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PubMed1990

The oligosaccharides of the glycoprotein pheromone of Volvox carteri f. nagariensis Iyengar (Chlorophyceae).

The sexuality-inducing glycoprotein of Volvox carteri f. nagariensis was purified from supernatants of disintegrated sperm packets of the male strain IPS-22 and separated by reverse-phase HPLC into several isoforms which differ in the degree of O-glycosylation. Total chemical deglycosylation with trifluoromethanesulphonic acid yields the biologically inactive core protein of 22.5 kDa. This core protein possesses three putative binding sites for N-glycans which are clustered in the middle of the polypeptide chain. The N-glycosidically bound oligosaccharides were obtained by glycopeptidase F digestion and were shown by a combination of exoglycosidase digestion, gaschromatographic sugar analysis and two-dimensional HPLC separation to possess the following definite structures: (A) Man beta 1-4GlcNAc beta 1-4GlcNAc; (B) (Man alpha)3 Man beta 1-4GlcNAc beta 1-4GlcNAc Xyl beta; (C) (Man alpha)2 Man beta 1-4GlcNAc beta 1-4GlcNAc; (D) (Man)2Xyl(GlcNAc)2. Xyl beta Two of the three N-glycosidic binding sites carry one B and one D glycan. The A and C glycans are shared by the third N-glycosylation site. The O-glycosidic sugars, which make up 50% of the total carbohydrate, are short (up to three sugar residues) chains composed of Ara, Gal and Xyl and are exclusively bound to Thr residues.

Carbohydrate Sequence↗
PubMed1994

An inducible arylsulfatase of Volvox carteri with properties suitable for a reporter-gene system. Purification, characterization and molecular cloning.

The multicellular green flagellate Volvox carteri synthesizes a periplasmic arylsulfatase in response to sulfur deprivation. The inducible enzyme has been purified to homogeneity and characterized. The corresponding gene and cDNA have been cloned. Determination of the sequence of genomic clones and comparisons to the cDNA sequence, revealed sixteen introns and seventeen exons that encode a 649-amino-acid polypeptide chain. Since the arylsulfatase enzyme is readily assayed using chromogenic substrates, but is not detectable in cells grown in sulfate-containing medium, the gene encoding arylsulfatase may be useful as a reporter gene in V. carteri. In addition, the highly regulated promoter of the arylsulfatase gene suggests its suitability as a tool for producing inducible expression vectors for cloned genes.

Amino Acid Sequence↗
PubMed1996

The evolutionary conservation of a novel protein modification, the conversion of cysteine to serinesemialdehyde in arylsulfatase from Volvox carteri.

A novel post-translational protein modification has recently been described in two human sulfatases, by which a cysteine is replaced by a serinesemialdehyde (2-amino-3-oxopropionic acid) residue [Schmidt, B., Selmer, T., Ingendoh, A. & von Figura, K. (1995) Cell 82, 271-278]. This cysteine is conserved among all known eukaryotic sulfatases. Here we report the presence of this modification in arylsulfatase from the green alga Volvox carteri. The evolutionary conservation of this novel protein modification between sulfatases of V. carteri and man lends further support to the assumption that this modification is required for the catalytic activity of sulfatases and may be present in all sulfatases of eukaryotic origin.

Alanine↗
PubMed1992

Inhibition of phototaxis in Volvox aureus by natural and simulated solar ultraviolet light.

Exposure to artificial UV wavelengths and the UV component of sunlight delays positive phototaxis in the green alga Volvox aureus. Broad band wavelength filters were used to modify the output from UV-B sources (280-320 nm) and natural sunlight. The delay in phototaxis by artificial UV is increased with exposure to shorter UV-B wavelengths. Natural sunlight experiments were performed with exposure to full sunlight and to its UV component only. The UV component present in summer sunlight produced long periods of inhibition in phototaxis and even lethality, while exposure to the total spectrum of sunlight had no significant effects on movement or survival. The data indicate that although this species of alga is well equipped to deal with present levels of UV exposure, increases in the short UV-B wavelengths in sunlight may force an alteration in patterns of photomovement.

Cell Movement↗
PubMed1997

The genetic program for germ-soma differentiation in Volvox.

Volvox carteri possesses only two cell types: mortal somatic cells and potentially immortal asexual reproductive cells called gonidia. Mutational analysis indicates that three categories of genes play central roles in programming this germ-soma division of labor: First the gls genes function during embryogenesis to cause asymmetric divisions that produce large and small cells. Then the lag genes act in the large cells (gonidial initials) to repress functions required for somatic development while the regA locus acts in the small cells (somatic initials) to repress functions required for reproductive development. Transposon tagging and DNA transformation have recently been used to recover and characterize the glsA and regA genes, and the sequences of these genes lead to testable hypotheses about how they play their roles in germ-soma differentiation.

