Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9.

In vitro recombination via restriction endonucleases and the in vivo genetic translocation of the Ap resistance (Apr) gene resulted in the construction of a new cloning vehicle, the plasmid pBR313. This vector was derived from a ColE1-like plasmid and, while it does not produce colicon E1, it still retains colicin E1 immunity. The Apr and tetracycline resistance (Tcr) markers carried in pBR313 were derived from the ampicillin transposon (TnA) of pRSF2124 and pSC101 respectively. During the construction of pBR313, the TnA component was altered and the Apr gene in pBR313 can no longer be translocated. This plasmid has a molecular weight of 5.8 Mdalton and has been characterized using thirteen restriction enzymes, six of which (EcoRI, SmaI, HpaI, HindIII, BamHI and SalI) cleave the plasmid at unique restriction sites. This allows the molecular cloning of DNA fragments generated by these six enzymes. The restriction sites for the latter three enzymes, HindIII, BamHI and SalI, are located in the Tcr gene(s). Cloning DNA fragments into these sites alters the expression of the Tcr mechanisms thus providing a selection for cells carrying recombinant plasmid molecules. An enrichment method for AprTcS cells carrying recombinant plasmid molecules is described.     

Effective method of oligonucleotide-controlled mutagenesis of DNA fragments

A procedure has been designed for changing specific nucleotides in a DNA sequence with efficiency. The method involves the use of the specially constructed cloning vectors pBRS1, pHS1, and pHS2. These plasmids are derivatives of pBR322 in which the EcoRI-HindIII region has been replaced by synthetic duplexes carrying SmaI, HpaI and XhoI sites, in addition to EcoRI and HindIII sites. The DNA fragment to be mutagenized is cloned in pHS1 (or pBRS1, or pHS2) using restriction sites close to the SmaI and HpaI sites. The recombinant plasmid obtained is digested with one of these enzymes to produce double-stranded DNA with blunt ends. This linear DNA is a substrate for exonuclease III (or T4 DNA polymerase). The digestion under controlled conditions produces duplex with protruding single-stranded 5'-regions which include the site of the desired mutation. The synthesis of DNA by DNA-polymerase I (Klenow's fragment), primed in part by the synthetic oligonucleotide containing the desired mutation, leads to the linear heteroduplex. The closed circular heteroduplex is formed by ligation. After transformation into E. coli, DNA replication generates homoduplexes, some of which contain the mutation. Colony hybridization with the same 32P-labeled oligonucleotide is used to select mutants. The yield of the mutants is 10-20%. This technique can be extended to replicative form of M13 vectors. It can be also applied to any DNA sequence which has a unique site of restriction endonuclease generating blunt ends.     

Restriction endonuclease mapping of adenovirus 35, a type isolated from immunocompromised hosts.

The DNA of adenovirus 35 (Ad35), a type recently associated with infections in immunocompromised hosts, was mapped by the use of BamHI, SmaI, PstI, EcoRI, and HpaI restriction endonucleases. In addition to standard mapping procedures, we used the in vitro adenovirus DNA replication system with origins at both physical ends of the linear molecule to determine the terminal fragments. Deletions of single restriction endonuclease sites in a group of closely related adenovirus isolates from patients with acquired immunodeficiency syndrome helped determine the location of some DNA fragments on the genome.     

Cloning of small DNA fragments containing the Escherichia coli tryptophan operon promoter and operator

A41-bp AluI restriction fragment from the trp promoter-operator region has been cloned into the PvuII site of pBR322, regenerating PvuII sites. Transformants were selected on media that allowed the selection of trp-operator-bearing plasmids. The cloned 41-bp fragment can be released from the vector by PvuII digestion, and it possesses a functional promoter and operator as demonstrated by in vivo tests. The 41-bp fragment contains several restriction sites: HincII, TaqI, RsaI, and a HpaI site that is located at the center of the operator sequence. Two new operator derivatives, symmetrical about the HpaI site, were prepared from the 41-bp fragment by joining two right-side, or two left-side PvuII-HpaI pieces together at the HpaI site. These derivatives showed in vivo operator activity. Plasmids containing up to five copies of the 41-bp trp-promoter-operator fragment have been constructed. These plasmids should be useful in preparing large amounts of the 41-bp fragment.     

Restriction enzyme cleavage map of Tn10, a transposon which encodes tetracycline resistance.

