Interallelic recombination is probably responsible for the occurrence
of a new a
s1
-casein variant found in the goat species
Claudia Bevilacqua
1,2,
*, Pasquale Ferranti
3,4
, Giuseppina Garro
3,4
, Cristina Veltri
1
, Raffaella Lagonigro
1
,
Christine Leroux
2
, Emilio Pietrola
`
1
, Francesco Addeo
3,4
, Fabio Pilla
1
, Lina Chianese
3
and Patrice Martin
2
1
Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Facolta
`

genomic level. The M protein displays the slowest elec-
trophoretic mobility of the a
s1
-Cas variants described so far.
MS and automated Edman degradation experiments
showed that this behavior was due to the loss of two phos-
phate residues in the multiple phosphorylation site (64S
P
-S
P
-
S
P
-S
P
-S
P
-E-70E) consecutively to a Ser fi Leu s ubstitution
at position 66 of the peptide chain (64S-S
P
-L-S
P
-S
P
-E-70E).
This was confirmed by sequencing a genomic DNA frag-
ment encompassing exon 9 where the 8th codon (TCG) was
shown to be mutated to TTG. Sequencing of amplified
genomic DNA segments spanning the 5¢ and 3¢ flanking
regions of each exon allowed us to identify 23 single nuc-

and j [3]. They have be en
mapped on chromosome 6 in cattle and goats [4,5]. The
a
s1
-casein locus (a
s1
-Cas) is characterized in the goat by a
polymorphism, the main feature of which is to be qualit-
ative as well as quantitative. Indeed, more than 11 alleles
have so far been characterized [6], distributed among seven
different classes of protein variants (a
s1
-CasA to a
s1
-CasG),
associated with four levels of expression ranging betwee n 0
(a
s1
-Cas0) and 3.5 g ÆL
)1
(a
s1
-CasA, B, and C) per allele.
Whereas the a
s1
-CasE variant, which is 199 amino-acid
residues in length, only differs from variants A, B and C by
single amino acid substitutions [7], the F variant displays an
internal deletion of 37 residues [8], leading to the loss of a
hydrophilic cluster of five contiguous phosphoseryl resi-

is considered to be the original t ype i n
goat because it shows the closest homology to its bovine
and ovine counterpart [6].
The distribution of these different alleles or variants has
been investigated in a great variety of breeds a nd popula-
tions [6,10–13]. Breeds from the Mediterranean a rea usually
display a high f requency of ‘strong’ alleles (mainly A and B).
However, local and now rare breeds generally do not follow
this rule and are often the source of rare ‘germoplasms’.
Three novel a
s1
-Cas variants (H, I and L) have been
identified by Chianese et al. [14] in southern Italian goat
populations. More recently, a further novel and rare
Correspondence to P. Martin, Laboratoire de Ge
´
ne
´
tique biochimique
et de Cytoge
´
ne
´
tique, INRA, Domaine de Vilvert,
78 352 Jouy-en-Josas, France. Fax: + 33 1 34 65 24 78,
Tel.: + 33 1 3 4 6 5 25 82, E-mail: mart [email protected]
Abbreviations: a
s1
-Cas, a
s1

-CasM allele. Extensive comparisons of
these sequences with those of previously characterized
alleles have allowed the identification of additional poly-
morphic sites, the arrangements (haplotypes) of which
strongly suggest an interallelic recombination (or a gene
conversion) event at the origin of the a
s1
-CasM allele. This
is, to our knowledge, t he first hypothesis o f a genomic
recombination event to account for genetic polymorphism
at a locus encoding a milk protein.
MATERIALS AND METHODS
Animals
A total of 147 individual milk samples were analysed from
Montefalcone goats, which are localized in southern Italy
(Molise r egion). Eight goats w ere used, as well as two bucks,
for peripheral blood (15–30 mL), which was subsequently
used for DNA extraction.
Casein preparation
Whole casein was prepared by acid precipitation of
individual skimmed milk as described by Aschaffenburg &
Drewry [17].
Gel electrophoresis
Vertical disc PAGE at pH 8.6, preparation of casein
samples and polyclonal antibodies against a
s1
-Cas, and
immunoblotting experiments were performed as described
elsewhere [18].
Preparation of polyacrylamide gel ultra-thin layers

