Figure 1.—

Analysis of the regulation of the cell size by the CLN1 and CLN2 genes. (A) The size of the cells in exponentially growing cultures on YPD medium of isogenic wild type (W303-1A), cln1 (JCY275), cln2 (JCY296), cln3 (MT244), cln1cln2 (JCY306), cln1cln3 (JCY223), and cln2cln3 (JCY317) was determined in a Coulter cell counter. (B) Wild type (W303-1A) transformed with a control vector (YCp50), or plasmids overexpressing either the CLN1 (ptetO:CLN1) or the CLN2 (ptetO:CLN2) genes under the control of the doxycycline-regulated tetO₂ promoter, were grown on synthetic medium, and cell sizes were determined. (C) Size of cln1cln2 cells (JCY306) transformed with the control vector (YCp50), the ptetO:CLN1, or the ptetO:CLN2 plasmid, in exponentially growing cultures in the presence of 0.05 μg/ml doxycycline. (D) Western analysis of Cln1p and Cln2p protein levels in extracts from the cln1cln2 strain (JCY306) bearing a centromeric plasmid containing a HA-tagged version of the CLN1 or CLN2 genes or the ptetO:CLN1 or ptetO:CLN2 centromeric plasmid, which expresses the HA-tagged version of CLN1 or CLN2 under the control of the tetO₂ promoter. The cells transformed with either the ptetO:CLN1 or the ptetOCLN2 plasmid were grown in the absence or in the presence of 0.05 μg/ml doxycycline, as indicated. Control extract from cells transformed with an empty vector was included (lane no tag). A nonspecific band that cross-reacts with the 12CA5 antibody is shown as the loading control. (E) Specific kinase activity in HA-immunoprecipitates from extracts of the cln1cln2 strain (JCY306) containing either the ptetO:CLN1 or the ptetO:CLN2 plasmid and grown in the presence of 0.05 μg/ml doxycycline.