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thesis entitled

IDENTIFICATION OF GENETIC COMPONENTS IN TURKEY
RYRS ASSOCIATED WITH PSE MEAT CHARACTERISTICS

presented by

HYO-JUNG YOON

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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*<SO and lesmin>5.8) and ten PSE meat turkeys (L*>52 and lesmm<S.8) were
selected based on the L* and lesm. Although the SNPs did not change the primary
structure of the B-RyR, four SNPs were associated with color (L‘). For those SNPs,
heterozygous birds have darker color (L*=49.30) than one type of homozygous birds
(L*=53.56) (p<0.0l). The lesmin between heterozygous and homozygous turkeys was
marginally different (lesmin=6.02, 5.78, respectively; p=0.06). Therefore, there
appeared to be an association between the four SNPs and meat quality traits, i.e., the
heterozygous turkeys for the mutations show better meat quality than one type of
homozygous turkeys.

There was no alternative splicing variant detected in cDNA regions of turkey [3-
RyR. Ribonuclease protection assay (RPA) was performed for an alternative splicing
region in turkey a-RyR to study quantitatively the effect of heat stress level on the pattern
and abundance of transcript variants. The assay showed that there are differences in
expression pattern of two alternative splicing transcript variants, full-length and AS-81,

between random-bred and commercial turkeys exposed to prolonged heat-stress.

Cepyright by
HYO-JUNG YOON
2006

ACKNOWLEDGMENTS

I would like to greatly thank my major advisor, Dr. Gale M. Strasburg for his
guidance, advices and patience. He has been always there for me and encouraged me
throughout this course of study. Without his support, I would not have come this far.

I gratefully acknowledge the members of my guidance committee: Drs. Alden M.
Booren, Matthew E. Doumit, Catherine W. Ernst and John E. Linz, who have shared their
knowledge to make this work possible. I deeply appreciate their kind advices and
valuable suggestions for my work.

I am grateful to my friends and graduate students in Michigan State University for
their support and friendship.

Finally, I would like to thank my parents for their love, support and

encouragement.

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................ ix

LIST OF FIGURES ........................................................................................................ xi

LIST OF APPENDICES ................................................................................................ xii

INTRODUCTION ........................................................................................................... 1
CHAPTER 1

Literature Review ........................................................................................................... 5

1. OVCI'VICW Of skeletal muscle structure and filIICtIOII .................................................... 5

1.1. Structure Of skeletal muscle .......................................................................... 5

1.2. Excitation-contraction coupling .................................................................... 8

2. Ryanodine receptors ................................................................................................... 10

2.1. Structure and functions of ryanodine receptors ........................................... 10

2.2. Factors affecting RyR activity ..................................................................... 17

2..21. Regulation by physiological agents .............................................. 17

2.2.2. Regulation by oxidation/phosphorylation ..................................... 21

2.2.3. Regulation Of RyR by associated proteins .................................... 24

2.3. RyR-related muscle diseases ........................................................................ 25

2H3 1 . RyR] -related mutations ................................................................. 26

2.3.1.1. Malignant hypertherrnia and central core disease ----------------------- 26

2.3.1.2. Porcine stress syndrome (p33) ................................................... 29

2.3.2. RyRZ-related mutations ............................................................................. 31

2.3.3. RyR3_related mutations ............................................................................. 32

3. Pale, SOfi and exudative (PSE) meat ............................................................................ 34

4. Single nuclwtide polymorphisms .............................................................................. 38

4.1. SNpS related with diseases .......................................................................... 41
4.2. SNPS in RYRS .............................................................................................. 45
5. Alternative SpIICIIIg .................................................................................................... 51
51. Heat stress and alternative splicing ............................................................. 51
5.2. Alternative Splicing in RyRs ....................................................................... 53
5.3. Alternative SpIICIl'Ig in turkey a-RyR .......................................................... 5 5
CHAPTER 2
Measurement of turkey meat quality indices and channel activity by [3H]-ryanodine
binding assay of turkey SR ........................................................................................... 57
1. Introduction ............................................................................................................... 57
2. Materials and methods .............................................................................................. 62
3. Results and discussion .............................................................................................. 66
4. Conclusion ............................................................................................................... 81
CHAPTER 3
Identification and analysis of single nucleotide polymorphisms of B-RyR associated with
meat quality trait .......................................................................................................... 32
1. IIItI’OdUCtIOII .............................................................................................................. 82
2. Materials and methods ............................................................................................ 87
3. Results and discussion ............................................................................................ 91
4. Conclusion ............................................................................................................ 109
CHAPTER 4

