The goal of this project is to find out how cells with dramatically different requirements for DNA synthesis control the expression of the enzyme ribonucleotide reductase. This enzyme plays a rate-limiting role in DNA synthesis: it controls the first step in de novo DNA synthesis, the reduction of ribonucleotides to deoxyribonucleotides. Reductase consists of two large (M1) and two small (M2) subunits, both of which are required for activity. Reductase activity is tightly coupled to the cell cycle in normal cells, being low in G1 and rising in S phase. Activity is undetectable in sea urchin oocytes and rises rapidly after fertilization. Recent work from my lab using a cloned M2 cDNA shows that this rise occurs as inactive, stored M2 mRNAs are translated after fertilization and suggest that the new M2 subunits join a pool of pre-existing M1 subunits. In contrast to this early translation-level control, late stage embryos appear to use transcriptional controls as well. This project has two immediate aims: (1) The first goal is to find out if (as we predict) the oocyte contains a stockpile of the large, M1 subunit, or if M1 mRNA is stored in the oocyte and translated after fertilization, just like M2 mRNA. To do this, presumptive M1 fractions will be isolated and tested in enzyme activity reconstitution experiments with M1. (2) Next, the number and kinds of M1 and M2 genes, their organization, and their expression in rapidly dividing embryonic cells vs. normal somatic cells will be determined. Our preliminary results indicate that there are two or three M2 genes in the genome. We plan to ask how these genes differ, and if different genes are expressed in rapidly dividing embryonic cells and somatic cells. M2 cDNA clones are already available; M1 cDNA clones will be constructed in an expression vector and selected using antibodies. The results from this work will help us to understand how cells with different division rates control the synthesis of reductase the first enzyme in the de novo DNA synthesis pathway.
