Comparative restriction analysis of chromosomal DNA of strains of Photobacterium leiognathi

Chromosomal DNA in 5 hereditary variants occurring in Photobacterium leiognathi population was subjected to restriction analysis. The variants differed in the levels and regulation of luminescence and colony morphology. Agarose electrophoresis of DNA fragments isolated after exposure to Hind II, Bam HI, Bgl I and Pst I restriction endonucleases revealed respectively 38, 28, 35 and 29 fragments equally distributed by their molecular weights. Electrophoregrams of the 5 strains were absolutely identical. After exposure of DNA of all the strains to PVu II, Xho II, Sal GI and Eco RI restriction endonucleases there were detected no fragments. The pleoiotropic genetic variation in these strains was not associated with large deletions or amplification of chromosomal DNA regions.     

Comparative restriction enzyme analysis of the genome in variola virus strains from the Russian collection

Comparative RFLP analysis was for the first time performed for 21 variola virus (VARV) strains of the Russian collection with 20 amplicons covering the total VARV genome. The amplicons were synthesized in the long polymerase chain reaction. A database useful as a reference for identifying VARV strains was generated. VARV strains isolated in different geographical regions were compared and proved to vary mostly in variable genome regions. Each of the dendrograms constructed included three clusters of African, Asian, and VARV-alastrim isolates. The VARV-alastrim isolates differed to the greatest extent from the other strains. VARV strains isolated during an ecdemic variola burst in Moscow (1960) grouped with Asian isolates. Polymorphism of VARV strains was for the first time observed for a single variola burst with a few affected patients.     

Use of a restriction endonuclease in analyzing the genomes from two different strains of vaccinia virus.

A restriction endonuclease from Haemophilus influenzae (Hind III) specifically cleaved vaccinia DNA into 14 fragments. The molecular weights of these fragments were determined by gel electrophoresis and ranged from 0.5 x 10(6) to 30 x 10(6). Hind III digestion of the DNA from the WR and CV-1 strains of vaccinia revealed a small molecular difference in one of the resulting fragments. The average molecular weight of the entire vaccinia genome was calculated to be 125 x 10(6).     

Molecular genetic analysis of vaccinia virus DNA polymerase mutants.

To further our understanding of the structure and function of the vaccinia virus DNA polymerase, we have performed fine genetic analysis of three mutants with lesions in the polymerase gene. By performing marker rescue analysis with DNA fragments of decreasing size, each lesion was localized to within 500 base pairs of DNA. The relevant regions of the mutant alleles were then cloned and subjected to DNA sequence analysis, which allowed the assignment of a single nucleotide and amino acid change to each mutant. As well as providing structure-function correlations germane to an understanding of polymerase activity, these data have provided insights into the frequency and possible mechanisms of viral homologous recombination.     

Genetic diversity of Azotobacter strains isolated from soils by amplified ribosomal DNA restriction analysis

Strains of Azotobacter mediate in the nitrogen fixation process by reducing of N₂ to ammonia. In this study, 50 strains were isolated from different rhizospheric soil in central Iran, by using soil paste-plate method. These strains were biochemically identified and characterized on differential LG medium based on morphological and physiological properties. Results obtained showed that identified strains were belonging to three species, namely A. chroococcum, A. vinelandii and A. beijernckii. In order to molecular analysis, the 16S rRNA gene was amplified using 27f and 1495r primers and PCR products were subsequently restricted with RsaI, HpaII and HhaI. Cluster analysis based on amplified ribosomal DNA restriction analysis were revealed intraspecific polymorphism and differentiated strains into two mains clusters, clusters A and B. Cluster A strains were related to the A. vinelandii, whereas cluster B strains were related to the A. chroococcum and A. beijerinckii. The results show that amplified ribosomal DNA restriction analysis is a powerful and discriminatory tool for the identification of members of the genus Azotobacter.     

