Molecular Cloning of MAPK Gene Family
Using
Synthetic Oligonucleotide Probe*
ZHOU Song, WANG Qin, CHEN Jing and CHEN Jiang-Ye
( State Key Laboratory of Molecular Biology, Shanghai Institute of
Biochemistry,the Chinese Academy of Sciences, Shanghai 200031, China
)
Abstract MAPK(mitogen activated protein
kinase) is a kind of Ser/Thr protein kinase. The MAPKs play an important role
in several different signal transduction pathways. The MAPKs may also have a
role in morphorgenesis of Candida albicans. An oligonucleotide probe
was used to screen novel MAPKs in C. albicans. All MAPKs shared high
homogeneity in their eleven kinase subdomains, especially subdoman VII and
VIII. In subdomain VII, nearly all MAPKs have the same KIDFGLAR sequence, and
the two known MAPKs in C. albicans： CEK1 and MKC1 have only one different nucleotide in that DNA
sequence. This probe was hybridized with C. albicans genomic DNA.
Under stringent conditions, the probe could only hybridize with CEK1
and MKC1 gene fragment. But when hybridized at 40 ℃ in non-SDS
solution, two novel bands appeared. This condition was used to screen SC5314
DNA library, and many positive clones with different hybridization density were
obtained. The strongest hybridization clones were identified to contain CEK1
and MKC1 gene. From the stronger positive hybridization clones, two
novel genes were identified. The first gene, named CRK1(CDC2-related
protein kinase 1), shared high homogeneity to MAPKs, but was not of them. It is
closest to SGV1 from S. cerevisiae (with homology 47%) and
PITALRE from human (with homology 41%), both of which are CDC2-related protein
kinases. The second gene called CEK2(Candida albicans extracelluar
signal-regulated kinase 2) is a novel MAPK of Candida albicans, which
shares the highest identity with CEK1 and its S. cerevisiae
homologs, FUS3 and KSS1, two redundant MAPKs in yeast
pheromone response and morphogenesis.
Key Words oligo nucleotides； MAP kinase gene； Candida albicans； morphogenesis
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