Genetic Markers for Genes Encoding Pit-1, GHRH-receptor, and IGF-II, and their Association with Growth and

Carcass Traits in Beef Cattle

Dissertation

Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Qun Zhao, B.S. ***** The Ohio State University 2002 Dissertation Committee: Professor Michael E. Davis, Advisor Professor Harold C. Hines Professor Keith M. Irvin Professor Steven K. St. Martin Professor Macdonald P. Wick Adviser Department of Animal Sciences Approved by

ABSTRACT

Growth and carcass traits are economically important traits of beef cattle and are under the control of multiple genes. Genetic markers for these candidate genes may be useful in marker-assisted selection. Three genes, Pit-1, growth hormone releasing hormone receptor (GHRH-R), and insulin-like growth factor II (IGF-II), were analyzed in this study. Pit-1 is a pituitary specific transcription factor and is able to positively regulate the expression of growth hormone, prolactin, and thyrotrophin subunit. GHRH-R is the receptor for growth hormone releasing hormone, which stimulates the secretion of growth hormone (GH) and regulates mammalian linear growth through its receptor. IGF-II has been shown to regulate pre-adolescent growth. Therefore, these three genes all influence growth and are important candidate genes. The experimental animals used in this study were Angus beef cattle, which were divergently selected for high or low blood serum insulin-like growth factor I (IGF-I) concentration. DNA was extracted from blood samples and the target DNA segments were amplified by PCR. Primers were designed based on DNA or mRNA sequences in Genbank. When amplification was achieved, SSCP (single strand conformation polymorphism) analysis was used to screen for mutations within the amplified segment. After an SSCP polymorphism was found, the PCR product of the two homozygous segments was sent for sequencing in order to determine the nature of the detected polymorphism. Proper restriction enzymes were determined for the polymorphisms

based on the DNA sequence and used to facilitate genotyping using PCR-RFLP (restriction fragment length polymorphism). Associations of the animal genotypes for each polymorphism with growth and carcass traits were analyzed using General Linear Model (GLM) procedures in the Statistical Analysis System (SAS). Three polymorphisms, Pit1I3H (HinfI), Pit1I3NL (NlaIII), and Pit1I3N ( ciI), were N detected in intron 3 of the Pit-1 gene. One polymorphism, Pit1I4N ( BstNI), w found as in intron 4 and one single nucleotide polymorphism (SNP) (Pit1I5) was found in intron 5. For the Pit1I3H polymorphism, genotypes CC, CD, and DD were detected with frequencies of .12, .49, and .39, respectively; there was an AAT deletion in allele C. For the Pit1I3NL polymorphism, genotypic frequencies of .12, .47, and .41 were observed for GG, GH, and HH genotypes, respectively; a G to C transition in allele G was observed. The Pit1I3N polymorphism was found to have genotypes MM, MN, and NN with frequencies of .12, .51, and .37, respectively. For the Pit1I4N polymorphism in intron 4, genotypes EE, EF, and FF were detected and had frequencies of .12, .47, and .41, respectively; a G to T transition was found in allele E. The previously reported polymorphism in exon 6, Pit1E6H (HinfI), was also studied and the frequencies of genotypes OO, OP, and PP were .11, .44, and .45, respectively. The intron 5 SNP, Pit1I5, was found to have genotypes AB and BB with frequencies of .09 and .91, respectively. No AA individuals were found. The A allele had a single nucleotide transition from G to T. No significant associations with growth or carcass traits were observed for Pit1I3H, Pit1I3NL, Pit1I3N, Pit1I4N, or Pit1I5. For Pit1E6H, a significant relationship was found with birth weight (P= .03), where genotype OO had the lowest mean. However, genotype OO was found to be superior for preweaning gain (P= .01)

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and weight gain during the 20-d period between weaning and the beginning of the postweaning test (P= .06). Significant effects of the Pit1E6H genotypes were observed on backfat thickness (P= .01) in the high IGF-I line, and on marbling score (P= .03) in the low line. One polymorphism, GHRHRE6N (NciI), was detected in exon 6 of the GHRH-R gene of Angus cattle. Genotypes AA, AB, and BB were observed with frequencies of .80, .19, and .01, respectively. Allele B has a point mutation from A to G in codon 189, which results in an amino acid change from Glutamine to Arginine. No significant associations of the genotypes with growth, carcass traits, or IGF-I concentrations were observed for this polymorphism. Another polymorphism was detected in the promoter region of the GHRH-R gene of Jersey cattle. Genotypes AA, AB, and BB were observed with frequencies of .44, .53, and .03, respectively. This polymorphism was due to a C to T mutation in allele B. Restriction enzyme AciI was used to genotype this SNP. Significant relationships were observed between genotypes of this polymorphism and relative value of milk production (P= .05) in Jersey cows. Animals with genotype AB tended to have the highest mean for this trait. Therefore, it is assumed that this polymorphism had an overdominant effect on milk production. An AciI polymorphism was observed in intron 8 of the I GF-II gene. Two alleles, A and B, and three genotypes, AA, AB, and BB, were observed. Genotypic frequencies of AA, AB, and BB were .15, .43, and .42, respectively. Sequencing results demonstrated a transition from T to G in allele A. Restriction enzyme AciI was used to genotype this SNP. Significant associations were found between genotypes and weight gain during

