Research Article Cloning, Sequencing, and the Expression...

Research ArticleCloning Sequencing and the Expression of the ElusiveSarcomeric TPM4120572 Isoform in Humans

Dipak K Dube Syamalima Dube Lynn Abbott Ruham Alshiekh-NasanyCharles Mitschow and Bernard J Poiesz

Department of Medicine SUNY Upstate Medical University 750 East Adams Street Syracuse NY 13210 USA

Correspondence should be addressed to Bernard J Poiesz poieszbupstateedu

Received 31 May 2016 Revised 22 July 2016 Accepted 10 August 2016

Academic Editor Jozsef Szeberenyi

Copyright copy 2016 Dipak K Dube et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In mammals tropomyosin is encoded by four known TPM genes (TPM1 TPM2 TPM3 and TPM4) each of which can generate anumber of TPM isoforms via alternative splicing andor using alternate promoters In humans the sarcomeric isoform(s) of eachof the TPM genes except for the TPM4 have been known for a long time Recently on the basis of computational analyses of thehuman genome sequence the predicted sequence of TPM4120572 has been posted in GenBank We designed primer-pairs for RT-PCRand showed the expression of the transcripts of TPM4120572 and a novel isoform TPM4120575 in human heart and skeletal muscle qRT-PCRshows that the relative expression of TPM4120572 and TPM4120575 is higher in human cardiac muscle Western blot analyses using CH1monoclonal antibodies show the absence of the expression of TPM4120575 protein (sim28 kDa) in human heart muscle 2D western blotanalyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass sim32 kD and abovein adult heart ByMass spectrometry we determined the amino acid sequences of the extracted proteins from these spots Spot ldquoGrdquoreveals the putative expression of TPM4120572 along with TPM1120572 protein in human adult heart

1 Introduction

Tropomyosin (TPM) is a coiled-coil dimer protein thatplays important role(s) in regulating muscle contractionin conjunction with other sarcomeric proteins like actintroponins tropomodulin and so forth TPM also providesstructural stability to actin filaments thereby modulatingcytoskeleton function In vertebrates there are four TPMgenes designated as TPM1 TPM2 TPM3 and TPM4 exceptfor zebrafish where six TPM genes are present [1ndash10] Eachof the four genes generates a large number of TPM isoformsvia alternative splicing andor using different promoters Inmammals the exon and intron organization of TPM genesare very similar if not identical [6] The rodent TPM4 genehas lost the use of exons 1a and 2 the high molecular weightisoforms encoding 284 amino acid residues such as TPM4120572and TPM4120573 are not expressed as they are in nonmammalianspecies It was speculated for a long time that the TPM4 genein humans as in rodents does not encode sarcomeric isoformwith exon 9ab [2 3] The first report on the expression of ahighmolecular weight TPM4 protein was reported in human

ovary tumor tissues in 2004 (accession number AK023385)This isoform defined as TPM4120573 contains exons 1a 2 3 4 56 7 8 and 9d (Figure 1) which differs from the sarcomericTPM4120572 isoform containing exon 9ab instead of 9d It is tobe noted that exon 9a but not 9d encodes the peptide thatis essential for binding TPM with troponins [2 3] essen-tial components of thin filament Recently two predictedsequences (derived from a genomic sequence) of the twodifferent isoforms of the human TPM4 gene containing exon9a have been reported in GenBank (on March 12 2015) Oneof the predicted isoforms is TPM4120572 (as in avian and amphib-ians) containing exons 1a 2 3 4 5 6b 7 8 and 9ab encoding284 amino acids (accession number XM 006722865) Theother one may encode a low molecular weight novel proteinwith 248 amino acids consisting of exons 1b 3 4 5 6 7 8 and9ab (accession number XM 0052600422) In this study forthe first time we confirmed the expression of these two newtranscripts viz TPM4120572 (or Tpm43 (8)) and TPM4120575 (Tpm44(8)) by RT-PCR with the RNA from human adult heart fetalheart and skeletal muscle using two primer-pairs designedfrom the predicted nucleotide sequences We confirmed

Hindawi Publishing CorporationMolecular Biology InternationalVolume 2016 Article ID 3105478 11 pageshttpdxdoiorg10115520163105478

2 Molecular Biology International

TPM41 1b2 3 4 5 6 7 8 9b9a 9d39d29d1

TPM4120572 (or Tpm 43)

TPM4120573 (or Tpm 41 as in [7])

TPM4120574 (or Tpm 42 as in [7])

TPM4120575 (or Tpm 44)P3(+)

P2(minus)

P2(minus)

P1(+)

Figure 1 Alternative splicing pattern of the TPM4 gene in humans Exon composition of the TPM4 gene is derived from the publisheddocuments (top) and the recently submitted data in GenBank Although the splice variants TPM4120573 (NM 001145160) and TPM4120574 (NM-0032902) are known for some time the cartoons of the splice variants of TPM4120572 and TPM4120575 are generated with the information of therecently submitted sequences of the corresponding predicted isoforms inGenBank as well as the results from the present study Exon locationsof primers P1(+) P2(minus) and P3(+) are shown

the sequences of both the isoforms by cloning and sequencingof the amplified products We also determined the relativeexpression of the two transcripts by qRT-PCR with RNAfrom human adult heart fetal heart and skeletal muscleusing isoform specific primer-pairs For protein expressionwe performed 1D and 2D western blot analyses with theextract from adult human heart using the exon 9a-specificmonoclonal antibody CH1 We found nine spots on westernblot and the protein was extracted from the spots for massspectrometry (LC-MSMS) analyses The results suggest thatspot G may have TPM4120572 along with TPM1120572 protein Tothe best of our knowledge this is the first report of TPM4120572protein expression in humans

2 Materials and Methods

Human adult and fetal cardiac tissue RNA were obtainedfrom Zyagen (San Diego CA) adult human heart proteinsamples were obtained from Imgenex (San Diego CA)

21 Conventional RT-PCR All PCR and post-PCR exper-iments were done by different personnel in in differentbuildings using separate equipment and separate ventilationso as to avoid carryover contamination For RT-PCR 05120583gof RNA in a total volume of 40 120583L was used to synthesizecDNAwith SuperScript II (Life Technologies Grand IslandNY) and oligo-dT primers following the manufacturerrsquosspecifications For each PCR 3 120583L of cDNA was used and

TPM4120572 and TPM4120575 RNA were amplified as previouslydescribed [11 12] The nucleotide sequences of the primer-pairs for amplification of TPM4120572 and TPM4120575 are givenin Table 1 It is to be noted that the negative primer foramplification of TPM4120572 and TPM4120575 is the sameThe positiveprimers for TPM4120572 and TPM4d are from exon 1a and exon1b respectively The PCR amplified DNAs were run in anagarose gel and the ethidium bromide stained bands wereexcised from the gel andDNAwas extracted from the excisedbands using a MinElute Gel extraction kit from Qiagen(Valencia CA) following the manufacturerrsquos direction Partof the DNA was used for sequencing and part of the gelextracted DNAs was ligated and cloned into the TA cloningvector (Life Technologies) following our published protocol[11 12] Positive clones were identified by PCR using theabove-mentioned specific primer-pairs previously describedVectors or constructs were grown in E coli and the DNAwas extracted using Qiagen Miniprep kit (Valencia CA)The isolated DNA was sequenced (Cornell University LifeScience Core Laboratories Center Ithaca NY) Each clonewas sequenced twice in both directions It is to be noted thatthe primer-pair we have used for TPM4120572 and TPM4120575 is veryspecific for the respective isoform

