Comparative Biochemistry and Physiology, Part D 4 (2009) 168–173

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Comparative Biochemistry and Physiology, Part D

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The sequence and phylogenesis of the α-globin genes of Barbary sheep(Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon)and Cyprus mouflon (Ovis aries ophion)

Monica Pirastru a, Chiara Multineddu a, Paolo Mereu a, Mara Sannai a, El Said el Sherbini b,Eleftherios Hadjisterkotis c, Andràs Nàhlik d, Paul Franceschi e, Laura Manca a, Bruno Masala a,⁎a Department of Physiological, Biochemical and Cell Sciences, and Center for Biotechnology Development and Biodiversity Research, University of Sassari, Italyb Department of Biochemistry, University of Mansura, Egyptc Ministry of the Interior, Nicosia, Cyprusd Institute of Wildlife Management, University of West Hungary, Sopron, Hungarye CEVAREN, Université de Corse, France

⁎ Corresponding author. Dipartimento di Scienze FisiolUniversità di Sassari. ViaMuroni, 25, 07100 Sassari, Italy.079228659.

E-mail address: masala@uniss.it (B. Masala).

1744-117X/$ – see front matter © 2009 Elsevier Inc. Aldoi:10.1016/j.cbd.2009.02.002

a b s t r a c t

a r t i c l e i n f o

Article history:

In order to investigate the p Received 15 December 2008Received in revised form 10 February 2009Accepted 10 February 2009Available online 15 February 2009

Keywords:Ammotragus lerviaCapra hircusOvis aries musimonOvis aries ophionα-globin geneα-globinSequencingPhylogeny

olymorphism of α-globin chain of hemoglobin amongst caprines, the linked Iαand IIα globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis ariesmusimon), and Cyprus mouflon (Ovis aries ophion) were completely sequenced, including the 5′ and 3′untranslated regions. European and Cyprus mouflons, which do not show polymorphic α globin chains, hadalmost identical α globin genes, whereas Barbary sheep exhibit two different chains encoded by twononallelic genes. Four different α genes were observed and sequenced in goat, validating previousobservations of the existence of allelic and nonallelic polymorphism. As in other vertebrates, interchromo-somal gene conversion appears to be responsible for such polymorphism. Evaluation of nucleotide sequencesat the level of molecular evolution of the Iα-globin gene family in the caprine taxa suggests a closerrelationship between the genus Ammotragus and Capra. Molecular clock estimates suggest sheep-mouflon,goat-aoudad, and ancestor-caprine divergences of 2.8, 5.7, and 7.1 MYBP, respectively.

© 2009 Elsevier Inc. All rights reserved.

1. Introduction

In nearly all mammals, the adult α chains of the hemoglobin (Hb)tetramers are encoded by two closely linked genes (Lauer et al., 1980;Zimmer et al., 1980; Schon et al., 1982; Hardison and Gelinas, 1986;Clegg, 1987) an exception being represented by rabbit (Cheng et al.,1987) which has a single gene. The nucleotide sequences of thetandem pair of α genes are highly similar to each other withinindividual species compared to α genes from other species, thussuggesting a gene family which arose from a single ancestral gene bygene duplications followed by concerted evolution (Zimmer et al.,1980; Michelson and Orkin, 1983; Hardison and Gelinas, 1986; Clegg,1987; Hurles, 2004). This mechanism has been shown to operate ondifferent pairs of genes encoding either the α and non-α chains, such

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as human α2 and α1 (Liebhaber et al., 1981) and Gγ and Aγ (Slightomet al., 1980), mouse βs and βt (Weaver et al., 1981), chicken αD and αA

(Dodgson et al., 1981), goat Iα and IIα (Schon et al., 1982), horse α2and α1 (Clegg, 1987), water buffalo Iα and IIα (Ferranti et al., 2001)and many other mammals (Cooper et al., 2006). As is the case inhumans, the α globins expressed by the linked genes sometimes haveidentical amino acid sequences. More often they differ at just one or afew residues, these changes frequently resulting in altered electro-phoretic mobilities of the Hb tetramers (Clegg et al., 1984; Pirastruet al., 2000; Ferranti et al., 2001). Variationmay also result from allelicpolymorphism at the two α loci (Clegg et al., 1984; Pirastru et al.,2000; Ferranti et al., 2001; Pieragostini et al., 2005). In nearly allspecies examined, the tandem organization of α genes is responsiblefor a gradient expression ofα chains production due to transcriptionalinterferences between the upstream (the most efficient) and down-stream (the less efficient) gene (Proudfoot, 1986).

