spn-F encodes a novel protein that affects oocyte patterning and bristle morphology in Drosophila

The anteroposterior and dorsoventral axes of the Drosophila embryo are established during oogenesis through the activities of Gurken (Grk), a Tgfalpha-like protein, and the Epidermal growth factor receptor (Egfr). spn-F mutant females produce ventralized eggs similar to the phenotype produced by mutations in the grk-Egfr pathway. We found that the ventralization of the eggshell in spn-F mutants is due to defects in the localization and translation of grk mRNA during mid-oogenesis. Analysis of the microtubule network revealed defects in the organization of the microtubules around the oocyte nucleus. In addition, spn-F mutants have defective bristles. We cloned spn-F and found that it encodes a novel coiled-coil protein that localizes to the minus end of microtubules in the oocyte, and this localization requires the microtubule network and a Dynein heavy chain gene. We also show that Spn-F interacts directly with the Dynein light chain Ddlc-1. Our results show that we have identified a novel protein that affects oocyte axis determination and the organization of microtubules during Drosophila oogenesis.     

The segment polarity gene armadillo encodes a functionally modular protein that is the Drosophila homolog of human plakoglobin

The Drosophila segment polarity gene armadillo is required for pattern formation within embryonic segments and imaginal discs. We have found that armadillo is highly conserved during evolution; it is 63% identical to human plakoglobin, a protein found in adhesive junctions joining epithelial and other cells. We have examined arm protein localization in a number of larval tissues and found that arm protein accumulation within cells shares many features with the accumulation of plakoglobin. We have compared the phenotype and molecular lesions responsible for the different arm mutations. Surprisingly, severely truncated proteins retain some function; the degree of function is strictly correlated with the length of the truncated protein, suggesting that the internally repetitive arm protein is modular in function. We present a possible model for the cellular role of arm.     

The three rows gene of Drosophila melanogaster encodes a novel protein that is required for chromosome disjunction during mitosis.

Zygotic expression of the three rows (thr) gene of Drosophila melanogaster is required for normal cell proliferation during embryogenesis. Mitotic defects in thr mutant embryos begin during mitosis 15, and all subsequent divisions are disrupted. Chromosome disjunction and consequently cytokinesis fail during these defective mitoses, although the initial mitotic processes (chromosome condensation, spindle assembly, metaphase plate formation, and cyclin degradation) are not affected. Despite the failure of chromosome disjunction and cytokinesis, later mitotic events (chromosome decondensation) and subsequent cell cycle progression continue. The thr gene has been isolated and shown to encode a 1209 amino acid protein that shares no extended sequence similarity with known proteins. thr mRNA is present as maternal mRNA that degrades at the time of cellularization. At this and all subsequent times during embryogenesis, zygotic expression correlates with mitotic proliferation. These observations, together with the observation that the zygotic phenotype of thr mutant embryos is influenced by the maternal genotype, suggest that the embryonic phenotype results from exhaustion of the maternal thr contribution and does not reflect a developmentally restricted requirement for thr function. Our results indicate that the novel thr product is required specifically for chromosome disjunction during all mitoses.     

The Drosophila ribbon gene encodes a nuclear BTB domain protein that promotes epithelial migration and morphogenesis.

During development of the Drosophila tracheal (respiratory) system, the cell bodies and apical and basal surfaces of the tracheal epithelium normally move in concert as new branches bud and grow out to form tubes. We show that mutations in the Drosophila ribbon (rib) gene disrupt this coupling: the basal surface continues to extend towards its normal targets, but movement and morphogenesis of the tracheal cell bodies and apical surface is severely impaired, resulting in long basal membrane protrusions but little net movement or branch formation. rib mutant tracheal cells are still responsive to the Branchless fibroblast growth factor (FGF) that guides branch outgrowth, and they express apical membrane markers normally. This suggests that the defect lies either in transmission of the FGF signal from the basal surface to the rest of the cell or in the apical cell migration and tubulogenesis machinery. rib encodes a nuclear protein with a BTB/POZ domain and Pipsqueak DNA-binding motif. It is expressed in the developing tracheal system and other morphogenetically active epithelia, many of which are also affected in rib mutants. We propose that Rib is a key regulator of epithelial morphogenesis that promotes migration and morphogenesis of the tracheal cell bodies and apical surface and other morphogenetic movements.     

