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Expression of Leishmania donovani cyclin 1 (LdCyc1) mRNA during the cell cycle of promastigotes is S-phase specific. Here, we show that the LdCyc1 protein is periodically expressed and the activity of its associated kinase varies during the cell cycle in line with its expression pattern. In addition, we have shown that LdCRK3, homologous to CRK3 from L. mexicana, is the cognate Cdk partner of LdCyc1 and that the activity of the complex is inhibited specifically by heat stable factor(s) from the parasite.

We have cloned and sequenced a DNA fragment (V1 kb) containing a complete open reading frame from a cDNA library of Leishmania donovani promastigotes. The alignment of the derived polypeptide sequence and the modeling studies revealed that the protein is highly homologous to the mammalian cyclins having conserved cyclin box and substrate-docking motif. Northern blot analysis of the RNA isolated from synchronized L. donovani promastigotes showed periodic expression of the message with maximum abundance at S-phase suggesting its involvement in the events related to the regulation of DNA replication. The results confirm that we have isolated a cyclin molecule from L. donovani (LdCyc1) which may play an important role in the regulation of the parasite cell cycle.

BackgroundThe selection of bacterial recombinants that harbour a desired insert, has been a key factor in molecular cloning and a series of screening procedures need to be performed for selection of clones carrying the genes of interest. The conventional cloning techniques are reported to have problems such as screening high number of colonies, generation of false positives, setting up of control ligation mix with vector alone etc.ResultsWe describe the development of a novel dual cloning/expression vector, which enables to screen the recombinants directly and expression of the gene of interest. The vector contains Green fluorescence protein (GFP) as the reporter gene and is constructed in such a way that the E. coli cells upon transformation with this vector does not show any fluorescence, but readily fluoresce upon insertion of a foreign gene of interest. The same construct could be easily used for screening of the clones and expression studies by mere switching to specific hosts.ConclusionsThis is the first vector reported that takes the property of colour or fluorescence to be achieved only upon cloning while all the other vectors available commercially show loss of colour or loss of fluorescence upon cloning. As the fluorescence of GFP depends on the solubility of the protein, the intensity of the fluorescence would also indicate the extent of solubility of the expressed target protein.

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