Analysis of western lowland gorilla (Gorilla gorilla gorilla) specificAlurepeats

Analysis of western lowland gorilla(Gorilla gorilla gorilla) specific Alu repeatsMcLain et al

McLain et al Mobile DNA 2013 426httpwwwmobilednajournalcomcontent4126

RESEARCH Open Access

Analysis of western lowland gorilla(Gorilla gorilla gorilla) specific Alu repeatsAdam T McLain1 Glenn W Carman1 Mitchell L Fullerton12 Thomas O Beckstrom1 William Gensler1Thomas J Meyer3 Christopher Faulk4 and Mark A Batzer1

Abstract

Background Research into great ape genomes has revealed widely divergent activity levels over time for Aluelements However the diversity of this mobile element family in the genome of the western lowland gorilla haspreviously been uncharacterized Alu elements are primate-specific short interspersed elements that have been usedas phylogenetic and population genetic markers for more than two decades Alu elements are present at high copynumber in the genomes of all primates surveyed thus far The AluY subfamily and its derivatives have been recognizedas the evolutionarily youngest Alu subfamily in the Old World primate lineage

Results Here we use a combination of computational and wet-bench laboratory methods to assess and catalog AluYsubfamily activity level and composition in the western lowland gorilla genome (gorGor31) A total of 1075 independentAluY insertions were identified and computationally divided into 10 subfamilies with the largest number of gorilla-specificelements assigned to the canonical AluY subfamily

Conclusions The retrotransposition activity level appears to be significantly lower than that seen in the human andchimpanzee lineages while higher than that seen in orangutan genomes indicative of differential Alu amplification in thewestern lowland gorilla lineage as compared to other Homininae

Keywords Primate Gorilla SINE Retrotransposon Mobile elements

BackgroundAlu elements are a family of primate-specific SINEs(Short INterspersed Elements) of approximately 300base pairs (bp) long and present in the genomes of allliving primates [1-3] Alu elements were derived from7SL RNA the RNA component of the signal recognitionparticle in the common ancestor of all living primates[4] In the past approximately 65 million years Aluelements have become widely distributed in primategenomes [15] Alu elements are now present at copynumbers of gt1000000 in all surveyed great ape genomes(Additional file 1) [1] Despite their high copy numberthe majority of Alu elements are genomic fossilsnon-propagating relics passed down over millions ofyears after earlier periods of replicative activity [16]It is hypothesized that a relatively small number of

lsquomasterrsquo elements are responsible for the continuedspread of all active subfamilies [78]As non-autonomous retrotransposons Alu elements

do not encode the enzymatic machinery necessary forself-propagation [12] This is accomplished by appropriatingthe replication machinery [29] of a much larger autonomousretrotransposon called LINE1 (L1) via a process termedtarget-primed reverse transcription (TPRT) [10-13]The effective use of SINEs as phylogenetic markers

was first demonstrated in 1993 in a study seeking toresolve relationships between Pacific salmonid species[14] Subsequent to this study SINE-based phylogeneticmethods have been applied across a wide range of speciesto determine evolutionary relationships [1516] Inparticular Alu elements have proven to be extremelyuseful tools for elucidating evolutionary relationshipsbetween primate species [117] The essentially homoplasyfree presence of an Alu element of the same subfamily at agiven locus between two or more primate species isalmost always definitive evidence of shared ancestry [18]The possibility of confounding events is very small and

Correspondence mbatzerlsuedu1Department of Biological Sciences Louisiana State University Baton RougeLA 70803 USAFull list of author information is available at the end of the article

copy 2013 McLain et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (httpcreativecommonsorglicensesby20) which permits unrestricted use distribution andreproduction in any medium provided the original work is properly cited The Creative Commons Public Domain Dedicationwaiver (httpcreativecommonsorgpublicdomainzero10) applies to the data made available in this article unless otherwisestated

easily resolved by the sequencing and examining of theelement in question [118] In the past 15 years Alu-basedphylogenetic methods have been used with great successto resolve evolutionary relationships among the Tarsiers[1920] New World [21] and Old World monkeys [22-24]gibbons [25] lemurs [2627] and great apes [28]In addition to phylogenetic applications Alu elements also

function as effective markers for the study of populationgenetics via examination of polymorphic elements betweenmembers of the same species [22930] Alu elements arealso linked to numerous genetic diseases and the insertionof an element at an importune genomic location can havegrave consequences for the individual involved [33132]Additionally Alu elements are thought to be a causal factorin genomic instability [33-36]Alu elements are classified in multiple major subfamilies

and numerous smaller derivative subfamilies based onspecific sequence mutations [37-40] All extant primatesshare older elements while all primate lineages examinedalso have younger lineage-specific subfamilies [41] Alusubfamily evolution is parallel not linear and varioussubfamilies have been found to be actively retrotransposingat the same time in all primate genomes surveyed eachprimate lineage thus possesses its own Alu subfamilies[14243]The AluJ subfamily is the most ancient Alu lineage

and was largely active from approximately 65 millionyears ago to approximately 55 million years ago at whichpoint AluS evolved and supplanted AluJ as the predominantactive subfamily [3741] Due to the antiquity of the lineageAluJ subfamilies are present in all extant primates includingStrepsirrhines [2744] AluS on the other hand evolvedfrom AluJ after the Strepsirrhine-Haplorrhine diver-gence and so is only found in New World and OldWorld primates [23745] The AluY subfamily subse-quently evolved from AluS in the Old World primatelineage and remains the predominant active subfamilyin catarrhines [14145]A number of AluY-derived subfamilies continue to be

active in great apes [1] and polymorphic lineage-specificAlu elements have been well documented between existinghuman populations [2] indicating a continued activity levelfor these mobile elements A rate of one new element inevery approximately 20 live births has been proposedas the current rate of Alu element activity in the extanthuman population but the large size of this populationcoupled with human generation time would make it verydifficult for new elements to come to fixation outside ofsmall population groups [4647] Research into Aluelement activity in Sumatran and Bornean orangutanshas indicated a comparatively low-level of continuedretrotransposition activity in these apes [48] suggestingsome alteration of the propagation of Alu within thislineage [49]

