Synchronisation of Giardia lamblia: identification of cell cycle stage-specific genes and a differentiation restriction point.
The intestinal parasite Giardia lamblia undergoes cell differentiations that entail entry into and departure from the replicative cell cycle. The pathophysiology of giardiasis depends directly upon the ability of the trophozoite form to replicate in the host upper small intestine. Thus, cell proliferation is tightly linked to disease. However, studies of cell cycle regulation in Giardia have been hampered by the inability to synchronise cultures. Here we report that Giardia isolates of the major human genotypes A and B can be synchronised using aphidicolin, a mycotoxin that reversibly inhibits replicative DNA polymerases in eukaryotic cells. Aphidicolin arrests Giardia trophozoites in the early DNA synthesis (S) phase of the cell cycle. We identified a set of cell cycle orthologues in the Giardia genome using bioinformatic analyses and showed that synchronised parasites express these genes in a cell cycle stage-specific manner. The synchronisation method also showed that during encystation, exit from the ordinary cell cycle occurs preferentially in G(2) and defines a restriction point for differentiation. Synchronisation opens up possibilities for further molecular and cell biological studies of chromosome replication, mitosis and segregation of the complex cytoskeleton in Giardia.
Reiner
David S
DS
Department of Pathology, University of California at San Diego, San Diego, CA 92103-8416, USA.
Ankarklev
Johan
J
Troell
Karin
K
Palm
Daniel
D
Bernander
Rolf
R
Gillin
Frances D
FD
Andersson
Jan O
JO
Svärd
Staffan G
SG
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects*; D002454:Cell Differentiation; D005434:Flow Cytometry; D015870:Gene Expression / Q000502:physiology; D018816:Genes, cdc / Q000502:physiology*; D005838:Genotype; D016829:Giardia lamblia / Q000254:growth & development*; D006801:Humans; D007421:Intestine, Small / Q000469:parasitology*
D016590:Aphidicolin