A restriction map of the bacteriophage T4 genome

Summary We report a detailed restriction map of the bacteriophage T4 genome and the alignment of this map with the genetic map. The sites cut by the enzymes BglII, XhoI, KpnI, SalI, PstI, EcoRI and HindIII have been localized. Several novel approaches including two-dimensional (double restriction) electrophoretic separations were used.     

Mapping of additional restriction enzyme cleavage sites on bacteriophage MB78 genome

A physical map of bacteriophage MB78 DNA indicating the cleavage sites for the enzymeBglII,ClaI,EcoRI,PvuII,SalI andSmaI comprising of a total of 34 cleavage sites have been constructed earlier. The cleavage sites for a few more restriction endonucleases likeApaI,AvaI,BglI,HindIII,KpnI andXhoI have now been mapped. A total of 72 cleavage sites on MB78 DNA are known by now. Relative positions ofEcoRI I and J fragments which could not be decided earlier has now been determined.     

Mapping of restriction sites in the attachment site region of bacteriophage lambda

Summary A fine structure map of theEcoRI fragment containing the lambda attachment-site region has been constructed. 38 different restriction endonucleases have been employed and 170 sites located in this fragment. In addition, sites in adjacent regions have been determined for several enzymes. Complete cleavage maps of the entire lambda genome have been obtained for endonucleasesBglII,BluI,KpnI,SacI,SacII,SalI andXbaI. The strategy employed for mapping included comparison of deletion and substitution mutants, analysis of mixed digests, and detailed analysis of subfragments.     

A physical map of bacteriophage T4 including the positions of strong promoters and terminators recognized in vitro

Summary We present a linearized physical map of the genome of bacteriophage T4. This map contains the cleavage sites for restriction enzymes SmaI, KpnI, SalI, BglII, XhoI, XbaI, ClaI, HaeII, EcoRI, and EcoRV. It also contains about 200 TaqI sites. The promoter sites recognized in vitro and a number of rho independent terminators have also been mapped.     

Restriction map of CELO virus DNA

A restriction map of chicken embryo lethal orphan (CELO) virus DNA was reported with ten restriction endonucleases (XbaI, XhoI, SalI, HindIII, EcoRI, BglI, KpnI, BamHI, PstI and SstI). CELO virus DNA was estimated by comparing CELO virus DNA fragments with marker DNA fragments to have a molecular weight of 29.3·10⁶.     

Chloroplast DNA differences between cultivated hop,Humulus lupulus and the related species H. japonicus

Chloroplast DNA (cpDNA) of Humulus Lupulus and H. japonicus was examined by restriction endonuclease analysis with BamHI, BanI, BclI, BstEII, DraI, EcoRI, EcoRV, HindIII, KpnI, PaeR7I, PstI, PvuII, SalI and XhoI. The restriction fragment patterns showed that the cpDNAs shared a large number of restriction sites. However, the chloroplast genomes of the two species could be distinguished by differences in restriction site and restriction fragment patterns in the PstI, PvuII, BclI, EcoRV, DraI and HindIII digests. On the basis of the complexity of restriction enzyme patterns, the enzymes PstI, PvuII, SalI, KpnI and XhoI were selected for mapping the chloroplast genomes. Single and double restriction enzyme digests of cpDNA from the two species were hybridized to cpDNA probes of barley and tobacco. The data obtained from molecular hybridization experiments were used to construct the cleavage site maps. Except for the PstI digest, the arrangement of cpDNA restriction sites was found to be the same for both species. An extra PstI site was present in H. lupulus. Three small insertions/deletions of about 0.8 kbp each were detected in the chloroplast genomes of the two species. Two of these insertions/deletions were present in the large and one in the small singlecopy region of the chloroplast genome. The cpDNA of Humulus was found to be a circular molecule of approximately 148 kbp that contains two inverted repeat regions of 23 kbp each, a small and a large single -copy region of approximately 20 kbp and 81 kbp, respectively. The chloroplast genome of hop has the same physical and structural organization as that found in most angiosperms.     

