Rebecca J. Van Beneden

An aryl hydrocarbon receptor (AHR) homologue from the soft-shell clam, Mya arenaria: evidence that invertebrate AHR homologues lack 2,3,7,8-tetrachlorodibenzo-p-dioxin and β-naphthoflavone binding

The aryl hydrocarbon receptor (AHR) mediates numerous toxic effects following exposure of vertebrate animals to certain aromatic environmental contaminants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To investigate possible effects of TCDD on invertebrates, a cDNA encoding an AHR homologue was cloned from the soft-shell clam, Mya arenaria. The predicted amino acid sequence contains regions characteristic of vertebrate AHRs: basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains and a glutamine-rich region. Phylogenetic analysis shows that the clam AHR sequence groups within the AHR subfamily of the bHLH-PAS family, in a clade containing AHR homologues from Drosophila melanogaster and Caenorhabditis elegans. AHR mRNA expression was detected in all tissue types tested: adductor muscle, digestive gland, foot, gill, gonad, mantle, and siphon. The in vitro-expressed clam AHR exhibited sequence-specific interactions with a mammalian xenobiotic response element (XRE). Velocity sedimentation analysis using either in vitro-expressed clam AHR or clam cytosolic proteins showed that this AHR homologue binds neither [(3)H]TCDD nor [(3)H]beta-naphthoflavone (BNF). Similarly, in vitro-expressed D. melanogaster and C. elegans AHR homologues lacked specific binding of these compounds. Thus, the absence of specific, high-affinity binding of the prototypical AHR ligands TCDD and BNF, is a property shared by known invertebrate AHR homologues, distinguishing them from vertebrate AHRs. Comparative studies of phylogenetically diverse organisms may help identify an endogenous ligand(s) and the physiological role(s) for this protein.

Expression of homologues for p53 and p73 in the softshell clam ( Mya arenaria ), a naturally-occurring model for human cancer

Homologues for human p53 (Hsp53) and p73 (Hsp73) genes were cloned and expression patterns for their corresponding proteins analysed in tissues from normal and leukemic softshell clams (Mya arenaria). These are the first structural and functional data for p53 and p73 cDNAs and gene products in a naturally occurring, non-mammalian disease model. Core sequence of the predicted clam p53 (Map53) and p73 (Map73) proteins is virtually identical and includes the following highly conserved regions: the transcriptional activation domain (TAD), MDM2 binding site, ATM phosphorylation site, proline rich domain, DNA binding domains (DBDs) II-V, nuclear import and export signals and the tetramerization domain. The core sequence is a structural mosaic of the corresponding human proteins, with the TAD and DBDs resembling Hsp53 and Hsp73, respectively. This suggests that Map53 and Map73 proteins may function similarly to human proteins. Clam proteins have either a short (Map53) or long (Map73) C-terminal extension. These features suggest that Map53 and Map73 may be alternate splice variants of a p63/p73-like ancestral gene. Map73 is significantly upregulated in hemocytes and adductor muscle from leukemic clams. In leukemic hemocytes, both proteins are absent from the nucleus and sequestered in the cytoplasm. This observation suggests that a non-mutational p53/p73-dependent mechanism may be involved in the clam disease. Further studies of these gene products in clams may reveal p53/p73-related molecular mechanisms that are held in common with Burkitts lymphoma or other human cancers.

Cloning of the p53 tumor suppressor gene from the Japanese medaka (Oryzias latipes) and evaluation of mutational hotspots in MNNG-exposed fish

A full-length cDNA clone of the medaka (Oryzias latipes) p53 tumor suppressor gene was isolated from a cDNA library from adult liver tissue, sequenced and characterized. Sequence analysis revealed a high degree of homology between putative functional domains of medaka p53 and p53 genes from other vertebrate taxa including rainbow trout (Oncorhynchus mykiss), frog (Xenopus laevis), chicken (Gallus gallus), rat (Rattus norvegicus), mouse (Mus musculus), hamster (Mesocricetus auratus), green monkey (Ceropithecus aethiops) and human (Homo sapiens). A single 1.9-kb p53 mRNA is expressed at a very low level in normal adult liver tissue. This transcript is similar in size to transcripts of p53 genes from other species. Preliminary screening of six MNNG-induced tumors in four adult medaka revealed no mutations within characteristic mutational hotspots encompassing conserved domains IV and V.