Domestic sheep (Ovis aries) and goat (Capra hircus) α globins havebeen characterized. Allelic and non-allelic variations are rathercommon in different sheep breeds amongst which three alleles ofthe Iα and twoof the IIα loci have been described (Vestri et al.,1983a,b;Rando et al., 1986; Hadjisterkotis et al., 1995). Chromosomes with

Fig. 1. AUT-PAGE of the dissociated globin chains showing the polymorphism at thelevel of both β and α globin chains. Lane 1: European mouflon; lane 2: Cyprus mouflon;lane 3: Ammotragus; lane 4: goat with two Iα and two IIα allelic chains.

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triplicated or quadruplicated loci due to unequal cross-over eventshave also been found rather frequently in sheep, these arrangementsclearly supporting the presence of the gradient expression of α chainsproduction (Vestri et al.,1987,1993,1994; Ristaldi et al.,1995). Goat Hbalso contains two non-allelic α chains differing at positions 19, 26, 113and 115: Iα has Gly, Ala, Leu and Asn, whereas IIα has Ser, Thr, His, andSer, respectively (Huisman et al., 1967, 1968; Garrick and Huisman,1968). A rarely observed allelic Iα chain (designated as IαB) has theAsp→Tyr substitution at position 75 (Huisman et al., 1968; Adamset al., 1969). Schon et al. (1982) described the complete DNA sequenceof two goatα-globin genes validating the structures established at theprotein level except at position 26 of the IIα gene which showed Alainstead of Thr. This finding supported the existence of an allele of theIIα gene. In a previouswork conducted on several goats from the islandof Sardinia, we identified eight allelic β-globin chains and fourdifferent α chains (Pirastru et al., 2000). The eight structural β-globingenes, which included the βA, βD, βDMalta, βE and four previouslyundescribed genes, have been completely sequenced (Pirastru et al.,2000, 2003). According to the electrophoretic mobility and quantity,the most common α chains were tentatively identified as being the Iαand IIα chains already described by Huisman et al. (1967, 1968),whereas the less common, temporarily designated as the IαF (fast) andIIαS (slow) were considered as being allelic chains. Three differenttandemassociations ofα chainswere observed: Iα-IIα, Iα-IIαS, and IαF-IIα, either in heterozygosity or in homozygosity. In recent work,conducted on a large number of goats belonging to the ItalianGarganica and Jonica breeds, a new allelic Iα chain (IαT) having theAla→Thr substitution at position 26 was detected by combined massspectrometric approaches (Pieragostini et al., 2005). These dataprovide evidence of the presence of, at least, three allelic Iα genesand two allelic IIα.

Previous research has resulted in conflicting evidence for theBarbary sheep (Ammotragus lervia).Wilson et al. (1970) described twoα globins, one of which being identical to goat Iα chain except atposition 74 (Glu in Barbary sheep, Asp in goat), and the other havingAla instead of Thr at position 26, and Glu instead of Asp at position 74,with respect to the goat IIα chain. Conversely, Blunt and Huisman(1975) observed two α globins, one of which being identical with thegoat Iα chain at all but the residue 82 where Asp or Asn is present ingoat, and Glu in Barbary sheep. The minor Barbary sheep IIα chaindiffers from goat IIα globin at positions 26 and 82. The first one isoccupied by Ala in Ammotragus and by Thr in goat, the second one isthe same present in Iα. Finally, the presence of a singleα chain, not yetinvestigated, has been observed in wild European (or Corsico-Sardinian) mouflon (O. aries musimon) (Naitana et al., 1990) and inCyprus mouflon (O. a. ophion) (Serreri et al., 2000).

With the aim to contribute to the knowledge of the α genesstructure and globin chain polymorphism amongst caprines, wedescribe the nucleotide sequence of the genes encoding the Iα and IIαglobins of Barbary sheep, Corsico-Sardinian and Cyprus mouflon andthe four α globins of goat previously described (Pirastru et al., 2000).Results are also evaluated at the level of molecular evolution of the α-globin gene family in the caprine taxa.

