K-12 and Salmonella typhimurium. The structural relationship of these genes was examined in eight other members of the family Enterobacteriaceae by genomic Southern blot hybridization. These genes are contiguous in all the strains examined, and specific restriction sites appear to be highly conserved, indicating the possible functional importance of the DNA sequences of which they are part.     

Restriction endonuclease analysis of the transducing bacteriophage lambda rif d18

The rif d18 bacteriophage carries essential parts of the E. coli genome which can not be mapped by conventional methods. The phage DNA was analysed with four restriction endonucleases (endo R. BamHI, Sall, HpaI and EcoRI) and a physical map was constructed.     

Restriction endonuclease analysis of chloroplast DNA from streptomycin-resistant mutants of Nicotiana plumbaginifolia.

Chloroplast DNA isolated from wild-type Nicotiana plumbaginifolia and 12 maternally inherited streptomycin-resistant mutants was digested with various restriction enzymes and the resultant patterns were compared. No gross structural alterations of the chloroplast genome were detected in any mutants; however, variant patterns owing to the gain or loss of a restriction site were found in three mutants, SR1007, SR1019, and SR1022. The variant patterns in SR1019 and SR1022 are identical and are the results of mutation in the psbG gene coding for a chloroplast membrane protein G, and that in SR1007 is due to mutation in the 16S rRNA gene. Inheritance of the variant patterns in mutants SR1007 and SR1019 was studied. The results showed that the variant patterns and streptomycin resistance were co-transmitted in reciprocal crosses.     

Restriction endonuclease activities in the legionellae

Fifteen legionellae isolates were studied for the presence of restriction endonucleases. At least three different specificities were found in nine of these isolates. One particular activity was present in two groups of isolates isolated from widely separated geographic areas.     

Restriction endonuclease fingerprinting analysis of Canadian isolates of Aeromonas salmonicida

Restriction endonuclease fingerprinting (REF) analysis was used to examine total cellular DNA prepared from 56 independent field isolates of the fish pathogen, Aeromonas salmonicida. DNA was digested singly with the restriction enzymes EcoRI and HindIII, and the resulting fragments separated by polyacrylamide gel electrophoresis and visualized by silver staining. The REF patterns of typical isolates of A. salmonicida subsp. salmonicida were distinct from those of A. hydrophila, A. salmonicida subsp. achromogenes, A. salmonicida subsp. masoucida, and atypical isolates of A. salmonicida subsp. salmonicida. Differences between strains of typical A. salmonicida subsp. salmonicida could also be distinguished. Canadian isolates examined could be assigned to 1 of 12 different groups (REF groups), with the majority of the isolates belonging to REF groups 1 and 5. REF group 1 strains were isolated from British Columbia and New Brunswick while REF group 5 isolates were found in Ontario. None of the European strains examined had REF patterns identical to those of Canadian isolates. Based on REF analysis, there was little genetic heterogeneity detected among 23 isolates from two short-term studies of naturally occurring infections. Several different REF groups were seen among A. salmonicida collected over a 10-year period from coho salmon from the Credit River. Consistent with earlier biochemical and hybridization studies, the REF data suggest that A. salmonicida is a clonal pathogen. REF analysis can, however, permit the identification of subgroups, which may be useful in epidemiological studies.     

Restriction endonuclease analysis of human globin genes in cellular DNA

Using samples of human cellular DNA digested with restriction endonucleases Eco RI, Hind III, Hinc II, Bam HI, Alu I, or Hae III, we were able to localize globin gene fragments separated by agarose gel electrophoresis. The fragments were transferred to nitro-cellulose filters and identified by hybridization to [³²P] cDNA for total adult globin mRNA. The α-globin gene fragments were specifically identified by their presence in normal controls and absence in DNA from homozygous α-thalassemia, a genetic disorder due to deletion of α-globin genes. In addition, the patterns with Hind III indicate a 4.1 kb distance between the centers of the normal duplicated α-globin gene loci.     

Restriction endonuclease map of Euglena gracilis chloroplast DNA.

A physical map of the Euglena gracilis chloroplast genome has been constructed, based on cleavage sites of Euglena gracilis chloroplast DNA treated with bacterial restriction endonucleases. Covalently close, circular chloroplast DNA is cleaved by restriction endonuclease SalI into three fragments and by restriction endonuclease BamHI into six fragments. These nine cleavage sites have been ordered by fragment molecular weight analysis, double digestions, partial digestions, and by digestion studies of isolated DNA fragments. A fragment pattern of the products of EcoRI restriction endonuclease digestion of Euglena chloroplast DNA is also described. One of these fragments has been located on the cleavage site map.     

