i . ..u 2...? .. . i : an. .1! . .o. .. ., INS}. 2‘»: , 1.. w .v. 1 any yawn .1 mo“; This is to certify that the thesis entitled IDENTIFICATION OF GENETIC COMPONENTS IN TURKEY RYRS ASSOCIATED WITH PSE MEAT CHARACTERISTICS presented by HYO-JUNG YOON has been accepted towards fulfillment of the requirements for the PhD. degree in Food Science Mm Major Professor’s Signature /0/13 / 0!, / I Date MSU is an Afiinnative Action/Equal Opportunity Institution LIBRARY E Michigan State University PLACE iN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 2/05 p:/ClRC/DateDue.indd-p.1 IDENTIFICATION OF GENETIC COMPONENTS IN TURKEY RYRS ASSOCIATED WITH PSE MEAT CHARACTERISTICS By Hyo-Jung Yoon A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 2006 ABSTRACT IDENTIFICATION OF GENETIC COMPONENTS IN TURKEY RYRS ASSOCIATED WITH PSE MEAT CHARACTERISTICS By Hyo-Jung Yoon The modern turkey industry has been successful in producing fast-growing birds with large breast muscle due to the application of intensive genetic selection. However, these improvements in growth rate and muscling have led to an increased incidence of pale, soft and exudative (PSE) meat. We hypothesized that 1) polymorphism(s) in the turkey skeletal muscle ryanodine receptor [3 isoform (Ii-RyR) result in channel activity differences associated with PSE incidence and 2) heat stress affects alternative splicing patterns and abundance of the RyR isoforms (or- and B-RyR) as an adaptive response in some birds, leading to differences in meat quality. Breast muscle tissues from eighty turkeys from two different genetic lines, i.e., genetically improved and random-bred lines, were obtained after exposure to heat stress for 5 or 7 days. The L“ values (objective measurement of lightness of color) and pH at 15 min postmortem of breast muscle were measured. To measure the channel activity of fi-RyR in turkeys, the [3H1-ryanedine binding assay was performed. Ryanodine binding activity of ,B-RyR of genetically improved turkeys was significantly increased (p=0.0008) as birds were exposed to longer heat stress while that of random-bred birds was not affected by heat stress level. .To test the hypothesis that polymorphisms in B-RyR are associated with altered channel activity, samples from sixteen turkeys were selected for sequence analysis based on the ryanodine binding activity of B-RyR. As a result of the screening of the entire B-RyR cDNA of the selected turkeys, ten synonymous single nucleotide polymorphisms (SNPs) were identified. None of the SNPs showed any significant correlation with ryanodine affinity of B-RyR. Thus, although the channel activity of ryanodine receptors as measured by [3 H] -ryanodine binding assay may suggest a mechanistic basis for differences in calcium fluxes, this assay may not be a good phenotype for identification of polymorphisms of the B-RyR gene. To investigate the correlation of those SNPs and meat quality traits, ten normal meat (L*5.8) and ten PSE meat turkeys (L*>52 and lesmm 1 uM) affinity ryanodine binding sites (Chu et al., 1990; Lai et al., 1988; Ogawa, 1994; Pessah et al., 1985). It is possible that there is cooperative interaction between the different ryanodine binding sites generating complex binding behavior. One interpretation suggests that ryanodine binding to the high affinity site depends on whether or not the RyR channel is in the open configuration (Chu et al., 1990). As the binding is dependent upon the functional state of the channel, the analysis of ryanodine binding to the channel protein provides important information about the activity of calcium channel and effects of modulators of channel function (Shoshan-Barmatz and Ashley, 1998). 2.2 Factors affecting RyR activity The activity of the calcium release channel protein is modulated by numerous factors, including a number of physiological agents (e.g., Ca2+, Mg2+, and ATP), various cellular processes (e. g., oxidation, phosphorylation, etc.), regulatory proteins (e.g., calmodulin, FK-506 binding proteins, etc.), and several pharmacological agents (e.g., ryanodine, caffeine, and ruthenium red). 2.2.1 Regulation by physiological agents 17 Cflosolic Ca2+ One of the most important physiological agents that modulate the channel activity of the RyRs is Ca2+ (Fleischer and Inui, 1989; Ebashi, 1991; Meissner, 1994; Coronado et al., 1994). Although Rle is known to be activated mechanically by a conformational change in DHPR, all RyR isoforms are activated by the binding of Ca2+. Rle activity displays a bell-shaped dependence on cytosolic Ca2+ concentration; low Ca2+ concentrations (low M range) activate the channel activity while higher concentrations (high uM to mM range) inhibit channel activity (Meissner et al., 1997). The response to high cytosolic Ca2+ concentrations of the Rle channel is different from that of RyR2 and RyR3 channels. The Rle channel is almost completely inhibited by 1 mM Ca2+, whereas substantially higher Ca2+ levels are required to inhibit the RyR2 and RyR3 channels (Chen et al., 1997; Bull et al., 1993; Chu et al., 1993). The reported half- maximal inhibiting concentrations for the RyR2 and RyR3 channels range from 2 to 10 mM (Chen et al., 1997). Because it is unlikely that such high cytopolasnric Ca2+ concentrations are ever reached in the cell, the physiological role of this very high Ca2+ inhibition of RyR2 and RyR3 channels is not yet clear. As these three isoforms are almost 70% identical in amino acid sequences, regions of diversity (D1, D2 and D3) could account for functional differences between 18 these isoforms. Consistent with this notion, there have been several reports suggesting that the Caz+-dependent activating and inactivating domains reside in the divergent regions. Du et al. (2000) reported that mutation of divergent region 1 altered both caffeine and Ca2+ sensitivity of Rle. Hayek et al. (1999) suggested D3 as a candidate for the Ca2+-dependent inactivation sites of RyR. Treves et al. (1993) generated an antibody to the amino acid sequences fiom a part of D1 region and found that this antibody could block Ca2+ dependent activation of Rle . Thus, this site was suggested as a potential Ca2+ activating site. These observations support the divergent regions as imparting some of the isoform specific characteristics in Ca2+-dependent activation/inactivation of RyRs. However, there have been studies supporting other Ca2+ binding sites in different regions in RyRs. Chen et al. (1998) found that a glutamic acid residue at position 3885 contributed to Cay-dependent activation of the channel by site-directed mutagenesis study. Therefore, the location of the Ca2+ activating/inactivating sites on RyRs is still not clear. ATP and Mg2+ Cytosolic Mg2+ is a potent RyR channel inhibitor. The Mg2+ action on single RyR channels is complicated and there have been multiple mechanisms suggested for this inhibiting effect of Mg2+. First, Mg2+ may competitively displace Ca2+ from the Ca2+ l9 activation site, shiffing the Ca2+ sensitivity of the channel. Second, Mg” may compete with Ca2+ at the high-Ca2+ inhibition site because the high-Ca2+ inhibition site does not discriminate between different divalent cations. Third, the channel protein may be sterically blocked as Mg2+ binds to a site near the conduction pathway (Copello et al., 2002; Laver et al., 1997; Meissner and Henderson, 1987). It has been reported that the Rle channel is substantially more sensitive to the action of Mg2+ than the RyR2 or RyR3 channels. In the presence of physiological levels of Mg”, the Rle channel required less Ca2+ to activate than the RyR2 or RyR3 channels (Copello et al., 2002; Jeyakumar et a1, 1998). In contrast to the effects of Mg”, adenine nucleotides (including ATP and ADP) strongly activate RyRs. At subnanomolar levels of Ca”, millimolar ATP increases the frequency of open events and decreases the duration of closed events (Smith et al. 1986). In the presence of micromolar Ca2+, millimolar ATP produces a synergism of activation that increases the duration of open events and allows the channel protein to remain open nearly 100% of the time. Thus, ATP is required to achieve the maximum open probability of RyR channels. The action of cytosolic ATP on single RyR channel function is also isoform specific. Free ATP is a much more effective activator of the 20 Rle channel than the RyR2 or RyR3 channels (Smith et al., 1985; Coronado et al., 1994). 2.2.2 Regulation by Oxidation/ Phosphorylation Various redox processes may also modulate single RyR channels. The RyR monomers contain 80-101 cysteine residues (Sun et al.,. 2001). Many of these cysteine residues are susceptible to modification by oxidants. In the rabbit Rle channel subunit, nearly half of the 101 cysteines are kept in a reduced (free thiol) state under conditions comparable with resting muscle. However, as the channel protein is exposed to oxidative stress such as introduction of high 02 tension or presence of oxidants, channel activity is greatly altered. Sun et al. (2001) demonstrated that the channel activity increases reversibly as the number of thiols is reduced (from 48~38 thiols/subunit) in response to the high 02 tension. More extensive oxidation (to ~13 thiols/subunit) inactivates the channel irreversibly, which may contribute to dysfunction of muscle. It has been suggested that nitric oxide (N 0) may directly modulate the redox regulation of the RyR channels. Low NO concentrations prevent oxidation of the RyR whereas higher concentrations promote oxidation, supporting a bifunctional effect of NO 21 on channel activity. The two effects of NO probably result from interaction with distinct sulfhydryls. Thus, there is a growing interest in identifying the cysteine residues that may be involved in RyR channel oxidation. Ogawa et al. (2002) proposed that redox states of critical sulflrydryls located on the cytoplasmic side of the Rle may alter both gating properties of the channel and responsiveness to channel modulators. There is also evidence that oxidants such as reactive oxygen intermediates (R015) and NO may prevent certain closely associated regulatory molecules, like calmodulin, from binding to the RyR channel. As a result of inhibiting the calmodulin binding to the RyR channel, the channel activity changes because calmodulin is also a regulator of the channel protein activity. This will be reviewed in section 2.2.3. Analysis of the RyR protein sequence reveals that it contains many consensus phosphorylation sites (Otsu et al., 1990; Suko et al., 1993 and Tunwell et al., 1996). Thus, the effects of exogenously applied kinases and phosphatases on RyR channel behavior have been extensively examined. It was reported that protein kinase A (PKA) increases the activity of the Rle channel, which is consistent with the observation that PKA significantly increases depolarization-induced Ca2+ release from skeletal muscle SR vesicles (Reiken et al., 2003). The capacity of PKA and calmodulin kinases to activate the RyR2 channel has also been studied (Wehren et al., 2004). However, there have 22 been contradictory results reported for calmodulin kinase. Some researchers report that PKA activates the RyR2 channels, whereas others report that PKA slightly decreases the open probability in the steady-state (P0) of the RyR2 channels. Marks et al. (2000) proposed that PKA hyperphosphorylation of RyR2 and Rle at Ser—2809/Ser—2843 results in activation of channels because PKA destabilizes channel opening via phosphorylation-induced dissociation of F KBP12.6. When FKBP12 is bound to Rle or RyR2, it inhibits the channel by stabilizing its closed state. Thus, the authors suggest that phosphorylation of the RyR channel at the binding site of F KBP12 will cause dissociation of FKBP12 and activate Ca2+ release fi'om the channel proteins. However, the PKA hyperphosphorylation/FKBP dissociation hypothesis has been challenged by Xiao et al., (2004). Using sequence-specific antibodies which recognize the phosphorylated and non-phosphorylated sites, they showed that a large proportion of the RyR2 channels appeared to be phosphorylated even prior to the addition of exogenous kinases, and FKBP 12.6 was found to bind to both wild-type RyR2 and mutant RyR2 which does not have a serine residue at the binding site. The reason for such contradictory and confusing results is unclear. The discrepancies between studies could arise fi'om the different experimental conditions used among different laboratories (Danila and Hamilton, 2004). 23 2.2.3 Regulation of RyR by associated proteins Many different proteins may be associated with and modulate the RyR channels. However, at the single channel level, only a few of these proteins have been thoroughly studied including calmodulin (CaM), calsequestrin, FK-506 binding proteins and DHPR (Fill and Copello, 2002). Calmodulin (CaM), a protein composed of four homologous Ca2+ binding sites, was the first polypeptide found to interact with RyR channels in studies using single RyR molecule incorporated into lipid bilayers. CaM appears to bind to the RyR at a stoichiometric ratio of 4 CaM molecules per RyR tetramer. CaM can either activate (at low Ca2+ levels, nM) or inhibit (at high Ca2+ levels, uM) the Rle and RyR3 channels (Chen et al., 1997; Fruen et al., 2000; Smith et al., 1989 and Tripathy et al., 1995). Hamilton et al. (2000) found that Ca2+ binding to sites 3 and 4 on CaM is responsible for its conversion from an activator to an inhibitor. For the RyR2 channels, however, only inhibitory effects of CaM have been reported. F ruen et al. (2000) suggested that apoCaM (Ca2+-free CaM) binds with much lower affinity to RyR2, and it does not increase RyR2 activity. It was reported that oxidation modifies Rle channel activity and its interaction with CaM. The binding sites for both apoCaM and Ca2+-CaM on RyR appear to be close to amino acids 3613-3643 (Zhang et al., 2003). Cysteine 3635 24 can form a disulfide bond with a cysteine on an adjacent subunit (amino acids 1975- 1999), and this leads to activation of the channel. CaM bound to Rle can prevent this oxidation-induced intersubunit cross-linking. Conversely, it has been also suggested that CaM may somehow protect the Rle channel from oxidative modifications during periods of oxidative stress because CaM binds at the site of intersubunit contact (Zhang et al., 1999). A potential role of CaM in modulation of the RyR2 channel during E-C coupling is not yet clear. The F KBP12 and F KBP12.6 proteins associate with the RyR], RyR2 and RyR3 proteins in apparently 4:1 stoichiometric proportions. Thus there are four FKBPs bound to each RyR channel complex. FKBP modulation of the Rle channel is generally accepted, yet there is some uncertainty as to the extent of this regulatory action (Wehrens et al., 2004 and Danila and Hamilton, 2004). These studies have been reviewed in section 2.2.2. 2.3 RyR-related muscle diseases Many muscle diseases are caused by mutations of proteins directly involved in the excitation-contraction-relaxation cycle, i.e., membrane proteins involved in voltage- sensing, signal transduction, Cay-release and Ca2+ uptake. Because of the central role 25 of RyRs in cellular calcium regulation, defects in RyR channels can lead to potentially fatal disorders in muscles. It has been shown that the mutated Rle and RyR2 isoforms are closely associated with the excessive Ca2+-release in malignant hyperthermia (MH)/central core disease (CCD) and cardiomyopathies, respectively (Jurkat-Rott et al., 2000; Marks, 2002; Laitinen et al., 2001). 2.3.1 Rle-related mutations 2.3.1.1 Malignant hyperthermia and central core disease Malignant hyperthermia (MH) has been recognized in humans since 1960 as an autosomal dominantly inherited muscle disease. MH-susceptible patients undergo uncontrollable skeletal muscle hyperrnetabolism when exposed to volatile halogenated anesthetics such as halothane. If not treated immediately with the highly effective Ca2+- release inhibitor dantrolene, up to 70% of the MH episodes result in fatalities (Jurkat-Rott et al., 2000; Froemming and Ohlendieck, 2001). Central core disease (CCD) is a non-progressive, human congenital myopathy in which patients typically present with diffuse muscle weakness, hypotonia during infancy, delayed motor development, and reduced muscle bulk (Shy and Magee 1956). The onset of the disease is early in childhood with hypotonia (flOppy infant syndrome), but 26 muscle strength usually improves during life, especially after continuous exercise (Froemming and Ohlendieck, 2001). Characteristics for CCD are areas of unstructured myofibrils in the myofibrillar cores and a lack of mitochondria (Quane et al., 1993). Moreover, CCD patients are generally considered to be at risk for MH. Genetic linkage analysis has revealed mutations in the Rle gene in human chromosomes in over 50 % of MH families (McCarthy et al., 1990; MacLennan et al., 1990; Quane et al., 1993). The mutated RyR] is believed to exhibit a prolonged ion channel opening time which increases cytosolic Cay-levels during excitation-contraction coupling. Prolonged excessive cytosolic Ca2+ concentration leads to accelerated muscle metabolism, glycogenolysis, ATP depletion, contracture development and ultimately to a disturbance of intra- and extracellular ion homeostasis with consequent muscle cell damage (Jurkat-Rott et al., 2000). CCD, which is closely related with MH, is also caused by mutations in the skeletal muscle Ca2+-release channel (Froemming and Ohlendieck, 2001). It has been proposed that myoplasmic Ca2+-overload due to a mutation in Rle is responsible for mitochondrial damage (Fardeau and Tome, 1994). If the Ca2+ concentration rises to very high levels, the mitochondria may participate in Cay-removal from central areas and could be damaged in the effort to protect the cell from Cay-induced cell necrosis 27 (Wrogemann and Pena, 1976). Loss of mitochondria from the center of the cell may lead to a reduction in ATP production and might be the underlying cause of the muscle weakness observed in central core disease (Froemming and Ohlendieck, 2001). In human ryrl , to date, 47 missense mutations are associated with MH and 25 are associated with central core disease (CDC) (The human gene mutation database, The Institute of Medical Genetics in Cardiff University, UK; http://www.hgrnd.cf.ac.uk/ac/ns/1/120359.html). Some of these mutations give rise to both MH and CCD. There are also 2 mutations showing association with multi- minicore disease (MCD), 1 with congenital myopathy, and l with core/rod disease (Dirksen and Avila, 2002; Marchant et al., 2004). Clustering of primary abnormalities occurs within the myoplasmic Rle-domain at the central receptor region (Region 2; amino acid residues 2162-2458) and at the extreme N-terminal region (Region 1; residues 35-614). Previously, only two point mutations (T4826I and R4893W) were identified for MH in the C-terminal region (Region 3, residues 4637-4898). However, recent discoveries of several mutations for CCD in this region suggested that the C-temrinal region of the Rle may represent a third mutation hot spot for MH (Monnier et al., 2000, 2001). Later, consistent with this notion, two more mutations for MH (G4942V, 28 P4973L) have been identified in the C-terminal region of Rle (Galli et al., 2002). All three of these hotspot regions are highly conserved across all known RyR isoforms. Further linkage analysis identified several additional possible mutation sites in other proteins for MH/CCD and the most important ones are linked to the DHPR (Rueffert et al., 2002). So far two mutations have been found within the gene encoding the al subunit of DHPR which is thought to directly interact with Rle during excitation-contraction coupling (Greenberg, 1999; Monnier et al., 1997). Thus, most of the unknown MH mutations are believed to be present in the Rle and DHPR genes although it cannot be excluded that abnormalities in other Ca2+-handling proteins are also associated with MH. 2.3.1.2 Porcine Stress Syndrome (PSS) Genetic susceptibility to MH is also present in pigs where it is identified as porcine stress syndrome (PSS). Crises of PSS are also brought on by physical and emotional stresses, including heat, exercise, mating, transportation and fear. As in human MH, muscle rigidity, tachycardia and fever are also triggered when PSS- susceptible pigs are exposed to inhalational anesthetics such as halothane (Harrison, 1972) 29 The high fatality rate of MH and its association with inferior quality pork have prompted the development of diagnostic tests for susceptibility to MH in swine. Halothane challenge testing was one of the earliest diagnostic tests used in the swine industry to detect MH susceptibility (Eikelenboom and Minkema, 1974). In this test, live pigs are physically restrained and forced to inhale 3 to 5% halothane in oxygen through a face mask for several minutes. Those pigs developing extensor muscle rigidity during the test are diagnosed as MH-susceptible. Major limitations of this test, however, include its low sensitivity (Nelson et al., 1983), the high number of fatalities which may occur, and the eventual discovery that the test only identifies homozygous stress-susceptible pigs. The genetic basis for PSS was found to be a single point mutation (Arg615Cys) in the gene coding for Rle (Fujii et al., 1991); this mutation has also been identified in human MH (Arg614Cys). Having discovered the PSS-susceptible gene, many pig breeders wanted to eliminate it or, at the very least, limit its propagation in their breeding populations. To exhibit PSS, an animal must have both recessive alleles (nn). Thus, the halothane test only detects those pigs carrying the homozygous mutant gene (nn) while the carriers with one copy of the mutant form (Nn, which are much more frequent in the population) remained undetected (O’Brien, 1987). A DNA-based test was 30 introduced to determine the exact genotype of pigs for the mutant gene (11) and to eliminate stress-susceptible pigs from breeding (Fujii et al., 1991, Foerster, 1992). In spite of the effort to remove stress-susceptible pigs from breeding, however, the PSE pork incidence climbed from 10.2 % to 15.5 % in the past 10 years (Kelley, 2003). This suggests that additional genetic and environmental factors other than the mutation identified in Rle are involved in PSE meat incidence. 