Joanne M Yeakley

Indiana University South Bend, South Bend, Indiana, United States

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Publications (27)235.64 Total impact

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    ABSTRACT: Recent breakthroughs in multiplexed SNP (single nucleotide polymorphism) genotyping technology have enabled global mapping of the relationships between genetic variation and disease. Discoveries made by such whole-genome association studies often spur further interest in surveying more focused subsets of SNPs for validation or research purposes. Here we describe a new SNP genotyping platform that is flexible in assay content and multiplexing (up to 384 analytes), and can serve medium- to high-throughput applications. The Illumina BeadXpress platform supports the GoldenGate Genotyping Assay on digitally inscribed VeraCode microbeads to allow streamlined workflow, rapid detection, unparalleled data reproducibility and consistency. Thus, it is a highly valuable tool for biomarker research and validation, pharmaceutical development, as well as the development of molecular diagnostic tests.
    Methods in Molecular Biology 02/2009; 496:129-42. DOI:10.1007/978-1-59745-553-4_10 · 1.29 Impact Factor
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    ABSTRACT: Molecular analyses of biological samples have traditionally been pursued in parallel, with those researchers studying genetic diversity having few technical approaches in common with those studying gene expression. Increasingly, scientists recognize the importance of integrating analytical technologies to further research, particularly into emerging fields such as epigenetics and the genetics of gene expression. In this chapter, we describe a suite of applications that take advantage of the Illumina® bead-based microarrays, all of which are read out on a single analytical instrument. The integration of whole genome genotyping, high throughput focused genotyping, whole transcriptome expression profiling, focused expression profiling of fresh or preserved tissues, allele-specific expression profiling and DNA methylation assays on the BeadArray™ Reader allows researchers to expand their perspectives, from whole genomes to single bases, from genetics to expression and on to epigenetics.
    04/2008: pages 10-24;
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    ABSTRACT: Formalin-fixed, paraffin-embedded (FFPE) tissues represent an invaluable resource for gene expression analysis, as they are the most widely available materials for studies of human disease. However, degradation of RNA during tissue fixation and storage makes FFPE-derived RNAs hard to work with using conventional microarray technology. Most gene expression studies done using FFPE tissues rely on quantitative RT-PCR (qPCR), but this approach has had limited success because of the short cDNA templates available in these samples. In this chapter, we describe the DASL (cDNA-mediated annealing, selection, extension, and ligation) Assay, a flexible, sensitive, and reproducible gene expression profiling system for parallel analysis of several hundred mRNA transcripts on the BeadArray platform. This technology is especially useful for determining cancer prognosis or therapy response, because it allows not only prospective studies but also retrospective analyses. Using the DASL Assay, gene expression analyses can be performed on routinely stored tumor specimens from patients with known outcomes.
    Methods in Molecular Biology 02/2008; 439:159-77. DOI:10.1007/978-1-59745-188-8_11 · 1.29 Impact Factor
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    ABSTRACT: The Down syndrome cell adhesion molecule (Dscam) gene has essential roles in neural wiring and pathogen recognition in Drosophila melanogaster. Dscam encodes 38,016 distinct isoforms via extensive alternative splicing. The 95 alternative exons in Dscam are organized into clusters that are spliced in a mutually exclusive manner. The exon 6 cluster contains 48 variable exons and uses a complex system of competing RNA structures to ensure that only one variable exon is included. Here we show that the heterogeneous nuclear ribonucleoprotein hrp36 acts specifically within, and throughout, the exon 6 cluster to prevent the inclusion of multiple exons. Moreover, hrp36 prevents serine/arginine-rich proteins from promoting the ectopic inclusion of multiple exon 6 variants. Thus, the fidelity of mutually exclusive splicing in the exon 6 cluster is governed by an intricate combination of alternative RNA structures and a globally acting splicing repressor.
