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p63/73 homologues in surf clam: Novel signaling motifs and implications for control of expression
Abstract
To understand the role of p53 gene family members during invertebrate embryonic development, we used polymerase chain reaction (PCR) to identify p63/73 homologues in the marine mollusc Spisula solidissima. Here, we report the sequences of two distinct p63/73-like homologues, both cloned from Spisula embryos. The first, Ssp63/73alpha is 2699 nucleotide (nt); the second, Spp63/73beta is 3920 nt. The nucleotide sequences of the two variants are nearly identical up to their stop codons but diverge in their 3'-untranslated regions (UTRs). The deduced amino acid sequence of both Ssp63/73 variants is 597 amino acids, coding for a protein with predicted molecular weight of approximately 68 kDa. We conclude that the two unique transcripts, containing 3' UTRs of variable lengths, represent tandem alternate polyadenylation sites for the Ssp63/73 gene. While alternative splicing has been well documented in the p63/73 gene family, this is the first report of alternate polyadenylation site choice as a control point for p63/73 gene expression in any species. In order to identify specific post-transcriptional as well as post-translational signals potentially involved in regulation of p63/73-like expression, we compared Ssp63/p73 nucleotide and Ssp63/73 deduced amino acid sequences to corresponding regions of other mammalian and nonmammalian p63 and p73 homologues. Within the Spisula 3' UTRs we identified multiple AU-rich elements (AREs) which may control translation activation. Within the deduced amino acid sequence, we identified potential sites for sumoylation, a post-translational process that has been identified in mammalian p63 and p73 proteins. Identification of these novel signaling sites provides information about potential mechanisms controlling expression of multiple p63/73 isoforms during development.
Supplementary resources (2)
Nucleotide Sequence
May 2003
... ath pathway ( Urist et al . , 2004 ) . 8 9 In invertebrate animals , p53 - like proteins have been discovered , such as CEP - 1 in the worm 10 Caenorhabditis elegans ( Lu and Abrams , 2006 ) , DMP53 in the fly Drosophila melanogaster , and 11 various homologs in different molluscan models ( Schmale and Bamberger , 1997 ; Kelley et al . , 2001 ; 12 Cox et al . , 2003 ; Muttray et al . , 2005 ; Goodson et al . , 2006 ; Muttray et al . , 2007 ) . Molluscan family 13 members , more so than CEP - 1 or DMP53 , show high sequence similarity to their vertebrate 14 counterparts . Thus , molluscan p53 isoform transcription may afford a relevant but simple model to 15 describe core properties of this ancient ...
... suggested as a biomarker in human cancers ( Concin et 11 al . , 2004 ) . 12 13 The expression of p53 proteins during haemic neoplasia has been studied in Mytilus edulis and the 14 soft - shell clam Mya arenaria ( Barker et al . , 1997 ; Kelley et al . , 2001 ; McGladdery et al . , 2001 ; 15 Stephens et al . , 2001 ; Jessen - Eller et al . , 2002 ; Cox et al . , 2003 , St - Jean , 2005 ) . Using an antibody to 16 the central DNA binding domain of the M . arenaria p53 / p73 , it was found that p53 and / or p63 / 73 is 17 expressed in the cytoplasm of leukaemic animals instead of the nucleus where it is required as a 18 transcription factor ( Kelley et al . , 2001 ) . In addition , a protein band with ...
... p63 / 73 was present only in leukaemic haemocytes of M . arenaria , while a band corresponding to p53 20 was present at equal levels in normal and leukaemic haemocytes ( Kelley et al . , 2001 ; Stephens et al . , 21 2001 ) . Using a cross - reactive antibody specific to the homodimerization domain of the surf clam 22 Spisula solidissima p63 / 73 ( Cox et al . , 2003 ) , it was shown that p63 / 73 was expressed at higher levels 23 in leukaemic haemocytes when compared to normal haemocytes of Mytilus edulis ( St - Jean et al . , 24 ACCEPTED MANUSCRIPT 5 2005 ) . Since these initial studies found that some p53 - family members were upregulated in end - stage 1 haemic neoplasia cells , we wanted to inv ...
Mussels of the genus Mytilus are widely used in environmental monitoring. They can develop a leukaemia-like disease, haemic neoplasia, which could be induced, in part, by environmental stressors. The molluscan p53 tumor suppressor gene family was previously shown to be involved in haemic neoplasia at the protein level. The purpose of this study was the quantification of molluscan p53-like isoforms at the mRNA level in mussels with haemic neoplasia compared to normal controls. The three isoforms monitored were a p53-like, a TAp63/73-like containing an intact transactivation (TA) domain, and an NH(2)-terminally truncated p63/73 isoform termed DeltaNp63/p73-like that lacks the full TA domain. Using a comprehensive data set of 62 individual Mytilus trossulus and reverse transcription real-time PCR, we found that both the p53 and the DeltaNp63/73 isoforms were up-regulated in neoplastic haemocytes compared to normal haemocytes (p<0.0001). In contrast, the mRNA levels of the non-truncated isoform TAp63/73 did not change significantly in mussels with the disease at alpha=0.01 (p=0.0141), in contrast to previous findings at the protein level. Correlations in mRNA levels between the truncated isoform and the full-length isoforms in normal haemocytes were lost in neoplastic haemocytes. The increase in mRNA concentration of the truncated DeltaNp63/73 isoform in molluscan haemic neoplasia is similar to observations in many human cancers and cell lines and underlines the phylogenetically ancient oncogenic role of this isoform.
... Evidence for p53 family genes was either extracted from Ensembl database or manually verified using BLAST (for the most recent assemblies available à ) (Altschul et al. 1990;Hubbard et al. 2009;Sayers et al. 2009). Evidence for p53 family genes in clams comes from gene isolation by C. Walker and others (Kelley et al. 2001;Cox et al. 2003) à Several species, such as lancelet and sea squirts, also have tentative intronless paralogs; these could be pseudogenes or candidate p53 family genes that are unlikely predecessors for mammalian p53-family and are not shown in the tree. One possible scenario is that they arose through independent duplication events with intron loss (Mourier and Jeffares 2003) The Origins and Evolution ...
... Thanks to Charles Walker and his colleagues (Kelley et al. 2001;Jessen-Eller et al. 2002;Cox et al. 2003;Bottger et al. 2008;Holbrook et al. 2009;Siah et al. 2009), we know a great deal about the p53 gene in clams. Mya arenaria, a common clam species, has a single p63/p73 hybrid-like gene but can splice the transcripts from this gene into a p53-like sequence and a p63/p73-like sequence. ...
... It is also clear that a genera of clams, such as the common surf clam, has been exploring evolutionary expansions of splice variants of the p63/p73 gene, producing a p97 and a p120 set of isoforms with common DNA binding domains. Interestingly, the p120 isoform is expressed only in the embryonic stages of clam development and appears to be involved in neuronal functions (like p73 in mice) (Jessen-Eller et al. 2002;Cox et al. 2003). ...
A common ancestor to the three p53 family members of human genes p53, p63, and p73 is first detected in the evolution of modern-day sea anemones, in which both structurally and functionally it acts to protect the germ line from genomic instabilities in response to stresses. This p63/p73 common ancestor gene is found in almost all invertebrates and first duplicates to produce a p53 gene and a p63/p73 ancestor in cartilaginous fish. Bony fish contain all three genes, p53, p63, and p73, and the functions of these three transcription factors diversify in the higher vertebrates. Thus, this gene family has preserved its structural features and functional activities for over one billion years of evolution.
... multiple isoforms of p63) and proteases involved in the destruction of proteins (e.g. caspase 3/7-1 and 3/7-2) [70][71][72][73]. Six isoforms of glutathione-S-transferase enzymes involved in the production of antioxidants were present in the upper 25% of M. galloprovincialis proteins with the most hydrogen bonds per amino acid [74]. ...
... M. galloprovincialis proteins that could exert a strong influence on cell fate during heat stress, including p63, DNA damage regulated protein (PDRP), and multiple caspase enzymes [72,73,83], had more hydrogen bonds and salt bridges per amino acid than M. trossulus proteins or other functional classes of M. galloprovincialis proteins. p63 is a member of the p53 family of transcription factors that serve as connection points for several stress response pathways and that govern the decision between cell survival and apoptosis by controlling the abundance of gene products that determine cell fate [70,71,73,84,85]. PDRP is a downstream target of p53, suggesting that this molecule also contributes to the balance between cell survival and death during environmental stress [73,86]. ...
Background:
Temperature exerts a strong influence on protein evolution: species living in thermally distinct environments often exhibit adaptive differences in protein structure and function. However, previous research on protein temperature adaptation has focused on small numbers of proteins and on proteins adapted to extreme temperatures. Consequently, less is known about the types and quantity of evolutionary change that occurs to proteins when organisms adapt to small shifts in environmental temperature. In this study, these uncertainties were addressed by developing software that enabled comparison of structural changes associated with temperature adaptation (hydrogen bonding, salt bridge formation, and amino acid use) among large numbers of proteins from warm- and cold-adapted species of marine mussels, Mytilus galloprovincialis and Mytilus trossulus, respectively.
Results:
Small differences in habitat temperature that characterize the evolutionary history of Mytilus mussels were sufficient to cause protein structural changes consistent with temperature adaptation. Hydrogen bonds and salt bridges that increase stability and protect against heat-induced denaturation were more abundant in proteins from warm-adapted M. galloprovincialis compared with proteins from cold-adapted M. trossulus. These structural changes were related to deviations in the use of polar and charged amino acids that facilitate formation of hydrogen bonds and salt bridges within proteins, respectively. Enzymes, in particular those within antioxidant and cell death pathways, were over-represented among proteins with the most hydrogen bonds and salt bridges in warm-adapted M. galloprovincialis. Unlike extremophile proteins, temperature adaptation in Mytilus proteins did not involve substantial changes in the number of hydrophobic or large volume amino acids, nor in the content of glycine or proline.