quadrupole mass spectrometer. The selectively precipitated
casein phosphopeptides were fractionated by RP-HPLC on
a 214TP54, 5 lm V yd ac C18, 25 0 · 2.1 mm i nternal
diameter column (Vydac, Hesperia, CA, USA). Solvent A
was 0.3 mL trifluoroacetic acid per L water. Solvent B was
0.2 mL trifluoroacetic acid per L acetonitrile. Samples
(500 lg) were dissolved in 200 lL w ater and injected on to
the HPLC column equilibrated in solvent A. A linear
gradient from 0% to 37% B was applied a t a flow rate of
0.5 m LÆmin
)1
over 60 min. The column effluent was split
1 : 25 to give a flow rate of % 4 lLÆmin
)1
into the
electrospray nebulizer. The bulk of the flow was run
throughthedetectorforpeakcollectionasmeasuredby
following A
220
. The ES-mass spectra were scanned from
1800 to 400 lm at a scan cycle of 5 s per scan. The source
temperature was 120 °C and the orifice voltage 40 V. Mass
values were reported as average masses. Signals recorded in
the mass spectra of peptides were associated with the
corresponding tryptic peptides on t he basis of the molecu lar
mass, taking into account the enzyme specificity and the
reported amino-acid sequence of a
s1
-Cas from different
species. Q uantitative a nalysis of components was performed

Tris/HCl, p H 9.0, 1% Triton X-100), 3 lL25m
M
MgCl
2
,2.5lL5m
M
dNTPs mixture, 0.5 lL (25 pmol)
each primer, 2 lL template DNA, and 0.25 lL(1.25U)
Taq polymerase ( Promega). To avoid evaporation ( with 480
thermal c ycler), the mixture was covered with 70 lL mineral
oil. After an initial denaturing step of 5 min (or 10 min) at
94 °C, the r eaction mixture was subjected to the f ollowing
three-step cycle which was repeated 35 times: denaturation
for 30 s (or 1 min) at 94 °C, annealing for 30 s (or 2 min) at
47–60 °C, and extension for 30–60 s (or 3 min) at 72 °C,
using the 2400 (or 480) thermal cycler. To estimate the
concentration of PCR products, 5 lL each reaction mixture
was analysed by elec trophoresis, i n the presence of ethidium
bromide (0.5 lLÆmL
)1
) in a 2% SeaKem (FMC) or Gibco
BRL Life Technologies agarose slab gel in T ris/borate/
EDTA (8.9 m
M
Tris, 8.9 m
M
boric acid, 0.2 m
M
EDTA,
pH 8.0) bu ffer.

s
complex. As the a
s1
-Cas and a
s2
-Cas
overlap in the same zone of the gel, the a
s1
-Cas composition
of each phenotype was analysed by immunostaining after
Table 1. Primers used in the p resent study. Each pair of primers
amplifies the target exon and its flanking regions (from 60 to 200
nucleotides upstream and downstream). Primers ending with U (upper)
and L (lower) are p ositioned 5¢ and 3¢ from the target exon, respect-
ively. Given the small size of introns 4 and 10, primers C45U/C45L
and C1011U/C1011L were designed to amplify t ogether e xons 4 and 5
and exons 10 and 11, respectively. Sequencing of exon 7 was performed
starting from a genomic DNA fragment produced by amplification
betweenC7UandC8L.Primersinitalicswereusedinthegenotyping
of allele M.
C1U
5¢ GAG AGG AAC TGA ACA GAA CAT TG 3¢
C1L 5¢ CAA CTG CGT ATT AGT GAA GAA TG 3¢
C2U 5¢ AAT CAA ATT TTA TTA TAA GAC C 3¢
C2L
5¢ AAT AGC TAA TTA GAG ACC AT 3¢
C3U 5¢ GGT GTC AAA TTT AGC TGT TAA A 3¢
C3L 5¢ GCC CTC TTC TCT AAA AAG GTT T 3¢
C4U 5¢ AAT GGA GAA TTT GTG TTC AA 3¢
C45U 5¢ TGA CTG TGT TTT TCA CTT CT 3¢

C9LM1 5¢ AAT CTT TAT TTT GTC TCT GAC AA 3¢
Fig. 1. Disc-PAGE at pH 8.6 of individual whole caprine casein samples
containing different a
s1
-Cas variants AA, FF and MF. Phenotypes are
indicated at the top of each lane. Staining was with ( A) Coomassie
Brilliant Blue and (B) polyclonal antibodies against a
s1
-Cas. a–e iden-
tify a
s1
-Cas bands of the MF sampl e in order o f increasing mobility
towards the anode.
Ó FEBS 2002 Interallelic recombination at the a
s1
-casein locus (Eur. J. Biochem. 269) 1295
transfer to NC paper with specific polyclonal antibodies
raised against a
s1
-Cas; the result is shown in Fig. 1B. The
new a
s1
-Cas phenotype (M/F) comprises at least five
components (a, b, c, d, and e). Two o f these (a and c)
appear to be shared with variants A and F, while
components e and d seem to be in common with the A
variant. Therefore, band b represents the only component
specific to t he M variant. The intensities of the bands in the
MF pattern indicate that variant M is a ‘strong’ variant like
variants A, B and C, i.e . it has a high level of expression.

