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Structure and expression of a single actin gene in Volvox carteri.

Southern blot analysis of Volvox carteri DNA indicated the presence of a single actin gene; the nucleotide sequence of that gene is reported here. In comparison with plant animal and fungal actins, the derived primary structure of 377 amino acids is highly conserved yielding similarity values of 79% to 94% (including non-identical conservative exchanges). In contrast, the intron structure of the gene is highly unusual: in addition to one intron in the 5' untranslated region (ten nucleotides upstream of the initiator ATG), it has eight introns in the coding region, only three of which are in locations where introns have previously been reported. Transcription starts 26 nucleotides downstream of the putative TATA box and 70 nucleotides downstream of a conspicuous CCAAT motif. A potential polyadenylation signal, TGTAA, is located 366 nucleotides downstream of the terminator TAA. Northern hybridization indicates that the actin gene is transcribed throughout the Volvox life cycle with only a slight depression during the release of juveniles from mother spheroids. This pattern of gene expression suggests that actin may assume various functional roles in the differentiation and growth of Volvox.

Actins↗

Structure, expression, and phylogenetic relationships of a family of ypt genes encoding small G-proteins in the green alga Volvox carteri.

In addition to the previously described gene yptV1 encoding a small G-protein we have now identified and sequenced four more ras-related ypt genes (yptV2-yptV5) from the green alga Volvox carteri. The four new genes encode polypeptides consisting of 203 to 217 amino-acid residues that contain the typical sequence elements (GTP-binding domains, effector domain) of the ypt/rab subgroup of the Ras superfamily. Comparison of the derived amino-acid sequences from the V. carteri ypt gene products and their Ypt homologs from other species revealed similarity values ranging from 60% to 85%, whereas intraspecies similarities were found to approach only 55%. The coding sequences are interrupted by 5-7 introns of variable size (70-1000 nucleotides) occupying different positions in the genes. Reverse-transcribed samples of stage-specific RNAs were PCR-amplified with primers specific to yptV1, yptV3, yptV4, and yptV5 to determine if yptV transcription might be restricted to either cell type or to a specific stage of the life cycle. These experiments demonstrated that each of these genes is expressed throughout the entire Volvox life cycle and in both the somatic and the reproductive cells of the alga. The transcription start sites of yptV1 and yptV5 were mapped by primer extension. Expression of recombinant yptV cDNA in E. coli yielded recombinant proteins that bound GTP specifically, demonstrating a property which is typical for small G-proteins. The derived YptV polypeptide sequences were used to group them into four distinct classes of Ras-like proteins. These are the first proteins of the Ras superfamily to be identified in a green alga. We discuss the possible role of the YptV-proteins in the intracellular vesicle transport of Volvox.

Amino Acid Sequence↗

Expression of highly active sex-inducing pheromone of Volvox carteri f. nagariensis in a mammalian cell system.

A cDNA fragment coding for the sex-inducing glycoprotein of Volvox carteri f. nagariensis was expressed in a mammalian cell system (baby hamster kidney (BHK) cells). The transfection product exhibited a specific biological activity intermediate between the natural pheromone of the strains Volvox carteri f. nagariensis and Volvox carteri f. weismannia. Immunoblot analysis showed that the sex-inducing activity was expressed as a set of three iso-glycoproteins (35, 34 and 31 kDa).

Cells, Cultured↗

L-Glutamic acid as a mediator of sexual morphogenesis in Volvox capensis.

In Volvox capensis the development of sexual individuals is in response to low concentrations (68 nM) of L-glutamic acid rather than to such species-specific glycoproteins as have been isolated in Volvox carteri or are believed to exist in a number of other species. V. capensis grows equally as well in light and in darkness in a medium supplemented with sodium acetate; however, L-glutamic acid is active as an inducer of the sexual form only in populations grown in the light. The site of action of L-glutamic acid and its biochemical role in the sexual response are unknown. Attempts to induce the sexual response by using the other L-amino acids, various analogs of glutamic acid, compounds of similar structure (e.g., gamma-aminobutyric acid), and intermediates of biochemical pathways known to involve L-glutamic acid (e.g., alpha-ketoglutarate or pyroglutamic acid) have been unsuccessful. L-Glutamic acid is produced by V. capensis as a natural product of the digestion of the glycoproteinaceous parental matrix at the time young spheroids escape. As a population increases, so does the level of L-glutamic acid produced at each succeeding generation until the threshold of sensitivity is reached and the induction of sexual forms is effected. This serves as a mechanism for ensuring the production of sexual spheroids and their zygotes, the only phase in the life cycle resistant to drying. Thus, Volvox is especially adapted to an existence in ephemeral pools of water resulting from seasonal rains.

