Isolation and expression of a gene specifying a cdc2-like protein kinase from the human malaria parasite Plasmodium falciparum.
A partially redundant oligonucleotide based on conserved protein sequences of cdk and cdc2-like proteins was used to isolate from genomic libraries of Plasmodium falciparum fragments of chromosome XIII carrying a 288-residue open-reading frame encoding a protein kinase sharing 57-58% identity with yeast p34cdc2. Based on sequence data, base composition and the striking similarity with other cdk and related proteins, four intervening sequences were identified. Their removal in vitro allowed expression of the gene, designated PfPK5, in Escherichia coli, the resulting product having kinase activity against casein and histone H1. Western blotting using a polyclonal antibody raised against the expressed protein showed that the kinase was located in the parasite's cytosol and was present in approximately constant amounts throughout the intra-erythrocytic asexual reproductive stage of the life cycle. The PSTAIRE region of the PfPK5 protein differs at three sites from that of p34cdc2, and the gene failed to complement cdc2/28 yeast mutants. However, Western blotting showed that the gene was not expressed in yeast, so this does not eliminate the possibility that it is the malarial version of cdc2.
Ross-Macdonald
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PB
National Institute for Medical Research, Mill Hill, London, England.
Graeser
R
R
Kappes
B
B
Franklin
R
R
Williamson
D H
DH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000235:genetics* / Q000378:metabolism; D002453:Cell Cycle; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D007438:Introns; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000737:chemistry; D010963:Plasmodium falciparum / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D016203:CDC2 Protein Kinase