A cleavage map of a recombinant plasmid carrying Tn10 was constructed for 13 different restriction enzymes. The Tn10 region of this plasmid contains cleavage sites for BamHI, AvaI, BglI, BglII, EcoRI, XbaI, HincII, HindIII, and HpaI. Restriction enzymes PstI, SmaI, KpnI, XhoI, SalI, and PvuI do not cleave within the Tn10 element. This map confirms the previously reported structure of this transposon; it is composed of a unique sequence (approximately6,400 base pairs long), which in part codes for the tetracycline resistance functions and is bounded by inverted repeats (approximately 1,450 base pairs long).     

Physical map of Pseudomonas aeruginosa phage phi kF77 genome. Localization of sites sensitive to restriction endonucleases

A physical map of the P. aeruginosa bacteriophage phi kF77 has been constructed using the restriction endonucleases SalI, HindIII, EcoRI, EcoRV, MuI, XbaI, ClaI. The phi kF77 DNA is resistant to cleavage by the restriction endonucleases BamHI, BglII, HpaI, PstI, PvuII, SmaI, XhoI.     

Improved vector, pHSG664, for direct streptomycin-resistance selection: cDNA cloning with G:C-tailing procedure and subcloning of double-digest DNA fragments

A plasmid cloning vector, pHSG664, has been constructed which is suitable for the direct-selection of transformed cells with recombinant DNAs. The plasmid contains the replicon and ApR-gene region of pUC9 ligated to the strA+ (rpsL+) gene derived from pNO1523 [Dean, Gene 15 (1981) 99-102]. The vector contains ten unique restriction sites: EcoRI, HpaI, PvuII, SphI, PstI, SmaI(XmaI), BamHI, SalI(AccI, HincII), XbaI and HindIII. Five sites (bold-face lettering) are located within the coding region of the strA+ gene. Any insertion at the five bold-faced sites or any nucleotide replacement involving the strA+ gene region and the other unique sites can be selected by Ap and Sm double selection in a strA- (SmR) strain such as E. coli HB101. Thus, this vector is useful for cDNA cloning at either the SphI or the PstI site with the G:C-tailing procedure, as well as for the cloning of double-digest DNA segments.     

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.     

Restriction mapping of the DNA of the Streptomyces temperate phage phi C31 and its derivatives

DNA of phi C31 propagated on Streptomyces lividans 66 contained no sites for the restriction enzymes BamHI, SalPI (=PstI) and XhoI; one for XbaI; three for HpaI; five for ClaI and KpnI; six for EcoRI; about 13 for HindIII; about 14 for BclI; and more than 15 for FspAI, HgiAI, SacI, SalGI and SmaI. A complete map of 20 sites (XbaI, HapI, ClaI, KpnI and EcoRI) was obtained using partial digestion and double digestion of DNA of the wild-type and deletion and insertion mutants. The total molecular size was estimated to be 41.2 kb.     

Physical map of the seven ribosomal RNA genes of Escherichia coli.

Escherichia coli DNA was digested with restriction endonucleases BamHI, PstI, EcoRI, SalI, HindIII, XhoI, BglII, SmaI, HpaI and with selected double and triple combinations of the same enzymes. The digests were electrophoresed and hybridized with 32P-labelled ribosomal RNA by using the Southern blotting technique. The resulting bands could be arranged into seven groups, and it was possible to construct a unique physical map of the seven rRNA genes (operons) of the bacterial chromosome. Mapping information obtained on several transducing phages and recombinant plasmids carrying rRNA genes, and mapping data published in the literature helped to determine the final map. The results suggest that phage lambda daroE152 carries a "hybrid" rRNA gene which was probably formed by recombination between two different chromosomal rRNA genes.     

Development of an all-fish gene cassette for gene transfer in aquaculture.

To develop an all-fish gene cassette suitable for gene transfer in aquaculture, the antifreeze protein (AFP) gene promoter from the ocean pout (Macrozoarces americanus) was analyzed for its ability to direct exogenous gene expression both in vitro and in vivo. The ocean pout AFP (opAFP) gene promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) was functionally analyzed in two fish cell lines and in Japanese medaka embryos. The opAFP gene promoter was active in these systems, as demonstrated by the transient expression of CAT activity. These results suggest that the opAFP gene promoter is useful for many other gene transfer experiments. To facilitate use of the opAFP gene promoter as a common and versatile vehicle for fish gene transfers, an expression vector, opAFP-V, was constructed by linking the 2.1-kb opAFP gene promoter, the 63-bp opAFP gene 5' untranslated sequence, and the 1.2-kb opAFP gene 3' sequence by two unique restriction sites, Bg/II and HpaI, respectively. Thus, genes of interest can be inserted into either the Bg/II site or the HpaI site depending on the length of their 5' untranslated sequence. The complete DNA sequence of opAFP-V was determined to facilitate future detailed analysis of integration and expression of the transgene.     

