Mitochondrial Biology

Malone Research Laboratory: Roonalika Wisidigama
Kohler Research Laboratory at UCLA

“YME1 gene regulation”

Precisely controlling when and where genes are turned on and off, known as controlling gene expression, is necessary for all cells to develop and function normally and avoid becoming cancerous or dying prematurely. This project focuses on how a gene called YME1 is turned on and off. YME1 is found in the inter-membrane space of mitochondria.  YME1 is a metalloprotease that when in a functional complex is called i-AAA.  The AAA (ATPase associated with diverse cellular activities) family of proteases are important for mitochondrial stasis, protecting the morphology of the organelle, and for mitochondrial genome integrity.  Scientists have also found that YME1 family members are responsible for a collection of paralytic diseases. For an example HSP – hereditary spastic paraplegia a neurodegenerative disorder which affects 1 in every 10,000 individuals, is caused by the paraplegin, a i-AAA protease family member. Understanding the on/off control of a gene is the first step in being able to manipulate its control for therapeutic reasons. Results gathered from our experiments will enhance our knowledge of how this particular gene is turned on and off in a normal cell.

This study focuses on how a gene called YME1 is turned on and off. We will isolate, subclone, and examine the functional activity of the YME1 gene control region DNA (the on/off switch), called a promoter from mouse cells that grow in a culture flask. The mouse YME1 gene coding sequence DNA will be used to find the 5’ genomic DNA sequences that constitute the YME1 gene promoter region using the National Center for Biotechnology Information (NCBI) mouse genome database website and Basic Local Alignment Search Tool-nucleotide (BLASTn) database website provided by the National Institutes of Health (NIH). We will use the known mRNA sequence for YME1 to locate the YME1 promoter DNA sequences using these databases and the MacVector bioinformatics computer program. DNA primers will be designed to amplify the 1 kilobase promoter region 5’ of the YME1 gene from genomic DNA using the polymerase chain reaction (PCR).

The amplified promoter DNA product will be sub-cloned into the pGL3 basic luciferase reporter vector that will be used to evaluate promoter activity. DNA sequencing will be employed to confirm insertion of the promoter region in the correct orientation in the pGL3 vector. The YME1 promoter-luciferase reporter vectors will be transiently co-transfected into a mouse cell line along with the pRLSV40 control vector for transfection efficiency. Dual luciferase reporter gene analysis will be performed using the Dual Luciferase Reporter Assay System to determine the activity of the YME1 promoter. Reporter vectors produce a quantifiable product that is equal to the amount of promoter activity when co-transfected into cells. In other words, this experiment will tell us if we have isolated the YME1 promoter and how good this promoter is at turning the YME1 gene on. Future experimentation will include determining the exact DNA sequences in the YME1 promoter that control the activity of the YME1 promoter. We will accomplish this goal by creating mutations in the DNA sequence of the YME1 promoter and then testing the mutated promoters for loss of activity using the experiments described above.