G2Cdb::Gene report

Gene id
Gene symbol
Tpm1 (MGI)
Mus musculus
tropomyosin 1, alpha
G00002006 (Homo sapiens)

Databases (9)

Curated Gene
OTTMUSG00000020411 (Vega mouse gene)
ENSMUSG00000032366 (Ensembl mouse gene)
22003 (Entrez Gene)
1206 (G2Cdb plasticity & disease)
Gene Expression
NM_024427 (Allen Brain Atlas)
22003 (Genepaint)
191010 (OMIM)
Marker Symbol
MGI:98809 (MGI)
Protein Sequence
P58771 (UniProt)

Synonyms (4)

  • TM2
  • Tm3
  • Tpm-1
  • alpha-TM

Literature (55)

Pubmed - other

  • A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

    Diez-Roux G, Banfi S, Sultan M, Geffers L, Anand S, Rozado D, Magen A, Canidio E, Pagani M, Peluso I, Lin-Marq N, Koch M, Bilio M, Cantiello I, Verde R, De Masi C, Bianchi SA, Cicchini J, Perroud E, Mehmeti S, Dagand E, Schrinner S, Nürnberger A, Schmidt K, Metz K, Zwingmann C, Brieske N, Springer C, Hernandez AM, Herzog S, Grabbe F, Sieverding C, Fischer B, Schrader K, Brockmeyer M, Dettmer S, Helbig C, Alunni V, Battaini MA, Mura C, Henrichsen CN, Garcia-Lopez R, Echevarria D, Puelles E, Garcia-Calero E, Kruse S, Uhr M, Kauck C, Feng G, Milyaev N, Ong CK, Kumar L, Lam M, Semple CA, Gyenesei A, Mundlos S, Radelof U, Lehrach H, Sarmientos P, Reymond A, Davidson DR, Dollé P, Antonarakis SE, Yaspo ML, Martinez S, Baldock RA, Eichele G and Ballabio A

    Telethon Institute of Genetics and Medicine, Naples, Italy.

    Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

    Funded by: Medical Research Council: MC_U127527203; Telethon: TGM11S03

    PLoS biology 2011;9;1;e1000582

  • Loss of the AE3 anion exchanger in a hypertrophic cardiomyopathy model causes rapid decompensation and heart failure.

    Al Moamen NJ, Prasad V, Bodi I, Miller ML, Neiman ML, Lasko VM, Alper SL, Wieczorek DF, Lorenz JN and Shull GE

    Department of Molecular Genetics, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.

    The AE3 Cl(-)/HCO(3)(-) exchanger is abundantly expressed in the sarcolemma of cardiomyocytes, where it mediates Cl(-)-uptake and HCO(3)(-)-extrusion. Inhibition of AE3-mediated Cl(-)/HCO(3)(-) exchange has been suggested to protect against cardiac hypertrophy; however, other studies indicate that AE3 might be necessary for optimal cardiac function. To test these hypotheses we crossed AE3-null mice, which appear phenotypically normal, with a hypertrophic cardiomyopathy mouse model carrying a Glu180Gly mutation in α-tropomyosin (TM180). Loss of AE3 had no effect on hypertrophy; however, survival of TM180/AE3 double mutants was sharply reduced compared with TM180 single mutants. Analysis of cardiac performance revealed impaired cardiac function in TM180 and TM180/AE3 mutants. TM180/AE3 double mutants were more severely affected and exhibited little response to β-adrenergic stimulation, a likely consequence of their more rapid progression to heart failure. Increased expression of calmodulin-dependent kinase II and protein phosphatase 1 and differences in methylation and localization of protein phosphatase 2A were observed, but were similar in single and double mutants. Phosphorylation of phospholamban on Ser16 was sharply increased in both single and double mutants relative to wild-type hearts under basal conditions, leading to reduced reserve capacity for β-adrenergic stimulation of phospholamban phosphorylation. Imaging analysis of isolated myocytes revealed reductions in amplitude and decay of Ca(2+) transients in both mutants, with greater reductions in TM180/AE3 mutants, consistent with the greater severity of their heart failure phenotype. Thus, in the TM180 cardiomyopathy model, loss of AE3 had no apparent anti-hypertrophic effect and led to more rapid decompensation and heart failure.

    Funded by: NHLBI NIH HHS: HL061974, HL081680, R01 HL061974, R01 HL061974-11, R01 HL081680; NIDDK NIH HHS: DK043495, R37 DK043495

    Journal of molecular and cellular cardiology 2011;50;1;137-46

  • Optimising experimental design for high-throughput phenotyping in mice: a case study.

    Karp NA, Baker LA, Gerdin AK, Adams NC, Ramírez-Solis R and White JK

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

    To further the functional annotation of the mammalian genome, the Sanger Mouse Genetics Programme aims to generate and characterise knockout mice in a high-throughput manner. Annually, approximately 200 lines of knockout mice will be characterised using a standardised battery of phenotyping tests covering key disease indications ranging from obesity to sensory acuity. From these findings secondary centres will select putative mutants of interest for more in-depth, confirmatory experiments. Optimising experimental design and data analysis is essential to maximise output using the resources with greatest efficiency, thereby attaining our biological objective of understanding the role of genes in normal development and disease. This study uses the example of the noninvasive blood pressure test to demonstrate how statistical investigation is important for generating meaningful, reliable results and assessing the design for the defined research objectives. The analysis adjusts for the multiple-testing problem by applying the false discovery rate, which controls the number of false calls within those highlighted as significant. A variance analysis finds that the variation between mice dominates this assay. These variance measures were used to examine the interplay between days, readings, and number of mice on power, the ability to detect change. If an experiment is underpowered, we cannot conclude whether failure to detect a biological difference arises from low power or lack of a distinct phenotype, hence the mice are subjected to testing without gain. Consequently, in confirmatory studies, a power analysis along with the 3Rs can provide justification to increase the number of mice used.

    Funded by: Wellcome Trust: WT077157/Z/05/Z

    Mammalian genome : official journal of the International Mammalian Genome Society 2010;21;9-10;467-76

  • Tropomyosin isoform expression regulates the transition of adhesions to determine cell speed and direction.

    Bach CT, Creed S, Zhong J, Mahmassani M, Schevzov G, Stehn J, Cowell LN, Naumanen P, Lappalainen P, Gunning PW and O'Neill GM

    Oncology Research Unit, The Children's Hospital at Westmead, Westmead, New South Wales 2145, Australia.

    The balance of transition between distinct adhesion types contributes to the regulation of mesenchymal cell migration, and the characteristic association of adhesions with actin filaments led us to question the role of actin filament-associating proteins in the transition between adhesive states. Tropomyosin isoform association with actin filaments imparts distinct filament structures, and we have thus investigated the role for tropomyosins in determining the formation of distinct adhesion structures. Using combinations of overexpression, knockdown, and knockout approaches, we establish that Tm5NM1 preferentially stabilizes focal adhesions and drives the transition to fibrillar adhesions via stabilization of actin filaments. Moreover, our data suggest that the expression of Tm5NM1 is a critical determinant of paxillin phosphorylation, a signaling event that is necessary for focal adhesion disassembly. Thus, we propose that Tm5NM1 can regulate the feedback loop between focal adhesion disassembly and focal complex formation at the leading edge that is required for productive and directed cell movement.

    Molecular and cellular biology 2009;29;6;1506-14

  • Disruption of striated preferentially expressed gene locus leads to dilated cardiomyopathy in mice.

    Liu X, Ramjiganesh T, Chen YH, Chung SW, Hall SR, Schissel SL, Padera RF, Liao R, Ackerman KG, Kajstura J, Leri A, Anversa P, Yet SF, Layne MD and Perrella MA

    Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, MA 02115, USA.

    Background: The striated preferentially expressed gene (Speg) generates 4 different isoforms through alternative promoter use and tissue-specific splicing. Depending on the cell type, Speg isoforms may serve as markers of striated or smooth muscle differentiation.

    To elucidate function of Speg gene isoforms, we disrupted the Speg gene locus in mice by replacing common exons 8, 9, and 10 with a lacZ gene. beta-Galactosidase activity was detected in cardiomyocytes of the developing heart starting at day 11.5 days post coitum (dpc). beta-Galactosidase activity in other cell types, including vascular smooth muscle cells, did not begin until 18.5 dpc. In the developing heart, protein expression of only Spegalpha and Spegbeta isoforms was present in cardiomyocytes. Homozygous Speg mutant hearts began to enlarge by 16.5 dpc, and by 18.5 dpc, they demonstrated dilation of right and left atria and ventricles. These cardiac abnormalities in the absence of Speg were associated with a cellular hypertrophic response, myofibril degeneration, and a marked decrease in cardiac function. Moreover, Speg mutant mice exhibited significant neonatal mortality, with increased death occurring by 2 days after birth.

    Conclusions: These findings demonstrate that mutation of the Speg locus leads to cardiac dysfunction and a phenotype consistent with a dilated cardiomyopathy.

    Funded by: NHLBI NIH HHS: HL60788, HL65639, K08 HL076286, R01 HL060788, R01 HL060788-09, R01 HL060788-10, R01 HL060788-11, R01 HL065639, R01 HL065639-03, R01 HL065639-04, R01 HL065639-05, R01 HL102897; NIGMS NIH HHS: GM53249, R01 GM053249, R01 GM053249-10, R01 GM053249-11, R01 GM053249-12

    Circulation 2009;119;2;261-8

  • Targeted deletion of Dicer in the heart leads to dilated cardiomyopathy and heart failure.

    Chen JF, Murchison EP, Tang R, Callis TE, Tatsuguchi M, Deng Z, Rojas M, Hammond SM, Schneider MD, Selzman CH, Meissner G, Patterson C, Hannon GJ and Wang DZ

    Carolina Cardiovascular Biology Center, Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC 27599, USA.

