Analysis of a swallow homologue from Drosophila pseudoobscura

We analyzed a functional homologue of the swallow gene from Drosophila pseudoobscura. The swallow gene of D. melanogaster plays an essential role in localizing bicoid mRNA in oocytes, and swallow mutant embryos show anterior pattern defects that result from the lack of localization of the bicoid morphogen. The pseudoobscura homologue rescues the function of swallow mutants when introduced into the genome of D. melanogaster, and its expression is similar to that of the melanogaster gene. The predicted pseudoobscura and melanogaster proteins are 49% identical and 69% conserved. The coiled-coil domain previously identified in the melanogaster swallow protein is strongly conserved in the pseudoobscura homologue, but the weak similarity of the melanogaster swallow protein to the RNP class of RNA-binding proteins is not conserved in the pseudoobscura homologue. These and other observations suggest a structural role for swallow in localizing bicoid mRNA, perhaps as part of the egg cytoskeleton. Received: 3 August 1999 / Accepted: 29 September 1999     

Profilin revealed in pollen nuclei: immuno-electron microscopy of high-pressure frozen Ledebouria socialis Roth (Hyacinthaceae)

 Cryoimmobilization by high-pressure freezing, combined with cryosubstitution and resin embedding, allowed accurate retention in situ of the small (12–15 kDa) water-soluble protein, profilin, in anthers of Ledebouria socialis Roth (Hyacinthaceae). The subcellular distribution of profilin was investigated by using post-embedding immunogold labelling with rabbit antisera raised against recombinant birch profilin (RP2) or birch COOH-terminal profilin peptide (RP3). The patterns observed in mature pollen grains are novel to eukaryotic organisms: profilin was consistently demonstrated within both the vegetative and generative nuclei, an addition to its well-known presence in the cytoplasm. Methodological and immunological aspects, as well as possible biological implications, of this finding are considered. Received: 17 March 1997 / Revision accepted: 10 July 1997     

Rethinking cytoskeleton in plant reproduction: toward a biotechnological future?

 Sexual reproduction in plants is intimately connected to the activity of the cytoskeletal apparatus in reproductive cells. Because of the ease with which the pollen tube can be studied, it has become a model for studying many aspects of cell physiology related to the cytoskeleton, such as movement of organelles and vesicles and cell division. However, information about cytoskeletal proteins is still insufficient for determining cytoskeletal functions during reproduction, especially in terms of cell-cell interactions. One reason may be that cytological and biochemical research on the cytoskeleton of pollen and the embryo sac has not been complemented by sufficient research activity at genetic and molecular levels, and few laboratories are currently involved in this work. This might be because of problems in identifying appropriate applied applications of the work that might attract more investigation. Received: 20 January 1999/ Revision accepted: 29 March 1999     

The Role of Formin Tails in Actin Nucleation,Processive Elongation,and Filament Bundling

Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements.     

Characterization of the Tribolium Deformed ortholog and its ability to directly regulate Deformed target genes in the rescue of a Drosophila Deformed null mutant

 We have analyzed the Tribolium castaneum ortholog of the Drosophila homeotic gene Deformed (Dfd) and determined its expression pattern during embryogenesis in this beetle. Tc Deformed (Tc Dfd) is expressed in the blastoderm and the condensing germ rudiment in a region that gives rise to gnathal segments. During germ band extension Tc Dfd is expressed in the mandibular and maxillary segments, their appendages, and the dorsal ridge. Comparison of insect Dfd protein sequences reveals several highly conserved regions. To determine whether common molecular features reflect conserved regulatory functions we used the Gal4 system to express the Tribolium protein in Drosophila embryos. When Tc Dfd is expressed throughout embryonic ectoderm under the control of P69B, the beetle protein autoregulates the endogenous Dfd gene. In addition, the Drosophila proboscipedia gene (a normal target of Dfd) is ectopically activated in the antennal and thoracic segments. We also compared the ability of the beetle and fly proteins to rescue defects in Dfd ^– mutants by expressing each throughout the embryonic during embryogenesis. Both proteins rescued Dfd ^– defects to the same extent in that they each restore the development of mouth hooks and cirri, as well as cause gain-of-function abnormalities of posterior mouth parts. As before, pb was ectopically activated in the antennal segment. This is the first demonstration of the ability of a heterologous homeotic selector protein to directly regulate a target gene independent of an endogenous Drosophila autoregulatory loop. Received: 11 December 1998 / Accepted: 8 March 1999     

