Skip to main content
SpringerLink
Log in

Detection and mapping of six miniF-encoded proteins by cloning analysis of dissected miniF segments

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

Summary

Various DNA subfragments were derived from miniF DNA by complete or partial PstI cleavage, and cloned in the plasmid vectors pBR322 or λdv1. The recombinant plasmids obtained were introduced into an Escherichia coli minicell-producing strain, and the plasmid-coded proteins were radiolabeled and analyzed by gel electrophoresis. Six miniF-encoded proteins, larger than 11 000 daltons, were detected and their coding regions were mapped on the F plasmid genome. Three of them were assigned by taking into account the known nucleotide sequences (Murotsu et al. 1981; K. Yoshioka, personal communication). The coding directions of some proteins were determined by inserting the lac promotor into one of the recombinant plasmids and analyzing the increase in production of the proteins. The coding direction of the five proteins analyzed so far was uniform. Comparison of these results with a functional map of miniF suggested possible roles of the proteins.

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

  • Bachman K, Ptashne M, Gilbert W (1976) Construction of plasmids carrying the cI gene of bacteriophage λ. Proc Natl Acad Sci USA 73:4174–4178

    Google Scholar 

  • Bergquist PL, Downard RA, Coughey PA, Gardner RC, Lane HED (1981) Analysis of mini-F plasmid replication by transposition mutagenesis. J Bacteriol 147:888–899

    Google Scholar 

  • Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heynecker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113

    Google Scholar 

  • Bonner WM, Laskey RA (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46:83–88

    Google Scholar 

  • Brown NL, Smith M (1977) The mapping and sequence determination of a single site in ΦX174 amber 3 replicative form DNA cleaved by restriction endonuclease PstI. FEBS Lett 65:284–287

    Google Scholar 

  • Clewell DB (1972) Nature of ColE1 plasmid replication in Escherichia coli in the presence of chloramphenico. J Bacteriol 110:667–676

    Google Scholar 

  • Cohen SN, Chang ACY, Hsu L (1972) Nochromosomal anitbiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA 69:2110–2114

    Google Scholar 

  • Davis BD, Mingioli ES (1950) Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol 60:17–28

    Google Scholar 

  • Dougan E, Sherratt D (1977) The transposon Tn1 as a probe for studying ColEl structure and function. Mol Gen Genet 151:151–160

    Google Scholar 

  • Ebbers J, Eichenlaub R (1981) Complementation of replication-deficient deletion derivatives of plasmid mini-F. J Bacteriol 147:736–743

    Google Scholar 

  • Eichenlaub R, Figurski D, Helinski DR (1977) Bidirectional replication from a unique origin in a mini-F plasmid. Proc Natl Acad Sci USA 74:1138–1141

    Google Scholar 

  • Eichenlaub R, Figurski D, Helinski DR (1977) Bidirectional replication from a unique origin in a mini-F plasmid. Proc Natl Acad Sci USA 74:1138–1141

    Google Scholar 

  • Eichenlaub R, Wehlman H (1980) Amber-mutants of plasmid mini-F defective in replication. Mol Gen Genet 180:201–204

    Google Scholar 

  • Greene PJ, Betlach MC, Goodman HM, Boyer HW (1974) The EcoRI restriction endonuclease. In: Wickner RB (ed) DNA replication. Marcel Dekker, Inc, New York, pp 87–103

    Google Scholar 

  • Guyer MS, Figurski D, Davidson N (1976) Electron microscope study of a plasmid chimera containing the replication region of the Escherichia coli F plasmid. J Bacteriol 127:988–997

    Google Scholar 

  • Hashimoto T, Matsubara K (1974) Transformation of Ca++−treated recA derivative of Escherichia coli K12 by λdv DNA. Jap J Genet 49:97–101

    Google Scholar 

  • Hayakawa Y, Matsubara K (1979) Construction and some properties of packageable plasmid F. Mol Gen Genet 169:107–112

    Google Scholar 

  • Hori H, Takata R, Muto A, Osawa S (1974) Ribosomal RNA synthesis in the F′14 episome-containing minicells of Escherichia coli. Mol Gen Genet 128:341–347

    Google Scholar 

  • Humphreys GO, Willshaw GA, Anderson ES (1975) A simple method for the preparation of large quantities of pure plasmid DNA. Biochim Biophys Acta 383:457–463

    Google Scholar 

  • Johnsrud L (1978) Contacts between Escherichia coli RNA polymerase and a lac operon promoter. Proc Natl Acad Sci USA 75:5314–5318

    Google Scholar 

  • Kingsbury DT, Helinski DR (1973) Temperature-sensitive mutants for the replication of plasmids in Escherichia coli: requirement for deoxyribonucleic acid polymerase I in the replication of the plasmid ColE1. J Bacteriol 114:1116–1124

    Google Scholar 

  • Klein RD, Selsing E, Wells RD (1980) A rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis. Plasmid 3:88–91

    Google Scholar 

  • Kline BC (1974) Mechanism and biosynthetic requirements for F plasmid replication in Escherichi coli. Biochemistry 13:139–146

    Google Scholar 

  • Kline BC, Lane D (1980) A proposed system for nomenclature for incompatibility genes of the Escherichia coli sex factor, plasmid F. Plasmid 4:231–232

    Google Scholar 

  • Kopylova-Sviridova TN, Soukovatitsin VV, Fodor I (1979) Synthesis of proteins coded by plasmid vectors of pCV series (Apr, Tcr) and their recombinant derivatives (pDm) in E. coli minicells. Gene 7:121–139

