Effect of New SNP Within Bovine Prolactin GeneEnhancer Region on Expression in the Pituitary Gland

P. Brym Æ T. Malewski Æ R. Starzynski ÆK. Flisikowski Æ E. Wojcik Æ A. Rusc ÆL. Zwierzchowski Æ S. Kaminski

Received: 12 January 2007 / Accepted: 30 May 2007 / Published online: 11 October 2007

� Springer Science+Business Media, LLC 2007

Abstract A new single nucleotide polymorphism was revealed using PCR–SSCP

and sequencing methods within the bovine prolactin distal promoter region

described as a functional enhancer. The A?G transition at position –1043 abolishes

the recognition site for Hsp92II restriction endonuclease, allowing for PCR–RFLP

genotyping. The application of real-time PCR revealed that the prolactin gene

expression level in the pituitary was higher in cattle with the AA genotype than in

those with the GG genotype. EMSA analysis, however, showed increased nuclear

protein binding to the sequence variant with G, suggesting a possible inhibition

event, in which the transcription factors Pit1, Oct1, and YY1 could be involved.

Keywords Bovine prolactin � SNP � Promoter � Gene expression

Introduction

Prolactin is a polypeptide hormone, synthesized and secreted not only in the anterior

pituitary gland but also produced by numerous other cells and tissues, including the

mammary gland (extrapituitary prolactin). In various classes of vertebrates more

than 300 actions and activities of this multifunctional hormone have been

P. Brym (&) � E. Wojcik � A. Rusc � S. Kaminski

Department of Animal Genetics, University of Warmia and Mazury, Olsztyn, Poland

e-mail: pawbrym@uwm.edu.pl

T. Malewski � R. Starzynski � K. Flisikowski � L. Zwierzchowski

Institute of Genetics and Animal Breeding, Polish Academy of Sciences,

Jastrzebiec, Wolka Kosowska, Poland

Present Address:T. Malewski

Museum and Institute of Zoology, Warsaw, Poland

123

Biochem Genet (2007) 45:743–754

DOI 10.1007/s10528-007-9115-9

reported (Bole-Feysot et al. 1998). Gene targeting experiments, however, proved its

irreplaceable role only in female fertility, mammary gland development, lactogen-

esis, and maintenance of milk secretion (galactopoiesis) (Horseman et al. 1997;

Ormandy et al. 1997). Therefore, the bovine prolactin gene (PRL) seems to be an

excellent candidate gene for linkage analysis with a QTL affecting reproduction and

milk performance traits. Extensive genetic polymorphism studies were carried out,

finding 20 SNPs within the bovine PRL structure gene sequence, although all of

them were silent mutations or located within introns (Sasavage et al. 1982; Brym

2004). Nevertheless, three independent groups confirmed statistically significant

associations between prolactin gene variants (identified by RsaI endonuclease) and

milk production traits in dairy cattle (Chung et al. 1996; Dybus 2002; Dybus et al.

2005; Brym et al. 2005). One can assume that the observed relations resulted from

linkage of the marker mutation used in the analysis and causative polymorphism in

PRL gene regulatory sequences. In the 50 flanking region of the bovine prolactin

gene, a distal regulatory element was found, which enhances the basal level of

expression of the reporter gene fivefold and functions independently of position and

orientation (Wolf et al. 1990). The postulated enhancer region extends from –1175

to –996 and displays considerable sequence similarity to equivalent regions in

human and rat prolactin gene promoters (Fig. 1).

Here we report a new single nucleotide polymorphism (A/G) found within the

bovine enhancer region at position –1043, which potentially affects prolactin gene

expression.

Materials and Methods

Animals and DNA Isolation

The analysis includes a total of 348 Polish Holstein Friesian cattle. Approximately

10 ml of blood was withdrawn from each animal by an authorized veterinarian into

tubes containing K2EDTA. DNA was isolated from leukocytes by a MasterPure

DNA Purification Kit (Epicentre) or by the method of Kanai et al. (1994). For some

samples, DNA isolation was performed from a commercial portion of semen by the

Fig. 1 ClustalW multiple alignment analysis of 180 nt DNA sequence postulated (Wolf et al. 1990) asan enhancer of the prolactin gene. Within this distal 50 prolactin element, high sequence similaritybetween bovine (GenBank acc. no. X16641), human (AL023883), and rat (J00766) sequences can beseen. Nucleotides are numbered according to the bovine prolactin gene transcription start site. Thepolymorphic site in the bovine sequence reported here is depicted by an arrow

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MasterPure DNA Purification Kit supplemented with additional DTT and proteinase

K treatment of sperm (Epicentre). All experimental procedures involving animals

were approved by the Local Ethics Commission (permit nos. 30/04 and 67/2001).