22 Real-Time Quantitative RT-PCR qRT-PCR analysis ofcDNA template was performed using the LightCycler 480Real-Time PCR System Reactions were carried out in a 384-well plate using the LightCycler 480 SYBR Green I Master kit

Molecular Biology International 3

Table 1 Nucleotide sequences of the primer-pairs used for conventional and qRT-PCR analyses

Primer Isoform Type of PCR Sequence

TPM4120572-F-1 (P1+) TPM4120572 Both conventionalamp qRT-PCR 51015840-CAGCCATGGAGGCCATCAAGA-31015840

TPM4120572-R-1 (P2minus) TPM4120572 Conventional 51015840-CACCATGTGAGAAGGACAGA-31015840

TPM4120572-R-2 TPM4120572 qRT-PCR 51015840-ACGAGCTGGATGCGTCGGT-31015840

TPM4120575-F-1 (P3+) TPM4120575 Conventionalamp qRT-PCR 51015840-ATGGCCGGCCTCAACTCCCTGG-31015840

TPM4d-R-1 TPM4d Conventional 51015840-CACCATGTGAGAAGGACAGA-31015840

TPM4d-R-2 TPM4d qRT-PCR 51015840-ACGAGCTGGATGCGTCGGT-31015840

18s rRNA-F 18s rRNA qRT-PCR 51015840-TGCTGCAGTTAAAAAGCTCGTA-31015840

18s rRNA-R 18s rRNA qRT-PCR 5-ACCAACAAAATAGAACCGCGG-31015840

Table 2 Key to 2D gel loading and sample preparation The sample was lyophilized and redissolved to 4mgmL in 1 1 diluted SDS boilingbuffer urea sample buffer before loading

Gel ID Sample 120583L loaded 120583g loaded TreatmentLF975 1 HT-801 Zyagen Human Heart 150 500 CoomassieLF975 2 HT-801 Zyagen Human Heart 150 500 Antitropomyosin

(Roche) Briefly each well contained a total volume of 10 120583Lreaction solution of which 2120583L was

cDNA template and 8 120583L were SYBR green mix (5 120583L1x SYBR green master mix 28 120583L of PCR-grade water and02 120583L of 10 120583M primer-pair) Primers for real-time PCR forTPM4120572 and TPM4120575 are listed in Table 1

In this case cDNA was made with an oligonucleotidefrom exon 9ab (51015840-CACCATGTGAGAAGGACAGA-31015840)that allowed us to make a gene specific cDNA correspondingto the mRNA containing exon 9b as in TPM4120572 and TPM4120575and would avoid any subsequent nonspecific PCR amplifica-tion For the amplification of TPM4120572 and TPM4120575 we haveemployed the positive primers from exon 1a and exon 1brespectively and the same negative primer located in exon3 Amplification in the absence of cDNA template was alsoevaluated to insure a lack of signal due to primer dimerizationand extension or carryover All samples and controls wereperformed in triplicate Data were analyzed using delta-delta CT (sample delta CT minus comparator delta CT)methods [13ndash15] Efficiencies (E) of PCR amplification weredetermined using dilution series of plasmids andor humanheart cDNAs generated with gene specific primer-pairs asdescribed by Nan et al [16] As a result the primer-pair wehave used from each of the isoforms is very specific and doesnot amplify other isoforms generated from the TPM4 or anyother TPM gene

23 Western Blot 10 120583g of protein from each sample (humanadult and fetal heart) was used for western blot analy-ses following our published protocol [17] Primary anti-bodies included TM311 (Sigma-Aldrich St Louis MO)and CH1 antisarcomeric tropomyosin (DSHB Universityof Iowa Iowa) Secondary antibody was sheep antimouseimmunoglobulin HRP (GE Healthcare Bio-Sciences Pitts-burgh PA)

24 2D Western Blot and Mass Spectrometry 2D (two-dimensional) western blot analyses of the commercial humanadult heart extract obtained from Zyagen (San Diego CA)were performed for us by Kendrick Labs Inc (MadisonWI)Two-dimensional electrophoresis was performed accordingto the carrier ampholine method of isoelectric focusing [1819] by Kendrick Labs Inc (Madison WI) as described byWang et al [20] The key to 2D Gel loading and samplepreparation is given in Table 2 The Coomassie blue-stainedPVDF membrane was desktop scanned destained in 100methanol rinsed briefly in Tween-20 Tris-buffered saline(TTBS) and blocked for two hours in 5 nonfat dry milk(NFDM) in TTBS The blot was incubated in primaryantibody (Anti-Tropomyosin-Clone CH1 (DevelopmentalStudies Hybridoma Bank) diluted 1 10000 in 2 NFDMTTBS) overnight and rinsed 3 times 10 minutes in TTBS Theblot was placed in secondary antibody (antimouse IgG-HRP(GE Healthcare Cat number NA931V Lot number 9621358)diluted 1 2000 in 2 NFDM TTBS) for two hours rinsed asabove treated with ECL and exposed to X-ray film For thedeveloped X-ray film when superimposed on the Coomassiestained human adult heart protein gel 9 spots (AndashI) wereexhibited (as in Figure 7(a)) Nine gel spots were excisedwashed and trypsinized following the published protocols[21ndash23] The resulting peptides were extracted twice with 5formic acid50mMammoniumbicarbonate50acetonitrileand oncewith 100 acetonitrile undermoderate shakingThepeptide mixture was then dried in SpeedVac solubilized in20120583L of 01 formic acid2 acetonitrile

25 LC-MSMS The peptides mixture was analyzed byreverse phase liquid chromatography (LC) and MS (LC-MSMS) using a nanoACQUITY UPLC (MicromassWatersMilford MA) coupled to a Q-TOF Ultima API MS (Micro-massWaters Milford MA) according to published proce-dures [22ndash24] Briefly the peptideswere loaded onto a 100120583m


1078

603

1 2 3 4 5 6 M

(a)

1 2 3 4 5 M

1078

603

(b)

Figure 2 Expression of TPM4 isoforms with exon 9ab in human heart and skeletal muscle by conventional RT-PCR (a) Expression ofTPM4120572 and TPM120575 in human fetal and adult hearts as determined TPM4120575 amplified with primer 1(+) and primer 2(minus) as shown in Figure 1and Table 1 The size of the amplified DNA is sim747 bp Lane 1 adult heart lane 2 fetal heart lane 3 primer control TPM4120572 amplified withprimer 1(+) and primer 2(minus) as shown in Figure 1 and Table 1The size of the amplified DNA is sim855 bp Lane 4 adult heart lane 5 fetal heartlane 6 primer control (b) Expression of TPM4120572 in human fetal heart adult heart and adult skeletal muscle TPM4120572 amplified with primer1(+) and primer 2(minus) as shown in Figure 1 and Table 1 The size of the amplified DNA is sim855 bp Lane 1 fetal heart lane 2 adult heart lane3 adult skeletal muscle (source 1) lane 4 adult skeletal muscle (source 2) lane 5 primer control

times 10mm nanoACQUITY BEH130 C18 17 120583mUPLC column(Waters Milford MA) and eluted over a 150-minute gradientof 2ndash80 organic solvent (ACN containing 01 FA) at aflow rate of 400 nLmin The aqueous solvent was 01 FA inHPLC water The column was coupled to a PicoTip EmitterSilicaTip nanoelectrospray needle (New Objective WoburnMA) MS data acquisition involved survey MS scans andautomatic data dependent analysis (DDA) of the top threeions with the highest intensity ions with the charge of 2+ 3+or 4+ ions The MSMS was triggered when the MS signalintensity exceeded 10 countssecond In survey MS scansthe three most intense peaks were selected for collision-induced dissociation (CID) and fragmented until the totalMSMS ion counts reached 10000 or for up to 6 seconds eachThe entire procedure used was previously described [22ndash24]Calibration was performed for both precursor and productions using 1 pmol GluFib (Glu1-Fibrinopeptide B) standardpeptide with the sequence EGVNDNEEGFFSAR and themonoisotopic doubly charged peak with119898119911 of 78584

