Endonuclease Cleavage Patterns Research Articles

Analysis of three salmonid herpesvirus DNAs by restriction endonuclease cleavage patterns.

Restriction endonuclease cleavage analysis of three salmonid herpesvirus (strain H-83, OO-7812 strain of Oncorhynchus masou virus (OMV) and Herpesvirus salmonis) DNAs was performed by using DNA extracted directly from virus-infected cells. Clear-cut cleavage patterns were obtained when DNA was extracted from approximately 1.1×105 cells infected with the viruses, and the patterns were unique for each herpesvirus. After passages of strain H-83 and OMV for 33 and 36 times in vitro, respectively, some fragments were noted to appear or disappear. However, no DNA fragment of H. salmonis changed during the 36th passage. During the second passage levels, strain H-83 and OMV showed high virulence in masu salmon (O. masou) fry (100% mortality by 27 days postinoculation), whereas the viruses showed a decrease in virulence even at more than 33 passage levels.

Sequence analysis in the E1 region of adenovirus type 4 DNA

Adenovirus type 4 (Ad4) is the sole member of adenovirus group E based on overall DNA sequence homology, restriction endonuclease cleavage patterns, and the size of capsid proteins. We cloned the BamHI-F fragment from the left end of Ad4 in pUC13-1 between the SalI and BamHI sites in order to carry out the structural analysis of the ElA region of Ad4. The complete sequence of the BamHI-F fragment (2042 bp) has been determined. From the DNA sequence, the splice sites for the putative 12 S and 13 S mRNAs, encoded by the E1A region of Ad4 were deduced. If protein synthesis initiates at the first available AUG triplet (position 575), these 12 S and 13 S mRNAs would code for polypeptides containing 226 and 257 amino acids, respectively. Comparison of Ad4- and Ad7-13 S mRNA-coded polypeptides indicates that there is 57% homology, whereas the homology is only 38% with Ad12 and 31 % with Ad2-13 S mRNA-coded polypeptides. The structural analysis in the E1 region of Ad4 also includes the coding region for the ElB 19-kDa protein. Ad4 and Ad7 shows 65% homology in the coding regions for E1B 19-kDa protein. Comparison of the DNA sequence of Ad4 with those of Ad2, Ad7, and Ad12 by using a dot matrix computer program and by Southern hybridization revealed that Ad4 bears a stronger homology with Ad7 than with Ad2 and Ad12 in this region. Hydropathy plots and alignments of the putative polypeptides coded by this region in Ad4 with those from the corresponding regions of different serotypes to reveal the highly conserved domains also support the above conclusion.

An in vivo cointegrate of two plasmids from incompatibility group X.

Formation of a recombinant plasmid designated pNH603 was observed when two plasmids from incompatibility group X, the multicopy plasmid pNH602 (a higher-copy-number deletion derivative of R6K) and the oligocopy plasmid R485, coexisted in a single Escherichia coli cell. According to its size and its restriction endonuclease cleavage pattern, plasmid pNH603 is a true cointegrate of pNH602 and R485. An insertion-sequence-like element coming from plasmid R485 is supposed to mediate the fusion of both replicons. The pNH603 copy number (1-2 per chromosome) indicates that the mechanism of replication of the low-copy-number plasmid is dominant in this cointegrate. No dissociation of pNH603 to parental plasmids was observed even in E. coli K-12 recA+ cells. On the other hand, deletion derivatives of four size classes originate from pNH603 in both recA+ and recA hosts. A miniplasmid designated pNH604, a representative of the most frequent 7 Mg/mol size class, was found, in a low number of copies per host chromosome.

Physical and genetic structure of the glpD-malT interval of the Escherichia coli K-12 chromosome

A transducing lambda phage carrying glpD''lacZ, glpR, and malT was isolated from a strain harboring a glpD''lacZ fusion. Comparison of restriction endonuclease cleavage patterns of DNA isolated from this phage with that of the previously cloned malT region (Raibaud and Schwartz 1980) facilitated the construction of recombinant plasmids carrying different portions of the glpD-malT region. Results of minicell analysis and complementation studies showed that this region of the chromosome encodes at least five polypeptides. These included the previously identified glpD, glpR, and malT gene products. In addition, two new structural genes of the glp regulon (glpE and glpG) located between the glpD and glpR genes were identified. Hybrid plasmids carrying glpD''lacZ and glpR''lacZ fusions were constructed. Restriction endonuclease cleavage analysis of these two plasmids demonstrated that glpD and glpR are divergently transcribed.

