Ovis aries POU1F1 gene: cloning, characterization and polymorphism analysis

Estela Bastos1,*, Ingrid Santos2,5, Isabelle Parmentier3, Jose Luis Castrillo4,Alfredo Cravador5, Henrique Guedes-Pinto1 & Robert Renaville31Centro de Genetica e Biotecnologia, Universidade de Tras-os-Montes e Alto Douro, Apdo. 1013, 5000-911Vila Real, Portugal; 2Departamento de Sistemas e Tecnicas de Producao Animal, Estacao ZootecnicaNacional, Fonte Boa, 2005-048Vale de Santarem, Portugal; 3Unite de Biologie Animale et Microbienne,Faculte Universitaire des Sciences Agronomiques, Bat. 92, B-5030Gembloux, Belgium; 4Centro de BiologıaMolecular Severo Ochoa (CSIC-UAM), Universidad Autonoma de Madrid, 28049Madrid, Spain; 5Fac-uldade de Engenharia dos Recursos Naturais (FERN), Universidade do Algarve, Campus de Gambelas, 8005-139Faro, Portugal; *Author for correspondence (Phone: +351-96-2715996; Fax: +351-259-350572;E-mail: ebastos@utad.pt)

Received 21 February 2005 Accepted 17 June 2005

Key words: gene cloning, ovine, POU1F1, polymorphism, sequencing

Abbreviations: BESS – Base excision sequence scanning; CPHD – Combined pituitary hormone deficiency;GH – Growth hormone; GHRH-R – Growth hormone releasing hormone receptor; MAS – Markerassisted selection; PCR – Polymerase chain reaction; POU proteins – proteins with domain first describedon PIT-1, OCT1,2 and UNC86; POUs – POU-specific domain; POUh – POU-homeodomain; PRL –Prolactin; RFLP – Restriction fragment length polymorphism; SNP – Single nucleotide polymorphism;STA – Serine/Threonine Activation Domain; TSHb – b subunit of thyroid-stimulating hormone

Abstract

POU1F1 (PIT-1/GHF-1) is a transcription factor with critical role in the transcriptional regulation ofmultiple genes in the pituitary and also important for the survival, differentiation and proliferation of threepituitary cell types. To understand the regulation of POU1F1 gene in Ovis aries we report its cloning,sequencing and characterization. The sequenced 5787 bp included six exons and two complete introns.Ovine POU1F1 gene has a high level of conservation with its bovine, human and rat counterparts showing98.2%, 91.2% and 86.2% of similarity at the coding level, respectively. All six exons were analyzed forpolymorphism detection in 100 animals of the Portuguese indigenous ovine breed ‘Churra da Terra Qu-ente’. One polymorphism was found at codon 58 in exon 2, in one allele of 4 animals leading to a changefrom cysteine to tyrosine (2% allelic frequency). In exon 3 two polymorphisms were detected: a G to Atransition altering a glycine to an asparagine at codon 89 in one allele of one animal (0.5% allelic fre-quency) and another G to A transition at codon 105 converting an alanine into a threonine in one allele of 3animals (1.5% allelic frequency). These polymorphisms might change the structure of the POU1F1 proteinand modify gene-expression. In intron 4, an A to G transition was detected in one allele of six animals (3%allelic frequency). Exons 1, 4 and 6 showed no polymorphisms.

Database GenBank accession numbers: AJ549204–549207

Genetica (2006) 126: 303–314 � Springer 2006DOI 10.1007/s10709-005-0034-6

Introduction

POU1F1 (also known as PIT-1 or GHF-1) isa tissue-specific transcription factor mainly ex-pressed in the anterior pituitary (Bodner et al.,1988; Ingraham et al., 1988). This protein was firstassociated with a critical role in the transcriptionalregulation of growth hormone (GH) and prolactin(PRL) genes (Lefevre et al., 1987; Nelson et al.,1988). It is also involved in the activation of the bsubunit of thyroid-stimulating hormone (TSHb)(Li et al., 1990), POU1F1 itself (Chen et al., 1990;McCormick et al., 1990) and growth hormonereleasing hormone receptor (GHRH-R) genes (Linet al., 1992). In addition to its role in gene acti-vation, POU1F1 is necessary for the differentia-tion, proliferation and survival of somatotrope,lactotrope and thyrotrope cells (Li et al., 1990).

