Isolation, characterization and expression of a cyclin fromLeishmania donovani

Abstract

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.

Leishmania donovanicyclin 1 (LdCyc1) forms a complex with cell cycle kinase subunit CRK3 (LdCRK3) and is possibly involved in S-phase-related activities

“…To characterize the periodic expression of LdCyc1 during the cell cycle, the synchronized L. donovani promastigote culture was prepared by arresting the exponentially growing cells at S‐phase with hydroxyurea (HU) for 15 h followed by release of the block (Banerjee et al , 2003). The cells were collected from such synchronously growing cultures at 1‐h intervals and processed for subsequent analyses.…”

“…The promastigote form of Leishmania donovani isolate AG83 (MHOM/IN/83/AG83) was grown in M199 medium containing 10 mM HEPES (pH 7.2–7.4) supplemented with 10% FBS, 100 U mL −1 Penicillin and 0.1 mg mL −1 streptomycin at 22°C with slow shaking as described previously (Banerjee et al , 2003).…”

“…CRK3 interacting cyclins have been identified in T. cruzi by the yeast two‐hybrid assay (Gomez et al , 2001). Recently, we have reported the characterization of a cyclin molecule LdCyc1 from Leishmania donovani (Banerjee et al , 2003). Thus, the presence of CRKs and cyclins suggests that the basic mechanism of cell cycle regulation is conserved in these organisms.…”

Ubiquitination of mRNA cycling sequence binding protein from Leishmania donovani (LdCSBP) modulates the RNA endonuclease activity of its Smr domain

“…The mRNA of S-phase cyclin LdCyc1 of L. donovani was found to contain a single copy of the conserved octamer sequence in its 5 0 -UTR [13,14], and in the present study, RNA electrophoretic mobility shift assay (REMSA) was performed to determine whether LdCSBP interacts with the UTR specifically. The slower moving bands in REMSA with the extract of Sf9 insect cell expressing LdCSBP and a 32 P-labeled RNA probe containing the 5 0 -UTR of LdCyc1 (Fig.…”

Trypanosomatid Cell Division Kinases

“…Leishmania mexicana CRK3 is known to be activated by CYC6 and CYCA in vitro [35,36], which by sequence analysis are predicted to be mitotic cyclins, and thus are likely to activate CRK3 at G 2 /M in vivo. Leishmania donovani CRK3 has also been reported to interact with, and be activated by, Cyc1 (the homolog of CYCA) both in vitro and in vivo, although in this case, Cyc1 was shown to be expressed during S phase [37,38]. The identified in vitro substrates of CRK3:Cyc1 include putative Ku70 and MYST family histone acetyl transferase homologs, which is consistent with an Sphase role for this CDK complex [39].…”