Identification of a new gene family specifically expressed in chicken embryonic stem cells and early embryo.

Chicken embryonic stem (CES) cells are pluripotent cells derived from chicken early blastoderm. In order to identify new genes specifically expressed in these pluripotent cells, we have used a gene trap strategy and cloned a novel gene family called cENS for chicken Embryonic Normal Stem cell gene. The cENS genes expression decreases after induction of CES cells differentiation in culture and is restricted in vivo to the very early embryo. We have characterized three different cENS genes. One, cENS-1, is composed of an open reading frame inserted between two terminal direct repeats which are the common point of the cENS genes. cENS-1 encodes a protein identical to cERNI, a recently described protein. cENS-2 is a truncated form of cENS-1. cENS-3 presents two adjacent open reading frames coding respectively for env and pol related proteins. The presence of conserved direct repeats, of retrovirus related genes and the absence of introns argue in favor of a retroviral origin of the cENS genes. In the cENS we identified a promoter region whose activity is strong in CES cells and decreases after induced differentiation showing a highly specific transcriptional activity specific of undifferentiated chicken embryonic stem cells.

Cloning of the mouse BTG3 gene and definition of a new gene family (the BTG family) involved in the negative control of the cell cycle.

It is well known that loss of tumor suppressor genes and more generally of antiproliferative genes plays a key role in the development of most tumors. We report here the cloning of the mouse BTG3 gene and show that its human counterpart maps on chromosome 21. This evolutionarily conserved gene codes for a 30 kDa protein and is expressed in most adult murine and human tissues analyzed. However, we demonstrate that its expression is cell cycle dependent and peaks at the end of the G1 phase. This gene is homologous to the human BTG1, BTG2 and TOB genes which were demonstrated to act as inhibitors of cell proliferation. Its description allowed us to define better this seven gene family (the BTG gene family) at the structural level and to speculate about its physiological role in normal and tumoral cells. This family is mainly characterized by the presence of two conserved domains (BTG boxes A and B) of as yet undetermined function which are separated by a non-conserved 20-25 amino acid sequence.

Identification and molecular analysis of BANP.

BTG3 belongs to a family of structurally related genes whose biochemical functions remain elusive. In order to investigate the mechanism underlying BTG3-mediated functions, we tried to identify BTG3 potential partners. The use of the yeast 'two-hybrid system', with BTG3 as bait, enabled us to isolate BANP (BTG3 Associated Nuclear Protein). Other commonly used protein-binding assays did not confirm this yeast interaction. However, BANP had never been described before, and this prompted us to further characterise this gene. In this paper, we present data on its molecular organization in mouse, then we speculate on the nature of this nuclear protein, and finally we localise BANP on the human chromosome 16q24 subregion; we discuss the fact that frequent loss of heterozygosity within this region has been observed in different tumours.

Map location on Agrobacterium root-inducing plasmids of homologies with the virulence region of tumor-inducing plasmids.

Southern-type hybridizations were carried out in order to identify sequence homologies with the pTi vir loci, on an agropine-type plasmid (pRiHRI) and a mannopine-type plasmid (pRi8196) of Agrobacterium rhizogenes. The localization of the sequences hybridizing with subcloned fragments containing vir A, B, G, C, and D from pTiAch5 indicated a similar linear organization of the pTi vir loci and their homologies on pRiHRI and pRi8196, though no homology was detected on both pRi with a 1.1-kb internal fragment of virD. No homology was detected either with the vir E locus on pRiHRI vir region, nor with the virF locus on both pRi vir regions. As on nopaline pTiC58, fragments bearing the homologies with virC and virG are closer together on both pRi than on octopine pTiAch5. A preliminary functional map of the pRiHRI vir region is deduced from this study.

IDS gene-pseudogene exchange responsible for an intragenic deletion in a Hunter patient.

Hunter disease or mucopolysaccharidosis type II is an X-linked disease caused by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The IDS gene (24 kb) contains nine exons and has been completely sequenced. A pseudogene (IDS-2 locus) distal to the functional IDS gene has recently been identified. This work reports the characterization of IDS gene alterations in two severely affected patients. Patient 1 has a partial deletion that removes exons I to VI and extends about 200 kb upstream of the IDS gene. Patient 2 has an internal deletion of exons IV, V, VI, and VII, which results from an IDS gene-pseudogene exchange between highly homologous regions. In the rearranged gene, the junction intron contains pseudogene intron 3- and intron 7-related sequences. An interchromosomal recombination is probably the cause of this rearranged X chromosome.

Identification of iduronate sulfatase gene alterations in 70 unrelated Hunter patients.

We studied 70 unrelated Hunter patients and found a gene alteration in every patient. The molecular heterogeneity was very important. Large gene rearrangements were identified in 14 patients. Forty-three different mutations were identified in the 56 other patients and 31 were not previously described. Deletions and insertions, splice site mutations were associated with a severe phenotype as nonsense mutations except Q531X. Only a few mutations were present in several patients making difficult genotype-phenotype correlations. Mutation identification allows accurate carrier detection improving prenatal diagnosis. The mother was not found to be a carrier in five cases among the 44 sporadic cases. Haplotype analysis demonstrated a higher frequency of mutations in male meiosis.

Interaction of BTG1 and p53-regulated BTG2 gene products with mCaf1, the murine homolog of a component of the yeast CCR4 transcriptional regulatory complex.

Both BTG1 and BTG2 are involved in cell-growth control. BTG2 expression is regulated by p53, and its inactivation in embryonic stem cells leads to the disruption of DNA damage-induced G2/M cell-cycle arrest. In order to investigate the mechanism underlying Btg-mediated functions, we looked for possible functional partners of Btg1 and Btg2. Using yeast two-hybrid screening, protein-binding assays, and transient transfection assays in HeLa cells, we demonstrated the physical in vitro and in vivo interaction of both Btg1 and Btg2 with the mouse protein mCaf1 (i.e. mouse CCR4-associated factor 1). mCaf1 was identified through its interaction with the CCR4 protein, a component of a general transcription multisubunit complex, which, in yeast, regulates the expression of different genes involved in cell-cycle regulation and progression. These data suggest that Btg proteins, through their association with mCaf1, may participate, either directly or indirectly, in the transcriptional regulation of the genes involved in the control of the cell cycle. Finally, we found that box B, one of two conserved domains which define the Btg family, plays a functional role, namely that it is essential to the Btg-mCaf1 interaction.

The leukemia-associated protein Btg1 and the p53-regulated protein Btg2 interact with the homeoprotein Hoxb9 and enhance its transcriptional activation.

BTG1 and BTG2 belong to a family of functionally related genes involved in the control of the cell cycle. As part of an ongoing attempt to understand their biological functions, we used a yeast two-hybrid screening to look for possible functional partners of Btg1 and Btg2. Here we report the physical and functional association between these proteins and the homeodomain protein Hoxb9. We further show that Btg1 and Btg2 enhance Hoxb9-mediated transcription in transfected cells, and we report the formation of a Hoxb9.Btg2 complex on a Hoxb9-responsive target, and the fact that this interaction facilitates the binding of Hoxb9 to DNA. The transcriptional activity of the Hoxb9.Btg complex is essentially dependent on the activation domain of Hoxb9, located in the N-terminal portion of the protein. Our data indicate that Btg1 and Btg2 act as transcriptional cofactors of the Hoxb9 protein, and suggest that this interaction may mediate their antiproliferative function.