Restriction endonuclease cleavage map of the DNA of JC virus.

A physical map of the sites cleaved by the following restriction endonucleases was derived for the DNA of JC virus, a human polyomavirus: EcoRI, HpaI, and PstI (one site each); HindII (four sites); and HindIII (three sites). By agarose gel electrophoresis of fragmented DNA, the size of full-length DNA of JC virus was estimated to be 5,125 +/- 105 base pairs (98 +/- 2% of the length of simian virus 40 DNA).     

Restriction endonuclease DNA analysis of Leptospira interrogans serovars Icterohaemorrhagiae and Copenhageni

Leptospira interrogans serovar icterohaemorrhagiae strains Ictero No. I and RGA and serovar copenhageni strains M20, Shiromizu and Shibaura were examined by restriction endonuclease DNA analysis. Fifteen endonucleases (AluI, BamHI, BglII, EcoRI, HaeIII, HhaI, HindIII, KpnI, PstI, SacI, SalI, SmaI, StyI, XbaI and XhoI) were used as the digesting enzymes. Strain Ictero No. I showed endonuclease cleavage patterns which differed from those of the other four strains only when it was digested with enzymes KpnI and HindIII. When digested with KpnI, an extra band of about 5.4 kb was clearly produced, and when digested with HindIII, an extra band of about 25 kb was produced. When the other 13 enzymes were used, no differences were found between the endonuclease cleavage patterns among the five strains. Moreover, strains RGA, M20, Shiromizu and Shibaura could not be distinguished by the restriction endonuclease DNA analysis using all 15 endonucleases. In addition, six newly isolated leptospires from patients with leptospirosis and from Rattus norvegicus were compared with the Ictero No. I and M20 strains, by restriction endonuclease DNA analysis using enzymes KpnI and HindIII. Three leptospires belonging to serovar icterohaemorrhagiae showed the same endonuclease cleavage patterns as the M20 strain. The other three strains, which belong to serovar copenhageni, showed almost the same endonuclease cleavage patterns as the M20 strain; only the Kai ima 702 strain produced an extra band which was not identical to the Ictero No. I-specific extra band when digested with HindIII. The leptospiral restriction endonuclease DNA analysis has revealed taxonomic structures that are unrecognized by serology alone.     

Specificity of the endonuclease activity of the baculovirus alkaline nuclease for single-stranded DNA

The Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) alkaline nuclease (AN) likely participates in the maturation of virus genomes and in DNA recombination. AcMNPV AN was expressed in a recombinant baculovirus as a His -tagged fusion and obtained in pure form (*AN) or as a (6)complex with the baculoviral single-stranded DNA-binding protein LEF-3 (*AN/L3). Both AN preparations possessed potent 5' --> 3'-exonuclease and weak endonuclease activities. Mutant *AN(S146A)/L3 with a change from serine to alanine at position 146 in a conservative motif was impaired in both activities. This proved that the endonuclease is an intrinsic activity of baculovirus AN. The AN endonuclease showed specificity for single-stranded DNA and converted supercoiled plasmid DNA (replicative form I, RFI) into the open circular form (RFII) by a single strand break. Plasmid DNA relaxed with topoisomerase I was resistant to *AN/L3 indicating that the partially single-stranded regions in negatively supercoiled molecules served as targets for the endonuclease. Unwinding the supercoiled DNA with ethidium bromide also made DNA resistant to AN/L3. In reactions with nicked circular DNA (RFII), AN and AN/L3 hydrolyzed exonucleolytically the broken strand or cut endonucleolytically the intact strand at the position opposite the nick (gap). When LEF-3 was added to the assay, the balance between the exonucleolytic and endonucleolytic modes of hydrolysis shifted in favor of the exonuclease. The data suggest that the AN endonuclease may digest the intermediates in replication and recombination at positions of structural irregularities in DNA duplexes, whereas LEF-3 may further regulate processing of the intermediates by AN via the endonuclease and exonuclease pathways.     