POU1F1 belongs to the large family of POU-domain proteins (name associated with the firstdescribed proteins with this domain: POU1F1;OCT1, 2 and UNC86), characterized by the pres-ence of two conserved regions: the POU-specificdomain (POUs) and the POU-homeodomain(POUh), responsible for high affinity DNA bind-ing to specific promoter regions. Transcriptionalactivation is achieved by a less conserved domainat the N terminus, rich in serine and threonineresidues (Serine/Threonine Activation Domain,STA) (Theill et al., 1989).

POU1F1 encoding cDNAs have been cloned inseveral mammalian species including bovine(Bodner et al., 1988), rat (Bodner et al., 1988; In-graham et al., 1988), mouse (Li et al., 1990), hu-man (Tatsumi et al., 1992), swine (Tuggle et al.,1993), rhesus monkey (Schanke et al., 1997), ovine(Thomas et al., 2000) and dog (Lantinga-vanLeeuwen et al., 2000). In birds, POU1F1 gene hasalready been cloned from turkey (Wong et al.,1992) and chicken (Tanaka et al., 1999) pituitary.The cDNA of this gene was also studied in somefish species, namely in chum salmon (Ono andTakayama, 1992), rainbow trout (Yamada et al.,1993), chinook salmon (Majumdar et al., 1996),Atlantic salmon (Lorens et al., 1996), giltheadseabream (Martinez-Barbera et al., 1997), ayu(Chiu et al., 2002) and zebrafish (Nica et al.,2004).

This gene has an important role in the controlof development and hormone gene-expression inthe pituitary. Several authors associate mutations

on this gene with dysfunction at the pituitary level.Namely, a point mutation in the POU-homeodo-main of Snell dwarf mouse POU1F1 gene or alarge deletion in the Jackson dwarf mouse genecauses hypoplasia of anterior pituitary and Com-bined Pituitary Hormone Deficiency (CPHD) ofGH, PRL and TSHb (Li et al., 1990). Recently,Nica et al. (2004) described zebrafish POU1F1null mutants lacking three pituitary cell types thatdevelop severe dwarfism.

Since 1992, multiple studies have reportedassociations between mutations within POU1F1gene and CPHD in humans. To date, 18 differentmutations were reported scattered over all the 6exons, excepting exon 2. Malgavia et al. (2003)presented a review of 15 of these mutations,including one new mutation detected in exon 4.Additionally, Blankenstein et al. (2001) observed amutation in exon 6; Hashimoto et al. (2003) de-scribed a mutation in exon 3 while Salemi et al.(2003) reported a mutation in exon 1.

Giving the importance of POU1F1 gene to thecontrol of gene expression of important hor-mones directly associated with growth processand milk production, it is sound to consider it asa good candidate gene for marker assisted selec-tion. In pig, Tuggle et al. (1993), Yu et al. (1993,1994) identified and analyzed PCR–RFLP poly-morphisms at the cDNA of POU1F1. Yu et al.(1995) established the association of these poly-morphisms with growth and carcass traits in pigsand found positive correlations. A similar resultwas obtained by Stancekova et al. (1999) in adifferent study. Sun et al. (2002) showed a sig-nificant effect of POU1F1 genotype on circulatinglevels of GH and PRL in neonatal Meishan pigsand Brunsch et al. (2002) suggested that there arecontributions of POU1F1 gene to the variation ofgrowth, meat quality and carcass compositiontraits in pigs.

Renaville et al. (1997) found a positive associa-tion of allele A of the POU1F1HinfI polymorphismwith milk and protein yields in Holstein-Friesiandairy cattle. Di Stasio et al. (2002) found no evi-dence of association of POU1F1 polymorphismswith meat production traits in Piemontese cattle.Recently, Zhao et al. (2004) detected polymor-phisms in introns 3 and 4 and in exon 6 of POU1F1and found no significant association between thesepolymorphisms and growth and carcass traits inAngus beef cattle.

