Grouping of adenoviruses and identification of restriction endonuclease cleavage patterns of adenovirus DNAs using infected cell DNA: simple and practical methods

Simple and practical methods for grouping of adenoviruses and for identification of restriction endonuclease cleavage patterns of viral DNA were established by using infected cell DNA. DNA homology groupings of adenoviruses could be examined by spot hybridization, and restriction endonuclease cleavage patterns of viral DNAs could be obtained by Southern blot hybridization, by using infected cell DNA. The method was very sensitive and allowed the identification of the cleavage pattern of viral DNA of the inoculum by means of cell DNA extracted from infected cells with undetectable cytopathic effect (CPE). In ethidium bromide-stained gels without Southern blot hybridization, the restriction endonuclease cleavage pattern of viral DNA could be detected precisely in spite of background staining due to cellular DNA. The preparation of infected cell DNA used in these procedures was technically much easier than that of viral DNA. These methods require only a small number of infected cells and allow many isolates to be investigated with ease.     

Intra-individual heterogeneity of rDNA allows the distinction between two closely related species in the Genus Helianthus

The Ribosomal DNAs of Helianthus annuus and H. argophyllus were analysed. Total DNA from single individuals of six cultivated lines, one wild ecotype ofH. annuus, and three ecotypes of H. argophyllus, were digested with various restriction enzymes. Hybridisation of Southern blots with sunflower ribosomal probes containing most of the interspacer regions (R3) or the 25 s coding region (R2) reveals different patterns from those expected: while no difference between H. annuus and H. argophyllus had been observed in previous rDNA RFLP analysis, our study clearly distinguished the two species on the basis of two different patterns when using R3 and BamHI, BstYI, or EcoRI/BamHI. Furthermore, the sum of the fragment weights of the BamHI restriction patterns was much greater than that of the rDNA entire unit-weight space. The co-existence of different rDNA units within single individuals is proposed as a model to explain these results. Four rDNA units were distinguished, which differed in their state of methylation and by the presence of mutations at two BamRI restriction sites. H. annuus individuals displayed two types of rDNA units while H. argophyllus individuals displayed four types.     

Localization of flagellin genes on the physical map of Methanococcus voltae

Methanococcus voltae DNA, digested individually with the restriction enzymes ApaI, SacII, BamHI, or EagI, was resolved by pulsed-field gel electrophoresis reproducing the previously published digestion patterns. Hybridization of a flagellin gene-specific probe to such gels dried down (unblots) resulted in the identification of one band per enzyme harboring the flagellin genes. These bands all overlapped, revealing that an approximately 15-kb BamHI/EagI DNA fragment should harbor the flagellin genes. Double digestion with BamHI and EagI resulted in the resolution of two bands in the 15-kb region of the gel. Separation of these two fragments prior to blotting and probing with a flagellar gene-specific probe revealed that one of these fragments possessed the flagellar sequences. The presence of an EagI restriction site in flaB3 localized the flagellin genes precisely at the junction of EagI fragments Ea2 and Ea5 at approximately the 1800-kb position of the physical map.     

Restriction analysis of lambda EMBL3 background recombinants: occurrence of lambda phages carrying a head to tail oriented left arm DNA sequence

Summary Eight representative recombinant background clones of λEMBL3 were analysed usingKpnI,BamHI,SalI,EcoRI andHindIII digestion. We found that λEMBL3 carries its own left arm in theBamHI cloning site. In this way, recombinant molecules were found to be generated which can grow onEscherichia coli strain NM539. In all cases analysed, the left arm DNA was inserted in a head to tail orientation. Seven clones carried a restoredBamHI site at thecos site-BamHI site connection. In the region where the inserted left arm and the right arm were ligated,BamHI cloning produces a large palindromic sequence consisting of two polylinkers. ThisBamHI site was incompletely cleaved in all cases analysed. We assume that a part of the λ DNA molecule in this region shows a cruciform structure prohibiting recognition or cleavage of this site by restriction endonucleaseBamHI.     

