Physical mapping of cleavage sites recognized by restriction endonucleases on the genome of bacteriophage T5

The DNA of bacteriophage T5 has been treated with restriction endonucleases EcoRi, HindIII, BamI, SmaI, PstI, SalI, KpnI and the electrophoretic pattern obtained in agarose gel has been analyzed in order to localize the specific cleavage sites on the T5 DNA. The localization of cleavage sites has been deduced from the electrophoretic pattern of double and partial digests, the digests of isolated restriction fragments and the digests of deletion mutant T5st(o) DNA.Four BamI cleavage sites have been found and localized on the physical map of T5 DNA at 0.21, 0.225, 0.685 and 0.725 fractional length. Endonuclease SmaI cleaves at 0.39, 0.59 and 0.69 fractional length. Endonuclease PstI cuts T5 DNA at 11 sites: 0.090, 0.210, 0.320, 0.510, 0.635, 0.670, 0.705, 0.770, 0.815, 0.840, 0.875 fractional length. Six KpnI cleavage sites have been mapped at 0.170, 0.215, 0.525, 0.755, 0.830, 0.850 fractional length. A complete cleavage map of the phage genome is presented for seven restriction enzymes.     

Survival and transfer in the human gut of poorly mobilizable (pBR322) and of transferable plasmids from the same carrier E. coli

We examined the survival of a host Escherichia coli K-12 bacterium containing two transferable plasmids (pLM2, pSL222-4) and one poorly mobilizable plasmid (pBR322), and the transfer of these three plasmids to endogenous bacteria in the human intestinal tract. The survival of this plasmid-carrying host organism in four human volunteers was 3.5 to 6 days at recovery rates of 10^?1 to 10^?4. This finding was similar to our previous survival data on the same organism bearing a single plasmid. The K-12 strain appeared to be under a strong selective disadvantage in the human gut, since, even when bearing a tetracycline-resistant plasmid, its titer did not increase despite the administration of tetracycline. Studies of transferability showed that, while the transfer-depressed incFII plasmid pSL222-4 transferred at a frequency of 10^?1 in culture, its transfer in the human gut was much less frequent. The number of new recipients per donor cell ingested was about 10^?5, which included new recipients arising by multiplication. The recovery of pSL222-4 transcipients was enhanced by the administration of tetracycline on day 6. Neither the transfer-repressed, broad host range incP plasmid pLM2, nor the plasmid pBR322, could be detected in any endogenous host bacteria. Using the transfer and mobilization frequencies obtained in culture and the number of new recipients of pSL222-4 in the intestinal tract, we estimated that any in vivo mobilization of pBR322 to a new recipient could not occur at a frequency higher than 10^?12.     

Expression of thymidylate synthetase activity in Bacillus subtilis upon integration of a cloned gene from Escherichia coli

The gene from Escherichia coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid, pER2, was effective in transforming both E. coli and Bacillus subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine-requiring strains of B. subtilis to thymine independence. Linearization of the chimeric plasmid, pER2, with restriction enzymes markedly diminished its ability to transform B. subtilis auxotrophs. The Thy+ transformants derived from the transformation of B. subtilis with pER2 DNA did not contain detectable extrachromosomal DNA as demonstrated by Southern hybridization patterns and centrifugation in CsCl gradients of DNA isolated from B. subtilis colonies transformed with the chimeric plasmid. We conclude that the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis, demonstrating that extensive homology is not required for the integration of foreign DNA. This is the first reported case of a gene from a Gram-negative bacterium functioning in a Gram-positive organism.     

Cloning and expression of the leucine gene from Thermus thermophilus in Escherichia coli

A pBR322-T.leu hybrid plasmid was constructed which contains a 3.75 Md HindIII-fragment derived from Thermus thermophilus HB27 chromosomal DNA. In the Escherichia coli host, this plasmid coded for the β-IPM dehy drogenase (product of leuB) activity, the optimal temperature of which was 80°C, suggesting that information on the thermostability of the enzyme lies in its structural gene. 10-day propagation of E. coli [pBR322-T.leu] at 37°C decreased the temperature optimum from 80°C to 75°C. This change, which was found to depend on the plasmid but not on the host cells, might be due to selection of some mutation at the non-restrictive temperature of 37°C. Our results suggest that the 3.75 Md HindIII-fragment of pBR322-T.leu carries a promoter of the thermophile, which could function in E. coli.     