Drosophila valois encodes a divergent WD protein that is required for Vasa localization and Oskar protein accumulation

valois (vls) was identified as a posterior group gene in the initial screens for Drosophila maternal-effect lethal mutations. Despite its early genetic identification, it has not been characterized at the molecular level until now. We show that vls encodes a divergent WD domain protein and that the three available EMS-induced point mutations cause premature stop codons in the vls ORF. We have generated a null allele that has a stronger phenotype than the EMS mutants. The vlsnull mutant shows that vls+ is required for high levels of Oskar protein to accumulate during oogenesis, for normal posterior localization of Oskar in later stages of oogenesis and for posterior localization of the Vasa protein during the entire process of pole plasm assembly. There is no evidence for vls being dependent on an upstream factor of the posterior pathway, suggesting that Valois protein (Vls) instead acts as a co-factor in the process. Based on the structure of Vls, the function of similar proteins in different systems and our phenotypic analysis, it seems likely that vls may promote posterior patterning by facilitating interactions between different molecules.     

OsBC1L4 encodes a COBRA-like protein that affects cellulose synthesis in rice

Plant morphogenesis is highly dependent on the regulation of cell division and expansion. The organization of the cellulose microfibrils in the cell wall is a key determinant of cell expansion. Previously, a dwarf mutant with fewer tillers, Osbc1l4 (Oryza sativa brittle culm 1 like 4), was identified by screening a rice T-DNA insertion mutant library. It is reported here that OsBC1L4 encodes a COBRA-like protein that exhibits typical structural features of a glycosylphosphatidylinositol-anchor protein. The T-DNA insertion in OsBC1L4 results in abnormal cell expansion. A decrease in cellulose content but the increase in pectin and starch contents was identified in Osbc1l4 mutants by measuring the content of wall components. OsBC1L4 was expressed in all tissues/organs examined, with a low level in leaves. OsBC1L4 protein is mainly located in the cell wall and plasma membrane. Correlation analysis indicated that the expression of OsBC1L4 was highly correlated to that of several primary wall-forming cellulose synthase genes (CESAs). Moreover, the expression level of several cellulose-related genes is increased in Osbc1l4 mutants, which suggests that a feedback mechanism may exist during cellulose synthesis.     

The barley stripe mosaic virus gamma b gene encodes a multifunctional cysteine-rich protein that affects pathogenesis.

Barley stripe mosaic virus contains seven genes, one of which specifies a 17-kD cysteine-rich protein, gamma b, that is known to affect virulence. To further characterize the role of gamma b in pathogenesis, we mutagenized sequences encoding amino acids within two clusters of cysteine and histidine residues in the cysteine-rich domain and a group of basic amino acids located between the clusters and determined the effects of these mutations on the symptom phenotype in barley. Three single amino acid substitutions in cluster 1 and two amino acid exchanges in the basic region caused bleached symptoms associated with pronounced elevations in accumulation of gamma b protein. In contrast, three single amino acid substitutions in cluster 2 and a mutation in the basic motif resulted in attenuated ("null") symptoms typical of those produced when the gamma b gene is deleted. Tissue infected with these "null" mutants accumulated slightly elevated amounts of the gamma b protein but significantly lower levels of coat protein and the putative movement protein beta b. Genetic complementation tests revealed that cluster 1 mutations are dominant over the wild-type gamma b gene, whereas those in cluster 2 are recessive. These results highlight the pivotal role of gamma b in pathogenesis and suggest that the two cysteine-rich clusters are functionally distinct and that they affect different aspects of disease development.     

The Drosophila tissue polarity gene starry night encodes a member of the protocadherin family.

The tissue polarity genes control the polarity of hairs, bristles and ommatidia in the adult epidermis of Drosophila. We report here the identification of a new tissue polarity gene named starry night (stan). Mutations in this essential gene alter the polarity of cuticular structures in all regions of the adult body. The detailed polarity phenotype of stan on the wing suggested that it is most likely a component of the frizzled (fz) pathway. Consistent with this hypothesis, stan appears to be downstream of and required for fz function. We molecularly cloned stan and found that it encodes a huge protocadherin containing nine cadherin motifs, four EGF-like motifs, two laminin G motifs, and seven transmembrane domains. This suggests that Stan functions in signal reception, perhaps together with Fz.     

A prostate-derived cDNA that is mapped to human chromosome 19 encodes a novel protein

The epithelial cells of prostate gland secrete various secretory products that play an important role in the growth and differentiation of prostate gland. These secretory products have also been implicated in neuroendocrine differentiation of benign prostatic hyperplasia and prostate malignancy. We have cloned a prostate-derived cDNA encoding a novel protein with a predicted molecular weight of 78 kDa (P(78)), and precisely mapped the cDNA sequence to chromosome 19. The P(78) gene has a complex genomic structure with 18 exons and 17 introns. The P(78) contains two conserved structural domains with limited similarity to domain D of synapsin I. The P(78) mRNA was expressed in various human cell lines. Western blot analysis using antibody specific for the P(78) revealed the presence of the P(78) protein in the prostate cancer cell lines with much lower level in metastatic prostate cancer cell lines compared to that in a primary prostate cancer cell line.     