Comparison of Three Methods for Epidemiological Typing of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and <Emphasis Type="Italic">C. coli</Emphasis>

A total of 168 Campylobacter strains (154 C. jejuni and 14 C. coli) isolated from human clinical samples and chicken meat were typed using Penner serotyping, randomly amplified polymorphic DNA (RAPD), and pulsed-field gel electrophoresis (PFGE) with four restriction enzymes (Sac II, Sal I, Sma I, Kpn I).The 168 strains were found to represent 13 different Penner-types and 72 different RAPD-types. However, the discriminatory potential of PFGE was dependent on the restriction enzymes used. The 168 strains were divided into 74 (Sac II), 73 (Sal I), 72 (Sma I) and 69 (Kpn I) types. The DNA of some strains was not digested by Sal I, Sma I and Kpn I. Although three RAPD-types were further subdivided by PFGE, RAPD showed good discriminatory power and a high level of agreement with PFGE patterns in terms of strain differentiation.To compare the similarities of PFGE patterns (Sac II) among the strains, a dendrogram was constructed based on the unweighted pair group method with averages (UPGMA). In most cases, DNA types of C. coli were different from those of C. jejuni. The similarities between human and meat isolates were less than 0.42 except for one outbreak in which the isolates from both patients and chicken meat showed the same DNA types.     

Physical mapping of the pea chloroplast DNA and localization of the ribosomal RNA genes

The closed circular DNA of pea chloroplast has been digested with restriction endonucleases SalI, SmaI, BamHI, XbaI, XhoI, HindIII, and EcoRI. A physical restriction map of pea ctDNA has been constructed by mapping the SalI and SmaI sites. The pea ctDNA has been found to contain one set of ribosomal RNA genes by Southern hybridization of restriction endonuclease digest, R-loop studies, and DNA-DNA heteroduplex mapping. The 23 S and 16 S RNA genes are confined to a DNA region of 3.0 and 1.5 kbp, respectively. The two rRNA chains are separated by a spacer region of 2.2 kbp.     

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.     

Organization of nuclear ribosomal DNA and species-specific polymorphism in closely related Fraxinus excelsior and F. oxyphylla

The ribosomal DNA repeat units of two closely related species of the genus Fraxinus, F. excelsior and F. oxyphylla, were characterized. The physical maps were constructed from DNA digested with BamHI, EcoRI, EcoRV and SacI, and hybridized with three heterologous probes. The presence or the absence of an EcoRV restriction site in the 18s RNA gene characterizes two ribosomal DNA unit types found in both species and which coexist in all individuals. A third unit type appeared unique to all individuals of F. oxyphylla. It carries an EcoRI site in the intergenic spacer. Each type of unit displayed length variations. The rDNA unit length of F. excelsior and F. oxyphylla was determined with EcoRV restriction. It varied between 11kb and 14.5kb in F. excelsior and between 11.8kb to 13.8kb in F. oxyphylla. Using SacI restriction, at least ten spacer length variants were observed in F. excelsior, for which a detailed analysis was conducted. Each individual carries 2–4 length variants which vary by a 0.3-kb step multiple. This length variation was assigned to the intergenic spacer. By using the entire rDNA unit of flax as probe in combination with EcoRI restriction, each species can be unambiguously discriminated. The species-specific banding pattern was used to compare trees from a zone of sympatry between the two species. In some cases, a conflicting classification was obtained from morphological analysis and the use of the species-specific rDNA polymorphism. Implications for the genetic management of both species are discussed.     

Physical mapping of plastid DNA variation among eleven Nicotiana species

Summary Plastid DNA of seven American and four Australian species of the genus Nicotiana was examined by restriction endonuclease analysis using the enzymes Sal I, Bgl I, Pst I, Kpn I, Xho I, Pvu II and Eco RI. These endonucleases collectively distinguish more than 120 sites on N. tabacum plastid DNA. The DNAs of all ten species exhibited restriction patterns distinguishable from those of N. tabacum for at least one of the enzymes used. All distinctive sites were physically mapped taking advantage of the restriction cleavage site map available for plastid DNA from Nicotiana tabacum (Seyer et al. 1981). This map was extended for the restriction endonucleases Pst I and Kpn I. In spite of variation in detail, the overall fragment order was found to be the same for plastid DNA from the eleven Nicotiana species. Most of the DNA changes resulted from small insertions/deletions and, possibly, inversions. They are located within seven regions scattered along the plastid chromosome. The divergence pattern of the Nicotiana plastid chromosomes was strikingly similar to that found in the genus Oenothera subsection Euoenothera (Gordon et al. 1982). The possible role of replication as a factor in the evolution of divergence patterns is discussed. The restriction patterns of plastid DNA from species within a continent resembled each other with one exception in each instance. The American species N. repanda showed patterns similar to those of most Australian species, and those of the Australian species N. debneyi resembled those of most American species.Abbreviations ims isonuclear male sterile - ptDNA plastid chloroplast DNA - Rubisco ribulosebisphosphate carboxylase/oxygenase - kbp kilobase pairs - LSU large subunit of Rubisco     

A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance.

