A physical map of the genome of temperate phage phi 3T.

A physical map of the genome of Bacillus subtilis bacteriophage phi 3T was constructed by ordering the fragments produced by cleavage of phi 3T DNA with restriction endonucleases AvaII (2 fragments), BglI (2 fragments), SmaI (3 fragments), BamHI (6 fragments), SalI (7 fragments), AvaI (7 fragments), SacI (12 fragments), PstI (14 fragments), and BglII (26 fragments). Two techniques were used to order the fragments: (1) Sets of previously ordered restriction fragments were isolated and redigested with the endonuclease whose cleavage sites were to be mapped. (2) Fragments located near the ends of the genome or near the ends of other restriction fragments were ordered by treating the DNA with lambda exonuclease prior to restriction endonuclease cleavage. The susceptibility of phi 3T DNA to 15 other restriction endonucleases is also reported.     

Use of pulsed-field agarose gel electrophoresis to size genomes of Campylobacter species and to construct a SalI map of Campylobacter jejuni UA580.

To determine the physical length of the chromosome of Campylobacter jejuni, the genome was subjected to digestion by a series of restriction endonucleases to produce a small number of large restriction fragments. These fragments were then separated by pulsed-field gel electrophoresis with the contour-clamped homogeneous electric field system. The DNA of C. jejuni, with its low G+C content, was found to have no restriction sites for enzymes NotI and SfiI, which cut a high-G+C regions. Most of the restriction enzymes that were used resulted in DNA fragments that were either too numerous or too small for genome size determination, with the exception of the enzymes SalI (5' ... G decreases TCGAG ... 3'), SmaI (5' .... CCC decreases GGG .... 3'), and KpnI (5' ... GGTAC decreases C .... 3'). With SalI, six restriction fragments with average values of 48.5, 80, 110, 220, 280, and 980 kilobases (kb) were obtained when calibrated with both a lambda DNA ladder and yeast Saccharomyces cerevisiae chromosome markers. The sum of these fragments yielded an average genome size of 1.718 megabases (Mb). With SmaI, nine restriction fragments with average values ranging from 39 to 371 kb, which yielded an average genome size of 1.726 Mb were obtained. With KpnI, 11 restriction fragments with sizes ranging from 35 to 387.5 kb, which yielded an average genome size of 1.717 Mb were obtained. A SalI restriction map was derived by partial digestion of the C. jejuni DNA. The genome sizes of C. laridis, C. coli, and C. fetus were also determined with the contour-clamped homogeneous electric field system by SalI, SmaI, and KpnI digestion. Average genome sizes were found to be 1.714 Mb for C. coli, 1.267 Mb for C. fetus subsp. fetus, and 1.451 Mb for C. laridis.     

Comparison of the physical maps and redundant ends of the chromosomes of phages 2C, SP01, SP82 and phi e

The physical map of 2C DNA (cf. following paper in this journal) was compared to the maps of SP01, SP82 and phi e (three other Bacillus subtilis phages containing hydroxymethyluracil in place of thymine in their DNA). The overall organization of the four genomes was remarkably similar, as indicated by the topology of HaeIII and SalI cleavage segments. The proof was gathered for the presence in the four phage DNAs of large redundant ends carrying a single HaeIII recognition site. The location of the latter proved identical for 2C and SP01, but was shifted in the DNAs of SP82 and phi e. Since the redundant end components of these hydroxymethyluracil genomes are colinear, as shown by cross-hybridization studies, the shifting of the HaeIII cleavage site is presumably due to two base substitutions, suppressing an endonuclease recognition site and establishing a new site elsewhere. Relatedness between the genomes of this family of viruses was evaluated from the fraction of conserved restriction fragments. According to these calculations, 6% base substitutions have occurred within the four viral DNAs, in the course of evolution. However, specific segments of 2C DNA were not present in SP01 and phi e DNA, as shown by cross-hybridization with restriction fragments. These data indicate the occurrence of deletions, in addition to base substitutions, as evolutionary mechanisms prevailing in the genomes of this family of phages.     

Physical map of the bacteriophage T5 genome based on the cleavage products of the restriction endonucleases SalI, SmaI, BamI, and HpaI.