The western lowland gorilla (Gorilla gorilla gorilla) asubspecies of the western gorilla (Gorilla gorilla) is acritically endangered great ape endemic to the forestsand lowland swamps of central Africa [5051] Westernlowland gorillas are gregarious living in family groupscomprised of a dominant male multiple females subadultmales and juvenile offspring [52] Western lowland gorillasare in danger of extinction due to human activity Theirwild population size is shrinking in the face of anthropo-genic pressure and diseases such as Ebola [50] Gorillas area close evolutionary relative of humans and the Pan lineageof chimpanzees and bonobos with the most widelyaccepted date for a common ancestor 6 to 9 millionyears ago [2853-55] though a date as early as 10 millionyears ago has been recently proposed [56]The genome of lsquoKamilahrsquo a female western lowland

gorilla living at the San Diego Zoo was initially assembledfrom 54 Gbp of capillary sequence and 1668 Gbp ofIllumina read pairs and further refined using bacterialartificial chromosome (BAC) and fosmid end paircapillary technology [57] This sequence is available fromthe Wellcome Trust-Sanger InstitutePrevious analyses of Alu elements in gorillas have been

limited to analysis in the context of wider researchprojects [2858-61] and have not focused specificallyon subfamily analysis Here we examine the westernlowland gorilla genome (build gorGor31) [57] to identifygorilla-specific AluY subfamilies and assess the activitylevels copy number and age of these subfamilies Ourfinal analysis resulted in the identification of 1075 Gorillaspecific Alu element insertions

Results and discussionComputational examination of the western lowlandgorilla genomeA total of 1085174 Alu elements were identified in thegenome of the western lowland gorilla (Additional file 1)Of these 286801 were identified as belonging to theancient AluJ subfamily and 599237 were identified asmembers of the AluS subfamily A total of 57427 elementswere too degraded or incompletely sequenced to be assigneda subfamily designation by RepeatMasker and were simplyidentified as lsquoAlursquo We identified 141709 members of theAluY subfamily This subfamily is of particular interest due toits relatively young age and known continued mobility inother great ape genomes [162] Approximately one-third(57458) of these putative AluY elements were gt250 bp inlength Gorilla-specific elements were subsequently identifiedby comparison of orthologous loci in the genomes ofhuman common chimpanzee and orangutan [63] Puta-tive unique gorilla-specific AluY insertions were estimatedat 4127 copies This number is similar (965) to the 4274gorilla-specific Alu elements identified using other ap-proaches [58] Individual examination demonstrated that

McLain et al Mobile DNA 2013 426 Page 2 of 10httpwwwmobilednajournalcomcontent4126

the majority of our 4127 loci were in fact shared insertionsThese loci were manually examined for gorilla specificityusing BLAT [64] This manual examination excluded 2858loci from further analysis due to the presence of sharedinsertions missed by Lift Over (2626 insertions) or the lackof orthologous flanking regions in the genomes of otherspecies that preclude PCR verification (232 insertions) Thisresulted in a total of 1269 likely gorilla-specific Aluinsertion loci for inclusion in subfamily structure analysisThese 1269 loci were analyzed for subfamily structure

using the COSEG program COSEG removed 194 probablegorilla-specific Alu insertions from the dataset due to thepresence of truncations or deletions in diagnostic regions ofthe element leaving 1075 probable gorilla-specific Aluinsertion loci for further analysis Additional file 2 COSEGthen divided the loci into 10 subfamilies based ondiagnostic mutations in the sequence of the individualAlu elements and provided subfamily consensus sequences(Figure 1) [43] The consensus sequences were thenaligned with known human AluY subfamilies from theRepBase database of repetitive elements [65] (Figure 2) Agorilla-specific nomenclature system was created to desig-nate subfamilies using the suffix lsquoGorillarsquo preceded by thesubfamily affiliation based on a comparison to identifiedhuman subfamilies (for example lsquoAluYc5a1_Gorillarsquo)Subfamilies were named in accordance with establishedpractice for Alu subfamily nomenclature [41] The firstidentified AluYc5-derived subfamily was for exampledesignated AluYc5a3_Gorilla The lsquoarsquo denotes the fact thatthis is the first Yc5-derived subfamily identified The lsquo3rsquodenotes the number of diagnostic mutations by which thisgorilla-specific subfamily differs from the human AluYc5consensus sequence [41] Subfamily age estimates werecalculated using the BEAST (Bayesian EvolutionaryAnalysis by Sampling Trees) program [66]

AluY subfamily activity in the western lowland gorillagenomeComputational and PCR analysis of the western lowlandgorilla genome has identified 1075 independent gorilla-specific AluY insertion loci Computational analysis of thisdataset indicates the presence of 10 distinct subfamiliesidentifiable by the presence of diagnostic mutations specificto each lineage The 1075 elements identified in our studyalmost certainly do not represent the total number of AluYspecific to western lowland gorilla genome Any loci underour arbitrary length of gt250 were excluded from our data-set It is also likely that a number of AluY loci are located inportions of the genome where sequence data is incompletewithin repeat regions for example Additionally some AluYloci were excluded when no orthologous genomic regionwas present in the species being used for comparisonThe largest newly identified gorilla-specific Alu sub-

family was designated as AluY_Gorilla This designation

was established via computational evaluation and manualalignment of the 759 elements assigned to this subfamilyThe consensus sequence for these elements wasfound to be 100 identical to the canonical AluY hu-man consensus sequence (Figure 2) This subset ofclassic AluY elements continued to propagate in theGorilla lineage after the divergence from the sharedcommon ancestor with the Homo-Pan lineage We assayedand verified a total of 135 loci from this subfamily via PCR(18) The 43 elements belonging to the AluYa1_Gorillasubfamily differ from the AluY consensus sequence by onediagnostic mutation at nucleotide position 133 We assayedand verified via PCR 21 elements in this subfamily (49)This sequence should not be confused with the Homo-PanAluYa subfamilyThe AluYa1b4 subfamily is derived from AluYa1_Gorilla