26 Data Processing and Protein Identification The raw datawere processed using ProteinLynx Global Server (PLGSversion 24) software as previously described by Darie et al[22]

3 Results

31 Detection of RNA Expression by RT-PCR and Cloning andSequencing of TPM4120572 and TPM4120575 cDNAs In order to detectthe expression of TPM4120572 and TPM4120575 in human striatedmuscles we used conventional RT-PCR with the RNA fromadult human heart and skeletal muscles The primer-pairswere designed from the predicted sequences as reportedin the GenBank (accession number XM 0067228651 forTPM4120572 and XM 0052600422 for TPM4120575) The nucleotidesequences of the primer-pairs designed are presented inTable 1

Conventional RT-PCR results presented in Figure 2(a)show that TPM4120575 is expressed in both fetal (lane 1) and adulthuman heart (lane 2)

Also the TPM4120572 is expressed in both fetal and adultheart as depicted in lanes 4 and 5 respectively Again TPM4ais expressed in fetal heart (lane 1 Figure 2(b)) adult heart(lane 2 Figure 2(b)) and also skeletal muscle (lanes 3 and4 representing two different sources of adult human skeletalmuscle RNA)

The amplified DNA from the ethidium stained band(s)on agarose gel was extracted and purified using a gelextraction kit from Ambion Inc Gel extracted DNA wasused subsequently for nucleotide sequence determinationas well as cloning into TA cloning vector (Invitrogen)Direct DNA sequence analyses confirmed the expressionof TPM4120572 and TPM4120575 in both heart and skeletal muscleDNA sequencing was also performed with the cDNA ofeach TPM4 isoform cloned in TA cloning vector Figures3 and 4 depict the nucleotide as well as deduced aminoacid sequences of TPM4120572 and TPM4120575 The sequences areidentical with the predicted sequences as submitted in Gen-BankWhen the amino acid sequences of TPM4120572 andTPM1120572(Acc number NP 001018005) are compared there are 96127percent similarity and 88028 percent identity between thetwo sarcomeric isoforms (best fit results not shown) Tothe best of our knowledge this is the first report on theexpression of TPM4120572 andTPM4120575 in human striatedmusclesComparisons of the amino acid sequences of TPM4120575 and theknown TPM4120574 indicate that both isoforms encode a 248-amino acid polypeptide with identical sequences except forexon 9 The exon compositions of TPM4120574 and TPM4120575 aregiven in Figure 1 TPM4120574 contains exons 1b 3 4 5 6b 7 8and 9d while TPM4120575 contains exon 9a instead of 9d

32 qRT-PCR Analyses to Determine the Relative Expressionof TPM4120572 and TPM4120575 in Human Fetal Heart Adult Heartand Adult Skeletal Muscle The results depicted in Figures 5and 2(b) confirm that TPM4120572 is expressed in human heartsand skeletal muscle Interestingly the expression level of bothisoforms is the least in skeletal muscle However the relativeexpression of TPM4120572 and TPM4120575 is higher in fetal heartcompared to adult heart and adult skeletal muscleThe resultsalso indicate that the relative expression of TPM4120572 is much


Translation start

TCGGCTTCAGCCATGGAGGCCATCAAGAAGAAAATGCAGATGCTGAAGTTGGACAAGGAG

1 ---------+---------+---------+---------+---------+---------+ 60M E A I K KK M Q M L K L D K E -

AATGCCATCGACCGCGCGGAGCAGGCGGAGGCGGATAAGAAAGCCGCTGAGGACAAGTGC

61 ---------+---------+---------+---------+---------+---------+ 120N A I D R A E Q A E A D K K A A E D K C -AAGCAGGTGGAGGAGGAGCTGACGCACCTCCAGAAGAAACTAAAAGGGACAGAGGACGAG

121 ---------+---------+---------+---------+---------+---------+ 180K Q V E E E L T H L Q K K L K G T E D E -CTGGATAAATATTCCGAGGACCTGAAGGACGCGCAGGAGAAGCTGGAGCTCACGGAGAAG

181 ---------+---------+---------+---------+---------+---------+ 240L D K Y S E D L K D A Q E K L E L T E K -AAGGCCTCCGACGCTGAAGGTGATGTGGCCGCCCTCAACCGACGCATCCAGCTCGTTGAG

241 ---------+---------+---------+---------+---------+---------+ 300K A S D A E G D V A A L N R R I Q L V E -GAGGAGTTGGACAGGGCTCAGGAACGACTGGCCACGGCCCTGCAGAAGCTGGAGGAGGCA

301 ---------+---------+---------+---------+---------+---------+ 360E E L D R A Q E R L A T A L Q K L E E A -GAAAAAGCTGCAGATGAGAGTGAGAGAGGAATGAAGGTGATAGAAAACCGGGCCATGAAG

361 ---------+---------+---------+---------+---------+---------+ 420E K A A D E S E R G M K V I E N R A M K -GATGAGGAGAAGATGGAGATTCAGGAGATGCAGCTCAAAGAGGCCAAGCACATTGCGGAA

421 ---------+---------+---------+---------+---------+---------+ 480D E E K M E I Q E M Q L K E A K H I A E -GAGGCTGACCGCAAATACGAGGAGGTAGCTCGTAAGCTGGTCATCCTGGAGGGTGAGCTG

481 ---------+---------+---------+---------+---------+---------+ 540E A D R K Y E E V A R K L V I L E G E L -GAGAGGGCAGAGGAGCGTGCGGAGGTGTCTGAACTAAAATGTGGTGACCTGGAAGAAGAA

541 ---------+---------+---------+---------+---------+---------+ 600E R A E E R A E V S E L K C G D L E E E -CTCAAGAATGTTACTAACAATCTGAAATCTCTGGAGGCTGCATCTGAAAAGTATTCTGAA

601 ---------+---------+---------+---------+---------+---------+ 660L K N V T N N L K S L E A A S E K Y S E -AAGGAGGACAAATATGAAGAAGAAATTAAACTTCTGTCTGACAAACTGAAAGAGGCTGAG

661 ---------+---------+---------+---------+---------+---------+ 720K E D K Y E E E I K L L S D K L K E A E -ACCCGTGCTGAATTTGCAGAGAGAACGGTTGCAAAACTGGAAAAGACAATTGATGACCTG

721 ---------+---------+---------+---------+---------+---------+ 780T R A E F A E R T V A K L E K T I D D L -GAAGATGAGTTATACGCTCAGAAGCTCAAGTACAAAGCTATCAGCGAGGAACTGGACCAC

781 ---------+---------+---------+---------+---------+---------+ 840E D E L Y A Q K L K Y K A I S E E L D H -GCTCTCAACGACATGACCTCTCTCTGAGAGGCAGCCAGGTCGCTGCCCTCTGTCCTTCTC

841 ---------+---------+---------+---------+---------+---------+ 900A L N D M T S L

Human TMP4120572

lowast

Figure 3 Nucleotide and deduced amino acid sequences of human TPM4120572 Deduced amino acid sequences are at the bottom of thenucleotide sequences Symbol of each amino acid residue is placed at the bottom of the first nucleotide of each codon lowast signifies Stop codon

higher compared to TPM4120575 in all the tissues tested Pleasenote the difference in scale for TPM4120572 and TPM4120575 shown inFigure 5

