title: Cloning and characterisation of a cysteine proteinase gene expressed in amastigotes of Leishmania (L.) amazonensis.
abstract: The present study describes the cloning and characterisation of a gene encoding a cysteine proteinase isoform, Llacys1, expressed in amastigote forms of Leishmania (L.) amazonensis. Recombinant clones containing the Llacys1 gene were isolated from genomic DNA by PCR amplification and screening of an amastigote cDNA library. Sequence analysis of the Llacys1 gene showed a high identity to sequence of Leishmania (L.) pifanoi Lpcys1, Leishmania (L.) major cpa, Leishmania (L.) mexicana LCPa, and Leishmania (L.) chagasi Ldccys2. The Llacys1 gene is present in a single copy per L. (L.) amazonensis haploid genome and was mapped on a chromosome of approximately 700 kb. Two transcripts of the Llacys1 gene were identified, one of 2.4 kb transcribed in both forms of L. (L.) amazonensis, and another of 1.6 kb weakly expressed in amastigotes. Related forms of Llacys1 gene exist in other species of Leishmania genus, including L. (L.) major, L. (L.) mexicana, L. (L.) chagasi and Leishmania (V.) braziliensis. The Llacys1 expression in Escherichia coli was obtained when the nucleotide sequence corresponding to the signal sequence was deleted, suggesting that this signal sequence was recognised by Escherichia coli and cleaved, generating a truncated protein.
authors.affiliation: Department of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Botucatu, 862, 6o andar, Brazil.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Lasakosvitsch
authors.forename: Luciana Girotto
authors.initials: LG
authors.lastname: Gentil
authors.forename: Márcia Regina Machado
authors.initials: MR
authors.lastname: dos Santos
authors.forename: José Franco
authors.initials: JF
authors.lastname: da Silveira
authors.forename: Clara Lúcia
authors.initials: CL
authors.lastname: Barbiéri
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics*; D016054:DNA, Protozoan / Q000235:genetics; D015870:Gene Expression; D017125:Genes, Protozoan / Q000235:genetics*; D007894:Leishmania mexicana / Q000235:genetics*; D008969:Molecular Sequence Data; D016053:RNA, Protozoan / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
chemical_list: D016054:DNA, Protozoan; D016053:RNA, Protozoan; D003546:Cysteine Endopeptidases