Conclusions:
Small shifts in organism temperature tolerance, such as that needed to cope with climate warming, may result from structural and functional changes to a small percentage of the proteome. Proteins in which function is dependent on large conformational change, notably enzymes, may be particularly sensitive to temperature perturbation and represent foci for natural selection. Protein temperature adaptation can occur through different types and frequencies of structural change, and adaptive mechanisms used to cope with small shifts in habitat temperature appear different from mechanisms used to retain protein function at temperature extremes.
... Although the gene sequence is not resolved for Mytilus ssp., it appears that Mytilus can similarly produce p53-and p63/p73-like isoforms and in addition an N-terminally truncated deltaN p63/p73-like isoform through alternative splicing (Muttray et al. 2007). The surf clam S. solidissima has additional splice variants, p97 and p120 (Cox et al. 2003), and interestingly the p120 is only expressed in the embryonic stages of clam development in response to stress and appears to be involved in neuronal development (similar to vertebrate p73) (Jessen-Eller et al. 2002). ...
... Interestingly, concentrations of a p63/73-like protein were also increased in neoplastic hemocytes in Mya and Mytilus (Kelley et al. 2001;Stephens et al. 2001), which was consistent with an increase observed in deltaNp63-p73 (but not TAp63/ p73) mRNA levels in those species (Muttray et al. 2008(Muttray et al. , 2012. Similarly, an antibody to the p73 HOMO-domain of the surfclam S. solidissima (Cox et al. 2003) detected an increased amount of p63/73 in neoplastic Mytilus hemocytes . Thus, the increase in p63/73-like proteins observed in Mya and Mytilus neoplastic hemocytes may be correlated with the increase in the truncated deltaNp63/73 mRNA. ...
Insights into the common mechanisms that likely exist in invertebrate and in vertebrate cancers will lead to a more coherent understanding of the fundamental processes that are altered during tumorigenesis and of tumor immunity. Invertebrates possess an innate immune system, primarily consisting of cellular and humoral defenses. In mollusks, hemocytes provide the first line of defense against foreign particles or organisms but lose their functionality when transformed into neoplastic cells. Neoplasia, or abnormal growth and proliferation of cells, has been described in many invertebrate species ranging from sponges to mollusks and arthropods. Occurrence of neoplasia in invertebrates appears to be by far not as common or as diverse in nature as it is in vertebrates, but some invertebrates even display cancers with metastatic potential, notably the bivalve mollusks. This chapter will focus on a cancer of the hemocytes in marine bivalves called disseminated neoplasia. Disseminated neoplasia in bivalves can be induced by a retrotransposon, appears to have contributing environmental factors, and is one of only four known cases of a transmissible cancers, not only between members of the same species but also between members of closely related species. This chapter will describe how disseminated neoplasia and the components of the innate immune system are linked by one of the central nodes in a wide signaling network that integrates DNA stability, apoptosis, and cell growth: the p53 tumor suppressor family.
... In addition, each member of the human p53 superfamily produces different protein isoforms that play critical roles in the regulation of biological processes and their abnormal expression contributes to tumorigenesis [78]. In mollusks, p53 superfamily coding sequences have been characterized for clams [79][80][81], mussels [82][83][84], cockles [85], oysters [86], and gastropods [87]. Interestingly, in some species, various isoforms have been detected, and these isoforms seem to arise from alternative splicing, as regions that overlap between different proteins from the same species are 100% identical [79,80,83,84]. ...
... In mollusks, p53 superfamily coding sequences have been characterized for clams [79][80][81], mussels [82][83][84], cockles [85], oysters [86], and gastropods [87]. Interestingly, in some species, various isoforms have been detected, and these isoforms seem to arise from alternative splicing, as regions that overlap between different proteins from the same species are 100% identical [79,80,83,84]. Examination of neoplastic hemocytes, using expression profiles and antibodies to p53 family members, has shown differential up-regulation of p53 gene in neoplastic cells compared with healthy ones [22,79,85,[88][89][90]. ...
Neoplasia-the abnormal growth of cells-is associated mainly with higher vertebrates (e.g., humans and mammals). However, neoplastic processes or cancers are ubiquitous among living organisms, with a high incidence of reported neoplasms in mollusks. Both benign and malignant cancers have been described in mollusks, but just two malignant neoplasms have raised scientific and industry concerns: gonadal neoplasia and disseminated or leukemia-like neoplasia. These cancers have been reported in either wildlife or captive populations; and as in humans, different causes have been suggested including genetic alterations, virus, retrotransposons, and pollutants. In this review, I give a general overview on neoplasia in mollusks with a focus on genes and molecular pathways involved in gonadal and disseminated neoplasia. Subsequently, I highlight some similarities between disseminated neoplasia and human cancers, particularly with leukemia, as well as the advantages of using mollusks affected by this disease as a model system to better understand cancer in humans. Finally, I discuss the feasibility of using mollusks to investigate tumorigenesis. As the field of marine genomics advances, I predict that comparative oncology will gain more attention in the years to come.
... The regulation of p53 function is tightly controlled through several mechanisms including p53 transcription and translation, protein stability and post-translational modification, as well as p53 location in the cell nucleus or cytoplasm (O'Brate et al., 2003, and references therein). Several authors have used various monoclonal and polyclonal antibodies raised against human and clam (Spisula, Mya) p53 family members to study the expression of the p53 family and to distinguish leukemic cells from normal cells (Kelley et al., 2001; Stephens et al., 2001; Jessen-Eller et al., 2002; Cox et al., 2003). We isolated haemocytes from two species of bivalve mollusc, Mytilus spp. ...
... p53 sequences revealed that unique and highly conserved sequence sites occur in the 3' untranslated regions (3' UTRs). Our previous reports suggest that the occurrence of cis-acting signaling sites in the 3' UTRs of the p53 gene family members in clams may control gene expression (Cox et al., 2003). Columbia. ...
The extent to which humans and wildlife are exposed to anthropogenic challenges is an important focus of environmental research. Potential use of p53 gene family marker(s) for aquatic environmental effects monitoring is the long-term goal of this research. The p53 gene is a tumor suppressor gene that is fundamental in cell cycle control and apoptosis. It is mutated or differentially expressed in about 50% of all human cancers and p53 family members are differentially expressed in leukemic clams. Here, we report the identification and characterization of the p53 gene in two species of Mytilus, Mytilus edulis and Mytilus trossulus, using RT-PCR with degenerate and specific primers to conserved regions of the gene. The Mytilus p53 proteins are 99.8% identical and closely related to clam (Mya) p53. In particular, the 3' untranslated regions were examined to gain understanding of potential post-transcriptional regulatory pathways of p53 expression. We found nuclear and cytoplasmic polyadenylation elements, adenylate/uridylate-rich elements, and a K-box motif previously identified in other, unrelated genes. We also identified a new motif in the p53 3'UTR which is highly conserved across vertebrate and invertebrate species. Differences between the p53 genes of the two Mytilus species may be part of genetic determinants underlying variation in leukemia prevalence and/or development, but this requires further investigation. In conclusion, the conserved regions in these p53 paralogues may represent potential control points in gene expression. This information provides a critical first step in the evaluation of p53 expression as a potential marker for environmental assessment.
... Genes coding for the P53 protein family have already been described in marine bivalves, e.g. Mya arenaria (Van Beneden et al., 1997) and Spisula solidissima (Cox et al., 2003). More recently, the identification of p53 genes in soft tissues of Mytilus spp. was reported (Ciocan and Rotchell, 2005;Muttray et al., 2005). ...
... Some further consideration on the newly identified mussel p53like gene should also be made. This gene is likely to belong to the p63/73 gene family that has already been described in bivalves (Cox et al., 2003), which in mammals is expressed several splicing variants. The full length gene variants-α and β include the p53-like transactivating (TA) domain that provides their function during proapoptosis, while ΔN genes lack the TA domain and instead behave as negative isoforms that dominate active variants (Moll et al., 2001). ...
In this study we describe the design and implementation of a novel low-density oligonucleotide microarray (the "Mytox-chip"). It consists of 24 mussel genes involving both normalizing elements and stress response related genes, each represented on the array with one or two different 50 mer oligonucleotide-probe reporters spotted in replicated samples on glass-activated slides. Target genes were selected on the basis of their potential involvement in mechanisms of pollutant and xenobiotic response. They are implicated in both basic and stress related cellular processes such as shock response, biotransformation and excretion, cell-cycle regulation, immune defense, drug metabolism, etc. The microarray was tested on mussels exposed to sublethal concentrations of mercury or a crude North Sea oil mixture. RNA samples were extracted from digestive glands of control and treated mussels for the synthesis of fluorescence labeled cDNAs to be used in dual color hybridizations. Transcription rates of two metallothionein iso-genes (mt10 and mt20), a p53-like gene and actin were quantitatively estimated also by real-time PCR to confirm microarray data. Significant alterations in the gene transcription patterns were seen in response to both treatments.
... El gen supresor de tumores p53 y su expresión proteica han sido muy estudiados debido a sus múltiples funciones en la regulación del ciclo celular, apoptosis, mantenimiento de la estabilidad genética y su alta implicación detectada en cánceres humanos (Oren, 2003). Se ha secuenciado el gen p53 en M. arenaria (Barker et al, 1997;Kelley et al., 2001), Spisula solidissima (Cox et al., 2003), M. edulis (Ciocan y Rotchell, 2005;Muttray et al, 2007), M. trossulus (Ciocan et al., 2006;Muttray et al, 2007), C. gigas (Farcy et al., 2008), M. galloprovincialis (Stifanic et al., 2009) y C. edule (Ruiz et al., 2013b). En fase avanzada de la enfermedad se detecta la expresión de la proteína p53 mutada en almejas M. arenaria; secuenciando el gen de la proteína mutada se observó, en dos de cinco almejas estudiadas, una transversión de citosina a guanina al final del exón 6, produciendo un cambio del aminoácido de ese codón de prolina a alanina (Barker et al, 1997). ...