and is a small and very likely young subfamily of 13elements that shared the diagnostic mutation at position133 of Ya1 but has also accrued four additional diagnosticmutations We assayed and verified via PCR seven elementsin this subfamily (54) A second identified AluY lineage ingorilla is the AluYc3_Gorilla subfamily We assayed andverified via PCR 20 of the 69 elements in this subfamily(29) The consensus sequence for the 69 members identi-fied in this subfamily is a 100 match to the human AluYc3subfamily consensus sequence (Figure 2)Two additional gorilla-specific AluYc-derived subfam-

ilies share the characteristic 12 bp deletion at positions87ndash98 that is a hallmark of human AluYc5 These twosubfamilies possess independent diagnostic mutations thatmake them distinct from the AluYc5 consensus sequenceThese two subfamilies are designated as AluYc5a3_Gorilla(55 elements identified) and AluYc5b2_Gorilla (46 elementsidentified) AluYc5a3_Gorilla has three additional diagnosticmutations differentiating it from the AluYc5 consensus as amark of identification In keeping with Alu subfamilynaming convention this subfamily has thus been deemedlsquoYc5a3rsquo lsquoarsquo as the first Yc5-like subfamily identified in thegorilla genome and lsquo3rsquo for the three diagnostic mutationsdifferentiating it from the canonical Yc5 consensus Weassayed and verified 27 members of this subfamily viaPCR (49) AluYc5b2 also shares the characteristic 12 bpdeletion of the human AluYc5 but has two independentdiagnostic mutations (Figure 2) We assayed and verifiedvia PCR 19 members of this subfamily (41) It isprobable that AluYc5a3_Gorilla and AluYc5b2_Gorilladerived from AluYc5 around the time of the HomoPan-Gorilla speciation eventA third lineage nearly identical to human AluYb3a2 was

identified as AluYb3a2b2_Gorilla (25 elements identified)This Alu subfamily contains two additional diagnosticmutations Termed AluYb3a2b2_Gorilla this lineage is anindependent evolution in the Gorilla gorilla gorilla genomeand not a derivative of the human-specific AluYb3a2 The

Figure 1 (See legend on next page)

AluYb lineage is human specific meaning any identical orapparently derived Alu lineages in other primate ge-nomes must be examples of independent evolution[67] This is confirmed by the lack of orthologs at thesame location in the human genome We assayed andverified 14 members of this subfamily via PCR (56)An additional subfamily present at only 17 copies andderived from AluYb3a2b2_Gorilla was identified andtermed AluYb3a2b2a2_Gorilla due to two diagnosticmutations separating these otherwise identical subfamiliesWe assayed and verified via PCR nine elements in this sub-family (53) The low copy number of these subfamiliescoupled with their lack of impairing point mutations evenwith the caveat that some subfamily members may havebeen overlooked leads us to posit that they are among theyoungest and potentially still active subfamilies in thewestern lowland gorilla genomeTwo additional subfamilies were identified that while

clearly AluY derived do not follow the consensus sequencesof established subfamilies available via RepBase The firsttermed AluY16_Gorilla is identified clearly by the presenceof an A-rich insert at position 219 followed by a 16 bp dele-tion and is present in 30 copies We assayed and verifiedvia PCR 10 members of this subfamily (33) The secondsubfamily apparently derived from the first and designatedAluY16a4_Gorilla is present in 18 copies and can be distin-guished from AluY16_Gorilla by a 20 bp deletion occurringafter the A-rich region at position 219 Seventeen elementsfrom this subfamily were assayed via PCR (94) with 100of these 17 being verified as gorilla-specific One locus(gorGor31 chrX74544052ndash74544324) lacked sufficientorthologous 5prime sequence in non-gorilla outgroups to suc-cessfully design a working primer but was included inthe total count based on computational verificationThe accumulation of non-diagnostic mutations in thesetwo subfamilies may indicate that they are more ancientApproximately 25 of the 1075 gorilla-specific AluY

elements computationally identified in this study wereverified by PCR with the remaining approximately 75verified by manual examination of computational data Itis important to note that we had no false positives in thisstudy and 100 of the elements computationally identifiedas gorilla-specific that were subsequently assayed via PCRwere confirmed to be in fact gorilla-specificOne means of identifying potential master elements

[7] is to look for subfamily members with mutation-free

polyA-tails [68] A possible source element for theAluY_Gorilla subfamily for instance was identified onchrX5135584ndash5135921 with a mutation-free 30 bppolyA-tail and intact promoter region A posited sourceelement for the AluYc5b2 subfamily was identified onchr917925753ndash17926051 also with a mutation-free 30 bppolyA-tail and intact promoter regionAluY retrotransposition rates appear to be lower in the

western lowland gorilla genome than in the human orchimpanzee genomes [69] while higher than that seenin the orangutan genome [4849] Factors influencing ratesof retrotransposition are myriad [146] Active retrotranspo-sons are frequently polymorphic within a population andare easily lost during events like speciation or populationbottlenecks [7071] The number of active elements andthe amplification rate of elements surviving such an eventcan vary greatly and impact overall retrotranspositionactivity in the host genomeA possible explanation for this lower activity level include

inhibition of retrotransposition in the Gorilla lineage by theinteraction of host factors such as members of the APOBECfamily of proteins with the enzymatic machinery of L1 [172]Interference with L1 and Alu retrotransposition by APOBEChas been documented [72-74] Analysis of the activity levelof Gorilla-specific L1 elements could elucidate this but hasnot yet been done Additionally environmental stress factorsmay impact retrotransposition rates [75] It is possible thatone or a combination of these retrotransposition-inhibitingfactors could be responsible for the lower level of AluYactivity in the western lowland gorilla genomeA median joining tree of relationships between gorilla-

specific AluY subfamilies was generated from a stepwisealignment [76] using the Network program (Figure 1)[4277] The tree generated supports the divergence of allgorilla-specific subfamilies from the AluY_Gorilla subfamilyand analysis of subfamily ages using BEAST places the datefor this subfamily divergence at the stem of the Gorillalineage Initial divergence of gorilla-specific subfamilies oc-curred shortly after the speciation event separating theGorilla lineage from the Homo-Pan lineage 6 to 9 millionyears ago [2853-55] and master elements have continued toproduce copies of each subfamily at varying rates since [7]