andM/FweresubjectedtoHPLCseparation(Fig.3).The
retention time of variant M was shorter than that of the
A variant while the relative percentage was the same.
The HPLC fractions were analysed by ES/MS, and t he
molecular m asses o f a
s1
-CasA, B, and F were in agreement
with the expected masses [ 7,9]. The molecular mass deter-
mined by ES/MS of the a
s1
-Cas components occurring in
the s ample containing M/F v ariants w as 23 134/23 214/23
294 Da (Fig. 4). A fter alkaline phosphatase hydrolysis, t he
molecular mass of the three main peaks shifted to the single
value of 22 734 Da, indicating the occurrence of three
a
s1
-Cas species carrying five, six, and seven phosphate
groups, respectively. A set of small HPLC peaks eluted
before the main a
s1
-Cas peak gave a molecular mass of
Fig. 2. 2D electrophoretic analysis of a whole casein sample prepared
from the milk of a single goat, heterozygous M/F at the a
s1
-Cas locus.
Disc-PAGE was performed in the first dimension followed by
UTLIEF in the second dimension. The UT LIEF pattern in th e pH
range 2.5–6.5 is shown on t he left. Staining was with polyclonal anti-
bodies raised against a

automated sequence analysis actually demonstrated that
peptide 62–79 (molecular mass 1833 Da and sequence
AGSSLSSEEIVPNSAQQK, where S indicates a phos-
phorylated serin e residue) contains the two substitutions
Ser66fiLeu an d Glu77fiGln, as compared with the B
2
variant. The substitution Ser fi Leu at position 66, first
makes this site unphosphorylatable and secondly impairs
the phosphorylation of Ser64 in the M variant. The
sequence determined is consistent with the molecular mass
measured for the native protein. The phosphorylated
residues a re therefore Ser46, 48, 65, 67, 68 (fully), and
Ser41 and Ser115 (partly), which originate in proteins with
five, six and seven phosphates/mol, explaining the hetero-
geneity of phosphorylation observed for the n ative protein
by ES/MS analysis (Fig. 4). Finally, peptide E96QLLR100,
diagnostic of the F v ariant, was present among the peptides
identified by Edman d egradation after tryptic digestion and
RP-HPLC fractionation, confirming the heterozygous sta-
tus (M/F) of the sample analysed.
Experimental strategy designed to analyse the new
a
s1
-Cas variant at the nucleotide level
To determine the coding sequence of a gene, there are at
least two possible strategies: it is possible to analyse it at
both the genomic level and messenger level. The most
straightforward option is undoubtedly mRNA extraction to
construct a cDNA molecule. The structure of the coding
region is then readily obtained by sequencing the cDNA.

the a
s1
-Cas [9] made this strategy possible. In addition, the
complete sequence of the bovine gene [28] was also available
and showed that the two genes display th e same o rganiza-
tion (number and sizing of exons) and 95% similarity at th e
exon sequence level. As goats and cattle are phylogenetically
close and known intron sequ ences in the goat show strong
similarity to their bovine counterparts, we designed prim-
ers upstream a nd downstream of each exon to amplify
and analyse genomic regions including flanking intron
Fig. 4 . Deconvoluted electrospray mass spectrum of caprine a
s1
-Cas
M variant.
Fig. 5. LC/ES/MS analysis of the tryptic dige st of the a
s1
-CasM vari-
ant. Th e purified protein was digested with a ppropr iate concentrations
of trypsin (see Materials and methods). The peptide mixture was
analyzed using a V ydac C18 column (250 · 2.1 mm, 5 lm), on-line
with a Platform mass spectrometer, as described in Materials and
methods. The peak of the variant p eptide is indicated by an arrow.
Ó FEBS 2002 Interallelic recombination at the a
s1
-casein locus (Eur. J. Biochem. 269) 1297
sequences, starting f rom both t he bovine and the g oat
sequences.
Analysis of the exon sequences at the genomic level
As the samples analysed were from goats that were

