Molecular characterization of a novel gene family (PHTF) conserved from Drosophila to mammals.

PHTF1 (putative homeodomain transcriptional factor; HGMW-approved symbol PHTF1) is a putative homeobox gene located at band 1p11-p13 of the human genome. We report here the cloning and sequencing of its mouse and Drosophila orthologs. The conservation between mouse and human proteins extends over the entire protein and is localized at the putative homeodomain and at the N- and C-terminal regions of Drosophila protein sequence. Blast searches allowed us to identify another member of the PHTF family, PHTF2, located at 7q11.23-q21 of the human genome. The strongest homologies between human PHTF1 and PHTF2 are localized to the domains that we already described in Drosophila, i.e., the putative homeodomain and the N- and C-terminal regions. The human and mouse genes display 98% similarity to one another, 56% similarity with the Drosophila gene, and 67% similarity with PHTF2, suggesting that phtf might define a novel gene family of highly divergent homeobox genes. Finally, Northern blot analysis showed that while PHTF1 is expressed mainly in testis, PHTF2 is predominantly expressed in muscle, suggesting that these two genes may have acquired different functions after their duplication and divergence.

Initiation of transcription of the erythroid promoter of the porphobilinogen deaminase gene is regulated by a cis-acting sequence around the cap site.

Although the erythroid-specific promoter of human porphobilinogen deaminase [PBGD] gene has no TATA box, transcription is initiated at a single nucleotide. Using 5' and 3' deletions and point mutations, we have identified an element, located around the initiation site, which is necessary and sufficient for 'in vitro' accurate initiation of transcription. This 15 bp element extends 1 bp 5' and 14 bp 3' from the initiation site. It is composed of two regions, a proximal region centred on the cap site and a distal region which bears homology with the TdT initiator element. We show that a nuclear factor, present both in erythroid and non erythroid cells, binds the distal PBGD initiator element. Lack of heat inactivation suggests that initiation of transcription mediated by this element is not TFIID dependent. By transfection into erythroid cells, we also show that the proximal PBGD initiator element is essential for the selection of the initiation site but not for the regulation of transcription of the PBGD erythroid promoter during erythroid differentiation.

Regulated expression of the overlapping ubiquitous and erythroid transcription units of the human porphobilinogen deaminase (PBG-D) gene introduced into non-erythroid and erythroid cells.

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.

Alternative transcription and splicing of the human porphobilinogen deaminase gene result either in tissue-specific or in housekeeping expression.

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.

Structure of the gene for human uroporphyrinogen decarboxylase.

Uroporphyrinogen decarboxylase, the fifth enzyme of the heme biosynthetic pathway, is an housekeeping enzyme whose activity is enhanced during erythropoietic differentiation. We have previously shown that this increased activity was in part accounted for by an enhanced transcription of the gene in erythropoietic tissues. To elucidate further the tissue specific regulation of an housekeeping gene we have isolated the human URO-D gene and determined its organization. The cloned gene comprises 10 exons spread over 3 Kb. Two transcriptional start sites were determined and analysis of 900 bp of the 5' flanking region suggests a very simple structural organization for the URO-D gene promoter. We also show that this gene is functional when transfected into mouse fibroblasts, and that its promoter is sensitive to a viral enhancer.

Molecular cloning and complete primary sequence of human erythrocyte porphobilinogen deaminase.

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 344 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.

Assignment of human uroporphyrinogen decarboxylase (URO-D) to the p34 band of chromosome 1.

A cDNA probe corresponding to mRNA encoding human uroporphyrinogen decarboxylase (URO-D) was used to determine the chromosomal localization of the URO-D gene in the human genome. In agreement with previous studies, we have found that the locus for URO-D is located on chromosome 1 in hybrid cell mapping panels. The use of in situ hybridization allowed us to map the URO-D locus to band 1p34.

Molecular cloning and nucleotide sequence of a complete human uroporphyrinogen decarboxylase cDNA.