    Cardiovascular disease is the leading cause of human morbidity and mortality. Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy associated with heart failure. Here, we report that cardiac-specific knockout of Dicer, a gene encoding a RNase III endonuclease essential for microRNA (miRNA) processing, leads to rapidly progressive DCM, heart failure, and postnatal lethality. Dicer mutant mice show misexpression of cardiac contractile proteins and profound sarcomere disarray. Functional analyses indicate significantly reduced heart rates and decreased fractional shortening of Dicer mutant hearts. Consistent with the role of Dicer in animal hearts, Dicer expression was decreased in end-stage human DCM and failing hearts and, most importantly, a significant increase of Dicer expression was observed in those hearts after left ventricle assist devices were inserted to improve cardiac function. Together, our studies demonstrate essential roles for Dicer in cardiac contraction and indicate that miRNAs play critical roles in normal cardiac function and under pathological conditions.

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;6;2111-6

  • Divergent regulation of the sarcomere and the cytoskeleton.

    Schevzov G, Fath T, Vrhovski B, Vlahovich N, Rajan S, Hook J, Joya JE, Lemckert F, Puttur F, Lin JJ, Hardeman EC, Wieczorek DF, O'Neill GM and Gunning PW

    Oncology Research Unit, The Children's Hospital at Westmead, Westmead, New South Wales 2145, Australia.

    The existence of a feedback mechanism regulating the precise amounts of muscle structural proteins, such as actin and the actin-associated protein tropomyosin (Tm), in the sarcomeres of striated muscles is well established. However, the regulation of nonmuscle or cytoskeletal actin and Tms in nonmuscle cell structures has not been elucidated. Unlike the thin filaments of striated muscles, the actin cytoskeleton in nonmuscle cells is intrinsically dynamic. Given the differing requirements for the structural integrity of the actin thin filaments of the sarcomere compared with the requirement for dynamicity of the actin cytoskeleton in nonmuscle cells, we postulated that different regulatory mechanisms govern the expression of sarcomeric versus cytoskeletal Tms, as key regulators of the properties of the actin cytoskeleton. Comprehensive analyses of tissues from transgenic and knock-out mouse lines that overexpress the cytoskeletal Tms, Tm3 and Tm5NM1, and a comparison with sarcomeric Tms provide evidence for this. Moreover, we show that overexpression of a cytoskeletal Tm drives the amount of filamentous actin.

    Funded by: NHLBI NIH HHS: HL 71952

    The Journal of biological chemistry 2008;283;1;275-83

  • Identification of transcripts with enriched expression in the developing and adult pancreas.

    Hoffman BG, Zavaglia B, Witzsche J, Ruiz de Algara T, Beach M, Hoodless PA, Jones SJ, Marra MA and Helgason CD

    Department of Cancer Endocrinology, BC Cancer Research Center, West 10th Ave, Vancouver, BC V5Z 1L3, Canada. bhoffman@bccrc.ca

    Background: Despite recent advances, the transcriptional hierarchy driving pancreas organogenesis remains largely unknown, in part due to the paucity of comprehensive analyses. To address this deficit we generated ten SAGE libraries from the developing murine pancreas spanning Theiler stages 17-26, making use of available Pdx1 enhanced green fluorescent protein (EGFP) and Neurog3 EGFP reporter strains, as well as tissue from adult islets and ducts.

    Results: We used a specificity metric to identify 2,536 tags with pancreas-enriched expression compared to 195 other mouse SAGE libraries. We subsequently grouped co-expressed transcripts with differential expression during pancreas development using K-means clustering. We validated the clusters first using quantitative real time PCR and then by analyzing the Theiler stage 22 pancreas in situ hybridization staining patterns of over 600 of the identified genes using the GenePaint database. These were then categorized into one of the five expression domains within the developing pancreas. Based on these results we identified a cascade of transcriptional regulators expressed in the endocrine pancreas lineage and, from this, we developed a predictive regulatory network describing beta-cell development.

    Conclusion: Taken together, this work provides evidence that the SAGE libraries generated here are a valuable resource for continuing to elucidate the molecular mechanisms regulating pancreas development. Furthermore, our studies provide a comprehensive analysis of pancreas development, and insights into the regulatory networks driving this process are revealed.

    Genome biology 2008;9;6;R99

  • Rescue of tropomyosin-induced familial hypertrophic cardiomyopathy mice by transgenesis.

    Jagatheesan G, Rajan S, Petrashevskaya N, Schwartz A, Boivin G, Arteaga GM, Solaro RJ, Liggett SB and Wieczorek DF

    Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0524, USA.

    Familial hypertrophic cardiomyopathy (FHC) is a disease caused by mutations in contractile proteins of the sarcomere. Our laboratory developed a mouse model of FHC with a mutation in the thin filament protein alpha-tropomyosin (TM) at amino acid 180 (Glu180Gly). The hearts of these mice exhibit dramatic systolic and diastolic dysfunction, and their myofilaments demonstrate increased calcium sensitivity. The mice also develop severe cardiac hypertrophy, with death ensuing by 6 mo. In an attempt to normalize calcium sensitivity in the cardiomyofilaments of the hypertrophic mice, we generated a chimeric alpha-/beta-TM protein that decreases calcium sensitivity in transgenic mouse cardiac myofilaments. By mating mice from these two models together, we tested the hypothesis that an attenuation of myofilament calcium sensitivity would modulate the severe physiological and pathological consequences of the FHC mutation. These double-transgenic mice "rescue" the hypertrophic phenotype by exhibiting a normal morphology with no pathological abnormalities. Physiological analyses of these rescued mice show improved cardiac function and normal myofilament calcium sensitivity. These results demonstrate that alterations in calcium response by modification of contractile proteins can prevent the pathological and physiological effects of this disease.

    Funded by: NHLBI NIH HHS: HL22231, HL22619, HL62426, HL71952, K01 HL67709

    American journal of physiology. Heart and circulatory physiology 2007;293;2;H949-58

  • Dilated cardiomyopathy mutant tropomyosin mice develop cardiac dysfunction with significantly decreased fractional shortening and myofilament calcium sensitivity.

    Rajan S, Ahmed RP, Jagatheesan G, Petrashevskaya N, Boivin GP, Urboniene D, Arteaga GM, Wolska BM, Solaro RJ, Liggett SB and Wieczorek DF

    Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati Medical Center, Cincinnati, OH 45267-0524, USA.

    Mutations in striated muscle alpha-tropomyosin (alpha-TM), an essential thin filament protein, cause both dilated cardiomyopathy (DCM) and familial hypertrophic cardiomyopathy. Two distinct point mutations within alpha-tropomyosin are associated with the development of DCM in humans: Glu40Lys and Glu54Lys. To investigate the functional consequences of alpha-TM mutations associated with DCM, we generated transgenic mice that express mutant alpha-TM (Glu54Lys) in the adult heart. Results showed that an increase in transgenic protein expression led to a reciprocal decrease in endogenous alpha-TM levels, with total myofilament TM protein levels remaining unaltered. Histological and morphological analyses revealed development of DCM with progression to heart failure and frequently death by 6 months. Echocardiographic analyses confirmed the dilated phenotype of the heart with a significant decrease in the left ventricular fractional shortening. Work-performing heart analyses showed significantly impaired systolic, and diastolic functions and the force measurements of cardiac myofibers revealed that the myofilaments had significantly decreased Ca(2+) sensitivity and tension generation. Real-time RT-PCR quantification demonstrated an increased expression of beta-myosin heavy chain, brain natriuretic peptide, and skeletal actin and a decreased expression of the Ca(2+) handling proteins sarcoplasmic reticulum Ca(2+)-ATPase and ryanodine receptor. Furthermore, our study also indicates that the alpha-TM54 mutation decreases tropomyosin flexibility, which may influence actin binding and myofilament Ca(2+) sensitivity. The pathological and physiological phenotypes exhibited by these mice are consistent with those seen in human DCM and heart failure. As such, this is the first mouse model in which a mutation in a sarcomeric thin filament protein, specifically TM, leads to DCM.

    Funded by: NHLBI NIH HHS: HL-062426, HL-22231, HL-71952, HL-79032, K01 HL-67709-4

    Circulation research 2007;101;2;205-14

  • Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria.

    Hoogaars WM, Engel A, Brons JF, Verkerk AO, de Lange FJ, Wong LY, Bakker ML, Clout DE, Wakker V, Barnett P, Ravesloot JH, Moorman AF, Verheijck EE and Christoffels VM

    Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.

    The sinoatrial node initiates the heartbeat and controls the rate and rhythm of contraction, thus serving as the pacemaker of the heart. Despite the crucial role of the sinoatrial node in heart function, the mechanisms that underlie its specification and formation are not known. Tbx3, a transcriptional repressor required for development of vertebrates, is expressed in the developing conduction system. Here we show that Tbx3 expression delineates the sinoatrial node region, which runs a gene expression program that is distinct from that of the bordering atrial cells. We found lineage segregation of Tbx3-negative atrial and Tbx3-positive sinoatrial node precursor cells as soon as cardiac cells turn on the atrial gene expression program. Tbx3 deficiency resulted in expansion of expression of the atrial gene program into the sinoatrial node domain, and partial loss of sinoatrial node-specific gene expression. Ectopic expression of Tbx3 in mice revealed that Tbx3 represses the atrial phenotype and imposes the pacemaker phenotype on the atria. The mice displayed arrhythmias and developed functional ectopic pacemakers. These data identify a Tbx3-dependent pathway for the specification and formation of the sinoatrial node, and show that Tbx3 regulates the pacemaker gene expression program and phenotype.

    Genes & development 2007;21;9;1098-112

  • p38-MAPK induced dephosphorylation of alpha-tropomyosin is associated with depression of myocardial sarcomeric tension and ATPase activity.

    Vahebi S, Ota A, Li M, Warren CM, de Tombe PP, Wang Y and Solaro RJ

    Department of Physiology and Biophysics, Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612-7342, USA.

