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Protein synthetic patterns during the asexual life cycle of Volvox carteri.

The polypeptide labeling patterns of somatic cells, gonidia (asexual reproductive cells), embryos, and juvenile spheroids of Volvox carteri cultures synchronized by a light/dark cycle were studied as a function of developmental stage and incubation condition. Specimens were exposed to 35SO=4 for 1-hr periods at selected intervals throughout the asexual life cycle; proteins were then extracted and analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography. Although sulfation accounts for more than half the 35S incorporated, the conditions of extraction and electrophoresis employed resulted in exclusion of most sulfated products and inclusion of nearly all products bearing incorporated sulfur amino acids in the electrophoretic analysis. Hence SDS-PAGE profiles reflect relative rates of synthesis of major polypeptides. The first phase of these studies involved examination of stage-specific differences in protein synthetic patterns. Because a single developmental stage exhibits different protein synthetic patterns in light and darkness, detailed developmental comparisons were made only on organisms or cells exposed to label in the light. They yielded the following results: Shortly after the completion of embryogenesis (while all cells are still linked by numerous cytoplasmic bridges) presumptive somatic cells and gonidia exhibit a nearly identical pattern of labeling of the major polypeptides. In just a few hours, however, as cytoplasmic bridges begin to break down, the synthetic patterns of the two cell types begin to diverge; with passing time this divergence becomes progressively greater. By the time gonidia are mature, the patterns of labeling of major polypeptides by somatic cells and gonidia exhibit far more differences than similarities. Embryos derived from these mature gonidia then exhibit numerous, reproducible, stage-specific changes in polypeptide labeling throughout embryogenesis. However, two glycoproteins that previous authors implicated in the control of the differentiative cleavage division are here shown to be labeled in the parental somatic cells, not in the embryos as was previously supposed; hence a central role for them in embryonic development seems highly unlikely. In the second phase of this study the effects of light on protein synthetic patterns of organisms at selected developmental stages were analyzed. At all stages marked, rapid, reversible changes in the pattern of labeling of major polypeptides occur when cultures are transferred from light to dark or vice versa, but these changes are most marked in juvenile spheroids at the end of the dark period during which they had completed their embryogenesis. Some, but by no means all, of the changes induced by light can be attributed to stimulated synthesis of chloroplast proteins, on both chloroplast and cytosol ribosomes. The proteins made at the beginning of one light period are not identical to those made at the end of the preceding light period...

Animals↗

Identification of cell-type-specific genes of Volvox carteri and characterization of their expression during the asexual life cycle.

Volvox carteri possesses two morphologically and functionally distinct cell types: somatic cells and gonidia (asexual reproductive cells). To define the developmental programs involved in the differentiation of these two cell types, we have isolated 31 nonhomologous cDNA clones that hybridized to RNAs that were significantly more abundant in one cell type than the other. Details of the cell-type- and stage-specificity of expression of the transcripts detected by these cDNAs (plus five genes previously characterized by others) were examined by Northern-blot analysis. Accumulation patterns for the 19 gonidial transcripts fell into two distinct classes: transcripts of one gene were maximally abundant in very early cleavage, whereas transcripts of the other 18 did not reach maximal abundance until quite late in gonidial development. Similarly, the 12 somatic-cell-specific transcripts fell into two categories: transcripts of 5 "early" somatic genes became abundant soon after the completion of embryogenesis, whereas transcripts of 7 "late" somatic genes were not detected until later developmental stages. Expression of 3 other genes (two involved in flagellar development and one that encodes an extracellular matrix component) was also found to be restricted largely to somatic cells. These studies indicate that phenotypic differences between somatic cells and gonidia can be at least partially explained by differential regulation of RNA accumulation, and that there appear to be multiple patterns of accumulation of cell-type-specific transcripts within each cell type.

Animals↗

Early and late gene expression programs in developing somatic cells of Volvox carteri.

