The simian varicella virus and varicella zoster virus genomes are similar in size and structure.

Simian varicella virus (SVV) DNA was purified from viral nucleocapsids and the molecular structure of the SVV genome was determined. SVV DNA was analyzed by agarose gel electrophoresis of BamHI, BglII, EcoRI, and PstI restriction endonuclease digests. SVV and varicella zoster virus (VZV) DNAs were demonstrated to have distinct restriction endonuclease profiles. Summation of the sizes of individual restriction endonuclease fragments indicate the size of SVV DNA is congruent to 121 kilobase pairs (kbp) or congruent to 76.8 megadaltons (Md). Electron microscopy, lambda exonuclease analysis, and Southern blot DNA hybridizations were utilized to determine the molecular structure of the SVV genome and to construct restriction endonuclease maps. The results indicate that SVV DNA consists of a long component (L, congruent to 100 kbp) covalently linked to a short component (S, congruent to 20 kbp) which is composed of a unique short sequence (Us, 5.3 +/- 0.7 kbp) bracketed by inverted repeat sequences (TRs and IRs, congruent to 7.2 kbp). The presence of 0.5 M PstI restriction endonuclease fragments indicates that the S component may invert relative to the L component and that the genome exists in two major isomeric forms. The findings demonstrate that the SVV and VZV genomes are similar in size and structure.     

Molecular cloning and physical mapping of the genome of fish lymphocystis disease virus

A defined and complete gene library of the fish lymphocystis disease virus (FLDV) genome was established. FLDV DNA was cleaved with EcoRI, BamHI, EcoRI/BamHI and EcoRI/HindIII and the resulting fragments were inserted into the corresponding sites of the pACYC184 or pAT153 plasmid vectors using T4 DNA ligase. Since FLDV DNA is highly methylated at CpG sequences (Darai et al., 1983; Wagner et al., 1985), an Escherichia coli GC-3 strain was required to amplify the recombinant plasmids harboring the FLDV DNA fragments. Bacterial colonies harboring recombinant plasmids were selected. All cloned fragments were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridization of recombinant plasmid DNA to viral DNA. This analysis revealed that sequences representing 100% of the viral genome were cloned. Using these recombinant plasmids, the physical maps of the genome were constructed for BamHI, EcoRI, BestEII, and PstI restriction endonucleases. Although the FLDV genome is linear, due to circular permutation the restriction maps are circular.     

Organization of the ribosomal RNA genes in Schistosoma mansoni

The organization of the rRNA genes of Schistosoma mansoni has been determined by Southern blot analysis of genomic DNA digested with restriction enzymes, by isolation of the entire repeat on a single fragment of about 11 kilobase pairs from a genomic DNA library constructed in bacteriophage lambda and by characterization of three cloned EcoRI fragments which span the entire repeat. The segments encoding both the large and small rRNA subunits have been identified using specific cloned yeast rDNA fragments as probes and EcoRI, HindIII and BamHI restriction enzyme maps of the rRNA genes were constructed. The ends of the RNAs have been precisely mapped on the genomic DNA by S1 protection experiments. Our data indicate that the rRNA genes are present as a tight cluster. The total length of the rDNA repeat is about 10 kilobase pairs. There appears to be no variation in the size of transcribed and non-transcribed spacer DNA. At the RNA level we have characterized and mapped a small gap in the 28S RNA molecule. The interruption causes the RNA to dissociate into two equal sized fragments when analyzed under denaturing conditions.     

Physical structure and molecular cloning of equine cytomegalovirus DNA

Restriction endonuclease (RE) mapping studies and molecular hybridization analyses were conducted to determine the molecular structure of the genome of equine cytomegalovirus (ECMV). The ECMV genome is a linear, double-stranded DNA with a molecular size of 126 +/- 0.6 MDa (189 kbp). A library of cloned BamHI, EcoRI, and HindIII fragments of the viral genome was used to construct RE maps. Individual 32P-labeled cloned DNA fragments were hybridized to Southern blots of viral genomic DNA digested to completion with BamHI, EcoRI, HindIII, or SalI. These analyses revealed that the ECMV genome consists of a 97-MDa unique long region which is bracketed by repeated sequences. At one terminus of the genome, a 21.3-MDa segment of repeated sequences with no apparent unique sequences was identified. At the other terminus, a 6-MDa unique region bracketed by 2.4-MDa repeat segments was identified. No submolar RE fragments were identified upon digestion of the ECMV genome with BamHI, EcoRI, HindIII, SalI, or other REs, including BclI, BglII, NruI, and XbaI. The genome possesses only two termini as judged by lambda exonuclease digestion and by T4 DNA polymerase end-labeling of the intact DNA followed by digestion with BamHI, EcoRI, HindIII, SalI, BclI, BglII, NruI, or XbaI. In addition, Southern blot analysis of DNA extracted from ECMV-infected rabbit kidney cells revealed that only one viral DNA fragment within the intracellular viral DNA pool contains fused genomic termini. Taken together, these observations indicate that the ECMV genome does not isomerize and suggest that the genome of ECMV may be unique among those of the herpesviruses and especially those of the betaherpesviruses (cytomegaloviruses) since it contains regions of extensive internal homology yet does not undergo isomerization. Lastly, the relatively small size of the viral genome indicates an evolutionary diversification among the cytomegaloviruses.     

