Under most conditions the induction of heme synthesis is required for terminal differentiation of murine erythroleukemia cells (MELC) in vitro. This project investigates the structure and expression of the genes encoding two heme biosynthetic enzymes that may function as regulatory sites for controlling the rate of heme synthesis in erythroid and/or hepatic mouse tissues: Delta-aminolevulinic acid synthase (EC. 2.3.1.37; ALAS) and ferrochelatase (EC. 4.99.1.1; FC). The objective is to develop the molecular probes and background information required for a detailed study of the molecular genetics of heme synthesis and of the subsequent role of heme in MELC differentiations. Rat cDNAs encoding FC and ALAS will be cloned and identified by immunological procedures developed for the cloning of low abundance mRNAs. The rat cDNAs will be used as probes to identify the homologous cloned cDNAs in a library of DMSO-induced MELC RNA. These cDNAs will be sequenced and used as probes (a) to investigate the structure and multiplicity of these genes in MELC and in DBA /2 mouse tissues, and (b) to isolate each FC and ALAS gene from a cosmid library of DBA/2 mouse DNA. RNA mapping techniques will be used to identify the transcription unit of each gene and to investigate whether differential gene expression could account for the different modes of regulation of heme synthesis in erythroid and hepatic mouse tissues. The regulated expression of these genes in inducible MELC will be examined by determining the step of enzyme biogenesis where amplification of gene expression occurs during differentiation. These studies will be augmented by examining the enzymatic and molecular basis of the heme-deficiencies of several DMSO-resistant MELC lines; one of these is known to have an FC deficiency while an ALAS deficiency has been suggested in another. As a prelude to longer term studies on the role of heme in differentiation, we will determine whether induction of heme synthesis is required for commitment of MELC to terminal differentiation or only for the subsequent expression of that commitment.
| Lathrop, J T; Timko, M P (1993) Regulation by heme of mitochondrial protein transport through a conserved amino acid motif. Science 259:522-5 |
