Philip Hieter
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Doctoral Student Supervision
Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Genotype-driven therapies are a new paradigm for cancer treatment. These approaches rely on identification of genetic vulnerabilities and genotype-linked therapeutic agents. One approach utilizes synthetic lethality (SL), which occurs when disruption of two gene products individually is non-lethal, but simultaneous disruption of both gene products results in lethality. A synthetic lethal target identified in our lab is the helicase DDX11, the human homolog of yeast CHL1. In yeast, CHL1 is a highly-connected synthetic lethal hub, that genetically interacts with many genes involved in processes often defective in tumours, such as sister-chromatid cohesion (SCC) and replication fork stability, and as such, would make a good synthetic lethal therapeutic target. The overarching goal of this research is to advance development of DDX11 inhibition as a synthetic lethal therapeutic. Previous work in our lab identified a genetic interaction between cohesin mutations and CHL1 in yeast. We first directly tested a potential genetic interaction between DDX11 and the cancer-mutated cohesin gene STAG2 in human cell lines and found that it did not result in synthetic lethality. We then conducted an unbiased screen for DDX11 genetic interactions in human cells and identified many genes involved in SCC, supporting the conserved role of DDX11, as well as supporting DDX11 inhibition as a potential SL-based therapy for tumours with cohesion defects. To date, only one SL-based drug has reached the clinic, PARP inhibitors, which trap PARP on the DNA creating a cytotoxic complex. Small molecule-induced trapping may represent a generalized mechanism for clinically relevant synthetic lethal interactions. We hypothesized that missense mutations that model such inhibitors can be utilized as an alternative to knock-out/knock-down based screens. As a proof-of principle, we expressed missense mutations in CHL1 that inhibited enzymatic activity but retained substrate binding, and found that these mutations elicited a dominant synthetic lethal phenotype consistent with the generation of cytotoxic intermediates. These results point to the utility of modeling trapping mutations in pursuit of more clinically relevant synthetic lethal interactions. Finally, we developed a biochemical method for high-throughput screening for DDX11 inhibitors. Together, this work contributes to the development of DDX11 inhibition as an anti-cancer therapeutic.
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Genome stability is essential for survival in all living organisms. Perturbations of genome stability can lead to cancer and other diseases in human. Dysfunction of genome care-taking genes and exposure to external genotoxins are the two major drivers of genomic instability. Here, we used a model organism, C. elegans to study these factors and how they interact to affect genome stability. We first generated a chemi-genetic interaction map as a preliminary reference for characterizing genes and agents. From there, we elucidated the mechanisms of action for an emerging anticancer therapeutic CX-5461. CX-5461 turned out to be a multi-modal agent that exhibits genotypic sensitivity, mutagenicity and photosensitivity. Next, we identified a few genetic interactions within and between double-strand break repair pathways in C. elegans, which have implications in the response to both endogenous and exogenous DNA damage. We also characterized a less-understood gene SMRC-1, an annealing helicase. Loss of SMRC-1 results in copy number variations, and SMRC-1 is synthetic lethal with a number of DNA damage response genes. Our work concludes that C. elegans is a great platform to untangle the sophisticated complexity in genome maintenance, and provide meaningful insights for prospective anticancer therapeutic development.
