Use of a fluorescent membrane probe to identify zooxanthellae in hospite among dissociated endoderm cell culture from coral

Summary. Preparation of homogeneous endoderm cells and culture is a prerequisite to understanding the cellular and molecular mechanism of endosymbiosis in the cnidarian-dinoflagellate association. During the cell isolation from the stony coral Euphyllia glabrescens, various amounts of symbiotic endoderm cells were found to release their symbionts (Symbiodinium spp., or zooxanthellae in generic usage) into the culture. Due to the bulky occupation by zooxanthellae inside the endoderm cell, the symbiotic endoderm cells, or zooxanthellae in hospite, are difficult to be distinguished from released zooxanthellae by microscopic examination. We now report a method for this identification using a fluorescent analogue of sphingomyelin, N-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C₅-DMB-SM). Incubation of symbiotic endoderm cells with C₅-DMB-SM–defatted bovine serum albumin (DF-BSA) complex results in bright fluorescent membrane staining. Nevertheless, the membrane staining of free-living or released zooxanthellae by this complex is significantly decreased or even diminished. This method has provided a fast and reliable assay to identify symbiotic endoderm cells and will greatly accelerate the progress of endosymbiosis research. Correspondence and reprints: National Museum of Marine Biology and Aquarium, 2 Houwan Road, Checheng, Pingtung, 944, Taiwan, R.O.C.     

Male-sterile chicory cybrids obtained by intergeneric protoplast fusion

Male-sterile chicory plants were obtained by fusion of chicory mesophyll protoplasts and hypocotyl protoplasts derived from male-sterile sunflower plants. The protoplasts of both species were fused by the PEG method and the products were selected manually and cultivated at very low density in a liquid medium. Three to twenty percent of the heterokaryocytes divided and evolved into microcalli, then into calli where budding could be induced. The mitochondrial genome of ten male-sterile or totally sterile plants was studied. Restriction endonuclease profiles of mitochondrial DNA and molecular hybridization with specific genes of the mitochondrial genome used as probes indicated that mitochondrial DNA rearrangement had occurred between sunflower and chicory and the intensity of the rearrangements correlated with the degree of sterility of the different plants.     

A mutator nuclear gene inducing a wide spectrum of cytoplasmically inherited chlorophyll deficiences in barley

Summary Some striped plants were observed in plots of a long-grain mutant barley grown at a field nursery. All of the plants of these plots, which were naturally self pollinated, were individually harvested, and most of their progenies (92.5%) segregated seedlings carrying chlorophyll deficiencies (CD) as determined by greenhouse analysis. The majority of the mutant seedlings (84.3%) showed a pattern of longitudinal chlorophyll sectors. The spectrum of CD was wide among the solid mutant seedlings and consisted of three main types (albina, viridis and discontinuous). In association with some CD types morphological changes were frequently observed. Non-CD-associated morphological changes and diminished seed-set were scarce and, so far, none of them has proved to be inherited. Analysis of CD in reciprocal crosses and backcrosses proved that while CD were transmitted cytoplasmically their induction was controlled by a single nuclear mutator gene, active when homozygous. In addition once the CD were induced, they were expressed independently of the nuclear constitution. The results suggest that the mutator gene induces diverse mutational events on chloroplast (cp) DNA. In barley, as in other monocots, nuclear genes which are inductors of cytoplasmic genetic changes have been reported. However, all of them produced a narrower spectrum of CD and had a more rapid sorting-out of the cytoplasmic mutants than what we observed. On this basis a distinction between chloroplast and mitochondrial (mt) mutator genes is proposed. Accordingly, the chloroplast mutator here described would be the first one reported for monocots. Increased knowledge on this subject can play a fundamental role in elucidating organelle heredity and its interactions with the nuclear genome. Moreover, this material could be a valuable source of variability of the otherwise conservative genetic information encoded in the chloroplast.Paper GEN 792, Institute of Genetics, CICA, INTA, Castelar     

