RESEARCH 2002
RESEARCH 2001
RESEARCH 2000

Julia K. Fangmann
Marquette University
Milwaukee, WI
Mentor: Dr. James Anderson 

All transfer RNAs (tRNA) contain a number of post-transcriptional modifications, which comprise a significant component of the tRNA processing events. Such modifications assist in forming the proper structure and impact the functional activity of tRNA. Mutations in mitochondrial tRNA have also been linked to a wide array of human diseases. For these reasons, it is important that tRNA modifying enzymes are studied further. A common type of modification is the addition of a methyl (CH3) to the ribose or base component of nucleotides within the tRNA sequence. Methyltransferases are the enzymes that transfer a methyl from a donor (often times AdoMet [S-adenosyl-L-methionine]) to a specific nucleotide in the sequence. Methyltransferases have been found and studied in all three domains of life: eukraya, eubacteria, and archaea; however, there are more methyltransferases whose activities have not been fully explored.

We hypothesized that a human gene (FLJ20628) is a potential tRNA m1A9 methyltransferase. The cDNA of the gene was obtained, and for the purpose of detection via two separate tags, it was PCR amplified using two different sets of primers. The two inserts FLJ1 and FLJ2 were ligated to pET15ba and pET11a, respectively. These products were transformed into bacterial cells. Once proper orientation of the inserts in the two different plasmids was determined, these positive colonies were used to obtain plasmid DNA for further analysis.

With the DNA of the positive clones in hand, we transformed a bacterial strain engineered specifically for the controlled expression of exogenous genes. The transformants were cultured under non-inducing conditions, and FLJ1 and FLJ2 expression was induced by addition of IPTG. Protein expression was allowed to continue for various amounts of time, and total proteins expressed were separated into soluble and insoluble extracts. After separating these samples via electrophoresis on a NuPAGE gel, it was determined that FLJ20628 protein expression was taking place at low levels. A Western blot was performed in order to confirm this result and detect the FLJ20628 protein possessing one of two separate epitopes.

FLJ1 did not show detectable expression of the FLJ20628 protein in either the soluble or insoluble extracts. In contrast, FLJ2 did show expression of the protein of interest in the insoluble portion, but not in the soluble fraction. Since further analysis of the protein and its activity requires expression of a soluble protein in large amounts, it will not be possible to continue this analysis until we achieve soluble expression of FLJ20628. Thus, the precise activity and specificity of the FLJ20628 protein is still unknown.