Abstract
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.
Highlights
We have isolated a genomic clone for rat ALAS using a rat liver cDNA clone (8)as a specific hybridization probe
Since the cDNA clone when used as a probe in Northern blot analysisdetectsa 2.3-kb ALAS mRNA in all rat tissues examined (B),this genomic clone represents the housekeeping ALAS gene.We have reported previously the structureof the chicken housekeeping ALAS gene which spans 6.9 kb and contains 10 exons (13)
A featureof the rathousekeeping ALAS genes is the presence in the 5”untranslated region of a single intron which is absent in the chicken gene
Summary
Ning 22 nucleotides downstream from theinitiationATG codon (Figs. 1 and 2). The oligonucleotide hybridized to the Isolation and Analysis of a Rat Housekeeping ALAS Genomic 1.4-kb PstI fragment in pRG-1, and this PstI fragment was. PlOlB1, and encoded the first N-terminal 20 amino acids of the rat contained the complete coding sequencfeor ALAS precursor liver ALAS precursorprotein (Fig. 2). This confirmed the proteinin a 2.0-kb insert.Usingthisclone as a specific identity of the isolatedgenomic clone. An pRG-1 further, the clonewasdigested with restriction en- examination of the nucleotidesequencein the immediate zymes and analyzedby Southern blotting using as probes theupstream region from these putative initiation sites did not three consecutive PstI fragments of 76, 1,800, and 190 bp, reveal sequence corresponding to any of the known control which comprise plOlBl (8).The 190-bp PstI fragment con- elements commonly found in eukaryotic gene promoters for taining sequence at the3'-end of the ALAS cDNA clone RNA polymerase I1 (28). The 76-bp PstI fragment of acceptor splice site located 21 bp upstream from the ATG
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