The periodic, seven-stripe pattern of the primary pair-rule gene even-skipped (eve) is initiated by crude, overlapping gradients of maternal and gap gene proteins in the early Drosophila embryo. Previous genetic studies suggest that one of the stripes, stripe 2, is initiated by the maternal morphogen bicoid (bcd} and the gap protein hunchback (hb), while the borders of the stripe are formed by selective repression, involving the gap protein giant (gt) in anterior regions and the Kriippel (Kr) protein in posterior regions. Here, we present several lines of evidence that are consistent with this model for stripe 2 expression, including in vitro DNA-binding experiments and transient cotransfection assays in cultured cells. These experiments suggest that repression involves a competition or short-range quenching mechanism, whereby the binding of gt and Kr interferes with the binding or activity of bcd and hb activators at overlapping or neighboring sites within the eve stripe 2 promoter element. Such short-range repression could reflect a general property of promoters composed of multiple, but autonomous regulatory elements.

To facilitate qualitative and quantitative analysis of glycosaminoglycans, we tagged the reducing end of lyase-generated disaccharides with aniline-containing stable isotopes ( 12 C 6 and 13 C 6 ). Because different isotope tags have no effect on chromatographic retention times but can be discriminated by a mass detector, differentially isotope-tagged samples can be compared simultaneously by liquid chromatography/mass spectrometry and quantified by admixture with known amounts of standards. The technique is adaptable to all types of glycosaminoglycans, and its sensitivity is only limited by the type of mass spectrometer available. We validated the method using commercial heparin and keratan sulfate as well as heparan sulfate isolated from mutant and wild-type Chinese hamster ovary cells, and select tissues from mutant and wild-type mice. This new method provides more robust, reliable, and sensitive means of quantitative evaluation of glycosaminoglycan disaccharide compositions than existing techniques allowing us to compare the chondroitin and heparan sulfate compositions of Hydra vulgaris, Drosophila melanogaster, Caenorhabditis elegans, and mammalian cells. Our results demonstrate significant differences in glycosaminoglycan structure among these organisms that might represent evolutionarily distinct functional motifs.Metazoans make several types of sulfated glycosaminoglycans (GAGs), 2 including keratan sulfate (KS), chondroitin sulfate/dermatan sulfate (CS/DS), and heparan sulfate/heparin (HS). Each type of chain consists of unique disaccharide units. KS consists of galactose (Gal) and GlcNAc ([Gal␤1, 4GlcNAc␤1,3] n ) with variable sulfation at C6 of either sugar. CS/DS assembles as a copolymer of GlcA␤1,3GalNAc␤1,4 and then undergoes various processing reactions, including C5 epimerization of a portion of GlcA units to iduronic acid in DS, O-sulfation at C2 and more rarely at C3 of the uronic acids, and O-sulfation at C4 and C6 of the GalNAc residues (1). HS is the most highly modified GAG, consisting initially of GlcA␤1,4GlcNAc␣1,4 units, which then undergo variable processing by GlcNAc N-deacetylation and N-sulfation, C5 epimerization of some GlcA units to iduronic acid, and O-sulfate addition to C2 of the uronic acids and C6 and more rarely at C3 of the glucosamine units (2). The arrangement of the modified residues along the chain creates binding sites for numerous growth factors, enzymes, and extracellular matrix proteins. The structural variation that can occur makes sulfated GAG chains one of the most complex classes of macromolecules found in nature.GAG fine structure is typically assessed by analyzing the disaccharide composition of an isolated mixture of chains. A number of techniques have been developed to accomplish this task that rely on chemical or enzymatic depolymerization of the chains into their constituent disaccharides, followed by separation via anion exchange chromatography, reversed-phase chromatography with ion pairing agents, or capillary electrophoresis. These techniques separate...

Decapentaplegic (Dpp) signaling. This contrasts with previous studies that suggested loss of ttv compromises only Hh signaling. Our results may contribute to understanding the biological basis of hereditary multiple exostoses (HME), a disease associated with bone overgrowth that results from mutations in EXT1 and EXT2, the human orthologs of ttv and sotv.

We demonstrate that dynein is required for nuclear anchoring and localization of cellular determinants during oogenesis. Strikingly, mutations in the kinesin motor also disrupt these processes and perturb dynein and dynactin localization. These results indicate that the activity of the two motors is interdependent and suggest a model in which kinesin affects patterning indirectly through its role in the localization and recycling of dynein.