Comparative analysis of west nile virus strains isolated from human and animal hosts using monoclonal antibodies and cDNA restriction digest profiles

The West Nile (WN) virus strains isolated in Bangui, Central African Republic (CAR), from patients with hepatitis were analysed comparatively with the prototype WN virus strain and 7 WN strains previously isolated from birds (2 strains), mosquitoes (3 strains) and ticks (2 strains) in CAR.The comparison was based on two techniques: an epitopic analysis by indirect immunofluorescence assay using a panel of 9 monoclonal antibodies to WN virus, and an analysis of HaeIII and TaqI restriction digest profiles of cDNA to infected cell RNA.Similar results were obtained with both techniques: the 3 human strains were found to be identical to each other and identical or very close to mosquito and tick strains, whereas prototype WN virus and bird strains were significantly different from the human strains.As “classical” infections due to WN virus without hepatic involvement were also reported during the period of isolation of the arthropod strains, we concluded that the same virus subtype may have been the cause of different infection patterns. A new definition of the disease spectrum of WN virus, including the possibility of liver involvement, should be established.Clearly, the Egyptian prototype WN virus represents a different topotype. Bird strains also appear to be different from human and arthropod strains, raising the question of their transmissibility and pathogenicity for man, and of the role of birds in the natural cycle of WN virus.     

Procedure for purification of intact DNA from vaccinia virus.

A procedure for the isolation of intact vaccinia DNA molecules by chromatography on hydroxyapatite in the presence of 6 M urea is described. When lysates of virions containing 0.5 to 10 microgram of DNA were employed, over 95% of the viral DNA could be recovered free of poteins. Vaccinia DNA molecules isolated in this manner sedimented at 68S in neutral sucrose gradients and had an average contour length of 62.3 micrometer when examined in an electron microscope, and the DNA could be cleaved with the restriction endonuclease EcoRI and BamHI. The results of these analyses showed that intact vaccinia DNA molecules of 120 X 10(6) to 130 X 10(6) molecular weight could be obtained by the procedures described.     

HindIII and Sst I restriction sites mapped on rabbit poxvirus and vaccinia virus DNA.

The DNAs of two closely related orthopoxviruses, rabbit poxvirus (RPV) and vaccinia virus (VV), were mapped by overlapping-fragment analysis using restriction endonucleases HindIII and Sst I. The exact arrangement of these fragments was accomplished by total digestion of isolated partial restriction products and by end-fragment determination. RPV and VV DNAs showed identical restriction patterns in an internal region comprising approximately 60% of the genome. The size, by electrophoretical analysis of the RPV DNA, was 118 X 10(6) daltons, some 6 X 10(6) daltons less than VV DNA. The two opposite terminal restriction fragments of RPV DNA cross-hybridized to each other.     

Differentiation of strains of bovine infectious rhinotracheitis virus using restriction analysis]

The virus of infectious rhinotracheitis of cattle (BHV-1) causes the respiratory diseases, encephalitis, vulvovaginitis, abortions, etc. in sensitive animals. The attempts to differentiate the viral strains by virology and serology methods were unsuccessful. The restriction analysis makes possible to divide the strains into the three main groups designated BHV-1.1, BHV-1.2 and BHV-1.3. Majority of authors note the lack of correlation between the restriction pattern of DNA and the syndromes caused by the strains of the first and second groups while all the representatives of the third group were isolated in case of meningoencephalitis. The restriction analysis of BHV-1 strains isolated in the USSR by restriction endonucleases EcoRI, BamHI, HpaI and ClaI is presented. The obtained results show the absence of correlation between the restriction pattern of the strain and the syndrome caused by the viruses of all three groups. The representatives of the group BHV-1.3 that are not associated with the neurological syndrome are isolated for the first time.     

Restriction enzyme mapping of vaccinia virus DNA.

The cleavage sites for the restriction enzymes Bg/I, HindIII, KpnI, SalI, SmaI, and XhoI were located, from primary data, on the DNA isolated from the WR strain of vaccinia virus. Bg/I and SmaI divide the DNA into five segments which can be isolated by sucrose gradient centrifugation. These large segments provide a convenient means to group segments produced by other enzymes. The construction of physical maps was initiated by identifying the segments at each end of the DNA and then finding segments which were adjacent to these terminal sections. This was accomplished by isolating large shear fragments which contained the covalently linked termini of the DNA. Most of the data needed to derive the maps were obtained by isolating segments produce by one enzyme and then cleaving these individual segments with a second enzyme.     

Mutational analysis of vaccinia virus topoisomerase identifies residues involved in DNA binding.