Fast and efficient egg collection and antibody staining from large numbers of Drosophila strains

 The genetic dissection of any developmental processes requires mutagenesis protocols and the subsequent phenotypic screen of the established mutant strains. Whereas external structures such as the Drosophila cuticle are relatively easy to score without the need of further manipulations, the analyses of internal structures such as the nervous system often requires the use of antibodies to detect abnormalities. Here we describe an improved method to: (a) simultaneously collect Drosophila eggs from large number of fly strains, (b) process them fast for antibody staining and (c) facilitate rapid subsequent screening. Received: 5 February 1997 / Accepted: 23 March 1997     

Specifying the introgressed regions from H. argophyllus in cultivated sunflower (Helianthus annuus L.) to mark Phomopsis resistance genes

A method based upon targetting of intro-gressed markers in a Phomopsis-resistant line (R) of cultivated sunflower, issuing from a H. argophyllus cross was used to mark the Phomopsis resistance regions. Our study was based upon 203 families derived from a cross between an inbred line susceptible to Phomopsis (S1) and the introgressed resistant line (R). Families were checked for Phomopsis resistance level in a design with replicated plots and natural infection was re-inforced by pieces of contaminated stems. Thirty four primers were employed for RAPD analysis. Out of 102 polymorphic fragments between (S1) and H. argophyllus, seven were still present in (R) suggesting that they marked introgressions of H. argophyllus into (R). The plants were scored for the presence or absence of 19 fragments obtained from five primers, and the relationships between the presence/absence of fragments in plants and Phomopsis resistance/susceptiblity in the progenies was determined by using an analysis of variance. We found that at least two introgressed regions, as well as favourable factors from sunflower, contributed to the level of Phomopsis resistance in cultivated sunflower. Received: 28 June 1996 / Accepted: 5 July 1996     

ribbon, raw, and zipper have distinct functions in reshaping the Drosophila cytoskeleton

rib and raw mutations prevent cells in a number of tissues from assuming specialized shapes, resulting in abnormal tubular epithelia and failure of morphogenetic movements such as dorsal closure. Mutations of zip, which encodes the nonmuscle myosin heavy chain, suppress the phenotypes of rib and raw, suggesting that rib and raw are not directly required for myosin function. Abnormal formation of the actin cytoskeletal structures underlying embryonic cuticular hairs suggests possible roles for rib and raw in organizing the actin cytoskeleton. The actin prehair structures are absent in rib mutants and abnormally shaped in raw mutants, indicating that the two genes have different functions required for organizing the actin cytoskeleton. Received: 4 December 1998 / Accepted: 26 January 1999     

The role of Ca2+ in progesterone-induced germinal vesicle breakdown of Xenopus laevis oocytes: the synergic effects of microtubule depolymerization and Ca2+