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

The human p53 tumour suppressor protein is inactivated in many cancers and is also a major player in apoptotic responses to cellular stress. The p53 protein and the two other members of this protein family (p63, p73) are encoded by distinct genes and their functions have been extensively documented for humans and some other vertebrates. The structure and relative expression levels for members of the p53 superfamily have also been reported for most major invertebrate taxa. The functions of homologous proteins have been investigated for only a few invertebrates (specifically, p53 in flies, nematodes and recently a sea anemone). These studies of classical model organisms all suggest that the gene family originally evolved to mediate apoptosis of damaged germ cells or to protect germ cells from genotoxic stress. Here, we have correlated data from a number of molluscan and other invertebrate sequencing projects to provide a framework for understanding p53 signalling pathways in marine bivalve cancer and stress biology. These data suggest that (a) the two identified p53 and p63/73-like proteins in soft shell clam (Mya arenaria), blue mussel (Mytilus edulis) and Northern European squid (Loligo forbesi) have identical core sequences and may be splice variants of a single gene, while some molluscs and most other invertebrates have two or more distinct genes expressing different p53 family members; (b) transcriptional activation domains (TADs) in bivalve p53 and p63/73-like protein sequences are 67-69% conserved with human p53, while those in ecdysozoan, cnidarian, placozoan and choanozoan eukaryotes are ≤33% conserved; (c) the Mdm2 binding site in the transcriptional activation domain is 100% conserved in all sequenced bivalve p53 proteins (e.g. Mya, Mytilus, Crassostrea and Spisula) but is not present in other non-deuterostome invertebrates; (d) an Mdm2 homologue has been cloned for Mytilus trossulus; (e) homologues for both human p53 upstream regulatory and transcriptional target genes exist in molluscan genomes (missing are ARF, CIP1 and BH3 only proteins) and (f) p53 is demonstrably involved in bivalve haemocyte and germinoma cancers. We usually do not know enough about the molecular biology of marine invertebrates to address molecular mechanisms that characterize particular diseases. Understanding the molecular basis of naturally occurring diseases in marine bivalves is a virtually unexplored aspect of toxicoproteomics and genomics and related drug discovery. Additionally, increases in coastal development and concomitant increases in aquatic pollutants have driven interest in developing models appropriate for evaluating potential hazardous compounds or conditions found in the aquatic environment. Data reviewed in this study are coupled with recent developments in our understanding the molecular biology of the marine bivalve p53 superfamily. Taken together, they suggest that both structurally and functionally, bivalve p53 family proteins are the most highly conserved members of this gene superfamily so far identified outside of higher vertebrates and invertebrate chordates. Marine bivalves provide some of the most relevant and best understood models currently available for experimental studies by biomedical and marine environmental researchers.

Transfer, methylation and spontaneous mutation frequency of ΦX174am3cs70 sequences in medaka (Oryzias latipes) and mummichog (Fundulus heteroclitus): Implications for gene transfer and environmental mutagenesis in aquatic species

Abstract This study describes the production of transgenic medaka ( Oryzias latipes ) and mummichog ( Fundulus heteroclitus ) containing multiple copies of the bacteriophage ΦX174 am3cs70 . This work is an initial approach for measuring mutations in aquatic species using the same gene target sequence in ish and laboratory mammals. The ΦX174 sequence is unique in that there is no detectable homology with chromosomal DNA of medaka, mummichog or mice. The authors have compared cytoplasmic injection of 1–2 cell embryos with linear single copy and catenated constructs of the phage DNA. The catenated construct results in greater efficiency of gene transfer for both species in terms of copies per cell. Analyses of DNA from founder transgenic fish with methylation sensitive (HpaII) and methylation insensitive (MspI) restriction enzyme isoschizmers indicates CpG methylation of the integrated ΦX174 sequence. This study also demonstrates the efficient rescue of live phage from the chromosomal DNA of founder fish in sufficient numbers to determine a spontaneous mutation frequency for reversion of am3 . A pooled sample of 20 μg DNA from four fish yielded 1.09 × 10 7 progeny phage with a spontaneous mutation frequency of 1.83 × 10 −7 . This spontaneous mutation frequency is similar to the spontaneous frequency for the same gene indictor recovered from transgenic mice. These results demonstrate that fish containing multiple copies of ΦX174 can be produced with no obvious detrimental effects and that the overall approach may be useful in basic and applied studies of environmental mutagenesis.

