title: Cloning and characterization of differentially expressed genes from in vitro-grown 'amastigotes' of Leishmania donovani.
abstract: Leishmanial parasites routinely undergo cyclic differentiation from promastigotes to amastigotes during their life cycle. This process involves both morphological and macromolecular changes. To study such changes, we used a axenic culture system which permits the continuous generation and cycling of Leishmania donovani from promastigotes to 'amastigotes' in vitro. cDNA libraries were constructed from poly(A)+ RNA isolated from both the pro- and amastigote forms. Using differential cDNA hybridization techniques, 3 unique cDNAs clones (P17, A41 and A45) were isolated from the amastigote library. To assess whether these clones were differentially expressed by the pro-or 'amastigotes' forms, they were hybridized to RNA isolated from each of these parasite forms in Northern and slot-blots. Results of these analyses showed that 'amastigotes' had approx. 2-fold higher levels of the A41 and A45 RNAs compared to the promastigotes. Conversely, promastigotes showed approx. 2-fold higher levels of the P17 RNA than 'amastigotes'. Nucleotide sequence analysis and comparison with those in Gene bank, revealed that the 3 cDNAs represent unique leishmanial genes. Comparison of the deduced amino acid sequences revealed that P17 open reading frame (ORF) had significant similarity with a soybean ribosomal protein S11; A41 ORF with a Bacillus subtilis spore germination gene (gerC) and A45 ORF with yeast stress-inducible protein (STI1). It is of interest to note that, of the 3 cDNAs identified, the A45-encoded protein was recognized by sera from patients with clinically active visceral leishmaniasis and was encoded by a single copy gene.
authors: Laboratory of Biochemistry, CBER, Food and Drug Administration, Bethesda, MD 20892.
authors: M
authors: M
authors: Joshi
authors: D M
authors: DM
authors: Dwyer
authors: H L
authors: HL
authors: Nakhasi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D015870:Gene Expression; D017125:Genes, Protozoan; D006801:Humans; D007893:Leishmania donovani / Q000235:genetics* / Q000254:growth & development; D007898:Leishmaniasis, Visceral / Q000469:parasitology; D008969:Molecular Sequence Data; D010641:Phenotype; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D016054:DNA, Protozoan; D012333:RNA, Messenger; D016053:RNA, Protozoan