Isolation of Trypanosoma brucei CYC2 and CYC3 cyclin genes by rescue of a yeast G(1) cyclin mutant. Functional characterization of CYC2.
Two Trypanosoma brucei cyclin genes, CYC2 and CYC3, have been isolated by rescue of the Saccharomyces cerevisiae mutant DL1, which is deficient in CLN G(1) cyclin function. CYC2 encodes a 24-kDa protein that has sequence identity to the Neurospora crassa PREG1 and the S. cerevisiae PHO80 cyclin. CYC3 has the most sequence identity to mitotic B-type cyclins from a variety of organisms. Both CYC2 and CYC3 are single-copy genes and expressed in all life cycle stages of the parasite. To determine if CYC2 is found in a complex with previously identified trypanosome cdc2-related kinases (CRKs), the CYC2 gene was fused to the TY epitope tag, integrated into the trypanosome genome, and expressed under inducible control. CYC2ty was found to associate with an active trypanosome CRK complex since CYC2ty bound to leishmanial p12(cks1), and histone H1 kinase activity was detected in CYC2ty immune-precipitated fractions. Gene knockout experiments provide evidence that CYC2 is an essential gene, and co-immune precipitations together with a two-hybrid interaction assay demonstrated that CYC2 interacts with CRK3. The CRK3 x CYC2ty complex, the first cyclin-dependent kinase complex identified in trypanosomes, was localized by immune fluorescence to the cytoplasm throughout the cell cycle.
Van Hellemond
J J
JJ
Wellcome Centre for Molecular Parasitology, University of Glasgow, Anderson College, Glasgow G11 6NU, Scotland, United Kingdom.
Neuville
P
P
Schwarz
R T
RT
Matthews
K R
KR
Mottram
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D002451:Cell Compartmentation; D018797:Cell Cycle Proteins; D056746:Cyclin G; D018844:Cyclin-Dependent Kinases / Q000302:isolation & purification / Q000378:metabolism; D016213:Cyclins / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000235:genetics*; D020798:Two-Hybrid System Techniques
C099912:CKS1 protein, Leishmania mexicana; D018797:Cell Cycle Proteins; C406804:Cyc3 protein, Trypanosoma brucei; D056746:Cyclin G; D016213:Cyclins; D005656:Fungal Proteins; D015800:Protozoan Proteins; D029702:Schizosaccharomyces pombe Proteins; C051655:Suc1 protein, S pombe; C402347:cyc2 protein, Paramecium tetraurelia; D018844:Cyclin-Dependent Kinases