Summary This paper reports a cleavage site map of Tn5 for restriction enzymes BamHI, BglI, BglII, HindII, HindIII, HpaI, SalI, AvaI, SmaI, XhoI, PstI, PvuII, HaeII and HaeIII that was determined by the analysis of restriction enzyme cleavage patterns of ColEl, two independent ColE1::Tn5 plasmids, and a ColE1::Tn5 deletion derivative. BalI, EcoRI, KpnI, and PvuI do not cleave Tn5. Construction and analysis of in vitro-generated deletions of a ColE1::Tn5 plasmid limit the sequences encoding neomycin resistance to a 1500-base-pair-long segment of Tn5. Insertion of DNA at a BglII site within this segment results in loss of the neomycin resistance phenotype. Since this BglII site lies in an inverted repeat region, sequences within this repeat seem to be involved in the expression of neomycin resistance.     

Restriction Map of Rachiplusia ou and Rachiplusia ou-Autographa californica Baculovirus Recombinants

The restriction sites of Rachiplusia ou nuclear polyhedrosis virus (RoMNPV) DNA were mapped for the endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI. Of the 60 DNA restriction sites of RoMNPV, 35 mapped in similar positions as compared to the restriction sites of Autographa californica nuclear polyhedrosis virus (AcMNPV) DNA. Two plaque-purified viruses, obtained from randomly picked plaques of a wild-type isolate of RoMNPV, were recombinants of RoMNPV and AcMNPV. The recombinants were shown to have RoMNPV and AcMNPV restriction fragments as well as structural polypeptides from each parental virus. Both recombinant viruses had a major RoMNPV capsid protein but were occluded in the AcMNPV polyhedrin protein.     

Chloroplast genome differences between Paspalum dilatatum Poir and the related species P. notatum Flugge

 Chloroplast DNA (cpDNA) of Paspalum dilatatum and P. notatum was digested singly or in combination with the restriction endonucleases PstI, PvuII, SalI, KpnI and XhoI. Data obtained from filter hybridization experiments with barley and wheat cpDNA probes were used to construct restriction site maps of the chloroplast genomes of the Paspalum species. The cpDNA fragments were ordered into a circular configuration of approximately 139.3 kbp that contained two inverted repeat regions of approximately 23 kbp and a small and large single-copy region of approximately 11 kbp and 83 kbp, respectively. The cpDNA maps showed that P. dilatatum and P. notatum shared a large number of restriction sites with the proportion of shared restriction sites S=0.90. No restriction site differences were detected in the KpnI maps. Eight species-specific restriction site differences that could be used to identify the cytoplasm of each Paspalum species were identified in the PstI, PvuII, SalI, and XhoI cleavage maps. The overall structural organization of the Paspalum cpDNAs is rather similar to those of most cpDNAs from other plants. The results presented in this study will be of value for exploring further phylogenetic relationships within the genus Paspalum. Received: 27 February 1997 / Accepted: 7 March 1997     

Structural organization of the nuclear ribosomal RNA genes in Cannabis and Humulus (Cannabaceae)

The structural organization of the nuclear ribosomal DNA (rDNA) of Humulus lupulus, H. japonicus and Cannabis sativa was determined by restriction site mapping. A high degree of DNA sequence similarity was evident in the coding regions of the rDNA repeats of the taxa and supports the placement of Cannabis and Humulus in one family, Cannabaceae. However, the presence of a BstEII site, an additional SacI site, absence of the SpeI site and positional differences of the SspI sites in the 25 S gene distinguished H. japonicus from H. lupulus. Humulus lupulus has an additional EcoRV site in the IGS region. A XhoI site in the 18S region of C. sativa distinguishes it from the two hop species. The diagnostic differences in the IGS of C. sativa include the EcoRI, HindIII and XhoI sites. These sites were not detected in the IGS of the two hop species.     