    Our objective in work presented here was to understand the mechanisms by which activated p38alpha MAPK depresses myocardial contractility. To test the hypothesis that activation of p38 MAPK directly influences sarcomeric function, we used transgenic mouse models with hearts in which p38 MAPK was constitutively turned on by an upstream activator (MKK6bE). These hearts demonstrated a significant depression in ejection fraction after induction of the transgene. We also studied hearts of mice expressing a dominant negative p38alpha MAPK. Simultaneous determination of tension and ATPase activity of detergent-skinned fiber bundles from left ventricular papillary muscle demonstrated a significant inhibition of both maximum tension and ATPase activity in the transgenic-MKK6bE hearts. Fibers from hearts expressing dominant negative p38alpha MAPK demonstrated no significant change in tension or ATPase activity. There were no significant changes in phosphorylation level of troponin-T3 and troponin-T4, or myosin light chain 2. However, compared with controls, there was a significant depression in levels of phosphorylation of alpha-tropomyosin and troponin I in fiber bundles from transgenic-MKK6bE hearts, but not from dominant negative p38alpha MAPK hearts. Our experiments also showed that p38alpha MAPK colocalizes with alpha-actinin at the Z-disc and complexes with protein phosphatases (PP2alpha, PP2beta). These data are the first to indicate that chronic activation of p38alpha MAPK directly depresses sarcomeric function in association with decreased phosphorylation of alpha-tropomyosin.

    Funded by: NHLBI NIH HHS: HL 062311, P01 HL062426, P01 HL62426, R01 HL64035, T32 HL07692-14-15

    Circulation research 2007;100;3;408-15

  • Tropomyosin isoforms localize to distinct microfilament populations in osteoclasts.

    McMichael BK, Kotadiya P, Singh T, Holliday LS and Lee BS

    Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, OH 43210, USA.

    Osteoclasts resorb bone through transient rearrangement of their cytoskeletons to create a polarized phenotype in which an apical ruffled membrane is surrounded by a ring of F-actin that creates a tight seal against bone substrate. This process, coupled with the capacity for rapid motility, necessitates the presence of a dynamic, multi-functional actin cytoskeleton. Tropomyosins are a large class of actin-binding proteins that can regulate microfilament stability and organization by recruiting other regulatory proteins to actin, or alternately, by inhibiting their binding. Tropomyosins are expressed from four distinct genes (alpha, beta, gamma, and delta) that are alternately spliced to produce over forty isoforms. In recent years, it has become clear that nonmuscle isoforms of tropomyosin may be differentially distributed among intracellular pools of F-actin possessing different functions. Here we have used Western analysis and immunocytochemistry coupled with confocal microscopy to identify the isoforms of tropomyosin expressed by osteoclasts, as well as their distributions within cells. Osteoclasts express at least seven isoforms with markedly different distributions. The products of the alpha gene (Tm-2, -3, and -5a/5b) are up-regulated during osteoclastogenesis, indicating potential cell-specific functions. Some isoforms (Tm-5a/5b, Tm-4) are specifically enriched within and around osteoclast attachment structures, the sealing zone and podosomes, whereas others are more abundant in internal regions of the cell. This compartmentalization of tropomyosins to specific actin structures within osteoclasts is likely to play a critical role in determining the dynamic properties of the actin cytoskeleton and thus osteoclast activity.

    Funded by: NIAMS NIH HHS: R01 AR051515; NIDDK NIH HHS: R01 DK052131

    Bone 2006;39;4;694-705

  • Changes in end-to-end interactions of tropomyosin affect mouse cardiac muscle dynamics.

    Gaffin RD, Gokulan K, Sacchettini JC, Hewett TE, Klevitsky R, Robbins J, Sarin V, Zawieja DC, Meininger GA and Muthuchamy M

    Cardiovascular Research Institute and Department of Systems Biology and Translational Medicine, College of Medicine, Texas A&M University System Health Science Center, TX 77843-1114, USA.

    The ends of striated muscle tropomyosin (TM) are integral for thin filament cooperativity, determining the cooperative unit size and regulating the affinity of TM for actin. We hypothesized that altering the alpha-TM carboxy terminal overlap end to the beta-TM counterpart would affect the amino-terminal association, which would alter the end-to-end interactions of TM molecules in the thin filament regulatory strand and affect the mechanisms of cardiac muscle contraction. To test this hypothesis, we generated transgenic (TG) mouse lines that express a mutant form of alpha-TM in which the first 275 residues are from alpha-TM and the last nine amino acids are from beta-TM (alpha-TM9aaDeltabeta). Molecular analyses show that endogenous alpha-TM mRNA and protein are nearly completely replaced with alpha-TM9aaDeltabeta. Working heart preparations data show that the rates of contraction and relaxation are reduced in alpha-TM9aaDeltabeta hearts. Left ventricular pressure and time to peak pressure are also reduced (-12% and -13%, respectively). The ratio of maximum to minimum first derivatives of change in left ventricular systolic pressure with respect to time (ratio of +dP/dt to -dP/dt, respectively) is increased, but tau is not changed significantly. Force-intracellular calcium concentration ([Ca2+]i) measurements from intact papillary fibers demonstrate that alpha-TM9aaDeltabeta TG fibers produce less force per given [Ca2+]i compared with nontransgenic fibers. Taken together, the data demonstrate that the rate of contraction is primarily affected in TM TG hearts. Protein docking studies show that in the mutant molecule, the overall carbon backbone is perturbed about 1.5 A, indicating that end-to-end interactions are altered. These results demonstrate that the localized flexibility present in the coiled-coil structures of TM isoforms is different, and that plays an important role in interacting with neighboring thin filament regulatory proteins and with differentially modulating the myofilament activation processes.

    Funded by: NHLBI NIH HHS: HL-60758

    American journal of physiology. Heart and circulatory physiology 2006;291;2;H552-63

  • MusTRD can regulate postnatal fiber-specific expression.

    Issa LL, Palmer SJ, Guven KL, Santucci N, Hodgson VR, Popovic K, Joya JE and Hardeman EC

    Muscle Development Unit, Children's Medical Research Institute, Wentworthville, NSW 2145, Australia.

    Human MusTRD1alpha1 was isolated as a result of its ability to bind a critical element within the Troponin I slow upstream enhancer (TnIslow USE) and was predicted to be a regulator of slow fiber-specific genes. To test this hypothesis in vivo, we generated transgenic mice expressing hMusTRD1alpha1 in skeletal muscle. Adult transgenic mice show a complete loss of slow fibers and a concomitant replacement by fast IIA fibers, resulting in postural muscle weakness. However, developmental analysis demonstrates that transgene expression has no impact on embryonic patterning of slow fibers but causes a gradual postnatal slow to fast fiber conversion. This conversion was underpinned by a demonstrable repression of many slow fiber-specific genes, whereas fast fiber-specific gene expression was either unchanged or enhanced. These data are consistent with our initial predictions for hMusTRD1alpha1 and suggest that slow fiber genes contain a specific common regulatory element that can be targeted by MusTRD proteins.

    Developmental biology 2006;293;1;104-15

  • BGEM: an in situ hybridization database of gene expression in the embryonic and adult mouse nervous system.

    Magdaleno S, Jensen P, Brumwell CL, Seal A, Lehman K, Asbury A, Cheung T, Cornelius T, Batten DM, Eden C, Norland SM, Rice DS, Dosooye N, Shakya S, Mehta P and Curran T

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States.

    Funded by: NINDS NIH HHS: 5R37NS036558, N01-NS-0-2331, R37 NS036558

    PLoS biology 2006;4;4;e86

  • Evaluation of candidate markers for the peritubular myoid cell lineage in the developing mouse testis.

    Jeanes A, Wilhelm D, Wilson MJ, Bowles J, McClive PJ, Sinclair AH and Koopman P

    Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.

    Despite the importance of peritubular myoid (PM) cells in the histogenesis of the fetal testis, understanding the origin and function of these cells has been hampered by the lack of suitable markers. The current study was aimed at identifying molecular markers for PM cells during the early stages of testis development in the mouse embryo. Expression of candidate marker genes was tested by section in situ hybridisation, in some instances followed by immunofluorescent detection of protein products. Collagen type-I, inhibinbetaA, caldesmon 1 and tropomyosin 1 were found to be expressed by early-stage PM cells. These markers were also expressed in subsets of interstitial cells, most likely reflecting their common embryological provenance from migrating mesonephric cells. Although not strictly specific for PM cells, these markers are likely to be useful in studying the biology of early PM cells in the fetal testis.

    Reproduction (Cambridge, England) 2005;130;4;509-16

  • Specific features of neuronal size and shape are regulated by tropomyosin isoforms.

    Schevzov G, Bryce NS, Almonte-Baldonado R, Joya J, Lin JJ, Hardeman E, Weinberger R and Gunning P

    Oncology Research Unit, The Children's Hospital at Westmead, Westmead NSW 2145, Australia.

    Spatially distinct populations of microfilaments, characterized by different tropomyosin (Tm) isoforms, are present within a neuron. To investigate the impact of altered tropomyosin isoform expression on neuronal morphogenesis, embryonic cortical neurons from transgenic mice expressing the isoforms Tm3 and Tm5NM1, under the control of the beta-actin promoter, were cultured in vitro. Exogenously expressed Tm isoforms sorted to different subcellular compartments with Tm5NM1 enriched in filopodia and growth cones, whereas the Tm3 was more broadly localized. The Tm5NM1 neurons displayed significantly enlarged growth cones accompanied by an increase in the number of dendrites and axonal branching. In contrast, Tm3 neurons displayed inhibition of neurite outgrowth. Recruitment of Tm5a and myosin IIB was observed in the peripheral region of a significant number of Tm5NM1 growth cones. We propose that enrichment of myosin IIB increases filament stability, leading to the enlarged growth cones. Our observations support a role for different tropomyosin isoforms in regulating interactions with myosin and thereby regulating morphology in specific intracellular compartments.

    Molecular biology of the cell 2005;16;7;3425-37

  • cTnT1, a cardiac troponin T isoform, decreases myofilament tension and affects the left ventricular pressure waveform.

    Nassar R, Malouf NN, Mao L, Rockman HA, Oakeley AE, Frye JR, Herlong JR, Sanders SP and Anderson PA

    Department of Pediatrics, Duke University, Durham, NC, USA.

