research article a new exon derived from a mammalian...

Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2013 Article ID 387594 9 pageshttpdxdoiorg1011552013387594

Research ArticleA New Exon Derived from a Mammalian Apparent LTRRetrotransposon of the SUPT16H Gene

Min-In Bae1 Yun-Ji Kim12 Ja-Rang Lee1 Yi-Deun Jung1 and Heui-Soo Kim1

1 Department of Biological Sciences College of Natural Sciences Pusan National University Busan 609-735 Republic of Korea2Department of Nanobiomedical Science ampWCU Research Center Dankook University Cheonan 330-714 Republic of Korea

Correspondence should be addressed to Heui-Soo Kim khs307pusanackr

Received 15 November 2012 Accepted 12 February 2013

Academic Editor Soraya E Gutierrez

Copyright copy 2013 Min-In Bae et al This 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

The SUPT16H gene known as FACTP140 is required for the transcription of other genes For transcription genes need to becomplexed with accessory factors including transcription factors and RNA polymerase II One such factor FACT interacts withhistones H2AH2B for nucleosome disassembly and transcription elongation The SUPT16H gene has a transcript and manyexpressed sequence tags (ESTs) We were especially interested in an MaLR-derived transcript (EST BX333035) that included a newexon introduced by a transposable element a mammalian apparent LTR retrotransposon (MaLR)TheMaLR was detected rangingfromhumans to galagos indicating theMaLR in the SUPT16H gene is integrated into the primate ancestor genome A new exonwascreated by alternative donor site provided by the MaLR The original transcript and the MaLR-derived transcript were expressedin various human rhesus monkey and other primate tissues Additionally we identified a new alternative transcript that includedthe MaLR but there was no significant difference in the expression of the original transcript and the MaLR-derived transcriptInterestingly the new alternative transcript and the MaLR-derived transcript had the MaLR sequence in the new exon but theyhad different structures by adopting different 31015840 splice sites From this study we verified transposable elements that contributed totranscriptome diversity

1 IntroductionTransposable elements (TEs) dispersed in the mammaliangenome are on the rise as completion of the whole humangenome sequence Contrary to our expectations TEsthought to be ldquojunk DNArdquo were estimated to account forapproximately 45 of the human genome (InternationalHuman Genome Sequencing Consortium 2001) TEs canbe categorized by various features such as the capacity tomobilize themselves DNARNA intermediation and theexistence of a long terminal repeat (LTR) Endogenous retro-viruses (ERVs) and long interspersed elements (LINEs) havegene-encoding enzymatic machinery that could mobilize toother genomic regions as autonomous elements Howevernonautonomous elements especially Alu elements which areclassified as short interspersed elements (SINE) and SINE-variable number tandem repeat-Alu-like sequences (SVA)mobilize using the reverse transcriptase of the LINE All ofthe TEs described above are integrated into the host genome

by their RNAs but the integration of DNA transposons ismediated by DNA These groups can be restructured by theexistence of LTRs on both ends ERVs and LTR retrotrans-posons have LTRs but SINEs and LINEs are non-LTRelements [1ndash3]

One group of retrotransposon-like elements mammal-ian apparent LTR retrotransposons (MaLRs) accounts forapproximately 365 and 482 including solitary LTRsof the human and mouse genome respectively [4] Thestructure of MaLRs is similar to that of ERVs with LTRsHowever MaLRs can be distinguished from other retro-viruses by some differences Typical ERVs consist of aninternal region gag pol and env with LTRs on both sidesLTRs are very important for integration and transcriptionbecause they function as a promoter element transcriptionstart site and polyadenylation signal and site [5] Comparedto ERVs MaLRs have short internal sequences and LTRsat the 51015840 and 31015840 ends but they do not have a transcription

2 International Journal of Genomics

initiation site reverse transcriptase and primer binding site[6] Recent studies have shown that TEs contributed toprimate genome evolution through gene structure variationand regulation by integrating into an intron gene-flankingregion and an exon [7 8] Additionally TEs that integrateinto introns could make alternative exons or cassette exonsin a process termed ldquoexonizationrdquo within human and mouseprotein-coding genes [9] in contrast TEs can make newintrons in a process called ldquointronizationrdquoThese events couldhelp with the understanding of transcriptome and proteomecomplexities [9ndash11] The exonization of the primate-specificAlu element is well established in the divergence of humanand chimpanzee genomes as a major mechanism of exoncreation [12 13] Lev-Maor et al (2003) reported that theAlu consensus sequence could provide a splice site leadingto exonization [14] In the case of LTRs Piriyapongsa etal (2007) reported that 256 of 1057 LTR retrotransposonsrelated to human genes were observed in protein-codingregions and 50 protein-coding exons of 45 genes werederived wholly from LTR retrotransposons Furthermorethey analyzed alternative exons of the interleukin 22 receptorthe alpha 2 gene (IL22RA2) which was derived from MaLRsthat provided splice sites indicating the important roles ofTEs in human evolution this result coincided with those ofprevious studies [1 5] Importantly these structural variationsof genes are associated with gene regulation and geneticdisease [15]