33 Western Blot Analyses with the Total Protein Extractsof Human Adult Heart In order to explore whether or notTPM4120572 andTPM4120575 proteins are expressed in human striated

muscles we have employed western blot analyses with theCH1monoclonal antibody that recognizes an epitope in exon9a In other words CH1 monoclonal antibody recognizes allknown sarcomeric TPM isoforms (TPM1120572 TPM1120581 TPM2120572TPM3120572 and TPM4120572) and the nonsarcomeric TPM4120575 havinga peptide encoded by exon 9a at the C-terminus end Eachof these sarcomeric isoforms contains 284 amino acids and


Translation start

GGCTTATGGCCGGCCTCAACTCCCTGGAGGCGGTGAAACGCAAGATCCAGGCCCTGCAGC

M A G L N S L E A V K R K I Q A L Q QAGCAGGCGGACGAGGCGGAAGACCGCGCGCAGGGCCTGCAGCGGGAGCTGGACGGCGAGC

Q A D E A E D R A Q G L Q R E L D G E RGCGAGCGGCGCGAGAAAGCTGAAGGTGATGTGGCCGCCCTCAACCGACGCATCCAGCTCG

E R R E K A E G D V A A L N R R I Q L VTTGAGGAGGAGTTGGACAGGGCTCAGGAACGACTGGCCACGGCCCTGCAGAAGCTGGAGG

E E E L D R A Q E R L A T A L Q K L E EAGGCAGAAAAAGCTGCAGATGAGAGTGAGAGAGGAATGAAGGTGATAGAAAACCGGGCCA

A E K A A D E S E R G M K V I E N R A MTGAAGGATGAGGAGAAGATGGAGATTCAGGAGATGCAGCTCAAAGAGGCCAAGCACATTG

K D E E K M E I Q E M Q L K E A K H I ACGGAAGAGGCTGACCGCAAATACGAGGAGGTAGCTCGTAAGCTGGTCATCCTGGAGGGTG

E E A D R K Y E E V A R K L V I L E G EAGCTGGAGAGGGCAGAGGAGCGTGCGGAGGTGTCTGAACTAAAATGTGGTGACCTGGAAG

L E R A E E R A E V S E L K C G D L E EAAGAACTCAAGAATGTTACTAACAATCTGAAATCTCTGGAGGCTGCATCTGAAAAGTATT

E L K N V T N N L K S L E A A S E K Y SCTGAAAAGGAGGACAAATATGAAGAAGAAATTAAACTTCTGTCTGACAAACTGAAAGAGG

E K E D K Y E E E I K L L S D K L K E ACTGAGACCCGTGCTGAATTTGCAGAGAGAACGGTTGCAAAACTGGAAAAGACAATTGATG

E T R A E F A E R T V A K L E K T I D DACCTGGAAGATGAGTTATACGCTCAGAAGCTCAAGTACAAAGCTATCAGCGAGGAACTGG

L E D E L Y A Q K L K Y K A I S E E L DACCACGCTCTCAACGACATGACCTCTCTCTGAGAGGCAGCCAGGTCGCTGCCCTCTGTCC

H A L N D M T S L

1 ---------+---------+---------+---------+---------+---------+ 60

61 ---------+---------+---------+---------+---------+---------+ 120

121 ---------+---------+---------+---------+---------+---------+ 180

181 ---------+---------+---------+---------+---------+---------+ 240

241 ---------+---------+---------+---------+---------+---------+ 300

301 ---------+---------+---------+---------+---------+---------+ 360

361 ---------+---------+---------+---------+---------+---------+ 420

421 ---------+---------+---------+---------+---------+---------+ 480

481 ---------+---------+---------+---------+---------+---------+ 540

541 ---------+---------+---------+---------+---------+---------+ 600

601 ---------+---------+---------+---------+---------+---------+ 660

661

721

---------+---------+---------+---------+---------+---------+ 720

---------+---------+---------+---------+---------+---------+ 780

TPM4120575

-

-

-

-

-

-

-

-

-

-

-

-

lowast

Figure 4 Nucleotide and deduced amino acid sequences of human TPM4120575 lowast signifies Stop codon

05

101520253035404550

Adult heart Fetal heart Skeletal muscle

Fold

diff

eren

ce

Tissue type

TPM4120572 expression

664 plusmn 021

4642 plusmn 016

1

(a)

0123456789

10


Fold

diff

eren

ce

Tissue type

1


554 plusmn 010

893 plusmn 0085

(b)

Figure 5 Relative expression of TPM4120572 and TPM4120575 in human adult and fetal hearts Fold changes of TPM4120572 and TPM4120575 in human adultheart fetal heart and skeletal muscle qRT-PCR was carried out in triplicate with isoform specific primer-pairs sequences of which are givenin Table 1 (a) TPM4120572 and (b) TPM4120575


M 1 2

Ponceau64 kD

39 kD

28 kD

(a)

CH139 kD

28 kD

(b)

TM311

51 kD

39 kD

(c)

Figure 6 Western blot analysis of protein extracts from humanadult and fetal hearts with CH1 and TPM311 antitropomyosinantibodies (a) Ponceau-stained blot lane 1 extract of adult heartlane 2 extract of fetal heart lane M molecular weight markers(b) Blots stained with CH1 monoclonal antibodies that recognizeall sarcomeric TPM isoforms as well as TPM4120575 with exon 9a C-terminus end (c) Blot stained with TM311 monoclonal antibodiesthat recognize all high molecular wt TPM isoforms with exon 1a atthe N-terminus end However it does not recognize TPM4120575 withexon 1b at the N-terminus end

is considered as high molecular wt tropomyosin AlthoughTPM4120575 encodes 248 amino acids and has different exon 1exon 9 is identical to TPM4120572 Hence if TPM4120575 protein isexpressed one should observe a signal at the sim28 kDa regionin the western blot We have employed another monoclonalantitropomyosin antibody TM311 which recognizes all highmolecular wt tropomyosin molecules (sim32 kDa and above)containing exon 1a As TPM4120575 unlike TPM4120572 contains exon1b instead of exon 1a one should not observe any sim28 kDasignal with TM311 antibodiesThe results depicted in Figure 6(staining with CH1 monoclonal antibodies) show strongsignal(s) at sim39 kDa region The results show the presence ofmost likely several sarcomeric isoforms for example TPM1120572TPM1120581 TPM2120572 and TPM3120572 in human hearts Withoutusing a TPM4 specific antibody one cannot infer that thesignal(s) at the sim39 kDa region indicates the presence ofTPM4120572 protein in human cardiac muscles In addition wedid not detect the presence of low molecular wt tropomyosinlike TPM4120575 If it was expressed in detectable quantitywe should have picked up a signal Western blot analyseswith TM311 confirm the expression of high molecular wtsarcomeric and nonsarcomeric TPM isoforms in adult andfetal heart tissues TM311 should not recognize lowmolecularwt TPM4120575 which contains exon 1b rather than exon 1a region[6]

34 2D Western Blot Analyses with Protein Extracts of AdultHumanHeart with CH1Monoclonal AntibodyThat Recognizes