A physical map of the bacteriophage T5 genome was constructed by ordering the fragments produced by cleavage of T5 DNA with the restriction endonucleases SalI (4 fragments), SmaI (4 fragments), BamI (5 fragments), and HpaI (28 fragments). The following techniques were used to order the fragments. (i) Digestion of DNA from T5 heat-stable deletion mutants was used to identify fragments located in the deletable region. (ii) Fragments near the ends of the T5 DNA molecule were located by treating T5 DNA with lambda exonuclease before restriction endonuclease cleavage. (iii) Fragments spanning other restriction endonuclease cleavage sites were identified by combined digestion of T5 DNA with two restriction endonucleases. (iv) The general location of some fragments was determined by isolating individual restriction fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme. (v) Treatment of restriction digests with lambda exonuclease before digestion with a second restriction enzyme was used to identify fragments near, but not spanning, restriction cleavage sites. (vi) Exonucleases III treatment of T5 DNA before restriction endonuclease cleavage was used to locate fragments spanning or near the natural T5 single-chain interruptions. (vii) Analysis of the products of incomplete restriction endonuclease cleavage was used to identify adjacent fragments.     

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.     

A method for generating selective DNA probes for the analysis of C-negative regions in human chromosomes

Linker-adapter polymerase chain reaction (LA-PCR) is among the most efficient techniques for whole genome DNA amplification. The key stage in LA-PCR is the hydrolysis of a DNA sample with restriction endonucleases, and the choice of a restriction endonuclease (or several endonucleases) determines the composition of DNA probes generated in LA-PCR. Computer analysis of the localization of the restriction sites in human genome has allowed us to propose an efficient technique for generating DNA probes by LA-PCR using the restriction endonucleases HaeIII and RsaI. In silico hydrolysis of human genomic DNA with endonucleases HaeIII and RsaI demonstrate that 100- to 1,000-bp DNA fragments are more abundant in the gene-rich regions. Applying in situ hybridization to metaphase chromosomes, we demonstrated that the produced DNA probes predominantly hybridized to the C-negative chromosomal regions, whereas the FISH signal was almost absent in the C-positive regions. The described protocol for generating DNA probes may be successfully used in subsequent cytogenetic analysis of the C-negative chromosomal regions.     

Restriction endonuclease map of Euglena gracilis chloroplast DNA.

A physical map of the Euglena gracilis chloroplast genome has been constructed, based on cleavage sites of Euglena gracilis chloroplast DNA treated with bacterial restriction endonucleases. Covalently close, circular chloroplast DNA is cleaved by restriction endonuclease SalI into three fragments and by restriction endonuclease BamHI into six fragments. These nine cleavage sites have been ordered by fragment molecular weight analysis, double digestions, partial digestions, and by digestion studies of isolated DNA fragments. A fragment pattern of the products of EcoRI restriction endonuclease digestion of Euglena chloroplast DNA is also described. One of these fragments has been located on the cleavage site map.     

Cleavage of adeno-associated virus DNA with Sali,Psti and Haeii restriction endonucleases.

Duplex AAV-2 DNA was digested with SalI, PstI or HaeII restriction endonucleases and the cleavage sites were mapped. SalI cleaves AAV DNA at 0.310 map units, PstI at 0.106, 0.422 and 0.914 and the five HaeII sites were mapped at 0.110. 0.156, 0.181, 0.536 and 0.600 map units. These cleavage products will be useful for the isolation of specific regions from the AAV DNA, located outside of the stably transcribed region of the genome, and will also help to map more complex restriction enzyme cleavages.     

Restriction endonuclease mapping of unintegrated viral DNA of B- and N-tropic BALB/c murine leukemia virus.