Here we identified a transcript that included an MaLRin the SUPT16H gene The SUPT16H gene also knownas FACTP140 is a component of the facilitates chromatintranscription (FACT) complex In eukaryotic cells the tran-scription of genes requires RNA polymerase II and tran-scription factors with altered chromatin structure [16] Inorder to initiate transcription DNA needs to be in an openstructure that is moderated by the alteration of chromatinwith accessory factors facilitating access to the DNA FACTspecifically interacts with histones H2AH2B to affect nucle-osome disassembly and transcription elongation [17] Asdescribed above the SUPT16H gene has an original transcriptconsisting of 26 exons However the integration of an MaLRcreated a new exon that was expressed transcriptionally(BX333035) Furthermore we found a new alternative tran-script including the MaLR-derived exon We assumed thetime of integration of the MaLR into the genome by PCRamplificationAll of the primates assayed had theMaLR in theSUPT16H gene which was a natural result of its conservationin the mammalian genome Additionally we compared thestructure and expression of 2 transcripts including MaLRThe expression patterns of the 2 transcripts in several tissuesof humans crab-eating monkey marmoset and squirrelmonkey were ubiquitous with no significant tissue specificityor species specificity Structurally the 2 transcripts includedthe exon-derived MaLR but they adopted different splicesites leading to exons of different sizes Taken together weshowed that the MaLR could regulate gene expression at thetranscriptional level that is TEs could provide transcriptomediversity resulting in the proteomersquos capacity for diversifica-tion which potentially occupies an important role in the hostgenome

2 Materials and Methods21 Computational Analysis To identify the sequencesrelated to the MaLR we scanned the human referencegenome sequence human ESTs and human RefSeq mRNAsUsing the MaLR consensus sequence as a query we screenednonredundant databases through BLAST version 2226+to identify the novel hybrid transcripts of the MaLRand SUPT16H [18] Then we aligned the transcripts ofthe SUPT16H gene to identify the precise splicing pat-terns TEs included in the SUPT16H transcripts and theirgenomic loci were identified using the RepeatMasker pro-gram (httpwwwrepeatmaskerorg) referencing a librarycontaining the consensus sequences from Repbase Update[19] Using the EST and RefSeq mRNAs we accuratelyreconstructed the SUPT16H gene structure To analyze thesequences of the various SUPT16H transcripts we alignedthem using the ClustalW program [20]

22 Preparing for DNA and RNA Using the TRIzol reagent(Invitrogen) we extracted the total RNA from the cerebrumcerebellum pituitary gland heart lung spleen liver pan-creas kidney and urinary bladder of the crab-eatingmonkeythe heart lung brain stomach liver kidney pancreas colonspleen and small intestine of the marmoset and the heartesophagus diencephalon small intestine lung pancreascerebrum colon and stomach of the squirrel monkey Thetotal RNA from human tissues including the adrenal glandcerebellum whole brain heart kidney liver lung testis tra-chea bone marrow fetal brain fetal liver placenta prostatesalivary gland skeletal muscle spinal cord thymus thyroidand uterus were purchased from Clontech We isolated themRNA from the total RNA by using PolyATract mRNAIsolation Systems (Promega)

We used the genomic DNA of the following species(1) hominoids common chimpanzee (Pan troglodytes)gorilla (Gorilla gorilla) orangutan (Pongo pygmaeus) andgibbon (Hylobates agilis) (2) Old World monkeys Japanesemonkey (Macaca fuscata) rhesus macaque (M mulatta)bonnet macaque (M radiata) mandrill (Mandrillus sphinx)mangabey (Cercocebus agilis) African green monkey(Chlorocebus aethiops) colobus (Colobus guereza) andlangur (Trachypithecus cristatus) (3) New World monkeysnight monkey (Aotus trivirgatus) squirrel monkey (Saimirisciureus) and common marmoset (Callithrix jacchus)and (4) prosimian ring-tailed lemur (Lemur catta) and agalago (Otolemur crassicaudatus) These genomic DNA wereisolated from the heparinized blood samples according to astandard protocol [21] After anticoagulant the buffy coatwas isolated carefully And then proteinase K and phenolequilibrated with Tris-Cl were treated for extraction Theviscous aqueous was transferred and ammonium acetate andethanol were added to form a precipitate Lastly the DNAprecipitate was washed dried and dissolved in TE (Tris andEDTA) Dr Takenaka (Primate Research Institute KyotoUniversity) kindly provided genomic DNA of primates afterconducting this procedure

23 PCR and Reverse Transcription (RT)-PCR AmplificationUsing the genomic DNA of humans and 17 primates the

International Journal of Genomics 3

chr14 q112

NM 007192

BX333035

5998400

3998400

LTRERVL-MaLR

UTRCDS

5 4 3 2 12526

Figure 1 Location and structure of the SUPT16H gene The SUPT16H gene is located on chromosome 14q112 and includes 26 exons Oneof transposable elements (TEs) distributed in genic region of the SUPT16H gene the mammalian apparent LTR retrotransposon (MaLR)is integrated in an intronic region between exon 2 and exon 3 Discriminatively EST (BX333035) has an alternative exonized MaLR Thestructure in the bottom is enlarged exon of BX333035 to show exonized MaLR clearly UTR untranslated region CDS coding sequence

MaLR in the SUPT16H gene was amplified to analyze theintegration time in the primate genome The primer pairincluded GS1 (51015840-AATTGGCGGAAAGGAGAAG-31015840) andGAS2 (51015840-GAGGCAGTCATTCCAGCTCT-31015840)

SUPT16H transcripts were analyzed by RT-PCR ampli-fication M-MLV reverse transcriptase (Promega) with anannealing temperature of 42∘C and an RNase inhibitor (Pro-mega) were used for the reverse transcriptions of the tran-scripts The SUPT16H transcripts were amplified with aprimer pair from GenBank (accession number NM001-033560) consisting of S1 (51015840-AATTGGCGGAAAGGA-GAAG-31015840) and AS1 (51015840-GAGGCAGTCATTCCAGCTCT-31015840) The SUPT16H transcripts related to the MaLR wereamplified with a primer pair from GenBank (BX 333035)consisting of S2 (51015840-TCCAGTTCTCTGTTCTGCCA-31015840)and AS2 (51015840-TGTGTGGAGCTTCAAGCAAG-31015840) The PCRsamples were subjected to an initial denaturation stepof 4min at 94∘C followed by 30 cycles of PCR at 40 sof denaturation at 95∘C 40 s of annealing at 55∘C and90 s of extension at 72∘C followed by a final extensionstep of 7min at 72∘C As a standard control GAPDH wasamplified with a primer pair consisting of GPH-S (51015840-GAG-CCCCAGCCTTCTCCATG-31015840) and GPH-AS (51015840-GAAATC-CCATCACCATCTTCCAGG-31015840) from human GAPDH(GenBank accession number NM002046)