All Sarcomeric Tropomyosin Isoforms including TPM4a andthe Newly Found TPM4120575 with Exon 9120572 Polypeptide at the C-Terminus End Two-dimensional western blot analyses wereperformed with commercially available human heart extractwith antisarcomeric antitropomyosin (CH1) antibodies Fig-ure 7(a) shows the Coomassie blue-stained PVDFmembranebefore western blotting and Figure 7(b) depicts the signalson the X-ray film exposed on the blot after being treatedwith ECL (see the method section) Figure 7(d) representsthe developed X-ray film superimposed on the top of theCoomassie blue-stained PVDFmembrane (as in Figures 7(a)and 7(b)) Nine spots were identified and marked as A BC D E F G H and I (Figures 7(a) and 7(b)) The resultsindicate that there are at least nine detectable CH1 positivetropomyosin molecules present in the extract from adulthuman hearts It is to be noted that each of these spotsrepresents highmolecular wt (32 kD and above) tropomyosinprotein However we have failed to detect the presence ofa low molecular wt CH1-positive tropomyosin protein witha molecular mass sim28 kDa like TPM4120575 in adult humanheart

In the next step each of the protein spots that wereseparated by 2D PAGE and stained by Coomassie dyewas excised and washed and the proteins from the gelwere treatedprocessed according to the published protocol(please see themethod section) for LS-MSMS analyses usingnanoACQUITY UPLC (MicromassWaters Milford MA)coupled to a Q-TOF Ultima API MS (MicromassWatersMilford MA) The raw data were processed using Protein-Lynx Global Server (PLGS version 24) software Mascot andPLGS database search provided a list of proteins for eachgel band To eliminate false positive results for the proteinsidentified by either one peptide or a mascot score lower than25 we verified the MSMS spectra that led to identificationof a protein In spot G a total of 53 peptide sequenceswere identified As shown in Figure 8 the identified peptidescover 76 of TPM1120572 (accession number gi63252898) and38 of TPM4120572 protein (accession number GI 578833543PREDICTED tropomyosin alpha-4 chain isoform X1 Homosapiens) It is to be noted that there are not very manydifferences between TPM1120572 and TPM4120572 In this study wefound three peptides containing a total of three amino acidresidues that are specific for TPM4120572 and eight peptidescontaining eighteen amino acids that are specific for TPM1120572The remainders of the identified peptides are identical in bothisoforms For TPM1120572 peptide in exons 1a 2 3 4 5 6 7 8and 9a was identified For TPM4120572 peptides in exons 1a 8and 9a were identified Some amino acid changes found inthe striated muscle TPM4120572 are also found in skeletal muscleTPM2120572 (gi I 42476296 isoform which is also expressed inheart) One of such changes is L19I Again 252T is also presentin TPM2120572 However the peptide of the TPM4120572 that we havesequenced is quite different from TPM2a peptideThis is truefor 284L The peptide containing 284L residue from TPM4120572that we have sequenced (asmarkedwith red color) is differentfrom human TPM2120572 (gi I42476296) The results indicate thepotential expression of TPM4120572 protein in human cardiactissues


(a) (b) (c)

(d)

E

F

G

(e)

Figure 7 2D gels of commercially available extracts of human adult heart (a) The Coomassie stained human adult heart protein across thegel Seven spots A B C D E F and G were identified which are not visible clearly (b) This panel represents the enlarged portion of the gelin (a) and shows clearly AndashE spots (c) The PVDF filter was stained with CH1 monoclonal antibody followed by treatment with secondaryantibody as mentioned under Materials and Methods and subsequently treated with ECL and exposed to X-ray film (d) Developed X-rayfilm was merged on the top of the Coomassie stained blot (e) Enlarged nine spots (AndashI) were marked on the PVDF filter (as in (a) and (b))and on the duplicate gel All the spots were marked and excised and were used for extraction of protein for subsequent mass spectrometricanalyses Extracted peptides from spots E F and G were analyzed for mass spectrometry as described in the method section

4 Discussion

In this study we have cloned and sequenced cDNAs oftwo novel TPM4 isoforms using RNA from human hearts(Figures 1ndash3) one is the sarcomeric isoform TPM4120572 thatencodes a protein with 284 amino acids and the other knownas TPM4120575 encodes a protein with 248 amino acids (Figures 1and 4)The transcripts of these two isoforms are expressed infetal heart adult heart and skeletal muscle The expressionlevel is somewhat higher in hearts compared to skeletalmuscle (Figures 2 and 5) We were unable to detect theexpression of TPM4120575 (a sim28 kD protein) in either adult orfetal hearts by western blot analyses using CH1 monoclonalantibody that recognizes an epitope in exon 9aHowever CH1antibody detected the expression of high molecular weightTPM proteins (sim32 kDa and above) in adult and fetal hearts(Figure 6) One of the limitations in tropomyosin research

is the unavailability of isoform specific antibodies Withoutthem it is practically impossible to pinpoint whether theTPM4120572 protein is present in the cluster of signals at the highmolecular wt region (32ndash40 kDa) as depicted in Figure 6 Inorder to gain further insight we have performed 2D westernblot analyses with CH1 antibody The gel extracted proteinsfrom various spots with positive signal were analyzed byLS-MSMS The results indicate that the spot G containsTPM4120572 protein along with TPM1120572 It is to be noted thatwe have also found the expression of TPM1120581 TPM2120572 andTPM3120572 in other spots (data not shown) Our future planis to further confirm the expression of TPM4120572 protein inhuman striated muscles in healthy and diseased ones Wehave compared the expression level of TPM4120572 transcripts infetal and adult human hearts and are planning to comparethe corresponding protein expression level in fetal and adulthuman hearts as well The fact that we found more peptides


1 MDAIKKKMQM LKLDKENALD RAEQAEADKK AAEDRSKQLE DELVSLQKKL

51 KGTEDELDKY SEALKDAQEK LELAEKKATD AEADVASLNR RIQLVEEELD

101 RAQERLATAL QKLEEAEKAA DESERGMKVI ESRAQKDEEK MEIQEIQLKE

151 AKHIAEDADR KYEEVARKLV IIESDLERAE ERAELSEGKC AELEEELKTV

201 TNNLKSLEAQ AEKYSQKEDR YEEEIKVLSD KLKEAETRAE FAERSVTKLE

251 KSIDDLEDEL YAQKLKYKAI SEELDHALND MTSI

TPM1120572

(a)

1 MEAIKKKMQM LKLDKENAID RAEQAEADKK AAEDKCKQVE EELTHLQKKL

51 KGTEDELDKY SEDLKDAQEK LELTEKKASD AEGDVAALNR RIQLVEEELD

101 RAQERLATAL QKLEEAEKAA DESERGMKVI ENRAMKDEEK MEIQEMQLKE

151 AKHIAEEADR KYEEVARKLV ILEGELERAE ERAEVSELKC GDLEEELKNV

201 TNNLKSLEAA SEKYSEKEDK YEEEIKLLSD KLKEAETRAE FAERTVAKLE

251 KTIDDLEDEL YAQKLKYKAI SEELDHALND MTSL

TPM1120572

(b)

Figure 8 Identification of amino acid sequences from the peptides extracted from spot G after 2D western blot analyses of adult heartextracts with CH1 antibodies Red color letters indicate the isolated peptides that have been sequenced by mass spectrometry (a) TPM1120572 and(b) TPM4120572 Three amino acid residues in exons 1a 8 and 9a are different in TPM4120572marked as bold In case of TPM1120572 76 (red marked) ofthe total 284 amino acid residues are being identified For TPM4120572 it is 38 It is to be noted that peptide SIDDLEDELYAQKLK in TPM1120572was identified by two precursor ions with 119898119911 of 59407 (3+) and 89006 (2+) ions On the other hand peptide TIDDLEDELYAQKLK inTPM4120572 was identified by one precursor ion with119898119911 of 88806 (2+)

specific to TPM1a than TPM4a in spot G is consistent withour bias that there is more of the former than the latter inhuman cardiomyocytes

