University of Khartoum

Molecular Features of the Leishmania donovani Cell Cycle

Molecular Features of the Leishmania donovani Cell Cycle

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Title: Molecular Features of the Leishmania donovani Cell Cycle
Author: Nahla Osman Mohamed Ali, Ali
Abstract: The protozoan parasite Leishmania donovani has a complex life cycle alternating between proliferative and non-proliferative stages adapted for life in different hosts; the sand fly vector and the phagolysosomal compartment mammalian macrophages. Cyclin-dependent kinases, the best characterized of which is cdc2, are the key regulators of the cell cycle of almost all the eukaryotes. Genes for cdc2-related kinase have been characterized from man trypanosomatids including Leishmania mexicana. One approach to the study (these proteins) was to clone genes by designing oligonucleotides to represent conserved regions of the proteins and using these as primers in the polymerase chain reaction (PCR). In this study, the same technique was used to isolate a cdc2-related kinase 3 gene (crk3) from L. donovani genomic DNA. A 900bp product from a PCR reaction was cloned (pQE60) and sequenced to confirm that it was derived from a crk3 gene. This fragment was then used to screen a L. donovani cDNA promastigote library to obtain clone which allow the determination of the complete gene sequence (full length gene). The gene, designated Ldcrk3 (L. donovani cdc2-related protein kinase3 gene), has between 77 to 94% amino acid identity with recently characterized Lmmcrk3 (Leishmania mexicana mexicana cdc2-related protein kinase3 gene), and between 38 to 46% amino acid identity with HsCDC2 (Human cdc2 protein kinase gene). Analysis of the predicted amino acid sequence revealed a number of features common to trypanosomatid (CRKs including the two substitutions in the conserved domain 'PSTAIR' box that involves in the cyclin binding. Another conserved feature is the presence of the 19-aa extension at the N-terminal. T14, Y15 and T16; the phospharylation sites which are important for the regulation of cdc2, are also present in the sequence. To further characterize the gene, the cloned PCR product was used as a probe in a southern blot. The result shows that there is one predominant band in each of the digests, except the Sal/I digest which has 2 bands as a result of an internal Sal/I site. This suggests that Ldcrk3 is a single copy gene.
URI: http://khartoumspace.uofk.edu/handle/123456789/13786
Date: 2015-06-22


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