2. Materials and methods

2.1. Detection and separation of globin chains

Blood samples were obtained in EDTA by Barbary sheep (A. lervia)reared in Mansoura Zoo (Egypt), goat (C. hircus), wild Corsico-Sardinian (O. aries musimon, also known as O. gmelini musimon)captured in different areas of Sardinia, Corsica and in Hungary, andCyprus mouflons (O. aries ophion, also known as O. gmelini ophion)captured in Cyprus. Dissociated globin chains were analyzed by theacid-urea-Triton X-100 gel electrophoresis (AUT-PAGE) as previouslydescribed (Masala et al., 1991; Manca et al., 1991). The percentage of

the globin components was determined by the reversed-phase highperformance liquid chromatography (RP-HPLC) using a Vydac C4

column (Masala and Manca, 1994).

2.2. Sequencing of α-globin genes

Genomic DNA was prepared from whole blood samples by astandardmethod. PCR and sequencing primers have been designed onthe basis of previously published goat Iα and IIα gene sequences(Schon et al., 1982). A fragment (861 bp in length) of the Iα gene,including the 5′ non-coding region, exon 1 and IVS I, was obtainedusing an upstream primer (GA8, 5′-GCATAGCGCCCCATCTTACAC-3′)that matches only the Iα gene from position −611 to −591, and adownstream primer (GA17, 5′-GGAACATCCTGGGTGAAGAGG-3′) thatanneals to both the duplicated genes at positions +230 to +250. Theremaining part of the gene was amplified by GA11, non-specific, 5′-AGAGAGAATCCACCATGGTGCTGTC-3′ (located from+26 to+50) andGA14, specific, 5′-TATTTCAGCTCTCCCCACCTC-3′, (from +944 to+964) primers. The GA9, 5′-GCATCTACCCAGCTCCACAAT-3′ (from−294 to −274) and GA4, 5′-AGGGTGAGGGCTGCCCAGAGAA-3' (from+921 to +942) primers, both specific, were used to selectivelyamplify the whole IIα gene. The amplification reactions included aninitial denaturation at 95 °C for 10 min, 30 cycles of denaturation at95 °C for 1 min, annealing at 63 °C (55 °C for the GA11/GA14 pair;65 °C for the GA9/GA4) for 30 s, and extension at 72 °C for 3min, and afinal extension at 72° for 7 min.

Direct sequencing of PCR-amplified segments of α genes wascarried out by means of automated capillary sequencing using theGA8, GA17 and GA14 primers for the Iα gene, GA9 and GA4 for the IIαgene, GA11, GA6, 5′-GGGCGTGCAGGTCACTCAGATC-3′, reverse (from+387 to +408), GA12 (5′-GATCTGAGTGACCTGCACGCCC-3′ (from+387 to +408), GA7 5′-CACTCAGACTTTATTCAAAGAT-3′, reverse(from +741 to +762) for both genes.

2.3. Phylogeny and molecular dating

A search for related nucleotide sequences of α globin genes in theGeneBank was performed by BLASTN (version 2.2.17) (Altschul et al.,1990). Sequences were aligned by means of CLUSTAL X 1.83(Thompson et al., 1997). Evolutionary relationships among α globingenes were estimated using Maximum Likelihood (ML) approach(Felsenstein, 1981) as implemented by TREEPUZZLE 5.2 (Schmidtet al., 2002) applied to the entire nucleotide sequences. ML distanceswere calculated using HKY (Hasegawa et al., 1985) model ofnucleotide evolution performed with gamma distribution of rate

Fig. 2. RP-HPLC chromatographic profiles: Panel A: goat showing Iα and IIα allelic chains and two adult β-globins (βA and the allelic βD-MALTA). Panel B: Barbary sheep showing Iα andIIα nonallelic chains. The βC peak refers to the pre-adult β globin.

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heterogeneity (Gu and Zhang, 1997). The divergence times wereestimated by applying a reference point based on fossil records: theemergence of the family Bovidae set at 18.5 MYBP with an intervalbetween 18 and 20 MYBP (Vrba and Schaller 2000). The calculationswere based on branch lengths from the ML tree. GenBank accessionnumbers for sequences are: donkey (Equus asinus) U69894; zebra (E.zebra) (U70194); E. hemionus (U70197); sheep (O. aries) Iα75Ly-s→Asp allele (X70213) and

IIα113 Leu→His allele (X70215); cow

(Bos taurus) IαN allele (AJ242799), IαY allele (AJ242797); waterbuffalo (Bubalus bubalis) Iα1 allele (AJ242731), Iα2 allele (AJ242732).