Algal Proteins↗
PubMed1976

Electron histochemistry and ultrastructural localization of carbohydrate-containing substances in the sheath of Volvox.

The location and characteristics of carbohydrate-containing structures within the intact sheath of Volvox were studied by 3,3'-diaminobenzidine tetrahydrochloride-osmium, colloidal iron, colloidal thorium, ruthenium red and periodic acid-silver methenamine staining. The sheath consists of external and internal fibrillar layers separated by a tripartite structure. The external layer reacts positively with 3,3'-diaminobenzidine tetrahydrochloride, colloidal iron, colloidal thorium and ruthenium red, indicating that it contains acid mucosaccharides. Staining in the external layer is abolished by Ba(OH)2 treatment. The tripartite structure and internal fibrillar layer contain periodic acid reactive groups which do not occur in the external layer. Under certain conditions, reactions between the cationic dyes and the internal material were also observed. It is postulated that the internal matrix of the sheath contains glycoproteins or a mixture of acid mucosaccharides and glycoproteins. Possible functions of the sheath material are discussed.

Animals↗
PubMed1991

The program for cellular differentiation in Volvox carteri as revealed by molecular analysis of development in a gonidialess/somatic regenerator mutant.

Development of a 'gonidialess'/'somatic regenerator' double mutant of Volvox carteri was analyzed with a number of cell-type-specific cDNA probes that had been identified in a previous study. Whereas in wild-type strains somatic cells and gonidia (asexual reproductive cells) constitute two distinct cell lineages, in this mutant all cells first differentiate as somatic cells and then redifferentiate as gonidia. During the initial period of somatic differentiation, we found that both gonidial and 'early' somatic transcripts were accumulated in the mutant, consistent with the idea that it is the regA gene product (which is defective in this mutant) that normally acts to suppress gonidial gene expression in somatic cells. Later in development, levels of early somatic transcripts fell abruptly, levels of the late somatic transcripts remained extremely low, and levels of gonidial transcripts rose as the cells redifferentiated. Thus it appears that in the mutant cells the gonidial program of development takes over and somatic differentiation is aborted before the stage at which late somatic genes are normally activated. These results provide molecular genetic support for a model which postulates that three types of genes (including the two that are defective in the strain studied here) are crucial for converting the sequential program of differentiation seen in more primitive volvocalean algae to the dichotomous program of germ-soma differentiation that occurs in wild-type V. carteri.

Blotting, Northern↗
PubMed1999

Actomyosin contraction of the posterior hemisphere is required for inversion of the Volvox embryo.

During inversion of a Volvox embryo, a series of cell shape changes causes the multicellular sheet to bend outward, and propagation of the bend from the anterior to the posterior pole eventually results in an inside-out spherical sheet of cells. We use fluorescent and electron microscopy to study the behavior of the cytoskeleton in cells undergoing shape changes. Microtubules are aligned parallel to the cell's long axis and become elongated in the bend. Myosin and actin filaments are arrayed perinuclearly before inversion. In inversion, actin and myosin are located in a subnuclear position throughout the uninverted region but this localization is gradually lost towards the bend. Actomyosin inhibitors cause enlargement of the embryo. The bend propagation is inhibited halfway and, as a consequence, the posterior hemisphere remains uninverted. The arrested posterior hemisphere will resume and complete inversion even in the presence of an actomyosin inhibitor if the anterior hemisphere is removed microsurgically. We conclude that the principal role of actomyosin in inversion is to cause a compaction of the posterior hemisphere; unless the equatorial diameter of the embryo is reduced in this manner, it is too large to pass through the opening defined by the already-inverted anterior hemisphere.

Actomyosin↗
PubMed1999

glsA, a Volvox gene required for asymmetric division and germ cell specification, encodes a chaperone-like protein.

The gls genes of Volvox are required for the asymmetric divisions that set apart cells of the germ and somatic lineages during embryogenesis. Here we used transposon tagging to clone glsA, and then showed that it is expressed maximally in asymmetrically dividing embryos, and that it encodes a 748-amino acid protein with two potential protein-binding domains. Site-directed mutagenesis of one of these, the J domain (by which Hsp40-class chaperones bind to and activate specific Hsp70 partners) abolishes the capacity of glsA to rescue mutants. Based on this and other considerations, including the fact that the GlsA protein is associated with the mitotic spindle, we discuss how it might function, in conjunction with an Hsp70-type partner, to shift the division plane in asymmetrically dividing cells.

Algal Proteins↗
PubMed1981

Inversion in Volvox tertius: the effects of con A.