Detection of Polymorphism and Genotyping

Primer3-designed PCR primers (forward 50-TTTTCTTAATGCAGCTCACTTGTC-

30 and reverse 50-TCTAGACAGTCATGTTCACTTTCCTT-30) were used to

amplify a 311 bp fragment (–1230 to –920) of the bovine PRL 50 flanking

sequence. The PCR was carried out in 25 ll of a mix containing 1.25 ll MasterAmp

20· PCR buffer (Epicentre), 0.75 mM MgCl2, 0.25 mM each dNTP, 4 ll 10·Enhancer (Epicentre), 20 pmol each primer, 0.8 U MasterAmp Tfl DNA polymer-

ase (Epicentre), ca. 200 ng of genomic DNA, and water up to 25 ll. The following

thermal profile was performed in a Mastercycler 5330 thermocycler (Eppendorf):

initial denaturation (94�C for 3 min), 35 cycles of denaturation (94�C for 30 s),

annealing (60�C for 30 s), extension (72�C for 30 s), and final synthesis (72�C for

5 min). Single-strand conformation polymorphism (SSCP) analysis of amplified

fragments was used to detect mutations (Orita et al. 1989). The PCR products were

mixed (1:2) with denaturation buffer (50 mM NaOH, 1 mM EDTA, 0.25% loading

buffer by Promega), denaturated at 85�C for 13 min, chilled on ice, and loaded on

8% polyacrylamide gels containing 1· TBE buffer. Electrophoresis was performed

in a Multiphor II Electrophoresis System (Pharmacia), at a constant temperature of

5�C, in 1· TBE buffer under the following conditions: 200 V, 20 mA for 30 min

(initial electrophoresis), and 375 V, 30 mA for 4 h. SSCP patterns on the gels were

visualized by silver staining. The PCR products representing different SSCP

patterns were sequenced with an ABI Prism 377 DNA Sequencer (Applied

Biosystems) and DYEnamic ET Terminator Cycle Sequencing Kit (Amersham

Biosciences), according to the procedure described by the manufacturers. The

detected substitution A?G at position –1043 abolishes the restriction site for

Hsp92II endonuclease, suitable for PCR–RFLP analysis. The overnight digestion of

10 ll of the PCR products with 2 U of restriction enzyme Hsp92II (Promega) at

37�C was followed by 3.5% agarose gel electrophoresis (PCR Low Melt Agarose,

Bio-Rad Laboratories). Both PCR–RFLP and PCR–SSCP methods were used

interchangeably for genotyping.

Computer-aided Analysis

The nucleotide sequence of the amplified 50 flanking region of the bovine prolactin

gene was analyzed for the presence of putative transcription factor binding sites with

TESS (Schug and Overton 1998) and MatInspector (Quandt et al. 1995) software.

Electrophoretic Mobility Shift Assay (EMSA)

The differences in transcription factor binding to alternative prolactin enhancer

sequence variants were analyzed by the EMSA technique. The bovine pituitaries

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were excised immediately after slaughter at the local abattoir and stored at –75�C

until use. Tissue homogenization, preparation of nuclei, and extraction of nuclear

proteins were conducted as described previously (Malewski et al. 2002), with one

modification: the solubilized proteins were dialyzed against 10 volumes of buffer

(10 mM Tris–HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, and 5%

glycerol) overnight at 8�C. The following chemically synthesized double-stranded

oligonucleotides were used as probes, with either A or G at position –1043

(underlined):