Isolation and characterization of a novel human papillomavirus type 6 DNA from an invasive vulvar carcinoma.

Human papillomavirus type 6 (HPV-6) DNA was detected in a rapidly growing vulvar verrucous carcinoma and two recurrent tumor samples. The viral DNA (HPV-6vc) was molecularly cloned and found to have a high degree of DNA sequence homology to HPV-6b DNA. Comparison of restriction endonuclease cleavage patterns between HPV-6b and HPV-6vc genomes and DNA sequencing analysis demonstrated an additional 106 bases in the HPV-6vc genome. These additional nucleotides were located in the noncoding region of the viral genome which contains the putative viral DNA replication and early gene transcriptional control elements. Seventy-four of the additional 106 nucleotides were found as one insert in the purine-thymidine-rich region 3' to the end of the L1 open reading frame. This 74-base-pair addition had homology with viral sequences immediately upstream to it and to poly(dG-dT) sequences found in the human genome including the conserved repeated sequences in human DNA (EC1) and in the human cardiac muscle actin gene. Two smaller inserts, 19 and 15 nucleotides, were found upstream from the transcriptional control elements and demonstrate homology with regions of human alpha and gamma interferon genes.

Antibiotic resistance and conjugative R plasmids in clinical isolates of Enterobacteriaceae in Peninsular Malaysia

Twenty-five strains of enterobacteria, isolated from man in Peninsular Malaysia and consisting of seven Enterobacter spp., five Escherichia coli, five Salmonella spp., four Klebsiella spp., two Shigella spp., one Proteus sp. and and one Providencia sp., were tested for antibiotic resistance and conjugative R plasmids. They were all sensitive to nalidixic acid and resistant to at least three antibiotics. The number of resistances ranged from 3 to 11 antibiotics, including cefoperazone and sissomicin (two) newly released antibiotics), in addition to common drugs of current use. Of the 25 isolates, 19 (76%) conjugally transferred, at varied frequencies, at least two resistance determinants. Results from equilibrium density gradient centrifugation, agarose gel electrophoresis and transformation experiments provided proof that the transferable resistances were plasmid-mediated. Restriction endonuclease cleavage patterns showed that the plasmids from Proteus strain K005 and Providencia strain K001 may be identical.

KBSH parvovirus: comparison with porcine parvovirus

We compared the molecular, antigenic, and pathogenic properties of KBSH parvovirus to those of porcine parvovirus (PPV) isolate NADL-8. KBSH, propagated in swine testes cells in culture, possessed two major capsid polypeptides of 83 and 64 kilodaltons that were similar in size to those of PPV. KBSH-infected cells also contained an 86-kilodalton nonstructural polypeptide that was identical in size to the PPV nonstructural polypeptide (NS-1). The KBSH polypeptides were structurally similar but not identical to the corresponding PPV polypeptides, as revealed by partial proteolysis mapping. Viral replicative-form DNA from KBSH-infected cells was similar in size to PPV replicative-form DNA and exhibited similar but not identical restriction endonuclease cleavage patterns to that of PPV replicative-form DNA. Antigenically, the two viruses were also very closely related. By using heterologous and homologous antisera, the two viruses were indistinguishable in hemagglutination inhibition and immunoprecipitation assays. However, pathogenically these viruses were dramatically different. NADL-8 caused fetal death when injected into swine fetuses in utero and viremia and high persisting antibody titers when administered orally to weaning-age swine. KBSH-inoculated fetuses were normal in appearance, and pigs orally exposed to KBSH failed to establish viremia and demonstrated only transient antibody titers. Thus, KBSH appears to be a PPV that is very closely related to a highly pathogenic PPV isolate, yet is itself nonpathogenic in swine. This reduced pathogenic potential of KBSH may be attributable to its poor ability to replicate in swine.