    Nature Structural & Molecular Biology 12/2007; 14(12):1134-40. DOI:10.1038/nsmb1339 · 11.63 Impact Factor
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    ABSTRACT: Virus-infected leaf tissues comprise a heterogeneous mixture of cells at different stages of infection. The spatial and temporal relationships between sites of virus accumulation and the accompanying host responses, such as altered host gene expression, are not well defined. To address this issue, we utilized Turnip mosaic virus (TuMV) tagged with the green fluorescent protein to guide the dissection of infection foci into four distinct zones. The abundance of Arabidopsis thaliana mRNA transcripts in each of the four zones then was assayed using the Arabidopsis ATH1 GeneChip oligonucleotide microarray (Affymetrix). mRNA transcripts with significantly altered expression profiles were determined across gradients of virus accumulation spanning groups of cells in and around foci at different stages of infection. The extent to which TuMV-responsive genes were up- or downregulated primarily correlated with the amount of virus accumulation regardless of gene function. The spatial analysis also allowed new suites of coordinately regulated genes to be identified that are associated with chloroplast functions (decreased), sulfate assimilation (decreased), cell wall extensibility (decreased), and protein synthesis and turnover (induced). The functions of these downregulated genes are consistent with viral symptoms, such as chlorosis and stunted growth, providing new insight into mechanisms of pathogenesis.
    Molecular Plant-Microbe Interactions 04/2007; 20(4):358-70. DOI:10.1094/MPMI-20-4-0358 · 4.46 Impact Factor
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    ABSTRACT: Heat shock protein 101 (HSP101) has been implicated in tobamovirus infections by virtue of its ability to enhance translation of mRNAs possessing the 5'Omega-leader of Tobacco mosaic virus (TMV). Enhanced translation is mediated by HSP101 binding to a CAA-repeat motif in TMV Omega leader. CAA repeat sequences are present in the 5' leaders of other tobamoviruses including Oilseed rape mosaic virus (ORMV), which infects Arabidopsis thaliana. HSP101 is one of eight HSP100 gene family members encoded by the A. thaliana genome, and of these, HSP101 and HSP98.7 are predicted to encode proteins localized to the cytoplasm where they could potentially interact with TMV RNA. Analysis of the expression of the HSP100s showed that only HSP101 mRNA transcripts were induced significantly by ORMV in A. thaliana. The induction of HSP101 mRNA was also correlated with an increase in its protein levels and was independent of defense-related signaling pathways involving salicylic acid, jasmonic acid, or ethylene. A. thaliana mutants lacking HSP101, HSP98.7, or both supported wild-type levels of ORMV replication and movement. Similar results were obtained for TMV infection in Nicotiana benthamiana plants silenced for HSP101, demonstrating that HSP101 is not necessary for efficient tobamovirus infection.
    Virus Research 11/2006; 121(1):33-41. DOI:10.1016/j.virusres.2006.03.013 · 2.83 Impact Factor
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    ABSTRACT: The expression of specific mRNA isoforms may uniquely reflect the biological state of a cell because it reflects the integrated outcome of both transcriptional and posttranscriptional regulation. In this study, we constructed a splicing array to examine approximately 1,500 mRNA isoforms from a panel of genes previously implicated in prostate cancer and identified a large number of cell type-specific mRNA isoforms. We also developed a novel "two-dimensional" profiling strategy to simultaneously quantify changes in splicing and transcript abundance; the results revealed extensive covariation between transcription and splicing in prostate cancer cells. Taking advantage of the ability of our technology to analyze RNA from formalin-fixed, paraffin-embedded tissues, we derived a specific set of mRNA isoform biomarkers for prostate cancer using independent panels of tissue samples for feature selection and cross-analysis. A number of cancer-specific splicing switch events were further validated by laser capture microdissection. Quantitative changes in transcription/RNA stability and qualitative differences in splicing ratio may thus be combined to characterize tumorigenic programs and signature mRNA isoforms may serve as unique biomarkers for tumor diagnosis and prognosis.
    Cancer Research 05/2006; 66(8):4079-88. DOI:10.1158/0008-5472.CAN-05-4264 · 9.28 Impact Factor
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    ABSTRACT: This chapter describes an accurate, scalable, and flexible microarray technology. It includes a miniaturized array platform where each individual feature is quality controlled and a versatile assay that can be adapted for various genetic analyses, such as single nucleotide polymorphism genotyping, DNA methylation detection, and gene expression profiling. This chapter describes the concept of the BeadArray technology, two different Array of Arrays formats, the assay scheme and protocol, the performance of the system, and its use in large-scale genetic, epigenetic, and expression studies.
