Progress has been on the first two of these aims during the current year. Efforts on the third requires completion of the first two. Efforts to localize the phosphorylation sites in GFAP of U-251 cells have placed one phosphorylation site within a 5 kd peptide whose amino terminus lies at amino acid residue 224. This localization is based on N-terminal sequencing of a peptide cleaved from GFAP by Staph. Based on the apparent molecular weight and protease specificity, this peptide is likely to contain 2-3 serine residues and 2 threonine residues.
Work continues to identify the specific amino acid residue that is phosphorylated.
Significant progress toward generation of a null mutation of the GFAP gene has been made. To accomplish this goal a double knockout of the GFAP gene is required in these diploid cells. Work is completed on the generation of two plasmid constructs for disruption of the gene. One of the constructs has produced a successful disruption of the GFAP gene by the more stringent criteria of a Southern analysis. However the quantitative analysis of this clone suggests some heterogeneity of the product.