3 Results and Discussion

31 Structure of the SUPT16H Gene with the Integration of aTransposable Element The SUPT16H gene located on chro-mosome 14q112 encodes accessory factors that may facilitateaccess to DNA by unpackaging the chromatin structure Itcontains 26 exons with several transposable elements suchas MaLRs (LTR) Alus (SINE) and L1s (LINE) distributedin the intronic regions The SUPT16H transcript related to

the MaLR (MaLR-derived transcript) has 3 exons and its lastexonwas created by the integration of theMaLR LTR elementin the opposite direction of the host gene (Figure 1)

MaLRs are considered to function in the mammaliangenome by providing alternative splice sites promotersand other cis-regulatory elements similar to other TEsthat have been conserved during primate evolution [6]For example RNF19 has an alternative transcript resultingfrom the exonization of MaLR and AluJo Specifically theMaLR in the first exon has promoter activity in the senseorientation in the African green monkey fibroblast cell lineCos7 and the human colorectal carcinoma cell line HCT116[22] As described above MaLRs are known to be presentin genomes of rodents to primates and humans indicatingthat they were distributed before the divergence of eutherianmammals In order to compare the integration time of theMaLR in the SUPT16H gene with that from previous reportswe conducted PCR amplification using the genomic DNAof human and other primates (Figure 2) We identified anamplicon that was 481 bp long in all of the primates thatwe assessed indicating that the MaLR in the SUPT16H geneintegrated into the genome of a common ancestor before thedivergence of the New World monkey and prosimians Inaddition from the sequencing alignment we also identified40 bp and 4 bp of specific sequences in Japanese macaque(Figure 2(b))These sequences were not detected in any otherprimate including the rhesus macaque which belongs to theOld World monkey family Thus these distinct sequencesdistinguish the Japanese macaque from other primates

32 Expressional Analysis of RefSeqmRNAand ESTBX333035of the SUPT16H Gene TEs distributed in the mammaliangenome have played important roles in gene regulationand evolution [23] One of the typical characteristics ofTEs which is that they affect the transcription of cellular


481bp

Hominoid Old World monkey Prosimian

M RHJMGIORGOCHHU M OGRTCMSQNMLACOAGMBMDBM

New Worldmonkey

(a)

10 1009080706050403020

110 200190180170160150140130120

210 300290280270260250240230220

310 400390380370360350340330320

410 500490480470460450440430420

510 530520

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NM

(b)

Figure 2 Phylogenetic extent and orthologous structure of theMaLR insertion locus in the primate lineage (a) Gel chromatographs showingresults of the PCR amplification of theMaLR-containing portion of an intron of SUPT16H in humans and 17 primates Species are abbreviatedas follows HU human CH chimpanzee GO gorilla OR orangutan GI gibbon JM Japanese macaque RH rhesus macaque BM bonnetmacaque MD mandrill MB mangabey AG African green monkey CO colobus LA langur NM night monkey SQ squirrel monkeyCM commonmarmoset RT ring-tailed lemur and OG galago M indicates a size marker (b)The alignment ofMaLR-containing sequencesin primates The black box and lines indicate MaLR sequences and Japanese-macaque-specific 40 bp and 4 bp sequences respectively


New alternative transcript

NM 0071921 2 3 45 25 26S AS

405bp

405bp

654bp

405bp654bp

405bp654bp

405bp654bp

654bp

HU

CR

MA

SQ

GAPDH

GAPDH

GAPDH

GAPDH

M 10987654321

(a)

10 1009080706050403020

110 200190180170160150140130120

210 300290280270260250240230220

310 400390380370360350340330320

410 500490480470460450440430420

510 530520 540 600590580570560550

610 700690680670660650640630620

710 800790780770760750740730720

810 900890880870860850840830820

910 1000990980970960950940930920

1010 110010901080107010601050104010301020

1110 11901180117011601150114011301120

New exon derived from MaLR

ERVL-MaLRNM 007192

Alternative transcriptBX 333035

ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


(b)

Figure 3 RT-PCR analysis of the SUPT16H transcript in human and primate tissues (a) The SUPT16H transcript is expressed broadly in allof the tissues assayed in humans crab-eating monkey marmoset and squirrel monkeyThe grey line indicates the position of the primer pair(S forward primer AS reverse primer) and the expected product size is 405 bp In this analysis a new alternative transcript was detectedthat was 654 bp long It was also expressed broadly Tissues assayed in humans M marker upper Lane 1 adrenal gland Lane 2 cerebellumLane 3 adult whole brain Lane 4 heart Lane 5 kidney Lane 6 liver Lane 7 lung Lane 8 testis Lane 9 trachea Lane 10 bone marrowlower Lane 1 fetal brain Lane 2 fetal liver Lane 3 placenta Lane 4 prostate Lane 5 salivary gland Lane 6 skeletal muscle Lane 7 spinalcord Lane 8 thymus Lane 9 thyroid and Lane 10 uterus Tissues assayed in crab-eatingmonkey Lane 1 cerebrum Lane 2 cerebellum Lane3 pituitary gland Lane 4 heart Lane 5 lung Lane 6 spleen Lane 7 liver Lane 8 pancreas Lane 9 kidney and Lane 10 urinary bladderTissues assayed in marmoset Lane 1 heart Lane 2 lung Lane 3 brain Lane 4 stomach Lane 5 liver Lane 6 kidney Lane 7 pancreasLane 8 large intestine Lane 9 spleen and Lane 10 small intestine Tissues assayed in squirrel monkey Lane 1 heart Lane 2 esophagusLane 3 interbrain Lane 4 small intestine Lane 5 lung Lane 6 pancreas Lane 7 cerebrum Lane 8 large intestine Lane 9 stomach andLane 10 interbrainGAPDH was amplified as a positive control in all instances (b)The alignment of the SUPT16H transcript new alternativetranscript and EST (BX333035) The alternative transcript has a cassette exon derived from the MaLR (grey box)