    Four isoforms of cardiac troponin T (cTnT), a protein essential for calcium-dependent myocardial force development, are expressed in the human; they differ in charge and length. Their expression is regulated developmentally and is affected by disease states. Human cTnT (hcTnT) isoform effects have been examined in reconstituted myofilaments. In this study, we evaluated the modulatory effects of overexpressing one cTnT isoform on in vitro and in vivo myocardial function. A hcTnT isoform, hcTnT(1), expressed during development and in heart disease but not in the normal adult heart, was expressed in transgenic (TG) mice (1-30% of total cTnT). Maximal active tension measured in skinned myocardium decreased as a function of relative hcTnT(1) expression. The pCa at half-maximal force development, Hill coefficient, and rate of redevelopment of force did not change significantly with hcTnT(1) expression. In vivo maximum rates of rise and fall of left ventricular pressure decreased, and the half-time of isovolumic relaxation increased, with hcTnT(1) expression. Substituting total cTnT charge for hcTnT(1) expression resulted in similar conclusions. Morphometric analysis and electron microscopy revealed no differences between wild-type (non-TG) and TG myocardium. No differences in isoform expression of tropomyosin, myosin heavy chain, essential and regulatory myosin light chains (MLC), TnI, or in posttranslational modifications of mouse cTnT, cTnI, or regulatory MLC were observed. These results support the hypothesis that cTnT isoform amino-terminal differences affect myofilament function and suggest that hcTnT(1) expression levels present during human development and in human heart disease can affect in vivo ventricular function.

    Funded by: NHLBI NIH HHS: HL-42250, HL-67385

    American journal of physiology. Heart and circulatory physiology 2005;288;3;H1147-56

  • Charged residue alterations in the inner-core domain and carboxy-terminus of alpha-tropomyosin differentially affect mouse cardiac muscle contractility.

    Gaffin RD, Tong CW, Zawieja DC, Hewett TE, Klevitsky R, Robbins J and Muthuchamy M

    Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, Texas A & M University System Health Science Center, 336 Reynolds Medical Building, College Station, TX 77843-1114, USA.

    Two important charge differences between the alpha- and beta-tropomyosin (TM) isoforms are the exchange of a serine residue in the inner-core region at position 229, and a histidine residue at the carboxy-terminal end at position 276, with glutamic acid and asparagine, respectively. We have recently shown that altering these two residues in alpha-TM to their beta-TM counterparts in transgenic (TG) mouse hearts causes a depression in both +dP/dt and -dP/dt and a decrease in calcium sensitivity. In this study, we address whether independent charge changes at these two residues in alpha-TM modulate cardiac function differentially. To test this hypothesis we generated two TG lines: alpha-TMSer229Glu and alpha-TMHis276Asn. Molecular analyses show that 98% of native alpha-TM is replaced by mutated protein in alpha-TM229 hearts whereas alpha-TM276 hearts show 82% replacement with the mutated protein. Isolated working heart data show that alpha-TM229 TG hearts exhibit a significant decrease in both +dP/dt (7%) and -dP/dt (8%) compared with nontransgenics (NTGs) and time to peak pressure (TPP) is also reduced in alpha-TM229 hearts. alpha-TM276 hearts show a decrease only in -dP/dt (14%) and TPP is increased. pCa(2+)-tension relationships in skinned fibre preparations indicate decreased calcium sensitivity in alpha-TM229 but no change in alpha-TM276 preparations. Force-[Ca(2+)](IC) measurements from intact papillary fibres indicate that alpha-TM276 fibres produce more force per given [Ca(2+)](IC) when compared to NTG fibres, while alpha-TM229 fibres produce less force per given [Ca(2+)](IC). These data demonstrate that changing charged residues at either the inner-core domain or the carboxyl end of TM alters sarcomeric performance differently, suggesting that the function of TM is compartmentalized along its length.

    Funded by: NHLBI NIH HHS: HL 60758, P01 HL 69779, P01 HL069779; NICHD NIH HHS: N01 HD 53229

    The Journal of physiology 2004;561;Pt 3;777-91

  • Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas.

    Watahiki A, Waki K, Hayatsu N, Shiraki T, Kondo S, Nakamura M, Sasaki D, Arakawa T, Kawai J, Harbers M, Hayashizaki Y and Carninci P

    Genome Science Laboratory, RIKEN, Wako main campus, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan.

    It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line-specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development.

    Nature methods 2004;1;3;233-9

  • Charged residue changes in the carboxy-terminus of alpha-tropomyosin alter mouse cardiac muscle contractility.

    Gaffin RD, Gokulan K, Sacchettini JC, Hewett T, Klevitsky R, Robbins J and Muthuchamy M

    Cardiovascular Research Institute and Department of Medical Physiology, College of Medicine, Texas A & M University System Health Science Center, College Station, TX 77843-1114, USA. marim@tamu.edu

    Striated muscle tropomyosin (TM) is an essential thin filament protein that is sterically and allosterically involved in calcium-mediated cardiac contraction. We have previously shown that overexpressing the beta-TM isoform in mouse hearts leads to physiological changes in myocardial relaxation and Ca(2+) handling of myofilaments. Two important charge differences in beta-TM compared to alpha-TM are the exchange of serine and histidine at positions 229 and 276 with glutamic acid and asparagine, respectively, imparting a more negative charge to beta-TM relative to alpha-TM. Our hypothesis is that the net charge at specific sites on TM might be a major determinant of its role in modulating cardiac muscle performance and in regulating Ca(2+) sensitivity of the myofilaments. To address this, we generated transgenic (TG) double mutation mouse lines (alpha-TM DM) expressing mutated alpha-TM at the two residues that differ between alpha- and beta-TM (Ser229Glu + His276Asn). Molecular analyses show 60-88% of the native TM is replaced with alpha-TM DM in the different TG lines. Work-performing heart analyses show that alpha-TM DM mouse hearts exhibit decreased rates of pressure development and relaxation (+dP/dt and -dP/dt). Skinned myofibre preparations from the TG hearts indicate a decrease in calcium sensitivity of steady state force. Protein modelling studies show that these two charge alterations in alpha-TM cause a change in the surface charges of the molecule. Our results provide the first evidence that charge changes at the carboxy-terminal of alpha-TM alter the functional characteristics of the heart at both the whole organ and myofilament levels.

    Funded by: NHLBI NIH HHS: HL-60758; NICHD NIH HHS: N01-HD-5-3229

    The Journal of physiology 2004;556;Pt 2;531-43

  • N terminus is essential for tropomyosin functions: N-terminal modification disrupts stress fiber organization and abolishes anti-oncogenic effects of tropomyosin-1.

    Bharadwaj S, Hitchcock-DeGregori S, Thorburn A and Prasad GL

    Departments of General Surgery and Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.

    Down-regulation of several key actin-binding proteins, such as alpha-actinin, vinculin, gelsolin, and tropomyosins (TMs), is considered to contribute to the disorganized cytoskeleton present in many neoplastic cells. TMs stabilize actin filaments against the gel severing actions of proteins such as cofilin. Among multiple TMs expressed in non-muscle cells, tropomyosin-1 (TM1) isoform induces stress fibers and functions as a suppressor of malignant transformation. However, the molecular mechanisms of TM1-mediated cytoskeletal effects and tumor suppression remain poorly understood. We have hypothesized that the ability of TM1 to stabilize microfilaments is crucial for tumor suppression. In this study, by employing a variant TM1, which contains an N-terminal hemagglutinin epitope tag, we demonstrate that the N terminus is a key determinant of tropomyosin-1 function. Unlike the wild type TM1, the modified protein fails to restore stress fibers and inhibit anchorage-independent growth in transformed cells. Furthermore, the N-terminal modification of TM1 disorganizes the cytoskeleton and delays cytokinesis in normal cells, abolishes binding to F-actin, and disrupts the dimeric associations in vivo. The functionally defective TM1 allows the association of cofilin to stress fibers and disorganizes the microfilaments, whereas wild type TM1 appears to restrict the binding of cofilin to stress fibers. TM1-induced cytoskeletal reorganization appears to be mediated through preventing cofilin interaction with microfilaments. Our studies provide in vivo functional evidence that the N terminus is a critical determinant of TM1 functions, which in turn determines the organization of stress fibers.

    Funded by: NIGMS NIH HHS: GM 63257

    The Journal of biological chemistry 2004;279;14;14039-48

  • The PTB interacting protein raver1 regulates alpha-tropomyosin alternative splicing.

    Gromak N, Rideau A, Southby J, Scadden AD, Gooding C, Hüttelmaier S, Singer RH and Smith CW

    Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.

    Regulated switching of the mutually exclusive exons 2 and 3 of alpha-tropomyosin (TM) involves repression of exon 3 in smooth muscle cells. Polypyrimidine tract-binding protein (PTB) is necessary but not sufficient for regulation of TM splicing. Raver1 was identified in two-hybrid screens by its interactions with the cytoskeletal proteins actinin and vinculin, and was also found to interact with PTB. Consistent with these interactions raver1 can be localized in either the nucleus or cytoplasm. Here we show that raver1 is able to promote the smooth muscle-specific alternative splicing of TM by enhancing PTB-mediated repression of exon 3. This activity of raver1 is dependent upon characterized PTB-binding regulatory elements and upon a region of raver1 necessary for interaction with PTB. Heterologous recruitment of raver1, or just its C-terminus, induced very high levels of exon 3 skipping, bypassing the usual need for PTB binding sites downstream of exon 3. This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor.

    The EMBO journal 2003;22;23;6356-64

  • Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention.

    Zambrowicz BP, Abuin A, Ramirez-Solis R, Richter LJ, Piggott J, BeltrandelRio H, Buxton EC, Edwards J, Finch RA, Friddle CJ, Gupta A, Hansen G, Hu Y, Huang W, Jaing C, Key BW, Kipp P, Kohlhauff B, Ma ZQ, Markesich D, Payne R, Potter DG, Qian N, Shaw J, Schrick J, Shi ZZ, Sparks MJ, Van Sligtenhorst I, Vogel P, Walke W, Xu N, Zhu Q, Person C and Sands AT

    Lexicon Genetics, 8800 Technology Forest Place, The Woodlands, TX 77381, USA. brian@lexgen.com

    The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in approximately 60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;24;14109-14

  • Altered signaling surrounding the C-lobe of cardiac troponin C in myofilaments containing an alpha-tropomyosin mutation linked to familial hypertrophic cardiomyopathy.