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the 20-d period between weaning and the beginning of the postweaning test (P= .05) and IGF-I concentration at d 28 of the 140-d postweaning test (P= .02). In addition, moderate relationships of the genotypes with preweaning gain, on-test weight, off-test weight, and weight at d 28 and 56 of the 140 postweaning test were observed. For all of these traits, genotype AB had a significantly higher mean than genotype AA. Significant relationships were also observed for ribeye area (P= .01) and yield grade (P= .03). Animals with AB genotype had the highest mean for ribeye area, whereas BB animals had the highest yield grades. In conclusion, the polymorphism Pit1E6H in the Pit-1 gene may be a useful marker for preweaning growth rate. Genotype OO was associated with lower birth weight and higher preweaning weight gain. The AciI polymorphism observed in the IGF-II gene may have a dominant effect on growth traits; the B allele was the favorable allele for growth rate. For carcass traits, genotype AB was the favorable genotype for both ribeye area and yield grade.

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Dedicated to my mother

My father

And my husband

ACKNOWLEDGMENTS

I would not have been able to complete this dissertation without help and encouragement from a lot of people around me. First of all, I have to give my great gratitude to my advisor, Dr. Michael E. Davis. He taught me a lot and helped to build up my academic background with his broad knowledge and generosity. He helped me through all of the difficulties for my research. His strong support and patience are what made this dissertation possible. Also, I would like to thank Dr. Harold C. Hines. He helped me with the molecular genetics problems I encountered during my research. He designed primers for some of my genes and advised me on all of my conference abstracts, posters, and, of course, this dissertation. Ms. Judy Riggenbach helped me a lot with her excellent and professional technical support for my research and also with her warm heart. Without her help, it would have been impossible to finish my experiments. I would also like to thank my dissertation committee members, Dr. Keith M. Irvin, Dr. Steven K. St. Martin, and Dr. Macdonald P. Wick. Their precious advices make this dissertation better. Finally, I would like to thank my parents. They encouraged me to take this challenge and helped me through difficulties with their support.

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My husband was with me all of the time. His understanding, and some real help on computer problems, made things easier for me. Thank you and I love you.

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VITA

Education Ph.D. student and research assistant, Animal Sciences The Ohio State University B.S., Veterinary Science Beijing Agriculture University

09/96-present

09/91-07/96

Memberships and Awards Member of American Society of Animal Science-08/00-present Graduate Student Alumni Research Award-April 2001 The Ohio State University Research associateship-09/96-present The Ohio State University Academic award for outstanding students-09/91-07/95 Beijing Agriculture University

PUBLICATIONS Hines, H. C., W. Ge, Q. Zhao, and M.E. Davis. 1998. Association of genetic markers in growth hormone and insulin-like growth factor-I loci with lactation traits in Holsteins. Animal Genetics 29 (Suppl. 1): 63. Zhao, Q., M.E. Davis, and H.C. Hines. 2000. Association of polymorphisms with growth rate in beef cattle. J. Anim. Sci. 78 (Suppl. 1): 77. two Pit-1

Zhao, Q., M.E. Davis, and H.C. Hines. 2001. Relationships of polymorphisms in the Pit-1 gene with growth traits in beef cattle. 2001 Research and Reviews. OARDC Special Circular 181: 35-40. Zhao, Q., M.E. Davis, and H.C. Hines. 2002. Two Pit-1 RFLPs and their association with growth traits in beef cattle. J. Anim. Sci. Vol. 80 (Suppl.2): 44.

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Zhao, Q., M.E. Davis, and H.C. Hines. 2002. An MspI polymorphism in the GHRHreceptor gene and its association with growth traits in Angus beef cattle. ISAG XXVIII International Conference on Animal Genetics. Section D052, 103. Zhao, Q., M.E. Davis, and H.C. Hines. 2002. Associations of an AciI polymorphism in the IGF-II gene with growth traits in beef cattle. Proceedings of the 7th World Congress on Genetics Applied to Livestock Production. CD-ROM communication n 11-44.

