Mutation and deletion analysis of GFRα-1, encoding the co-receptor for the GDNF/RET complex, in human brain tumours

Glial cell line-derived neurotrophic factor (GDNF) plays a key role in the control of vertebrate neuron survival and differentiation in both the central and peripheral nervous systems. GDNF preferentially binds to GFRα-1 which then interacts with the receptor tyrosine kinase RET. We investigated a panel of 36 independent cases of mainly advanced sporadic brain tumours for the presence of mutations in GDNF and GFRα-1. No mutations were found in the coding region of GDNF. We identified six previously described GFRα-1 polymorphisms, two of which lead to an amino acid change. In 15 of 36 brain tumours, all polymorphic variants appeared to be homozygous. Of these 15 tumours, one also had a rare, apparently homozygous, sequence variant at codon 361. Because of the rarity of the combination of homozygous sequence variants, analysis for hemizygous deletion was pursued in the 15 samples and loss of heterozygosity was found in 11 tumours. Our data suggest that intragenic point mutations of GDNF or GFRα-1 are not a common aetiologic event in brain tumours. However, either deletion of GFRα-1 and/or nearby genes may contribute to the pathogenesis of these tumours

FLP-mediated Intermolecular recombination in the Cytoplasm of Drosophila embryos.

We show that when a heat-shock-driven gene that encodes the yeast FLP recombinase is injected into preblastoderm Drosophila embryos, it promotes intermolecular recombination between two coinjected plasmids that bear the specific recombination target sequence, FRT. Minimal, 34-bp FRT sites in the two plasmids are sufficient for their cointegration. The reaction is efficient enough to produce detectable recombinants when one of the plasmids is present in as little as 1000 molecules per embryo. This is comparable to the concentration of unique chromosomal sites, raising the possibility that integration of injected plasmid DNA into FRT-bearing fly chromosomes may also be achievable. Since integrants might be stabilized against the reverse excision reaction if the recombinase could be provided in a sharp pulse, it is encouraging that efficient plasmid cointegration is also achieved when in vitro synthesized FLP RNA rather than DNA is injected into the embryos

FLP-MEDIATED INTERMOLECULAR RECOMBINATION IN THE CYTOPLASM OF DROSOPHILA EMBRYOS

We show that when a heat-shock-driven gene that encodes the yeast FLP recombinase is injected into preblastoderm Drosophila embryos, it promotes intermolecular recombination between two coinjected plasmids that bear the specific recombination target sequence, FRT. Minimal, 34-bp FRT sites in the two plasmids are sufficient for their cointegration. The reaction is efficient enough to produce detectable recombinants when one of the plasmids is present in as little as 1000 molecules per embryo. This is comparable to the concentration of unique chromosomal sites, raising the possibility that integration of injected plasmid DNA into FRT-bearing fly chromosomes may also be achievable. Since integrants might be stabilized against the reverse excision reaction if the recombinase could be provided in a sharp pulse, it is encouraging that efficient plasmid cointegration is also achieved when in vitro synthesized FLP RNA rather than DNA is injected into the embryos

The EGF-CFC family: novel epidermal growth factor-related proteins in development and cancer

The EGF-CFC gene family encodes a group of structurally related proteins that serve as important competence factors during early embryogenesis in Xenopus, zebrafish, mice and humans. This multigene family consists of Xenopus FRL-1, zebrafish one-eyed-pinhead (oep), mouse cripto (Cr-l) and cryptic, and human cripto (CR-1) and criptin. FRL-1, oep and mouse cripto are essential for the formation of mesoderm and endoderm and for correct establishment of the anterior/posterior axis. In addition, oep and cryptic are important for the establishment of left-right (UR) asymmetry, In zebrafish, there is strong genetic evidence that oep functions as an obligatory cc-factor for the correct signaling of a transforming growth factor-beta (TGF beta)-related gene, nodal, during gastrulation and during UR asymmetry development. Expression of Cr-l and cryptic is extinguished in the embryo after day 8 of gestation except for the developing heart where Cr-l expression is necessary for myocardial development. In the mouse, cryptic is not expressed in adult tissues whereas Cr-l is expressed at a low level in several different tissues including the mammary gland, In the mammary gland, expression of Cr-l in the ductal epithelial cells increases during pregnancy and lactation and immunoreactive and biologically active Cr-l protein can be detected in human milk. Overexpression of Cr-l in mouse mammary epithelial cells can facilitate their in vitro transformation and in vivo these Cr-l-transduced cells produce ductal hyperplasias in the mammary gland. Recombinant mouse or human cripto can enhance cell motility and branching morphogenesis in mammary epithelial cells and in some human tumor cells. These effects are accompanied by an epithelial-mesenchymal transition which is associated with a decrease in beta -catenin function and an increase in vimentin expression. Expression of cripto is increased several-fold in human colon, gastric, pancreatic and lung carcinomas and in a variety of different types of mouse and human breast carcinomas. More importantly, this increase can first be detected in premalignant lesions in some of these tissues. Although a specific receptor for the EGF-CFC proteins has not yet been identified, oep depends upon an activin-type RIIB and RIB receptor system that functions through Smad-e. Mouse and human cripto have been shown to activate a ras/raf/MAP kinase signaling pathway in mammary epithelial cells. Activation of phosphatidylinositol 3-kinase and Akt are also important for the ability of CR-1 to stimulate cell migration and to block lactogenic hormone-induced expression of p-casein and whey acidic protein. In mammary epithelial cells, part of these responses may depend on the ability of CR-1 to transactivate erb B-4 and/or fibroblast growth factor receptor 1 through an src-like tyrosine kinase