Restriction endonuclease analysis of ribosomal DNA from Saccharomyces cerevisiae

Summary Temperature-sensitive mutants of Escherichia coli that are unable to grow at high temperature can be obtained among those selected for resistance to streptovaricin or rifampicin at low temperature (Yura et al., 1970). One of these mutants (KY5323) that was supposed to carry a single mutation affecting both rifampicin resistance and temperature sensitivity was further investigated. Using purified RNA polymerase preparations obtained from the mutant and the wild type, it was found that the activity for DNA chain elongation is more sensitive to heat treatment than that for RNA chain initiation or DNA binding, and that the mutant enzyme is significantly more labile than the wild-type enzyme with respect to RNA chain elongation, when heat treatment is carried out at high salt concentration. These results, taken together with those of the enzyme reconstitution experiments, strongly suggest that the subunit of the polymerase is directly involved in both RNA chain initiation and elongation reactions. Enzyme reconstitution experiments using isolated subunits derived from the mutant and the wild-type polymerases demonstrate that the alteration of subunit is primarily responsible for both rifampicin resistance and thermolability of the mutant enzyme. In addition, the results suggested the apparent alteration of both and subunits in this mutant. Extensive transduction experiments provided genetic evidence that are consistent with the view that the strain KY5323 carries a second mutation affecting the subunit, beside the primary mutation affecting the subunit. The hypothetical subunit mutation seems to modify quantitatively the rifampicin resistance caused by the subunit mutation.     

Restriction endonuclease analysis of the genomes of virulent and avirulent Marek's disease viruses

The DNAs from virulent strain BC-1 and avirulent strains C2(A) of Marek's disease virus (MDV) were compared by electrophoresis on 0.5% agarose gels of the products obtained with the restriction endonucleases Bam H1, Sal 1, and Sma 1. The patterns of the fragments of the DNAs from these two strains were very similar, but showed some significant differences in the number and mobility of the DNA bands. The DNA fragments obtained with the restriction endonucleases, and especially Sma 1, were mostly present in equal molar ratios, but a few of those obtained with Bam H1 and Sal 1 were present in submolar amounts. In addition, several fragments obtained with Bam H1 and Sal 1 were present in greater than molar quantities, suggesting the presence of reiterated sequences in MDV DNA. The terminal fragments of MDV DNA were identified by their sensitivity to lambda 5'-exonuclease. The terminal fragments obtained with Bam H1 A and Sal 1B showed heterogeneous electrophoretic mobility and contained sequences with high content of guanosine and cytosine, suggesting the presence of reiterated sequences at the end of the MDV DNA molecule.     

Restriction endonuclease mapping of the colicin Ib plasmid

Summary A cleavage site map of the colicin Ib plasmid (ColIb) has been determined for the enzymes Sall, XhoI, and HindIII by analysis of partial digests, double digests, DNA-DNA hybridization, and Tn5-induced insertion mutants. The site of the colicin gene has been determined by probing with cloned DNA coding for colicin production, as well as by analysis of a colicin negative ColIb:Tn5.     

Restriction endonuclease analysis of chloroplast DNA in interspecies somatic Hybrids of Petunia

Summary Restriction endonuclease cleavage pattern analysis of chloroplast DNA (cpDNA) of three different interspecific somatic hybrid plants revealed that the cytoplasms of the hybrids contained only cpDNA of P. parodii. The somatic hybrid plants analysed were those between P. parodii (wild type) + P. hybrida (wild type); P. parodii (wild type)+P. inflata (cytoplasmic albino mutant); P. parodii (wild type) + P. parviflora (nuclear albino mutant). The presence of only P. parodii chloroplasts in the somatic hybrid of P. parodii + P. inflata is possibly due to the stringent selection used for somatic hybrid production. However, in the case of the two other somatic hybrids P. parodii + P. hybrida and P. parodii + P. parviflora it was not possible to determine whether the presence of only P. parodii chloroplasts in these somatic hybrid plants was due to the nature of the selection schemes used or simply occurred by chance. The relevance of such somatic hybrid material for the study of genomic-cytoplasmic interaction is discussed, as well as the use of restriction endonuclease fragment patterns for the analysis of taxonomic and evolutionary inter-relationships in the genus Petunia.     

A single PCR-restriction endonuclease analysis for rapid identification of Malassezia species

AIMS: The present study describes a system based on PCR and restriction endonuclease analysis (REA) to distinguish the seven currently recognized Malassezia species. METHODS AND RESULTS: Fifty-five representative yeast isolates were examined. A single primer pair was designed to amplify the large subunit ribosomal RNA (LSU rRNA) gene of the seven Malassezia species, and identification was achieved by digestion of the PCR products with three restriction endonucleases: BanI, HaeII and MspI. A specific restriction endonuclease analysis pattern was determined for each species investigated. Moreover, PCR-REA allowed the detection and characterization of mixtures of several Malassezia species. CONCLUSION: PCR-REA of only the LSU rRNA gene is a reliable and rapid method to distinguish all Malassezia species. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR-REA represents a considerable saving in time over currently available identification procedures. This method should be evaluated on clinical material directly.