    Burkart EM, Arteaga GM, Sumandea MP, Prabhakar R, Wieczorek DF and Solaro RJ

    Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, 835 S Wolcott Avenue, MC 901, Room E-202 MSB, Chicago, IL 606127342, USA.

    A region of interaction between the near N-terminal of cardiac troponin I (cTnI) and the C-lobe of troponin C (cTnC), where troponin T (cTnT) binds, appears to be critical in regulation of myofilament Ca(2+)-activation. We probed whether functional consequences of modulation of this interface influence the function of tropomyosin (Tm) in thin filament activation. We modified the C-lobe of cTnC directly by addition of the Ca(2+)-sensitizer, EMD 57033, and indirectly by replacing native cTnI with cTnI-containing Glu residues at Ser-43 and Ser-45 (cTnI-S43E/S45E) in myofilaments from hearts of non-transgenic (NTG) and transgenic (TG) mice expressing a point mutation on alpha-Tm (E180G) linked to familial hypertrophic cardiomyopathy. Introduction of cTnI-S43E/S45E induced a significantly greater reduction in tension in TG myofilaments compared to NTG controls. Furthermore, the effect of EMD 57033 to restore Ca(2+)-sensitivity was higher in TG compared to NTG fiber bundles containing cTnI-S43E/S45E and compared to TG or NTG fiber bundles containing native TnI. Our results indicate that alterations in regions of interaction among the N-terminal of cTnI, the C-lobe of cTnC, and the C-terminus of cTnT are important in the regulation of myofilament activity. Although levels of phosphorylation at protein kinase C-dependent sites were the same in TG and NTG myofilaments, our data indicate that the effects of phosphorylation were more depressive in TG hearts.

    Funded by: NHLBI NIH HHS: F32 HL10409, P01 HL 22619, P01 HL 62426, R01 HL64035; PHS HHS: T3207692

    Journal of molecular and cellular cardiology 2003;35;10;1285-93

  • Subtractive hybridisation screen identifies sexually dimorphic gene expression in the embryonic mouse gonad.

    McClive PJ, Hurley TM, Sarraj MA, van den Bergen JA and Sinclair AH

    The sex of most mammals is determined by the action of SRY. Its presence initiates testis formation resulting in male differentiation, its absence results in ovary formation and female differentiation. We have used suppression subtraction hybridisation between 12.0-12.5 days postcoitum (dpc) mouse testes and ovaries to identify genes that potentially lie within the Sry pathway. Normalised urogenital ridge libraries comprising 8,352 clones were differentially screened with subtracted probes. A total of 272 candidate cDNAs were tested for qualitative differential expression and localisation by whole mount in situ hybridisation; germ cell-dependent or -independent expression was further resolved using busulfan. Fifty-four genes were identified that showed higher expression in the testis than the ovary. One novel gene may be a candidate for interactions with WT1, based on its localisation to Sertoli cells and map position (16q24.3).

    Genesis (New York, N.Y. : 2000) 2003;37;2;84-90

  • A mouse model of familial hypertrophic cardiomyopathy caused by a alpha-tropomyosin mutation.

    Prabhakar R, Petrashevskaya N, Schwartz A, Aronow B, Boivin GP, Molkentin JD and Wieczorek DF

    Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0524, USA.

    Familial hypertrophic cardiomyopathy, a disease caused by mutations in cardiac contractile proteins, is characterized by left and/or right ventricular hypertrophy, myocyte disarray, fibrosis, and cardiac arrhythmias that may lead to premature sudden death. Five distinct point mutations within alpha-tropomyosin are associated with the development of familial hypertrophic cardiomyopathy. Two of these mutations are found within a troponin T binding site, located at amino acids 175 and 180. In this study, we analyze a transgenic mouse model for one of the mutations that occur at codon 180: a substitution of a glutamic acid for a glycine. These mice develop severe cardiac hypertrophy, substantial interstitial fibrosis, and have an increased heart weight/ body weight ratio. Results show that calcium-handling proteins associated with the sarcoplasmic reticulum exhibit decreased expression. These alterations in gene expression, coupled with the structurally-altered tropomyosin, may contribute to the demonstrated decreased physiological performance exhibited by these transgenic mice. A DNA hybridization microarray analysis of the transgenic vs. control ventricular RNAs shows that 50 transcripts are differentially expressed by more than 100% during the onset of the hypertrophic process, many of which are associated with the extracellular matrix. This study demonstrates that mutations within tropomyosin can be severely disruptive of sarcomeric function, triggering a hypertrophic response coupled with a cascade of alterations in gene expression.

    Funded by: NHLBI NIH HHS: HL22619, HL54912

    Molecular and cellular biochemistry 2003;251;1-2;33-42

  • BayGenomics: a resource of insertional mutations in mouse embryonic stem cells.

    Stryke D, Kawamoto M, Huang CC, Johns SJ, King LA, Harper CA, Meng EC, Lee RE, Yee A, L'Italien L, Chuang PT, Young SG, Skarnes WC, Babbitt PC and Ferrin TE

    Department of Pharmaceutical Chemistry, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA.

    The BayGenomics gene-trap resource (http://baygenomics.ucsf.edu) provides researchers with access to thousands of mouse embryonic stem (ES) cell lines harboring characterized insertional mutations in both known and novel genes. Each cell line contains an insertional mutation in a specific gene. The identity of the gene that has been interrupted can be determined from a DNA sequence tag. Approximately 75% of our cell lines contain insertional mutations in known mouse genes or genes that share strong sequence similarities with genes that have been identified in other organisms. These cell lines readily transmit the mutation to the germline of mice and many mutant lines of mice have already been generated from this resource. BayGenomics provides facile access to our entire database, including sequence tags for each mutant ES cell line, through the World Wide Web. Investigators can browse our resource, search for specific entries, download any portion of our database and BLAST sequences of interest against our entire set of cell line sequence tags. They can then obtain the mutant ES cell line for the purpose of generating knockout mice.

    Funded by: NCRR NIH HHS: P41 RR001081, P41 RR01081; NHLBI NIH HHS: U01 HL066621, U01 HL66621

    Nucleic acids research 2003;31;1;278-81

  • Interacting QTLs for cholesterol gallstones and gallbladder mucin in AKR and SWR strains of mice.

    Wittenburg H, Lammert F, Wang DQ, Churchill GA, Li R, Bouchard G, Carey MC and Paigen B

    Jackson Laboratory, Bar Harbor, Maine 04609, USA.

    We employed quantitative trait locus (QTL) mapping in a backcross between gallstone-susceptible SWR/J and gallstone-resistant AKR/J inbred mice to identify additional susceptibility loci for cholesterol gallstone formation. After 12 wk of feeding the mice a lithogenic diet, we phenotyped 330 backcross progeny for gallstones, gallbladder mucin accumulation, liver weight, and body weight. Marker-based regression analysis revealed significant single QTLs associated with gallstone formation on chromosome 9 and the liver weight/body weight ratio on chromosomes 5 and X. A search for gene pairs detected significant gene-gene interactions for mucin accumulation between loci on chromosomes 5 and 11 and suggestive gene-gene interactions linked to gallstone formation between the QTL on chromosome 9 and loci on chromosomes 6 and 15. These findings uncover new QTLs for cholesterol gallstones, reveal independent loci for mucin accumulation, and demonstrate the importance of considering gene-gene interactions in cholesterol cholelithiasis. According to standard nomenclature, the gallstone QTL on chromosome 9 is named Lith5.

    Funded by: NIDDK NIH HHS: DK-34854, DK-36588, DK-51568, DK-52911, DK-54012

    Physiological genomics 2002;8;1;67-77

  • Construction of long-transcript enriched cDNA libraries from submicrogram amounts of total RNAs by a universal PCR amplification method.

    Piao Y, Ko NT, Lim MK and Ko MS

    Developmental Genomics and Aging Section, Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.

    Here we report a novel design of linker primer that allows one to differentially amplify long tracts (average 3.0 kb with size ranges of 1-7 kb) or short DNAs (average 1.5 kb with size ranges of 0.5-3 kb) from a complex mixture. The method allows one to generate cDNA libraries enriched for long transcripts without size selection of insert DNAs. One representative library from newborn kidney includes 70% of clones bearing ATG start codons. A comparable library has been generated from 20 mouse blastocysts, containing only approximately 40 ng of total RNA. This universal PCR amplification scheme can provide a route to isolate very large cDNAs, even if they are expressed at very low levels.

    Genome research 2001;11;9;1553-8

  • Impaired motor coordination in mice that lack punc.

    Yang W, Li C and Mansour SL

    Department of Human Genetics, University of Utah, 15 N 2030 E, Salt Lake City, UT 84112, USA.

    The punc gene, encoding a member of the neural cell adhesion molecule family expressed in the developing central nervous system, limbs, and inner ear, was identified. To extend studies of the normal expression pattern of punc and to determine its function, a mouse strain bearing a lacZ/neo insertion in a 5' coding exon was created. The complex pattern of punc expression in embryos from embryonic day 9.5 (E9.5) to E11.5 was mimicked accurately by beta-galactosidase (beta-Gal) activity. As development proceeded, the distribution of beta-Gal activity was increasingly restricted, finally becoming confined to the brain and inner ear by E15.5. In the adult, beta-Gal activity was detected in several regions of the inner ear and brain and was particularly strong in the cerebellar Bergmann glia. Genetic analysis of this null allele demonstrated that punc is not required for normal embryogenesis. Interestingly, comparisons of beta-Gal activity and punc transcripts in heterozygous and homozygous mutant individuals demonstrated that punc is negatively autoregulated in some tissues. Adult punc-deficient mice were overtly normal and had normal hearing. Compared with control littermates, however, homozygous mutants had significantly reduced retention times on the Rotarod, suggesting a role for Bergmann glia-expressed Punc in the cerebellar control of motor coordination.

    Funded by: NIDCD NIH HHS: R01 DC002043, R01-DC02043

    Molecular and cellular biology 2001;21;17;6031-43

  • PKA accelerates rate of force development in murine skinned myocardium expressing alpha- or beta-tropomyosin.

