During erythroid differentiation the activities of most, if not all, of the heme biosynthetic enzymes are increased. The mechanisms underlying these increases are unknown, but most recent data suggest that a mechanism of coordinate activation may be involved. For at least two of the heme biosynthetic genes activation appears to result in the production of erythroid- specific mRNAs. Superimposed on the general activation of these enzymes is a feedback control circuit in which heme acts to regulate the net flux of porphyrin substrates to the terminal enzyme in the pathway (ferrochelatase, FC) by modulating the expression of the first enzyme in the pathway (delta- aminolevulinic acid synthase, ALAS). This project investigates the structure and regulated expression of the murine ALAS and FC genes, with the ultimate goals of understanding the molecular mechanisms and genetic signals underlying coordinate activation, feedback repression of ALAS, and the coordination of heme and apoprotein synthesis during erythroid differentiation. cDNA libraries constructed from differentiated murine erythroleukemia cells (MELC) and porphyric liver will be screened with a heterologous ALAS cDNA probe to identify cDNAs encoding two DBA/2 mouse ALAS mRNAs that are differentially inducible in MELC and liver. The structures of these mRNAs will be determined by cDNA sequencing. Putative bovine liver and MELC FC cDNA clones will be verified by in vitro translation of hybridization-selected mRNA and DNA sequencing. Each of the murine ALAS and FC cDNAs will be used as probes in RNA blotting experiments to examine the diversity and physiological expression of ALAS and FC mRNAs iN a variety of DBA/2 mouse tissues. A combination of DNA blotting and cosmid cloning will then be used to isolate each of the FC and ALAS genes from the DBA/2 genome. RNA mapping techniques and DNA sequencing will be used to deduce the structure and coding potential of each gene. The structure and expression of these genes will be examined in MELC and Hepa lclc7 hepatoma cells to determine whether these cell lines can be used as models for the physiological expression of these genes in erythroid and hepatic tissues, respectively. These systems will then be exploited to investigate the molecular mechanisms and genetic signals involved in the regulated expression of each gene.
| Lathrop, J T; Timko, M P (1993) Regulation by heme of mitochondrial protein transport through a conserved amino acid motif. Science 259:522-5 |