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Jiang et al. (2004) detected a single nucleotidepolymorphism (A fi T transversion) in the POUdomain of chicken POU1F1 gene, changing thecodon 299 from asparagine (Asn) to isoleucine(Ile). The distribution of allele frequencies differedsignificantly between meat-type and layer-typechickens and revealed a positive relationship be-tween genotype A/A and body weight at 8 weeksof age. These authors considered that this SNP inPOU1F1 gene is a potential molecular marker forearly growth rate in chicken.

Apart from the sequence of ovine POU1F1mRNA and the localization of this gene on ovinechromosome 1 (1q21–22) (Woollard et al., 2000),there is a lack of information about the basicmolecular function of POU1F1 in these species.Thomas et al. (2000) found that, in opposition toneonatal pigs, in sheep the POU1F1 mRNA levelswere not correlated to GH mRNA levels, sug-gesting the need to further investigate the factorscontrolling synthesis and secretion of ovine GH.

‘Churra da Terra Quente’ is an interestingPortuguese indigenous sheep breed not onlybecause of its economic importance to theNortheastern region of Portugal, but also becauseof its variability. This breed is the result of crossesbetween two Portuguese breeds (‘Badana’ and‘Mondegueira’) that occurred in 19th century.There is a remarkable variability in the daily milkproduction that ranges from 0.25 to 1.5 l. Thisvariation is not only associated with managementdifferences but also to possible genetic variability.Evaluation of the genetic diversity for 6 genes in 40animals of this Portuguese indigenous breed wasperformed by Bastos et al. (2001). Single-strandconformation polymorphism (SSCP) detectionwas optimized, starting from genomic DNA andPCR amplification of seven fragments: exon 1 ofthe a-lactalbumin gene; exons 10 and 11 of theas1-casein gene; exon 7 of the b-casein gene; exon4 of the j-casein gene; exon 4 and 5 of the growthhormone gene and exon 6 of the growth hormonereceptor gene. Only j-casein and growth hormonereceptor showed to be monomorphic. a-lactalbu-min and as1-casein exons showed three confor-mational patterns, b-casein and growth hormoneexon 4 showed two electrophoretic patterns andgrowth hormone exon 5 showed five conforma-tional patterns. These data provided evidence that‘Churra da Terra Quente’ has a high geneticvariability.

Until now, to our best knowledge, no poly-morphisms have been analyzed in POU1F1 gene inOvis aries. As a first step towards understandingthe regulation of POU1F1 gene in this species, wehave cloned and sequenced a total of 5787 bp,which includes 6 exons, 2 complete introns (introns4 and 5) and 3 partial introns (introns 1, 2 and 3)from the Portuguese indigenous ovine breed‘Churra da Terra Quente’. With the information ofthe intronic sequence adjacent to each exon, it waspossible to analyze the entire sequence of all the sixexons and detect and characterize naturallyoccurring polymorphisms in this ovine breed.

Material and methods

Animals and DNA extraction

For cloning and sequencing O. aries POU1F1 genewe used a DNA sample from an adult female ofthe Portuguese indigenous ovine breed ‘Churra daTerra Quente’ belonging to a flock from itsNational Sheep Breeders Association (ANCO-TEQ). For polymorphism analysis, we used100 DNA samples from the same flock. DNA wasextracted by a high salt concentration protocol(Montgomery and Sise, 1990), with minor modi-fications from the original method. The source ofnucleated cells was the leukocytes from a 10 mlblood sample.

PCR amplification for sequencing

In order to sequence the ovine POU1F1 gene, sixgenomic fragments containing the beginning of anexon and the end of the following exon were pre-pared. Comparing the partial sequences from thehuman POU1F1 gene (GenBank accession num-bers D12887-D12892) with the mRNA sequencesfrom sheep (GenBank accession number U88399)and from cattle (GenBank accession numberX12657), specific primers were manually designed(Table 1), allowing the amplification, by PCR, ofsix fragments covering the entire region of thegene, from exon 1 to exon 6 (Figure 1).

