www.elsevier.com/locate/cbpb

Comparative Biochemistry and Physio

The alpha chains of goat hemoglobins: Old and

new variants in native Apulian breeds

Elisa Pieragostini a,*, Rosario Rullo b, Andrea Scaloni b, Grazia Bramante a, Aldo Di Luccia c

aDepartment of Engineering and Management of the Agricultural, Livestock and Forest Systems�University of Bari, 70100 Bari, ItalybISPAAM�National Research Council, 80147 Napoli, Italy

cDepartment of Animal Production�University of Bari, 70100 Bari, Italy

Received 18 January 2005; received in revised form 29 April 2005; accepted 5 May 2005

Available online 11 July 2005

Abstract

Blood samples were collected from 324 goats belonging to the native Apulian breeds Garganica and Jonica; 60 Alpine goats were also

sampled to serve as a comparison. Hemoglobin phenotypes were analyzed with isoelectric focusing in a pH range of 6.7–7.7. Heterogeneity

of globin chains was evidenced both by AUT-PAGE and RP-HPLC. The primary structure of four alpha globins was analyzed by combined

mass spectrometry approaches. Two of these globins had never been sequenced before. One was a new alpha variant, an allele of the HBA1A

gene from which it differed for the mutation A26T and has been registered with a low frequency only in Apulian breeds; the other was a

globin encoded by the HBA2 locus, whose primary structure was previously derived from the corresponding gene. The two alleles recorded

at the HBA2 locus presented a different frequency in the three breeds but may be considered to be generally rather common. Notwithstanding

the sample size no goat was found to exhibit HbA1B. The Authors discuss their findings in the light of the results reported by other

researchers and argue that, in spite of what had been inferred in pioneer works on goat hemoglobins, HBA1B is not a common allele.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Goat hemoglobin; a-chain; Primary structure; Non allelic variants; Gene expression; Gene frequency; Apulian native breeds; Supernumerary

haplotypes; CpG site; Hot spot

1. Introduction

In Apulia, the region covering the heel of the boot-

shaped Italian peninsula, there are 8 recognized native

breeds of farm animals. The rather harsh pedoclimatic

conditions as well as the selective pressure of endemic

hemotropic parasites have yielded genetic pools that are

generally rustic and tolerant to the diseases caused by these

parasites. Phenotypic variability induced by parasites is a

matter of fact, as impressively exemplified by the high

number of hemoglobinopathies in human populations living

in malaria-endemic areas (Evans and Wellems, 2002). The

past decade has registered remarkable advances in the study

1096-4959/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.cbpc.2005.05.005

* Corresponding author. Dipartimento PROGESA-Facolta di Agraria, via

Amendola 165/A, 70126 Bari, Italy. Tel.: +39 080 5442842; fax: +39 080

5442504.

E-mail address: pierelis@agr.uniba.it (E. Pieragostini).

of hemoglobin polymorphism in Apulian livestock (Pier-

agostini et al., 1994; Scaloni et al., 1998; Pieragostini et al.,

2000) and a special focus has been the peculiar hemato-

logical features of native breeds which seem to be related to

their general ability to thrive in endemic TBD areas

(Pieragostini and Petazzi, 2000). In particular, extra alpha

genes were found both in sheep and horses (Pieragostini et

al., 2002, 2003).

The alpha globin genes are a relevant example of gene

families which have arisen from a single gene by gene

duplication events (Hardison, 1998). In mammals, non-

allelic loci produce unequal amounts of alpha globin which

may give rise to quantitative polymorphism with the

upstream gene being the most efficient; in particular, as

observed in sheep, the percent gene efficiencies in the aa

and aaa gene haplotypes, from the 5Vto the 3Vend, are about32–18 and 30–14–6, respectively (Vestri et al., 1991). A

