Construction of Candida albicans Two-hybrid Library and Screening for Proteins Interacting with Crk1.
In order to study interactions among proteins involved in Candida albicans morphorgenesis, C.albicans genomic DNA was digested with Sau3AI and fused to activating domain(AD) of LEXA to construct an AD-fused C.albicans genomic DNA expression library. The library contained 7.7x10(4) independent clones and included 1.2x10(5) kb DNA which was 10 fold of the C.albicans whole genomic DNA. The CRK1 gene encodes a CDC2-related protein, that was involved in morphorgenesis of C.albicans. The Crk1 fused to DNA binding domain of LexA was used as the bait to screen the LexA library. Among 9x10(5) transformants, eight Leu and LacZ double positive clones were selected. With restriction enzyme analysis, three kinds of fragments were identified. The clones NJ1, NJ2, NJ3 were sequenced and analyzed with BLAST program. It was found that NJ1, NJ2, NJ3 were homologs of S.cerevisiae gene STI1, RET2 and NFI1, respectively. The kinase domain of Crk1(Crk1N)interacted weakly with protein encoded by NJ1, while the noncatalytic domain of Crk1(Crk1C) interacted strongly with proteins encoded by NJ2 and NJ3. The interaction of NJ3 and Crk1C was verified by the coimmunoprecipition of NJ3-HA with Crk1C-LexA. These proteins may be involved in maturation, transport, localization, and activity regulation of the Crk1.
Ni
Jian
J
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China. jychen@sunm.shcnc.ac.cn
Chen
Xi
X
Yang
Tao
T
Chen
Jiang-Ye
JY