Showing papers by "Philip A. Beachy published in 1996"

Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function.

Abstract: Targeted gene disruption in the mouse shows that the Sonic hedgehog (Shh) gene plays a critical role in patterning of vertebrate embryonic tissues, including the brain and spinal cord, the axial skeleton and the limbs. Early defects are observed in the establishment or maintenance of midline structures, such as the notochord and the floorplate, and later defects include absence of distal limb structures, cyclopia, absence of ventral cell types within the neural tube, and absence of the spinal column and most of the ribs. Defects in all tissues extend beyond the normal sites of Shh transcription, confirming the proposed role of Shh proteins as an extracellular signal required for the tissue-organizing properties of several vertebrate patterning centres.

Cholesterol Modification of Hedgehog Signaling Proteins in Animal Development

Abstract: Hedgehog (Hh) proteins comprise a family of secreted signaling molecules essential for patterning a variety of structures in animal embryogenesis. During biosynthesis, Hh undergoes an autocleavage reaction, mediated by its carboxyl-terminal domain, that produces a lipid-modified amino-terminal fragment responsible for all known Hh signaling activity. Here it is reported that cholesterol is the lipophilic moiety covalently attached to the amino-terminal signaling domain during autoprocessing and that the carboxyl-terminal domain acts as an intramolecular cholesterol transferase. This use of cholesterol to modify embryonic signaling proteins may account for some of the effects of perturbed cholesterol biosynthesis on animal development.

Overexpression of a Rrp1 transgene reduces the somatic mutation and recombination frequency induced by oxidative DNA damage in Drosophila melanogaster

Abstract: Recombination repair protein 1 (Rrp1) includes a C-terminal region homologous to several DNA repair proteins, including Escherichia coli exonuclease III and human APE, that repair oxidative and alkylation damage to DNA. The nuclease activities of Rrp1 include apurinic/apyrimidinic endonuclease, 3'-phosphodiesterase, 3'-phosphatase, and 3'-exonuclease. As shown previously, the C-terminal nuclease region of Rrp1 is sufficient to repair oxidative- and alkylation-induced DNA damage in repair-deficient E. coli mutants. DNA strand-transfer and single-stranded DNA renaturation activities are associated with the unique N-terminal region of Rrp1, which suggests possible additional functions that include recombinational repair or homologous recombination. By using the Drosophila w/w+ mosaic eye system, which detects loss of heterozygosity as changes in eye pigmentation, somatic mutation and recombination frequencies were determined in transgenic flies overexpressing wild-type Rrp1 protein from a heat-shock-inducible transgene. A large decrease in mosaic clone frequency is observed when Rrp1 overexpression precedes treatment with gamma-rays, bleomycin, or paraquat. In contrast, Rrp1 overexpression does not alter the spot frequency after treatment with the alkylating agents methyl methanesulfonate or methyl nitrosourea. A reduction in mosaic clone frequency depends on the expression of the Rrp1 transgene and on the nature of the induced DNA damage. These data suggest a lesion-specific involvement of Rrp1 in the repair of oxidative DNA damage.

pH-Dependent Enhancement of DNA Binding by the Ultrabithorax Homeodomain†

Abstract: Ultrabithorax (Ubx) and Deformed (Dfd) proteins of Drosophila melanogaster contain homeodomains (HD) that are structurally similar and recognize similar DNA sequences, despite functionally distinct genetic regulatory roles for Ubx and Dfd. We report in the present study that Ubx-HD binding to a single optimal target site displayed significantly increased affinity and higher salt concentration dependence at lower pH, while Dfd-HD binding to DNA was unaffected by pH. Results from studies of chimeric Ubx−Dfd homeodomains showed that the N- and C-terminal regions of the Ubx-HD are required for this pH dependence. The increase in binding affinity at lower pH was greater for the Ubx optimal binding site than for other DNA binding sites, indicating that subtle sequence alterations in DNA binding sites may influence pH-dependent behavior. These data demonstrate enhanced DNA binding affinity at lower pH for the Ubx-HD in vitro and suggest the potential for significant discrimination of DNA binding sites in vivo.

Method of using hedgehog polypeptides to regulate neuronal cell growth

Abstract: The present invention provides two novel polypeptides, referred to as the “N” and “C” fragments of hedgehog, or N-terminal and C-terminal fragments, respectively, which are derived after specific cleavage at a Gly↓Cys Phe site recognized by the autoproteolytic domain in the native protein. Methods of identifying compositions which affect hedgehog activity based on inhibition of cholesterol modification of hedgehog protein are described. Also provided are methods of use of the N and C fragments.