DEAD-box protein HEL64 from Trypanosoma brucei: subcellular localization and gene knockout analysis.
RNA helicases are molecules that play a central role in the control of ribonucleic acid metabolism. Only two putative RNA helicase genes of the DEAD-box family have been identified in the protozoan parasite Trypanosoma brucei brucei. One of these genes is HEL64, a single-copy gene of unknown function. In this study we conducted targeted gene-disruption experiments of the HEL64 locus with the aim of identifying a phenotype that would suggest a function for the encoded protein. It is likely that HEL64 is an essential gene in insect-stage trypanosomes, since all attempts to create a HEL64 double-allele knockout cell line failed. Instead, we obtained a mutant derived from an unusual recombination event that nonetheless contained a functional copy of the gene. One allele of HEL64 is sufficient for the survival of the parasite. Single-allele knockout mutants showed no gross change in cell morphology and multiplied with a cell-doubling time identical to that of wild-type trypanosomes. Though HEL64 has high sequence homology with the nuclear DEAD-box protein p68, it is localized in the cytosol of trypanosomes and, thus, cannot be a homologue of p68 as previously suggested. Since the protein did not cross-hybridize with an anti-eIF-4A antibody, we excluded the possibility that HEL64 might be a homologue of the translation initiation factor eIF-4A. Although the function of HEL64 remains unknown, the present data indicate that the encoded DEAD-box protein plays an important role during the insect life-cycle stage of the parasite.
Missel
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Qiagen GmbH, Max-Volmer-Strasse 4, Hilden, Germany.
Lambert
L
L
Nörskau
G
G
Göringer
H U
HU
D000483:Alleles; D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D015139:Blotting, Southern; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D003429:Cross Reactions; D039601:Eukaryotic Initiation Factor-4A; D018390:Gene Targeting; D017125:Genes, Protozoan; D009154:Mutation; D010448:Peptide Initiation Factors / Q000276:immunology; D010957:Plasmids / Q000235:genetics; D020365:RNA Helicases / Q000032:analysis* / Q000235:genetics* / Q000276:immunology; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D000913:Antibodies, Protozoan; D010448:Peptide Initiation Factors; D039601:Eukaryotic Initiation Factor-4A; D020365:RNA Helicases