2.3.2 RyR2-related mutations It has been reported that defects in cardiac ion channels, e.g. the potassium channels, sodium channels and cardiac Ca2+-release channel (RyR2), can lead to ventricular arrhythmias which constitute one of the major causes of sudden cardiac- related death. To date, mutations in the human RyR2 gene have been associated with two different forms of cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2) (Marks, 2002; Laitinen et al., 2001; George et al., 2003; Wehrens and Marks, 2003). CPVT, a rare disorder with a very high mortality rate, is an autosomal- dominant, genetic arrhythmogenic disorder characterized by stress-, emotion-, or physical 31 exercise-induced bidirectional ventricular tachycardia (Laitinen et al., 2001) and ARVD2 is characterized by exercise-induced sudden cardiac death (Marks, 2002). So far at least 21 mutations in RyR2 that are linked to stress-induced, sudden cardiac death have been discovered (Marks, 2002). Just as almost all the MH/CCD mutations are clustered in three highly conserved regions of RyR, all arrhythmogenic mutations found to date are located in the corresponding regions in RyR2. Based on their similar locations as the MH/CCD mutations, it was hypothesized that the disease- associated RyR2 mutations are likely to cause the Cay-channel to open spontaneously or to increase the sensitivity of the channel to activation by Ca2+, which was supported by the channel activity study of heterologously expressed RyR2 channels carrying one of the reported mutations (J iang et al., 2002). Figure 1-5 shows hot spots of Rle and RyR2 mutations related with muscle disease. 2.3.3 RyR3-related mutations Several mutations including five point mutations and a single amino acid deletion have been identified in the N-terminal region of human RyR3 gene (Marziali et al., 1996; Leeb and Brenig, 1998). In addition, several alternative splice sites of the RyR3 gene 32 MH/CCD Hotspot ** NIH/CCD MH/CCD Hotspot Hotspot ** [ :2: RyR2 1=12 l L=ml] —(500 aa) FKBP12 93M Hap-J binding site Binding site Transmembrane region Figure 1-5. Linear RyR2 protein sequence. Asterisks indicate site of reported RyR2 mutation. White boxes show MH/CCD hot spots in Rle. (Adapted from Allen, 2002) 33 (human, mink and mouse) have been reported (reviewed in section 5.2). However, no pathology or physiological significance has yet been linked to mutations in RyR3. 3. Pale, soft, and exudative (PSE) meat The term PSE was first coined to describe a meat quality problem in pork and thus, the basis of this condition has been extensively studied in swine (W ismer-Pederson, 1959; Bendall and Wismer-Pederson, 1962). Environmental stressors are known to induce the hypermetabolic condition associated with development of PSE meat; reduction of stresses prior to and during the slaughter process decreases the incidence of PSE pork. However, it is important to note that genetics also plays a key role in an animal’s susceptibility to these stresses; i.e., genetically stress-susceptible animals yield a higher incidence of PSE meat than genetically normal animals (Lundstrom et a1, 1989; Offer 1991). In swine, PSE meat is ofien associated with Porcine Stress Syndrome (PSS) or malignant hyperthermia (MI-I) which is characterized by excessive heat and lactic acid produced as a result of spontaneous muscle contractions and rapid anaerobic glycolysis (Mitchell and Heffron, 1982). During the first several hours postmortem, muscle of a stress-susceptible pig undergoes a rapid decrease of the pH while the carcass temperature is still high (Mitchell and Heffron, 1982), which induces denaturation of muscle proteins. 34 The protein denaturation results in a decrease in protein solubility and water holding capacity (W ismer-Pederson, 1959) which are characteristics of PSE meat (Cassens et al., 1975). Skeletal muscle metabolism is regulated in part by the calcium ion concentration in the cytoplasm (Ebashi and Endo, 1968). An abnormally high concentration of calcium ions accelerates muscle contraction and glycolysis, which generates excessive heat and lactic acid in muscle leading to the development of PSE meat (Gronert et al., 1980; Cheah et al., 1984). It was suggested that the calcium release mechanism in skeletal muscle is involved in malignant hyperthermia and that a defect in the calcium release channel might be the basic defect in animals, including humans, with MH (Mickelson et al., 1986; McLennan et a1, 1990). Fujii et al. (1991) identified a single point mutation, Arg615Cys, in the skeletal calcium release channel (Rle) in swine as an ultimate cause responsible for the accelerated calcium release associated with PSS in pigs, as well as development of PSE meat; this mutation corresponds to one of the point mutations, Arg614Cys, found in human MH. Reports of PSE turkey meat are more recent (Sosnicki et a1, 1991; Sosnicki et al., 1996; McKee et a1, 1996; McKee and Sams, 1997). In turkey as in pork, the incidence of PSE meat appears to have increased in concert with intensive genetic selection for 35 lean, heavily muscled, rapidly growing animals (Toelle et al., 1991). Sosnicki et al. (1996) and Pietrzak et al. (1997) reported that there are many striking similarities in postmortem conversion of muscle to meat between PSE turkey and PSE pork; i.e., a reduction in protein functionality is associated with an elevated muscle temperature and rapid pH decline leading to the occurrence of PSE meat. In addition, as environmental stressors increase the incidence of PSE pork, the incidence of PSE turkey is more frequent among birds that have been exposed to severe anti-mortem stressors, e.g., heat, humidity and transportation, etc., resulting in accelerated postmortem glycolysis (McCurdy et al., 1996; McKee and Sams, 1997). McCurdy et al. (1996) used color score measurements (L values) during a year to assess the frequency of the PSE problem in a total 45 turkey flocks. As high L values (lighter color) were correlated with poor water holding capacity on the basis of their study, they used a cut-off L value > 50 to classify turkey breasts as PSE. They observed that the highest average L-value in the summer and the lowest in the winter, which implicates heat stress as a contributing factor. It is important to note that they also observed considerable flock-to-flock variation, with several flocks having as little as 1% PSE meat and one flock with as much as 29% of birds above this cut-off. These results agree with observations reported by the turkey industry. The turkey industry has indicated that the magnitude of the ‘light meat 36 problem’ can range from 5 to 30% depending on the flock. This suggests that there is a genetic basis predisposing some turkeys to the development of PSE meat. These similarities in development of PSE pork and PSE turkey could be explained by a genetic defect of the calcium release channel which alters the channel activity during the development of PSE turkey (Owens et al., 2000). Although there are similarities in many aspects between the development of PSE pork and PSE turkey, it may be overly simplistic to assume that the specific mutation associated with development of PSE pork would also be associated with PSE turkey. A key difference between turkey and pig skeletal muscle is that there are two ryanodine receptor isoforms (or and B or Rle and RyR3) in avian skeletal muscle while there is only one major isoform (Rle) in mammalian skeletal muscle (Airey et al., 1993). Therefore, there could be alteration(s) in either or both isoforms which could contribute to the PSE turkey meat problem. Besides, no phenotype for stress-susceptibility in live turkeys has been identified (Wheeler et al., 1999; Owens et al., 2000) while the halothane challenge test can detect the specific point mutation (Arg615Cys) in homozygous stress- susceptible pigs (Eikelenboom and Minkema, 1974). To avoid further propagation of genetic contribution to PSE meat in breeding flocks, attempts were made to develop a halothane test as a possible detection method for PSE-susceptible turkeys (Wheeler et al., 37 1999; Owens et al., 2000). However, the halothane test was not able to detect a relationship between the response of the birds to the anesthetic halothane and the occurrence of PSE-like meat (Wheeler et al., 1999). In contrast, Owens et al. (2000) reported that the incidence of PSE was significantly higher in halothane-sensitive turkeys compared with halothane-non-responding turkeys when they were subjected to heat stress prior to slaughter. Thus, Fletcher (2002) concludes that halothane susceptibility is only a limited predictor of meat quality in turkey. Moreover, unlike in swine, the incidence of PSE meat seems to be uniformly distributed among all major lines in turkeys. Therefore it is essential to develop a systematic approach for study of the PSE turkey problem instead of simply applying the information on PSE pork to provide a fimdamental understanding of the molecular mechanisms involved in meat quality problems in turkey. 4. Single nucleotide polymorphisms A single nucleotide polymorphism (SNP) is a simple DNA sequence change within a gene among alleles; i.e., a single nucleotide replaces one of the three nucleotides at a specific location in the gene sequence. Within a population, SNPs are usually defined as the less common variant with a frequency of at least 1%, but for some 38 purposes rarer variants are important as well. It is important to note that there are variations between populations, so a SNP that is common enough for inclusion in one group may be much rarer in another. Variations in the DNA sequences of a species can have a major impact on how they develop diseases and respond to environmental insults such as pathogens, chemicals, drugs, etc. Therefore, SNPs are of great value for biomedical research and in developing pharmaceutical products or medical diagnostics. As SNPs are inherited and do not change much from generation to generation, following them during population studies is straightforward. They are also used in some forms of genealogical DNA testing. SNPs may be found within coding sequences of genes, non-coding regions of genes, or in the intergenic regions between genes. In fact, most SNPs are found outside of the coding region because only a small portion of DNA sequence codes for the proteins in many species. SNPs in noncoding regions are also believed to be less likely to create dysfunctional proteins. SNPs in within a coding region may or may not change the amino acid sequence of the protein that is produced, due to redundancy in the genetic code. A SNP in which both forms lead to the same amino acid sequence is termed synonymous; if different amino acids are encoded they are non-synonymous. SNPs that 39 are found in non-coding sequences of genes may still have consequences for gene splicing, transcription factor binding, or the sequence of non-coding RNA (Kwok, 2003). SNPs are useful for finding genes contributing diseases in two ways. Some SNP alleles are the actual DNA sequence variants that cause differences in gene function or regulation directly related with disease processes. However, most SNP alleles are usefirl as genetic markers that can be used to find the functional SNPs due to associations between the marker SNPs and the functional SNPs. The most obvious type of fimctional SNP is a non-synonymous SNP that produces a protein with a different amino acid sequence. Some SNPs found at splice sites may be also functional as they result in alternative spliced transcript variants that express proteins that differ in the exons they contain (Krawczak et al., 1992). Some SNPs in promoter regions are known to be functional by affecting the regulation and expression of proteins (El-Omar et al., 2000; Ligers et al., 2001). Assigning SNPs directly causing diseases or differences in function and regulation of proteins, however, requires caution as many SNPs may all be associated with a disease or phenotype, even though only one or a few may directly affect the phenotype. Although major studies for SNPs have been carried out in human genomes, the study of SNPs is also important in crop and livestock breeding programs. Animal 40 breeders are interested in the association between alleles and traits of interest and have started applying marker-assisted selection to improve the quality and performance of their livestock. The halothane gene (Hal 1843) for porcine stress syndrome (Fujii et al., 1991) is the first marker used in pig breeding as the gene affects meat quality traits. The cattle industry has been actively conducting research on identification of SNPs associated with milk production, meat quality traits and diseases (Stasio et al., 2005). In poultry science, the sequence of the entire chicken genome was recently released (Hiller et al., 2004) and more than 1 million SNPs were reported. More thorough and extensive studies are continuing to develop useful markers for animal breeding. 4.1 SNPs related with diseases Common diseases such as cancer, stroke, cardiovascular diseases, diabetes, Alzheimer’s and psychiatric disorders run in families and are influenced by many genes as well as environmental and lifestyle factors. Inheritance can also play an important role in susceptibility to a drug or to an environmental factor. Therefore it is a fundamental goal of biomedicine to identify the genetic contributors to human health. Although most SNPs are not responsible for a disease state and only serve as biological 41 markers for pinpointing a disease on the human genome map, some SNPs may actually affect disease development. One well-known example of the association between SNPs and diseases is illustrated by apolipoprotein B gene (ApoE) in human. Apolipoprotein E is a major component of very low-density lipoproteins (V LDL) whose function is to remove excess cholesterol from the blood and carry it to the liver for processing (Nicodemus et al., 2004). The ApoE gene is located on the long arm (q) of human chromosome 19 at position 13.2 and is known to be associated with Alzheimer’s disease (AD). The gene contains two SNPs that result in three possible alleles; E2, E3 and E4. The most common allele, E3, produces a protein in which the 112th amino acid is cysteine and the 158th amino acid is arginine. In the E2 protein, the 112th and 158th amino acids are both cysteines while both amino acids are arginine in the E4 protein. It was revealed that an individual who inherits at least one E4 allele fi'om parents will have a greater risk of developing AD as the change of one amino acid in the E4 protein alters its structure and flmction enough to make disease development more likely. On the other hand, those inheriting the E2 allele are less likely to develop AD (Martin et al., 2000; Nicodemus et al., 2004). Still, those SNPs are not absolute indicators of AD development because 42 some people with two risky E4 genes may never get AD and a few people with two protective E2 genes develop the disease. Schizophrenia is also a highly heritable disease and thus, there have been considerable research efforts aimed at identification of susceptibility genes. So far, there are a large number of candidate genes suggested for the illness. Among them, the genes involved in dopaminergic pathways have been extensively studied because altered dopamine function is thought to cause several symptoms of schizophrenia (Kukreti et al., 2006). The dopamine D2 receptor (DRD2) is one of the dopamine receptors and a good candidate gene for schizophrenia because all antipsychotic medications are either antagonists or partial agonists of the DRD2, which is the primary site of action for these medications (J onsson et al., 2003). Thus, a large number of researchers have attempted to find association of DRD2 polymorphisms to validate the influence of DRD2 on the disease etiology (Missale et al., 2006; Scharfetter, 2004). There are, to date, nine SNPs discovered in the coding region of human DRD2: three non-synonymous SNPs and six synonymous SNPs. Functional polymorphisms at DRD2 include the missense variant C/G in exon 7, which results in a Ser/Cys substitution at position 311 (Itokawa et al., 1993) and the synonymous SNP, C/T in exon 7, which produces Pro at position 319 (Cargill et al., 1999). The nonsynonymous SNP, Ser/Cys C/G variant impairs 43 dopamine-mediated inhibition of forskolin-induced adenylate cyclase activity, (Cravchik et al., 1996) and the synonymous SNP, Pro 319 C/T variant affects DRD2 transcript stability and translational efficiency (Duan et al., 2003). Moreover, other polymorphisms also have been studied and suggested as possible functional SNPs associated with schizophrenia (Hashimoto et al., 2005). The story of SNPs associated with hypertension is more complicated. There are more than 170 candidate genes and a large number of SNPs in those genes have been reported. The candidate genes express proteins with various functions including apolipoproteins, channel proteins, transporters, growth factors and fat and lipid regulators, etc. For complex and multifactorial diseases such as hypertension, SNPs are useful because they can be used as markers throughout the genome and they make it easy to perform large-scale genotyping by high-throughput methods. So far, various association studies with hypertension have been performed (Okuda et al., 2002). The Met235Thr variant of the human angiotensiongen gene is an example showing association with an increased risk of hypertension. Studies to identify association between SNPs found in candidate genes and hypertension are still underway. These studies have provided insight into the nature of human sequence variation. However, it is not absolutely clear at present whether these variations identified so far are 44 truly significantly associated with diseases or not. Moreover, it is likely that SNP alone may not be sufficient to predict the risk of disease susceptibility and it is possible that most complex, common disorders are caused by the combinational effects of multigenes and environmental factors (multifactorial). Nevertheless, these variants may provide a starting point for further inquiry. 