Contribution of sponge genes to unravel the genome of the hypothetical ancestor of Metazoa (Urmetazoa)

Recently the term Urmetazoa, as the hypothetical metazoan ancestor, was introduced to highlight the finding that all metazoan phyla including the Porifera (sponges) are derived from one common ancestor. Sponges as the evolutionarily oldest, still extant phylum, are provided with a complex network of structural and functional molecules. Analyses of sponge genomes from Demospongiae (Suberites domuncula and Geodia cydonium), Calcarea (Sycon raphanus) and Hexactinellida (Aphrocallistes vastus) have contributed also to the reconstruction of the evolutionary position of Metazoa with respect to Fungi. Furthermore, these analyses have provided evidence that the characteristic evolutionary novelties of Metazoa, such as the extracellular matrix molecules, the cell surface receptors, the nervous signal transduction molecules as well as the immune molecule existing in Porifera, share high sequence and in some aspects also functional similarities to related polypeptides found in other metazoan phyla. During the transition to Metazoa new domains occurred; as one example, the formation of the death domain from the ankyrin is outlined. In parallel, domanial proteins have been formed, such as the receptor tyrosine kinases. The metazoan essentials have been defined by analyzing and comparing the sponge sequences with the related sequences from the metazoans Homo sapiens, Caenorhabditis elegans and Drosophila melanogaster, the fungus Saccharomyces cerevisiae and the plant Arabidopsis thaliana. The data revealed that those sponge molecules grouped to cell adhesion cell recognition proteins are predominantly found in Protostomia and Deuterostomia while they are missing in Fungi and Viridiplantae. Moreover, evidence is presented allowing the conclusion that the sponge molecules are more closely related to the corresponding molecules from H. sapiens than to those of C. elegans or D. melanogaster. Especially surprising was the finding that the Demospongiae are provided with elements of adaptive immunity.     

Glycosylation sites identified by solid-phase Edman degradation: O-linked glycosylation motifs on human glycophorin A

The human red blood cell sialoglycoprotein, glycophorin A (GpA),contains a ‘mucin-like’ extensively O-glycosylatedextracellular domain which carries the MN blood group antigens.We have revised the sites of O-glyccsylation in the extracellulardomain of GpA by automated solid-phase Edman degradation, whichallowed positive identification and quantitation of O-glycosylatedSer and Thr residues, as well as the single N-glycosylationsite. One N-linked and 16 O-linked sites were identified. Carbohydratewas absent on Ser 1, Ser14, Ser15, Ser23, Thr28 and Thr58 inGpA. We propose that the glycosyltransferases present in erythrocytesrecognize specific flanking sequences around potential O-glycosylationsites. All 16 O-glycosylation sites are explained on the basisof four motifs. Three motifs are associated with Thr-glycosylation:Xaa—Pro—Xaa—Xaa where at least one Xaa = Thr;Thr—Xaa—Xaa—Xaa where at least one Xaa = Thr;Xaa—Xaa—Thr—Xaa where at least one X = Argor Lys. The fourth motif is associated with Ser-glycosylation:Ser—Xaa—Xaa—Xaa where at least one Xaa = Ser.These simple rules explain the glycosylation (or lack of it)on 21 of 22 Ser/Thr in the extracellular domain of GpA. glycophorin A O-glycosylation motif solid-phase Edman degradation     

The response of tomato roots (Lycopersicon esculentum Mill.) to iron deficiency stress: alterations in the pattern of protein synthesis

Dicotyledon plants adapt to iron (Fe) deficiency through a seriesof reactions that increase the ability of the plant to assimilateFe and to increase the efficiency of Fe utilization. In an attemptto gain an insight into these adaptive processes, the specificchanges in protein synthesis associated with the onset of theFe deficiency response in tomato roots (Lycopersicon esculentumMill cv. Rutgers) have been investigated. Roots were grown underFe—sufficient and —deficient conditions, and thepattern of protein synthesis was analysed by in vitro translationof root mRNA and by in vivo labelling of root proteins. Polypeptideswere resolved by two—dimensional polyacrylamide gel elec—trophoresis.Seven polypeptides were identified by in vitro translation,whose synthesis was significantly increased during Fe deficiency.The increase was probably specific to Fe deficiency in thatthe polypep—tide synthesis was not increased during phosphatedeficiency stress, was less prominent following prolonged Fedeficiency and was decreased following re—supply of Feto the hydroponic medium. The pattern of in vitro translation of mRNA isolated from Fe—deficientroots was compared to the results obtainedin vivo followingradiolabelling of proteins. In these analyses, eight polypeptideswere identified, tentatively including the seven polypeptidespreviously identified by in vitro translation. All polypeptideswere characterized with regard to molecular mass and pl andtheir localization in the cell, whether being membrane boundor soluble. It is suggested that members of this group of polypeptidesare involved in the response of the root to Fe deficiency: althoughtheir functions remain to be identified. Key words: In vitro protein synthesis, iron, iron deficiency, root, 2-dimensional PAGE     

Review: How was metazoan threshold crossed? The hypothetical Urmetazoa.

