BSCI 415

Laboratory in Molecular Genetics

SUMMARY OF PROJECTS:

Mutagenesis Project: Students will perform a genetic screen for phenotypic mutants in the nematode C. elegans and the plant Arabidopsis thaliana. In both, a rapid life-cycle allows production of the M2 generation in which mutants can be screened and subjected to genetic analysis. Students will screen for developmental. Behavioral mutants will also be screened in C. elegans. Further, students will design their own screens for conditional or response mutants. The mutants will be analyzed through crosses with tester strains and other student’s mutants to determine dominance/recessive relationships, allelism and perform initial mapping. Databases of mutant phenotypes, the C. elegans genome and journal publications will be used by the students to place their mutants into the existing knowledge.

PCR cloning Project: In order to study a specific gene sequence, students will use PCR to amplify and sequence one of their own genes –CYP2D6. The gene is a member of a large gene family (cytochrome P450) that is important in detoxification of environmental chemicals and carcinogens, and in the turnover of medications. The students will use the human genome database to identify the specific gene and determine sequence polymorphisms they possess. Comparison to the databases and literature analysis of polymorphisms in this gene will be used to illustrate the potential and limitations of human population genetics and genetic counseling.

Transformation Project: Students will transform Arabidopsis using Agrobacterium carrying constructs that will help define the function of a cloned Arabidospsis gene. Arabidopsis offers a simple dip-method transformation that results in transgenic seed and so does not require tissue culture. The students will recover transformants by plating seed on kanamycin selection and test for GUS expression in seedlings with qualitative and quantitative assays. One type of gene construct to be used is a fusion of the promoter of a gene to the GUS reporter gene. Expression of GUS in the transgenic plants will indicate the location and timing of expression of the gene. A second use of transformation will be to illustrate the use of overexpression of a gene to determine function. Genes encoding transcription factors will be overexpressed by fusing their coding region to the strong CaMV 35S promoter. Phenotypes that develop in the transformed plants will help define a function for the transcription factor.

Mapping Project: Students will map a phenotypic mutation onto the Arabidopsis genome map using molecular markers (CAPS, SSLP and InDel). Since time does not allow the students to create a F2 mapping population in the semester, seeds will be provided that serve as this mapping population that represent a mutation isolated in a previous year’s course. Students will prepare DNA from approximately 40 mutant progeny. PCR-based molecular markers will be used to map the mutation using the mapping population. Using the Arabidopsis genome database, students will position the gene on the map with the mapping data.