genes by exonization which creates an alternatively splicedexon by integration into exonic or intronic regions hasbeen reported in many studies [24 25] Actually thesealternative transcripts are transient and flexible according tothe cellular environment and stimuli However alternativelyspliced variants can be stabilized and increased withoutselection and exonization can lead to not only transcriptomediversity but also proteome diversity [2 10] Furthermorethese events occur differently or specifically according tothe tissue developmental stage and disease thus alternativetranscripts are used to mine disease diagnostic markers [926]

To confirm whether the MaLR sequences are expressedand could regulate transcription we conducted RT-PCRanalysis of the RefSeq mRNA (original transcript) andMaLR-derived transcript (BX333035) from several humantissues Firstly original transcript was expressed broadly inhuman tissues It did not show strong expression or specificexpression in particular tissuesWe analyzed its expression intissues of other primates including the crab-eating monkeycommon marmoset and squirrel monkey (Figure 3) Likethe expression in humans it was ubiquitously expressed inthe tissues of these primates Because we identified japanesemonkey-specific sequences we conducted RT-PCR analysis


S ASBX333035

169bp

169bp

169bp

169bp

169bp

HU

CR

MA

SQ

GAPDH

GAPDH

GAPDH

GAPDH

M 10987654321 M 20191817161514131211

Figure 4 RT-PCR analysis of BX333035 in human and primate tissues The grey line indicates the position of the primer pair (S forwardprimer AS reverse primer) and the expected product size is 169 bp Tissues assayed in humans M marker Lane 1 adrenal gland Lane2 cerebellum Lane 3 adult whole brain Lane 4 heart Lane 5 kidney Lane 6 liver Lane 7 lung Lane 8 testis Lane 9 trachea Lane 10bone marrow Lane 11 fetal brain Lane 12 fetal liver Lane 13 placenta Lane 14 prostate Lane 15 salivary gland Lane 16 skeletal muscleLane 17 spinal cord Lane 18 thymus Lane 19 thyroid and Lane 20 uterus Tissues assayed in crab-eating monkey Lane 1 cerebrum Lane2 cerebellum Lane 3 pituitary gland Lane 4 heart Lane 5 lung Lane 6 spleen Lane 7 liver Lane 8 pancreas Lane 9 kidney and Lane10 urinary bladder Tissues assayed in marmoset Lane 1 heart Lane 2 lung Lane 3 brain Lane 4 stomach Lane 5 liver Lane 6 kidneyLane 7 pancreas Lane 8 large intestine Lane 9 spleen and Lane 10 small intestine Tissues assayed in squirrel monkey Lane 1 heart Lane2 esophagus Lane 3 interbrain Lane 4 small intestine Lane 5 lung Lane 6 pancreas Lane 7 cerebrum Lane 8 large intestine Lane 9stomach and Lane 10 interbrain GAPDH was amplified as a positive control in all instances

in rhesus macaque samples to compare the expression in OldWorldmonkeys However we could not detect a difference inthe SUPT16H expression between the japanese monkey andrhesus macaque (data not shown)

Gene expression is a process that produces RNA orproteins through transcription splicing and translationTheinitiation of gene expression was regulated by chromatinstructure The SUPT16H gene is a subunit of the FACTcomplex that acts as a chromatin-specific transcription elon-gation factor and interacts with histone H2AH2B to disas-semble nucleosomes and promote transcription elongation[17] Namely the broad expression of SUPT16H in humanand other primate tissues indicated its essential function forgene expression Next we analyzed the expression of MaLR-derived transcript As shown in Figure 4 a transcript relatedto the MaLR was also expressed broadly in human tissuesThis product was also detected in tissues of crab-eatingmonkey commonmarmoset and squirrelmonkeyTherewashardly any difference in the expression of 2 transcripts whichindicatedMaLR did not affect the expression of the SUPT16Hgene

33 Identification of anAlternative Transcript and Its CreationFrom the RT-PCR analysis we identified a new alternativetranscript Its product was longer than that of the originaltranscript because alternative splicing retained an intronicregionThis intronic region was derived from the MaLRTheMaLR in the intron was not spliced and exonized (exon 21015840)wholly between exons 2 and 3 (Figure 3) Comparing thenew alternative transcript and the MaLR-derived transcript(BX 333035) we identified specific features of alternativesplicing Entire sequences of the new exon of the alternativetranscript identified in RT-PCRwere derived from theMaLRIn addition this exon was not created by canonical splicingsites As shown in Figure 5 the MaLR provided both typicalalternative splice sites the acceptor site (AG) and a nucleotide(G) of the donor site (GT) Although these splicing sites weretypical sequences recognized by the spliceosome the splicingpattern could be changed In case of the transcript related tothe MaLR (BX 333035) the MaLR in the intronic region wasalso exonized to form a new alternative transcript Strangelythe pattern of splicing was different Only 1 site the acceptorsite was recognized by the spliceosome and the donor site



