The Full Globin Repertoire of Turtles Provides Insights into Vertebrate Globin Evolution and ...

18 Pages·2015·1.66 MB·English

GBE
The Full Globin Repertoire of Turtles Provides Insights into
Vertebrate Globin Evolution and Functions
Kim Schwarze, Abhilasha Singh and Thorsten Burmester*
InstituteofZoology,DepartmentofBiology,UniversityofHamburg,Germany
*Correspondingauthor:E-mail:[email protected].
Accepted:June 9, 2015
D
o
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n
Abstract lo
a
d
e
Globinsaresmallhemeproteinsthatplayanimportantroleinoxygensupply,butmayalsohaveotherfunctions.Globinsoffera d
uniqueopportunitytostudythefunctionalevolutionofgenesandproteins.Wehavecharacterizedtheglobinrepertoireoftwo fro
m
differentturtlespecies:theChinesesoftshellturtle(Pelodiscussinensis)andthewesternpaintedturtle(Chrysemyspictabellii).Inthe h
genomesofbothspecies,wehaveidentifiedeightdistinctglobintypes:hemoglobin(Hb),myoglobin,neuroglobin,cytoglobin,globin ttp
s
E,globinX,globinY,andandroglobin.Therefore,alongwiththecoelacanth,turtlesaresofartheonlyknownvertebrateswithafull ://a
c
globinrepertoire.Thisfactallowsforthefirsttimeacomparativeanalysisoftheexpressionofalleightglobinsinasinglespecies. ad
e
PhylogeneticanalysisshowedanearlydivergenceofneuroglobinandglobinXbeforetheradiationofvertebrates.Amongtheother m
ic
globins,cytoglobindivergedfirst,andthereisacloserelationshipbetweenmyoglobinandglobinE;thepositionofglobinYisnot .o
u
resolved.TheglobinEgenewasselectivelylostinthegreenanole,andthegenescodingforglobinXandglobinYweredeletedin p
.c
chicken.Quantitativereal-timereversetranscriptionpolymerasechainreactionexperimentsrevealedthatmyoglobin,neuroglobin, om
andglobinEarehighlyexpressedwithtissue-specificpatterns,whichareinlinewiththeirrolesintheoxidativemetabolismofthe /g
b
e
striated muscles, the brain, and the retina, respectively. Histochemical analyses showed high levels of globin E in the pigment /a
epitheliumoftheeye.GlobinEprobablyhasamyoglobin-likeroleintransportingO2acrossthepigmentepitheliumtosupplyin rticle
themetabolicallyhighlyactiveretina. -a
b
s
Key words: gene duplication, globins, neuroglobin, oxygen, retina. tra
c
t/7
/7
/1
Introduction 8
the best known and thoroughly investigated globins. Since 9
6
Globinsaresmallhemeproteinsthatarewellknownfortheir 2000,however,sixadditionalglobintypeshavebeendiscov- /63
1
abilitytobindmolecularoxygen(O ),butalsoothergaseous ered,whichmostlyhavepoorlydefinedfunctions(Burmester 2
2 7
ligands. Globins are widespread in the animal kingdom. As andHankeln2014).Threeofthese“novel”globinsarewide- 4 b
y
hemoglobins (Hbs) and myoglobins (Mbs), these proteins spread among the jawed vertebrates (Gnathostomata): 1) g
u
serveforthetransportandstorageofO forrespiratorypur- Neuroglobin (Ngb), which is nerve specific (Burmester et al. e
2 s
poses.However,globinsalsohaveotherfunctions,andmay 2000); 2) cytoglobin (Cygb), which is mainly expressed in t o
n
be involved in the production and decomposition of nitric fibroblast but also some nerve cells (Kawada et al. 2001; 0
5
oxide (NO), the detoxification of reactive oxygen species Burmester et al. 2002; Trent and Hargrove 2002); and 3) M
a
(ROS),orintracellularsignaling(BurmesterandHankeln2014). androglobin (Adgb), which is predominantly expressed in y 2
0
Globinsareaclassicalmodelsystemtostudytheevolution the testes (Hoogewijs et al. 2012). Three other globins have 1
9
ofgenesandproteins(Goodmanetal.1987,1989;Hardison a more restricted taxonomic distribution and phylogenetic
1996, 1998; Wajcman et al. 2009; Burmester and Hankeln analyses suggest that the genes have been secondarily lost
2014). Vertebrates possess eight distinct globin types that in some vertebrate taxa (Hoffmann et al. 2011; Schwarze
differ in structure, evolutionary history, and probably also and Burmester 2013; Schwarze et al. 2014): 1) Globin X
function (Burmester and Hankeln 2014). Hb, which serves (GbX), which is a membrane-bound globin in the nervous
forthetransportofO intheredbloodcells(Dickersonand system,hasnotbeenfoundinbirdsandmammals(Roesner
2
Geis 1983), and Mb, which facilitates O supply within the etal. 2005; Blank, Wollberg, etal. 2011);2)globinY(GbY)
2
striatedmuscles(WittenbergBAandWittenbergJB1989),are has yet poorly defined expression patterns and taxonomic
(cid:2)TheAuthor(s)2015.PublishedbyOxfordUniversityPressonbehalfoftheSocietyforMolecularBiologyandEvolution.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestrictedreuse,
distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
1896 GenomeBiol.Evol.7(7):1896–1913. doi:10.1093/gbe/evv114 AdvanceAccesspublicationJune15,2015
GBE
TurtleGlobinRepertoire
distribution(Fuchsetal.2006);and3)globinE(GbE)isaneye- (ATP) production (Lutz and Nilsson 1997; Staples and Buck
specific globin found in birds, some reptiles, and the coela- 2009). In anoxia-tolerant turtles, the neuronal activity is
canth (Kugelstadt et al. 2004; Hoffmann et al. 2011; reducedbychannelandspikearrestduringoxygen-deficient
SchwarzeandBurmester2013). periods (Hochachka 1986; Hylland et al. 1997).
