Physical mapping of strain AD169 human cytomegalovirus DNA

The whole human cytomegalovirus strain AD169 genome was cloned into plasmid pAT153 in the form of 25 HindIII fragments. Double and triple digestions of the recombinant plasmids with restriction endonucleases BamHI, BglII, ClaI, DraI, EcoRI, EcoRV, HindIII, HpaI, KpnI, PaeR7, PstI, SphI and XbaI yielded a detailed restriction map of human cytomegalovirus DNA. Knowing the exact position of numerous restriction sites in the viral DNA molecule, we have been able to examine very closely the heterologous region between the long and the short segments of the human cytomegalovirus genome.     

Comparative restriction analysis of HindIII-F-fragments of DNA from 4 strains of vaccinia virus

The localization of KpnI, SacI, XhoI, AvaI, PstI, BglI, BamHI, EcoRI, PmiI, SalI, BglII, restriction endonuclease cleavage sites in HindIII-F-fragments of DNA from vaccinia strains WR, Copenhagen, LIVP and neurovaccine has been detected. The fragments have been shown to differ in the number of AvaI, EcoRI and BamHI sites. The fragments also differ from the analogue of Tian Tan vaccinia strain in the pattern of restriction by AvaI, XhoI, PstI, EcoRI and BamHI endonucleases.     

Physical map of Pseudomonas aeruginosa phage phi kF77 genome. Localization of sites sensitive to restriction endonucleases

A physical map of the P. aeruginosa bacteriophage phi kF77 has been constructed using the restriction endonucleases SalI, HindIII, EcoRI, EcoRV, MuI, XbaI, ClaI. The phi kF77 DNA is resistant to cleavage by the restriction endonucleases BamHI, BglII, HpaI, PstI, PvuII, SmaI, XhoI.     

Physical map of the Rhodobacter sphaeroides bacteriophage phi RsG1 genome and location of the prophage on the host chromosome.

We constructed a physical map of the 50-kilobase-pair (kb) DNA of the temperate Rhodobacter sphaeroides bacteriophage phi RsG1, with the relative positions of the cleavage sites for the nine restriction endonucleases KpnI, HindIII, XbaI, ClaI, BclI, EcoRV, EcoRI, BglII, and BamHI indicated. Using biotinylated phi RsG1 DNA as a probe in hybridization studies, we detected homologies with virus DNA and fragments of restriction endonuclease-digested host chromosomal DNA but not with plasmid DNA. This indicates that the prophage is integrated into the host chromosome. In addition, the use of specific probes such as the 10.4-kb BglII A fragment and the 2.65-kb BamHI H fragment allowed the determination of the position of phage attachment site (attP).     

Restriction enzyme cleavage map of Tn10, a transposon which encodes tetracycline resistance.

A cleavage map of a recombinant plasmid carrying Tn10 was constructed for 13 different restriction enzymes. The Tn10 region of this plasmid contains cleavage sites for BamHI, AvaI, BglI, BglII, EcoRI, XbaI, HincII, HindIII, and HpaI. Restriction enzymes PstI, SmaI, KpnI, XhoI, SalI, and PvuI do not cleave within the Tn10 element. This map confirms the previously reported structure of this transposon; it is composed of a unique sequence (approximately6,400 base pairs long), which in part codes for the tetracycline resistance functions and is bounded by inverted repeats (approximately 1,450 base pairs long).     

Biochemical characterization of three nucleopolyhedrovirus isolates of Spodoptera exigua and Mamestra brassicae