    Patel JR, Fitzsimons DP, Buck SH, Muthuchamy M, Wieczorek DF and Moss RL

    Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA. jrpatel@physiology.wisc.edu

    In myocardium, protein kinase A (PKA) is known to phosphorylate troponin I (TnI) and myosin-binding protein-C (MyBP-C). Here, we used skinned myocardial preparations from nontransgenic (NTG) mouse hearts expressing 100% alpha-tropomyosin (alpha-Tm) to examine the effects of phosphorylated TnI and MyBP-C on Ca2+ sensitivity of force and the rate constant of force redevelopment (k(tr)). Experiments were also done using transgenic (TG) myocardium expressing approximately 60% beta-Tm to test the idea that the alpha-Tm isoform is required to observe the mechanical effects of PKA phosphorylation. Compared with NTG myocardium, TG myocardium exhibited greater Ca2+ sensitivity of force and developed submaximal forces at faster rates. Treatment with PKA reduced Ca2+ sensitivity of force in NTG and TG myocardium, had no effect on maximum k(tr) in either NTG or TG myocardium, and increased the rates of submaximal force development in both kinds of myocardium. These results show that PKA-mediated phosphorylation of myofibrillar proteins significantly alters the static and dynamic mechanical properties of myocardium, and these effects occur regardless of the type of Tm expressed.

    Funded by: NHLBI NIH HHS: HL-54912/PO1 HL-2261, K08 HL-03134, P01 HL-47053

    American journal of physiology. Heart and circulatory physiology 2001;280;6;H2732-9

  • A mutation in alpha-tropomyosin(slow) affects muscle strength, maturation and hypertrophy in a mouse model for nemaline myopathy.

    Corbett MA, Robinson CS, Dunglison GF, Yang N, Joya JE, Stewart AW, Schnell C, Gunning PW, North KN and Hardeman EC

    Muscle Development Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia.

    Nemaline myopathy is a hereditary disease of skeletal muscle defined by a distinct pathology of electron-dense accumulations within the sarcomeric units called rods, muscle weakness and, in most cases, a slow oxidative (type 1) fiber predominance. We generated a transgenic mouse model to study this disorder by expressing an autosomal dominant mutant of alpha-tropomyosin(slow) previously identified in a human cohort. Rods were found in all muscles, but to varying extents which did not correlate with the amount of mutant protein present. In addition, a pathological feature not commonly associated with this disorder, cytoplasmic bodies, was found in the mouse and subsequently identified in human samples. Muscle weakness is a major feature of this disease and was examined with respect to fiber composition, degree of rod-containing fibers, fiber mechanics and fiber diameter. Hypertrophy of fast, glycolytic (type 2B) fibers was apparent at 2 months of age. Muscle weakness was apparent in mice at 5-6 months of age, mimicking the late onset observed in humans with this mutation. The late onset did not correlate with observed changes in fiber type and rod pathology. Rather, the onset of muscle weakness correlates with an age-related decrease in fiber diameter and suggests that early onset is prevented by hypertrophy of fast, glycolytic fibers. We suggest that the clinical phenotype is precipitated by a failure of the hypertrophy to persist and therefore compensate for muscle weakness.

    Human molecular genetics 2001;10;4;317-28

  • Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray.

    Tanaka TS, Jaradat SA, Lim MK, Kargul GJ, Wang X, Grahovac MJ, Pantano S, Sano Y, Piao Y, Nagaraja R, Doi H, Wood WH, Becker KG and Ko MS

    Laboratory of Genetics and DNA Array Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224-6820, USA.

    cDNA microarray technology has been increasingly used to monitor global gene expression patterns in various tissues and cell types. However, applications to mammalian development have been hampered by the lack of appropriate cDNA collections, particularly for early developmental stages. To overcome this problem, a PCR-based cDNA library construction method was used to derive 52,374 expressed sequence tags from pre- and peri-implantation embryos, embryonic day (E) 12.5 female gonad/mesonephros, and newborn ovary. From these cDNA collections, a microarray representing 15,264 unique genes (78% novel and 22% known) was assembled. In initial applications, the divergence of placental and embryonic gene expression profiles was assessed. At stage E12.5 of development, based on triplicate experiments, 720 genes (6.5%) displayed statistically significant differences in expression between placenta and embryo. Among 289 more highly expressed in placenta, 61 placenta-specific genes encoded, for example, a novel prolactin-like protein. The number of genes highly expressed (and frequently specific) for placenta has thereby been increased 5-fold over the total previously reported, illustrating the potential of the microarrays for tissue-specific gene discovery and analysis of mammalian developmental programs.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;16;9127-32

  • Mouse model of a familial hypertrophic cardiomyopathy mutation in alpha-tropomyosin manifests cardiac dysfunction.

    Muthuchamy M, Pieples K, Rethinasamy P, Hoit B, Grupp IL, Boivin GP, Wolska B, Evans C, Solaro RJ and Wieczorek DF

    Department of Molecular Genetics, Division of Cardiology, University of Cincinnati College of Medicine, OH 45267-0524, USA.

    To investigate the functional consequences of a tropomyosin (TM) mutation associated with familial hypertrophic cardiomyopathy (FHC), we generated transgenic mice that express mutant alpha-TM in the adult heart. The missense mutation, which results in the substitution of asparagine for aspartic acid at amino acid position 175, occurs in a troponin T binding region of TM. S1 nuclease mapping and Western blot analyses demonstrate that increased expression of the alpha-TM 175 transgene in different lines causes a concomitant decrease in levels of endogenous alpha-TM mRNA and protein expression. In vivo physiological analyses show a severe impairment of both contractility and relaxation in hearts of the FHC mice, with a significant change in left ventricular fractional shortening. Myofilaments that contain alpha-TM 175 demonstrate an increased activation of the thin filament through enhanced Ca2+ sensitivity of steady-state force. Histological analyses show patchy areas of mild ventricular myocyte disorganization and hypertrophy, with occasional thrombi formation in the left atria. Thus, the FHC alpha-TM transgenic mouse can serve as a model system for the examination of pathological and physiological alterations imparted through aberrant TM isoforms.

    Funded by: NHLBI NIH HHS: HL-22231, HL-54912

    Circulation research 1999;85;1;47-56

  • p57Kip2 expression is enhanced during mid-cardiac murine development and is restricted to trabecular myocardium.

    Kochilas LK, Li J, Jin F, Buck CA and Epstein JA

    Division of Pediatric Cardiology, Children's Hospital of Philadelphia, Pennsylvania, USA.

    During embryonic development the heart is required to grow in size and cell number, undergo complex morphologic alterations, and function to circulate the blood. Between embryonic d 10.5 (E10.5) and E11.5, cardiac myocytes undergo rapid cell division, resulting in doubling of cardiac mass, while metabolic requirements are increased and contraction force is enhanced. Accelerated cardiomyocyte differentiation is accompanied by a significant increase in trabeculation of ventricular myocardium. Many single gene mutations in the mouse result in a "thinned myocardium" and embryonic lethality between E10.5 and E13.5 secondary to heart failure. This is the case in the Splotch mouse in which a mutation of the Pax3 gene results in neural crest and cardiac defects. Nevertheless, the molecular events governing these important developmental steps remain largely unknown. Here, we describe the use of suppression subtractive hybridization to identify mRNA transcripts whose expression is enhanced during this critical period in normal hearts. These genes encode functions related to maturation of the contractile apparatus, cardiomyocyte differentiation, altered cellular metabolism, and transcriptional regulation. One of the genes that we identified, p57Kip2, encodes a cyclin-dependent kinase inhibitor of the p21 family. We show that p57Kip2 is normally expressed in the inner trabecular layer of the developing heart. In Splotch embryos, expression of p57Kip2 is expanded to encompass the entire thickness of the myocardium. This result and further structural analysis suggests that the myocardial defect of Splotch embryos is associated with precocious cardiomyocyte differentiation.

    Funded by: NHLBI NIH HHS: HL03267, HL47670, HL515333

    Pediatric research 1999;45;5 Pt 1;635-42

  • Tissue-specific expression and mapping of the Cox7ah gene in mouse.

    Jaradat SA, Ko MS and Grossman LI

    Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

    We have isolated and examined the gene for the heart isoform of cytochrome c oxidase subunit VIIa (COX VIIa-H) in mouse, an isoform gene previously thought to be lacking in rodents. Interspecies amino acid comparisons indicate that mouse COX VIIa-H protein displays 82.5 and 70.9% identity with the bovine and human heart isoforms of COX VIIa, but only 53.7% identity with the paralogous mouse liver isoform (COX VIIa-L). Expression in adult mouse tissues is limited to heart and skeletal muscle, as found in other species. In the early mouse embryo, Cox7al was the exclusive isoform expressed and Cox7ah mRNA was not detectable until day 17 postcoitum. That the mouse Cox7ah gene characterized in this study is orthologous to the human COX7AH gene was also suggested by its mapping to mouse chromosome 7, to a conserved region syntenic with the human chromosome location of COX7AH, 19q13.1. As a result, all three COX heart isoform genes in mouse group to chromosome 7. Interestingly, mapping of the mouse Cox7al to chromosome 9 suggests a new syntenic region between the mouse and the human genomes.

    Funded by: NICHD NIH HHS: HD32243; NIGMS NIH HHS: GM48517

    Genomics 1998;49;3;363-70

  • Tropomyosin in preimplantation mouse development: identification, expression, and organization during cell division and polarization.

    Clayton L and Johnson MH

    Department of Anatomy, University of Cambridge, Downing Street, Cambridge, CB2 3DY, United Kingdom. lc@mole.bio.cam.ac.uk

    Tropomyosin is an actin-binding cytoskeletal protein which has been extensively characterized in a variety of cell types and tissues, with the exception of very early developmental stages during which cellular polarization first occurs. We have identified five polypeptides in mouse preimplantation conceptuses which show many of the characteristics of tropomyosin. They form the major portion of the heat-stable cytoskeletal protein fraction of blastomeres and have the characteristic isoelectric and SDS-PAGE migration characteristics on 1-D and 2-D gels. All five polypeptides were synthesized in late 2- and 4-cell, and all 8-cell stages, with three of the five polypeptides showing lower synthetic levels in fertilized eggs and early 2-cell conceptuses. These heat-stable proteins showed specific differences from proteins isolated from mouse 3T3 fibroblasts by the same method, namely higher Mr isoforms were not represented, also some of the isoforms can be labeled by incorporation of [14C]proline. The cellular distribution of tropomyosin in early stage conceptuses was examined using monoclonal and affinity-purified polyclonal antibodies. Tropomyosin becomes associated both with the blastomere cortex postfertilization and with the cleavage furrow during cytokinesis. The interphase cortical association is uniform until the 8-cell stage, when tropomyosin becomes associated with the developing apical pole and is excluded from the basolateral cortex. This polar localization is inherited along with the pole at the 8- to 16-cell division, but experiments in which cell division is artificially prolonged show that tropomyosin localization does not represent a permanent marking of the pole. We conclude that the early mouse conceptus contains a unique and specific set of tropomyosins which respond to polarizing signals.