In situ hybridization was used to resolve details of the temporal and spatial aspects of expression of a number of cell-type-specific genes of Volvox carteri. In confirmation of earlier results obtained by Northern-blot analysis, this study revealed that accumulation of transcripts of somatic genes was largely restricted to the somatic-cell lineage. In extension of the previous study, two distinct gene expression programs during somatic-cell development were more fully defined: "early" somatic genes were expressed in the somatic-cell lineage even before visible differentiation began, whereas "late" somatic genes were not expressed until after somatic cells had been visibly differentiated for more than a day and had already reached the presenescent-adult stage of development. We postulate that products of the early somatic genes may be involved in generalized somatic functions that are required throughout somatic-cell development, whereas the late somatic gene products may be involved in more specialized processes that characterize mature, parental somatic cells. The most significant finding of the present study is that transcripts of early somatic genes are significantly more abundant in presumptive somatic cells than in presumptive gonidia during embryonic stages, while the two cell types are linked by a network of cytoplasmic bridges. The fact that the two cell lineages can establish and maintain different transcript populations despite the extensive cytoplasmic continuity that exists between them is somewhat surprising and must be taken into account in future attempts to elucidate the way in which dichotomous differentiation is initiated in the organism.

Animals↗

Cyclic AMP as an intraspheroidal differentiation signal in Volvox carteri.

The action of the macromolecular inducer glycoprotein on sexual reproduction in the green alga Volvox carteri can be modified by altering the external (intraspheroidal) cAMP concentration. Direct proof for the presence of cAMP in the spheroids is given. Protein binding assay and HPLC-fluorimetric analysis independently demonstrate the existence of cAMP in the matrix, cells, and culture medium. Its concentration is higher in sexual cultures, pointing to a transmitting function in sex induction. The presence in the matrix of other members of a protein phosphorylation system suggests an induction-specific signal cascade in this plant.

Cell Differentiation↗

The sexual inducer of Volvox carteri. Primary structure deduced from cDNA sequence.

The primary structure of the sexual inducer of Volvox carteri f. nagariensis has been deduced by cloning and sequence analysis of cDNA. The sexual inducer contains 208 amino acids including a signal sequence. A total of six potential N-glycosylation sites are found within the polypeptide chain. At the genomic level, the sexual inducer protein is encoded in five exons.

Algal Proteins↗

Reverse transcriptase families and a copia-like retrotransposon, Osser, in the green alga Volvox carteri.

By using the polymerase chain reaction (PCR) we have isolated and sequenced two distinct families of reverse transcriptase (RT) sequences from the genome of the colonial alga, Volvox carteri. Probing a genomic library with these RT clones revealed copia-like retrotransposons. One of these elements, named Osser, is 4,875 bp long, bordered by 197-bp identical long terminal repeats (LTRs), and shows the typical organization of retrotransposons belonging to the copia-Tyl group. This is the first complete copia-like retrotransposon sequence described in a green alga.

Amino Acid Sequence↗

A novel glycosphingolipid that may participate in embryo inversion in Volvox carteri.

Mouse monoclonal antibody 4-I-244 detects a developmentally regulated antigen in embryos of Volvox carteri and inhibits specifically the morphogenetic process of inversion (the process by which the embryo turns inside out). Antigen 4-I-244 was chemically characterized as a complex phytosphingolipid containing the neutral sugars xylose, galactose, and glucose as well as inositol and phosphate.

Antibodies, Monoclonal↗

Stage-specific hypermutability of the regA locus of Volvox, a gene regulating the germ-soma dichotomy.

Mutation at the regA locus confers on somatic cells of Volvox (which otherwise undergo programmed death) ability to redifferentiate as reproductive cells. Stable mutations at the regA locus, but not at other loci, were induced at high frequency when embryos at one particular stage were exposed to either UV irradiation, novobiocin, nalidixic acid, bleomycin, 4-hydroxyaminoquinoline-1-oxide, 5-bromodeoxyuridine, or 5-fluorouracil. All treatments led to some mutations that were not expressed until the second generation after treatment. The sensitive period was after somatic and reproductive cells of the next generation had been set apart, but before they had undergone cytodifferentiation. Hypermutability occurs in presumptive reproductive cells (in which regA is normally not expressed) somewhat before regA normally acts in somatic cells. We postulate that hypermutability of regA in the reproductive cells at this time reflects a change of state that the locus undergoes as it is inactivated.

Bleomycin↗

Organization and transcription of Volvox histone-encoding genes: similarities between algal and animal genes.

The nucleotide sequences of two non-allelic histone H2A-H2B gene loci of the green alga Volvox carteri have been determined. Each locus contains a divergently arranged H2A-H2B gene pair. The encoded proteins differ in one (H2A) and 16 positions (H2B), respectively. The coding regions are separated by short intercistronic segments (256 bp and 298 bp) containing TATA boxes and a central tandem repeat of a conserved 20-bp element as the putative histone-specific transcription signals. The 3'-untranslated regions exhibit a characteristic 3'-palindrome and weakly conserved spacer elements. Transcription in one gene locus was shown to initiate 48 bp upstream from H2A and 59 bp upstream from H2B. Contrary to higher plants, V. carteri histone mRNAs are nonpolyadenylated. S1 mapping and Northern-blotting experiments indicated that V. carteri histone mRNAs are terminated at the 3'-palindrome by the same mechanism that operates in vertebrates and sea urchins.