ThefunctionsoftheglobinsotherthanHbandMbarestill Nevertheless, the central nervous system and particularly
poorly understood (Burmester and Hankeln 2014). The high the visual system (Wong-Riley 2010) still have a relatively
expression levels of GbE in the retina of chicken and similar high ATP turnover.
O2-binding kinetics suggest that this protein has an Mb-like A main advantage ofthe turtles isthat they are—in con-
function(Blank,Kiger,etal.2011).Severallinesofevidence trast to the coelacanth—available for experimental studies.
suggestthatNgbisinvolvedintheoxidativemetabolismofthe Knowledge of the tissue-specific distribution of the various
neurons(Hankelnetal.2005;BurmesterandHankeln2009). globinsmayfirstprovideimportant cluesaboutglobin func- Do
w
On the one hand, Ngb may enhance local O2 supply to the tions,andsecondhelptounderstandbetterhowthedifferent nlo
mitochondria,ontheotherhand,Ngbmayprotecttheneu- globins contribute to the hypoxia adaptation of turtles. ad
rtoonsspefrcoifimcoexnizdyamtiveesa(Sncdhmotihdetresttarel.ss2e0s0.4C)y,gmbamybayepinrvoovlivdeedOin2 TthheerCefhoinree,sehesroeftswheellotbutratilneeadndthethefuwllegsltoebrinnpraeipnetretdoirteusrtloef. ed from
theNO metabolism (Avivi et al. 2010; Hundahl et al. 2013), We obtained the tissue-specific expression patterns of each h
mayprotectthecellsfromROS(Fangetal.2011;Singhetal. ttp
globininbothspecies;wespecificallyfocusedonthelocaliza- s
2014), or may function in signaling pathways (Reeder et al. tion of GbE, for which no data outside the chicken existed ://a
c
2011).GbXharborsN-terminalacylationsites(myristoylation a
(Blank,Kiger,etal.2011). d
e
atGly2andpalmitoylationatCys3),bywhichitisanchoredin m
the membrane (Blank, Wollberg, et al. 2011). GbX may be ic.o
u
involved in the protection of the membrane lipids or an p
Materials and Methods .c
unknown signaling process (Burmester and Hankeln 2014). o
m
AstheAdgbisspecificallyexpressedinthetestis,afunction SequenceandSyntenyAnalyses /g
b
inspermatogenesismaybeassumed(Hoogewijsetal.2012). The globin genes of the Chinese softshell turtle P. sinensis e/a
FitosrfuGnbcYti,otnh.ereisstilltoolittleinformationtospeculateabout wetearel. 2id0e1n3ti)fiaevdaiilnabtleheatgEeNnoSmEMeBaLss(hemttpb:l/y/wPwelwSi.ne_n1s.e0m(bWl.oarngg, rticle-a
b
Thefirstgnathostomevertebratehadeightdifferenttypes s
of globins with distinct functions (Burmester and Hankeln last accessed June 3, 2015) employing the BLAST algorithm trac
2014). The loss of specific globin genes in some vertebrate (Altschul et al. 1990). The globin repertoire of the western t/7
painted turtle C. picta bellii along with additional positional /7
lineagesmaybeexplainedbythefactthatitsspecificfunction /1
had become unnecessary or that another (globin) gene had information were obtained from the genome assembly 896
ChrPicBel3.0.1 (Shaffer et al. 2013), as available at /6
takenoveritsrole.Mostvertebratestodaydonotharborthe 3
Pre-ENSEMBL (http://pre.ensembl.org, last accessed June 3, 1
full globin repertoire (Burmester and Hankeln 2014), 27
2015). Because of the lack of annotation in Pre-ENSEMBL 4
hampering a comparative analysis of globin patterns in a b
single species. The only vertebrate so far with a full set of database, the GenBank identifiers (http://www.ncbi.nlm.nih. y g
globin genes was the coelacanth Latimeria chalumnae gov, last accessed June 3, 2015) were used for the painted ues
(Schwarze and Burmester 2013), which was therefore re- turtle. In addition to the gene models taken from ENSEMBL t on
ferredtoasa“globinfossil.”Becauseofthelackofmaterial, and GenBank, gene predictions were carried out manually 05
andwithGenScan(http://genes.mit.edu/GENSCAN.html,last M
functional studies were not possible with the coelacanth.
a
However, analyses of the genomes of the Chinese softshell accessedJune3,2015)andAugustus(http://bioinf.uni-greifs- y 2
turtle(Pelodiscussinensis)(Wangetal.2013)andthewestern wald.de/augustus/, last accessed June 3, 2015). Protein se- 01
9
painted turtle (Chrysemys picta bellii) (Shaffer et al. 2013) quences were predicted by translation with the web-based
identified copies of all eight vertebrate globin genes in both toolprovidedattheExPASyMolecularBiologyServer(http://
species. www.expasy.org, last accessed June 3, 2015). Partial globin
Turtles are one of the oldest reptile groups; some turtles sequences were completed by RACE (rapid amplification of
show specific adaptations that allow them to survive ex- cDNA ends) (see below). For gene synteny analysis, the se-
treme conditions, for example, hypoxia or even anoxia quences and gene order were obtained from the genome
(Krivoruchko and Storey 2010; Shaffer et al. 2013). During assemblies of the chicken Gallus gallus (build 2.1,
hibernation, many freshwater turtles survive at the bottom Annotationrelease102),fromthegreenanoleAnoliscaroli-
offrozenlakesunabletobreathe,orinthemudundertotal nensis (AnoCar2.0, Annotation release 101), and from the
anoxic conditions (Ultsch 2006). This tolerance is mainly coelacanth L. chalumnae (LatCha1.0) available at the
based on the ability to reduce their energy consumption NCBI website (http://www.ncbi.nlm.nih.gov/mapview/, last
along with increasing anaerobic adenosine triphosphate accessedJune3,2015).