The origin of Metazoa remained--until recently--the most enigmatic of all phylogenetic problems. Sponges [Porifera] as "living fossils", positioned at the base of multicellular animals, have been used to answer basic questions in metazoan evolution by molecular biological techniques. During the last few years, cDNAs/genes coding for informative proteins have been isolated and characterized from sponges, especially from the marine demosponges Suberites domuncula and Geodia cydonium. The analyses of their deduced amino acid sequences allowed a molecular biological resolution of the monophyly of Metazoa. Molecules of the extracellular matrix/basal lamina, with the integrin receptor, fibronectin and galectin as prominent examples, cell-surface receptors (tyrosine kinase receptors), elements of nerve system/sensory cells (metabotropic glutamate receptor), homologs/modules of an immune system [immunoglobulin-like molecules, SRCR- and SCR-repeats, cytokines, (2-5)A synthetase], as well as morphogens (myotrophin) classify the Porifera as true Metazoa. As "living fossils", provided with simple, primordial molecules allowing cell-cell and cell-matrix adhesion, as well as processes of signal transduction as known in a more complex manner from higher Metazoa, sponges also show peculiarities. Tissues of sponges are rich in telomerase activity, suggesting a high plasticity in the determination of cell lineages. It is concluded that molecular biological studies with sponges as models will not only help to understand the evolution to the Metazoa but also the complex, hierarchical regulatory network of cells in higher Metazoa [reviewed in Progress in Molecular Subcellular Biology, vols. 19, 21 (1998) Springer Verlag]. The hypothetical ancestral animal, the Urmetazoa, from which the metazoan lineages diverged (more than 600 MYA), may have had the following characteristics: cell adhesion molecules with intracellular signal transduction pathways, morphogens/growth factors forming gradients, a functional immune system, and a primordial nerve cell/receptor system.     

Beware of mis-assembled genomes

With hundreds of genomes now in GenBank, researchers might beforgiven for assuming that genome sequence data are correct,at least at a large scale. Certainly there might be errors atsome small rate, perhaps 1 in 50 000 or 100 000 bases (Schmutz et al., 2004;Read et al., 2002), but at a large scale thesegenomes are put together correctly, are not they? Well, notalways. We have been looking at the assemblies of large genomes forseveral years now, and for every ‘draft’ genomewe look at, we find hundreds—and sometimes thousands—ofmis-assemblies. These include regions where a genome is incorrectlyre-arranged as well as places where large chunks of DNA sequenceare simply     

A COMPARATIVE STUDY OF THE MORPHOLOGY OF THE PULMONATE SNAIL PSEUDOTACHEA LITTURATA (PFEIFFER) AND OTHER SPECIES OF PSEUDOTACHEA, IBERUS AND CEPAEA

A population of Pseudotachea litturata (Pfeiffer, 1851) fromTarifa (Càdiz, Spain) has been studied. The morphologicalresults are compared with those from P. splendida, Iberus gualtierianus,I. alonensis, I. marmoralus, I. guiraoanus and four speciesof the genus Cepaea using, as an exploratory method, the Wagnerparsimony procedure and 18 characters of the shell, genitalsystem and karyotype have been analysed. According to this methodit seems that the taxonomical position of P. litturata in thegenus Pseudotachea is confirmed, and agrees with the phylogeneticalrelationships in this group of species. The genus Cepaea seemsto be well established, although two species groups can be distinguished:C. nemoralis—C. hortensis and C. syluatica—C. vindobonensis.These differ mainly in chromosome number, diverticulum lengthand degree of shell polymorphism. Although the present resultsdo not allow us to clarify the current taxonomical problemswithin the genus Iberus, the species studied seem to belongto a natural group (Received 15 September 1987; accepted 1 January 1988)     