4.2 SNPs in RyRs The human skeletal tyrl gene is located on the long arm (q) of chromosome 19 and consists of 153,832 bases (15114 bases in coding region). There are a total of 434 SNPs reported to date in human ryrI (NCBI). In the coding region of ryrI, 47 SNPs have been identified (12 nonsynonymous and 35 synonymous SNPs). More than half of the nonsynonymous SNPs on this list are associated with muscle diseases (Table 1-1, Table 1-2). One example is the first mutation reported in this gene, a nonsynonymous SNP C1840T which results in an Arg/Cys substitution at position 614 (Gillard et al., 1991). This mutation is known to be associated with development of human malignant hyperthermia (MH). None of synonymous SNPs has shown association with diseases so far. 45 Table 1-1. Synonymous SNPs“ in the coding region of human Rle Region dbSNP Protein Region dbSNP Protein allele residue allele residue exon_7 C 9 T Asp191 exon_45 C 9 T Hi52420 exon_7 A 9 G Leu198 exon_47 G 9 A Ala2500 exon_10 G 9 A Ala291 exon_47 G 9 A Va12509 exon_ll C 9 A Ile338 exon_47 C 9 T Pr02528 exon_ll T 9 C Ala359 exon_49 C 9 T Arg2624 exon_12 C 9 T Thr406 exon_50 G 9 A Thr2569 exon_15 G 9 A Ser556 exon_51 T 9 C Ile2706 exon_19 C 9 T Pro762 exon_51 T 9 C Asp2730 exon_24 G 9 A Thr 981 exon_53 G 9 A Glu2779 exon_24 G 9 A Thr]981 exon_55 T 9 C Ser2863 exon_24 C 9 T Asn 993 exon_62 A 9 G Pro3062 exon_24 G 9 A Ala1014 exon_66 G 9 A Leu3230 exon_26 C 9 T Ile1152 exon_67 C 9 T Asp3396 exon_28 T 9 C Leu1286 exon_92 G 9 A Pro4488 exon_28 C 9 T Prol369 exon_92 G 9 A Pro4501 exon_29 C 9 T Prol423 exon_93 C 9 T Gly4539 exon_37 A 9 G Lys2013 exon_98 A 9 C Thr4752 exon_37 A 9 G Ly52013 exon_104 C 9 A Ile4964 exon_44 C 9 T Pr02366 *Provided in NCBI dbSNP database with the genelD 6261 46 Table 1-2. Nonsynonymous SNPs* in the coding region of human RyR] , Amino Region dbSNP Protein acid allele resrdues , , posrtlon exon_ll G 9 A Gly 9 Arg 341 exon_17 C 9 T Arg 9 Cys 614 exon_18 C 9 T Thr 9 Met 656 exon_39 C 9 T Arg 9 Cys 2163 exon_39 G 9 A Arg 9 His 2163 exon_40 C 9 T Thr 9 Met 2206 exon_45 G 9 A Arg 9 His 2435 exon_46 C 9 T Arg 9 Cys 2458 exon_50 C 9 T Arg 9 Trp 2676 exon_63 C 9 T Ala 9 Va] 3118 exon_79 C 9 G Gln 9 Glu 3756 exon_91 G 9 C Ala 9 Pro 4345 47 *Provided in NCBI dbSNP database with the geneID 6261 Human cardiac ryr2 is located on chromosome 1 and has 790,951 bases in the entire gene (14901 bases in coding region). There are more than 3,000 SNPs reported to date in the ryr2 gene in NCBI list. In the coding region, a total 17 SNPs have been identified (6 nonsynonymous and 11 synonymous, Table 1-3). Although there are about 30 missense mutations in human ryr2 known to be associated with cardiovascular diseases, none of these SNPs, whether nonsynonymous or synonymous, in the coding region has been reported as associated with cardiac diseases. Human RyR3 is located on chromosome 15 and consists of 555,142 bases in the entire gene (14610 bases in coding region). A total of 2412 SNPs have been reported to date in ryr3 gene and 24 of them have been found in the coding region (9 nonsynonymous and 15 synonymous, Table 1-4). None of these SNPs, however, has known to be associated with any human disease. As described above, there have been extensive results for SNPs in human RyRs. On the contrary, reports of SNPs in animal RyRs are only limited to those used in animal- model studies such as rat and mouse. So far, the only exception among animals that have reported SNPs information in RyRs is chicken as the chicken genome project was completed in 2004 (Hiller et al.). A total of 276 SNPs in introns have been found in 48 Table 1-3. SNPs* in the coding region of human RyR2 dbSNP Protein Codon Amino acid Region Function , , , , , allele mndue positron positron exon_lS synonymous C 9 T Ser 3 453 exon_l6 nonsynonymous G 9 A Val 9 Ile l 507 exon_19 synonymous C 9 T His 3 621 exon_20 nonsynonymous A 9 G Asn 9 Ser 2 658 exon_24 synonymous C 9 T Phe 3 921 exon_26 synonymous A 9 G Ser 3 991 exon_37 nonsynonymous A 9 C Glu 9 Asp 3 1682 exon_37 synonymous A 9 G Lys 3 1800 exon_37 nonsynonymous G 9 A Gly 9 Ser 1 1886 exon_45 synonymous T 9 C Leu 3 2302 exon_48 nonsynonymous T 9 C Leu 9 Pro 2 2426 exon_51 synonymous C 9 T His 3 2602 exon_65 synonymous T 9 G Ser 3 3106 exon_73 synonymous C 9 T Thr 3 3501 exon_76 synonymous C 9 T Ser 3 3592 exon_85 nonsynonymous G 9 A Asp 9 Asn 1 3853 exon_90 synonymous G 9 A Ser 3 4209 ’Provided in NCBI dbSNP database with the geneID 6262 49 Table 1-4. SNPs” in the coding region of human RyR3 . dbSNP Protein Codon Amino acid Region Function . , . _ . allele resrdue positron positron exon_l nonsynonymous G 9 T Glu 9 Ter 1 6 exon_ll nonsynonymous T 9 C Ile 9 Thr 2 358 exon_ll synonymous C 9 T Asp 3 370 exon_12 synonymous G 9 A Gln 3 401 exon_13 synonymous C 9 T Ser 3 423 exon_14 nonsynonymous G 9 A Val 9 Ile 1 494 exon_18 synonymous G 9 C Arg 3 643 exon_19 nonsynonymous A 9 G Ile 9 Val 1 731 exon_20 synonymous G 9 C Leu 3 801 exon_20 synonymous T 9 A Ile 3 834 exon_22 nonsynonymous A 9 G Asn 9 Ser 2 898 exon_25 synonymous G 9 A Gly 3 1048 exon_26 synonymous T 9 C Tyr 3 1080 exon_35 nonsynonymous C 9 T Arg 9 Cys 1 1641 exon_35 synonymous G 9 A Leu 3 1657 exon_44 synonymous G 9 A Gly 3 2239 exon_45 nonsynonymous G 9 A Gly 9 Glu 2 2270 exon_52 synonymous C 9 T Tyr 3 2616 exon_63 synonymous C 9 T Pro 3 2999 exon_63 nonsynonymous A 9 G Ile 9 Va] 1 3000 exon_73 synonymous A 9 G Lys 3 3426 exon_78 synonymous C 9 T Phe 3 3604 exon_79 synonymous A 9 G Ala 3 3627 exon_89 nonsynonymous C 9 A Asn 9 Lys 3 4007 *Provided in NCBI dbSNP database with the geneID 6263 50 chicken tyr3, but no SNP was detected in the coding region. Also, there is no information provided for SNPs in ryrl in chicken. 5. Alternative Splicing 5.1 Heat stress and alternative splicing Alternative mRNA splicing is the term used to describe the regulatory process of differential inclusion or exclusion of regions of the pre-mRNA; this process represents an important source of protein diversity in higher eukaryotes. Johnson et al. (2003) reported that at least 74% of genes in the human genome undergo alternative splicing, suggesting that alternative splicing is one of the most significant components of the functional complexity of the human genome. Recent studies indicate that 70—88% of alternative splices change the protein product (Kan et al., 2001; Modrek et al., 2001), and the majority of these changes appear to be functionally interesting, such as replacement of the amino or carboxy terminus, or in-frame addition and removal of a functional unit (Modrek et al., 2001). Moreover, it has been reported that errors in alternative splicing can cause a variety of disease states (Caceres and Komblihtt, 2002; Black, 2003). There are a number of environmental stresses which alter gene expression through the alternative splicing of pre-mRNA (Shukla et al., 1990; Bournay et al., 1996). For 51 example, heat stress has been reported to regulate the alternative splice pattern of pre- mRNA of many genes. The mouse heat shock protein 47 (HSP47) gene expresses three HSP47 transcript variants and one of these alternatively spliced mRNAs was detected only after heat shock (Takechi et al., 1994). Denegri et al. (2001) reported that the adenovirus E1 A gene in HeLa cells generated five different ElA transcript variants (three major and two minor isoforms) under unstressed condition and heat shock at 42°C for 1 hr induced an increase in the relative abundance of one major transcript variant and a concomitant decrease of the other two major transcript variants. This effect was even more evident after 1 and 2 h of recovery at 37°C, but at longer times (6 h of recovery) the splicing pattern of EIA transcripts was similar to that observed in unstressed cells. Stress experienced by meat-producing animals prior to slaughter influences the physicochemical components involved in conversion of muscle to meat. Stressors that trigger a physiological response can be short term (loud noise, unfamiliar environment, fighting, electric prod goading) or long term (sickness, dehydration, malnourishment, hot/cold stress). Among these, environments of high temperatures and humidity are detrimental to the productivity of commercial animal agriculture (Fuquay, 1981). The importance of heat stress to US livestock industries is increasing with time because 52 animals genetically selected for fast grth tend to produce more body heat due to their greater metabolic activity (Leenstra and Cahaner, 1992; Berong and Washbum, 1998). 5.2 Alternative splicing in RyRs There have been a few examples of alternative splicing for Rle, RyR2 and RyR3 identified so far. Kimura et a1. (2002) found a significant increase of an alternatively spliced isoform of RyR] in myotonic dystrophy type 1 (DMl) patients. The alternative splicing results in the deletion of 5 amino acids at a modulatory region of the protein. Insertion of five amino acids (Ala-Gly-Asp-Ala-Gln) after residue 3479 in rabbit Rle (Zorzato et al., 1994) or deletions of Ala(3481)-Gln(3485) and Val(3865)- Asn(3 870) in mouse RyR] (F utatsugi et al., 1995) are near the putative phosphorylation sites and binding sites for Ca2+, calmodulin and ATP. In human RyR2, insertions of ten amino acids between residues 1479 and 1480 and eight amino acids between residues 3715 and 3716 were found (Tunwell et a1, 1996). Compared to Rle and RyR2, there have been more RyR3 alternative splicing cases reported (Table 1-5). Jiang et al. (2003), and Leeb and Brenig (1998) characterized several rabbit and human RyR3 splice variants. Deletion of His(4406)-Lys(4434) in rabbit RyR3 encompasses a predicted transmembrane helix and this splice variant cannot form a firnctional calcium release 53 Table 1-5. Examples of alternative splice sites of the RYR3 gene Location Type Species Ref. (nucleotide#) 2359-2575 Removal of 217 bp rabbit Jiang et al., 2003 6812 Addition of 3 bp rabbit Jiang et al., 2003 8714-8763 Removal of 50 bp rabbit Jiang et al., 2003 10012-10026 Removal of 15 bp mink Marziali et a1, 1996 10021-10035 Removal of 15 bp rabbit Jiang et al., 2003 11146-11163 Removal of 18 bp mink Marziali et a1, 1996 1 1153-11170 Removal of 18 bp rabbit Jiang et al., 2003 11569-11650 Removal of 82 nucleotides human Leeb and Brenig, leads to truncated protein 1998 13216-13302 Removal of 87 bp rabbit Jiang et al., 2003 13799-13800 341bp intron is not removed mouse Miyatake et al., and leads to truncated protein 1996 14371-14523 Removal of 153 bp rabbit Jiang et al., 2003 54 channel (RyR3) when expressed alone in HEK293 cells. However, the spliced product and the wild type RyR3, upon co-expression, combine to form functional mixed heteromeric channels with clearly altered activation of Ca2+-release (Jiang et al., 2003). It is important to note that most splice variants are identified in either the putative modulatory or transmembrane domain. Therefore, alternative splicing of RyRs may have important and unique functional roles in regulating the activity of these proteins. 5.3 Alternative splicing in turkey aRyR Chiang et al. (2004) reported the first avian RyR transcript variants. They identified at least seven alternatively spliced transcript variants of turkey orRYR clustered in three different cDNA regions. The first alternative splicing region (ASRl) was found in exon 13 (according to the human RyR] genomic DNA structure) of aRyR corresponding to the 3’-end of mutation hot spot 1 of human Rle. This region encompasses two alternatively spliced variants (81 bp and 193 bp deletions; Chiang et al., 2004), which introduces a 27-amino acid deletion (81 bp deletion), and a premature stop codon (193 bp deletion), respectively. The first region has been proposed to serve as part of the protein-protein contact site of Rle with the DHPR. They also suggest that 55 there may be an effect of heat stress on the pattern and abundance of those three transcript variants (Chiang et al., 2004). Continuing studies (Chiang et al., 2005) identified a second alternative splicing region (ASR 2) which spans exon 24 to exon 29. ASR2 encompasses at least 4 different transcript variants including deletions of 1043 bp, 401 bp, 454 bp and 268 bp (amino acid residues #1055-1402, #1255-1388, #1258-1409 and #1313-1402), respectively. With the exception of the transcript variant carrying a 401 bp deletion (removal of exon 28), the other three transcript variants occur at aberrant alternative splicing sites. ASR2 is located in divergent region 2 and each of these deletions introduced a premature stop codon in the coding region. The third alternative splicing region (ASR3) resides in the C-terminal region of orRyR, which is in divergent region 1. One transcript variant, carrying a deletion of 816 bp, was identified and this transcript variant is predicted to result in a deletion of 272 amino acids (residues #4214-4485) encoded by exon 91 of orRyR in the human Rle sequence (Chiang et al., 2005). 56 Chapter 2 Measurement of turkey meat quality indices and channel activity by [311]- Ryanodine binding assay of turkey SR 1. Introduction It is widely accepted that a mutation in the calcium release channel protein in skeletal muscle (ryanodine receptor skeletal isoform, Rle) is one of the basic defects associated with malignant hyperthernria (MH) in animals and humans, (Mickelson et al., 1986; McLennan et a1, 1990). This defect leads to an abnormally high concentration of calcium ions in the cytoplasm postmortem, which accelerates muscle contraction and glycolysis, generating excessive heat and accumulation of lactate and H“ in muscle leading to the development of PSE meat (Gronert et al., 1980; Cheah et al., 1984). Fujii et al. (1991) identified a single point mutation, Arg615Cys, in the skeletal calcium release channel (Rle) in swine as the molecular basis for accelerated calcium release associated with porcine stress syndrome (PSS) in pigs, as well as development of PSE meat. This mutation corresponds to one point mutation, Arg614Cys, found in human MH. In turkey as in pork, the incidence of PSE meat appears to have increased in concert with intensive genetic selection for lean, heavily muscled, rapidly growing animals (Toelle et al., 1991). Sosnicki et a1. (1996) and Pietrzak et al. ( 1997) reported 57 that there are many striking similarities in postmortem conversion of muscle to meat between PSE turkey and PSE pork. A reduction in protein functionality is associated with an elevated early postmortem muscle temperature and rapid pH decline, leading to the development of PSE meat. In addition, just as environmental stressors increase the incidence of PSE pork, the development of PSE turkey appears to be influenced by environmental factors, most notably heat stress of birds prior to slaughter (McKee and Sams, 1997). These similarities in development of PSE pork and PSE turkey suggest a common underlying mechanism based on abnormal calcium regulation in the development of PSE turkey (Owens etal., 2000). Despite the similarities in development of PSE pork and PSE turkey, it is simplistic to assume that the specific mutation associated with development of PSE pork would also be found in turkey. Moreover, there are significant physiological differences in the Ca2+-release mechanism between turkey and pig. A key difference is the presence of two ryanodine receptor isoforms (or and B, or Rle and RyR3, respectively) in avian skeletal muscle while there is only one major isoform (Rle) in mammalian skeletal muscle (Airey et al., 1993). Therefore, there could be alteration(s) in either or both isoforms that could contribute to the PSE turkey meat problem. In addition, no definitive phenotype for stress-susceptibility in live turkeys has been identified that could 58 be associated with PSE meat (Wheeler et al., 1999; Owens et al., 2000). In contrast, a positive halothane challenge test is associated with the specific point mutation (Arg615Cys) in homozygous stress-susceptible pigs (Dietze et al., 1989; Iaizzo and Lehmann—Horn, 1989). Moreover, unlike in swine, the incidence of PSE meat seems to be uniformly distributed among all major lines in turkeys. Thus, it is essential to develop a systematic approach to identify the genetic basis for development of PSE turkey. Mickelson et al. (1988) utilized the [3H] ryanodine binding assay to characterize differences in calcium release activity of sarcoplasmic reticulum (SR) vesicles isolated from the skeletal muscle of malignant hyperthermia susceptible (MHS) pigs and normal pigs. In their report, SR vesicles from Mil-susceptible pigs showed higher affinity for ryanodine than normal pigs (Kd = 92 vs. 265 nM, respectively), which suggests that calcium channel activity, as measured by the ryanodine binding assay, may be altered due to altered primary structure of Rle, or a post-translational modification resulting in altered channel activity. Later studies demonstrated that a mutation in ryrl was associated with the defect. Thus, the ryanodine binding assay is a useful tool to characterize the calcium release activity of RyRs, particularly with respect to differences due to primary structure of the proteins. 