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Humanized yeast offer a valuable resource with which to model and study human biology. Using cross-species complementation, model organisms like the budding yeast, Saccharomyces cerevisiae, can be utilized to measure the impact of tumor-specific mutations and screen for genetic vulnerabilities of genes overexpressed in cancer. To this end, we performed three parallel screens, one-to-one complementation screens for essential and nonessential yeast genes implicated in chromosome instability (CIN) and a pool-to-pool screen that queried all possible essential yeast genes for rescue of lethality by all possible human homologs. Our work identified 65 human cDNAs that can replace the null allele of essential yeast genes, including the nonorthologous pair yRFT1/hSEC61A1. For the nonessential yeast genes, 20 human-yeast complementation pairs were determined to be replaceable in 44 assays that test rescue of chemical sensitivity and/or CIN defects. For five human-yeast complementation pairs expressing human cDNAs encoding hLIG1, hSSRP1, hPPP1CA, hPPP1CC and hPPP2R1A, we introduced 45 tumor-specific missense mutations and assayed for growth defects and sensitivity to DNA-damaging agents in yeast. This set of human-yeast gene complementation pairs allows human genetic variants to be readily characterized in yeast, generating a prioritized list of somatic mutations that could contribute to chromosome instability in human tumors. We also selected a human-yeast pair expressing hFEN1, which is frequently overexpressed in cancer and is an anti-cancer therapeutic target, to perform synthetic dosage lethal (SDL) screens using ectopic overexpression of wild-type and catalytically inactive hFEN1 in yeast. The SDL screens identified homologous recombination (HR) repair mutants as synthetic dosage lethal with overexpression of catalytically-inactive hFen1. The SDL interactions were dependent on binding of hFen1 to DNA suggesting that toxicity was a result of catalytically inactive hFen1 becoming trapped on DNA and resulting in DNA damage. Our study establishes the utility of using cross-species complementation and ectopic overexpression to generate human-yeast genetic interaction networks and to model protein-inhibitor interactions using genetic approaches. Overall, these data establish the utility of this cross-species experimental approach.
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DNA:RNA hybrid formation is emerging as a significant cause of genomic instability in biological systems ranging from bacteria to mammals. However, the scope of cellular pathways that prevent DNA:RNA hybrids and the genomic loci prone to hybrid formation are unclear. In Saccharomyces cerevisiae, DNA:RNA hybrids were found to be prevalent in various RNA processing, DNA repair and kinetochore mutants. In particular, mRNA cleavage and polyadenylation factors were demonstrated to maintain genome integrity by preventing transcription-dependent DNA:RNA hybrid formation. Genome-wide profiling of DNA:RNA hybrids showed that highly transcribed genes are prone to hybrid formation in the absence of hybrid-mitigating enzymes. Furthermore, the hybrid profiles highlight various genetic features prone to hybrid formation and suggest potential functions for DNA:RNA hybrids in antisense transcription regulation. Together, these findings elucidate previously unrecognized pathways that mitigate DNA:RNA hybrid formation as well as the characteristics of hybrid prone genomic regions.
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Genome instability has been observed in mutants involved in various aspects of transcription and RNA processing. The prevalence of this mechanism among essential chromosome instability (CIN) genes remains unclear. In this thesis, it is shown that RNA biogenesis mutants exhibit elevated sensitivity to DNA damaging agents. A secondary screen for increased Rad52 foci in CIN mutants, representing ~25% of essential genes, identified seven essential subunits of the mRNA cleavage and polyadenylation (mCP) machinery. Genome-wide analysis of fragile sites by ChIP-chip of phosphorylated-H2A in these mutants supported a transcription-dependent mechanism of DNA damage characteristic of RNA:DNA hybrids known as R-loops, which were subsequently observed in mCP mutants. Among the CIN mutants with elevated Rad52 foci levels were the GPN proteins, a poorly-characterized and deeply evolutionarily conserved family of three paralogous small GTPases, Gpn1, 2 and 3. The founding member, GPN1/NPA3/XAB1, is proposed to function in nuclear import of RNA polymerase II along with a recently described protein called Iwr1. Here, it is shown that the previously uncharacterized protein Gpn2 binds both Gpn3 and Npa3/Gpn1, and that temperature-sensitive alleles of Saccharomyces cerevisiae GPN2 and GPN3 exhibit genetic interactions with RNA polymerase II mutants, hypersensitivity to transcription inhibition and defects in RNA polymerase II nuclear localization. Importantly, previously unrecognized RNA polymerase III localization defects were observed in GPN2, GPN3 and IWR1 mutant backgrounds but no localization defects of unrelated nuclear proteins or of RNA polymerase I were found. In this study, it was shown that the nuclear import defect of iwr1Δ, but not the GPN2 or GPN3 mutant defects, is partially suppressed by fusion of a nuclear localization signal to the RNA polymerase II subunit Rpb3. These data, combined with strong genetic interactions between GPN2 and IWR1 suggest that the GPN proteins function upstream of Iwr1 in RNA polymerase II and III biogenesis. We propose that the three GPN proteins execute a common function in RNA polymerase assembly and subsequent transport. These findings demonstrate how mRNA cleavage and polyadenylation and proper RNA polymerase assembly contribute to maintenance of genome integrity and may be relevant to certain human cancers.