3. Results

3.1. Detection and quantitation of globin chains

The electrophoretic patterns and polymorphism of globin chains ofthe four examined species are shown in Fig. 1. The two mouflonspecies have no polymorphic α chains, Barbary sheep shows onlynonallelic polymorphism whereas goat shows both allelic and non-allelic polymorphism. The Iα, IαF (fast), IIα and IIαS (slow)nomenclature refers to that previously described on the basis of thepercent level and according to the electrophoretic mobility (Pirastruet al., 2000).

The RP-HPL chromatogram of globin chains is showed in Fig. 2. Inboth goat (panel A) and Barbary sheep (panel B) the underexpressedIIα globins elute at a shorter retention timewith respect to the Iα and tothe β-chains. The Iα/ IIα ratio, obtained by integrating the absorbance

Fig. 3. Alignment of the deduced amino acid sequence of the Iα globins and of goat IαT alleleO.a.: sheep.

at 220 nm, resulted 67:33 and 75:25 in goat and Barbary sheep,respectively, supporting the presence of the already described gradientof expression.

3.2. Sequencing of α-globin genes

The α globin gene sequences obtained in this study, as assessed inthe region ∼600 (upstream to the cap site) to ∼964 (3′ untranslatedregion) of the Iα gene, and −294 to +942 of the IIα gene, weredeposited in the GenBank database under the following AccessionNos.: Barbary sheep (A. lervia) Iα EU938072, IIα EU938076; goat(C. hircus) Iα EU938069, IαT allele EU938074, IIα EU938075, IIαT alleleEU938078; European mouflon (O. aries musimon) Iα EU938073, IIαEU938077; Cyprus mouflon (O. a. ophion) Iα EU938070, IIα EU938071.

The noncoding sequences of the α genes in the species examined,which include those of O. aries described by Ristaldi et al. (1995), agreewith those previously obtained by Schon et al. (1982) in goat. In fact, thelinked pairs of genes were highly similar to each other (99%) not only intheir coding regions, but in their IVSs and the immediate 5′ and 3′flanking sequences as well, extending 123 bp 5′ to the ATG initiationcodon and139bp3′ to the TAA stop codon. This homology,which coversapproximately 900bp, andwhichessentially defines the extentof a geneconversion unit (Schon et al., 1982), ends rapidly in both the 5′ and 3′directions beyond these two points, and declines to backgroundsequence homology (26%) in the remaining sequenced area.

The IVS I of the IIα gene resulted 101 bp in length and that of Iαresulted 108 bp long in goat and 101 bp in Barbary sheep and Ovis, this

. A.l.: Ammotragus, C.h.: goat, O.a.m.: Corsico-Sardinian mouflon, O.a.o.: Cyprus mouflon,

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difference being due to a 7-bp sequence (CGCCGGG) which isrepeated twice in tandem in goat Iα, but is present only once inBarbary sheep andmouflon Iα and in IIα. The IVS II of both Iα and IIα is104 bp in length in all animals. A few nucleotide substitutions wereobserved in the two IVS, the sequence homology being 95.2% in bothgenes. Such homology decreases to 64.1–66.7% in the 5′-UTR region,and to 84.1–84.6% in the 3′-UTR region and to 49.7 and 56% in thenoncoding regions extending further in both the 5′ and 3′ directions,respectively. Coding regions of the five genera differ at codons 74 and104 of the Iα gene, and at codons 19, 74, 104, 113 and 115 of the IIαgene giving rise to amino acid replacements. Moreover, synonymouscodons are present at positions 52, 79, 98, 118, 120 and 137 of the Iαand at positions 52, 79, 89 and 98 of the IIα. Both goat IαT (Pieragostiniet al., 2005) and IIαT (Huisman et al., 1967) alleles are due to theGCA→ACA substitution (corresponding to the Ala→Thr change)occurring at codon 26 in genes showing the otherwise identicalsequence with respect to the Iα and the IIα genes.