 By monitoring ⁴⁵Ca²⁺ influx and efflux from oocytes a transient increase followed by a transient decrease in the Ca²⁺-content of progesterone-treated oocytes was observed. Chelation of intracellular Ca²⁺ with EGTA or BAPTA-type buffers inhibited progesterone-induced GVBD. Buffers with a mid-range K_d (∼1.5 μm) were most effective in inhibiting GVBD whereas buffers with a K_d above or below this value were less effective. These observations indicate that intracellular Ca²⁺, probably in the form of a localized release, is required for progesterone-induced oocyte maturation. However, Ca²⁺ alone was insufficient to induce GVBD. When the effects of nocodazole and taxol upon this Ca²⁺-requirement were tested, we observed that taxol-induced microtubule polymerization not only delayed progesterone-induced GVBD but also completely inhibited it in combination with BAPTA-AM. Conversely, nocodazole-induced microtubule depolymerization in combination with ionophore A23187 not only accelerated progesterone-induced GVBD, but also induced GVBD in the absence of progesterone. The combined treatment of oocytes with nocodazole and InsP_3, or with cold treatment and ionophore A23187 also induced GVBD in the absence of progesterone. Thus, Ca²⁺ and microtubule depolymerization synergistically promote GVBD. In both nocodazole- and cold-treated oocytes, the GV was displaced to the periphery of the oocyte and underwent GVBD when treated with A23187. However, when the GV was displaced to the cortex by a centrifugal force under conditions that would not cause microtubule depolymerization and the oocyte was treated with A23187, oocytes did not undergo GVBD. Received: 19 January 1996 / Accepted: 21 May 1996     

An IQGAP-related gene is activated during tentacle formation in the simple metazoan Hydra

Differentiation of body column epithelial cells into tentacle epithelial cells in Hydra is accompanied by changes in both cell shape and cell-cell contact. The molecular mechanism by which epithelial cells acquire tentacle cell characteristics is unknown. Here we report that expression of a Hydra homologue of the mammalian IQGAP1 protein is strongly upregulated during tentacle formation. Like mammalian IQGAP, Hydra IQGAP1 contains an N-terminal calponin-homology domain, IQ repeats and a conserved C terminus. In adult polyps a high level of Hydra IQGAP1 mRNA is detected at the basis of tentacles. Consistent with a role in tentacle formation, IQGAP1 expression is activated during head regeneration and budding at a time when tentacles are emerging. The observations support the previous hypothesis that IQGAP proteins are involved in cytoskeletal as well as cell-cell contact rearrangements. Received: 25 January 2000 / Accepted: 2 May 2000     

The yan gene is highly conserved in Drosophila and its expression suggests a complex role throughout development

 Competence for cell fate determination and cellular differentiation is under tight control of regulatory genes. Yan, a nuclear target of receptor tyrosine kinase (RTK) signaling, is an E twenty six (ETS) DNA-binding protein that functions as a negative regulator of cell differentiation and proliferation in Drosophila. Most members of RTK signaling pathways are highly conserved through evolution, yet no yan orthologues have been identified to date in vertebrates. To investigate the degree of yan conservation during evolution, we have characterized a yan homologue from a sibling species of D. melanogaster, D. virilis. Our results show that the organization, primary structure and expression pattern of yan are highly conserved. Both genes span over 20 kb and contain four exons with introns at identical positions. The areas with highest amino acid similarity include the Pointed and ETS domain but there are other discrete regions with a high degree of similarity. Phylogenetic analysis reveals that yan’s closest relative is the human tel gene, a negative regulator of differentiation in hematopoetic precursors. In both species, Yan is dynamically expressed beginning as early as stage 4/5 and persisting throughout embryogenesis. In third instar larvae, Yan is expressed in and behind the morphogenetic furrow of the eye imaginal disc as well as in the laminar precursor cells of the brain. Ovarian follicle cells also contain Yan protein. Conservation of the structure and expression patterns of yan genes strongly suggests that regulatory mechanisms for their expression are also conserved in these two species. Received: 3 November 1998 / Accepted: 9 December 1998     