Unintegrated linear and closed circular DNAs of B- and N-tropic endogenous BALB/c murine leukemia virus (MuLV) were extracted from newly infected mouse cells and cleaved with EcoRI, XhoI, PvuI, HindIII, SalI, XbaI, KpnI, SmaI, and PstI restriction endonucleases. The DNA fragments were separated by electrophoresis and analyzed by the Southern blot hybridization procedure. EcoRI did not cleave the two genomes. A physical map of 15 cleavage sites on B- and N-tropic genomes was constructed with the other restriction endonucleases. Identical cleavage sites of B- and N-tropic MuLV DNAs were found with all these enzymes. However, the N-tropic linear genome was found to lack about 75 base pairs at each end of the molecule. PstI, KpnI, and SmaI recognize a cleavage site at both ends of the linear molecules. And sequences derived from the 5' end of the RNA genome were found in the third left end of the linear DNA and at its extreme right-end terminus, suggesting the presence of redundant sequences. Two species of closed circular viral DNA were observed. The larger species has the same size as the linear molecule and appears to be a circularized form of linear DNA. The smaller species contains sequences common to both the linear and the larger circular viral DNA but seems to be deleted from sequences present at either one or both ends of the linear DNA. Therefore, the general structure of the linear and circular DNA species of these B- and N-tropic endogenous BALB/c MuLV appears analogous to the structure found with other retroviruses.     

Map of restriction sites on bacteriophage T4 cytosine-containing DNA for endonucleases bamHI, BglII, KpnI, PvuI, SalI, and XbaI.

A complete map of the cleavage sites of restriction endonucleases BamHI, BglII, KpnI, PvuI, SalI, and XbaI was determined for the cytosine-containing DNA of a bacteriophage T4 alc mutant. The 56 sequence-specific sites were assigned map coordinates based on a least-squares analysis of measured fragment lengths. Altogether, the lengths of 118 fragments from single and double enzyme digestions were measured by electrophoresis of the fragments in agarose gels. DNA fragments of known sequence or DNA fragments calibrated with fragments of known sequence were used as standards. The greatest deviation between an experimentally measured fragment length and its computed map coordinates was 3.0%; the average deviation was 0.8%. The total length of the wild-type T4 genome was calculated to be 166,200 base pairs.     

Simian adenovirus genome. II. Identification of specific fragments of viral DNA possessing transforming activity

The antigenic variant of simian adenovirus 7 (SA7) DNA was cleaved by restriction endonucleases EcoRI, XbaI, BamHI, SalI. The resulted digests of viral DNA were tested for transforming activity using the "calcium" technique. It was shown that BamHI. XbaI and SalI digests transformed primary baby rat kidney cells as well as native viral DNA. The transforming activity of separated BamHI and SalI fragments was tested also. The viral DNA fragments with transforming activity (BamHI-B and SalI-B) were localised on the left of the physical map of the viral genome. It was also shown that fragment-transformed cell lines were able to form colonies in 0.33% agarose medium.     

Physical mapping of a large-plaque mutation of adenovirus type 2.

We have developed a simple method based on cotransfection of overlapping DNA restriction fragments for construction of recombinants of adenovirus type 2 (Ad2) and Ad5. When Ad2 DNA digested with restriction endonuclease EcoRI was cotransfected with Ad5 DNA digested with SalI, recombination occurred between Ad2 EcoRI-A (map position 0 to 59) and Ad5 SalI-A (map position 45 to 100). Analysis of the recombinant DNAs by digestion with EcoRI or BamHI restriction endonucleases indicated that, as expected, recombination had occurred in overlapping sequences (map position 45 to 59) between the Ad2 EcoRI-A fragment and the Ad5 SalI-A fragment. By using this method, several recombinants were constructed between a large-plaque (lp) mutant of Ad2 and wild-type Ad5. Cleavage of the recombinant genomes with restriction endonucleases BamHI, EcoRI, and HindIII revealed that the lp mutation is located within the left 41% of Ad2 genome.     

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.     

Physical and genetic maps of the genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

A restriction map of the chromosome of the cyanobacterium Anabaena sp. strain PCC 7120 was generated by the determination of the order of restriction fragments of the infrequently cleaving restriction endonucleases AvrII, SalI, and PstI. These restriction fragments were resolved by the pulsed homogeneous orthogonal field gel electrophoresis system of pulsed-field gel electrophoresis (I. Bancroft and C. P. Wolk, Nucleic Acids Res. 16:7405-7418, 1988). Other infrequently cutting restriction endonucleases (AhaII, Asp718, AsuII, BanII, BglII, BssHII, FspI, NcoI, NruI, SphI, SplI, SstII, and StuI) were identified that could prove useful for higher-resolution mapping. The chromosome was found to be 6.4 megabases in size and circular. Three apparently circular large plasmids (410, 190, and 110 kilobases) were also identified. A genetic map was constructed by hybridization with gene-specific probes. Genes encoding components of the photosynthetic electron transport chain were not within a single tight cluster.     