Cockle Cerastoderma edule is a species of great commercial interest in Galicia. It is one of the bivalve species with more annual catches and, therefore, has a high economic and ecological importance in this region. Pathological studies associated with cockle mortality events in Galicia detected high prevalence of a pathological condition known as disseminated neoplasia. Knowledge of cancer in molluscs is scarce. This study aims to deep in the knowledge about the epidemiology of this disease, its etiology and the biochemical and physiological changes it causes. A thorough knowledge of this disease is required to develop strategies to fight it.
... arenaria, GenBank Accession Nos. AF253323 and AF253324) (23), three for surf clam (Spisula solidissima, AF285104, AY289767, and AY289768) (24,25), one for squid (Loligo forbesi, U43595), and one for oyster (Crassostrea rhizophorae, AY442309). ...
Mussels are susceptible to a wide range of environmental toxicants, including carcinogens, and thus are often employed as bioindicator species. To elucidate the molecular aetiology of such neoplastic damage, we have cloned Mytilus edulis homologues of the vertebrate ras proto-oncogene, and p53 tumor suppressor gene. The M. edulis ras cDNA encodes a predicted protein of 184 amino acids. The DNA sequence analysis with vertebrate ras sequences demonstrates that the M. edulis ras cDNA is highly conserved in regions of functional importance, including mutational hot spots. The partial p53 sequence also demonstrates that M. edulis p53 is highly conserved in two regions of functional importance and that these regions also include four of the five mutational hot spots for this gene. In contrast, the M. edulis p53 sequence shows little similarity to the other published invertebrate p53-like sequences. The cancer gene sequences characterized herein will allow development of specific biomarkers of genotoxic damage.
... Las mutaciones del gen p53 son las más estudiadas debido a su función reguladora del ciclo celular y de la apoptosis y a su alta frecuencia de mutación en cánceres humanos. Se ha secuenciado el gen p53 en Mytilus edulis (MUTTRAY et al. 2005; CIOCAN y ROTCHEL 2005), Mytilus trossolus (MUTTRAY et al. 2005), Spisula solidissima (COX et al. 2003), Crassostrea rhizophorae (Genbank Accession Number AY442309) y Crassostrea gigas (Genbank Accession Number AM236465). En células neoplásicas de Mya arenaria se ha detectado una transversión de citosina a guanina en el final del exon 6 (BARKER et al. 1997). ...
... The tumor-suppressor protein p53 regulates genes involved in progression through the cell cycle, DNA repair, senescence or apoptosis in response to cell stress; thus dysregulation or dysfunction of p53 can result in uncontrolled cellular proliferation; inactivation of p53 occurs in almost all cancers (Oren, 2003). A p53-like gene has been characterized in a number of mollusk species, including M. arenaria (Barker et al., 1997;Kelley et al., 2001), Spisula solidissima (Cox et al., 2003), M. edulis (Ciocan and Rotchell, 2005;Muttray et al., 2007), M. trossulus (Ciocan et al., 2006;Muttray et al., 2007), M. galloprovincialis (Stifanic et al., 2009), and C. edule (Ruiz et al., 2013b). The structure and phylogeny of p53 family in mollusks was reviewed by Walker et al. (2011); various isoforms have been detected, all seem to derive from the same gene through different splicing or other transformations. ...
Two types of prevalent neoplastic diseases have been described in marine bivalves of commercial interest: disseminated neoplasia (DN) and gonadal neoplasia. The first involves the excessive proliferation of abnormal cells with unknown origin (probably of hemic source in some cases/species), disseminating through the circulatory system and infiltrating the connective tissue of various organs; the second consists of an abnormal proliferation of undifferentiated germinal cells of the gonad. These two types of bivalve neoplasia fit the criteria of malignant tumors: pleomorphic and undifferentiated cells, rapid and invasive growth, abundance of mitotic figures, metastasis and progressive development often resulting in the death of the affected individual. Different causes have been suggested regarding etiology: genetic alterations, virus, retrotranspons, and contaminants, although it could depend on the mollusk species; evidence of horizontal transmission of clonal cancer cells as the cause of DN spreading in clam Mya arenaria populations has been recently reported. In some species and populations, the neoplastic disorders affect only a few individuals, but in others reach high prevalence. Among the diagnostic methods, DN has been detected by histology and cytologic examination of hemolymph, and with developed specific antibodies. Recently, flow cytometry has also been applied, allowing detecting DNA quantity alteration. Several studies reported many genes and pathways critically involved in neoplastic transformation in Mya arenaria, Mytilus spp. and Ostrea edulis. These genetic studies will allow the development of diagnosis by PCR which can be used in biomonitoring studies.
Copyright © 2015. Published by Elsevier Inc.
... The tumor-suppressor protein p53 regulates genes involved in progression through the cell cycle, DNA repair, senescence or apoptosis in response to cell stress; thus dysregulation or dysfunction of p53 can result in uncontrolled cellular proliferation ; inactivation of p53 occurs in almost all cancers (Oren, 2003 ). A p53-like gene has been characterized in a number of mollusk species, including M. arenaria (Barker et al., 1997; Kelley et al., 2001), Spisula solidissima (Cox et al., 2003), M. edulis (Ciocan and Rotchell, 2005; Muttray et al., 2007), M. trossulus (Ciocan et al., 2006; Muttray et al., 2007), M. galloprovincialis (Stifanic et al., 2009), and C. edule (Ruiz et al., 2013b ). The structure and phylogeny of p53 family in mollusks was reviewed by Walker et al. (2011); various isoforms have been detected, all seem to derive from the same gene through different splicing or other transformations . ...
... Furthermore, according to our previous and present studies, p63 is the only gene of the p53 family present in molluscs and vertebrate p63 nomenclature is fully applicable to all known molluscan p63 expression isoforms (Štifanić et al., 2009). Complete DNA coding sequences of different p63 isoforms (Kelley et al., 2001;Jessen-Eller et al., 2002;Cox et al., 2003;Muttray et al., 2005;Goodson et al., 2006;Muttray et al., 2007;Farcy et al., 2008;Estévez-Calvar et al., 2013) and only one complete genomic sequence (Mya arenaria p63/p73 and p53 gene; GenBank accession no. FJ041332) are at present publicly available in GenBank. ...
... Alteration of P53 was also found in soft-shell clams in advanced stages of the disease (Barker et al., 1997). The use of a set of monoclonal and polyclonal antibodies raised against clam (Spicula solidissima, M. arenaria) P53 protein allowed to distinguish neoplastic cells from normal cells in M. arenaria (Cox et al., 2003;Kelley et al., 2001;Stephens et al., 2001). Up-regulation of P53 protein expression has been reported in M. edulis affected by DN . ...
Disseminated neoplasia (DN) is a pathological condition reported for several species of marine bivalves throughout the world, but its aetiology has not yet been satisfactorily explained. It has been suggested that chemical contamination could be a factor contributing to neoplasia. The aim of the present study was to compare cell and tissue biomarkers and the transcription level of cancer-related genes in cockles (Cerastoderma edule) affected by DN with those of healthy cockles in relation to chemical contaminant burdens. For this, cockles were collected from a natural bed in Cambados (Ria de Arousa, Galicia) in May 2009. The prevalence of DN was 12.36% and 3 degrees of DN severity were distinguished. No significant differences in metal accumulation, non-specific inflammatory responses and parasites were observed between healthy and DN-affected cockles. Lysosomal membrane stability was significantly reduced in cockles affected by DN, which indicates a poorer health condition. Very low frequencies of micronuclei were recorded and no significant differences were detected between DN severity groups. Haemolymph analyses showed a higher frequency of mitotic figures and binucleated cells in cockles affected by moderate and heavy DN than in healthy ones. Neoplastic animals showed significantly higher transcription levels of p53 and ras than healthy cockles and mutational alterations in ras gene sequence were detected. Low concentrations of metals, polycyclic aromatic hydrocarbons, polychlorinated biphenyls and phthalate esters were measured in cockles from Cambados. In conclusion, cockles affected by DN suffer a general stress situation and have altered patterns of cancer-related gene transcription. Further studies are in progress to elucidate mechanisms of carcinogenesis in this species.
... The mouse monoclonal antibody Mab-1E10, previously described and characterized (Reinisch et al., 1983), was raised from existing hybridomas and affinity purified by CedarLane Laboratories (Burlington, ON); this new batch of 1E10 antibody was successfully compared to the previously produced 1E10. The rabbit polyclonal p63/73 homodomain antibody was previously raised against the Spisula solidissima p63/73 homodimerization domain and characterized by Cox et al. (2003). Antibodies were purchased from the following sources: mouse monoclonal anti-human p53 (SC-99) (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-actin (A2066) (Sigma, Oakville, ON), tetramethylrhodamine goat anti-mouse (T2762) and anti-rabbit (T2769) secondary antibodies (Invitrogen, Burlington, ON), and goat anti-mouse (81-6522) and anti-rabbit (62-6122) secondary antibodies conjugated to alkaline phosphatase (Zymed Laboratories, San Francisco, CA). ...
