The cyanobacterium Anabaena, which utilizes both photosynthesis and nitrogen fixation, is used for studying the differentiation in cells. It is an ideal organism because of its simple genetic makeup. In the course of investigating origins of differentiation within the multicellular filaments of Anabaena, genes dmnA, dmnB, and dmnC-all coding for methyltransferases-were isolated (Matveyev, Young, Meng, Elhai, submitted).

Studies of amino acid sequences of over 50 methylases have led researchers to identify three general groups: 5mC-methyltransferases (cytosine), N6mA-methyltransferases (adenine), and N4mC-methyltransferases (cytosine). The three groups share a common 3-D structure, and the N6mA and N4mC methylases appear to have closely related sequences.

One of the newly discovered methyltransferases, DmnA, is highly unusual due to the fact that its amino acid sequence shows little similarity tothose of the three methylase groups. This methyltransferase recognizes the sequence GATC and methylates the adenine. Through my summer research, I hoped to use NMR to determine the 3-D structure of DmnA and look for similarities to the structures of the three methylase groups. NMR couldshow that while amino acid sequences for a methylase differ, the tertiary structures are similar and thus permit the methylating function. If DmnA were found to have an entirely different 3-D structure from the three methyltransferase groups, we might designate its sequence and structure as a fourth category of methylases.

My goals for the research were the following: (1) isolate DmnA from E. coli in which the gene had been cloned; (2) purify the DmnA using affinity column chromatography; and (3) prepare DmnA for NMR spectroscopy performedby Neel Scarsdale at Virginia Commonwealth University. I was well aware that the ten weeks would be spent mostly isolating and purifying the DmnA samples. Much of my methodology and approach for isolation and purification resulted from consulting faculty--Jeff Elhai and Suzanne O'Handley--and primary literature on the purification of methyltransferases from bacteria.

Most of my research time dealt with the isolation of DmnA. The strain, pUR146, was a pBluescript vector to which the dmnA gene had been added antiparallel to lacZ. An attempt of electroporation to transform pUR146into the BL21(DE3) strain failed, most likely due to a faulty vial. In another effort, competent BL21 cells were prepared and transformed with pUR146, and were administered to ampicillin plates for selection. Using a modified purification protocol, cells were prepared under specified conditions for sonication which would release DmnA. After sonication, SDS-PAGE was utilized, but no particular banding resulted between control and experimentals, therefore signifying that DmnA had not been isolated.

In another experiment to isolate DmnA, primers were designed for 3' and 5' ends of dmnA in pUR147 (identical to pUR146 with dmnA parallel to lacZ) so that a PCR-generated fragment could be ultimately cut and inserted into pET11b. This design would result in dmnA under the control of the T7 promoter in pET11b. PCR was performed on pUR147 using the selected primers, and a DNA agarose gel was used to confirm the amplification of the desired fragment. A pGEMT vector was used to clone the amplified PCR fragment containing dmnA. The pGEM vector and pET11b were both cut with restriction enzymes BamHI and NdeI so that the pGEM fragment could ultimately be ligated into pET11b. DNA agarose gels were run to check on fragment sizes before isolation and ligation. Unfortunately, only the pET11b vector cut correctly with the restriction enzymes. The pGEM-dmnA vector showed cutting which did not match expected fragment lengths. These results came at the end of my tenth week, thus bringing my research to an end. I realized through many failed tests and different methodologies that isolation of a methylase was not so simple. Currently, my research awaits continuation to successfully isolate DmnA. I believe my approach with the pGEM vector could be repeated with slight modifications to result in successful isolation of DmnA.

My summer research, funded by Merck, proved to be a valuable and exciting experience. As a biology major and premedical student, I never had considered researching in a lab. I am very grateful to Jeff Elhai for approaching me and informing me of this opportunity. Not only did I learn how to read research articles efficiently, but I also learned how experimental procedures--such as SDS-PAGE, sonication, and electrolysis--worked. I also have a new appreciation for the hard work and numerous experiments which researchers must use. My biology classes only briefly explained these methods, whereas working in the lab taught me how to operate the tests and why they worked. This information was useful for my upper division biology classes which required a broad understanding of methodologies.

I enjoyed my ten weeks of summer research. While my future in the field remains to be determined, this research opportunity greatly enriched my biological background, and I would recommend it to anyone with a sincere interest in the sciences.