recent investigation on Apulian native sheep compared the

logy, Part B 142 (2005) 18 – 27

Hemoglobin Tetramers Globin chain

responsible

for the Hb

name

References

Protein sequence Nucleotide

sequence

HbA a2Ah2

A IaA Huisman et al.,

1968

Schon et al.,

1982HbB a2Bh2

A IaB

HbC a2Ah2

C Ch Huisman et al.,

1967

Haynes et al.,

1980a2Bh2

C

HbD a2Ah2

D hD Adams et al.,

1968

Pirastru et al.,

2000

HbDMalta a2Ah2

D Malta hD Malta Bannister et al.,

1979

Pirastru et al.,

2000

HbE a2Ah2

E hE Wrightstone

et al., 1970

Pirastru et al.,

2000

HbF a2Ag2 g Kleinschmidt and

Braunitzer, 1982

Haynes et al.,

1980a2Bg2

E. Pieragostini et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 18–27 19

total amount of a�globin chains with the total h�chains and

found that the a /h ratio appears to be linearly related to the

number of a�globin genes; specifically, the more genes an

individual possesses, the greater the imbalance. The related

hematological pattern, which mimics a thalassemia-like

syndrome, was suggested to impart some protection against

endemic TBD parasites. This is analogous to what happens

in thalassemic red blood cells where malaria parasite growth

is unimpaired (Nagel, 1990). In Apulia, the relatively high

frequency of individuals characterized by unusual extra

a�globin genes may then be taken as a telltale signature of a

positive selection (Pieragostini et al., 2003).

Based on these considerations, we have focussed our

attention on the alpha globin genetic system in Apulian

native goat breeds. Unfortunately since it was first reported,

hemoglobin polymorphism in goat populations appeared to

be a complex issue.

The first biochemical studies were performed in the late

sixties and seventies, directly on the primary structure of

five h-and three a-globin chains (as reviewed by Garrick

and Garrick, 1983). Investigations into fetal hemoglobin

highlighted the existence of monomorphism at the gamma

gene level while heterogeneity was related to the presence of

polymorphic alpha globins (Wilson et al., 1969); analo-

gously, hemoglobin switching to HbC in adults during

anemic episodes is known to conceal polymorphism at the

beta globin level because of the substitution of all beta

variants by the beta C globin chain encoded by the dormant

gene Ch. In the eighties research by the Cincinnati Group

thoroughly explored the structure and evolution of the h-globin gene clusters and of the relative developmentally

regulated genes, hF, Ch and hA (Lingrel et al., 1985). The

primary structure of a fourth a-globin was deduced from the

nucleotide sequence (Schon et al., 1982) followed by the

nucleotide sequence of the embryonic alpha globin gene

(zeta) and by the evolutionary analysis of the complete

alpha globin cluster (Wernke and Lingrel, 1986).

Parallel investigations undertaken by European research-

ers with high resolution techniques provided new insights

into hemoglobin polymorphism in goat populations (Tucker

et al., 1983; Braend et al., 1987a,b). Using IPG electro-

phoresis, which is a very sensitive technique, the HbA band

was found to be a mixture of tetramers, differing for the beta

chains produced by the hA2, hA4, hA6, hA8 beta alleles

(Braend et al., 1987a) and simply defined on the basis of

their different electrophoretic mobility. In the nineties no

steps were taken to identify the electrophoretic variants

found so far but in 2003 Pirastru et al. analyzed the DNA

sequences of four new beta genes (hL125E, hK104R, L125V,

hQ87H, K104R, hK104R), possibly encoding for the beta globin

allele described by Braend et al. (1987a).

Thus, present knowledge of goat hemoglobin may be

summarized into two essential points. 1) As to the alpha

globin system, the protein sequence of four non-allelic

alpha-chains has been described in the literature; two of the

chains are encoded by the 5Vupstream gene-IaA and IaB-and

the other two by the 3Vdownstream gene (Huisman et al.,

1967; Schon et al., 1982). 2) For the beta globin system, the

protein sequence of four allelic (hA, hE, hD and hD Malta)

and two non-allelic globin genes (hF and Ch) can be

numbered. Only seven of the possible tetramers resulting

from the combination between such a heterogeneity of alpha

and beta globins are identified by a name, i.e., HbA, HbB,

HbE, HbD, HbDMalta, HbF and HbC; the former two

derived their name from the respective alpha globin, all the

others from the respective beta globin. These pieces of

information are catalogued according to the nomenclature

for polypeptide chains as described by Huisman and

Schroeder (1971) and listed in the following scheme.

