Identification of regulatory sequences in the gene for 5-aminolevulinate synthase from rat.

The housekeeping enzyme 5-aminolevulinate synthase (ALAS) regulates the supply of heme for respiratory cytochromes. Here we report on the isolation of a genomic clone for the rat ALAS gene. The 5'-flanking region was fused to the chloramphenicol acetyltransferase gene and transient expression analysis revealed the presence of both positive and negative cis-acting sequences. Expression was substantially increased by the inclusion of the first intron located in the 5'-untranslated region. Sequence analysis of the promoter identified two elements at positions -59 and -88 bp with strong similarity to the binding site for nuclear respiratory factor 1 (NRF-1). Gel shift analysis revealed that both NRF-1 elements formed nucleoprotein complexes which could be abolished by an authentic NRF-1 oligomer. Mutagenesis of each NRF-1 motif in the ALAS promoter gave substantially lowered levels of chloramphenicol acetyltransferase expression, whereas mutagenesis of both NRF-1 motifs resulted in the almost complete loss of expression. These results establish that the NRF-1 motifs in the ALAS promoter are critical for promoter activity. NRF-1 binding sites have been identified in the promoters of several nuclear genes encoding mitochondrial proteins concerned with oxidative phosphorylation. The present studies suggest that NRF-1 may co-ordinate the supply of mitochondrial heme with the synthesis of respiratory cytochromes by regulating expression of ALAS. In erythroid cells, NRF-1 may be less important for controlling heme levels since an erythroid ALAS gene is strongly expressed and the promoter for this gene apparently lacks NRF-1 binding sites.

Characterisation of cis-acting DNA sequences required for the expression of the chicken 5-aminolevulinate synthase gene in Xenopus oocytes.

In this paper, we have constructed deletion and deletion-insertion mutations of the chicken 5-aminolevulinate synthase 5' flanking region and examined the expression of these constructs in microinjected Xenopus oocytes. Utilising this assay, we have delimited the boundary of the 5' flanking region required for expression to be 80 bp upstream from the transcription initiation site. A second major focus of this study has been to define the role of known putative cis-acting sequences in regulating 5-aminolevulinate synthase gene expression. Expression of the insertion-deletion mutants demonstrated that only a TATA box at position -28, and a single GC box at position -78 was necessary for expression of the 5-aminolevulinate synthase gene in Xenopus oocytes. This result is unusual in view of the current state of knowledge of the function of cis-acting sequence elements in transcriptional regulation.

5-Aminolevulinate synthase is at 3p21 and thus not the primary defect in X-linked sideroblastic anemia.

The gene for 5-aminolevulinate synthase (ALAS) has been mapped to 3pter-3q13.2 by Southern blot hybridization analysis of a mouse/human hybrid cell panel. In situ hybridization maps the gene to 3p21, distal to the common fragile site at 3p14.2 (FRA3B). The mapping of this gene to an autosome makes it improbable that it is the site of the primary defect in X-linked sideroblastic anemia.

Regulation of 5-aminolevulinate synthase mRNA in different rat tissues.

cDNA clones for rat liver 5-aminolevulinate synthase have been isolated and used to examine mRNA levels in different rat tissues. Northern hybridization analysis of total RNA from various rat tissues showed the presence of a single 5-aminolevulinate synthase mRNA species of estimated length 2.3 kilobases. Primer extension and RNase mapping studies indicated that the mRNA is identical in all tissues. Highest basal levels were seen in liver and heart. Administration of hemin to rats reduced the basal level of this mRNA only in liver but the heme precursor, 5-aminolevulinate (or its methyl ester), repressed the basal levels in liver, kidney, heart, testis, and brain. The drug 2-allyl-2-isopropylacetamide increased the mRNA level in liver and kidney only while human chorionic gonadotropin hormone elevated the level in testis. Administration of the heme precursor 5-aminolevulinate prevented these inductions. Nuclear transcriptional run-off experiments in liver cell nuclei showed that 2-allyl-2-isopropylacetamide and 5-aminolevulinate exert their effect by altering the rate of transcription of the 5-aminolevulinate synthase gene. The results indicate that a single 5-aminolevulinate synthase mRNA is expressed in all tissues and that its transcription is negatively regulated by heme.

