Proteoglycan UDP-galactose:beta-xylose beta 1,4-galactosyltransferase I is essential for viability in Drosophila melanogaster

Heparan and chondroitin sulfates play essential roles in growth factor signaling during development and share a common linkage tetrasaccharide structure, GlcAbeta1,3Galbeta1,3Galbeta1,4Xylbeta1-O-Ser. In the present study, we identified the Drosophila proteoglycan UDP-galactose:beta-xylose beta1,4-galactosyltransferase I (dbeta4GalTI), and determined its substrate specificity. The enzyme transferred a Gal to the -beta-xylose (Xyl) residue, confirming it to be the Drosophila ortholog of human proteoglycan UDP-galactose:beta-xylose beta1,4-galactosyltransferase I. Then we established UAS-dbeta4GalTI-IR fly lines containing an inverted repeat of dbeta4GalTI ligated to the upstream activating sequence (UAS) promoter, a target of GAL4, and observed the F(1) generation of the cross between the UAS-dbeta4GalTI-IR fly and the Act5C-GAL4 fly. In the F(1), double-stranded RNA of dbeta4GalTI is expressed ubiquitously under the control of a cytoplasmic actin promoter to induce the silencing of the dbeta4GalTI gene. The expression of the target gene was disrupted specifically, and the degree of interference was correlated with phenotype. The lethality among the progeny proved that beta4GalTI is essential for viability. This study is the first to use reverse genetics, RNA interference, to study the Drosophila glycosyltransferase systematically.     

Identification of the Drosophila core 1 beta1,3-galactosyltransferase gene that synthesizes T antigen in the embryonic central nervous system and hemocytes

T antigen (Galbeta1-3GalNAcalpha1-Ser/Thr), the well-known tumor-associated antigen, is a core 1 mucin-type O-glycan structure that is synthesized by core 1 beta1,3-galactosyltransferase (C1beta3GalT), which transfers Gal from UDP-Gal to Tn antigen (GalNAcalpha1-Ser/Thr). Three putative C1beta3GalTs have been identified in Drosophila. However, although all three are expressed in embryos, their roles during embryogenesis have not yet been clarified. In this study, we used P-element inserted mutants to show that CG9520, one of the three putative C1beta3GalTs, synthesizes T antigen expressed on the central nervous system (CNS) during embryogenesis. We also found that T antigen was expressed on a subset of the embryonic hemocytes. CG9520 mutant embryos showed the loss of T antigens on the CNS and on a subset of hemocytes. Then, the loss of T antigens was rescued by precise excision of the P-element inserted into the CG9520 gene. Our data demonstrate that T antigens expressed on the CNS and on a subset of hemocytes are synthesized by CG9520 in the Drosophila embryo. In addition, we found that the number of circulating hemocytes was reduced in third instar larvae of CG9520 mutant. We, therefore, named the CG9520 gene Drosophila core 1 beta1,3-galactosyltransferase 1 because it is responsible for the synthesis and function of T antigen in vivo.     

Identification and characterization of a Drosophila melanogaster ortholog of human beta1,4-galactosyltransferase VII

Drosophila melanogaster is widely considered to be an attractive model organism for studying the functions of the carbohydrate moieties of glycoconjugates produced by higher eukaryotes. However, the pathways of glycoconjugate biosynthesis are not as well defined in insects as they are in higher eukaryotes. One way to address this problem is to identify genes in the Drosophila genome that might encode relevant functions, express them, and determine the functions of the gene products by direct biochemical assays. In this study, we used this approach to identify a putative Drosophila beta4-galactosyltransferase gene and determine the enzymatic activity of its product. Biochemical assays demonstrated that this gene product could transfer galactose from UDP-galactose to a beta-xylosyl acceptor, but not to other acceptors in vitro. The apparent K(m) values for the donor and acceptor substrates indicated that this gene product is a functional galactosyltransferase. Additional assays showed that the enzyme is activated by manganese, has a slightly acidic pH optimum, and is localized in the insect cell Golgi apparatus. These results showed that Drosophila encodes an ortholog of human beta4-galactosyltransferase-VII, also known as galactosyltransferase I, which participates in proteoglycan biosynthesis by transferring the first galactose to xylose in the linkage tetrasaccharide of glycosaminoglycan side chains.     