Evolution of sensory structures in basal metazoa

Cnidaria have traditionally been viewed as the most basal animalswith complex, organ-like multicellular structures dedicatedto sensory perception. However, sponges also have a surprisingrange of the genes required for sensory and neural functionsin Bilateria. Here, we: (1) discuss "sense organ" regulatorygenes, including; sine oculis, Brain 3, and eyes absent, thatare expressed in cnidarian sense organs; (2) assess the sensoryfeatures of the planula, polyp, and medusa life-history stagesof Cnidaria; and (3) discuss physiological and molecular datathat suggest sensory and "neural" processes in sponges. We thendevelop arguments explaining the shared aspects of developmentalregulation across sense organs and between sense organs andother structures. We focus on explanations involving divergentevolution from a common ancestral condition. In Bilateria, distinctsense-organ types share components of developmental-gene regulation.These regulators are also present in basal metazoans, suggestingevolution of multiple bilaterian organs from fewer antecedentsensory structures in a metazoan ancestor. More broadly, wehypothesize that developmental genetic similarities betweensense organs and appendages may reflect descent from closelyassociated structures, or a composite organ, in the common ancestorof Cnidaria and Bilateria, and we argue that such similaritiesbetween bilaterian sense organs and kidneys may derive froma multifunctional aggregations of choanocyte-like cells in ametazoan ancestor. We hope these speculative arguments presentedhere will stimulate further discussion of these and relatedquestions.     

Mineral nutrition, of cultured chlorophyllous cells of tobacco VII. Effects of the NH4/NO3 ratio and of Cl in the media on the growth and ionic balance of cells

NG, a strain of cultured tobacco cells of Nicotiana glutinosahad high growth rates and carboxylate contents (C—A) of100 to 130 meq/100 g of dry cells on media containing 42 meqNO₃/liter as the sole N source. (C—A) is the amount ofinorganic cations minus inorganic anions in meq per 100 g ofdry cells. NG, cultured on media containing NH₄ 10+NO₃ 42 in meq/liter,had lower growth rates and lower (C—A) values as comparedwith NG on media containing NO₃ as the sole N source. NG, cultured on media containing NH₄ 30+NO₃ 42 in meq/liter,had high growth rates and (A—C) values of 22 to 53 meq/100gof dry cells. In this case, the (A—C) content may correspondto organic cations, basic organic N compounds such as free asprotein-bound basic amino acids. The easily absorbed Cl mayhave been required maintain good growth conditions such as ionicbalance and a favorable pH in the cells. Thus cultured cells of Nicotiana glutinosa may have physiologicaladaptability against variations in a relatively wide range of|C—A| contents [|C—A| being the absolute valuesof (C—A)]. (Received May 15, 1980; )     

Physical Analysis of Tissue Mechanics in Amphibian Gastrulation

During morphogenesis, intercellular attachments constrain cellmobility so that embryonic tissues may (i) deform as solid sheetsof tightly bound cells, (ii) disperse to migrate as separatecells or (iii) flow as multicellular liquids (in which cellsremain aggregated yet can still slide past one another). Bymodelling deep germ layers as multicellular liquids in amphibiangastrulation, Davis and I have predicted, and then confirmedexperimentally, (i) the area-invariance of deep-germ-layer surfacetensions in vitro, (ii) spontaneous cell slippage in deformeddeep—germ—layer cell aggregates and (iii) correlationsof tissue surface tensions with tissue positioning in deep-germ-layercell-sorting and aggregate—fusion experiments. Liquid—tissueflow involves intercalations of interior cells into expandingtissue interfaces (or withdrawal of surface cells from shrinkingtissue interfaces). Tissue surface tensions are macroscopicreflections of the microscopic, tissue—specific adhesivedifferentials which direct these cell translocations. Such adhesivedifferentials may act independently of, or together with, activecell—shape changes, chemotaxis, contact guidance and/orhaptotaxis in controlling embryonic tissue rearrangements. Deepcell intercalations in vivo occur throughout amphibian gastrulation:during ectodermal epiboly; during marginal—zone extensionand convergence (and therefore blastopore closure); during mesodermalinvolution; and probably during the anterior spreading and axialextension (and therefore dorsal convergence) of involuted mesoderm.Tissue—surface—tension measurements may help determine(i) which of these cell—intercalation processes are activeand which are passive, (ii) the specific contributions of variousmicroscopic cell properties to the regulation of liquid—likegerm—layer assembly and (iii) similarities and differencesbetween in vitro and in vivo control mechanisms governing amphibiangastrulation.     