50-ACTTTAGAGCCC(A/G)TGAAAGATGAA-30

30-TGAAATCTCGGG(T/C)ACTTTCTACTTGAGGG-50

An additional single-stranded end 30-GAGGG-50 (italics) in the reverse strand

(originally not occurring in the prolactin gene sequence) was added for efficient

labeling with [alpha-32P] dCTP (ICN) using Klenow enzyme (Random Primer DNA

Labeling Kit, Sigma) according to the manufacturer’s protocol. The probes were

purified from unbound nucleotides using Sephadex G-25 spin columns (Sigma). The

labeled probes (ca. 100,000 cpm) were incubated for 30 min at room temperature in

a reaction mix containing 2 ll buffer (10 mM Tris–HCl pH 7.5, 50 mM NaCl,

1 mM DTT, 1 mM EDTA, 5% glycerol), 5 lg nuclear proteins, and water up to

20 ll. For EMSA competition experiments, unlabeled ‘‘cold’’ double-stranded

oligonucleotide competitors in 10·, 50·, and 100· molar excess were added to the

same reaction mix and incubated for 30 min. The subsequently labeled probes A or

G (ca. 100,000 cpm) were added and incubated for an additional 30 min. Unlabeled

probes with A or G at position –1043 were used as competitors (cross-competition),

and specific competitors containing transcription factor binding sites for Pit1, Oct1,

YY1, and GATA were designed according to the Transfac database. The following

double-stranded oligonucleotides were utilized (only sense strand is shown): Pit1 50-TTAGATGGATAATTTAGATGGATAATTTAG-30, Oct1 50-GATCATGCAAA

TGATCATGCAAAT-30, YY1 50-CGCTCCGCGGCCATCTTGGCGGCTGGT-30,GATA 50-GGGGTGTGATAAGGATGATAAGGATGATAAGGA-30. After DNA-

nuclear protein binding, 2 ll loading buffer (50% glycerol, 0.25% bromophenol

blue) was added to each sample, and the reaction mixtures were applied on a pre-run

6% nondenaturing polyacrylamide gel in 0.25· TBE. The electrophoresis was

performed at room temperature at 100 V for 4 h. The EMSA gels were dried in a

Gel Dryer 543 (Bio-Rad) and subjected to autoradiography with a Kodak Imaging K

Screen. The autoradiograms were scanned with a Molecular Imager FX, and

densitometry was performed with Quantity One software (Bio-Rad).

Real-Time PCR

Quantification of prolactin gene expression in bovine pituitaries of cattle carrying

PRL genotypes AA, AG, and GG was conducted by real-time PCR. We took

advantage of experimental material described in detail previously (Oprzadek et al.

2003) The young bulls used were the progeny of AI Holstein sires and were

slaughtered at the age of 15 months, after 24 h fasting. The pituitaries were

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removed immediately after slaughter, frozen in liquid nitrogen, and stored at –80�C

until used for RNA isolation. Total RNA from male bovine pituitaries (each

genotype was represented by three pituitaries) was isolated with the GenElute

Mammalian Total RNA Kit (Sigma). The quality of isolated RNA was verified by

electrophoresis in 2% agarose gel (Sigma). Then RNA was treated with DNase I

(Sigma) and reverse transcribed with an Enhanced Avian HS RT-PCR Kit (Sigma)

according to a protocol described by the manufacturer. Real-time PCR was

performed with the LightCycler apparatus and LightCycler software 3.5, based on

the second derivative maximum method (Roche). The reaction mix contained 5 ll

SYBR Green Taq ReadyMix for Quantitative PCR (Sigma), 1 ll cDNA, 10 pmol

each primer, and water up to 10 ll; it was transferred to a glass capillary, sealed,

centrifuged, and placed into the rotor of the LightCycler apparatus. The following

primers were used to amplify the cDNA fragment encompassing parts of exons 4

and 5 of the bovine prolactin gene (according to GenBank AF426315): forward

RTprl1 50-GAGCTTGATTCTTGGGTTGC-30, reverse RTprl2 50-TGTGGGC

TTAGCAGTTGTTG-30. The internal reference standard was a housekeeping b-

actin gene, which was amplified with primers forward Bvbact1 50-GAGAAGC

TGTGCTACGTCGC-30 and reverse Bvbact2 50-CCAGACAGCACTGTGTTGGC-

30. The following thermal profile was applied: initial denaturation (95�C for

10 min), 45 cycles of four segment amplification with denaturation (95�C for 10 s),

annealing (58�C for 15 s), elongation (72�C for 20 s), and fluorescence measure-

ment (82�C for 4 s). Subsequently, a melting step was performed consisting of

denaturation (95�C for 2 s), annealing (58�C for 5 s), slow heating with a rate of

0.1�C for 1 s up to 95�C, with continuous fluorescence measurement, and finally

cooling to 35�C. On the basis of obtained crossing point (CT) values, the relative

gene expression between genotypes was analyzed by the 2–DDCT method (Livak and

Schmittgen 2001).

