Novel Purification of Archaeal Rnase P Protein, Pfu Rpp38, for Nmr Studies

Dr. Kenneth Murray, Ph.D. Assistant Professor of Biology Ribonuclease P (RNase P) is an essential and ubiquitous ribonucleoprotein enzyme primarily responsible for cleaving 5' leader sequences during tRNA maturation. RNase P comprises one essential RNA, and one protein subunit in eubacteria, five proteins in archaea, and ten in humans. Due to its homology to human RNase P, its higher stability, and simpler structure; extensive studies have been conducted utilizing the enzyme from the archaeal hyperthermophile, Pyrococcus furious (Pfu). Previous studies identified only four protein subunits associated with the archaeal RNase P. This fourprotein reconstituted particle, however, had an optimal temperature of 55°C, compared to the optimal 70°C of the wild type RNase P. Additional probing of the organism's genome database revealed a fifth RNase P protein subunit, RPP38. To facilitate further investigations of Pfu RNase complexes, we sought to develop a protocol for the purification ofRPP38. Our results, presented herein, represent the first known expression.purification protocol developed for RPP38. Briefly, we synthesized an N-terminal6x-His RPP38 fusion construct, reengineered to contain a Tobacco Etch Virus (TEV) protease cleavage site. Purification was achieved via immobilized metal affinity chromatography and reversed phase high performance liquid chromatography. Following purification the 6X-His affinity tag was removed via TEV cleavage, thus regenerating the native RPP38 protein. Purity and identity of RPP38 were confirmed by sodium dodecylsulfate - polyacrylamide gel electrophoresis and mass spectrometry, respectively. Our work is expected to contribute to our understanding ofRNase P function and tRNA maturation by providing an efficient, facile technique to express and purify Pfu RNase protein RPP38 as a means to facilitate structural and functional analyses.

An evaluation of a curriculum response to the State of Florida mandate for computer literacy at a large comprehensive high school in Dade County, Florida

Minimum Student Performance Standards in Computer Literacy and Science were passed by the Florida Legislature through the Educational Reform Act of 1983. This act mandated that all Florida high school graduates receive training in computer literacy. Schools and school systems were charged with the task of determining the best methods to deliver this instruction to their students. The scope of this study is to evaluate one school's response to the state of Florida's computer literacy mandate. The study was conducted at Miami Palmetto Senior High School, located in Dade County, Florida. The administration of Miami Palmetto Senior High School chose to develop and implement a new program to comply with the state mandate - integrating computer literacy into the existing biology curriculum. The study evaluated the curriculum to determine if computer literacy could be integrated successfully and meet both the biology and computer literacy objectives. The findings in this study showed that there were no significant differences between biology scores of the students taking the integrated curriculum and those taking a traditional curriculum of biology. Student in the integrated curriculum not only met the biology objectives as well as those in the traditional curriculum, they also successfully completed the intended objectives for computer literacy. Two sets of objectives were successfully completed in the integrated classes in the same amount of time used to complete one set of objectives in the traditional biology classes. Therefore, integrated curriculum was the more efficient means of meeting the intended objectives of both biology and computer literacy.