A unique gene for 5-aminolevulinate synthase in chickens. Evidence for expression of an identical messenger RNA in hepatic and erythroid tissues.

Reports to date have led to the conclusion that there are isozymes for 5-aminolevulinate synthase in the liver and erythroid tissue of chicken. Indeed, the existence of a multigene family for chicken 5-aminolevulinate synthase has been proposed. We find no evidence to support these proposals. In this work we show that 5-aminolevulinate synthase mRNA from chicken liver and reticulocytes is identical as determined by RNase mapping and primer extension studies and that the 5-aminolevulinate synthase protein from these tissues is the same size as judged by immunoblot analysis. We also show that a single mRNA species for 5-aminolevulinate synthase is present in chicken liver, reticulocytes, brain, and heart and an avian erythroblastosis virus-transformed chicken erythroblast cell line. Southern analysis shows the presence of only one gene copy for 5-aminolevulinate synthase in the chicken haploid genome. Overall, these results lead to the conclusion that in chickens 5-aminolevulinate synthase is encoded by a unique gene and is expressed as a single mRNA species in all tissues.

Nucleotide sequence of the chicken 5-aminolevulinate synthase gene.

5-Aminolevulinate synthase, the first and rate-controlling enzyme of heme biosynthesis, is regulated in the liver by the end-product heme. To study this negative control mechanism, we have isolated the chicken gene for ALA-synthase and determined the nucleotide sequence. The structural gene is 6.9 kb long and contains 10 exons. The transcriptional start site for ALA-synthase was determined by primer extension analysis. A fragment of 291 bp from the 5' flanking region including 34 bp of the first exon shows promoter activity when introduced upstream of a chicken histone H2B gene and injected into the nuclei of Xenopus laevis oocytes.

Control of 5-aminolevulinate synthase in animals.

The proposed mechanism by which hepatic ALV-synthase mitochondrial levels are regulated is outlined in Fig. 2. ALV-synthase catalyzes the first and rate-limiting step in the heme pathway and is normally present in low amounts. A cytosolic, regulatory free heme pool tightly controls the amount of ALV-synthase in two ways. In the primary mechanism of regulation, heme is proposed to inhibit the synthesis of ALV-synthase mRNA. Most likely this would be mediated through the action of specific heme-binding protein(s) which recognize regulatory control regions of the ALV-synthase gene. Gene activity therefore is significantly repressed most of the time. When there is an increased demand for heme by newly synthesized cellular hemoproteins, the free heme pool is reduced, leading to a derepression of ALV-synthase mRNA synthesis. Once the need for increased heme synthesis is satisfied, inhibitory heme levels build up again. When drugs such as phenobarbital are administered to animals, there is a rapid induction in the liver of both cytochrome P-450 and ALV-synthase. It is proposed that the heme pool governing ALV-synthase levels is lowered by the increased heme demand due to cytochrome P-450 apoprotein formation. The primary event in the drug induction of ALV-synthase is therefore the increased synthesis of cytochrome P-450 apoprotein. However, the mechanism by which this occurs is unknown, although drugs do increase the synthesis of mRNA for cytochrome P-450 (Fig. 2). (There is evidence that for the aromatic hydrocarbons a specific cytosolic receptor exists.) In the acute hepatic porphyria diseases, uncontrolled synthesis of hepatic ALV-synthase occurs. The various forms are characterized by reduced levels of one of the heme pathway enzymes other than ALV-synthase. Attacks of the disease are commonly precipitated by drugs which induce cytochrome P-450, and the uncontrolled accumulation of ALV-synthase which accompanies these attacks results from the combined action of the block in the heme pathway and the increased cytochrome P-450 levels. A major challenge which now exists is to understand at the molecular level how the genes for ALV-synthase and cytochrome P-450 are regulated in the liver and other tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Complete nucleotide sequence of hepatic 5-aminolaevulinate synthase precursor.