Divergence dates of gorilla-specific AluY subfamiliesBEAST analysis of individual subfamily ages suggests nodelay or change in transposon activity in western

(See figure on previous page)Figure 1 Analysis of gorilla-specific Alu subfamilies (A) A schematic diagram of a tree of evolutionary relationships of the four genera inFamily Hominidae (great apes) based on divergence dates of 6 to 9 million years ago for the Gorilla-HomoPan speciation event [2853-55](B) A pie chart showing a color-coded distribution of Gorilla-specific AluY subfamilies AluY_Gorilla is the largest subfamily representing slightlyless than three-fourths of the total copy number identified (C) A stepwise analysis of the relationships between Gorilla-specific AluY subfamiliesgenerated from a Network analysis of the consensus sequences for each subfamily The color of the dots representing each subfamily are correlatedwith the colors in the pie chart in Figure 1B

lowland gorilla following the divergence of the Gorillaand Homo-Pan lineages The age of the gorilla-specificlineages ranges from 65-671 million years ago based ona baseline divergence of 7 million years ago for the mostrecent common ancestor of Gorilla and Homo-Pan Thisindicates that all of the identified subfamilies originatedaround the time of the speciation event that separatedthese two lineages This result is consistent with the ongoingpropagation of these subfamilies before during and after thespeciation event at a relatively constant rate This indicatesthat the lsquomaster genesrsquo [7] from which these subfamilies arederived already existed and were retrotranspositionally activeprior to the aforementioned speciation event and haveremained active subsequently Examination of Alu elementsindicates retrotranspositionally active elements are relativelyrare and that most Alu activity is the result of a small num-ber of lsquomasterrsquo copies engaging in retrotranspositional activityover time [7] Our results suggest that the 10 gorilla-specificAluY subfamilies identified in this study diverged and are stilldiverging from master elements already present in the gen-ome of the common ancestor of the Gorilla and Homo-Panlineages A table listing each subfamily the lsquomaster genersquo orancestral Alu subfamily from which it was likely derived the divergence from the consensus sequence of the masterelement copy number and suggested age of the most recentcommon ancestral element are available in the Additionalfiles section of this paper as Additional file 3

ConclusionsAluY subfamily activity appears to be greatly reduced inthe western lowland gorilla genome when compared to

the human and chimpanzee genomes The level ofactivity seen while not as low as that observed in thegenome of the orangutan is consistent with a changein host surveillance or intrinsic retrotransposition capacityAlu subfamily age estimates provide further support for themaster gene model of Alu retrotransposition with a rela-tively low number of ancient lineages responsible for on-going retrotranspositional activity The 1075 lineage specificAluY insertion loci and the 10 subfamilies identified shouldprovide future researchers with a rich source of geneticsystems for conservation biology and evolutionary genetics

MethodsComputational methodologyThe genome of the Western lowland gorilla (Gorillagorilla gorilla) was downloaded and analyzed for thepresence of Alu elements using an in-house installation ofthe RepeatMasker program [62] The Gorilla gorilla gorillagenome is available for download and analysis via thewebsite of the Wellcome Trust-Sanger Institute [78] Theresulting dataset was parsed into separate files based onthe Alu subfamily designations assigned by RepeatMaskerThe file containing elements designated as members ofthe AluY subfamily was then further parsed to remove84251 elements under the length of 250 bp using theestimation that shorter elements were likely to be olderelements present in multiple species and therefore notuseful for our analysis The lsquoFetch Sequencesrsquo functionfrom the online version of the Galaxy suite of programs[6379-81] was then used to retrieve the individual DNAsequence present at each of these loci using the gorilla

Figure 2 Alu sequence alignment The consensus sequence for the AluY subfamily is shown at the top with western lowland gorilla-specificAlu subfamilies listed below The dots below the consensus denote the same base with insertions and deletions noted by dashes and mutationswith the appropriate bases The consensus sequences for the AluYa1 AluYc1 and AluYc5 subfamilies included for comparative purposesSubfamily-specific diagnostic mutations are highlighted in yellow Lineage-specific deletions are highlighted in red AluY_Gorilla is 100 identicalto the AluY consensus sequence The shared 12-bp deletion identifying the AluYc5-derived Gorilla subfamilies is located at position 86 The 16-bpand 20-bp deletions identifying the AluY16_Gorilla and AluY16a4_Gorilla subfamilies are visible at positions 228 and 232

genome build gorGor31 and the Lift Over function wasused to examine these loci for gorilla lineage specificity bycomparison to the closely related genomes of human(Homo sapiens hg19) chimpanzee (Pan troglodytes pan-Tro2) and Sumatran orangutan (Pongo pygmaeus abeliiponAbe2) An additional 200 bp of flanking sequence oneach side of the loci assayed was included in this analysisfor validation of orthologous loci between the nineprimate species considered in this study (Table 1)

Loci selected for verification were examined for fur-ther evidence of gorilla-specificity using the BLAST-LikeAlignment Tool (BLAT) available at the UCSC GenomeBrowser website [82] Putative gorilla-specific loci werecompared to the available genomes of three other primatespecies human (hg19) chimpanzee (panTro2) and orang-utan (ponAbe2) [6483] Elements found to be absent inthese species and with sufficient orthologous flankingacross species were marked for PCR primer design andexperimental validation Loci determined to be sharedinsertions as well as those lacking sufficient orthologousflanking for effective primer design were removed fromfurther consideration [64]The COSEG program [84] designed to identify repeat

subfamilies using significant co-segregating mutations wasthen run on the remaining putative gorilla-specific inser-tions to identify and group specific subfamilies togetherCOSEG ignores non-diagnostic mutations during ana-lysis providing an accurate representation of relation-ships between subfamilies of elements by ignoringpotentially misleading mutational events [43] COSEGuses a minimum subfamily size of 50 elements as thedefault setting We arbitrarily defined subfamilies asgroups of gt10 elements to increase the detail of sub-family structure resolved A subset of a minimum of10 of each identified subfamily was then chosen forverification using locus-specific PCR with a total of279 loci assayed and verified (Figure 1)A multi-species alignment comprised of the species

listed above was created for each locus using BioEdit[85] Oligonucleotide primers for the PCR assays weredesigned in shared regions flanking each putative gorilla-specific element chosen for experimental verification usingthe Primer3Plus program [86] These primers were thentested computationally against available primate genomesusing the in-silico PCR tool on the UCSC GenomeBioinformatics website [83]