In particular, the findings concerning HbB appear to be

controversial because HbDMalta and HbB exhibit a similar

electrophoretic behavior and, in the absence of other

analytical investigations or biological trials (such as

response to anemic stress or developmental investigations),

one cannot exclude that HbDMalta (Bannister et al., 1979)

may have been misidentified as HbB. In other instances,

details from quantitative evaluations offer indications as to

the nature of the recorded polymorphism; in fact, due to the

already mentioned characteristic differences in the average

levels of globin production in a�loci, the relative densities

of the electrophoretic bands can deduce many genotypes.

Hence, even supernumerary haplotypes, where present, may

be detected. The aim of this work, therefore, is to expand the

evidence provided so far by thoroughly investigating alpha

globin polymorphism in two native Apulian goat breeds, the

Garganica and the Jonica, and by evaluating the results in

the light of literature.

2. Materials and methods

2.1. Animals

Blood samples were collected from 324 individual goats

(Capra hircus) belonging to the Garganica and Jonica

E. Pieragostini et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 18–2720

breeds and reared in pure breeding on different farms. A

total of 60 individual samples from a flock of over 1000

pure Alpine goats were also analyzed to serve as a

comparison since this breed is present throughout the world.

Blood samples were processed through standard procedures

as described in Di Luccia et al. (1991).

2.2. Isoelectric focusing

Lysates were analyzed by isoelectric focusing (IEF) in a

narrow pH range (6.7–7.7) as already described by Di

Luccia et al. (1991) to assess the Hb phenotypes. Gel slabs

were scanned with a computerized Ultroscan XL enhanced

laser densitometer equipped with gelscan 2.0 software from

Pharmacia-LKB (Uppsala-Sweden).

2.3. Reverse phase HPLC

Globin chain separation was performed by a reverse

phase HPLC procedure described by Manca et al. (1991).

Hemoglobin solutions were brought to a final concentration

of 70–100 Ag and filtered through 0.45 Am HV filters

(Millipore, Bredford, MA). Fifteen AL of globin solution

were then applied onto a large pore Vydac C4 column

250�4.60 mm, (300A) (The Separation Group, Hesperia,

USA). The chromatogram was developed with a linear

gradient between solvent B (60% acetonitrile containing

0.1% trifluoroacetic acid ) and solvent A (20% acetonitrile

containing 0.1% trifluoroacetic acid ) at a flow rate of 1 mL/

min. The gradient was 48–65% developer B in 85 min. The

HPLC equipment was a Beckman Gold System.

2.4. Acid-Urea Triton X-100 polyacrylamide gel electro-

phoresis (AUT-PAGE)

Acid-Urea Triton X-100 polyacrylamide gel electro-

phoresis (AUT-PAGE) of the globin chains was carried

out according to Di Luccia et al. (1991).

2.5. Structural analysis

Electrospray mass spectra of intact globins were

recorded by using an API-100 single quadrupole mass

spectrometer (Applied Biosystems, USA) equipped with an

atmospheric pressure ionization source, as reported else-

where (Scaloni et al., 1998). Mass calibration was

performed by means of the multiply charged ions from a

separate injection of horse heart myoglobin (molecular

mass 16,951.5 Da). All masses have been reported as

average values.

Trypsin and endoproteinase Asp-N digestions of indi-

vidual globin chains were performed in 0.4% ammonium

bicarbonate, pH 8, at 37 -C, overnight, using an enzyme to

substrate ratio of 1 :100 (w/w).

Peptide mixtures were loaded onto the MALDI target

using the dried droplet technique and a-cyano-4-hydroxy-

cinnamic as a matrix and analysed with a Voyager-DE PRO

mass spectrometer (Applied Biosystems, Framingham,

USA). Internal mass calibration was performed with

peptides deriving from protease autoproteolysis. The mass

spectra were acquired in either reflectron or linear mode

with delayed extraction. Post-source decay fragment ion

spectra were acquired for intense signals after isolation of

the appropriate precursor by using timed ion selection, as

already reported (Scaloni et al., 1998).

Edman degradation was performed using a Procise 491

protein sequencer (Applied Biosystems, Foster City, USA)

equipped with a 140 C microgradient apparatus and a 785A

UV detector for the automated identification of phenyl-

thiohydantoin amino acids.

2.6. Statistics

To reduce bias in the quantitative evaluation of gene

products and consequent differential gene expressions, only

homozygous phenotypes at the HbA2 locus were selected

from the data set obtained by densitometric evaluation of the

Hb bands, and least square means with their standard

deviations were calculated.

