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[Evolutionary reorganizations of ontogenesis in related species of coenobial volvocine algae].

The evolutionary aspects of ontogenesis in green volvocine algae have been considered on the basis of the author's and published data, as well as the information on taxonomy, phylogeny, and ecology of this group. Analysis of the rate, diurnal rhythm, and light/dark control of cell divisions in various species, as well as experiments with the nucleic acid and protein synthesis inhibitors made it possible to elucidate cellular mechanisms underlying evolutionary rearrangements of asexual development in the genus Volvox.

Cell Division↗

How a sex pheromone might act at a concentration below 10(-16) M.

The sex-inducing pheromone of Volvox carteri is a glycoprotein that triggers development of males and females at a concentration below 10(-16) M. Evidence is presented for the existence of a novel mechanism of signal amplification operating within the extracellular matrix of this multicellular organism. A family of 70 kDa matrix glycoproteins denoted pherophorins bear a C-terminal domain being homologous to the sex-inducing pheromone. Under the influence of the pheromone, this domain is liberated by highly specific proteolysis.

Algal Proteins↗

Identification of an ARF type low molecular mass GTP-binding protein in pea (Pisum sativum).

The ARF class of low molecular mass GTP-binding proteins, originally detected as cholera toxin-activated ADP-ribosylation factors (ARF) in mammalian cells, is now known to participate in intracellular membrane vesicle trafficking. We identified a GTP-binding protein in pea plumules which resembles ARF in several respects. Like mammalian ARF, the pea protein had an apparent molecular mass of 21 kDa and was distributed mainly (approximately 97%) in the cytosol, with 1% and 1.5% occurring in the Golgi and microsomal fractions, respectively. In comparison, small GTP-binding proteins in the 26-30 kDa range were enriched in membranous fractions. The 21 kDa protein crossreacted strongly with an ARF class I antibody prepared against a mammalian ARF, but did not crossreact with ARF 5 (of class II) antibodies. An anti-Volvox yptl antibody did not show immunoreactivity to the 21 kDa pea protein, although it crossreacted strongly with a 28 kDa pea plumule protein. Our results strongly suggest that the 21 kDa pea protein is an analog of the ARF protein localized in the cytosol of mammalian cells. As far as we are aware, this is the first observation of an ARF protein in plants.

ADP-Ribosylation Factors↗

The survival of slow reproducers.

Multicellularity, and the attendant segregation of the germ line, entails the loss of reproductive capacity by the soma: in Volvox carteri, less than 1 cell in 100 contributes to the next generation. However, compensatory advantages are unlikely to be very large (Koufopanou & Bell, 1993. Proc. R. Soc. Lond. (B) 254,107-113). Somewhat similarly, sex implies the generation of males, hence a dramatic reproductive slowdown (Barton & Charlesworth, 1998. Science281, 1986-1990); yet, a compensating (two-fold) advantage of sex has not been found. Here, I try to evaluate the actual cost of maintaining slow reproductive cycles, namely cycles that necessitate the production of "dead end" units such as somatic cells or males. In a quantitative model for the competition of individuals with different, heritable reproductive rates, this cost turns out to be unexpectedly small, and may even sometimes become irrelevant. The bases for this are made fairly clear: thus, when all enjoy high fecundity (e.g. a long reproductive life) the handicap of a slower reproduction vanishes; alternatively, a slight separation of ecological niches may be sufficient for survival of slower but otherwise unchanged reproducers; and finally, inherent to slow reproduction is a low rate of destabilizing genetic change. These facts are largely independent of the formal model details, and are supported by direct computer simulations. They give a quantitative basis for analysing the evolution and prevalence of slow life cycles. The implications of these findings for the evolution of multicellularity are briefly discussed.

Animals↗

Domain conservation in several volvocalean cell wall proteins.