Physical mapping of BglII, BamHI, EcoRI, HindIII and PstI Restriction fragments of bacteriophage P1 DNA

Summary A cleavage map of bacteriophage P1 DNA was established by reciprocal double digestion with various restriction endonucleases. The enzymes used and, in parenthesis, the number of their cleavage sites on the P1clts genome are: PstI (1), HindIII (3), BglII (11), BamHI (14) and EcoRI (26). The relative order of the PstI, HindIII and BglII sites, as well as the order of 13 out of the 14 BamHI sites and of 17 out of the 26 EcoRI sites was determined. The P1 genome was divided into 100 map units and the PstI site was arbitrarily chosen as reference point at map unit 20.DNA packaging into phage heads starts preferentially at map unit 92 and it proceeds towards higher map units. The two inverted repeat sequences of P1 DNA map about at units 30 and 34.     

Physical characterization of plasmids from Streptomyces kasugaensis MB273

Plasmid DNA of molecular weight 6.8 × 10⁶ was isolated from Streptomyces kasugaensis MB273. The plasmid DNA showed a single CsCl-ethidium bromide density gradient centrifugation, in neutral sucrose gradient centrifugation, and in agarose gel electrophoresis. When this DNA was digested with BamHI or SalI endonucleases, an unexpected number of fragments were found on agarose gel electrophoresis. Molecular weight summation of fragments obtained from double restriction enzyme digestions suggested that the plasmid DNA was a mixture of two different plasmids. This was confirmed by constructing recombinant plasmids between S. kasugaensis plasmid DNA and pBR322, and then by isolating two plasmids after SalI endonuclease treatment followed by sucrose gradient centrifugation. One of the plasmids (pSK1) had a single recognition site for BamHI, EcoRI, and SalI, and three sites for BglII. The other plasmid (pSK2) had a single recognition site for EcoRI and BglII, two recognition sites for BamHI, and no cleavage site for SalI. The cleavage maps of these plasmids were constructed using several restriction endonucleases.     

Genetic differentiation between laboratory lines of the musk shrew (Suncus murinus,insectivora) based on restriction endonuclease cleavage patterns of mitochondrial DNA

The cleavage patterns of mitochondrial DNA (mtDNA) by 17 restriction endonucleases were compared between eight lines of musk shrews derived from different wild-caught stocks. Enzymatic digestion byBamHI,PvuII,XbaI, andXhoI showed a cleavage pattern common to all lines that were from five Japanese islands (Nag, Ize, OKI, TKU, and Tr), Bangladesh (BAN), Sri Lanka (SRI), and Java (Bog), and every line lacked cleavage sites forSalI andSmaI. Different cleavage patterns were detected by the remaining 11 enzymes. Within the BAN line, the presence of at least two types of mtDNAs was proved by six enzymes and was not contradictory to the maternal pedigrees going up to the wild ancestors of the stock. More than 30 cleavage sites of the shrew mtDNA were mapped by double-digestion methods. Nucleotide diversities of mtDNA were calculated from these maps and were estimated to be less than 0.5% among Japanese and Bog lines but to be 3.8% between BAN and the other seven lines and 2.3% within the BAN line. These results indicate that BAN shrews differentiate from the other lines to the intersubspecific extent reported in mice previously.     

Studies of viable T4 bacteriophage containing cytosine-substituted DNA (T4dC phage)

Summary Digestion of non-glucosylated and cytosine-substituted T4 phage (T4dC) DNA with SalI restriction endonuclease showed that the DNA had nine SalI-sensitive sites. There were eight SalI sites in DNA from a strain which had a deletion in the rII-denB-ndd region. The comparison of two digestion patterns indicated that one of the SalI-sensitive sites was present in the deleted region and that the SalI-F fragment (8.4x10⁶ daltons) was located adjacent to the SalI-C or SalI-D fragment (15.5x10⁶ daltons) on the T4 chromosome. The DNA gave no detectable cleavage product when digested with BamHI endonuclease alone, while, when digested successively with BamHI and SalI, the DNA yielded two new digestion products in place of one fragment formed by SalI alone. The BamHI-sensitive site was in the SalI-A fragment (25.2x10⁶ daltons). The usefulness of this information for making cleavage maps of T4 phage chromosome is discussed.     