Amino Acid Sequence↗

The nitrate reductase-encoding gene of Volvox carteri: map location, sequence and induction kinetics.

The nitrate reductase (NR) structural gene (nitA) of Volvox carteri has been cloned and characterized. There is a single copy of this gene in the genome, and RFLP (restriction-fragment length polymorphism) analysis assigns it to the previously defined nitA/chlR locus on linkage group IX, 20-30 cM from the two beta-tubulin-encoding loci. Determination of the 5871-nt sequence of the coding region of genomic clones, and comparisons to a cDNA sequence, revealed ten introns and eleven exons that encode a 864-aa polypeptide. Detailed comparisons with higher-plant and fungal NRs indicate that, whereas the aa sequence is strongly conserved within functional domains for the flavin adenine dinucleotide-, heme- and molybdenum-pterin cofactor-binding sites, substantial differences in the aa sequence occur in the N-terminal end and the two inter-domain regions. Two potential transcription start points 439 and 452 nt upstream from the start codon and a polyadenylation signal 355 nt downstream from the stop codon have been identified by primer-extension analysis and cDNA sequencing, respectively. Accumulation of the nitA transcript is both induced by nitrate and repressed by ammonium and urea: after the organism is transferred from ammonium to nitrate as the nitrogen source, a 3.6-kb NR transcript is readily detectable on Northern blots by 10 min, reaches maximum abundance by 30 min, and then rapidly declines to an intermediate level that is subsequently maintained. Substantial induction by nitrate is observed at the end of the dark portion of the daily light/dark cycle, but the inductive response peaks in the first hour of the light period.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

The yptV1 gene encodes a small G-protein in the green alga Volvox carteri: gene structure and properties of the gene product.

Small G-proteins encoded by ras-like genes are ubiquitous in eukaryotic cells. These G-proteins are believed to play a role in central processes, such as signal transduction, cell differentiation and membrane vesicle transport. By screening genomic and cDNA libraries of the colonial alga, Volvox carteri f. nagariensis, with ypt DNA probes from Zea mays, we have identified the first member of a ypt gene family, yptV1, within a green alga. The 1538-bp yptV1 gene of V. carteri consists of nine exons and eight introns and has three potential polyadenylation sites 210, 420 and 500 bp downstream from the UGA stop codon. The derived 203-amino-acid polypeptide, YptV1, exhibits 81% similarity with Ypt1 from mouse, with the corresponding genes sharing four identical intron positions. Recombinant YptV1 (reYptV1) produced in Escherichia coli retains the ability to bind GTP after SDS-PAGE and immobilization on nitrocellulose. Immunological studies using polyclonal antibodies against reYptV1 indicate that the protein is present in the membrane fraction of a V. carteri extract and is expressed throughout the whole life-cycle of the alga. Similar to other Ras-like proteins, YptV1 contains two conserved C-terminal cysteine residues suggesting post-translational modification(s), such as isoprenylation or palmitoylation, required for membrane anchoring. The presumptive role of YptV1 in cytoplasmic vesicle transport is briefly discussed.

Amino Acid Sequence↗

Two histone H1-encoding genes of the green alga Volvox carteri with features intermediate between plant and animal genes.

Southern hybridization indicated the presence of at least two and possibly four histone H1-encoding genes occurring as singlets in the Volvox carteri genome. Two of these genes, H1-I and H1-II, have been cloned and characterized. Their coding sequences are each interrupted by three introns, but only the position of the second intron is identically conserved in both H1-I and H1-II. The encoded 260-amino-acid (aa) (H1-I) and 240-aa (H1-II) polypeptides possess the typical tripartite organization of animal H1 histones, with variable N- and C-terminal domains flanking a conserved 'globular' DNA-binding domain. Extensive differences in their variable regions suggest that H1-I and H1-II (62% identity) represent two isotypes with different functions. A prominent KAPKAP-KAA motif in the H1-I N-terminal region, similarly seen in single H1 variants of a mosquito and a nematode, has a putative function in packing condensed subtypes of chromatin. Different from higher plants, but like animals, the H1 genes of V. carteri possess a typical 3' palindrome for mRNA processing, resulting in non-polyadenylated mRNAs. Transcription initiates 33 nucleotides (nt) (H1-I) and 26 nt (H1-II) downstream of typical TATA boxes. A putative 20-bp conserved enhancer element upstream of each TATA box closely resembles the consensus sequence associated with the nucleosomal histone-encoding genes in V. carteri [Müller et al., Gene 93 (1990) 167-175] and suggests stringent regulation. Accordingly, transcription of H1 was shown to be restricted to late embryogenesis, when new flagella are produced. We discuss the inferred accessory role of histone H1 proteins in stabilizing axonemal microtubules, as has been recently observed in sea urchin flagella [Multigner et al., Nature 360 (1992) 33-39].