Based on our previous work demonstrating that (SerPro)x epitopes are common to extensin-like cell wall proteins in Chlamydomonas' reinhardtii, we looked for similar proteins in the distantly related species C. eugametos. Using a polyclonal antiserum against a (SerPro)10 oligopeptide, we found distinct sets of stage-specific polypeptides immunoprecipitated from in vitro translations of C. eugametos RNA. Screening of a C. eugametos cDNA expression library with the antiserum led to the isolation of a cDNA (WP6) encoding a (SerPro)x-rich multidomain wall protein. Analysis of a similarly selected cDNA (VSP-3) from a C. reinhardtii cDNA expression library revealed that it also coded for a (SerPro)x-rich multidomain wall protein. The C-terminal rod domains of VSP-3 and WP6 are highly homologous, while the N-terminal domains are dissimilar; however, the N-terminal domain of VSP-3 is homologous to the globular domain of a cell wall protein from Volvox carteri. Exon shuffling might be responsible for this example of domain conservation over 350 million years of volvocalean cell wall protein evolution.

Algal Proteins↗

Actin gene structure in two Artemia species, A. franciscana and A. parthenogenetica.

Genomic clones coding for actin have been isolated from two species of the crustacean Artemia, A. parthenogenetica and A. franciscana. The Act211 isoform gene was isolated from A. parthenogenetica, and the two other isoform genes, Act302 and Act403, were isolated from A. franciscana. The comparison of the nucleotide sequence of genomic and cDNA clones showed an interspecific divergence of 4% in translated and 6.1% in untranslated regions. However, the establishment of the partial structure of the Act211 gene in A. franciscana and of the Act302 gene in A. parthenogenetica suggests their similarity in the two species. The Act211 gene is divided into four exons, the Act302 gene into six exons, and the Act403 gene into seven exons. The three genes have introns in the 5' untranslated region and between codons 41 and 42. The Act211 and 403 genes have one common intron in codon 168. The Act302 and 403 genes have common introns between codons 121-122, 246-247, and within codon 301. While introns in the 5' untranslated region and between codons 41-42 and 121-122 are present in many organisms, the introns in positions 168 and 246-247 had only been found previously in actin genes from the nematode Onchocerca volvulus and the green alga Volvox carterii, respectively. The intron in position 301 had not been reported before. The transcription initiation sites of these three genes as well as the nucleotide sequences of the promoter regions have been also determined.

Actins↗

Modeling light-induced currents in the eye of Chlamydomonas reinhardtii.

Rhodopsin-mediated electrical events in green algae have been recorded in the past from the eyes of numerous micro-algae like Haematococcus pluvialis, Chlamydomonas reinhardtii and Volvox carteri. However, the electrical data gathered by suction-pipette techniques could be interpreted in qualitative terms only. Here we present two models that allow a quantitative analysis of such results: First, an electrical analog circuit for the cell in suction pipette configuration is established. Applying this model to experimental data from unilluminated cells of C. reinhardtii yields a membrane conductance of about 3 Sm(-2). Furthermore, an analog circuit allows the determination of the photocurrent fraction that is recorded under experimental conditions. Second, a reaction scheme of a rhodopsin-type photocycle with an early Ca(2+) conductance and a later H(+) conductance is presented. The combination of both models provides good fits to light-induced currents recorded from C. reinhardtii. Finally, it allowed the calculation of the impact of each model parameter on the time courses of observable photocurrent and of inferred transmembrane voltage. The reduction of the flash-to-peak times at increasing light intensities are explained by superposition of two kinetically distinct rhodopsins and by assuming that the Ca(2+)-conducting state decays faster at more positive membrane voltages.

Algorithms↗

Evolutionary correlation between linker histones and microtubular structures.

Histones of the H1 group (linker histones) are abundant components of chromatin in eukaryotes, occurring on average at one molecule per nucleosome. The recent reports on the lack of a clear phenotypic effect of knock-out mutations as well as overexpression of histone H1 genes in different organisms have seriously undermined the long-held view that linker histones are essential for the basic functions of eukaryotic cells. In an attempt to resolve the paradox of an abundant conserved protein without a clear function, we re-examined the molecular and phylogenetic data on linker histones to see if they could reveal any correlation between the features of H1 and the functional or morphological characteristics of cells or organisms. Because of an earlier demonstration that in sea urchin the chromatin-type histone H1 is also found in the flagellar microtubules (Multigner et al. 1992), we focused on the correlation between the features of H1 and those of microtubular structures. A phylogenetic tree based on multiple alignment of over 100 available HI sequences suggests that the first divergence of the globular domain of H1 (GH1) resulted in branching into separate types characteristic for plants/Dictyostelium and for animals/ascomycetes, respectively. The GH1s of these two types differ by a short region (usually 5 amino acids) placed at a specific location within the C-terminal wing subdomain of GH1. Evolutionary analysis of the diversification of H1 mRNA into cell-cycle-dependent (polyA-) and independent (polyA+) forms showed a mosaic occurrence of these two forms in plants and animals, despite the fact that the H1 proteins of plants and animals belong to two well-distinguished groups. However, among organisms from both animal and plant kingdom, only those with H1 mRNA of a polyA- type have flagellated gametes. This correlation as well as the demonstration that in Volvox carteri the accumulation of polyA- mRNA of H1 occurs concurrently with the production of new flagella (Lindauer et al. 1993), suggests a direct link between polyA- phenotype of histone H1 mRNA and flagellogenesis.