Evidence for the existence of a universal crown-gall tumor initiation enhancer

Tumor initiation of different dicotyledonous plant species inoculated with Agrobacterium tumefaciens B₆ has been studied in vivo and in vitro. The tumor formation in weakly susceptible plants can be strongly enhanced by exogenously applied active extract fractions derived from highly susceptible Helianthus cotyledons. It is found that highly susceptible plants (Kalanchoë, Lycopersicon and Pinto beans) contain an active tumor initiation enhancer which is clearly similar to the compound(s) found in Helianthus cotyledons. No activity can be detected in extracts derived from weakly susceptible plants (Coleus, Phaseolus) or in those obtained from crown-gall tumor tissues.Abbreviations HS high susceptibility for tumor initiation - LS low susceptibility for tumor initiation - PEF purified active extract fraction     

A simple and efficient method to identify replacements of P-lacZ by P-Gal4 lines allows obtaining Gal4 insertions in the bithorax complex of Drosophila

The functional replacement of one gene product by another one is a powerful method to study specificity in development and evolution. In Drosophila, the Gal4/UAS method has been used to analyze in vivo such functional substitutions. To this aim, Gal4 lines that inactivate a gene and reproduce its expression pattern are required, and they can be frequently obtained by replacing pre-existing P-lacZ lines with such characteristics. We have devised a new method to quickly identify replacements of P-lacZ lines by P-Gal4 lines, and applied it successfully to obtain Gal4 insertions in the Ultrabithorax and Abdominal-B Hox genes. We have used these lines to study the functional replacement of a Hox gene by another one. Our experiments confirm that the abdominal-A gene can replace Ultrabithorax in haltere development but that it cannot substitute for Abdominal-B in the formation of the genitalia.     

Expression of the<Emphasis Type="Italic"> lacZ</Emphasis> reporter gene in sporophytes of the seaweed<Emphasis Type="Italic"> Laminaria japonica</Emphasis> (Phaeophyceae) by gametophyte-targeted transformation

The seaweed Laminaria japonica (Phaeophyceae) has a two-generation life cycle consisting of haploid gametophytes and diploid sporophytes. Female and/or male gametophytes were transformed using particle bombardment and the histological LacZ assay was performed on sporophytes generated by either parthenogenesis or inbreeding. Female gametophyte-targeted transformation resulted in similar lower efficiencies in both parthenogenetic and zygotic sporophytes, and only a chimeric expression pattern was observed. Male gametophyte-targeted transformation led to a higher efficiency, with 3.5% of the zygotic sporophytes stained completely blue (all-blue), implying the integration of lacZ at the one-cell stage. Polymerase chain reaction analysis using primers specific for a lacZ-vector juncture fragment and subsequent blotting indicated the presence of the introduced gene in the sporophytes. The method reported here has a potential for seaweed transformation using spore-based bombardment followed by the developmental process.Abbreviations DPR Detected positive rate - ER Expression rateCommunicated by F. Sato     

Use of a promoter-specific probe to identify two loci from the Rhizobium meliloti nodulation regulon

The nodulation regulon of Rhizobium meliloti AK631 includes several operons (nodABC, hsnABC, hsnD, efn locus) which have in common a consensus promoter sequence called the nod box. A synthetic nod box probe was used to identify two additional nod boxes, n4 and n5, which were subcloned for study. By constructing lac fusions, we show that n4 and n5 sponsor induction of downstream regions as previously shown for n1-nodABC and n2-hsnABC. Using site-directed Tn5 mutagenesis, we find that the n5 locus plays a significant role in nodulation of alfalfa and sweetclover, whereas the n4 locus is important for alfalfa, but not for sweetclover. Hybridization data suggest that the n5 locus is conserved among Rhizobium species. In contrast, the n4 locus seems to be unique to Rhizobium meliloti strains, in agreement with the host-specific phenotype of n4 locus mutants. Thus, the use of a promoter probe allows us to identify nodulation genes which may be overlooked by standard methods such as random Tn5 mutagenesis.     

Development of thoracic curvature inDrosophila melanogaster

Summary The influence of muscle development on thorax morphogenesis has been investigated inDrosophila melanogaster. The development of an indirect flight muscle, the dorsal longitudinal muscle (DLM), has been thought to be responsible for the formation of the distinct thoracic curvature. Using aDrosophila mutant (sr/Df(3)sr) in which the DLM is completely missing, we have shown that a normally curved thorax still is produced. Such results indicate that an external structure (epidermis) is capable of developing wholly independent of an absent internal structure (muscle).     

