Skip to main content
SpringerLink
Log in

Molecular cloning and in vitro transcription of Bacillus subtilis plasmid in Escherichia coli

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Summary

A composite plasmid (pAT2010) has been constructed in vitro from RSF2124 and Bacillus subtilis IFO3022 plasmid (pAT1060) by covalent joining of the two DNA molecules by means of Escherichia coli DNA ligase through the cohesive ends generated by restriction endonuclease RI (EcoRI) cleavage. The composite plasmid was selected by transformation of E. coli C600 r −m− with the ligated mixture affer enrichment for composite plasmid by preparative agarose gel electrophoresis, and plating of the transformants on a medium containing ampicillin and colicin El. Treatment of the composite plasmid with EcoRI yielded two fragments corresponding to the linear forms of the parental plasmids.

The composite plasmids replicated as biologically functionally units in E. coli, and expressed genetic information carried by RSF2124. In the presence of chloramphenicol, the composite plasmids continued to replicate and the copy number gradually increased. Such nature of replication in the presence of chloramphenicol is characteristic to RSF2124 derived from colicin El factor, and so it is suggested that the replicator of RSF2124 is functional in the composite plasmid.

The composite plasmid was found to synthesize mRNA of B. subtilis plasmid in cell-free extracts of E. coli, by hybridization of the mRNA to the original plasmid DNA of pAT1060.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bedbook, J.R., Ausubel, F.M.: Recombination between bacterial plasmids leading to the formation of plasmid multimers. Cell 9, 707–716 (1976)

    Google Scholar 

  • Bennet, G.N., Schweingruber, M.E., Brown, K.D., Squires, C., Yanofsky, C.: Nucleotide sequence of region preceding trp mRNA initiation site and its role in promoter and operator function. Pro. nat. Acad. Sci. (Wash.) 73, 2351–2355 (1976)

    Google Scholar 

  • Bonner, J., Kung, G., Bekhor, I.: A method for the hybridization of nucleic acid molecules at low temperature. Biochemistry 6, 3650–3653 (1967)

    Google Scholar 

  • Chang, A.C.Y., Cohen, S.N.: Genome construction between bacterial species in vitro: replication and expression of Staphylococcus plasmid genes in Escherichia coli. Pro. nat. Acad. Sci. (Wash.) 71, 1030–1034 (1974)

    Google Scholar 

  • Clewell, D.B.: Nature of ColEl plasmid replication in the presence of chloramphenicol. J. Bact. 110, 667–676 (1972)

    Google Scholar 

  • Cohen, S.N., Chang, A.C.Y.: A method for selective cloning of eukaryotic DNA fragments in Escherichia coli by repeated transformation. Molec. gen. Genet. 134, 133–141 (1974)

    Google Scholar 

  • Collins, C.J., Jackson, D.A., Devries, F.A.J.: Biochemical construction of specific chimeric plasmids from ColEl DNA and unfractionated Escherichia coli DNA. Proc. nat. Acad. Sci. (Wash.) 73, 3838–3842 (1976)

    Google Scholar 

  • Davis, R.W., Simon, M.N., Davidson, N.: In: Methods in enzymology (Grossman, L., Moldave, K., eds.), Vol 21, pp. 413–438. New York: Academic Press 1971

    Google Scholar 

  • Foulds, J., Barrett, C.: Characterization of Escherichia coli mutants tolerant to bacteriocin JF246: two new classes of tolerant mutants J. Bact. 116, 885–892 (1973)

    Google Scholar 

  • Gillespie, D., Spiegelman S.: A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. molec. Biol. 12 829–842 (1965)

    Google Scholar 

  • Gottesman, M.M., Hicks, M.L., Gellert, M.: Genetics and function of DNA ligase in Escherichia coli. J. molec. Biol. 77, 531–547 (1973)

    Google Scholar 

  • Hershfield, V., Boyer, H.W., Yanofsky, C., Lovett, M.A., Helinski, D.R.: Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA. Proc. nat. Acad. Sci. (Wash.). 71, 3455–3459 (1974)

    Google Scholar 

  • Humphreys, G.P., Willshaw, G.A., Anderson, E.S.: A simple method for the preparation of large quantities of pure plasmid DNA. Biochim. biophy. Acta (Amst.) 383, 457–463 (1975)

    Google Scholar 

  • Kikuchi, A., Elseviers, D., Gorini, L.: Isolation and characterization of lambda transducing bacteriophage for argF argI, and adjacent genes. J. Bact. 122, 727–742 (1975)

    Google Scholar 

  • Lederberg E.M., Cohen, S.N.: Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J. Bact. 119, 1072–1074 (1974)

    Google Scholar 

  • Lovett, P.S.: Plasmid in Bacillus pumilus and the enhanced sporulation of plasmid-negative variants. J. Bact. 115, 291–298 (1973)

    Google Scholar 

  • Lovett, P.S., Bramucci, M.G.: Plasmid deoxyribonucleic acid in Bacillus subtilis and Bacillus pumilus J. Bact. 124, 484–490 (1975)

    Google Scholar 

  • Lovett, P.S., Duvall, E.J., Keggins, K.M.: Bacillus pumilus plasmid pPL10: properties and insertion into Bacillus subtilis 168 by transformation. J. Bact. 127, 817–828 (1976)