Three nucleopolyhedroviruses (NPVs), of different geographical origins, were biochemically characterized and compared. Two that were isolated from Spodoptera exigua (Se-UZB and Se-SP3) in Uzbekistan and Spain were SeMNPV type, and the third which was isolated from Mamestra brassicae (Mb-PL) in Poland was MbMNPV type. The Spanish isolate Se-SP3 showed restriction endonuclease (REN) profiles that were closely related to two previously described Spanish strains Se-SP1 and Se-SP2, but had some unique and characteristic REN fragments. On the other hand, comparison between the Uzbekian (Se-UZB) and the Spanish (Se-SP1, Se-SP2, and Se-SP3) isolates from S. exigua showed unrelated REN profiles. However, the Pst I and Bgl II profiles of Se-UZB and Mb-PL were identical, and very similar to the REN profiles of the MbMNPV strain, which constitutes the active component of Mamestrin^® (NPP, Nagueres, France), a commercial bioinsecticide. It is therefore very likely that the Se-UZB samples were cross-infected by the Polish strain (Mb-PL). This work presents two new strains of SeMNPV and MbMNPV, called Se-SP3 and Mb-PL, respectively, which were surveyed in two distant areas.     

Isolation and characterization of a temperate bacteriophage from Streptomyces galilaeus

A new temperate actinophage from Streptomyces galilaeus ATCC 31133 was purified after that strain was crossed with S. peucetius ATCC 29050. Sensitive hosts became lysogenized and yielded turbid plaques of 2 to 3 mm in diameter. Host-range analysis indicated that 16 of 27 Streptomyces strains tested were sensitive to infection on solid medium. S. lividans and S. coelicolor A3(2) were among those not infected by this new actinophage. The new actinophage, designated phi SPK1, belongs to the Bradley group B morphological type, the pH optimum for infection is 6.75 to 7.0, it is not efficiently induced by mitomycin C or UV irradiation, it has a circular chromosome of 35.8 +/- 0.5 kilobase pairs in length containing overlapping (cohesive) ends, and the G+C content of its DNA was calculated from the buoyant density of 1.7240 to be 69 mol%. The DNA of phage phi SPK1 was cleaved by the restriction endonucleases ApaI, AluII, EcoRI, PvuII, and SalI, but, in all cases except that with EcoRI, treatment yielded greater than 20 restriction fragments. No sites were detected for BamHI, BclI, BglII, ClaI, HindIII, MluI, PstI, SmaI, SphI, SstI, XbaI, or XhoI.     

Isolation and characterization of a temperate bacteriophage from Streptomyces galilaeus.

A new temperate actinophage from Streptomyces galilaeus ATCC 31133 was purified after that strain was crossed with S. peucetius ATCC 29050. Sensitive hosts became lysogenized and yielded turbid plaques of 2 to 3 mm in diameter. Host-range analysis indicated that 16 of 27 Streptomyces strains tested were sensitive to infection on solid medium. S. lividans and S. coelicolor A3(2) were among those not infected by this new actinophage. The new actinophage, designated phi SPK1, belongs to the Bradley group B morphological type, the pH optimum for infection is 6.75 to 7.0, it is not efficiently induced by mitomycin C or UV irradiation, it has a circular chromosome of 35.8 +/- 0.5 kilobase pairs in length containing overlapping (cohesive) ends, and the G+C content of its DNA was calculated from the buoyant density of 1.7240 to be 69 mol%. The DNA of phage phi SPK1 was cleaved by the restriction endonucleases ApaI, AluII, EcoRI, PvuII, and SalI, but, in all cases except that with EcoRI, treatment yielded greater than 20 restriction fragments. No sites were detected for BamHI, BclI, BglII, ClaI, HindIII, MluI, PstI, SmaI, SphI, SstI, XbaI, or XhoI.     

A restriction map of Xenopus laevis mitochondrial DNA

The mitochondrial DNA from Xenopus laevis is a 17.4 x 10(3)-base-pair circular DNA molecule. The mapping of this DNA, using 19 different restriction endonucleases is reported here. The sites are as follows: 1 for BamHI, PstI, SacI, SalI, BalI; 2 for BglII, SacII, EcoRI, ClaI, 3 for XhoI, 4 for AvaI, XbaI, PvuII, 5 for HindIII, 6 for HhaI, BclI, HpaI, 10 for AvaII and 11 for HincII. The same sites (except for one of the two ClaI sites) are observed in the molecule cloned in pBR322 DNA. The fragments corresponding to 62 cleavage sites have all been ordered and precisely located. They provide suitable conditions for further investigations connected with the study of replication and nucleotide sequence determination of this molecule.     

