Molecular Cloning of MAPK
Gene Family Using Degenerate PCR
CHEN Jing, NI Jian, CHEN Jiang-Ye*
( State Key Laboratory of Molecular Biology, Shanghai Institute of
Biochemistry, the Chinese Academy of Science, Shanghai 200031, China
)
Abstract MAPK(Mitogen-activated
protein kinase) pathways play an important role in morphorgenesis of Candida albicans. According to
the conserved amino acid sequence of the known MAPKs, two degenerate primers
attaching to subdomain VIB and IX were designed to screen novel MAPKs in C. albicans. The PCR was
performed under non-strigent conditons. 100 PCR fragments were sequenced and
among 25 novel gene fragments, two novel MAPK gene fragments were obtained.
Using these two PCR fragments as probes to screen a Candida albicans genomic DNA
library, two novel MAPK genes
designated CSK1(Candida albicans
sporulation-related MAP kinase1) and CEK2(Candida albicans extracellular
signal-regulated kinase 2) were cloned. These two genes share high similarity
with three cloned MAPK genes： CEK1 and MKC1 and CaHOG1. The CSK1 gene is 1
193 bp in length, containing a 92 bp intron, coding for a 367 aa protein. The CSK1 shares
highest similarity with SMK1, with homology 55.3% in nucleotide
sequence and 50% in amino acid sequence. SMK1 encodes a MAPK involved in
the sporulation pathway in S. cerevisiae. In vitro kinase activity assay showed
that the Csk1 kinase exhibited a phosphorylation ability when using MBP as a
substrate but not histone H1.
Key words C.albicans； degenerate PCR； MAPK gene； CSK1
Received:
October 26, 1999 Accepted: December 27, 1999
This work was supported by National Natural Science Foundation
of China(No. 39625009, No. 39970016) and Special Foundation of the chinese
Academy of Sciences(No. STZ-02-03)
* Corresponding author: Tel, 86-21-64374430-251; Fax,
86-21-64338357; e-mail, [email protected]