Animals↗

Vision in microalgae.

Flagellate green algae such as Chlamydomonas and related genera are guided by their eyes to places where light conditions are optimal for photosynthetic growth. These eyes constitute the simplest and most common visual system found in nature. The eyes contain optics, photoreceptors and the elementary components of a signal-transduction chain. Rhodopsin serves as the photoreceptor, as it does in animal vision. Upon light stimulation, its all-trans-retinal chromophore isomerizes into 13-cis and activates a photoreceptor channel which leads to a rapid Ca2+ influx into the eyespot region. At low light levels, the depolarization activates small flagellar currents which induce in both flagella small but slightly different beating changes resulting in distinct directional changes. In continuous light, Ca2+ fluxes serve as the molecular basis for phototaxis. In response to flashes of higher energy the larger photoreceptor currents trigger a massive Ca2+ influx into the flagella which causes the well-known phobic response. The identification of proteins contributing to this signalling system has just begun with the isolation and cloning of the opsins from Chlamydomonas and Volvox. These plant opsins are highly charged, are not typical seven-helix receptors, and are believed to form a protein complex with the photoreceptor channel. In Spermatozopsis, a G-protein has been found which interacts either directly with the rhodopsin or with the rhodopsin-ion channel complex. By using insertional mutagenesis, genes coding for proteins that are involved in signalling have been tagged. One of them is connected to the flagellar channel and crucial for the flagellar action potential. Elucidation of photoreception in flagellated algae will provide deeper insight into the development of visual systems, starting from single-celled organisms and moving up through higher animals.

Amino Acid Sequence↗

Cell wall biogenesis in Chlamydomonas: molecular characterization of a novel protein whose expression is up-regulated during matrix formation.

In the unicellular eukaryote Chlamydomonas, disruption of cell-matrix interactions by treatment with a periplasmic matrix metalloproteinase, g-lysin, activates a signal transduction pathway that results in the rapid synthesis and secretion of matrix molecules, followed by their assembly into a new matrix. I have identified and partially characterized several cDNA clones for transcripts that are dramatically up-regulated following treatment of cells with g-lysin. Here I report the complete nucleotide sequence and preliminary characterization of a matrix-related molecule termed Mrp47. The cDNA clone for Mrp47 contained an insert of 2.5 kb, corresponding to a transcript of 3.0 kb that is encoded by a single-copy gene. Sequence analysis indicated that Mrp47 cDNA contains an open reading frame (ORF) that encodes a 46-kDa polypeptide. The putative polypeptide is unusually rich in the amino acids proline, alanine and serine, with prolines clustered together in a 30-amino acid N-terminal region and a 80-amino acid C-terminal region. Further analysis of the predicted amino acid sequence suggested that Mrp47 is likely to be a secreted glycoprotein. Southern hybridization analysis indicated that Mrp47 is encoded by a single-copy gene in the Chlamydomonas genome. Database searches suggested that Mrp47 shows homology to other proline-rich proteins including a surface glycoprotein in Volvox and verprolin from yeast.

Algal Proteins↗

Molecular characterization of two light-induced, gamete-specific genes from Chlamydomonas reinhardtii that encode hydroxyproline-rich proteins.

Gametic differentiation in Chlamydomonas reinhardtii is a two-step process, which is controlled by the sequential action of the two extrinsic signals, nitrogen starvation and blue light. The gamete-specific genes GAS28 and GAS29 are expressed in the late phase of gametogenesis. Their light-induced expression is restricted to cells that have completed the first, nitrogen starvation-activated, phase of differentiation. A comparison of the two genes revealed striking similarities as well as differences. Their most prominent shared feature is an extended sequence homology of over 90% in their 5'-untranslated regions, suggesting a role in translational regulation. GAS28 and GAS29 both encode hydroxyproline-rich proteins (HRGPs) of very similar sizes that exhibit typical features of volvocalean cell wall constituents. GAS28 shows a high degree of homology with the Volvox pherophorin gene family, suggesting a relationship between these genes.

