HindII, HindIII, and HpaI restriction fragment maps of bacteriophage lambda DNA

The site-specific restriction endonucleases isolated from Hemophilus influenzae strains Rc (HincII) and Rd (HindII + III), and Hemophilus parainfluenzae (HpaI) were used to digest bacteriophage lambda DNA into 34, 40, and 15 specific fragments, respectively. The sites cleaved by each of these enzymes were localized on the lambda physical map and the fragments resulting from these cleavages were electrophoretically identified on gels by (1) analysis of the digestion profiles of deletion and transducing derivatives of lambda; and (2) digesting individual fragments produced by one restriction endonuclease with another restriction endonuclease. This paper presents the HindII, HindIII, and HpaI restriction fragment maps for the entire lambda genome, and the data used to derive these maps for the region of the lambda genome between the attachment site (at 57.3% lambda) and the right vegetative end (100% lambda). The data for mapping the left arm of lambda may be found in the accompanying paper (Robinson and Landy, 1977).     

Construction of an EcoRI restriction map of Mycoplasma pneumoniae and localization of selected genes.

A restriction map of the genome of Mycoplasma pneumoniae, a small human pathogenic bacterium, was constructed by means of an ordered cosmid library which spans the complete bacterial chromosome. The positions of 143 endonuclease EcoRI restriction fragments were determined and aligned with the physical map. In addition, restriction sites for the rare-cutting enzymes XhoI (25 sites), ApaI (13 sites), NotI (2 sites), and SfiI (2 sites) were included. The resulting map consists of 185 restriction sites, has a mean resolution of 4.4 kbp, and predicts a genome size of 809 kbp. In addition, several genes were identified and mapped to their respective genomic EcoRI restriction fragments.     

Sizing and mapping of the genome of Campylobacter coli strain UA417R using pulsed-field gel electrophoresis.

Agarose-immobilized chromosomal DNA from the nalidixic-acid-resistant Campylobacter coli strain UA417 and its streptomycin-resistant (StrR) derivative, UA417R, were digested with the restriction enzymes SalI (GTCGAC) and SmaI (CCCGGG). The sizes of the resulting fragments were determined using pulsed-field gel electrophoresis. The two genomes showed similar restriction patterns of seven and 13 fragments for the two respective enzymes and the total genome size was determined to be approx. 1.7 Mb. Analysis of partial digestion fragments, as well as Southern-blot hybridization, were used to construct a physical map of the C. coli UA417R genome. Natural transformation studies using DNA fragments extracted from UA417R, as well as the erythromycin-resistant (EryR) C. coli strain UA585, were used to locate the StrR and EryR resistance markers on the genomic map.     

Nonspecific primer and PCR generated hybridization probes for physical ordering large restriction fragments in complex genome of S. aureus.

Pulsed field gel electrophoresis (PFGE) allows separation of large restriction fragments from bacterial genome. Restriction fragments obtained by digestion of Staphylococcus aureus DNA with rare cutting enzymes (Sma I, and Csp I) were separated by PFGE. To arrange the physical order of the fragments generated by digestion with one enzyme, probes were prepared by nonspecific priming and polymerase chain reaction (PCR), using individual fragments of the other enzymatic digest as a template. Probes were then used for Southern hybridization to the PFGE separated fragment distribution of the two infrequent cleaving enzymes (Sma I and Csp I). Using probes generated from four Sma I fragments and five Csp I fragments as individual templates, a partial physical order of Csp I fragments of the genome of S. aureus ISP8 has been determined in relation to a previously published Sma I map of S. aureus genome.     

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     

Chromosome map of the thermophilic archaebacterium Thermococcus celer.

A physical map for the chromosome of the thermophilic archaebacterium Thermococcus celer Vu13 has been constructed. Thirty-four restriction endonucleases were tested for their ability to generate large restriction fragments from the chromosome of T. celer. Of these, the enzymes NheI, SpeI, and XbaI yielded the fewest fragments when analyzed by pulsed-field electrophoresis. NheI and SpeI each gave 5 fragments, while XbaI gave 12. The size of the T. celer chromosome was determined from the sum of the apparent sizes of restriction fragments derived from single and double digests by using these enzymes and was found to be 1,890 +/- 27 kilobase pairs. Partial and complete digests allowed the order of all but three small (less than 15 kilobase pairs) fragments to be deduced. These three fragments were assigned positions by using hybridization probes derived from these restriction fragments. The positions of the other fragments were confirmed by using hybridization probes derived in the same manner. The positions of the 5S, 16S, and 23S rRNA genes as well as the 7S RNA gene were located on this map by using cloned portions of these genes as hybridization probes. The 5S rRNA gene was localized 48 to 196 kilobases from the 5' end of the 16S gene. The 7S RNA gene was localized 190 to 504 kilobases from the 3' end of the 23S gene. These analyses demonstrated that the chromosome of T. celer is a single, circular DNA molecule. This is the first such demonstration of the structure of an archaebacterial chromosome.     

