RESUMO
The Entamoeba histolytica Ehcp112 and Ehadh112 genes that encode the EhCPADH complex are separated by a non-coding 188pb region. Their proximity suggests a coordinated expression regulation for both genes. Here, we studied the structure and function of 996 bp (p996CAT) upstream the ATG start codon of the Ehadh112 gene. The p996CAT plasmid drove CAT transcription with a 78% of the activity showed by actin promoter. Deletion of 330 bp at the 5' end of p966CAT to produce the p776CAT plasmid, decreased activity to 40% in relation to actin promoter and to 50% of p996CAT, suggesting the presence of a silencer in this region. Interestingly, deletion of other 297 bp to the p776CAT to generate the p469CAT plasmid, augmented activity in 2.5-fold compared with p776CAT construction, showing the presence of a proximal enhancer promoter. Transcription initiation sites (-69 and -150 bp), TATA like box, GAAC, and Inr elements, as well as putative DNA binding motifs, were mapped in the -1 to -469 bp core promoter region.
Assuntos
Entamoeba histolytica/genética , Lectinas/genética , Glicoproteínas de Membrana/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas de Protozoários/genética , Animais , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , DNA de Protozoário/química , Lectinas/fisiologia , Glicoproteínas de Membrana/fisiologia , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas/genética , Proteínas de Protozoários/fisiologia , TransfecçãoRESUMO
Ehcp112 encodes the Entamoeba histolytica EhCP112 cysteine protease that is part of the EhCPADH complex. By in silico analysis we identified putative transcription factor-binding sites along 837 bp upstream the Ehcp112 gene ATG codon. A TATA-like motif (TATATAAA) was located at -36 to -29 bp, a GAAC box (GAACC) was found at -10 to -14 bp and an Inr sequence (TTCAAC) at -8 to -2 bp. These tripartite promoter elements are in non-canonical positions, downstream the transcription initiation site (-280 bp). We cloned four Ehcp112 promoter fragments in pBSCAT-ACT plasmid to obtain pI (355 bp), pII (681 bp), pIII (833 bp), and pIV (554 bp) constructs. In transfected trophozoites, only pIII drove CAT activity with 44% efficiency in relation to actin promoter activity. Our results showed the presence of a distal and weak promoter in the Ehcp112 gene. The active DNA region is inside the open reading frame of the Ehrab B gene, suggesting that expression of both genes could be coordinately regulated.