Identification and characterization of a Pantoea citrea gene encoding glucose dehydrogenase that is essential for causing pink disease of pineapple.

Pantoea citrea, a member of the family Enterobacteriaceae, causes pink disease of pineapple, whose symptom is characterized by the formation of pink to brown discolorations of the infected portions of the pineapple fruit cylinder upon canning. Molecular genetic approaches were applied to elucidate the mechanism responsible for this fruit discoloration. A P. citrea mutant strain, CMC6, defective in its ability to cause pink disease and fruit discoloration, was generated by nitrosoguanidine mutagenesis. A DNA fragment that restored these activities was isolated by screening a genomic cosmid library of P. citrea. A large open reading frame of 2,361 bp, identified by nucleotide sequencing of a subclone of the complementing DNA, showed high similarities to identified genes encoding glucose dehydrogenase (GDH) in Escherichia coli, Acinetobacter calcoaceticus, and Gluconobacter oxydans. The predicted amino acid sequence of GDH of P. citrea was identical to known GDHs in these bacteria by 54, 44, and 34%, respectively. GDH of P. citrea has a predicted molecular mass of 86.2 kDa, contains a conserved binding domain for the cofactor pyrroloquinoline quinone, and possesses GDH activity as demonstrated by biochemical assay. GDH is the key branch point enzyme leading to the biosynthesis of gluconate, which in turn serves as the substrate leading to the formation of 2-ketogluconate, 2,5-diketogluconate, 6-phosphogluconate, and 2-keto-6-phosphogluconate. Addition of gluconate to CMC6 restores the juice- and fruit-discoloring activity. Although the pigments formed by heating (or canning) have not been identified, it is clear that GDH is one of the enzymes required for pigment formation leading to pink disease.     

Identification and characterization of a gene encoding a kinesin-like protein in Drosophila

We identified and sequenced a cDNA clone encoding a kinesin-like protein from Drosophila. The predicted product of this cDNA has a carboxy-terminal domain that is substantially similar to the motor domain of kinesin heavy chain. The amino-terminal domain is unlike that found in previously identified kinesins or kinesin-like proteins. Analyses of this new sequence suggest that the maximal motor unit in the kinesin superfamily may be as little as 350 amino acids, and that the existence of both kinesin and kinesin-like molecules must be an evolutionarily ancient feature of eukaryotes. We also tested some of the biochemical properties of the protein encoded by this cDNA and found them to be similar to those of kinesin. Finally, the clone we isolated appears to correspond to the non-claret disjunctional (ncd) gene, which when mutant causes defects in meiotic and early embryonic mitotic chromosome segregation, and whose recently determined sequence predicts a kinesin-like domain.     

Drosophila E-cadherin is essential for proper germ cell-soma interaction during gonad morphogenesis

In most animal species, germ cells require intimate contact with specialized somatic cells in the gonad for their proper development. We have analyzed the establishment of germ cell-soma interaction during embryonic gonad formation in Drosophila melanogaster, and find that somatic cells undergo dramatic changes in cell shape and individually ensheath germ cells as the gonad coalesces. Germ cell ensheathment is independent of other aspects of gonad formation, indicating that separate morphogenic processes are at work during gonadogenesis. The cell-cell adhesion molecule Drosophila E-cadherin is essential both for germ cell ensheathment and gonad compaction, and is upregulated in the somatic gonad at the time of gonad formation. Our data indicate that differential cell adhesion contributes to cell sorting and the formation of proper gonad architecture. In addition, we find that Fear of Intimacy, a novel transmembrane protein, is also required for both germ cell ensheathment and gonad compaction. E-cadherin expression in the gonad is dramatically decreased in fear of intimacy mutants, indicating that Fear of Intimacy may be a regulator of E-cadherin expression or function.     

