Study of the promotional region of gene PHEX: a gene with expression modified in the infection for the Mycobacterium
Leprosy is a chronic infectious disease caused by Mycobacterium leprae. Although the global number of leprosy registered cases has decreased since 1985, Brazil is still the fifth country in prevalence of the disease in the world, presenting an incidence of more than 49.000 new cases per year. Peripheral nerve damage is the most important consequence of leprosy leading to sensitiveness and motor loss of the infected nerves. Another important pathological aspect is the frequent incidence of bone alterations that contribute to the physical deformities that are hallmarks of the disease. Recent studies focused on the interaction of M. leprae with human cells have shown that M. leprae is able to down regulate the expression of PHEX. This gene codes for a membrane endopeptidase related to the phosphate metabolism, which is involved in the mineralization of the bone extracellular matrix. The modulation of PHEX gene expression by the M. leprae points PHEX as a possible molecular agent of the bone alterations seen in leprosy. In order to define the mechanisms used by M. leprae to inhibit PHEX expression, studies on the regulation of PHEX gene expression are required. These studies have been hampered by the fact that the promoter region of the human PHEX gene has not been experimentally characterized. The aim of the present work was to identify and characterize the promoter region of the human PHEX gene. By using bioinformatic tools, the analysis of a sequence of 4498 bp located in front of PHEX open reading frame allowed the identification of a region of 1046 nucleotides that probably corresponds to the promoter of this gene. This region as well as a smaller region containing half of the putative promoter (546 bp) were amplified by PCR using specific primers. The fragment of 546 bp was successfully cloned in the pGL3-Basic vector (-500 clone) and the cloning of the fragment of 1046 bp is in progress. The potential regulating function of the region of 546 bp was analyzed using luciferase gene reporter assays. The -500 clone was not able to generate luminosity in osteoblasts and Schwann cells, indicating that the portion of 546 bp of the putative promoter is not sufficient for the induction of human PHEX gene expression. A comparison of our results with those obtained with the murine promoter, in which luciferase expression was achieved with fragments shorter than 546 bp, suggests that the minimum region necessary for the induction of PHEX expression in vivo varies considerably in human and mouse.
Advisor:Antonio Jorge Tempone; Maria Cristina Vidal Pessolani
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Mycobacterium leprae PHEX Leprosy
Date of Publication:07/27/2006