Structure-function analysis of the EGF-CFC family member Cripto identifies residues essential for nodal signalling.

Cripto is the founding member of the family of EGF-CFC genes, a class of extracellular factors essential for early vertebrate development. In this study we show that injection of Cripto recombinant protein in mid to late zebrafish Maternal-Zygotic one-eyed pinhead (MZoep) blastulae was able to fully rescue the mutant phenotype, thus providing the first direct evidence that Cripto activity can be added extracellularly to recover oep-encoded function in zebrafish early embryos. Moreover, 15 point mutations and two deletion mutants were generated to assess in vivo their functional relevance by comparing the ability of cripto wild-type and mutant RNAs to rescue the zebrafish MZoep mutant. From this study we concluded that the EGF-CFC domain is sufficient for Cripto biological activity and identified ten point mutations with a functional defective phenotype, two of which, located in the EGF-like domain, correspond to loss-of-function mutations. Finally, we have developed a three-dimensional structural model of Cripto protein and used it as a guide to predict amino acid residues potentially implicated in protein-protein interaction.

A truncated form of teratocarcinoma-derived growth factor-1 (cripto-1) mRNA expressed in human colon carcinoma cell lines and tumors.

The human teratocarcinoma-derived growth factor (TDGF)-1 gene encodes a 188-amino acid protein, cripto-1. The TDGF-1 gene is overexpressed in the majority of human primary colorectal carcinomas and hepatic metastases, in breast carcinomas and in testicular nonseminoma germ cell embryonal carcinomas. In the human embryonal carcinoma cell line NTERA-2 clone D1, a 2-kb TDGF-1 mRNA transcript is expressed. The present study shows that a 1.7-kb mRNA transcript lacking the first two exons of the TDGF-1 gene is expressed in the human colon carcinoma cell line GEO. This shorter mRNA is the only TDGF-1 transcript that is present in the majority of primary human colorectal carcinomas and hepatic metastases and in adult human tissues such as the pancreas, heart, stomach, mammary gland, skeletal muscle, liver and placenta. In contrast, in the kidney, brain, testis, ovary and spleen, the longer 2-kb TDGF-1 mRNA transcript is expressed. The putative shorter protein starts at a CUG codon 129 nucleotides downstream of the starting AUG codon of the longer protein. These data indicate the potential for differential transcriptional regulation of the TDGF-1 gene in different normal and tumor tissues.

Membrane-anchorage of Cripto protein by glycosylphosphatidylinositol and its distribution during early mouse development.

cripto is the original member of the family of EGF-CFC genes, recently recognized as novel extracellular factors essential for vertebrate development. During the early stages of mouse gastrulation, cripto mRNA is detected in mesodermal cells; later, cripto mRNA is detected only in the truncus arteriosus of the developing heart. Here we describe the in vivo distribution of Cripto protein throughout mouse embryo development and show that cripto mRNA and protein colocalize. By means of immunofluorescence analysis and biochemical characterization, we show that Cripto is a membrane-bound protein anchored to the lipid bilayer by a glycosylphosphatidylinositol (GPI) moiety. We suggest that presentation of Cripto on the cell surface via a GPI-linkage is important in determining the spatial specificity of cell-cell interactions that play a critical role in the early patterning of the embryo.

Assignment of human teratocarcinoma derived growth factor (TDGF) sequences to chromosomes 2q37, 3q22, 6p25 and 19q13.1.

The human teratocarcinoma derived growth factor 1 (TDGF1) gene maps on chromosome (Chr) 3p21.3. One pseudogene (TDGF3) maps on Chr Xq21-->q22. We now report the nucleotide sequence and chromosome location of three additional TDGF pseudogenes. The three new sequences (TDGF2, TDGF4 and TDGF5) are truncated at the 5' end and have accumulated several point mutations, deletions and insertions. TDGF2, TDGF4 and TDGF6 map on Chrs 2q37, 6p25 and 3q22, respectively. Finally, Southern blot analysis of DNA from normal individuals shows a highly variable restriction pattern of the TDGF sequences.

Upregulation of the angiogenic factors PlGF, VEGF and their receptors (Flt-1, Flk-1/KDR) by TSH in cultured thyrocytes and in the thyroid gland of thiouracil-fed rats suggest a TSH-dependent paracrine mechanism for goiter hypervascularization.

Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) represent two closely related angiogenic growth factors active as homodimers or heterodimers. Since goiters of the thyroid gland are extremely hypervascular, we investigated the expression of PlGF, VEGF and their receptors, Flt-1 and Flk-1/KDR, in a small panel of human goiters from patients with Graves's disease, in an animal model of thyroid goitrogenesis and in in vitro cultured thyroid cells. Here we report that the mRNA expression of PlGF, VEGF and their receptors is markedly enhanced in biopsies of goiters resected from Graves's patients. In vivo studies demonstrated that in the thyroid gland of thiouracil-fed rats, increased mRNA and protein expression of PIGF, VEGF, Flt-1 and Flk-1/KDR occurred subsequent to the rise in the serum thyroid stimulating hormone (TSH) levels and in parallel with thyroid capillary proliferation. In vitro studies confirmed the existence of such TSH-dependent paracrine communication between thyroid epithelial cells and endothelium since the conditioned medium collected from TSH-stimulated thyrocytes acquired mitogenic activity for human umbilical vein endothelial (HUVE) cells. Altogether, these data suggest that PlGF and VEGF, released by thyrocytes in response to the chronic activation of the TSH receptor pathway, may act through a paracrine mechanism on thyroid endothelium.

Mapping of the mouse Tdgf1 gene and Tdgf pseudogenes.

Teratocarcinoma-derived growth factor-1 (Tdgf1), a member of the "EGF family" of growth factors, is expressed during mouse gastrulation in the forming mesoderm and later in the truncus arteriosus of the developing heart. In humans, TDGF1 is highly expressed in germ cell tumors and in colon and mammary carcinomas. In mouse, one gene (Tdgf1) and two pseudogenes (Tdgf1-ps1 and Tdgf1-ps2) have been isolated and characterized. Tdgf1 corresponds to the gene expressed in F9 teratocarcinoma cells. Tdgf1-ps1 and Tdgf1-ps2 are two intronless sequences with all the characteristics of retroposons. In the present paper, we assign the chromosomal location for Tdgf1, Tdgf1-ps1, and Tdgf1-ps2 sequences to Chromosomes (Chrs) 9, 16, and 17, respectively. Two previously described mouse mutants, scant hair (sch) and fur deficient (fd), map near the Tdgf1 gene. Analysis of their DNA coding region provided no evidence that Tdgf1 could be the responsible gene for these phenotypes. Finally, analysis of the DNA from several Mus musculus strains and from Mus spretus mice revealed a highly variable restriction pattern and the absence of the Tdgf1-ps1 genomic sequence from the Mus spretus genome.

Placenta growth factor-1 is chemotactic, mitogenic, and angiogenic.

The placental-derived growth factor (PIGF) is a dimeric glycoprotein showing a high degree of sequence similarity to the vascular endothelial growth factor. Alternative splicing of the PIGF primary transcript gives rise to two forms, named PIGF-1 and PIGF-2, which differ only in the insertion of a highly basic 21-amino acid stretch at the carboxyl end. The presence of the PIGF mRNA in thyroid, placenta, lung, and goiter has indicated the tissues where this factor functions. However, the role of PIGF in vascular development has not yet been clearly established. In the present study, we described the purification of PIGF-1 from overexpressing eukaryotic cells and then measured the angiogenic activity of the purified PIGF-1 in vivo in the rabbit cornea and the chick chorioallantoic membrane assays. In both in vivo assays, PIGF-1 induced a strong neovascularization process that was blocked by affinity-purified anti-PIGF-1 antibody. In the avascular cornea, PIGF-1 induced angiogenesis in a dose-dependent manner and seemed to be at least as effective (if not more effective) than vascular endothelial growth factor and basic fibroblast growth factor under the same conditions and at the same concentration. PIGF-1 was shown to induce cell growth and migration of endothelial cells from bovine coronary postcapillary venules and from human umbilical veins. In these two in vitro assays, PIGF-1 seemed to have a comparable effect to that of vascular endothelial growth factor and basic fibroblast growth factor on the cultured microvascular endothelium (eg, capillary venule endothelial cells). In summary, this is the first study to demonstrate that PIGF-1 can induce angiogenesis in vivo and stimulate the migration and proliferation of endothelial cells in vitro.

Neovascularization in human germ cell tumors correlates with a marked increase in the expression of the vascular endothelial growth factor but not the placenta-derived growth factor.