Epidemiological markers of Shigella sonnei infections: R-plasmid fingerprinting, phage-typing and biotyping

In 1980, the number of Shigella sonnei strains isolated in Sicily increased markedly. Approximately 80% of the isolates belonged to phage-type 3 and showed the same antibiotic resistance pattern, suggesting that an epidemic had been going on for several months. Plasmid analysis of strains isolated at various times in different places supported this view. Agarose gel electrophoresis of plasmid preparations from seven selected phage-type 3 isolates showed the presence of two plasmids, of 80 megadaltons (Mdal) and 55 Mdal, respectively. In addition, all but one harboured a 2.8-Mdal plasmid, while a 30-Mdal and a 47-Mdal plasmid were, respectively, present in two other isolates. The 80-Mdal plasmid was self-transmissible to Escherichia coli K12, which acquired "en bloc" the resistance patterns of the donor strains. All the self-transmissible R plasmids fell into the incompatibility group I1 and showed a similar endonuclease cleavage pattern. An S. sonnei strain which was isolated during the same period, but did not belong to phage-type 3, exhibited a totally different plasmid pattern. We can conclude that phage-typing and R-plasmid characterization (i.e. incompatibility group, molecular weight and endonuclease cleavage pattern) represent the most reliable methods for epidemiologic study of S. sonnei isolates.

Genomic and phenotypic analyses of avian ureaplasma strains.

Ureaplasmas isolated from avian sources were compared to the two established Ureaplasma species, U.urealyticum from humans and U.diversum from cattle, by the metabolic inhibition test, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell protein patterns, restriction endonuclease cleavage patterns of genomic DNA and by [3H] DNA-DNA hybridization. All seven unclassified avian ureaplasma strains tested were serologically related to each other but were different from the two established bovine and human Ureaplasma species. The antigenic differences were also evident in Western blot analysis. The [3H] DNA-DNA hybridization data further confirmed these results by showing that avian ureaplasmas were genetically related to each other but distinct from the established Ureaplasma species. The restriction endonuclease cleavage patterns revealed variations among the avian strains that were not apparent by the serological and biochemical techniques. Our results support the notion that the avian ureaplasmas are distinct, are unrelated to the two established Ureaplasma species and merit consideration as a new species.

Application of "Hirt supernatant" DNA to the molecular epidemiology of cytomegalovirus infections.

Human cytomegalovirus DNA was isolated from infected cells by the Hirt method (B. Hirt, J. Mol. Biol. 26:365-369, 1967). The restriction endonuclease cleavage patterns of DNA obtained in this manner were comparable with those of DNA extracted from purified virions. The "Hirt supernatants" were satisfactory for identifying individual cytomegalovirus strains by their DNA fingerprinting.

Association of human papillomavirus subtype and clinical course in respiratory papillomatosis.

The relationship of human papillomavirus type 6 (HPV-6) subtypes to the clinical manifestations of respiratory papillomatosis was investigated. DNA was isolated from biopsy specimens of 21 patients and the viral genome analyzed by molecular hybridization. Four subtypes, designated HPV-6c through HPV-6f, were distinguishable by restriction endonuclease cleavage patterns of the viral genome. Patient records were reviewed to identify associations between viral subtype and sex, race, age at onset of papilloma, duration of disease, frequency of operations, history of tracheotomy and anatomical extension of papilloma. HPV-6c was present in 13 cases which were characterized by extensive anatomical spread of disease, higher frequency of operations, and a need for tracheotomy. HPV-6d occurred in 4 black patients with juvenile onset papilloma. HPV-6c was found in 2 white patients--1 with juvenile onset and 1 with adult onset papilloma. HPV-6f was identified in 2 white patients with adult onset disease.

Partial characterization of DNA from five entomopoxviruses

Extensive genomic heterogeneity was detected in the restriction endonuclease cleavage patterns of DNA from five entomopoxvirus isolates and vaccinia virus, strain WR. An 8.2 kilobase pair extra-chromosomal element was detected in Amsacta moorei entomopoxvirus and a 22 kilobase pair extra-chromosomal DNA element was isolated from Choristoneura biennis EPV. The extent of DNA base sequence homology was determined by Southern hybridization of HindIII and BamHI DNA restriction fragments of C. biennis EPV DNA and A. moorei EPV DNA with (α 32P)-labeled A. moorei EPV DNA. Methylation of 5′-C mCGG-3′ sequences was not detected in the DNA of A. moorei, C. biennis, E. auxiliaris, M. sanguinipes, and A. conspersa entomopoxviruses after cleavage of the viral DNAs with MspI and HpaII restriction endonucleases. Based upon the DNA base sequence homology data presented here, the five entomopoxviruses used in this study appear to be unrelated.

Isolation of a tetracycline-resistance plasmid excised from a chromosomal DNA sequence in Bacillus subtilis.