During the development of Volvox tertius spheroids, a single-celled gonidium enlarges and undergoes multiple incomplete cleavages to give an embryo which is 'inside-out' with respect to the adult organism. A morphogenetic movement, termed 'inversion', turns this hollow ball of cells 'inside-out' through a hole, the phialopore. In V. tertius this phialopore possesses 4 inwardly directed lips. Normal inversion was studied in vitro in slide chambers and involved cell-shape changes accompanied by the production of pseudopodia and the bending backwards of the phialopore lips. 100 micrograms/ml Con A specifically and reversibly blocked inversion. Despite the inhibitory effect on cell division, the blocking of inversion was not due to the blocking of the last cell division some 50-100 min prior to inversion. Neither did the first cell-shape change from pear- to spindle-shape appear blocked. A feature of inhibition by Con A was the enhanced production of pseudopodia by embryos blocked at inversion, and the abnormal production of pseudopodia by embryos blocked at earlier stages. Non-inverting embryos showed internal flagella. We suggest that the Con A block to inversion, which may be reversed by alpha-methyl mannoside, arises from the prevention of backwards-bending of the phialopore lips. Fluorescein-isothiocyanate-Con A bound to embryo and cell coat, ane more strongly to the embryo at pre-inversion. SDS-polyacrylamide gel analysis of proteins isolated from embryos showed 4 glycoprotein bands, but Con A binding to these bands could not be demonstrated.

Cell Division↗
PubMed1976

Nucleic acid and protein differences in Volvox carteri cell types.

Nucleic acids in Volvox carteri have different radioactive labelling patterns in the two cell types, reproductive (gonidia) and somatic. Incorporation of 32P into DNA is maximum during gonidial division in both whole spheroids and isolated somatic and reproductive cells. The most active period of radioactive incorporation into the rRNA of gonidia and somatic cells is during spheroid enlargement, after which the terminally differentiated somatic cells show a steady, continuous decline. Incorporation into the gonidial rRNA declines markedly during late stages of gonidial division but increases during daughter spheroid growth. Polyarylamide gel electrophoresis profiles of cellular proteins are distinctive for spheroids at different developmental stages and also for the isolated cell types.

Cell Division↗
PubMed1991

Genetic and cytological control of the asymmetric divisions that pattern the Volvox embryo.

The highly regular pattern in which approximately 2000 small somatic cells and 16 large reproductive cells (or 'gonidia') are arranged in a typical asexual adult of Volvox carteri can be traced back to a stereotyped program of embryonic cleavage divisions. After five symmetrical divisions have produced 32 cells of equal size, the anterior 16 cells cleave asymmetrically, to produce one small somatic cell initial and one larger gonidial initial each. The gonidial initials then cease dividing before the somatic cell initials do. The significance of the visibly asymmetric divisions is underscored by genetic and experimental evidence that differences in size--rather than differences in cytoplasmic quality--are causally important in activating the programs that cause small cells to become mortal somatic cells and large cells to differentiate as reproductive cells. A number of loci, including at least five mul ('multiple gonidia') loci, appear to be responsible for determining where and when asymmetric divisions will occur, since mutations at these loci result in modified temporal and/or spatial patterns of asymmetric division in one or more portions of the life cycle. But the capacity to divide asymmetrically at all appears to require a function encoded by the gls (gonidialess) locus, since gls mutants fail to execute any asymmetric divisions. Second-site suppressors of gls that have been identified may encode other functions required for asymmetric division. Cytological and immunocytochemical studies of dividing embryos are being undertaken in an attempt to elucidate the mechanisms by which cell-division planes are established--and shifted--under the influence of such pattern-specifying genes. Studies to date clearly indicate a central role for the basal body apparatus, and particularly its microtubular rootlets, in establishing the orientation of both the mitotic spindle and the cleavage furrow; but it remains to be determined how behavior of the division apparatus becomes modified during asymmetric division.

Animals↗
PubMed1979

Effect of tetrahydrocannabinol and ethidium bromide on DNA metabolism and embryogenesis in Volvox.

The effects of delta 9-tetrahydrocannabinol (THC) and ethidium bromide (EB) on the developmental life cycle and DNA metabolism of Volvox carteri have been investigated. THC, previously shown to interfere specifically with cytoplasmic DNA (cDNA) in this organism, was used at different concentrations and at different times during the life cycle. The morphological consequences observed were found to be dependent on the nature and time of treatment. This study also indicates that ethidium bromide induces degradatin of cDNA similar to that mediated by THC. However, unlike THC, it also causes the cessation of nuclear DNA synthesis. The consequences of EB treatment on morphological development are different from those observed with THC. A correlatin of these observations with the biochemical results presented suggests possible models in which the amounts and proportions of nuclear and cytoplasmic DNA play a role in the regulation of embryogenesis in this organism.

Chlorophyta↗
PubMed1980

Sexual spheroid formation in Volvox carteri f. nagariensis lyengar induced by phenolic extracts.