Amino Acid Sequence↗

Volvox carteri alpha 2- and beta 2-tubulin-encoding genes: regulatory signals and transcription.

Microtubules (MT) carry out several specialized morphogenetic functions in the multicellular green alga Volvox carteri (Vc), in addition to functions also executed in its closest unicellular relative, Chlamydomonas reinhardtii (Cr). To find out if these differences in morphogenetic complexity are reflected in tubulin (Tub) differences, we have compared the Vc alpha tub and beta tub genes with their Cr counterparts. The Vc genome contains two alpha tub and two beta tub genes. We report here the sequences of the alpha 2tub and beta 2tub genes, and thus complete the set of four tub sequences. The two alpha tub and two beta tub genes code for identical 451 (alpha) and 443 (beta) amino acid (aa) polypeptides; they differ from the Cr homologs in two (alpha) and one (beta) residues, respectively. Silent nucleotide (nt) exchanges between sibling genes are much more frequent in Vc than in Cr (12 vs. 2%), probably owing to a more stringent codon bias in the latter alga. Transcription of alpha 2tub and beta 2tub starts with an A, 26 bp (alpha 2) or 25 bp (beta 2) downstream from the TATA box. A 16-bp promoter element upstream and a G + C-rich sequence downstream from the TATA box are conserved in all tub of both species. Moreover, a 28-bp element of conserved sequence, and hence of possible functional significance, was found at similar locations in the 5' untranslated region (UTR) of all four alpha tub. A conserved TGTAA downstream from the translation stop codon represents the algal poly(A)-addition signal (in both Vc and Cr).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

A kinesin, invA, plays an essential role in volvox morphogenesis.

In Volvox carteri adults, reproductive cells called gonidia are enclosed within a spherical monolayer of biflagellate somatic cells. Embryos must "invert" (turn inside out) to achieve this configuration, however, because at the end of cleavage the gonidia are on the outside and the flagellar ends of all somatic cells point inward. Generation of a bend region adequate to turn the embryo inside out involves a dramatic change in cell shape, plus cell movements. Here, we cloned a gene called invA that is essential for inversion and found that it codes for a kinesin localized in the cytoplasmic bridges that link all cells to their neighbors. In invA null mutants, cells change shape normally, but are unable to move relative to the cytoplasmic bridges. A normal bend region cannot be formed and inversion stops. We conclude that the InvA kinesin provides the motile force that normally drives inversion to completion.

Amino Acid Sequence↗

Translational regulation of protein synthesis, in response to light, at a critical stage of Volvox development.

In Volvox cultures synchronized by a light-dark cycle, juveniles containing presumptive somatic and reproductive cells are produced during the dark, but their cells do not differentiate until after the lights come on. The pattern of protein synthesis changes rapidly after the lights come on. Action spectra and effects of photosynthesis inhibitors indicate that this protein synthetic change is not simply a consequence of renewed flow of energy from illuminated chloroplasts. Actinomycin, at a level adequate to block the response to heat shock, has virtually no effect on the response of the same cells to light; furthermore, RNAs isolated from unilluminated and illuminated juveniles yield indistinguishable in vitro translation products. We conclude, therefore, that this effect of light is exerted almost exclusively at the translational level, generating one of the most striking examples of translational regulation yet described.

Animals↗

Cooperation and conflict in the evolution of individuality. IV. Conflict mediation and evolvability in Volvox carteri.