The PCR primers used for sequencing eachfragment of ovine POU1F1 gene are described inTable 1, together with the final concentration ofdNTPs and MgCl2 used in each reaction. Frag-ments 1, 2 and 3 were obtained using a 50 ll PCR

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reaction volume with 200 ng of DNA, 16 pmol ofeach primer and 2.6 U of a long template TaqDNA polymerase (Expand Long Template PCRSystem, Roche), while fragments 4, 5 and 6 wereproduced using a 25 ll final reaction volume,50 ng of DNA, 8 pmol of each primer and 1 U ofTaq DNA polymerase (Amersham Biosciences).All PCR reactions were performed with a Gene-cycler (Biorad). For fragments 1, 2 and 3, after aninitial denaturation step at 93�C for 2 min, twosets of cycles were performed, followed by a finalelongation of 7 min at 68�C. For fragments 1 and3, 10 cycles were performed with denaturation at93�C for 10 s, annealing at 60�C for 30 s, exten-sion at 68�C for 3 min, followed by 20 additionalcycles with the same conditions and a 20 s incre-ment of the extension time per cycle. For fragment2, 10 cycles were performed using 94�C for 10 s,

60�C for 30 s, 68�C for 6 min followed by 20additional cycles using the same conditions butwith a 20 s increment of the extension time percycle. For fragments 4, 5 and 6, amplification wasperformed at 95�C for 5 min, followed by 30 cyclesof 95�C for 30 s, 60�C for 30 s, 72�C for 30 s and afinal extension of 72�C for 10 min. All PCRreactions were repeated at least twice in bothstrands before cloning in order to confirm thesequencing result. The strategy used for sequenc-ing Ovis aries POU1F1 gene is presented inFigure 1.

Cloning and sequencing

The six PCR products were separated on agarosegels and purified by GenElute agarose spin col-umns (Sigma) after excision from the gel, and

Table 1. Sequences of the primers used for the sequencing of O. aries POU1F1 gene

Primer sequence [dNTP] [MgCl2]

Fragment 1 1F 5¢-TGTGGGAATGAGTTGCCAACC-3¢ 1.4 mM 2.25 mM

(Ex1-In1-Ex2) 2R 5¢-CCATCACGCCATAGGTCG-3¢Fragment 2 2F 5¢-CAGGACTTCATTATTCTGTT-3¢ 2 mM 4 mM

(Ex2-In2-Ex3) 3R 5¢-TCTTCTCACTTTAAACTCATTGGC- 3¢Fragment 3 3F 5¢-TGAGTTTCCTGACCACACGC-3¢ 1.4 mM 2.25 mM

(Ex3-In3-Ex4) 4R 5¢-CTTGCTCAGCTTCCTCCAGC-3¢Fragment 4 4F 5¢-AGCTCTGGCAGCTGTGCATG-3¢ 0.2 mM 1.5 mM

(Ex4-In4-Ex5) 5R 5¢-CATTTGCACCAACTTTCTCATTG-3¢Fragment 5 5F 5¢-CAATGAGAAAGTTGGTGCAAATG-3¢ 0.2 mM 1.5 mM

(Ex5-In5-Ex6) 6R 5¢-CCAAACCCTCACCACTTCTTTC-3¢Fragment 6 6F 5¢-AAGACGCCCTGGAGAGACAC-3¢ 0.2 mM 1.5 mM

(Ex6 final) 6Rf 5¢-AGATAGAGGGAAAGATATAGTGAAAGGGACAG-3¢

The exon (Ex) and intron (In) composition of the six fragments is indicated in parenthesis as well as the identification of each primer as

mentioned in Figure 1. Final concentrations of dNTPs and MgCl2 are presented for each fragment.

1F 2R

Intron V Intron IVIntron I Intron II Intron III

2 (~ 8 kb)

2F 3R

4 (0.9 kb)

4F 5R

6(0.46 kb)

6Rf6F

1 (~ 4 kb) Fragment Fragment Fragment

Fragment Fragment Fragment

3 (~ 3 kb)3F 4R

5 (1.2 kb)5F 6R

Ex 1 142 bp

ATG

Ex 2A78 bp

Ex 2 72 bp

Ex 3 225 bp

Ex 4 165 bp

Ex 5 Ex 6 211 bp

TAG

Figure 1. Schematic diagram of the O. aries POU1F1 gene, with the position of the PCR primers (arrowheads) used for the ampli-

fication of each fragment. Introns are shown as double lines and exons (Ex) as boxes with the corresponding length. The starting

point given for exon 1 is the initiation codon (ATG) and the end of exon 6 is the stop codon (TAG).