Substances extracted with a phenolic reagent mixture form spheroids of volvox carteri f. nagariensis have been shown to induce the formation of sexual spheroids. Analysis of the cultures from which the phenolic extracts were prepared implicated the glycoprotein sex hormone as the inducing substance present in potent extracts. Phenolic extracts were shown to interact with calf-thymus histones such that, when the two were mixed, the extract-histone complex formed and the induction potency increased 100-fold or more. Assays of both effects are described. Differences in the physical properties and activities of the sex hormone present in male sexual fluids and the inducing substance in phenolic extracts are discussed with reference to some effects the extraction process may have on the inducer.

Chlorophyta↗
PubMed1980

A method for studying the effect of inhibitors on the development of the isolated gonidia of Volvox tertius.

During asexual reproduction in Volvox tertius, a single cell, the gonidium, contained within the parental spheroid, undergoes enlargement followed by multiple cleavages. A method has been developed for the isolation and maintenance of gonidia in vitro in paraffin chambers. In these chambers development of the gonidium to a multicellular spheroid and subsequent inversion was apparently normal. The paraffin chambers allowed the monitoring of the effects of inhibitors on development without the possible interference of the parental spheroid. Enlargement and division of isolated gonidia was prevented by actinomycin D, cycloheximide and streptomycin. Division but not enlargement was inhibited by 5-fluorouracil, mitomycin C and ethidium bromide. Colchicine at high concentrations prevented inversion, as did streptomycin, cycloheximide and actinomycin D.

Anti-Bacterial Agents↗
PubMed1980

Some properties of male-inducing phermones from Volvox aureus M5.

The differentiation of male colonies in Volvox aureus M5 may be induced by chemical substances (phermones) found in culture fluids and in phenol extracts from whole colonies. The behaviour of the phermones from the two systems are similar in that they can survive phenol extraction under appropriate conditions, are activated by the addition of calf thymus histone, and are heat labile. The phermones differ in respect to the degree of heat sensitivity. A hypothetical model is proposed to account for these observations.

Animals↗
PubMed1981

Changes in somatic cell structure during senescence of Volvox carteri.

Senescence of the terminally differentiated somatic cells of the green alga, Volvox carteri f. weismannia, was investigated by light, fluorescence, and electron microscopy. Viability of the somatic cell population, as determined by trypan blue or erythrosin B exclusion, showed a sharp reduction beginning 144 h after the somatic cells had lost the ability to divide. This increased mortality rate was correlated at the light microscopic level with a retraction of the somatic cell cytoplasm, a reduction in chloroplast autofluorescence (and total chlorophyll content), and a decline in the number of vacuoles which could be localized with 9-aminoacridine fluorescence microscopy. Nuclear fluorescence with acridine orange remained unaffected during this time. Lipid bodies increased in older cells, and total lipid analysis showed a sharp increase beginning 96 h after the somatic cells had stopped dividing. Electron microscopic comparison between young (48--72 h) and old (168 h) somatic cells showed a disorganization of chloroplast structure, a decline in the number of cytoplasmic ribosomes, and, substantiating the light microscopy, and accumulation of lipid bodies in the cytoplasm of the older cells. The results demonstrate progressive changes in somatic cell structure with age and are suggestive of cells under nutrient stress even though they are in nutrient medium. Therefore senescence and death of the V. carteri somatic cells may be caused, in part, by an inability to take up or utilize nutrients present in the culture medium.

Aging↗
PubMed1995

Purification and characterization of a cAMP-binding protein of Volvox carteri f. nagariensis Iyengar.

Two cAMP-binding proteins, cbp1 and cbp2, were purified from the cytoplasm of the green alga Volvox carteri. Both proteins have a native molecular mass of 90 kDa as determined by gel filtration. cbp2 was purified to apparent electrophoretic homogeneity, having a subunit molecular mass of 42 kDa as determined by SDS/PAGE. The cbp1 preparation contains a 42-kDa and a 44-kDa band. The cAMP-binding activity is not associated with protein kinase activity. Tryptic peptides of cbp2 were sequenced by automated Edman degradation. Two pairs of peptides differ in one amino acid only, thus pointing to the presence of isoforms of cbp2. Both binding proteins differed from the cAMP-specific phosphodiesterases of V. carteri with respect to charge, molecular mass and binding affinity to N6-cAMP-agarose. Reverse-phase chromatography of the bound ligand revealed that the two binding proteins hydrolyse cAMP to 5' AMP. The binding specificity of purified cbp1 and cbp2 was probed by a set of modified cAMP derivatives. Both proteins bind cAMP strictly specifically in the anti conformation; position 1 and 6 of the adenine moiety and at least one of the exocyclic O atoms of the ribose cyclic phosphate moiety are essential. 3-Isobutyl-1-methylxanthine is an effective inhibitor of binding but the natural methylxyanthines are not. At present it is not clear whether cbp1 and cbp2 are individual proteins or isoforms of one another.

Amino Acid Sequence↗