    Methods in Enzymology 02/2006; 410:57-73. DOI:10.1016/S0076-6879(06)10003-8 · 2.19 Impact Factor
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    ABSTRACT: Plant viruses elicit the expression of common sets of genes in susceptible hosts. Studies in Arabidopsis (Arabidopsis thaliana) and tomato (Lycopersicon esculentum) indicate that at least one-third of the genes induced in common by viruses have been previously associated with plant defense and stress responses. The genetic and molecular requirements for the induction of these stress and defense-related genes during compatible host-virus interactions were investigated with a panel of Arabidopsis mutant and transgenic plants defective in one or more defense signaling pathways. pad4, eds5, NahG, npr1, jar1, ein2, sid2, eds1, and wild-type Columbia-0 and Wassilewskija-2 plants were infected with two different viruses, cucumber mosaic virus and oilseed rape mosaic virus. Gene expression was assayed by a high-throughput fiber-optic bead array consisting of 388 genes and by RNA gel blots. These analyses demonstrated that, in compatible host-virus interactions, the expression of the majority of defense-related genes is induced by a salicylic acid-dependent, NPR1-independent signaling pathway with a few notable exceptions that did require NPR1. Interestingly, none of the mutant or transgenic plants showed enhanced susceptibility to either cucumber mosaic virus or oilseed rape mosaic virus based on both symptoms and virus accumulation. This observation is in contrast to the enhanced disease susceptibility phenotypes that these mutations or transgenes confer to some bacterial and fungal pathogens. These experimental results suggest that expression of many defense-related genes in compatible host plants might share components of signaling pathways involved in incompatible host-pathogen interactions, but their increased expression has no negative effect on viral infection.
    Plant physiology 04/2005; 137(3):1147-59. DOI:10.1104/pp.104.056028 · 7.39 Impact Factor
  • Clinical Chemistry 01/2005; 50(12):2384-6. DOI:10.1373/clinchem.2004.037432 · 7.77 Impact Factor
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    ABSTRACT: We recently developed a sensitive and flexible gene expression profiling system that is not dependent on an intact poly-A tail and showed that it could be used to analyze degraded RNA samples. We hypothesized that the DASL (cDNA-mediated annealing, selection, extension and ligation) assay might be suitable for the analysis of formalin-fixed, paraffin-embedded tissues, an important source of archival tissue material. We now show that, using the DASL assay system, highly reproducible tissue- and cancer-specific gene expression profiles can be obtained with as little as 50 ng of total RNA isolated from formalin-fixed tissues that had been stored from 1 to over 10 years. Further, tissue- and cancer-specific markers derived from previous genome-wide expression profiling studies of fresh-frozen samples were validated in the formalin-fixed samples. The DASL assay system should prove useful for high-throughput expression profiling of archived clinical samples.
    American Journal Of Pathology 12/2004; 165(5):1799-807. DOI:10.1016/S0002-9440(10)63435-9 · 4.60 Impact Factor
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    ABSTRACT: We report a flexible, sensitive, and quantitative gene-expression profiling system for assaying more than 400 genes, with three probes per gene, for 96 samples in parallel. The cDNA-mediated annealing, selection, extension and ligation (DASL) assay targets specific transcripts, using oligonucleotides containing unique address sequences that can hybridize to universal arrays. Cell-specific gene expression profiles were obtained using this assay for hormone-treated cell lines and laser-capture microdissected cancer tissues. Gene expression profiles derived from this assay were consistent with those determined by qRT-PCR. The DASL assay has been automated for use with a bead-based 96-array matrix system. The combined high-throughput assay and readout system is accurate and efficient, and can cost-effectively profile the expression of hundreds of genes in thousands of samples.