The protein sequences encoded by the two loci are shown in Figs. 3and 4. By comparing the Iα and IIα sequences it appears that: 1) thetwo chains in Barbary sheep are the same as previously predicted byWilson et al. (1970) and differ at positions 19, 113, and 115; 2) Barbarysheep and goat α chains are identical except at position 74 which isrespectively occupied by Glu or Asp; 3) both goat Iα and IIα chainshave an allele carrying the Ala→Thr substitution at position 26, thuscorresponding to the IαT and IIαT; 3) goat IαF and IIαS chains,previously observed in our laboratory (Pirastru et al., 2000), have Thror Ala at position 26, thus corresponding to the IαT (Pieragostini et al.,2005) and to the IIα chain described by Schon et al. (1982),respectively; 4) Iα and IIα globins of Corsico-Sardinian and Cyprusmouflon and sheep are identical to each other; 5) the Iα of the genusOvis has the same sequence of goat with the additional Ser→Thrchange at 104, whereas IIα has additional substitutions at positions 19,104, 113, and 115.

3.3. Phylogeny and molecular dating

The multiple alignments assembled with Clustal X of the Iα-globingenes, subjected to phylogenetic analysis, inferred the ML tree shownin Fig. 5. The maximum distancewas 2.5% observed by comparing Oviswith Capra and A. lervia, whereas within genus distances were lower(0–0.7%). A goat-aoudad split of 5.7 MYBP has been estimated,whereas the divergence ancestor/caprines was dated at 7.1 MYBP.Divergence time between sheep andmouflonwas set at 2.8MYBP. Dueto the limited number of published sequences, phylogenetic analysison IIα-globin genes and on amino acid sequences were not statisticallysignificant.

Fig. 4. Alignment of the deduced amino acid sequence of the IIα globins and of goat IIαT alleleO.a.: sheep.

4. Discussion

The observation of the high overall nucleotide sequence homologyof the human Gγ and Aγ globin genes (Slightomet al.,1980),whichwasfollowed by a similar observation at the level of goat (Schon et al.,1982) and horse (Clegg, 1987) α globin genes, including flanking andintervening sequences, provided strong evidences for the role of geneconversion in maintaining sequence identity between closely linkedpairs of genes. Elevated Hb polymorphism described both in goat andsheep (Schon et al.,1982; Vestri et al.,1983a; Hadjisterkotis et al.,1995;Pirastru et al., 2000; Pieragostini et al., 2005) which seems to be loweror even absent in strictly related species, offers additional opportunityto investigate the evolution of these genes in caprines. In this work wehave determined the complete nucleotide sequence of four goat αglobin genes (allelic and nonallelic) and of three related species:Barbary sheep (A. lervia), European mouflon (O. aries musimon) andCyprus mouflon (O. aries ophion). Results, also compared with ninedifferentmammalian species and the only complete sequence of sheepso far described (Ristaldi et al., 1995), support and extend the aboveprevious observations. In fact, the two linked α genes showed a highsequence homology to each other and among related species (99%) notonly in their coding regions, but in their intervening sequences andtheir immediate 5′ and 3′ flanking sequences aswell, extending 123 bp5′ to the ATG initiation codon and 139 bp 3′ to the TAA stop codon. Thesequence homology at the level of coding regions is thus responsiblefor the rather high similarity of the encoded amino acid sequences. Infact, α-globins expressed by the linked pair of genes sometimes haveidentical amino acid sequences, as observed in wild European andCyprus mouflons, whereas in others they differ at just one or a fewresidues, resulting in altered electrophoretic mobilities of theassociated Hbs. The protein sequences encoded by the nonallelic αgenes of Barbary sheep indicate that three amino acids distinguish thetwo globins at positions 19 IαGly/IIαSer, 113 IαLeu/IIαHis, and 115IαAsn/IIαSer. Comparison of α globins of Barbary sheep and goatindicates that both Iα and IIα genes differ only at position 74 which isoccupied by Glu and Asp, respectively.

Variation resulting from both nonallelic and allelic polymorphismsat the two α loci is well illustrated in domestic sheep and goat (Vestriet al.,1983a,b; Pieragostini et al., 2005). Pieragostini et al. (2005) foundthat bothnonallelic Iα and IIα genes of goat have an allelewhich carriesthe Ala→Thr substitution at position 26, thus identified as the IαT andIIαT, respectively, the former being a newly described allele whereasthe latter resulting the same described by Huisman et al. (1968). Ourresults on protein sequence encoded by the α genes, agrees with theabove observations. It is of some interest that the IαB allele, havingthe Asp→Tyr substitution at position 75 (Huisman et al., 1967; Adams

. A.l.: Ammotragus, C.h.: goat, O.a.m.: Corsico-Sardinian mouflon, O.a.o.: Cyprus mouflon,

Fig. 5. Phylogenetic tree of the Iα globin genes as reconstructed by TREEPUZZLE 5.2 using HKY likelihood model with gamma distributed rates (parameter α is estimated fromdataset: 0.74 (S.E. 0.14); number of gamma categories: four). ⁎Denotes the Iα75Lys→Asp allele.