Unusually infrequent cleavage with several endonucleases and physical map construction of Bacillus subtilis bacteriophage phi 1 DNA.

HaeIII, BalI, StuI, BamHI, SlaI, and EcoRII did not cut the genome of Bacillus subtilis phage phi 1 at all, whereas ThaI, BglII, EcoRI, SalI, and Bsu1247I cut the genome once or twice. The physical map of the phi 1 genome was constructed with the latter restriction endonucleases.     

Characterization of a cloned ribosomal fragment from mouse which contains the 18S coding region and adjacent spacer sequences.

The large EcoRI fragment of mouse ribosomal genes containing parts of the non-transcribed spacer, the external transcribed spacer located at the 5' end of the precursor molecule and about two thirds of the 18S sequence has been cloned in bacteriophage lambda gtWES. A physical map of the DNA was constructed by cleavage with several restriction endonucleases and hybridization of the restriction fragments of the recombinant DNA with labelled 18S and 45S rRNA. The orientation of the inserted fragment as well as the length of the 18S sequence was determined by electron microscopy of R-loop containing molecules. The absence of hybridization of the cloned fragment to other fragments in the genome shows that the non-transcribed spacer does not have a significant length of sequences in common with other sequences in the genome.     

Cleavage maps of a tetracycline plasmid from Staphylococcus aureus

The cleavage maps of a Staphylococcus aureus plasmid, pSN1 (2.75 megadaltons), conferring tetracycline resistance, were determined. Cleavage maps are given for HpaI and HindIII restriction endonucleases by using the single HpaII site as a reference point. Nucleases EcoRI, BamHI, SalI, and HaeIII have no sites on this plasmid.     

Restriction endonuclease mapping of the hepatitis B viral genome isolated from Taiwan

The Eco RI fragment of hepatitis B virus (HBV) DNA isolated from human blood plasma Dane particles were inserted into plasmid pUC8 Eco RI site and transformed into E. coli JM103 host. Two recombinants pTWL1 and pTWL2 were found to carry 3.2 kbp fragment and proved to have HBV genome by Southern hybridization method. The 1.4 kbp Bam HI fragment which carried the hepatitis B viral surface antigen (HBsAg) gene, obtained via Bam HI digestion of Dane particles DNA which was made fully double stranded by endogenous DNA polymerase reaction, was also inserted into plasmid pUC8 Bam HI site. Four recombinant clones, pTWS1, pTWS2, pTWS3, and pTWS4 were found. Only one of the clones pTWS1 carried the HBsAg gene in a correct orientation with respect to the lac promoter sequence. The physical mapping of HBV DNA was performed with several restriction endonucleases. Our results indicated that the HBV DNA insert contains unique XbaI and HpaI cleavage sites and lacks the cleavage sites for the HindIII, SmaI, KpnI, SalI, and SstI endonucleases. The locations of Bam HI, BglII, and HincII endonucleases cleavage sites within the cloned HBV DNA of the pTWL1 plasmid were similar to that HBV DNA of adw and adw2 subtypes.     

The molecular characterization of the genome of Muraena helena L. I. Isolation and hybridization of two MboI-restricted DNA fractions.

To investigate the genome of the anguilliform fish Muraena helena at the molecular level we characterized total DNA by agarose gel electrophoresis after cleavage with AluI, HaeIII, MboI, and DdeI restriction endonucleases. Subsequently, we isolated the DNA from two specific electrophoretic fractions to be used as probes for Southern and in situ hybridization experiments. One such fraction showed an electrophoretic pattern typical of highly repetitive DNA localized in the centromeres of many chromosomes. The other fraction was shown to be located in the nucleolar organizer region, partially coincident with 45S rDNA, and to be composed of highly repetitive sequences.