Aspergillus nidulans swoK encodes an RNA binding protein that is important for cell polarity

The Aspergillus nidulans swoK1 mutant is defective in polarity maintenance when grown at restrictive temperature (38 degrees C). Upon germination, the mutant extends a primary germ tube that swells to an enlarged, non-uniform cell with pronounced wall thickenings. The mutant is fully restored to wild-type growth when transformed with a plasmid containing the AN5802.2 ORF as designated in The Broad Institute A. nidulans sequence database. Genetic mapping places swoK in the same region of chromosome I, as that occupied by An5802.2 on the physical map. swoK is predicted to encode a protein that contains an N-terminal RRM (RNA Recognition Motif) and a highly repetitive C-terminus with numerous RD/DR and RS/SR dipeptides. We hypothesize that SwoK participates in one of the known functions of SR proteins (those that contain SR/RS repeats): mRNA maturation through the spliceosome and or transport of mRNAs out of the nucleus to sites of protein translation.     

Opaque7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis in maize endosperm

In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins, the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7 mutant was produced by the reduction of protein body size and number caused by a decrease in the α-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting α-ketoglutaric acid and oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for the genetic manipulation of storage protein contents in cereal seeds.     

Antibody to a human DNA repair protein allows for cloning of a Drosophila cDNA that encodes an apurinic endonuclease.

The cDNA of a Drosophila DNA repair gene, AP3, was cloned by screening an embryonic lambda gt11 expression library with an antibody that was originally prepared against a purified human apurinic-apyrimidinic (AP) endonuclease. The 1.2-kilobase (kb) AP3 cDNA mapped to a region on the third chromosome where a number of mutagen-sensitive alleles were located. The cDNA clone yielded an in vitro translation product of 35,000 daltons, in agreement with the predicted size of the translation product of the only open reading frame of AP3, and identical to the molecular size of an AP endonuclease activity recovered following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Drosophila extracts. The C-terminal portion of the predicted protein contained regions of presumptive DNA-binding domains, while the DNA sequence at the amino end of AP3 showed similarity to the Escherichia coli recA gene. AP3 is expressed as an abundant 1.3-kb mRNA that is detected throughout the life cycle of Drosophila melanogaster. Another 3.5-kb mRNA also hybridized to the AP3 cDNA, but this species was restricted to the early stages of development.     

PDIP38 is a novel mitotic spindle-associated protein that affects spindle organization and chromosome segregation

In order to maintain genomic integrity during mitosis, cells assemble the mitotic spindle to separate sister chromosomes to the two daughter cells. A variety of motor- and non motor-proteins are involved in the organization and regulation of this complex apparatus. DNA polymerase δ-interacting protein 38 (PDIP38) is a highly conserved protein and has so far been shown to be a cytoplasmic and nuclear protein. Cell cycle dependent nuclear localization and the interaction with DNA polymerase δ and proliferating cell nuclear antigen (PCNA) indicate a role for PDIP38 in DNA modification and/or proliferation. Here, we show for the first time that PDIP38 localizes to the mitotic spindle throughout mitosis. Using anti-PDIP38 antibody injections and siRNA silencing, we demonstrate that PDIP38 loss-of-function causes problems with spindle organization, aberrant chromosome segregation, and multinucleated cells. Taken together, the data indicate different roles for PDIP38 in safeguarding a proper cell division at various stages of the cell cycle, including DNA synthesis and repair, organization of the mitotic spindle and chromosome segregation.     

DrhoGEF3 encodes a new Drosophila DH domain protein that exhibits a highly dynamic embryonic expression pattern

The Rho GTPases regulate many different cellular and developmental processes, and activation of Rho GTPase signalling is mediated through interaction with the Dbl homology (DH) protein domain. We describe the expression pattern of DrhoGEF3 (cytological position 61B1-B3), which encodes a new member of the DH domain protein family from Drosophila and is a homologue of the human protein hPEM-2. During gastrulation and germ band extension, DrhoGEF3 exhibits a segmented expression pattern. DrhoGEF3 is subsequently expressed in the visceral mesoderm, at the sites of muscle attachment and in specific groups of sub-epidermal cells. The possible function of such a dynamically expressed signalling molecule is discussed.