Amino Acid Sequence↗

Gulose as a constituent of a glycoprotein.

The aldohexose gulose was identified as a constituent of a hydroxyproline-rich glycopeptide derived from the glycoprotein SSG 185. This glycoprotein is part of the extracellular matrix of the green alga Volvox carteri. The gulose residue occupies a terminal position in the corresponding saccharide.

Amino Acid Sequence↗

Pathogenic Entamoeba histolytica: cDNA cloning of a histone H3 with a divergent primary structure.

Entamoeba histolytica has an unusual nuclear structure characterized by a low degree of chromatin condensation and the absence of stainable metaphase chromosomes. Although nucleosome-like particles were observed, no information about histones was available so far. In this paper we describe a cDNA clone with significant homology to H3 histones that was isolated from a library of pathogenic E. histolytica. The complete cDNA encodes a 15-kDa polypeptide, which like the histone sequence from Volvox carteri is shorter by one residue than the human homologue. The amino acid sequence has only 69% identity with human H3.3 histone and 67% identity with the human H3.1 histone. This is the highest degree of sequence divergence observed for any eukaryote H3 histone sequence. Our results indicate that this divergence may contribute to the unusual chromatin structure of E. histolytica.

Amino Acid Sequence↗

Intracellular translocation of a 28 kDa GTP-binding protein during osmotic shock-induced cell volume regulation in Dunaliella salina.

The primary aim of this study was to determine if small GTP-binding proteins play a role in the conspicuous and much-examined volume control process in Dunaliella salina. We confirmed the previous identification by Rodriguez et al. (Rodriguez Rosales, M.P., Herrin, D.L. and Thompson, G.A., Jr. (1992) Plant Physiol. 98, 446-451) of small GTP-binding proteins in the green alga Dunaliella salina and revealed the presence of at least five such proteins, having molecular masses of approx. 21, 28, 28.5, 29 and 30 kDa. These proteins were concentrated largely in the endoplasmic reticulum (ER) and in an intermediate density organelle fraction (GA) containing mainly Golgi vesicles, mitochondria and flagella. The chloroplast fraction and plasma membrane contained the 28-kDa GTP-binding protein exclusively, while the cytosol contained both the 28-kDa component and small amounts of a 21-kDa GTP-binding protein. Immunodetection analysis showed that the D. salina 28-kDa protein cross-reacted strongly with a polyclonal antibody raised against a Volvox carteri yptV1 type GTP-binding protein. This antibody was utilized for quantitative GTP-binding protein measurements as described below. Certain anti-GTP-binding protein antibodies derived from non-plant sources, namely, monoclonal antibodies raised against yeast and mouse ypt1 GTP-binding proteins, cross-reacted not only with the D. salina 28-kDa protein but also the 29-kDa component. The 30-kDa GTP-binding protein of D. salina did not bind the antibodies mentioned above but did cross-react with an anti-yeast ypt1 polyclonal antibody. None of the D. salina GTP-binding proteins reacted positively with polyclonal antibodies raised against SEC4, rab1 or rab6 proteins. When D. salina cells were subjected to hypoosmotic swelling by abruptly reducing the NaCl concentration of their medium from 1.7 M to 0.85 M, the increase in cell surface area was accompanied by a substantial translocation of the 28-kDa GTP-binding protein from the ER and GA fractions to the plasma membrane, chloroplast and cytosolic fractions, as determined by quantitative [32P]GTP binding and [125I]antibody binding on nitrocellulose blots. This translocation increased the content of the 28-kDa component in the plasma membrane, chloroplast and cytosol by 3-4-fold. No net movement of the 30-kDa GTP-binding protein from either the ER or GA fractions was observed following hypoosmotic shock.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Size↗

On the origins and early evolution of multicellularity.

In this paper an attempt is made to consider the significance of recent data on the organization and development of Volvox, a multicellular spheroidal green alga, for the unsolved problem of metazoan origins. A brief analysis is made of differences and similarities in some trends and principles during the establishment of metazoan and volvocalean multicellularity.