exon of the A alle le, which is deleted in the F allele, is
mutatedtoTintheMallele, giving rise to a Ser fi Leu
substitution.
Analysis of the intronic flanking sequences
The flanking intronic regions directly upstream and down-
stream of each exon were sequenced over 50–200 nucleo-
tides a nd the complete sequences of introns 4, 7, and 10 w ere
determined for alleles A, F,andM.Inthisway,20further
polymorphic sites were identified besides the f our polymor-
phic exon nucleotides (Fig. 7). In addition, an RsaI
restriction site was found between exon 6 and exon 8 of
alleles F and M, which is lacking in the A allele, giving a
total of 25 polymorphic sites useful for phylogenetic allele
comparisons. Taking into account these data, it is worth
noting that in the 5¢ part of the gene, up to exon 8, the
nucleotide combination (haplotype ) observed f or the M
allele is identical with t hat shown by the F allele. In contrast,
in its 3¢ part, beyond exon 8, the haplotype of the M allele is
identical with that of the A allele, e xcept at the polymorphic
sitelocatedinexon9.
In addition, intron 5 was completely sequenced starting
from genomic DNA isolated from blood of two goats,
genotyped as M/F and F/F at the a
s1
-Cas locus. Compared
with the bovine sequence, a deletion spanning nucleotides
376 t o 594 was observed f or both goats. The deleted region
in this intron did not match any known sequence in the
EMBL databank. S ubsequently, the existence of this
deletion was confirmed by PCR for six goats of different

(TTGA instead of TCGA).
To discriminate between the M and the F alleles, we took
advantage of the presence o f an 11-bp insertion in intron 9
of the F allele, which is lacking in the M allele. Thus, using
two primers, C9UM1 (forward) and C9LM1 (reverse),
located just upstream from exon 9 and 82 nucleotides
downstream of the 11-bp insertion site, respectively, a
238-bp DNA fragment was yielded by PCR starting from
the M allele, whereas the F allele gives a 248-bp fragment
(Fig. 8 , Step IIA).
Individuals analysed here, which allowed the M allele to
be characterized were heterozygous M/F. Consistent with
our structural results, they gave the two fragments (238 and
248 bp) as shown for one of them at F ig. 8, Step IIB (lane
1). I t is worth noting that the third band observed with this
sample is due to the occurrence of a heteroduplex structure.
this was confirmed by analysing an amplification product
from the mix of samples F/F and X/X (Fig. 8, Step IIB,
lane 4).
DISCUSSION
We report the identification and the molecular character-
ization of a new allele, named M, occurring at the a
s1
-Cas
locus in the goat. This novel allele, characte rized by the
transition CfiT at position 23 in the 9th exon of the gene,
was found in the Montefalcone breed, at v ery l ow frequency
(< 2%) after phenotypic analysis of 147 individual m ilk
samples. All goats bearing the M variant were s hown to be
heterozygous (M/F and M/B).

variants, must be considered a ‘strong’ variant, given the
intensity of the isoelectrofocusing bands and the surface of
the relevant peak in RP-HPLC.
Fig. 6. Nucleotide sequence of the expected a
s1
-CasM cDNA obtained by genomic e xon sequencing analysis: c omparison with its A and F allele
counterparts. Numbering begins with the first nucleotide of the first exon ( up) and the first amino-acid residue of the mature M protein (down).
Dashes indicate nucleotides identical with those of the M all ele. The stop codon is symbolized by ***. Numbers in vertical framed arrows indicate
the position of the introns. T he boxes i ndicate amino-acid substitutions.
Ó FEBS 2002 Interallelic recombination at the a
s1
-casein locus (Eur. J. Biochem. 269) 1299
Unexpectedly, placing variant M in the phylogeny
(Fig. 9) proposed by Grosclaude and Martin [6] turned
out to be rather difficult. Indeed, a comparison of the
different variants at the peptide sequence level suggests a
hybrid structure for the M protein. T aking into account
amino-acid combinations at the polymorph ic residues
(haplophenotypes), the M variant, with a proline and
glutamine residue at position 16 and 77, respectively,
could be placed in both lineages (A and B) arising from
the putative ancestral protein B
1
. This possible dual
membership strongly suggests the invo lvement of a
recombination/gene conversion event between alleles from
the two lineages. This hypothesis was strengthened by
genomic sequence d ata. Although a mutation-driven
convergence c annot be excluded, an interallelic rec ombi-
nation/gene conversion event seems to be the most