Table 1 DNA sample data of all species examined in this study

Taxonomic name Common name Origin ID number

Gorilla gorilla gorilla Western lowland gorilla Coriell1 AG05251

Homo sapiens Human HeLa ATCC2 HeLa CCL-2

Pan troglodytes Common chimpanzee IPBIR3 NS06006

Pan paniscus Bonobo IPBIR3 PR00661

Pongo pygmaeus Bornean orangutan Coriell1 AG05252A

Pongo abelii Sumartran orangutan Coriell1 GM06213A

Nomascus leucogenys Northern white-cheeked gibbon Carbone Lab4 NLL606

Macaca mulatta Rhesus macaque Coriell1 NG07098

Chlorocebus aethiops African green monkey ATCC2 CCL701Coriell Institute for Medical Research 403 Haddon Avenue Camden NJ 08103 USA2From cell lines provided by American Type Culture Collection (ATCC) PO Box 1549 Manassas VA 20108 USA3Integrated Primate Biomaterials and Information Resource (IPBIR) httpccrcoriellorgSectionsCollections4Laboratory of Dr Lucia Carbone Oregon Health amp Science University Beaverton Oregon httpcarbonelabcom

Figure 3 Phylogenetic assay of a western lowland gorilla-specificAlu insertion (Primer Pair Gor112) An agarose gel chromatograph ofthe gorilla specific Alu insertion Gor112 The filled site is approximately550 bp (lane 7) and the empty site is 250 bp (lanes 3 to 6 and 8 to 11)Lanes (1) 100 bp DNA ladder (2) negative control (3) human (4)bonobo (5) common chimpanzee (6) northern white-cheeked gibbon(7) western lowland gorilla (8) Sumatran orangutan (9) Borneanorangutan (10) Rhesus macaque (11) green monkey (12) empty(13) 100 bp DNA ladder

Subfamily age estimates were calculated using the BEASTprogram [6687] BEAST has previously been used to esti-mate dates of divergence using transposon data [88] Foreach subclade the consensus sequence for each subfamilywas determined from the COSEG output [43] The progeni-tor element was determined by RepeatMasker analysis ofeach consensus sequence Elements were aligned using theSeaView software program and MUSCLE algorithm [7689]The progenitor element was then used as an out-group toroot the tree of each subclade BEAST was calibrated with abaseline divergence date of 7 million years ago for the splitbetween the Gorilla and Homo-Pan lineages A divergencedate of 7 million years ago is within the generally accepted6 to 9 million years ago range for this divergence[2853-55] BEAST was run with the following parametersSite Heterogeneity = lsquogammarsquo Clock = lsquostrict clockrsquo SpeciesTree Prior = lsquobirth death processrsquo Prior for Time of MostRecent Common Ancestor (tmrca) = lsquoNormal distributionrsquowith mean of 70 million years and 10 standard deviationrsquoucldmean = lsquouniform modelrsquo with initial rate set at 0033Length of Chain = lsquo10000000rsquo all other parameters wereleft at default settingsThe Network program [90] was run on gorilla-specific

AluY subfamily consensus sequences to generate a stepwisetree of relationships between identified subfamilies [4277]The consensus sequences for the gorilla-specific AluYsubfamilies were aligned using the MUSCLE algorithm [76]and converted to the rdf file format using the DNAspprogram [91] The rdf file was then imported to Networkand a median-joining analysis was run The resultingoutput file demonstrating evolutionary relationshipsbetween subfamilies is presented in Figure 1C

PCR and DNA sequencingTo verify gorilla-specificity locus specific PCR wasperformed with a nine-species primate panel comprised ofDNA samples from the following species Western lowlandgorilla (Gorilla gorilla gorilla) Human HeLa (Homo sapiens)Common chimpanzee (Pan troglodytes) Bonobo (Panpaniscus) Bornean orangutan (Pongo pygmaeus) Sumatranorangutan (Pongo abelii) Northern white-cheeked gibbon(Nomascus leucogenys) Rhesus macaque (Macaca mulatta)African green monkey (Chlorocebus aethiops) Informationon all DNA samples used in PCR analysis is listed in Table 1PCR amplification of each locus was performed in

25 μL reactions using 15 ng of template DNA 200 nMof each primer 200 μM dNTPs in 50 mM KCl 15 mMMgCl2 10 mM TrisndashHCl (pH 84) and 2 units of TaqDNA polymerase PCR reaction conditions were asfollows an initial denaturation at 95degC for 1 min followedby 32 cycles of denaturation at 95degC annealing at thepreviously determined optimal annealing temperature(60degC with some exceptions) and extension at 72degCfor 30 s each followed by a final extension of 72degC

for 1 min PCR products were analyzed to confirmgorilla specificity of all loci on 2 agarose gelsstained with 025 ug ethidium bromide and visualizedwith UV fluorescence (Figure 3) A list of all 279 assayedloci corresponding primer pairs and optimal annealingtemperatures for each are available as Additional file 4 inthe Additional files for this study Additionally all PCRtested loci containing unidentified bases in the originalsequence data were subjected to chain-terminationsequencing to verify bp composition [92] Sequencedata generated from this project for gorilla-specificAluY subfamilies has been deposited in GenBankunder the accession numbers (KF668269-KF668278)

Additional files

Additional file 1 Enumeration of Alu elements in ape genomes TheRepeatMasker program was run on the ape genomes currently availablefor download via Genbank An in-house Perl script was then used to tallyAlu elements by total copy number and total copy number per each ofthe three major subfamilies

Additional file 2 A complete listing of all 1075 verified gorilla-specificAluY insertions