Characterization of viral DNAs from cells infected with chicken anaemia agent: sequence analysis of the cloned replicative form and transfection capabilities of cloned genome fragments

Summary The viral DNAs induced by the unclassified animal virus, chicken anaemia agent (CAA), during replication in MDCC-MSB 1 cells have been investigated. Analyses after S₁ nuclease, restriction endonuclease and denaturation treatments indicated that infected cell extracts contained genome-size, single-stranded DNA (M_r 2.3 kb), closed and open circular, double-stranded replicative form (RF) DNAs (M_r 2.3 kbp) and a population of smaller doublestranded DNAs (M_r 0.8 kbp). Recombinant plasmids containing 2.3 kbp CAA RF fragments cloned at the PstI, BamHI and EcoRI sites failed to transfect MDCC-MSB 1 cells. However, one plasmid, which contained two 2.3 kbp CAA RF fragments ligated in tandem at the PstI site, and cloned 2.3 kbp PstI, BamHI and EcoRI fragments, excised from their respective plasmids by restriction endonuclease digestion, were capable of transfection.The nucleotide sequence of the circular genome (2298 bp) of the Cux-1 isolate of CAA has indicated the presence of three overlapping open reading frames (ORFs) of 52 kDa, 24 kDa and 13 kDa on one strand. The existence of these ORFs was corroborated by analyses of partial sequences from three other isolates. The non-coding region of the CAA genome contained sequences with putative regulatory function. These results are discussed in relation to the rolling circle model of DNA replication.     

Isolation and characterization of recombinant lambda gt11 bacteriophages expressing four different Mycobacterium intracellulare antigens.

Four bacteriophages expressing different immunoreactive recombinant Mycobacterium intracellulare antigens were isolated from a lambda gt11 library with monoclonal antibodies to M. intracellulare. These four antibodies reacted with native M. intracellulare proteins of 54, 43, 40/38, and 22 kilodaltons. Southern blot hybridizations with DNA probes prepared from insert fragments of these bacteriophages confirmed the M. intracellulare derivation of the inserts. The physical maps of the immunoreactive phages were deduced by restriction enzyme digestions. The molecular weights of the expressed recombinant antigens were determined by Western (immuno-) blotting.     

Limited diversity of the immunoglobulin A1 protease gene (iga) among Haemophilus influenzae serotype b strains.

Immunoglobulin A1 (IgA1) proteases are thought to be important virulence factors in certain bacterial infections, including meningitis, and may have potential usage in vaccines. In this study, we compared the locations of EcoRI, BamHI, and PstI restriction endonuclease sites in the IgA1 protease gene (iga) region of whole-cell DNA from 76 Haemophilus influenzae strains. The analysis was performed by using isolated fragments of the cloned iga gene, which encodes the IgA1 protease originating from a H. influenzae serotype d strain, as probes in Southern blot experiments. All strains, including three without detectable IgA1 protease activity, had DNA sequences with a high degree of homology to the iga probes. The numbers and sizes of the DNA fragments hybridizing with the probes indicated that only three strains, none of which was of serotype b, had more than one iga gene. The iga restriction fragment length patterns of 60 clinical isolates of serotype b were of only four distinct types, which correlated with previously observed clusters of multilocus genotypes (electrophoretic types). This correlation supports the concept of the clonal population structure of H. influenzae. Three of the iga gene restriction types, which appear to represent 98% of the H. influenzae serotype b population, encode IgA1 proteases that were inhibited by antisera to any one of these types and therefore could form the basis for the development of a vaccine against H. influenzae meningitis.     

