Article Title: A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division
Figure Lengend Snippet: Expression profiles and interactions between D cyclins, CDKG1 and MAT3/RBR during the cell cycle. ( A ) Ethidium bromide stained agarose gels showing amplification of cDNAs made with RNA samples taken from synchronized cultures in a 14 hr light/10 hr dark cycle at four stages: daughter cells (1 hr light), post-commitment cells (10 hr light), S/M phase cells (1 hr dark) and post-mitotic cells (4 hr dark). Primers used are listed in Supplementary file 2 and the number of PCR amplification cycles used for each reaction is displayed on the right. GBLP is an internal control. ( B ) Profiles of CDKG1, CDKB1 and GBLP mRNAs as described in ( A ), except the pre-commitment sample was from 4 hr light. ( C ) Interaction of CYCD3 AxAxA mutant with CDKG1 and MAT3. Mutated CYCD3 (LxCxE→AxAxA) was fused to the Gal4 activation domain (AD) and tested in an Y2H assay with MAT3/RBR and CDKG1. Empty indicates vector-only with no fusion protein. Growth of two independent co-transformants is shown, and the relative strength of interaction is indicated by -, no interaction, +, weak interaction, and +++, very strong interaction. ( D ) Bar graph shows relative kinase activity of IVT CDKG1, CDKG1 kd and CDKG1+CYCD3 using GST-MAT3 as a substrate. The amount of 32 P-labeled GST-MAT3 from each reaction was normalized to the value from lane 2. Data are expressed as the mean of three independent experiments. Error bar: S.D. ( E ) Immunoprecipitation (IP) from whole cell extracts, fractionation by SDS-PAGE and detection of CDKG1 by Western blotting using polyclonal antisera raised against full length CDGK1. All strains used were synchronized in S/M phase. Lanes 1,2 are concentrated input fractions prior to IP from indicated strains. Anti-HA (lanes 3,4) or anti-CDKG1 (lanes 5–8) antibodies were used for IPs as indicated and the supernatant (lanes 3,5,7) or pellet (lanes 4,6,8) fractions probed using anti-CDKG1. The anti-CDKG1 antibody detects a single band near the predicted molecular weight of 44 kDa that is not present in cdkg1-2 mutants. ( F ) Schematic of the regulatory hierarchy for the Chlamydomonas mitotic sizer pathway (left side) and the metazoan G1 control pathway (right side). Both pathways integrate internal and/or external signals for cell cycle progression through D-cyclin dependent CDKs that phosphorylate RB-related proteins. DOI: http://dx.doi.org/10.7554/eLife.10767.009
Article Snippet: CDKG1 antibody generation pET28a-CDKG1 or pET28a-CDKG1∆N (missing residues 1–92) were made by PCR amplification from pGEM-T-CDKG1 using primer sets described in and ligation into pET28a (EMD Millipore, Billerica, MA) at NdeI/EcoRI sites. pGST-MAT3 was generated as described previously ( ).
Techniques: Expressing, Staining, Amplification, Polymerase Chain Reaction, Mutagenesis, Activation Assay, Y2H Assay, Plasmid Preparation, Activity Assay, Labeling, Immunoprecipitation, Fractionation, SDS Page, Western Blot, Molecular Weight