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.     

The detailed physical map of the temperate phage 16-3 of Rhizobium meliloti 41

Restriction cleavage maps for enzymes EcoRI, BamHI, PstI, PvuII, XbaI and EcoRV of Rhizobium meliloti temperate phage 16-3 have been established. Together with the earlier maps (HindIII, KpnI, HpaI, BglII) 98 restriction sites, 'evenly' distributed, have been mapped along the phage genome, including the so far unmarked silent region of the chromosome. All the restriction maps have been fitted to each other by computer optimalization. Beyond for conventional techniques a computer program (PMAP) for physical mapping of linear DNA has been employed which made the experimentation, in several cases, extremely efficient.     

In vitro phenotypic markers of a poliovirus recombinant constructed from infectious cDNA clones of the neurovirulent Mahoney strain and the attenuated Sabin 1 strain.

Infectious cDNA corresponding to the entire genome of the attenuated Sabin strain of type 1 poliovirus has been inserted into EcoRI site of bacterial plasmid pBR325. Two consecutive PstI fragments (nucleotide positions 1814 to 3421) of the infectious cDNA of the Sabin 1 strain were replaced by the corresponding DNA fragments prepared from an infectious DNA clone of the genome of the virulent Mahoney strain of poliovirus type 1. The exchanged segment encodes capsid protein VP1 and part of capsid protein VP3, a region in which a large number of amino acid differences between the attenuated Sabin and the parental, neurovirulent Mahoney strain cluster. The recombinant virus was obtained by DNA transfection of HeLa S3 cells, and several in vitro phenotypes of the virus were compared with those of the parental viruses. The recombinant virus was recognized by a neutralizing monoclonal antibody specific to the Mahoney strain. Growth of the Sabin strain of poliovirus has been shown to be quite dependent upon the bicarbonate concentration (d marker). The growth of the recombinant virus, however, was not highly dependent upon the concentration of bicarbonate in cell culture media, and thus resembled that of the Mahoney strain. On the other hand, the temperature-sensitive multiplication (rct marker) and the small-plaque morphology of the recombinant virus corresponded to the phenotype of the Sabin 1 strain. The in vitro recombination of infectious cDNA clones of genomic RNA and subsequent analysis of the growth properties of the recombinant virus have allowed us to correlate specific mutations in the genome of an RNA virus with certain biological characteristics of that virus.     

Localization of canonical single-strand breaks in DNA of the Pseudomonas aeruginosa bacteriophage phi kF77

Bacteriophages phi k of P. aeruginosa were characterized by the presence of T4 DNA-ligase-repaired, single-chain breaks in their genome. A restriction map was constructed for one of these phages (phi kF77) with restriction endonucleases SalI, HindIII, EcoRI, MluI, XbaI and ClaI. phi kF77 DNA was resistant to the cleavage by BamHI, BglII, HpaI, PstI, PvuII and XhoI endonucleases. Single-chain breaks were mapped by means of electron microscopy of partially denatured DNA molecules, electrophoretic studies of denatured DNA and S1-analysis. Four major nicks were thus located which were revealed in 33 to 83% of DNA molecules. On the basis of mutual hybridization of single-strand DNA fragments it was shown that all nicks are located in one of the phi kF77 DNA chains. S1-treated hybrids of 32P-labeled single-strand fragments with intact DNA chain were used for DNA orientation. The physical map of phi kF77 DNA was constructed.     

Epstein-Barr virus intrastrain recombination in oral hairy leukoplakia.