59 Based on the success of the ryanodine binding assay in pig skeletal muscle, it was hypothesized that this assay may be useful in predicting genetic differences in turkey RyR related with PSE meat. Wang et al. ( 1998) conducted a [3H] ryanodine binding study revealing that there are differences in the calcium channel activity of two different genetic lines of turkeys, i.e. a random-bred and a commercial line. These authors showed that the SR vesicles from commercial turkeys exhibited a higher mean affinity for ryanodine when compared to that fiom genetically unimproved turkeys (Kd=12.2 vs. 20.5 nM, respectively). Subsequently, after optinrizing the ryanodine binding assay conditions for turkey SR, Zhang (2000) showed that differences in ryanodine binding could be grouped as high affinity or low affinity lines. Eight random-bred turkey samples were compared with seventeen commercial turkey samples (Group I) obtained from a slaughter facility and with five commercial turkey samples (Group H) obtained fi'om the same plant two years later (Zhang, 2000). The SR vesicles from Group I turkeys showed two-fold higher ryanodine affinity compared to the unimproved turkeys (K; = 8.4 vs. 16.0 nM, respectively). Group II turkeys, however, showed similar calcium channel properties to those of random-bred turkeys (Kd = 19.1 vs. 16.0 nM, respectively; Zhang, 2000). This 60 suggests that there is a diversity of the calcium channel activity between the random-bred and commercial turkey populations and even within the commercial turkey population. Analysis of ryanodine binding data is more complex in turkeys than mammals. Unlike mammals, birds, fish, amphibians and reptiles generally have two RyR isoforms expressed in equal abundance in skeletal muscle. Thus, it was difficult to determine which isoform could contribute to the abnormal activity of the calcium release channel protein. A recent study of RyR activity in frog SR vesicles showed that the a-RyR channel activity in frog skeletal muscle is almost completely suppressed when a- and B- RyR isoforms coexist in SR (Murayama and Ogawa, 2001). To determine whether this observation is unique to frog or it if also occurs in mammalian muscle, these researchers investigated [3H] ryanodine-binding of RyR] and RyR3 in bovine diaphragm SR vesicles (Murayama and Ogawa, 2004). Again, the activity of Rle was markedly lower than that of RyR3 under the same binding assay conditions, indicating selective suppression of Rle as observed with frog or-RyR Therefore, although there are two RyR isoforms in turkey skeletal muscle, the ryanodine binding data primarily reflect li-RyR activity since a-RyR activity is likely almost completely suppressed. On this basis, we suggest that the ryanodine binding assay may be a plausible tool to correlate differences in ryanodine binding with differences in B-RyR primary structure or in regulation of its activity. The 61 objective of this study was to test the hypothesis that differences in ryanodine binding may be correlated with meat quality indices. 2. Materials and Methods 2.1. Animals A total of eighty turkey skeletal muscle samples were used for this project. Those turkeys were obtained fiom a commercial line (Hybrid turkeys, Hybrid, Inc., Kitchener, ON) and from a random-bred control population (RBCl, Dr. Karl Nestor, Ohio Agricultural Research and Development Center). Turkeys were raised to market weight (approximately 10 kg for random-bred turkeys and 17 kg for commercial turkeys) from hatchlings at the MSU poultry farm and slaughtered on Aug 29 and Aug 31, 2000. Twenty birds from each line were slaughtered after they were exposed to five days of heat stress (12 hours of 95 °F, 12 hours of 80 °F), twenty from each line were subjected to two more days of stress and then slaughtered under commercial condition at the MSU meat laboratory. Turkeys were electrically stunned, their necks were cut and blood was collected during exsanguination of birds for purification of genomic DNA. The pectoralis major muscle from one side of the carcass was collected within 5 minutes postmortem, and was immediately sectioned, snap-frozen in liquid nitrogen and stored at 62 -80°C for SR purification and total RNA extraction. The remaining carcass was processed under commercial conditions. The remaining pectoralis major muscle was used to evaluate meat quality indices, including the pH of the muscle at 15 nrin post mortem and the color (L*, lightness of color) of the meat at 24 h postmortem. 2.2. Chemicals Ryanodine was purchased from Wako Chemical USA, Inc. (Richmond, VA) and radioactive ryanodine was purchased from New England Nuclear Life Science (Boston, MA). Protease inhibitors, phenylmethylsulfonyl fluoride (PMSF), aprotinin, benzamidine, pepstatin, leupeptin were purchased from Sigma-Aldrich (St. Louis, M0). 2.3. Measurement of B-RyR ryanodine binding activity Purificagion of Crude Sarcoplasmic reticulum vesicles (CSR) Crude skeletal muscle SR was isolated from breast muscle samples of the eighty turkeys by procedures described in the MS dissertation of Zhang (2000). Two hundred grams of frozen muscle cubes were thawed at 4°C for 20 min, incubated with 5 volumes of Buffer I (0.1 M NaCl, 5 mM Tris-maleate, pH 7.0), for 5 min, and homogenized in a Waring blender for 2 min with intervals of 15 sec. on and 15 sec. off. After 30 min of 63 centrifugation at 3300 x gmax, the supernatant was collected and filtered through a 16- layer cheesecloth. The pellet was discarded, and the supernatant was centrifuged for 30 min at 16,000 x gmax. This pellet was collected, resuspended with 20 mM Buffer II (0.6 M KCl, 5 mM Tris-maleate, pH 7.0), and gently homogenized in a Dounce homogenizer. The homogenized suspension was centrifuged for 40 min. at 150,000 x gm“ using a Beckman Ti-7O rotor. The pellet was collected and suspended in 10 % sucrose solution and gently homogenized in a Dounce homogenizer. The crude SR vesicles obtained by this step were flash-frozen in liquid nitrogen and stored at -80 °C. During the crude SR (CSR) vesicles purification, the following protease inhibitors were added to each buffer to yield the final concentration noted: PMSF at 0.2 mM, and aprotinin, benzamidine, pepstatin and leupeptin at 1 ug/mL. B-mercaptoethaol was added to Buffer I and II to yield a final concentration of 30 mM. CSR served as the material for analysis of ryanodine binding activity. CSR protein concentration was determined by the method of Lowry using bovine serum albumin as a standard (Lowry et al., 1951). Ryanodine binding assays Fifty pg of CSR protein was incubated in buffer containing 10 mM MOPS (pH 7.2), a CaClz-EGTA-nitrilotriacetic acid buffer to give a specific [Ca2+]fme of 10 M, 0.1 64 M NaCl, 0.5 M sucrose. [3H] ryanodine (2-70 nM as final concentration) was added to each tube. Triplicate samples of each [3 H] ryanodine concentration were incubated for 6 h at 37 °C, and filtered through Whatrnan GF/B filters that were pre-soaked in washing buffer (10 mM MOPS, 0.1 M NaCl, 50 MM CaClz, pH 7.0). Filters were washed three times with 5 mL of ice-cold washing buffer, and the amount of [3H] ryanodine retained on the filter was determined by scintillation counting. Non-specific binding of ryanodine was determined by adding unlabeled ryanodine at 1000 times the [3H]- ryanodine in the incubation buffer. Specific ryanodine binding was determined by subtracting the non-specific binding from the total binding. The K, (ryanodine binding affinity) and Bmax (maximum binding capacity) values for ryanodine binding were obtained by Scatchard analysis. _St_atistical An_alvsis The General Linear Model procedure of SAS (SAS Inst. Inc., Cary, NC, 2000) was used to analyze data. Least square means of meat quality traits and K, were compared using mixed model analysis of variance procedures. The model statement included the fixed effects of genetic line, heat stress level and the interactions of the two factors. 65 3. Results and Discussion 3.1. Effects ofJenetic lines and heat stress levels on meat quality indices of turkeys The meat quality indicators including pH at 15 min postmortem (pHISmin) and color (L*) at 24 hr postmortem of turkey breast muscle were measured for eighty turkeys from two different genetic lines, i.e., genetically improved and random-bred lines after they were subjected to heat stress for 5 or 7 days. Thus, there are four subgroups of turkeys used in this study: 1) 5-day heat stressed random bred turkeys (5R); 2) 7-day heat stressed random bred turkeys (7R); 3) 5-day heat stressed commercial turkeys (5C); and 4) 7-day heat stressed commercial turkeys (7C). L“ and pHrsm values obtained from the individual samples are listed in Appendix 1. According to the statistical analysis of the fixed effect of genetic lines, random- bred turkeys had significantly darker color (L*=50.58 i 0.38) than commercial turkeys (L*=52.99 d: 0.34) (p<0.0001, Table 2-1 (a)), but there was no significant difference in lesmin between two genetic lines. However, the effect of heat stress was highly significant: 5-day heat stressed birds had higher lesmin (pHrsmin =6.14 :I: 0.03) than 7-day heat stressed birds (pHrsmin =5.85 :t 0.03) (p<0.0001, Table 2-1 (b)). There was no significant difference in color between turkeys exposed to different heat-stress levels. 66 Analysis of the interaction of effects of two factors on color and lemm for 4 subgroups showed that turkeys in SR group had higher lesmin than 5C turkeys (thsmin = 6.21 :t 0.05 vs. 6.07 d: 0.04, respectively, p=0.0377). However, for 7-day heat stressed birds, there was no significant difference in pHISmin between two different genetic lines, 7R and 7C (pHrsmin = 5.85 d: 0.05 vs. 5.86:1: 0.03, respectively, p=0.93) (Table 2-1 (c)). Color and pH are important meat quality traits and, in many cases, they are used as indicators of PSE meat (Barbut, 1993). Protein denaturation occurs in early postmortem stages which generate low pH combined with high muscle temperature (Bendall and Wismer-Pederson, 1962; Mitchell and Heffron, 1982). Pale meat is the result of denaturation of muscle proteins, which results in scattering of light (Swatland, 1989). Therefore, pale color and low early postmortem pH are two primary indicators of PSE meat development. Analysis of meat quality data in this study showed that random-bred turkeys had significantly darker color than commercial turkeys, but heat stress levels did not affect color of the meat. Le Bihan-Duval et al. (2001) reported that color parameters appeared to be the most heritable traits when they measured meat quality traits for chicken. Therefore, it is not surprising that there is a statistically significant difference in color between two different genetic lines of birds in this study. 67 Table 2-1. Analysis of color (L*) and early postmortem pH (pHISmin) of random-bred and commercial turkeys. (a) Least square means and standard errors of color (L*) and pHISmtn: Fixed effects of genetic lines on color and pHrsmtn Random-bred commercial Color (L*) 50.58 a 0.38a 52.99 i 0.341) pH15min 6.03 a 0.05c 5.96 a: 003° W Different letters within a row indicate significant differences (p<0.05) (b) Least square means and standard errors of color and pHISmin: Fixed effects of heat stress levels on color and pHISmin 5-day HS 7-day HS Color (1)) 51.72 s: 0.41“ 51.84 a: 0.40a pH15min 6.14 e 0.03b 5.85 :1: 0.03c “bf Different letters within a row indicate significant differences (p<0.05) (c) Least square means and standard errors of color and pHISmm: Effects of interaction of genetic lines and heat stress levels on meat quality traits Group" 5R 5C 7R 7C Color (L*) 50.56 r 0.551‘ 52.87 a: 0.49b 50.58 at 0.52‘ 53.10 i 0.48b prim... 6.21 r 0.05a 6.07 :1: 0.04b 5.85 a 0.05c 5.86m 0.03c ‘b'c'd Different letters within a row indicate significant differences (p<0.05) *5R: 5-day heat-stressed random-bred, 5C: 5-day heat-stressed commercial, 7R: 7-day heat-stressed random-bred and 7C: 7-day heat-stressed commercial turkeys 68 There was no significant difference in pH 15min between two genetic lines of turkeys, but there was a highly significant effect of heat stress on thsmtn. Analysis of the effect of the interaction of two factors (genetic lines and heat stress levels) on lesmin for 4 subgroups showed that turkeys in SR group had higher leSmin than 5C turkeys while there was no significant difference in lesmin between 7R and 7C turkeys. This suggests that random-bred turkeys are more heat-resistant than commercial turkeys for a limited period of heat stress, but if exposure to the heat-stress is prolonged, even random- bred turkeys became as susceptible to heat stress as commercial turkeys and show rapid pH drop after slaughter. Afier death of the animal, anaerobic metabolism reduces the pH from about 7.2 in muscle to an ultimate pH of approximately 5.4 in meat and stiffness develops (rigor mortis). It has been commonly observed that antemortem stress factors induce rapid pH decline yielding more PSE meat. Heat stress is one prominent antemortem environmental factor that causes a rapid pH decline (McKee and Sams, 1997), which is consistent with the results from this study. In fast-growing turkeys, the rate of pH decline in pectoral muscle was about twice the rate of slow-growing lines. It is well known that, with the increase in growth rate and muscle size of turkeys, there has been an increase in incidence of PSE and commercial turkeys originally have the problem of 69 muscles that outgrow their life support systems (Mills, 1998; Dransfield and Sosnicki, 1999). It was suggested that lack of oxygen may trigger muscle damage in heat-stressed broilers (Mills, 1998) and faster growing birds experience more severe panting due to a local anoxia and this disturbs the acid-base balance in the blood leading to muscle damage. Sandercock et al. (2006) also reported the effect of acute heat stress on indices of respiratory thermoregulation and skeletal muscle damage in broiler chickens. They proposed that acute heat stress altered antemortem acid-base balance and induced changes in breast muscle glycolytic metabolism as indicated by lower muscle pH immediately post slaughter. In faster growing birds, these systems are more likely nearer a physiological limit and therefore, less tolerant to heat stress. This explains the difference in lesmin between SR and 5C groups. As previously described, however, even random-bred birds showed limited resistance under prolonged heat stress although they are more tolerant to heat stress. 3.2 Analysis of ryanodine binding_activity in birds of differing_genetic lines subiected to varying heat stress The ryanodine binding assay was conducted in parallel on turkey SR samples based on the following assumptions: 1) Differences in ryanodine binding affinity reflect difl'erences in primary structure; 2) Differences in primary RyR structure lead to altered 70 Ca2+-release properties; 3) Turkey meat quality is strongly correlated with the Ca2+- release activity of RyRs in skeletal muscle; and 4) The ryanodine binding activity measured under the binding assay conditions in this study represents the activity of B- RyR (Murayama and Ogawa, 2001; 2004). The [3H] ryanodine binding conditions optimized by Zhang (2000) were used for this study to analyze the turkey crude SR (CSR) from random-bred and commercial turkeys. Representative binding data from one individual turkey are shown in Figure 2- 1. The equilibrium binding constants (K; and BM) of [3 H] ryanodine binding of CSR can be obtained by Scatchard analysis (Figure 2-1 (b)). From this graph, K, is the negative reciprocal of the slope of the Scatchard plot and the intercept on the abscissa is Bmax. The results of the binding assay for eighty turkeys are shown in Appendix I. Least square means of ryanodine affinity (Kd) of CSR from four groups of turkeys are presented in Table 2-2. First, the fixed effects of genetic lines and heat stress levels on meat quality and K, were analyzed (Table 2-2 (a) and (b)). Then, the effect of the interaction of two factors on ryanodine affinity (Kd) was analyzed for 4 subgroups of turkeys, i.e. 5R, 5C, 7R and 7C (Table 2-2 (c)). The statistical analysis of fixed effects showed that there was no significant difference in ryanodine binding activity of B-RyR between two genetic lines (p=0.077) 71 1.00 «— N fl-t- --~ ~ — - ._n ._.(..im-fi 0.90 ' 0.80 " 0.70 L 0.60 0.50 -. 0.40 " 0.30 . Bound (pmol/mo SR protein) 0.20 r. 0.10 t ’0 0.00 0.00 0.04 —- ~ . 0.02 '* Bound/Free (pmol/mo/nM) 0.01 '* 0.00 0.00 50.00 0.20 Bound (pmol/ma SR protein) 100.00 150.00 Free Ry (nM) l j 0.60 0.80 200.00 250.00 Figure 2-1. (a) Specific ryanodine binding by turkey CSR. (b) Scatchard plot of ryanodine binding by turkey CSR. 72 Table 2-2. Effects of genetic lines and heat stress levels on ryanodine affinity (a) Least square means and standard errors of ryanodine affinity (Kd) of [3H] ryanodine binding in crude SR from turkeys (random-bred and commercial) Group Random-bred turkeys Commercial turkeys Kd(nM) 37.14:t 1.22 41.12i 1.98 p=0.077 (b) Least square means and standard errors of ryanodine affinity (Kd) of [3H] ryanodine binding in crude SR from turkeys (5—day and 7-day heat- stressed) Group 5-day HS 7-day HS Kd (nM) 42.38 :t 1.983 35.87 :1: 1.21b at”, Different letters within row indicate significant differences (p<0.005) (c) Least square means and standard errors of ryanodine affinity (Kd) of [3H] ryanodine binding in crude SR from turkeys: Effects of interaction of genetic lines and heat stress levels Group“ 5R 5C 7R 7C Kd (nM) 38.25 i: 1.96a 46.52 :1: 3.06b 36.02 :1: 1.53a 35.72 :t 1.92“ a” Different letters within row indicate significant differences (TKO-001) ’5R: 5-day heat-stressed random-bred, 5C: 5-day heat-stressed commercial, 7R: 7- day heat-stressed random-bred and 7C: 7-day heat-stressed commercial turkeys 73 while the ryanodine binding activity of ,B-RyR of 7-day heat stressed turkeys was significantly higher (Kd=35.87i1.21nM, p<0.005) than that of 5-day heat stressed turkeys (Kd=42.38:l:1.98 nM). When the interaction of two factors (genetic lines and heat stress levels) was considered, it was observed that heat stress level did not affect Kd (38.25 i 1.96 vs. 36.02 i 1.53 nM, 5-day HS vs. 7-day HS, respectively. p=0.49) in random-bred group while heat stress level greatly influences Kd (p<0.001) of commercial group (46.52 :t 3.06 vs. 35.72 i: 1.92 nM, 5-day HS vs. 7-day HS, respectively). High ryanodine affinity represented by low Kd value is correlated with increased open state probability of channel proteins. This suggests that there will be more Ca2+- release through RyR to the sarcoplasm during the conversion of muscle to meat, potentially leading to increased incidence of PSE meat due to accelerated glycolysis and rapid pH drop. Therefore, turkeys with higher ryanodine affinity (lower Kd) would likely have higher probability of yielding PSE meat compared with turkeys with lower ryanodine affinity. The ryanodine binding activity measured in this study is thought to represent the activity of the B-RyR only as the activity of or-RyR is almost completely suppressed (Murayama and Ogawa, 2001). There was a wide variation in the ryanodine affinity of turkey SR samples (Kd 20~74 nM; Appendix I) obtained in this study, which contrasts with previous results 74 obtained by Wang (1999) and Zhang (2000). In their studies, although there was variation among different harvest groups, the data tended to cluster within the same group. In addition, overall Kd values obtained in this study are higher than those fi'om previous studies. Wang et al. (1999) showed that the K, data of the heavy SR fi'actions from commercial turkeys and random-bred turkeys as 12.2 nM and 20.5 nM, respectively. In Zhang’s study (2000), the K. values of HSR from three different groups of turkeys were still present in the range of 8.4 nM ~ 19.] nM. When Zhang used the same binding assay technique for crude SR as used in this study, however, the K. values increased to around 22~25 nM (~20%). Theoretically, the Kd values should not vary between SR fiaction used (HSR or CSR) while the maximum binding, Bmax could be different. However, HSR and CSR are different in terms of not only the amount of RyRs but also in amount of other proteins present in the preparation such as myosin and the Ca2+ pump. The HSR is enriched in junctional membrane vesicles that contain RyRs, calsequestrin, triads, terminal cistemae, etc. while the CSR is composed of HSR and light SR (LSR). The LSR is mainly longitudinal tubule membrane vesicles enriched in the Ca2+ pump protein. Also, the amount of myosin, was greater in CSR than HSR based on SDS-PAGE analysis. The presence of other proteins may affect the channel activity by interacting with certain 75 functional sites in RyRs causing higher Kd values for CSR. Another important variation in experimental conditions between previous studies and the current study is the exposure of birds to acute heat stress. Turkey samples for the previous studies were obtained from unstressed turkeys, while samples for the current study were collected from 5- or 7- day heat stressed birds. However, it is not clear why heat-stressed birds in this study showed lower ryanodine affinity than unstressed birds. Although Wang (1999) and Zhang (2000) reported that one group of commercial turkeys has higher channel activity than random-bred birds, Zhang (2000) also showed that the channel activity measured for the other group from the same commercial line was not different fiom that of random-bred birds. She suggested that those two commercial groups might have come from different genetic subpopulations. In this study, there was no difference in channel activity between commercial turkeys and random-bred turkeys at 7-days of heat stress. However, at 5 days of heat stress, the 5R group has higher affinity for ryanodine than 5C, suggesting a higher open state probability of the channel in the former group. The commercial turkeys used in this study are fiom a different commercial line (Hybrid) than the commercial line used in Zhang’s study. So, the commercial turkeys from Hybrid line may have similar channel activity to random-bred turkeys obtained from Ohio State University. 