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ligation was performed promptly. For fragments1, 2 and 3 ligation was done with pCR-XL-TOPOvector and for fragments 4, 5 and 6 with pCR 2.1-TOPO vector, according to the supplier’s recom-mendations (respectively TOPO XL PCR andTOPO TA Cloning kits, Invitrogen Corporation).The selection of recombinant colonies was madeby blue/white screening with TOPO-TA cloningkit while only recombinant colonies grew with theTOPO-XL cloning kit. Plasmid DNA was purifiedwith GenElute Plasmid Miniprep kit (Sigma).Clones were sequenced according to the dide-oxynucleotide chain termination method using theT7 sequencing kit and following the supplier’srecommendations (Amersham Biosciences). Driedgels were exposed to autoradiographic film (Ko-dak). In order to validate the sequencing results, asecond set of sequencing was made on bothstrands at an independent enterprise (MWG-Bio-tech). Sequences were analyzed with the GeneticsComputer Group (GCG) program package fromthe Belgian Embonode (BEN). All the sequenceswere submitted to EMBL Database and areavailable under the accession numbers AJ549204-549207.

PCR amplification for polymorphism detectionby BESS-T Scan

After analysing all the sequences, primers locatedin the intron regions were used to completelyscreen the exons in order to detect polymorphisms.The sequences of the wild type fragments areshown in Figure 3 (as part of the results of thesequencing strategy). The PCR primers used forpolymorphism detection are shown in boxes; exonregions are shaded in grey, considering the initia-tion codon as the beginning of exon 1 and the stopcodon as the end of exon 6. For exon 2, thealternative exon 2A is underlined.

BESS-T (Base Excision Sequence Scanning-Thymine) scan (Biozym) was the chosen approachfor polymorphism detection.

All PCR reactions were first optimized. Reac-tions for fragments BESS1, 2, 4, 5 and 6 wereprepared in a final volume of 25 ll containing100 ng of DNA; 1.5 mM of MgCl2; 200 lM ofdNTPs and 16 lM of dUTP, 8 pmol of each pri-mer (forward primer unlabelled and reverse primerlabelled with Cy5 and the inverse) and 1 U of TaqDNA polimerase (Promega). For BESS3 fragment

amplification the final concentration of MgCl2(2 mM), and primers (12 pmol) was increased. 25cycles were performed with 30 s for each step:denaturation (95�C), annealing (60�C for BESS1,57�C for BESS2 and 55�C for BESS3, 4, 5 and 6)and extension (72�C).

Following the confirmation of the amplifica-tion, by agarose gel electrophoresis, 5 ll of eachPCR reaction were combined with 1 ll of enzymemix (containing uracil N-glycosylase and endo-nuclease IV) and 3 ll ddH2O, according to sup-plier’s recommendations. After a 30 minincubation at 37�C, 5 ll of stop loading buffer(95% formamide and 10 mM EDTA) were added,followed by heat inactivation of the enzymes at75�C for 5 min. From this mixture, 3 ll wereloaded onto an 8% polyacrylamide sequencing gelcontaining 8 M urea and analyzed using AlfEx-press 2.00 Alfwin-sequence analyser (AmershamBiosciences). The electropherogram shows theadenine present in the sequence when reverse pri-mer is labelled and the thymine when forwardprimer is labelled with Cy5.

All the polymorphisms detected by this meth-odology were confirmed by direct sequencing ofthe PCR product by dideoxynucleotide chain ter-mination method.

Polymorphism detection by SNaPshot methodology

Following BESS T-Scan analysis and confirmationof the polymorphism by sequencing, we optimizedSNaPshot methodology (Epicentre) to rapidlygenotype the polymorphism detected in exon 2.