Ribonuclease protection assay of alternative spliced transcript variants of turkey ot-RyR
mid screening of turkey B_RyR for Splice variants .................................................. l 1 1

vii

1. Inn-oduction ........................................................................................................... 1 l 1

2. Materials and methods .......................................................................................... 1 18
3. Results and discussion .......................................................................................... 124
4. COIICIUSIOD ............................................................................................................ 1 3 1
REFERENCES ........................................................................................................... 1 3 3
APPENDICES ........................................................................................................... 148

viii

LIST OF TABLES

Table 1-1. Synonymous SNPs in the coding region of human Rle ------------------------- 46
Table 1-2. Nonsynonymous SNPs in the coding region of human Rle ------------------- 47
Table 1.3. SNPs in the coding region of human RyR2 ............................................... 49
Table 1_4 SNPS in the coding region Of human RyR3 ............................................... 50
Table 1-5. Examples of alternative splice sites of the RyR3 gene ------------------------------ 54

Table 2-1. Analysis of color and early postmortem pH of random-bred and commercial
turkeys ......................................................................................................................... 68

Table 2-2.Analysis of ryanodine affinity of turkeys of different subgroups --------------- 73

Table 2—3. Meat quality data and ryanodine binding activity of PSE meat and normal
meat turkeys ................................................................................................................. 79

Table 2-4. Number of PSE meat and normal meat turkeys from each different subgroup
....................................................................................................................................... 80

Table 3-1 . Primers used for sequencing cDNA regions of B-RyR ------------------------------- 90

Table 3-2. Meat quality traits (L* and lesmin) and ryanodine affinity (Kd) of turkeys with
low Kd and high Kd values ............................................................................................ 93

Table 3-3. Ten synonymous single nucleotide polymorphisms identified in B-RyR 94

ix

Table 3-4. Meat quality data and ryanodine binding activity of PSE meat and normal
meat turkeys ................................................................................................................... 96

Table 3-5. Ten synonymous SNPs identified in normal meat and PSE meat turkeys 97

Table 3-6. Mean values of meat quality indices (UI and lesmin) and ryanodine affinity
(Kd) among three difi‘erent genotypes Of individual SNPS ............................................ 98

Table 3-7. Meat quality indices (L* and lesmin) and ryanodine affinity of SNPs 1,5,8
and 9 .............................................................................................................................. 99

Table 3_8. Haplotype analysis Of ten SNPS identified .................................................. 102

Table 3-9. Meat quality indices and ryanodine affinity data among B-RyR genotypes
........................................................................................................................................ 1 03

Table 4-1. The ratio between full length transcript variant and AS-8l for selected turkeys
........................................................................................................................................ 1 27

LIST OF FIGURES

Figure 1_1. Structure Of skeletal muscle ....................................................................... 7
Figure 1-2. Proteins involved in excitation-contraction coupling -------------------------------- 12
Figure 1_3. Divergent region between RyR] and RyR2 ............................................... 14
Figure 1_4. Triad mOdel for a muscle With two RyR isoforms. .................................... 16
Figure 1_5. Linear RyR2 protein sequence. .................................................................. 33
Figure 2_1. Ryanodine binding assay data .................................................................... 73

Figure 4-1. Three alternative splicing regions identified in turkey a-RyR by RT-PCR

Figure 4-2. Locations and sequences of the alternative splice junctions in turkey a-RyR

....................................................................................................................................... 115

Figure 4-3. Comparison of alternative splicing regions and divergent regions in turkey or-
RyR ............................................................................................................................... 1 16

Figure 4-4. mRNA species representing three alternatively spliced forms of ASR] region
of turkey a-RyR ............................................................................................................ 121