The alpha gene frequencies were estimated assuming

equilibrium conditions and statistically assessed with the

v2 test.

3. Results

3.1. Isoelectric focusing of hemoglobins

Polyacrylamide gel isoelectric focusing in a narrow pH

range (6.7–7.7) of goat hemoglobins carrying different a

and h globin chains showed various hemoglobin band

patterns (Fig. 1). Two main electrophoretic zones were

recognized in the gel; Fig. 1 clearly shows that each major A

and/or B zone consists of stronger forward bands and one or

two weaker backward bands.

A more complex hemoglobin pattern was observed in

lanes 4 and 5 where the minimal difference of isoelectric

points observed (0.04 pH units) suggested the presence of

globin variants bearing non-charged amino acid substitu-

tions. Resolving power reduction towards the cathode was

due to gradient drifting for an electro-osmotic effect.

3.2. Densitometric evaluation of Hb bands

Table 1 shows the output of the HBA1 and HBA2 genes

as estimated from the concentrations of different alpha

globin chains on the basis of the densitometric evaluation of

their relevant Hb PAGIF bands (Fig. 1). The values

observed have been compared to the values expected on

the basis of those reported in the literature and those of

previous experiences with sheep and buffalo (Pieragostini et

al., 2003; Iorio et al., 2004).

Table 1

Output of alpha I and alpha II genes in goat as compared with buffalo and

sheep

Specie N Ia IIa Ia / IIa Reference

Goat 120a 32.02T2.59 18.19T1.44 Present paper

21 36.32T2.73b – Deduced from

Braend et al., 1987b

Buffalo 362 32.56T3.12 17.50T2.6 1.86 Iorio et al., 2004

Sheep 30 32.20T2.68 17.60T1.91 1.83 Pieragostini et al.,

2003

a To reduce bias, only densitometric data of homozygotes at alpha 2 locus

were considered.b This finding refers to the so-called reverse type that supposedly takes

into account the expression level of a single Ia gene.

Fig. 1. Polyacrylamide gel isoelectric focusing in narrow pH range, 6.7–

7.7, of goat hemoglobins carrying different a and h globin chains. Sample

1: two non-allelic alpha globins (IaA and IIaT) and one beta globin (hA )

result in two hemoglobin bands, Ia2Ah2

A and IIa2Th2

A; sample 2: two non-

allelic alpha globins (IaA and IIaT) and two beta globins (hA and hDMalta)

result in a twin couple of hemoglobin bands, the former couple migrating

in the zone A (Ia2Ah2

A and IIa2Th2

A) and the latter one in zone B (Ia2Ah2

DMalta

and IIa2Th2

DMalta); sample 3: one non-allelic alpha globins (IaA), two allelic

alpha globins (IIaA and IIaT) and two beta globins (hA and hD) result in a

twin triplet of hemoglobin bands, the former triplet migrating in the zone

A (Ia2Ah2

A, IIa2Th2

A and IIa2Ah2

A) and the latter one in zone B (Ia2Ah2

D, IIa2Th2

A

and IIa2Ah2

D); sample 4: two allelic alpha globins (the wild type IaA and the

new variant IaT), a non-allelic alpha (IIaA) and two beta (hA and hE) result

in six hemoglobins all migrating in the zone A; sample 5: the same alpha

globins as in sample 4 (IaA, IaT and IIaA) but different beta globins (hE

and hD) result in six hemoglobins among which the three tetramers

containing the hE globin migrate in the zone A, the others containing the

hD globin in the zone B.

E. Pieragostini et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 18–27 21

No patterns showing unusual quantitative polymorphism

were found, suggesting that only common duplicate alpha

arrangements were present in the sampled populations.