GT

AGGT

Exon 3AG

LTRERVL-MaLR

EST (BX333035)

1 2 2998400 3 4 5 25 26

Exon 2

Exon 2 GT

LTRERVL-MaLR

AG New exon

Exon derived from MaLR

Figure 5 Comparison of structure between MaLR-derived transcript (BX333035) and new alternative transcript Both MaLR-derivedtranscript and new alternative transcript have a cassette exon derived from theMaLR in the intron of SUPT16HTheMaLR could be exonizedby the acceptor site (AG) in its sequence One difference between the 2 transcripts is the length of the cassette exon The new alternativetranscript has an alternative exon derived from the MaLR only because the MaLR has not only an acceptor site but also a donor site (GT)However the MaLR-derived transcript has an elongated cassette exon that was formed by combining the new exon and the original exon

was skippedThus the new exon was elongated Additionallythe elongated exon included MaLR-derived sequences andsequences of intron and exon 3 of the original and newalternative transcript In spite of the different splicing patternthe new alternative transcriptwas expressed broadly in tissuesof humans and other primates similar to the original andMaLR-related transcripts (Figures 3 and 4)

Alternative splicing is a common mechanism by whichmost genes are processed [27 28] In a previous reportapproximately 60 of human genes have at least one alterna-tive transcript [29] Alternative splicing can be controlled bygenetic mutation and epigenetic alteration [30 31] GenerallyAG and GT are recognized by the spliceosome as the splicingsite However the spliceosome can recognize splicing sitescreated by mutation and cryptic splicing sites leading toalternative splicing [32] In addition to point mutations theintegration of transposable elements can induce alternativesplicing as genetic mutations [2 25] If the 2 transcriptsshowed different and tissue-specific expression patternswe could assume that tissue-specific factors contributed toalternative splicing however both transcripts were expressedbroadly in tissues of humans and primates [33] Alternativelywe could guess that the chromatin structure affected alterna-tive splicing The mechanism of recognizing splice sites canbe altered by epigenetic regulation [34] Various mechanismscan be considered to understand the differences betweenthe 2 transcripts Compared to original transcript the newexon is very unstable and temporary First we could expectthis new exon was created by cryptic splicing site Howeverthe new exon includes intronic sequences by the typical

splice site AG-GT (31015840ndash51015840) in the new alternative transcriptand AG (31015840) in the MaLR-derived transcript existing in theintron If so we could guess that the acceptor site and donorsite of the new exon could be marked as a weak splicesite by epigenetic regulation such as histone modificationsand DNA methylationan although it was the typical splicesite In a previous study histone modification was reportedas important factor for alternative splicing Among histonemodifications such as H3K36me3 H3K79me1 H2BK5me1H3K27me1 H3K27me2 and H3K27me3 enriched in exonthe donor site of the new exon could be recognized weakly bythe spliceosome with H3K36me3 which is found in weaklyexpressed exon [35 36] Additionally the DNA methyla-tion and the CG content in the new exonic region couldaffect the expression of the 2 transcripts Zhou et al (2012)reported that exclusive exons have lower levels of CG andmethylated CG whereas retained intron has higher levels[31]

4 Conclusion

We suggest that the MaLR integrated into an intron ofthe SUPT16H gene before the divergence of New Worldmonkeys and prosimians and it played a biological role in theevolution of the SUPT16H gene during primate evolution byproviding an alternative splice site Although further analysesare required to conclusively elucidate the functions of thetranscriptional variants of the SUPT16H gene in primategenomes the results of this study will help us understand thecharacteristics of the transposable element insertion driving


the transcript diversification and its effect on the evolution ofthe host gene

Authorsrsquo Contribution

M I Bae and Y J Kim contributed equally to this work

Acknowledgment

All the authors contributed to this study and declared that noconflict of interests existsThis research was supported by theBio-Scientific Research Grant funded by the Pusan NationalUniversity (PNU-2008-101-20080596000)

References

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[3] J Lee J Ha S-Y Son and K Han ldquoHuman genomic deletionsgenerated by SVA-associated eventsrdquo Comparative and Func-tional Genomics vol 2012 Article ID 807270 7 pages 2012

[4] P K Mandal and H H Kazazian Jr ldquoSnapShot vertebratetransposonsrdquo Cell vol 135 no 1 pp 192ndash192e1 2008

[5] J Piriyapongsa N Polavarapu M Borodovsky and J McDon-ald ldquoExonization of the LTR transposable elements in humangenomerdquo BMC Genomics vol 8 article 291 2007

[6] A F A Smit ldquoIdentification of a new abundant superfamily ofmammalian LTR-transposonsrdquo Nucleic Acids Research vol 21no 8 pp 1863ndash1872 1993

[7] R EMills E A Bennett R C Iskow et al ldquoRecently mobilizedtransposons in the human and chimpanzee genomesrdquoAmericanJournal of Human Genetics vol 78 no 4 pp 671ndash679 2006

[8] R Cordaux and M A Batzer ldquoThe impact of retrotransposonson human genome evolutionrdquo Nature Reviews Genetics vol 10no 10 pp 691ndash703 2009

[9] N Sela B Mersch A Hotz-Wagenblatt and G Ast ldquoCharac-teristics of transposable element exonization within human andmouserdquo PLoS ONE vol 5 no 6 Article ID e10907 2010

[10] R Sorek ldquoThe birth of new exons mechanisms and evolution-ary consequencesrdquo RNA vol 13 no 10 pp 1603ndash1608 2007