76 Interestingly, ryanodine affinity of commercial birds was strongly influenced by heat stress levels (46.52 vs. 35.72 nM, 5C vs. 7C, respectively, p<0.001) while random- bred birds that are known to be more heat-stress resistant were not affected by heat stress levels (38.25 vs. 36.02 nM, 5R vs. 7R, respectively, p=0.49). Based on this result, we suggest that genetically improved birds tend to yield more PSE meat than random-bred turkeys under heat stress as higher ryanodine binding activity is associated with higher PSE incidence. 3.3. Correlation of B-RvR ryanodine bindinme meat quality indices To test the hypothesis that the meat quality is correlated with ryanodine affinity of SR samples, the meat quality data and Kd values were subjected to SAS analysis. However, there was no correlation found between ryanodine affinity and individual meat quality indices (p > 0.], data not shown). To determine the appropriate and more conservative threshold values for normal and PSE meat, reports fi-om previous studies on PSE turkey were reviewed. The reference values for PSE and normal turkeys vary depending on the study. Owens et al. (2000) used L“ values higher than 53 as an indicator of PSE meat while McCurdy et al. (1996) used L>50/51 as PSE indicator and L<42 as dark meat indicator. Pietrzak et al. 77 (1997) and Sosnicki et al. (1997) reported higher L* values for PSE turkey breast meat (szornin < 5.8) than for normal meat (szomin > 5.8). Considering reports from literature reviews and values obtained from the current study, the threshold values of color (L*) used in this study are L*>52 and L*<50 for PSE and normal meat, respectively. For pH, pHrsm =5.8 was used as the threshold to segregate birds. Based on this segregation, it was observed that there are only 10 PSE turkeys and 10 normal turkeys (Table 2-3) in the current study and the remaining 60 turkeys fall into an intermediate range of color and pH. The incidence of PSE meat was highest in group 7C which is more heat-stressed commercial group and was lowest in group 5R which is less heat-stressed random-bred turkeys (Table 2-4). When the statistical analysis was performed to compare the channel activity of PSE and normal meat turkeys, i.e., 10 PSE meat turkeys with L*>52 and pHISmin<5-8 and 10 normal meat turkeys with L*<50 and pH 15min>5.8, the channel activity of B-RyR fi'om PSE meat turkeys was significantly higher than that from normal meat turkeys (Kd= 31.79 :1: 2.39 nM vs. 41.24 i 3.16" nM, respectively, p=0.025). Higher ryanodine binding activity is associated with accelerated postmortem glycolytic metabolism of skeletal muscle leading to increased incidence of PSE meat, which is consistent with the results obtained. 78 Table 2-3. Meat quality data and ryanodine binding activity of PSE meat and normal meat turkeys" Group ID pH] 5mm color (L‘) K, 291 1 6.63 47.42 38.64 31-13 6.42 48.53 30.03 2915 6.31 47.45 44.05 2921 6.28 46.68 37.04 2937 6.01 49.72 51.28 Normal 29-03 5.96 47.18 41.15 31-26 5.90 49.56 26.46 31.03 5.90 45.01 39.22 29.40 5.86 49.72 59.52 2934 5.86 49.08 45.05 LSmean** 6.11 :1: 0.09 ' 48.04 a: 0.49 ' 41.24 :1: 3.16‘ 31-33 5.80 55.13 28.65 31-28 5.79 56.58 27.1 31-25 5.76 55.07 21.01 31-36 5.75 52.03 24.57 31-19 5.74 52.24 30.86 PSE 31-32 5.73 54.95 29.85 3122 5.73 53.69 41.67 3120 5.72 53.45 30.67 29-08 5.71 56.25 39.22 3142 5.64 54.31 44.25 LSmean** 5.74 at 0.01 " 5437 e 0.49 " 31.79 :1: 2.39” *PSE meat: L*>52 and pHrsm<5.8, and normal meat: L*<50 and pH15m>5.8 "Values are least square means 3: S.E. 3‘” Different letters within column indicate significant differences (p<0.05) 79 Table 2-4. Number of PSE meat and normal meat turkeys fi‘om each subgroup: i.e., 5- day heat-stressed random-bred (5R), 7-day heat-stressed random-bred (7R), 5-day heat-stressed commercial (5C) and 7-day heat-stressed commercial (7C) turkeys.* PSE Normal 5R 0 5 7R 3 2 5C 1 2 7C 6 1 *PSE meat: L*>52 and pH15m1n<5.8, and normal meat: L*<50 and pHrsm>5.8 80 4. Conclusion The data presented in this chapter show differences in ryanodine binding activity which could be explained by a mutation in B-RyR, by alternative splicing in the primary structure of B-RyR or by post-translational modification of [i-RyR. Focusing on identification of genetic polymorphisms, ryanodine affinity data obtained in this study will be used to select turkeys for screening the B-RyR gene to detect genetic variation(s) associated with incidence of PSE meat in turkey. 81 Chapter 3 Identification and analysis of single nucleotide polymorphisms of B-RyR associated with meat quality traits 1. Introduction The increasing incidence of pale, soft and exudative (PSE) meat over the past several decades is believed to be associated with intensive genetic selection for heavily muscled aninmals (Lengerken et al., 2002). In pigs, the incidence of PSE meat was closely associated with porcine stress syndrome (PSS), an inheritable skeletal muscle disorder found with highest frequency in heavily muscled pigs (Louis et al., 1993). The genetic basis of PSS was later determined to be a point mutation in the skeletal muscle calcium release channel (Fujii et. al., 1991). Because there are many striking similarities between pork and turkey PSE meat problems, we hypothesized that there is a genetic component(s) associated with the channel activity of turkey calcium release channels that may cause the PSE meat problems in turkey. The [3H] ryanodine binding assay provides an assessment of the open-state probability of ryanodine receptors (RyRs) or calcium release channels that may be associated with PSE incidence. Mickelson et al. (1988) reported that sarcoplasmic reticulum (SR) vesicles from PSS-susceptible pigs showed higher affinity for ryanodine 82 than normal pigs (Kd = 92 vs. 265 nM, respectively), which suggests that calcium channel activity, determined by the ryanodine binding assay of SR vesicles, may be altered due to the defect in Rle gene. Using the ryanodine binding msay, Wang et a1. (1998) and Zhang (2000) revealed that there are differences in the ryanodine affinity of the calcium channel proteins from two different genetic lines of turkeys, random-bred and commercial lines. These studies suggested that there may be a genetic component(s) in turkey RyRs associated with the PSE meat problem. The ryanodine binding affinity measured in this study represents the activity of B-RyR only because the activity of or-RyR is almost completely suppressed (Murayama and Ogawa, 2001). Therefore, differences in ryanodine binding could result from genetic polymorphisms in B-RyR gene, alternative splicing of B-RyR, post- translational modification of B-RyR, or modification of other regulatory proteins. Although these studies showed that there are differences in ryanodine binding between calcium channel proteins from two different genetic lines of turkeys, neither of these studies included analysis of association of ryanodine binding data with meat quality data, which is necessary to establish a link to the PSE meat problem. In the current study, using a larger number of turkeys, the channel activity of SR vesicles and meat 83 quality traits (L* and pHISmtn) from two different genetic lines, i.e. commercial and random-bred turkeys were measured (Chapter 2). A total of eighty turkeys from two different genetic lines, i.e., genetically improved and random-bred lines, were obtained after exposure to heat stress for 5 or 7 days. The L* values (objective measurement of lightness of color) at 24 hrs postmortem and pH at 15 min postmortem of breast muscle were measured. To measure the channel activity of RyRs in turkeys, the [3H] ryanodine binding assay was performed. This study showed that calcium channel activity of RyRs of commercial turkeys increased significantly (p=0.0008) as birds were eXposed to longer heat stress while that of random- bred birds was not affected by heat stress level. Based on this result, we suggested that genetically improved birds tend to yield more PSE meat than random-bred turkeys under heat stress as higher channel activity is associated with higher PSE incidence. A single nucleotide polymorphism (SNP) is one genetic component that could alter the channel activity of RyRs. A SNP is a DNA sequence variation among alleles. Variations in the DNA sequences of a species can have a major impact on the risk for developing disease and the response to environmental insults. Therefore, identification of SNPs is useful for finding genes that contribute to disease. Some SNP alleles are functional SNPs that cause differences in gene function or regulation directly related with 84 disease processes through changes in protein sequence or RNA stability. However, most SNP alleles are useful as genetic markers that can be used to find the flmctional SNPs due to associations between the marker SNPs and the functional SNPs (Kwok, 2003). Although major studies for SNPs have been carried out in the human genome, the study of SNPs is also important in crop and livestock breeding programs. Animal breeders are interested in the association between alleles and traits of interest and have started applying marker-assisted selection to improve the quality and performance of their livestock (Van der Steen et al., 2005). Fujii et al. (1991) reported eighteen SNPs in the skeletal muscle calcium release channel gene (ryrI). One of these SNPs (C1843T, called Hal-1843) causes the amino acid change (Arg615Cys) in the skeletal muscle calcium release channel (Rle) resulting in the accelerated calcium release associated with porcine stress syndrome (PSS) in pigs. This mutation corresponds to one point mutation, Arg614Cys, found in human MH and was the first marker used in pig breeding stock as the genetic component underlying the PSE pork problem. Despite efforts to remove stress-susceptible pigs from breeding, the PSE pork incidence climbed from 10.2 % to 15.5 % over the past 10 years (Kelley, 2003). Thus, the Hal-1843 alone is not suflicient to predict the risk of PSE susceptibility in pigs. It is likely that a complex 85 disease such as PSS or inferior meat quality such as PSE meat is caused by the combined effects of multigenes and environmental factors (multifactorial). Several muscle diseases are caused by mutations of proteins directly involved in the excitation-contraction-relaxation cycle and RyRs play a central role in cellular calcium regulation. Thus, polymorphism(s) in RyR channels can lead to potentially fatal disorders in muscles. It has been shown that the mutated Rle and RyR2 isoforms are closely associated with excessive Cay-release observed in malignant hyperthermia (MH)/central core disease (CCD) and cardiomyopathies, respectively. For example, in human RyR], to date, 47 missense mutations are associated with MH and 25 are associated with central core disease (CDC). Some of these mutations give rise to both MH and CCD (The human gene mutation database, The Institute of Medical Genetics in Cardiff University, UK; http://wwwhgmdcfacuk/ac/ns/l/120359.htm1). Thus, mutation(s) in turkey or- or B-RyR may be linked to the excessive Ca2+ release that is associated with PSE meat development in turkeys. The turkey B-RyR protein sequence is almost identical to the chicken B-RyR sequence (> 98%) and shows high sequence homology with the human RyR3 (87.7%). There are no SNPs identified in the coding region of chicken B-RyR to date. However, for human RyR3, 2412 SNPs have been reported in the ryr3 gene and 24 of them have 86 been found in the coding region (9 nonsynonymous and 15 synonymous). None of these SNPs in human ryr3 is known to be associated with any human disease. Therefore, identification of SNPs in turkey B-RyR associated with channel activity or meat quality will be the first report of phenotype-related SNPs in RyR3 (or B-RyR). To test the hypothesis that polymorphisms in the fl-RyR alter the channel activity and further, that they may be associated with inferior meat quality, turkeys were selected based on meat quality data and Kd values obtained from the ryanodine binding assay described in Chapter 2. In this study, screening of B-RyR cDNA in selected turkeys and statistical analysis are introduced. 2. Materials and Methods 2.1. Animals 2.1.1. Identification of SNPs in the coding region of turkey B-RyR Muscle samples from sixteen turkeys were selected based on their Kd values obtained from the ryanodine binding assay from Chapter 2. Seven birds were chosen with high K, (Average Kd=57.45 :1: 4.07 nM) and nine birds with low Kd (Average =28.25 :1: 1.17 nM). The entire coding regions of B-RyR from those turkey samples were screened for polymorphism(s). 87 2.1.2. Determination of SNPs associated with turkey meat quality traits Ten muscle sample from turkeys classified as PSE (L*>52 and pHISmin<5.8) and ten samples from turkeys classified as normal (L*<50 and pHrsmtn>5.8) were selected to determine whether there is an association between meat quality and SNPs. 2.2. Isolation of total RNA from turkey skeletal muscle Frozen turkey skeletal muscle tissue was ground into a fine powder in a pre- chilled mortar filled with liquid N2. To a 50-mL Corex tube, 0.5 g of muscle tissue was quickly transferred and 5 mL of TRIzol® reagent (Invitrogen; Carlsbad, CA) was added. Each sample was homogenized immediately using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) with two bursts of 10 see. each. Afier incubating samples at room temp for 5 min, homogenates were centrifuged at 12,000 x g for 5 min. at 4°C . Following centrifugation, the supernatant was decanted into a new Corex tube and 2 mL of l-bromo-3 -chloropropane was added and the tube was vortexed lightly for 3 seconds until the solution was well-mixed and uniformly pink. The sample was incubated for 2 min at room temperature, and the mixture was then centrifuged at 12,000 x g for 15 min at 4 °C. The upper aqueous phase was transferred to a fresh tube and total RNA was precipitated by the addition of 2.5 mL of isopropanol. The mixture was incubated at 4 88 °C for 15-20 min. After centrifugation at 12,000 x g for 10 min at 5 °C , the supernatant was decanted and the pellet was washed with 5 mL of pre-chilled 75% ethanol. The tube was centrifuged at 12,000 x g for 10 min. at 5°C and the resulting pellet was resuspended in nuclease-free water to a concentration of approximately 1 mg/mL. The quantity and quality of the total RNA were determined spectrophotometrically. Purified RNA samples were kept in a -80 °C freezer until use. 2.3. Reverse-Transcription Polymerase Chain Reaction All primers (listed in Table 3-1) for this objective were synthesized based on the B-RyR cDNA sequence completed by Chiang (2001, Appendix II). Reverse transcription (RT) and amplification of cDNA was performed using the Access RT-PCR kit according to the manufacturer’s protocol (Promega, Madison, WI). Following amplification, PCR products were loaded on to a 0.9 % agarose gel for analysis. The product band of the expected size was cut from the gel and the amplified cDNA was isolated from the gel slice using a Quiagen PCR purification kit (Quiagen, Inc., Valencia CA) according to the manufacturer’s protocol. Purified PCR products were sequenced in both strands of DNA at the MSU-DOE DNA sequencing facility. 89 .mzmln no HOE<§CVHOHwo0H52 and thsmin<5.8) and 10 normal meat turkeys (L*<50 and pH15mm>5.8), were selected for screening. The meat quality indices and ryanodine affinity for selected turkeys are shown in Table 3-4. The entire coding regions of 0-RyR of these turkeys were screened for the ten SNPs identified in the previous section. The ten SNPs identified in the previous study were detected in the twenty turkeys representing the differing meat quality characteristics and are listed in table 3-5. To determine the significance of these SNPs, least square means of meat quality traits (L* and lesmin) by genotype were analyzed for each SNP. The statistical analysis showed that some of the SNPs appeared to be associated with color (L*) and lesmin (table 3-6). For SNP 1, 5, 8 and 9 (Table 3-7), the heterozygous genotype g/a-t/c-t/c-c/t was associated with significantly darker color (L* = 49.30 :1: 0.97, p<0.01) and marginally higher pHrsmtn (pH1 5min = 6.02 i 0.10, p = 0.06) than one of homozygous genotypes gg- tt-tt-cc (L* = 53.56 :1: 0.94, pHrsmin= 5.77 :1: 0.04). The other homozygous genotype 95 Table 3-4. Meat quality data and ryanodine binding activity of PSE meat and normal meat turkeys“ Group Turkey ID pHISmin color (L’) Kd (nM) 291 1 6.63 47.42 38.64 31 13 6.42 48.53 30.03 2915 6.31 47.45 44.05 2921 6.28 46.68 37.04 2937 6.01 49.72 51.28 Normal 2903 5.96 47.18 41.15 3126 5.90 49.56 26.46 3103 5.90 45.01 39.22 2940 5.86 49.72 59.52 2934 5.86 49.08 45.05 LSmean** 6.11 :1: 0.09 ' 48.04 :1: 0.49 ° 41.24 a: 3.16” 3133 5.80 55.13 28.65 3128 5.79 56.58 27.1 3125 5.76 55.07 21.01 3136 5.75 52.03 24.57 31 19 5.74 52.24 30.86 PSE 3132 5.73 54.95 29.85 3122 5.73 53.69 41.67 3120 5.72 53.45 30.67 2908 5.71 56.25 39.22 3112 5.64 54.31 44.25 LSmean** 5.74 :1: 0.01 " 5437 :1: 0.49 ‘ 31.79 :1: 2.39' *PSE meat: L*>52 and pHrsm<5 .8, and normal meat: L*<50 and pH15m>5.8 ”Values are least square means t SE. ”’W'e'f Different letters within column indicate significant differences (p<0.05) 96 S. S S. S. S <6 S. S. S. 3.. R3 3% men can mma m... S S an S S. <6 S. <6 S. 3.3 San Se 2 _ m mme S. S S. an S <6 S. me S 3.. 8.2 2.2 3 3... man we on S S. on S S m.» S. mm 2.2 Sen .2 83 m2 mm on S S. S S S ma 6.6 mm 8.3. .38 .63 a: 3.. ma 6.6 S S. on S S ma 6.6 an $5... 2.3 NE 8; 3.. S. 6.6 S as 6e S S m.» S. we 3.... 3.2 m3 2: mma ma 6.6 S we S S S ma .6 mm 2.2 3.: ME Sm mma ma 6.6 S an S S S S. S. m.» E ”new a? S: mma S. S on a.» S S. S. S S. S. 8.: 8.3 c5 a: man S. S <6 S. on S. S. we on ma 8.? .34.... can ES .852 S. S <6 S. 6.6 S. S. S. S. we mean «he. can 8% 3:82 we S <6 a» S S. <6 ma 6.6 S. 3.3 8.9. an 8: .282 S. S no an S S. <6 S. 6e we 2.; 2.2. can 88 3582 S. S S. S. S S. S. S. S. S. 3.2 See Se :3 3:52 we S S. an S <6 .