Resynthesis of Brassica carinata by protoplast fusion and recovery of a novel cytoplasmic hybrid

Brassica carinata (2n=34, BBCC), was synthesized by fusing dark grown etiolated hypocotyl protoplasts of B. nigra (2n=16, BB) with green mesophyll protoplasts of B. oleracea (2n=18,CC) using polyethylene glycol. Heterokaryons could be microscopically distinguished from the parental types by their dark green chloroplasts in the colourless hypocotyl protoplast background. The mean heterokaryotic fusion frequency estimated on the basis of this morphological distinction was about 16%. A total of 626 calli were obtained, of which 92 regenerated shoots after transfer to zeatin (2 mg/l) supplemented MS medium. Of these, 81 calli differentiated into plants morphologically similar to naturally occurring B. carinata and 11 calli yielded plants resembling parental types. Meiosis in seven hybrid plants showed the chromosome number to be 2n=34 the sum of B. nigra and B. oleracea chromosomes. Molecular confirmation of the amphidiploid nature of hybrids was obtained by probing with a B. juncea derived genomic clone. The use of chloroplast and mitochondrial specific gene probes, revealed that one plant was a cytoplasmic hybrid having cp DNA sequences of both B. oleracea and B. nigra and mt DNA sequences of B. nigra.Abbreviations PEG Polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA Naphthaleneacetic acid - IBA Indole-3-butyric acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962)     

Contribution of the geneextramacrochaetae to the precise positioning of bristles inDrosophila

We examine the effect of mutations in theextramacrochaetae (emc) gene on the positioning of macrochaetes on the notum ofDrosophila. We show that, inemc mutants, most of the precursor cells appear earlier than in wild-type individuals, consistent with an antagonizing effect ofemc on the action of the proneural genesachaete andscute. We also show that reducingemc function affects the position of three bristles and/or of their precursors, but has no marked effect on the positioning of the other bristles.     

Genomic imprinting of chromatin inDrosophila melanogaster

During gametogenesis, chromosomes may become imprinted with information which facilitates proper expression of the DNA in offspring. We have used a position effect variegation mutant as a reporter system to investigate the possibility of imprinting inDrosophila melanogaster. Genetic crosses were performed in which the variegating gene and a strong modifier of variegation were present either within the same parental genome or in opposite parental genomes in all possible combinations. Our results indicate that the presence of the variegating chromosome and a modifier chromosome in the same parental genome can alter the amount of variegation formed in progeny. The genomic imprinting we observed is not determined by the parental origin of the variegating chromosome but is instead determined by the genetic background the variegating chromosome is subjected to during gametogenesis.     

Cytoplasmic reorganization accompanies the deposition of a bipolar cell wall in the large-celled red alga Anotrichium tenue