Journal Article↗

The relationship between cell size and cell fate in Volvox carteri.

In Volvox carteri development, visibly asymmetric cleavage divisions set apart large embryonic cells that will become asexual reproductive cells (gonidia) from smaller cells that will produce terminally differentiated somatic cells. Three mechanisms have been proposed to explain how asymmetric division leads to cell specification in Volvox: (a) by a direct effect of cell size (or a property derived from it) on cell specification, (b) by segregation of a cytoplasmic factor resembling germ plasm into large cells, and (c) by a combined effect of differences in cytoplasmic quality and cytoplasmic quantity. In this study a variety of V. carteri embryos with genetically and experimentally altered patterns of development were examined in an attempt to distinguish among these hypotheses. No evidence was found for regionally specialized cytoplasm that is essential for gonidial specification. In all cases studied, cells with a diameter > approximately 8 microns at the end of cleavage--no matter where or how these cells had been produced in the embryo--developed as gonidia. Instructive observations in this regard were obtained by three different experimental interventions. (a) When heat shock was used to interrupt cleavage prematurely, so that presumptive somatic cells were left much larger than they normally would be at the end of cleavage, most cells differentiated as gonidia. This result was obtained both with wild-type embryos that had already divided asymmetrically (and should have segregated any cytoplasmic determinants involved in cell specification) and with embryos of a mutant that normally produces only somatic cells. (b) When individual wild-type blastomeres were isolated at the 16-cell stage, both the anterior blastomeres that normally produce two gonidia each and the posterior blastomeres that normally produce no gonidia underwent modified cleavage patterns and each produced an average of one large cell that developed as a gonidium. (c) When large cells were created microsurgically in a region of the embryo that normally makes only somatic cells, these large cells became gonidia. These data argue strongly for a central role of cell size in germ/soma specification in Volvox carteri, but leave open the question of how differences in cell size are actually transduced into differences in gene expression.

Animals↗

Histone genes of Volvox carteri: DNA sequence and organization of two H3-H4 gene loci.

Two Volvox genomic clones each containing a pair of histone H3-H4 genes were sequenced. In both loci the H3 and H4 genes show outwardly divergent polarity, their coding regions being separated by short intercistronic sequences containing TATA boxes and a conserved 14-bp element. The 3' untranslated regions contain a characteristic motif with hyphenated dyad symmetry otherwise only found associated with animal histone genes. Derived amino acid sequences of histones H3 and H4 are highly conserved and identical between the two sets. The Volvox H3 genes both contain one intron whose relative position is shifted by one basepair. Sequence comparisons led to a new interpretation of intron sliding. The Volvox H3 gene structure combines the exon-intron organization of fungal H3 and vertebrate H3.3 genes with a termination signal typical for animal H3.1 genes. These features are discussed in view of histone gene evolution.

Alleles↗

Evidence for an incomplete dolichyl-phosphate pathway of lipoglycan formation in Volvox carteri f. nagariensis.

Crude membrane fractions from Volvox carteri in the presence of detergent and metal complexing agent catalyze the transfer of glucose from dolichyl phosphate glucose to branched dolichyl diphosphate chitobiosyl pentamannoside Dol-PP-(GlcNAc)2-(Man)5, a known intermediate of the lipid-mediated pathway of N-glycosylation of proteins, resulting in the formation of Dol-PP-(GlcNAc)2-(Man)5-(Glc)1. Under the various conditions tested, neither Dol-P-Man nor other known mannosyl donors of the nucleoside-activated or lipid-activated type can serve as donor molecules for the elongation of the lipid-linked heptasaccharide. On the other hand, calf liver microsomes in similar experiments mannosylated the heptasaccharide further with Dol-P-Man up to a nonamannoside, Dol-PP-(GlcNAc)2-(Man)9. A direct glucosylation of the acceptor, however, with Dol-P-Glc failed in this system. The (GlcNAc)2-(Man)5-(Glc)1, obtained after mild acid hydrolysis of the above glycolipid is not significantly split by an unspecific alpha-glucosidase from yeast. However, Volvox microsomes liberated most of the glucose indicating a specific glucosidase in the membranes of the alga. This enzyme does not act on (GlcNAc)2-(Man)9-(Glc)1, the usual protein-linked carbohydrate intermediate of trimming processes of N-glycosidic glycoproteins. The data on glycolipid formation let us postulate that in Volvox the normal N-glycosylation pathway differs from that found in higher plants and animals either by a lack of evolution or by mutation in the genes coding for the mannosyl transferases involved.

