A comparative study on the haemoglobin polymorphism of domestic sheep of the islands of Chios, Cyprus and Sardinia

Camp. Biochem. Physiol. Vol. 112A. Nos. 314, pp. 541-552, 1995 Copyright 0 1995 Elsevier Science Inc. Printed in Great Britain. All rights reserved 0300-%29/95 $9.50+ .OO

Pergamon 0300-9629(95)02026-G

A comparative study on the haemoglobin polymorphism of domestic sheep of the islands of Chios, Cyprus and Sardinia E. Hadjisterkotis,

* L. Manca,‘f S. Naitana$ and B. Masalat

*Game and Fauna Service, Ministry of Interior, Cyprus; tIstituto di Fisiologia Generale e Chimica Biologica, Universita degli Studi di Sassari, Via Muroni 25, I-07100 Sassari, Italy; and SIstituto di Fisiologia degli Animali Domestici, Universita degli Studi di Sassari, Via Vienna 2, I-07100 Sassari, Italy. Different alleles at the two linked or-globin loci are present in sheep: 1~i13Le”and ua113Le”, 1~8Ala,ll3Leu 1~lSAsp,ll3Leu , and ua113=. Triplicated &gene haplotypes are also common. Six different I(;-globins (A, B, E, G, H, and I) contribute to the Hh polymorphism. A comparative study on the biochemical polymorphism of (Y- and /?-globins in pure sheep breeds of the Mediterranean islands of Chios, Cyprus, and Sardinia is described. Cyprus and Sardinia breeds have the lower frequency of cu-globin variants. Chios sheep possess the high 0.40 frequency of the 1~8Ala~113Leu-1x~113His arrangement. No a-gene triplications were observed amongst Chios and Cyprus breeds. None showed the 1cx15Asp*113Le” variant. The pB allele largely predominates; the frequency of the PA was lower (f = 0.03) amongst Sarda sheep that also possess the /3’ variant. A novel /lB-globin variant was observed in the Chios breed. Both natural and artificial selection may be the cause for these differences. Key words: Domestic sheep; a-Globin chain allele; Haemoglobin Comp. Biochem.

polymorphism;

Lysate.

Physiol. 112A, 547-552, 1995.

Introduction As in most vertebrates, the cr-globin gene is duplicated in sheep (Ovis aries) and the two loci have been designated as ‘cz (the 5’ located gene) and **CX (the 3’ located gene) (Vestri et al., 1980, 1983a, 1991; Rando et al., 1986). Different alleles at both loci have been described: the most common and identicals 1~i’3Le” and 15$13Leu,

the

I (y 8Ala.l13Leu,

I(ylSAsp,113Leu,

and

the

(Huisman et al., 1968; Vestri er al., 1980, 1983a, 1983b; Vestri and Salmaso, 1981). A simplified nomenclature of these allelic chains is: czLeu,aAla, aAsp, and aHis. It has been estimated that, of the 50% a-globin con-

11a113His

fo: B. Masala, Istituto di Fisiologia Generale e Chimica Biologica, Universita degli Studi di Sassari, Via Muroni 25, I-07100 Sassari, Italy. Tel. + 79.228650; FAX + 79.228615. Received 1 August 1994; accepted 7 November 1994. Correspondence

tributed by each chromosome, the output of the 5’ located gene is 32% whereas that of the 3’ gene is 18% (Vestri et al., 1983a, 1983b). A gradient of decreasing expression has been clearly shown also in multiple a-gene haplotypes (triplications and quadruplications) (Vestri et al., 1991, 1993). The most illustrative examples are the ‘~Asp-llczLeu-lll~Hisarrangement, in which the percentage gene efficiency is approximately 32: 12: 6 and the I~Asp_Il~Leu_III~Leu_Iv~His arrangement with the 32 : 12 : 5 : I efficiency. The different globins can be easily distinguished by biochemical methods so that their relative amounts are indicative of the a-globin loci arrangement. Three common haemoglobins due to different @globins (the adult Hb A and Hb B and the pre-adult Hb C) have been described earlier in domestic sheep (Garrick and Garrick, 1983). The Hb C is produced under anaemia

547

E. Hadjisterkotis

548

conditions only by sheep of the Hb A type, suggesting a physical linkage of the PC gene to the PA. In fact, two different chromosomal arrangements of the p-globin gene cluster have been found by Garner and Lingrel (1989): the A-haplotype in which the PA-gene is structurally linked to the PC-gene and the recently diverged B-haplotype in which the PB-gene lacks this linkage. Three variants of the PB globin (pE, PHI p’) and a variant of the PA ( PG) have been described (John, 1979; Kilgour et al., 1990; Manta et al., 1993a, 1993b). A comparison of this noticeable polymorphism in different sheep breeds, and particularly in pure breeds that remained isolated from others, may be of importance for studies on physiological adaptation, evolutionary interpretations, and natural or artificial selection. In this article, we refer to a comparative study of the biochemical Hb polymorphism in domestic sheep of pure breeds from the three Mediterranean islands of Chios, Cyprus, and Sardinia.

