Restriction endonuclease mapping and mutagenesis of the F sex factor replication region.

The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of beta-lactamase production. By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that approximately 1.9 X 10(6) daltons of the 6.0 X 10(6) dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.     

The control region of the F sex factor DNA transfer cistrons: Restriction mapping and DNA cloning

Summary A restriction endonuclease map of EcoRI fragment f6 of F sex factor DNA was constructed and aligned with pre-existing physical and genetic maps. Results of genetic complementation tests and analysis of proteins synthesized in minicells from PstI and BglI₁ sub-fragment clones, or from a specific BglII fragment deletion, have allowed mapping of the locations of the origin of DNA transfer and many of the transfer genes known to lie on f6. The proteins detected account for 78% of the coding capacity of fragment f6.     

Restriction endonuclease mapping of the colicin Ib plasmid

Summary A cleavage site map of the colicin Ib plasmid (ColIb) has been determined for the enzymes Sall, XhoI, and HindIII by analysis of partial digests, double digests, DNA-DNA hybridization, and Tn5-induced insertion mutants. The site of the colicin gene has been determined by probing with cloned DNA coding for colicin production, as well as by analysis of a colicin negative ColIb:Tn5.     

Restriction endonuclease mapping of pSC101 and pMB9

Summary A restriction endonuclease analysis of the plasmids pSC101 and pMB9 has allowed a determination of the alterations that occurred in the tetracycline resistance locus during the construction of pMB9 from pSC101. The genes for four of the polypeptides involved in tetracycline resistance have been positioned on the restriction endonuclease map of pSC101.     

Restriction endonuclease mapping of six novel deletions of the factor VIII gene in hemophilia A

Summary Hemophilia A is an X-linked disease of blood coagulation caused by deficiency of factor VIII. Using cloned cDNA, genomic and synthetic oligonucleotide factor VIII probes, we have identified six novel partial gene deletions in patients with severe hemophilia A. We have previously reported six other deletions of the factor VIII gene. The number of gross molecular defects (deletions, insertions) in the factor VIII gene in our series of 240 patients is 17 (3 insertions and 2 complicated deletions will be described elsewhere). No association was observed between the size or location of the deletions and the presence of inhibitors to factor VIII. No deletion breakpoint hotspots have been identified by restriction analysis. The parental origin of several of the deletions was determined.     

The control region of the F sex factor DNA transfer cistrons: Physical mapping by deletion analysis

Summary A technique has been developed which allows the isolation of random deletions extending from unique restriction enzyme sites in plasmid DNA molecules. The method involves transformation of E. coli cells with linear plasmid DNAs generated by restriction enzyme cleavage. We have used this technique to map DNA transfer genes in the tra control region of F sex factor DNA. Deletions within EcoRI fragment f6 of F DNA have been isolated and used to assign physical locations to tra genes by a combination of genetic complementation tests, restriction enzyme analysis, DNA heteroduplexing and the analysis of the proteins synthesised in minicells and in vitro. Deletion analysis has also allowed the identification of the traK gene product.     

Restriction endonuclease mapping of the root-inducing plasmid of Agrobacterium rhizogenes 1855

The root-inducing plasmid of the agropine type Agrobacterium rhizogenes 1855 was mapped by means of the restriction endonuclease EcoRI. The circular arrangement of the more than 60 fragments generated by this enzyme was established by electrophoretic analysis of pBR322 clones harboring overlapping segments of pRi1855 derived by partial digestion with EcoRI. A large region of the plasmid comprising the T-DNA was mapped with two additional enzymes, BamHI and HindIII, by means of Southern blot hybridizations between the fragments generated by the three enzymes.     

Restriction mapping of T4 bacteriophage late gene region which contains the origins of DNA replication

DNAs of lambda T4 recombinants 596-27 (genes 50-5), 596-30 (genes 50-8), 596-29 (genes 50-12), 591-16 (genes 6-8), 591-1 (genes 9-12), 596-13 (genes 13-16), 596-17 (genes 18-20) and 596-11 (genes 25-29) were mapped with the use of EcoRI, HindIII, SmaI, SalI and BamHI restriction enzymes. T4 dcDNA was digested with HindIII restriction endonuclease and resulting fragments were cloned into HindIII lambda vector 761. The recombinants 761-7, 761-17, 761-19, 761-24, 761-44, 761-50, 761-55 contained the region of genes 25-48 and 761-42, 761-26 and 761-16 contained a single HindIII-fragment with genes 6-12 in both orientations. Data obtained with the DNA of the latter recombinants allowed to show the correctness of the map established earlier which did not contain a full set of overlapping sequences. As a result of the experiments reported, the position of EcoRI and HindIII recognition sites in the region of genes 50-20 and 25-48 was determined and in the region of genes 25-48 BglII and XhoI restriction sites were mapped. The location of a single BamHI restriction site in the region of gene 8 was also established.     