Cations↗

The developmentally regulated ECM glycoprotein ISG plays an essential role in organizing the ECM and orienting the cells of Volvox.

Volvox is one of the simplest multicellular organisms with only two cell types, yet it has a surprisingly complex extracellular matrix (ECM) containing many region-specific morphological components, making Volvox suitable as a model system for ECM investigations. ECM deposition begins shortly after inversion, which is the process by which the embryo turns itself right-side-out at the end of embryogenesis. It was previously shown that the gene encoding an ECM glycoprotein called ISG is transcribed very transiently during inversion. Here we show that the developmentally controlled ISG accumulates at the bases of the flagella right after inversion, before any morphologically recognizable ECM structures have yet developed. Later, ISG is abundant in the 'flagellar hillocks' that encircle the basal ends of all flagella, and in the adjacent 'boundary zone' that delimits the spheroid. Transgenic Volvox were generated which express a truncated form of ISG. These transgenics exhibit a severely disorganized ECM within which the cells are embedded in a highly chaotic manner that precludes motility. A synthetic version of the C-terminal decapeptide of ISG has a similar disorganizing effect, but only when it is applied during or shortly after inversion. We postulate that ISG plays a critical role in morphogenesis and acts as a key organizer of ECM architecture; at the very beginning of ECM formation ISG establishes an essential initial framework that both holds the somatic cells in an adaptive orientation and acts as the scaffold upon which the rest of the ECM can be properly assembled, assuring that somatic cells of post-inversion spheroids are held in orientations and locations that makes adaptive swimming behavior possible.

Amino Acid Sequence↗

Polyadenylated RNA of Volvox: isolation and partial characterization.

Polyadenylated RNA from Volvox carteri has been isolated and partially characterized. Electrophoretic profiles of total cellular poly(A)-associated RNA of Volvox spheroids indicate a hetero-disperse distribution of size classes with the range extending from an apparent sedimentation value of approximately 10S to greater than 38S. The radioactive labelling kinetics of this material are typical for rapidly-turning-over RNA. The profiles of poly(A) RNA from different cell types show marked differences in average migration rate. Terminally-differentiated somatic cells contain a greater proportion of material of higher molecular weight than either gonidia (germ cells) or cleaving embryos. The poly(A) segments associated with cellular RNA, obtained by selective RNase digestion are heterogeneous in size as determined by gel electrophoresis with the largest tracts estimated to be 75-80 nucleotides long. Gonidia and embryos display the greatest degree of size heterogeneity, while somatic cells show predominantly the largest classes of poly(A) tract. It is apparent that gross changes in poly(A) RNA metabolism accompany development and cellular differentiation in Volvox.

Centrifugation, Density Gradient↗

Orthologs and paralogs of regA, a master cell-type regulatory gene in Volvox carteri.

The multicellular green alga Volvox carteri forma nagariensis has only two cell types: terminally differentiated somatic cells and reproductive cells. The regA gene maintains the terminally differentiated state of the somatic cells, apparently by repressing transcription of genes required for chloroplast biogenesis and thereby preventing cell growth. Because the RegA protein sequence bore no obvious motifs, we are attempting to identify regions of functional importance by searching for strongly conserved domains in RegA orthologs. Here we report the cloning and characterization of regA from the most closely related known taxon, V. carteri f. kawasakiensis. Given the closeness of the relationship between these two formas, their regA genes are surprisingly different: they differ in the number of introns and by several lengthy indels, and they encode proteins that are only 80% identical. We also serendipitously discovered a paralogous gene immediately upstream of each regA locus. The two regA genes, both upstream paralogs and several genes in Chlamydomonas (the closest unicellular relative of Volvox) encode a conserved region (the VARL domain) that contains what appears to be a DNA-binding SAND domain. This discovery has opened up a new avenue for exploring how regA and the terminally differentiated state that it controls evolved.

Algal Proteins↗

Swapped green algal promoters: aphVIII-based gene constructs with Chlamydomonas flanking sequences work as dominant selectable markers in Volvox and vice versa.

Production of transgenic organisms is a well-established, versatile course of action in molecular biology. Genetic engineering often requires heterologous, dominant antibiotic resistance genes that have been used as selectable markers in many species. However, as heterologous 5' and 3' flanking sequences often result in very low expression rates, endogenous flanking sequences, especially promoters, are mostly required and are easily obtained in model organisms, but it is much more complicated and time-consuming to get appropriate sequences from less common organisms. In this paper, we show that aminoglycoside 3'-phosphotransferase gene (aphVIII) based constructs with 3' and 5' untranslated flanking sequences (including promoters) from the multicellular green alga Volvox work in the unicellular green alga Chlamydomonas and flanking sequences from Chlamydomonas work in Volvox, at least if a low expression rate is compensated by an enforced high gene dosage. This strategy might be useful for all investigators that intend to transform species in which genomic sequences are not available, but sequences from related organisms exist.