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Cancer is a multigenic disease. The genetic distinctness of cancer cells offers aweakness that can be exploited: for example, nearly all cancers carry mutations in processesrelating to the maintenance of genomic stability. As this is an essential process, this presentsa weakness that can be leveraged towards inviability – a concept known as syntheticlethality. The ideal cancer therapeutic would have a broad spectrum, but genetic techniquesin human cells are not sufficiently developed to identify the spectrum of synthetic lethalinteractions of sets of genome stability genes easily. The use of model organisms canfacilitate the identification of second-site targets for the development of anticancertherapeutics, and allows the construction of synthetic lethal interaction networks. This has thepotential to identify “hub” genes having synthetic lethal interactions with many cancermutatedorthologs. If these synthetic lethal interactions are found to be conserved in humancells, these highly connected hub genes are potential targets for therapeutic development.Assembly of a synthetic lethal interaction network of yeast orthologs of 10 genes mutated incolorectal cancer, based on data in Saccharomyces cerevisiae, previously identified five suchsynthetic lethal hub genes in yeast. In this thesis, the evolutionary conservation of thisnetwork is interrogated in mammalian cells. The interactions between orthologs of colorectalcancer CIN genes in yeast were found to be highly conserved in human cells. A highthroughputassay to screen for small-molecule inhibitors of the protein encoded by one suchgene, FEN1, was developed and used to identify 13 compounds that inhibited FEN1 in vitrowith IC50 values in the low-micromolar range or less. These compounds were applied to cellsbearing mutation in the tumor suppressor CDC4, and two compounds were found to yieldselective killing of CDC4-deficient cells. Finally, yeast genetic techniques were used to characterize CTF4, a second highly connected hub gene within the colon cancer CIN genenetwork, and to expand the therapeutic range of cancers that could be selectively killed byinhibitors of Ctf4/WDHD1 or Rad27/FEN1. Taken together, these data demonstrate theconsiderable power of applying model organisms genetics to the discovery of new anticancertherapeutic targets.
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Chromosome instability (CIN) is characterized by the loss or gain of large portions of DNA and is characteristic of ~85% of solid tumours. Sequencing of the human orthologues of ~200 genes that cause CIN in yeast identified mutations in approximately 25% of tumours tested. Mutations in cohesin genes and CDC4 represented the two major mutational categories identified. Large scale genetic interaction networks in model organisms can provide insight into the biology underlying tumour mutations and can identify potential therapeutic targets. This approach is based on the concept of synthetic lethality (SL); cell death resulting from the combination of two sub-lethal mutations. Therapies that take advantage of SL distinguish a cancer cell from a normal cell based on their genetic background. This thesis investigated genetic interactions of three cohesin genes, SMC1, SCC1, and SCC2, using high throughput synthetic genetic array (SGA) methods in S. cerevisiae. The overlay of these three genome wide SGA screens and validation using growth curve analysis found that sub-optimal cohesin requires the presence of proteins that mediate replication fork progression. The protruding vulva assay was developed to identify genetic interactions in the somatic cells of C. elegans. It was used to test whether the cohesin interactions were conserved in a multicellular animal. 80% of the validated interactions identified with cohesin in yeast are conserved in C. elegans. Additional fork mediators, namely the pme/PARP family of genes was found to interact with him-1/SMC1 in both C. elegans and human cells. Human cells depleted of SMC1 by siRNA are selectively sensitive to the PARP inhibitor olaparib, currently being evaluated in phase II clinical trials. Additional genetic interaction testing found that CDC4 has a distinct genetic interaction profile from that of cohesin, suggesting different mutational consequences. Work in C. elegans with the lin-23 mutant suggested LIN-23 is involved in controlling CYE-1/Cyclin E levels. lin-23 mutants and human CDC4-/- cells are unable to properly respond to alkylating DNA damage, suggesting CDC4 is important for the DNA damage response. Keywords: colon tumours, chromosome instability, cohesin, CDC4, genetic interactions, SGA, replication fork, PARP.