    Funded by: Wellcome Trust

    Experimental cell research 1998;238;2;450-64

  • Molecular and physiological effects of alpha-tropomyosin ablation in the mouse.

    Rethinasamy P, Muthuchamy M, Hewett T, Boivin G, Wolska BM, Evans C, Solaro RJ and Wieczorek DF

    Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinatti Medical Center, OH 45267-0524, USA.

    Tropomyosin (TM) is an integral component of the thin filament in muscle fibers and is involved in regulating actin-myosin interactions. TM is encoded by a family of four alternatively spliced genes that display highly conserved nucleotide and amino acid sequences. To assess the functional and developmental significance of alpha-TM, the murine alpha-TM gene was disrupted by homologous recombination. Homozygous alpha-TM null mice are embryonic lethal, dying between 8 and 11.5 days post coitum. Mice that are heterozygous for alpha-TM are viable and reproduce normally. Heterozygous knockout mouse hearts show a 50% reduction in cardiac muscle alpha-TM mRNA, with no compensatory increase in transcript levels by striated muscle beta-TM or TM-30 isoforms. Surprisingly, this reduction in alpha-TM mRNA levels in heterozygous mice is not reflected at the protein level, where normal amounts of striated muscle alpha-TM protein are produced and integrated in the myofibril. Quantification of alpha-TM mRNA bound in polysomal fractions reveals that both wild-type and heterozygous knockout animals have similar levels. These data suggest that a change in steady-state level of alpha-TM mRNA does not affect the relative amount of mRNA translated and amount of protein synthesized. Physiological analyses of myocardial and myofilament function show no differences between heterozygous alpha-TM mice and control mice. The present study suggests that translational regulation plays a major role in the control of TM expression.

    Funded by: NHLBI NIH HHS: HL-22231, HL-46826, HL-54912

    Circulation research 1998;82;1;116-23

  • Assignment of a disintegrin and metalloproteinase domain 10 (Adam10) gene to mouse chromosome 9.

    Yamazaki K, Mizui Y, Sagane K and Tanaka I

    Tsukuba Research Laboratories, Eisai Co., Ltd., Ibaraki, Japan. k5-yamazaki@eisai.co.jp

    Genomics 1997;46;3;528-9

  • Targeted ablation of the murine alpha-tropomyosin gene.

    Blanchard EM, Iizuka K, Christe M, Conner DA, Geisterfer-Lowrance A, Schoen FJ, Maughan DW, Seidman CE and Seidman JG

    Howard Hughes Medical Institute, Boston, Mass., USA.

    We created a mouse that lacks a functional alpha-tropomyosin gene using gene targeting in embryonic stem cells and blastocyst-mediated transgenesis. Homozygous alpha-tropomyosin "knockout" mice die between embryonic day 9.5 and 13.5 and lack alpha-tropomyosin mRNA. Heterozygous alpha-tropomyosin knockout mice have approximately 50% as much cardiac alpha-tropomyosin mRNA as wild-type littermates but similar alpha-tropomyosin protein levels. Cardiac gross morphology, histology, and function (assessed by working heart preparations) of heterozygous alpha-tropomyosin knockout and wild-type mice were indistinguishable. Mechanical performance of skinned papillary muscle strips derived from mutant and wild-type hearts also revealed no differences. We conclude that haploinsufficiency of the alpha-tropomyosin gene produces little or no change in cardiac function or structure, whereas total alpha-tropomyosin deficiency is incompatible with life. These findings imply that in heterozygotes there is a regulatory mechanism that maintains the level of myofibrillar tropomyosin despite the reduction in alpha-tropomyosin mRNA.

    Circulation research 1997;81;6;1005-10

  • Genetic mapping of 262 loci derived from expressed sequences in a murine interspecific cross using single-strand conformational polymorphism analysis.

    Brady KP, Rowe LB, Her H, Stevens TJ, Eppig J, Sussman DJ, Sikela J and Beier DR

    Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

    We have demonstrated previously that noncoding sequences of genes are a robust source of polymorphisms between mouse species when tested using single-strand conformation polymorphism (SSCP) analysis, and that these polymorphisms are useful for genetic mapping. In this report we demonstrate that presumptive 3'-untranslated region sequence obtained from expressed sequence tags (ESTs) can be analyzed in a similar fashion, and we have used this approach to map 262 loci using an interspecific backcross. These results demonstrate SSCP analysis of genes or ESTs is a simple and efficient means for the genetic localization of transcribed sequences, and is furthermore an approach that is applicable to any system for which there is sufficient sequence polymorphism.

    Funded by: NHGRI NIH HHS: HG00941, HG00951

    Genome research 1997;7;11;1085-93

  • Dynamin genes Dnm1 and Dnm2 are located on proximal mouse chromosomes 2 and 9, respectively.

    Klocke R, Augustin A, Ronsiek M, Stief A, van der Putten H and Jockusch H

    Developmental Biology Unit W7, University of Bielefeld, Germany.

    Dynamins, microtubule-binding GTPases, are encoded by at least three genes in mammals. Two distinct gene-specific cDNAs were used to analyze the segregation of dynamin genes Dnm1 and Dnm2 in a mouse interspecies backcross. The nervous system-expressed gene Dnm1 was localized to Chr 2 between the genes for vimentin and nebulin, within a chromosomal region of conserved synteny to human chromosome 9q, consistent with the localization of the human dynamin-1 gene by FISH (see accompanying paper by Newman-Smith et al., 1997, Genomics 41:286-289). The ubiquitously expressed Dnm2 gene was found to be closely linked to the intercellular adhesion molecule-1 gene, Icam1, in a region with homologies to human chromosomes 19p, 8q, and 11q. Potential relations of both loci to disease genes are discussed.

    Genomics 1997;41;2;290-2

  • Anti-oncogenic effects of tropomyosin: isoform specificity and importance of protein coding sequences.

    Braverman RH, Cooper HL, Lee HS and Prasad GL

    Laboratory of Cellular Oncology, NCI, Bethesda, Maryland 20892, USA.

    Suppression of muscle type isoforms of tropomyosin (TM) is a common biochemical event in malignantly transformed cells. To evaluate the role of TM proteins and isoform specificity in cellular transformation, cDNAs that consist of coding sequences of TM1 (product of beta gene) and TM2 (product of alpha gene), but lacking untranslated regions (UTRs), have been expressed separately in DT (v-Ki-ras transformed NIH3T3) cells, and elevated levels of the corresponding proteins were detected. DT cells which over express TM2 manifest growth in soft agar. Elevated levels of TM1 protein in DT cells resulted in flattened cell morphology and complete abolition of anchorage independent growth. Tumorigenesis in athymic nude mice was observed in the absence of transduced TM1 mRNA. Thus, expression of TM1 protein is sufficient for tumor suppression: the UTRs of TM1 are not required for the tumor suppressive effects. Expression of TM2 protein, on the other hand, has no effect on the transformed phenotype of DT cells. These data indicate that isoforms 1 and 2 of TMs perform distinct physiological roles.

    Oncogene 1996;13;3;537-45

  • Molecular diversity of myofibrillar proteins: gene regulation and functional significance.

    Schiaffino S and Reggiani C

    Department of Biomedical Sciences, University of Padova, Padua, Italy.

    Myofibrillar proteins exist as multiple isoforms that derive from multigene (isogene) families. Additional isoforms, including products of tropomyosin, myosin light chain 1 fast, troponin T, titin, and nebulin genes, can be generated from the same gene through alternative splicing or use of alternative promoters. Myofibrillar protein isogenes are differentially expressed in various muscle types and fiber types but can be coexpressed within the same fiber. Isogenes are regulated by transcriptional and posttranscriptional mechanisms; however, specific regulatory sequences and transcriptional factors have not yet been identified. The pattern of isogene expression varies during muscle development in relation to the different origin of myogenic cells and primary/secondary fiber generations and is affected by neural and hormonal influences. The variable expression of myofibrillar protein isoforms is a major determinant of the contractile properties of skeletal muscle fibers. The diversity among isomyosins is related to the differences in the parameters of chemomechanical transduction as ATP hydrolysis rate and shortening velocity. Troponin and tropomyosin isoforms determine the variable sensitivity to calcium, whereas titin isoforms dictate the elastic properties of muscle fibers at rest. Both myosin and troponin isoforms contribute to the differences in the resistance to fatigue of muscle fibers.

    Funded by: Telethon: 343, A.012

    Physiological reviews 1996;76;2;371-423

  • Contractile protein mutations and heart disease.

    Vikstrom KL and Leinwand LA

    Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309-0347, USA. vikstrom@stripe.colorado.edu

    Mutations in several muscle structural proteins (the myosin heavy chain, alpha tropomyosin, cardiac troponin T and myosin binding protein C) result in a genetically dominant heart disease, hypertrophic cardiomyopathy. Biochemical data from studies of mutant myosin suggest a dominant-negative mechanism for inheritance of this disease. The most likely primary defect is sarcomere dysfunction, which is followed by the major clinical symptoms.

    Current opinion in cell biology 1996;8;1;97-105

  • Chromosome mapping of nine tropomyosin-related sequences in mice.

    Gariboldi M, Manenti G, Dragani TA and Pierotti MA

    Division of Experimental Oncology A, Istituto Nazionale Tumori, Milan, Italy.