\o S... on me 632 9.2. So :2 .4852 S. S. S as S. S S ma 6.6 m.» 8.8 one. an cm: 35.52 S. S. S we S S S ma S. we 3: fine. a... 2.3. .282 S. S S. we S S. S an S. S. 8.8 m3... Sc 2: .252 S. S S. we S on 6e S. S as 2.3. as: .3 2% 3:52 2.95 aim 22m 52m cazm Ezm Ezm 82m 82m 32m 6. L eerie e are. 955 mace—.3 SSE mmm can :38 BEE: E coat—BE mmZm mace—.809? Ed. .mrm 2pm... 97 Table 3-6. Mean values of meat quality indices (L* and pHrsm) and ryanodine affinity (Kg) among three different genotypes of individual SNPs color pH Kg SNP # Nucleotide (Amino acid) 1" 2i 3* 1* 2§ 3* 1* 2’ 3* 1 a/g (V) 51.26'Lb 53.56' 49.30” 604°‘1 577° 6.02d 32.53i 36.31i 37.47i 2 c/t(S) 50.90' 47.45° 52.81' 592° 631° 5.86° 384914405i 29.48i 3 a/g(K) 51.26°~f 53.00° 49.39‘ 604° 585° 598° 2.53‘3561‘3830i 4 ch (1.) 51.26°-f 53.00° 50.70f 604° 585° 598° 82.53i35.61i 38.30i 5 c/t(H) 51.26“ 53.56' 49.30” 604°d 578° 6.02d 32531136303747i 6 c/t (N) 51.96' 50.99° 50.82‘ 580° 602° 596° 404313203i 39.89i 7 a/g (A) 51.96‘ 50.99' 50.82' 580° 602° 596° 40431320313989i 8 c/t(S) 51.26“ 53.56' 49.30b 604°‘1 578° 6.02°’32.53*36.31‘37.47i 9 U0 (N) 51.26“ 53.56’ 49.30b 604°“ 578° 6.02‘l 32.53‘3631‘3747i 10 a/g (K) 5207*“ 53.558 49.30h 593° 579° 6.02°35.58‘35.55i 37.47i ‘genotype aIa for SNPs 1, 3, 7, 10; do for SNPs 2, 4, 5, 6, 8; t/t for SNP 9 §genotype g/g for SNPs 1, 3, 7, 10; t/t for SNPs 2, 4, 5, 6, 8; c/c for SNP 9 *genotype a/g for SNPs 1, 3, 7, 10; c/t for SNPs 2, 4, 5, 6, 8; t/c for SNP 9 a"""fi’e'f’g'h’iDifferent letter within a row of each phenotype indicate significant differences (“p=0.0099, °’dp=0.0640, °‘fp=0.0325, “p=0.0137) 98 Table 3-7. Meat quality indices (L* and pHrsmin ) and ryanodine affinity of SNPs l, 5, 8and9 Genotype SNP 1-5-8-9 a/a-c/c-c/c-t/t g/g-t/t-t/t-c/c a/g-c/t-cIt-t/c n 2 8 10 Color (L*) 51.26 :1: 3.85":b 53.56 i 0.94a 49.30 :1: 0.97b p1115min 6.04 :t 028°d 5.77 :t 004° 6.02 :1: 0.10d K4 (nM) 32.53 :1: 11.64" 36.31 :1: 320° 37.47 :1: 3.17° £”"”°""eDifferent letters within a row indicate significant differences c°p=0.0099, °r° p=0.06 ) aa-cc-cc-tt was detected only for a small number of turkeys (n=2), so the statistical power was insufficient to determine the significance. For this group of SNPs, the heterozygous genotype (g/a-t/c-t/c-c/t) showed better meat quality than one of the homozygous genotypes (gg-tt-tt-cc). The frequency of the heterozygous genotype in random-bred turkeys was 70% and that in commercial turkeys was 10% while the frequency of the homozygous genotype (gg-tt-tt-cc) with poor meat quality in random-bred turkeys was 10% and that in commercial turkeys was 70%. This implies that a larger population of random-bred turkeys tends to have the heterozygous genotype with better meat quality while most commercial turkeys have the homozygous genotype with the inferior meat quality. Likewise, in PSE turkey meat, the frequency of the heterozygous genotype with the better meat quality was 30% and the frequency of the homozygous genotype associated with the inferior meat quality (gg-tt-tt-cc) was 60% while the frequency of the heterozygous genotype was 70% and that of the homozygous genotype (gg-tt-tt-cc) was 20% in normal turkeys. SNPs 3, 4 and 10 also showed a significant difference in L‘ value (p<0.05) between heterozygous genotype and one homozygous genotype as listed in table 3-6. However, there was no significant difference detected in terms of lesmin between two 100 genotypes. Other SNPs, e.g., SNP 2, 6 and 7 did not show any significantly different values between different genotypes. 3.3. Haplogpe and genotype analysis of identified SNPs Haplotype is a set of closely linked alleles (genes or DNA polymorphisms) inherited as a unit (http://www.nhgri.nih.gov/DIR/VIP/Glossary/pub_glossary.cgi). To specify the genetic information descending through a pedigree and to visualize the gene flow through a pedigree, haplotype analysis for given individuals was performed. Haplotypes constructed using SAS haplotype analysis program revealed a total of eight haplotypes in twenty birds (T able 3-8). Based on the haplotype analysis, there were eleven possible genotypes for the selected turkeys. Color (L"‘), lesmin and K; within the observed genotypes are listed in Table 3-9. Comparison of least square means of meat quality traits (L* and thsmm) and K; by genotype indicated several significant differences among genotypes. For example, genotype G presents a significantly darker color (L*#7 .91) than genotype J (L*=56.68, p=0.0419) and genotype E has higher ryanodine affinity (Kd=21.01) than genotype F (Kd=52.29, p=0.0048). However, given the large number of genotypes and distribution 101 323. w o w w a a a w a w a 25295 SEE. m o c m . . . m c m e oebeaem Ram... m o c e o c . m c m c 883...: 2536 m o c m o H a w o w m 252%: 9.32. e . o m c o o e . e e 252%: cows... e c o w . o c m o e m 252%: vooood e c o m . ... o e o e m 8.33% Sana—d a a o a o o o a o m _ cacao—mam Soeoeeoe eazm 32m mazm Ezm cmzm 32m Ezm 82m 82m 32m BEES: maZm :8 mo max—Ea cacao—am: .wrm 28d. 102 Table 3-9. Meat quality indices and ryanodine affinity data among B-RyR genotypes Genotypes“ A B C D E F G H I J K F (4/4) (5/5) (6/6) (7/7) (1/4) (1/6) (1/7) (3/7) (4/6) (6/7) (7/8) Color” 47.45 53.69 53.09 52.02 55.07 49.40 47.91 48.53 51.35 56.58 54.95 pH" 6.31 5.73 5.78 5.83 5.76 5.86 6.06 6.42 5.94 5.79 5.73 Kd (nM)" 44.05 41.67 31.12 47.77 21.01 52.29 35.90 31.03 32.18 27.10 29.85 *the numbers in parentheses refers to the haplotype listed in table 3-8. ”mean values if there are more than one animal in the same genotype group 103 of birds among the genotypes, analysis will need to be conducted on a large number of birds to determine the possible significance and relationship to meat quality. 3.4. Synonymous SNPwsociated with meat quality or diseases The ten synonymous SNPs identified in the B-RyR coding region in the current study have not been previously reported in any species including chicken and human RyR3. In the mammalian skeletal muscle isoform (Rle) in pigs, 18 SNPs were reported (Fujii et al., 1991). One of the SNPs in their study was a non-synonymous SNP (C1843T) that induced Arg615Cys point mutation in Rle; the rest of the SNPs were all synonymous. The synonymous SNPs did not attract interest because they do not result in a change in coding for amino acids. Allison (2004) identified 22 synonymous SNPs of Rle among six pigs, including the 13 SNPs reported by F ujii et a1. (1991). Based on the haplotype analysis of the 6 pigs, he chose a cDNA region containing 4 SNPs of Rle for screening more pigs (n=22) and reported that pigs with one genotype (n=3) had a lower halothane sensitivity, a lower ryanodine binding activity and a higher initial pH, compared to other genotype (n=8). When he increased the number of animals (n=41) for association studies, however, no statistical differences were 104 observed among genotypes except the halothane sensitivity and mobility status of pigs in two genotypes was consistent with preliminary observations with 22 pigs. To date, there have been a considerable number of reports of synonymous SNPs in the coding regions of other proteins associated with phenotypic characteristics. Volcik et al. (2004) evaluated the synonymous SNPs in exon 6 of the Jumonji gene as a variation associated with higher risk for a congenital heart defects (CHD) in infants. Capon et al. (2004) also reported that a synonymous SNP (C971T) in the coding region of the comeodesmosin gene leads to increased mRNA stability and demonstrates association with psoriasis across diverse ethnic groups. As shown in these studies, some synonymous SNPs identified in the current study may alter the stability of mRN A transcripts or change the translational efficiency which could lead to altered meat quality traits. However, it is highly possible that the SNPs may not be functional SNPs and instead, they could be useful as genetic markers for allelic variants associated with meat quality traits. 3.5. Absence of association between SNPs and ryanodine binding affinity It was revealed that the color (L*) of meat was strongly affected by genetic lines of turkeys in the current study (described in Chapter 2). The random-bred birds have 105 significantly darker color (L*=50.58) than commercial turkeys (L’=52.99) (p<0.0001). This is consistent with the results obtained from the analysis of SNPs as the variations (SNPs l, 5, 8 and 9) were associated with significant difference in color between two genotypes. However, the rate of pH decline was not affected by genetic lines, but was correlated with heat stress levels. The same variations (SNPs 1, 5, 8 and 9) only showed a marginal relationship with the initial pH (p=0.064) in turkeys. None of the SNPs identified was correlated with the ryanodine binding affinity of the B-RyR as described in Table 3-8. The heat stress level appeared to affect the early postmortem pH decline and the channel activity of B-RyR as measured by the ryanodine binding assay. As described in Chapter 2, turkeys exposed to a longer period of heat stress showed a faster pH decline and higher ryanodine affinity than turkeys experiencing less heat stress. Exposure to acute heat stress for a longer period may have induced a post-translational modification of the channel protein or associated proteins leading to greater Ca2+ release and a faster pH decline, predisposing the meat to become PSE. This phenomenon should result in increased light scattering of the surface of meat to yield paler meat. However, even in the same genetic line of turkeys, the heat stress level was not associated with color difference among meat samples in this study. Ryanodine binding activity difference of 106 the B-RyR was observed only within the commercial turkey line when exposed to different heat stress levels. Therefore, it is possible that there are differences in extent to which the B-RyR is regulated in acute heat stress between random-bred birds and commercial birds. One possible explanation for the difference in the initial pH and the channel activity could be that the commercial birds tend to experience more severe oxidative stress under acute heat stress than random-bred birds and thus, the calcium channel protein of the commercial birds is more oxidized compared with that of random-bred birds. Lin et al. (2006) investigated the extent of lipid peroxidation, activities of superoxide dismutase and total antioxidant power in plasma, liver and heart tissues of heat-stressed broiler chickens. They suggested that the physiological changes such as elevated body temperature in heat-stressed chickens are also involved in the induction of oxidative stress. As a result of the in-vivo oxidation of the protein, cysteine residues can be oxidized to form disulfide bonds that alter the activity of the B-RyR. Then, the excessive amount of Ca2+ released through the leaky channel could accelerate postmortem metabolism causing the increased rate of pH drop. Although this explains the heat stress effects on the initial pH, it cannot elucidate the difference in the channel activity between turkeys with different heat stress levels because reducing agents such as 107 mercaptoethanol were used to reduce the disulfide bonds under the experimental conditions to isolate CSR and to perform the ryanodine binding assay. Therefore, the channel activity difference observed under this experimental condition would not be due to the in-vivo oxidation of cysteine residues. Phosphorylation of the channel protein is another post-translational modification that may alter channel activity when birds are stressed. Analysis of the RyR protein sequence reveals many consensus phosphorylation sites, and thus, various kinases and phosphatases could affect the behavior of the RyRs (Otsu et al., 1990; Suko et al., 1993 and Tunwell et al., 1996). Although there have been contradictory results from different research groups, it seems obvious that there are effects of phosphorylation of RyR on the activity of the protein (Marks et al., 2002; Danila and Hamilton, 2004). This could explain the channel activity difference detected for birds with different heat stress levels. Thus, we suggest that alternative splicing, or post-translational modification of the protein or other regulatory proteins may be one of the major factors altering the ryanodine binding activity of the ,B-RyR and the initial pH of the meat in the current study. Hence the channel activity measured by the [3 H] ryanodine binding assay may not be a good phenotype to represent polymorphisms of the ,B-RyR gene identified in this study as originally proposed. 108 Results of the present study indicate that some variations (SNPs l, 5, 8 and 9) in genotype of the B-RyR were strongly associated with color (L*) of the breast muscle. Therefore, the SNPs identified in this study may be functional SNPs directly regulating the color or genetic markers associated with the actual functional SNPs correlated with color of the meat. In addition, the thsmin was marginally associated with the SNPs (1, 5, 8 and 9). Because of the limited number of animals, further investigation with a larger number of turkeys is necessary to determine whether the SNPs are linked with meat quality. 4. Conclusion As a result of the screening of the entire ,6 -RyR cDNA of the selected turkeys, ten synonymous single nucleotide polymorphisms (SNPs) were identified. None of the SNPs showed any significant correlation with channel activity of the ,B-RyR. Although the SNPs did not change the primary structure of the ,B-RyR, seven SNPs were strongly associated with color (L‘) of the meat and four SNPs among the seven SNPs were marginally associated with the pHrsmtn. Therefore, there appeared to be an association between some SNPs and meat quality traits, i.e., the heterozygous turkeys for the mutations show better meat quality than homozygous turkeys. 109 The SNPs identified in this study may be functional SNPs directly regulating the meat quality or genetic markers associated with the actual ftmctional SNPs correlated with the meat quality. Further study with increased number of animals is required to evaluate potential association between meat quality and genetic components identified in the current study. 110 Chapter 4 Ribonuclease protection assay of alternatively spliced transcript variants of turkey a-RyR and screening of turkey B-RyR for splice variants 1. Introduction Alternative mRNA splicing is the term used to describe the regulatory process involving differential inclusion or exclusion of regions of pre-mRNA and is an important source of protein diversity and functional complexity of proteins in higher eukaryotes (Johnson et al. 2003; Kan et al., 2001; Modrek et al., 2001; Black, 2003). Alternative splicing patterns for a given protein may change with developmental stage and life cycle, with tissue localization, or as a result of the influence of environmental factors on the organism (Strehler and Zacharias, 2001). Moreover, it has been reported that errors in alternative splicing can cause a variety of disease states (Caceres and Komblihtt, 2002; Black, 2003). Many environmental stressors alter gene expression through the alternative splicing of pre-mRNA (Shukla et al., 1990; Bournay et al., 1996). Among them, heat stress has been reported to regulate the alternative splice pattern of pre-mRNA of many genes such as adenovirus ElA gene (Denegri et al. 2001) and mouse heat shock protein 47 gene (Takechi et al., 1994). 111 The involvement of RyR] in porcine stress syndrome and its association with PSE meat are well documented (Mickelson et al., 1986; 1988). The central role of RyR in modulating postmortem muscle metabolism and the association of a mutant form of RyR with PSE meat raises the question of the possible involvement of differentially expressed alternatively spliced RyR transcript variants in development of PSE meat. There have been a few examples of alternative splicing for mammalian Rle , RyR2 and RyR3 identified so far. Kimura et al. (2002) found a significant increase in an alternatively spliced isoform of Rle in myotonic dystrophy type 1 (DMl) patients. Insertion of five amino acids (Ala-Gly-Asp-Ala-Gln) after residue 3479 in rabbit Rle (Zorzato et al., 1994) or deletions of Ala(3481)-Gln(3485) and Val(3865)-Asn(3870) in mouse Rle (Futatsugi et al., 1995) have been reported. In human RyR2, insertions of ten amino acids between residues 1479 and 1480 and eight amino acids between residues 3715 and 3716 were found (Tunwell et a1, 1996). Compared to Rle and RyR2, there have been more RyR3 alternative splicing cases reported. Jiang et al. (2003), and Leeb and Brenig (1998) characterized several rabbit and human RyR3 splice variants. Most of the splice variants identified in each isoform lead to changes in either the putative modulatory or transmembrane domain of the protein. Therefore, alternative splicing of 112 RyRs may have important and unique functional roles to regulate the activity of the channel proteins. Chiang et al. (2004) reported the first avian RyR transcript variants. They found three alternative splicing regions in turkey a-RyR cDNA using the reverse-transcription polymerase chain reaction (RT-PCR) Gigure 4-1). The first alternative splicing region (ASRl) was found in exon 13 (based on the human Rle genomic DNA structure) of a- RyR corresponding to the 3’-end of mutation hot spot 1 of human RyR] (Figure 4-2). This region encompasses two alternative splicing variants: an 81-bp-deletion (AS-81) which introduces a 27-amino-acid deletion and a l93-bp-deletion (AS-193) which generates a premature stop codon (Chiang et al., 2004). The first alternative splicing region has been proposed to serve as part of the protein-protein contact site of Rle with the DHPR. Alternative splicing in this region could alter regulation of channel activity by the DHPR. The locations of the other two alternative splicing regions (ASR 2 and ASR 3) coincide with divergent regions 2 (DR2) and 1 (URI) in Rle (Figure 4-3). Chiang et al. (2005) identified a second region (ASR 2) which spans exon 24 to exon 29 and encompasses at least 4 different transcript variants including deletions of 1043 bp, 401 bp, 454 bp and 268 bp. These deletions correspond to amino acid residues #1055-1402, 113 0 1000 2000 3000 4000 5000 A.A. all F ASR 1 ASR 2 ASR 3 AS-81 AS-1043 AS-193 AS401 AS454 AS-370 AS-268 AS-816 Figure 4-1. Three alternative splicing regions identified in turkey a-RyR by RT-PCR (Adapted from Chiang et al., 2005) 114 .968 :3 Ho w§£U 89¢ 3Eme$ 33% £5 E @569 madam—mm 03358:. 28852 moez BEE momma 5 2m 32.258 :02: 05 Ea moxon 05 S 850:... Pa 88268 :98 BE. .8523 0.8 muouoesm 32% 05 9593 8:268 Boa 965 e5. 02820:: 2:. .MSMS >83: 5 message 82% 033833 05 no 88259... use 332804 .mé 0.33m Nvm—rcmm~< no: 0 ~— — ..:.—3:3,...— eesfioe ram“ «mm m mazcuwmwiws own So 3... .:owe m «5.55% we 3a.... mama. Angina“ Va uouo m~ name 2 none 3.32.22 4. _ 2 e l m _ .8523. @5on was www MenzqquWEwS owe 39 Saiowm 23.25:... mo 8a.... Ermd. .51; 3 :38 < 2 :38 S .85 <....— 2 U HES..— m A ~.....m m m 2: emac— ==h wiofi wee www «3.53 www.5w6 own 39 Saiowm w «8.5.3:» mo 03.... 3;“ 3 none 2 :88 2 no: . «02.9.0 20:05.... 03:5 023532 0:32 115 0 l000 2000 3000 4000 5000 A.A. a-RyR %-% % 0R2 0R3 DR1 I l l ASR‘I ASR2 A8R3 Figure 4-3. Comparison of alternative splicing regions (ASR) and divergent regions (DR) in turkey a-RyR (Adapted from Chiang et al., 2005). 