Isolation of Trypanosoma brucei CYC2 andCYC3 Cyclin Genes by Rescue of a Yeast G1Cyclin Mutant

107

183

Cyclin-dependent protein kinases are among the key regulators of eukaryotic cell cycle progression. Potential functions of the five cdc2-related kinases (CRK) in Trypanosoma brucei were analyzed using the RNA interference (RNA i ) technique. In both the procyclic and bloodstream forms of T. brucei, CRK1 is apparently involved in controlling the G 1 /S transition, whereas CRK3 plays an important role in catalyzing cells across the G 2 /M junction. A knockdown of CRK1 caused accumulation of cells in the G 1 phase without apparent phenotypic change, whereas depletion of CRK3 enriched cells of both forms in the G 2 /M phase. However, two distinctive phenotypes were observed between the CRK3-deficient procyclic and bloodstream forms. The procyclic form has a majority of the cells containing a single enlarged nucleus plus one kinetoplast. There is also an enhanced population of anucleated cells, each containing a single kinetoplast known as the zoids (0N1K). The CRK3-depleted bloodstream form has an increased number of one nucleus-two kinetoplast cells (1N2K) and a small population containing aggregated multiple nuclei and multiple kinetoplasts. Apparently, these two forms have different mechanisms in cell cycle regulation. Although the procyclic form can be driven into cytokinesis and cell division by kinetoplast segregation without a completed mitosis, the bloodstream form cannot enter cytokinesis under the same condition. Instead, it keeps going through another G 1 phase and enters a new S phase resulting in an aggregate of multiple nuclei and multiple kinetoplasts in an undivided cell. The different leakiness in cell cycle regulation between two stagespecific forms of an organism provides an interesting and useful model for further understanding the evolution of cell cycle control among the eukaryotes.

Section: Methodsmentioning

confidence: 99%

The Involvement of Two cdc2-related Kinases (CRKs) in Trypanosoma brucei Cell Cycle Regulation and the Distinctive Stage-specific Phenotypes Caused by CRK3 Depletion

Wang

2004

107

183

“…S100 lysates (prepared as described previously (9)) were incubated with 50 l of control, p12 cks1 or p13 suc1 beads for 1 h at 4°C. Beads were washed extensively with lysis buffer and used for histone H1 kinase assays as described previously (9).…”

Section: Methodsmentioning

confidence: 99%

“…CRK3⅐CYC6 is required for progression through the G 2 /M phase boundary into mitosis, but the function of CRK3⅐CYC2 remains undefined. The cyclin box homology region of CYC2 exhibits 38% identity to PREG1 from Neurospora crassa and 25% identity to PHO80 from Saccharomyces cerevisiae at the amino acid level (9). PHO80 is one of 10 cyclins that bind to the CDK PHO85.…”

mentioning

confidence: 99%

“…Based on sequence similarity to other known cyclins, the T. brucei cyclins can be grouped into three classes: the PREG1/ PHO80-like cyclins (CYC2, CYC4, CYC5, and CYC7), the mitotic-like cyclins (CYC3, CYC6, and CYC8), and the transcriptional cyclins (CYC9). It is thought that CRK3 is the functional trypanosome CDK1 (8), and this kinase has been shown to interact with two trypanosome cyclins, CYC2 (9) and CYC6 (7) to form active kinase complexes in vivo. CRK3⅐CYC6 is required for progression through the G 2 /M phase boundary into mitosis, but the function of CRK3⅐CYC2 remains undefined.…”

mentioning

confidence: 99%

See 1 more Smart Citation

The Trypanosoma brucei Cyclin, CYC2, Is Required for Cell Cycle Progression through G1 Phase and for Maintenance of Procyclic Form Cell Morphology

Hammarton

Engstler

Mottram

2004

Self Cite

CYC2 is an essential PHO80-like cyclin that forms a complex with the cdc2-related kinase CRK3 in Trypanosoma brucei. In both procyclic and bloodstream form T. brucei, knock-down of CYC2 by RNA interference (RNAi) led to an accumulation of cells in G 1 phase. Additionally, in procyclic cells, but not in bloodstream form cells, CYC2 RNAi induced a specific cell elongation at the posterior end. The G 1 block, as well as the posterior end elongation in the procyclic form, was irreversible once established. Staining for tyrosinated ␣-tubulin and morphometric analyses showed that the posterior end elongation occurred through active microtubule extension, with no repositioning of the kinetoplast. Hence, these cells can be classified as exhibiting the "nozzle" phenotype as has been described for cells that ectopically express TbZFP2, a zinc finger protein that is involved in the differentiation of the bloodstream form to procyclic form. Within the tsetse fly, procyclic trypanosomes differentiate to elongated mesocyclic cells. However, although mesocyclic trypanosomes isolated from tsetse flies also show active microtubule extension at the posterior end, the kinetoplast is coincidentally repositioned such that it always lies approximately midway between the nucleus and posterior end of the cell. Thus, in the procyclic form CYC2 has dual functionality and is required for both cell cycle progression through G 1 and for the maintenance of correct cell morphology, whereas in the bloodstream form only a role for CYC2 in G 1 progression is evident.

“…However, the molecular identity of such transport proteins has been evasive, precluding molecular studies on this essential cellular function. To identify a bona fide pyruvate transporter, a T. brucei bloodstream form cDNA library, previously used to clone other trypanosomal genes (21,24,25), was transformed into the pyruvate auxotrophic mutant of S. cerevisiae JMY75 (20). The JMY75 yeast mutant, deficient in pyruvate kinase, malic enzyme, and pyruvate transporter, can use pyruvate as an efficient carbon source only if the pyruvate transport deficiency conferred by the jen1⌬ mutation is complemented by expression of a functional pyruvate permease.…”

Section: Cloning Of a Pyruvate Transporter Gene By Geneticmentioning

confidence: 99%

Molecular Identification and Characterization of an Essential Pyruvate Transporter from Trypanosoma brucei

Sánchez

2013

Background: Pyruvate transport is vital for growth and survival of T. brucei bloodstream form. Results: TbPT0 is a unique monocarboxylate transporter with preference for pyruvate and essential for bloodstream form viability. Conclusion: TbPT0 represents the first molecular identification of essential pyruvate transporters in T. brucei. Significance: Unique T. brucei pyruvate transporters provide a potential drug target against the bloodstream form parasites.