TY - JOUR
T1 - Fate of transforming DNA following uptake by competent Bacillus subtilis. III. Formation and properties of products isolated from transformed cells which are derived entirely from donor DNA
AU - Dubnau, David
AU - Cirigliano, Carol
N1 - Funding Information:
We acknowledge valuable discussions with Drs Issar Smith, LeonardMindich and Barbara Scher, and with Mrs Rosa Davidoff-Abelson, and the expert secretarial assistance of Annabel Howard. This work was supported by National Science Foundation grant No. BG-18146 awarded to one of us (D.D.).
PY - 1972/2/28
Y1 - 1972/2/28
N2 - High molecular weight DNA labeled with [3H]thymine and in some cases deuterium oxide as well, was used to transform competent Bacillus subtilis cultures. Lysates were prepared from these transformed cultures and the properties of products derived entirely from the donor DNA were investigated. Three kinds of radioactive products were identified: 5′-TMP and metabolic derivatives, SSF‡ ‡ Abbreviations used: SSF, DNA species consisting of single-stranded fragments; DSF, DNA species consisting of double-stranded fragments. and DSF. SSF consisted of single-stranded fragments with an approximate molecular weight of 1 to 2 × 105 daltons. DSF consisted of duplex fragments with an initial molecular weight of 9 × 106 daltons. The 5′-TMP was released quantitatively into the medium and could be detected starting about one minute after the addition of transforming DNA to the cells. It is tentatively concluded that the release of 5′-TMP is due to an exonuclease which functions outside the cell permeability barrier. Our evidence suggests that DSF is produced by endonucleolytic cleavage across both strands of the donor molecule. The endonuclease also presumably functions outside the permeability barrier since DSF is susceptible to extracellular nuclease. SSF does not appear to be an artifactual degradation product of higher molecular weight single-stranded DNA which has been generated during isolation. SSF has no transforming activity for a donor marker, and the activity of DSF is anomalously low compared to DNA with the same single strand molecular weight generated by the action of pancreatic deoxyribonuclease. Conversion of donor DNA to acid-soluble products and to SSF and DSF therefore explains the eclipse phenomenon in B. subtilis.
AB - High molecular weight DNA labeled with [3H]thymine and in some cases deuterium oxide as well, was used to transform competent Bacillus subtilis cultures. Lysates were prepared from these transformed cultures and the properties of products derived entirely from the donor DNA were investigated. Three kinds of radioactive products were identified: 5′-TMP and metabolic derivatives, SSF‡ ‡ Abbreviations used: SSF, DNA species consisting of single-stranded fragments; DSF, DNA species consisting of double-stranded fragments. and DSF. SSF consisted of single-stranded fragments with an approximate molecular weight of 1 to 2 × 105 daltons. DSF consisted of duplex fragments with an initial molecular weight of 9 × 106 daltons. The 5′-TMP was released quantitatively into the medium and could be detected starting about one minute after the addition of transforming DNA to the cells. It is tentatively concluded that the release of 5′-TMP is due to an exonuclease which functions outside the cell permeability barrier. Our evidence suggests that DSF is produced by endonucleolytic cleavage across both strands of the donor molecule. The endonuclease also presumably functions outside the permeability barrier since DSF is susceptible to extracellular nuclease. SSF does not appear to be an artifactual degradation product of higher molecular weight single-stranded DNA which has been generated during isolation. SSF has no transforming activity for a donor marker, and the activity of DSF is anomalously low compared to DNA with the same single strand molecular weight generated by the action of pancreatic deoxyribonuclease. Conversion of donor DNA to acid-soluble products and to SSF and DSF therefore explains the eclipse phenomenon in B. subtilis.
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U2 - 10.1016/0022-2836(72)90318-X
DO - 10.1016/0022-2836(72)90318-X
M3 - Article
C2 - 4622632
AN - SCOPUS:0015526574
SN - 0022-2836
VL - 64
SP - 9
EP - 29
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 1
ER -