A note on ryanodine receptor gene (ryr 1) occurrence in the anserine genome

The PCR products of the ryrl gene amplified from anserine genomic DNA were analysed. We used the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to investigate the anserine ryanodine receptor (ryr 1) gene. The anserine fragment of the ryrl gene was the same size as exon 17 of the porcine ryrl gene.


INTRODUCTION
The typical avian karyotype consists of a few distinguishable macrochromosomes and a high number of very small microchromosomes (Ladjali et al., 1993;Rodionov, 1996). The presence of numerous cytogenetically indistinguishable mi-crochromosomes complicates the mapping of genes and other DNA fragments and integrating the genetic maps of the karyotype. Henschel and Louw (1978) reported malignant hyperthermia (MH) in birds. MH is linked to mutations in the ryanodine receptor which is a calcium release channel protein on the intracellular Ca 2+ -store in skeletal muscle. The human and rabbit ryanodine receptor genes (RYR1) have been mapped (MacKanzie et al., 1990) and sequenced Phillips et al., 1996), and the porcine ryrl gene has been localised (Davies et al., 1988); cDNAs have been cloned (Fujii et al., 1991). The RYR1 gene is composed of 106 exons, of which two are alternatively spliced (Phillips et al., 1996). In studies on the genetic basis of malignant hyperthermia (MH), the al843C-^T mutation in the porcine ryrl gene corresponding to an Arg 615 to Cys alteration has been identified (Fujii et al., 1991) and linked to malignant hyperthermia (MH) with a lod score of 102 for 9 max. = 0.0 . The corresponding human RYR1 mutation, Arg 614 to Cys, was found to be expressed in about 2% of MH families (Gillard et al., 1992).
The primary aim of this study was to determine whether the ryrl gene occurs in the anserine genome.

Methods
In this study, consensus primers were designed based on the porcine ryanodine receptor (ryrl) cDNA sequences where natural polymorphism occurs, and used to amplify a fragment of exon 17 of the anserine ryanodine receptor gene. The PCR reactions were performed in a 25 ml mix containing: 0.1 mg genomic DNA, 10 pmol primer RYR1F 5 ? -Cy5-GTTCCCTGTGTGTGTGCAATGGTG-3', which corresponds to porcine ryrl cDNA nucleotides 1811 to 1834, and 10pmolRYRlR5 f -(GCCAGGGAGCAAGTTCTCAGTAAT-3') in which the last 24 nucleotides are complementary to ryrl cDNA nucleotides 1861 to 1884, dNTPs fc 300 JIM, 2.5 [Ltl 10 x PCR buffer and 0.5U Taq DNA Polymerase. Thermocycling was performed by initial denaturation for 5 min at 94°C, 30 cycles of 45 s denaturation at 92°C, 45 s annealing at 55°C, 60 s hybridisation at 70°C and 7 min final extension at 70°C. For automatic analysis of PCR products on ALFexpress™ Sequencer (Pharmacia LKB) primers were labelled at the 5'end with Cy5. Electrophoresis was performed on 10% polyacrylamide gel at 30°C using the following conditions: 25 W, 60 mA, 200V. Allele size was quantified using Fragment Manager V 1.2® (Pharmacia Biotech) software. Because the 1843C->T mutation changes, aHin?l site to a//giAl site the PCR-RFLP method was used. The PCR-RFLP for detection of mutant alleles was performed using HiriPl enzyme (10,000 U/ml, Biolabs) as follows: 10 ml of the amplified products were digested with//zwPl (10 units) for 3 h at 37°C in the appropriate buffer containing BSA.

RESULTS AND DISCUSSION
The goose PCR product was of the same size as the porcine one (74 bp) (Figure 1). In the analyzed geese material, no individuals with substitution of T for C at nucle-

CONCLUSIONS
This analysis shows that PCR based on primers for a known porcine cDNA sequence may be suitable for the homologous gene sequence of other genomes. Molecular techniques applied in this study permitted the analysis of a fragment of the ryrl gene in the goose. It seems that the gradual mapping of the goose genome should make the identification of genes responsible for genetic defects feasible. Creation of marker maps of goose genomes may help in explaining their evolution.
This study may be a model for other genes, especially for those species which, despite being closely related to one another, display cytogenetic differences. It seems that localization and identification of animal disease genes are the most important aspects of animal gene mapping.