Tài liệu Báo cáo Y học: Interallelic recombination is probably responsible for the occurrence of a new as1-casein variant found in the goat species potx

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 ` di Agraria dell’Universita ` del Molise, Campobasso, Italy; 2 Laboratoire de Ge ´ ne ´ tique biochimique et de Cytoge ´ ne ´ tique, INRA, Domaine de Vilvert, Jouy-en-Josas, France; 3 Dipartimento di Scienza degli Alimenti, Facolta ` di Agraria, le Universita ` di Napoli ‘Federico II’, Portici, Italy; 4 Istituto di Scienze dell’Alimentazione del CNR, Avellino, Italy The a s1 -casein (a s1 -Cas) locus in the goat is characterized by a polymorphism, the main feature of which is to be qualit- ative as well as quantitative. A systematic analysis performed in an autochthon sou thern Italy breed identified a new rare allele (M), which was characterized at both the protein and 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- leotide polymorphisms and two i nsertion/deletion events in the coding as well as the noncoding regions. A comparison o f specific haplotypes defined for each of the a s1 -CasF, A and M alleles indicates that the M allele probably arises from interallelic recombination between alle les A and B 2 , followed by a C fi T transition a t nucleotide 23 of the ninth exon. The region encompassing the recombination break point was putatively located between nucleotide 86 upstream and nucleotide 40 downstream of exon 8. Interallelic recombi- nation therefore appears to be a possible means of gener- ating alle lic diversity at the a s1 -Cas locu s, at least i n the goat. The previously proposed molecular phylogeny must now be revised, possibly starting from t wo ancestral a llelic lineages. Keywords: a s1 -casein gene; allelic recombination; genetic polymorphism; g oat milk. Caseins comprise the main protein fraction of ruminant milk. They are encoded by four tightly linked genes [1], clustered in a 250-kb g enomic DNA segment [ 2] in the following order: a s1 , b, a s2 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- dues: 64Ser P -Ser P -Ser P -Ser P -Ser P -Glu-70Glu. This deletion arises from the outsplicing of three exons (9, 10 and 11) during the processing of primary transcripts, probably because of a single nucleotide dele tion occurring within the first (exon 9 ) unspliced exon [9]. M ore recently, the B alle le has been split up in to four alleles giving rise to the synthesis of four protein variants B 1 ,B 2 ,B 3 ,andB 4 , which differ as a result of amino-acid substitutions [6]. These substitutions have no effect on th e net charge of the protein, which therefore makes the relevant variants indistinguishable on PAGE. Variant B 1 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 in@jouy.inra.fr Abbreviations: a s1 -Cas, a s1 -casein; UTLIEF, ultra-thin-layer isoelectric focusing; LC/ES /MS, liquid chromatography/electrospray/ mass spectrometry; ACRS-PCR, amplified created restriction site-PCR. *Present address: I NSERM E9925, Interactions de l’e ´ pithe ´ lium intestinal avec le syste ` me immunitaire, Faculte ´ Necker-Infants Malades, 156, rue de Vaugirard, 75 743 Paris Cedex 15, F rance. (Received 29 August 2001, revised 17 December 2 001, accepted 9 January 2002) Eur. J. Biochem. 269, 1293–1303 (2002) Ó FEBS 2002 variant, named M, was detected in the Molisane Montefal- cone goat breed [15], which was shown, in addition, to display a rather high frequency of the F allele [16]. In this paper, we report t he characterization of this new variant at both the protein and genomic level. The complete amino-acid sequence of the M variant has been determined. Starting from genomic DNA, we amplified, by PCR, the coding regions (exons) a nd their intron fl anking regions, which have been subsequently sequenced. Such a dual approach has made it possible to identify the mutation specific for the 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 (0.25 mm) and isoelectric focusing (UTLIEF) were carried out as recommended by EEC Regulation no. 690/92 [19]. The pH gradient in the range 2.5–6.5 was obtained by mixing Ampholine (Pharmacia LKB) 2.5–5, 4.5–5.4, and 4–6.5 in the volume ratio 1.6 : 1.4 : 1. 