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J Biol Chem, Vol. 275, Issue 12, 8315-8323, March 24, 2000

Isolation of Trypanosoma brucei CYC2 and CYC3 Cyclin Genes by Rescue of a Yeast G₁ Cyclin Mutant
FUNCTIONAL CHARACTERIZATION OF CYC2^*

Jaap J. Van Hellemond^§, Philippe Neuville^§, Ralph T. Schwarz^¶, Keith R. Matthews, and Jeremy C. Mottram^**

From the  Wellcome Centre for Molecular Parasitology, University of Glasgow, Anderson College, Glasgow G11 6NU, Scotland, United Kingdom, ^¶ Zentrum fur Hygiene und Medizinische Mikrobiologie, Philipps-Universitat, 35037 Marburg, Germany, and  School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom

Two Trypanosoma brucei cyclin genes, CYC2 and CYC3, have been isolated by rescue of the Saccharomyces cerevisiae mutant DL1, which is deficient in CLN G₁ 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·CYC2ty complex, the first cyclin-dependent kinase complex identified in trypanosomes, was localized by immune fluorescence to the cytoplasm throughout the cell cycle.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AJ242519 and AJ242520, respectively.

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