    Google Scholar 

  • Kupersztoch-Portnoy YM, Lovett MA, Helinski DR (1974) Strand and site specificity of the relaxation event for the relaxation complex of the antibiotic resistance plasmid R6K. Biochemistry 13:5484–5490

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Google Scholar 

  • Lane HED (1981) Replication and incompatibility of F and plasmids in the IncFI group. Plasmid 5:100–126

    Google Scholar 

  • Laskey RA, Mills AD (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem 56:335–341

    Google Scholar 

  • Levy SB (1971) Physical and functional characteristics of R-factor deoxyribonucleic acid segregated into Escherichia coli minicells. J Bacteriol 108:300–308

    Google Scholar 

  • Lovett MA, Helinski DR (1976) Method for the isolation of the replication region of a bacterial replicon: construction of a mini-F′km plasmid. J Bacteriol 127:982–987

    Google Scholar 

  • Lugtenberg B, Meijers J, Peters R, van der Hoek P (1975) Electrophoretic resolution of the ‘major outer membrane protein’ of Escherichia coli K12 into four bands. FEBS Lett 58:254–258

    Google Scholar 

  • Matsubara K (1976) Genetic structure and regulation of plasmid λdv. J Mol Biol 102:427–439

    Google Scholar 

  • Matsubara K, Kaiser AD (1968) λdv: an autonomously replicating DNA fragment. Cold Spring Harbor Symp Quant Biol 33:769–775

    Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, New York

    Google Scholar 

  • Murotsu T, Matsubara K, Sugisaki H, Takanami M (1981) Nine unique repeating sequences in a region essential for replication and incompatibility of the mini-F plasmid. Gene 15:257–271

    Google Scholar 

  • Murotsu T, Tanaka H, Imaji M, Koga H, Matsubara K, Horiuchi T (1977) Purification and properties of a λ operator-binding protein which is expected to be autorepressor (tof protein) from E. coli carrying λdv plasmid. Mol Gene Genet 157:139–147

    Google Scholar 

  • Ogura T, Miki T, Hiraga S (1980) Copy-number mutants of the plasmid carrying the replication origin of the Escherichia coli chromosome: evidence for a control region of replication. Proc Natl Acad Sci USA 77:3993–3997

    Google Scholar 

  • Radloff R, Bauer W, Vinograd J (1967) A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc Natl Acad Sci USA 57:1514–1521

    Google Scholar 

  • Roozen KJ, Fenwick RG, Curtiss III R (1971) Synthesis of ribonucleic acid and protein in plasmid-containing minicells of Escherichia coli K-12. J Bacteriol 107:21–33

    Google Scholar 

  • Seelke RW, Kline BC, Trawick JD, Ritts GD (1982) Genetic studies of F plasmid maintenance genes involved in copy number control, incompatibility and partitioning. Plasmid 7:163–179

    Google Scholar 

  • Sharp PA, Hsu MT, Ohtsubo E, Davidson N (1972) Electron microscopic heteroduplex studies of sequence relations among plasmids of Escherichia coli. J Mol Biol 71:471–497

    Google Scholar 

  • Skurray RA, Guyer MS, Timmis K, Cabello F, Cohen SN, Davidson N, Clark AJ (1976) Replication region fragments cloned from Flac + are identical to EcoRI fragment f5 of F. J Bacteriol 127:1571–1575

    Google Scholar 

  • Sutcliffe JG (1979) Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp Quant Biol 43:77–90

    Google Scholar 

  • Timmis K, Cabello F, Cohen SN (1975) Cloning, isolation, and characterization of replication regions of complex plasmid genomes. Proc Natl Acad Sci USA 72:2242–2246

    Google Scholar 

  • Tolun A, Helinski DR (1981) Direct repeats of the F plasmid incC region express F incompatibility. Cell 24:687–694

    Google Scholar 

  • Wehlmann H, Eichenlaub R (1980) Plasmid mini-F encoded proteins. Mol Gen Genet 180:205–211

    Google Scholar 

  • Wehlmann H, Eichenlaub R (1981) Analysis of transcripts from plasmid mini-F by electron microscopy of R loops. Plasmid 5:259–266

    Google Scholar 

Download references

Author information

Author notes

  1. Tsutomu Nishizawa

    Present address: Central Research Laboratory, Wakunaga Pharmaceutical Company, Ltd., 729-64, Hiroshima, Japan

  2. Yasuhiko Hayakawa

    Present address: Department of Virology, Research Institute for Microbial Diseases, Osaka University, 565, Suita, Japan

Authors and Affiliations

  1. Laboratory of Molecular Genetics, Osaka University Medical School, Kita-ku, 530, Osaka, Japan

    Nobuhisa Komai, Tsutomu Nishizawa, Yasuhiko Hayakawa, Tomoaki Murotsu & Kenichi Matsubara

Authors
  1. Nobuhisa Komai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tsutomu Nishizawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yasuhiko Hayakawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tomoaki Murotsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kenichi Matsubara
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by T. Yura

Rights and permissions

Reprints and permissions

About this article

Cite this article

Komai, N., Nishizawa, T., Hayakawa, Y. et al. Detection and mapping of six miniF-encoded proteins by cloning analysis of dissected miniF segments. Mol Gen Genet 186, 193–203 (1982). https://doi.org/10.1007/BF00331850

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00331850

Keywords

Search

Navigation