Mitochondrial DNA from Podospora anserina

Summary EcoRI fragments of the 94 kilobase mitochondrial DNA (mtDNA) from young, wild type Podospora anserina were cloned into the EcoRI site of the E. coli plasmid vector pBR325. A complete EcoRI clone bank was developed, containing all 16 of the EcoRI fragments from the native mtDNA. Restriction endonuclease maps for the enzymes SalI, XhoI, BamHI, EcoRI, BglII, and HaeIII were constructed from the analysis of single, double, and triple restriction digests of cloned and native mtDNA. In constructing the maps data were refined by extensive Southern analysis of the native genome hybridized to cloned DNA probes. Restriction maps were analyzed and permitted us to locate the origin of mtDNA derived from senescent cultures.Both the large and small rRNA genes were then localized on these restriction maps using Southern and Northern blot analysis. We have shown the large rRNA locus to lie within a 10.8 kb region of EcoRI fragments E5 and E7, and the small rRNA locus to lie on a 5 kb subfragment of EcoRI fragment E1. The limit of separation between these two loci was determined to be between 6 and 9 kb.Surprisingly, when electrophoresed in agarose-CH₃HgOH gels, the large rRNA was found to be 3.8 kb long, 500 bases longer than that from the very closely related Neurospora crassa, making it the largest rRNA yet described.     

Isolation and characterization of a new ruminal bacteriophage lytic toStreptococcus bovis

A new bacteriophage, designated F4, was isolated from the ruminal fluid of a calf. The host range of F4 phage was limited to five strains ofStreptococcus bovis out of ten tested on which clear plaques 0.6–1.2 mm in diameter were found. Bacteriophage F4 had an elongated head 75 nm long and 33 nm wide with a noncontractile flexible tail 100 nm in length on average. This phage is defective in the generation of plaques at low multiplicities of infection. Its genome consists of double-stranded linear DNA of 60.38 kb lacking cohesive ends. The F4 DNA was analyzed with 13 restriction enzymes. The restriction enzymes that did not cleave it wereBamHI,EcoRI,PvuI, andSmaI. The circular restriction map was constructed with four restriction endonucleases (XbaI,EcoI,SalI, andBglI).     

The genome of Bacillus subtilis phage SPP1: the arrangement of restriction endonuclease generated fragments.

Summary SPP1 DNA was cleaved by the restriction endonucleases, BglI, BglII, EcoRI, KpnI, SmaI, and SalI. The molecular weights of the DNA fragments obtained by single enzyme digestion or by consecutive digestion with two enzymes were determined by electron microscopic measurements of contour length and by gel electrophoresis. The major fragments from the six digests could be ordered to give a consistent restriction map of SPP1. The electropherograms of several digests indicated that certain fragments occurred in less than stoichiometric amounts or were heterogeneous in size. Such bands carried a major part of radioactivity, when SPP1 DNA was terminally labelled with P³² prior to degradation by restriction enzymes. These results, and studies of the effect of exonuclease III treatment on restriction enzyme patterns define the terminal restriction fragments. All data obaained support the conclusion drawn in the preceding paper (Morelli et al., 1978b) that the SPP1 genome is terminally redundant and partially circularly permuted.Part of this work is from the doctoral dissertations to be submitted to Stanford University¹ and the Freie Universität Berlin²     

Plasmids from bacterial endosymbionts of hump-killer paramecia

Six isolates ofCaedibacter taeniospiralis, collected from four continents, were screened for plasmid DNA. Plasmid DNA species containing between 41.5 and 49.5 kilobase pairs (kb) were observed in all strains. Physical maps of plasmids were constructed by determining relative positions of the restriction endonuclease (BamHI,SalI,XhoI,SacI,PstI,AvaI, andEcoRI) recognition sequences in each plasmid. The physical map of the smallest plasmid (41.5 kb), pKAP30, is reflected in each of the plasmids isolated from the other strains ofC. taeniospiralis. Plasmid DNA from three of the isolates (strains 51 and 116 both from Indiana and strain 169 from Japan) each contain 43 kb, where 41.5 kb appear to be identical to pKAP30 (obtained from the Australian strain, A30). The extra 1.5 kb present in pKAP51, pKAP116, and pKAP169 is included as a single polynucleotide sequence. The 1.5-kb inclusion is located at apparently identical positions in pKAP116 and pKAP169 and at a totally different position in pKAP51. The two remaining plasmids, pKAP47 (from California strain 47) and pKAP298 (from Panama strain 298), both contain 49 kb to include a continuous 41.5-kb sequence that is apparently identical to pKAP30. The results indicate that the polynucleotide sequences of these plasmids are highly conserved and that the observed variations among them may be accounted for by transposable elements.     

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.