Application of multivariate analyses of enzymic data to classification of members of the Actinobacillus-Haemophilus-Pasteurella group.

Outer membrane vesicles and fragments from Actinobacillus actinomycetemcomitans, Actinobacillus lignieresii, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica, and Pasteurella multocida were isolated and examined semiquantitatively for 19 enzyme activities by using the API ZYM micromethod. The enzyme contents of vesicles and fragments were compared with the enzyme contents of whole cells of the same organisms. Enzymic data were analyzed by using principal-component analysis and soft independent modeling of class analogy. This technique allowed us to distinguish among the closely related organisms A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. A. actinomycetemcomitans was divided into two groups of strains. A. lignieresii fell outside or on the border of the A. actinobacillus class. A. ureae, H. influenzae, H. parainfluenzae, P. haemolytica, and P. multocida fell outside the A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus classes.     

Methylase activities from Haemophilus influenzae that protect Haemophilus parainfluenzae transforming deoxyribonucleic acid from inactivation by Haemophilus influenzae endonuclease R.

Specific methylases that have the properties of deoxyribonucleic acid (DNA) modification enzymes have been isolated from Haemophilus influenzae strain Rd. Two activities ((Methylase IIa and methylase III) were found to protect transforming DNA of H. parainfluenzae from the action of H. influenzae restriction enzymes. To determine the specificty of the protection, a procedure based on biological activity was developed for the separation and purification of the restriction endonucleases from H. influenzae strain Rd. Two endonuclease R activities presumably corresponding to Hind II and Hind III (P. H. Roy and H. O. Smith, 1973; H. O. Smith and K. W. Wilcox, 1970) were characterized by differences in their chromatographic properties, ability to attack T7 DNA, and inactivation of the transforming activity of different markers of H. parainfluenzae DNA. One endonuclease R enzyme (Hind II) attacked T7 DNA and was found to inactivate the dalacin resistance marker (smaller than 0.01% activity remaining) with only a slight effect on the streptomycin resistance marker (83% activity remaining). Methylase IIa treatment protected 40% of the dalacin resistance marker of H. parainfluenzae DNA from inactivation by Hind II. The other restriction activity (Hind III) was inert towards T7 DNA and inactivated the streptomycin resistance marker of H. parainfluenzae DNA (smaller than 0.01% activity remaining) without any effect on the dalacin resistance marker. The methylation of H. parainfluenzae DNA accomplished by methylase III protected 60% of the transforming activity of the streptomycin resistance marker of H. parainfluenzae DNA from the action of Hind III.     

Derivation of a physical map of chloroplast DNA from Nicotiana tabacum by two-dimensional gel and computer-aided restriction analysis

A combined approach was used to derive a detailed physical map of Nicotiana tabacum chloroplast DNA for the restriction enzymes SalI, SmaI, KpnI, and BamHI. Complete maps for the restriction enzymes SalI, SmaI, and KpnI were derived by using two-dimensional agarose gel analysis of fragments obtained by reciprocal double digestion of chloroplast DNA. We have characterized a complete cloned library of N. tabacum chloroplast DNA which contains overlapping restriction fragments resulting from partial digestion by BamHI. With these clones and existing data, we used a novel computer-aided analysis to derive a detailed map for the enzyme BamHI. A comparison and compilation of all published N. tabacum chloroplast DNA restriction maps is presented. Differences between ours and a previously published SmaI and BamHI restriction map are discussed.     

Cleavage map of bacteriophage phiX174 RF DNA by restriction enzymes.

phiX RF DNA was cleaved by restriction enzymes from Haemophilus influenzae Rf (Hinf I) and Haemophilus haemolyticus (Hha. I). Twenty one fragments of approximately 25 to 730 base pairs were produced by Hinf I and seventeen fragments of approximately 40 to 1560 base pairs by Hha I. The order of these fragments has been established by digestion on Haemophilus awgyptius (Hae III) and Arthrobacter luteus (Alu I) endonuclease fragments of phiX RF with Hinf I and Hha1. By this method of reciprocal digestion a detailed cleavage map of phiX RF DNA was constructed, which includes also the previously determined Hind II, Hae III and Alu I cleavage maps of phiX 174 RF DNA (1, 2). Moreover, 28 conditional lethal mutants of bacteriophage phiX174 were placed in this map using the genetic fragment assay (3).     

