Molecular cloning and sequence analysis of mutant alleles of the fission yeast cdc2 protein kinase gene: implications for cdc2+ protein structure and function.
The cdc2+ gene function plays a central role in the control of the mitotic cell cycle of the fission yeast Schizosaccharomyces pombe. Recessive temperature-sensitive mutations in the cdc2 gene cause cell cycle arrest when shifted to the restrictive temperature, while a second class of mutations within the cdc2 gene causes a premature advancement into mitosis. Previously the cdc2+ gene has been cloned and has been shown to encode a 34 kDa phosphoprotein with in vitro protein kinase activity. Here we describe the cloning of 11 mutant alleles of the cdc2 gene using two simple methods, one of which is presented here for the first time. We have sequenced these alleles and find a variety of single amino acid substitutions mapping throughout the cdc2 protein. Analysis of these mutations has identified a number of regions within the cdc2 protein that are important for cdc2+ activity and regulation. These include regions which may be involved in the interaction of the cdc2+ gene product with the proteins encoded by the wee1+, cdc13+ and suc1+ genes.
Carr
A M
AM
Department of Biochemistry, University of Oxford, UK.
MacNeill
S A
SA
Hayles
J
J
Nurse
P
P
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D016203:CDC2 Protein Kinase; D002455:Cell Division; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D005800:Genes, Fungal; D006801:Humans; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics*; D010957:Plasmids; D011487:Protein Conformation; D011494:Protein Kinases / Q000235:genetics*; D015183:Restriction Mapping; D004718:Saccharomycetales / Q000235:genetics*; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature; D014170:Transformation, Genetic
D010750:Phosphoproteins; D011494:Protein Kinases; D016203:CDC2 Protein Kinase