Identification of highly conserved genes: SNZ and SNO in the marine sponge Suberites domuncula: their gene structure and promoter activity in mammalian cells(1).

Recently, we reported that cells from the sponge Suberites domuncula respond to ethylene with an increase in intracellular Ca(2+) level [Ca(2+)](i), and with an upregulation of the expression of (at least) two genes, a Ca(2+)/calmodulin-dependent protein kinase and the potential ethylene-responsive gene, termed SDSNZERR (A. Krasko, H.C. Schröder, S. Perovic, R. Steffen, M. Kruse, W. Reichert, I.M. Müller, W.E.G. Müller, J. Biol. Chem. 274 (1999)). Here, we describe for the first time that also mammalian (3T3) cells respond to ethylene, generated by ethephon, with an immediate and transient, strong increase in [Ca(2+)](i). Next, the promoter for the sponge SDSNZERR gene was isolated from S. domuncula. It was found that the SDSNZERR gene is positioned adjacent to the SNZ-related gene (SNZ-proximal open reading frame) (SDSNO) and linked, as in Saccharomyces cerevisiae, in a head-to-head manner. Until now, neither homologues nor orthologues of these two genes have been identified in higher metazoan phyla. The full-length genes share a bidirectional promoter. 3T3 cells were transfected with this promoter; the activity of the SDSNZERR promoter was strong and twice as high as that of the SV40 promoter, while the SDSNO promoter was less active. Surprisingly, the activity of the SDSNZERR promoter could not be modulated by ethylene or salicylic acid while it is strongly upregulated, by 4-fold, under serum-starved conditions. It is concluded that the modulation of the level of [Ca(2+)](i) by ethylene in mammalian cells is not correlated with an upregulation of the ethylene-responsive gene SDSNZERR. The data indicate that in mammalian cells, the activity of the SDSNZERR promoter is associated with the repression of serum-mediated growth arrest.

Modulation of intracellular calcium and proliferative activity of invertebrate and vertebrate cells by ethylene.

BACKGROUND: Ethylene is a widely distributed alkene product which is formed enzymatically (e.g., in plants) or by photochemical reactions (e.g., in the upper oceanic layers from dissolved organic carbon). This gaseous compound was recently found to induce in cells from the marine sponge Suberites domuncula, an increase in intracellular Ca2+ level ([Ca2+]i) and an upregulation of the expression of two genes, the potential ethylene-responsive gene, SDERR, and a Ca2+/calmodulin-dependent protein kinase. RESULTS: Here we describe for the first time, that besides sponge cells, mammalian cell lines (mouse NIH-3T3 and human HeLa and SaOS-2 cells) respond to ethylene, generated by ethephon, with an immediate and strong, transient increase in [Ca2+]i level, as demonstrated using Fura-2 imaging method. A rise of [Ca2+]i level was also found following exposure to ethylene gas of cells kept under pressure (SaOS-2 cells). The upregulation of [Ca2+]i was associated with an increase in the level of the cell cycle-associated Ki-67 antigen. In addition, we show that the effect of ethephon addition to S. domuncula cells depends on the presence of calcium in the extracellular milieu. CONCLUSION: The results presented in this paper indicate that ethylene, previously known to act as a mediator (hormone) in plants only, deserves also attention as a potential signaling molecule in higher vertebrates. Further studies are necessary to clarify the specificity and physiological significance of the effects induced by ethylene in mammalian cells.

Cloning and expression of new receptors belonging to the immunoglobulin superfamily from the marine sponge Geodia cydonium.