Simplified haplotype formulae strongly suggest that the
allele that provided the 3¢ part of the recombinant allele ( M)
is the A allele (Fig. 10). In c ontrast, one can wonder whether
the donor allele o f the 5¢ part is the F allele or another allele
belonging to t he same B allelic lineage (excluding B
1
and C),
as they share the same simplified haplotype formula, up to
exon 8. To reach a definite conclusion, the complete
sequence o f the 5¢ region of each allele would be required,
because no differences have been found in the available
sequences (exons and intron-flanking regions).
If our recombination hypothesis is correct, t he break
point should b e located between nucleotide 86 upstream
and nucleotide 40 downstream f rom exon 8, and the cross
over should have been accompanied by a reciprocal
exchange. One can therefore expect to find the reciprocal
recombinant allele among the alleles so far described. The
structural features of such a recombinant allele should be
an A-type sequence in the 5¢ part followed by a B-type
(B
2
/B
3
/B
4
or F) sequence in the 3¢ part. The only a llele
found so far gathering such characteristics is allele B
1
,

might result from an interallelic recombi-
nation between alleles A and B
2
, which can therefore be
Fig. 7. Polymorphisms occurring at 25 sites in the goat a
s1
-CasA, F and M alleles. The position of each polymorphic site is identified and
numbered relative t o the nearest exon. Intro nic nucleotides are p receded by a ‘–’ or ‘+’ when they are upstream or downstream, repectively
(e.g. )11/1 corresponds to the nucleotide located 11 nucleotides upstream from the first exon). Polymorphic sites in an exon sequence are
identified without a sign (e.g. 8/4 identifies the 8th nucleotide of the 4th exon). RsaI/6–8 indicates the loss (–) or gain (+) of an RsaI
restriction site within the DNA fragment spanning exon 6 to exon 8. Mutations specific for alleles M and F atposition23inthe9thexon
are highlighted. The symbol D indicates the nucleotide deletion in allele F [6]. The hatched boxes, identified by i7-e8-i8, encompass the
putative recombination region.
1300 C. Bevilacqua et al.(Eur. J. Biochem. 269) Ó FEBS 2002
Fig. 8. Genotyping the M allele at the a
s1
-Cas locus. Step I: ACRS-PCR using the primers pair MWU and C9LM* yields a 265/266-bp fragment,
whatever the allele. Amplicons a re then submitted to restriction by TaqI(A).TheTaqI restriction site (TCGA) created in exon 9 is ind icated.
Nucleotides C and A* correspond t o the mut ation characteristic for a llele M and s ubstitution introduced within the primer C9LM*, respectively.
Fragments generated are finally analysed by agarose gel (2% Metaphore + 2% agarose) electrophoresis (B). Lane 1, M olecular mass marker
(pBR322 digested by Hae III); lane 2, nondigested PCR product; lanes 3–5, homozygous X/X, heterozygous M/F and heterozygous X/F samples,
respectively, where X represents an allele different from F, B , E ,andC. Sizes (in b p) of DNA fragments are given on the right of the gel.
Step I I: AS-PCR to discriminate between alleles F and M. (A). Amplification between primers C9UM1 and C9LM1 generates DNA fragments of
characteristic size for the allele. (B) Agarose gel (2% Metaphore + 2% agarose) analysis of amplicons from heterozygous M/F (lane 1),
homozygous X/X (lane 2), homozygous F/F (lane 3), F/F + X/X mix (lane 4), with X different f rom F, B, E,andC. Lane 5 shows a molecular mass
marker (pBR322, HaeIII d igested). Sizes (in bp) of DNA fragments are given on the l eft of the gel.
Ó FEBS 2002 Interallelic recombination at the a
s1
-casein locus (Eur. J. Biochem. 269) 1301
considered representatives of two ancestral allelic lineages.

Fig. 9. Phylogeny proposed by Grosclaude and Martin [6] for the
a
s1
-Cas alleles and differences between the corresponding variants. The
phylogenetic t ree proposed is based on the existence of a single
ancestra l allele ( B
1
), which was considered to be the o riginal o ne give n
its close similarity to its ovine and bovine a
s1
-Cas counterparts.
Fig. 10. A new phylogenetic tree integrating the possible interallelic recombination between two allelic lineages. The four alleles (B
2
, A, B
1
,andW)
putatively involved in the recombination event are schematically represented as a chain of six boxes (mimicking exons) on w hich are indicated
polymorphic a mino-acid residues and their position in t he p eptide chain. A sim plified haplotype formula is thus provided (e.g. HPS
P
ERT and
HLS
P
QRT for alleles B
2
and A , r espect ively) . The RsaI polymorphic restriction site a nd insertio ns o ccurring, resp ectively, bet ween e xons 6 and 8
and within intron 9 are indicated. Alleles d eriving from t hese four ‘potentially recombinant’ alleles (boxed) are circled. A rrows indicate a possible
pathway of evolution to alleles associated with high (black) or r educed (red) amounts of casein synthesized. The M allele is derived f rom allele W by
a single n ucleotide transition CfiT (nucleotide 23/exon 9) leading to the o ccurrence o f a leucine r esidue (allele M) instead of the Ser (putative allele
W) in the multiple phosphorylation site of a
s1

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