Fig. 3.

Gcf1 is a DNA-binding protein. GCF1 was expressed in E. coli as native or GST-fusion protein. (a) In contrast to uninduced cultures, crude protein extracts obtained from IPTG-induced cells contain a polypeptide (arrow) with a molecular mass corresponding to that of the GST–Gcf1 fusion protein. (b) Native (thrombin-cleaved) Gcf1p and GST–Gcf1p after purification on glutathione-agarose. (c) Gcf1 (40, 80, 120, 160, 200, 240, 280, 320 and 360 ng; lanes 2–10) protein was added to 300 ng NdeI-digested mtDNA of C. albicans. Lane 1, NdeI-digested mtDNA of C. albicans without addition of Gcf1p. Purified GST was used as a negative control (data not shown). (d) GST–Gcf1p (50 ng) was incubated with 40 ng terminally labelled fragments of mtDNA of C. albicans obtained by restriction with a combination of BamHI and PvuII in the absence or presence of nonlabelled mtDNA, as indicated. The numbers above the lanes indicate the molar excess of the competitor DNA. B, BamHI; P, PvuII.