    Google Scholar 

  • Magdalene, S., Gill, R., Falkow, S.: The generation of a ColEl-Apr cloning vehicle which allows detection of inserted DNA. Molec. gen. Genet. 142, 239–249 (1975)

    Google Scholar 

  • Meagher R.B., Tait, R.C., Betlach, M., Boyer, H.W.: Protein expression in E. coli minicells by recombinant plasmids. Cell 10, 521–536 (1977)

    Google Scholar 

  • Meselson M., Yuan, R.: DNA restriction enzyme from E. coli. Nature (Lond.) New Biol. 217, 1110–1114 (1968)

    Google Scholar 

  • Miller, D.L., Gubbins, E.J., Pegg III, E.W., Donelson, J.E.: Transcription and translation of cloned Drosophila DNA fragments in Escherichia coli. Biochemistry 16, 1031–1038 (1977)

    Google Scholar 

  • Modrich, P., Anraku, Y., Lehman, I.R.: Deoxyribonucleic acid ligase: isolation and physical, characterization of the homogenous enzyme from Escherichia coli. J. biol. Chem. 248 7495–7501 (1973)

    Google Scholar 

  • Morrow, J.F., Cohen, S.N., Chang, A.C.Y., Boyer, H.W., Goodman, H.M., Helling, R.B.: Replication and transcription of eukaryotic DNA in Escherichia coli. Proc. nat. Acad. Sci. (Wash.) 71, 1743–1747 (1974)

    Google Scholar 

  • Ratzkin, B., Carbon, J.: Functional expression of cloned yeast DNA in Escherichia coli. Proc. nat. Acad. Sci. (Wash.) 74, 487–491 (1977)

    Google Scholar 

  • Sakakibara, Y., Tomizawa, J.: Replication of colicin El plasmid DNA in cell extracts. Proc. nat. Acad. Sci. (Wash.) 71, 802–806 (1974)

    Google Scholar 

  • Sens, D., James, E.: Regulation of argF mRNA synthesis. performed in vitro. Biochem. biophys. Res. Commun. 64, 169–174 (1975)

    Google Scholar 

  • Sharp, P.A., Sugden, B., Sambrook, J.: Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide gel electrophoresis. Biochemistry 12, 3055–3063 (1973)

    Google Scholar 

  • Struhl, K., Cameron, J.R., Davis, R.W.: Functional genetic expression of eukaryotic DNA in Escherichia coli. Proc. nat. Acad. Sci. (Wash.) 73, 1471–1475 (1976)

    Google Scholar 

  • Tanaka, T., Kuroda, M., Sakaguchi, K.: Isolation and characterization of four plasmids from Bacillus subtilis. J. Bact. 129, 1487–1494 (1977)

    Google Scholar 

  • Tanaka, T., Weisblum, B.: Construction of a colicin El-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid. J. Bact. 121, 354–362 (1975)

    Google Scholar 

  • Timmith, K., Cabello, F., Cohen, S.N.: Utilization of two distinct modes of replication by a hybrid plasmid constructed in vitro from separate replicons. Proc. nat. Acad. Sci. (Wash.) 71, 4556–4560 (1974)

    Google Scholar 

  • Thomas, M., Cameron, J.R., Davis, R.W.: Viable molecular hybrids of bacteriophage lambda and eukaryotic DNA. Proc. nat. Acad. Sci. (Wash.) 71, 4579–4583 (1974)

    Google Scholar 

  • Uozumi, T., Hoshino, T., Miwa, K., Horinouchi, S., Beppu, T., Arima K.: Restriction and modification in Bacillus species: genetic transformation of bacteria with DNA from different species, part I. Molec. gen. Genet. 152, 65–69 (1977)

    Google Scholar 

  • Wensink, P.C., Finnegan D.J., Donelson, J.E., Hogness, D.S.: A system for mapping DNA sequences in the chromosomes of Drosophila melanogasten. Cell 3, 315–325 (1974)

    Google Scholar 

  • Wu, M., Holmes, D.S., Davidson, N., Cohn R.H., Kedes, L.H.: The relative positions of sea urchin histone genes on the chimeric plasmid pSp2 and pSp17 as studied by electron microscopy. Cell 9, 163–169 (1976)

    Google Scholar 

  • Yoshimori, R.N.: A genetic and biochemical analysis of the restriction and modification of DNA by resistance transfer factors. Ph. D. Thesis. University of California 1971

  • Zubay, G., Chambers, D., Cheong, L. Cell-free studies on the regulation of the lac operon. In: The lactose operon (Beckwith J.R., Zipser, D., eds.), pp. 375–391. New York: Cold Spring Harbor Laboratory 1970

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Argricultural Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

    Sueharu Horinouchi, Takeshi Uozumi, Takayuki Hoshino, Akio Ozaki, Sadayo Nakajima, Teruhiko Beppu & Kei Arima

Authors
  1. Sueharu Horinouchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Takeshi Uozumi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Takayuki Hoshino
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Akio Ozaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sadayo Nakajima
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Teruhiko Beppu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kei Arima
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by F. Kaudewitz

Rights and permissions

Reprints and permissions

About this article

Cite this article

Horinouchi, S., Uozumi, T., Hoshino, T. et al. Molecular cloning and in vitro transcription of Bacillus subtilis plasmid in Escherichia coli . Molec. Gen. Genet. 157, 175–182 (1977). https://doi.org/10.1007/BF00267395

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00267395

Keywords

Search

Navigation