Physical mapping of homologous segments of mitochondrial episomes from S male-sterile maize

Restriction endonuclease maps of two double-stranded plasmid-like DNAs, 6180 and 5175 bp each, isolated as linear molecules from the mitochondria of S-type cytoplasmic male-sterile maize were prepared. Twelve cleavage sites were mapped in each using HindIII, XhoI, EcoRI, SacI, XbaI, SalI, BamHI, and BstEII. BamHI does not cleave S-1 DNA and SalI does not cleave S-2 DNA. A 1150-bp homologous sequence in addition to the 200-bp terminal inverted repeats was terminally oriented on both DNAs by reciprocal hybridization and heteroduplex analysis.     

Restriction Fragment Length Polymorphism Analysis of the Mitochondrial DNA of Fusarium oxysporum f. sp. ciceris

Seven isolates of Fusarium oxysporum f. sp. ciceris, representing pathogenic races 1 , 2, 3, and 4 from India and 0, 5, and 6 from Spain, were assayed for restriction fragment length polymorphisms (RFLPs) in the mitochondrial DNA,(mt DNA). The mt DNA fraction of total fungal DNA was purified and digested with the restriction endonucleases Bam HI, Bgl II, Eco RI. Kpn I, Sac I, Sal I, Sma I, and Xho I. The mt DNA is a circular molecule of 40.5 kb. No RFLP in the mt DNA was detected among the seven races of F. o. ciceris. The identical restriction patterns of mt DNA indicates an extensive conservation in the gene composition of mt DNA without sequence variation, and suggests that mt DNA of F. o. ciceris may not be responsible for pathogenic diversity. The restriction map of mt DNA from the race 6 isolate Fo 8272 was constructed by digestion of the mt DNA with five restriction enzymes: Eco RI, Kpn I, Sac I, Sal I, and Xho I, either singly or in selected pairs.     

Mitochondrial DNA polymorphism in native Philippine cattle based on restriction endonuclease cleavage patterns

An analysis of patterns of cleavage of mtDNA by restriction endonucleases was performed for nine individuals from the Philippine population of native cattle. MtDNA polymorphisms were detected in the restriction patterns generated by the following six enzymes,BamHI,BglII,EcoRV,HindIII,PstI, andScaI. The restriction patterns showing polymorphisms were distributed nonrandomly among the nine individuals examined from the Philippine population of native cattle, indicating the existence of two separate types of mtDNA. These two types of mtDNA are very different from each other, at the level of subspecies. Since the native Philippine cattle are considered to represent an admixture of European and Indian cattle, the two types of mtDNA must be derived from the mtDNAs of both varieties. The polymorphic sites in mtDNA have been located on a restriction map, and the nucleotide substitutions at some of the sites have also been estimated.     

Human mitochondrial DNA types in Finland

Summary Variation in mitochondrial DNA (mtDNA) in a sample of 110 Finns was analyzed with six restriction enzymes, AvaII, BamHI, HaeII, HinII, HpaI, and MspI, by using total blood cell DNA probed with mouse mtDNA. Two new enzyme morphs were observed, one for HaeII and one for HindII. Double-digestion experiments indicated that the BamHI morphs 2 and 3 result from base changes leading to AvaII morphs 3 and 9, respectively. Of the ten different mtDNA types observed, defined by restriction fragment patterns, seven have been previously described in Caucasoid populations. The three new Finnish mtDNA types can be derived from Caucasoid lineages by single restriction site changes. The results were used to reconstruct a phylogenetic tree for Caucasoid mtDNA types defined by the enzymes used. The frequencies of mtDNA types were used to compute genetic distances between Finns, Italians, and Israeli Jews. The frequencies of both enzyme morphs and mtDNA types show that the Finnish population is highly homogeneous.     