We have cloned and sequenced a full-length cDNA coding for human uroporphyrinogen decarboxylase. The deduced 367-amino acid sequence is consistent with the molecular weight, the partial amino acid sequence of cyanogen bromide peptides, and the total amino acid composition of the purified 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. We have also demonstrated that the level of uroporphyrinogen decarboxylase mRNA is markedly increased in tissues or cell lines of erythroid origin and that this is due to a tissue-specific transcriptional activation of the uroporphyrinogen decarboxylase gene.

Line-restricted hemoglobin synthesis in chick embryonic erythrocytes.

The presence of embryonic hemoglobin in early definitive erythrocytes was checked by indirect immunofluorescence assay, using specific antibodies raised against embryonic Hb P. As positive control we used anti-Hb A which reacted with the alpha A chain shared by the minor embryonic Hb E and the adult Hb A. The assay was performed using blood smears from embryos between 6 and 15 days of incubation and yolk sac sections from embryos between 4 and 6 days. Hb P was never detected in the definitive line in circulating erythrocytes or in maturing erythroblasts still sequestered in the blood islands of the yolk sac. The expression of the 'specific' embryonic genes is thus restricted to the primitive line (as the 'specific' adult beta gene is restricted to the definitive line), and the hemoglobin switch is the result of the progressive substitution of the primitive line by the definitive one.

Erythropoiesis and haemoglobin ontogeny in the turtle Emys orbicularis L.

The erythropoietic sites and developmental patterns of haemoglobins have been investigated during ontogeny of Emys orbicularis. The yolk-sac blood islands seem to be the unique erythropoietic site during most of embryonic life. Bone marrow haemopoiesis is first observed in young turtles aged one year. The cortical haemopoietic layer of the liver appears involved mainly in granulopoiesis. There is no morphologically well-defined series of primitive or definitive erythrocytes. Rather there is a gradual shift in size from a mean length of 17.4 micrometer in embryos to 19.9 micrometer in the adult. However the size of erythrocytes is highly variable at all stages. Three haemoglobins of adult type and three haemoglobins of embryonic type have been identified by electrophoretic separation. It seems that one haemoglobin is synthesized during the whole life. Embryonic haemoglobins persist for more than a year after hatching while the typically adult haemoglobins appear shortly before hatching.

Developmental relationships between vitelline and intra-embryonic haemopoiesis studied in avian 'yolk sac chimaeras'.

Using the quail-chick marker technique, cells have been traced in the haemopoietic organs of 149 developing chimaeras composed of a quail embryo with a chick yolk sac. The existence of intraembryonic stem cell relaying yolk sac stem cells, previously demonstrated with this system, is confirmed. Thymus, bursa of Fabricius and bone marrow are preferentially populated by intraembryonic stem cells. The spleen shows a transitory phase of colonization by chick yolk sac stem cells at 11--12 days of incubation. From 7 days onwards the yolk sac receives quail stem cell emigrating from the embryo.

Origin of erythropoietic stem cells in avian development: shift from the yolk sac to an intraembryonic site.

Haemopoietic stem cells are currently considered as deriving exclusively from the embryonic yolk sac in birds and mammals. A new experimental approach has been devised: quail embryonic bodies are grafted on to chick yolk sacs before establishment of circulation, and allowed to develop. The proportions of quail and chick erythrocytes in the circulating blood are assayed through a differential immunohaemolysis technique. Up to 10 days of incubation, grafted quails have 80 to 95% chick erythrocytes. Between 10 and 13 days, quail erythrocytes appear in increasing proportions making up as much as 80% of the red blood cells in some embryos. Morphological analysis reveals that chick cells colonize transiently the spleen of grafted embryos between 10 and 12 days, and undergo erythropoiesis in that organ. However at 13 days chick cells have disappeared from the spleen of grafted embryos. It is concluded that yolk sac stem cells are capable of colonizing intraembryonic organs, but do not do permanently and that there is an intraembryonic source of definitive erythropoietic stem cells.