Embryonic retinoic acid synthesis is essential for heart morphogenesis in the mouse

Retinoic acid (RA), the active derivative of vitamin A, has been implicated in various steps of cardiovascular development, but its contribution to early heart morphogenesis has not been clearly established in a mammalian system. To block endogenous RA synthesis, we have disrupted the gene encoding RALDH2, the first retinaldehyde dehydrogenase whose expression has been detected during early mouse post-implantation development. We describe here the heart abnormalities of the RA-deficient Raldh2 mutants that die in utero at gestational day 10.5. The embryonic heart tube forms properly, but fails to undergo rightward looping and, instead, forms a medial distended cavity. Expression of early heart determination factors is not altered in mutants, and the defect in heart looping does not appear to involve the Nodal/Lefty/Pitx2 pathway. Histological and molecular analysis reveal distinct anteroposterior components in the mutant heart tube, although posterior chamber (atria and sinus venosus) development is severely impaired. Instead of forming trabeculae, the developing ventricular myocardium consists of a thick layer of loosely attached cells. Ultrastructural analysis shows that most of the ventricular wall consists of prematurely differentiated cardiomyocytes, whereas undifferentiated cells remain clustered rostrally. We conclude that embryonic RA synthesis is required for realization of heart looping, development of posterior chambers and proper differentiation of ventricular cardiomyocytes. Nevertheless, the precise location of this synthesis may not be crucial, as these defects can mostly be rescued by systemic (maternal) RA administration. However, cardiac neural crest cells cannot be properly rescued in Raldh2(-/- )embryos, leading to outflow tract septation defects.     

Interaction of Par-6 and Crumbs complexes is essential for photoreceptor morphogenesis in Drosophila

Apicobasal cell polarity is crucial for morphogenesis of photoreceptor rhabdomeres and adherens junctions (AJs) in the Drosophila eye. Crumbs (Crb) is specifically localized to the apical membrane of photoreceptors, providing a positional cue for the organization of rhabdomeres and AJs. We show that the Crb complex consisting of Crb, Stardust (Sdt) and Discs-lost (Dlt) colocalizes with another protein complex containing Par-6 and atypical protein kinase C (aPKC) in the rhabdomere stalk of photoreceptors. Loss of each component of the Crb complex causes age-dependent mislocalization of Par-6 complex proteins, and ectopic expression of Crb intracellular domain is sufficient to recruit the Par-6 complex. We also show that the absence of Par-6 complex proteins results in severe mislocalization and loss of Crb complex. We further demonstrate that Dlt directly binds to Par-6, providing a molecular basis for the mutual dependence of the two complexes. These results suggest that the interaction of Crb and Par-6 complexes is required for the organization and maintenance of apical membranes and AJs of photoreceptors.     

Eyes closed, a Drosophila p47 homolog, is essential for photoreceptor morphogenesis.

Starting with a mutation impacting photoreceptor morphogenesis, we identify here a Drosophila gene, eyes closed (eyc), as a fly homolog of p47, a protein co-factor of the p97 ATPase implicated in membrane fusion. Temporal misexpression of Eyc during rhabdomere extension early in pupal life results in inappropriate retention of normally transient adhesions between developing rhabdomeres. Later Eyc misexpression results in endoplasmic reticulum proliferation and inhibits rhodopsin transport to the developing photosensitive membrane. Loss of Eyc function results in a lethal failure of nuclear envelope assembly in early zygotic divisions. Phenotypes resulting from eyc mutations provide the first in vivo evidence for a role for p47 in membrane biogenesis.     

Drosophila tumor suppressor Merlin is essential for morphogenesis of mitochondria during sperm formation

Drosophila Merlin, an ortholog of the merlin protein encoded by the human Neurofibromatosis 2 (NF2) gene, is important for the regulation of cell proliferation and differentiation in the eye and wing. Also, it has been shown to be involved in male fertility in flies. In the present study, we formation using the comparative light and electron microscopic research of merlin mutants (mer3 and mer4) and ectopic expression of the Mer+ construct. Our work defines specific functions for Merlin in the mitochondria association and aggregation during the nebenkern formation and unfurling mitochondrial derivates during spermatid elongation. Possible role of Merlin as an adaptor protein that can link mitochondria with cytoskeleton is discussed.     

Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation

Bacteria inhabiting biofilms usually produce one or more polysaccharides that provide a hydrated scaffolding to stabilize and reinforce the structure of the biofilm, mediate cell-cell and cell-surface interactions, and provide protection from biocides and antimicrobial agents. Historically, alginate has been considered the major exopolysaccharide of the Pseudomonas aeruginosa biofilm matrix, with minimal regard to the different functions polysaccharides execute. Recent chemical and genetic studies have demonstrated that alginate is not involved in the initiation of biofilm formation in P. aeruginosa strains PAO1 and PA14. We hypothesized that there is at least one other polysaccharide gene cluster involved in biofilm development. Two separate clusters of genes with homology to exopolysaccharide biosynthetic functions were identified from the annotated PAO1 genome. Reverse genetics was employed to generate mutations in genes from these clusters. We discovered that one group of genes, designated psl, are important for biofilm initiation. A PAO1 strain with a disruption of the first two genes of the psl cluster (PA2231 and PA2232) was severely compromised in biofilm initiation, as confirmed by static microtiter and continuous culture flow cell and tubing biofilm assays. This impaired biofilm phenotype could be complemented with the wild-type psl sequences and was not due to defects in motility or lipopolysaccharide biosynthesis. These results implicate an as yet unknown exopolysaccharide as being required for the formation of the biofilm matrix. Understanding psl-encoded exopolysaccharide expression and protection in biofilms will provide insight into the pathogenesis of P. aeruginosa in cystic fibrosis and other infections involving biofilms.     

patufet, the gene encoding the Drosophila melanogaster homologue of selenophosphate synthetase, is involved in imaginal disc morphogenesis

Proliferation in imaginal discs requires cell growth and is linked to patterning processes controlled by secreted cell-signalling molecules. To identify new genes involved in the control of cell proliferation we have screened a collection of P-lacW insertion mutants that result in lethality in the larval/pupal stages, and characterized a novel gene, patufet (ptuf). Inactivation of ptuf by a P element insertion in the 5′ untranslated region leads to aberrant imaginal disc morphology characterized by a reduction in mass of discs and disorganisation of disc cells where no folding or patterning can be detected. Moreover, apoptotic cells can be observed in these small and abnormal mutant discs. To examine the role of ptuf we have studied its clonal behaviour in genetic mosaics generated by mitotic recombination. The mutation causes reduced cell viability, smaller cell size and stops vein differentiation. Non-autonomous effects, such as abnormal differentiation of wild-type cells surrounding the clones, are also observed. We have cloned the ptuf gene of Drosophila melanogaster and found that it encodes a selenophosphate synthetase, which is the first identified in insects. Mutant flies transformed with the full-length cDNA show complete reversion of lethality and disc phenotype. Northern blot analysis and in situ hybridization indicate that the ptuf gene is expressed in imaginal discs as well as at different stages of development. The synthesis of selenoproteins by the selenophosphate synthetase, the role of selenoproteins in the maintenance of the oxidant/antioxidant balance of the cell and its possible implications in imaginal disc morphogenesis are discussed.     

Cloning of cDNA encoding the membrane-bound form of bovine beta 1,4-galactosyltransferase

UDPgalactose: N-acetyl-D-glucosamine 4-beta-D-galactosyltransferase (EC 2.4.1.38) (GalT) is a Golgi-membrane-bound enzyme that participates in the biosynthesis of the oligosaccharide structures of glycoproteins and glycolipids. Synthetic DNA oligomers representing segments of the published partial cDNA sequence for bovine GalT were used as molecular probes to isolate from bovine-liver cDNA libraries overlapping cDNA clones that span 1728 nucleotides and potentially code for the entire polypeptide chain of bovine galactosyltransferase. The cDNA sequence for bovine GalT reveals a 1206-base-pair open reading frame that codes for 402 amino acids, including a presumptive N-terminal membrane anchoring domain of 20 hydrophobic amino acids. The colinearity between the cDNA sequence and 29 non-overlapping amino acid residues which were positively identified by N-terminal sequencing of two polypeptides isolated from the soluble form of the enzyme was consistent with the translation frame and confirmed the authenticity of the cDNA clones. The finding of an N-terminal hydrophobic segment which serves as the membrane anchor and signal sequence suggests that the C-terminal region of the GalT polypeptide is oriented within the lumen of the Golgi membranes. This conclusion is in agreement with previous biochemical studies which indicated that the 51-kDa and 42-kDa soluble forms of the enzyme which encompass the C-terminal 324 and 297 amino acid residues of the entire GalT polypeptide, respectively, include the catalytic site.