Intensive farming of potatoes in Prince Edward Island (PEI) relies on the repeated and widespread application of fertilizers and pesticides. In PEI the main potato farming areas are in close proximity and drain directly to estuaries. Runoff from high agricultural activity watersheds could impact benthic organism health in the depositional zone of downstream estuaries. The estuarine filter feeder Mya arenaria (soft-shell clam) could be particularly vulnerable to both particle-adsorbed and water soluble contaminants. M. arenaria is susceptible to haemocytic leukemia. In May 2009, we established that heavily proliferated leukemia (HPL) prevalence was generally higher in PEI estuaries located downstream of high intensity potato farming (Dunk and Wilmot estuaries) watersheds than in estuaries downstream of lower intensity areas. Using Mab-1E10 based immunocytochemistry we observed that leukemic haemocytes from the Dunk and Wilmot estuaries were 1E10 negative whereas those from the Ox/Sheep estuary (low potato farming intensity) were 1E10 positive. The expression of genes in the p53 tumour suppressor pathway enabled us to differentiate groups of leukemic and normal M. arenaria, validating our diagnoses. In October 2009, we confirmed that HPL prevalence was elevated in the Dunk and Wilmot estuaries compared to reference (Souris River). Moreover, leukemia prevalence declined with distance from the river mouths along transects through the Dunk and Wilmot estuaries. The pesticides ß-endosulfan and α-endosulfan were detected in surface sediments from the Dunk and Wilmot estuaries, but not in sediments from either the Souris River or several other lower intensity potato farming watersheds. Our study provides evidence of an association between intensity of potato farming and prevalence of clam leukemia at downstream estuaries in PEI.
... 1.3 and 1.4). These residues are also present in p53 sequences of the Pacific oyster Crassostrea gigas (Farcy et al., 2008) and the surf clam Spisula solidissima (Cox et al., 2003). In molluscs, the putative Mdm2 binding site is identical for both p53-and p63/73-like proteins. ...
The physiological mechanisms that regulate adaptive plasticity of clonal organisms are key to their success in changing environments. Here, we review the mechanisms that regulate morphological plasticity of colonial hydrozoans. There is a heritable, genetic basis to colony form, but environmentally-induced plasticity and self-reinforcing developmental physiology explain much of total phenotypic variance. Morphological development of colonial hydrozoans emerges from interactions among (1) behaviors which drive gastrovascular transport, (2) architecture of the gastrovascular system that determines hydrodynamic characteristics of vascular flow, and, (3) gene products that vary in response to physiological signals provided by gastrovascular transport. Several morphogenetic signaling mechanisms have been identified, including, reactive oxygen species and nutrient concentrations in the hydroplasm, and hydromechanical forces associated with gastrovascular transport. We present a conceptual model of the interacting forces that drive hydrozoan morphological development. Several avenues for future research are suggested by the synthesis of information from prior studies of hydrozoans. Elucidating the morphogenetic signaling pathways responsive to metabolites or hydromechanical forces and the epigenetic effect of vascular architecture on colony form may give new insight into the self-maintenance of indeterminately growing and continuously developing vascular systems.
... 1.3 and 1.4). These residues are also present in p53 sequences of the Pacific oyster Crassostrea gigas (Farcy et al., 2008) and the surf clam Spisula solidissima (Cox et al., 2003). In molluscs, the putative Mdm2 binding site is identical for both p53-and p63/73-like proteins. ...
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.
... p53 family in development p53, p63 and p73 are conserved during evolution between species. For example, p63 gene has been identified at least in mouse, human, Xenopus, zebrafish, chick and a marine mollusc (Lu et al. 2001; Lee and Kimelman 2002; Bakkers et al. 2002; Yasue et al. 2001; Cox et al. 2003). Mice engineered to have p53 deletions have multiple types of tumors (increased risk of tumorigenesis) (Donehower et al. 1992). ...
... Homologues for human p53 and the p53 family have been cloned in several bivalves, e.g. Mya arenaria (Kelley et al. 2001), Spisula solidissima (Cox et al. 2003), Crassostrea rhizohorae (GenBank accession no. AY442309), Crassostrea gigas (GenBank AM236465), Mytilus edulis (Ciocan & Rotchel 2005, Muttray et al. 2005 and Mytilus trossulus (Muttray et al. 2005), and have shown highly conserved regions. ...
High prevalence of disseminated neoplasia has been found in cockles Cerastoderma edule of Galicia (NW Spain). Disseminated neoplasia has been associated with high mortalities of various bivalve species. In vertebrates, proteins such as p53 and heat shock proteins (HSPs) play important roles in carcinogenesis. The protein p53 has been detected in neoplastic cells of bivalve molluscs such as Mytilus edulis, Mytilus trossulus, Mya arenaria, Spisula solidissima, Crassostrea rhizophorae and Crassostrea gigas. In this study, western blotting analyses were used to test the expression of Hsp70, Hsp90 and mutant p53 proteins in the cells and plasma of the haemolymph of cockles showing various intensities of neoplasia. Disseminated neoplasia was previously diagnosed by examination of stained haemolymph monolayers with light microscopy. In the present study, mutant p53 was detected in haemolymph cells of cockles diagnosed as affected by moderate and heavy neoplasia intensity, whereas it was not detected in cockles with either no or light neoplasia. The higher the neoplasia intensity, the higher the levels of Hsp70 and Hsp90. These proteins were not found in plasma. The results reveal the possible association between p53 and HSPs in neoplastic cells of cockles, which could prevent p53 from carrying out its functions, as occurs in human cancers.
... p53 superfamily coding sequences have been published for six mollusc species, two in the soft-shell clam Mya arenaria (Kelley et al. 2001), five each for the mussels Mytilus edulis and Mytilus trossulus, including the only known deltaN isoforms in invertebrates (Muttray et al. 2005;Muttray et al. 2007), two in the Hawaiian bobtail squid Euprymna scolopes (Goodson et al. 2006), four in the surf clam Spisula solidissima (Jessen-Eller et al. 2002;Cox et al. 2003), and one in the Pacific oyster Crassostrea gigas (Farcy et al. 2008). Interestingly, whereas within some species a number of different proteins have been identified, it appears that all isoforms may arise from alternate splicing as regions that overlap between different proteins from the same species are 100% identical as seen in Mytilus trossulus and Mytilus edulis (Muttray et al. 2007), Mya arenaria (Kelley et al. 2001), Euprymna scolopes (Goodson et al. 2006), and possibly S. solidissima (Jessen-Eller et al. 2002). ...
The origin of the p53 superfamily predates animal evolution and first appears in unicellular Flagellates. Invertebrate p53 superfamily members appear to have a p63-like domain structure, which seems to be evolutionarily ancient. The radiation into p53, p63, and p73 proteins is a vertebrate invention. In invertebrate models amenable to genetic analysis p53 superfamily members mainly act in apoptosis regulation in response to genotoxic agents and do not have overt developmental functions. We summarize the literature on cnidarian and mollusc p53 superfamily members and focus on the function and regulation of Drosophila melanogaster and Caenorhabditis elegans p53 superfamily members in triggering apoptosis. Furthermore, we examine the emerging evidence showing that invertebrate p53 superfamily proteins also have functions unrelated to apoptosis, such as DNA repair, cell cycle checkpoint responses, compensatory proliferation, aging, autophagy, and innate immunity.
... Some of these mutations have been shown to result in dysfunctional p53 proteins thus contributing to the deregulation of cell proliferation and the progression of cancer [20]. Deduced amino acid sequences and proteins homologous to mammalian p53, p63 and p73 have been identified in clams and mussels [21][22][23][24][25]. It has been suggested that there is only one gene in mollusks from which p53-like and p63/p73-like mRNA isoforms are expressed, although several conflicting papers have been published [26][27][28]. ...
Several bivalve species, including mussels (Mytilus spp.) and clams (Mya spp.), are susceptible to a leukemia-like disease called haemic neoplasia that has been known to decimate whole populations. Previous studies of molecular processes associated with late stages of this disease have implicated analogs of the p53 tumour suppressor protein family in disease etiology. We detected synonymous single nucleotide polymorphisms (SNPs) in the coding region sequence of p53-like cDNA from Mytilus trossulus (bay mussel) that differ between normal and neoplastic haemolymph. SNPs were located at positions 182, 392 and 821 bp. Most (94%) of the late leukemic animals sampled from cages in Burrard Inlet (British Columbia, Canada) had the same p53-like genotype, C182T G392G C821T, whereas 75% of the healthy animals were homozygous at positions C182C and T821T, independent of the genotype at the 392 bp position. As well, we detected an increased number of allelic variants in the leukemic animals that may arise from separate somatic mutation events in haemocyte precursors or from additional p53-like gene copies in polyploidy. Therefore, detection of these SNPs may provide a useful genetic biomarker for efficient monitoring of mussel population health.
... Its sister proteins p63 and p73 have important functions in embryonic development (Yang et al. 2002). Homologous proteins have been isolated in a number of invertebrates (Schmale et al. 1997; Kelley et al. 2001; Cox et al. 2003; Muttray et al. 2005; Goodson et al. 2006; Muttray et al. 2007); functional domains exhibit a high degree of similarity to their vertebrate homologues. We have shown previously that p53 mRNA and an N-terminal truncated isoform, ΔNp63/73, were expressed during late-stage haemic neoplasia in M. trossulus at levels higher than those seen in normal cells (Muttray et al. 2008). ...
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.
... As already discussed by other authors (Goodson et al., 2006), this suggests we deal here with alternative sequences originating from the same gene, which is not surprising given that all p53 family genes are able to express alternative products (MurrayZmijewski et al., 2006). Protein alignments showed all the domains, motifs and conserved residues already discussed by other authors (Kelley et al., 2001; Jessen-Eller et al., 2002; Cox et al., 2003; Muttray et al., 2005; Goodson et al., 2006;). The facts that drew our attention were: ...
Genes of the p53 family are known to be critical regulators of the cell cycle. They have already been established as possible biomarkers. Elaborate regulation mechanisms result in numerous cDNA and protein isoforms being expressed from each gene of the p53 family. Their similarity caused an often misleading nomenclature in non-vertebrate species. The aim of the present work is a clarification of the nomenclature of molluscan p53 family sequences, an essential prerequisite for reliable interpretation of gene expression and protein function studies. Here, we report five partial cDNA and one partial genomic p63 sequences, all originating from two Mytilus galloprovincialis individuals. DNA, deduced protein sequences, and the exon/intron architecture were analyzed and compared to p53, p63 and p73 sequences from other organisms. Along with our sequences, we analyzed all similar molluscan sequences found in the GenBank database. The analysis showed our cDNA sequences code for the TAp63gamma isoform of the p63 protein, and identified all other molluscan p53 family sequences as p63 genes or their expression isoforms. Our results also indicate p63 as the ancestral gene of the p53 family as well as the only gene of the family present in non-chordate metazoan species.