DNA restriction endonuclease cleavage patterns,DNA sequence similarity and phenotypical characteristics in some strains of Lactobacillus helveticus and Lactobacillus jugurti

Physiological characteristics, deoxyribonucleic acid (DNA) base composition (% guanine + cytosine; % GC), DNA sequence similarity (% DNA-DNA hybridization) and DNA restriction endonuclease cleavage patterns of two strains of Lactobacillus helveticus and four strains of Lactobacillus jugurti were examined.All the strains investigated were closely related genetically, having DNA-DNA hybridization values ranging from 89–100%. Nevertheless, these strains can be differentiated from one another on the basis of the digestion of their DNA by specific restriction endonucleases, such as Bam HI, Eco RI and Hind III. The DNA of these strains shows clear, reproducible and distinct cleavage patterns. Cleavage patterns of DNA from strains L. jugurti S.35.19 and S.36.2 were found to be similar. These findings suggest that fingerprinting of DNA by restriction endonuclease cleavage might provide, in addition to the conventional methods, a useful tool for the characterization of closely related microorganisms at the strain level.     

Comparative macromolecular analysis of the cytoplasms of normal and cytoplasmic male sterile Brassica napus

Summary Chloroplast (cp) and mitochondrial (mt) compartments of normal (N) and cytoplasmic male sterile (cms) lines of Brassica napus have been characterized and compared on the basis of cp and mt DNA restriction enzyme analysis and in vitro protein synthesis by isolated mitochondria. Cytoplasmic male sterility of B. napus (rape) comes from cms Raphanus sativus (radish) through intergeneric crosses.Cp DNAs isolated from N and cms lines had distinct restriction patterns with Sal I, Kpn I and Sma I enzymes. The size of the two cp DNAs measured from the restriction patterns was found to be identical and of about 95 × 10⁶ d. N and cms lines of B. napus were characterized by specific mt DNAs, as shown from Sal I, Kpn I, Pst I and Xho I cleavage patterns. The small number of well-separated restriction fragments obtained with Sal I enabled us to determine precisely mt DNA sizes. The values of 136.5 and 140.3 × 10⁶ d, obtained from restriction patterns with N and cms DNAs respectively, are smaller than any of those previously obtained from studies on other genera. With molecular hybridization experiments, it was possible to distinguish N and cms lines by the different locations of rRNA genes on the cp and mt DNAs.Two lines of B. napus are characterized by specific mt translation products formed in isolated mitochondria.     

A restriction enzyme cleavage map of the histidine utilization (hut) genes of Klebsiella aerogenes and deletions lacking regions of hut DNA

Summary The histidine utilization (hut) operons of Klebsiella aerogenes were cloned into pBR322. The hut genes are wholly contained on a 7.9 kilobase pair fragment bounded by HindIII restriction sites and expression of hut is independent of the orientation of the fragment with respect to pBR322. A restriction map locating the 27 cleavage sites within hut for the enzymes, HindIII, PvuII, SalI, BglII, KpnI, PstI, SmaI, AvaI, and BamHI was deduced. Several of the cleavage sites for the enzymes HaeIII and HinfI were also mapped. A set of deletion plasmids was isolated by removing various restriction fragments from the original plasmid. These deletions were characterized and were used to assist in mapping restriction sites. This physical characterization of hut DNA opens the way for genetic and molecular analysis of the regulation of hut gene expression in vitro as well as in vivo.     

