The cramped gene of Drosophila is a member of the Polycomb-group, and interacts with mus209, the gene encoding Proliferating Cell Nuclear Antigen

We have isolated and molecularly characterized the cramped (crm) gene of Drosophila melanogaster, and show that it can be classified as a Polycomb-group (Pc-G) gene. crm mutants exhibit typical Pc-G mutant phenotypes, reminiscent of ectopic homeotic gene expression, with additional sex comb teeth found on mesothoracic and metathoracic legs, and proximodistal transformations of the tarsal segments. crm encodes an 693 amino acids protein, with no significant homology to known proteins. We used polyclonal antibodies raised against bacterially expressed truncated CRM protein to show that the crm gene product is localized to the nucleus during embryogenesis. This nuclear localization appears to be restricted to S-phase nuclei, as CRM immunostaining disappears at mitosis. We found that this cell-cycle-dependent staining pattern was identical to that of Proliferating Cell Nuclear Antigen (PCNA). Furthermore, we provide evidence for co-localization of CRM and PCNA proteins in salivary gland polytene nuclei, and for a genetic interaction between crm and mus209, the Drosophila gene encoding PCNA. Together, our data suggest that these two proteins are involved in a common regulatory pathway and highlight possible interactions between Pc-G-mediated silencing and DNA replication in Drosophila.     

A polysomal ribonuclease involved in the destabilization of albumin mRNA is a novel member of the peroxidase gene family

We have purified an approximately 60 kDa endoribonuclease from Xenopus liver polysomes with properties expected for a messenger RNase involved in the estrogen-regulated destabilization of serum protein mRNAs (Dompenciel et al., 1995, J Biol Chem 270:6108-6118). The present report describes the cloning of this protein and its identification as a novel member of the peroxidase gene family. This novel enzyme, named polysomal RNase 1, or PMR-1 has 57% sequence identity with myeloperoxidase, and like that protein, appears to be processed from a larger precursor. Unlike myeloperoxidase, however, PMR-1 lacks N-linked oligosaccharide, heme, and peroxidase activity. Western blot and immunoprecipitation experiments using epitope-specific antibodies to the derived protein sequence confirm the identity of the cloned cDNA to the protein originally isolated from polysomes. The 80 kDa pre-PMR-1 expressed in a recombinant baculovirus was not processed to the 60 kDa form in Sf9 cells and lacks RNase activity. However, the baculovirus-expressed mature 60-kDa form of the enzyme has RNase activity. The recombinant protein is an endonuclease that shows selectivity for albumin versus ferritin mRNA. While it does not cleave at consensus APyrUGA elements, recombinant PMR-1 generates the same minor cleavage products from albumin mRNA as PMR-1 purified from liver. Finally, we show estrogen induces only a small increase in the amount of PMR-1. This result is consistent with earlier data suggesting estrogen activates mRNA decay through a posttranslational pathway.     

The role of selectins in Drosophila eye and bristle development

Mutations in the furrowed (fw) gene of Drosophila result in defects in the development of the eye and mechanosensory bristles. The eyes are reduced in size, have furrows or crevices in the retina, and show a disturbed patterning of ommatidia. In addition, the ommatidia have an altered morphology and often contain abnormal numbers of cells. The bristles show altered structure and polarity, and are occasionally duplicated or missing. These results suggest that the product of thefw gene is involved in cell determination events within sensory organs. Thefw gene has been cloned and shown to encode a protein homologous to vertebrate selectins. Like selectins, Fw contains a lectin-binding domain, ten complement binding repeats, and a transmembrane domain. The Fw protein is expressed in the larval imaginal discs where it might mediate carbohydrate-protein interactions necessary for proper development of a subset of adult sensory organs.     

ClpE, a novel member of the HSP100 family, is involved in cell division and virulence of Listeria monocytogenes

We identified, in the facultative intracellular pathogen Listeria monocytogenes, a previously unknown Clp ATPase, unique among the HSP100 proteins because of the presence of a short N-terminal region with a potential zinc finger motif. This protein of 726 amino acids is highly homologous to ClpE of Bacillus subtilis, and is a member of a new subfamily of HSP100/Clp ATPases. The clpE gene is transcribed as a monocistronic mRNA from a typical consensus sigma A promoter. clpE is not stimulated by various stresses, but is upregulated in a clpC mutant. This is the first example of cross-regulation between Clp ATPases. By constructing a clpE mutant of L. monocytogenes, we found that ClpE is required for prolonged survival at 42 degrees C and is involved in the virulence of this pathogen. A double mutant deficient in both ClpE and ClpC was avirulent in a mouse model and completely eliminated in the liver. Electron microscopy studies did not show any morphological alterations in clpE or clpC mutants. In the clpE-clpC double mutant, however, cell division was affected, indicating that ClpE acts synergistically with ClpC in cell septation. These results show that the Clp chaperones play a crucial role in both cell division and virulence of L. monocytogenes.     