3' Flanking Region↗

Characterization of a heat-shock-inducible hsp70 gene of the green alga Volvox carteri.

The green alga Volvox carteri possesses several thousand cells, but just two cell types: large reproductive cells called gonidia, and small, biflagellate somatic cells. Gonidia are derived from large precursor cells that are created during embryogenesis by asymmetric cell divisions. The J domain protein GlsA (Gonidialess A) is required for these asymmetric divisions and is believed to function with an Hsp70 partner. As a first step toward identifying this partner, we cloned and characterized V. carteri hsp70A, which is orthologous to HSP70A of the related alga Chlamydomonas reinhardtii. Like HSP70A, V. carteri hsp70A contains multiple heat shock elements (HSEs) and is highly inducible by heat shock. Consistent with these properties, Volvox transformants that harbor a glsA antisense transgene that is driven by an hsp70A promoter fragment express Gls phenotypes that are temperature-dependent. hsp70A appears to be the only gene in the genome that encodes a cytoplasmic Hsp70, so we conclude that Hsp70A is clearly the best candidate to be the chaperone that participates with GlsA in asymmetric cell division.

Algal Proteins↗

Developmental changes in the sensitivity of Volvox to ultraviolet light.

The response of Volvox to ultraviolet irradiation was analyzed. Young individuals isolated from a synchronous culture were exposed to UV light (120 J/m2) and subjected to variable length periods of dark following irradiation. The major effect of the UV treatment was the inability of the gonidia present in the colonies at the time of irradiation to continue and complete the developmental program. Individuals show a heightened sensitivity to UV for a limited period immediately following inversion and are insensitive at other stages of development. The cytotoxic effect of UV during this interval is completely reversed by the immediate exposure to white light and is increased with longer periods of dark treatment prior to exposure to white light. The temporal profile of the sensitivity defines a smooth curve in which the maximal sensitivity occurs three hours after inversion. The response to higher doses of UV (up to 500 J/m2) is a nonlinear increase in cytotoxicity and is disproportionately greater in those individuals just prior to the period of maximal sensitivity than those later in development. The results suggest that Volvox has at least two pathways for the repair of UV damage and that one of these, the principal dark repair pathway, is temporarily deficient in the gonidia of young individuals.

Animals↗

Repetitious structure and transcription control of a polyubiquitin gene in Volvox carteri.

Southern analysis indicated the presence of at least four ubiquitin gene loci in the Volvox carteri genome. Three of these, a polyubiquitin gene described here and a non-segregating ubiquitin gene pair, were assigned to two different linkage groups by RFLP mapping; the non-polymorphic fourth gene locus remained unassigned. The polyubiquitin gene was cloned and its 2,116-bp sequence determined. It contains six exons each interrupted by an intron at Gly35, and it encodes a pentameric polyubiquitin polypeptide consisting of five runs of 76 identical amino-acid residues and a C-terminal extension of one leucine. The five tandem repeats of coding units plus introns exhibit an unusually high degree of overall sequence identity indicating an efficient process of gene homogenization in this region of the V. carteri genome. S1 mapping revealed two closely-spaced transcription starts, 24 and 28 nucleotides downstream from a putative TATA sequence. Preceding the TATA box are two 14-bp conserved heat-shock elements (HSEs) and two octameric sequences closely resembling an yesat HSE. Consistent with a 1.6-kb transcript seen on Northern blots are two polyadenylation signals (TGTAA) located 99 bp and 169 bp downstream from the TGA translational stop. The polyubiquitin gene was transcribed throughout the Volvox life cycle with peaks in the 1.6-kb mRNA levels during pre-cleavage, cleavage, and post-inversion. In contrast, an 0.6-kb monoubiquitin transcript was abundant only at the pre-cleavage stage suggesting a different type of gene control. Heat shock increased the level of polyubiquitin mRNA, whereas the level of monoubiquitin mRNA was down-regulated.

Amino Acid Sequence↗

Nucleus-encoded, plastid-targeted acetolactate synthase genes in two closely related chlorophytes, Chlamydomonas reihardtii and Volvox carteri: phylogenetic origins and recent insertion of introns.