3.3. Electrophoretic and chromatographic analyses of

globins

The results of the AUT-PAGE and RP-HPLC analyses of

two hemoglobin phenotypes relevant for the purposes of our

interests are shown in Fig. 2. Both separation techniques are

primarily based on hydrophobicity and well define the

tetramer composition, displaying the differences between

the a and h globins. Fig. 2A shows the AUT-PAGE and RP-

HPLC globin pattern of one of the phenotypes exhibiting

the new alpha variant. As to the AUT-PAGE, it can be

observed that, at the alpha globin zone, two major bands, as

well as a minor one are detectable. The IaA globin chain is

slower than the new alpha variant, attesting a primary

structure change with a decrease in hydrophobicity. The IEF

results excluded the substitution of a charged amino acid so

the effect recorded can be ascribed to a neutral to polar

amino acid substitution. RP-HPLC analysis of the above

samples confirmed the results obtained by AUT-PAGE.

In the beta globin zone, a co-migration of hA ed hDmalta

can be observed since in an acid environment the Asp

residue at position 69 in the hDmalta is not charged and

behaves like a neutral or polar amino acid. Conversely, these

two beta chains were well resolved by RP-HPLC (Fig. 2B).

RP-HPLC analysis of the above samples confirmed the

results obtained by AUT-PAGE. Furthermore, direct molec-

ular mass measurement by ESI-MS unambiguously ascer-

tained the nature of each globin component. Globin identity,

defined by this combined approach, definitively demon-

strated the occurrence of a new a-globin variant in goat

hemolysates, together with the ubiquitous absence of IaB.

As to the genes responsible for different products, the

evaluation of the relative peak areas and retention times

confirmed that the slower-migrating proteins are encoded by

the upstream a-globin gene while the faster-migrating

proteins are encoded by the downstream a-globin gene.

3.4. Structural analysis

AMALDI-TOF peptide mass mapping approachwas used

to determine the primary structure of this new variant and

confirmed the nature of the other a-globins observed.

Analysis of tryptic digests highlighted the strict structural

homology between IaA and the new variant, suggesting that

the amino acid differences were confined to peptide 17–31,

as shown in Fig. 3A and B. These results were confirmed by

independent mass mapping analysis on endoprotease Asp-N

digests (data not shown). Post-source decay MALDI-TOF

analysis of the mutated peptide at m/z 1465.6 definitively

demonstrated the occurrence of the A26T substitution in this

new a-globin variant (Fig. 3C). These results were confirmed

by Edman degradation analysis on electroblotted protein

samples. The nature of this amino acid replacement was in

perfect accordance with the relative migration observed in

AUT-PAGE and RP-HPLC. Table 2 summarizes the data

obtained for the different goat a-globins reported in this study

and compares them with those reported in the literature.

3.5. Nomenclature

On the basis of the structure analyses and in agreement

with what had been suggested in previous papers on

sheep and buffalo (Pieragostini et al., 2003; Iorio et al.,

2004) and in the guidelines for gene nomenclature in

Fig. 2. AUT-PAGE and HPLC specific chromatographic profile: in panel A one non-allelic alpha globins (IIaA), two allelic alpha globins (IaA and IaT) and two

beta globins (hA and hD); in panel B, two allelic alpha globins (IIaT and IIaA), a non-allelic alpha (IaA) and two beta (hA and hDMalta).

E. Pieragostini et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 18–2722

ruminants (Andresen et al., 1995), we suggest placing the

superscript on the right with the capital letter indicating

the point mutation distinguishing the alleles to describe

the as-yet unnamed goat alpha genes (Table 3). As to the

nomenclature for polypeptide chains we followed the

same pattern as described by Huisman and Schroeder

(1971).

3.6. Gene frequencies

Table 4 shows the estimated gene and genotype

frequencies at the HBA1 and HBA2 loci in the Garganica

and Jonica breeds and compares them with those of the

Alpine goats. In Alpine goats HBA1 appears to be

monomorphic for HBA1A, while in Garganica and Jonica

Fig. 3. Mass spectrometric characterization of the new a-globin variant isolated in Apulian goat hemolysates. (Panel A) MALDI-TOF peptide mapping analysis

of IaA following trypsin digestion. (Panel B) MALDI-TOF peptide mapping analysis of IaT following trypsin digestion. (Panel C) Post-source decay MALDI-

TOF analysis of the mutated peptide 17–31 occurring in IaT.

E. Pieragostini et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 18–27 23

goats the polymorphism concerned only the additional

presence of very rare HBA1T alleles, recorded with a

frequency value of 0.0028 (Garganica) and 0.0106 (Jonica).