Cloning and physical mapping of Yaba monkey tumor virus DNA

The physical map positions for the BamHI, EcoRI, and SalI restriction fragments of Yaba monkey tumor pox virus DNA were determined using cloned virus DNA fragments as probes for hybridization as well as analyzing the secondary digests of larger DNA restriction fragments. Digests of EcoRI A and B fragments and SalI A and B fragments with BamHI allowed for the orientation of most of the BamHI restriction map. These secondary digest products were confirmed and the map positions for the EcoRI fragments were established using cloned BamHI fragments. Yaba monkey tumor virus DNA was cloned using the plasmid vector pBR322.     

Physical maps of SfMNPV baculovirus DNA and its genomic variants

Spodoptera frugiperda MNPV was plaque-purified, and the viral DNA from the plaque-purified isolates was analyzed with restriction endonuclease enzymes. Seven distinct variants were identified when the DNA of the isolates were analyzed by EcoRI and HindIII. The DNAs of the SfMNPV predominant type (prototype) and the variants were mapped with BamHI, BglII, BstEII, EcoRI, HindIII, KpnI, and PstI by multiple enzyme digestion and blot hybridization. The cleavage sites generated by the seven restriction enzymes were ordered, and the sites were assigned map coordinates using a least-squares procedure. Since Autographa californica MNPV-E2 EcoRI fragment I, which contains the polyhedrin gene, hybridized with SfMNPV EcoRI fragment P, the physical map of SfMNPV was oriented with EcoRI P on the left, with site 1 being the EcoRI site between fragments F and P. The calculated genome size was 121.76 kilobase pairs or 80.36 X 10(6) Da. The DNA from each variant was compared to the DNA of the prototype for insertions, deletions, and new restriction sites. Physical maps were generated for each of the variants. The differences between the variant and the prototype were confined to four regions in the SfMNPV genome representing less than 16% of the prototype genome.     

N-methyl-N-nitrosourea-induced T-lymphomas of AKR/J mice contain somatically acquired ecotropic-like murine leukemia proviruses

We have studied somatically murine leukemia proviral integrations in primary N-methyl-N-nitrosourea (MNU)-induced thymic lymphomas of AKR/J mice. The majority of MNU-induced lymphomas contain newly acquired murine leukemia proviral sequences. In contrast to spontaneous AKR/J lymphomas which contain multiple integrations of mink cell focus-forming recombinant proviruses, MNU-induced lymphomas contain ecotropic-related proviruses. This conclusion was based on the demonstration that EcoRI- and PvuII-digested DNA from MNU-induced lymphomas contains new 3' proviral-cellular junction fragments that hybridize with the ecotropic-specific pAKV-4 and pAKV-5 hybridization probes. Also, EcoRI/PstI double digests of DNA from MNU-induced lymphomas revealed that the acquired proviruses do not contain an internal 3' EcoRI site characteristic of mink cell focus-forming recombinant viruses. The proviral integration patterns suggest that MNU-induced lymphomas are clonal or oligoclonal in nature. This conclusion is supported by comparison of proviral integration patterns in lymphomas obtained from thymus and spleen of individual mice, and by analyses of T-cell receptor beta-chain gene rearrangements. The frequent occurrence of ecotropic-related proviral sequences in MNU-induced lymphomas suggests that these newly acquired proviruses may play a role in tumor development.     

Use of λgt11 to identify antigenic components of equine herpesvirus 4

A library of the equine herpesvirus 4 (EHV-4) genome was constructed in the gt11 expression vector. Recombinant bacteriophage expressing EHV-4 antigens as beta-galactosidase fusion proteins were detected with rabbit antiserum raised against EHV-4 virions and convalescent horse serum. EHV-4 DNA sequences contained in the immunopositive recombinants were used as hybridization probes for mapping the genes encoding the antigens on the viral genome. The DNA sequence of the probes was determined. Screening the library with rabbit antiserum led to the identification of 40 recombinants, 26 of which were further characterized. Determination of the DNA sequence of the EHV-4 inserts revealed that 23 of the recombinants encode an identical portion of glycoprotein gB. Two of the recombinants encode a portion of the previously unidentified EHV-4 homologue of the EHV-1 immediate early protein. The EHV-4 insert of the remaining recombinant encodes a portion of the previously unidentified EHV-4 homologue of herpes simplex virus 1 (HSV-1) UL36, a tegument protein. Screening the library horse serum led to the identification of three recombinants, one of which encodes the same gB sequence as the gB recombinant recognized with the rabbit serum. The other two contain overlapping sequences that encode a portion of EHV-4 gX.     