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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Genome stability is crucial for the proper functioning of all living organisms. In eukaryotes, genome stability is safeguarded by an intricate network of mechanisms that detect DNA damage and mediate its repair. This network, termed the DNA damage response, ensures the integrity of the genome and its accurate transmission to daughter cells. Defects in DNA damage response mechanisms can result in unrepaired DNA damage, ultimately leading to the onset of genetic diseases. Several proteins have been shown to participate in the DNA damage response, including cohesin, a multi-protein complex that functions together with a collection of cohesion auxiliary factors. The full extent to which cohesin participates in the DNA damage response to maintain genome stability remains to be defined and is of great importance since cohesin mutations are associated with a variety of cancer types. Genetic interaction networks can provide insights into the involvement of genes in biological processes and can identify potential targets for therapeutic inhibition. This approach exploits the concept of synthetic lethality, which is a type of genetic interaction in which two individual genetic perturbations are viable but combining the perturbations results in lethality. Another type of genetic interaction, termed synthetic cytotoxicity, results in severe sensitivity to genotoxic agents that induce DNA damage.We used the synthetic genetic array methodology in Saccharomyces cerevisiae to generate synthetic lethal and synthetic cytotoxic interaction networks using cohesin mutations as query genes to identify DNA damage response factors that genetically interact with the cohesin complex. Retesting studies using spot assays found that cells expressing hypomorphic cohesin alleles were dependent on factors involved in DNA damage checkpoint, HR inhibition, and DNA damage tolerance, and that cohesin-defective cells rely on translesion synthesis for viability upon replication stress. Using missense mutations that abolish the catalytic activity of translesion synthesis polymerases in a manner to mimic the effect of therapeutic inhibition, we showed that dominant mutant forms of Rad30 and Rev3 are synthetic cytotoxic with several DNA damage response mutants, including cohesin. Overall, these data highlight the utility of yeast to expand our knowledge of functional genomics and to identify potential targets for anti-cancer treatments.
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Cancer therapy is changing. Whole genome sequencing technologies are advancing at an unprecedented pace, opening new opportunities for the genotype-driven personalized treatment of cancer. Synthetic Lethality (SL) based therapeutics have emerged as promising approaches to target cancer-specific somatic mutations, by targeting a second gene that is required for viability in the presence of a tumor-specific mutation. The targetable set of SL partner genes can be expanded by screening for a conditional SL interaction, in which loss of function of two genes results in sensitivity to low doses of a DNA-damaging agent, a concept we have called Synthetic Cytotoxicity (SC). SC also has the potential to expand the number of genotypes that can be treated with existing chemotherapeutics and to improve the efficacy of these therapeutics. In contrast to SL and SC negative genetic interactions, Phenotypic Suppression (PS) describes a genetic interaction in which the double mutant cell is more fit than anticipated based on the fitness of each single mutant. The model organism, Saccharomyces cerevisiae was used to screen for SC interactions with cohesin-mutated genes, with the aim of identifying cross-species candidate genes that could be followed up in subsequent studies as SL-based cancer-drug targets. The cohesin complex is frequently mutated across a wide range of tumors and is conserved from yeast to man. We used Synthetic Genetic Array (SGA) technology, a high-throughput genetic method available in yeast, to screen cohesin-mutated strains for synthetic lethal genetic interactions against an array of 310 deletions affecting mainly DNA damage response genes. The screens were done in the presence and absence of four clinically-relevant genotoxic agents. We screened and analyzed 4,650 potential genetic interactions, identifying hundreds of negative and positive interactions, belonging to conserved biological pathways, and potentially relevant to cancer. Using ScanLag, a new validation method, we re-tested and validated several genetic interactions that represent potential therapeutic candidates. These strong SL, SC and PS interactions can be further analyzed in mammalian cells to potentially inform and improve individual cancer therapies as personalized medicine treatments, and lead to the discovery of new pathways or candidates for anti-cancer treatments.
