1. Fig 4: Alignment of SAM Homologues: Conserved bases shown in yellow for sequence that we analyzed. In green for predicted conserved bases. Fig 5: The evolutionary tree above was produced using the Clustal W alignment program and the protein coding sequence for SAM synthetase in different organisms. Because SAM functions in required biosynthetic pathways, it is an ubiquitous protein. We propose it would be useful as an alternative to cytochrome b in molecular ecology studies. “SAM I AM”: A Highly Conserved Methyl Transfer Pathway N. Anbarasan, V. Benyo, S. Briddell, A. Bolton, D. Chen, G. Deo, J. Gluck, A. Harrison, V. Kakarla, G. Lynch, N. Mittal, A. Patel, J. Pepper, A. Petrelli, M. Singh, and A. Verri Rutgers Preparatory School, Somerset, New Jersey ABSTRACT The goal of our project was to clone genes from Artemia franciscana using a cDNA library and analyze the proteins for which our DNA coded. We analyzed plasmids using restriction digest and PCR amplification. We sent 64 of these inserts to be sequenced, and analyzed the sequences using BLAST alignment software to determine conservation of genes between Artemia franciscana and other eukaryotes. Analysis of the 17AP7.06 clone revealed high homology with the gene for S-adenosyl methionine synthetase. METHODS Fig 1: Flow chart of experimental procedures RESULTS 1. Isolation of DNA from of A. franciscana clones and confirmation of insert sizes: Independent clones from A. franciscana were grown overnight in LB-medium with ampicillin and plasmid DNA was isolated by GFX Micro Plasmid mini prep. Plasmid DNA was cut with PvuII restriction enzyme. The size of the insert was determined through agarose gel analysis. Alternatively, the DNA was used as a template and amplified with forward and reverse primers designed from vector. PCR reactions were carried out using Taq polymerase and PCR reaction buffer with 30 cycles of 94  C for 1 minute; 50  C for 1 minute; 72  for 1 minute. 2. Sequences of A. franciscana clones: Clones containing inserts ranging from 300-1600bp (including 17AP7.06) were used for sequencing. DNA Sequencing was performed at GE Healthcare after amplification with TempliPhi. Sequencing data obtained was analyzed with Chromas. The sequences were compared with database sequences using NCBI BLAST. Fig 2: Agarose gel showing PCR amplified fragment from 17AP7.06 clone. M: 1-kB Plus DNA marker, U: Uncut 17AP7.06 plasmid DNA, C: PvuII digest 17AP7.06 DNA, P: PCR amplification of 17AP7.06 insert. Clone 17AP7.06 was found to have excellent homology (E value: 3 e-93) to S-adenosyl methionine synthetase. Fig 3: SAM is synthesized from ATP and methionine. SAM functions in biosynthesis by adding methyl groups to electron rich atoms, such as oxygen, nitrogen, and benzene rings. ( Berg, J., Tomoczko, J. and Stryer, L. "S- Adenosylmethionine Is the Major Donor of Methyl Groups." Biochemistry. 2002. W. H. Freeman. 2 Jun 2007.) DISCUSSION S-Adenosylmethionine Synthetase catalyzes the formation of SAM Found in nearly every organism One form is conserved within bacteria and eukarya, while the other is conserved within archaea 77% identity between our sequence and the Drosophila homolog, 68% identity to human homolog SAM Function: Most common methylating agent in living creatures. Transfers methyl groups (-CH 3 ) to electron rich atoms. In RNA: SAM is involved with the “capping” of eukaryotic messenger RNA. Co-product, S-adenosylhomocysteine is an inhibitor of most methylases Ameliorates symptoms of arthritis, liver cirrhosis, Down syndrome, and depression. A correlation between low levels of SAM in the brain and the existence of Alzheimer’s disease. Now available as daily dietary supplements as an over the counter drug. ACKNOWLEDGMENTS : We thank Drs. William Sofer, Drew Vershon, Marty Nemeroff and Mrs. Susan Coletta of Waksman Institute; the scientists at GE Healthcare, and Mrs. Jacque Lamb for their help in our research. Fig.6: 3-Dimentional Crystal Structure of SAM Synthetase : The red region in the structure above represents the 220 amino acids coded in our sequence. The purple region consists of approximately 160 amino acids in the amino terminus that is not present in our sequence. The structure is shown complexed with SAM.