Identification of DeltaN isoform and polyadenylation site choice variants in molluscan p63/p73 -like homologues

Author Posting. © The Author(s), 2006. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Marine Biotechnology 9 (2007): 217-230, doi:10.1007/s10126-006-6045-1.The p53 family of transcription factors has been implicated in many vertebrate cancers. Altered p53 and p73 protein expression observed in leukemic cells of mollusks 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 mollusks Mytilus trossulus (bay mussel) and Mytilus edulis (blue mussel). These species, widely used for environmental assessment, develop a haemic neoplasia (leukemia) that is frequently fatal. The correlation between expression of p53 and its close relative p73 and onset of molluskan 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 trans-activation (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 over-expression serves as a robust marker for mammalian cancer. In addition, we report on the occurrence of alternate polyadenylation sites in the molluskan p63/p73: one proximal and one distal site, which differ by 1260 nt. We hypothesize that differential expression of various molluskan 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 molluskan 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 molluskan p53 gene family members have acquired an intronic promoter or splicing mechanism, either by invention that predates the evolutionary split of deuterostoms 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. This supports the notion that alternative splicing, intronic promoter usage and polyadenylation site choice may lead to expression of distinct isoforms originating from one common gene.A.F.M. was supported by the Greater Vancouver Regional District, BC, Canada and a Collaborative Research and Development Grant from the Natural Science and Engineering Research Council of Canada (#CRDP J 323120 –05). R.L.C. was supported by Environmental Protection Agency grant # R82935901 and National Institute of Health grant #1R21ES012273-01

Identification and phylogenetic comparison of p53 in two distinct mussel species (Mytilus)

Author Posting. © The Authors, 2005. This is the author's version of the work. It is posted here by permission of Elsevier B. V. for personal use, not for redistribution. The definitive version was published in Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 140 (2005): 237-250, doi:10.1016/j.cca.2005.02.011.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.AFM was supported by the Greater Vancouver Regional District, BC, Canada, and RLC was supported by STAR grant R82935901 from the Environmental Protection Agency (USA)

Low Variation in a Y-Chromosomal Growth Hormone Pseudogene Relative to its Functional Autosomal Progenitor Gene in Chinook Salmon

<div><p></p><p>Most fish species do not have single-chromosome-based sex determination or display cytologically distinguishable sex chromosomes. The selective forces acting on homologous sequences in diploid autosomal versus haploid sex-chromosomal regions are expected to be distinct and thus to differentially influence genetic variation. In Chinook Salmon <i>Oncorhynchus tshawytscha</i>, the Y chromosome possesses a growth hormone pseudogene (<i>ghp</i>) that is linked to the sex-determination locus and is derived from the functional autosomal growth hormone 2 gene (<i>gh2</i>). Thus, examining these two paralogues provides a model with which to study the forces affecting the persistence of genetic variation between sex-linked and autosomal loci among individuals in Chinook Salmon populations. We characterized single-nucleotide polymorphisms in a 1.6-kb contiguous homologous region in <i>gh2</i> and <i>ghp</i> in 315 individuals from 19 Chinook Salmon populations ranging from Russia to Alaska, British Columbia, and California. The <i>ghp</i> sequence was highly similar among individuals and populations, with variant haplotypes being detected in only 5.4% of individuals and restricted to just two populations. In contrast, <i>gh2</i> variants from the most common haplotype were found in 46.7% of individuals. We detected more sites of variation in <i>ghp</i> (nine positions, five haplotypes) than in <i>gh2</i> (three positions, five haplotypes), but these were restricted to just four genotypes for <i>ghp</i>, compared with nine for <i>gh2</i>. Selection may have caused a single Y chromosome to become fixed among most populations in this species, while the variation at the <i>gh2</i> locus is maintained under diploid autosomal conditions. Since the <i>ghp</i> locus variants are not likely to be directly associated with strong functions, other linked Y<i>-</i>chromosomal loci may have important functions that, when selected, cause Y chromosomes to either be damaged and rapidly eliminated or to have enhanced fitness and sweep through the species. Whether such loci include the sex-determination locus itself or other functional loci is not yet known.</p><p>Received January 15, 2015; accepted June 3, 2015</p></div