Chick embryo liver mitochondrial matrix protein, 5-aminolaevulinate synthase, is synthesised initially as a larger cytosolic precursor. In this report we present the complete nucleotide sequence of a cDNA clone coding for the precursor together with corresponding confirmatory amino acid sequence of peptides derived from purified mature mitochondrial enzyme. The deduced amino acid sequence shows that the precursor consists of mature enzyme of 579 amino acids and an N-terminal extension of 56 amino acids. The latter presequence is highly basic in character as found with other mitochondrial preproteins.

Electron microscopic studies on liver 5-aminolaevulinate synthase.

The structure of chick embryo liver 5-aminolaevulinate synthase has been examined by electron microscopic studies using negative staining. From the different projections of the enzyme particles observed in electron micrographs, a model for the enzyme molecule has been proposed. In this model, an enzyme molecule consists of two curved and identical subunits associated in opposite polarities. From the dimensions of an enzyme molecule subunit measured from electron micrographs, the relative molecular mass of each subunit is estimated to be 70 000.

Molecular cloning of hepatic 5-aminolevulinate synthase.

Hepatic 5-aminolevulinate synthase was induced in chick embryos by administration of the porphyrinogenic drugs 2-allyl-2-isopropylacetamide and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. A cDNA library was constructed from drug-induced liver mRNA and clones containing sequences coding for 5-aminolevulinate synthase were identified by hybrid-selected translation. The identity of these clones was confirmed by comparing DNA sequence data with the amino acid sequence of peptides from purified 5-aminolevulinate synthase. From Northern blot analysis the size of the mRNA for 5-aminolevulinate synthase was estimated to be 2800 base pairs, approximately 600 base pairs more than that required to code for the primary translation product of relative molecular mass 74000.

Effect of lead ions on chick-embryo liver mitochondrial delta-aminolaevulinate synthase.

Pb2+ activated native chick-embryo liver mitochondrial delta-aminoaevulinate synthase (EC 2.3.1.37). This result contradicted with the inhibitory effect observed by earlier workers who used degraded enzyme preparations. Enzyme activation was biphasic. An initial activation phase was observed with Pb2+ concentrations up to 200 microM, and a secondary phase with concentrations from 200 microM to at least 2mM. Maximum primary activation was 2.5-fold at 200 microM-Pb2+, with a further 2-fold activation observed at 2mM-Pb2+. Primary activation was not affected by a 10-fold molar excess of dithioerythritol, but the secondary activation was abolished by dithioerythritol. Secondary-phase activation was lost upon increasing time of incubation of the enzyme with Pb2+. The implications of these findings are discussed with reference to lead poisoning and the mechanism of delta-aminolaevulinate synthase.

Effect of heme on the activity of chick embryo liver mitochondrial delta-aminolevulinate synthase.

We have examined the effect of heme on the activity of native delta-aminolevulinate synthase isolated from drug-induced chick embryo liver mitochondria. The enzyme was not inhibited by concentrations of heme up to 1 mM and this finding makes it improbable that heme acts physiologically to control mitochondrial delta-aminolevulinate synthase activity.

Evidence that 2-allyl-2-isopropylacetamide, phenobarbital and 3,5-diethoxycarbonyl-1,4-dihydrocollidine induce the same cytochrome P450 mRNA in chick embryo liver.