Biological Evolution↗

Structure of the three beta-tubulin-encoding genes of the unicellular alga, Polytomella agilis.

The quadriflagellate, unicellular, colorless alga, Polytomella agilis, contains several distinct microtubule arrays. To study the genetic basis of microtubule heterogeneity in P. agilis, we characterized its tubulin(Tub)-encoding genes (tub). The three beta tub genes detected in blots of P. agilis DNA were isolated from a genomic library. The structure and organization of the genes were examined by restriction mapping and nucleotide (nt) sequencing. S1 nuclease protection studies showed that all three genes are expressed. The predicted amino acid (aa) sequences are more than 98% conserved with the Chlamydomonas reinhardtii and Volvox carteri beta-Tubs, underscoring the close phylogenetic relationship of these species. Evolutionary divergence among the P. agilis genes is demonstrated by differences in intron number, nt sequences in noncoding regions, and silent nt substitutions in the coding regions. However, the proteins encoded by the beta 1 and beta 3 tub genes are identical; the beta 2 gene product differs by one conservative aa substitution. These results are in striking contrast to the C-terminal aa diversity reported within beta tub gene families in animal, higher plant and fungal systems. The data support the hypothesis that those tub genes whose products assemble into axonemal microtubules are subject

Amino Acid Sequence↗

Analysis of a family of ypt genes and their products from Chlamydomonas reinhardtii.

Small G-proteins encoded by the ras-like ypt genes are ubiquitous in eukaryotic cells. They have been shown to play an essential role in membrane vesicle transport. We have isolated four ypt genes, yptC1, yptC4, yptC5 and yptC6, from Chlamydomonas reinhardtii (Cr) genomic and cDNA libraries. Three of them, yptC1, yptC4 and yptC5, are close homologues of ypt genes previously found in the multicellular alga Volvox carteri (Vc), the fourth, yptC6, is new. Each yptC gene is present as a single copy in the genome. Comparisons of genomic and cDNA sequences revealed that the coding regions are interrupted by five (yptC5), six (yptC6), seven (yptC4) and eight (yptC1) introns, respectively. Cr ypt genes and the closely related Vc ypt genes have identical exon-intron structures, but the corresponding intron sequences are completely different. Polyadenylation is signalled by UAUAA, UGUAG and UGUAA. The deduced amino acid (aa) sequence of YptC6 exhibited 79% identity with HRab2; YptC1, YptC4 and YptC5 exhibited over 90% identity with their Vc homologues. Primary structures of the 9-aa 'effector domain' and the contiguous 'helix3-loop7' motif (approx. 30 aa) are 'diagnostic' features for functional assignment. Recombinant YptC proteins, overproduced in Escherichia coli and purified to near homogeneity, displayed strong and specific binding of GTP, but not of GMP or ATP. The four Cr Ypt proteins showed immunochemical cross reactions to their Vc counterparts. Moreover, Western blots demonstrated at least six types of Ypt in both Cr and Vc, suggesting that these Ypt are used for household functions responsible for vesicle transport rather than for cellular differentiation.

Amino Acid Sequence↗

Cloning and characterization of the actin-encoding gene of Chlamydomonas reinhardtii.

The genomic and complementary DNA sequences were determined for the unique actin-encoding gene in Chlamydomonas reinhardtii (Cr). The deduced amino acid (aa) sequence of this actin was similar to most known actin sequences, with the highest identity (98.1%) being with that of Volvox carteri actin. The Cr actin-encoding gene has one intron in the 5'-untranslated region and eight introns in the coding region. The latter eight introns occur at the same positions as those in the V. carteri actin-encoding gene. The 5'-upstream region contains four short stretches of sequence similar to the so-called 'tub box', a characteristic sequence proposed to be responsible for the regulation of synthesis of various axonemal proteins upon deflagellation and during the cell cycle. Southern blot analysis indicated that the Cr genome has only a single actin-encoding gene. An antibody specific for the 11-aa peptide corresponding to the N-terminal sequence of this actin was found to react with a 43-kDa protein associated with flagellar inner-arm dynein. These findings indicate that a single actin functions in both the cytoplasm and flagella of this organism.

Actins↗