Hairy-related proteins include the Drosophila Hairy and Enhancer of Split proteins and mammalian Hes proteins. These proteins are basic helix-loop-helix (bHLH) transcriptional repressors that control cell fate decisions such as neurogenesis or myogenesis in both Drosophila melanogaster and mammals. Hairy-related proteins are site-specific DNA-binding proteins defined by the presence of both a repressor-specific bHLH DNA binding domain and a carboxyl-terminal WRPW (Trp-Arg-Pro-Trp) motif. These proteins act as repressors by binding to DNA sites in target gene promoters and not by interfering with activator proteins, indicating that these proteins are active repressors which should therefore have specific repression domains. Here we show the WRPW motif to be a functional transcriptional repression domain sufficient to confer active repression to Hairy-related proteins or a heterologous DNA-binding protein, Gal4. This motif was previously shown to be necessary for interactions with Groucho, a genetically defined corepressor for Drosophila Hairy-related proteins. Here we show that the WRPW motif is sufficient to recruit Groucho or the TLE mammalian homologs to target gene promoters. We also show that Groucho and TLE proteins actively repress transcription when directly bound to a target gene promoter and identify a novel, highly conserved transcriptional repression domain in these proteins. These results directly demonstrate that Groucho family proteins are active transcriptional corepressors for Hairy-related proteins and are recruited by the 4-amino acid protein-protein interaction domain, WRPW.Basic helix-loop-helix (bHLH) transcription factors control cell fate decisions, such as myogenesis or neurogenesis, in many animal species (9-11, 21, 33, 34, 38, 60, 80, 81). These proteins can be classified into two groups, the activator bHLH proteins and the repressor bHLH proteins, on the basis of biological function (56). Remarkably, the DNA binding specificities of the activator and repressor bHLH proteins directly correlate with their biological functions (56). The activator proteins, such as MyoD or the proteins of the Achaete-Scute complex, promote differentiation by binding to class A binding sites and activating transcription (21,50,52,53,56,77,81). The repressor bHLH proteins are Hairy-related proteins such as Hairy, the proteins of the Enhancer of Split [E(spl)] complex, and the homologous mammalian Hes proteins (2,19,23,32,41,43,56,61,62,70,71). These proteins antagonize the activator proteins and prevent differentiation by binding to specific class B or C sites and repressing transcription (2,32,54,56,70,73,75). Thus, Hairy-related proteins are distinct from the emc and Id HLH repressors, which lack basic regions and repress by forming non-DNA-binding heterodimers with the activator bHLH proteins (7,17,22,25,76,77). Drosophila neurogenesis is regulated by both activator and repressor bHLH genes (9-11, 33, 34, 38). The activators are proneural genes (33) and include daughterless (13), the four genes of the ach...

Sensory organ formation in Drosophila is activated by proneural genes that encode basic-helix-loop-helix (bHLH) transcription factors. These genes are antagonized by hairy and other proline-bHLH proteins, hairy has not been shown to bind to DNA and has been proposed to form inactive heterodimers with proneural activator proteins. Here, we show that hairy does bind to DNA and has novel DNA-binding activity: hairy prefers a noncanonical site, CACGCG, although it also binds to related sites. Mutation of a single CACGCG site in the achaete [ac) proneural gene blocks hairy-mediated repression of ac transcription in cultured Drosopbila cells. Moreover, the same CACGCG mutation in an ac minigene transformed into Drosopbila creates ectopic sensory hair organs like those seen in bairy mutants. Together these results indicate that hairy represses sensory organ formation by directly repressing transcription of the ac proneural gene.

The lesswright (lwr) gene encodes an enzyme that conjugates a small ubiquitin-related modifier (SUMO). Since the conjugation of SUMO occurs in many different proteins, a variety of cellular processes probably require lwr function. Here, we demonstrate that lwr function regulates the production of blood cells (hemocytes) in Drosophila larvae. lwr mutant larvae develop many melanotic tumors in the hemolymph at the third instar stage. The formation of melanotic tumors is due to a large number of circulating hemocytes, which is approximately 10 times higher than those of wild type. This overproduction of hemocytes is attributed to the loss of lwr function primarily in hemocytes and the lymph glands, a hematopoietic organ in Drosophila larvae. High incidences of Dorsal (Dl) protein in the nucleus were observed in lwr mutant hemocytes, and the dl and Dorsal-related immunity factor (Dif) mutations were found to be suppressors of the lwr mutation. Therefore, the lwr mutation leads to the activation of these Rel-related proteins, key transcription factors in hematopoiesis. We also demonstrate that dl and Dif play different roles in hematopoiesis. dl primarily stimulates plasmatocyte production, but Dif controls both plasmatocyte and lamellocyte production.