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Cancer is due to an accumulation of mutations in constellations of genes that cause uncontrolled proliferation and evasion of apoptotic pathways. In addition, mutations that cause genome instability, another hallmark of cancer, predispose cancer progenitor cells to accumulating the large number of mutations and chromosome aberrations that are observed in cancer cells. Genome instability is either due to mutations that cause an increased mutation rate (mutator phenotype) or increases in aberrations to chromosome number or structure (chromosome instability). Recent work has cataloged nearly all genes in the budding yeast, Saccharomyces cerevisiae, that cause a chromosome instability (CIN) phenotype due to reduction-of-function mutations and gain-of-function mutations, with the ultimate goal of translating the results found in yeast to human cancer. To investigate the effects of gene dosage on mutation rate, we systematically overexpressed ~85% of the yeast genome in a CAN1 forward-mutation screen and identified 5 genes that when overexpressed conferred a strong mutator phenotype, several of which have been associated with cancer. Overexpression of MPH1, the yeast ortholog of Fanconi Anemia gene FANCM, resulted in the strongest mutator phenotype. MPH1 was further investigated to gain insight into the mechanisms which lead to its dosage mutator phenotype.
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Polyploidy is the process of genome doubling that gives rise to organisms with multiple sets of chromosomes. Expression patterns and levels of genes duplicated by polyploidy, termed homeologs, can change and gene silencing can occur after polyploidy. Alternative splicing (AS) creates multiple mature mRNAs from a single type of precursor mRNA. AS can change the level of gene expression by degradation of transcripts with premature stop codons, as well as create new protein isoforms. Little is known about how AS changes after a polyploidization event, either within a few generations after polyploidy or over evolutionary time, and what effects AS changes have on gene expression in polyploids. In this project, the evolution of alternative splicing patterns after genome duplication in allotetraploid Brassica napus and a synthetic allotetraploid B. napus was examined by RT-PCR assays of a set of 31 duplicated genes. Since genes can show different patterns of AS in different organ types and under different abiotic stresses, two different organ types (leaf and cotyledon), and two different abiotic stresses (heat and cold) were used. Comparing the AS patterns between the two homeologs in B. napus revealed that 18% of the gene pairs show AS in only one homeolog. In contrast 33% of the gene pairs in the synthetic allotetraploid showed AS in only one homeolog. Gene silencing was observed for 6% and 9% of genes in B. napus and synthetic B. napus, respectively. These results indicate that there are many changes in AS in both the synthetic B. napus and natural B. napus after polyploidy, but more AS changes occurred in the synthetic polyploid. The PASTICCINO gene showed partitioning of two AS events between the homeologs in the synthetic allopolyploid, suggesting subfunctionalization of AS forms. Results from this project indicate that AS patterns can change rapidly after polyploidy and suggest that changes in AS patterns are a major phenomenon in allopolyploids.
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Cancer is a leading cause of death worldwide. This somatic cell genetic disease is characterized by progressive accumulation of mutations in multiple genes. An important characteristic of cancer cells is an increased rate of gains and losses of chromosomes, termed Chromosomal Instability (CIN). One of the frequently mutated genes in a variety of cancers is FBXW7 (F-Box and WD repeat domain-containing 7), encoding the substrate-recognition component of a ubiquitin ligase complex. Fbxw7 targets a number of oncoproteins such as, Cyclin E, c-Myc, Notch1 and Aurora A for ubiquitin mediated degradation. Inactivation of FBXW7 has been linked to CIN in cancer cell lines. The majority of cancer-associated mutations in FBXW7 are monoallelic, missense substitutions whose phenotypic effects are difficult to predict. Interestingly, most of the mutations in FBXW7 cluster at three mutational hotspots, Arg465, Arg479 and Arg505. Located at β propeller-tip, these residues are critical for interaction with the Fbxw7 substrates. This study investigates the functional consequences of the substitutions at these residues. We individually tested the functional status of the R465C, R479Q and R505C variants of FBXW7 in three colorectal cancer cell lines in an HCT116 background. These cell lines had both, one or none of the alleles of FBXW7 inactivated by homologous recombination. Our data shows that the cell lines producing R465C, R479Q or R505C variants of FBXW7 failed to degrade Cyclin E, one of the major targets of FBXW7. These cell lines also exhibited a CIN phenotype, observed as an increase in the frequency of abnormal anaphases. These results show that mutations R465C, R479Q and R505C occurring in FBXW7 cause loss of function of the protein and act as dominant negative mutations.