A cDNA encoding a receptor tyrosine kinase (RTK) was previously cloned and expressed from the marine sponge (Porifera) Geodia cydonium. In addition to the two intracellular regions characteristic for RTKs, two immunoglobulin (Ig)-like domains are found in the extracellular part of the sponge RTK. In the present study it is shown that no further Ig-like domain is present in the upstream region of the cDNA as well as of the gene hitherto known from the sponge RTK. Two different full-length cDNAs have been isolated and characterized in the present study, which possess two Ig-like domains, one transmembrane segment, and only a short intracellular part, without a TK domain. The two deduced polypeptides were preliminarily termed sponge adhesion molecules (SAM). The longer form of the SAM, GCSAML, encodes a deduced aa sequence, GCSAML, which comprises in the open reading frame 505 amino acids (aa) and has a calculated Mr of 53911. The short form, GCSAMS, has 313 aa residues and an Mr of 33987. The two Ig-like domains in GCSAML and GCSAMS are highly similar to the corresponding Ig-like domains in the RTKs from G. cydonium; the substitutions on both the aa and nt level are restricted to a few sites. Phylogenetic analyses revealed that the Ig-like domain 1 is similar to the human Ig lambda chain variable region, while the Ig-like domain 2 is related more closely to the human Ig heavy chain variable region. Transplantation experiments (autografting) were performed to demonstrate that the level of expression of the two new genes, GCSAML and GCSAMS, is upregulated during the self/self fusion process. Immunohistochemical analyses using antibodies raised against the two Ig-like domains demonstrate a strong expression in the fusion zone between graft and host. This finding has been supported by northern blotting experiments that revealed that especially GCSAML is strongly upregulated after autografting (up to 12-fold); the expression of GCSAMS reaches a value of 5-fold if compared with the controls. The results presented here demonstrate that the expression of the new molecules described, comprising two Ig-like domains, is upregulated during the process of autograft fusion.

Promoter and exon-intron structure of the protein kinase C gene from the marine sponge Geodia cydonium: evolutionary considerations and promoter activity.

We report the gene structure of a key signaling molecule from a marine sponge, Geodia cydonium. The selected gene, which codes for a classical protein kinase C (cPKC), comprises 13 exons and 12 introns; the introns are, in contrast to those found in cPKC from higher Metazoa, small in size ranging from 93 nt to 359 nt. The complete gene has a length of 4229 nt and contains exons which encode the characteristic putative regulatory and catalytic domains of metazoan cPKCs. While in the regulatory domain only one intron is in phase 0, in the catalytic domain most introns are phase 0 introns, suggesting that the latter only rarely undergo module duplication. The 5'-flanking sequence of the sponge cPKC gene contains a TATA-box like motif which is located 35-26 nt upstream from the start of the longest sequenced cDNA. This 5'-flanking sequence was analyzed for promoter activity. The longest fragment (538 nt) was able to drive the expression of luciferase in transient transfections of NIH 3T3 fibroblasts; the strong activity of the sponge promoter was found to be half the one displayed by the SV40 reference promoter. Deletion analysis demonstrates that the AP4 site and the GC box which is most adjacent to the TATA box are the crucial elements for maximal promoter activity. The activity of the promoter is not changed in 3T3 cells which are kept serum starved or in the presence of a phorbol ester. In conclusion, these data present the phylogenetically oldest cPKC gene which contains in the 5'-flanking region a promoter functional in the heterologous mammalian cell system.

Origin of the metazoan bodyplan: characterization and functional testing of the promoter of the homeobox gene EmH-3 from the freshwater sponge Ephydatia muelleri in mouse 3T3 cells.

Porifera [sponges] represent the lowest metazoan phylum, probably already existing prior to the 'Cambrian explosion'. Based on amino acid sequences deduced from cDNAs that code for structural proteins, the monophyly of Metazoa was established. Now we analyzed for the first time a promoter of a sponge gene for its activity in a heterologous cell system from higher Metazoa. The promoter of the homeobox gene EmH-3 was cloned and sequenced from a genomic library of the freshwater sponge Ephydatia muelleri. For the determination of functional promoter activity, transient transfection experiments in mouse NIH 3T3 cells were performed; the promoter was fused with the luciferase reporter gene. The data revealed that a 401 nt long promoter fragment, comprising several binding elements for metazoan transcription factors, showed the highest activity, while the 175 bp long promoter segment, comprising solely the TATA- and Cap boxes, showed only 25% of that activity. This result demonstrates that the sponge promoter is activated by factors present in mammalian cells and supports the view that Porifera, together with the other metazoan phyla, are of monophyletic origin.

Evolutionary analysis of G-proteins in early metazoans: cloning of alpha- and beta-subunits from the sponge Geodia cydonium.