    Genome Research 06/2004; 14(5):878-85. DOI:10.1101/gr.2167504 · 13.85 Impact Factor
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    ABSTRACT: Cold acclimation is the major process that prepares plants for freezing tolerance. In addition to extensive transcription regulation by cold-inducible master transcription factors, oxidative stress signaling has been postulated to play a role in freezing tolerance. Activation of oxidative signaling through the expression of an active mitogen-activated protein kinase kinase kinase provided benefits in transgenic tobacco at freezing temperature bypassing cold acclimation. Because involvement of the mitogen-activated protein kinase cascade in oxidative stress signaling is evolutionarily conserved in eukaryotes from yeast to mammals, we tested the effect of expressing a heterologous tobacco mitogen-activated protein kinase kinase kinase (Nicotiana PK1), which can mimic H(2)O(2) signaling, in a major cereal crop. We demonstrate that low-level but constitutive expression of the Nicotiana PK1 gene enhances freezing tolerance in transgenic maize plants that are normally frost sensitive. Our results suggest that a new molecular approach can be designed to genetically enhance freezing tolerance in important crops.
    Proceedings of the National Academy of Sciences 04/2004; 101(9):3298-303. DOI:10.1073/pnas.0308095100 · 9.81 Impact Factor
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    ABSTRACT: Many genetic diseases are caused by mutations in cis-acting splicing signals, but few are triggered by defective trans-acting splicing factors. Here we report that tissue-specific ablation of the splicing factor SC35 in the heart causes dilated cardiomyopathy (DCM). Although SC35 was deleted early in cardiogenesis by using the MLC-2v-Cre transgenic mouse, heart development appeared largely unaffected, with the DCM phenotype developing 3-5 weeks after birth and the mutant animals having a normal life span. This nonlethal phenotype allowed the identification of downregulated genes by microarray, one of which was the cardiac-specific ryanodine receptor 2. We showed that downregulation of this critical Ca2+ release channel preceded disease symptoms and that the mutant cardiomyocytes exhibited frequency-dependent excitation-contraction coupling defects. The implication of SC35 in heart disease agrees with a recently documented link of SC35 expression to heart failure and interference of splicing regulation during infection by myocarditis-causing viruses. These studies raise a new paradigm for the etiology of certain human heart diseases of genetic or environmental origin that may be triggered by dysfunction in RNA processing.
    The EMBO Journal 03/2004; 23(4):885-96. DOI:10.1038/sj.emboj.7600054 · 10.75 Impact Factor
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    ABSTRACT: The human transcriptome is marked by extensive alternative mRNA splicing and the expression of many closely related genes, which may be difficult to distinguish using standard microarray techniques. Here we describe a sensitive and specific assay for parallel analysis of mRNA isoforms on a fiber-optic microarray platform. The method permits analysis of mRNA transcripts without prior RNA purification or cDNA synthesis. Using an endogenously expressed viral transcript as a model, we demonstrated that the assay readily detects mRNA isoforms from as little as 10-100 pg of total cellular RNA or directly from a few cells. Multiplexed analysis of human cancer cell lines revealed differences in mRNA splicing and suggested a potential autocrine mechanism in the development of choriocarcinomas. Our approach may be useful in the large-scale analysis of the role of alternative splicing in development and disease.
    Nature Biotechnology 05/2002; 20(4):353-8. DOI:10.1038/nbt0402-353 · 39.08 Impact Factor
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    ABSTRACT: The SR superfamily of splicing factors and regulators is characterized by arginine/serine (RS)-rich domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS domain-mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of the single nonessential SR protein-specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in vivo using the two-hybrid assay. Strikingly, all RS domain-mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein-specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Delta yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression assay, we further showed that most LexA-SR fusion proteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain-containing proteins within the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.
    The Journal of Cell Biology 06/1999; 145(3):447-55. · 9.69 Impact Factor
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    ABSTRACT: Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain-containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain-containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH2 terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.