Notwithstanding the sample size and the considerable

Table 2

Structural properties of goat a�globins

Globin Mutation Exp. Mass (Da)

IaA

IaT A26TIaB D75YIIaA G19S; L113H; N115SIIaT G19S; A26T; L113H; N115S

degree of variability recorded, no subjects carrying the

HBA1B allele were found.

In the HBA1 locus, HBA2T seemed to be the most

common allele in the Apulian breeds whereas in the Alpine

Theor. Mass (Da) Reference

15033.5T0.2 15033.2 Huisman et al., 1968

15063.4T0.3 15063.2 Present paper

– 15081.3 Huisman et al., 1968

15060.1T0.3 15060.2 Huisman et al., 1968

15089.9T0.2 15090.2 Present paper

Table 3

Nomenclature of alpha globin genes and products

HBA1 Locus HBA1 Locus

Gene Symbol HBA1A HBA1B HBA1T HBA2A HBA2T

Globin symbol IaA IaB IaT IIaA IIaT

E. Pieragostini et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 18–2724

goats both HBA2T and HBA2A were present with almost

the same frequency.

The alleles and genotypes in each breed sample were in

HW equilibrium.

4. Discussion

The main information provided by our results concern: a)the lack of evidence that alpha globin extra genes are

present; b) the identification of a new alpha globin variant

and the primary structure of the globin encoded by the alpha

globin gene sequenced by Schon et al. (1982); c) the

quantitative data to deduce the output of alpha genes and to

assign the new variant to the appropriate gene; and d) the

allele frequencies at the alpha loci.

4.1. Alpha globin genes and chains

In Table 2, the values recorded in goats have been

compared with the other species for the HbA1 and HbA2

gene outputs. Based on the size of the data set and the fact

that the range of variability records is comparable to that

reported previously for sheep and buffalo with the same

duplicated gene arrangements (Pieragostini et al., 2003;

Iorio et al., 2004) the values in Table 2 may confidently be

defined as appropriate levels of gene expression for goats,

and more generally, the alpha gene expression gradient for

goats is confirmed to be close to a 38% output reduction of

the 5Vupstream gene.

On the basis of the quantitative evaluation of the PAGIF

bands and the RP-HPLC peaks, it can be inferred that the

new alpha variant found is encoded by an allele of the

HBA1A gene from whose product it differs for the

substitution of alanine with threonine at position 26. This

result is consistent with the results obtained through previous

analytical approaches with the side chain hydrophobicity

values for alanine and threonine amounting to 0.616 and

0.359, respectively (Black and Mould, 1991). The difference

of 0.257 between the two justifies the electrophoretic and

chromatographic behavior of the corresponding proteins.

Table 4

Estimated gene and genotype frequencies both HB1A and HB2A loci in Gargani

Breed Herds Genotypes at HBA1 locus Genotypes at H

AA AT AA AT

Garganica 5 182 1 13 63

Ionica 7 141 3 8 19

Alpine 1 60 0 17 25

The substitution of alanine with threonine at position 26

is present not only in the newly detected Ia variant but also

in the sequence of the globin chain encoded by one of the

alleles at the IIa locus. Yet, considering the differences in the

amino acid sequences of the two chains, this is not the case

of a tandem repetition of individual gene copies. Thus the

phenomenon cannot be included amongst the genomic

mechanisms of turnover capable of achieving sequence

homogeneity, such as gene conversion or unequal crossing-

over between individual copies of the gene family. The

presence of the same mutation in the two non-allelic genes

in goats is more likely to be associated with independent

events since the change of alanine to threonine at position

26 involves a CpG dinucleotide which is a mutation hot-

spot. According to Perutz (1990), however, the presence of

the highly mutagenic CpG dinucleotide is a necessary but

not a sufficient condition for a mutation to occur. Only

CpGs methylated in the presence of a specific methylase

(Roy and Weissbach, 1975) act as hot spots because

deamination converts 5-methylcytosine to thyamine which

then pairs with adenine.

Position 26, as may be observed by analyzing the amino

acid sequences of mammalian a-globin chains present in the

data bank (ExPASy Proteomics Server: http://www.expasy.

org/), is constantly occupied by an alanine residue. The

evidence reported above suggests that only goat Ia-and Ia-

globin genes contain methylated CpG, thus producing the

Ala26Thr mutation. However, one cannot exclude that this

conservative substitution exists in other species because it

may have escaped detection since it does not interfere with

structure and is not electrophoretically visible by conven-

tional methods.