G-protein-coupled (seven-transmembrane segment)-receptors represent a major group of metazoan receptors, involved in transduction of extracellular signals. The G-proteins, which are made up of Galpha/beta/gamma-subunits, link the receptors to the effector system(s). To analyze the phylogenetic relationships among the metazoan alpha-subunits of G-proteins, cDNAs of alpha-subunits were isolated from Geodia cydonium, a marine sponge belonging to the lowest metazoan phylum, Porifera. One encodes a putative isotype of a stimulator of the adenylyl cyclase (Galpha s), another one a putative inhibitor of the adenylyl cyclase (Galpha i/o) and the third one a putative activator of phospholipase C (Galpha q). In addition one putative beta-subunit was cloned from the same species. The deduced amino acid sequences of the sponge Galpha s -(putative Mr 44749), the Galpha i/o -(Mr 41064) and the Galpha q subunits (Mr 41363) were found to display high similarity with the corresponding sequences from higher Metazoa, and are only distantly related to those of slime mold, yeast or plants. Of lower similarity are the sequences of the beta-subunits among animals and plants, thus not allowing robust grouping. These data demonstrate that the phylogenetic relationships, obtained from analyses of the alpha subunits from metazoan G-proteins, support the conclusion that all metazoan phyla, including the Porifera are of monophyletic origin.

Molecular cloning and primary structure of a Rhesus (Rh)-like protein from the marine sponge Geodia cydonium.

In humans, the 30,000 M(r) Rhesus (Rh) polypeptide D (RhD) is a dominant antigen (Ag) of the Rh blood group system. To date, an Rh-like protein has been found in chimpanzees, gorillas, gibbons, and rhesus monkeys. Related to the 30,000 M(r) Rh Ag protein are two polypeptides of 50,000 M(r), the human 50,000 M(r) Rh Ag and the RhD-like protein from Caenorhabditis elegans. The function of all these proteins is not sufficiently known. Here we characterize a cDNA clone (GCRH) encoding a putative 57,000 M(r) polypeptide from the marine sponge Geodia cydonium, which shares sequence similarity both to the RhD Ag and the Rh50 glycoprotein. The sponge Rh-like protein comprises 523 aa residues; hydropathy analysis hints at the presence of ten transmembrane domains. An N-terminal hydrophobic cleavage signal sequence is missing, suggesting that the first membrane-spanning domain of the sponge Rh-like protein acts as a signal-anchor sequence. The sponge Rh-like protein, like the human Rh50, lacks the CLP motif which is characteristic of the 30,000 M(r) RhD. In addition, the hydropathy profile of the sponge Rh-like protein is of a similar size and shape as that of human Rh50. This data indicates that the RhD and its structurally related Rh50 glycoprotein, which are highly immunogenic in humans, share a common ancestral molecule with the G. cydonium Rh-like protein.

Cathepsin, a major protease of the marine sponge Geodia cydonium: purification of the enzyme and molecular cloning of cDNA.

Sponges are suspension-feeders that are devoid of body cavities. Phagocytosis is the major route of nutrition in these animals. In an attempt to understand protein digestion, cathepsin was identified in crude extracts from the sponge Geodia cydonium. This enzyme was purified from lysosomes by a two-step procedure--pH precipitation and FPLC separation--to apparent homogeneity; it showed an M(r) of 26,000. Inhibitor as well as substrate studies showed that the sponge cathepsin belongs to the subfamily L of these cysteine proteases. The complete cDNA coding for cathepsin L was isolated and characterized. The deduced aa sequence contains 322 residues, has an M(r) of 36,085, and shows the characteristic signatures known from other cathepsins of the L subfamily: e.g., cleavage site for the proregion, the ERFNIN motif, and the conserved regions forming the catalytic triad of cysteine proteases. Phylogenetic analyses revealed that the sponge sequence groups with the cathepsin L subfamily and branches off first from the other metazoan members. The sponge sequence shows high homology to that isolated from Dictyostelium discoideum and only low similarity to the protozoan cathepsins L from Paramecium tetraurelia and Tetrahymena thermophila. From the data presented it is concluded that cathepsin L is the major digestive protease in sponges.