Additional file 3 Estimated age of gorilla-specific Alu subfamiliesbased on BEAST analysis The BEAST program was run on eachgorilla-specific subfamily with a baseline divergence age of 7 million yearsago to determine the age of the subfamilies the most likely progenitor orancestral element and the divergence from the consensus sequence ofthe ancestral subfamily

Additional file 4 All primer pairs used in this study listed withchromosomal location of the locus assayed and optimal annealingtemperature

AbbreviationsBAC Bacterial artificial chromosome BEAST Bayesian evolutionary analysissampling trees BLAST Basic local alignment search tool BLAT Blast-likealignment tool bp Base pair Chr Chromosome Gpb Gigabase-pairLINE Long interspersed element PCR Polymerase chain reaction SINE Shortinterspersed element TPRT Target-primed reverse transcription TSDTarget-site duplication UCSC University of California Santa Cruz

Competing interestsThe authors declare that they have no competing interests

Authorsrsquo contributionsATM and MAB designed the research and wrote the paper ATM GWC MLFTOB and WG performed the experiments ATM GWC MLF TOB and WGdesigned the PCR primers ATM CF and TJM performed the computationalanalyses All authors read and approved the final manuscript

AcknowledgementsThe authors wish to thank G Cook JA Walker S Herke and MK Konkel forall of their helpful advice during the course of this project Special thanks goto Sydney Szot (szottigerslsuedu) for the primate illustrations We thankthe American Type Culture Collection The Coriell Institute for MedicalResearch the Integrated Primate Biomaterials and Information Resource andDr Lucia Carbone (httpcarbonelabcom) for providing the DNA samplesused in this study This research was supported by National Institutes ofHealth Grant RO1 GM59290 (MAB) ATM was supported in part by aLouisiana Board of Regents Graduate Fellowship and the Louisiana StateUniversity Graduate School Dissertation Fellowship MLF was supported bythe Louisiana Biomedical Research Network with funding from the NationalCenter for Research Resources (Grant Number P20GM103424) and by theLouisiana Board of Regents Support Fund

Author details1Department of Biological Sciences Louisiana State University Baton RougeLA 70803 USA 2Department of Bioengineering Clemson UniversityClemson SC 29634 USA 3Department of Behavioral Neuroscience OregonHealth amp Science University Portland OR 97239 USA 4Department ofEnvironmental Health Sciences University of Michigan Ann Arbor MI 48109USA

Received 19 September 2013 Accepted 23 October 2013Published 22 November 2013

References1 Konkel MK Walker JA Batzer MA LINEs and SINEs of primate evolution

Evol Anthropol 2010 19236ndash2492 Batzer MA Deininger PL Alu repeats and human genomic diversity

Nat Rev Genet 2002 3370ndash3793 Cordaux R Batzer MA The impact of retrotransposons on human

genome evolution Nat Rev Genet 2009 10691ndash7034 Ullu E Tschudi C Alu sequences are processed 7SL RNA genes

Nature 1984 312171ndash1725 Roy-Engel AM Batzer MA Deininger PL Evolution of human retrosequences

Alu Encyclopedia of Life Sciences Chichester John Wiley amp Sons Ltd 20086 Cordaux R Lee J Dinoso L Batzer MA Recently integrated Alu

retrotransposons are essentially neutral residents of the human genomeGene 2006 373138ndash144

7 Deininger PL Batzer MA Hutchison CA 3rd Edgell MH Master genes inmammalian repetitive DNA amplification Trends Genet 1992 8307ndash311

8 Han K Xing J Wang H Hedges DJ Garber RK Cordaux R Batzer MA Underthe genomic radar the stealth model of Alu amplification Genome Res2005 15655ndash664

9 Dewannieux M Esnault C Heidmann T LINE-mediated retrotranspositionof marked Alu sequences Nat Genet 2003 3541ndash48

10 Luan DD Korman MH Jakubczak JL Eickbush TH Reverse transcription ofR2Bm RNA is primed by a nick at the chromosomal target site amechanism for non-LTR retrotransposition Cell 1993 72595ndash605

11 Luan DD Eickbush TH RNA template requirements for targetDNA-primed reverse transcription by the R2 retrotransposable elementMol Cell Biol 1995 153882ndash3891

12 Cost GJ Feng Q Jacquier A Boeke JD Human L1 element target-primedreverse transcription in vitro EMBO J 2002 215899ndash5910

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14 Murata S Takasaki N Saitoh M Okada N Determination of thephylogenetic relationships among pacific salmonids by using shortinterspersed elements (SINEs) as temporal landmarks of evolutionProc Natl Acad Sci U S A 1993 906995ndash6999

15 Shedlock AM Okada N Sine insertions powerful tools for molecularsystematics Bioessays 2000 22148ndash160

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17 Minghetti PP Dugaiczyk A The emergence of new DNA repeats and thedivergence of primates Proc Natl Acad Sci U S A 1993 901872ndash1876

18 Ray DA Xing J Salem AH Batzer MA SINEs of a nearly perfect characterSyst Biol 2006 55928ndash935

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20 Schmitz J Ohme M Zischler H SINE insertions in cladistic analyses andthe phylogenetic affiliations of tarsius bancanus to other primatesGenetics 2001 157777ndash784

21 Ray DA Xing J Hedges DJ Hall MA Laborde ME Anders BA White BRStoilova N Fowlkes JD Landry KE Chemnick LG Ryder OA Batzer MAAlu insertion loci and platyrrhine primate phylogeny Mol Phylogenet Evol2005 35117ndash126

22 Xing J Wang H Han K Ray DA Huang CH Chemnick LG Stewart CBDisotell TR Ryder OA Batzer MA A mobile element based phylogeny ofOld World monkeys Mol Phylogenet Evol 2005 37872ndash880

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24 Li J Han K Xing J Kim HS Rogers J Ryder OA Disotell T Yue B Batzer MAPhylogeny of the macaques (cercopithecidae macaca) based on Aluelements Gene 2009 448242ndash249

25 Meyer TJ McLain AT Oldenburg JM Faulk C Bourgeois MG Conlin EMMootnick AR De Jong PJ Roos C Carbone L Batzer MA An Alu-basedphylogeny of gibbons (hylobatidae) Mol Biol Evol 2012 293441ndash3450