Neoangiogenesis is a prerequisite for tumor growth and metastasis. In germ cell cancer patients with the disease limited to the testicle (stage A), tumor-associated neovascularization is predictive of metastatic disease (stage B). To investigate the molecular mechanisms underlying neovascularization in human germ cell tumors (GCTs), we analysed the expression of two angiogenic growth factors, vascular endothelial growth factor (VEGF) and placenta growth factor (P1GF), and of their receptors (FLT-1) and Flk-1/KDR) in a panel of testicular tumors. In this study we show a marked increase in VEGF expression in 36/44 (81.8%) primary testicular-derived GCTs, as compared to normal testis, that significantly correlates with a high density of intratumor microvessels (r = 0.72461, P < 0.001; n = 24). As determined by RT - PCR and/or Western blot, the predominant VEGF isoforms expressed in GCTs are the VEGF121 and VEGF165, which are more efficiently secreted by the cells, and thus more active in eliciting angiogenesis. Conversely, in the case of PIGF, only a weak correlation with the vascular density of tumors is observed (r = 0.26599, P < 0.05; n = 24). Northern blot analysis also revealed significant up-regulation of VEGF/ PIGF receptors in highly vascularized germ cell tumors, compared to normal testes. These findings suggest that VEGF may act in a paracrine manner to induce neovascularization, oedema extravasation and cyst formation in human germ cell tumors. The correlation between VEGF expression and the vascular density of tumors, suggest that the evaluation of VEGF expression may be of help in predicting patients at risk for metastatic diseases. Finally, we demonstrate that VEGF up-regulation may occur at the RNA level since no gene amplification is observed; conversely, in in vitro models such as the embryonal stem cell line NTERA-2 and the choricarcinoma JEG-3 cell line, VEGF (but not PIGF) mRNA expression is regulated by hypoxic stress.

Characterization of the mouse Tdgf1 gene and Tdgf pseudogenes.

Cripto protein is a member of the "EGF family" of growth factors present in colon tumors and in human and mouse undifferentiated teratocarcinoma cells. During gastrulation in the mouse, cripto-encoding transcripts are expressed in the forming mesoderm and later in the truncus arteriosus of the developing heart. As a necessary step prior to investigating the in vivo role of cripto through gene disruption, we have isolated all the genomic cripto-related sequences in the mouse. One gene (Tdgf1) and two pseudogenes (Tdgf2 and Tdgf3) have been isolated and characterized. The mouse Tdgf1 (coding for cripto), like the human gene, is divided into six exons. Comparison of the human and mouse genomic sequences reveals that mouse exons 1 and 3 are shorter than the corresponding human exons. The pseudogene Tdgf2 corresponds to about 1 kb of the mRNA and contains five base substitutions in the coding region that represent both silent and replacement substitutions. The pseudogene Tdgf3 corresponds only to the coding portion of Tdgf. Many mutations have been introduced in this pseudogene, suggesting its early origin. Alignments of the Tdgf3, human and mouse mRNA sequences, shows that this pseudogene has retained the 33 nucleotides of the human exon 3 that are missed in the Tdgf1 gene. Taken together, these data suggest that Tdgf3 is derived from an ancestral gene and that the human and mouse genes are probably evolving separately.

The placenta growth factor gene of the mouse.

Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) are angiogenic factors containing the 8-cysteine motif of platelet-derived growth factor (PDGF). Both PlGF and VEGF are mitogens for endothelial cells in vitro and promote neoangiogenesis in vivo. In addition, PlGF strongly potentiates the proliferative and the permeabilization effects exerted by VEGF on the vascular endothelium. We have now isolated the cDNA coding for mouse Plgf by screening a mouse heart cDNA library with the human PlGF sequence as probe. The human PlGF protein has two forms, PlGF-1 and PlGF-2, that arise from alternative splicing of a single gene mapping on Chromosome (Chr) 14; the isolated mouse Plgf cDNA encodes the longer of these two forms (PlGF-2). We show that the mouse Plgf-2 mRNA is the only transcript present in the normal tissues analyzed. Mouse Plgf-2 is a 158-amino-acid-long protein that shows 78% similarity (65% identity) to the human PlGF-2. Computer analysis reveals a putative signal peptide and three probable N-glycosylation sites, two of which are also conserved in human PlGF. The mouse Plgf gene was isolated and characterized; the gene is encoded by 7 exons spanning a 13-kb DNA interval. Finally, we have mapped the mouse Plgf gene to Chr 12, one cM from D12Mit5, and the human PlGF gene to 14q24, using both FISH and genetic crosses.

Utilization of cellobiose and other beta-D-glucosides in Agrobacterium tumefaciens.

Agrobacterium tumefaciens strain C58 was able to utilize carbon from cellobiose and some other beta-D-glucosides as efficiently as from glucose. beta-D-glucoside utilization was partially inducible and the induction was subject to catabolite repression by glucose, independently of the presence of cyclic AMP in the medium. It was also independent of Ti plasmid-encoded functions. beta-D-glucosides were hydrolysed by a single, cytoplasmic and constitutively expressed beta-glucosidase, which was active on non-phosphorylated substrates and insensitive to glucose inhibition.

Organization of Lrp-binding sites upstream of ilvIH in Salmonella typhimurium.