Gliadin polymorphism in wild and cultivated einkorn wheats

To study the relationships between different species of the Einkorn group, 408 accessions of Triticum monococcum, T. boeoticum, T. boeoticum ssp. thauodar and T. urartu were analyzed electrophoretically for their protein composition at the Gli-1 and Gli-2 loci. In all the species the range of allelic variation at the loci examined is remarkable. The gliadin patterns of T. monococcum and T. boeoticum were very similar to one another but differed substantially from those of T. urartu. Several accessions of T. boeoticum and T. monococcum were shown to share the same alleles at the Gli-1 and Gli-2 loci, confirming the recent nomenclature that considers these wheats as different subspecies of the same species, T. monococcum. The gliadin composition of T. urartu resembled that of the A genome of polyploid wheats more than did T. boeoticum or T. monococcum, supporting the hypothesis that T. urartu, rather than T. boeoticum, is the donor of the A genome in cultivated wheats. Because of their high degree of polymorphism the gliadin markers may help in selecting breeding parents from diploid wheat germ plasm collections and can be used both to search for valuable genes linked to the gliadin-coding loci and to monitor the transfer of alien genes into cultivated polyploid wheats. Received: 8 July 1996 / Accepted: 12 July 1996     

The mapping of phytochrome genes and photomorphogenic mutants of tomato

The map positions of five previously described phytochrome genes have been determined in tomato (Lycopersicon esculentum Mill.) The position of the yg-2 gene on chromosome 12 has been confirmed and the classical map revised. The position of the phytochrome A (phy A)-deficient fri mutants has been refined by revising the classical map of chromosome 10. The position of the PhyA gene is indistinguishable from that of the fri locus. The putative phyB1-deficient tri mutants were mapped by classical and RFLP analysis to chromosome 1. The PhyB1 gene, as predicted, was located at the same position. Several mutants with the high pigment (hp) phenotype, which exaggerates phytochrome responses, have been reported. Allelism tests confirmed that the hp-2 mutant is not allelic to other previously described hp (proposed here to be called hp-1) mutants and a second stronger hp-2 allele (hp-2 ^j ) was identified. The hp-2 gene was mapped to the classical, as well as the RFLP, map of chromosome 1. Received: 24 May 1996 / Accepted: 14 June 1996     

Flow cytometric analysis of the chromosomes and stability of a wheat cell-culture line

A rapidly growing, long-term suspension culture derived from Triticum aestivum L. (wheat) was synchronized using hydroxyurea and colchicine, and a chromosome suspension with chromosomes was made. After staining with the DNA-specific fluorochromes Hoechst 33258 and Chromomycin univariate and bivariate flow-cytometry histograms showed 15 clearly resolved peaks corresponding to individual chromosome types or groups of chromosomes with similar DNA contents. The flow karyotype was closely similar to a histogram of DNA content measurements of Feulgen-stained chromosomes made by microdensitometry. We were able to show the stability of the flow karyotype of the cell line over a year, while a parallel subculture had a slightly different, stable, karyotype following different growth conditions. The data indicate that flow cytometric analysis of plant karyotypes enables accurate, statistically precise chromosome classification and karyotyping of cereals. There was little overlap between individual flow-histogram peaks, so the method is useful for flow sorting and the construction of chromosome specific-recombinant DNA libraries. Using bivariate analysis, the AT:GC ratio of all the chromosomes was remarkably similar, in striking contrast to mammalian flow karyotypes. We speculate about a fundamental difference in organization and homogenization of DNA sequences between chromosomes within mammalian and plant genomes. Received: 24 April 1996 / Accepted: 24 May 1996     

Rapid preparation of a panel of polyclonal antibodies to Drosophila segmentation proteins

 We describe a method for rapidly raising a panel of high quality polyclonal antibodies from bacterially expressed proteins. Approximately 1²/₃ days of preparation is required per protein. One step that speeds up the procedure is the visualization of purified bands by precipitated sodium dodecyl sulfate (SDS). Antigenicity of the purified recombinant proteins may be increased by precipitation in double-distilled water. The results of using the serums obtained for fluorescent staining of Drosophila embryos are shown. Received: 2 February 1998 / Accepted: 19 February 1998