Correspondence Comment on “Conservation of Cancer Genes in the Marine Invertebrate Mytilus edulis” The p53 protein is a well-known and highly conserved tumor suppressor (1), and genomic or cDNA sequences have been reported from many animal taxa in sequence databases. More recently, the p53 protein has been expanded to a family containing various other homologues such as p63, p73, and C- and N-terminally truncated isoforms of each protein. Databases provide helpful information on both gene and protein sequences and are therefore frequently used by researchers from many fields. However, errors in DNA and protein sequences submitted to sequence databanks have been found in the past (2). It is important to recognize these sequence errors in cases where polymorphisms and protein isoforms exist and where conserved domains are used to align new sequences to previously annotated sequences. Ciocan and Rochette (3) described partial cDNA sequences of p53 in the mussel Mytilus edulis. Concurrently, our group described complete p53 cDNA sequences in Mytilus edulis and the closely related mussel species Mytilus trossulus (4). In the latter study, p53 amino acid sequences of the two different Mytilus sp. were almost 100% homologous. However, despite both laboratories using a similar experimental approach the Mytilus edulis p53 cDNA sequence obtained in ref 4 was quite different from that reported in ref 3. Ciocan and Rotchell (3) obtained a single 480 bp product using 3′RACE with a degenerate p53 primer based on the alignment of p53 sequences in zebra fish (Danio rerio) and barbel (Barbus barbus). They performed two separate alignments of the partial p53 sequence and concluded, based on amino acid homology, that the new M. edulis p53 sequence was more closely related to the fish than to the invertebrate counterparts. 5′RACE extension of their partial sequence was unsuccessful. Muttray et al. (4) similarly performed 3′ and 5′RACE, but with degenerate primers based on other known molluscan p53 and p73 (Mya arenaria, Spisula solidissima, Loligo forbesi). A single comprehensive alignment of the complete cDNA with thirty-three p53 sequences from across all known-to-date taxa placed this sequence in the invertebrate branch of a p53 phylogram (5). Our laboratory was curious as to where the two sequences differed and why they aligned to different branches of the p53 phylogenetic tree. We therefore performed an alignment of the M. edulis p53 by Muttray et al. (4) (Genbank Accession number AY579472, from now on referred to as Mep53a) and the partial M. edulis p53 sequence by Ciocan and Rotchell (3) (AY705932, from now on referred to as Mep53b) to Mollusca and Ostariophysi, and one representative of each other known p53 branch, such as Oncorhynchus mykiss (Neoteleostei), Homo sapiens (Mammalia), Xenopus laevis (Amphibia), and Drosophila melanogaster (Insecta) (Figure 1). Mep53a has 62-100% identity with other mollusks and 30-38% with vertebrates, whereas, Mep53b has 31-37% identity with other mollusks and 47-78% with vertebrates, which is unexpected. The differences in sequence between Mep53a and Mep53b are clearly above average polymerase error inherent in the sequencing reaction. 10.1021/es062950b CCC: $37.00 Published on Web 05/27/2007
2007 American Chemical Society
The origins of the mussels used for the experiments are different for the two laboratories: Muttray et al. (4) obtained M. edulis from a shellfish grower (Island Scallops, Qualicum Beach, British Columbia) who had previously obtained certified M. edulis from Prince Edward Island (Canada, Atlantic coast). Subsequently, these mussels were confirmed to be M. edulis by ITS restriction fragment length polymorphism (unpublished data, our laboratory). Ciocan and Rochette collected M. edulis from the English Channel near Brighton, UK. However, even though there may be some sequence divergence between the two M. edulis populations, it can reasonably be expected that any difference would be less than the difference seen between M. edulis and M. trossulus (96.6% and 99.8% sequence identity at the nucleic acid and protein level, respectively) or their hybrids. We then considered the possibility that different homologues of the p53 family were sequenced by the two laboratories. Kelley et al. (6) showed for the first time that there are at least two isoforms present in softshell clam Mya arenaria, p53 and p73-like. Our laboratory recently isolated p63/p73-like and DeltaNp63/p73-like cDNA sequences in both M. edulis and M. trossulus (7). In both cases, the core DNA binding domain of the molluscan p53, p63/p73-like sequences are identical in each species, suggesting that the isoforms are produced from one gene in mollusks (8). This would exclude the possibility that the Mep53b partial sequence, which