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Chromosome instability (CIN) is a hallmark of cancer cells and could, in theory, be exploited in the design of cancer therapeutics. Tumor cells harboring CIN mutations may be dependent on certain DNA repair pathways for viability. Thus, inhibition of specific DNA repair enzymes may enhance the CIN phenotype to an intolerable level, or may sensitize cells to DNA damage stress. To test this hypothesis, I focused on the CIN gene ATM, which is often mutated in human tumors. I hypothesized that knockdown of certain second site DNA repair genes would selectively kill ATM-deficient cells resulting in synthetic lethality (SL), or sensitize ATM-deficient cells to a sub-lethal dose of DNA damaging agent resulting in synthetic cytotoxicity (SC). The goal of this research is to use budding yeast as a model system to identify candidate SL or SC interaction partner genes for ATM with/without sub-lethal doses of DNA damaging agents, using mutations in the yeast ATM homologues, TEL1 and MEC1. I tested for interactions with TEL1 and MEC1 in a small matrix of three DNA repair genes (RAD27, TDP1 and TPP1) and four DNA damaging agents (hydroxyurea, 5-fluorouracil, bleomycin, and camptothecin). I also performed a genome-wide screen for interactions between TEL1 and ~5000 non-essential genes, both in the presence and absence of low doses of camptothecin. I discovered one SL interaction with MEC1 and fourteen SC interactions with TEL1. Most of the SC interaction partner genes are involved in DNA repair and show sensitivity specifically to camptothecin. These data provide a rationale for testing specific combination therapies for selective killing of cancer cells bearing ATM mutations. Specifically, the Shu complex, Ku complex, Rrm3, Rad27 and CK2β subunits can be further tested as potential combination therapeutic targets, together with a sub-lethal dose of camptothecin, to kill ATM-deficient cancer cells.
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Polyploidy is the process of genome doubling that gives rise to organisms with multiple sets of chromosomes. Expression patterns and levels of genes duplicated by polyploidy, termed homeologs, can change and gene silencing can occur after polyploidy. Alternative splicing (AS) creates multiple mature mRNAs from a single type of precursor mRNA. AS can change the level of gene expression by degradation of transcripts with premature stop codons, as well as create new protein isoforms. Little is known about how AS changes after a polyploidization event, either within a few generations after polyploidy or over evolutionary time, and what effects AS changes have on gene expression in polyploids. In this project, the evolution of alternative splicing patterns after genome duplication in allotetraploid Brassica napus and a synthetic allotetraploid B. napus was examined by RT-PCR assays of a set of 31 duplicated genes. Since genes can show different patterns of AS in different organ types and under different abiotic stresses, two different organ types (leaf and cotyledon), and two different abiotic stresses (heat and cold) were used. Comparing the AS patterns between the two homeologs in B. napus revealed that 18% of the gene pairs show AS in only one homeolog. In contrast 33% of the gene pairs in the synthetic allotetraploid showed AS in only one homeolog. Gene silencing was observed for 6% and 9% of genes in B. napus and synthetic B. napus, respectively. These results indicate that there are many changes in AS in both the synthetic B. napus and natural B. napus after polyploidy, but more AS changes occurred in the synthetic polyploid. The PASTICCINO gene showed partitioning of two AS events between the homeologs in the synthetic allopolyploid, suggesting subfunctionalization of AS forms. Results from this project indicate that AS patterns can change rapidly after polyploidy and suggest that changes in AS patterns are a major phenomenon in allopolyploids.
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