Vaccinia DNA topoisomerase catalyzes the cleavage and re-joining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(C/T)CCTT downward arrow. The 314 aa protein consists of three protease-resistant structural domains demarcated by protease-sensitive interdomain segments referred to as the bridge and the hinge. The bridge is defined by trypsin-accessible sites at Arg80, Lys83 and Arg84. Photocrosslinking and proteolytic footprinting experiments suggest that residues near the interdomain bridge interact with DNA. To assess the contributions of specific amino acids to DNA binding and transesterification chemistry, we introduced alanine substitutions at 16 positions within a 24 aa segment from residues 63 to 86(DSKGRRQYFYGKMHVQNRNAKRDR). Assays of the rates of DNA relaxation under conditions optimal for the wild-type topoisomerase revealed significant mutational effects at six positions; Arg67, Tyr70, Tyr72, Arg80, Arg84 and Asp85. The mutated proteins displayed normal or near-normal rates of single-turnover transesterification to DNA. The effects of amino acid substitutions on DNA binding were evinced by inhibition of covalent adduct formation in the presence of salt and magnesium. The mutant enzymes also displayed diminished affinity for a subset of cleavage sites in pUC19 DNA. Tyr70 and Tyr72 were subjected to further analysis by replacement with Phe, His, Gln and Arg. At both positions, the aromatic moiety was important for DNA binding.     

Silver staining of DNA restriction fragments for the ultrarapid identification of pseudorabies virus strains.

Restriction endonuclease analysis of pseudorabies virus DNA has been used to study various virus strains. To make use of a rapid technique for the identification of viral strains, studies have been undertaken to facilitate purification of the DNA from viral particles present in cytoplasmic fractions. The ultrasensitive photochemical silver staining of nucleic acid, described by Beidler et al. (Analytic Biochemistry 1982: 126) has been adapted and applied to the detection of pseudorabies virus restriction fragments. In a period of 5 h more than 1 microgram of DNA can be extracted from a 25 cm2 plastic flask containing infected cells and purified without ultra centrifugation. Low molecular weight DNA was separated from high molecular weight DNA by polyacrylamide gel electrophoresis. The restriction fragments were selectively visualized by silver staining which can detect 0.025 micrograms of total pseudorabies virus DNA. The electrophoretic DNA pattern of vaccine and wild strains has been studied using these techniques and the results agree with previously published data.     

High-frequency homologous recombination in vaccinia virus DNA.

A recombinant vaccinia virus genome was constructed in which the viral thymidine kinase (tk) gene was placed between direct repeats of a 1.5-kilobase-pair DNA sequence of heterologous origin. When forced to replicate in tk- cells in the presence of methotrexate (i.e., under tk+-selective conditions), the recombinant maintained its tk+ phenotype. Under nonselective conditions, however, the tk gene was frequently excised by both inter- and intramolecular recombination events because the repeated sequences provided substantial targets for homologous DNA recombination. Unique DNA products of intramolecular recombination were detected in the cytoplasm of infected cells soon after the onset of viral DNA replication, and their appearance was blocked by inhibitors of DNA synthesis. During repeated passage of the virus under nonselective conditions, the tk+ fraction decreased with first-order kinetics at a rate that reflected the frequency of recombination per cycle of virus replication. Eventually, a residual population of stable tk+ viruses remained, and analyses of the genome structures of individual members of this population showed that some of them appeared to be the products of nonhomologous DNA recombination.     

Immunogenicity analysis following human immunodeficiency virus recombinant DNA and recombinant vaccinia virus Tian Tan prime-boost immunization

This study assessed and compared the immunogenicity of various immunization strategies in mice using combinations of recombinant DNA(pCCMp24) and recombinant attenuated vaccinia virus Tian Tan(rddVTT-CCMp24).Intramuscular immunization was performed on days 0(prime) and 21(boost).The immunogenicity of the vaccine schedules was determined by measuring human immunodeficiency virus(HIV)-specific binding antibody levels and cytokine(interleukin-2 and interleukin-4) concentrations in peripheral blood,analyzing lymphocyte proliferation capacity against HIV epitopes and CD4 + /CD8 + cell ratio,and monitoring interferon-gamma levels at different times post-immunization.The results showed that pCCMp24,rddVTT-CCMp24 and their prime-boost immunization induced humoral and cellular immune responses.The pCCMp24/rddVTT-CCMp24 immunization strategy increased CD8 + T cells and induced more IFN-γ-secreting cells compared with single-shot rDNA.The prime-boost immunization strategy also induced the generation of cellular immunological memory to HIV epitope peptides.These results demonstrated that prime-boost immunization with rDNA and rddVTT-CCMp24 had a tendency to induce greater cellular immune response than single-shot vaccinations,especially IFN-γ response,providing a basis for further studies.     

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.