GenomeBiol.Evol.7(7):1896–1913. doi:10.1093/gbe/evv114 AdvanceAccesspublicationJune15,2015 1897
GBE
Schwarzeetal.
MultipleSequenceAlignmentandPhylogenetic constructed with 400–500bp fragments of the respective
Reconstructions globin. Fragments of 100bp fragments of each globin were
amplifiedbyqRT-PCR(seebelow).Forthesoftshellturtle,we
Todeterminetheorthologyoftheturtleglobingenesidenti-
usedtheqRT-PCRprimersalsotoconstructstandardplasmids.
fiedinthisstudy,theywereaddedtoarecentlypublisheddata
The PCR products were cloned into the pGEM-T/JM109
setofvertebrateglobingenes(Schwarzeetal.2014).Multiple
system (Promega, Mannheim, Germany) and sequenced by
sequence alignments of the protein sequences were carried
a commercial service (GATC, Konstanz, Germany). Missing
out with different algorithms and ranked with MUMSA
30 and 50 ends of cDNAs were obtained by RACE using the
(Lassmann and Sonnhammer 2005). We used MAFFT with
GeneRacer Kit (Invitrogen, Carlsbad, CA) according to
the FFT-NS-i, L-INS-i, and G-INS-i models (Katoh and Toh
themanufacturer’sinstructions.
2008; Katoh et al. 2009), MUSCLE (Edgar 2004),
D
PROMALS3D (Pei et al. 2008), and T-coffee (Notredame o
w
et al. 2000). The MAFFT L-INS-i algorithm received the best QuantitativeReal-TimeReverseTranscriptionPolymerase nlo
ChainReaction a
MUMSAscoreandwasusedforphylogeneticreconstructions. d
e
aTnhedmGoassctuaeplp2ro0p0r8ia)tweamsosdeelelcotfedambiynoPraoctiTdesetvo(Alubtaiosnca(lLGet;aLel. TtihmeateexdprbeyssqioRnT-oPfCgRl.oWbinemdeetsesremnigneerdRtNhAesgl(ombRinNAmsR)NwAerleeveesls- d from
2005) applying the Akaike Information Criterion. from brain, eye, muscle, heart, kidney, liver, intestine, and http
IwmitphlemMernBtaaytieosn 3o.2f.3ph(yHlougeelsneentbicecaknaalynsdis Rwoansqupisetrfo2r0m0e1d; lpuaningt,eedacthurftrleosm.BthloeoCdhsiunbessaemsopfltesshwelletruerutleseadnfdrothmretewwoewsteesrtn- s://ac
a
Ayresetal.2012)withtheLGmodelofaminoacidsubstitu- ernpaintedturtles.TheAdgbmRNAexpressionlevelwasob- de
m
tion (Le and Gascuel 2008). Two independent runs with tained from two western painted turtles. qRT-PCR ic
four simultaneous chains and 5,000,000 generations were amplification(40cycles:95(cid:3)Cfor15s,60(cid:3)Cfor15s,72(cid:3)C .ou
p
performed.Thetreesweresampledevery1,000thgeneration. for30s,detectionatlaststep)wascarriedoutonanABI7500 .c
o
The final average standard deviation of split frequencies real-timePCRsystemusingtheABIPowerSYBRGreenmaster m
/g
was <0.01. Convergence was further analyzed by estimat- mix (Applied Biosystems, Darmstadt, Germany). Experiments b
e
i1n.g00.tThhee ppoostteenrtioiarlprsocbalaebilirteiedsuwcteiorenesftaimctaotre,dwonhicthhe fiwnaasl cwDeNreApaemrfoourmnteedqausivtarilpelnictaotfes37in.5amvgoltuomtaelRoNfA20amnldw2i0th0anMfinoafl /article
3,000trees. each intron-spanning oligonucleotide (supplementary table -ab
s
S1,SupplementaryMaterialonline).Negativecontrolswithout tra
c
RNAExtractionandcDNACloning cDNAwereincluded.Successandspecificityofamplification t/7
were evaluated using dissociation curve analyzes. We calcu- /7
Theturtlesusedinthisstudywereobtainedfromapetshop. /1
latedthemRNAcopynumberwithastandardcurvemethod, 8
OneChinesesoftshellturtleandthreewesternpaintedturtles, 96
each2yearsold,wereusedinthisstudy.Allanimalhandling inwhichduplicatesofrecombinantplasmidswithknowncopy /63
numberrepresentingeachglobincDNAwererunusingserial 1
weredoneincompliancewiththeguidelinesoftheGerman 2
dilutions 10-fold (107–102). The samples were normalized 74
AnimalWelfareAct.Theanimalsweresacrificed,tissueswere b
accordingto1mgoftotalRNA. y
collectedandstoredinRNAlater(Qiagen,Hilden,Germany)at g
u
(cid:2)20(cid:3)C. Total RNA from each tissue sample (brain, eye, es
muscle, heart, kidney, liver, intestine, lung, and blood) was SDS-PAGEandWesternBlotting t o
n
extracted using peqGOLD Trifast (PEQLAB, Erlangen, Frozen eye samples of the Chinese softshell turtle and the 05
Germany) and Crystal RNA Mini Kit (Biolab Products, chicken, respectively, were each homogenized in 10mM M
a
y