The 251 bp fragment containing exon 2 wasamplified using the same conditions as describedfor BESS-T method, except that primers were notlabelled and dUTP was not used. The resultingPCR product was purified with the High purePCR purification kit (Roche Diagnosis). Posteriorreactions were performed using the SNaPshot kitaccording to the supplier’s recommendations. Thereaction mixture consisted of 3 ll of purified PCRtemplate, 5 ll of SNaPshot ready reaction premix,1 ll ddH2O and 1 ll (0.2 lM) of SNaP reverseprimer (5¢-ATGACTGGTTTCCGTAAT GA-3¢),which has the 3¢ extremity one nucleotide beforethe mutation point. After 25 cycles of 96�C for10 s, 50�C for 5 s and 60�C for 30 s, the amplifiedmixture was treated with CIP (Calf IntestinalPhosphatase) to remove the 5¢ phosphoryl groups

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of the unincorporated fluorescent ddNTPs,avoiding their co-migration with the fragment ofinterest. This reaction was performed at 37�C for30 min, followed by heat inactivation of the en-zyme at 75�C for 15 min. The electrophoresis wasperformed on the ABI PRISM 3100 genetic ana-lyser (Applied Biosystems) using GeneScan 120LIZ size standard.

Results

Cloning and sequencing

Six genomic fragments of the ovine POU1F1 gene,each spanning an intron of unknown length, wereamplified using specific primers located in the ex-ons and were subsequently cloned and sequenced.The amplification strategy is presented in Figure 1,with the approximate length of each fragmentobtained and the position of the primers used ineach case (Table 1).

Figure 2(a and b) shows the results of theamplification of the six POU1F1 gene fragments.

Introns 1, 2 and 3 appeared to be very long,yielding PCR products of approximately 4, 8 and3 kb, for fragments 1, 2 and 3, respectively (Fig-ure 2a). As the main aim of this work was to se-quence the regions adjacent to the coding sequenceof POU1F1 gene in order to allow the subsequentscreening for polymorphisms in the entire exons,introns 1, 2 and 3 were only partially sequenced.The electrophoretic separation of the PCR prod-ucts for fragments 4, 5 and 6 (Figure 2b) shownthe lengths of 0.9, 1.2 and 0.46 kb, respectively.Sequencing of these fragments led to the estab-lishment of the complete nucleotide sequences ofexons 4, 5 and 6 and introns 4 and 5.

A total of 5787 bp of O. aries POU1F1 genewere sequenced. The sequences were submitted toEMBL with accession numbers AJ549204-549207.

Polymorphism detection

Polymorphism detection was performed in 100animals of the ‘Churra da Terra Quente’ breed.Figure 3 shows the sequence and length of the six

Figure 2. Agarose gels stained by ethidium bromide with the PCR amplified and purified ovine POU1F1 genomic fragments used

for the sequencing purpose. (a) Fragment 1 (Ex1-In1-Ex2) has approximately 4 kb, fragment 2 (Ex2-In2-Ex3) about 8 kb and frag-

ment 3 (Ex3-In3-Ex4) around 3 kb. (b) The length of fragment 4 (Ex4-In4-Ex5), fragment 5 (Ex5-In5-Ex6) and fragment 6 (Ex6

and a short sequence downstream the stop codon) was 0.9 kb, 1.2 kb and 0.46 kb, respectively.

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fragments amplified for the detection of poly-morphism in O. aries POU1F1 gene, after theorganization of all the sequence information ob-tained previously. The position of the detected

polymorphisms in BESS 2, 3 and 5 fragments,described in detail below, is shaded in black.

We first focus on the analysis of the BESS2fragment. Figure 4 shows the result of BESS-T

Figure 3. Sequence and length of the analyzed fragments for the detection of polymorphisms in each exon of O. aries POU1F1

gene. Primers are shown in boxes; exon regions are shaded in grey, considering the initiation codon as the beginning of exon 1 and

the stop codon as the end of exon 6. For exon 2, the alternative exon 2A is underlined. The position of the detected polymor-

phisms in BESS 2, 3 and 5 fragments is shaded in black.

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Scan polymorphism analysis and SNaPShotpolymorphism genotyping of the 251 bp PCRproduct containing exon 2.