26 McLain AT Meyer TJ Faulk C Herke SW Oldenburg JM Bourgeois MGAbshire CF Roos C Batzer MA An alu-based phylogeny of lemurs(infraorder lemuriformes) PLoS One 2012 7e44035

27 Roos C Schmitz J Zischler H Primate jumping genes elucidatestrepsirrhine phylogeny Proc Natl Acad Sci U S A 2004 10110650ndash10654

28 Salem AH Ray DA Xing J Callinan PA Myers JS Hedges DJ Garber RKWitherspoon DJ Jorde LB Batzer MA Alu elements and hominidphylogenetics Proc Natl Acad Sci U S A 2003 10012787ndash12791

29 Batzer MA Stoneking M Alegria-Hartman M Bazan H Kass DH Shaikh THNovick GE Ioannou PA Scheer WD Herrera RJ African origin ofhuman-specific polymorphic Alu insertions Proc Natl Acad Sci U S A 19949112288ndash12292

30 Perna NT Batzer MA Deininger PL Stoneking M Alu insertionpolymorphism a new type of marker for human population studiesHum Biol 1992 64641ndash648

31 Deininger PL Batzer MA Alu repeats and human disease Mol Genet Metab1999 67183ndash193

32 Hancks DC Kazazian HH Jr Active human retrotransposons variation anddisease Curr Opin Genet Dev 2012 22191ndash203

33 Cook GW Konkel MK Major JD 3rd Walker JA Han K Batzer MA Alu pairexclusions in the human genome Mob DNA 2011 210

34 Hedges DJ Deininger PL Inviting instability transposable elementsdouble-strand breaks and the maintenance of genome integrityMutat Res 2007 61646ndash59

35 Konkel MK Batzer MA A mobile threat to genome stability the impact ofnon-LTR retrotransposons upon the human genome Semin Cancer Biol2010 20211ndash221

36 Cook GW Konkel MK Walker JA Bourgeois MG Fullerton ML Fussell JTHerbold HD Batzer MA A comparison of 100 human genes using an aluelement-based instability model PLoS One 2013 8e65188

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38 Slagel V Flemington E Traina-Dorge V Bradshaw H Deininger P Clusteringand subfamily relationships of the Alu family in the human genomeMol Biol Evol 1987 419ndash29

39 Willard C Nguyen HT Schmid CW Existence of at least three distinct Alusubfamilies J Mol Evol 1987 26180ndash186

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41 Batzer MA Deininger PL Hellmann-Blumberg U Jurka J Labuda DRubin CM Schmid CW Zietkiewicz E Zuckerkandl E Standardizednomenclature for Alu repeats J Mol Evol 1996 423ndash6

42 Cordaux R Hedges DJ Batzer MA Retrotransposition of Alu elementshow many sources Trends Genet 2004 20464ndash467

43 Price AL Eskin E Pevzner PA Whole-genome analysis of Alu repeatelements reveals complex evolutionary history Genome Res 2004142245ndash2252

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45 Kapitonov V Jurka J The age of Alu subfamilies J Mol Evol 1996 4259ndash6546 Cordaux R Hedges DJ Herke SW Batzer MA Estimating the

retrotransposition rate of human Alu elements Gene 2006 373134ndash13747 Xing J Zhang Y Han K Salem AH Sen SK Huff CD Zhou Q Kirkness EF

Levy S Batzer MA Jorde LB Mobile elements create structural variationanalysis of a complete human genome Genome Res 2009 191516ndash1526

48 Locke DP Hillier LW Warren WC Worley KC Nazareth LV Muzny DM YangSP Wang Z Chinwalla AT Minx P Mitreva M Cook L Delahaunty KDFronick C Schmidt H Fulton LA Fulton RS Nelson JO Magrini V Pohl CGraves TA Markovic C Cree A Dinh HH Hume J Kovar CL Fowler GRLunter G Meader S Heger A et al Comparative and demographicanalysis of orang-utan genomes Nature 2011 469529ndash533

49 Walker JA Konkel MK Ullmer B Monceaux CP Ryder OA Hubley R Smit AFBatzer MA Orangutan Alu quiescence reveals possible source elementsupport for ancient backseat drivers Mob DNA 2012 38

50 Strier KB Primate behavioral ecology 3rd edition Boston MA Allyn andBacon 2007

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56 Langergraber KE Prufer K Rowney C Boesch C Crockford C Fawcett KInoue E Inoue-Muruyama M Mitani JC Muller MN Robbins MM SchubertG Stoinski TS Viola B Watts D Wittig RM Wrangham RW Zuberbuhler KPaabo S Vigilant L Generation times in wild chimpanzees and gorillassuggest earlier divergence times in great ape and human evolutionProc Natl Acad Sci U S A 2012 10915716ndash15721

57 Scally A Dutheil JY Hillier LW Jordan GE Goodhead I Herrero J Hobolth ALappalainen T Mailund T Marques-Bonet T McCarthy S Montgomery SHSchwalie PC Tang YA Ward MC Xue Y Yngvadottir B Alkan C Andersen LNAyub Q Ball EV Beal K Bradley BJ Chen Y Clee CM Fitzgerald S Graves TAGu Y Heath P Heger A et al Insights into hominid evolution from the gorillagenome sequence Nature 2012 483169ndash175

58 Ventura M Catacchio CR Alkan C Marques-Bonet T Sajjadian S Graves TAHormozdiari F Navarro A Malig M Baker C Lee C Turner EH Chen LKidd JM Archidiacono N Shendure J Wilson RK Eichler EE Gorilla genomestructural variation reveals evolutionary parallelisms with chimpanzeeGenome Res 2011 211640ndash1649

59 Lee J Han K Meyer TJ Kim HS Batzer MA Chromosomal inversionsbetween human and chimpanzee lineages caused by retrotransposonsPLoS One 2008 3e4047

60 Sen SK Han K Wang J Lee J Wang H Callinan PA Dyer M Cordaux RLiang P Batzer MA Human genomic deletions mediated byrecombination between Alu elements Am J Hum Genet 2006 7941ndash53