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 a-RyR 125

xi

LIST OF APPENDICES

Appendix 1. Meat quality data and ryanodine affinity of turkey muscle samples -------- 154
Appendix II. CDNA sequence 0f turkey B'RYR ............................................................ 156
Appendix III. ASR 1 region in turkey a-RyR .............................................................. 167
Appendix IV. ................................................................................................................. 168

xii

INTRODUCTION

Turkey consumption has more than doubled since 1975 as consumers have
recognized the health benefits of turkey as a low fat, high protein food source (National
Turkey Federation, 2002). The increasing demand, especially for processed products, by
consumers has shified turkey sales from whole birds to further processed products
(Woelfel et al., 2002). To meet consumer demand for breast meat, the turkey industry has
successfully engaged in intensive genetic selection for rapid growth and heavy breast
muscling. As a result, today's commercial turkeys grow twice as fast, and twice as large,
as their ancestors. However, these dramatically rapid strides have led to an increased
incidence of meat quality defects, especially the occurrence of pale, sofi and exudative
(PSE) meat (Wheeler et a1, 1999; Pietrzak et al., 1997). Meat products processed from
PSE turkeys have soft texture, poor cohesiveness, and poor juiciness, which results in
substantial economic losses to the turkey industry (Sosnicki, 1993). Although processors
have tried various methods along the processing continuum to reduce the PSE incidence,
success has been limited due to the lack of absolute understanding of the causes of the
turkey PSE problem.

The term PSE was first coined to describe a meat quality problem in pork and
thus, this condition has been extensively studied in swine (Msmer-Pederson, 1959;
Bendall and Wismer-Pederson, 1962). Environmental stressors are known to induce the
hypermetabolic condition associated with development of PSE meat. 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 al, 1989; Offer 1991). In swine, PSE
meat is often associated with Porcine Stress Syndrome (PSS) or malignant hyperthermia
(MH) which is characterized by excessive heat and lactic acid produced as a result of
spontaneous muscle contractions and rapid anaerobic glycolysis (Mitchell and Hef‘fron,
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 Hefi‘ron, 1982), which induces denaturation of muscle proteins. The protein
denaturation results in the decrease of protein solubility and water holding capacity
(Msmer-Pederson, 1959) which are characteristic 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 P88 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 lean,
heavily muscled, rapidly growing animals (Toelle et al., 1991). Sosnicki et al. (1996) and
Pietrzak et a1. (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, 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 that there is also 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, we cannot assume that the specific mutation associated with
development of PSE pork is applicable to turkey because there are also differences
between turkey and pig. A key difference between turkey and pig skeletal muscle is that
there are two ryanodine receptor isoforms (a and [3) expressed in approximately equal
abundance 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. In
addition, no phenotype for stress-susceptibility in live turkeys has been identified
whereas the halothane challenge test can detect the specific point mutation (Arg615Cys)

in homozygous stress-susceptible pigs. 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.

Studies fi'om our laboratory showed that there are significant differences in
ryanodine binding activity among genetically improved (commercial) and unimproved
(random-bred) turkeys (Wang et al., 1999; Zhang, 2000), which support the hypothesis
there is a genetic basis for the incidence of PSE meat. Recently, our group reported that
there are at least two aRyR genotypes which show correlation with meat quality indices
and that there are aRyR transcript variants generated by alternative splicing (Chiang et al.,
2004). The pattern and abundance of the variants may change as an adaptive response to
heat stress. The results support the hypothesis that there is a genetic component related to
turkey meat quality and that there may be sub-population of turkeys that are more
susceptible than other birds to the effects of heat stress.

In this project, we proposed to identify genetic determinants of altered calcium
release channel activity leading to the incidence of turkey PSE meat. The specific
objectives of this project were: 1) to identify the genotypic differences in B-RyR which
are correlated to the altered B-RyR channel activity of turkey skeletal muscle; 2) to
characterize the effect of varying levels of heat stress on the alternative splice patterns of
B-RyR of turkey skeletal muscle; and 3) to determine whether differences in expression
pattern of a-RyR are associated with heat stress and turkey meat quality. Genetic and
molecular variations are expected to modify the primary structure of both receptor
isoforms, leading to altered channel activity which can be associated with PSE incidence

in turkeys.