Figure 4a shows BESS-T Scan result with partof the adenines present in the analyzed fragment.As it is evidenced by the arrow, there is an extraadenine in the third sample, corresponding to apoint mutation. Comparison of the sequence readfrom the peaks with the POU1F1 nucleotide se-quence, led to the assignment of this point muta-tion to nucleotide 150 of the analyzed fragment. Inorder to confirm this polymorphism, the PCR

product of the polymorphic animal was sequenced.This polymorphism, consisting of a G fi A tran-sition at codon 58, leads to a change from cysteineto tyrosine. Figure 4b shows the result of SNaP-Shot analysis, revealing a homozygous animal(genotype GG, left side) and a heterozygous ani-mal (genotype GA, right side). Four out of a totalof 100 animals of ‘Churra da Terra Quente’ breedshowed the mutant allele on the heterozygousform (2% allelic frequency). As we used a reverseSNaPShot primer and thus analyzed the comple-mentary strand, a single peak appeared in the GG

Figure 4. Polymorphism detection by: (a) BESS-T Scan: Electropherogram showing part of the adenine peaks present in the

BESS2 PCR product. The arrow shows the extra adenine in the third sample. The PCR reaction was performed using the Cy5 la-

belled BESS2-R primer; (b) SNaPshot: After amplification of the same 251 bp BESS2 fragment, without Cy5 labelling, the PCR

product was labelled using SNaPShot ready reaction premix (containing fluorescent ddNTPs) and a specific SNaP reverse primer.

The electrophoresis was performed with the ABI PRISM 3100 genetic analyser. The peaks marked with asterisks correspond to the

internal LIZ size standard. The left picture shows a homozygous animal (genotype GG) and the right picture shows a heterozy-

gous animal (GA). (As we analyzed the complementary strand, we visualize a single cytosine for the animal with genotype GG and

a cytosine and a thymine for the animal with genotype GA).

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genotype corresponding to a cytosine. For GAanimal two peaks were revealed corresponding toa cytosine and a thymine. In both pictures, thefour peaks marked with asterisks correspond tothe LIZ internal size standard.

Fragments BESS1, 4 and 6 showed no poly-morphisms.

Two polymorphisms were detected in fragmentBESS3: a G to A transition altering a glycine to anasparagine at codon 89 in one allele of one animal(0.5% allelic frequency), and another G to Atransition at codon 105 converting an alanine intoa threonine in one allele of 3 animals (1.5% allelicfrequency).

A polymorphism (A to G transition) was de-tected in fragment BESS5 in the base 93, corre-sponding to the intron 4 region, in one allele of 6animals (3% allelic frequency). No polymorphismswere found in exon 5.

Discussion

The POU1F1 gene has an essential role in thecontrol of pituitary development and in the regu-lation of GH and PRL expression. It is thereforean interesting candidate gene for marker assistedselection (MAS). In order to identify naturallyoccurring polymorphisms in the coding region ofthis gene in O. aries, information about the se-quence of the intron regions immediately adjacentto each exon was necessary to allow the design ofspecific primers.

The 5787 bp sequenced in the present workincludes the complete six exons, two complete in-trons (introns 4 and 5) and part of each of introns1, 2 and 3. The considerable increment of infor-mation obtained allowed the screening of poly-morphisms in all the exons of the POU1F1 gene,using primers located in the intron regions.

The nucleotide coding sequence (876 bp) and thepredicted protein sequence (291 amino acids) werecompared with bovine, human and rat sequences.This analysis showed that the ovine POU1F1 cod-ing region has a high level of similarity with theirbovine, human and rat counterparts (98.2%, 91.2%and 86.2%, respectively) and that the amino acidlevel similarity is very high (99%, 96.2%and93.1%,respectively). The publishedmRNA sequence of thePOU1F1 of O. aries (GenBank accession numberU88399) (Thomas et al., 2000) differs in 7 bp withthe sequence determined in the present work (99%

similarity). The predicted protein sequence wasfound to differ from the published one (GenBankaccession number P79364) in 3 amino acids (99% ofsimilarity).

The structure of the gene admits an alternativesecond exon 2, named exon 2A, as shown in Fig-ure 1. This extra exon (78 bp) was first describedin rats (Theill et al., 1992) and humans (Delhaseet al., 1995) and is considered responsible for analternative mRNA transcript (named Pit2 orPit1b). In the present work, the sequencing of thegenomic DNA revealed that this exon, underlinedin BESS2 fragment in Figure 3, is present at thesame position in sheep and shows 88.5% of simi-larity with the human sequence. Using RT-PCRwith RNA extracted from sheep pituitary we havefound the same alternative splicing transcript forPOU1F1 gene (data not published). We are pres-ently investigating the possible implications of thisvariant on the transcriptional control of GH andPRL gene-expression.

