Deduccion In-Sílico de la secuencia de la proteína hepática metalotioneina en muestras de Eleutherodactylus Johnstonei en el Valle del Cauca, Cali- Colombia

Abstract

According to global historical reports, the presence of heavy metals has increased dramatically since the beginning of the industrial revolution. Some of these metals such as cadmium, mercury and lead, have been linked to death and degradation of ecosystems by eco- toxicity of flora and fauna, and on the other hand are related to human poisonings ( acute and chronic ), degenerative damage generated during embryonic development, youth and neurological at different levels. Many of the focus that concentrate these metals are industrial centers, the garbage collection centers (without proper management and recycling) and gold mining currently. It becomes necessary to propose monitoring systems of heavy metals, control and waste management. The knowledge of hepatic protein Metallothionein and understanding its primary function as heavy metal chelating agent, is useful to allow the analysis of contamination levels and consequences that can result after bioaccumulation in living organisms. In the environment the presence and levels of heavy metals are unknown; some organisms can contribute to the discovery of new concepts and patterns of contamination that we face. This study aims to isolate the complementary DNA Metallothionein liver protein that is expressed in environments highly disturbed and contaminated by heavy metals. To achieve this objective we propose the use of invasive frog Eleutherodactylus johnstonei to analyze their molecular aspects, evidencing the presence of this protein and thus establish in 2013 as a bioindicator organism for the presence of heavy metals in areas of the city Cali. The methodology is routed for total RNA isolation, reverse transcription and sequence amplification by PCR. The isolation of total RNA from liver Eleutherodactylus johnstonei was refined until the point of obtain good quality RNA and qualities verified by electrophoresis of denaturing gels to RNA and display the two bands characteristic of ribosomal RNA. Using bioinformatic training of eight sequences alignment of metallothionein vertebrate , the use of a tool for designing primers and the screening ( by Blastn search) resulting design primers, were obtained three pairs of degenerate primers designed to vertebrate metallothioneins non-mammals. The reactions of reverse transcription and polymerase chain reaction (RT -PCR) made in this study totaled 54 reactions that were employed in combination with the degenerate primers designed to metallothionein. When were verified the PCR reactions on agarose gels, did not show well defined bands ( amplicons ), except for the reaction in which the cDNA was carried out using poly dT primer , whose product was subsequently amplified by PCR using DG2F and DG1R primers . However the sequence obtained by subjecting a Blastn search, this had not similarity to metallothionein. Also, the sequence readable only had 25 bases and the other portion was not possible to read. For this study, the results suggest that the RT- PCR reactions must be modified and temperature variables primers nesting or redesigning primers to reinforce the strategy of obtaining the sequence