Figure 2
Kinesin Gene Disruptions. (A). Schematics of constructs, showing details of the homologous regions and relevant enzymes used to target recombination and to confirm disruption. The position of the bsrrcassette used for selection is also shown. ATG indicates the start of the protein-coding region. (B). Southern blot comparisons of wild-type AX-2 control (WT), kif8, kif10, kif11, and kif4 knockout (KO) DNAs. DNA was digested with the indicated enzymes and probed with the entire amplified kinesin gene fragment. Since we were unable to detect kif4 mRNA in wild type cells (see text), we include multiple digests in this panel to demonstrate disruption. All resulting DNA fragments are as predicted from the wild-type and recombination sequences. (C). Northern analysis of AX-2, kif8, kif10 and kif11 knockout cells. Top panel shows mRNA hybridization, bottom panel shows a loading control (4.1 kb 26S rRNA). Note the abundant level of kinesin message in wild-type cells, but the complete absence of message in the disrupted clones.