Materials and Methods Sample collection

and preparation

Blood samples were obtained from sheep of pure breeds of the islands of Chios (N = 66), Cyprus (N = 24), and Sardinia (N = 258), drawn into evacuated tubes containing EDTA as anticoagulant, washed 3 times with isotonic saline and lysed with 0.05% KCN to a final concentration of 0.7-1.0 g/d1 Hb.

Fig. 1. Isoelectric

et al.

Isoelectric

focusing

of haemoglobins

Lysates were routinely analysed by isoelectric focusing (IEF) of native Hb tetramers on thin layer 5% polyacrylamide slab gels in 6.7-7.7 pH range (Masala and Manta, 1991). Globin chain analysis

Dissociated globin chains of the same lysates were separated in polyacrylamide gels in the presence of acetic acid, 8M urea, and Triton X-100 (AUT-PAGE) as described previously for human globin chain separation (Alter et al., 1980). This method has also been used in nonhuman globin chain separation, including normal and variant chains that differ in neutral amino acid substitutions (Braend et al., 1987; Naitana et al., 1990; Di Luccia et al., 1991; Masala et al., 1991; Manta et al., 1993a). Globin chain composition was also studied by the reversed-phase high-performance liquid chromatography (RPHPLC) as already described (Manta et al., 1991; Masala and Manta, 1994). Percentages of globins were calculated by the chromatography data system ChromStar (BrukerFranzen Analytik, Bremen) connected to a personal computer.

Results Figure 1 shows the IEF pattern of lysates containing different a-chain variants in sheep of both the. Hb A and Hb B phenotypes, whereas Fig. 2 shows the dissociated globin

focusing of native Hb A (anodal) and Hb B (cathodal) tetramers a-globin chain alleles.

containing the

Hb polymorphism

549

of Chios, Cyprus, and Sardinia sheep

chains of selected lysates separated by the AUT-PAGE method. The presence of an undescribed P-chain variant migrating slightly anodal with respect to the pB chain was observed. This abnormal Hb has the same isoelectric point as the Hb B in the IEF gel. A conservative amino acid substitution, either of the neutral-to-neutral or charged-to-charged type, that occurred on a pB chain, may be the cause for this behaviour. Because the mutated Hb was found only in sheep of the Chios breed, it will be temporarily referred to as the Hb Chios. The sample analysed displayed 24 pBWhios and 2 pAlpChios heterozygotes and 3 homozygotes for the abnormal chain. Figure 3 shows the HPLC chromatogram of a lysate containing three different a-chains concurrently with the PA and pB chains. In accordance with data previously reported (Vestri et al., 1991, 1993), determination of the percentages of the different globins allowed a precise identification of the a-globin gene arrangement. These are listed in Table 1. Amounts of the crHischain as low as 1% can be detected by means of this HPLC method, thus enabling the identification of the quadru-

Fig. 2. AUT-PAGE of dissociated globin chains. By IEF, samples no. 1,2,4, and 5 resulted from the Hb BB phenotype and no. 3 resulted from the Hb AB phenotype. 2: heterozygote for the p’ and the PB chains; 4: homozygote for the novel px chain: 5: heterozygote for the px and the pB chains. Allelic a chains are also indicated.

,HEME

His 17%)

I

20

1

1

1

40 TIME IN MINUTES

I

60

I

I

80

I

I

100

(lml/min)

Fig. 3. HPLC chromatogram of the lysate of a sheep of the Hb AB phenotype, which is also the carrier of the three allelic a-globins. An asterisk denotes the position of the Px-globin chain.

550

E. Hadjisterkotis Table 1. Percentages

et a/.

rt SD of a-globins detected by HPLC in lysates from sheep with different haplotypes Percent cx-globins in lysate

Haplotype

n

I~Leu_lI~Leu/lcYLeu_Il(yLeu IQLeu_IlaLeu/l(lLeu_II~His IclLeu_lI(yH~s/IQLeu_llaHis laLeu_II~Leu~I(yAla_II~His I,Ala_II,His~I(yAla_II,His IaLeu_IIaHis[ICyAIa_IIaHis

I ~Leu_ll(XLeu/IaLeu_II~Lcu_IIl(yHa I(yLeu_II(XHis/laLeu_IIaLeu_III(IHis IaLeu_IIaLeu_lIIaHis/IaLeu_IIaLeu_IIIaHis