When Bacillus subtilis GSY908 (recE4-) (H. C. Spatz and T. A. Trautner, 1971, Mol. Gen. Genet. 113, 174-190) protoplasts were infected with Staphylococcus aureus plasmid pNS1 specifying tetracycline resistance (Tcr) (N. Noguchi et al., 1983, Gene 21, 105-112), which was modified such that it either could not replicate or did not carry a functional Tcr gene, a plasmid with a molecular weight of 3.1 X 10(6) (4.9 kb) was generated in Tcr phenotypes. This plasmid, named Tcr pNS1981, exhibited completely different restriction endonuclease cleavage patterns to pNS1 and showed only negligible sequence homology in hybridization experiments. Southern hybridization experiments revealed that pNS1981 arises by excision of a B. subtilis chromosomal DNA sequence. No sequence corresponding to pNS1 was detectable on the chromosome of pNS1981-maintaining B. subtilis. The production of pNS1981 was also observed in B. subtilis RM125 (r-Mm-Mrec+) (T. Uozumi et al., 1977, Mol. Gen. Genet. 152, 65-69.) with almost the same frequency as B. subtilis GSY908. Since the recipient B. subtilis Marburg 168 derivatives stated above are sensitive to Tc, the results indicate that information essential for Tcr is under negative regulatory control in the integrated state on the chromosome. Restriction endonuclease analysis suggested that pNS1981 is essentially the same as pBC16, formerly found in B. cereus (K. Bernhard, H. Schrempf, and W. Goebel, 1978, J. Bacteriol. 133, 897-903).

Molecular comparison of virulence plasmids in Shigella and enteroinvasive Escherichia coli

Virulent isolates of Shigella dysenteriae and Shigella boydii harboured a 140 Mdal plasmid which was either absent or deleted in spontaneously avirulent strains. Together with previous data concerning S. sonnei, S. flexneri and enteroinvasive Escherichia coli, the present results established the general role of extrachromosomal elements in the virulence of such enteroinvasive species. Among different species, these virulence plasmids showed unrelated endonuclease cleavage patterns, whereas hybridization experiments showed that homologous sequences were present throughout the molecules. These plasmids may therefore have derived from a common ancestor molecule which overcame evolutionary alterations in restriction sites. Furthermore, intraspecies and intraserotype comparison of these plasmids by endonuclease cleavage demonstrated highly conserved sequences. The consequences of these data for evolution, epidemiology and diagnosis of Shigella and enteroinvasive E. coli are discussed.

Characterization of Two HPV-3 Related Papillomaviruses from Common Warts That Are Distinct Clinically from Flat Warts or Epidermodysplasia Verruciformis

We have recently identified two unusual human papillomavirus (HPV) isolates while engaged in an ongoing study of wart disease in meat handlers and veterinarians. The papillomas from which these two viruses were isolated clinically resembled verruca vulgaris rather than either flat warts or epidermodysplasia verruciformis (EV). These two previously uncharacterized HPVs were molecularly cloned and characterized with respect to known HPVs. The genomes of the two viruses exhibited dramatically different restriction endonuclease cleavage patterns but were found to have significant sequence homology to each other, as well as to HPV-3 and a new virus isolated from a patient with EV. Neither of the two new HPV isolates exhibit detectable sequence homology under stringent conditions of hybridization or share similar restriction endonuclease cleavage patterns with previously characterized HPV types 1,2,4,5,6b, or a previously isolated HPV from meat handlers.

Amplification of rDNA and type I sequences in Drosophila males deficient in rDNA.

rDNA magnification is a heritable change in rDNA content that occurs in D. melanogaster males when chromosomes deficient in rDNA are placed together for several generations. We have examined the restriction endonuclease cleavage pattern of the rDNA from an X chromosome undergoing magnification, and find no evidence for the selective amplification of either uninterrupted rDNA units or those containing insertion sequences. In addition, we observe an amplification of rDNA in the first generation of extremely bobbed male progeny to a level exceeding that of wild-type flies, but that reduces to the wild-type level in subsequent generations. The type I rDNA insertion elements also occur as tandem arrays, independently of rDNA. Southern hybridizations indicate that the majority of these sequences are located in the heterochromatin surrounding the nucleolus organizer on the X chromosome, and we find that they, too, amplify transiently in the first generation of magnifying males.

Inheritance of extrachromosomal rDNA in Physarum polycephalum.