In laboratory lymphoblastoid cell lines and in natural human infections, Epstein-Barr virus (EBV) strains have been identified by DNA restriction fragment length polymorphisms of the BamHI H fragment. Multiple, heterogeneous BamHI H fragments have been detected in oral hairy leukoplakia (HLP), raising the question of EBV coinfection with multiple strains. To investigate whether the heterogeneous BamHI H fragments represent different EBV strains or recombinant variants of the same strain, EBV DNA from HLP lesions was analyzed to characterize the viral strains and determine the source of possible recombinant variants. Clones of heterogeneous BamHI H fragments from a single HLP lesion were determined to have strain identity on the basis of sequence identity of the EBNA-2 genes. Intrastrain homologous recombination within the IR2 internal repeat region and nonhomologous recombination of other sequences accounted for the heterogeneity of the BamHI H fragments. PCR amplification from additional HLP specimens detected similar recombinant variants. A possible example of site-specific recombination joining the BamHI Y portion of the EBNA-2 gene to sequences within the BamHI S fragment was also detected in multiple HLP specimens. These data indicate that intrastrain recombination during productive replication confounds the use of restriction fragment length polymorphism analysis of the BamHI Y and H fragments to identify EBV strains in HLP. In patients with permissive epithelial EBV infections, EBV strains could be more accurately distinguished by sequence identity or divergence within known regions of genetic strain variation.     

XbaI, PstI, and BglII restriction enzyme maps of the two orientations of the varicella-zoster virus genome.

Cleavage of varicella-zoster virus DNA with the restriction endonucleases PstI, XbaI, and BglII resulted in 18, 22, and 20 fragments, respectively. Based on the molecular weights and molarities of these fragments, a molecular weight of 84 x 10(6) could be calculated for the varicella-zoster virus genome. In both the XbaI and the BglII patterns, four 0.5 M fragments were identified. The arrangement of the fragments was determined by molecular hybridization techniques, and the terminal fragments were identified by lambda exonuclease digestion. The 0.5 M fragments, of which two were located at the same terminus of the genome, contained repeated sequences: one terminally and one inverted internally. These results were in agreement with the existence of two equimolar subpopulations of the varicella-zoster virus genome, differing in the relative orientation of a short region of unique sequences. This region was bounded by the repeated sequences. From the molecular weights of the submolar fragments, a maximal molecular weight of 5 x 10(6) for the repeated region and a minimal molecular weight of 3.5 x 10(6) for the short unique sequence could be calculated.     

Human immunodeficiency virus type 1 intersubtype (B/E) recombination in a superinfected chimpanzee.

Genetic characterization of a large number of human immunodeficiency virus type 1 (HIV-1) isolates indicates that at least 10% of all strains have mosaic genomes generated by recombination between viruses of the same or different subtypes or clades. What is not known, however, is the time between infection with the first and second HIV-1 strains as well as the time between infection with the second strain and the recombinational event. After 32 months of infection with HIV-1(LAI(IIIB)), a chimpanzee was inoculated intravenously and became infected with a subtype E strain, HIV-1(90CR402). With PCR amplification, DNA heteroduplex analysis, and DNA sequencing, both parental strains and two distinct recombinant proviruses were found in genomic DNA from lymph node tissue obtained 24 weeks after exposure to HIV-1(90CR402). These results show (i) that antiviral immune responses established by long-term infection with an HIV-1 subtype B strain did not prevent infection by a subtype E strain and (ii) that both strains actively replicated and produced sufficient quantities of virus to coinfect the same cell(s), resulting in recombinant viruses.     

Histone genes from Xenopus laevis: molecular cloning and initial characterization

Histone DNA sequences, were detected in Eco RI fragments of total Xenopus laevis DNA, by hybridization with 32P-labeled h22-DNA, a histone gene repeat unit of the sea urchin Psammechinus miliaris. The about 6 kb-size class, which was found to hybridize, was subsequently integrated into the E. coli plasmid pCR1. A clone was isolated that contains a 5.8 kb EcoRI fragment hybridizing with h22-DNA. A physical map was constructed using the restriction endonucleases BamHI, PstI, HincII, BglII, XbaI, PvuII, XhoI, AvaI, SmaI, HinfI and HpaII. The fragment was not cleaved by KpnI, AvaI, SalI and HindIII. Using this restriction map we were able to determine the gene order by hybridization with purified gene probes derived from h22-DNA. The gene order was found to be H3, H4, H2A and H2B. The localization of the H1 gene was not possible, probably due to its greater evolutionary divergence. Part of the sequence of the H3-gene is presented providing unambiguous evidence on the identity, map position and polarity of this gene.     