Physical maps of bovine papillomavirus type 1 and type 2 genomes.

Physical maps of bovine papillomavirus type 1 and type 2 (BPV-1 and BPV-2) DNA were constructed from analysis of the electrophoretic mobilities of restriction endonuclease cleavage fragments from dual digests. BPV-1 DNA was sensitive to Hind III, HindIII, EcoRI, HpaI, AND BamHI, with all but HindII yielding single scissions. BPV-2 DNA was resistant to EcoRI, and HindIII had one cleavage site whereas HpaI, BamHI, and HindII yielded multiple fragments. Of four BPV-1 isolates examined, DNA from one isolate was resistant to HindIII, and another DNA isolate was resistant to BamHI. The three BPV-2 isolates examined were uniformly sensitive to the restriction endonucleases employed.     

Physical map of the Agrobacterium rhizogenes strain 8196 virulence plasmid

Virulence of Agrobacterium rhizogenes, agent of hairy root disease, is conferred by large plasmids called Ri (root-inducing) plasmids. We have determined the BamHI fragment map of pRi8196, MW 143 Mda, principally by analysis of recombinant plasmids containing overlapping BamHI partial-digest fragments. Clones containing solitary BamHI inserts of remaining unmapped fragments were used to probe a series of Southern-blotted, pRi8196-derived EcoRI, PstI, HindIII, SalI, or SmaI digests. Continguous hybridized bands represented complements of EcoRI, PstI, HindIII, SalI, or SmaI fragments which bridged the unmapped BamHI fragments. We present, in addition, a detailed map of the core T-DNA region with respect to the restriction endonucleases SalI, EcoRI, HpaI, and HindIII.     

Restriction endonuclease mapping of the proviral DNA of the exogenous RIII murine mammary tumor virus

Cellular DNA containing integrated murine mammary tumor virus (MuMTV) was isolated from FeI/C6 feline kidney cells and CCL64 mink lung cells infected with milkborne RIII MuMTV. By using restriction enzyme HpaI, intact RIII MuMTV provirus (length, 8.7 kilobases [kb]) was excised from the cellular DNA. Subsequent restriction endonuclease analysis of this HpaI fragment with KpnI, HindIII, EcoRI, BamHI, BglII, PstI, SstI, SalI, and XhoI enabled us to construct a map of the RIII virus genome. A comparison of this map with the maps of the GR and C3H MuMTV's revealed that there are greater sequence differences between the RIII virus and the GR and C3H MuMTV proviruses than there are between the GR and C3H proviruses. The following are features of the restriction map unique to the RIII provirus: the presence of three BamHI and two EcoRI cleavage sites, a HpaI cleavage site in the terminal 3'-5' repeat unit of the provirus, and the absence of an XhoI cleavage site. Another distinguishing feature of the RIII provirus is that the sizes of some of the restriction fragments produced by cleavage of the RIII provirus with PstI are different from the sizes of the fragments obtained by PstI cleavage of the GR and C3H proviruses. Like the GR proviral DNA, the RIII proviral DNA has three SstI (SacI) cleavage sites, whereas the C3H provirus has only two SstI sites. HpaI digestion of MuMTV-infected mink lung cell DNA revealed only one class of provirus (an 8.7-kb fragment); however, we observed several minor classes of RIII proviral DNA in addition to the major class of provirus DNA in infected cat kidney cells. PstI digestion of the HpaI 8.7-kb fragments from both feline and mink cells generated a 3.7-kb DNA fragment identical in size to a PstI-generated fragment that has been found in GR and C3H milkborne virus-infected cells. Although a fragment similar in size to the milkborne 3.7-kb PstI fragment has been found as an endogenous component in many C3H and GR mouse tissues, we did not observe such an endogenous fragment in the RIII mouse strain. Therefore, the 3.7-kb fragment may be useful as a marker for the milkborne RIII MuMTV provirus in RIII mice.     

