This proposal is intended to study the molecular processes which control the differentiation of hematopoietic cells and to apply such basic knowledge within the field of pediatric hematology- oncology. The first phase of this proposal will be a research project in the field of eukaryotic gene trans-regulation. Animal cells infected with Herpes Simplex Virus type 1 accumulate proteins in three kinetic classes. Immediate early (IE) viral proteins are required for the expression of both delayed early (DE) and late (L) viral genes. Evidence indicates that IE proteins induce DE and L gene expression by affecting both transcription initiation and a subsequent rate-limiting step in mRNA accumulation. The subject of this project is this second step in control of DE and L gene expression. Frog oocytes will be microinjected with either the DE gene, thymidine kinase, or the L gene, glycoprotein C, and with artificial mRNAs which encode IE proteins. RNA initiation will be assayed by primer extension using as primer a genomic probe near but not including the 5' terminus of the gene to be assayed. mRNA accumulation will be assayed by S1 analysis using as a probe a genomic fragment which encompasses the 3' RNA polyadenylation site. Artificial mRNAs will be created from a cDNA library from virally infected animal cells treated with cycloheximide to increase IE mRNA production. Desired clones will be selected with IE genomic 3' terminus probes, ligated to proper 5' genomic restriction fragments, capped with guanylyltransferase, and placed in SP-6 RNA expression vectors. By varying the concentrations and combinations of IE mRNAs used in the microinjection protocols, I hope to verify the existence of the second rate-limiting step in control of DE and L gene expression and to determine how IE proteins might differentially regulate DE and L gene expression. The second phase will investigate hematopoiesis, perhaps by identifying lineage specific RNA transcripts and analyzing their DNA and protein controlling elements within the cell.