To elucidate the functional role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in neuronal cells, we studied the phenotypic effects of overexpression of the CaM kinase II wild-type alpha subunit and a mutant enzyme alpha isoform (Ala-286), in which formation of the Ca(2+)-independent form by autophosphorylation is markedly suppressed by replacement of Thr-286 with Ala, using Neuro2a (Nb2a) and NG108-15 neuroblastoma cell lines. The cDNAs inserted into the EcoRI site of pEF321 expression vector were introduced into Nb2a and NG108-15 cells with pEF321-neo (neo). Stable clones were obtained by G418 selection. The specific activities of CaM kinase II in alpha and Ala-286 transfectants were two to four times higher than those in non-transfectants and in cells transfected with neo alone. Indirect immunofluorescence using a monoclonal antibody specific to the CaM kinase II alpha isoform revealed that CaM kinase II was mainly localized in the perikaryal and dendritic cytoplasm of the alpha and Ala-286 transfectants. Immediately after plating, Nb2a and NG108-15 cells transfected with neo, alpha and Ala-286 cDNAs appeared round. Several hours after plating, alpha transfectants showed cell flattening and initiation of neurite outgrowth, and thereafter extended numerous long and branching neurites. Numerous filopodia protruded from flat growth cones, some of which were accompanied by extensive veil formation. Non- and neo transfectants remained round. In Ala-286 transfectants, however, the phenotypic changes were remarkably less than in alpha transfectants.(ABSTRACT TRUNCATED AT 250 WORDS)

The primary structures of the precursors of urotensin II (UII)-a and -7, neuropeptide hormones of the caudal neurosecretory system of the carp, Cyprinus carpio, have been determined by analyzing the nucleotide sequences of cloned DNAs complementary to the mRNAs encoding them.A cDNA library, constructed with poly(A)+RNA in the preterminal spinal cord, was screened using 3ZP-labeled synthetic oligonucleotldes representing all possible cDNA sequences corresponding to the pentapeptlde common to all forms of carp UII (UII-a, -0, and 9). Twenty out of 39 positive clones were analyzed with the restriction endonucleases, Hi&III and PvuII, and classified into 4 groups. Nucleotide sequence analysis of 4 clones representing each group revealed that 2 clones encode the precursor of UIk and the other 2 that of UII-y. Both precursors consist of 125 amino acid residues, and UI1-a and -"/ exist at the carboxyl-termini preceded by Arg-Lys-Arg. The homology in both nucleotide and amino acid sequences between the precursors of UII-a and -7 is more than 90%, suggesting that the genes were generated from a common ancestral gene by duplication. There is no sequence homology between the precursors of UII and urotensin I, another peptlde hormone of the caudal neurosecretory system, nor between the precursors of UII and somatostatin-14. RNA transfer blot analysis indicated that mRNAs encoding the precursors of UI1-u and 9 are present in the spinal cord but not in the brain, intestine, liver, or kidney of the carp. In sifu hybridization using 32P-labeled synthetic oligonucleotide complementary to common sequence of mRNAs for both UII-ol and --y has detected the UII-producing neurons in the caudal spinal cord of the carp.The caudal neurosecretory system in the posterior spinal cord of elasmobranch and teleost fishes, defined by Enami (1959), synthesizes and releases at least 2 neurohormones, urotensins I and 11 (UI and UII). Although the architecture of this system, similar to the hypothalamoneurohypophysial system, and its universal presence in fish suggest that these peptides have an important role in their physiology, a coherent function of caudal neurosecretion has not been established. Urotensin I is a 41 amino acid residue peptide (Ichikawa et al., 1982; homologous to mammalian corticotropin-releasing factor (CRF; see Ichikawa, 1985). The elucidation ofthe primary structure of the precursor of carp UI (Ishida et al., 1986) has shown a sequence homology, as well as an organizational sim-

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Ciliary zonules are responsible for changing the curvature of a lens in the dioptric focus of an eye. Present established theory is based on the relaxation of zonular superficial fasciculi affixed to the capsular surface, thereby inducing the change of anterior-and posterior lens curvature causing spontaneous liquid movement of lens material. To achieve precise focusing at any distance, a more active functional organization should exist. The present studies were performed to determine not only the surface attachment but also the intracapsular affix of zonules on monkey eyes. In addition, the development of focusing in newborn and presbyopia is analyzed. Histology was prepared by conventional and molecular immunofluorescence stainings on the compositions of zonules with fibrillin-1 (FBN 1) and lens capsule with collagen IV (COL IV), and in situ hybridization (ISH) analyses on frozen sections. Superficial circumferential attachments of zonule were found radially oriented between ciliary processes and anterior/posterior lens capsules forming a triangular figure. Two functional intralayer integrations were found above them; anteriorposterior crossed fibers over the equator and radial fibers distributed toward the anterior or posterior polar areas. These fibers were bound tightly to the deep layer connective tissues close to the lens epithelium. Fine zonular fibers were aggregated, gradually forming bundles and bifurcated again on the way to the capsule. The lateral striped staining pattern in bundles suggested their elastic nature. Response of a-helixes of collagen IV immunostaining was more positive on a-1,2,4 than a-3,5,6 on anterior-and posterior lens capsules. Newborn eyes revealed not fascicular but fine membranous zonules on the lens surface and small ciliary processes. ISH analysis revealed high synthetic expression of FBN 1 mRNA in cytoplasm of nonpigmented epithelial cells of ciliary processes. The synthetic expression of FBN 1 declined with aging.

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