    Tropomyosins are a group of actin-binding proteins expressed as different isoforms in muscle and non-muscle cells. Two tropomyosin loci have already been mapped in the mouse genome, on Chromosomes (Chrs) 6 and 9. By using a human cDNA fragment of tropomyosin non-muscle isoform (TPM3) gene that maps on human Chr 1q, and a mapping panel from a murine interspecific cross, we mapped nine distinct tropomyosin-related loci in the mouse genome, on seven different chromosomes: Chrs 3, 4, 6, 7, 14, 17, and X.

    Mammalian genome : official journal of the International Mammalian Genome Society 1995;6;4;273-7

  • Assignment of the human skeletal muscle alpha-tropomyosin gene (TPM1) to band 15q22 by fluorescence in situ hybridization.

    Eyre H, Akkari PA, Wilton SD, Callen DC, Baker E and Laing NG

    Department of Cytogenetics and Molecular Genetics, Women's and Children's Hospital, North Adelaide, Australia.

    A sequence-tagged site (STS) was developed for the human skeletal muscle alpha-tropomyosin gene (TPM1) and used to isolate a genomic clone, lambda TPM1.1, containing part of the TPM1 gene. Fluorescence in situ hybridization of this clone to metaphase chromosome spreads localised TPM1 to chromosome band 15q22. This localisation in humans is consistent with that recently described for the mouse.

    Cytogenetics and cell genetics 1995;69;1-2;15-7

  • Subtractive cDNA cloning as a tool to analyse secondary effects of a muscle disease. Characterization of affected genes in the myotonic ADR mouse.

    Schleef M, Zühlke C, Schöffl F and Jockusch H

    University of Bielefeld, Developmental Biology Unit, Germany.

    In myotonic ADR mice that are homozygous for a defect in the muscular chloride channel gene adr/Clc-1, the hyperexcitability of fast muscles is associated with secondary changes in gene expression and fibre type composition. cDNA clones derived from a set of genes down regulated in fast muscles of the myotonic ADR mouse were isolated by a subtractive cloning procedure. A total of 1200 clones were analysed for high expression in fast muscle of wild type and low expression in mutant mouse. Differential transcript levels were verified by northern blot hybridizations. The identities of the corresponding transcripts were determined by sequencing as myosin heavy chain IIB, alpha-tropomyosin, troponin C, a Ca2+ ATPase and parvalbumin mRNAs. Of these, mRNAs for parvalbumin and myosin heavy chain IIB were drastically downregulated in myotonic muscle (to < 10% of control). A full length cDNA clone for skeletal muscle alpha-tropomyosin was homologous to the mouse fibroblast tropomyosin isoform 2, except for the portion encoding the alpha-tropomyosin specific amino acids 258-284. A cDNA derived from the 1100 nucleotide parvalbumin transcript was cloned and the sequence for the as yet unknown 3' extended trailer, generated by alternative polyadenylation, was determined.

    Neuromuscular disorders : NMD 1994;4;3;205-17

  • Chromosomal localization and genomic cloning of the mouse alpha-tropomyosin gene Tpm-1.

    Schleef M, Werner K, Satzger U, Kaupmann K and Jockusch H

    Developmental Biology Unit, University of Bielefeld, Federal Republic of Germany.

    In vertebrates, the alpha-tropomyosin gene, Tpm-1, codes for at least 9 tropomyosin isoforms that are expressed by tissue-specific alternative splicing. Using interspecies backcrosses, we have localized Tpm-1 on mouse chromosome 9, cen-Cyp1a2-Tpm-1-Mod-1-Mylc-Scn5a, near the d-se region. The restriction fragment length variant used for chromosomal assignment, as well as other restriction fragments hybridizing to a 3'-specific alpha-tropomyosin cDNA probe in genomic Southern blots, was investigated by cloning 17.5-kb of Tpm-1. The same restriction patterns were observed, proving the identity of the mapped and the cloned gene. The identity was supported by sequencing the 3' end of the gene.

    Genomics 1993;17;2;519-21

  • Developmental analysis of tropomyosin gene expression in embryonic stem cells and mouse embryos.

    Muthuchamy M, Pajak L, Howles P, Doetschman T and Wieczorek DF

    Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.

    Tropomyosins (TMs) comprise a family of actin-binding proteins which play an important role in the regulation of contractility in muscle (cardiac, skeletal, and smooth) and nonmuscle cells. Although they are present in all cells, different isoforms are characteristic of specific cell types. In vertebrates, there are four different TM genes (alpha-TM, beta-TM, TM30, and TM4), three of which generate alternatively spliced isoforms. This study defines the expression patterns of these isoforms during murine embryogenesis, using both in vivo and in vitro conditions. The embryonic stem cell culture system, which has been shown to mimic different stages of mouse embryonic development, including the differentiation of primitive organ systems such as the myocardium, is used for our in vitro analysis. Our results demonstrate that several TM isoforms are expressed in specific developmental patterns, often correlated with the differentiation of particular tissues or organs. Surprisingly, other TMs, such as the striated muscle beta-TM and smooth muscle alpha-TM, are expressed constitutively. This study also demonstrates that there is an excellent correlation between the expression patterns of the TM isoforms observed in developing embryonic stem cells and mouse embryos. In addition, a quantitative molecular analysis of TM isoforms was conducted in embryonic, neonatal, and adult cardiac tissue. Our results show for the first time that the alpha- and beta-TM striated muscle transcripts are present in the earliest functional stages of the heart, and these TM isoforms are identical to those present throughout cardiac development.

    Funded by: NHLBI NIH HHS: HL 46826; NIAMS NIH HHS: AR 39423

    Molecular and cellular biology 1993;13;6;3311-23

  • Protein interaction cloning in yeast: identification of mammalian proteins that react with the leucine zipper of Jun.

    Chevray PM and Nathans D

    Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

    To identify proteins that interact with Jun or Fos we have used the protein interaction cloning system developed by S. Fields and O.-K. Song [(1989) Nature (London) 340, 245-246] to clone mammalian cDNAs encoding polypeptides that interact with the dimerization and DNA-binding motif (bZIP; basic domain leucine zipper motif) of Jun. For this purpose, yeast cells lacking GAL4 activity but expressing a GAL4 DNA-binding domain-Jun bZIP fusion protein were transformed with a mouse embryo cDNA plasmid library in which the cDNA was joined to a gene segment encoding the GAL4 transcriptional activation domain. Several transformants exhibiting GAL4 activity were identified and shown to harbor plasmids encoding polypeptides predicted to form coiled-coil structures with Jun and/or Fos. One of these is a bZIP protein of the ATF/CREB protein family--probably the murine homolog of TAXREB67. Two others encode polypeptides with predicted potential to form coiled-coil structures, and seven other isolates encode segments of alpha- or beta-tropomyosin, classical coiled-coil proteins. The tropomyosin polypeptides were found to interact in the yeast assay system with the bZIP region of Jun but not with the bZIP region of Fos. Our results illustrate the range of protein interaction cloning for discovering proteins that bind to a given target polypeptide.

    Funded by: NCI NIH HHS: P01 CA16519-16; NIGMS NIH HHS: GM07309

    Proceedings of the National Academy of Sciences of the United States of America 1992;89;13;5789-93

  • Coordinate induction of fibronectin, fibronectin receptor, tropomyosin, and actin genes in serum-stimulated fibroblasts.

    Ryseck RP, MacDonald-Bravo H, Zerial M and Bravo R

    European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.

    From a collection of more than 80 nonoverlapping clones, isolated by differential screening of a lambda cDNA library prepared from serum-stimulated cells in the presence of cycloheximide, we have identified four clones that encoded for components of the cytoskeleton and extracellular matrix. DNA sequencing of clones B2, V58, TT1, and P38 demonstrated that they corresponded to beta-actin, alpha-tropomyosin, fibronectin, and the beta-subunit of fibronectin receptor. All four mRNA levels showed a detectable increase 30 min after stimulation and remained at high levels for at least 8 h. The half-lives of these mRNAs were found to be very long in contrast to those of other growth factor-inducible genes. An increase in transcription was observed for the four genes. Actin and fibronectin showed nearly maximal increase at 15 min, while fibronectin receptor and tropomyosin reached their maximum transcription at 1 h. These results demonstrated that four interacting components of the cytoskeleton and extracellular matrix are rapidly induced in stimulated quiescent cells, possibly reflecting part of the coordinate changes in gene expression that occur during embryogenesis and wound healing.

    Experimental cell research 1989;180;2;537-45

  • Isolation and characterization of a cDNA that encodes mouse fibroblast tropomyosin isoform 2.

    Takenaga K, Nakamura Y, Tokunaga K, Kageyama H and Sakiyama S

    Division of Chemotherapy and Biochemistry, Chiba Cancer Center Research Institute, Japan.

    We isolated and characterized a cDNA clone encoding tropomyosin isoform 2 (TM2) from a mouse fibroblast cDNA library. TM2 was found to contain 284 amino acids and was closely related to the rat smooth and skeletal muscle alpha-TMs and the human fibroblast TM3. The amino acid sequence of TM2 showed a nearly complete match with that of human fibroblast TM3 except for the region from amino acids 189 to 213, the sequence of which was identical to those of rat smooth and skeletal muscle alpha-TMs. These results suggest that TM2 is expressed from the same gene that encodes the smooth muscle alpha-TM, the skeletal muscle alpha-TM, and TM3 via an alternative RNA-splicing mechanism. Comparison of the expression of TM2 mRNA in low-metastatic Lewis lung carcinoma P29 cells and high-metastatic D6 cells revealed that it was significantly less in D6 cells than in P29 cells, supporting our previous observations (K. Takenaga, Y. Nakamura, and S. Sakiyama, Mol. Cell. Biol. 8:3934-3937, 1988) at the protein level.

    Molecular and cellular biology 1988;8;12;5561-5

Gene lists (6)

Gene List Source Species Name Description Gene count
L00000001 G2C Mus musculus Mouse PSD Mouse PSD adapted from Collins et al (2006) 1080
L00000008 G2C Mus musculus Mouse PSP Mouse PSP adapted from Collins et al (2006) 1121
L00000060 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus (ortho) 748
L00000062 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus 984
L00000070 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list (ortho) 1461
L00000072 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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