116 #1255-1388, #1258-1409 and #1313-1402, respectively. Each of these deletions introduces a premature stop codon in the coding region. The third alternative splicing region (ASR3) resides in the C-terminal region of the a-RyR, which is in divergent region 1. One transcript variant comprising a deletion of 816 bp was identified, and this transcript variant would result in a deletion of 272 amino acids (residues #4214-4485) encoded by exon 91 of a-RyR in the corresponding human Rle sequence. The overlap of the alternative splicing region and the divergent region suggests that these regions of protein sequence are highly variable, and both alternative splicing and sequence variation may be responsible for altered RyR activity. Although there have been several alternatively spliced transcript variants (ASTVs) identified in turkey skeletal muscle a-RyR, there has been no report of ASTVs in turkey B-RyR. In the current study, the entire cDNA of B-RyR was screened for the presence of ASTVs, which could be associated with development of PSE turkey. Subsequently, the expression of AS-81 and AS-193 fi'om a—RyR was quantified to determine the expression pattern of each ASTV. We hypothesize that there is a difference in the expression pattern of ASTVs in the ASR 1 region of a-RyR fiom different subgroups of turkeys (5R: 5-day heat stressed random bred, 5C: 5—day heat stressed commercial, 7R: 7-day heat stressed random-bred 117 and 7C: 7-day heat stressed commercial turkeys). For B-RyR, we hypothesize that there are alternative splicing variants associated with ryanodine binding affinity which can be further correlated with meat quality. 2. Materials and Methods 2.1. Animals 2.1.1. Screening of B-RyR for alternatively spliced transcript variants Sixteen turkeys were selected for analysis based on their Kd values obtained from the ryanodine binding assay: i.e., seven birds with high K. (Average Kd=57.45 i 4.07 nM) and nine birds with low Kd (Average Kd=28.25 i 1.17 nM). The entire coding region of the B-RyR of those turkey samples was screened for alternative splicing in B- RyR. 2.1.2. Quantification of alternatively spliced transcript variants in the turkey a- RyR A total of twenty-nine turkeys were selected based on their color (L*), early postmortem pH (lesmin) and ryanodine binding aflinity (Kd). This is the combined group of the first sixteen turkeys, i.e. seven birds with high K. (Average Kd=57.45 :1: 4.07 nM) and nine birds with low Kd (Average Kd=28.25 i 1.17 nM) and twenty turkeys 118 selected for PSE meat and normal meat samples, i.e., ten PSE meat (L*>52 and lesmin<5.8) and ten normal meat (L*<50 and pH15m>5.8) samples. 2.2. Isolation of total RNA fiom turkey skeletal muscle Frozen turkey skeletal muscle tissue was ground into a fine powder in a pre- chilled mortar filled with liquid N2. To a SO-mL Corex tube, 0.5 g of muscle tissue was quickly transferred and 5 mL of TRIzol® reagent (Invitrogen; Carlsbad, CA) was added. Each sample was homogenized immediately using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) with two bursts of 10 sec. each. Afier incubating samples at room temp for 5 min, homogenates were centrifuged at 12,000 x g for 5 min. at 4°C. Following centrifugation, the supernatant was decanted into a new Corex tube, 2 mL of 1-bromo-3-chloropropane was added, and the tube was vortexed gently until the solution was well-mixed and uniformly pink. The sample was incubated for 2 min at room temperature, and the mixture was then centrifuged at 12,000 x g for 15 min at 4°C. The upper aqueous phase was transferred to a flesh tube and total RNA was precipitated by the addition of 2.5 mL of isopropanol. The mixture was incubated at 4C for 15-20 min. After centrifugation at 12,000 x g for 10 min at 4°C, the supernatant was decanted and the pellet was washed with 5 mL of pre-chilled 75% ethanol. The tube was 119 centrifuged at 12,000 x g for 10 min. at 5 °C and the resulting pellet was resuspended in nuclease-free water to a concentration of approximately 1mg/mL. The quantity and quality of the total RNA were determined spectrophotometrically. Purified RNA samples were kept in a -80°C freezer until use. 2.3. Ribonuclease protection assay A probe was designed for each ASTV fragment such that protected fragments of the specific size would be obtained following digestion with RNase. 2.3. 1. Probe synthesis The RPA probe was synthesized by generating an RT-PCR fi'agment fiom total RNA isolated from turkey breast muscle tissue with primers based on the strategy shown in the diagrams in Figure 4-4 (forward primer, 5’- CTGCACCAGGAGGGCCACATGGACGA—3 ’; reverse primer, 5’- GTGGTTCTCCTGGATGATGTTCAGAAC-B ’) and cloning the fragment into pGEM-T Easy Vector (Promega). The cDNA sequence of the amplified region is shown in Appendix III. Anti-sense RNA was synthesized from NcoI-cut cDNA with SP6 RNA polymerase and labeled with biotinylated CTP“ according to the manufacturer’s 120 ASTV Full length probe is 701 nts. with partial vector sequence 193 bp —fi Full length 116 b 81b22:21:11?:Ertzizzgzxzzrzz; -:-:-:-:-:-:-:-:-:-:-:-:-:42'9'2‘5-‘54-1-2-32-:-:.:-:-:-:-:-:-:-: (601 hp) p $35.; -------------------- pm T515333:':-.-:11:1:433193;!!23533533331 ‘2':f'I'I'Z'Z'lf‘Z‘Z'Z' '''''''''''''' pf‘:':‘2'1'Z'I'Z':':':':':':‘:': Expected fragment size: 610 nts. protected 811) d letio ........................................... " e " 116 hp ====+>>>112his“2225222322232:2:2:2:192.hpzszzzszzrzzszzzszsrez2252222232. (520 hp) .................................................................................... Expected fragment size: 116 nts. and 404 nts. l93b d Ietio ...... " ° " 116 bp :::::2:s:2:s:2:3:2:2:222:2:;z;9zzhhzs:22322:3:s:s:2:s:s:;:;:;:;:;:; (m m ..................................... Expected fiagment size: 116 nts. and 292 nts. Figure 4-4. mRNA species representing three alternatively spliced forms of ASR] region of turkey a-RyR. 121 instruction (MAXIscript kit, Ambion, TX). The reaction mixture was subjected to electrophoresis on a 4% (w/v) polyacrylamide/8.0 M urea gel and the probe was eluted into elution buffer provided in the MAXIscript kit. The concentration of the labeled RNA probe was determined spectrophotometrically and the probe was stored at -80°C until use. 2.3 .2. Ribonuclease Protection Assay (RPA) The RPA assay was performed with RPA III Kit (Ambion, TX) according to the manufacturer’s guidelines. Briefly, total RNA (10 pg) and the labeled RNA probe (10 ug) were mixed in a 1.5-mL microfuge tube. The probe and sample RNA were co- precipitated by adding 5 M ammonium acetate to a final concentration to 0.5 M, and 2.5 volumes of ethanol. The tube was placed at -20°C in a freezer for 15 min and then centrifuged at maximum speed in a microcentrifuge (13,000 rpm) for 15 min. After removing the supernatant, the pellet was washed with pre-chilled 70 % ethanol. The pellet was resuspended in 10 uL of hybridization buffer included in the RPA III Kit and the hybridization mixture was incubated at 90-95°C for 3~4 minutes to denature the RNA and aid its solubilization. The contents of the tube were incubated at 45 °C overnight to allow hybridization of the probe with its complementary sequence in the sample RNA. 122 The remaining single stranded RNA was digested at 37°C for 30 min with 150 uL of the RNase A/RNase T1 mixture diluted in RNase Digestion III Buffer (1:100 dilution). The RNase was inactivated after the digestion was completed by adding 225 uL of RNase Inactivation/Precipitation III solution fi'om the kit. The mixture was precipitated and the pellet containing the RNA fi'agments hybridized to the probe was dissolved in sample buffer. The hybridized fragments were analyzed on a 4 % (w/v) polyacrylamide/8 .0 M urea electrophoresis gel. Following electrophoresis, the RN A-probe hybrid was transferred to a positively-charged nylon membrane by electroblotting and the non- isotopic probe was visualized by the Streptavidin-Alkaline Phosphatase conjugate system (BrightStar BioDetect Kit, Ambion, TX). Quantification of the levels of the products was accomplished using a FluorChem imaging system (Alpha Innotech, San Leandro, CA). Turkey B-actin was used as an internal control because B-actin is known as a housekeeping gene. However, the ratio of transcript variants (combined amount of full- length and AS-8 lvariants) to B-actin was consistent among birds. Thus, B-actin was not included in calculation to obtain the ratio of full-length nanscript variant to AS-81. All measurements were conducted in duplicate. 2.4. Statistical Analysis 123 The General Linear Model procedure of SAS (SAS Inst. Inc., Cary, NC, 2000) was used to analyze data. Least square means of the ratio of the full-length and 81-bp deleted fragments were compared for turkeys in different groups. 3. Results and Discussion 3.1. Analysis of ASTVs in tugaw a-IQR using RPA According to the previous results from RT-PCR analysis of a—RyR sequence in our lab, there are three transcript variants generated by alternative splicing in the ASR] region. However, RT-PCR is not an effective tool to quantify the expression level of each ASTV. The ribonuclease protection assay (RPA) is an extremely sensitive procedure for the detection and quantification of ASTV in a complex sample mixture of total RNA (Takechi et al., 1994; Friedberg, 1990). Therefore, in the current study, RPA was used to detect and quantify ASTVs in the ASR] region of turkey a—RyR to test the hypothesis that expression pattern of ASTVs in the region is regulated by heat stress and that expression pattern is correlated with meat quality indicators. The result of RPA is shown in Figure 4-5. Previous RT-PCR analysis showed that there are three transcript variants in ASR] of a-RyR: full-length, AS-81 and AS-193. Upon analysis of the presence and amount of each ASTV using RPA, fragment sizes of 124 Turkey ID ASTV Fragment size 2901 3132 2908 2911 3112 3113 (bases) - - V: | l >.- I Figure 4-5. A sample image of the RPA of mRNA from turkey skeletal muscle tissue using a biotinylated RNA probe complementary to ASR 1 region in turkey or-RyR. Fragment sizes of 610 bases and 404 bases were observed that correspond to the full- length and AS-81 transcript variant, respectively. Turkey B-actin was used as an internal control. (- full-length 610 (- AS-8l 404 (- B-actin 215 125 610 bases and 404 bases were observed that corresponded to the fitll-length and AS-81 transcript variant, respectively. However, no fragment corresponding to AS-193 (292 bases) was detected in any sample. A possible explanation for this discrepancy is that when RT-PCR conditions were altered, the full-length and AS-81 were always detected, but AS-193 was not consistently present. The absence of a band in the RPA assay corresponding to AS-193 suggests that this variant may be an artifact of RT-PCR procedure. Thus, it appears that there are two ASTVs, full-length and AS-81 as the major transcript variants in this region of a-RyR. The relative intensities of the RPA bands of the full-length and AS-81 isoforms were measured in order to determine whether altered patterns of transcript variant expression could occur as a result of heat stress, and to determine whether there are differences in splicing between the two turkey lines. Splice patterns of turkey lines exposed to different levels of heat stress were determined by analysis of the least square means of the ratios of full length to AS-81 transcript variants. As shown in Table 4-1, there were no differences between random-bred and commercial turkeys when both groups were exposed to five days of heat stress. However, after turkeys were subjected to a longer heat-stress (7 days), the ratio between full-length and AS-81 transcript variants of random-bred birds was significantly lower than that of commercial birds 126 Table 4-1. The ratio of full-length transcript variant to AS-81 for selected turkeys. Random-bred Commercial Turkey ID Ratio" Turkey ID Ratio 2901 0.81 1 0.30 2908 1.44 1 0.12 2903 1.20 1 0.77 2910 0.63 1 0.28 2911 1.44 1 0.40 2912 0.89 1 0.00 2915 2.08 1 0.44 2918 1.16 1 0.35 5'day baa" 2921 0.90 1 0.11 2924 0.45 1 0.08 stressed 2929 1.38 1 0.38 2934 1.32 1 0.07 2933 0.97 1 0.23 2940 1.68 1 0.78 2937 1.87 1 0.35 LSmean” 1.33 :1: 0.13“’c LSmean” 1.08 i 0.14“"; 3103 1.17 1 0.57 3112 1.68 1 0.27 3105 0.63 1 0.11 3118 1.73 1 0.33 3113 0.92 1 0.20 3120 1.32 1 0.18 3119 0.93 1 0.12 3122 1.30 1 0.65 7433’ 1163" 3125 1.41 1 0.23 3126 1.15 1 0.06 stressed 3133 1.09 1 0.78 3128 1.29 1 0.18 3132 1.73 1 1.20 3136 1.56 1 0.24 LSmean" 1.02 1 0.15“ LSmean" 1.47 1 0.13“,“ *obtained fi'om the duplicated measurements of RPA for each turkey RNA sample. Ratio for each turkey sample is expressed as average value :1: SD. "LSmean 3: standard error obtained from the General Linear Model procedure of the SAS analysis: '1’” Different letters within rows represent a significant difference (p=0.0342). °‘d Different letters within columns represent a marginal difference (p=0.0519). 127 (p=0.034). For the random-bred turkeys, there was not an effect of heat stress on the relative abundance of those two fragments (p>0.1). In contrast, commercial turkeys seemed to have a higher ratio between full-length and AS-81 transcript variants when they were exposed to a longer heat stress (p=0.052). The data suggest that there may be a trend that random-bred turkeys have a constant ratio of full-length/AS-Sl while commercial turkeys undergo produce less of the AS-8l transcript variant. Based on these results, we suggest that there are differences in expression patterns of two ASTVs between random-bred birds and commercial birds under different heat stress levels. As Table 4-1 shows, there was significant variation in some of the sample measurements. This problem is mainly due to the difficulty in determining the boundary of each band during the quantitative analysis of each band image on the nylon membrane. Only a slight change of the position of each boundary could generate a big change in the final results obtained from quantitative analytical tools. This problem could be reduced by increasing numbers of birds in each group or by increasing the number of measurements for each bird. Since all turkeys used in this study were heat-stressed, and there is only a two-day difference between heat stress levels, it is difficult to conclusively ascribe differences in transcript variant expression to heat. More systematic studies including unstressed birds, 128 together with different periods of stress, will be necessary to more conclusively determine the mechanism by which alternative splicing is regulated by heat stress. Although the mRNA for AS—81 is repeatedly observed, it is unknown whether this message is translated into protein. Jiang et a1. (2003) reported that there are as many as seven ASTVs of rabbit RyR3. They used RPA to reveal that one of these splice variants was highly expressed in smooth muscle tissues, but not in skeletal muscle, the heart, or the brain. Although this splice variant did not form a functional Ca2+ release channel when expressed alone in HEK293 cells, it was able to form functional heteromeric charmels when co-expressed with the wild type RyR3. Therefore, it is possible that the splice variants observed in turkey a-RyR are able to form functional channels that modulate channel activity of RyR. The factors influencing expression of the different variants are not clear but our results suggest a possible role for heat stress. There are a number of environmental stressors which alter gene expression through the alternative splicing of pre-mRN A (Shukla et al., 1990; Bournay et al., 1996). Among them, heat stress has been reported to regulate the alternative splice pattern of pre-mRNA of many genes. For example, the mouse heat shock protein 47 (HSP47) gene expresses three HSP47 transcript variants and one of these alternatively spliced mRN As was detected only after heat shock (Takechi et 129 al., 1994). Denegri et al. (2001) also reported five different adenovirus ElA transcript variants that showed different splicing patterns under heat-shock conditions. It is likely that different genetic lines of turkeys use alternative splicing with different extents as an adaptive tool to heat stress based on the results in this study. Although the ratio of full-length to AS-81 is not correlated with meat quality traits (p>0.1, data not shown), further studies including other alternative splicing regions (ASR-2 and ASR-3) could reveal the whole figure of relationship between meat quality and alternative splicing patterns in turkey a-RyR. 3.2. Screening of tu’rkey B-RVR for ASTVs Independently from the or-RyR, as the channel activity of the B-RyR was measured in chapter 2 and the entire cDNA region of the B-RyR was sequenced for the same turkeys, the B-RyR was also screened for any possible alternatively spliced products. Compared to Rle and RyR2, there have been more isoforms of RyR3 due to alternative splicing characterized in other species including rabbit and human (J iang et al., 2003 and Leeb and Brenig, 1998). Since the turkey B-RyR in the skeletal muscle is orthogous to the RyR3 in mammalian smooth muscle tissue, it seemed likely that there would be several transcript variants in the turkey B-RyR. However, after screening sixteen turkeys in the current study, there were no alternatively spliced transcript variants 130 detected in B-RyR. This result suggests that the coding region of B-RyR pre-mRNA in turkey skeletal muscle does not yield any ASTVs even after the birds are exposed to environmental stressors such as acute heat stress. In contrast, a-RyR comprises several ASTVs, some of which are likely to result in expression of fimctional a-RyR channel protein with varying activity. As Sorrentino and Reggiani (1999) suggested, co-expression of two isoforms, (1- and B-RyR, may be relevant to adapt the modality of Ca2+ release to regulation of specific cellular functions. Also, in the two-component model suggested by O’Brien (1995), each isoform must be influential in the calcium release activity of both RyRs. It is not clear why there are more various modifications in the a-RyR while the B-RyR coding region is well-conserved except several synonymous SNPs. The current study optimized the RPA conditions available for turkey a-RyR to quantify mRNA levels of various transcript variants. Also, this study suggests that there are differences in abundance of transcript variants expressed in the ASRl region of the or- RyR of turkeys obtained fi'om different genetic lines under prolonged heat stress conditions. To confirm this, further studies are needed. 4. Conclusion 131 Previous studies using RT-PCR suggested the possibility of the alternative splicing of a-RyR as an adaptive response of turkeys under heat stress. The RPA method was employed to quantify the amount of firll-length and AS-81 transcript variants. The results revealed that at least one transcript variant (AS-193) discovered by RT-PCR was not detected by RPA. This suggests that AS-193 identified by RT-PCR could be an artifact of the RT-PCR procedure. 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Commun. 203: 1725-1730. 147 Appendix 1: Meat quality data and ryanodine affinity of turkey muscle samples Group Turkey ID P111521"; L“ Kd (anD Bmax * 29-01 6.24 50.31 50.50 1.65 29-03 5.96 47.18 41.15 1.94 29-05 6.26 46.10 - - 2907 6.03 50.28 45.25 1.72 29-09 5.86 51.03 29.33 1.36 29-11 6.63 47.42 38.64 1.30 Random- 29-13 6.29 50.58 45.66 1.53 bred 29-15 6.31 47.45 44.05 1.09 29-17 6.39 52.95 - - 5-day 29-19 6.40 51.94 36.50 1.71 heat- 29-21 6.28 46.68 37.04 1.42 stressed 29-23 6.28 53.18 - - 29-25 6.17 52.84 39.22 1.65 turkeys 29-27 6.08 50.30 33.44 1.65 29-29 6.62 54.27 25.71 1.83 29-31 6.20 51.98 44.84 2.24 29-33 6.01 51.33 31.55 1.80 29-35 5.80 51.67 25.58 1.66 29-37 6.01 49.72 51.28 3.44 29-39 6.30 54.05 30.49 1.74 2902 6.26 52.83 34.84 1.05 29-04 6.05 54.35 68.49 1.06 29-06 6.17 56.44 31.85 1.50 29-08 5.71 56.25 39.22 0.99 29-10 6.25 51.14 69.44 2.41 29-12 5.85 51.77 74.00 1.76 . 