Joshua Andersland
College of St. Scholastica
Duluth, MN
Faculty Mentor: Dr. James Anderson

Transfer ribonucleic acid (tRNA) interprets the genetic information found in messenger RNA (mRNA) during protein synthesis. Many of the nucleotides in tRNA are post-transcriptionally modified, and these modifications play roles in tRNA structure and function. Thus far, at least 80 different modified nucleosides have been identified with unique enzymes catalyzing most of these. One such modification enzyme, the 1-methyladenosine (m¹A) methyltransferase (Mtase), catalyzes the formation of m¹A at position 58 of several tRNAs. The m¹A Mtase from Saccharomyces cerevisiae (yeast) is novel among tRNA modification enzymes in that it is composed of two-subunits, Gcd10p and Gcd14p, and each protein is essential for cell viability.

BLAST (Basic Local Alignment Search Tool) database searches using the Gcd10p and Gcd14p amino acid sequences have revealed the presence of homologues in a variety of eukaryotes, suggesting that the two-subunit structure of this enzyme has been maintained throughout evolution. Notably, the m¹A modification has been demonstrated to be an important determinant in the replication of HIV. Therefore, cloning and studying the human m¹A Mtase is potentially relevant for drug development in the fight against HIV infection. 

Previously, Dr. Anderson cloned the human homologue of the yeast GCD10 gene, HuGCD10, into a plasmid for expression in yeast. To complement those studies, the human homologue of the yeast GCD14 gene, HuGCD14 will be cloned. Several expressed sequence tags (ESTs) from human fetal lung containing HuGCD14 sequences were found during database searches, which led us to believe that fetal lung mRNA would provide an excellent source for the successful cloning of HuGCD14. To expedite the cloning of the HuGCD14 gene, we conducted reverse transcription (RT) using mRNA from human fetal lung. In addition, we used human kidney cell mRNA as another test in our RT experiments when we encountered difficulties using the fetal lung mRNA. After making the first strand cDNA using RT, we attempted to amplify the RT products using polymerase chain reaction (PCR) and oligonucleotides specific for HuGCD14.

In addition to cloning HuGCD14, HuGCD10 will be cloned into a bacterial vec or for expression studies involving the co-expression of HuGcd10p and HuGcd14p. Toward this goal, HuGCD10 was obtained by PCR amplification from a plasmid known to contain the gene and inserted into the bacterial expression vector. Further tests were done to determine the inducibility and solubility of the recombinant HuGcd10p. Future studies will include the sub-cloning of HuGCD14 into the HuGCD10 expression plasmid to co-express them and determine whether they form a complex that possesses m¹A Mtase activity.