Drosophila crumbs is required to inhibit light-induced photoreceptor degeneration

Mutations in the human transmembrane protein CRB1 are associated with severe forms of retinal dystrophy, retinitis pigmentosa 12 (RP12), and Leber's congenital amaurosis (LCA). The Drosophila homolog, crumbs, is required for polarity and adhesion in embryonic epithelia and for correct formation of adherens junctions and proper morphogenesis of photoreceptor cells. Here, we show that mutations in Drosophila crumbs result in progressive, light-induced retinal degeneration. Degeneration is prevented by expression of p35, an inhibitor of apoptosis, or by reduction of rhodopsin levels through a vitamin A-deficient diet. In the dark, rhabdomeres survive but exhibit morphogenetic defects. We demonstrate that it is the extracellular portion of the Crumbs protein that is essential to suppress light-induced programmed cell death, while proper morphogenesis depends on the intracellular part. We conclude that human and Drosophila Crumbs proteins are functionally conserved to prevent light-dependent photoreceptor degeneration. This experimental system is now ideally suited to study the genetic and molecular basis of RP12- and LCA-related retinal degeneration.     

Cytotype control of Drosophila P element transposition: the 66 kd protein is a repressor of transposase activity

Drosophila P transposable elements encode two proteins, an 87 kd transposase protein and a 66 kd protein that has been hypothesized to repress transposition. We have made germline transformants carrying modified P element derivatives that encode only the 66 kd protein and shown that these elements repress transposase activity in both the germline and the soma. The position of these elements in the genome quantitatively affected their ability to negatively regulate transposase and to express the 66 kd protein. Single 66 kd element-containing strains did not exhibit the maternal inheritance of P cytotype characteristic of P strains. However, we demonstrated that a true P strain produced higher levels of the 66 kd protein during oogenesis than single 66 kd P elements. Thus, the expression of the 66 kd repressor during oogenesis may be a major determinant of the maternal effect of P cytotype.     

Heterochromatin protein 1 is involved in control of telomere elongation in Drosophila melanogaster

Telomeres of Drosophila melanogaster contain arrays of the retrotransposon-like elements HeT-A and TART. Their transposition to broken chromosome ends has been implicated in chromosome healing and telomere elongation. We have developed a genetic system which enables the determination of the frequency of telomere elongation events and their mechanism. The frequency differs among lines with different genotypes, suggesting that several genes are in control. Here we show that the Su(var)2-5 gene encoding heterochromatin protein 1 (HP1) is involved in regulation of telomere length. Different Su(var)2-5 mutations in the heterozygous state increase the frequency of HeT-A and TART attachment to the broken chromosome end by more than a hundred times. The attachment occurs through either HeT-A/TART transposition or recombination with other telomeres. Terminal DNA elongation by gene conversion is greatly enhanced by Su(var)2-5 mutations only if the template for DNA synthesis is on the same chromosome but not on the homologous chromosome. The Drosophila lines bearing the Su(var)2-5 mutations maintain extremely long telomeres consisting of HeT-A and TART for many generations. Thus, HP1 plays an important role in the control of telomere elongation in D. melanogaster.     

Human placental 15-hydroxy-prostaglandin dehydrogenase activity is under fetal genetic control

Placental 15-hydroxy-prostaglandin dehydrogenase (PGDH type I) was measured in 33 placentae obtained from 1 trizygotic, 7 dizygotic and 8 monozygotic pregnancies. PGDH activity ranged from 0.33 to 4.62 nmol PGF2 alpha metabolized per mg placental protein per min, which was within the range observed in singleton pregnancies. Expressing PGDH activity per mg DNA, offered no advantage over expressing it per mg total protein. PGDH activity differed significantly between the placentae of 6 of the 9 genetically non-identical placental pairs. The placentae of genetically identical twins, on the other hand, showed no difference in PGDH activity between the pairs. The data indicate that the genetic constitution of the fetus determines placental PGDH activity. They also provide us with the first evidence that the variation in prostaglandin catabolizing capacity of the human placenta is not entirely dictated by the maternal endocrine environment, but is under fetal control.     

