Nucleic Acids Research: VOLUME 40 DATABASE ISSUE JANUARY 1, 2012

We have cloned and sequenced a cDNA clone coding for human erythrocyte porphobilinogen deaminase. It encompasses the translated region, part of the 5' and the 3' untranslated regions. The deduced 3tt amino acid sequence is consistent with the molecular weight and the partial amino-acid sequence of the NH2 terminal of the purified erythrocyte enzyme. Southern analysis of human genomic DNA shows that its gene is present as a single copy in the human genome and Northern analysis demonstrates the presence of a single size species of mRNA in erythroid and non-erythroid tissues and in several cultured cell lines. Quantitative determinations indicate that the amount of PBG-D mRNA is modulated both by the erythroid nature of the tissue and by cell proliferation, probably at the transcriptional level. INTRODUCTION Porphobilinogen deaminase (PBG-D, EC t-3-1.8) is the third enzyme of the biosynthetic pathway leading to the production of heme. It catalyses the head to tail condensation of four molecules of the raonopyrrole porphobilinogen, to form the linear tetrapyrrole, hydroxymethylbilane which is then converted by uroporphyrlnogen III synthase to uroporphyrinogen III (1). In humans, deficiency in its activity is responsible for a dominant hereditary disease: Acute Intermittent Porphyria (A.I.P.) (2). Studies using antibodies specifically directed against human erythrocyte PBG-D show that inactive cross reacting immunological material is present in about 20$ of patients with this disorder but absent from the remainder (3). Therefore Acute Intermittent Porphyria appears to be, at the molecular level, a heterogeneous disorder. Although widely distributed in tissues, the enzymes of the heme biosynthetic pathway are particularly active In the liver where heme synthesis is regulated mainly by the activity of 6-aminolevulinic acid synthase (1) and in bone marrow, where the activity of all the enzymes of the pathway progressively augment during erythroid differentiation. Thus the control of > IRL Press Limited, Oxford, England. 5955 Nucleic Acids Research heme biosynthesis in erythroid cells and in hepatocytes appears to be different and a recent report (5) suggests that Porphobilinogen Deaminase may be the primary enzyme controlling heme synthesis during erythropoietin induced erythroid differentiation. Furthermore, Grandchamp et al. (6) have shown that in Mouse Erythroleukemia Cells (M.E.L.), capable of undergoing in vitro erythroid differentiation, induction by DMSO is followed by an increase in the copy number of Porphobilinogen Deaminase mRNA. This process precedes accumulation of a and B globin mRNAs, indicating a concerted and time dependent regulation. This paper reports the cloning and analysis of a cDNA sequence complementary to human erythrocyte PBG-D mRNA. It contains a reading frame of 1029 bp which encodes for 311) amino-acids, a 5' non coding region of 81 bp and a complete 3' non coding region of 266 bp excluding the poly(A) tail. Using this cDNA as a hybridization probe, we confirm that PBG-D is encoded by a single copy gene per haploid genome and show by Northern blot analysis that there is only one size species of mRNA in erythroid and non-erythroid tissues and in cultured cell lines. Furthermore we demonstrate that the concentration of PBG-D mRNA is modulated in tissues undergoing erythroid differentiation in a manner that is different to that in immortalized cell lines where the PBG-D gene seems to have the same rate of transcription irrespective of the cell phenotype. MATERIALS AND METHODS General procedures A previous communication (7) described the methods used for in Yitro translation in a messenger-dependent rabbit reticulocyte cell-free system, PBG-D immunoprecipitation from in vitro translation products, and SDSpolyacrylamide gel electrophoresis. Cell culture The different cell lines studied were a gift from Dr. U. Testa. Cells were grown in suspension culture in a modified RPMI 16<1O medium containing 10$ heat inactivated newborn fetal calf serum plus 2% human serum, in a humidified atmosphere with 5% CO2. All cell lines were grown exponentially to a density of 10^ to 10& cells per ml. Plasaid Isolation, insert purification and nick translation Plasmids containing rat or human PBG-D cDNA inserts were prepared from chloramphenicol treated bacteria by thermal denaturation and sedimentation of chromosomal DNA (8), followed by treatment with RNAase and proteinase K and extraction with buffered phenol. The recombinant plasmids were further