The induction of cytochrome P450 in chick embryo liver has been studied using three different porphyrinogenic drugs, 2-allyl-2-isopropylacetamide, 3,5-diethoxycarbonyl-1,4-dihydrocollidine and phenobarbital. Pulse-labelling studies have shown that for each drug the cytochrome P450 synthesized either in ovo or in a wheat germ translation system reacted immunologically with antibody raised against the purified 2-allyl-2-isopropylacetamide-induced enzyme (Mr = 50000). To investigate whether this is due to the three drugs inducing the same protein or different proteins with common immunological determinants, nucleic acid hybridization studies have been carried out using a recently characterised 2-allyl-2-isopropylacetamide-induced cytochrome P450 cloned cDNA probe [Brooker, J. D. et al. (1982) Eur. J. Biochem. 129, 325-333]. It has been shown that the mRNA induced by each drug hybridizes with this probe and all are of similar size. The melting profile of the mRNA . cDNA hybrids indicates that the mRNAs induced by the three drugs have at least 98% homology with the cDNA probe. Restriction endonuclease digestions of total chick embryo genomal DNA and a chick cytochrome P450 genomal clone indicates that the cytochrome P450 gene homologous with the cDNA probe is represented in the genome only once. These results strongly suggest that the three drugs cause increased levels of the same cytochrome P450 mRNA, possibly due to enhanced expression of the same gene. Results are also presented which show that other cytochrome-P450-inducing drugs, 3-methylcholanthrene, beta-naphthoflavone or pregnenolone-16 alpha-carbonitrile do not increase the level of the 2-allyl-2-isopropylacetamide-inducible mRNA but rather reduce it to a level which was lower than that of the untreated controls.

Hemin inhibits transfer of pre-delta-aminolevulinate synthase into chick embryo liver mitochondria.

Pulse labelling studies in chick embryo livers show that hemin prevents the transfer of drug induced pre-delta-aminolevulinate synthase from the cytosol into the mitochondria, leading to an accumulation of precursor in the cytosol. No effect of hemin was observed on the transfer of pre-pyruvate carboxylase into mitochondria. These results eliminated a general toxic effect of hemin on mitochondrial import of proteins and are consistent with the view that hemin specifically inhibits the transfer of ALA synthase.

Purification of 5-aminolaevulinate synthase from liver mitochondria of chick embryo.

5-Aminolaevulinate synthase from chick-embryo liver mitochondria has, for the first time, been purified to homogeneity in its native non-degraded form by molecular sieve chromatography, chromatofocusing and affinity chromatography. The enzyme has a minimum molecular weight of 68000 as determined by sodium dodecylsulphate/polyacrylamide gel electrophoresis and a specific activity of 35000 units/mg of protein. This result conflicts with the previous report of Whiting, M.J. and Granick, G. [(1976) J. Biol. Chem. 251, 1340-1346] that the chick embryo enzyme has a molecular weight of 49000. We show here that the purified form can be degraded proteolytically to a smaller form of molecular weight around 50000 while retaining full enzymatic activity. It seem evident, therefore, that the enzyme isolated by Whiting & Granick (1976) was degraded. We have further established by pulse-labelling studies and immunoprecipitation that the enzyme isolated by our new and rapid procedure has the same minimum molecular weight as that which exists in vivo.

Evidence for a cytosolic precursor of chick embryo liver mitochondrial delta-aminolevulinate synthase.

Following the recent demonstration [Borthwick, I.A., Srivastava, G., Brooker, J.D., May, B.K. and Elliott, W.H. (1982) Eur. J. Biochem. in press] that chick embryo liver mitochondrial delta-aminolevulinate synthase has a minimum molecular weight of 68,000 (rather than the hitherto accepted value of 49,000), we have shown that the primary translation product of delta-aminolevulinate synthase mRNA is a protein of molecular weight 74,000. This protein has for the first time been shown to occur in the cytosol fraction of drug-treated chick embryo livers. This form does not occur in mitochondria nor does the smaller mitochondrial form occur in the cytosol. It is concluded that the 74,000 molecular weight protein is a precursor which is processed during transport into the mitochondria. In vivo labelling experiments are consistent with this conclusion.