[11] N Sela B Mersch N Gal-Mark G Lev-Maor A Hotz-Wagenblatt and G Ast ldquoComparative analysis of transposedelement insertion within human and mouse genomes revealsAlursquos unique role in shaping the human transcriptomerdquoGenomeBiology vol 8 no 6 article R127 2007

[12] L Lin S Shen A Tye et al ldquoDiverse splicing patterns ofexonized Alu elements in human tissuesrdquo PLoS Genetics vol4 no 10 Article ID e1000225 2008

[13] S Shen L Lin J J Cai et al ldquoWidespread establishment andregulatory impact of Alu exons in human genesrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 108 no 7 pp 2837ndash2842 2011

[14] G Lev-Maor R Sorek N Shomron andGAst ldquoThe birth of analternatively spliced exon 31015840 splice-site selection in Alu exonsrdquoScience vol 300 no 5623 pp 1288ndash1291 2003

[15] J L Goodier and H H Kazazian Jr ldquoRetrotransposons revis-ited the restraint and rehabilitation of parasitesrdquo Cell vol 135no 1 pp 23ndash35 2008

[16] R Belotserkovskaya S Oh V A Bondarenko G OrphanidesV M Studitsky and D Reinberg ldquoFACT facilitates tran-scription-dependent nucleosome alterationrdquo Science vol 301no 5636 pp 1090ndash1093 2003

[17] G Orphanides W H Wu W S Lane M Hampsey andD Reinberg ldquoThe chromatin-specific transcription elongationfactor FACT comprises human SPT16 and SSRP1 proteinsrdquoNature vol 400 no 6741 pp 284ndash288 1999

[18] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[19] J Jurka ldquoRepbase Update a database and an electronic journalof repetitive elementsrdquoTrends in Genetics vol 16 no 9 pp 418ndash420 2000

[20] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[21] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratoryManual Cold SpringHarbor Laboratory Press ColdSpring Harbor New York NY USA 1989

[22] J-W Huh D-S Kim H-S Ha et al ldquoCooperative exonizationof MaLR and AluJo elements contributed an alternative pro-moter and novel splice variants of RNF19rdquo Gene vol 424 no1-2 pp 63ndash70 2008

[23] M Warnefors V Pereira and A Eyre-Walker ldquoTransposableelements insertion pattern and impact on gene expressionevolution in hominidsrdquo Molecular Biology and Evolution vol27 no 8 pp 1955ndash1962 2010

[24] N J Bowen and I K Jordan ldquoExaptation of protein codingsequences from transposable elementsrdquoGenomeDynamics vol3 pp 147ndash162 2007

[25] I Vorechovsky ldquoTransposable elements in disease-associatedcryptic exonsrdquoHumanGenetics vol 127 no 2 pp 135ndash154 2010

[26] Q Yi and L Tang ldquoAlternative spliced variants as biomarkers ofcolorectal cancerrdquo Current Drug Metabolism vol 12 no 10 pp966ndash974 2011

[27] E T Wang R Sandberg S Luo et al ldquoAlternative isoformregulation in human tissue transcriptomesrdquoNature vol 456 no7221 pp 470ndash476 2008

[28] Q Pan O Shai L J Lee B J Frey and B J Blencowe ldquoDeepsurveying of alternative splicing complexity in the human tran-scriptome by high-throughput sequencingrdquo Nature Geneticsvol 40 no 12 pp 1413ndash1415 2008

[29] D L Black ldquoMechanisms of alternative pre-messenger RNAsplicingrdquo Annual Review of Biochemistry vol 72 pp 291ndash3362003

[30] J P Venables ldquoAberrant and alternative splicing in cancerrdquoCancer Research vol 64 no 21 pp 7647ndash7654 2004

[31] Y Zhou Y Lu and W Tian ldquoEpigenetic features are signifi-cantly associated with alternative splicingrdquo BMCGenomics vol13 p 123 2012

[32] M Burset I A Seledtsov and V V Solovyev ldquoAnalysisof canonical and non-canonical splice sites in mammaliangenomesrdquoNucleic Acids Research vol 28 no 21 pp 4364ndash43752000


[33] T W Nilsen and B R Graveley ldquoExpansion of the eukaryoticproteome by alternative splicingrdquoNature vol 463 no 7280 pp457ndash463 2010

[34] R F Luco M Allo I E Schor A R Kornblihtt and T MistelildquoEpigenetics in alternative pre-mRNA splicingrdquo Cell vol 144no 1 pp 16ndash26 2011

[35] P Kolasinska-Zwierz T Down I Latorre T Liu X S Liuand J Ahringer ldquoDifferential chromatinmarking of introns andexpressed exons by H3K36me3rdquo Nature Genetics vol 41 no 3pp 376ndash381 2009

[36] R Andersson S Enroth A Rada-Iglesias C Wadelius and JKomorowski ldquoNucleosomes are well positioned in exons andcarry characteristic histone modificationsrdquo Genome Researchvol 19 no 10 pp 1732ndash1741 2009

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


initiation site reverse transcriptase and primer binding site[6] Recent studies have shown that TEs contributed toprimate genome evolution through gene structure variationand regulation by integrating into an intron gene-flankingregion and an exon [7 8] Additionally TEs that integrateinto introns could make alternative exons or cassette exonsin a process termed ldquoexonizationrdquo within human and mouseprotein-coding genes [9] in contrast TEs can make newintrons in a process called ldquointronizationrdquoThese events couldhelp with the understanding of transcriptome and proteomecomplexities [9ndash11] The exonization of the primate-specificAlu element is well established in the divergence of humanand chimpanzee genomes as a major mechanism of exoncreation [12 13] Lev-Maor et al (2003) reported that theAlu consensus sequence could provide a splice site leadingto exonization [14] In the case of LTRs Piriyapongsa etal (2007) reported that 256 of 1057 LTR retrotransposonsrelated to human genes were observed in protein-codingregions and 50 protein-coding exons of 45 genes werederived wholly from LTR retrotransposons Furthermorethey analyzed alternative exons of the interleukin 22 receptorthe alpha 2 gene (IL22RA2) which was derived from MaLRsthat provided splice sites indicating the important roles ofTEs in human evolution this result coincided with those ofprevious studies [1 5] Importantly these structural variationsof genes are associated with gene regulation and geneticdisease [15]