A member of the Ran-binding protein family, Yrb2p, is involved in nuclear protein export

Yeast cells mutated in YRB2, which encodes a nuclear protein with similarity to other Ran-binding proteins, fail to export nuclear export signal (NES)-containing proteins including HIV Rev out of the nucleus. Unlike Xpo1p/Crm1p/exportin, an NES receptor, Yrb2p does not shuttle between the nucleus and the cytoplasm but instead remains inside the nucleus. However, by both biochemical and genetic criteria, Yrb2p interacts with Xpo1p and not with other members of the importin/karyopherin beta superfamily. Moreover, the Yrb2p region containing nucleoporin-like FG repeats is important for NES-mediated protein export. Taken together, these data suggest that Yrb2p acts inside the nucleus to mediate the action of Xpo1p in at least one of several nuclear export pathways.     

The dynamics of neurogenic signalling underlying bristle development in Drosophila melanogaster

We have examined expression of the neurogenic gene, Delta (Dl), and the regulatory relationships between the Delta-Notch signalling pathway and the proneural gene, achaete, during microchaeta development in Drosophila. Delta is expressed in all microchaeta proneural cells and microchaeta sensory organ precursors (SOPs) and is expressed dynamically in SOP progeny. We find that Delta expression in microchaeta proneural cells is detected prior to the onset of achaete expression and arises normally in the absence of achaete/scute function, indicating that initial Delta expression in the notum is not dependent on proneural gene function. Activation of the Delta-Notch pathway results in loss of Delta protein accumulation, suggesting that Delta expression is regulated, in part, by Delta-Notch signalling activity. We find that Delta signalling is required for correct delineation of early proneural gene expression in developing nota. Within microchaeta proneural stripes, we demonstrate that Delta-Notch signalling prohibits adoption of the SOP fate by repressing expression of proneural genes.     

Nucleotide sequence of 5''-upstream region and expression of a silkworm gene encoding a new member of the attacin family

The morphometric and morphological changes in the mesothelial cell population were studied in rabbits in peritoneal dialysis with lactate and bicarbonate buffer solution. During dialysis the mesothelial population underwent radical changes in morphology and morphometric analysis showed a significant increase in cell size. Light microscope examination showed two types of changes: hyperplasia of the mesothelial cell with diameters of up to 80 microns, nucleus proportional to the cytoplasm, a large nucleole giving an owl's eye appearance and cytoplasm rich in granular material. The second change was multiple nuclei and arrest of cell division. Nuclear division occurred, but no separation of the cytoplasm. The cells became larger than 200 microns, packed with nuclei and relatively little cytoplasm. Electron microscopy confirmed that the hyperplastic cells had perfect structure whereas the polynucleate cells contained vacuoles and little cytoplasmic reticulum. Immunohistochemistry using monoclonal antibodies SK2-27 and SK 60-61 specific to cytokeratins 14, 16, 17 and 8, 18, respectively, identified the cells as mesothelial. The changes were related to the glucose content of the peritoneal dialysis solution. Glucose is therefore the bioincompatible agent that modifies the mesothelium during peritoneal dialysis, causing it to become hyperplastic or blocking replication.     

SOX20, a new member of the SOX gene family, is located on chromosome 17p13

SOX genes share a high sequence identity with the HMG box present in the testis determining gene SRY. We have identified a HMG box-like sequence motif on six contiguous cosmids, which cross-hybridize to a SOX9 cDNA probe. A data base search revealed a high similarity of the deduced amino acid sequence to the human SOX12 and the murine Sox16 HMG domains. The cosmids were assigned to chromosome 17p13 by FISH analysis.     

DHR3, an ecdysone-inducible early-late gene encoding a Drosophila nuclear receptor, is required for embryogenesis

Response to the steroid hormone ecdysone in Drosophila is controlled by genetic regulatory hierarchies that include eight members of the nuclear receptor protein family. The DHR3 gene, located within the 46F early-late ecdysone-inducible chromosome puff, encodes an orphan nuclear receptor that recently has been shown to exert both positive and negative regulatory effects in the ecdysone-induced genetic hierarchies at metamorphosis. We used a reverse genetics approach to identify 11 DHR3 mutants from a pool of lethal mutations in the 46F region on the second chromosome. Two DHR3 mutations result in amino acid substitutions within the conserved DNA binding domain. Analysis of DHR3 mutants reveals that DHR3 function is required to complete embryogenesis. All DHR3 alleles examined result in nervous system defects in the embryo.