Although the expression of multiple isoforms oftropomyosin has long been known in vertebrates includinghumans the precise function of each of the isoformsgenerated by various TPM genes is not well understood Forexample the expressions of TPM1120572 (the sarcomeric isoformof the TPM1 gene) and TPM2120572 (sarcomeric isoform of theTPM2 gene) in human hearts have been known for a longtime However the precise role(s) of the two sarcomericTPM proteins in cardiac muscle contraction have not beenelucidatedMore recent published results suggest that TPM1120572makes up about 90 of the total sarcomeric TPM proteinin human hearts TPM2120572 and TPM1120581 (another sarcomericisoform of the TPM1 gene) may constitute the remaining 10percent of the TPM protein Peng et al [25] using top-downmass spectrometry confirmed that TPM1120572 constitutes 90 ofthe total sarcomeric tropomyosin in human heartsThey alsodetected a low amount of TPM2120572 and TPM1120581 expression inhuman hearts There was no mention about the expressionof TPM4120572 in this paper Marston et al also reported theexpression of TPM3120572 in human heart [26] These authorsreported that the level of expression of TPM1120572 and TPM1120581was 95 and 4 respectively of the total sarcomerictropomyosin in human hearts In addition a low level ofexpression of TPM2120572 TPM3120572 and TPM1120573 (smooth muscletype tropomyosin) was also detected Again there was nomention about the expression of TPM4120572

Researchers from different laboratories using transgenicmouse models have delineated the tentative function ofTPM1120572 and TPM2120572 in mammalian hearts [27ndash29] In theadult healthy mouse heart the protein expression level of

TPM2120572 is less than 2 When there was 55ndash60 of TPM2120572present in the myocardium of transgenic mouse heart thetime of relaxation maximum rate of relaxation and sensi-tivity to calcium changed significantly Further altering ofTPM2120572 to an even higher level in the heart of transgenicmicewill cause postnatal death in between 10 and 14 days [27ndash29]TPM3120572 is not expressed in mouse hearts [4] High ectopicexpression level of TPM3120572 (40ndash60) can lead to hyperdy-namic effect on systolic and diastolic function and decreasedcalcium sensitivity [30] As TPM3120572 is not expressed inmouseheart it is not possible to perform knockoutknockdownexperiments for TPM3120572 Again overexpression of TPM1120581in a cardiac-specific manner in transgenic mouse leads to adilated cardiomyopathy (DCM) like syndrome Interestinglythe expression level of TPM1120581 protein has been foundto be significantly higher in hearts from DCM and heartfailure patients The functional analysis of TPM1120581 providesa possible mechanism for the consequences of the TPMisoform switch observed in DCM and heart failure patients[31]

The functional aspect of TPM3120572 in human cardiac tissuesis not known yet Our finding of the potential expression ofTPM4120572 in human heart muscles makes the situation morecomplicated The most important as well as most difficultquestion yet to be addressed is whether minute expressionof the sarcomeric TPM proteins such as TPM1120581 TPM2120572TPM3120572 and TPM4120572 plays any significant role in cardiaccontractility andor cardiac function

As rodents with truncated TPM4 genes are not capable ofproducing sarcomeric TPM4120572 protein the in vivo functionof TPM4120572 using knockdownknockout technology is notpossibleHowever one can generate transgenicmouse ectopi-cally overexpressing human TPM4120572 protein in a cardiac


specific manner and find out whether ectopic expression isaltering the cardiac morphology andor cardiac contractilityAnother approach researchers have used for assigning thefunction of a protein is to look for its association with anyhuman disease(s) For example various TPM1 and TPM2mutations have been implicated in hypertrophic cardiomy-opathy (HCM) including familial hypertrophic cardiomy-opathy (FHC) in humans [31] Similarly several missensemutations in TPM2 and TPM3 have been implicated innemalinemyopathy that suggest the functional importance ofthese genes or particular isoform in cellular function To thebest of our knowledge no mutation in TPM4 gene has beenimplicated in any diseases of striated muscles in humansHowever one cannot rule out that this possibility was notconsidered before because of the belief that the TPM4 genein humans as in rodents does not produce the sarcomericisoform TPM4120572

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The work was supported by a grant from the Departmentof Medicine and Barbara Kopp Cancer Research Fund toBernard J Poiesz The authors would like to thank Dr CostalC Darie Associate Professor Clarkson University PotsdamNew York for his kind help in explaining the details of LC-MSMS analysis Authors are thankful to Ms Lori Spicer forher assistance for the preparation of the manuscript

References

[1] P Gunning G OrsquoNeill and E Hardeman ldquoTropomyosin-based regulation of the actin cytoskeleton in time and spacerdquoPhysiological Reviews vol 88 no 1 pp 1ndash35 2008

[2] J P Lees-Miller and D M Helfman ldquoThe molecular basis fortropomyosin isoform diversityrdquo BioEssays vol 13 no 9 pp429ndash437 1991

[3] S V Perry ldquoVertebrate tropomyosin distribution propertiesand functionrdquo Journal of Muscle Research and Cell Motility vol22 no 1 pp 5ndash49 2001

[4] K Pieples and D F Wieczorek ldquoTropomyosin 3 increasesstriated muscle isoform diversityrdquo Biochemistry vol 39 no 28pp 8291ndash8297 2000

[5] M F Pittenger J A Kazzaz and D M Helfman ldquoFunc-tional properties of non-muscle tropomyosin isoformsrdquo Cur-rent Opinion in Cell Biology vol 6 no 1 pp 96ndash104 1994

[6] G Schevzov S P Whittaker T Fath J J-C Lin and P WGunning ldquoTropomyosin isoforms and reagentsrdquo BioArchitec-ture vol 1 no 4 pp 135ndash164 2011

[7] M A Geeves S E Hitchcock-DeGregori and P W GunningldquoA systematic nomenclature for mammalian tropomyosin iso-formsrdquo Journal of Muscle Research and Cell Motility vol 36 no2 pp 147ndash153 2015

[8] D E Fleenor K H Hickman G J Lindquester and RB Devlin ldquoAvian cardiac tropomyosin gene produces tissue-specific isoforms through alternative RNA splicingrdquo Journal ofMuscle Research and Cell Motility vol 13 no 1 pp 55ndash63 1992

[9] S Hardy NTheze D LepetitM-R Allo and PThiebaud ldquoTheXenopus laevis TM-4 gene encodes non-muscle and cardiactropomyosin isoforms through alternative splicingrdquo Gene vol156 no 2 pp 265ndash270 1995

[10] B J Spinner R W Zajdel M D McLean et al ldquoCharac-terization of a TM-4 type Tropomyosin that is essential formyofibrillogenesis and contractile activity in embryonic heartsof theMexican axolotlrdquo Journal of Cellular Biochemistry vol 85no 4 pp 747ndash761 2002

[11] A Thomas S Rajan H L Thurston et al ldquoExpression of anovel tropomyosin isoform in axolotl heart and skeletalmusclerdquoJournal of Cellular Biochemistry vol 110 no 4 pp 875ndash8812010

[12] H L Thurston S Prayaga A Thomas et al ldquoExpression ofNkx25 in wild type cardiac mutant and thyroxine-inducedmetamorphosed hearts of the mexican axolotlrdquo CardiovascularToxicology vol 9 no 1 pp 13ndash20 2009

[13] M W Pfaffl ldquoA new mathematical model for relative quantifi-cation in real-time RT-PCRrdquoNucleic Acids Research vol 29 no9 article e45 pp 2002ndash2007 2001