Resting membrane potential and action potential of Nitella expansa protoplasts

Inside negative membrane potentials were observed for protoplastsobtained from Nitella expansa leaf internodal cells in mediacontaining 1 to 100 mM CaCl₂ using the microelectrode technique.The potential values were less negative than the membrane potentialof intact N. expansa leaf internodal cells. In addition, anaction potential consisting of two components—a fast componentand a slow component—was induced by electrical stimulationfor the protoplasts as well as the intact cells. (Received December 18, 1979; )     

Starting Points for a Developmental Genetics of Nerve Patterns

SYNOPSIS. The genie control of the development of neural morphologyand specific neural connections is little known, primarily becauseof a paucity of neurological mutants in animal models suitablefor experimental manipulation. However, by using cell markers—suchas tetraploidy, a large cell marker whose development is describedhere—the development of single retinal cells in tetraploidembryos, the growth of marked polyclones in chimeric eyes, andthe central connections of various chimeric eyes have been analyzedto identify the strategies used in molding eye morphology andestablishing precise central connections. Theoretical modelsand computer simulations were used to draw inferences aboutthe information used to accomplish the identified strategies.This analysis resulted in predictions about the sorts of mutantgenes which might be found to affect neural ontogeny.     

Ethylene-bis-Nitrourethane: Investigation of the Plant Growth Stimulant Action of Analogous Compounds

The results of a systematic attempt to relate the growth-stimulatingfunction of compounds analogous to ethylene-bis-nitrourethaneto their chemical structure and properties showed that, withthe exception of a series of nitraminocarboxylic acids, NO₂NH(CH₂)COOH,activity was confined to compounds of the form R—N(NO₂)—X—N(NO₂)—R,where X is an alkylene or substituted alkylene chain. It issuggested that active compounds are converted to dinitramines(R = H) in the plant, and that the acidic nature of the —NHNO₂groups is of importance. The oil/water partition coefficientsand the geometrical structure of the molecules are also shownto influence their growth-stimulating activity.     

Development and Histochemistry of Style and Stigma, and Pollen-Pistil Interaction in Madhuca indica (Sapotaceae)

The histological and histochemical changes during the developmentof the stigma and style of Madhuca indica of the family Sapotaceaehave been investigated. The stigma is of the wet type and thestyle is open. The stigma secretion, present from stage I onwardsis lipophilic. But the protein, polysaccharides and variousenzymes are secreted only by the third, receptive stage. Thestigma has a stellate cavity opening into the stylar canals.The exudate along with various constituents is present up tostage VI of flower maturity. The pollen—pistil interactionstudies show a strong self-incompatibility response. The pollentubes show coiling and clubbing in the stigmatic cavity andstylar canals, even in the case of compatible cross pollination.Various methods are successfully employed to overcome self-incompatibility. Madhuca indica, Mahuda, development, histochemistry, pollen—pistil interaction, self-incompatibility, style and stigma     

Freezing avoidance mechanism of primordial shoots of conifer buds

Excised winter buds of very hardy fir supercooled to —30or — 35?C, though primordial shoots excised from thesewinter buds (freezing point: about —5.5?C) supercooledonly to —12 to — 14?C. Also, excised primordialshoots did not tolerate freezing, but were rather resistantto desiccation. Differential thermal analysis (DTA) of primordialshoots revealed that the capability of supercooling increasedwith decreasing water content and that no exotherm could bedetected in the primordial shoots with a water content belowabout 20%. When excised whole buds were cooled very slowly,the exotherm temperature shifted markedly to a lower value andthe exotherm became much smaller. Also, masses of needle icewere observed, mainly beneath the crown of the primordial shoot.From these results, it may be concluded that most of the waterin primordial shoots gradually migrates out through the crownand freezes as the temperature decreases (extraorgan freezing),which enables primordial shoots to survive at very low temperatures.Winter buds of Abies balsamea held at — 20?C for 30 daysand then slowly cooled down to —50 or —60?C remainedalive. Thus, there seems to be no low temperature limit to thisfrost avoidance mechanism, if the primordial shoots can resistintensive freeze-dehydration. Low temperature exotherms wereobserved in all genera which belong to Abietoideae and Laricoideaeof Pinaceae, all of which have a crown in the primordial shoots,but not in other conifers. ¹ Contribution No. 2037 from the Institute of Low TemperatureScience. (Received June 25, 1979; )     

