0000000000082763
AUTHOR
Claudia Koziol
Cloning of Hsp70 genes from the marine sponges Sycon raphanus (Calcarea) and Rhabdocalyptus dawsoni (Hexactinellida). An approach to solve the phylogeny of sponges
The phylogenetic relationships among the three classes of the Porifera—Demospongiae, Calcarea and Hexactinellida—are still unresolved, despite the use of molecular analyses of rRNA. To determine whether phylogenetic resolution of these classes is possible based on genes coding for specific proteins, in the present study the genes for the 70 kDa heat shock protein [Hsp70] were isolated fromRhabdocalyptus dawsoni[Hexactinellida] and fromSycon raphanus[Calcarea], and compared to that previously isolated from the demospongeGeodia cydonium. The gene fromR. dawsoniis 2021 bp long and encodes a predicted Hsp70 of Mr77,697; the protein comprises the characteristic sites of eukaryotic, cytoplasmic H…
Phenylalanine hydroxylase from the sponge Geodia cydonium: implication for allorecognition and evolution of aromatic amino acid HYDROXYLASESfn1fn1Thesequence reported here is deposited in the EMBL\GenBank data base (Accession no. Y16353).
Abstract The prophenoloxidase activating system is a defense system, frequently reported both in protostomes and in deuterostomes. The final product of the phenoloxidase activity is melanin which is ubiquitously present throughout the metazoan kingdom. The melanin synthesis pathway starts with the amino acid [aa] phenylalanine which is converted to tyrosine by the phenylalanine hydroxylase [PAH]. We show that after allo-transplantation in the marine sponge Geodiacydonium PAH is upregulated in the grafts. Enzyme determination studies revealed that PAH activity increases by three-fold two d after transplantation and reaches its maximum after 3 d (by 3.7-fold). This finding was supported by de…
Diagnosis of sublethal stress in the marine sponge Geodia cydonium: application of the 70 kDa heat-shock protein and a novel biomarker, the Rab GDP dissociation inhibitor, as probes
Abstract Sponges (Porifera) are among the major phyla inhabiting the marine hard-substrate benthos, both in respect to the number of species and their biomass. Hence reliable biomarkers need to be developed to monitor the environmental load in those animals. Recently, the cDNA for the heat shock protein HSP70 has been isolated from the sponge Geodia cydonium and found to be a reliable indicator for temperature stress. In the present study, we have isolated the Rab GDP-dissociation inhibitor (GDI), which has previously been shown to be a key element in the intracellular traffic system. The 1521 bp long cDNA, encoding sponge GDI, has been isolated and analyzed. The deduced aa sequence ( M r =…
Sponges (Porifera) model systems to study the shift from immortal to senescent somatic cells: the telomerase activity in somatic cells.
Abstract Sponges (Porifera) represent the lowest metazoan phylum, characterized by a pronounced plasticity in the determination of cell lineages. In a first approach to elucidate the molecular mechanisms controlling the switch from the cell lineage with a putative indefinite growth capacity to senescent, somatic cells, the activity of the telomerase as an indicator for immortality has been determined. The studies were performed with the marine demosponges Suberites domuncula and Geodia cydonium . It was found that the activity for the telomerase in the tissue of both sponges is high; a quantitative analysis revealed that the extract from S. domuncula contained 10.3 TPG units per 5000 cell e…
Immediate early response of the marine sponge Suberites domuncula to heat stress: Reduction of trehalose and glutathione concentrations and glutathione S-transferase activity
The marine sponge Suberites domuncula was used to identify early markers for thermal stress. Cubes from sponges have been kept for 30 min at 31°C (10 °C higher than the ambient temperature). After this treatment the sponge cubes were kept again at 21°C. To demonstrate that the animals reacted to the elevated temperature, the expression of heat shock protein (HSP) was determined. Using an antibody raised against HSP70, it was found by Western blotting that the animals specifically express a 45 kDa polypeptide after heat treatment. It was shown that even after 10 min of heat treatment the steady-state concentration of trehalose drops by 40% from a base level of 13 nmol/mg protein. The activit…
Evolution of Early Metazoa: Phylogenetic Status of the Hexactinellida Within the Phylum of Porifera (Sponges)
The evolution of the Metazoa from their protozoan ancestors is one of the greatest puzzles of phylogeny (Willmer 1994; Cavalier-Smith et al. 1996). The emergence of multicellular animals has been explained by two major theories: the syncytial theory (Hadzi 1963) - origin from a multinucleated ciliate - and the colonial theory (Haeckel 1868) - origin from a colonial flagellate - both of which assume a di(poly)phyletic origin of the Metazoa. Numerous attempts to resolve whether the Metazoa are of mono- or polyphyletic origin have sought evidence from a wide variety of developmental and morphological data such as body symmetry, type of development (protostome vs. deuterostome), type of body ca…
