Expression of Ets2, a proto-oncogene and transcription factor, occurs in a variety of cell types. During murine development it is highly expressed in newly forming cartilage, including in the skull precursor cells and vertebral primordia. Ets2 is located on human chromosome 21 (ref. 8) and is overexpressed in Down's syndrome (trisomy 21). Here we generate transgenic mice to investigate the consequences of overexpression of Ets2. We find that mice with less than 2-fold Ets2 overexpression in particular organs develop neurocranial, viscerocranial and cervical skeletal abnormalities. These abnormalities have similarities with the skeletal anomalies found in trisomy-16 mice and humans with Down's syndrome, in which the gene dosage of Ets2 is increased. Our results indicate that Ets2 has a role in skeletal development and implicate the overexpression of Ets2 in the genesis of some skeletal abnormalities that occur in Down's syndrome.

Previous analysis of the amdS gene of Aspergillus nidulans has identified multiple regulatory circuits mediated by trans-acting regulatory genes, cis-acting mutations have been identified and shown to specifically affect individual regulatory circuits. Fine-structure genetic mapping of the amdS regions showed that these cis-acting mutations occur in a complex controlling region adjacent to the amdS structural gene. The amdS gene was cloned by differential hybridization, using cDNA probes derived from a high-level-producing strain and from a strain with a large amdS deletion mutation. RNA blotting experiments showed that a single RNA species of 1,600 to 1,700 base pairs is transcribed from the amdS gene. DNA blotting experiments on a large number of amdS mutant strains, including deletions and translocations, allowed the genetic and physical maps of the gene to be correlated. The controlling region of the gene is situated at the 5' end of the gene and the direction of transcription is toward the centromere of chromosome III. The regulatory mutations in the controlling region were found to be due to small-scale alterations in the DNA rather than to large-scale rearrangements resulting in gene fusions.

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The amdS gene of Aspergillus nidulans, which encodes an acetamidase enzyme, is positively regulated by the trans-acting genes amdR, facB, amdA, and areA. Sequence changes in several cis-acting mutations in the 5' region of the gene which specifically affect amdS regulation were determined. The amdI9 mutation, which results in increased facB-dependent acetate induction, is due to a single-base change at base pair -210 relative to the start point of translation. The amdI93 mutation, which abolishes amdR-dependent omega-amino acid induction, is a deletion of base pairs -181 to -151. The amdI66 mutation, which causes increased gene activation in strains carrying amdA regulatory gene mutations, is a duplication of base pairs -107 to -90. Transformation of A. nidulans can generate transformants containing multiple integrated copies of plasmid sequences. When these plasmids carry a potential binding site for a regulatory gene product, growth on substrates whose catabolism requires genes activated by that regulatory gene can be reduced, apparently because of titration of the regulatory gene product. Introduction of 5' amdS sequences via cotransformation into strains of various genotypes was used to localize sequences apparently involved in binding of the products of the amdR, amdA, and facB genes. The position of these sequences is in agreement with the positions of the specific cis-acting mutations. Consistent with these results, a transformant of A. nidulans derived from a plasmid deleted for sequences upstream from -111 was found to have lost amdR- and facB-mediated control but was still regulated by the amdA gene. In addition, amdS expression in this transformant was still dependent on the areA gene.