Differences in susceptibility to restriction by E. coli B between various heteroduplex molecules of bacteriophage FD DNA

Fragments of B-modified bacteriophage fd sB1^o sB2 RF DNA were prepared with the help of purified endonuclease R from Haemophilus parainfluenzae (Hpa II). These were hybridized with unmodified circular single stranded fd DNA. The resulting partial heteroduplex molecules were assayed for infectivity on competent cells of B-restricting and non restricting strains of E. coli. There of such heteroduplexes originating from neighbouring fragments on the physical map of fd RF DNA were shown to be more resistant to Eco B restriction than six others and the unmodified control. It is suggested that the three corresponding vicinal fragments contain essential parts of the Eco B recognition site on this phage DNA.     

Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome.

A physical map of the Methanobacterium thermoautotrophicum Marburg chromosome was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by NotI, PmeI, and NheI. The order of the fragments was deduced from Southern blot hybridization of NotI fragment probes to various restriction digests and from partial digests. The derived map is circular, and the genome size was estimated to be 1,623 kb. Several cloned genes were hybridized to restriction fragments to locate their positions on the map. Genes coding for proteins involved in the methanogenic pathway were located on the same segment of the circular chromosome. In addition, the genomes of a variety of thermophilic Methanobacterium strains were treated with restriction enzymes and analyzed by pulsed-field gel electrophoresis. The sums of the fragment sizes varied from 1,600 to 1,728 kb among the strains, and widely different macrorestriction patterns were observed.     

Characterization of fall armyworm mitochondrial DNA (Lepidoptera: Noctuidae)

Mitochondrial DNA from the fall armyworm, Spodoptera frugiperda (J.E. Smith), was cloned and characterized using restriction enzyme mapping. Genome size is approximately 16.3 kilobase (Kb), consistent with that of most animals. Three fragments, 8.1 Kb, 6.2 Kb, and 2.0 Kb, were produced by digestion with restriction enzyme Xbal and cloned into a pUC19 vector. Twenty-nine restriction enzymes were used to generate a detailed physical restriction enzyme map of the three cloned fragments. These data represent the first detailed characterization of a lepidopteran mitochondrial genome. © 1992 Wiley-Liss, Inc.     

Restriction map of E. coli shuttle plasmid (p# GTE5) with secretive function

The shuttle plasmid (p# GTE5) DNA with secretive function was extracted by the alkali lysozyme method from E. coli RRI strain. Its molecular weight is 4.5 Md and DNA size is 6.9 Kb. Restriction fragments of plasmid was obtained by single and double enzymes complete digestion using five different restriction endonucleases. The restriction map of shuttle plasmid (p# GTE5) was established for the enzymes EcoRI, BglII, pstI, PvuII, and TaqI.     

Construction of the temperature-sensitive vectors pLUCH80 and pLUCH88 for delivery of Tn917::NotI/SmaI and use of these vectors to derive a circular map of Listeria monocytogenes Scott A, a serotype 4b isolate.

A physical map of Listeria monocytogenes Scott A was generated by the pulsed-field technique of contour-clamped-homogeneous-electric-field (CHEF) electrophoresis. The circular genome of this serotype 4b strain contains 12 AscI fragments (38 to 790 kb), 5 NotI fragments (55 to 1,400 kb), 3 SrfI fragments (110, 1,110, and 2,000 kb), and 2 SfiI fragments (1,320 and 1,920 kb). Summation of individually sized fragments derived by digestion of Scott A genomic DNA with each of these four enzymes provided an average estimated genome length of 3,210 +/- 60 kb. Efforts to assemble the macrorestriction map benefited greatly from the construction and use of pLUCH80 and pLUCH88, temperature-sensitive vectors for delivering transposon Tn917::NotI/SmaI to the chromosome of Scott A. As another component of this study, the positions of four known virulence genes (inlA, mpl, hly, and prf) and three L. monocytogenes-specific sequences (lisM44, lisM51, and lisM52) were localized on the physical map of Scott A by hybridization. Probes prepared from lisM44, lisM51, and the four virulence genes hybridized within a cluster on a 150-kb fragment of the Scott A genome that overlaps part of the NotI-B and AscI-D fragments. The lisM52 probe hybridized with the AscI-F2 (120-kb) fragment of Scott A, which is separated from the NotI-B-AscI-D region by about 300 kb. These results established the first physical and genetic map of a serotype 4b strain of L. monocytogenes and provided further insight on this important food-borne pathogen at the genome level.     

