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Please direct all comments, questions, and suggestions to biobzc@hofstra.edu
Course Design
The laboratory exercises for this course begin with a phenotypic characterization of the mutant that will be used throughout the course and a computer simulation of the use of P-transposable elements to create mutants in Drosophila. The phenotypic characterization includes behavioral testing of flies for the presence of flight mutants and determination of reporter gene expression. One of the lines assigned for testing has a P-element insertiion in the gene for myosin heavy chain protein (Mhc mutant). The students need not know the identity of this mutant. Each group will be assigned at least one other P-insertion line of flies that will be used for an independent research project. The students use the lacZ reporter gene to determine the expression pattern of genes in the vicinity of the P-element insertion for each mutant line uncovered in the flight-testing. The expression patterns in both embryos and cryostat sections of adults are determined. The results of these experiments indicate the normal expression pattern of the gene that has been mutated in each of the mutant lines. (See the Introduction to Enhancer Trap Analysis for a discussion of the caveats associated with the use of reporter gene expression.)
As an out-of-class assignment, the students use a computer simulation to genetically map the Mhc mutant to a specific region on one of the chromosomes. This is followed by a database search for known mutants, genes and clones in that chromosomal area.
Over the next four laboratory sessions the students obtain a plasmid rescue clone for each of their mutant lines. In the process of obtaining these clones they will prepare chromosomal DNA, digest the DNA with restriction endonucleases, ligate DNA, transform E. coli by electroporation, isolate colonies and carry out a small-scale plasmid DNA preparation. The plasmid rescue fragment can then be sequenced using primers from the cloning vector.
The plasmid rescue clone that students are expected to obtain from the Mhc mutant has already been sequenced and is available at this site in Sample Results. This sequence is used to obtain information from the Drosophila genome database. The sequence can be mapped to an exact location within the sequenced genome. The fragment isolated from the Mhc mutant contains a large segment of the coding region for the Mhc gene, although the actual P-element insertion site is 5' to the transcription start site. Various online molecular analysis tools can be used to find the predicted protein and to identify other genes and mutants that have been mapped to this chromosomal location (see Links).
At the same time as they are working on the common mutant (Mhc ), the students should also be working alone or in groups on an unknown mutant. Once a plasmid rescue fragment is obtained for the unknown, it can be sequenced and analyzed. A blast search of the National Center for Biotechnology Information (NCBI) and Berkeley Drosophila Genome Project (BDGP) databases (see Links).can be carried out to determine if the clones from the unknown mutants are homologous to previously sequenced genes, ESTs, P1, cosmid and other clones in Drosophila or any other species. The database search will lead to the identification of the location of the P-element insertion within the sequenced genome. Within the sequenced genome, each P-element will be inserted in one of the following:
1) a known Drosophila gene,
2) a candidate gene (CG….) with sequence homology to another Drosophilagene that maps to a different location in the genome or to a gene in another species,
3) a candidate gene without sequence homology to any other gene
4) non-coding DNA.
Students work alone or in groups to plan your own experiments for further investigation of their unknown mutant based on the results of their database analysis.
The plasmid rescue clone from the Mhc mutant is used as a tool to learn another series of techniques. The clone can be labeled and used as a probe in Genomic Southern Analysis, Northern Analysis and a cDNA library screen. The cDNA can be cloned into an expression vector and the protein can be expressed and isolated. RT-PCR of adult and embryonic mRNA can used to demonstrate alternative splicing.
The Syllabus included in this web site includes all of the above-mentioned experiments. A Schedule of Laboratories is also included as well as Protocols for all of the experiments. In most of the proposed laboratory sessions, parts of several different experiments are performed during a single laboratory session. This gives the students experience in planning and organizing their work in a way that mimics more closely the experience of a research laboratory.