Here we identified a transcript that included an MaLRin the SUPT16H gene The SUPT16H gene also knownas FACTP140 is a component of the facilitates chromatintranscription (FACT) complex In eukaryotic cells the tran-scription of genes requires RNA polymerase II and tran-scription factors with altered chromatin structure [16] Inorder to initiate transcription DNA needs to be in an openstructure that is moderated by the alteration of chromatinwith accessory factors facilitating access to the DNA FACTspecifically interacts with histones H2AH2B to affect nucle-osome disassembly and transcription elongation [17] Asdescribed above the SUPT16H gene has an original transcriptconsisting of 26 exons However the integration of an MaLRcreated a new exon that was expressed transcriptionally(BX333035) Furthermore we found a new alternative tran-script including the MaLR-derived exon We assumed thetime of integration of the MaLR into the genome by PCRamplificationAll of the primates assayed had theMaLR in theSUPT16H gene which was a natural result of its conservationin the mammalian genome Additionally we compared thestructure and expression of 2 transcripts including MaLRThe expression patterns of the 2 transcripts in several tissuesof humans crab-eating monkey marmoset and squirrelmonkey were ubiquitous with no significant tissue specificityor species specificity Structurally the 2 transcripts includedthe exon-derived MaLR but they adopted different splicesites leading to exons of different sizes Taken together weshowed that the MaLR could regulate gene expression at thetranscriptional level that is TEs could provide transcriptomediversity resulting in the proteomersquos capacity for diversifica-tion which potentially occupies an important role in the hostgenome

2 Materials and Methods21 Computational Analysis To identify the sequencesrelated to the MaLR we scanned the human referencegenome sequence human ESTs and human RefSeq mRNAsUsing the MaLR consensus sequence as a query we screenednonredundant databases through BLAST version 2226+to identify the novel hybrid transcripts of the MaLRand SUPT16H [18] Then we aligned the transcripts ofthe SUPT16H gene to identify the precise splicing pat-terns TEs included in the SUPT16H transcripts and theirgenomic loci were identified using the RepeatMasker pro-gram (httpwwwrepeatmaskerorg) referencing a librarycontaining the consensus sequences from Repbase Update[19] Using the EST and RefSeq mRNAs we accuratelyreconstructed the SUPT16H gene structure To analyze thesequences of the various SUPT16H transcripts we alignedthem using the ClustalW program [20]

22 Preparing for DNA and RNA Using the TRIzol reagent(Invitrogen) we extracted the total RNA from the cerebrumcerebellum pituitary gland heart lung spleen liver pan-creas kidney and urinary bladder of the crab-eatingmonkeythe heart lung brain stomach liver kidney pancreas colonspleen and small intestine of the marmoset and the heartesophagus diencephalon small intestine lung pancreascerebrum colon and stomach of the squirrel monkey Thetotal RNA from human tissues including the adrenal glandcerebellum whole brain heart kidney liver lung testis tra-chea bone marrow fetal brain fetal liver placenta prostatesalivary gland skeletal muscle spinal cord thymus thyroidand uterus were purchased from Clontech We isolated themRNA from the total RNA by using PolyATract mRNAIsolation Systems (Promega)

We used the genomic DNA of the following species(1) hominoids common chimpanzee (Pan troglodytes)gorilla (Gorilla gorilla) orangutan (Pongo pygmaeus) andgibbon (Hylobates agilis) (2) Old World monkeys Japanesemonkey (Macaca fuscata) rhesus macaque (M mulatta)bonnet macaque (M radiata) mandrill (Mandrillus sphinx)mangabey (Cercocebus agilis) African green monkey(Chlorocebus aethiops) colobus (Colobus guereza) andlangur (Trachypithecus cristatus) (3) New World monkeysnight monkey (Aotus trivirgatus) squirrel monkey (Saimirisciureus) and common marmoset (Callithrix jacchus)and (4) prosimian ring-tailed lemur (Lemur catta) and agalago (Otolemur crassicaudatus) These genomic DNA wereisolated from the heparinized blood samples according to astandard protocol [21] After anticoagulant the buffy coatwas isolated carefully And then proteinase K and phenolequilibrated with Tris-Cl were treated for extraction Theviscous aqueous was transferred and ammonium acetate andethanol were added to form a precipitate Lastly the DNAprecipitate was washed dried and dissolved in TE (Tris andEDTA) Dr Takenaka (Primate Research Institute KyotoUniversity) kindly provided genomic DNA of primates afterconducting this procedure

23 PCR and Reverse Transcription (RT)-PCR AmplificationUsing the genomic DNA of humans and 17 primates the


chr14 q112

NM 007192

BX333035

5998400

3998400

LTRERVL-MaLR

UTRCDS

5 4 3 2 12526










481bp



New Worldmonkey

(a)

10 1009080706050403020

110 200190180170160150140130120

210 300290280270260250240230220

310 400390380370360350340330320

410 500490480470460450440430420

510 530520

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NM

(b)




NM 0071921 2 3 45 25 26S AS

405bp

405bp

654bp

405bp654bp

405bp654bp

405bp654bp

654bp

HU

CR

MA

SQ

GAPDH

GAPDH

GAPDH

GAPDH

M 10987654321

(a)