... arenaria, GenBank Accession Nos. AF253323 and AF253324) (23), three for surf clam (Spisula solidissima, AF285104, AY289767, and AY289768) (24,25), one for squid (Loligo forbesi, U43595), and one for oyster (Crassostrea rhizophorae, AY442309). ...
Mussels are susceptible to a wide range of environmental toxicants, including carcinogens, and thus are often employed as bioindicator species. To elucidate the molecular aetiology of such neoplastic damage, we have cloned Mytilus edulis homologues of the vertebrate ras proto-oncogene, and p53 tumor suppressor gene. The M. edulis ras cDNA encodes a predicted protein of 184 amino acids. The DNA sequence analysis with vertebrate ras sequences demonstrates that the M. edulis ras cDNA is highly conserved in regions of functional importance, including mutational hot spots. The partial p53 sequence also demonstrates that M. edulis p53 is highly conserved in two regions of functional importance and that these regions also include four of the five mutational hot spots for this gene. In contrast, the M. edulis p53 sequence shows little similarity to the other published invertebrate p53-like sequences. The cancer gene sequences characterized herein will allow development of specific biomarkers of genotoxic damage.
... It is interesting to note that outside vertebrates, p53-like sequences have only been found as single genes. Noteworthy, while some of the invertebrate p53-like sequences reported to date appear more similar in domain organization (i.e., lacking a SAM domain) to the vertebrate p53 (e.g., the p53-like sequences from the fly Drosophila melanogaster [Ollmann et al. 2000], the nematode Caenorhabditis elegans [Schumacher et al. 2001], and the unicellular amoeba Entamoeba histolytica [Mendoza et al. 2003]), others (i.e., the p53-like sequences from mollusks; Cox et al. 2003; Muttray et al. 2005) are more similar to the vertebrate p63/ p73 counterparts (i.e., they contain a SAM domain). Considering the phylogenetic distribution of p53-like sequences, the structural and functional differences between the p53 and p63/p73 paralogs, and the recently emerging picture of cooperative and internecine interactions among the p53 family members, several questions arise. ...
The p53 tumor suppressor plays the leading role in malignancy and in maintaining the genome's integrity and stability. p53 belongs to a gene family that in vertebrates includes two additional members, p63 and p73. Although similar in sequence, gene structure, and expression potential, the three p53 members differ in domain organization (in addition to the transactivation, DNA-binding, and tetramerization domains, p63 and p73 encode a sterile alpha motif, SAM, domain) and functional roles (with p63 and p73 assuming additional key roles in development). It is interesting to note that outside vertebrates, p53-like sequences have only been found as single genes, of either the p53 or the p63/p73 type (i.e., without or with a SAM domain, respectively). In this paper, we report that the diversification of this family is not restricted to the vertebrate lineage, as both a p53- and a p63/p73-type sequence are present in the unicellular choanoflagellate, Monosiga brevicollis. Furthermore, multiple independent duplication events involving p53-type sequences took place in several other animal lineages (cnidarians, flat worms, insects). These findings argue that selective factors other than those associated with the evolution of vertebrates are also relevant to the diversification of this family. Understanding the selective pressures associated with the multiple independent duplication events that took place in the p53 family and the roles of p53-like proteins outside vertebrates will provide further insight into the evolution of this very important family. In addition, the presence of both a p53 and a p63/73 copy in the unicellular M. brevicollis argues for its suitability as a model system for elucidating the functions of the p53 members and the mechanisms associated with their functional diversification.
... Moreover, human p53 and p73 sequences are not identical where they overlap, so that human p53 is more similar to mouse p53 than to human p73. In molluscs, several authors highlighted the putative existence of p53, p63 and p73 isoforms (Jessen-Eller et al., 2002; Stephens et al., 2001; Kelley at al, 2001; Cox et al., 2003; Muttray et al., 2007). Surprisingly, the data thus far (Mya sp. and Mytilus sp.) show that the central DNA binding domain is 100 percent conserved between all members of the p53 family within one species. ...
Like other sessile filter-feeding molluscs, oysters may be exposed in the natural environment to a variety of contaminants. Long-term exposure to pollutants may be one factor affecting prevalence of cancerous-like disorders, such as neoplasia. Environmentally induced alterations in p53 protein expression, in relation to leukemia, have been reported in various mollusc species inhabiting polluted water, suggesting that p53 proteins can also be used as a marker for environmental research. This work reports the cloning and sequencing of a p53-like cDNA in the mollusc bivalve Crassostreagigas. The deduced amino acid sequences of p53 shared a high degree of homology with the homologues from other mollusc species, including typical eukaryotic p53 signature sequences. We examined the p53 transcription expression pattern during the annual cycle in oyster gills and whole soft tissues in four locations along the French coasts. Real-time PCR analysis suggested that strong variations at p53 mRNA level are probably synchronized with the seasonal cycle at the four locations investigated.
The purpose of this chapter is to review the neoplasms (or tumors) affecting different species of clams. We include as "clams" any bivalve mollusc other than oysters, mussels and scallops. Two types of neoplasm have been described in clams: disseminated neoplasms and germinoma (one histotype of gonadal neoplasms). Disseminated neoplasm, the type prevalent in bivalve molluscs, including clams, involves the extensive proliferation of circulating abnormal cells (neoplastic cells) of unknown origin, through the tissues of the organisms. Germinomas comprise the abnormal proliferation of altered immature germ cells (neoplastic cells). Some clam species and populations present epidemic levels of neoplasms (Mya arenaria, Mercenaria spp., Cerastoderma edule and Venerupis aurea), while others are less affected (Venerupis decussata (=Ruditapes decussatus), Ensis magnus (=E. arcuatus) and Ensis siliqua). The etiology of neoplasms is unknown, some studies suggesting the implication of an infectious agent and other factors in the case of disseminated neoplasms. By contrast, only stress factors seem to be involved in gonadal neoplasms. In recent years, many studies have been carried out focusing on the molecular basis of this disease.
Disseminated neoplasia (DN), an oyster disease resembling leukaemia, has been reported in a number of species of marine bivalve molluscs. The disease is characterised by a proliferation of abnormal circulating cells of unknown origin resulting in the invasion of tissues and organs, frequently with a fatal end of the affected individuals. To obtain a more comprehensive view of bivalve cancer processes, suppressive subtracted hybridisation (SSH) and quantitative RT-PCR (q-PCR) approaches were combined to investigate changes in the transcriptome of Ostrea edulis haemolymph cells associated to DN. Two SSH libraries were constructed and 587 expressed sequence tags (ESTs) were sequenced, obtaining 329 ESTs which showed expression changes in neoplastic process. Transcription expression analyses (q-PCR) were done for a total of 24 genes that could be relevant in neoplastic process, including genes with role in the regulation of cell cycle, apoptosis or chromosomal defects. Most of those genes had not been reported in association with cancer in non-vertebrate organisms. The over-expression and under-expression of some of those genes in DN-affected oysters was in agreement with observations in vertebrate cancer. The results herein reported contribute to cancer understanding in bivalve molluscs.
Author Posting. © National Research Council Canada, 2005. This article is posted here by permission of National Research Council Canada for personal use, not for redistribution. The definitive version was published in Canadian Journal of Fisheries and Aquatic Sciences 62 (2005): 2055-2066, doi:10.1139/F05-119. Evaluating patterns of expression of p53-related proteins in cells is a novel approach in defining environmentally linked diseases. We have examined the induction of haemocytic leukemia in Mytilus edulis by municipal and industrial contaminants in Pictou Harbour, Nova Scotia, Canada. We used a murine monoclonal antibody, 1E10, as a diagnostic reagent to detect leukemic cells. We first characterized the reactivity of 1E10 with both normal and leukemic Mytilus haemocytes by confocal microscopy. We then compared p53 gene family expression (p53, p63–p73, and p97) in normal versus leukemic haemocytes using a panel of monoclonal and polyclonal antibodies to p53 family proteins. The immunochemical data demonstrate that haemocytic leukemia cells of M. edulis differentially express p63–p73 and p97–p120 proteins. We subsequently used 1E10 to diagnose haemocytic leukemia in 500 M. edulis previously deployed 6 months earlier in Pictou Harbour. In the field, Mytilus caged near untreated municipal wastewater and bleached kraft pulpmill effluents have a significantly greater chance of developing haemocytic leukemia than do mussels exposed to reference sites. This research was funded in part by the Pictou Harbour Biomonitoring Project, Fisheries and Oceans Canada, and Environment Canada.
Within hours of hatching, the squid Euprymna scolopes forms a specific light organ symbiosis with the marine luminous bacterium Vibrio fischeri. Interactions with the symbiont result in the loss of a complex ciliated epithelium dedicated to promoting colonization of host tissue, and some or all of this loss is due to widespread, symbiont-induced apoptosis. Members of the p53 family, including p53, p63, and p73, are conserved across broad phyletic lines and p63 is thought to be the ancestral gene. These proteins have been shown to induce apoptosis and developmental morphogenesis. In this study, we characterized p63-like transcripts from mRNA isolated from the symbiotic tissues of E. scolopes and described their role in symbiont-induced morphogenesis. Using degenerate RT-PCR and RACE PCR, we identified two p63-like transcripts encoding proteins of 431 and 567 amino acids. These transcripts shared identical nucleotides where they overlapped, suggesting that they are splice variants of the same gene. Immunocytochemistry and Western blots using an antibody specific for E. scolopes suggested that the p53 family members are activated in cells of the symbiont-harvesting structures of the symbiotic light organ. We propose that once the symbiosis is initiated, a symbiont-induced signal activates p53 family members, inducing apoptosis and developmental morphogenesis of the light organ.