Chapter 1

LITERATURE REVIEW

1. Overview of Skeletal Muscle Structure and Function

1.1 Structure of Skeletal Muscle

Skeletal muscle is composed of a highly organized hierarchical arrangement of
organelles, cells, bundles of cells and supporting tissues that give rise to the organ. A
whole muscle is surrounded by a strong connective tissue sheath called the epimysium
that attaches the muscle to the tendons. Parts of connective tissue from this outer sheath
(perimysium) extend into the body of the muscle, subdividing it into columns known as
fascicles (Aberle et al., 2001).

Dissection of a muscle fascicle under a microscope reveals that it is composed of
a bundle of elongated, multinucleated cells called muscle fibers or myofibers. Each
muscle fiber is surrounded by a cell membrane, or sarcolemma, enveloped by a thin
connective tissue layer called the endomysium. Within each muscle fiber there are
tubes of regularly arranged contractile proteins known as myofibrils. One distinctive
feature of the microscopic appearance of skeletal and cardiac muscle fibers is their

pattern of striation which is due to the alignment of myofibrils within the muscle fiber.

The striations are produced by alternating bands of proteins comprising the myofibril that

give rise to the alternating light and dark bands that appear to cross the width of the fiber.

The dark bands are known as A (from anisotropic) bands and the light bands are called I

(from isotropic) bands. Also, there are thin, dark lines, called Z lines (from

Zwischenscheibe, meaning between discs) or disks at the center of the 1 bands (Figure 1-

1) (Rogers, 1983).

Myofibrils are composed of numerous contractile proteins organized into

structures called myofilaments. These proteins, which occupy 80~90% of the volume in

a myofiber, are responsible for the transduction of chemical energy into mechanical work

during muscle contraction and relaxation. Each sarcomere, which is the repeating unit

of the myofibril, contains thick filaments and thin filaments, which are anchored to the Z-

line. The thin filament is composed of actin, and the regulatory proteins tropomyosin and

troponin. The thick filament is composed primarily of myosin. Myosin molecules

consist of four polypeptide “light chain” and two “heavy chains” which fold into a

structure with two globular heads and a long rod-like tail. The myosin heads can bind to

actin in the thin filament, forming crossbridges. It is this actin-myosin interaction that is

the molecular basis for force generation and movement in muscle cells. When muscle

cells contract, the thick and thin filaments do not change their size. Instead, the

2 line

 
  

M line

\A band /

i

 

Figure 1-1. Structure of skeletal muscle (Adapted from Rogers, 1983)

interaction between the myosin heads and actin pulls the thin filaments past the thick
filaments. Thus, the sarcomere which is 2.5 to 3pm long in relaxed muscle,
progressively shortens and develops tension as the muscle contracts by this sliding

filament mechanism (Fox, 2005).

1.2 Excitation-Contraction Coupling

The process by which an action potential triggers a muscle to contract is known as
excitation-contraction coupling. Cylindrical invaginations of the sarcolemma form
structures called transverse tubules (T-tubules) which penetrate the myofiber at periodic
intervals (Figure 1-1). Between the T-tubules is nestled a membrane-bound
compartment called the sarc0plasmic reticulum (SR) which surrounds the myofibrils and
serves as a reservoir of Ca2+ ions. The proteins of the SR/T-tubule membrane system
are responsible for regulation of Ca2+ concentration in the cytoplasm of the muscle fiber
in response to neural signalling (de Meis, 1981; Rtlegg, 1992).

Because T-tubules are distributed along the entire length of the myofiber, the
action potential originating at the neuromuscular junction, is conducted into the interior
of the muscle cell via the T—tubules and this signaling pathway is ultimately responsible

for calcium release from the SR. The mechanism by which depolarization of the T-

 

tubule is communicated to the SR involves two membrane-bound calcium channel
proteins, the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR) located
in the T-tubules and the SR, respectively. These proteins are located at the triad
junction where two sarcoplasmic reticuli meet with one T-tubule.