Results

The SSCP method was used to identify nucleotide sequence polymorphism within

the 50 flanking region of the bovine prolactin gene. First, specific PCR products of a

desirable size (311 bp) were obtained, involving the whole distal regulatory element

acting as an enhancer. Subsequently, the PCR products were denatured and

subjected to polyacrylamide gel electrophoresis to find sequence variation. Three

SSCP patterns were observed (Fig. 2). DNA samples representing the SSCP

variants were sequenced, and the location and nature of the mutation underlying the

SSCP polymorphism were precisely identified. A substitution A?G at position

–1043 was found (Fig. 2). Sequencing results were confirmed with the PCR–RFLP

method and the Hsp92II restriction endonuclease. Digestion of the 311 bp PCR

product with the enzyme resulted in four restriction fragments of 111, 102, 88, and

10 bp for the AA genotype; three restriction fragments of 213, 88, and 10 bp for the

GG genotype; and five restriction fragments of 213, 111, 102, 88, and 10 bp for the

AG heterozygotes (Fig. 2).

Biochem Genet (2007) 45:743–754 747

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Using the PCR–RFLP and PCR–SSCP methods, we found 183 animals with the

AA genotype, 149 with AG, and 16 with GG. The estimated allele frequencies were

0.74 for A and 0.26 for G.

A computer analysis of the –1230 to –920 fragment of bovine prolactin gene

using TESS and MatInspector programs showed putative binding sites for several

transcription factors. The polymorphism at position –1043 is located within or close

to putative binding sites for pituitary tissue-specific Pit1, among others, and

ubiquitous Oct1 and YY1.

The effect of the mutation on interaction with nuclear protein was studied by an

electrophoretic mobility shift assay. The radiolabeled oligonucleotide probes A and

G differing at the mutation site were subjected to binding with nuclear proteins,

extracted from bovine pituitaries, and nondenaturing polyacrylamide gel electro-

phoresis. Two DNA-protein complexes with retarded migration were observed

(Fig. 3). Densitometry scanning showed that the probe with G binds about 20%

more pituitary nuclear protein than the probe with nucleotide A. The difference

Fig. 2 Genotyping of the SNP at position –1043R (A/G) within the bovine prolactin enhancer region. (a)SSCP polymorphism of 311 bp silver-stained PCR products. Three SSCP patterns are visible,corresponding to AA, AG, and GG genotypes. (b) Direct sequencing of three PCR products withdifferent SSCP patterns indicates the mutation (A/G) at position –1043. Sequence I occurs in the AAgenotype, II in AG, and III in GG. The double peak in sequence II, marked by the base N, shows bothnucleotides A and G in the heterozygote. (c) Restriction analysis of PCR products containing mutation–1043R. PCR products digested with Hsp92II on 3.5% agarose gel stained with ethidium bromide. LaneP, undigested 311 bp PCR product. Genotype AA lanes, restriction fragments of 111, 102, and 88 bp.Genotype GG lane, restriction fragments 213 and 88 bp. Genotype AG lane, restriction fragments 213,111, 102, and 88 bp. A restriction fragment of 10 bp, common to all the genotypes, is not visible. Lane M,molecular weight marker UX 174/HaeIII

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approached statistical significance (P £ 0.057). Moreover, cross-competition exper-

iments (Fig. 3) showed that unlabeled G competitor displaced the protein complex

1, formed with labeled probe A, less efficiently than did the unlabeled A competitor.

Competition analysis with the application of gradual molar excess of unlabeled

oligos specific for Pit1, YY1, and Oct1 confirmed the possible participation of these

transcription factors in both DNA-protein complexes 1 and 2, although less efficient

displacement of nuclear protein from complex 2 was observed for the Pit1-specific

competitor (Fig. 3). As expected, the oligo competitor for the GATA transcription

factor did not compete for nuclear protein binding, as no binding sites for GATA TF

were localized within the utilized probe (Fig. 3).