Human nucleotide sequences related to the transforming gene of a murine sarcoma virus: studies with cloned viral and cellular DNAs

A recombinant plasmid, pI26, has been constructed by cloning into pBR322 a transforming gene of murine sarcoma virus (a Moloney strain, clone 124, MSV) synthesized by detergent-treated virions. From this plasmid a XbaI-HindIII fragment has been isolated which contains only mos-specific sequences. This mos-specific probe has been used for screening a human gene library cloned in bacteriophage λ Charon 4A. Of these, 19 clones have been isolated containing mos-related sequences. By physical mapping and molecular hybridization it has been shown that these sequences are neighboured by DNA regions related to Moloney murine leukemia virus. Recombinant phages have also been found containing human inserts related to MLV, not to the mos gene. The possible existence of murine-like endogenous retroviruses in the normal human genome, including that of a sarcoma type, is discussed. By Northern blotting, expression of the cellular c-mos gene has been detected in mouse liver treated with a hepatocarcinogen. The general significance of the suggested model for evaluating the relationship between chemical carcinogenesis and oncogene expression is discussed.     

Isolation of cloned ribosomal protein genes from the yeast Saccharomyces carlsbergensis

A colony bank of yeast dna obtained by cloning HindIII-generated fragments of total yeast nuclear DNA in Escherichia coli K-12 with the vector pBR322, was screened with a radioactive RNA probe enriched for a subset of ribosomal protein mRNAs. The selected recombinant DNA molecules were hybridized with poly(A)-containing mRNA under R-loop conditions. From the DNA-RNA hybrids the respective mRNAs were melted off and translated in vitro in a rabbit reticulocyte cell-free system. The translational products were analyzed by immunoprecipitation with antibodies raised against ribosomal proteins. The identity of the ribosomal protein gene products was further established by electrophoresis on two-dimensional gels. At least 15 recombinant DNA molecules were shown to contain ribosomal protein genes. Four of them, i.e. Y65, Y89, Y113 and Y138, have been characterized preliminarily.     

Physical and genetic organization of the IncN-group plasmid pCU1

A restriction endonuclease-cleavage map of the IncN group plasmid pCU1 was constructed. Deletion mutants of the plasmid were obtained by in vivo or in vitro methods. Comparison of the restriction maps of these mutants to that of pCU1 enables one to assign the known functions of the plasmid to particular regions on the plasmid DNA. For different enzymes, the number and distribution of restriction sites on pCU1 is compared to that of other IncN and related plasmids.     

Cloning a promoter that puts the expression of tetracycline resistance under the control of the regulatory elements of the mer operon

We have sub-cloned from the Eco RI-H fragments of the IncFII plasmid R100 a 260-bp EcoRI fragment, using the promoter-cloning vehicle, pBRH4, (The Inc FII plasmid codes for the mer operon, and pBRH4 expresses tetracycline resistance only when the deleted tet promoter has been replaced by another sequence that can serve as a promotor). With the 260-bp fragment inserted, the derivative plasmid, pFB4, directs the expression of tetracycline resistance only if there is a second plasmid in the strain that carries the merR-positive regulatory element. Under these conditions, the level of tetracycline resistance is directly proportional to the concentration of Hg2+ present in the medium. The 260-bp fragment also allows low-level constitutive expression of tet resistance when transactivated with merR mutants that have a "micro-constitutive" phenotype. The 260-bp mer promoter fragment contains a single HincII site; there is also but one HincII site in the EcoRI-H fragment of R100 from which the promoter fragment was derived. Restriction analysis of purified Eco RI-H DNA shows that the single HincII site is at 550 bp from the "right"terminus of the IS1b element, which is also present in the EcoRI-H fragment. Because of its biological activity and its location within the "H" fragment, this promoter is very likely a promoter for the structural genes of the operon.     

Cloning vehicles for the homologous Bacillus subtilis host-vector system

A series of Bacillus subtilis plasmids was constructed which carry either the leu region or both the leu and the dihydrofolate reductase (DHFR) regions of the B. subtilis chromosome. The DHFR-coding gene was derived from a trimethoprim resistant (Tmpr) B. subtilis strain, and cells harboring the DHFR plasmid showed resistance to trimethoprim (Tmp). One such leu+tmpr plasmid, pTL12, was found to be useful for cloning DNA fragments at the BamHI, EcoRI, BglII and XmaI sites. It was also shown that insertion of DNA fragments at the BamHI and XmaI sites of pTL12 inactivated the leuA gene function (insertional inactivation) but not tmpr, indicating that cells carrying recombinant plasmids can be detected easily by selecting Leu-Tmpr colonies. Combination of B. subtilis 168 and plasmid pTL12 should serve as an efficient homologous cloning system in B. subtilis.