[14] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[15] J S Yuan A Reed F Chen and C N Stewart Jr ldquoStatisticalanalysis of real-time PCR datardquo BMC Bioinformatics vol 7article 85 2006

[16] C Nan S Dube A Matoq et al ldquoExpression of sarcomerictropomyosin in striated muscles in axolotl treated with Shz-1a small cardiogenic moleculerdquo Cardiovascular Toxicology vol15 no 1 pp 29ndash40 2015

[17] S Dube L Panebianco A A Matoq et al ldquoExpression ofTPM1120581 a novel sarcomeric isoform of theTPM1 gene inmouseheart and skeletal musclerdquoMolecular Biology International vol2014 Article ID 896068 9 pages 2014

[18] PHOrsquoFarrell ldquoHigh resolution two-dimensional electrophore-sis of proteinsrdquoThe Journal of Biological Chemistry vol 250 no10 pp 4007ndash4021 1975

[19] A Burgess-Cassler J J Johansen D A Santek J R Ide and NCKendrick ldquoComputerized quantitative analysis of coomassie-blue-stained serum proteins separated by two-dimensionalelectrophoresisrdquo Clinical Chemistry vol 35 no 12 pp 2297ndash2304 1989

[20] J Wang D K Dube J White Y Fan J M Sanger and J WSanger ldquoClock is not a component of Z-bandsrdquo Cytoskeletonvol 69 no 12 pp 1021ndash1031 2012

[21] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[22] C C Darie K Deinhardt G Zhang H S Cardasis M VChao and T A Neubert ldquoIdentifying transient protein-proteininteractions in EphB2 signaling by blue native PAGE and massspectrometryrdquo Proteomics vol 11 no 23 pp 4514ndash4528 2011

[23] I Sokolowska A G Woods M A Gawinowicz U Roy andC C Darie ldquoIdentification of potential tumor differentiationfactor (TDF) receptor from steroid-responsive and steroid-resistant breast cancer cellsrdquoThe Journal of Biological Chemistryvol 287 no 3 pp 1719ndash1733 2012


[24] D S Spellman K Deinhardt C C Darie M V Chao andT A Neuebrt ldquoStable isotopic labeling by amino acids incultured primary neurons application to brain-derived neu-rotrophic factor-dependent phosphotyrosine-associated signal-ingrdquo Molecular and Cellular Proteomics vol 7 no 6 pp 1067ndash1076 2008

[25] Y Peng D Yu Z Gregorich et al ldquoIn-depth proteomic anal-ysis of human tropomyosin by top-down mass spectrometryrdquoJournal of Muscle Research and Cell Motility vol 34 no 3 pp199ndash210 2013

[26] S B Marston O Copeland A E Messer et al ldquoTropomyosinisoform expression and phosphorylation in the human heart inhealth and diseaserdquo Journal ofMuscle Research andCellMotilityvol 34 no 3-4 pp 189ndash197 2013

[27] M Muthuchamy I L Grupp G Grupp et al ldquoMolecularand physiological effects of overexpressing striated muscle 120573-tropomyosin in the adultmurine heartrdquoTheJournal of BiologicalChemistry vol 270 no 51 pp 30593ndash30603 1995

[28] M Muthuchamy L Pajak P Howles T Doetschman and DF Wieczorek ldquoDevelopmental analysis of tropomyosin geneexpression in embryonic stem cells and mouse embryosrdquoMolecular andCellular Biology vol 13 no 6 pp 3311ndash3323 1993

[29] M Muthuchamy P Rethinasamy and D F WieczorekldquoTropomyosin structure and function new insightsrdquo Trends inCardiovascular Medicine vol 7 no 4 pp 124ndash128 1997

[30] K Pieples G Arteaga R John Solaro et al ldquoTropomyosin 3expression leads to hypercontractility and attenuates myofila-ment length-dependent Ca2+ activationrdquo American Journal ofPhysiologymdashHeart and Circulatory Physiology vol 283 no 4pp H1344ndashH1353 2002

[31] S Rajan G Jagatheesan C N Karam et al ldquoMolecular andfunctional characterization of a novel cardiac-specific humantropomyosin isoformrdquo Circulation vol 121 no 3 pp 410ndash4182010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


TPM41 1b2 3 4 5 6 7 8 9b9a 9d39d29d1

TPM4120572 (or Tpm 43)

TPM4120573 (or Tpm 41 as in [7])

TPM4120574 (or Tpm 42 as in [7])

TPM4120575 (or Tpm 44)P3(+)

P2(minus)

P2(minus)

P1(+)

Figure 1 Alternative splicing pattern of the TPM4 gene in humans Exon composition of the TPM4 gene is derived from the publisheddocuments (top) and the recently submitted data in GenBank Although the splice variants TPM4120573 (NM 001145160) and TPM4120574 (NM-0032902) are known for some time the cartoons of the splice variants of TPM4120572 and TPM4120575 are generated with the information of therecently submitted sequences of the corresponding predicted isoforms inGenBank as well as the results from the present study Exon locationsof primers P1(+) P2(minus) and P3(+) are shown

the sequences of both the isoforms by cloning and sequencingof the amplified products We also determined the relativeexpression of the two transcripts by qRT-PCR with RNAfrom human adult heart fetal heart and skeletal muscleusing isoform specific primer-pairs For protein expressionwe performed 1D and 2D western blot analyses with theextract from adult human heart using the exon 9a-specificmonoclonal antibody CH1 We found nine spots on westernblot and the protein was extracted from the spots for massspectrometry (LC-MSMS) analyses The results suggest thatspot G may have TPM4120572 along with TPM1120572 protein Tothe best of our knowledge this is the first report of TPM4120572protein expression in humans

2 Materials and Methods

Human adult and fetal cardiac tissue RNA were obtainedfrom Zyagen (San Diego CA) adult human heart proteinsamples were obtained from Imgenex (San Diego CA)

21 Conventional RT-PCR All PCR and post-PCR exper-iments were done by different personnel in in differentbuildings using separate equipment and separate ventilationso as to avoid carryover contamination For RT-PCR 05120583gof RNA in a total volume of 40 120583L was used to synthesizecDNAwith SuperScript II (Life Technologies Grand IslandNY) and oligo-dT primers following the manufacturerrsquosspecifications For each PCR 3 120583L of cDNA was used and

TPM4120572 and TPM4120575 RNA were amplified as previouslydescribed [11 12] The nucleotide sequences of the primer-pairs for amplification of TPM4120572 and TPM4120575 are givenin Table 1 It is to be noted that the negative primer foramplification of TPM4120572 and TPM4120575 is the sameThe positiveprimers for TPM4120572 and TPM4d are from exon 1a and exon1b respectively The PCR amplified DNAs were run in anagarose gel and the ethidium bromide stained bands wereexcised from the gel andDNAwas extracted from the excisedbands using a MinElute Gel extraction kit from Qiagen(Valencia CA) following the manufacturerrsquos direction Partof the DNA was used for sequencing and part of the gelextracted DNAs was ligated and cloned into the TA cloningvector (Life Technologies) following our published protocol[11 12] Positive clones were identified by PCR using theabove-mentioned specific primer-pairs previously describedVectors or constructs were grown in E coli and the DNAwas extracted using Qiagen Miniprep kit (Valencia CA)The isolated DNA was sequenced (Cornell University LifeScience Core Laboratories Center Ithaca NY) Each clonewas sequenced twice in both directions It is to be noted thatthe primer-pair we have used for TPM4120572 and TPM4120575 is veryspecific for the respective isoform