10 2 20 8 3 14 1 1

aLeU

100 81.5 63.6 49.9 32.0 93.4 76.1 87.1

* 1.4 ? 1.3 + 2.2 ” 1.1 ‘: 0.6

aMa

31.9 ? 2.6 63.2 f 1.4 32.7 t 1.1 -

aHis -

18.5 36.4 18.1 36.8 35.2 6.6 23.9 12.9

” f f f t 2

1.4 1.3 1.1 1.4 1.9 0.6

ple a-gene arrangement (Vestri et al., 1993). Rando et al., 1986). Due to this high degree None of the lysates, however, showed aHis of homology, repeated unequal crossovers are a common phenomenon that would prevent chain levels lower than 6%. the divergence of the two duplicated loci. As As shown in Table 2, Cyprus and Sardinia breeds have a (relatively) lower frequency of in the case of man (Goossens et al., 1980), variants whereas Chios breed has a high 0.40 crossover occurs fairly frequently in many sheep breeds and is also the cause of (at least) frequency of the ~y*‘~-ty~~~ arrangement. None showed the LY*~P mutation. The aLeu-aLe”-aHis 7 different haplotypes, including 2 types of triplications, a quadruplication, and a (never triplication, which is present in the Sardinian breed, was not observed among Chios and Cy- detected) single a-locus chromosome (Rando prus sheep. No quadruplications were ob- et al., 1986; Vestri et al., 1991). This study served. The PB globin predominates in all extends data obtained in sheep breeds from breeds, the PA gene frequency being lower (f North Europe and Italy indicating that the dif= 0.03) among Sardinian sheep, which also ferent cY-globin haplotypes are widespread but possess the previously described 0.08 fre- irregularly distributed (Vestri et al., 1981, quency of the pi chain (Manta et al., 1993a). 1983b). It has been observed that the “‘aHis In the Chios breed, the novel P-globin chain gene associated with the triplicated haplotype, has been observed with a rather high 0.24 gene although not detected in North European sheep, is relatively common in 4 Italian breeds frequency. including Sarda, which evolved completely isolated from the others (Vestri et al., 1981, Discussion 1983b; Rando et al., 1986). Our result indiIt is assumed that the loci coding for adult cates that the triplicated haplotype does not a-globin chains were in the duplicated state represent a characteristic of the Mediterraprior to mammalian radiation (-300 Myr ago) nean breeds because it appears to be absent (Zimmer et al., 1980) and that concerted evo(or rare) in the two, similarly isolated, breeds lution is responsible for the high similarity ex- of Chios and Cyprus. Cyprus sheep seem to isting between the duplicated Q genes of man, possess only the aA’a-~Hishaplotype whereas apes, goat and sheep (Zimmer et al., 1980; Chios sheep show a frequency of this arrangeLiebhaber et al., 1981; Schon et al., 1982; ment unusually higher than the aLe”-czHiSfrom which it is considered to have originated. The aAsp gene, which has been found only in triplicated and quadruplicated arrangements in the Table 2. The frequency of a-globin gene arrangements Altamurana, Sopravvissana, and Bergamasca and @globin alleles in three different sheep breeds Italian breeds and in Yugoslavian breeds Cyprus Sardinia Chios (Huisman et al., 1968; Vestri et al., 1983b, (n-l 24) (n = 258) Haplotype/allele (n = 66) 1991), was absent. I,Leu_II,Leu 0.55 0.88 0.86 The PA allele is more common in 4 North IdLeu_IIaH~s 0.05 0.08 European breeds than in Italian sheep. In the i ,AIa_ll,Hir 0.40 0.12 0.02 IaLeu_IIaLeu_II1aH~r 0.04 former, the average frequency is 0.48 whereas in the Italian it is 0.13, the lower (f = 0.03) 0.11 0.17 0.03 being observed in Sarda breed and the higher 0.64 0.83 0.89 0.08 (f = 0.26) in Maremmana (Vestri et al., 1981; 0.24 Manta et al., 1993a; this paper). This study

Hb polymorphism

of Chios, Cyprus, and Sardinia sheep

indicates that this gene also has a low frequency in other Mediterranean breeds. Of interest is the observation of a novel variant of the /3 chain having a somewhat high 0.18 gene frequency. Though preliminary, data collected shows that the variant tetramer is “electrophoretically silent” with respect to Hb B, and that the globin has only slightly different properties than the pB chain. This suggests that the variant is due to a conservative amino acid substitution that occurred on a pB chain. This observation increases the number of different sheep P-chain variants to seven. As already pointed out in describing sheep Hb I (Manta et al., 1993a, 1993b) and buffalo Hb BS (Di Luccia er al., 1991), it is reasonable that the application of biochemical methods such as those described in this study will increase knowledge on Hb polymorphism. The different physiological properties of the high-oxygen affinity Hb A (linked to the production of the somewhat higher oxygen affinity Hb C) and of the low-oxygen affinity Hb B, suggested that natural selection cannot be excluded in reducing the PA gene in domesticated sheep (Masala ef al., 1991). Nonetheless, it should also be considered that a reproductive advantage of the Hb B type sheep has already been suggested (Evans and Turner, 1965) and that sheep have been the object of a high selective human pressure. Therefore, both natural and artificial selection could be responsible for the low Hb A frequency in Mediterranean sheep breeds, as well as for the differences observed at the level of a-globin haplotypes. Acknowledgements-This

work was supported by funds by the R. A. S. (Progetto Ambiente) and by MI U. R. S. T. (60%). The authors exnress their thanks to Mr. I. Chimonides, Head of the Athalassa Government Farm, for allowing them to collect blood samples from Cyprus local and Chios sheep, and Mr. A. Rousias, Head of the Pafos Government Farm, for allowing them to collect samples from Chios sheep.

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