Identification and nucleotide sequence of the regions of Autographa californica nuclear polyhedrosis virus genome carrying insertion elements derived from Spodoptera frugiperda

A morphology mutant of the baculovirus Autographa californica nuclear polyhedrosis virus called M5 was previously shown to synthesize two size classes of viral DNA, one of which had a deletion of 42% of the genome. It was hypothesized that the presence of the smaller M5 circular DNA resulted from the specific deletion of the region located between two sites carrying short DNA insertions. These sites have now been identified by DNA-DNA hybridization using cloned EcoRI fragments of M5 DNA. One cloned M5 EcoRI fragment was found to correspond to the deletion junction fragment where sequences from the insertion site in the 2.6-map unit region were covalently linked to sequences from the insertion site in the 46-map unit region. The 2.6- and 46-map unit regions of Wt AcMNPV DNA were sequenced. Potential long open reading frames which would be disrupted by the M5 inserts were detected. Nucleotide sequence analysis of the same regions of M5 DNA revealed the presence of almost identical inserts of 290 bp. The primary sequence of the inserts revealed characteristics similar to the termini of transposons. Hybridization studies suggested that the inserts were derived from repetitive elements of the Spodoptera frugiperda host cell genome.     

The characterization of Gardnerella vaginalis DNA using non-radioactive DNA probes

DNA restriction profiles of various Gardnerella vaginalis isolates, generated by BamHI, EcoRI, PstI and other restriction enzymes, varied considerably. Only a few DNA fragments were identified as common in ethidium bromide fluorescence profile and Southern-blot hybridization patterns (employing a digoxigenin-labelled G. vaginalis DNA probe and an enzyme-linked immunoassay detection method). While the efficiencies of Southern-blot hybridization appeared inconsistent, in dot-blot assays, DNA from each isolate hybridized readily, enabling the detection of at least 10 ng DNA. A 5.7-kb DNA fragment from G. vaginalis ATCC 14018 genomic library, cloned in the BamHI site of pBR322, could replace the total genomic DNA probe. This specific DNA fragment was present in different sizes in 12 analysed G. vaginalis strains, describing a restriction fragment length polymorphism. In control studies, none of the DNA from bacteria other than G. vaginalis (including some genitourinary tract residents) hybridized with the G. vaginalis total or specific DNA probes. Non-radioactive G. vaginalis DNA probes can thus form the basis of a useful detection method for further studies of this organism.     

Physical map of SeMNPV baculovirus DNA: an AcMNPV genomic variant

A physical map of the plaque-purified SeMNPV-25 baculovirus DNA was constructed with HindIII, EcoRI, PstI, SstII, BstEII, BamHI, KpnI, and SmaI by multiple-enzyme digestion and DNA-DNA hybridization. The orientation of the physical map was to the AcMNPV polyhedrin gene, and a total of 104 restriction sites were ordered. The genome size was 131.89 kilobase pairs (87.05 X 10(6) Da). The physical maps of SeMNPV-25 DNA and AcMNPV-E2 DNA were compared, and they were similar except in four regions. Since the differences between the physical maps of SeMNPV-25 and AcMNPV-E2 were reconciled, the BstEII and SstII fragments for AcMNPV-E2 were also ordered. In addition, reiterated sequences in the SeMNPV-25 genome were identified and located on the physical map.     

Molecular cloning and expression of Neisseria meningitidis class 1 outer membrane protein in Escherichia coli K-12

A genomic library of meningococcal DNA from a clinical isolate of Neisseria meningitidis was constructed in the expression vector lambda gt11. Outer membrane complex was prepared from the same strain and used to immunize rabbits to raise polyclonal anti-outer membrane complex serum. The amplified library was probed with this polyclonal serum, and seven expressing recombinants were isolated; further investigations indicated these to be identical. The expressed meningococcal gene in these recombinants was fused to vector beta-galactosidase and shown to encode epitopes present on the 42-kilodalton class 1 outer membrane protein. Estimation of the size of the recombinant fusion protein suggests that up to 40 kilodaltons of protein-coding sequence is present. The lambda gt11 recombinant contains a 3.4-kilobase DNA insert, which has been recloned into a plasmid and characterized by restriction endonuclease analysis. A restriction fragment from the insert, representing the protein-coding region hybridizes to a single 2.2-kilobase XbaI fragment from the homologous strain and to similar-sized XbaI fragments in other strains of meningococci, expressing antigenically distinct class 1 proteins.