Some Complicating Factors in the Study of the Calcium Metabolism of Teleosts

A number of factors which may complicate studies dealing withthe calcium metabolism of teleosts are pointed out. Thus, lowenvironmental levels of calcium markedly affect both gill RNAand sodium metabolism in dilute sea water, and the absence ofmonovalent cations affects calcium metabolism in dilute environments.Hypophysectomy is not always followed by the disappearance ofcortisol from the plasma, and prolactin—which is requiredfor the survival of either hypophysectomized or intact animalsin a divalent ion-free environment—stimulates cortisolproduction. It is also pointed out that Fundulus kansae can be maintainedin divalent ion-free environments for long periods, and thatcalcium is mobilized from internal reservoirs such as acellularbone and scales. Failure to survive in such environments isdue to the regulatory effect of environmental calcium on sodiummetabolism rather than on calcium metabolism per se. Finally,it is pointed out that while one parameter of calcium metabolism—suchas plasma calcium levels in male fish—may remain constantthroughtout the year, other parameters such as calcium uptakemay vary considerably. Seasonal differencees in responses tomammalian calcitonin have also been noted.     

Ecological Bases of Hormone--Behavior Interactions: The "Emergency Life History Stage"

SYNOPSIS. Superimposed upon seasonal changes in morphology,physiology and behavior, are facultative responses to unpredictableevents known as labile (i.e., short-lived) perturbation factors(LPFs). These responses include behavioral and physiologicalchanges that enhance survival and collectively make up the "emergency"life history stage. There is considerable evidence that glucocorticosteroids,and other hormones in the hypothalamo—pituitary—adrenal(HPA) cascade, initiate and orchestrate the emergency life historystage within minutes to hours. This stage has a number of sub—stagesthat promote survival and avoid potential deleterious effectsof stress that may result from chronically elevated levels ofcirculating glucocorticosteroids over days and weeks. Thesesub—stages may include: redirection of behavior from anormal life history stage to increased foraging, irruptivetypemigration during the day, enhanced restfulness at night, andelevated gluconeogenesis. Once the perturbation passes, glucocorticosteroidsmay also promote recovery. Additional evidence from birds indicatesthat glucocorticosteroid responses to a standardized capture,handling and restraint protocol are modulated both on seasonaland individual levels. Field work reveals that these changesin responsiveness to LPFs have ecological bases, such as reproductivestate, body condition etc., that in turn indicate differenthormonal control mechanisms in the HPA cascade.     

The molecular basis for the evolution of the metazoan bodyplan: extracellular matrix-mediated morphogenesis in marine demosponges

Molecular data on development/differentiation and on comparative genomics allow insights into the genetic basis of the evolution of a bodyplan. Sponges (phylum Porifera) are animals that are the (still extant) stem group with the hypothetical Urmetazoa as the earliest common ancestor of all metazoans; they possess the basic features of the characteristic metazoan bodyplan also valid for the animals of the crown taxa. Here we describe three homeobox genes from the demosponge Suberites domuncula whose deduced proteins (HOXa1_SUBDO, HOXb1_SUBDO, HOXc1_SUBDO) are to be grouped with the Antennapedia class of homeoproteins (subclasses TIx-Hox11 and NK-2). In addition, a cDNA encoding a LIM/homeobox protein has been isolated which comprises high sequence similarity to the related LIM homeodomain (HD) proteins in its LIM as well as in its HD domains. To elucidate the potential function of these proteins in the sponge a new in vitro system was developed. Primmorphs which are formed from dissociated cells were grown on a homologous galectin matrix. This galectin cDNA was cloned and the recombinant protein was used for the preparation of the matrix. The galectin/polylysine matrix induced in primmorphs the formation of channels, one major morphogenetic process in sponges. Under such conditions the expression of the gene encoding the LIM/homeobox protein is strongly upregulated, while the expression of the other homeobox genes remains unchanged or is even downregulated. Competition experiments with galactosylceramides isolated from S. domuncula were performed. They revealed that a beta-galactosylceramide, named Sdgal-1, prevented the expression of the LIM gene on the galectin matrix, while Sdgal-2, a diglycosylceramide having a terminal alpha-glycosidically linked galactose, caused no effect on the formation of channels in primmorphs or on LIM expression. This study demonstrates for the first time that an extracellular matrix molecule, galectin, induces a morphogenetic process in sponges which is very likely caused by a LIM/homeobox protein. Furthermore, a new model is introduced (galectin-caused channel formation in sponge primmorphs) to investigate basic pathways, thus allowing new insights into the functional molecular evolution of Metazoa.