Pulsed Field Gel Electrophoresis Analysis of Borrelia burgdorferi Sensu Lato Isolated in Japan and Taxonomic Implications with Lyme Disease Spirochetes

Genomic DNAs of Borrelia burgdorferi sensu lato isolates obtained in Japan sharing different rRNA gene ribotypes were digested with rare-cutter restriction endonucleases and the fragments obtained were separated by pulsed field gel electrophoresis (PFGE). The sizes of large restriction cleavage bands with MluI endonuclease were quite similar among isolates in each ribotype group. On the other hand, the PFGE profiles obtained with the other enzymes (NruI, Sal I or SplI) were rather divergent, and Japanese isolates were distinguishable from the United States and European isolates. The Japanese isolates classified as ribotypes group II (Borrelia garinii) and III (B. afzelii) showed different PFGE patterns from that of European isolates. The isolates grouped into ribotype IV revealed distinctively different PFGE profiles. These results indicate that the Japanese isolates may be genetically divergent and distinct from the United States and European isolates.     

A specific viral DNA sequence is stably integrated in herpesvirus oncogenically transformed cells

The integration patterns of viral DNA sequences in three hamster embryo cell lines independently derived by transformation with equine herpesvirus type 1 (EHV-1) have been investigated by DNA blot hybridization analyses for the restriction enzymes Eco RI, Bgl II, Xba I and Bam HI with ³²P-labeled selected DNAs from a collection of cloned EHV-1 restriction enzyme fragments as probes. These EHV-1-transformed cell lines contained subgenomic portions of the viral genome in an integrated state at multiple sites in the host genome. At least one copy of a viral DNA sequence mapping colinearly from 0.32 to 0.38 map units within the EHV-1 genome was common among these three EHV-1 transformed cell lines. The 0.32–0.38 viral DNA sequence was maintained stably even after 125 cell passages, whereas sequences from other positions in the EHV-1 genome were lost progressively during continued cell passage. The significance of the findings that these oncogenically transformed cell lines harbor a specific region of the EHV-1 genome is discussed with regard to stable maintenance of the oncogenically transformed state.     

Mapping of BamHI and SmaI DNA restriction sites on the genome of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica

The DNA of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica (AcNPV), has been analyzed with restriction endonucleases BamHI and SmaI. The molecular weight of the BamHI fragments, SmaI fragments, and BamHI + SmaI fragments has been determined. The molecular weight of AcNPV DNA is calculated to be about 82 million. A presumptive physical map of the BamHI and SmaI restriction sites on the AcNPV genome has been constructed.     

Sequence analysis of Vicia faba highly repeated DNA: the BamHI repeated sequence families

Cleavage of Vicia faba nuclear DNA with the restriction endonuclease BamHI yielded discrete size classes of 250, 850, 900, 990, 1 150, 1 500 and 1 750 bp of highly repetitive DNA. Each of these sequence families comprised about 3% of the total genomic DNA. Some sequence members from each sequence family were cloned in pBR322 and their primary structures determined. Computer analyses of nucleotide sequences suggested the existence of about 60 bp sequence periodicity within the repeating unit of the 990 bp sequence family, though the extent of homology among the surmised shorter subrepeat units was very low. With other BamHI sequence families, however, the data did not show any clear internal sequence periodicity. The repeat units of the 850 bp and 1 750 bp sequence families contained nucleotide sequences homologous to the 250 bp family sequence. No sequence relationship between or among other sequence families was observed. There was 13–25% sequence variation among 6 cloned members of the 250 bp family and probably also among those of other BamHI repeat families. DNA sequences homologous to these V. faba BamHI repeat families were detected in Pisum sativum DNA by Southern blot hybridization. Furthermore, very weak cross-hybridization was observed with plant DNAs from Phaseolus vulgaris, Triticum aestivum, Cucumis sativus and Trillium kamtschaticum.     

Integrated map of the Schizosaccharomyces pombe genome

A restriction map of the entire Schizosaccharomyces pombe genome was constructed using two restriction enzymes (BamHI and PstI) that recognize 6 bp. The restriction map contains 420 minimally overlapping clones (miniset) and has 22 gaps. We located 126 genes, marker fragments of DNA (NotI and SfiI linking clones), and 36 transposable elements by hybridization to unique restriction fragments. Received: 21 November 1996; in revised form: 3 March 1997 / Accepted: 27 March 1997