22 Real-Time Quantitative RT-PCR qRT-PCR analysis ofcDNA template was performed using the LightCycler 480Real-Time PCR System Reactions were carried out in a 384-well plate using the LightCycler 480 SYBR Green I Master kit





















1078

603

1 2 3 4 5 6 M

(a)

1 2 3 4 5 M

1078

603

(b)




3 Results







Translation start

















841 ---------+---------+---------+---------+---------+---------+ 900A L N D M T S L

Human TMP4120572

lowast






Translation start














H A L N D M T S L

1 ---------+---------+---------+---------+---------+---------+ 60

61 ---------+---------+---------+---------+---------+---------+ 120

121 ---------+---------+---------+---------+---------+---------+ 180

181 ---------+---------+---------+---------+---------+---------+ 240

241 ---------+---------+---------+---------+---------+---------+ 300

301 ---------+---------+---------+---------+---------+---------+ 360

361 ---------+---------+---------+---------+---------+---------+ 420

421 ---------+---------+---------+---------+---------+---------+ 480

481 ---------+---------+---------+---------+---------+---------+ 540

541 ---------+---------+---------+---------+---------+---------+ 600

601 ---------+---------+---------+---------+---------+---------+ 660

661

721

---------+---------+---------+---------+---------+---------+ 720

---------+---------+---------+---------+---------+---------+ 780

TPM4120575

-

-

-

-

-

-

-

-

-

-

-

-

lowast


05

101520253035404550


Fold

diff

eren

ce

Tissue type


664 plusmn 021

4642 plusmn 016

1

(a)

0123456789

10


Fold

diff

eren

ce

Tissue type

1


554 plusmn 010

893 plusmn 0085

(b)



M 1 2

Ponceau64 kD

39 kD

28 kD

(a)

CH139 kD

28 kD

(b)

TM311

51 kD

39 kD

(c)







(a) (b) (c)

(d)

E

F

G

(e)


4 Discussion










TPM1120572

(a)







TPM1120572

(b)










Competing Interests


Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















1078

603

1 2 3 4 5 6 M

(a)

1 2 3 4 5 M

1078

603

(b)




3 Results







Translation start

















841 ---------+---------+---------+---------+---------+---------+ 900A L N D M T S L

Human TMP4120572

lowast






Translation start














H A L N D M T S L

1 ---------+---------+---------+---------+---------+---------+ 60

61 ---------+---------+---------+---------+---------+---------+ 120

121 ---------+---------+---------+---------+---------+---------+ 180

181 ---------+---------+---------+---------+---------+---------+ 240

241 ---------+---------+---------+---------+---------+---------+ 300

301 ---------+---------+---------+---------+---------+---------+ 360

361 ---------+---------+---------+---------+---------+---------+ 420

421 ---------+---------+---------+---------+---------+---------+ 480

481 ---------+---------+---------+---------+---------+---------+ 540

541 ---------+---------+---------+---------+---------+---------+ 600

601 ---------+---------+---------+---------+---------+---------+ 660

661

721

---------+---------+---------+---------+---------+---------+ 720

---------+---------+---------+---------+---------+---------+ 780

TPM4120575

-

-

-

-

-

-

-

-

-

-

-

-

lowast


05

101520253035404550


Fold

diff

eren

ce

Tissue type


664 plusmn 021

4642 plusmn 016

1

(a)

0123456789

10


Fold

diff

eren

ce

Tissue type

1


554 plusmn 010

893 plusmn 0085

(b)



M 1 2

Ponceau64 kD

39 kD

28 kD

(a)

CH139 kD

28 kD

(b)

TM311

51 kD

39 kD

(c)







(a) (b) (c)

(d)

E

F

G

(e)


4 Discussion










TPM1120572

(a)







TPM1120572

(b)










Competing Interests


Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Translation start

















841 ---------+---------+---------+---------+---------+---------+ 900A L N D M T S L

Human TMP4120572

lowast






Translation start














H A L N D M T S L

1 ---------+---------+---------+---------+---------+---------+ 60

61 ---------+---------+---------+---------+---------+---------+ 120

121 ---------+---------+---------+---------+---------+---------+ 180

181 ---------+---------+---------+---------+---------+---------+ 240

241 ---------+---------+---------+---------+---------+---------+ 300

301 ---------+---------+---------+---------+---------+---------+ 360

361 ---------+---------+---------+---------+---------+---------+ 420

421 ---------+---------+---------+---------+---------+---------+ 480

481 ---------+---------+---------+---------+---------+---------+ 540

541 ---------+---------+---------+---------+---------+---------+ 600

601 ---------+---------+---------+---------+---------+---------+ 660

661

721

---------+---------+---------+---------+---------+---------+ 720

---------+---------+---------+---------+---------+---------+ 780

TPM4120575

-

-

-

-

-

-

-

-

-

-

-

-

lowast


05

101520253035404550


Fold

diff

eren

ce

Tissue type


664 plusmn 021

4642 plusmn 016

1

(a)

0123456789

10


Fold

diff

eren

ce

Tissue type

1


554 plusmn 010

893 plusmn 0085

(b)



M 1 2

Ponceau64 kD

39 kD

28 kD

(a)

CH139 kD

28 kD

(b)

TM311

51 kD

39 kD

(c)







(a) (b) (c)

(d)

E

F

G

(e)


4 Discussion










TPM1120572

(a)







TPM1120572

(b)










Competing Interests


Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Translation start














H A L N D M T S L

1 ---------+---------+---------+---------+---------+---------+ 60

61 ---------+---------+---------+---------+---------+---------+ 120

121 ---------+---------+---------+---------+---------+---------+ 180

181 ---------+---------+---------+---------+---------+---------+ 240

241 ---------+---------+---------+---------+---------+---------+ 300

301 ---------+---------+---------+---------+---------+---------+ 360

361 ---------+---------+---------+---------+---------+---------+ 420

421 ---------+---------+---------+---------+---------+---------+ 480

481 ---------+---------+---------+---------+---------+---------+ 540

541 ---------+---------+---------+---------+---------+---------+ 600

601 ---------+---------+---------+---------+---------+---------+ 660

661

721

---------+---------+---------+---------+---------+---------+ 720

---------+---------+---------+---------+---------+---------+ 780

TPM4120575

-

-

-

-

-

-

-

-

-

-

-

-

lowast


05

101520253035404550


Fold

diff

eren

ce

Tissue type


664 plusmn 021

4642 plusmn 016

1

(a)

0123456789

10


Fold

diff

eren

ce

Tissue type

1


554 plusmn 010

893 plusmn 0085

(b)



M 1 2

Ponceau64 kD

39 kD

28 kD

(a)

CH139 kD

28 kD

(b)

TM311

51 kD

39 kD

(c)







(a) (b) (c)

(d)

E

F

G

(e)


4 Discussion










TPM1120572

(a)







TPM1120572

(b)










Competing Interests


Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


M 1 2

Ponceau64 kD

39 kD

28 kD

(a)

CH139 kD

28 kD

(b)

TM311

51 kD

39 kD

(c)







(a) (b) (c)

(d)

E

F

G

(e)


4 Discussion










TPM1120572

(a)







TPM1120572

(b)










Competing Interests


Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)

(d)

E

F

G

(e)


4 Discussion










TPM1120572

(a)







TPM1120572

(b)










Competing Interests


Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








TPM1120572

(a)







TPM1120572

(b)










Competing Interests


Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Competing Interests


Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Cloning, Sequencing, and the Expression...

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Transcript of Research Article Cloning, Sequencing, and the Expression...