Homogeneous preparations of actin cleaved into two fragments, the N-terminal 9- and C-terminal 36-kDa peptides, were achieved by proteolysis of G-actin with subtilisin at 23 degrees C at a 1:1000 (w/w) ratio of enzyme to actin. The subtilisin cleavage site was identified by sequence analysis to be between Met-47 and Gly-48. Although under nondenaturing conditions the two fragments remained associated to one another, the cleavage affected macromolecular interactions of actin. The rates of cleaved actin polymerization by MgCl2, KCl, and myosin subfragment 1 (S-1) were slower and the critical concentrations for this process were higher than in intact protein. Intact and cleaved actin formed morphologically indistinguishable filaments and copolymerized in the presence of MgCl2. The affinity of actin for S-1 was decreased by about 10-fold due to subtilisin cleavage, but the S-1 ATPase activity was activated to the same Vmax value by both intact and cleaved actins. DNase I inhibition measurements revealed lower affinity of cleaved actin for DNase I than that of intact protein. These results are discussed in terms of actin's structure.
The product of the zygotically active decapentaplegic (dpp) gene appears to function as a morphogen that specifies positional information in the dorsal half of the Drosophila embryo. The dorsal-specific transcription of dpp is the key step in establishing a morphogen gradient. We demonstrate here that multiple regions
Abstract. Subtilisin cleaved actin was shown to retain several properties of intact actin including the binding of heavy meromyosin (HMM), the dissociation from HMM by ATE and the activation of HMM ATPase activity. Similar Vm~x but different Km values were obtained for acto-HMM ATPase with the cleaved and intact actins. The ATPase activity of HMM stimulated by copolymers of intact and cleaved actin showed a linear dependence on the fraction of intact actin in the copolymer. The most important difference between the intact and cleaved actin was observed in an in vitro motility assay for actin sliding movement over an HMM coated surface. Only 30% of the cleaved actin filaments appeared mobile in this assay and moreover, the velocity of the mobile filaments was "-,30% that of intact actin filaments. These results suggest that the motility of actin filaments can be uncoupled from the activation of myosin ATPase activity and is dependent on the structural integrity of actin and perhaps, dynamic changes in the actin molecule.
The Drosophila melanogaster decapentaplegic (dpp) gene encodes a transforming growth factor -related cell signaling molecule that plays a critical role in dorsal/ventral pattern formation. The dpp expression pattern in the Drosophila embryo is dynamic, consisting of three phases. Phase I, in which dpp is expressed in a broad dorsal domain, depends on elements in the dpp second intron that interact with the Dorsal transcription factor to repress transcription ventrally. In contrast, phases II and III, in which dpp is expressed first in broad longitudinal stripes (phase II) and subsequently in narrow longitudinal stripes (phase III), depend on multiple independent elements in the dpp 5-flanking region. Several aspects of the normal dpp expression pattern appear to depend on the unique properties of the dpp core promoter. For example, this core promoter (extending from ؊22 to ؉6) is able to direct a phase II expression pattern in the absence of additional upstream or downstream regulatory elements. In addition, a ventral-specific enhancer in the dpp 5-flanking region that binds the Dorsal factor activates the heterologous hsp70 core promoter but not the dpp core promoter. Thus, the dpp core promoter region may contribute to spatially regulated transcription both by interacting directly with spatially restricted activators and by modifying the activity of proteins bound to enhancer elements.The transforming growth factor  gene superfamily encodes a large set of cell signaling proteins with diverse roles in development, cell growth, and differentiation (21). These factors are synthesized as long precursors, which are processed to generate ϳ100-amino-acid polypeptide factors. The signals generated by these secreted factors are transmitted by membrane-bound receptor serine/threonine kinases (22). Because several members of the transforming growth factor  superfamily contribute to spatially regulated developmental phenomena, a full understanding of the processes that are controlled by these factors will require an understanding of the mechanisms that control their spatially restricted synthesis and release.The product of the decapentaplegic (dpp) gene is one of several known transforming growth factor -related factors in Drosophila melanogaster (23). Among its many roles in both embryonic and larval development, Dpp acts as a morphogen that helps to determine cell fate on the dorsal side of the embryo (8). The expression pattern of dpp in the developing embryo is controlled at the level of transcription and is extremely dynamic, consisting of three distinct phases (30). In phase I expression, which is first detected in the syncytial blastoderm (ϳ1.5 h postfertilization), dpp is transcribed in the dorsal 40% of the embryo, a region that includes the anlagen of the amnioserosa and the dorsal epidermis ( Fig. 1A and D). Phase I expression is dependent upon the maternally encoded Dorsal morphogen, which binds to elements in the dpp second intron to repress dpp expression in the ventral 60% of the blastoderm embryo (13)....
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