In response to nerve stimulation, action potentials transverse a muscle fiber via
the T-tubule and the conformation of the DHPR is altered by the voltage change inducing
the release of calcium ions from the SR through the RyR. Release of Ca2+ from the SR
results in a rise in intracellular Ca2+ concentration, which activates the troponin complex.
The conformational change in troponin due to binding of Ca2+ causes tropomyosin to
move deeper into the thin filament groove, exposing the myosin binding sites on actin
and thereby enabling actomyosin crossbridge formation to initiate the contraction of the
muscle. A conformational change in the actin-myosin complex causes the actin and
myosin filaments slide past each other thereby shortening the sarcomere length, and
resulting in muscle contraction. These cycles occur as long as the cytosolic Ca2+ remains
elevated. Muscle relaxation occurs when the neural signal ceases and Ca2+ is pumped
back into the SR through the Ca2+ pump. Cytosolic Ca2+ concentrations are lowered,
Ca2+ is released from troponin and actomyosin interaction are blocked by return of

tropomyosin to its original position along the filament (Aberle et al., 2001 ).

The mechanism of activation of the calcium release channel has been most
extensively studied in vertebrate skeletal and cardiac muscle. It is widely accepted that
there are two distinct modes to activate the calcium channel proteins: depolarization-
induced Ca2+ release (DICR) and Ca2+-induced Ca2+ release (CICR). The former,
DICR, is the primary mechanism in skeletal muscle contraction and is triggered by the
conformational change of the DHPR in response to the change in voltage across the T-
tubule membrane. In this mode, the mechanical coupling hypothesis for skeletal muscle
suggests that the SR RyR is physically linked to the DHPR. However, in the latter case,
CICR, the activation of the calcium channel is caused by an influx of extracellular Ca2+
through the DHPR to the cytoplasm. The local increase of calcium ion concentration
triggers opening of RyR through binding of Ca2+ to the cytoplasmic domain of RyR. In
this mode, tight association of RyR with DHPR is not required (Meissner et al., 1994; Wu

and Hamilton, 1998).

2. Ryanodine Receptors
2.1 Structure and Functions of Ryanodine Receptors (RyRs)
The SR calcium release channel was initially identified in SR vesicles of rabbit

skeletal muscle (Lai et al., 1989) and named as ryanodine receptor protein mle)

10

because of its high affinity for ryanodine, a neutral plant alkaloid (J enden and Fairhurst,

1969). These proteins, which are large homotetramers with subunit molecular mass of

565 kDa, bind ryanodine specifically. RyRs are found in most cell types but they are

concentrated in striated muscle. In striated muscle, RyRs are located at the triadic

junctions between SR terminal cistemae and sarcolemmal transverse tubule (1‘ -tubules)

(Figure 1-2).

Based on the analysis of Rle primary structure, the monomeric subunit is

predicted to have a short cytoplasmic C-terminal domain and between four to ten

membrane-spanning regions in the C-terminal one-fifth of the molecule (Takeshima et a1,

1989). The N-terminal four-fifths of the molecule form the cytoplasmic “foot” structure

that spans the gap between the SR and the T-tubule membranes where the voltage-

sensing dihydropyridine receptors (DHPR) are localized (Figure 1-2).

RyRs have been identified in various animals by molecular cloning of their

cDNAs. Mammalian RyR has three genetically distinct isoforms: Rle is

predominantly found in skeletal muscle, RyR2 is predominantly found in cardiac muscle,

and RyR3 was originally identified in mammalian brain but later found in various kinds

of cells (Sierralta et al., 1996). In mammals, RyR3 is expressed also in skeletal muscle,

although at levels that are 20- to 50-fold lower than those of the Rle (Ottini et al.,

11

, , Extracellular Space
"\,'. Taxi
/ DHPR

(voltage sensor)

  
 
   

Cytoplasm

Figure 1-2. Proteins involved in excitation-contraction coupling. SR,
sarcoplasmic reticulum, DHPR, dihydropyridine receptor, RyR], skeletal ryanodine
receptor, CaM, calmodulin. Greek letters represent DHPR subunits (adapted fi‘om
Hamilton et al., 2000)

1996). These three RyR isoforms show strong sequence homology (approximately
70%) (Dulhunty and Pouliquin, 2003; Mattle et al., 1994) while there are three short
regions with high sequence diversity. These regions, called divergent (D) regions (D1:
residues 4250-4627, D2: residues 1302-1406 and D3: residues 1864-1925, Figure 1-3),
may impose functional differences between the isoforms. For the same isoform,
sequence homology among species is even higher (~85%).