The amdS gene of Aspergillus nidulans, which encodes an acetamidase enzyme, is positively regulated by the trans-acting genes amdR, facB, amdA, and areA. Sequence changes in several cis-acting mutations in the 5' region of the gene which specifically affect amdS regulation were determined. The amdI9 mutation, which results in increased facB-dependent acetate induction, is due to a single-base change at base pair -210 relative to the start point of translation. The amdI93 mutation, which abolishes amdR-dependent omega-amino acid induction, is a deletion of base pairs -181 to -151. The amdI66 mutation, which causes increased gene activation in strains carrying amdA regulatory gene mutations, is a duplication of base pairs -107 to -90. Transformation of A. nidulans can generate transformants containing multiple integrated copies of plasmid sequences. When these plasmids carry a potential binding site for a regulatory gene product, growth on substrates whose catabolism requires genes activated by that regulatory gene can be reduced, apparently because of titration of the regulatory gene product. Introduction of 5' amdS sequences via cotransformation into strains of various genotypes was used to localize sequences apparently involved in binding of the products of the amdR, amdA, and facB genes. The position of these sequences is in agreement with the positions of the specific cis-acting mutations. Consistent with these results, a transformant of A. nidulans derived from a plasmid deleted for sequences upstream from -111 was found to have lost amdR- and facB-mediated control but was still regulated by the amdA gene. In addition, amdS expression in this transformant was still dependent on the areA gene.

Previous analysis of the amdS gene of Aspergillus nidulans has identified multiple regulatory circuits mediated by trans-acting regulatory genes, cis-acting mutations have been identified and shown to specifically affect individual regulatory circuits. Fine-structure genetic mapping of the amdS regions showed that these cis-acting mutations occur in a complex controlling region adjacent to the amdS structural gene. The amdS gene was cloned by differential hybridization, using cDNA probes derived from a high-level-producing strain and from a strain with a large amdS deletion mutation. RNA blotting experiments showed that a single RNA species of 1,600 to 1,700 base pairs is transcribed from the amdS gene. DNA blotting experiments on a large number of amdS mutant strains, including deletions and translocations, allowed the genetic and physical maps of the gene to be correlated. The controlling region of the gene is situated at the 5' end of the gene and the direction of transcription is toward the centromere of chromosome III. The regulatory mutations in the controlling region were found to be due to small-scale alterations in the DNA rather than to large-scale rearrangements resulting in gene fusions.

Studies of the development and differentiation of early mammalian embryos have been severely limited by the paucity of material. Such studies have been largely restricted to the examination of abundant genes/proteins or to developmental expression studies of known genes for which DNA sequence data are available, allowing the use of reverse transcription and polymerase chain reaction amplification (RT‐PCR). To eliminate the need for hundreds or thousands of oocytes or embryos in the construction of representative cDNA libraries, we describe a technique for generating and cloning cDNA using small caesium chloride gradient centrifugation to isolate total RNA from oocytes or embryos, followed by RT‐PCR of mRNA from this total RNA. Total RNA was isolated from 70 mouse blastocysts. A portion of the cDNA generated (equivalent to seven blastocysts) was cloned, yielding a mouse blastocyst cDNA library of 1 million clones. We show that the library is representative in that it contains β‐actin, intracisternal A‐type particles, tissue plasminogen activator, and B1 and B2 repetitive elements in frequencies comparable with published data from conventionally constructed libraries and estimates of mRNA abundance from expression studies. Furthermore, DNA sequencing of 22 clones chosen at random and compared with DNA sequence databases shows that approximately half are novel sequences. These data demonstrate that representative cDNA libraries can be constructed in situations where cell numbers are limiting and will facilitate the isolation of novel and interesting clones. © 1996 Wiley‐Liss, Inc.

A monoclonal antibody that interferes with the EDTA-resistant adhesion of Dictyostelium discoideum slug cells recognised a carbohydrate epitope on four major antigens (95, 90, 35 and 30 kDa) in slug cells. The 35 and 30 kDa antigens were specific for stalks and spores, respectively. The 30 kDa antigen was identified as the cell surface glycoprotein, PsA. Cyclic AMP, acting via cell surface receptors, induced only the 90 kDa slug cell antigen. Slug cell adhesion proteins may be involved in cell-sorting and the glycosylation of the 95 and 90 kDa antigens appeared to be abnormal m a mutant defective in cell-sorting. Previously, a 150 kDa glycoprotein has been strongly implicated in slug cell adhesion and the present work suggests that additional glycoprotein(s) are involved.