2D ge l electrophoresis (PAGE in the first dimension followed by UTLIEF in the second) has been described elsewhere [18]. Enzymatic hydrolyses Trypsin (Boehringer Mannheim) hydrolysis was carried out in 0.4% NH 4 HCO 3 ,pH8.5,at37°C, for 4 h, in a substrate/enzyme ratio of 5 0 : 1 (w/w). Dephosphorylation with calf intestine alkaline phosphatase (Boehringer Mann- heim) was performed in the same buffer by using 1 mU enzyme/mg casein at 37 °C for 18 h; these c onditions have been previously shown to p roduce complete d ephosphory- lation of the sample [ 20]. Reactions were stopped by freeze- drying. Liquid chromatography/mass spectrometry analysis of proteins and peptides The whole caprine casein samples were fractionated by the procedure of J aubert and M artin [21], modified by Ferranti et al . [22]. Liquid chromatography/electrospray/mass spectrometry (LC/ES/MS) was performed using a HP1100 modular system on-line connected to a Platform (Micromass) single 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 by integration of the multiple charged ions of the single species [22]. Sequence analysis Automated Edman degradation was performed using a n Applied Biosystems mode l 477A Protein Sequencer with on-line phenylthiohydantoinyl amino acid (Pth-Xaa)- HPLC analyzer. Phosphorylated peptides were modified by the procedure of Ferranti et al .[20]. Genomic DNA preparation Goat genomic DNA was prepared f rom leucocytes i solated from the plasma fraction of EDTA-anticoagulated periph- eral blood samples, as described previously [23,24]. Oligonucleotides Intronic primers used either for amplification from genomic DNA or f or sequencing o f amplified DNA fragments were provided by Genosys Biotechnologies Inc. (Cambridge, UK) and Primm (Milano, Italy). Their sequences are given in Table 1, together with those used for genotyping. PCR conditions In vit ro am plification was performed with the t hermostable DNA polymerase of Thermus aquaticus (Taq polymerase) using either a 480 or a 2400 thermal cycler (PerkinElmer), essentially as described [25]. A typical 50-lL reaction 1294 C. Bevilacqua et al.(Eur. J. Biochem. 269) Ó FEBS 2002 mixture consisted o f 5 lL10·PCR buffer (500 m M KCl, 100 m M 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. For genotype a s1 -CasM, using the amplified created restriction site (ACRS)-PCR procedure [26], experimental conditions are essentially the s ame as those mentioned b efore except for the primer c oncentration (50 pmolÆ50 lL )1 reac- tion mix) and the concentration of the agarose slab gel used to visualize the PCR products was 4% (2% Gibco-BRL and 2% high-resolution agarose FMC). Sequencing of amplified genomic DNA fragments PCR products were either d irectly sequenced or sequenced after cloning (fragments amplified between primers C9U and C9L) into SmaI-digested pUC18 plasmid vector, using fluorescent Cycle Sequencing (AmpliTaq FS, Dye Termi- nator Cycle Sequencing Kit; PerkinElmer) with an ABI 377A or an ABI 310 DNA sequencer. RESULTS PAGE analysis and immunoblotting of whole casein Figure 1A shows t he typical electrophoretic patterns yielded, in polyacrylamide gel at pH 8.6, by the new a s1 -Cas phenotype, subsequently shown to be a heterozygous M/F (M being the new variant), in comparison with two reference phenotypes AA (lane 1) and FF (lane 2). This new phenotype is characterized, under t hese conditions, by t he presence of a protein band with a slower mobility (lane 3, *) occurring within the a 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¢ C45L 5¢ GCT TTG TTA ATT CTG CAG TA 3¢ C6U 5¢ CCT TTT CCA GAA GTG TTT AGA AAG 3¢ C6L 5¢ CAT ACC ACC TTA ATT TTC GTA TT 3¢ C7U 5¢ CAT GAA GCA ATA TAT CTG CTC C 3¢ C7L 5¢ TGG TCA ACA TAC ATG TTG CAT C 3¢ C8U 5¢ CTT CAG TTA GCC TGG TAG GTA 3¢ C8L 5¢ TGG CAC AAC ATT GTA CAT TCT TGG G 3¢ C9U 5¢ GTA TGG AAG TGT GGA ATA GTT T 3¢ C9L 5¢ GGA CAC CAC AGA TAT CCA ATA G 3¢ C1011U 5¢ CAT AAA ACT AAC AAT ACA TGT 3¢ C1011L 5¢ TAG CAG ATA TTG AAA AGG AG 3¢ C12U 5¢ CCA GTG AAT ATT CAG GAC TGA T 3¢ C12L 5¢ AGG CTC TAG CAT GAT TTG ATG T 3¢ C13U 5¢ GCA TTT TTA TTT TGA ATG TAA A 3¢ C13L 5¢ TAG TTC AAA TGC ACA TCT TAT 3¢ C14U 5¢ GGC AGA GAA TAC GTT TAT ACT AA 3¢ C14L 5¢ TCT CAG ATT GAC TAC TAC AAC TT 3¢ C15U 5¢ CAT GAA AAG CAT TTC AAA AA 3¢ C15L 5¢ TAA AAA ACA GTG GTT ACC AA 3¢ C16U 5¢ CTA AAG AGT ACA CTA TCC TCA C 3¢ C16L 5¢ TTG CTG TGG TTG CCT ATC CTA 3¢ C17U 5¢ TGA TTT CTC ATA CAC TGT TG 3¢ C17L 5¢ TTG ATA AGG CAA CAA TAT GC 3¢ C18U 5¢ GTC CCA ACT TGA AAT CCT GAT C 3¢ C18L 5¢ CAA GTT TAT AGT CTA CAC GTT GTA C 3¢ C19U 5¢ CTT AGC ATC TTC CAT GGC TTG ATC 3¢ C19L 5¢ ATA CAC ACA AAC TCA CAA GG 3¢ MWU 5¢ CAA CAT ATT TTA AAT AAA ATT GAC AAT 3¢ C9LM* 5¢ ATA AAA ATG GTA TAC CTC ACT TGT*C 3¢ C9UM1 5¢ TAA CAA TGA TTC TCT TTC TTT TAG 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. However, as the intensities of three apparently homologous components (a, c, and e) in the AA and MF profile were different, further heterogeneity of the PAGE components may be suspected. To understand t he high degree of heterogeneity observed with goat a s1 -Cas and to try to explain the difference in band intensities, further electrophoretic experiments were carried out, including UTLIEF analysis and 2D electro- phoresis followed by staining with polyclonal a ntibodies. In UTLIEF (results not shown) the a s1 -CasM/F phenotype comprised at least seven major components, two of which were in common with variant F. Using 2D electrophoresis (Fig. 2), at least two main spots surrounded by a number o f minor components differing in their pI were found in each PAGE band. This large microheterogeneity, which a lso occurs for other casein phenotypes (results not shown), m ay be attributable to nonallelic a s1 -Cas forms generated by defective mRNA splicing an d to differently phosphorylated a s1 -Cas chains, as reported by Ferranti et al .[20]. MS and sequence analyses To determine the molecular m ass of the new variant (M), whole c asein s of individual milks o f the phenotypes A/A, F/F, 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 s1 -Cas. Fig. 3. RP-HPLC a nalysis of casein fractions from goats of different genotypes F/F (A), M/F (B) and A/A (C) at the a s1 -Cas locus. 1296 C. Bevilacqua et al.(Eur. J. Biochem. 269) Ó FEBS 2002 18 715/18 795/18 875 Da (18 555 Da after a lkaline phos- phatase hydrolysis), corresponding to that expected for the F variant. This result is the first evidence for the heterozy- gous status (M/F) of the individual goat milk analysed. In addition to this, t he HPLC profile confirms that the M variant is abundantly expressed. Thus, as previously mentioned, we were working with a mixture of two unresolvable variants, one of which (M) a ccounts for more than 80% of the whole a s1 -Cas. This overrepresentation of the M variant allowed us to continue the molecular characterization with such a material. The a s1 -Cas fraction containing the M variant was digested with trypsin, and the resulting peptide mixture analysed by LC/ES/MS (Fig. 5). The peptide sequence determined for t he M variant was identical w ith t hat yielde d by variant B 2 (from the published sequence [6,7]) except for two substitutions located in peptide 62–79. MS and 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. In our situation, however, such a strategy was not possible. Given t he low number of animals in the popula- tion, it was not possible to slaughter individuals of interest. In addition, as the a nimals were from private flocks bred in mountain m eadows, it was not possible to make mammary tissue biopsy samples under appropriate hygienic condi- tions. To overcome this, we tried to extract mammary mRNA from milk somatic cells, using the technique first de scribed by Martin et al. [27]. Unfortunately, we could not obtain enough material to synthesize cDNA. However, as expected from the phen otypic analysis (at the p rotein level), the f ew animals yielding in their milk the a s1 -CasM variant were exclusively heterozygous M/F a t the a s1 -Cas locus. There- fore, analysis o f their transcripts could have been rather difficult because of the occurrence of at least nine different forms of messenger arising from the F allele [9]. Finally, t o integrate this new allele into the phylogenetic t ree proposed by Grosclaude & M artin [6], we also n eeded to obtain information ab out relevant noncoding regions in which specific and informative mutations are localized. For these reasons, we d ecided to analyse the sequence of the M allele at the genomic level. After amplification of each exon an d i ts intron-flanking regions, amplified genomic DNA fragments were sequenced. The knowled ge of the structural organization of the goat gene encoding 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 heterozygous (M/F)atthea s1 -Cas locus, to discriminate between the two alleles and therefore determine the 19 exon sequences coming from the M allele, sequence