Cloning and characterisation of a cysteine proteinase gene expressed in amastigotes of Leishmania (L.) amazonensis.
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.
Lasakosvitsch
Fernanda
F
Department of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Botucatu, 862, 6o andar, Brazil.
Gentil
Luciana Girotto
LG
dos Santos
Márcia Regina Machado
MR
da Silveira
José Franco
JF
Barbiéri
Clara Lúcia
CL
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
D016054:DNA, Protozoan; D016053:RNA, Protozoan; D003546:Cysteine Endopeptidases