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et al., 1969), despite large screenings on different goat breeds (Pirastruet al., 2000, 2003; Pieragostini et al., 2005), remains unobservedamong Mediterranean breeds. The lower level of polymorphism inBarbary sheep andmouflonswith respect to goat and sheepmay be anindication of an older origin of these species. This is also suggested bythe phylogenetic tree calculated on Iα gene. The topology of the tree(shown in Fig. 5) indicates two separated branches containing distinctclusters of species. The α globin genes of A. lervia and C. hircus aregrouped together and separated from those of theOvis genus. Topologyof both branches indicates that caprines most probably share acommon ancestor that appeared 7.1 MYBP. The closer relationshipsof A. lervia and C. hircus has been already observed both at the level ofβglobin gene (Manca et al., 2006) and mitochondrial DNA (Hassaninet al.,1998; Ropiquet andHassanin, 2005;Mereuet al., 2008) sequencing.

Acknowledgements

This work was funded by the European Community (INTERREG IIIfunds), and the Consorzio per gli Studi Universitari nella SardegnaCentrale.

References

Adams,H.R.,Wrightstone, R.N.,Miller, A., Huisman, T.H.J.,1969.Quantitationofhemoglobinalpha chains in adult and fetal goats; gene duplication and the production of polypep-tide chains. Arch. Biochem. Biophys. 132, 223–236.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignmentsearch tool. J. Mol. Biol. 215, 403–410.

Blunt, M.H., Huisman, T.H.J., 1975. The hemoglobin of sheep. In: Blunt, M.H. (Ed.), TheBlood of Sheep. Springer-Verlag, Berlin, pp. 153–183.

173M. Pirastru et al. / Comparative Biochemistry and Physiology, Part D 4 (2009) 168–173

Cheng, J., Raid, L., Hardison, R.C., 1987. Block duplication of a ζ-ζ-α-τ-gene set in rabbitα-like globin gene cluster. J. Biol. Chem. 262, 5414–5421.

Clegg, J.B., 1987. Gene conversions in the horseα-globin gene complex. Mol. Biol. Evol. 4,492–503.

Clegg, J.B., Goodbourn, S.E., Braend, M., 1984. Genetic organization of the polymorphicequine α globin locus and sequence of the BII α1 gene. Nucleic Acids Res. 12,7847–7858.

Cooper, S.J.,Wheeler,D.,De Leo,A., Cheng, J.F.,Holland,R.A.,MarshallGraves, J.A.,Hope, R.M.,2006. The mammalian αD-globin gene lineage and a new model for the molecularevolution of α-globin gene clusters at the stem of the mammalian radiation. Mol.Phylogenet. Evol. 38, 439–448.

Dodgson, J.B., McCune, K.C., Rusling, D.J., Krust, A., Engel, J.D., 1981. Adult chicken α-globin genes αA and αD: no anemic shock α-globin exists in domestic chickens.Proc. Natl. Acad. Sci. U. S. A. 78, 5998–6002.

Felsenstein, J., 1981. Evolutionary trees from DNA sequences: a maximum likelihoodapproach. J. Mol. Evol. 17, 368–376.

Ferranti, P., Facchiano, A., Zappacosta, F., Vincenti, D., Rullo, R., Masala, B., Di Luccia, A.,2001. Primary structure of α-globin chains from river buffalo (Bubalus bubalis L.)hemoglobins. J. Protein Chem. 20, 171–179.

Garrick,M.D.,Huisman,T.H.J.,1968.Geneduplicationof thealphachainof goathemoglobin:evidence from a homozygous mutant. Biochim. Biophys. Acta 168, 585–587.

Gu, X., Zhang, J., 1997. A simple method for estimating the parameter of substitution ratevariantion among sites. Mol. Biol. Evol. 28, 1106–1113.

Hadjisterkotis, E., Manca, L., Naitana, S., Masala, B., 1995. A comparative study on thehemoglobin polymorphism of domestic sheep of the island of Chios, Cyprus, andSardinia. Comp. Biochem. Physiol. 112A, 547–552.