Restriction and electron microscopic analyses of deletion derivatives thermosensitive with respect to maintaining plasmid pEG1

Temperature-independent deletion mutants of temperature-sensitive in self-maintenance plasmid pEG-1, derived from R-factor RP4, are mapped using the restriction endonucleases PstI, SmaI, EcoRI, BamHI and the heteroduplex analysis. The pEG1 derivatives under study are found to have deletions in the area of ampicillin transposon Tn1 and nearby genes. The left and the right ends of these deletions boundaries are localized between 37.5 MD and 7.6 MD in the RP4 map. Thus far, the area of plasmid RP4 (37.5-7.6 MD) with Tn1 and, presumably, inc gene(s) in it does not have any genes needed for stable maintenance of R-factor. A conclusion is made that the gene RP4, which carries the mutation determining thermosensitive character of pEG1 maintenance and of inhibition of the cell growth, is localized between the EcoRI site and transposon Tn1 at a distance less than 1.0 MD from the latter.     

Tn10-derived transposons active in Bacillus subtilis.

Small derivatives of the Escherichia coli transposon Tn10, comprising IS10 ends and a chloramphenicol resistance gene, were introduced in Bacillus subtilis on a thermosensitive plasmid, pE194. In the presence of the Tn10 transposase gene fused to signals functional in B. subtilis, these derivatives transposed with a frequency of 10(-6) per element per generation. They had no highly preferred insertion site or region, as judged by restriction analysis of the chromosomal DNA, and generated auxotrophic and sporulation-deficient mutants with a frequency of about 1%. These results suggest that Tn10 derivatives might be a useful genetic tool in B. subtilis and possibly other gram-positive microorganisms.     

Plasmid vehicles for direct cloning of Escherichia coli promoters.

A multicopy plasmid cloning vehicle, pGA22, which carries genes for ampicillin resistance (Apr), tetracycline resistance (Tcr), chloramphenicol resistance (Cmr), and kanamycin resistance (Kmr) has been constructed. This plasmid has five unique sites for restriction endonucleases EcoRI, PstI, XhoI, SmaI, and SalI within antibiotic resistance genes. pGA22, which is 5.1 megadaltons in size, has a low copy number (probably fewer than 10 per genome), is capable of relaxed replication, and is mobilized by F-factor at a frequency of 10(-5). A series of promoter-cloning vehicles, pGA24, pGA39, and pGA46, has been developed from pGA22. In these plasmids the natural promoter for Tcr has been removed and has been replaced by small deoxyribonucleic acid fragments carrying unique sites for several restriction endonucleases. Cells carrying these vectors are sensitive to tetracycline unless insertional activation of the Tcr occurs by cloning a promoter-carrying deoxyribonucleic acid fragment in one of the unique sites adjacent to the 5' end of Tcr. In this way, promoters carried on a HindIII-generated deoxyribonucleic acid fragment can be inserted at the HindIII site of plasmid pGA24, pGA39, or pGA46. A promoter in fragments generated by digestion with restriction endonuclease XmaI or PstI or by any restriction endonucleases which generate flush ends, such as SmaI, PvuII, HpaI, HincII, or HaeIII, can be clones in plasmid pGA39. Plasmid pGA46 can be used to detect a promoter fragment carried on a BglII, BamHI, MboI, or PstI fragment. We also describe a plasmid, pGA44, with a unique KpnI site in the rifampin resistance gene rpoB.     

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.     

H2, a temperate bacteriophage isolated from Bacillus amyloliquefaciens strain H

Bacillus amyloliquefaciens strain H is lysogenic for a large temperate phage we call H2. H2 has a polyhedral head 85 nm in diameter and a tail of about 17 x 434 nm. H2 lysogenizes Bacillus subtilis between the tyrA and metB genes, and gives specialized transduction of metB and, at lower frequencies, of ilvD and ilvA. The phage carries a thymidylate synthase gene and converts thymine auxotrophs of B. subtilis to prototrophy. The H2 genome is a linear DNA molecule about 129 kb in length. DNA extracted from phage particles grown in B. subtilis is not cut by the restriction endonucleases HaeIII, Fnu4HI, Bsp1286I, and BamHI; the latter enzyme is produced by B. amyloliquefaciens strain H. The prophage in lysogenic B. subtilis cells can be cut by these enzymes. We have isolated H2 mutants that carry the transposon Tn917, or a mutation resulting in clear-plaque morphology, or both.