Lrp, a major regulatory protein in Escherichia coli, controls the expression of numerous operons, including ilvIH. Lrp binds to six sites upstream of ilvIH, and Lrp binding is required for ilvIH expression. We show here that an Lrp-like protein is also present in Salmonella typhimurium. This protein can bind both E. coli and S. typhimurium ilvIH DNA, as can E. coli Lrp. Methidiumpropyl-EDTA footprinting studies were performed with purified E. coli Lrp and S. typhimurium ilvIH DNA. Six binding sites were defined, three of them being similar to corresponding sites in E. coli, and three being organized differently. A consensus derived from six S. typhimurium sites is compatible with that derived from a similar analysis of E. coli sequences.

Absence of acetohydroxy acid synthase III in Salmonella typhimurium is due to early termination of translation within the ilvl gene.

The cryptic ilvlH locus of Salmonella typhimurium has genetic information for two distinct subunits of acetohydroxy acid synthase III. We show that the ilvH-encoded subunit is normally translated and the lack of activity is due to early termination of translation within the promoter-proximal ilvl gene. Analysis of the 5' region of the operon led to identification of the promoter and the amino-terminal part of ilvl. Expression of this gene in a mutant producing acetohydroxy acid synthase is due to a transversion which creates a UUA (leucine) codon in the place of a UGA (stop) codon present in position 12 of the wild-type coding region.

Enhanced acetohydroxy acid synthase III activity in an ilvH mutant of Escherichia coli K-12.

The acetohydroxy acid synthase III isozyme, which catalyzes the first common step in the biosynthesis of isoleucine, leucine, and valine in Escherichia coli K-12, is composed of two subunits, the ilvI and ilvH gene products. A missense mutation in ilvH (ilvH612), which reduced the sensitivity of the enzyme to the end product inhibition by valine, also increased its specific activity and lowered the Km for alpha-acetolactate synthesis. The mutation increased the sensitivity of acetohydroxy acid synthase III to dialysis and heat treatment and reduced the requirement for thiamine pyrophosphate addition to the assay mixture for activity. A strain carrying the ilvH612 mutation grew better than a homologous ilvH+ strain in the presence of leucine. The data indicate that this is a consequence of a more active acetohydroxy acid synthase III isozyme rather than the result of an alteration of the leucine-mediated repression of the ilvIH operon.

The ilvIH operon of Escherichia coli K-12. Identification of the gene products and recognition of the translational start by polypeptide microsequencing.

The ilvI and ilvH gene products were identified physically by electrophoretic analysis of in vivo-labelled polypeptides produced in minicells from plasmids carrying the wild-type ilvIH operon of Escherichia coli K-12 and derivatives of it. An analysis of the distribution of methionine residues in the amino-terminal portion of micro-quantities of the ilvI product eluted from gel showed that the translational start of the ilvI gene is the promoter-proximal one of three putative methionine codons predicted from the DNA sequence.

IlvHI locus of Salmonella typhimurium.

In Escherichia coli K-12, the ilvHI locus codes for one of two acetohydroxy acid synthase isoenzymes. A region of the Salmonella typhimurium genome adjacent to the leucine operon was cloned on plasmid pBR322, yielding plasmids pCV47 and pCV49 (a shortened version of pCV47). This region contains DNA homologous to the E. coli ilvHI locus, as judged by hybridization experiments. Plasmid pCV47 did not confer isoleucine-valine prototrophy upon either E. coli or S. typhimurium strains lacking acetohydroxy acid synthase activity, suggesting that S. typhimurium lacks a functional ilvHI locus. However, isoleucine-valine prototrophs were readily isolated from such strains after mutagenesis with nitrosoguanidine. In one case we found that the Ilv+ phenotype resulted from an alteration in bacterial DNA on the plasmid (new plasmid designated pCV50). Furthermore, a new acetohydroxy acid synthase activity was observed in Ilv+ revertants; this enzyme was similar to E. coli acetohydroxy acid synthase III in its lack of activity at low pH. This new activity was correlated with the appearance in minicells of a new polypeptide having an approximate molecular weight of 61,000. Strains carrying either pCV49 or pCV50 produced a substantial amount of ilvHI-specific mRNA. These results, together with results from other laboratories, suggest that S. typhimurium has functional ilvB and ilvG genes and a cryptic ilvHI locus. E. coli K-12, on the other hand, has functional ilvB and ilvHI genes and a cryptic ilvG locus.

Acetohydroxy acid synthase isoenzymes of Escherichia coli K12 and Salmonella typhimurium.

In Escherichia coli K12 and in Salmonella typhimurium the first step common to the biosynthesis of isoleucine, leucine and valine is catalyzed by an intriguing system of isoenzymes. Two of these are normally expressed, while the genetic determinant for a third one is transcribed, but not translated as an active polypeptide. We analyze here the significance of this system in the light of the most recent results.