Hardison, R.C., Gelinas, R.E., 1986. Assignment of orthologous relationships amongmammalian α-globin genes by examining flanking regions reveals a rapid rate ofevolution. Mol. Biol. Evol. 3, 243–261.

Hasegawa, M., Kishino, H., Yano, T.A., 1985. Dating of the human–ape splitting by amolecular clock of mitochondrial DNA. J. Mol. Evol. 22, 160–174.

Hassanin, A., Pasquet, E., Vigne, J.-D.,1998.Molecular systematics of the subfamily Caprinae(Artiodactyla, Bovidae)asdetermined fromcytochromeb sequences. J.Mamm.Evol. 5,217–236.

Huisman, T.H.J., Wilson, J.B., Adams, H.R., 1967. The heterogeneity of goat hemoglobin:evidence for the existence of two nonallelic and one allelicα chain structural genes.Arch. Biochem. Biophys. 121, 528–530.

Huisman, T.H.J., Brandt, G., Wilson, J.B., 1968. The structure of goat hemoglobins. II.Structural studies of the α chains of the hemoglobins A and B. J. Biol. Chem. 243,3675–3686.

Hurles,M., 2004.Gene duplication: the genomic trade in spare parts. PLoSBiol. 2, 900–904.Lauer, J., Shen, C.K., Maniatis, T., 1980. The chromosomal arrangement of human α-like

globin genes: sequence homology and α-globin gene deletions. Cell 20, 119–130.Liebhaber, S.A., Goossens, M., Kan, Y.W., 1981. Homology and concerted evolution at the

α1 and α2 loci of human α-globin. Nature 290, 26–29.Manca, L., Masala, B., Ledda, S., Naitana, S., 1991. Separation of caprine globin chains by

reversed-phase high-performance liquid chromatography: evidence for the presenceof a silent beta B-globin allele in Sardinian sheep. J. Chromatogr. 563, 158–165.

Manca, L., Pirastru,M.,Mereu, P.,Multineddu, C., Olianas, A., Sherbini, E.-S., Franceschi, P.,Pellegrini, M.G., Masala, B., 2006. Barbary sheep (Ammotragus lervia): the structureof the adult β-globin gene and the functional properties of its hemoglobin. Comp.Biochem. Physiol. B 145, 214–219.

Masala, B., Manca, L., 1994. Detection of globin chains by reversed-phase high-performance liquid chromatography. Methods Enzymol. 231, 21–44.

Masala, B., Manca, L., Cocco, E., Ledda, S., Naitana, S., 1991. Kinetics of the ontogenic andreversible hemoglobin switching in mouflon (Ovis musimon) and in sheep ×mouflon hybrids. Comp. Biochem. Physiol. A 100, 675–680.

Mereu, P., Palici di Suni, M., Manca, L., Masala, B., 2008. Complete nucleotide mtDNAsequence of Barbary sheep (Ammotragus lervia). DNA Sequence 19, 241–245.

Michelson, A.M., Orkin, S.H., 1983. Boundaries of gene conversionwithin the duplicatedhuman α-globin genes. Concerted evolution by segmental recombination. J. Biol.Chem. 258, 15245–15254.

Naitana, S., Ledda, S., Cocco, E., Manca, L., Masala, B., 1990. Hemoglobin phenotype of theEuropean mouflon sheep living on the Island of Sardinia. Anim. Genet. 21, 67–75.

Pieragostini, E., Rullo, R., Scaloni, A., Bramante, G., Di Luccia, A., 2005. The alpha chains ofgoat hemoglobins: old and new variants in native Apulian breeds. Comp. Biochem.Physiol. B 142, 18–27.

Pirastru, M., Palici di Suni, M., Vacca, M., Franceschi, P., Masala, B., Manca, L., 2000. Thehemoglobin polymorphism in Sardinian goats: nucleotide sequence and frequencyof βA, βD, βD-MALTA, and βE globin genes. In: Di Prisco, G., Giardina, B., Weber, R.(Eds.), Hemoglobin Function in Vertebrates. Molecular Adaptations in Extreme andNon-extreme Environments. Springer Verlag, Berlin, pp. 97–108.

Pirastru, M., Manca, L., Masala, B., 2003. Characterization of four novel variants of goatβA-globin gene. Biochem. Genet. 41, 209–217.