data were compared with those from a homozygous F/F goat genomic DNA sample. All the sequences yielded were unambigu- ously determined except that corresponding to the PCR fragment encompassing the ninth exon in which a single nucleotide deletion has been shown to occur in the F allele [9]. This m akes the s equence chromatogram unreadable from that point for the DNA template amplified from the heterozygous M /F sample. To overcome this pro blem, t he amplified fragment was cloned. Of the 10 clones sequenced, four displayed a typical F exon-9 sequence, and five showed the same sequence, which was different from that of the F allele, with a 33-nucleotide exon 9. Taken together, the exonic sequence data allowed us to construct a sequence corresponding to the c omplete cDNA of the M allele. This sequence is given in Fig. 6, where it is c ompared w ith that of alleles F and A. Only four polymorphic nucleotides were identified, t hree of which yielded amino-ac id substitutions: (a) the transition TfiC on the second nucleotide of the third codon within exon 4, leading to a LeufiPro substitution at position 16 of the peptide chain, as compared with the A variant; (b) a transversion GfiC on the first nucleotide of the l ast exon-10 codon, leading to a GlufiGln substitution at position 77 o f the peptide chain, as compared with the F variant; (c) the deoxycytidyl phosphate residue at position 2 3 in the 9th 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 a s1 -Cas genotypes (A/A, F/F, M/F) from different Italian breeds (Montefalcone, Teramana, Garganica, Girgentana, and Sarda) and for s ix sheep of different I talian breeds (Comisana, Gentile di Puglia, and Valle del Belice). These results: (a) confirm the difference in size (% 200 bp) previously reported [23] between goat (% 450 bp) and cattle (641 bp) intron 5, and (b) show that the ovine intron 5 is also shorter than the cattle one. This could be expected, given the phylogenetic proximity between sheep and goats. Genotyping of the M allele The genotyping procedure designed consists of two steps. The first one is an ACRS-PCR technique [26], the principle of which is to create a TaqI restriction site (TCGA) by using a mismatching primer (C9LM*) which allows both the F and M alleles to be discriminated from all the others (Fig. 8, Step IA). These two alleles will be subsequently distin- guished after a second amplification which allows discrim- ination between the alleles on the basis of the f ragment sizes (Fig. 8 , Step IIA). In the first step, a 266-bp (265 bp for the F allele) DNA fragment is amplified between primers MWU and C9LM* with every allele. After digestion with TaqI, the 265/266-bp fragment is split into two fragments (240 and 26 bp) for each allele e xcept the M and F alleles, for wh ich no TaqIsite has been created (Fig. 8, Step IB), because of mutations (deletion o r substitution) occurring a t position 23 in exon 9 (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). Interestingly, the mutation s pecific for the M allele affects the same nucleotide as that which is deleted in the F allele, and shown to be responsible for the internal deletion of 37 amino-acid residues in the F variant, as a consequence of t he 1298 C. Bevilacqua et al.(Eur. J. Biochem. 269) Ó FEBS 2002 skipping of three successive exons during the course of the processing of the primary transcripts [9]. At the peptide level, the CfiT transition, which leads to a Ser66 fi Leu66 substitution, gives rise to the loss of two of the five phosphate groups clustered in the multiple phosphorylation site of the a s1 -Cas. This loss explains t he lower ele ctro- phoretic mobility of the M protein compared with the other caprine a s1 -Cas variants described so far. This situation is similartothatobservedinsheep,withthea s1 -CasD variant (previously called the Welsh variant). Actually, this ovine variant has only two phosphoserine residues instead of five in the homologous region of the a s1 -CasA and C variants [22]. However, whereas the structural alteration in the D (Welsh) variant is associated with a reduction in milk casein content [29,30], the M variant, like the goat a s1 -CasA and 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 plausible. Intronic sequences relative to A, F and M alleles (Fig. 7) strongly support such a notion. Indeed, a detailed comparative analysis