The long-term objectives of our studies are to determine the biochemical mechanisms which regulate the biogenesis of liver delta-aminolevulinate synthase (ALA synthase). A relatively large number of agents induce the synthesis of the enzyme in liver, and elevated activities of ALA synthase in man have been associated with hereditary hepatic porphyrias. The proposed studies will focus mainly on the mechanism by which hemin regulates the synthesis of the enzyme in embryonic chick livers. Initially, a cDNA for ALA synthase mRNA will be prepared by molecular cloning, and the cDNA will be used as a probe in subsequent studies. The possible effects of hemin on the transcription of ALA synthase mRNA and on the maturation of the messenger will be investigated then by RNA.cDNA hybridization analyses. The possibility that hemin blocks translation of ALA synthase mRNA will be examined also by measurements on the distributions of the messenger in polysomes. The effects of hemin on the binding of pre-ALA synthase to isolated outer mitochondrial membranes and on the translocation of pre-ALA synthase into mitochondria will be studied in cell-free preparations; this will be to determine if hemin can block directly the biogenesis of ALA synthase at the level of translocation of the protein into mitochondria. Finally, the nucleotide sequence of ALA synthase mRNA will be determined by sequencing cloned cDNA prepared with ALA synthase mRNA serving as the template.