The filamentous red alga Anotrichium tenue C. Aghard (Naegeli) (formerly Griffithsia tenuis C. Aghard; Baldock, 1976, Aust. T. Bot. 24, 509–593) has large (1–2 mm long), cylindrical, multinucleate cells that exhibit a daily, cyclic redistribution of chloroplasts. Chloroplasts accumulate in the mid-region of each growing cell during the day; consequently, filaments appear banded with a light apical end-band, a dark mid-band and a light basal end-band in each growing cell. Chloroplasts disperse at night so that the bands are no longer visible and the cells appear evenly pigmented. Anotrichium tenue also has a type of cell elongation, known as bipolar band growth, in which new material is added to the microfibrillar part of the wall in bands located at the apical and basal poles of elongating cells. This site of wall growth corresponds to the position of the light-colored end-bands present during the day. Here we examine the structural relationship between the cytoplasmic bands and the wall-growth bands. Our results show that, in addition to the previously described bipolar wall bands, there is a non-microfibrillar wall band in the mid-region of the cell. This wall component apparently branches from near the top of the microfibrillar outer wall and terminates near but not at the bottom of the cell. It contains nodules of sulphated polysaccharide material secreted from a band of vesicles, which co-localize with the chloroplasts in the mid-band. The outer wall appears to enclose the entire cell. Nuclei do not redistribute with the chloroplasts or wall vesicles into the mid-band but remain evenly distributed throughout the cytoplasm. Each wall component grows by a different mechanism. We show that two types of wall growth, diffuse and the bipolar-type of tip growth, occur in the same cell and we propose that the observed segregation of the cytoplasm supports localized growth of the unique inner wall component. Additionally, we show that A. tenue is an excellent model for study of the role and mechanism of cytoplasmic compartmentalization and cell polarity during plant cell growth.We wish to thank Dr. Richard Cloney (University of Washington and Dr. Tom Schroeder (Friday Harbor Laboratories, Friday Harbor, Wash.) for helpful discussions and critical review of this work. We also thank Dr. Susan Waaland (University of Washington) for sharing her original observations on the chloroplast banding phenomenon in Anotrichium tenue. We are grateful to the Friday Harbor Laboratories for the use of their space and facilities. This research was supported by funds from the Washington Sea Grant Program (awarded to J.R.W.) and by the Developmental Biology Training Grant, predoctoral fellowship, National Institutes of Health, No. HD07183 to A.W.S.     

Altered establishment of cell lineages in theCaenorhabditis elegans embryo after suppression of the first cleavage supports a concentration-dependent decision mechanism

Summary In the early embryo ofCaenorhabditis elegans five somatic cell lineages and a germ cell lineage are established by a series of unequal cleavages in the germline. We suppressed first cleavage by means of cold, mechanical pressure or centrifugation. Thereafter, with the second attempt of the zygote to divide, four blastomeres were generated simultaneously in a tetrapolar cleavage. Cell division pattern, segretation of germline-specific granules, and terminal differentiation of such manipulated embryos were analysed. Instead of six, only from one to five visible cell lineages were established before the germline prematurely aborted from its typical pattern of unequal cleavage. The absence of germline-specific cleavage appears to accompany the abnormal segregation of germline-specific granules. While muscle differentiation was detected even in embryos expressing only one cell lineage, in general, gut differentiation became visible only if a separate gut lineage had been generated. We hypothesize that the potential for differential cleavage is lost in manipulated embryos because a cytoplasmic control factor is diminished as a result of the retarded soma/germline separation. According to this hypothesis, after manipulation, a concentration-dependent decision mechanism leads to: a reduced number of unequal germline cleavages or even none at all, the establishment of fewer distinct cell lineages, and limited cellular differentiation.     

Partial hyperploidy of the X-chromosome inDrosophila hydei leading to duplication of male gonadal and genital structures

Summary Introduction of two doses of the X-chromosomalw^mCo duplication next to a normal X-chromosome in males ofD. hydei leads to duplication of testis tissue and structures derived from the male genital disc. The effect of this partial hyperploidy of the X-chromosome seems restricted to the male. We tentatively conclude that this part of the X-chromosome contains some factor(s) which may specifically affect the reproductive system and analia of males.     

Improved metabolic action of a bacterial lysine decarboxylase gene in tobacco hairy root cultures by its fusion to arbcS transit peptide coding sequence

The gene of a bacterial lysine decarboxylase (ldc) fused to arbcS transit peptide coding sequence (tp), and under the control of the CaMV 35S promoter, was expressed in hairy root cultures ofNicotiana tabacum. The fusion of theldc to the targeting signal sequence improved the performance of the bacterial gene in the plant cells in many respects. Nearly all transgenic hairy root cultures harbouring the35S-tp-ldc gene contained distinctly higher lysine decarboxylase activity (from 1.5 to 30 pkat LDC per mg protein) than those which had been transformed with constructs in which the gene had been directly cloned behind the CaMV 35S promoter. The higher enzyme activity led to the accumulation of up to 0.7% cadaverine on a dry mass basis. In addition, part of the cadaverine pool was used for increased biosynthesis of anabasine, an alkaloid which was hardly detectable in control cultures. The best line contained anabasine levels of 0.5% dry mass, which could further be enhanced by feeding of lysine.