научная статья по теме DELETION/INSERTION POLYMORPHISM OF THE PRION PROTEIN GENE (PRNP) IN POLISH RED CATTLE, POLISH WHITE-BACKED CATTLE AND EUROPEAN BISON (BISON BONASUS L., 1758) Биология

Текст научной статьи на тему «DELETION/INSERTION POLYMORPHISM OF THE PRION PROTEIN GENE (PRNP) IN POLISH RED CATTLE, POLISH WHITE-BACKED CATTLE AND EUROPEAN BISON (BISON BONASUS L., 1758)»

ГЕНЕТИКА, 2009, том 45, № 4, с. 519-525

ГЕНЕТИКА ЖИВОТНЫХ

УДК 575.174.015.3:599.735.5

Deletion/Insertion Polymorphism of the Prion Protein Gene (PRNP) in Polish Red Cattle, Polish White-backed Cattle and European Bison (Bison bonasus L., 1758)

© 2009 U. Czarnik1, G. Grzybowski2, T. Zabolewicz1, J. Strychalski1, S. Kaminski1

1 Department of Animal Genetics, University of Warmia and Mazury in Olsztyn, Olsztyn, 10-719 Poland; e-mail:czar@uwm.edu.pl 2Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, Wolka Kosowska, 05-552 Poland Received April 03, 2007

The aim of the present study was to identify deletion/insertion polymorphism of the bovine prion protein (PRNP) gene within the promoter sequence (23 bp indel), intron 1 (12 bp indel) and the 3' end untranslated region (14 bp indel). The experiment was performed on three groups of animals protected under a genetic resources conservation program: 139 Polish Red (PR) cows, 79 Polish White-backed cows and 50 European bison (Bison bonasus L., 1758). White-backed cattle were characterized by a higher frequency of ins/del heterozygotes and a relatively lower frequency of ins/ins homozygotes within the promoter sequence region (23 bp indel), compared to Polish Red cattle. At the polymorphic locus of intron 1 (12 bp indel) the genetic structure of both cattle populations was similar. Monomorphism, expressed by the occurrence of one genotype variant in each of the analyzed sequence regions, was observed in European bison. Five haplotypes were found in Polish White-backed cows, four haplotypes in Polish Red cows and only one in analyzed group of bison. Differences between the observed and expected number of PRNP haplotypes were recorded in Polish Red cattle.

The pathogenic agent of TSEs (Transmissible Spongiform Encephalopathies) is conformational transformation of the prion protein. Physiologically, the protein PrPc is a tetramer containing domains with the alpha-helix structure, while the pathogenic form PrPSc comes into being by the unfolding of two alpha-helixes that are transformed into the beta-helix structure [1]. The deformed prion protein is accumulated in the brain, forming deposits of amyloid. The process is accompanied by the activation of astrocytes [2], leading to disturbances in normal electrical conductivity in the brain and, in consequence, to death. The conservative structure of the prion protein gene (PRNP) in various species [3], and especially the occurrence of familial forms of transmissible spongiform encephalopathies (TSE) in humans, indicate that the etiology of BSE in cattle may be genetic. The so called functional mutations, showing a relationship with BSE susceptibility and incubation time, are searched for in the bovine PRNP gene localized within chromosome BTA13 at position q17 [4]. Deletion of 24 nucleotides within the ORF region and transition within the 3' flanking fragment of the third exon were detected first [5], and further research allowed to identify polymorphic microsatellite loci [6] and many point mutations [5, 7]. In studies directed towards the identification of the genetic etiology of BSE, whose main part is the determination of relationships between the structure and functions of the PRNP gene, particular attention is paid to insertion/deletion (indel) polymorphism within three regions of the nucleotide sequence: 23 bp insertion in the promoter, 12 bp insertion in intron 1 and 14 bp in-

sertion in the end untranslated region (3' UTR). Sander et al. [7] demonstrated that sequence polymorphism within these regions may be the factor enabling to differentiate between clinically healthy and infected animals. Differences in transcription level, dependent on promoter sequence polymorphism, also support the hypothesis that BSE susceptibility in cattle may have a genetic basis [8]. The possibility of BSE transmission to humans [9, 10], as well as the fact that the etiopatho-genesis of TSE diseases and the nature of their infectious agents in humans and animals have not been explicitly explained to date, make it necessary to continue research on BSE-affected and unaffected populations. Recent results show that molecular markers of BSE susceptibility in cattle can be found on chromosome BTA 5 [11] and BTA 17 [12]. Since today cases of BSE are very rare, studies on the genetic etiology of this disease must be based on an indirect analysis. It is essential to identify PRNP gene sequence polymorphism in many cattle breeds in order to determine to what degree it reflects interbreed differences and, in consequence, functional differences within the PRNP gene. The occurrence frequency of mutated fragments of the PRNP gene has been determined only in a few populations of Holstein-Friesian cattle selected towards milk performance improvement [7, 13-15]. It has been found that Holstein-Friesians, similarly as BSE-affected German cattle, are characterized by a high frequency of the 23 bp deletion allele in the promoter sequence and the 12 bp deletion allele in intron 1.