The p53 family of transcription factors has been implicated in many vertebrate cancers. Altered p53 and p73 protein expression observed in leukemic cells of molluscs suggests that these transcription factors might be involved in invertebrate cancers as well. Here, we fully characterize the mRNA of four novel p53-like variants in the bivalve molluscs Mytilus trossulus (bay mussel) and Mytilus edulis (blue mussel). These species, widely used for environmental assessment, develop a hemic neoplasia (leukemia) that is frequently fatal. The correlation between expression of p53 and its close relative p73 and onset of molluscan leukemia was documented previously. We report the sequences of two distinct and novel p63/p73-like mRNAs, amplified by polymerase chain reaction (PCR) from both species. One of the p63/p73-like isoforms contains a 360 nt truncation in the 5' coding region. Based on this truncation and concomitant lack of a transactivation (TA) domain, we designate this variant as a DeltaNp63/p73-like isoform: the first to be reported in an invertebrate species. In mammalian species, DeltaNp73 potently inhibits the tumor-suppressive function of p73 and p53, and its overexpression serves as a robust marker for mammalian cancer. In addition, we report on the occurrence of alternate polyadenylation sites in the molluscan p63/p73: one proximal and one distal site, which differ by 1260 nt. We hypothesize that differential expression of various molluscan p63/p73-like isoforms, controlled in part by polyadenylation site choice variation, may help to interpret the apparently opposing roles of this gene in the development of cancer. Overall, this research further illustrates the utility of the molluscan model for studies involving the molecular mechanisms of oncogenesis in naturally occurring populations. The data presented here require a revisiting of hypotheses regarding evolution of the p53 gene family. Current hypotheses indicate that (1) the protostome gene family does not contain an intronic promoter for DeltaN expression and (2) p53 gene duplication did not occur in protostomes. Our characterization of DeltaN p63/73 in mussel suggests that molluscan p53 gene family members have acquired an intronic promoter or splicing mechanism, either by invention that predates the evolutionary split of deuterostomes from protostomes, or by parallel evolution. Our data also show that Mytilus p53, p63/p73, and DeltaNp63/p73 are identical in their core regions with variation limited to their C- and N-terminals, supporting the notion that alternative splicing, intronic promoter usage, and polyadenylation site choice may lead to expression of distinct isoforms originating from one common gene.
The tumor suppressor p53 is mutated in approximately 50% of all human cancer cases worldwide. It is commonly assumed that the phylogenetic history of this important tumor suppressor has been thoroughly studied; however, few detailed studies of the entire extended p53 protein family have been reported, and none comprehensively and simultaneously consider functional, molecular, and phylogenetic data. Herein we examine a diverse collection of reported p53-like protein sequences, including representatives from the arthropods, nematodes, and protists, with the goal of answering several important questions. First, what evidence supports these highly divergent proteins being true homologues to the p53 family? Second, is the inferred overall family phylogeny concordant with known structures and functions? Third, does the extended p53 family possess recognizable conserved sites outside of the within-chordate, highly-conserved DNA-binding domain? Our study shows that the biochemical and functional evidence of p53 homology for nematodes, arthropods, and protists is inconsistent with their implied phylogenetic relationship within the overall family. Although these divergent sequences are always reported as functionally similar to human p53, our results confirm and extend the hypothesis that p63 is a far more appropriate protein for comparison. Within these divergent sequences, we find minimal conservation within the DNA-binding domain, and no conservation elsewhere. Taken together, our findings suggest that these sequences are not bona fide homologues of the extended p53 family and provide baseline criteria for the future identification and characterization of distant p53-family homologues.
The tumour-suppressor gene p53 is frequently mutated in human cancers and is important in the cellular response to DNA damage. Although the p53 family members p63 and p73 are structurally related to p53, they have not been directly linked to tumour suppression, although they have been implicated in apoptosis. Given the similarity between this family of genes and the ability of p63 and p73 to transactivate p53 target genes, we explore here their role in DNA damage-induced apoptosis. Mouse embryo fibroblasts deficient for one or a combination of p53 family members were sensitized to undergo apoptosis through the expression of the adenovirus E1A oncogene. While using the E1A system facilitated our ability to perform biochemical analyses, we also examined the functions of p63 and p73 using an in vivo system in which apoptosis has been shown to be dependent on p53. Using both systems, we show here that the combined loss of p63 and p73 results in the failure of cells containing functional p53 to undergo apoptosis in response to DNA damage.
Two-hybrid screening in yeast with p73α isolated SUMO-1 (small ubiquitin-likemodifier 1), the enzyme responsible for its conjugation, Ubc-9, and a number of novel SUMO-1-interacting proteins, including thymine
DNA glycosylase, PM-Scl75, PIASx, PKY, and CHD3/ZFH. A subset of these proteins contain a common motif,hhXSXS/Taaa, where h is a hydrophobic amino acid and a is an acidic amino acid, that is shown to interact with SUMO-1 in the two-hybrid system. We show here that p73α, but not
p73β, can be covalently modified by SUMO-1. The major SUMO-1-modified residue in p73α is the C-terminal lysine (Lys627). The sequence surrounding this lysine conforms to a consensus SUMO-1 modification siteb(X)XXhKXE, whereb is a basic amino acid. SUMO-1-modified p73 is more rapidly degraded by the proteasome than unmodified p73, although SUMO-1
modification is not required for p73 degradation. SUMO-1 modification does not affect the transcriptional activity of p73α
on an RGC-luciferase reporter gene in SK-N-AS cells. Instead, SUMO-1 modification may alter the subcellular localization of
p73, because SUMO-1-modified p73 is preferentially found in detergent-insoluble fractions. Alternatively, it may modulate
the interaction of p73 with other proteins that are substrates for SUMO-1 modification or which interact with SUMO-1, such
as those identified here.
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
The mRNAs of transiently expressed genes frequently contain an AU-rich sequence in the 3' untranslated region. We introduced a 51 nucleotide AT sequence from a human lymphokine gene, GM-CSF, into the 3' untranslated region of the rabbit beta-globin gene. Our experiments demonstrate that this caused the otherwise stable beta-globin mRNA to become highly unstable in vivo. The instability conferred by the AU sequence in the mRNA was partially alleviated by treatment of the cells with cycloheximide. We propose that the AU sequences are the recognition signal for an mRNA processing pathway which specifically degrades the mRNAs for certain lymphokines, cytokines, and proto-oncogenes.
The polypeptides of the human erythrocyte membrane were analyzed by polyacrylamide gel electrophoresis in 1% sodium dodecyl sulfate. Six major bands (I-VI) together make up over two-thirds of the protein staining profile. Component III (mol wt 89,000) predominates in the ghost membrane; it constitutes 30% of the protein and numbers over 106 chains/ghost. Components I and II form a slow-moving doublet (approximate mol wt 250,000) containing 25% of the protein. The molar amounts of I + II, IV (mol wt 77,500), V (mol wt 41,300), and VI (mol wt 36,200) are similar, falling in the range 3.4-4.6 × 105 chains/ghost. Four bands were recognized in gels stained by the periodic acid-Schiff procedure. A broad Schiff-positive zone just behind the tracking dye corresponds to membrane lipids. Three bands of lower mobility are sialoglycoproteins. The most prominent of these has an apparent molecular weight of 83,500 and contains at least 57% of the sialic acid of ghosts. The Schiff-positive bands were not colored by protein stains. Sialidase treatment of ghosts selectively increased the mobilities of the sialoglycoproteins without affecting the protein-staining profile. Attempts to produce subunits from the large polypeptides by treatment with various denaturing agents were unsuccessful. Normally, no polypeptides of size less than 15,000 were seen in ghost electrophorograms. However, heating ghosts with low levels of sodium dodecyl sulfate and high levels of salt produced diffuse bands of low average molecular weight. This highly variable effect is attributed to degradation by proteinases. Components I, II, and V were solubilized by incubating ghosts at low ionic strength. Component VI was released by washing with buffered saline at concentrations above 0.1 M. Both elution procedures were rapid (15 min), complete, and selective; they were also conservative in that new bands were not created and the electrophorograms of released and retained material were complementary. The eluted material contained negligible sialic acid and no Schiff-positive lipids. Two classes of membrane protein were distinguished by their response to the elution procedures. Components I, II, V, and VI compose one class. They make up 30-35 % of the protein and are tenuously related to the membrane, possibly by predominantly ionic bonds. The second class, which includes components III, IV, and the sialoglycoproteins, together with various minor components, constitutes 65-70% of the protein. These polypeptides are tightly bound; their properties may reflect participation in hydrophobic protein-protein and protein-lipid interactions.
AU-rich sequence motifs (specifically sequences containing reiterations of AUUUA) are found in the 3' untranslated region of mammalian mRNAs encoding cytokines, adhesion molecules, and protooncogenes. Because these AU-rich elements (3'AURE) have been observed to reduce the stability and translational efficiency of transcripts that contain them, and because many of these transcripts accumulate in cells exposed to inflammatory stimuli, we reasoned that mRNAs with 3'AURE may be highly conserved and that the AURE is a marker of mRNAs that are inducible by environmental stressors. To test this hypothesis, we developed a polymerase chain reaction (PCR) strategy to isolate specifically mRNAs with 3'AURE. We first validated the effectiveness of this approach by selectively amplifying two mRNAs containing 3'AURE from interleukin 1 (IL-1)-induced human endothelial cells, then used the same primers in reverse transcriptase-PCR of sea urchin RNA, and used the radiolabeled reaction products to screen a cDNA library prepared from endotoxin-exposed sea urchin coelomocytes. We identified 124 positive clones and isolated a 1608-base-pair fragment that contains an AU-rich consensus sequence upstream from a poly(A) tail. This sea urchin transcript hybridizes with immobilized poly(A)(+)-selected RNA prepared from living coelomocytes maintained in vitro for 8.5-13 h but not with RNA prepared from freshly harvested coelomocytes. Our results provide support for the growing body of evidence that 3' AURE are both conserved and functional and indicate further that isolation and short-term in vitro culture of sea urchin coelomocytes is sufficient to induce the expression of transcripts containing 3'AURE.