The continued well being of evolutionary individuals (units of selection and evolution) depends upon their evolvability, that is their capacity to generate and evolve adaptations at their level of organization, as well as their longer term capacity for diversifying into more complex evolutionary forms. During a transition from a lower- to higher-level individual, such as the transition between unicellular and multicellular organisms, the evolvability of the lower-level (cells) must be restricted, while the evolvability of the new higher-level unit (multicellular organism) must be enhanced. For these reasons, understanding the factors leading to an evolutionary transition should help us to understand the factors underlying the emergence of evolvability of a new evolutionary unit. Cooperation among lower-level units is fundamental to the origin of new functions in the higher-level unit. Cooperation can produce a new more complex evolutionary unit, with the requisite properties of heritable fitness variations, because cooperation trades fitness from a lower-level (the costs of cooperation) to the higher-level (the benefits for the group). For this reason, the evolution of cooperative interactions helps us to understand the origin of new and higher-levels of fitness and organization. As cooperation creates a new level of fitness, it also creates the opportunity for conflict between levels of selection, as deleterious mutants with differing effects at the two levels arise and spread. This conflict can interfere with the evolvability of the higher-level unit, since the lower and higher-levels of selection will often "disagree" on what adaptations are most beneficial to their respective interests. Mediation of this conflict is essential to the emergence of the new evolutionary unit and to its continued evolvability. As an example, we consider the transition from unicellular to multicellular organisms and study the evolution of an early-sequestered germ-line in terms of its role in mediating conflict between the two levels of selection, the cell and the cell group. We apply our theoretical framework to the evolution of germ/soma differentiation in the green algal group Volvocales. In the most complex member of the group, Volvox carteri, the potential conflicts among lower-level cells as to the "right" to reproduce the higher-level individual (i.e. the colony) have been mediated by restricting immortality and totipotency to the germ-line. However, this mediation, and the evolution of an early segregated germ-line, was achieved by suppressing mitotic and differentiation capabilities in all post-embryonic cells. By handicapping the soma in this way, individuality is ensured, but the solution has affected the long-term evolvability of this lineage. We think that although conflict mediation is pivotal to the emergence of individuality at the higher-level, the way in which the mediation is achieved can greatly affect the longer-term evolvability of the lineage.

Adaptation, Physiological↗

A senescence-associated S-like RNase in the multicellular green alga Volvox carteri.

Asexual individuals of the green alga Volvox carteri consist of only two cell types: somatic and reproductive cells. The somatic cells are terminally differentiated, post-mitotic cells which undergo gradual senescence leading to cell death in every generation. To elucidate the self-degrading process of macromolecules associated with senescence, we attempted to clone an RNase whose mRNA accumulation is increased during senescence. The corresponding cDNA clone VRN1, encoding an S-like RNase of V. carteri, is the first T(2)/S-like RNase to be cloned from green algae. Semi-quantitative RT-PCR analysis revealed that a relative amount of VRN1 mRNA is more than three-fold higher in the senescent somatic cells than in young somatic cells when the mRNA of ribosomal protein S18 is used as an internal standard. VRN1 mRNA is not induced by phosphate starvation, indicating that its accumulation during senescence is not due to a self-induced defect in utilizing phosphates. Similar regulation has been reported for RNS3, which encodes the S-like RNase that is induced in senescent leaves of Arabidopsis thaliana. These observations imply that VRN1 may promote RNA degradation during senescence of somatic cells in V. carteri, and that its regulation has similarity with that of certain senescence-associated RNases in higher plants.

Amino Acid Sequence↗

Cloning and characterization of novel extensin-like cDNAs that are expressed during late somatic cell phase in the green alga Volvox carteri.

Asexual individuals of the green alga Volvox carteri consist of two cell types, somatic and reproductive cells. The somatic cells are terminally differentiated post-mitotic cells which undergo gradual senescence leading to cell death in every generation. To understand the gene expression programs associated with senescence of somatic cells, we cloned two cDNAs, LSG1 and LSG2, that are preferentially expressed during this late developmental stage. These two cDNAs were deduced to encode Pro-rich motifs characteristic of extensin proteins that are components of the extracellular matrix. LSG1 also resembled genes encoding plant pathogenesis-related protein 1 (PR-1), while LSG2 showed similarities with genes encoding matrix metalloproteinases, including a gamete lytic enzyme of Chlamydomonas. We also found that S9, one of the late somatic cDNAs previously cloned by Tam and Kirk (Dev. Biol. 145 (1991) 51), was deduced to encode a protein with a composition similar to LSG2. The expression of PR-1 and a matrix-metalloproteinase-encoding gene has been shown to be induced during senescence in higher plants. These results indicate that some of the late somatic genes in V. carteri are related to the senescence-associated genes in higher plants.

Amino Acid Sequence↗