In addition, we showed a strong conservationof a 61 bp region at the 3¢-end of intron 1 in hu-man, rat, mouse and sheep. This fact suggests afunctional important role, namely the recognitionof this region by DNA-binding transcription fac-tors (enhancer or silencer) or the formation of aconserved RNA secondary structure important forthe splicing or the stability of POU1F1 mRNAs.Another possible explanation is that this sequenceforms a new alternative exon (new exon 2C).

The BESS-T Scan methodology, used for thepolymorphism detection, is a very powerful toolthat allows a simple and efficient identification of aspecific difference between multiple DNA samples.SNaPShot is a very reliable technique to perform aposterior genotyping of the identified mutation.

The search for nucleotide changes at thePOU1F1 gene revealed polymorphisms in exons 2and 3 and in intron 4.

In exon 2, a transition from guanine to adeninewas detected, resulting in an amino acid changefrom cysteine to tyrosine in the codon 58. Four outof a total of 100 animals of ‘Churra da TerraQuente’ breed showed the mutant allele in theheterozygous form (2% allelic frequency). Thisamino acid change occurs in the transactivationdomain (STA) and might therefore have animportant effect on the tertiary structure of thePOU1F1 protein and on its ability to regulatedownstream genes. Until now, there are no reports

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of mutations associated with CPHD or other dis-order in this codon or in this exon in humans.There is no single nucleotide polymorphism de-scribed in any species.

In exon 3, two different polymorphisms weredetected: a G to A transition converting a glycine toan asparagine at codon89 in one allele of one animal(0.5% allelic frequency) and another G to A tran-sition at codon 105 converting an alanine into athreonine in one allele of 3 animals (1.5% allelicfrequency). These two polymorphisms are locatedin the region between the transactivation domainand thePOUsdomain, namedCHG,which is rich incharged aminoacids. De la Hoya et al. (1998) pro-posed a model for the 3D structure and suggestedthat both STA and CHG regions should have aninterface for directing contacts with surfaces of co-activators and general transcription factors. In hu-mans there are nomutations reported in this region.Mutations have been described in codons 135, 143and 145; these codons belong to another region(POUs domain), which is very important for thebinding to the target genes. There is also a report inhumans of a single nucleotide polymorphism incodon 84 (SNP rs4988460), near to the position ofthe polymorphism detected in ovine, showing thatthis region is polymorphic.

In intron 4, an A to G transition was detectedin one allele of 6 animals (3% allelic frequency).This intron is not polymorphic in humans, but ishighly polymorphic in chicken, where five SNPshave been reported.

In order to anticipate the implications of thepolymorphisms detected on the structure ofPOU1F1, protein secondary structure predictionwas performed by JPRED method (Cuff andBarton, 2000) and two protein sequence analysisservers: PredictProtein (Rost et al., 2003) and PSA(Stultz et al., 1997). Three short helix structureswere predicted in C58Y variant (aas #57–63),G89D variant (aas #83–90), and A105T variant(aas#102–120). In addition, all the three amino-acid changes (tyrosine, asparagine and threonine)increase the negative charge of the trans-activationdomain of POU1F1.

Acknowledgements

The authors wish to thank ANCOTEQ (Asso-ciacao Nacional de Criadores de Ovinos Churra

da Terra Quente) and DRATM (DireccaoRegional de Agricultura de Tras-os-Montes) forgenerously providing sheep blood samples. Thiswork was financed by the EC - III FrameworkProgramme for Research and TechnologicalDevelopment, co-financed by the European SocialFund (ESF), by Portuguese funding from MCES(Ministerio da Ciencia e do Ensino Superior)(PRAXIS XXI 3/3.2/CA/1991/95) and the Na-tional Fund for Scientific Research, B-1000 Brus-sels, Belgium (# 24524.01). E.B. and I.C.S. thankFCT (Fundacao para a Ciencia e a Tecnologia)and ESF (EC-III Framework Programme) fortheir Ph.D. grants (BD-1365/2000 and BD/18061/98, respectively). J.L.C. and Centro de BiologiaMolecular Severo Ochoa (CBMSO) have bene-fited from an institutional grant from FundacionRamon Areces (Spain).

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