4.2. Frequency data

The focal point of a discussion on frequency data is the

comparison between populations to measure genetic dis-

tances or check latitudinal or altitudinal trends so that the

adaptive significance of genes, such as beta B in sheep

(Agar et al., 1972; Pieragostini et al., 1994), may be

accounted for. Unfortunately, there are not many data in the

literature to compare with the variability recorded in this

investigation on goats.

As to frequency values at the HBA1 and HBA2 loci, the

results concerning the HBA2 alleles in native Apulian goats,

where IIaT is the most frequent form, are very similar to

those reported by Pirastru et al. (2003) in Sardinian breeds

ca and Jonica breeds compared to Alpine one

BA2 locus Alleles at HBA1 locus Alleles at HBA2 locus

TT HBA1A HBA1T HBA2A HBA2T

107 0.997 0.0028 0.2432 0.7568

90 0.989 0.0106 0.1736 0.8264

18 1 0 0.4917 0.5083

E. Pieragostini et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 18–27 25

and by Nyamsamba et al. (2003) in Mongolian goats whereIIaT practically seems to be the only form present. By

contrast both alleles are equally present in the Alpine goat

population-at least as far as our sample is concerned. As to

the HBA1 alleles, the IaT variant seems to be very similar

both for analytical behavior and frequency to that found in

Sardinian goats (Pirastru et al., 2003) but the most intriguing

point is the absence of HbA1B (Table 4). Since the findings

on HbA1B in various populations, as already pointed out,

seem to be rather controversial, we reconsidered our results

in the light of those found in the relevant literature. It is

worth remembering that in 1979 Bannister et al. highlighted

that the Ia chain variant HbB was absent in Maltese goats.

Unfortunately, several of the investigations inferring the

occurrence of HbB on the basis of electrophoretic evidence

did not confirm the identification with structural techniques

or biological trials (Nguyen and Bunch, 1980; Tunon et al.,

1987; Igarashi et al., 2000). In other cases, such as in Sartore

et al. (1981) or Fesus et al. (1983), the conclusions drawn by

the Authors are not entirely corroborated by well-grounded

arguments. Sartore et al. (1981) analysed 398 adults and 568

kids of the Saanen breed by starch gel electrophoresis and

found both HbA and HbB in the adults while the kids were

monomorphic for HbA; considering that the frequency

value assumed for HBA1B was 0.11 in the adults, it is rather

surprising that no HBA1B genes were found in such a large

sample of kids. Analogously, in their evaluation of

hemoglobin polymorphism in native Hungarian breeds

Fesus et al. (1983) took into account the presence of a

certain number of HbB goats but, in the mean time,

concluded that all the newborn kids exhibited a single type

of hemoglobin. In the erythrocytes of newborn kids only

foetal hemoglobin can be found, thus polymorphic alpha

genes, where present, should be evident while polymor-

phism at the beta locus is known to become apparent only

after the start of adult beta chain synthesis. Hence, whether

the lack of HbB in the kids studied was a sampling effect or

the HbB in the adults was an effect of misidentification

based on the analytical system remains a moot question.

In other cases an initial misidentification of HbB was

recognized and corrected by the biological trials. Tucker et

al. (1983) bled their supposed AB and BB goats but, rather

surprisingly, they found them to produce an HbC identical

to that produced by AA goats. Real biallelism at HBA1

would have produced two different tetramers, namely

a2ACh2 and a2

BCh2. The presence of the same band proved

that the polymorphism was due to allelism at the h locus

and particularly that the HbB band (a2Bh2

A) was mistaken for

that of the HbD (a2Ah2

D). In particular, IaB differing from

IaA for the amino acid substitution D75Y and hD and hD

differing from hA, respectively for a D21H and D69G, it

implies that the corresponding hemoglobins exhibit almost

the same electrophoretic mobility at alkaline pH. Thus, it is

possible that Hb D and DMalta may be mistaken for HbB

when there is no unambiguous identification. In the case of

Hb D the confusion can be generated only at pH above 7

because titration and the loss of charge of the His residue—

whose pKa is 6-can be observed in an alkaline environment.

