Grant Proposal

Undergraduate Research Project

Project Title: Functional demonstration of the role of a Schizophyllum commune gene by complementation in yeast.

Total Amount Requested: $ 500

Student Signatures:______

______

Faculty Mentor Signature: ______

Date: Aug. 31, 2001

Recombinant DNA technology has demonstrated that a gene from one organism can be inserted into another organism. In most cases the recipient organism will replicate, transcribe, and translate this foreign gene as it does all the genes in its genome. This phenomenon has shown to be an extremely useful tool in the study of the genetics, molecular biology, and biochemistry of organisms. In our case, a gene from Schizophyllum commune could be inserted into a plasmid and then be introduced into the yeast, Saccharomyces cerevisiae. The S. cerevisiae strain used in the procedure would be a mutant strain whose genome is missing the gene homologous to the S. commune gene.

A gene we’ve isolated from Schizophyllum commune shows sequence similarity to a gene called UMP1 in yeast. We hypothesize that the gene we’ve isolated from S. commune is functionally the same as the yeast gene. To test this hypothesis, we will insert the putative S. communeUMP1 gene into mutant S. cerevisiae cells in which the normal UMP1 gene has been deleted. We will then look for restoration of normal UMP1 function in the transformed cells.

It has been shown that ump1∆ mutant S. cerevisiae cells are hypersensitive to canavanine. Canavanine is an arginine analog. It gets inserted into proteins during translation and causes them to misfold. The misfolded proteins are then sent to the proteasome for degradation.

The UMP1 gene codes for a proteasome maturation factor. Proteasome maturation factor is required for a functional proteasome. If defective, cells can’t destroy misfolded proteins. This reduces the apparent concentration of functional proteins leading to cell death.

A putative S. communeUMP1 gene will be obtained from a pBluescript plasmid, in which it has been previously cloned by members of our lab. The gene will be removed from pBluescript utilizing a double digest method with the restriction enzymes EcoRI and XhoI. The gene will then be ligated into a similarly digested YEP vector, which contains a yeast promoter. The recombinant plasmid will then be sequenced to establish that proper insertion has occurred.

Parallel experiments to demonstrate the tractability of standard yeast transformation protocols will also be done. Ultimately, the engineered YEP/UMP1 plasmid will be transformed into yeast cells and they will be screened for canavanine resistance.

Budget

1. / Media/Plates (300 plates of media at $.10 per petri dish + $.10 chemicals per dish)
Chemical Reagents (antibiotics, tryptophan, electrophoresis gel materials) / $ 60
$51
2. / Yeast Strains (5 strains at $10 each- from Carolina Biological) / $ 50
3. / Restriction enzymes (2,000 units each of EcoR1 and Xho1 = $35)
DNA ligase (200 units = $50) / $ 85
4. / DNA sequencing (12 reactions at $12 each) / $ 144
5. / Plasmid Prep Materials (Qiagen, kit for 25 preps) / $ 110

Total

/ $ 500

Budget Justification

  1. This amount is primarily for the purchase of canavanine, which is required in the selective media.
  2. Yeast strains for this project must be purchased from the American Type Culture Collection.
  3. Restriction enzymes and DNA ligase are required to engineer the YEP/ump1 plasmids that we are making.
  4. Once the plasmid constructs are made, it’s necessary to determine that the inserted ump1 gene is in the right reading frame to be translated correctly. This can only be done by DNA sequencing.
  5. This is for Qiagen kits, used for plasmid preparation.