The aim of the present study was to identify deletion/insertion polymorphism within the promoter se-

Fig. 1. Insertion/deletion polymorphism (23 bp) within the promoter sequence of the PRNP gene. Ins/ins (+/+) homozygotes - lanes 3, 7; del/del (-/-) homozygotes - lanes 1, 4; ins/del (+/-) heterozygotes - lanes 2, 5, 6, 8; molecular weight marker (PhiX/Hae III) - lane 9.

quence (23 bp), intron 1 (12 bp) and the 3' end untranslated region (14 bp) of the PRNP gene in endemic breeds of Polish Red cattle, Polish White-backed cattle and European bison, i.e. in populations known for their natural adaptation ability with low level of production traits, in which no BSE cases have been recorded to date.

MATERIALS AND METHODS

The experiment was performed on three groups of animals protected under a national genetic resources conservation program for natural and breeding populations: 139 Polish Red (PR) cows raised at the Research Station for Ecological Agriculture and Preservation of Native Breeds, Polish Academy of Sciences in Popiel-no, and by individual farmers organized at the Center for Preservation of Native Breeds in the Province of Malopolska, 79 Polish White-backed cows raised at the Experimental Station in Uhrusk, Agricultural University in Lublin, forming a founding herd used for the purposes of a national restitution program, and 50 lowland European bison (Bison bonasus L., 1758) of the Bia-lowieza line, harvested or designed to cull.

Genomic DNA was isolated from whole blood samples using a Master Pure™ Genomic DNA Purification Kit (Epicenter, USA). The concentration and purity of DNA preparations were controlled spectrophotometri-cally (GeneQuant, Pharmacia, USA). In order to analyze the polymorphism of bovine PRNP gene sequences, three fragments of the PRNP gene were amplified (GenBank accession code: AJ 298878):

I) within the promoter - position from 47784 bp to 47883 bp;

II) within intron 1 - position from 49686 bp to 49777 bp;

III) within 3' UTR - position from 67976 bp to 68072 bp.

PCR primers were used for amplification, as described by Sander et al. [7]. The PCR mixtures contained: 1.25 |il buffer 20 x Master Amp™ Tfl Buffer (Epicenter Technology), 3.0 |l 10 x Master Amp.

TM PCR Enhancer (Epicenter Technology), 1.5 |l 10 x nucleotide solution dNTF (Epicenter Technology), (dATP, dGTP, dCTP, dTTP) each at a concentration of 2 mM, 2.0 |l of a 25 mM MgCl2 solution, 1.0 |l solution of primers at a concentration of 100 pM/|l, 0.5 U polymerase Tfl Master Amp TM(Epicenter Technology) at a concentration of 1 U/| l, 1 | l DNA at a concentration of 100 ng/ml and H2O to a volume of 25 |l. A "touch down" thermal program was used [16].

The PCR products were separated by 2.5% agarose gel electrophoresis (AmpliSize, Bio-Rad). The size of the amplified fragments was verified against the molecular weight marker PhiX 174/Hae III, applying the Fluor STM Multimager System (Bio-Rad).

Differences between the observed and expected frequency of the PRNP genotypes were verified using the chi-square test. Haplotype frequency was reconstructed based on genotype data, using the expectation-maximization (EM) algorithm implemented in software Arlequin v.3 [17].

RESULTS

The efficiency of nucleotide sequence amplification was at the same level in all groups of animals, which indicates that the amplified fragments of the PRNP gene in cattle and European bison are identical. As a result of the amplification of a fragment of the PRNP gene encompassing a 23 bp mutation within the promoter region, three genotypes were identified. Ins/ins (+/+) and del/del (-/-) homozygotes showed one distinct band, 123 or 100 bp in length, respectively, whereas ins/del (+/-) heterozygotes showed two bands, 123 and 100 bp in length (Fig. 1).

Three genotypes were also identified within the in-tron 1 region, ins/ins (+/+) homozygotes, del/del (-/-) homozygotes and ins/del (+/-) heterozygotes, based on two DNA bands, 103 bp and 91 bp in length (Fig. 2).

Two genotypes were identified within the 3' end UTR region (14 bp), ins/ins (+/+) homozygotes and ins/del (+/-) heterozygotes, based on two DNA bands, 97 bp and 83 bp in length (Fig. 3).

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+/- +/- +/+ -/- +/+ +/- +/+ +/- M

Fig. 2. Insertion/deletion polymorphism (12 bp) within the intron 1 sequence of the PRNP gene. Ins/ins (+/+) homozygotes - lanes 3,5, 7; del/del (-/-) homozygotes - lane 4; ins/del (+/-) heterozygotes - lanes 1, 2,6,8; molecular weight marker (PhiX/Hae III) - lane 9.

Table 1 presents the frequencies of genotypes and alleles for particular indels and the state of genetic equilibrium.

It was found that White-backed cattle were characterized by a higher frequency of ins/del heterozygotes and a relatively lower frequency of ins/ins homozygotes within the promoter region, compared to a randomly tested group of Polish Red cattle. At the polymorphic locus of intron 1 (12 bp) the genet

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