Go¨denstorf, Germany) according to manufacturer’s instruc- Tris–HCl, pH 7.4, 10mM NaCl, 5mM MgCl2, 1mM dithio- 20
tions. Samples were treated with on-column RNase-free threitol(DTT),1mMPefablocSCproteaseinhibitor(CarlRoth) 1
9
DNase (Qiagen) and the integrity of the RNA was assessed andCompleteproteaseinhibitormix(RocheAppliedScience)
by denaturating gel electrophoreses. Reverse transcription using the Sonopuls HD2070 Ultrasonic Homogenizer
(RT) of 750ng total RNA was performed with the RevertAid (Bandelin, Berlin, Germany). After 10min centrifugation at
H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, 10,000(cid:4)gat4(cid:3)C,theproteinconcentrationsofthesuper-
Bonn, Germany) with oligo-(dT) -primer according to natants were determined using the fluorometric quantifica-
18
manufacturer’s instructions. Gene-specific oligonucleotides tion with the Qubit 2.0 (Life Technologies, Darmstadt,
(supplementary table S1, Supplementary Material online) Germany).Atotalof50mgproteinand35mgrecombinantly
were used for amplification of selected turtle globin cDNAs. expressed chicken GbE protein were denatured in 65mM
Forthepaintedturtleglobins,differentoligonucleotideswere Tris–HCl, pH 6.8, 1% sodium dodecyl sulfate (SDS), 5% b-
usedtoconstructeitherthestandardplasmidsorforamplifi- mercaptoethanol,10%glycerolat95(cid:3)Cfor5min,andsepa-
cationinquantitativereal-timeRTpolymerasechainreaction rated on a 15% SDS-polyacrylamide gel electrophoresis
(qRT-PCR) (see below). The standard plamids were (PAGE). Proteins in the polyacrylamide gel were visualized
1898 GenomeBiol.Evol.7(7):1896–1913. doi:10.1093/gbe/evv114 AdvanceAccesspublicationJune15,2015
GBE
TurtleGlobinRepertoire
withCoomassieBrilliantBlue.TheGbEbandintheturtleeye ribonuclease A (0.18 Kunitz unit/ml, Roth, Karlsruhe,
proteinlanewascutoutfromtheSDS-Gel.Proteinsequenc- Germany) in 10mM Tris pH 8.0, 0.5M NaCl, 0.5mM EDTA
ing was carried out by static electrospray ionization tandem buffer(30minat37(cid:3)C),rinsed5minin2(cid:4)SSC(+1mMDTT)
massspectrometryattheDepartmentofClinicalChemistryof atroomtemperaturetwice,10minin1(cid:4)SSC(+1mMDTT)
the University of Medical Center Hamburg Eppendorf, atRT,10minin0.5(cid:4)SSC(+1mMDTT)atRT,and30minin
Germany. 0.1(cid:4)SSC(+1mMDTT)at60(cid:3)C.
Proteins were transferred onto a 0.22mm nitrocellulose Slideswereequilibrated5mininPBS/0.1%Tween-20and
membrane. Nonspecific binding sites were blocked for 1h buffer B (100mM Tris–HCl, 150mM NaCl, pH 7.5, 0.5%
with2.5% nonfat dry milk inTBS(10mM Tris–HCl, pH7.4, blocking reagent; Roche Diagnostics) before incubating
140mMNaCl)anddetectionwasperformedfor2hatroom with alkaline phosphatase-coupled antidigoxigenin antibody
temperaturewithaffinity-purifiedpolyclonalanti-GbEantibo- (Roche Diagnostics) diluted 1:5,000 in buffer B for 2h at Do
w
dies(Blank,Kiger,etal.2011),diluted1:1,000in2.5%milk/ 37(cid:3)C. Two 15min washes in 100mM Tris–HCl, 150mM n
lo
TBS. Membranes were washed four times in TBS for 5min NaCl, pH 7.5, followed by 15min incubation in 100mM a
d
e
and incubated 1h with the goat antirabbit antibody Tris–HCl, 100mM NaCl, 50mM MgCl , pH 9.5 removed d
coupled with alkaline phosphatase (1:20,000 in TBS; unbound antibodies. The visualization 2of the probes was fro
m
Jackson Immunoresearch Laboratories, West Grove, PA, performed with the NBT/BCIP substrate system for about h
111-055-003).Thevisualizationoftheproteinbandswasper- 16h. The reaction was stopped by washingin 100mM Tris, ttp
s
formedwiththeNBT/BCIPsubstratesystem. 1mMEDTA,pH7.4for15min.Afterthreewashesfor10min ://a
c
in PBS, the nuclei were stained with Hoechst dye 33258 ad
e
(0.3mg/ml; Calbiochem, Darmstadt, Germany) for 15min at m
InSituHybridization ic
RT. Slides were rinsed 30s in 95% ethanol; air dried and .o
Afresheyewasobtainedfromanadultfemalechickenand u
embeddedin1(cid:4)PBS/Glycerin(1:9),coveredwithacoverslip p
stored at (cid:2)80(cid:3)C before cryosection. The pGEMT-plasmids and fixed by nail polish. Sections were analyzed with an .com
containing GbE and NgbcDNA, respectively, of the Chinese Olympus BX51 research microscope, and the images were /g
