
	1999 Summer Scholars Program

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Cloning and Study of the Pol I Largest Subunit

By Jeffery C. Giering
Mentor: Dr. Craig Pikaard
Department of Biology, Washington University, St. Louis, MO

Understanding the growth and development of the cell is vital to our understanding of life, for what is life if not growth and development? The structures that play one of the most important roles are the ribosomes. These complexes of ribosomal ribonucleic acids (rRNA) and proteins perform a critical function, namely the translation of messenger RNA (mRNA) into protein.

The rRNAs within ribosomes perform two functions. First, they act as a sort of skeleton around which the proteins that form the functional ribosome bind. Second, they are important for messenger RNA (mRNA) recognition, without which the ribosomes would be incapable of producing proteins. It is the production of the rRNA that is of most interest to Dr. Craig Pikaard's lab, the transcription of rRNA genes by RNA Polymerase I (pol I).

It is currently understood that there are an equal number of rRNA genes within the genome of every cell in an organism. However, it has been observed that not all cells in an organism produce rRNA at an equal rate. This makes sense, because cells that are not growing and dividing as rapidly as others do not need as much protein nor as many ribosomes to translate these proteins. This means that the cell must possess a mechanism for controlling the number of transcriptionally active rRNA genes and even one to control the number of one of the transcription factors, pol I.

It was my objective to determine whether or not the enzyme pol I is synthesized in equal quantities in all cells and to make a tool along the way that could be used to test other factors that could effect rRNA transcription. Rather than study all of the components of the enzyme, it was far simpler to examine a subunit that was specific to pol I, the largest subunit. The entire process envisioned was to insert the promoter for the transcription of the pol I largest subunit upstream of the coding region for a reporter gene, a gene whose product is clearly visible. The next step would be to insert this recombinant gene into the genome of a plant and infer the relative amount of the subunit from the observed activity of the reporter. For instance, deepening shades of blue in the presence of a particular chemical would indicate a greater activity of the GUS reporter gene. It is expected that the activity of the reporter will be highest in rapidly dividing cells, where ribosomes are most needed. If this is the case, then the reporter gene system used will be a beneficial tool for further studies that seek to determine how hormones and other growth regulators affect RNA polymerase I synthesis and, thus, the transcription of rRNA and ribosome production.

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