An invertebrate mdm homolog interacts with p53 and is differentially expressed together with p53 and ras in neoplastic Mytilus trossulus haemocytes

The mussel Mytilus trossulus can develop a neoplasia of the haemolymph, which occurs with high frequency (up to 40%) in nature. Associated with this disease are pro-apoptotic tumor-suppressor protein p53 isoforms, which are highly conserved between molluscs and vertebrates. The vertebrate wildtype p53 protein is maintained at low levels by the MDM2 protein in non-stressed cells to prevent undesired apoptosis. Identification of a putative invertebrate MDM-like homolog suggests early evolution of this mechanism of p53 regulation. The M. trossulus MDM homolog consists of a conserved NH(2)-terminal p53 binding domain, an acidic domain with highly conserved phosphorylation sites, and a highly conserved C-terminal RING-finger Zn-binding domain. Although BLAST queries predict this homologue to be more similar to vertebrate MDM2 than to MDM4, phylogenetic analysis suggests that it may be an ancestral form to both vertebrate MDM genes. Using yeast-two-hybrid assays and pull-down assays, we show that this molluscan MDM is able to bind to its p53 counterpart. We also show that MDM expression levels are directly correlated with p53 expression levels in healthy and in neoplastic haemocytes, but not with other p53 isoforms or with the proto-oncogene RAS. The combination of expression levels of five gene transcripts (p53, mdm, ras, Np63/73, and TAp63/73) is significantly correlated with late-stage haemic neoplasia in M. trossulus.

A new cytochrome P450 (CYP30) family identified in the clam, Mercenaria mercenaria.

A full-length clone with sequence similarity to genes in the cytochrome P450 superfamily was isolated from a cDNA library prepared from female Mercenaria mercenaria gonadal tissue. This clone was isolated while screening an expression library with an antibody prepared against a peptide sequence within the ligand-binding region of the murine Ah receptor. Comparison of the predicted amino acid sequence of this clone to those of other cytochrome P450 genes indicated that the closest overall sequence similarity (38%) was to proteins predicted from genes in the CYP3 family. Northern blots indicated the presence of a transcript of the appropriate size (3.0 kb) with homology to the clam cytochrome P450. In vitro translation of the cDNA clone produced a 50.7-kDa protein as determined by SDS-polyacrylamide gel electrophoresis. The in vitro translated protein was not recognized on Western blots by two polyclonal antibodies specific for members of the CYP3 family. Since the degree of similarity to existing cytochrome P450 families was below the 40% level required for membership, and the expressed protein was not recognized by CYP3-specific antibodies, this clam cytochrome P450 cDNA has been placed in a new family, cytochrome P450 30 (CYP30).

Soft-shell clam (Mya arenaria) p53: a structural and functional comparison to human p53.