b
softshellturtlewereconstructedasdescribedabove.Theplas- e
combinedusingAdobePhotoshopCS512.0.4. /a
mids with chickenNgband GbE cDNAwere obtained in an rtic
earlier study (Blank, Kiger, et al. 2011). Digoxigenin-labeled le
Immunofluorescence -a
antisenseandsensemRNAprobesweregeneratedusingthe b
s
DIG RNA Labeling Kit (Roche Diagnostics, Mannheim, Thecut,fixed,andbleachedeyeslicesfromtheadultchicken tra
c
Germany)withlinearizedplasmidsastemplatesaccordingto (describedabove)wererehydratedin1(cid:4)TBS,andnonspecific t/7
/7
themanufacturer’sinstructions. bindingsiteswereblockedfor30minwith1%bovineserum/ /1
8
Frozen eye samples were cut at 16mm thickness using a 0.1% Triton X-100/TBS. In immunohistochemistry (IHC), 9
6
CryostatCM1950(Leica,Wetzlar,Germany)andmountedon sections were incubated overnight at 4(cid:3)C in freshly affinity- /63
1
poly-L-lysineslides(FisherScientific,Schwerte,Germany).The purified polyclonal anti-GbE antibodies (Blank, Kiger, et al. 27
4
sectionswerefixedonicefor20minin4%paraformaldehyde 2011), diluted 1:2,500 in 1% bovine serum/0.1% Triton b
y
in phosphate buffered saline (PBS) (140mM NaCl, 2.7mM X-100/TBS. For immunofluorescence (IF), the commercial g
u
KCl, 8.1mM Na HPO , 1.5mM KH PO , pH 6.9) and antibodies citrate synthetase (Abcam, Cambridge, UK) e
2 4 2 4 s
bleached for 30min in 3% H O /1% KOH. Sections were and ATP synthase beta (Thermo Scientific) were diluted t o
2 2 n
neutralized with 1% acetic acid, rinsed twice in 1(cid:4)PBS 1:1,000and1:200,respectively,andincubatedovernightat 0
5
5min each, and then acetylated for 10min in 0.5% acetic 4(cid:3)C. Slides were washed three times in TBS for 10min. M
a
y
anhydride in 0.1M triethanolamine. After washing in PBS IncubationwiththesecondaryantibodyinIHCwasperformed 2
0
twice for 5min, slides were dehydrated in a graded ethanol with a goat antirabbit antibody coupled with alkaline 1
9
series(70%,90%,95%,and100%,3mineach)andairdried phosphatase (1:20,000 in TBS; Jackson Immunoresearch
atroomtemperature. Laboratories)for2hatroomtemperature.ForIFexperiments,
Hybridizationwasperformedoncoverslippedslidessealed thesecondaryantibodyusedwasadonkeyantirabbitF(ab)
2
by DPX new (Merck Chemicals, Darmstadt, Germany) with fragment coupled to Cy3 (1:2,000 in TBS; Jackson
probe mix (400ng/ml probe, 2.5mg/ml tRNA, 50mM DTT) Immunoresearch Laboratories). In IHC, the visualization
mixed1:5inhybridizationbuffer(50%deionizedformamide, of the protein was performed with the NBT/BCIP substrate
10%dextransulfate,1(cid:4)Denhardt’ssolution,300mMNaCl, system with 1ml/ml levamisole (Sigma-Aldrich, Munich,
10mM Tris–HCl pH 8.0, 1mM ethylenediaminetetraacetic Germany) for 10min. Slides were rinsed 30s in 95% etha-
acid [EDTA] pH 8.0) at 58(cid:3)C, overnight. Posthybridization nol;airdriedandembeddedin1(cid:4)PBS/glycerol(1:9),covered
slides were washed four times in 4(cid:4)saline sodium citrate with a coverslip, and sealed with nail polish. IF slides were
(SSC) (20(cid:4)SSC: 3M NaCl, 0.3M sodium citrate, pH 7.0), washed three times 10min in PBS and nuclei staining was
5min at room temperature each, before being treated with performed using Hoechst dye 33258 (0.3mg/ml,
GenomeBiol.Evol.7(7):1896–1913. doi:10.1093/gbe/evv114 AdvanceAccesspublicationJune15,2015 1899
GBE
Schwarzeetal.
Calbiochem) for 15min at room temperature in the dark. aminoacids,whichhave92.5%identityand96.9%similarity.
Dried sections were embedded in Mowiol (Calbiochem). The genomic organization of Ngb in the turtle genomes
Sections were analyzed with an Olympus BX51 research showeda conservedsynteny withthe homologouschromo-
microscope, and the images were combined using Adobe somal regions of chicken and Anolis (supplementary fig.S1,
PhotoshopCS512.0.4. SupplementaryMaterialonline).Inturtles,theanalysisofthe
Ngbregionwasrestrictedtothe30-sideoftheNgbgenedue
Results to the fragmentary assembly. The covered region is strictly
conserved (supplementary fig. S1, Supplementary Material
IdentificationandAnalysisofTurtleGlobinGenes
online).