Restriction endonuclease mapping and cloning of Mycobacterium intracellulare plasmid pLR7

A restriction map of Mycobacterium intracellulare plasmid pLR7 was developed. This 15.3-kb plasmid had unique sites for BamHI, HindIII, and XbaI. Various large fragments of pLR7 were cloned into pBR322 or pHP34 and propagated in Escherichia coli. A hybrid pLR7 ::pBR322 plasmid carrying the complete pLR7 sequence was constructed by joining the plasmids at their HindIII sites. The construction of these hybrids will facilitate the analysis and manipulation of pLR7 and may allow the development of this plasmid as a model system for genetic analysis in mycobacteria.     

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.     

Restriction endonuclease mapping of a Rhizobium leguminosarum Sym plasmid

A circular map of the Sym plasmid pRle1001a corresponding to a genome size of 150 × 10⁶ D was established using the restriction endonucleases HpaI, SmaI, and KpnI. The map was derived from the results obtained by hybridizing individual HpaI fragments to blotted SmaI and KpnI digests of the plasmid. A complete map of the HpaI fragments was constructed in this way. Regions of homology with the structural nif-genes D and H of Klebsiella pneumoniae, with the Sym plasmid of Rhizobium trifolii 5, and with an octopine and nopaline Ti plasmid of Agrobacterium tumefaciens were mapped. A large area of plasmid pRle1001a, which carries nif structural genes, is highly conserved in the Sym plasmid of R. trifolii 5. It is assumed that this could be an area carrying genes involved in various functions that play a role in the process of symbiotic nitrogen fixation.     

Nucleotide sequence of the traD region in the Escherichia coli F sex factor

The complete nucleotide sequence has been determined of a 3635-bp region, extending from the HpaI site in traT, at F coordinate 90.3 kb, to beyond the end of traD, of the F sex factor plasmid of Escherichia coli K-12. This region contains the C-terminal coding part of traT and the entire traD gene. An open reading frame (ORF) of 2148 bp within the sequence confirms that traD encodes an 81.4-kDa cytoplasmic membrane protein. The TraD protein has several regions with an unusually high pI (greater than 10), suggesting that they may correspond to the DNA-binding domains. Several other ORFs were detected within the region including the gene (ORF1) for a 26.3-kDa protein and ORF2, probably corresponding to traI, which continues to the end of the sequence. An ORF for an 8.5-kDa protein preceded by an excellent promoter and ribosome-binding site is present in the region following traD but on the opposite strand. This promoter is thought to correspond to the major RNA polymerase binding site in this region, implying that traI does not have its own promoter. The lack of a typical terminator following traD and ORF1 and the translational coupling provided by overlapping stop and start codons is consistent with this conclusion.     

Restriction endonuclease mapping of the HI2 incompatibility group plasmid R478.

A restriction map of the 272-kb IncHI2 plasmid R478 was constructed by using the enzymes ApaI, XbaI, SalI, and XhoI. The map was derived from cloned restriction fragments from R478 inserted into cosmid and plasmid vectors as well as from double-digestion analysis of R478 and R478 miniplasmids. All previously known resistance determinants were cloned from R478, and their positions were located on the restriction map. A region involved in incompatibility was cloned and mapped. The location of a previously unreported arsenite resistance gene was also determined. The genes encoding tellurite resistance, colicin B resistance, and phage inhibition were found to be associated with a 6.7-kb SalI fragment of R478.     

Restriction endonuclease mapping of three plasmids from Bacillus thuringiensis var. israelensis

Restriction endonuclease cleavage site maps have been constructed of plasmids pTX14-1, pTX14-2, and pTX14-3 from Bacillus thuringiensis var. israelensis (Bti).     

Restriction endonuclease BamHI cleaves bacteriophage Mu DNA within cistrons E and F

We have cleaved phage Mu DNA with restriction endonucleases EcoRI and BamHI and have cloned three specific DNA fragments from the middle of the Mu genome into vector plasmid pBR322. By marker rescue experiments, we have determined that the two BamHI cleavage sites in Mu DNA occur within cistrons E and F.     

Identification and restriction endonuclease mapping of the threonine operon regulatory region.

A DNA fragment carrying the thrP region of Escherichia coli has been identified and characterized. Starting from a secondary site lysogen of bacteriophage λ, where the position of the prophage is either within thrP or between thrP and thrP structural genes (Gardner et al., 1974; Gardner &; Smith, 1975) it has been possible to isolate transducing phages which carry bacterial DNA from either side of the prophage. By Int-Xis mediated site-specific recombination we have generated recombinant transducing phages which carry an intact thrP region. A phage carrying a regulatory mutation, which maps in the thrP region, has also been constructed.Mapping with several restriction endonucleases has allowed us to construct a map of the cleavage sites of the thrP region. A 1700 base-pair HaeIII fragment carrying the secondary attachment site (attΔOΔ′) was isolated and its position was localized within a 180 base-pair TaqI-HhaI restriction fragment. The location of attΔOΔ′ in the HaeIII fragment suggests that the entire thrP region is carried by this fragment.