Development of Molecular Tools for the Analysis of Heavy Metal Tolerance in Lancelets (2011 - Present)

Investigator:  Thomas Sorger, Roger Williams University

Abstract:  The specific aims of this project are (i) to train undergraduate students to design and carry out studies in molecular toxicology by (ii) having them design and test primer pairs useful for the amplification and sequencing of seven genes associated with tolerance to heavy metals in two species of lancelet.

The hypothesis driving this project is that there is  an association between exposure to heavy metals and the operation of two adaptive mechanisms:  (a) changes in the expression of one or ore among seven 'metal response genes' (biochemical/ physiological adaptation); (b) changes in the allele frequencies of one or more among seven 'metal response genes' (population genetic adaptation).  Lancelets may be useful indicator species for heavy metal pollution, and the ultimate foal of this project is to develop the molecular tools (PCR primers and SNP probes) that will enable us to detect microevolution and/or physiologic adaptation to heavy metals.

The training of students will take place in several stages, and at each stage students will be expected to understand the experimental strategy and be able to interpret both positive and negative results.  Beginning with seven "metal response genes' from the one species of lancelet whose genome has been sequenced, students will learn to (i) identify the region(s) of each gene that has been most conserved over the course of animal evolution; (ii) design PCR primers that will be useful in two different species of lancelet:  Branchiostoma floridae (USA) and Branchiostoma lanceolatum (Mediterranean); (iii) purify genomic DNA and RNA from several orgainisms from each species and determine the quantity and quality of each preparation; (iv) prepare cDNA from several individual and design PCR reactions that optimize the detection of specific products; (v) analyze these products by agarose gel electrophoresis, and (vi) prepare samples of the PCR products for DNA sequencing.  In addition, students will learn how to apply mathematical models ('F-statistics and the 'neutral theory of molecular evolution') in order to interpret differences in allele frequencies for these seven 'metal response genes' in planned studies of natural populations exposed to heavy metal pollution.