The existence of the HbB seems to be unquestionable.

Huisman et al., 1967, 1968 extensively documented

biallelism at the Hb1A locus. Electrophoretic, chromato-

graphic and structural analyses and biological trials pro-

vided evidence for the existence of the A and B alpha

variants. On the basis of these results Huisman et al. (1967)

suggested that goat hemoglobin polymorphism was similar

to that of the buffalo in that it was due to variations in the

alpha globin genetic system. The only weak point in this

argument is that structure analyses of the alpha chain variant

HbB were all performed sampling the blood of a single goat,

namely Huisman’s goat n-5 of an unidentified breed, while

the analytical procedures and subsequent results seem to be

indisputable.

Subsequently John and Barnabas (1978) sampled goats

from a local slaughterhouse in India and found goats that

were phenotypically HbA, HbAB and HbB. The identi-

fication was based on starch gel electrophoresis of

hemoglobins and DEAE-Sephadex chromatographic sepa-

ration of globins.

Actually, Braend might have found an HbB goat when

analyzing an experimental herd in Norway. In their paper of

1987b, Braend et al. described the presence of ‘‘hemoglobin

reverse phenotypes’’ detected by IEF with Immobiline gels

at a pH gradient of 6.9–7.5. The crucial point is that this pH

range reveals only tetramers which normally migrate into

the A zone, while potential Ia2Bh2

A (amounting to 30% of the

total Ia2h2 concentration) may remain undetected. In this

case, the zone A may be characterized by weaker forward

bands (30% of the Ia2Ah2 tetramers) and stronger backward

bands (40% of the IIa2h2 tetramers). This view seems to be

supported precisely by the findings reported in Braend et al.

(1987a). In this paper, Braend, together with Tucker and

Clarke, examined 150 Norwegian goats by PAGIF in a pH

range of 6–8 and found 5 HbAD individuals; in order to

resolve the variability recorded in the A zone, the remaining

HbA goats were analyzed only by Immobiline gels and

curiously no reverse phenotypes were found! It is our

opinion that Braend et al. (1987b) simply did not consider

the possibility of a IaB allele, chose a system a priori which

did not permit the detection of HbB tetramers and were then

biased in evaluating the results of the screening.

Finally, Johnson et al. (2002) found HbB in Omani goats.

Although there are no structural analyses or biological trials,

the densitometric data of the Hb bands suggested that in one

of the three breeds the IaB gene was rather frequent.

Evidence of this conclusion was provided by the AB

phenotypes which exhibited two-band patterns differing in

the relative band intensities (one with 67% A and 33% B

and the other with 67% A and 33% B). The analytical

procedure they used does not permit separation of different

Ia and IIa in the A zone. Thus, considering the data in Table

2, the A band is supposedly composed by the tetramers

encoded both by the Ia genes and IIa genes, with relative

E. Pieragostini et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 18–2726

efficiencies of about 32 and 18, respectively. This entails

that the truly AB Omani goats are only those exhibiting the

67:33 band pattern; 67 being equal to the percent gene

efficiencies of both IIa genes plus that of the single IaA

genes (�36%+�32%), while 33% is equal to the percent

gene efficiencies of the single IaB gene. The goats

exhibiting the 33:67 band pattern reasonably seem to be

BB where 33 is equal to the percent gene efficiencies of twoIIa genes, while 67 is equal to the percent gene efficiencies

of the two IaB genes. The same type of misinterpretation the

results may have occurred in Canatan and Boztepe (2000)

who did not find BB goats in their populations, though a

relatively high number of heterozygotes was recorded in

both cases.

The above considerations suggest that the IaB is not a

common allele. Thus, in contrast with the conclusions

drawn by Huisman et al. (1967), hemoglobin polymorphism

in goats is not similar to that of the buffalo but to that of the

sheep where the rare allele, IaD, has been found only in

some European breeds (Tucker, 1981) and particularly in

Apulian native sheep (Pieragostini et al., 1994, 2003)

although the suggested similarities with sheep do not

include the presence of supernumerary haplotypes for the

time being.

Acknowledgements

We are indebted to Dr Athina Papa for her accuracy in

revising the English of the manuscript. This research has

been funded by the Italian Ministry for University.

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