The tumor suppressor p53 regulates genes involved in progression through the cell cycle, DNA repair, senescence or apoptosis in response to cell stress. Dysregulation of p53 can result in uncontrolled cellular proliferation. Invertebrate homologues to human p53 (Hsp53) have been identified, including a putative p53 gene (Map53) from the soft-shell clam (Mya arenaria). Predicted sequences for human and clam p53 proteins exhibit conservation in key domains. In light of this similarity, and the apparent dysregulation of Map53 under morphologically aberrant/pathologic conditions, we tested the hypothesis that the two proteins function in a similar manner. Plasmids expressing either Hsp53 or Map53 were introduced by transient transfection into the p53-null H1299 cell line. Functionality was assessed by monitoring the p53/mdm2 feedback loop and expression of p53-mediated downstream markers of growth arrest and apoptosis under non-stressed conditions. Hsp53 spontaneously induced markers of growth arrest, while Map53 expression induced neither cell arrest nor apoptosis. The difference in downstream activation is not likely the result of cytosolic sequestration since Map53, like Hsp53, localized almost exclusively to the nucleus. Functional similarity was observed in regulation by human MDM2, suggesting that the clam may have an mdm2 homologue. Protein modeling identified an apparent MDM2 binding site in Map53, supporting the observation of a potential Map53/MDM2 interaction. Significant amino acid differences present in the Map53 tetramerization domain may potentially affect p53 protein/protein interactions. Taken together, these data suggest that the Map53 shares some functional similarity with human p53 as well as with other invertebrates, positioning the mollusk at a critical juncture in evolution of this gene family.

Halogenated aromatic hydrocarbon-binding proteins identified in several invertebrate marine species

Abstract The vertebrate aryl hydrocarbon receptor (AhR) is a cytosolic protein which binds halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). In vertebrates, the AhR-ligand complex, in association with other proteins, binds specific DNA sequences and modifies the expression of a number of genes. Most of the toxic effects of halogenated aromatic hydrocarbon exposure are mediated through this receptor. A similar receptor system has not yet been identified in invertebrates. The current study investigated whether proteins which specifically bind halogenated aromatic hydrocarbons were present in marine invertebrates. We used the photoaffinity TCDD-analog, 2- azido -3-[ 125 I] iodo -7,8- dibromodibenzo -p- dioxin ([ 125 I]N 3 Br 2 DpD), to detect the presence of cytosolic proteins which specifically bound this ligand. Specific binding was defined as labeling which could be competed off by an excess of unlabeled ligand. Eleven species of marine invertebrates were examined which represented six different phyla: Cnidaria, Mollusca, Annelida, Arthropoda, Chordata, and Echinodermata. Cytosols prepared from gill and gonad in the soft-shell clam ( Mya arenaria ), the eastern oyster ( Crassostrea virginica ), and hard-shell clam ( Mercenaria mercenaria ), as well as the hepatopancreas of the blue crab ( Callinectes sapidus ), contained proteins in the same size range (28–39 kDa) which were specifically labeled with the dioxin analog. No proteins of the size expected for vertebrate Ah receptors (95–146 kDa) were seen in any of the invertebrates. The biological function of these dioxin-binding proteins is not yet known.

Proteomic Analysis of Arsenic-Exposed Zebrafish (Danio rerio) Identifies Altered Expression in Proteins Involved in Fibrosis and Lipid Uptake in a Gender-Specific Manner

The zebrafish (Danio rerio) was used to investigate protein expression in the liver following arsenic exposure. Several disorders have been linked to arsenic exposure, including cancer, diabetes, and cardiovascular disease. The mechanisms of arsenic toxicity are poorly understood. Prior studies have described altered gene expression, inflammation, and mitogenic signaling in acute or chronic exposure models. A proteomic approach was employed to investigate arsenic-induced alteration in the zebrafish liver proteome following a 7-day exposure to 50 ppb sodium arsenite. Over 740 unique proteins were identified, with fewer than 2% showing differential expression. Molecular pathway analysis software identified lipid metabolism and transport as potential molecular targets. Immunoblots were used to confirm protein expression changes, whereas qPCR was employed to investigate gene expression changes. Overall, 25 proteins were differentially expressed in a gender-specific manner, 11 in males and 14 in females. Of these 25, a single protein, hydroxysteroid dehydrogenase like 2, showed decreased expression in both males and females following arsenic exposure. These findings indicate that protein expression is altered following arsenic exposure. The changes presented here seem to be most prevalent in lipid transport and metabolic pathways, suggesting a potential increase in fibrosis in males and decreased lipid accumulation and uptake in females.