29-14 6.37 52.1 1 34.97 1.40 “mm“ 29-16 6.02 52.57 50.51 1.74 May 29-18 6.34 53.74 59.17 1.61 beam 29-20 5.97 53.98 36.76 1.82 guessed 29-22 6.29 53.50 30.12 0.76 29-24 6.05 51.38 27.47 0.88 turkeys 29-26 5.87 51.09 49.02 1.77 29-28 6.20 57.69 36.76 0.94 29-30 6.11 53.43 39.53 1.60 29-32 5.96 51.19 43.86 1.56 29-34 5.86 49.08 45.05 1.94 29-36 6.05 53.81 51.02 1.68 29-38 6.18 51.38 48.78 1.48 29-40 5.86 49.72 59.52 2.20 " BM (pmol/mg SR protein) 148 Commercial 7-day heat-stressed turkeys (pmong SR protein) 149 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 Appendix H. cDNA sequence of turkey B-RyR* ATGGCTGAAG GGGGTGAAGG AGGAGAAGAT GAAATACAGT TGATGACGAA GGAAGTTTTG GAACCAACGT CTTTGTCCTT CCAACACAGG ACTTTGCTGT GTATTTAACT TTGATGTGGG ATACACCCTG TGATGATCTT CCATGTCAAA TGGAATGTAC TCTTGGTGGG CAaTTCCTTC GAAACTGGAG CCTTCGAATA GTCTTCGACA GGTCTTGTAA TTGTTTCAGA AACGTGACAT GTCTGTTTTG ACCAGATGCT TACACCAGGA CATGAAGAAT CAGCCAGTTT CTGTTGAGGA GTAGTACTTC CTTGGCAGCT CAgAAGCTAA GAACAGTCTC GGATAATGCC ATGGCCATGC TGCTTAACAT ACTACGAGAA CTTCTAAACA ATCCTGGTTA TGGAAGTATT ATCCTACATG CATGTAGTAC TACAGATCAG GAGCAGGTGT AGTTGGAGCG TATAACAACA TGTTGGACAG GCATCAAAGG TGATGGAATG TTCAACATGT AAATCAGCAC AGGTCATATG CACAGGCTGC ATAAGTGGGA GATGGCTCAg AGTGTGTTTC GAGGGACTTG ATATGTTCCT TGTCTGTAAG AGTGAAGGGG TATACTACTT CATCAAGATC AATGCAGCAG AAGATCAgAA GTGTGTCCTC CAGGTGGACG CTCAGGAAGC GTTTTTTCCA AATGATTCAC TCGAGCGAGA GAAGTAACAT GGAATGTACT AGAAAAGTCA AACTAAAAGA GGAGTCCCAG AGCCAGTGCC GCTTAGGGCT GATGATGGTC TCGAATCATT ACAACAGAAC ACTCTGCAAG 150 CAGTATTCAC GAAATCGCTT CCAGACCTTT AGCCCTTCAA CAGCTCAAGG CGACATTCAT CCAGACTGAT GTGAGGCATG GGAGAAAAAG TGAAAGATAC CCTCCTTCAT AATATTACAG TGGTCATGAT AGCAGAAGAA TCTTTGTGGA CAGATGGGGA TGGCTTTGAA GACACAACAT AAAGCAGGAT AAATAAAATA TTATGGCTGA CCTGAAAAGA TAACCTTACA CGCAATACTA ACTATCACCC ACCTGATCAA TCCTACGAAC AAAGAACAAA GTGCTTTTTG GCATCTGTAA GAGATGCTGA TGGTCACAGA TCAgTGAAAT AAACTTGCAT CTGGTGGACG TTCGAATAGG CTGCATCTCT GCAGACACTT AAGGATATCT GAGTGTTTGA AGTACTTTAT GAGTAGAACC CAGCCTTTCC TGATGATGAA CTTCTGCTTT TCTACTCTTA CGGTGATTCA CTTATAAAGC AAGGTTATAT AAGATGTCAA CAAGCTTATT ATTGCCCTGC GTaCTTCCAg 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 CCTCCTGGAG CCTCAAAAAC TTTTGAATTG GCAGAAATTG TCTCCTTTAT GTACTCAGTT CTGGAATCTT AAGTCCAGAA TTTCACTGTT TGCTCTCTTT gATCTGtGAC TgATTAATGA GAGGGCTCTG GGTTGATCCA CTTCTACTTC GGCAATGGCG TCTGTGGTCT TGCTGTCATC AGCATTTCAT CTTCTGTACT TAAAAGCTCG CCTCCTGCTG GATGAAATTG GAGATTTGCT TGtCCTATAg CAgGGATAaa TAGAgCTGGG CATCCTTGTC AAGACCTGGA AGACAAAACC CATCGATCGT CAAGAGAAGA GAACTGCTTG CTCCAGTAAC CCTCAGGTAT GCTTTAAATG GGACAAGCAT GTGTGTGTAA AATCTACTGC TGTGACAAGC CACAATACAA TTCTTGACGG AGGTTATGCC TTGGAGATGA GGTCGTGTAC TGATGATGTA TCCGCATTAA GAAGGGTTTT TTTCTTACTG GCTATGCTCC GAACCAGTGA GGGTACgACA ACACCAGTCA cTAGCAGaAA CTGGACAtAT TTGTGGAAtT ACATGAGGAT TATTCAAAGA TTAAATGACT AAATAGTACA CTGCTCTCAT CTTGATTGGC TTTGGAAGTG TGATAGCTGA GGGCGCAATT TGGAGTTGCA CAAGAAGAGA aTAAGGCCAA GAAGTGGTAC CAGAACCCAC CCTTATCCTG CCTATATTCC CCAGAGCTGT GTTAGCTGCT TGGTCAGCCT TCTTCCCTGT GGTGGACGTC CTGTTATGAA AAGAATATAA CAATTCCTCT gAtTGCTCTT AtATCCATGA GGCaAGAtac cTCAAaGTta 151 AAGCAAAACA TGAGGGAATG ACAACAGTGC GCATGGAAAG TCGTGGCAAC TAATAAGCAA TTGCACTGTA GGAGCATATA ACAAGGTTCT GTTCGTGCCA CTTACTTTtG ACATATTTTT TTTGAATTAA CCATTTACGG GAGGTGGAGA TTTGGTTTTG GGCATCTGTC GCTTGGATTT GTACAAGGAA TGTAAGCTTA ATGGAGAGTT gCCTTGCTTC GAgAGATTCT CCCAAGCTTC CCTTTTCATC ACTGTGGGgA ggGATgATAA CCTgAGacAg AGCTTCGGTC CTGGCACTAG AGCTCATTTT AAATTTTAAA AGAAACAATT ATTGGACAGA TCTTGATTGA AAATCTATTA TGATGTGCTT ATCAAAACTT CAAACACGTT GGGTGTTGCT TAATTGATCA GTTGGGTGGG AGGATGGGGT ATGGCCTTCA AATCAACATT AGGTGTGCCC TGTTTGAGAA TCAGCAGGTG TAAATTTCTG CAAAAGAAAA GATGGAgTGA ATTTATACCT TTGAAAAgAT ATgAATAAaA TAAAAGGCAT AgaAGAATtA 2751 2801 2851 2901 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 3751 3801 3851 3901 3951 4001 4051 4101 4151 TaATCtaCAA GTCACAtTGT AAGCTTCCTA TGATCTTTCT AACTAGCAGA GGATGGACCT GCTAGTGCCA ATAGCCTCCG GAGCCACCTG TGACAAGATA GAAAGTGGTA GGCTGGGCCA CCAAGCATTT GTGGGTTTTT ATAAACTTGG TATAACCAGT GTGGTTTTGT AACCTTGGAA TCAAGAAGGT TGTGGTTTAG CCTCACATAG GTTAAAAGTT TGATATATTG TTTGGCGTGG ACACAGCCAA GGATATTTGC CCAGACTATC AGTGACAGTT AGCGCAGTAA ATGTCAACAg TCATGCTAAT AAAATTATAT GAAGTGAAAT AAATGCACAT ATGGCATTCA TATGCATTAC TGAAGCTGTT ACCAAGAAAT CGTTTTTTCA TTTTGAATTT GGCCAGGCTG GTTTTTGAAG TGGACGAAGT ATGACAAATC AAAGGTTCAG TCCAATTTGC TGGATGCCAG TTTGAACCTT TAAACGCTTA AGATATGGAG ACTCACAAAA TCGTTTGAGT GTGTGGAAAA GAATTTCCTG TGGCCAAGAC ATTTTTACAG ACTTTAGGAG TTGCTATATG AAACCCTcaa CCAGCAGCTG CATGTCAAAT TGTTACCTTC AATGTCTGGG GCAGGATCTT TAGATGAACG AGGACATTTG AGCTGACCAA GAGTAGAACA GAAGCTGTAA TCGACCTGAC GAAGCAAAGG TGGCAACCGG AATTATCTTT AACTTGCATT TCACTGGGTC TACATTCAAG TTGCAGTGAA CCAACATTTG AATTGATGGA CACTTGGTAC ATGCCCATTG TGCCTCATCT CTTCTTCTAC CCATCTTCTG TGAGAATTTT ATGAGAGAGG GTTTGGGGAG 152 AACGCtTTTG aGGAAGATCT GGTTATAAAC TCAAGAATTT CAAAAGACAG AAGAACAAAC TACTAAAAAA CAGGCTATGG ACATTGGAAA GTCTTATGCA CAGGTGGAGA ATTGAACTGG CCAGCGTTGG GAGATGTGGT ACTCTGAATG TGCTGACTTT TAGCTCAGAT TATTATACCA CATGAACCGA TTAATGTGCC ACCATTGAGA ACAAAACAGC AGTTCCGTTC GATGCTCTTC CACATATTTT TCtGGGTTGG GACATAAATA CAGGGTTCAT GAGATATAAC GCCCtTGgAT TAAAAaAGTC CTGCCCCTCT CTAGTTGACA AATAAAGCAA GTAATCCTCG TCAAACAGAG TTATAATATT AAGTCAGCAT GTGAAGTCTG TATGCGTGTT GAGCTGATGA CATCAAGGCA TGGATGCATG GAGAGTTGCT GGAATAGAAA TGGACGTATG TGTGTGGTCT GATGTCGCTA AAAAAATCAT GTCCACCTCG AATTCTGATA ATCATTCAAC AAAAACGAAA TATTCCTTAC TTGGGTAACA AAAACTGTAC GAAAGTGTGA TGCTAATTCT 4201 4251 4301 4351 4401 4451 4501 4551 4601 4651 4701 4751 4801 4851 4901 4951 5001 5051 5101 5151 5201 5251 5301 5351 5401 5451 5501 5551 CAGAGaTCAG CCTGGCTACT CTTGTTATCA CAGCCTACAA TACCATGCCG TTCCTCAGTG TGGAGTAGGA TGAACGTCAT CACTTCATAT GAACAAGAAG CTCAGTTTGT ATGTAGATAT GGACTCCTAC CCATGCCAAG TTACAGAGGA CATGGTTTAC TTTTTCTACT GCCCAGAAAT ACAGAGGCGG ACATATTGCA TTATAATGGG ATTGATCCCA AGAGAAGGAA AAAAGGCAGT AGATTACCAG CTGTGACTGC aCCACTATGT CTCATGCAAG GTCGCAGTAA GGAATGTTGT GGTTGAACCA GCACTAACTT TTATCAGCAG TCCCCCTCGC TGCCTAACAG GGCTGGGTTG CCCAGAGGAG ATTTGATGAA GCTCTAGGTA ATCTCAGTTA GTTCTGGATT CAAGCCAAGC AACTCGAACT CCGGAGTAGG CCTTGCTTTA TCCCCTTGaC TaCAGTGCAG TTCCAGTTTG AGTTTTTGAT ATGTCTTTGG GAAGTTACAC AAAAGAAACA AATCTGGTAA GAGCTGCAAC ATCAAAACTG CCTTGGACAT TGTTGATTTA CATTCACTGC AACACAAAAC AATTCAGTTT CAATTTTTAA CTTGATGTGC CTTTCTAAAA TGCAGTGTTT AACAGATGTG GTTTCACTAT ACACTAGAGT TTTTACACAA CTATGACTTG TCATGATGAA ATTAAATTAT GCTTAGCACT TTGTGACCTC ACACTTAAAT TGGTTCTCAT TTCCTGTTCT GATGATGATG AGATAACAAG AAGTTGAAGA AAAACACCTA GCTTCAGATG ATAGAGTGGA CAATATAATC GTCTGCTGCT 153 GAAATTGGAT CAATGGAAAA TTCTTCCTGC GAACTTGGTA AAGTGAGGAA AAACAATTAC GTAGAAACAG GGAACCACTG TGGATATTTT CACACTTTAA TGCTTATGCA TAGATAATCA CTCATTAGTA CAATGAATTT ATCCTGATGA TGTCTCAAAC GGaaGAACAT CCAAAGCGAT ATACGAGATC TAAACTCATA TGAAGCAGGT GAAGAAACAG AAAAGCTGTA CAAAGGGCTT TGCCACTTAC AGCGATTGtA AGAAGTACAG TTGaCTGCCA GCTTTGTTGA GAGCTTGGCA AGCTTTTGTA AATTAAAGAA AGAAATCCTG ACCTGTTTTA AGCGCGTGAG CAGATGATGG GGAACTATGT AACTTTATAG CTTTGTAGCC GTATTTACCT TTCATTTGGA ATTATTCCAG AACAAAGAAG CAAGCTTTAA CAGACATCCA AAGTATGCTG CTGTTGGAGG GCAACATTGC GTTAATCCTC AAGAGAGGAC GAAGCTGGAG ATTGCAGACA TCAATTATTT TCATTTGCAG GTATAATGAA AGAAGACTAA 5601 5651 5701 5751 5801 5851 5901 5951 6001 6051 6101 6151 6201 6251 6301 6351 6401 6451 6501 6551 6601 6651 6701 6751 6801 6851 6901 6951 7001 GGaATTTCGA AACTGGGAGA GACTTCCATG GGAAGAGGAA TATACAAAAT GAAGAAGATA GGTGCGCTGG TTATGTACAC CAAGCTCTGA CATTAATCTG GAATGGGAAA ATGAACAACA CATGCATGAG AATCTCAGAT TGCTACTTCT TCTCAGTTAC TGAGAGGTTC AATGAGCTTG TTACCTGGCA GATATCCAGA TTCCTAAGAT AAGTGTTGTT AGCTTAGAGG ATACGGATCT GAGAGAAGGT ATGCAATCAT GCACCAGAAA CAGATCAATC TAAGTATACC TCTCCTCCAC GGATTGTCCC ATGATCTTCT GAGGAAGATT CAAAGGTCCA AATCGCCTAC TCCCAGGAAG TCTGCTGCGT GGAAAGCATA CTTGCTGCAC AGAAGAGGAA AGGtTTTTTA ACAGTTATGG CGTTTTTCCT GTCGAATTAG TTACTGGAAA GACACCACTT CACTAGCACT GGTTGGGGCC CATTGGGTGG TTGCAGTTTT GTAAAGCTTC AGAAGGAGGA CAGAGAATCC GATGAAGAGG GTCATTTTAC TGCACCTTAT CTTCGATCTC ACTAAAGCTG AAGAACAGAT TGTCCAGAAG AATTCACTGT CCTCCTTGAC CCAAAACCAG TACACTGAAG ATCAGATTCA AGGCAATATG CACTATTAGT TGGGCCAGAT CTGCTAATGA TCAGCATCCT ATGTGATGGT AAGATGGTGG TCGTCAAAAC ACAGCAGCGT GATGTTGCAG GGAAGAACCA TGCAGAGATG AATCCAATAG TGTTAACAGT TTATTCGACG AATGGATTGC TTCTCGTGAC AGGAGGAGGA TCTGCTCTCA TCAAAGTGGA TAGTGCCAAC TCAACAGTTA 154 TAATATGCTG AGATTCGGGA GGTATTCCAC TGGCAAGCTT AAAAAATAGA GAACTCATAT AGATCCAGAA ATAGCATTGG GCTGGCTCTG TCGCTCACTT TTAATGGATT AACTTAATGA GAATGTGCTT CAAGCTGTTG CAAAAAGCCA TGGGTTGGCA CAGCCTCTGT GATCTTGATA TCCAGTGTTG AGGGTGAACG GAAAGTGTTG ACCAGAGTGC TGGCAGCTAT CTTCCCTCAC GGAGATCGTA TAGATTTACT AAAGGTGAAG TGAAGATTTG ACAAAGATGG CTGAATTTTC TGAACTATAT TAGAAGAGGA CGTTCATTAA GCCCAGAGAA CCCAAACTAT TTGGTTCGGA TGAGCTACTT TGAAGGATAC CTCAGTGTAA AGGAGATATC GAGTCTTGGG GGAGGAGATA TCGATTTCTG TGTTTGAGCA TTTCCTTCAA AATGGACAAC AAGTTGTTAC CTAGCTAAAG TTACCTGTCA AAGAAAATGC TTTGGACCAG GCAAGAAGCT AAGGATATAA CACATGGGCA TGGACGCTGT CTATTCGAAT GTTGGGATTA CACTGTAAAT 7051 7101 7151 7201 7251 7301 7351 7401 7451 7501 7551 7601 7651 7701 7751 7801 7851 7901 7951 8001 8051 8101 8151 8201 8251 8301 8351 8401 GAGCCAGACA ACTCTTTTTG ACCTACTTGA GATACAGTTT TATTTGCTCA CTGGAACAGA TATAGGTTAT TGAAGAATGC AACAGCTATT TGTAAAATGC ATATTATTGT ATGAATTACA TCTCATAAGA AAGTGCTATA GATCAACATT CCCAAACCCA TATTGTCAGC AGACTAATAA ACTCATCCAT AATTTATCGT GGTGGAGCCT CGTGAAAATG ATATAGCCCA TTCAGGGAAT AAAAAGAAGA ATTGGTACCT AAAAGGCACA TCTCGGGGTC TGTCTGCAAA GACCGTGTGT AGTTGGATTT CTCTAAGTAC GCTGtGTTTC ACATCATGCC CCAAAGGACG CTGCTTGCTA GAGAAGGCTA CACTCAAGCT CTACCTTCAG TTTAACTGAA AGTATGATCC GCTGGGGCTT GGAAAAGCAG TCAACACAGC AAATATGCTG TGGGTGGAAA TAATAAGACC TGGCCTGTGA CGAAAGGACC AAAAGCTCCG GCACCACTTG GGTTGAGGTA AAATGGAGTT TACGACACAT AGAGCTGTTT TGAATGACAT TTTCTGTCCT ATGGCATTAA TTACCAGATT CACAGAAGCA CGTTACTCAA TCTCTTGTTG TTCCCTTACA TCTGCCACCA GTTTTTGATG TCTGACAAAT GAATGGGAAG AAACTTTTTT AGAGCTCTTT TGCCTCCTGA ACCTCAGTGG AAACCTTGTA AGCATTCTCA TACGGTGTTT TTTCAAAACT GAGAATCCCT AAGGAAGGGG CAGCATATCT ATCTCACTAA ATGGCAGAAA GGAAAGCAAA TGACAGCCAA AAATTCCTTC GGATTTGGAT 155 GATCATAAGG GGACCAAAGC tAAgAGCTTC GCTCTTGCAC AAGATGTGCT ACTCCATGCT AAAGCACAAC CTTACGTCCC TGCCCTTACT CACTATGAAC CTATGGAATT GGGGAATATT AGAATGGCCT TTATTTAGAT ATCCAGAAGG CTTCCTGAAA TGATAAATGG CACTGGATGA TTAACTGAGA GAAAACCATG GCGAAGGAAT CAGTCTAGCC CGTGGTACTT ACTACCATAA GGTGGAGGCA GGAGAAATCA AAGTGAATGG GCTTCATCCA CACCAATGGT TTCCTCCTTC TGCCTCTCTG TAAaCaGATA CCCCTCTTTT TCACACAATA GAGACACTAT TCTATGCTCC CAATGAATAC AGTGTTGGAA GCAGCAGAAG TGATTCCTTG TGCCCTGCCT ACACGAATTA AAATTTTGAT AGCTGGAGTA GCTTTTGATA AAATACAAAG AGGAAAAAGA TTGGCCATGG GTTACATCAG AGGGAAATGG TCTAGGGAAC TATATGGGCC GTCATCCTTT CGGGACCGTG CATAATCATA TGGAAAAGAG 8451 8501 8551 8601 8651 8701 8751 8801 8851 8901 8951 9001 9051 9101 9151 9201 9251 9301 9351 9401 9451 9501 9551 9601 9651 9701 9751 9801 9851 GTTTGCCTTT AAGAGTTCAT AAATCCCCGC ATTAGTTGAT CAACAAAAAC ATGGTAGCAA CTCAATTTTT TAGCTCAGTC GTTAAAGCTG AAAAACTTCA AAATCAAAGG CCTGTCTTGA TGATTTACTT GCCTCTATTC CCTGCACTTG ATTCCTTGAA CAAAAAGTGC GAAATGTGTC TAATTTAGCA AGGTTATCTT GGGTCTGAGA ATCTGAGCAT CCAATCTGGG GCTCAACCTA TATTCCAACA AAGAGGAGCA CTACTTATTC CTATCCAATG AGAAACCAGA AAGTTTCTGA TGCACATTTG ATGACCAAGA CAATACTTTA ACTCAGTAGC GCCTGTTCTG GGTAGTGATT TCTGGAtACT GACTACGTGC GAAAATCTTA TGTTTCACAG CGTCAATTTT CTGGGTGATG TCTTGGGACT GTGAATGCTT CCTTCTCTCA AAGAGAAAGA CAGAAATCCC GAGTCTGGAG ACCCATGCTG GTGTTCCTGA CTGAGCATCA AATAGATGAA TCATCAGCAA CTGGAGAAAT ACTGAGAGCA TCGATGAGTT CTGATACGTT TGCAGATTCT AGAAAATTTT GAAGCCATTG AATAAAATTC CAAATCACTG AGTGGATATG CAAACTAGCT CTACAACAAT CGAACTGTTA TTTTTTTGAA AACTTGGAAA AACATCAATT TGAGCATATT TACAAGTTTC GGGAAGAACA AGCATCTTTT ACCACTACAA GCAATTTTAG TCAGCTGGAT CAAGGTATAC TGCAATTATT AAGTGCTGGC TTCTGGGAAA GCATCTTGGA AGCCAGACCT TGAAGAAGAA GACAGTAAAA TGCTGTTCTT ACGTAGACAA GATGAACTGT 156 AAAATATGTT TTACTAGTGG TTTGCCAAAG CTTGTACTTC CATCAAATAA GCTCTTGTTA GGTGAGCTGC TGAAATCAGG AATGCAGCTG ATTTACACAT ATACTACAGT TCACAGTATC ATGTTACAGA TCTACGTTGA GCAGCAGCCA CCCATTGTCT GTATGCCTGA GGACTAATAA TGAAATGCCT TGTCCTACTG CCTTGCTGTA TATTCTGAAA TGAAAAGAAT GATCTGCTGA AGCTATAAAG GTGACACTCA TGTAGAGACC CAACAGAGCC TTCGAATGGT GACTCTGCTC AAAAACTGAA TTCTTTTACC CTGTCTTCTC GGAAAAGGAA GACATAGGAT CTGCACATCT ATCTGAGCTA AAGATCTGGA TCACGAACAC AGCATTGCTA ATTTTGGAGT ATTCTTTGTA AAGGCAACGT TTCCAGTAGC GTCTTCAACA TACAGTAGAG AGGAAATTAA CACGTAATTG GTGGGAACGA CGATGATAAC ATCATTAACA TGCAGTTTAT AAACTCACTT ATTGTGATGG AGAAGCTGAG TCTATGCCTT AACTGGCTAA AGCTGAAGTT 9901 9951 10001 10051 10101 10151 10201 10251 10301 10351 10401 10451 10501 10551 10601 10651 10701 10751 10801 10851 10901 10951 11001 11051 11101 11151 11201 11251 TTTATCCTGT TGTCATACAG AAAGCAAGAT GAGAGAAAGA CTTGATTGTA GTACTCCAGG CATAAGGACA TCTGCAGGAA ATAAAGACAT GAACGTGTGC ACAACCACTG AACGCAAAAG TTACCCAGGC TTGGATAGAA TGGCTACGTC GAACAACCTG TCTCACAGAG CTGCCATGAT GACAAAGAGA TTATCAGCAA AGATGATCAG CTAAAACTTG GAAAATGCTA GTCTTTCTGG GAGAGACAAA TCTCATCGTA CTCGAGATCT GATTTTCAAA GGTGCAAGTC AATGAAATCA GTCTAAAGCC AATCAAAACG GCTGCACTTA AGATCAAGAG CTGATGAAGA AAGTCTGATG TCTGAAGAGT AGAGGATATC AGATCAAAGA AGCTGTTGTT ACCGTTCTAT ACAGAGGAAT TCCAAAAAAA aCCCaCTTCA AGGAGCAAAC GGCAAAGAGC AGACATTTGA GCTCGCTTAC TGCAAGCAAA GTATTGCTAT GACTACTTGA TTTGATGCAG ATAAAGCAGA CGTGAGCGTG ATTTAGATTT ATTATCTACG ACATAATTTC ACAATTTGGC ATGCAAGTAA CAGGGGGGAT AAAAGATGCT CTCATCTCAT AGTCAAAGAG ATCCAGCTGT GATGAGCCTC AGCTGCTCTG AAGCTGTTTG GCTTGTTTTA TAACCTCTTC ATTCATTTGA GAGGAAGAAG TCAGATTATC TAGAAGATGA TGTCAGGAAG GGAGAAAGAA ATGATCGTGG GGTCATACAG TCTAAATGGT AGGAGAAAAA TCCTGCAGTG AGGCCTGGGA GTGAAAAAGT TTACAACTGC CACTCAGATG 157 AAAAGAGAAG ATTTTTAACA AGTCTGGAGG TTGTACTCCA TCCTATTGGC TGGCTAAGAC CATATaCGTA GAAATGGCAG CTGACCCTGA TATCATCTGG GCACAAACTT GAATGGCGCC CTCCATGGCT GGAGAAACTA AAGAGGAAGA CTCTATTTTA TCCCTTATAT AAGAAGAAGA ATGGAAAAGC AGCTGCTGAG GACCCATGGT GGGAATACAA GGATGCTGGA TTCTTGACTT ATGGTTACTG GTTGCAGCAT TCTGTGAAGG GGCAACACCA AACAAAATTT GGAGATACCA TCAAGATCAA TACAAACATC TTGAATATGT TAGATATAGC ACAAtTTACA TTAAATCTAT GAAAAATGTG ACCAGGTTGA CTGTCAAAAC GTTATACAAC ATCAGAACTA GTTCAGGATT TACAGAGAAA GTCGCAATGC ATTGCCTATG GGAGGAGGAA AGAGAACTCT ATGGTCCTTC TGTTGAAACA TAGTTCAACA TTCTTTCAAA GAATGCGTTT AAGAAGGAAC GATGAATTTA GCATAACAAT CAACAGTGAA 11301 11351 11401 11451 11501 11551 11601 11651 11701 11751 11801 11851 11901 11951 12001 12051 12101 12151 12201 12251 12301 12351 12401 12451 12501 12551 12601 12651 12701 TATTATCATT GTGACTTCTA CAACGTAACT CCTTACCGAA CTCATAGTAG AATATGCAGA GGAACTGCTA TTGAAGGTAA CTTGTAGAAT GTTTCTCAAA ACCCAGATGG GAGGCTCAAA TACGGAAGCT GATTCCATGA ACAAACCTTT TGAACCTGCA TTGAAATAAT AGTGAATCCA AAGGCAATTC TGGAGCTCTT GCATCCCAAA AGAAGAGCCT AGTCCCTGGA AAGAACGTCG ACAGTACAGG TTTTCTCTTT ACTATAGTAT TGGACTGGTT ACATGCCTGA AGTACAGTTG TTGGTACTAT TCTCTAAAGC TATATACAGG GCTGTGGGAT TGAAACTTTC GaTCTGCTAA CGTTGTAAAT CATCTAGCAA TTAAAAGATT TAAGGGCATC AACAATATAT GATGAAAATG ACCAGCCAAA CAGAGCATAT GAAAGTGTTC GGGTGGAGCC GTAGAATGCA ATATTTGATG CGTGAACTTC TCTCTGAGAC TGCTACATTG ATCTCCTTCA CCAACTTTTT AAAGTCAGAA TTTCTGGATT GGGGAATTTT GAAGGAGCCA CCCAACGCAG ACTACCTCTT TCAGGAAAAG TCTGGCTGTC GACCTTGCAT GCAGTTGTTG ACAGGACTCT AGGATATGGT GGAACAATTG CGTAGAATTA TAACTAACTC ATTTCAAAGA ACAATCAGAG ATATGTTCAA GATATTGGCT GCCTAATGAC TTAATTACTT AAGAAAATAG GTGGGAAAAG TTGTAAACGA TGTGAAGACA TGATTCAGCT TGGATATCGG GCTTTTGCAA TAAAATGGTT AGATGACAGT CTATTTGTAG CCAGATTCTT AAAATATTAA TTTGGAATCC 158 GCGTCTTCAG AGTTTATTGA ACCAAACAAA CGGTAACCAA GATTTCTTCA GCTCAGATTG TGTGATGTTG GAAAACAAAT ATCTTGAAGT GGATGCTTTC AGGATTTCCA ATTGAATTCC CTACATTGAT TTAATGTAGC TCACGCCTTC TGAACCATAC AAAGAGTTTA CCACAGGTGA AGGTGGAGAG CGATCTTTGA GAGAGACCTG AGATGATGAG TGGCCTGTGC ACTGTGAAGA AAAAGAGATG GGGTGTTCCA TGGAGCACCG AGTTACTAAA ATGATGATGT GAATCAATCA TGAATCAGGA TATTCAATTC CAGAGTCTGG TGTATTTGCC AACTGCTTAA TTGTCATTAC GGTCGATACA TTTTTGACAT AAGGAGCATG GAAGTCAATG TGTTGTCATG TTTGTAGAAA AGTTTTGCTA AGAGCTTACT CTTGGCCGTA CTTTGAAATC AGGAATCCAA CAAGAAAAAA AATGCAGTTA AGGAAGAGGA GAAGAAGAAA TGCAGTCAAG ACCTAAGGAA GTGAAAGTGT ACTGTTTTTT TATTTGGGGG ATACTAGGGG CACAGAAGCA 12751 12801 12851 12901 12951 13001 13051 13101 13151 13201 13251 13301 13351 13401 13451 13501 13551 13601 13651 13701 13751 13801 13851 13901 13951 14001 14051 14101 GAAAAAACTG TGTAAAGGGT TTCCTACTAA GGAGATATTG CAATGTTCGT CTGAAGCAGA GAGAAACAGG AAAAACAAAG TGGCTGTTAT ATGCTTCACT ATTTGTTGCA AAGAGCCACT GAAGAAGAGG TACAGGTTAC TCATCTCCTT GTTGTATTCA ATTATACaTA ATCGCCTGGT TTTGTAAAAC GCGTATAGCA CAGTGGAAGA TCCATTGATA TAATTCATTT ACTACAACAA TTCAAGACAT GCTTGTGCTT tTGtGGCATT GATGAACCAG AAGGTGCTGA GAAAAGGGAG GAAAGAAGGT CAGAAATACT AAGAAAAAAG AAGAAAAGTA ATAAAGCAAA AAAAAGAAGC GGCTAATTTC ACCTGGCAAG TTTGCCATCA TGATGAAGTG AAGAGGAGGA ATGGCACCTA TGtCTGTGTG AGAgAGAAAA ACTGAACAAC TATAAACACA GAAAGGTTAT GAACTTTTGG GAGCGAACCA CAAAGTACCA TTGTATTTGG CTTCTTTTTT TGCGAACCAT ACTGTAGGAC CAaCTTCtTc ATATGAAATG GCATGGCATT AAGCTGACAT GGCTCAAAAC TGGCTCTGAT GATTACAGAC GAAGCTGAGA GGAAGAACAC GAAGGCATGG TTCAAAGCTT GAACTTTTAT ACTTTATTCT GAGGAAGACT AGGAATGGTA CTCTCAGAGC ATTGGATACT GGAGGTAGCT CATCTGAAGA CCGTCCTTTC TAACAAGTAT GTTTGGACAA GAAGCGGCAT CATTTGGAAG CTTGGTACAC GCTGCTCATC TTTGTCCTCT TCCTGGCTGT CGCaAATTCT TGATGACATG 159 AGAGATGAAC AATTTCAGAT ATGGTCATGA ATCCAGTCTT ACCTGAAACT AGGCTGACAT TCTGAGCAGC ACAAAAAATT TGGAAATATA AACTTACGGT GCTATTTTAC CTAATCTCTG TTTTTTGTTT ACTGGCAGTT ATTGCTTAAA AGGAAgCTGG TGATATTAAA CTAACAATTA GGAGACTTGT AAATGCCCTT CTCTTGTATC CTTGGTGTTG AACTATGTCT TGCTGGACAT GTAACTCACA AGTAGTGTAT ACAaTaAAAG ATGACGTGTT TTGTGCAGTT ATTTTTGGCA CGCTGGACTT CTCTGGAAAA GCAAAAGACG GGAAGATGGT AGGAAGAGGG GAGAAACCTG TCAAACAAAA TTCTTGCTCT AAGGTGACAG GAACTCTTTT TGGAAGAAAG ATTCATACTA GGtCCCTTTG AAtTTGATGG GGACAGTGGG CTGGGATAAA ACGGAGCAGA GATTTTAGTC ATGGTTAAGT TTTTCACTGA ATCCTTGGAC CGCTATGGGT ATGGCAAACA CtTTACACTG tgaagaTGAA ACCTATTCCA 14151 14201 14251 14301 14351 14401 14451 14501 14551 14601 *cDNA sequence of turkey B-RyR was completed by Dr. Wen Chiang (Michigan CATGTATGTG ATCCTGCTGG TTTTTCTTCT TATTGACGCT ATATGGAGAC ACAACTCCAC AAACTACCTG ATACTGGCCA GATTTCTTCC CGGCAATCGA State University) GGTGTAAGAG AGACCCTTAT TTGTCATTGT TTTGGTGAAT CAAATGCTTT ATGGCTTTGA TTCTTCTTAA GGAGTCATTT CAGCAGGAGA ATTCCCGCGG CTGGTGGTGG GAAATTTATC CATCCTACTG TAAGAGACCA ATTTGTGGAA AACACATACT TGTACCTCAT GTGTGGAAGA CTGCTTCCGG 160 CATTGGGGAT GAATTGTCTT GCCATTATTC GCAAGAACAA TTGGCAACGA CTGCAGGAAC AAACAAGGAT TGTACCAAGA AAACAGTATG GAAATTGAGG CGACATCACA AAGGTCTGAT GTGAGAGAAG CTATTTTGAC ACAACTTGGC GAGACTGAGC AAGATGCTGG AGGATCAACT 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 Appendix HI. ASR 1 region in turkey or-RyR“ GACCCCCGGA GCCTCGGGGC CCTGGGGCTG ACGACGCGCT CGgATGATTT GGACGCGCTG TGCCCATCGC CGCGCCCCGC CTCCCTGCGG TGGTGCTGAA TTCGCCGAGT CAACCTCCTC ACTGCGCCCT CGGCTGGAGG CGAGAGCCCC tcatctccct ctctgctctc tctcatcacc GATCAAATAT TGTGGCTCAC ATGAAACGGA GTCGCTCAGC ACAGCACGGC AGCTCTCGTG CGCCGTCATC ACACCGAGCT CgcCGCCAGG CTGCATCGAC TCGCCGGGGA TATGAGCTGC GTTCTCCACC CGTCGTCAGG GAGGtTCTGA gctggacaaa tgtgtgtctg gaaaatctgc GGGGAGTCGC CTACGCTGCT GGCCCAtCCT CGCTCGCAGG GGGGCTCTAC GCCGTGGCGG CTCAGCCTGC GCAGCACGAG ACCTCTTCCA CGGCTGAACG GGAGGCGGCG TGGCGTCGCT AACCTGGACT GATCCTGGAG ACATCatcca cacggccgca caatgctgtg tcccgcgacg TGTGCTTCGT GCTGACACCA GCACCAGGAg GCGAGgAGTC GGGAGCTTCA CGGTGCGGGG GGGATCTGAT CAGCGCCAGA GCAGGAGGGG TGTACAGCAC GCCGCCTGGA GATCCGGGGG GGCTGGTCAG GTGCTTTACT ggagaaccac accataaggt gccgttcgtt cgacctcctg GCAGCACGCG AAGCGCTGCG GGCCACATGG GCAGgCGGCG TCCgGAGCCT AACGCGGCTC CGCTTATTTC ACCGCCTGCG ATGATCTCCC GGCCGCGCAC AGGAGATCGT AACCGAACCA CAAACTGGAC GCGTCCTGAT atcaagtcca cctgaacgtg ccaaccaaaa ctgcagaccg *cDNA sequence of turkey B-RyR was completed by Dr. Wen Chiang (Michigan State University) 161 Appendix IV This study with was originally designed to identify the differences in meat quality between random-bred and commercial turkeys under different heat stress levels. To create stressful environment possibly inducing PSE meat in turkeys, turkeys were exposed to five days or seven days of heat stress (12 hours of 95 °F, 12 hours of 80 °F) based on the personal communication with Dr. A. R. Sams at Texas A&M University. 162