10 1009080706050403020

110 200190180170160150140130120

210 300290280270260250240230220

310 400390380370360350340330320

410 500490480470460450440430420

510 530520 540 600590580570560550

610 700690680670660650640630620

710 800790780770760750740730720

810 900890880870860850840830820

910 1000990980970960950940930920

1010 110010901080107010601050104010301020

1110 11901180117011601150114011301120


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


(b)





S ASBX333035

169bp

169bp

169bp

169bp

169bp

HU

CR

MA

SQ

GAPDH

GAPDH

GAPDH

GAPDH

M 10987654321 M 20191817161514131211







GT

AGGT

Exon 3AG

LTRERVL-MaLR

EST (BX333035)

1 2 2998400 3 4 5 25 26

Exon 2

Exon 2 GT

LTRERVL-MaLR

AG New exon






4 Conclusion






Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


chr14 q112

NM 007192

BX333035

5998400

3998400

LTRERVL-MaLR

UTRCDS

5 4 3 2 12526










481bp



New Worldmonkey

(a)

10 1009080706050403020

110 200190180170160150140130120

210 300290280270260250240230220

310 400390380370360350340330320

410 500490480470460450440430420

510 530520

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NM

(b)




NM 0071921 2 3 45 25 26S AS

405bp

405bp

654bp

405bp654bp

405bp654bp

405bp654bp

654bp

HU

CR

MA

SQ

GAPDH

GAPDH

GAPDH

GAPDH

M 10987654321

(a)

10 1009080706050403020

110 200190180170160150140130120

210 300290280270260250240230220

310 400390380370360350340330320

410 500490480470460450440430420

510 530520 540 600590580570560550

610 700690680670660650640630620

710 800790780770760750740730720

810 900890880870860850840830820

910 1000990980970960950940930920

1010 110010901080107010601050104010301020

1110 11901180117011601150114011301120


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


(b)





S ASBX333035

169bp

169bp

169bp

169bp

169bp

HU

CR

MA

SQ

GAPDH

GAPDH

GAPDH

GAPDH

M 10987654321 M 20191817161514131211







GT

AGGT

Exon 3AG

LTRERVL-MaLR

EST (BX333035)

1 2 2998400 3 4 5 25 26

Exon 2

Exon 2 GT

LTRERVL-MaLR

AG New exon






4 Conclusion






Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


481bp



New Worldmonkey

(a)

10 1009080706050403020

110 200190180170160150140130120

210 300290280270260250240230220

310 400390380370360350340330320

410 500490480470460450440430420

510 530520

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NMSQ

CM

HUCHGOORGIJMRH

NM

(b)




NM 0071921 2 3 45 25 26S AS

405bp

405bp

654bp

405bp654bp

405bp654bp

405bp654bp

654bp

HU

CR

MA

SQ

GAPDH

GAPDH

GAPDH

GAPDH

M 10987654321

(a)

10 1009080706050403020

110 200190180170160150140130120

210 300290280270260250240230220

310 400390380370360350340330320

410 500490480470460450440430420

510 530520 540 600590580570560550

610 700690680670660650640630620

710 800790780770760750740730720

810 900890880870860850840830820

910 1000990980970960950940930920

1010 110010901080107010601050104010301020

1110 11901180117011601150114011301120


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


(b)





S ASBX333035

169bp

169bp

169bp

169bp

169bp

HU

CR

MA

SQ

GAPDH

GAPDH

GAPDH

GAPDH

M 10987654321 M 20191817161514131211







GT

AGGT

Exon 3AG

LTRERVL-MaLR

EST (BX333035)

1 2 2998400 3 4 5 25 26

Exon 2

Exon 2 GT

LTRERVL-MaLR

AG New exon






4 Conclusion






Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



NM 0071921 2 3 45 25 26S AS

405bp

405bp

654bp

405bp654bp

405bp654bp

405bp654bp

654bp

HU

CR

MA

SQ

GAPDH

GAPDH

GAPDH

GAPDH

M 10987654321

(a)

10 1009080706050403020

110 200190180170160150140130120

210 300290280270260250240230220

310 400390380370360350340330320

410 500490480470460450440430420

510 530520 540 600590580570560550

610 700690680670660650640630620

710 800790780770760750740730720

810 900890880870860850840830820

910 1000990980970960950940930920

1010 110010901080107010601050104010301020

1110 11901180117011601150114011301120


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


ERVL-MaLRNM 007192


(b)





S ASBX333035

169bp

169bp

169bp

169bp

169bp

HU

CR

MA

SQ

GAPDH

GAPDH

GAPDH

GAPDH

M 10987654321 M 20191817161514131211







GT

AGGT

Exon 3AG

LTRERVL-MaLR

EST (BX333035)

1 2 2998400 3 4 5 25 26

Exon 2

Exon 2 GT

LTRERVL-MaLR

AG New exon






4 Conclusion






Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


S ASBX333035

169bp

169bp

169bp

169bp

169bp

HU

CR

MA

SQ

GAPDH

GAPDH

GAPDH

GAPDH

M 10987654321 M 20191817161514131211







GT

AGGT

Exon 3AG

LTRERVL-MaLR

EST (BX333035)

1 2 2998400 3 4 5 25 26

Exon 2

Exon 2 GT

LTRERVL-MaLR

AG New exon






4 Conclusion






Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



GT

AGGT

Exon 3AG

LTRERVL-MaLR

EST (BX333035)

1 2 2998400 3 4 5 25 26

Exon 2

Exon 2 GT

LTRERVL-MaLR

AG New exon






4 Conclusion






Acknowledgment


References













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgment


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 a new exon derived from a mammalian...

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