Increased Expression of Integrin and Receptor Tyrosine Kinase Genes During Autograft Fusion in the SpongeGeodia cydonium
Recently cDNAs coding for cell surface molecules have been isolated from sponges. The molecules for alpha-integrin, galectin, and receptor tyrosine kinase (RTK), obtained from the marine sponge, Geodia cydonium, have been described earlier. In the present study also the cDNA for one putative beta-integrin has been identified from G. cydonium. The deduced aa sequence comprises the characteristic signatures, found in other metazoan beta-integrin molecules; the estimated size is 95,215 Da. To obtain first insights into the molecular events which proceed during autograft fusion, the expressions of these genes were determined on transcriptional and translational level. The cDNAs as well as antib…
Combinatory effects of temperature stress and nonionic organic pollutants on stress protein (hsp70) gene expression in the freshwater spongeEphydatia fluviatilis
This is the first documentation of a heat shock protein (hsp) response in sponges. Subjecting the freshwater sponge Ephydatia fluviatilis to temperature stress (18 to 33 C; 2 h) resulted in an increased expression (>10 times) of the M{sub r}70,000 (hsp70). The induction of hsp70 could be demonstrated on the level of gene expression and by quantification of the hsp70 protein. Temperature stress also resulted in a 25% reduction of sponge cell proliferation. A mixture of nonionic organic compound was extracted from water from the polluted Schwarzbach River (S. Hesse, Germany) by adsorption onto XAD-7 resin. Concentrations of this Schwarzbach River water extract at two and four items ambient le…
Primmorphs generated from dissociated cells of the sponge Suberites domuncula: a model system for studies of cell proliferation and cell death
Sponges (Porifera) represent the lowest metazoan phylum; they have been shown to be provided with the characteristic metazoan structural and functional molecules. One autapomorphic character of sponges is the presence of high levels of telomerase activity in all cells (or almost all cells, including somatic cells). In spite of this fact previous attempts to cultivate sponge cells remained unsuccessful. It was found that dissociated sponge cells do not replicate DNA and lose their telomerase activity. In addition, no nutrients or metabolites have been detected that would stimulate sponge cells to divide. In the present study we report the culture conditions required for the formation of mult…
17β-Estradiol-dependent regulation of chaperone expression and telomerase activity in the marine sponge Geodia cydonium
It is known that species belonging to the lowest metazoan phylum, the Porifera (sponges), do not develop tumors. Sponge cells share with tumor cells of higher animals at least one characteristic; they contain high levels of telomerase activity, suggesting that they possess a high proliferation capacity. This assumption, however, has not been substantiated experimentally. In addition, sponges show a specific bauplan, leading us to postulate that they undergo apoptosis to replace a given set of cells at a given time. In the present study, 17β-estradiol (βE2) was used as a defined agent to assess its effect on both the telomerase activity and the process of apoptosis in the marine sponge Geodi…
Induction of DNA strand breaks and expression of HSP70 and GRP78 homolog by cadmium in the marine sponge Suberites domuncula
The marine sponge Suberites domuncula was used as a bioindicator to study the effects of cadmium on the occurrence of DNA strand breakage and on the induction of the expression of the stress biomarkers, heat shock protein 70 (HSP70) and glucose-regulated protein 78 (GRP78) homolog. The cDNA encoding GRP78 homolog from S. domuncula was isolated and characterized. The GRP78 cDNA has a length of 2.1 kb and displays characteristic features of the HSP70 family ; it encodes an aa sequence of M-r 72, 000. Exposure of S. domuncula to 1 mg/L of cadmium chloride for 24 h caused a strong(16.6-fold) increase in cadmium content to 7.7 mu g/g wet weight of sponge tissue ; after an incubation period of 6 …
Towards an understanding of the molecular basis of immune responses in sponges: The marine demospongeGeodia cydonium as a model
The phylogenetic position of the phylum Porifera (sponges) is near the base of the kingdom Metazoa. During the last few years, not only rRNA sequences but, more importantly, cDNA/genes that code for proteins have been isolated and characterized from sponges, in particular from the marine demosponge Geodia cydonium. The analysis of the deduced amino acid sequences of these proteins allowed a molecular biological approach to the question of the monophyly of the Metazoa. Molecules of the extracellular matrix/basal lamina, with the integrin receptor, fibronectin, and galectin as prominent examples, and of cell-surface receptors (tyrosine kinase receptor), elements of sensory systems (crystallin…
A homolog of the putative tumor suppressor QM in the sponge Suberites domuncula: downregulation during the transition from immortal to mortal (apoptotic) cells