The globin gene sequences of the Chinese softshell turtle
D
(P. sinensis) and the western painted turtle (C. picta bellii) o
wereidentifiedinthepublishedgenomeassemblies(Shaffer GlobinX wn
lo
etal.2013;Wangetal.2013)andwithRACEifrequired.The In the softshell turtle, no GbX gene was annotated or pre- ad
e
globin repertoire of the Chinese softshell turtle comprised dicted, and therefore identified with Augustus on contig d
the full set of vertebrate globins with three Hba and two JH211032 at the position 890205-873421. The GbX of the from
Hb(cid:2) genes, and one of each Mb, Ngb, GbE, GbY, GbX, painted turtle was misannotated as cytoglobin-1-like h
and Adgb gene (fig. 1 and supplementary table S2, (XM_005293187.1). Like the GbX genes of amphibians and ttps
Supplementary Material online). The western painted turtle fishes(Roesneretal.2005;Blank,Wollberg,etal.2011),both ://a
c
has a higher quality in genome assembly and annotation. It GbX genes of the turtles harbor four introns at positions ad
e
harbors the same gene set and has an additional Hb(cid:2) gene m
B12.2, G7.0, H10.0, and E10.2. For synteny analysis of the ic
(figs. 1 and 2B and supplementary table S3, Supplementary GbXregion,weincludedtheGbX1contigofthecoelacanth. .o
u
Material online). Supplementary tables S2 and S3, p
Despite the lack of GbX in chicken, the gene synteny in the .c
Supplementary Material online, summarize the names, the homologous regions is partially conserved (fig. 2A). The om
lengths, the accession numbers, and the genomic positions /g
comparison of the turtle, coelacanth GbX1, and Anolis GbX b
e
of the globins of the softshell turtle and the painted turtle, contigs shows a conservation of the neighboring genes, /a
respectively. CHP2-DLL3-TIMM50-SUPT5H-GbX-PLEKHG-MED29-PAF1. In rticle
thecoelacanth,theorientationoftheregionwiththegenes -ab
Androglobin s
GbX and PLEKHG is inversed. The synteny analysis of the tra
c
Adgb has a modular structure that possesses an N-terminal globin genes verified the orthology of the identified turtle t/7
calpain-like domain, an internal, circularly permuted globin globingenestothechicken,coelacanth,andAnolis(fig.2A) /7
/1
domain, and an IQ calmodulin-binding motif (Hoogewijs (Opazo,Lee,etal.2015). 89
6
etal.2012).TheAdgbgeneofthesoftshellturtleisannotated /6
3
asXM_006111304.1;inthepaintedturtle,5Adgbtranscripts 1
2
Cytoglobin 7
areannotatedwiththeaccessionnumbersXM_005280422.1 4
b
toXM_005280426.1,andcodefor1,596aminoacidprotein. There are three transcript predictions for P. sinensis Cygb y g
The protein sequences of the turtle Adgbs display 88.3% (XM_006136484–XM_006136486) in the databases, which ue
s
identity and 92.6% similarity (supplementary table S4, differintheir30codingexon.However,sequencecomparisons t o
n
Supplementary Material online). Synteny analyses of the withotherCygbproteinsshowedthatprobablyonlytranscript 0
5
Adgb region shows a high conservation of the neigboring variantX3(XM_006136486.1)iscorrect.Inthepaintedturtle M
a
genes EPM2A-FBXO3O-GRM1-RAB32-ADGB-STXBP5- genome, six splice variants are annotated (XM_008163872, y 2
SAMD5 between the chicken, green anole, and turtle XM_005297538, XM_008163873, XM_005297539, 01
9
genome sequences (supplementary fig. S1, Supplementary
XM_005297540,andXM_008163874),with—basedonthe
Materialonline).
comparison with the orthologous sequences—variant X5
(XM_005297540.2) probably being correct. Cygb is the
Neuroglobin
most highly conserved globin, displaying 97.8% identity
TheNgbgenesofthesoftshellturtleandthepaintedturtleare between the two turtles, and 84.9% and 84.4% identity
annotated as XM_006117796.1 and XM_005301643.2, with the chicken Cygb (supplementary table S4,
respectively (supplementary tables S2 and S3, Supplementary Material online). The genomic organization
Supplementary Material online). The Ngb genes have three of Cygb is conserved in the homologous regions of chicken
exons at position B12.2 (i.e., the intron is found between and Anolis and shows that Cygb is located between
codon positions 2 and 3 in the 12th codon of globin helix the genes PRPSAP1-SPHK1-UBE20-AANAT-RHBDF2 and
B), G7.0, and E11.0, which are typical for Ngb (Burmester ST6GALNAC-MXRA7-JMJD6-METTL23 (supplementary fig.
et al. 2004). Both turtle globins code for proteins of 160 S1,SupplementaryMaterialonline).