61 Hormozdiari F Konkel MK Prado-Martinez J Chiatante G Herraez IH Walker JANelson B Alkan C Sudmant PH Huddleston J Catacchio CR Ko A Maliq MBaker C Great Ape Genome Project Marques-Bonet T Ventura M Batzer MAEichler EE Rates and patterns of great ape retrotransposition Proc Natl AcadSci U S A 2013 11013457ndash13462

62 RepeatMasker open-30 [httpwwwrepeatmaskerorg]63 Giardine B Riemer C Hardison RC Burhans R Elnitski L Shah P Zhang Y

Blankenberg D Albert I Taylor J Miller W Kent WJ Nekrutenko A Galaxya platform for interactive large-scale genome analysis Genome Res 2005151451ndash1455

64 Kent WJ BLATndashthe BLAST-like alignment tool Genome Res 200212656ndash664

65 Jurka J Kapitonov VV Pavlicek A Klonowski P Kohany O Walichiewicz JRepbase update a database of eukaryotic repetitive elementsCytogenet Genome Res 2005 110462ndash467

66 Drummond AJ Rambaut A BEAST bayesian evolutionary analysis bysampling trees BMC Evol Biol 2007 7214

67 Carter AB Salem AH Hedges DJ Keegan CN Kimball B Walker JA Watkins WSJorde LB Batzer MA Genome-wide analysis of the human Alu Yb-lineageHuman Genomics 2004 1167ndash178

68 Roy-Engel AM Salem AH Oyeniran OO Deininger L Hedges DJ Kilroy GEBatzer MA Deininger PL Active Alu element ldquoA-tailsrdquo size does matterGenome Res 2002 121333ndash1344

69 Hedges DJ Callinan PA Cordaux R Xing J Barnes E Batzer MA Differentialalu mobilization and polymorphism among the human and chimpanzeelineages Genome Res 2004 141068ndash1075

70 Hedges DJ Batzer MA From the margins of the genome mobileelements shape primate evolution Bioessays 2005 27785ndash794

71 Belancio VP Hedges DJ Deininger P Mammalian non-LTR retrotransposonsfor better or worse in sickness and in health Genome Res 2008 18343ndash358

72 Schumann GG APOBEC3 proteins major players in intracellular defenceagainst LINE-1-mediated retrotransposition Biochem Soc Trans 200735637ndash642

73 Hulme AE Bogerd HP Cullen BR Moran JV Selective inhibition of Aluretrotransposition by APOBEC3G Gene 2007 390199ndash205

74 Bogerd HP Wiegand HL Hulme AE Garcia-Perez JL OrsquoShea KS Moran JVCullen BR Cellular inhibitors of long interspersed element 1 and Aluretrotransposition Proc Natl Acad Sci U S A 2006 1038780ndash8785

75 Farkash EA Kao GD Horman SR Prak ET Gamma radiation increasesendonuclease-dependent L1 retrotransposition in a cultured cell assayNucleic Acids Res 2006 341196ndash1204

76 Edgar RC MUSCLE multiple sequence alignment with high accuracy andhigh throughput Nucleic Acids Res 2004 321792ndash1797

77 Bandelt HJ Forster P Rohl A Median-joining networks for inferringintraspecific phylogenies Mol Biol Evol 1999 1637ndash48

78 Wellcome trust-sanger institute gorilla genome homepage[httpwwwsangeracukresourcesdownloadsgorilla]

79 Blankenberg D Von Kuster G Coraor N Ananda G Lazarus R Mangan MNekrutenko A Taylor J Galaxy a web-based genome analysis tool forexperimentalists Curr Protoc Mol Biol 2010 Chapter 1911ndash21

80 Goecks J Nekrutenko A Taylor J Galaxy a comprehensive approach forsupporting accessible reproducible and transparent computationalresearch in the life sciences Genome Biol 2010 11R86

81 Galaxy [httpgalaxyprojectorg]82 UCSC genome browser [httpgenomeucscedu]83 Kent WJ Sugnet CW Furey TS Roskin KM Pringle TH Zahler AM Haussler D

The human genome browser at UCSC Genome Res 2002 12996ndash100684 COSEG [httpwwwrepeatmaskerorgCOSEGDownloadhtml]85 Hall TA BioEdit a user-friendly biological sequence alignment editor and

analysis program for windows 9598NT Nucleic Acids Symp Ser 19994195ndash98

86 Untergasser A Nijveen H Rao X Bisseling T Geurts R Leunissen JAPrimer3Plus an enhanced web interface to Primer3 Nucleic Acids Res2007 35W71ndash74

87 BEAST [httpbeastbioedacuk]88 Hellen EH Brookfield JF The diversity of class II transposable elements in

mammalian genomes has arisen from ancestral phylogenetic splitsduring ancient waves of proliferation through the genome Mol Biol Evol2013 30100ndash108

89 Gouy M Guindon S Gascuel O SeaView version 4 a multiplatformgraphical user interface for sequence alignment and phylogenetic treebuilding Mol Biol Evol 2010 27221ndash224

90 NETWORK [httpwwwfluxus-engineeringcomsharenethtm]91 Librado P Rozas J DnaSP v5 a software for comprehensive analysis of

DNA polymorphism data Bioinformatics 2009 251451ndash145292 Sanger F Air GM Barrell BG Brown NL Coulson AR Fiddes CA Hutchison CA

Slocombe PM Smith M Nucleotide sequence of bacteriophage phi X174DNA Nature 1977 265687ndash695

doi1011861759-8753-4-26Cite this article as McLain et al Analysis of western lowland gorilla(Gorilla gorilla gorilla) specific Alu repeats Mobile DNA 2013 426

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Abstract

Background

Results

Conclusions

Background

Results and discussion

Computational examination of the western lowland gorilla genome

AluY subfamily activity in the western lowland gorilla genome

Divergence dates of gorilla-specific AluY subfamilies

Conclusions

Methods

Computational methodology

PCR and DNA sequencing

Additional files

Abbreviations

Competing interests

Authorsrsquo contributions

Acknowledgements

Author details

References