The world of archaea: genome analysis,evolution and thermostable enzymes

Pyrococcus sp. KOD1 is a newly isolated hyperthermophilic archaeon from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan. A physical map of the KOD1 chromosome was constructed using pulsed-field gel electrophoresis of restriction fragments generated by AscI, PacI and PmeI. The order of the AscI fragments was deduced from Southern hybridization using the AscI, PmeI and PacI fragments as a probe. The derived physical map indicates that KOD1 possesses a circular-form genome and its size was estimated to be 2036 kb. Several cloned genes were hybridized to restriction fragments to locate their positions on the physical map. Some genes involved in the central dogma were located on the restricted segment of the genome. Novel characteristics of KOD1 enzymes are also introduced in this article.     

A combined genetic and physical map of the Streptomyces coelicolor A3(2) chromosome.

The restriction enzymes AseI (ATTAAT), DraI (TTTAAA), and SspI (AATATT) cut the Streptomyces coelicolor A3(2) chromosome into 17, 8, and 25 fragments separable by pulsed-field gel electrophoresis (PFGE). The sums of their lengths indicated that the chromosome consists of about 8 Mb of DNA, some 75% more than that of Escherichia coli K-12. A physical map of the chromosome was constructed for AseI and DraI, using single and double digests, linking clones, cross-hybridization of restriction fragments, and locations of genetically mapped genes, insertion sequences, prophages, and the integrated SCP1 and SLP1 plasmids on the physical map. The physical map was aligned with the previously established genetic map, revealing that the two long opposite quadrants of the genetic map that are almost devoid of markers (the silent regions at 3 o'clock and 9 o'clock) are indeed physically long rather than being hot spots for genetic exchange. They must therefore contain long stretches of DNA different in function from the remainder of the genome. Consistent with this conclusion are the locations of significant deletions in both of the silent regions. Of these, a 40-kb deletion in the 9 o'clock region accompanied or followed integration of the SCP1 linear plasmid to produce the NF fertility state. PFGE analysis of Streptomyces lividans 66, a close relative of S. coelicolor A3(2), was hampered by the previously described susceptibility of its DNA to degradation during electrophoresis. However, ZX7, a mutant derivative of S. lividans lacking the DNA modification responsible for this degradation, yielded good PFGE preparations. Not more than 7 of the 17 S. coelicolor AseI fragments could be shared by the S. lividans strain.     

Gene localization, size, and physical map of the chromosome of Streptococcus pneumoniae.

A physical map of the Streptococcus (Diplococcus) pneumoniae chromosome, which is circular and 2,270 kbp in circumference, has been constructed. The restriction enzymes ApaI, SmaI, and SacII were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). The digests produced 22, 20, and 29 fragments, respectively. The order of the fragments was deduced from Southern blot hybridization of isolated labeled fragments to separated fragments of the various restriction digests. Genetic markers were correlated with the physical map by transformation of recipient cells with FIGE-isolated DNA fragments derived from genetically marked S. pneumoniae strains. In addition, markers were mapped by the hybridization of cloned genes to FIGE-separated restriction fragments. Six rRNA gene (rrn) clusters were mapped by hybridization to rrn-containing fragments of Haemophilus influenzae.     

Derivation of a restriction map of bacteriophage T3 DNA and comparison with the map of bacteriophage T7 DNA.

The DNA of bacteriophage T3 was characterized by cleavage with seven restriction endonucleases. AvaI, XbaI, BglII, and HindIII each cut T3 DNA at 1 site, KpnI cleaved it at 2 sites, MboI cleaved it at 9 sites, and HpaI cleaved it at 17 sites. The sizes of the fragments produced by digestion with these enzymes were determined by using restriction fragments of T7 DNA as molecular weight standards. As a result of this analysis, the size of T3 DNA was estimated to be 38.74 kilobases. The fragments were ordered with respect to each other and to the genetic map to produce a restriction map of T3 DNA. The location and occurrence of the restriction sites in T3 DNA are compared with those in the DNA of the closely related bacteriophage T7.