Most avian, amphibian and piscine skeletal muscles display the presence of two
RyR isoforms, originally termed a and B (Airey et al., 1990, 1993). In contrast with
mammalian skeletal muscle, or and B RyR are often found at approximately equimolar
ratio in skeletal muscles of fish, amphibians and birds. or and B RyRs are most similar
in primary structure to the mammalian Rle and RyR3 isoforms, respectively (Ottini et
al., 1996). The sequence similarity suggests that orRyR functions by DICR while BRyR
operates through the CICR mechanism. This hypothesis is supported by the fact that the
Crooked Neck Dwarf mutant chicken, which does not express orRyR but does express
BRyR, fails to develop normal DICR on electrical or neuronal stimulation (Airey et al.,
1993). Also, the purified BRyR of bullfrog is approximately 20 times as sensitive to
Ca2+-activation as aRyR (Murayarna and Ogawa, 1992), which supports CICR as the

likely mechanism for B-RyR. Under Cay-dependent ryanodine binding assay

13

 

Amino Acids

1000 2000 3000 4000 5000

| J |

 

D2 D3 D1

Figure 1-3. Divergent region between Rle and RyR2.
(Adapted from Hamilton, 2005)

conditions, binding of ryanodine by the or isoform is almost completely suppressed in the
presence of BRyR in sarcoplasmic reticulum (SR) vesicles (Murayama and Ogawa,
2001)

O’Brien et a1. (1995) suggested a two-component model for the Ca2+ release
mechanism in non-mammalian vertebrate skeletal muscle. In this model, depolarization
of the T-tubule triggers Ca2+ release through the orRyR which is physically coupled to the
DHPR, and then, the local increase in Ca2+ concentration triggers channel opening of B-
RyR by CICR. Using electron microscopy, F elder and Franzini-Armstrong (2002)
suggested that RyR3 of frog skeletal muscle are segregated in parajunctional locations
while Rle occupy the junctional domains of the SR (Figure 1-4) interacting directly
with DHPR. This model may describe the distribution pattern of two isoforms, a and
BRyR, co-expressed in avian skeletal muscle. Studies on the expression of two isoforms
of RyRs in developing skeletal muscles have led to the hypothesis that combinations of
various Ca2+ release channels may be relevant to adapt the modality of Ca2+ release to
regulation of specific cellular frmctions (Sorrentino and Reggiani, 1999).

The alkaloid ryanodine binds with high affinity to RyR proteins. Binding
studies indicate that each RyR tetramer contains a single high-affinity binding site for

ryanodine (K; < 50 nM; Lai et al., 1988). Some ryanodine binding studies suggest the

15

 

Figure 1-4. Triad model of a muscle with two RyR isoforms. A double row of
orthogonally placed junctional feet (dark profiles) occupies the gap between T tubule
('IT) and SR vesicles (SR). Light-colored parajunctional feet are located in the
adjacent SR membrane, which faces toward the myofibrils. They are separated by a
small zigzag strip from the junctional feet and are arranged in a zigzag pattern.
(adapted from F elder and Franzini-Amstrong et al., 2002)

existence of additional lower (K, > 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

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2.4. Statistical Analysis

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; by genotype

were analyzed. The model statement included the fixed effects of genotype. For

haplotype analysis, the Haplotype procedure of SAS (SAS Inst. Inc., Cary, NC, 2004)

was used.

3. Results and Discussion

3.1. Identification of SNPs in B-RvR of turkeys selectedflsed on the ganodine

binding affiniy

In our previous study (Chapter 2), different genetic lines of birds were identified

with differences in ryanodine binding under different levels of heat stress. We

hypothesized that differences in ryanodine binding are due to a polymorphism(s) in B-

RyR which alters channel activity. Altered channel activity, in turn, may lead to PSE

meat incidence.