at 25 polymorphic sites, including 23 single nucleotide polymorphisms, spanning a large part of t he transcription unit provides a haplotype formula allowing each allele to be precisely characterized. Thus, the M allele unequivocally appears to be a hybrid structure made o f F -type allele se quences in its 5¢ part followed by A-type allele sequences in its 3¢ part (except the transition CfiT at nucleotide 23 in the ninth exo n). Following such a scheme, a recombination event would have occurred around exon 8 ( Fig. 7). However, t he genomic sequences analysed do not allow us to distinguish whether the mechanism by w hich allele M originates is consecutive to a double (gene conversio n) or to a single (recombination) cross over. However, as o ver t he 10 kb separating exon 8 from the end of the transcription unit there are no sequence clues indicating a sec ond cross o ver, it see ms most likely that the M allele originated in an interallelic recombination event. Gene conversion events, which usually account for exchanges over short sequence tracts [31], have been mainly described and intensively investigated as mechanisms generating allelic diversity in highly polymorphic genetic systems, such as the loci encoding the class-II cell surface antigens of the major histocompatibility complex in humans [32,33]. Both mechanisms have also been thought to account for genetic disorders in humans, such as sporadic Alzheimer disease cases [34] and diabetic pathology involving the gene encoding insulin [35]. 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 , with a B 2 simplified haplotype formula in its 3¢ part (Fig. 10). This confirms our assumption and suggests that the M allele probably r esults from an interallelic re com- bination event involving alleles A and B 2 whereas the reciprocal event might have given B 1 . However, w ith a leucine residue at position 66, it is clear that the M allele does not arise directly from the recombination event between alleles A and B 2 . It probably is derived from an intermediate hybrid allele (B 2 :A), putatively W, not yet identified, which was subsequently mutated at nucleotide 23 of the ninth exon. Because of its close similarity to its bovine and ovine a s1 -Cas counterparts, allele B 1 was considered to be the ancestral allele in the goat [6]. The results reported here indicate that 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. The reciprocal proposal, i.e. B 1 and W are parental a lleles, the recombinant products of which are A and B 2 , cannot be ruled out (Fig. 10). The latter proposal is, however, less plausible, given the low frequencies at which alleles B 1 and M have been found in the g oat populations analysed so far. It is worth noting that both alleles are characteristic of l ocal breeds, Poitevine (France) and Montefalcone (Italy), respectively. Nevertheless, whatever the hypothesis retained, the existence of two ancestral allelic lineages seems to be the most likely scenario. Thus, interallelic recombination between two alleles may be responsible for the generation of four possible allelic lineages (represented by A, B 2 , B 1 , and W ), one of which (W/M) i s r evealed by this work. The high polymorp hism of the goat a s1 -Cas system provides further evidence that a llelic diversity can arise from multiple pathways, including shuffling of polymorphic sequences generated by point mutations, through interallelic recombi- nation events. 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 -Cas. The new phylogeny has been e nriched with three novel variants (H, I and L ) reported in 1997 by Chianese et al. [14]. 1302 C. Bevilacqua et al.(Eur. J. Biochem. 269) Ó FEBS 2002 [...]... 249, 1–7 21 Jaubert, A & Martin, P (1992) Reverse-phase HPLC analysis of a goat casein Identification of as1- and as2-casein genetic variants Lait 72, 235–247 22 Ferranti, P., Malorni, A. , Nitti, G., Laezza, P., Pizzano, R., Chianese, L & Addeo, F (1995) Primary structure of ovine as1-caseins: localization of phosphorylation sites and characterization of genetic variants A, C and D J Dairy Res 62, 281–296... 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CTT AGC ATC TTC CAT GGC TTG ATC 3¢ C19L 5¢ ATA CAC ACA AAC TCA CAA GG 3¢ MWU 5¢ CAA CAT ATT TTA AAT AAA ATT GAC AAT 3¢ C9LM* 5¢ ATA AAA ATG GTA TAC CTC ACT. 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