In the acellular slime mold Physarum polycephalum, the several hundred genes coding for rRNA are located on linear extrachromosomal DNA molecules of a discrete size, 60 kilobases. Each molecule contains two genes that are arranged in a palindromic fashion and separated by a central spacer region. We investigated how rDNA is inherited after meiosis. Two Physarum amoebal strains, each with an rDNA recognizable by its restriction endonuclease cleavage pattern, were mated, the resulting diploid plasmodium was induced to sporulate, and haploid progeny clones were isolated from the germinated spores. The type of rDNA in each was analyzed by blotting hybridization, with cloned rDNA sequences used as probes. This analysis showed that rDNA was inherited in an all-or-nothing fashion; that is, progeny clones contained one or the other parental rDNA type, but not both. However, the rDNA did not segregate in a simple Mendelian way; one rDNA type was inherited more frequently than the other. The same rDNA type was also in excess in the diploid plasmodium before meiosis, and the relative proportions of the two rDNAs changed after continued plasmodial growth. The proportion of the two rDNA types in the population of progeny clones reflected the proportion in the parent plasmodium before meoisis. The rDNAs in many of the progeny clones contained specific deletions of some of the inverted repeat sequences at the central palindromic symmetry axis. To explain the pattern of inheritance of Physarum rDNA, we postulate that a single copy of rDNA is inserted into each spore or is selectively replicated after meiosis.

Inheritance of extrachromosomal rDNA in Physarum polycephalum.

In the acellular slime mold Physarum polycephalum, the several hundred genes coding for rRNA are located on linear extrachromosomal DNA molecules of a discrete size, 60 kilobases. Each molecule contains two genes that are arranged in a palindromic fashion and separated by a central spacer region. We investigated how rDNA is inherited after meiosis. Two Physarum amoebal strains, each with an rDNA recognizable by its restriction endonuclease cleavage pattern, were mated, the resulting diploid plasmodium was induced to sporulate, and haploid progeny clones were isolated from the germinated spores. The type of rDNA in each was analyzed by blotting hybridization, with cloned rDNA sequences used as probes. This analysis showed that rDNA was inherited in an all-or-nothing fashion; that is, progeny clones contained one or the other parental rDNA type, but not both. However, the rDNA did not segregate in a simple Mendelian way; one rDNA type was inherited more frequently than the other. The same rDNA type was also in excess in the diploid plasmodium before meiosis, and the relative proportions of the two rDNAs changed after continued plasmodial growth. The proportion of the two rDNA types in the population of progeny clones reflected the proportion in the parent plasmodium before meoisis. The rDNAs in many of the progeny clones contained specific deletions of some of the inverted repeat sequences at the central palindromic symmetry axis. To explain the pattern of inheritance of Physarum rDNA, we postulate that a single copy of rDNA is inserted into each spore or is selectively replicated after meiosis.

A comparative analysis of restriction enzyme digests of the DNA of herpes simplex virus isolated from genital and facial lesions.

The restriction endonuclease (RE) cleavage patterns of the DNA of herpes simplex virus (HSV) genital isolates from two geographically distinct groups and from one group of facial isolates were examined. Eleven of 21 genital isolates from females, 1 of 27 genital isolates from males and all 17 of the facial isolates were HSV type 1 (HSV-1). The groups of isolates of the same serotype could not be distinguished by significant differences in the frequency of variable restriction endonuclease sites or molecular weight of variable length fragments. Simultaneous consideration of two or more variable sites has disclosed some which are apparently correlated in the HSV-1 isolates, and which could provide a useful marker for phylogenetic relationships. However, most pairs showed no correlation, while certain sites appeared more closely correlated in the genital than in the facial isolates. All 39 HSV-2 isolates could be distinguished from each other on the basis of a combination of variable RE sites and variable length fragments. Bg/II RE sites appeared less variable than other RE sites.

Restriction endonuclease cleavage analysis of adenovirus type 8: two new subtypes from patients with epidemic keratoconjunctivitis in Sapporo, Japan.

We studied the restriction endonuclease cleavage patterns of DNAs of adenovirus type 8 (Ad 8) isolated from epidemic keratoconjunctivitis cases. DNAs of 25 Ad 8 isolates collected during the period from 1975 to 1981 in Sapporo were subjected to enzymatic cleavage with PstI, BamHl , HindIII and SalI. On the basis of the cleavage patterns, the isolates were divided into two subtypes, A and B, both different from Trim strain used as the Ad 8 prototype strain. Subtype A was prevalent in the period from 1975 to 1978, while subtype B in the period from 1976 to 1981.