Summary
The effects of restriction in vivo by competent B. subtilis R cells and in vitro by purified endonuclease BsuR on transformation and transfection with native and denatured DNA were investigated.
The results show that transformation by either native, or denatured DNA is not affected by restriction, whereas transfection both with native and denatured SPP1 DNA is severely restricted.
In contrast to the results obtained in vivo, the biological activity of native and denatured transforming DNA is destroyed by BsuR in vitro, as is the transfecting activity of native and denatured SPP1 DNA. The sensitivity of denatured DNA, either with mixtures of the complementary strands or with separated single strands1 alone, is significantly lower than that of native DNA.
The results are discussed in the context of possible mechanisms underlying the different responses of transforming and transfecting DNA to in vivo restriction by B. subtilis R cells.
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Abbreviations
- EGTA:
-
ethyleneglycolbis-(aminoethylether)tetraacetic acid
- m+ :
-
modified
- m- :
-
non-modified
- moi:
-
multiplicity of infection
- r+ m+ :
-
restricting and modifying
- r- m- :
-
mon-restricting and non-modifying
- SSC:
-
0.15M NaCl+0.015 M trisodium citrate
- SDS:
-
sodium dodecyl sulphate
References
Behrens, B., Lüder, G., Behncke, M., Trautner, T.A.: The genome of B. subtilis phage SPP1. Physical arrangement of phage genes. Mol. Gen. Genet. 175, 351–357 (1979)
Blakesley, R.W., Wells, R.D.: “Single-stranded” DNA from ϕX174 and M13 is cleaved by certain restriction endonucleases. Nature 257, 421–422 (1975)
Boyer, H.W.: DNA restriction and modification mechanisms in bacteria. Annu. Rev. Microbiol. 25, 153–176 (1971)
Bron, S., Hörz, W.: Purification and properties of the Bsu endonuclease. In: Methods in enzymology, nucleic acids and protein synthesis. (L. Grossman, ed.), Vol. 65, pp. 112–132. New York: Academic Press 1980
Bron, S., Luxen, E., Trautner, T.A.: Restriction and modification in B. subtilis. The role of homology between donor and recipient DNA in transformations and transfection. Mol. Gen. Genet. 179, 111–117 (1980)
Bron, S., Murray, K., Trautner, T.A.: Restriction and modification in B. subtilis. Purification and general properties of a restriction endonuclease from strain R. Mol. Gen. Genet. 143, 13–23 (1975)
Bron, S., Venema, G.: Ultraviolet inactivation and excision-repair in Bacillus subtilis. I. Construction and characterization of a transformable eightfold auxotrophic strain and two ultravioletsensitive derivates. Mutat. Res. 15, 1–10 (1972)
Burgess, R.R., Travers, A.A.: DNA-dependant polymerase. In: Prog. nucl. acid res, (Catoni, G.L. and Davies, D.R., eds.) Vol. 2, pp. 851–863. New York: Harper and Row 1972
Canosi, U., Trautner, T.A., Bron, S.: Restriction and modification in B. subtilis. Transformation with plasmid DNA. Mol. Gen. Genet. (1980) (submitted)
Davidoff-Abelson, R., Dubnau, D.: Conditions affecting the isolation from transformed cells of Bacillus subtilis of high-molecular-weight single-stranded DNA of donor origin. J. Bacteriol. 116, 146–153 (1973)
Dishman, B.R.: The role of molecular configuration in transfection of Bacillus subtilis with bacteriophage DNA. Ph. D. Thesis, Houston, 1972
Greene, P.J., Poonian, M.S., Nussbaum, A.L., Tobias, L., Garfin, D.E., Boyer, H.W., Goodman, H.M.: Restriction and modification of a self-complementary octanucleotide containing the Eco RI substrate. J. Mol. Biol. 99, 237–261 (1975)
Gromkova, R., Goodgal, S.H.: On the role of restriction enzymes of Haemophilus in transformation and transfection. In: Mechanisms in recombination (R.F. Grell, ed.), pp. 209–215. New York: Plenum Press 1974
Heininger, K., Hörz, W., Zachau, H.G.: Specificity of cleavage by a restriction nuclease from Bacillus subtilis. Gene 1, 291–303 (1977)
Horiuchi, K., Zinder, N.D.: Site-specific cleavage of single-stranded DNA by a Haemophilus restriction endonuclease. Proc. Natl. Acad. Sci. USA 72, 2555–2558 (1975)
Kahmann, R.: Das Verhalten von SPP1 DNA nach Transfektion von B. subtilis. Ph. D. Thesis, Freien Universität, Berlin, 1974
Loveday, K.: Fate of ϕe transfecting DNA in Bacillus subtilis. Ph.D. Thesis, Massachusetts Inst. Technology, Cambridge, Massachusetts, 1976
Loveday, K., Fox, M.S.: The fate of bacteriophage ϕe transfecting DNA. Virology 85, 387–403 (1978)
Piechowska, M., Fox, M.S.: Fate of transforming deoxyribonucleate in Bacillus subtilis. J. Bacteriol. 108, 680–689 (1971)
Riva, S., Polsinelli, M., Falaschi, A.: A new phage of B. subtilis with infectious DNA having separable strands. J. Mol. Biol. 35, 347–356 (1968)
Soltyk, A., Shugar, D., Piechowska, M.: Heterologous DNA uptake and complexing with cellular constituents in competent Bacillus subtilis. J. Bacteriol. 124, 1429–1438 (1975)
Spatz, H.Ch., Trautner, T.A.: One way to do experiments on gene conversion? Mol. Gen. Genet. 109, 84–106 (1970)
Studier, F.W.: Conformational changes of single-stranded DNA. J. Mol. Biol. 41, 189–197 (1969)
Stuy, J.H.: Restriction enzymes do not play a significant role in Haemophilus homospecific or heterospecific transformation. J. Bacteriol. 128, 212–220 (1976)
Trautner, T.A., Pawlek, B., Bron, S., Anagnostopoulos, C.: Restriction and modification in B. subtilis. Biological aspects. Mol. Gen. Genet. 131, 181–191 (1974)
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Communicated by J. Schell
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Bron, S., Luxen, E., Venema, G. et al. Restriction and modification in B. subtilis . Molec. Gen. Genet. 179, 103–110 (1980). https://doi.org/10.1007/BF00268451
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DOI: https://doi.org/10.1007/BF00268451