Many genes have been described and characterized which result in alternative polyadenylation site use at the 3'-end of their mRNAs based on the cellular environment. In this survey and summary article 95 genes are discussed in which alternative polyadenylation is a consequence of tandem arrays of poly(A) signals within a single 3'-untranslated region. An additional 31 genes are described in which polyadenylation at a promoter-proximal site competes with a splicing reaction to influence expression of multiple mRNAs. Some have a composite internal/terminal exon which can be differentially processed. Others contain alternative 3'-terminal exons, the first of which can be skipped in some cells. In some cases the mRNAs formed from these three classes of genes are differentially processed from the primary transcript during the cell cycle or in a tissue-specific or developmentally specific pattern. Immunoglobulin heavy chain genes have composite exons; regulated production of two different Ig mRNAs has been shown to involve B cell stage-specific changes in trans -acting factors involved in formation of the active polyadenylation complex. Changes in the activity of some of these same factors occur during viral infection and take-over of the cellular machinery, suggesting the potential applicability of at least some aspects of the Ig model. The differential expression of a number of genes that undergo alternative poly(A) site choice or polyadenylation/splicing competition could be regulated at the level of amounts and activities of either generic or tissue-specific polyadenylation factors and/or splicing factors.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons,
a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST
programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering
the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program
that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining
statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using
this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration
as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST
is used to uncover several new and interesting members of the BRCT superfamily.
The p63 gene, a homologue of the tumour-suppressor p53, is highly expressed in the basal or progenitor layers of many epithelial tissues. Here we report that mice homozygous for a disrupted p63 gene have major defects in their limb, craniofacial and epithelial development. p63 is expressed in the ectodermal surfaces of the limb buds, branchial arches and epidermal appendages, which are all sites of reciprocal signalling that direct morphogenetic patterning of the underlying mesoderm. The limb truncations are due to a failure to maintain the apical ectodermal ridge, a stratified epithelium, essential for limb development. The embryonic epidermis of p63-/- mice undergoes an unusual process of non-regenerative differentiation, culminating in a striking absence of all squamous epithelia and their derivatives, including mammary, lacrymal and salivary glands. Taken together, our results indicate that p63 is critical for maintaining the progenitor-cell populations that are necessary to sustain epithelial development and morphogenesis.
We report the sequence of the guinea pig p53 cDNA. The comparative analysis of the coding and noncoding regions of p53 cDNAs of all available complete vertebrate sequences has allowed us to single out new conserved signals possibly involved in p53 functional activity. We have focused our attention on the most variable region of the protein, the proline (P)-rich domain, suggested to play a fundamental role in antiproliferative pathways. In this domain we have identified the PXXXXP repeated motif and singled out a common consensus sequence that can be considered a signature for mammalian p53: PXXXXPX{0,4}PX{0,9}PA(T,P,I,)(S,P)WPL. We have demonstrated the significance of the PXXXXP motif in SH3-binding protein and suggested its structure to be a loop. Also, the 5' and 3' untranslated regions (UTRs) of the guinea pig were sequenced, and this study represents the first detailed structural analysis of the UTRs of the p53 mRNAs available in literature. The 5' UTR of guinea pig (233 nt) can be folded into a stable secondary structure resembling that predicted in mouse. The 3' UTR of guinea pig is 771 nt long and shows higher similarity with human than with rodent sequences, having a region of about 350 nt that is deleted in rat and mouse. In the 3' UTR we have identified the presence of a mammalian-wide interspersed repeat sequence and of a cytoplasmic polyadenylation element, which could be involved in translational activation by promoting polyadenylation of mRNA, providing information about a possible mechanism of regulation of p53 expression mediated by the 3' UTR of the mRNA. The observations presented here could open new avenues to targeted mutations and experimental approaches useful in investigating new regulation mechanisms of p53 translation, activity, and stability.
The 3' untranslated region of human p53 mRNA represses translation both in vitro and in vivo. Here, we identify a cis-acting 66-nucleotide U-rich sequence in the human p53 mRNA 3' untranslated region that mediates translational repression. Using UV cross-linking, we detect a 40 kDa protein that interacts specifically with the p53 3'UTR containing the repressor element. Enhanced translation of p53 mRNA contributes to the accumulation of p53 protein in cells exposed to gamma-radiation and could be a consequence of relieving the inhibition mediated by the repressor element.
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 Burkitt's lymphoma or other human cancers.
The cell-cycle checkpoint protein p53 both directs terminal differentiation and protects embryos from DNA damage. To study invertebrate p53 during early development, we identified three differentially expressed p53 family members (p53, p97, p120) in the surf clam, Spisula solidissima. In these mollusks, p53 and p97 occur in both embryonic and adult tissue, whereas p120 is exclusively embryonic. We sequenced, cloned, and characterized p120 cDNA. The predicted protein, p120, resembles p53 across all evolutionarily conserved regions and contains a C-terminal extension with a sterile alpha motif (SAM) as in p63 and p73. These vertebrate forms of p53 are required for normal inflammatory, epithelial, and neuronal development. Unlike clam p53 and p97, p120 mRNA and protein levels are temporally expressed in embryos, with mRNA levels decreasing with increasing p120 protein (R(2) = 0.97). Highest surf clam p120 mRNA levels coincide with the onset of neuronal growth. In earlier work we have shown that neuronal development is altered by exposure to polychlorinated biphenyls (PCBs), a neurotoxic environmental contaminant. In this study we show that PCBs differentially affect expression of the three surf clam p53 family members. p120 mRNA and protein are reduced the most and earliest in development, p97 protein shows a smaller and later reduction, and p53 protein levels do not change. For the first time we report that unlike p53 and p97, p120 is specifically embryonic and expressed in a time-dependent manner. Furthermore, p120 responds to PCBs by 48 hr when PCB-induced suppression of the serotonergic nervous system occurs.
Methylation at CpG dinucleotides in genomic DNA is a fundamental epigenetic mechanism of gene expression control in vertebrates. Proteins with a methyl-CpG-binding domain (MBD) can bind to single methylated CpGs and most of them are involved in transcription control. So far, five vertebrate MBD proteins have been described as MBD family members: MBD1, MBD2, MBD3, MBD4 and MECP2.
We performed database searches for new proteins containing an MBD and identified six amino acid sequences which are different from the previously described ones. Here we present a comparison of their MBD sequences, additional protein motifs and the expression of the encoding genes. A calculated unrooted dendrogram indicates the existence of at least four different groups of MBDs within these proteins. Two of these polypeptides, KIAA1461 and KIAA1887, were only present as predicted amino acid sequences based on a partial human cDNA. We investigated their expression by Northern blot analysis and found transcripts of ~8 kb and ~5 kb respectively, in all eight normal tissues studied.
Eleven polypeptides with a MBD could be identified in mouse and man. The analysis of protein domains suggests a role in transcriptional regulation for most of them. The knowledge of additional existing MBD proteins and their expression pattern is important in the context of Rett syndrome.
In coastal locations, marine invertebrates, primarily molluscs, develop fatal leukemias in their blood or hemolymph. In the clam Mya arenaria, non-adhesive, mitotic, spherical leukemia cells replace adhesive, motile, normal hemocytes as leukemia progresses. End-stage leukemia cells express a unique antigen, IE10, while normal cells express the 2A4 marker. The goals of this work were to further differentiate the normal and leukemia specific antigens relative to protein structure, determine if other protein distinctions exist, and examine p53 gene family expression in both cell types. Recognized by the monoclonal antibody 2A4, normal cells express a 185-kDa glycoprotein that may have multiple forms. Detected by the monoclonal antibody 1E10, leukemic cells express a very hydrophobic 252-kDa glycoprotein that is likely to be a transmembrane protein with spectrin/dystrophin-like characteristics. After normalization to the major cytoskeletal protein actin, sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals major distinguishing protein and glycoprotein differences between the two cell types. Most obvious is the near-absence of tubulin in the non-mitotic normal hemocytes. We have also characterized the expression of p53 gene family members in normal and end-stage leukemia cells, finding shifts in expression of the p53 gene homologues p73 and p97 coincident with leukemia-specific protein synthesis.
The crucial role of the non-coding portion of genomes is now widely acknowledged. In particular, mRNA untranslated regions are involved in many post-transcriptional regulatory pathways that control mRNA localization, stability and translation efficiency. We review in this paper the major structural and compositional features of eukaryotic mRNA untranslated regions and provide some examples of bioinformatic analyses for their functional characterization. (C) 2001 Published by Elsevier Science B.V.
Tubulin is the major protein found in the membrane/periaxonemal matrix fraction of mature sea urchin embryonic cilia but its distribution and possible function during ciliary assembly are unknown. Hypertonic salt may be used to deciliate the embryos, allowing synchronous regrowth of cilia and subsequent deciliation of the regenerating embryos at various times. During the earliest stages of regeneration, the amounts of tubulin in the axoneme and membrane/matrix fractions are nearly equal, but the proportion of tubulin in the axoneme fraction increases coincident with the quasi-linear growth phase while the membrane/matrix tubulin remains constant. Antibodies to tyrosinated and detyrosinated alpha-tubulin show that both the membrane/matrix and axonemal tubulin fractions are primarily unmodified (i.e. tyrosinated) at the earliest stages of regeneration but are progressively and equally detyrosinated coincident with regeneration, approaching a final level of 50% C-terminal Glu. A monoclonal antibody to acetylated alpha-tubulin reveals that both tubulin fractions are equally and maximally acetylated at relatively early stages of regeneration. In contrast, three-times-repolymerized tubulin from either unfertilized eggs or midgastrula embryos is primarily tyrosinated (greater than 97%) and not detectably acetylated. These data suggest that membrane/matrix tubulin is a precursor to axonemal tubulin and that acetylation and detyrosination may be involved in partitioning tubulin among cytoplasmic, ciliary membrane/matrix, and 9 + 2 compartments.