Human placental 15-hydroxy-prostaglandin dehydrogenase activity is under fetal genetic control

Placental 15-hydroxy-prostaglandin dehydrogenase (PGDH type I) was measured in 33 placentae obtained from 1 trizygotic, 7 dizygotic and 8 monozygotic pregnancies. PGDH activity ranged from 0.33 to 4.62 nmol PGF_2α metabolized per mg placental protein per min, which was within the range observed in singleton pregnancies. Expressing PGDH activity per mg DNA, offered no advantage over expressing it per mg total protein. PGDH activity differed significantly between the placentae of 6 of the 9 genetically non-identical placental pairs. The placentae of genetically identical twins, on the other hand, showed no difference in PGDH activity between the pairs. The data indicate that the genetic constitution of the fetus determines placental PGDH activity. They also provide us with the first evidence that the variation in prostaglandin catabolized capacity of the human placenta is not entirely dictated by the maternal endocrine environment, but is under fetal control.     

A rho-binding protein kinase C-like activity is required for the function of protein kinase N in Drosophila development

The Rho GTPases interact with multiple downstream effectors to exert their biological functions, which include important roles in tissue morphogenesis during the development of multicellular organisms. Among the Rho effectors are the protein kinase N (PKN) proteins, which are protein kinase C (PKC)-like kinases that bind activated Rho GTPases. The PKN proteins are well conserved evolutionarily, but their biological role in any organism is poorly understood. We previously determined that the single Drosophila ortholog of mammalian PKN proteins, Pkn, is a Rho/Rac-binding kinase essential for Drosophila development. By performing "rescue" studies with various Pkn mutant constructs, we have defined the domains of Pkn required for its role during Drosophila development. These studies suggested that Rho, but not Rac binding is important for Pkn function in development. In addition, we determined that the kinase domain of PKC53E, a PKC family kinase, can functionally substitute for the kinase domain of Pkn during development, thereby exemplifying the evolutionary strategy of "combining" functional domains to produce proteins with distinct biological activities. Interestingly, we also identified a requirement for Pkn in wing morphogenesis, thereby revealing the first postembryonic function for Pkn.     

Dlg protein is required for junction structure, cell polarity, and proliferation control in Drosophila epithelia

The Discs large (Dlg) protein of Drosophila is the prototypic member of a growing family of proteins termed membrane-associated guanylate kinase homologs (MAGUKs). The MAGUKs are composed of a series of peptide domains that include one or three DHR/PDZs, an SH3, and a region homologous to guanylate kinase (GUK). We have previously shown that the product of this gene, the Dlg protein, is localized at the septate junctions between epithelial cells, and that mutations in the gene cause neoplastic overgrowth of the imaginal discs. The dlg locus is therefore defined as a tumor suppressor gene. In this paper, we show that the Dlg protein is localized on the cytoplasmic face of the septate junction and is required for the maintenance of this structure. It is also required for proper organization of the cytoskeleton, for the differential localization of membrane proteins, and for apicobasal polarity of epithelial cells. However, these other functions can be uncoupled from Dlg's role as a tumor suppressor since mutations in two domains of the protein, the SH3 and GUK, cause loss of normal cell proliferation control without affecting the other functions of the protein. These results suggest that, besides regulating cellular proliferation, the Dlg protein is a critical component of the septate junctions and is required for maintaining apicobasal polarity in Drosophila epithelium.     