    The Journal of Cell Biology 03/1998; 140(4):737-50. · 9.69 Impact Factor
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    ABSTRACT: Multiple copies of the hexamer TGCATG have been shown to regulate fibronectin pre-mRNA alternative splicing. GCATG repeats also are clustered near the regulated calcitonin-specific 3' splice site in the rat calcitonin/CGRP gene. Specific mutagenesis of these repeats in calcitonin/CGRP pre-mRNA resulted in the loss of calcitonin-specific splicing, suggesting that the native repeats act to enhance alternative exon inclusion. Mutation of subsets of these elements implies that alternative splicing requires a minimum of two repeats, and that the combination of one intronic and one exonic repeat is necessary for optimal cell-specific splicing. However, multimerized intronic repeats inhibited calcitonin-specific splicing in both the wild-type context and in a transcript lacking endogenous repeats. These results suggest that both the number and distribution of repeats may be important features for the regulation of tissue-specific alternative splicing. Further, RNA containing a single repeat bound cell-specific protein complexes, but tissue-specific differences in protein binding were not detected by using multimerized repeats. Together, these data support a novel model for alternative splicing regulation that requires the cell-specific recognition of multiple, distributed sequence elements.
    Proceedings of the National Academy of Sciences 12/1997; 94(23):12343-7. DOI:10.1073/pnas.94.23.12343 · 9.81 Impact Factor
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    ABSTRACT: Pre-mRNA splicing requires a large number of RNA-binding proteins that have one or more RNA-recognition motifs (RRMs). Among these is the SR protein family, whose members are essential for splicing and are able to commit pre-mRNAs to the splicing pathway with overlapping but distinct substrate specificity. Some SR proteins, such as SC35, contain an N-terminal RRM and a C-terminal arginine/serine-rich (RS) domain, whereas others, such as SF2/ASF, also contain a second, atypical RRM. Although both the RRMs and the RS domain of SR proteins are required for constitutive splicing, it is unclear which domain(s) defines their substrate specificity, and whether two RRMs in a given SR protein function independently or act coordinately. Using domain swaps between SC35 and SF2/ASF and a functional commitment assay, we demonstrate that individual domains are functional modules, RS domains are interchangeable, and substrate specificity is defined by the RRMs. The atypical RRM of SF2/ASF does not appear to function alone in splicing, but can either activate or suppress the splicing specificity of an N-terminal RRM. Therefore, multiple RRMs in SR proteins act coordinately to achieve a unique spectrum of pre-mRNA substrate specificity.
    Proceedings of the National Academy of Sciences 05/1997; 94(8):3596-601. DOI:10.1073/pnas.94.8.3596 · 9.81 Impact Factor
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    ABSTRACT: Serine/arginine-rich (SR) proteins are essential for pre-mRNA splicing, and modify the choice of splice site during alternative splicing in a process apparently regulated by protein phosphorylation. Two protein kinases have been cloned that can phosphorylate SR proteins in vitro: SRPK1 and Clk/Sty. Here, we show that these two kinases phosphorylate the same SR proteins in vitro, but that SRPK1 has the higher specific activity toward ASF/SF2. SRPK1, like Clk/Sty, phosphorylates ASF/SF2 in vitro on sites that are also phosphorylated in vivo. Tryptic peptide mapping of ASF/SF2 revealed that three of the phosphopeptides from full-length ASF/SF2 phosphorylated in vitro contain consecutive phosphoserine-arginine residues or phosphoserine-proline residues. In vitro, the Clk/Sty kinase phosphorylated Ser-Arg, Ser-Lys, or Ser-Pro sites, whereas SRPK1 had a strong preference for Ser-Arg sites. These results suggest that SRPK1 and Clk/Sty may play different roles in regulating SR splicing factors, and suggest that Clk/Sty has a broader substrate specificity than SRPK1.
    Journal of Biological Chemistry 11/1996; 271(40):24569-75. DOI:10.1074/jbc.271.40.24569 · 4.60 Impact Factor

Publication Stats

2k Citations
235.64 Total Impact Points


  • 2006
    • Indiana University South Bend
      • Department of Biological Sciences
      South Bend, Indiana, United States
  • 2005
    • Iowa State University
      Ames, Iowa, United States
  • 1993–2005
    • University of California, San Diego
      • • Department of Cellular and Molecular Medicine (CMM)
      • • Department of Medicine
      San Diego, California, United States
  • 1999
    • Harvard University
      • Department of Molecular and Cell Biology
      Cambridge, Massachusetts, United States
  • 1998
    • Massachusetts Institute of Technology
      • Department of Biology
      Cambridge, MA, United States
  • 1996
    • Samuel Lunenfeld Research Institute
      Toronto, Ontario, Canada
  • 1992
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States