Analysis of the quantity of specific prolactin mRNA in pituitaries with different

genotypes was performed with real-time PCR. Total RNA from nine pituitaries

representing AA, AG, and GG prolactin promoter genotypes (three for each

genotype) was isolated and reverse transcribed. Real-time analysis was repeated

seven times and results were averaged. Using the 2�DDCT method and b-actin gene

Fig. 3 (a) The representative result of an effect of A/G polymorphism in bovine prolactin enhancerregion at position –1043 on pituitary nuclear proteins binding with the electrophoretic mobility shift assaytechnique. Lane 1, free probe. Lanes 2–9, successive binding to probes A and G of total nuclear proteinsfrom four different pituitaries. Two slow-migrating complexes are marked. Lanes 9 and 10, cross-competition of unlabeled A and G probes at 100· molar excess with radiolabeled probe A. Lanes 12–14,competition with 10·, 50·, and 100· molar excess of Pit 1 competitor. Lanes 15–17, competition with10·, 50·, and 100· molar excess of YY1 competitor. Lanes 18 and 19, competition with 100· molarexcess of Oct1 competitors, probes A and G, respectively. Lane 20, competition with negative competitorGATA. (b) Densitometry estimates of DNA-protein complexes and the difference between probe A andprobe G. The more intensive DNA-protein complexes can be seen for probe G; the difference approachesstatistical significance (P £ 0.057, N = 26). The competition experiments were repeated three times withdifferent competing probes, always giving similar results

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as an internal standard, we showed 4.45-fold more prolactin mRNA in pituitaries

with the AA genotype in comparison with the GG genotype (Fig. 4). These results

suggested that the studied polymorphic site could influence the expression of the

bovine prolactin gene.

Discussion

Prolactin is a common mediator of the immunoneuroendocrine network, where

nervous, endocrine, and immune systems communicate with each other. The gene

encoding prolactin is a well-acknowledged candidate for a quantitative trait marker

in farm animals. Prolactin’s importance at lactogenesis seems to be absolute

irrespective of species, but whether it has a role in mammogenesis and maintenance

of milk secretion in dairy ruminants remains open to debate. Some data suggest that

lactation in cattle and goats bred for milk production is less dependent on prolactin,

emphasizing instead the role of growth hormone in maintaining milk yield (Flint

and Knight 1997). Other data indicate that dairy ruminant mammary tissue has

developed a high avidity for any prolactin that is present in the circulation and can

respond adequately even to very low levels of the hormone and small changes in its

concentration (Knight 2001). Additionally, local production of the prolactin in

mammary tissue and other extrapituitary prolactin can influence the results of

experiments based only on the measurements of the prolactin secretion in the

pituitary or its total blood concentration in relation to lactation status and yield. On

the other hand, some reports confirm statistically significant associations between

bovine prolactin gene variants and milk production traits in dairy cattle (Chung

et al. 1996; Dybus 2002; Dybus et al. 2005; Brym et al. 2005). It is noteworthy that

no missense mutations were found within the PRL gene (Sasavage et al. 1982; Brym

2004), and the observed statistical relation probably resulted from causative

mutation in the prolactin gene regulatory sequences influencing quantitatively

Fig. 4 Real-time PCR detection of the prolactin transcript in pituitaries with different AA and GGgenotypes. The relative gene expression between genotypes was analyzed by the 2–DDC

T method (Livakand Schmittgen 2001). An internal reference standard (a housekeeping b-actin gene) was utilized fornormalization of the amounts of template RNA in each sample. Values represent averages of seven real-time experiments with RNA derived from three animals of each genotype. The difference betweengenotypes AA and GG was statistically significant at P £ 0.05

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prolactin gene expression. Generally, prolactin impact on milk yield and content is

still enigmatic and requires further study.

Prolactin gene expression has been extensively studied in only two species,

humans and rats, mostly using pituitary tumor cell lines. It was concluded that in the

pituitary, prolactin synthesis and secretion was regulated by a number of factors,

including dopamine, thyrotropin-releasing hormone (TRH), estradiol, glucocorti-

coids, epidermal growth factor (EGF), Ca2+, cAMP, and phorbol esters. These

factors modify prolactin gene transcription through numerous transcription factors

interacting with composite response elements of the prolactin 50 flanking promoter

region. In the pituitary involvement of tissue-specific Pit1 and ubiquitous C/EBP,

AP1, Ets, GR, ER, Oct1, YY1, and Sp1 factors have been emphasized (Freeman

et al. 2000, Evans et al. 1978; Day et al. 1998; Jacobs and Stanley 1999).

