The overall objective of our study is to describe how the genetic information of mammalian cells is expressed, as well as to determine how this expression is regulated. During the last granting period our efforts have been directed toward showing that the 16s rna which we have isolated from nucleated erythroid cells is a precursor to globin mRNA, and a description of its sequence organization. During the next granting period we will continue our characterization of the globin mRNA precursor. Our studies with ribonuclease H suggest that the globin mRNA sequences within the precursor are not contiguous. During the next granting period this will be established using another approach. The globin mRNA precursor will be hybridized to full-length globin cDNA and the hybrid exposed to RNase A. The RNase will degrade RNA which is not hybridized to the cDNA. If the globin mRNA sequence is interrupted by silent RNA in the globin mRNA precursor, then these silent regions will not hybridize with the globin cDNA and will form loops. These loops will be sensitive to RNase and when resistant material is analyzed, one should obtain two fragments if one insert occurs, and three fragments if two inserts occur. Authentic globin mRNA hybridized to its cDNA will serve as a control. In this case, a continuous hybrid between the globin mRNA sequence and its cDNA will occur, and the complete RNA will be resistant to nuclease. When this material is analyzed by denaturing polyacrylamide gels, only one RNA will be obtained. Also during the upcoming year, we will search for an enzyme or enzyme system which is capable of processing the globin mRNA precursor. Extracts from various cells will be prepared and incubated with the 16s RNA precursor.
NIH ExPorter
NIH ExPorter