Isolation of recombinant cDNAs encoding chicken erythroid delta-aminolevulinate synthase.

Abstract

We report the isolation of cDNA clones encoding 6-aminolevulinate synthase (ALA synthase; EC 2.3.1.37), the first enzyme in the heme biosynthetic pathway in animal cells. The gene was isolated from a chicken erythroid cDNA library prepared in the bacteriophage X fusion/ expression vector gtll, using rabbit antibody raised against the relatively abundant chicken liver enzyme. The chicken liver and red cell ALA synthase isozymes share substantial crossreactivity to the antibody, thereby allowing isolation of the erythroid-specific gene by using the heterologous antibody in immune screening of the red cell cDNA library. Preliminary analysis documenting the tissue specificity of transcription indicates that the enzyme is encoded by a highly homologous set of messages, which appear to differ in size in various avian tissues. From analysis using strand-specific RNA probes, it appears that the different ALA synthase mRNAs detected may be transcribed from a family of genes that are closely related in nucleotide sequence and are each regulated in a developmentally specific manner.8-Aminolevulinate synthase (ALA synthase; EC 2.3.1.37), the first enzyme in the biosynthetic pathway leading to heme production, catalyzes the condensation of glycine and succinyl-CoA to form 8-aminolevulinic acid (1). The mRNA for the enzyme is encoded in the nucleus, from which it is transported to the cytoplasm, where it is translated into an enzymatically active precursor form. The ALA synthase preenzyme is processed into the mature mitochondrial enzyme (a proteolytic cleavage product of the preenzyme) as it traverses the mitochondrial membrane, where it finally occupies its normal cellular compartment (2).Various studies have previously shown that both the ALA synthase gene and the enzyme are subject to a complex array of regulatory interactions. Thus, the enzyme form found in the liver is responsive to feedback regulation by the end product (heme) and can be induced by a variety of chemical effectors of hepatic porphyria, notably 3,5-dicarbethoxy-1,4-dihydrocollidine and allylisopropylacetamide. Furthermore, this induction appears to be regulated at both the transcriptional and translational levels (3-11). In the liver, heme also appears to directly interfere with the translocation of the preenzyme from the cytoplasmic to the mitochondrial compartments, causing abnormal increases in the cytoplasmic levels of the preenzyme (12, 13).In contrast to these observations in hepatic tissue, the erythroid form of the enzyme is unresponsive to chemicals that normally induce porphyria, and this isozyme does not appear to accumulate in the cytoplasm of erythrocytes on treatment of animals with the same porphyrogenic agents (14-18). Instead, the biosynthesis of ALA synthase in erythroid cells appears to be strongly stimulated by chemical agents normally used for induction of anemia [e.g., phenylhydrazine (G. Kikuchi and M. Hasegawa, personal communication)]. Thus the enzyme activity appears to be regulated in a cell-specific manner. In keeping ...

Conditional expression of the ubiquitous transcription factor MafK induces erythroleukemia cell differentiation.

“…The expression of mRNA for erythroid S-aminolevulinate synthase, which is essential for erythroid cell differentiation (27,30,31) (5,7,22). As shown in Fig.…”

Molecular and Genetic Characterization of Ferrochelatase.

“…In non-erythroid cells, the rate of heme synthesis is controlled by the first pathway enzyme, ALA synthase, at transcriptional and posttranscriptional levels (Yamauchi et al 1980;Yamamoto et al 1982Yamamoto et al , 1983Yamamoto et al , 1988. In differentiating erythroid cells, ALA synthase is not present in the same regulatory mechanism, and a diferent type of ALA synthase is present (Yamamoto et al 1985;Riddle et al 1989). Differentiation occurs after ferrochelatase is induced, even though the other enzymes of the heme biosynthetic pathway are induced earlier (Sassa 1976).…”

Erythropoietin Gene Expression: Developmental-Stage Specificity, Cell-Type Specificity, and Hypoxia Inducibility

“…The Epo gene has been cloned from a variety of mammals as well as fish and frogs (Miyake et al 1977;Jacobs et al 1985;McDonald et al 1986;Shoemaker and Mitsock 1986;Chou et al 2004;Nogawa-Kosaka et al 2010). However, even though anemic chicken plasma has historically exhibited high erythropoietic activity, avian Epo genes have not yet been identified (Yamamoto et al 1985). The human Epo amino acid sequence exhibits an overall sequence homology of 80% to mouse Epo but less than 33% of the fish Epo protein (Chu et al 2008).…”