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A 114-base-pair promoter fragment of the human porphobilinogen deaminase gene functioned in an erythroid-specific manner in transient transfection experiments. Site-directed mutagenesis of the binding site for the erythroid-specific transcription factor (NF-E1) or an adjacent CACCC motif abolished the promoter activity. Increasing the spacing between these sites progressively reduced promoter activity, but there was no evidence that a critical alignment of the two factors on the DNA helix was required.

We have studied the erythroid-specific promoter of the human gene coding for Porphobilinogen Deaminase (PBGD) by DNaseI footprinting, gel retardation and methylation interference assays. We show that this promoter, which is inducible during MEL cell differentiation, contains three binding sites for the erythroid-specific factor NF-E1 and one site for a second erythroid-specific factor, which we name NF-E2. NF-E1 is a factor that also binds the promoter and the enhancer (present in the 3' flanking region) of the human beta-globin gene. NF-E2 has not yet been described and although it binds to a sequence containing the Ap1 consensus, it appears to be different from Ap1.

Porphobilinogen deaminase [PBGD; porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] is a cytosolic enzyme involved in the heme biosynthetic pathway. Two isoforms of PBGD, encoded by two mRNAs differing solely in their 5' end, are known: one is found in all cells and the other is present only in erythroid cells. We have previously shown that the human PBGD is encoded by a single gene and have now cloned and characterized this gene, which is split into 15 exons spread over 10 kilobases of DNA. We demonstrate that the two mRNAs arise from two overlapping transcription units. The first one (upstream) is active in all tissues and its promoter has some of the structural features of a housekeeping promoter; the second, located 3 kilobases downstream, is active only in erythroid cells and its promoter displays structural homologies with the beta-globin gene promoters.

In parsley (Petroselinum crispum), phenylalanine ammonia-lyase (PAL) is encoded by 4 structurally similar genes. The nucleotide sequence of a near full-length cDNA and the deduced amino acid sequence of PAL-4 are presented and compared with the corresponding sequences of PAL-1, a previously described representative of the gene family. Transformation of Escherichia coli cells with PAL-1 or PAL-4 cDNA yielded catalytically active PAL, suggesting that the catalytic center of the enzyme is formed spontaneously rather than by a plant-specific mechanism.

The gene hemD taking part in the formation of uroporphyrinogen III from porphobilinogen was mapped by two- and three-factor transduction crosses in Bacillus subtilis. This gene codes uroporphyrinogen III cosynthase. The gene hemD is linked to the hemA locus and is located between the hemA and pheA loci.

Data from four sets of recombinant inbred strains confirm that variation at a single genetic locus is responsible for the previously observed differences in the rate of histidase synthesis in inbred mice. Linkage testing stocks were used to demonstrate linkage with steel (Sl) on chromosome 10.

We have determined the mutation in a patient with acute intermittent porphyria. The mRNA coding for porphobilinogen deaminase was reverse transcribed then the cDNA was enzymatically amplified in vitro. Upon sequencing of a polymerase chain reaction product of abnormal size we found that this fragment lacked exon 12 of the gene. We analysed a genomic fragment containing exon 12 and determined that the patient was heterozygous for a point mutation G A at the last position of exon 12. We propose that this base change is responsible for an abnormal processing of the mutant allele such that exon 12 is missing in the mature mRNA. The resulting aberrant mRNA encodes a truncated protein which is inactive but stable and can be detected using antibodies directed against the normal enzyme.