Extrapituitary expression of the prolactin gene could be regulated by other tissue-

specific transcription factors and additional or alternative tissue-specific promoters

(Freeman et al. 2000; Van de Weerdt et al. 2000).

There are only a few reports of details about the bovine prolactin gene promoter

region. Deletion analysis indicated that the first 250 bp of the bovine 50 flanking

region from the transcription initiation site is sufficient for both stimulation and

inhibition of the CAT reporter gene (Camper et al. 1985). The other distal

regulatory element was found from position –1175 to –996, which enhances the

basal level of expression of the reporter gene fivefold and functions independently

of position and orientation. The deletion analysis of the enhancer region shows that

sequences –1124 to –985 are necessary and sufficient for enhancer activity (Wolf

et al. 1990). Both the sequences of the first 250 bp and the distal enhancer region

show high DNA identity (80%) between cattle, rat, and human species, in contrast

to the rest of the prolactin promoter regions (Fig. 1).

In the 50 regulatory region of the bovine PRL gene, nucleotide sequence

variation was found by means of the SSCP method (Hart et al. 1993; Zhang et al.

1994). The nature of the observed polymorphism was revealed by Klauzinska

(2002), and deletion of the TGTG repeat at position –877 and substitution of C/T

at position –924 were connected with differential nuclear protein binding

(probably GR and AP1, respectively) by the EMSA method. The current study

reports on the identification of a new mutation (A/G) at position –1043 within the

sequence necessary for enhancer activity. In silico transcription factor binding

analysis suggested that the polymorphism revealed was located within or close to

sequences recognized by Pit1, Oct1, and YY1 transcription factors. Using the

EMSA method with cross-competition and densitometry measurements, we

showed quantitative differences in nuclear proteins binding to alternative enhancer

variants. More DNA-protein complexes were formed with the sequence containing

G. The competition experiments with oligonucleotides specific for Pit1, Oct1,

YY1, and GATA (as a negative competitor) confirmed the possible contribution of

Pit1, Oct1, and YY1 in the complexes observed on the gel. The synergistic acting

of the above-mentioned transcription factors could not be excluded. Since the

level of prolactin transcripts measured with real-time PCR in animals with the GG

genotype was decreased about fourfold compared with the AA genotype, a

possible inhibition event could occur.

Biochem Genet (2007) 45:743–754 751

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The transcription factor Yin Yang 1 (YY1) is known to have a fundamental role

in normal biologic processes such as embryogenesis, differentiation, replication, and

cellular proliferation. YY1 exerts its effects on genes involved in these processes via

its ability to initiate, activate, or repress transcription, depending on the context in

which it binds. Mechanisms of action include direct activation or repression,

indirect activation or repression via cofactor recruitment, and activation or

repression by disruption of binding sites or conformational DNA changes (Gordon

et al. 2006). YY1 represses involucrine gene expression in keratocytes (Alvarez-

Salas et al. 2005) and alpha-myosin heavy chain in myocytes (Mariner et al. 2005).

Oct-1 is involved in the transcriptional repression of the Von Willebrand factor gene

in human umbilical vein endothelial cells (HUVECs) and HeLa cells (Schwachtgen

et al. 1998), human GnRH receptor gene in ovarian OVCAR-3, placental JEG-3,

gonadotrope-derived alphaT3-1 cells (Cheng et al. 2002), and human neuropeptide

Y gene (Mayer et al. 2003). YY1 and Oct1 Pit1 in some circumstances can repress

gene expression, as shown by Scully et al. (2000).

Because of substantial participation of familial material within analyzed

individuals, the observed allele frequencies (0.74 for A and 0.26 for G) could be

underestimated for allele A and overestimated for allele G compared to a random

unrelated population. It looks as though the selection toward improvement of milk,

fat, and protein yields increases the frequency of allele A associated with more

efficient transcription of the prolactin gene.

In humans, T/G substitution at a similarly located position –1149 affects

prolactin expression in lymphocytes and is associated with systemic lupus

erythematosus disease (Stevens et al. 2001).

We are aware that the small number of available pituitaries with the GG genotype

limits the possibility of unequivocal conclusions. Further studies on more material

are needed with additional confirmation of results, both at the mRNA and the

protein level. Furthermore, the possible associations of the described polymorphism

with production traits and the occurrence of preferable haplotypes within the

prolactin gene are of great interest.

Acknowledgement This work was financially supported by UWM grant no. 0105–0804.

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