Characterization of chitinase gene from lutzomyia longipalpis:alternative splicing description and search for promoter sequence / Caracterização de gene de quitinase de Lutzomyia longipalpis: descrição de processamento alternativo e busca por seqüência promotora

AUTOR(ES)
DATA DE PUBLICAÇÃO

2009

RESUMO

Leishmaniasis is a disease caused by Leishmania protozoa transmitted by the bite of infected sand flies. Current methods used to combat this illness have been shown to be inefficient, and better knowledge of aspects related to Leishmania-sand fly interaction are necessary for the development of new controlling methods. A cDNA codifying for a midgutspecific chitinase of Lutzomyia longipalpis, nominated LlChit1A, was previously identified in our laboratory using differential display RT-PCR (DDRT-PCR). It was found that the LlChit1 gene had high transcription levels 3 days after blood meal, which indicated a putative role on peritrophic matrix (PM) degradation. A LlChit1A fragment, was used for screening a L. longipalpis genomic library, which led to the isolation of a clone called LlChit1G, containing the chitinase gene. The PCR amplification and sequencing of this gene revealed 4 introns which interrupt the LlChit1A cDNA sequence. RT-PCR showed that the LlChit1 gene is also expressed in larvae where it transcribed two new forms of splicing, called LlChit1B and LlChit1C. These two transcripts possess early stop codons in the last intron, interrupting the translation of the chitin binding domain (CBD) codified by the last exon. These putative enzymes possibly act in the digestion of chitin rich food, as previously observed for others insect chitinases without this functional domain. The flanking region 5 (FR5) present in the genomic clone was also sequenced, evidencing a possible minimum promoter. Curiously, the initial part of this 5 UTR sequence was identical to the initial part of a Phlebotomus papatasi orthologous gene. This may be an indication of a promoter system conserved among sandflies, with possible performance in the control of PM thickness, which seems to occur exactly at the moment and place of Leishmania attachment to the epithelium of the midgut. The use of inverted PCR allowed the sequencing of a region upstream the chitinase gene, which is not present in the genomic clone. Bioinformatics analyzes suggested the participation of ecdysone on the expression control of this gene. The annotation for ESTs codifying complete and partial chitinase like proteins from L. longipalpis and P. papatasi provided the identification of 4 new genes from the first specie and 5 new genes from the later. These genes codify for protein conserved domains with high similarity to the catalytic domain of family 18 glycosylhydrolases. Phylogenetic trees were also constructed.

ASSUNTO(S)

alternative splicing chitinase psychodidae biologia molecular processamento alternativo regiões promotoras genéticas quitinase psychodidae genetic promoter regions

ACESSO AO ARTIGO

Documentos Relacionados