To investigate the molecular pathology in acute intermittent porphyria (AIP), the nature of the defective porphobilinogen (PBG)-deaminase was determined in erythrocyte lysates from 165 AIP heterozygotes from 92 unrelated families representing 20 different ethnic or demographic groups. Immunologic and physicokinetic studies revealed the occurrence of four classes of PBG-deaminase mutations. In the majority of families studied, the amount of immunoreactive enzyme protein corresponded to the amount of enzymatic activity, indicating the absence of cross-reacting immunologic material (CRIM) produced by the mutant allele. In 78 of these CRIM-negative families (designated type 1), the affected heterozygotes had half-normal PBG-deaminase activity. In three families (designated CRIM-negative type 2), symptomatic patients had increased urinary excretion of delta-aminolevulinic acid and PBG, and normal levels of erythrocyte PBG-deaminase activity. In contrast, noncatalytic, immunoreactive protein was expressed in heterozygotes from 11 families, about one-eighth of those studied, consistent with mutations in the structural gene for PBG-deaminase. Two types of CRIM-positive mutations were identified: the type 1 mutation had a CRIM/activity ratio of approximately 1.7 and a crossed-immunoelectrophoretic profile in which all the enzyme intermediates were increased, with the B or monopyrrole-enzyme intermediate predominant (B greater than A much greater than C congruent to D greater than E). The mutation altered both the kinetic and stability properties of the noncatalytic immunoreactive enzyme protein. The second CRIM-positive mutation, type 2, had markedly increased levels of noncatalytic immunoreactive protein (CRIM/activity ratio approximately 5.7). Crossed-immunoelectrophoresis revealed markedly increased amounts of the substrate-bound intermediates, B, C, D, and E (B greater than C greater than D greater than E much greater than A). The accumulation of these noncatalytic enzyme intermediates presumably resulted from the enhanced binding and/or defective release of substrate molecules. The conformation of these enzyme-substrate intermediates apparently rendered the complexes more resistant to intraerythrocyte proteolysis. These findings provide evidence for the presence of different allelic mutations in the structural gene for PBG-deaminase and document molecular genetic heterogeneity in AIP.

The porphobilinogen deaminase gene encodes the third enzyme of the heme biosynthetic pathway. This gene is expressed in a tissue-specific manner and gives rise to two isoenzymatic forms encoded by mRNA species differing in their 5' extremity. Recent studies in human demonstrated that the tissue-specific expression of the porphobilinogen deaminase gene is determined in erythropoietic cells, by the utilization of a specific promoter situated 3' to the housekeeping promoter used in other cell types. This results, through differential splicing, in the mutually exclusive presence of either exon 1 or exon 2 in mature mRNAs. Here, we report the cloning and sequencing of the porphobilinogen deaminase gene from mouse. The overall organization of the mouse gene is similar to that of the human one. In the housekeeping promoter, only a short stretch of homology is found including two potential Sp1 binding sites; in contrast, more extensive similarity appears in the erythroid-specific promoter including two motifs also found in globin gene, a CACCC box, and a recently described Ery F1 consensus binding sequence. We derived a set of single-stranded probes corresponding to different parts of the mouse gene to carry out a detailed analysis of the transcriptional unit in various cell types, using a run-on transcription assay on isolated nuclei. In liver cells, the first (non-erythropoietic) exon is more actively transcribed than parts of the gene situated downstream, suggesting that the elongation of transcripts is blocked within the 5' part of the first intron. In erythropoietic cells, the downstream promoter becomes activated; surprisingly, the initiation of transcription is also enhanced from the upstream (housekeeping) promoter and most of the transcripts initiated at the housekeeping promoter stop downstream of the first exon, between the two promoters.

The complete nucleotide sequence of the Rhodosporidium toruloides gene coding for the enzyme phenylalanine ammonia-lyase (PAL) has been determined. The primary structure of PAL was deduced from the nucleotide sequence of the two cDNA clones, pPAL1 and pPAL2, which covered the entire amino acid-coding sequence. Comparison of cDNA and genomic sequences of pal revealed the presence of six introns. The nucleotide sequences of these introns were compared to those from other fungi. The primary amino acid sequence of the enzyme exhibits only 30.8% identity with the determined primary sequence of PAL from Phaseolus vulgaris. Upstream from the structural gene there is a stretch of C + T-rich DNA similar to that found upstream from a number of Neurospora and Saccharomyces cerevisiae genes. In the case of S. cerevisiae, these C + T-rich sequences are thought to be involved in the transcription of highly expressed genes.

Two cis-acting sequences, recognized by two erythroid-specific trans-acting factors, are involved in the regulation of the erythroid promoter of the human gene coding for porphobilinogen deaminase (PBGD). The first region, located at -70, binds the erythroid factor NF-E1, and point mutations within this region abolish the induction of transcription of this promoter during murine erythroleukemia (MEL) cell differentiation. The second region, located at -160, binds the erythroid-specific factor NF-E2 and the ubiquitous factor AP1. Using UV cross-linking, we show that NF-E2 has a higher molecular weight than AP1, demonstrating that NF-E2 is not an erythroid-specific degradation product of AP1. By point mutagenesis of the NF-E2/AP1 binding site, we define mutations that abolish binding of either NF-E2 alone or AP1 and NF-E2 together. Regulation of transcription of the PBGD erythroid promoter is abolished by those mutations, suggesting that NF-E2 but not AP1 is necessary for correct regulation of this promoter in erythroid cells.