We describe a staining technique, using Ponceau S in very mild conditions, by which proteins can be visualized on nitrocellulose replicas without being permanently fixed to the membrane itself, thus allowing subsequent procedures such as immunoblotting or preparative elution of the proteins to be performed. This staining technique can detect 250 to 500 ng protein, which is essentially the same sensitivity seen for Coomassie blue staining of proteins on nitrocellulose. The Ponceau S staining technique was used to locate proteins on nitrocellulose replicas for subsequent in situ radioiodination and trypsin digestion, followed by separation of the resultant digests in two-dimensional peptide analysis. Staining proteins with Ponceau S did not interfere with either the radioiodination or trypsin digestion, as indicated by essentially identical peptide patterns being obtained for the internal protein p26 from equine infectious anemia virus, regardless of whether the digests were prepared from polyacrylamide gel slices or nitrocellulose sections. The combination of preparation of radioiodinated tryptic digests on nitrocellulose and subsequent two-dimensional analysis is sensitive enough to detect peptide additions and deletions occurring in the surface antigen gp90 recovered from two antigenically distinct strains of equine infectious anemia virus. Thus these procedures provide a relatively simple, inexpensive, and highly reproducible technique for the analysis of as little as 250 nanograms of protein after separation by electrophoresis in polyacrylamide gels.
Two modifications to Western blots which enhance immunochemical recognition have been developed. The first is transfer in carbonate buffer at pH 9.9, rather than the more commonly used Tris-glycine buffer at pH 8.3. This alteration improved the recognition of four of the five subunits of Escherichia coli F1-ATPase by monoclonal antibodies, the smaller subunits showing the greatest effects. Recognition of dinitrophenyl groups attached to the subunits by polyclonal antibodies was improved by the carbonate buffer only for the smallest ATPase subunit, epsilon. The second modification was incubation of the gel in mild buffers, designed to promote the renaturation of proteins, before the electrophoretic transfer step. The most effective buffer was 20% glycerol in 50 mM Tris-HCl, pH 7.4. Improvements in the signal obtained with monoclonal antibodies to all the subunits of ATPase were obtained by this procedure. As the subunits vary markedly in size, isoelectric point, and other properties, this method should be useful for most proteins. The fate of the 15,000-Da epsilon subunit, labeled with 125I, was followed through a blotting experiment. As long as no sodium dodecyl sulfate was added to the transfer buffer, epsilon was bound to nitrocellulose efficiently in either Tris-glycine or carbonate buffer. However, the epsilon was retained much more strongly during the subsequent incubation steps if the transfer was done in the carbonate buffer. The binding of epsilon to the nitrocellulose was even more stable when the gel had been treated with the buffered glycerol solution before transfer. These results indicate that the conditions under which epsilon subunit first encounters the nitrocellulose markedly affect the stability of binding during subsequent steps. The F1-ATPase was partially fragmented by treatment with proteases and then run on a gel and either transferred immediately in Tris-glycine buffer or else treated with the buffered glycerol solution and transferred in the carbonate buffer. The second blot gave stronger recognition of residual alpha subunit and fragments by an anti-alpha monoclonal antibody, with the largest improvement for the smaller fragments. This result suggests that the modified procedure may be particularly useful in enhancing the detection of small proteins.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment
of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order
to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are
varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific
gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather
than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced
gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new
program, CLUSTAL W which is freely available.
The cytoplasmic life of an mRNA revolves around the regulation of its localization, translation and stability. Interactions between the two ends of the mRNA may integrate translation and mRNA turnover. Regulatory elements in the region between the termination codon and poly(A) tail - the 3' untranslated region - have been identified in a wide variety of systems, as have been some of the key players with which these elements interact.
We describe a gene encoding p73, a protein that shares considerable homology with the tumor suppressor p53. p73 maps to 1p36, a region frequently deleted in neuroblastoma and other tumors and thought to contain multiple tumor suppressor genes. Our analysis of neuroblastoma cell lines with 1p and p73 loss of heterozygosity failed to detect coding sequence mutations in remaining p73 alleles. However, the demonstration that p73 is monoallelically expressed supports the notion that it is a candidate gene in neuroblastoma. p73 also has the potential to activate p53 target genes and to interact with p53. We propose that the disregulation of p73 contributes to tumorigenesis and that p53-related proteins operate in a network of developmental and cell cycle controls.
We describe the cloning of p63, a gene at chromosome 3q27-29 that bears strong homology to the tumor suppressor p53 and to the related gene, p73. p63 was detected in a variety of human and mouse tissues, including proliferating basal cells of epithelial layers in the epidermis, cervix, urothelium, and prostate. Unlike p53, the p63 gene encodes multiple isotypes with remarkably divergent abilities to transactivate p53 reporter genes and induce apoptosis. Importantly, the predominant p63 isotypes in many epithelial tissues lack an acidic N terminus corresponding to the transactivation domain of p53. We demonstrate that these truncated p63 variants can act as dominant-negative agents toward transactivation by p53 and p63, and we suggest the possibility of physiological interactions among members of the p53 family.
p53 plays an essential pro-apoptotic role, a function thought to be shared with its family members p73 and p63. Here, we show
that p73 is primarily present in developing neurons as a truncated isoform whose levels are dramatically decreased when sympathetic
neurons apoptose after nerve growth factor (NGF) withdrawal. Increased expression of truncated p73 rescues these neurons from
apoptosis induced by NGF withdrawal or p53 overexpression. In p73–/– mice, all isoforms of p73 are deleted and the apoptosis
of developing sympathetic neurons is greatly enhanced. Thus, truncated p73 is an essential anti-apoptotic protein in neurons,
serving to counteract the pro-apoptotic function of p53.
Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants that accumulate to toxic levels in the food chain. Using Spisula solidissima (surf clam) embryos as a developmental model, it was shown that Aroclor 1254 specifically targets two neuronal structures during embryonic development. Embryos were exposed to 1, 10), or 100 ppm Aroclor 1254 or an acetone vehicle control posthatching for 24, 48, and 72 h. Embryos labeled with a serotonin antibody or a neural antigen antibody and a rhodamine-conjugated secondary antibody were viewed by confocal microscopy. The cerebropleural ganglion showed a decrease both in serotonin production and in the size of the serotonin-synthesizing region upon exposure to 10 and 100 ppm Aroclor 1254. These decreases were detectable as early as 48 h postfertilization. When exposed to 100 ppm Aroclor 1254, the primitive neural plexus, which coordinates the movements of the mouth and velum, showed a delay in onset and cessation of expression of a molluscan-specific neural antigen. Exposure to Aroclor 1254 did not affect the overall growth and morphology of the embryos. In addition, analyses of total protein profiles and heat-shock protein 70 levels showed that exposure to Aroclor 1254 did not trigger protein degradation or cause a stress or shock response. These results show that exposure of Spisula embryos to Aroclor 1254 specifically targets neurogenesis while having no effect on the overall growth of the embryo.
The discoveries of the p53 homologs, p63 and p73, have both fueled new insights and exposed enigmas in our understanding of the iconic p53 tumor suppressor. Although the pivotal role of p53 in cancer pathways remains unchallenged, because p63 and p73 are now implicated in stem cell identity, neurogenesis, natural immunity and homeostatic control. Despite their seemingly separate tasks, there are hints that the p53 family members both collaborate and interfere with one another. The question remains, therefore, as to whether these genes evolved to function independently or whether their familial ties still bind them in pathways of cell proliferation, death and tumorigenesis.
p53 controls crucial stress responses that play a major role in preventing malignant transformation. Hence, inactivation of p53 is the single most common genetic defect in human cancer. With the recent discovery of two close structural homologs, p63 en p73, we are getting a broader view of a fascinating gene family that links developmental biology with tumor biology. While unique roles are apparent for each of these genes, intimate biochemical cross-talk among family members suggests a functional network that might influence many different aspects of individual gene action. The most interesting part of this family network derives from the fact that the p63 and p73 genes are based on the "two-genes-in-one" idea, encoding both agonist and antagonist in the same open reading frame. In this review, we attempt to present an overview of the current status of this fast moving field.
An anti-apoptotic role for the p53 family member p73 Towbin, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications
- C D Pozniack
- S Radinovic
- A Yang
- F Mckeon
- D R Kaplan
- F D H Miller
- T Staehelin
- J Gordon
Pozniack, C.D., Radinovic, S., Yang, A., McKeon, F., Kaplan, D.R., Miller, F.D., 2000. An anti-apoptotic role for the p53 family member p73 Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U. S. A. 76, 4350 – 4354.
Spisula p63/p73 alpha variant; Ssp63/73h, Spisula 63/73 beta variant; SUMO, small ubiquitin-related modifier; TA, transactivation domain; TI, transactivation-inhibitory domain; UTR, untranslated region. * Corresponding author
- Ssp
Ssp63/73a, Spisula p63/p73 alpha variant; Ssp63/73h, Spisula 63/73 beta variant; SUMO, small ubiquitin-related modifier; TA, transactivation domain; TI, transactivation-inhibitory domain; UTR, untranslated region. * Corresponding author. Tel.: +1-508-289-7517; fax: +1-508-540-6902.
On the shoulders of giants: p63, p73 and the rise of p53
- Yang
p63 and p73 are required for p53-dependent apoptosis in response to DNA damage
- Flores
Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- Salinovich
Sequence determinants in human polyadenylation site selection
- Legendre
An anti-apoptotic role for the p53 family member p73 during developmental neuronal cell death
- Pozniack
p63 is essential for regenerative proliferation in limb, cranio-facial and epithelial development
- Yang
p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage
- Yuan