Proudfoot, N.J., 1986. Transcriptional interference and termination between duplicatedα-globin gene constructs suggests a novel mechanism for gene regulation. Nature322, 562–565.

Rando, A., Ramunno, L., Masina, P., 1986. Variation in the number of α-globin loci insheep. Mol. Biol. Evol. 3, 168–176.

Ristaldi, M.S., Casula, S., Rando, A., Vestri, R., 1995. Sheep α-globin gene sequences:implications for their concerted evolution and for the down-regulation of the 3′ genes.J. Mol. Evol. 40, 349–353.

Ropiquet, A., Hassanin, A., 2005. Molecular evidence for the polyphyly of the genusHemitragus (Mammalia, Bovidae). Mol. Phylogenet. Evol. 36, 154–168.

Schmidt, H.A., Strimmer, K., Vingron, M., von Haeseler, A., 2002. TREE-PUZZLE: amaximum likelihood phylogenetic analysis using quartets and parallel computing.Bioinformatics 18, 502–504.

Schon, E.A., Wernke, S.M., Lingrel, J.B., 1982. Gene conversion of two functional goat α-globin genes preservesonlyminimalflankingsequences. J. Biol. Chem. 257, 6825–6835.

Serreri, E., Hadjisterkotis, E., Naitana, S., Rando, A., Manca, L., Masala, B., 2000. Theorganization of the β-globin gene cluster and the nucleotide sequence of the β-globin gene of Cyprus Mouflon (Ovis gmelini ophion). In: Di Prisco, G., Giardina, B.,Weber, R. (Eds.), Hemoglobin Function in Vertebrates. Molecular Adaptations inExtreme and Non-extreme Environments. Springer Verlag, Berlin, pp. 109–120.

Slightom, J.L., Blechl, A.E., Smithies, O., 1980. Human fetal Gγ- and Aγ-globin genes:complete nucleotide sequences suggest that DNA can be exchanged between theseduplicated genes. Cell 21, 627–638.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. TheClustalX windows interface: flexible strategies for multiple sequence alignmentaided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882.

Vestri, R., Giordano, P.C., Bernini, L.F., 1983a. Duplication of the hemoglobin α-chaingene in sheep: characterization of a new α-chain variant present in animalspossessing the IαLeu and the IIαHis chains. Biochem. Genet. 21, 25–35.

Vestri, R., Giordano, P.C., Bernini, L.F., 1983b. Different quantitative expression of thehemoglobin α-chain in sheep. Biochem. Genet. 21, 1089–1099.

Vestri, R., Masina, P., Rando, A., Testa, A., Di Gregorio, P.,1987. Triplication ofα-globin genesis responsable for inusual α113Leu/α113His-globin chain ratios in sheep. Biochem.Genet. 25, 611–619.

Vestri, R., Pieragostini, E., Manca, L., Masala, B., 1993. Detection by reversed-phase HPLCof trace amounts of the IIα113His globin chain in a sheep homozygous for aquadruple α-globin gene chromosome. Int. J. Biochem. 25, 1939–1941.

Vestri, R., Pieragostini, E., Ristaldi, M.S., 1994. Expression gradient in sheepαα andαααglobin gene haplotypes: mRNA levels. Blood 83, 2317–2322.

Vrba, E.S., Schaller, G.B., 2000. Phylogeny of Bovidae (Mammalia) based on behavior,glands and skull morphology. In: Vrba, E.S., Shaller, G.B. (Eds.), Antelopes, Deer, andRelatives: Fossil Record, Behavioral Ecology, Systematics, and Conservation. YaleUniversity Press, New Haven, CT, pp. 203–222.

Weaver, S., Comer, M.E., Jahn, C.L., Hutchison III, C.L., Edgell, M.H., 1981. The adult β-globin genes of the “single” type mouse C57BL. Cell 24, 403–411.

Wilson, J.B., Wrightstone, R.N., Huisman, T.H.J., 1970. Haemoglobin α chain duplicationin Barbary sheep, Ammotragus lervia, Pallas, 1777. Nature 226, 354–355.

Zimmer, E.A., Martin, S.L., Beverly, S.M., Kan, Y.W.,Wilson, A.C.,1980. Rapid duplication andloss of genes coding for the alpha chains of hemoglobin. Proc. Natl. Acad. Sci. U. S. A. 77,2158–2162.