Based on our recent determinations of the nucleotide sequences of the L-aspartate ammonia-lyase genes from Escherichia coli and Pseudomonas fluorescens, primary structures of the two L-aspartate ammonia-lyases and fumarate hydratases from Bacillus subtilis and E. coli (N-terminal partial sequence) were compared by computer analysis. These four enzymes exhibited a significant homology of at least 37%, implying that L-aspartate ammonia-lyase and fumarate hydratase share a common evolutionary origin. To authors' knowledge, this feature appears to be the first example showing that two kinds of enzymes catalyzing different types of reactions, albeit similar, share such a high degree of sequence homology.

A 1.1-kilobase promoter fragment of the bean (Phaseolus vulgaris L.) phenylalanine ammonia-lyase (EC 4.3.1.5) gene PAL2 was translationally fused to the beta-glucuronidase reporter gene and transferred to tobacco by Agrobacterium tumefaciens-mediated leaf disk transformation. The distribution of beta-glucuronidase activity in these transgenic plants is very similar to that of endogenous PAL2 transcripts in bean, with very high levels in petals; marked accumulation in anthers, stigmas, roots, and shoots; and low levels in sepals, ovaries, and leaves. Histochemical analysis of the spatial pattern of beta-glucuronidase activity showed that the PAL2 promoter is highly active in the shoot apical meristem, the zone of cell proliferation immediately adjacent to the root apical meristem, and in the early stages of vascular development at the inception of xylem differentiation. Wounding and light evoke specific changes in the spatial pattern of beta-glucuronidase activity in stems, including induction in the epidermis. These data indicate that the PAL2 promoter transduces a complex set of developmental and environmental cues into an integrated spatial and temporal program of gene expression to regulate the synthesis of a diverse array of phenylpropanoid natural products.

Octopine and nopaline are two arginine-derived opines synthesized in plant cells transformed with octopine or nopaline plasmids. Utilization in Agrobacterium tumefaciens is mediated by Ti plasmid regions called occ or noc (octopine or nopaline catabolism), and recent experiments showed that noc in pTiC58 codes for a pathway from nopaline to L-proline. The last enzyme is ornithine cyclodeaminase (OCD), an unusual protein converting L-ornithine directly into L-proline. We investigated whether octopine plasmid pTiAch5 also harbors a gene for OCD. The results revealed an ocd gene which is induced by octopine and maps in the occ region. DNA sequence analysis and comparison with the gene from pTiC58 showed that the two genes are related (69% homology in DNA and deduced amino acid sequence), and antiserum against OCD(C58) also reacted with OCD(Ach5). The enzyme activity was characterized, and a comparison with OCD(C58) showed that the properties are similar, but not identical. Differences were detected in the regulation of enzyme activity by L-arginine and L-proline and in the response to varying ratios of NAD+/NADH. It is proposed that this reflects different mechanisms for integration of opine catabolism into general metabolism.

The human gene coding for porphobilinogen deaminase (PBG-D) is transcribed into two distinct transcription units giving two mRNAs. These units originate from two adjacent promoters distant of 3 kilobase pairs. The upstream promoter is active in all cell types, whereas the downstream promoter is active only in erythroid cells. We have studied the expression of this gene either after introduction of the corresponding human chromosome into murine erythroid cells using somatic hybrids or after transfection into both erythroid and non-erythroid cells. Using somatic hybrids, we showed that activation of the erythroid-specific promoter of the PBG-D gene did not reduce the rate of initiation of the ubiquitous promoter. Transfection experiments in erythroid cells showed that the PBG-D erythroid transcription unit, controlled by the PBG-D erythroid promoter, was correctly transcribed and regulated. Furthermore, we found that the PBG-D erythroid promoter alone was sufficient for correct expression and regulation of a reporter gene during erythroid differentiation. When the human PBG-D gene was transfected into non-erythroid cells, only the ubiquitous promoter was active. Deletion of the ubiquitous promoter did not lead to any activation of the erythroid promoter, suggesting that its inactivity in non-erythroid cells was not due to promoter occlusion but to a strict erythroid specificity.