Characterization of restriction endonuclease maps of hepatitis B viral DNAs

The HBV DNA isolated from Dane particles of 9 patients' plasma was cloned into the EcoRI or BamHI site of the pUC8 plasmids. Two plasmids with full length HBV DNA and four plasmids containing the HBV surface antigen gene were obtained. Based on our cloned HBV DNA and a comparison with 7 complete sequences and 5 restriction endonuclease patterns of HBV DNA published by others, we can recognize common restriction sites shared by different subtypes (adw, adr, ayw, and adyw): (1) a HincII site in the S gene, (2) a BamHI site in the X region, and (3) two BglII sites in the C gene. In addition adw has specific sites for HincII, BamHI, and PstI in the pre-S region. A unique XhoI site is present in the pre-S region in all subtypes except for adw.     

A new selective phage cloning vector, lambda 2001, with sites for XbaI, BamHI, HindIII, EcoRI, SstI and XhoI

An improved bacteriophage lambda cloning vector, lambda 2001, has been constructed. The phage includes a 34-bp polylinker oligonucleotide which introduces cleavage sites for XbaI, SstI, XhoI, EcoRI, HindIII and BamHI, and can accommodate 10-kb to 23-kb fragments. Inserts that destroy the BamHI or XhoI cloning sites may be recovered by excision at flanking sites in the polylinker sequence. Insertion of foreign DNA into lambda 2001 generates phage with a Spi- phenotype. The recombinant phage are able to grow on P2 lysogens but the parental vector phages are not. In the course of this work, the polylinker sequence was also introduced into M13mp8. This produced a new vector, M13mp12, with cloning sites for EcoRI, SmaI, XbaI, SstI, XhoI, BamHI, and HindIII.     

Restriction endonuclease mapping of the proviral DNA of the exogenous RIII murine mammary tumor virus

Cellular DNA containing integrated murine mammary tumor virus (MuMTV) was isolated from FeI/C6 feline kidney cells and CCL64 mink lung cells infected with milkborne RIII MuMTV. By using restriction enzyme HpaI, intact RIII MuMTV provirus (length, 8.7 kilobases [kb]) was excised from the cellular DNA. Subsequent restriction endonuclease analysis of this HpaI fragment with KpnI, HindIII, EcoRI, BamHI, BglII, PstI, SstI, SalI, and XhoI enabled us to construct a map of the RIII virus genome. A comparison of this map with the maps of the GR and C3H MuMTV's revealed that there are greater sequence differences between the RIII virus and the GR and C3H MuMTV proviruses than there are between the GR and C3H proviruses. The following are features of the restriction map unique to the RIII provirus: the presence of three BamHI and two EcoRI cleavage sites, a HpaI cleavage site in the terminal 3'-5' repeat unit of the provirus, and the absence of an XhoI cleavage site. Another distinguishing feature of the RIII provirus is that the sizes of some of the restriction fragments produced by cleavage of the RIII provirus with PstI are different from the sizes of the fragments obtained by PstI cleavage of the GR and C3H proviruses. Like the GR proviral DNA, the RIII proviral DNA has three SstI (SacI) cleavage sites, whereas the C3H provirus has only two SstI sites. HpaI digestion of MuMTV-infected mink lung cell DNA revealed only one class of provirus (an 8.7-kb fragment); however, we observed several minor classes of RIII proviral DNA in addition to the major class of provirus DNA in infected cat kidney cells. PstI digestion of the HpaI 8.7-kb fragments from both feline and mink cells generated a 3.7-kb DNA fragment identical in size to a PstI-generated fragment that has been found in GR and C3H milkborne virus-infected cells. Although a fragment similar in size to the milkborne 3.7-kb PstI fragment has been found as an endogenous component in many C3H and GR mouse tissues, we did not observe such an endogenous fragment in the RIII mouse strain. Therefore, the 3.7-kb fragment may be useful as a marker for the milkborne RIII MuMTV provirus in RIII mice.