1900 GenomeBiol.Evol.7(7):1896–1913. doi:10.1093/gbe/evv114 AdvanceAccesspublicationJune15,2015
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FIG.1.—AlignmentofglobinsoftheChinesesoftshellturtle(Pelodiscussinensis;Psi)andthewesternpaintedturtle(Chrysemyspictabellii;Cpi).The 74
secondarystructureofhumanMbissuperimposedintheupperrow,witha-helicesdesignatedAthroughH,theglobinconsensusnumberingisgivenbelow b
y
thesequences.Conservedresiduesareshaded(100%conservation,black;75%,darkgray).Theconservedhistidineandphenylalanineresiduesrequiredfor gu
e
oxygenbindingaremarked.NotethatPsiHbAaharborsaglutamineinsteadofthehistidineathelixpositionE7. s
t o
n
0
5
Myoglobin online), which is conserved among amniotes (Schwarze and M
TheMbgenesofthesoftshellturtleandthepaintedturtleare Burmester2013). ay 2
0
annotated as ENSPIG00000008494 and XM_005300826.2, 1
GlobinE 9
and XM_005301643.2, respectively (supplementary tables
S2 and S3, Supplementary Material online). The coding se- The GbE gene (ENSPSIG00000017186) resides on contig
quences of the turtle Mb genes each translate into proteins JH209952 of the softshell turtle genome and codes for
of 154 amino acids. The Mb proteins of the turtles share a protein with 151 amino acids. In the painted turtle
88.3% of their amino acids and have 92.9% similarity genome, the GbE was misannotated as cytoglobin-1-like
(supplementary table S4, Supplementary Material online). (XM_005293060.2). The translation of the gene results in
Comparison with the chicken Mb showed 75.3% and 151aminoacidprotein. ThetwoturtleGbEproteinsdisplay
76.6% identity on the protein level. The Mb chromosomal 96.0% identity and 98.7% similarity. Comparison with
region shows the syntenic gene order HMGXB4-TOM1- chickenGbEresultedinidentityscoresof85.4%.Evaluation
HMOX1-MCM5-RASD2-Mb-(APOL3-like)-RBFOX2-MYH9-TX oftheGbEgenesyntenyshowsthegeneorderofLUC7L-GbE
N2-FOXRED2(supplementaryfig.S1,SupplementaryMaterial (fig. 2B), which is also conserved in the chicken and the
GenomeBiol.Evol.7(7):1896–1913. doi:10.1093/gbe/evv114 AdvanceAccesspublicationJune15,2015 1901
GBE
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FIG.2.—ConservedsyntenyoftheGbX(A),GbE(B),Hb(cid:3)/GbY(C),andHb(cid:2)(D)chromosomalregionsinthegenomesofturtle,chicken,andgreen st o
anole.Globingenesareshowninblack;conservedneighboringgenesareshadedindarkgrayandgeneswithoutorthologsinthehomologousregionsare n
0
shadedinlightgray.HbG(markedwithanasterisk)isonlypresentinthepaintedturtle.Otherwise,thetwoturtlespecieshavethesamegeneconfiguration. 5
M
BecauseofthelackofGbXinchicken,thecoelacanthwasincludedintheanalysis.SyntenyanalysesoftheMb,Cygb,andNgbchromosomalregionsare a
y
giveninsupplementaryfigureS1,SupplementaryMaterialonline. 2
0
1
9
coelacanth (Schwarze and Burmester 2013). In the Anolis ENSPSIG00000012161.Exons1and2ofHbAdareannotated
genome, the homologous region is present, although asHbM(ENSPSIG00000012157);thethirdexonwasretrieved
Anolis has lost the GbE gene (fig. 2B). The absence of GbE by RACE. Two additional globin gene fragments
ofAnoliswasverifiedbyamplificationandsequencingofthe (ENSPSIG00000011874 and ENSPSIG00000011856), each
genomicregionbetweentheLUC7LandPOLDIP3byPCR. having the first and second exons, were annotated on the
samecontig.CompletionofthesequencesbyRACEillustrated
thatthesetwosequences,includingtheuntranslatedregions,
TheHemoglobinGenes
wereidentical,suggestingeitherarecentgeneduplicationor
ThethreeHbagenesofthesoftshellturtleresideonasingle an assembly error. This sequence was identified as HbAa.
contig (JH209131; fig. 2C and supplementary table S2, Notably, HbA(cid:3) possesses a glutamine instead of the distal
Supplementary Material online). HbZ is annotated as histidine at position E7 (fig. 1). The two Hb(cid:2) genes (HbB1
1902 GenomeBiol.Evol.7(7):1896–1913. doi:10.1093/gbe/evv114 AdvanceAccesspublicationJune15,2015
GBE
TurtleGlobinRepertoire
[ENSPSIG00000004624] and HbB2 [ENSPSIG00000005077]) Supplementary Material online. Adgb was excluded from
arelocatedoncontigJH210967. the analysis because of its highly divergent sequence
ThethreeHbagenesofthepaintedturtleresideonasingle (Hoogewijsetal.2012).NgbandGbXproteinswereassumed
contig (JH584800) in head-to-tail orientation (fig. 2C and as the outgroup because they split from the clade that
supplementary table S3, Supplementary Material online). includes the other globins before the divergence of
When compared withthe putative orthologs inthe softshell Protostomia and Deuterostomia (Roesner et al. 2005; Blank
turtle, HbZ was found to be the highest conserved Hba and Burmester 2012; Hoffmann, Opazo, Storz 2012). We
(91.5% identity) (supplementary table S4, Supplementary found the Cygb proteins as a paraphyletic assemblage at
Material online). The HbAd and HbAa of the two turtles the base of the other globin types, with the agnathan
share 89.4% and 86.6%, respectively, of the amino acids. Cygbs diverging first. However, the support was low (0.65
All analyzed Hb(cid:3) have 1:1 orthologs in the chicken Bayesianposteriorprobability[PP]). Thenextcladeisformed Do
w
genome,whichdisplaybetween82.4%and90.1%identity bytheagnathanHbsandMbs(aHbandaMb)(cf.Schwarze n
lo
(supplementary table S4, Supplementary Material online). In et al. 2014). The clade that includes vertebrate Hba and b, a
d
contrast to the softshell turtle, the painted turtle possesses Mb, GbE, and GbY received high support (0.99 PP). Like in ed
