Molecular Biology

Abstract

Stable and transient transfection in Plasmodium has been use to study gene function and gene regulation, becoming a powerful tool to better understand the biology of this parasite. This technique has helped to elucidate the mechanisms involved in virulence, imune evasion, drug resistance, cell invasion, stage differentiation and others. Plasmodium vivax, the parasite responsible for about 80% of Brazilian malaria cases, and which causes great socio-economical burden for this region cannot be cultured continuously in vitro precluding the use of transfection technology to carry functional studies of this human malaria parasite. To functionally characterize promoters and elements responsible for the control of gene expression in P. vivax, we constructed vectors having the luciferase reporter gene under the control of the promoter regions of the P. vivax msp1 and dhfr genes and used them in heterologous transient transfections in P. falciparum. Strikingly, none of the constructs were able to drive expression of the luciferase gene unlike positive controls which included promoter regions from P. berghei and P. chabaudi. These data seem to indicate that unlike other Plasmodia spp, P. vivax promoters are unable to recruit the transcriptional machinery of P. falciparum. As the AT-content of the pvmsp1 and pvdhfr promoter regions is GC-rich, present efforts are guided to construct a luciferase reporter plasmids with an AT-rich promoter region from P. vivax which closer resembles promoter regions of P. falciparum. Supported by FAPESP and CNPq BM2 -PLASMODIUM VIVAX: PRIMARY STRUCTURE AND ALLELE POLYMORPHISM OF THE PVMDR1 GENE AMONG ISOLATES FROM BRAZIL, PAPUA AND MONKEY-ADAPTED STRAINS.Martha-Sá J+, Nomura T++, Wellems TE++ & del Portillo HA+@. +Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo SP, Brazil. ++ National Institute of Allergy and Infectious Diseases, NIH, USA @correspondence (hernando@icb.usp.br).Despite intensive efforts at eradication and control, malaria remains a major public health problem. Yearly, it is estimated that 300-500 million people worldwide are afflicted with a death toll of 1.7-2.4 millions mostly in children below five years old. The emergence of Plasmodium falciparum resistance to widely used antimalarial drugs such as chloroquine as worsened this scenario and made malaria control and treatment even more difficult. Drug resistance is also now emerging in Plasmodium vivax, the most widely distributed human malaria and responsible for 70-80 million clinical yearly cases. Multidrug resistance genes (mdr) from Plasmodium are proposed to be involved in certain forms of drug resistance, including resistance to mefloquine, chloroquine and quinine. These genes have been reported and analyze for P. falciparum but not for P. vivax.To identify P. vivax mdr genes, degenerate oligonucleotides were used in PCR amplifications of P. vivax DNA and a unique 4.5 kB fragment cloned and sequenced. In silico analysis revealed that this sequence shared 70% simila...