Acetolactate synthase (ALS) catalyzes the first committed step in the synthesis of branched-chain amino acids. In green plants and fungi, ALS is encoded by a nuclear gene whose product is targeted to plastids (in plants) or to mitochondria (in fungi). In red algae, the gene is plastid-encoded. We have determined the complete sequence of nucleus-encoded ALS genes from the green algae Chlamydomonas reinhardtii and Volvox carteri. Phylogenetic analyses of the ALS gene family indicate that the ALS genes of green algae and plants are closely related, sharing a recent common ancestor. Furthermore, although these genes are clearly of eubacterial origin, a relationship to the ALS genes of red algae and cyanobacteria (endosymbiotic precursors of plastids) is only weakly indicated. The algal ALS genes are distinguished from their homologs in higher plants by the fact that they are interrupted by numerous spliceosomal introns; plant ALS genes completely lack introns. The restricted phylogenetic distribution of these introns suggests that they were inserted recently, after the divergence of these green algae from plants. Two introns in the Volvox ALS gene, not found in the Chlamydomonas gene, are positioned precisely at sites which resemble "proto-splice" sequences in the Chlamydomonas gene.

Acetolactate Synthase↗

Synthesis and characterization of lipid-linked mannosyl oligosaccharides in Volvox carteri f. nagariensis.

Particulate membrane fractions from Volvox carteri catalyze the transfer of mannose from GDP-mannose to dolichyl diphosphate-[14C]chitobiose to form lipid-linked oligosaccharides up to a dolichyl diphosphate-chitobiose-(mannose)5 structure. Mannosylation of the chitobiosyl lipid requires divalent cations and detergents as solubilizing agents. Depending on the nature of the detergent, the oligosaccharide pattern differs markedly: With deoxycholate or the zwitterionic detergent 3-14 a lipid-linked trisaccharide accumulates. The nonionic Triton X-100, however, gives rise to a spectrum of compounds up to a heptasaccharide. Enzyme digestion of the tri- and pentasaccharide structure, obtained after mild acid hydrolysis of the corresponding [14C]glycolipids, revealed that the first mannose is bound via a beta-glycosidic linkage to the chitobiosyl core, whereas the outer mannose residues are linked as alpha-mannosides. Our studies indicate that, in agreement with recent findings in other organisms, the innermost alpha-mannosidic residues are donated directly from GDP-mannose. The structure of oligosaccharides synthesized by Volvox membranes is thus consistent with results from other eucaryotic species, suggesting a common pathway of N-glycosylation of glycoproteins.

Chemical Phenomena↗

Reproductive cell specification during Volvox obversus development.

Asexual spheroids of the genus Volvox contain only two cell types: flagellated somatic cells and immotile asexual reproductive cells known as gonidia. During each round of embryogenesis in Volvox obversus, eight large gonidial precursors are produced at the anterior extremity of the embryo. These cells arise as a consequence of polarized, asymmetric divisions of the anteriormost blastomeres at the fourth through nine cleavage cycles, while all other blastomeres cleave symmetrically to yield somatic cell precursors. Blastomeres isolated from embryos at any point between the 2-cell and the 32-cell stage cleaved in the normal pattern and produced the same complement and spatial distribution of cell types as they would have in an intact embryo. This result indicates that intrinsic features control the cleavage patterns and developmental potentials of blastomeres, and rules out any significant role for cell-cell interactions in gonidial specification. When substantial quantities of anterolateral cytoplasm were deleted from uncleaved gonidia or 4-cell stage blastomeres, the cell fragments frequently regulated and embryos were produced with the expected number of asymmetrically cleaving cells and gonidial precursors at their anterior ends. However, when anterior cytoplasm was deleted from 8-cell stage blastomeres, the depleted cells frequently failed to cleave asymmetrically and produced no gonidial precursors. Furthermore, when compression was used to reorient cleavage planes at the fourth division cycle, so that anterior cytoplasm was transmitted to more than the normal number of cells, those cells receiving a significant amount of such cytoplasm cleaved asymmetrically to produce supernumerary gonidial precursors. Together, these last two experiments indicate that blastomeres in the V. obversus embryo acquire (at least by the end of the third cleavage cycle) a polarized organization in which anterior cytoplasm plays a causal role in the process of reproductive-cell specification.

Animals↗

The sexual inducer of Volvox carteri. Its large-scale production and secretion by Saccharomyces cerevisiae.

The DNA sequence coding for the sexual inducer glycoprotein of Volvox carteri and its N-terminal signal peptide was placed under the control of the repressible acid phosphatase promoter of the yeast Saccharomyces cerevisiae in a yeast-E. coli shuttle vector. Yeast transformed by this construct synthesized and secreted into the culture medium biologically active inducer in amounts two to three orders of magnitude higher than observed in the Volvox system.

Animals↗