Cell-specific expression of kidney androgen-regulated protein messenger RNA is under multihormonal control

Kidney androgen-regulated protein (KAP) gene expression is under androgenic control in the epithelial cells of the proximal convoluted tubule in the mouse kidney. In Tfm/Y androgen receptor-deficient mice, KAP mRNA was detected by in situ hybridization in a subpopulation of these cells only in the S3 segment of the proximal tubules in the outer medulla. Treatment of Tfm/Y animals with testosterone caused a partial induction of KAP mRNA levels, while dihydrotestosterone had no effect. These data suggested that the androgen receptor-independent induction of KAP gene expression in these animals was mediated by an estrogenic metabolite of testosterone, since dihydrotestosterone cannot be aromatized to an estrogenic form. Estrogen treatment of Tfm/Y mice caused an increase in KAP gene expression similar to that observed with testosterone. However, ovariectomy of normal female mice did not eliminate KAP gene expression in the S3 cells and, in fact, resulted in a slight increase. Adrenalectomy in combination with castration had no effect on KAP mRNA levels in S3 cells. However, hypophysectomy alone completely eliminated this cell-specific component of KAP gene expression. These results indicate that KAP gene expression is subject to cell-specific regulation in different segments of the proximal tubule and that this regulation is mediated by hormones of both gonadal and pituitary origin.     

The early light-induced protein is also produced during leaf senescence of Nicotiana tabacum

To better understand the genetic controls of leaf senescence, a tobacco (Nicotiana tabacum L. cv. SR1) mRNA that is up-regulated during senescence was isolated by the cDNA-amplified restriction fragment polymorphism method and the cDNA was cloned. The mRNA coded for the early light-induced protein (ELIP), a member of the chlorophyll a/b-binding protein family that has been implicated in assembly or repair of the photosynthetic machinery during early chloroplast development and abiotic stress. A protein antigenically recognized by antibodies to ELIP appeared during senescence with kinetics similar to those of its mRNA. The mRNA, designated ELIP-TOB, was detected earlier when senescence was enhanced by leaf detachment and treatment with 1-amino-cyclopropane-1-carboxylic acid, and was detected later when senescence was retarded by benzyladenine. However, no ELIP-TOB mRNA was seen in the dark even though senescence was accelerated under these conditions. Furthermore, water stress and anaerobiosis stimulated the appearance of ELIP-TOB mRNA before losses of chlorophyll could be detected. We discuss the conditions that may lead to the up-regulation of ELIP-TOB during senescence and speculate as to the role of the gene product in this terminal phase of leaf development. Received: 18 May 2000 / Accepted: 24 June 2000     

The functioning of the Drosophila CPEB protein Orb is regulated by phosphorylation and requires casein kinase 2 activity

The Orb CPEB protein regulates translation of localized mRNAs in Drosophila ovaries. While there are multiple hypo- and hyperphosphorylated Orb isoforms in wild type ovaries, most are missing in orb(F303), which has an amino acid substitution in a buried region of the second RRM domain. Using a proteomics approach we identified a candidate Orb kinase, Casein Kinase 2 (CK2). In addition to being associated with Orb in vivo, we show that ck2 is required for orb functioning in gurken signaling and in the autoregulation of orb mRNA localization and translation. Supporting a role for ck2 in Orb phosphorylation, we find that the phosphorylation pattern is altered when ck2 activity is partially compromised. Finally, we show that the Orb hypophosphorylated isoforms are in slowly sedimenting complexes that contain the translational repressor Bruno, while the hyperphosphorylated isoforms assemble into large complexes that co-sediment with polysomes and contain the Wisp poly(A) polymerase.     

Highly conserved ENY2/Sus1 protein binds to Drosophila CTCF and is required for barrier activity

Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers for the spreading of repressive chromatin. Drosophila insulator protein dCTCF marks active promoters and boundaries of many histone H3K27 trimethylation domains associated with repressed chromatin. In particular, dCTCF binds to such boundaries between the parasegment-specific regulatory domains of the Bithorax complex. Here we demonstrate that the evolutionarily conserved protein ENY2 is recruited to the zinc-finger domain of dCTCF and is required for the barrier activity of dCTCF-dependent insulators in transgenic lines. Inactivation of ENY2 by RNAi in BG3 cells leads to the spreading of H3K27 trimethylation and Pc protein at several dCTCF boundaries. The results suggest that evolutionarily conserved ENY2 is responsible for barrier activity mediated by the dCTCF protein.