three Hb(cid:2) genes. Phylogenetic analysis and sequence com- other recent analyses (Hoffmann, Opazo, Hoogewijs, et al. fro
m
parisonrevealedthatHbB1andHbB2haveorthologsinboth 2012; Hoffmann, Opazo, Storz 2012; Schwarze and h
turtlespecies,butthesoftshellturtlelosttheorthologofHbG Burmester 2013; Burmester and Hankeln 2014; Schwarze ttps
(oritisnotrepresentedinthecurrentassembly).Althoughthe et al. 2014; Opazo, Lee, et al. 2015), two monophyletic ://a
c
HbB1 and HbB2 genes have orthologs in chicken, HbG is clades that include a- and b-Hb chains on the one hand, ad
e
duplicatedinchickenand,therefore,thereisno1:1ortholog and GbE and Mb on the other hand received high m
totheturtleHbG(figs.2Dand3).TheturtleandchickenHb(cid:2) support. In this analysis, GbY was found as sister group of ic.o
u
genes have no 1:1 orthologs in the human genome the clade comprising GbE and Mb, but the support value p
.c
(Hoffmann et al. 2010) (fig. 3). The Hb genes of the turtle waslow(0.60PP). om
areseparatedontwodifferentclusters(fig.2CandD).Gene /g
b
syntenyanalysesoftheHb(cid:3)regionidentifiedMPG-NPRL3and ExpressionPatternofGlobinsinTurtleTissues e/a
(GbY)-TMEM8a-MPRL28astheneighboringgenes.Thegene rtic
order is highly conserved among vertebrates (fig. 2C). WequantifiedthemRNAlevelsofMb,Ngb,Cygb,GbE,GbY, le-a
ThenumbersofHbgenesmaydifferbetweencloselyrelated GbX,andAdgbbymeansofqRT-PCRinbrain,eye,muscle, bs
species, which is due to a high rate of lineage-specific gene heart,kidney,liver,intestine,lung,andbloodoftheChinese trac
duplication (Hoffmann et al. 2008) (fig. 3). The Hb(cid:2) genes softshell turtle (P. sinensis) and the western painted turtle t/7/7
are embedded in a cluster of olfactory receptor genes (OR) (C. picta bellii). The mRNA levels were normalized according /1
8
(fig.2D). to the total RNA content used for cDNA synthesis and the 96
standard curve methodwas usedtocalculate absolute copy /63
1
numbers. 2
GlobinY 7
4
EvaluationoftheglobinmRNAlevelsrevealedcharacteristic b
TheGbYgeneofthesoftshellturtle(XM_006124603.1)was y
expression patterns. In both species, Mb, Ngb, and GbE g
identified on contig JH209131. In Ensembl, the exon–intron showed high mRNA levels in specific tissues, whereas Cygb, ue
s
boundaries of the genes (ENSPSIG00000011829) are misan- GbY, and GbX showed a low to moderate expression in a t o
n
notated. The GbY gene of the painted turtle broad range of tissues. In the softshell turtle, Mb is highly 0
5
(XM_005306194.2) resides on contig JH584800. In both expressed in muscle and heart tissues with 108 copies per M
a
species,thecurrentannotation(cytoglobin-1-like)isincorrect. mgtotalRNA,followedbytheamountof107copiespermg y 2
Thepredictedproteinsare80.5%identical(91.6%similarity). 0
RNA measured in the eye (fig. 4A). In other tissues, the Mb 1
9
Inbothgenomes,GbYisadjacenttotheHb(cid:3)-cluster(fig.2C), expressionrangedbetween101and105copiespermgRNA.A
asitwasfoundinplatypus,thegreenanole,Xenopus,andthe
very similar pattern of Mb expression was found in painted
coelacanth (Hoffmann et al. 2010, 2011; Schwarze and
turtle,with,however,slightlylowerMbmRNAlevels(fig.4A).
Burmester2013).
Asexpected,wedetectedveryhighNgbmRNAlevelsinthe
brain(~2(cid:4)106copiespermgRNA);however,Ngbwasalso
PhylogenyofTurtleandOtherVertebrateGlobins moderatelyexpressedintheeyeandthekidney(105),butlow
The amino acid sequences of 163 vertebrate globins were in the lung in both species (fig. 4B). GbE showed the most
usedtoconstructamultiplesequencealignment(supplemen- distinct tissue specificity (fig. 4C). We measured a very high
tary table S5, Supplementary Material online). A Bayesian GbE content with 108 copies per mg RNA in the eye of
analysis with the LG model was used and the resulting tree thesoftshellturtle and2(cid:4)107inthe paintedturtle, respec-
is displayed in figure 3. The full tree without collapsed tively.Inallothertissues,theGbEmRNAlevelswereatleast
branches is given in supplementary figure S2, 1,000-foldlower.
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FIG.3.—Bayesianphylogenetictreeofvertebrateglobins.TheChinesesoftshellturtleglobinsaregreen;theglobinsofthewesternpaintedturtlearered.
Thenumbersatthenodescorrespondtotheposteriorprobabilities.Thenodeswithoutnumbersaresupportedby1.0Bayesianposteriorprobability.Thebar
represents0.5PAMdistance.Thecommonnamesofthespeciesaregiven(Schwarzeetal.2014).SeesupplementarytableS5,SupplementaryMaterial
online,forfurtherinformation.ThefulltreewithoutcollapsedbranchesisgiveninsupplementaryfigureS2,SupplementaryMaterialonline.
1904 GenomeBiol.Evol.7(7):1896–1913. doi:10.1093/gbe/evv114 AdvanceAccesspublicationJune15,2015
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FIG.4.—QuantificationofmRNAlevelsofallvertebrateglobins(exceptHb)indifferenttissuesoftheChinesesoftshellturtle(gray)andthewestern
paintedturtle(black).AbsolutecopynumbersofmRNAwereobtainedusingqRT-PCRexperiments.Mb(A),Ngb(B),andGbE(C)showedatissue-specific
expressionpatterninstriatedmuscle,neuronaltissues+kidneyandeye,respectively.GbX(D),Cygb(E),andGbY(F)showedawidespreadexpressionin
differenttissueswithlowerlevels.Adgb(G),whichwasonlytestedinwesternpaintedturtle,isexpressedinalltissuesanalyzed.Logarithmicscaledfiguresare
giveninsupplementaryfig.S3,SupplementaryMaterialonline.
GenomeBiol.Evol.7(7):1896–1913. doi:10.1093/gbe/evv114 AdvanceAccesspublicationJune15,2015 1905