Abstract The activation of components of the transcription factors such as AP-1 or c-jun is essential for a physiological response of metazoan cells during aging. The activity of such proto-oncoproteins is under enzymatic control. The function of c-jun is additionally modulated by the QM protein. Here, we studied the expression of the gene, encoding the QM-like protein in the sponge Suberites domuncula . These animals contain high levels of telomerase in their somatic cells. To understand the switch from telomerase-positive immortal cells to telomerase-negative mortal cells which undergo apoptosis, the expression of the QM-like gene was measured in this system. The cDNA, termed QMSD , encod…
Prolidase in the Marine Sponge Suberites domuncula: Enzyme Activity, Molecular Cloning, and Phylogenetic Relationship
: The enzyme prolidase hydrolyzes the peptide bond that involves the imino nitrogen of proline or hydroxyproline; hence, it catalyzes the final step in collagen degradation. From mammals it is known that this enzyme plays a major role in the recycling of proline for collagen synthesis and can be considered to be essential for the control of cell growth. The dominant organic exoskeleton in sponges, especially in Demospongiae, is collagen and the collagen-related spongin. Here we demonstrate that crude extracts of the demosponge Suberites domuncula contain prolidase or prolidase-like activity. The complementary DNA encoding the putative prolidase was cloned from a library of the same animal. …
Cloning of sponge heat shock proteins: evolutionary relationships between the major kingdoms
In the present study we have cloned from sponges (Porifera) those molecules which are involved in the protection of organisms against physiological and stress conditions; the inducible heat shock protein Mr 70,000, hsp70, from the marine sponge Geodia cydonium, its interacting hsp40, a DnaJ-like protein (from G. cydonium) and the constitutively expressed counterpart the glucose-regulated protein Mr 78,000, GRP78 from Suberites domuncula. Alignments of the sequences revealed that the deduced aa sequences of all sponge hsp's share high homology to other metazoan sequences, and are separated from related sequences from plants and fungi (hsp70, GRP78, DnaJ) as well as Bacteria (DnaK, the hsp70 …
Apoptosis in marine sponges: a biomarker for environmental stress (cadmium and bacteria)
The marine demosponge Suberites domuncula is abundantly present on muddy sand bottoms, both in the open sea and in harbors. In the present study it is shown that exposure of S. domuncula to cadmium (CdCl2) in concentrations ranging from 0.01 to 5.0 g ml−1 for up to 5 d results in apoptotic fragmentation of DNA. Kinetics experiments revealed that after 24 h a significant increase of DNA fragmentation already occurred. Besides cadmium a second stimulus was identified to also cause apoptosis in this species, namely exposure to heat-treated Escherichia coli. In order to support the finding that both cadmium and E. coli induce apoptosis in the sponge, expression of the apoptotic gene MA-3 was st…
Induction of heat-shock (stress) protein gene expression by selected natural and anthropogenic disturbances in the octocoral Dendronephthya klunzingeri
Previously it was found that the expression of selected heat-shock proteins is upregulated in corals after exposure to elevated temperature. We published that HSPs are suitable markers in sponges to monitor the degree of environmental stress on these animals. In the present study the heat-shock proteins (HSPs) with a molecular weight of 90 kDa have been selected to prove their potential usefulness as biomarkers under controlled laboratory conditions and in the field. The studies have been performed with the octocoral Dendronephthya klunzingeri from which the cDNA coding for HSP90 was cloned first. The expression of the HSP90 gene is upregulated by thermal stress; treatment of the animals fo…
Sponges (Porifera) Molecular Model Systems to Study Cellular Differentiation in Metazoa
Evolution is a gradual process whereby primarily new genes are formed either by gene duplication (Ohno 1970) or exon shuffling (Gilbert 1978). New proteins can also be produced by overlapping genes, alternative splicing or gene sharing (Li and Graur 1991). These facts imply that (1) proteins found in a given phylum contain elements or modules which are present already in ancestral protein(s) of members of phylogenetically older phyla and (2) that new combinations of such modules create proteins that possess new functions.
Stress Response in Marine Sponges: Genes and Molecules Involved and Their use as Biomarkers
Sponges (Porifera) are sessile filter feeders that are able to accumulate compounds from the surrounding water and thus are highly exposed to environmental stress by pollutants of both anthropogenic and natural origin. However, these animals possess a number of protective mechanisms against environmental stress, such as the expression of heat-shock proteins, the induction of the multi xenobiotic resistance mechanism, and the apoptotic elimination of cells. In the last years, a number of genes involved in the stress response of sponges have been cloned and characterized. In thischapter, the various molecular mechanisms by which cells of the lowest multicellular organisms—the marine sponges—r…