To test these related hypotheses, muscle samples from turkeys with significant

difference in ryanodine binding were selected for screening for polymorphism(s) of B-

RyR cDNA. Samples fi'om sixteen turkeys were chosen based on their Kd values

91

 

obtained from the ryanodine binding assay: i.e., the seven birds with the highest Kd
values (Average Kd=57.45 a: 9.98 nM) and the nine birds with the lowest Kd values
(Average Kd=28.25 i 3.32 nM). Table 3-2 shows the meat quality indices and the
ryanodine affinity of B-RyR of the selected turkey samples. The entire cDNA regions
of the B-RyR of those sixteen turkeys were screened for polymorphism(s) in BRyR.

Sequencing analysis revealed ten SNPs, which were located within exons 19, 64,
67, 73, 82, 89, and 99 according to the chicken B-RyR genomic DNA structure (See
Table 3-3). None of the SNPs resulted in a change of amino acid coding, and therefore
would not change protein sequence.

To determine whether the SNPs could be used as markers for allelic variants that
could be possibly related with ryanodine binding activity of B-RyR, least square means of
K; values by genotype were analyzed. However, none of the SNPs showed any
significant relationship with the ryanodine binding activity of the B-RyR of the selected
sixteen turkeys. Therefore, the channel activity as measured by the [3 H] ryanodine
binding assay may not be a good phenotype to represent polymorphisms of the turkey [3-
RyR gene identified in this study. Since the differing ryanodine binding of B-RyR is not
caused by polymorphism, we suggest that other mechanisms such as alternative splicing

or post-translational modification of B-RyR may contribute to the difference.

92

Table 3-2. Meat quality traits (L* and pHrsmin) and ryanodine aflinity (Kd) of turkeys

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

with low Kr and high K, values
Turkey
PHlsmin L" Kd (nM)
11)
3125 5.76 55.07 21.01
2929 6.62 54.27 25.71
3128 5.79 56.58 27.1
2924 6.05 51.38 27.47
3133 5.8 55.13 28.65
Low K“ 3132 5 73 54 95 29 85
(F9) . . .
3113 6.42 48.53 30.03
2933 6.01 51.33 31.55
3105 6.1 51.96 32.89
LSmcan 6.038 53.24 b 28.256
S.E. 0.104 0.857 1.17
3112 5.64 54.31 44.25
2901 6.24 50.31 50.5
2937 6.01 49.72 51.28
, 3118 5.89 53.28 53.48
High K.
2918 6.34 53.74 59.17
(n=7)
2910 6.25 51.14 69.44
2912 5.85 51.77 74
LSmean 6.03“ 52.04b 57.45d
S.E. 0.098 0.67 4.07

 

 

 

 

 

‘b’c'd Different letters within column indicate significant differences (p<0.05)

93

Table 33. Ten synonymous single nucleotide polymorphisms (SNPs) identified

 

 

 

 

 

 

 

 

 

 

 

 

in B-RyR

SNP [1) Nucleotide #* Location“ Base Amino acid
1 2352 Exon 19 AIG Val
2 9111 Exon 64 TIC Ser
3 9681 Exon 67 AIG Lys
4 9762 Exon 67 CIT Leu
5 10434 Exon 73 CIT His
6 11094 Exon 82 TIC Asn
7 11097 Exon 82 G/A Ala
8 12159 Exon 89 CIT Ser
9 12675 Exon 89 TIC Asn
10 14076 Exon 99 AIG Lys

 

 

 

 

 

 

 

*nucleotide number is assigned based on cDNA sequence of turkey B-RyR
”exon numbers are determined based on the chicken B-RyR genomic DNA

sequence (NCBI)

94

3.2. Analysis of individual SNPs identified in PSE and normal meat turkey
marks

To see if the B-RyR SNPs are correlated with meat quality traits, 10 PSE meat
turkeys (L*>52 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

 

 

 

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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

 

 

 

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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

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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. Quantitative analysis of the two ASTVs, firll length
and AS-81 in the ASR] region showed that there are differences in the expression pattern
of two ASTVs between random-bred and commercial turkeys exposed to prolonged heat-
stress. However, more studies involving larger numbers of birds are required to more
conclusively determine the significance of the alternative splicing mechanism of heat
stressed turkeys. As a result of screening of the entire cDNA, there was no alternative

splicing variant detected in turkey B-RyR.

132

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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