contains the DNA binding domains IV and V, is one of the p53 family homologues in M. edulis. This then leaves us with the question of whether DNA contamination could possibly have contributed to the dissimilarity in the Mep53a and Mep53b sequences. For that, it may be helpful to recount our group’s experience with DNA contamination during the isolation of mussel p53: We initially attempted to obtain a Mytilus p53 sequences in a laboratory that has extensively worked on p53/p73 in the clam species Mya arenaria and Spisula solidissima. Despite commonly used precautions and controls, we initially obtained a p53 nucleic acid sequence highly similar to a Spisula solidissima p63/p73-like sequence (AY289767) (9). We then repeated the experiments in our Vancouver-based laboratory using new stock solutions and primers and obtained the published Mytilus p53 sequence (AY579472), which is different from both Mya arenaria p53/p73 and Spisula solidissima p63/p73 in the core DNA binding as well as other domains. Polymerase chain reaction contamination is not uncommon ((10) and others). We have to conclude that contamination by DNA from other species regularly used in a laboratory is a very real possibility and has to be carefully managed and controlled. We suggest that this may have been the cause for a M. edulis partial p53 sequence grouping with the vertebrate Ostariophysi p53 in a phylogram, instead of with Mollusca (invertebrate) as would have been expected. To cite the conclusion of a previous, third-party, review of another sequence database error (11): “Many genes in the databanks are represented by more than one entry. We would, therefore, recommend that database users consult more than one entry for comparison in order to receive reliable information on any gene sequence analysis.” VOL. 41, NO. 13, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Multiple alignment of DNA binding domains IV, V and tetramerization domain of p53 from mollusks M. edulis (Mep53a and b), M. trossulus (Mtp53), M. arenaria (Map53), and L. forbesi (Lfp53), from ostariophysi representatives B. barbus (Bbp53) and D. rerio (Drp53), from neoteleostei representative O. latipes (Olp53), from vertebrate groups H. sapiens (Hsp53) and X. laevis (Xlp53), and from D. melanogaster (Dmp53). Boxshade 3.21 (www.ch.embnet.org) was used to shade blocks of sequences where more than 0.3 of sequences must be identical (black) or similar (gray) for shading. DNA binding domains (DBD) and tetramerization domain are indicated. 4830
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Literature Cited (1) Mills, A. A. p53: Link to the past, bridge to the future. Genes Dev. 2006, 19, 2091-2099. (2) Roberts, L. Finding DNA sequencing errors. Science 1991, 252, 1255-1256. (3) Ciocan, C. M.; Rotchell, J. M. Concervation of cancer genes in the marine invertebrate Mytilus edulis. Environ. Sci. Technol. 2005, 39, 3029-3033. (4) Muttray, A. F.; Cox, R. L.; St-Jean, S. D.; van Poppelen, P.; Reinisch, C. L. Identification and phylogenetic comparison of p53 in two distinct mussel species (Mytilus). Comput. Biochem. Physiol., Part C: Pharmacol., Toxicol. Endocrinol. 2005, 140, 237-250. (5) Page, R. D. M. TREEVIEW: An application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 1996, 12, 357-358. (6) Kelley, M. L.; Winge, P.; Heaney, J. D.; Stephens, R. E.; Farell, J. H.; Van Beneden, R. J.; Reinisch, C. L.; Lesser, M. P.; Walker, C. W. Expression of homologues for p53 and p73 in the softshell clam (Mya arenaria), a naturally-occurring model for human cancer. Oncogene 2001, 20, 748-758. (7) Muttray, A. F.; Cox, R. L.; Reinisch, C. L.; Baldwin, S. A. Identification of DeltaN isoform and polyadenylation site
(8)
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choice variants in molluskan p63/p73-like homologues. Mar. Biotechnol. 2007, 9 (2), 217-230. Van Beneden, R. J.; Walker, C. W.; Laughner, E. S. Characterization of gene expression of a p53 homologue in the softshell clam (Mya arenaria). Mol. Mar. Biol. Biotechnol. 1997, 6, 116-122. Cox, R. L.; Stephens, R. E.; Reinisch, C. L. p63/73 homologues in surf clam: novel signaling motifs and implications for control of expression. Gene 2003, 320, 49-58. Imrie, F.; Karim, S. N.; Goudie, R. B. Error in reported frequency of dominant T-cell receptor V gamma 8 gene rearrangements in T-cell lymphomas. J. Pathol. 1992, 166, 417-418. Grasemann, H.; Drazen, J. M.; Yandava, C. N. Protein Sequence of the Human Neuronal Nitric Oxide Synthase (Type I NOS): An Error in the Sequence Database. Nitric Oxide 1997, 1, 441.
Annette F. Muttray* and Susan A. Baldwin Department of Chemical and Biological Engineering University of British Columbia 2360 East Mall Vancouver, BC, V6T 1Z3, Canada ES062950B
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