FIELD OF STUDY Major Field: Animal Sciences

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TABLE OF CONTENTS

Abstracti Dedication...v Acknowledgments.....vi Vita..viii List of Tables...xiii Chapters: 1. Introduction.1 2. Literature Review5 Pit-1/Growth Hormone Factor I...5 Pit-1 introduction....5 Biological function of Pit-1.6 Physiological effect.7 DNA binding and target gene activation.8 Pit-1 gene structure...12 Pit-1 gene expression....13 Growth Hormone Releasing Hormone Receptor...15 GHRH and GHRH-R introduction....15 GHRH-R biological function and physiological effect.....16 GHRH-R characterization.....18 GHRH-R ligand binding and signaling properties........19

GHRH-R gene structure...20 GHRH-R gene expression....23 Insulin-like growth factor II (IGF-II)...25 IGFs introduction.....25 Biological function of IGF-II...26 Receptors and binding proteins....28 Structure of IGF-II protein and IGF-II gene....29 IGF-II gene expression ...31 Imprinting of IGF-II gene....32 3. Associations of Polymorphisms in the Pit-1 Gene with Growth and Carcass Traits in Angus Beef Cattle....35 Abstract....35 Introduction......36 Materials and Methods... 38 Results and Discussion.....46 Conclusions......51 References........52

4. Associations of Polymorphisms in the Growth Hormone Releasing Hormone Receptor Gene with Growth and Carcass Traits in Angus Beef Cattle...72 Abstract....72 Introduction......73 Materials and Methods.....74

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Results...79 Discussion.....81 Conclusions...82 References.........83 5. Associations of Polymorphisms in the Promoter region of the Growth Hormone Releasing Hormone Receptor Gene with Milk Production in Jersey Cattle....93 Abstract....93 Introduction......94 Materials and Methods.....96 Results and Discussion ........99 Conclusions.........100 References.......100 6. Associations of Polymorphisms in the Insulin-like Growth Factor II Gene with Growth and Carcass Traits in Angus Beef Cattle..104 Abstract......104 Introduction....105 Materials and Methods...107 Results....112 Discussion..113 Conclusions....115 References..116 8. Bibliography..124

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LIST OF TABLES

Table 3.1 3.2 3.3

Page Sequences of Primers and Lengths of Their Products..54 Polymorphisms and Their Sequence Characteristic.....55 Genotypic Frequencies of Polymorphisms by High and Low IGF-I Selection Lines ..56

3.4

Analysis of Linkage Disequilibrum for the Five Diallelic Loci: Pit1I3H, Pit1I3N, Pit-1I3NL, Pit1I4N, and Pit1E6H..57

3.5 3.6 3.7 3.8 3.9 3.10

Least-squares Means and Standard Errors by Pit1I3H Genotype.......58 Least-squares Means and Standard Errors by Pit1I3NL Genotype.....59 Least-squares Means and Standard Errors by Pit1I3N Genotype...60 Least-squares Means and Standard Errors by PitI4N Genotype.........61 Least-squares Means and Standard Errors by Pit1E6H Genotype......62 Least-squares Means and Standard Errors by Pit1E6H Genotype in High IGF-I Line..63

3.11

Least-squares Means and Standard Errors by Pit1E6H Genotype in Low IGF-I Line...64

3.12 3.13

Least-squares Means and Standard Errors by Pit1I5 Genotype......65 Least-squares Means and Standard Errors by Pit1I5 Genotype in High IGF-I Line..66

3.14

Least-squares Means and Standard Errors by Pit1I5 Genotype in Low IGF-I Line........67

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3.15 Least-squares Means and Standard Errors for Carcass Traits by Pit1E6H Genotypes across Selection Lines.....68 3.16 Least-squares Means and Standard Errors for Carcass Traits by Pit1E6H Genotypes in High Selection Line....69 3.17 Least-squares Means and Standard Errors for Carcass Traits by Pit1E6H Genotypes in Low Selection Line.....70 3.18 Least-squares Means and Standard Errors for Carcass Traits by Pit1I5 Genotypes across Selection Lines.......71 4.1 4.2 Genotypic Frequencies for NciI Polymorphism by IGF-I Selection Line....86 Least-squares Means and Standard Errors for Growth Traits by GHRHRE6N Genotypes Across Lines..87 4.3 Least-squares Means and Standard Errors for Growth Traits by GHRHRE6N Genotypes in High Line..88 4.4 Least-squares Means and Standard Errors for Growth Traits by GHRHRE6N Genotypes in Low Line......89 4.5 Least-squares Means and Standard Errors for Carcass Traits by GHRHRE6N Genotypes across Selection Lines.....90 4.6 Least-squares Means and Standard Errors for Carcass Traits by GHRHRE6N Genotypes in High Selection Line..91 4.7 Least-squares Means and Standard Errors for Carcass Traits by GHRHRE6N Genotypes in Low Selection Line..92 5.1 Least-squares Means and Standard Errors for Milk Merit by Genotypes of the GHRHRP Polymorphism..103

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6.1

Genotypic Frequencies for IGFIIE9 (AciI) Polymorphism by IGF-I Selection Line..119

6.2

Least-squares Means and Standard Errors by IGFIIE9 Genotypes across the two Selection Lines......120

6.3

Least-squares Means and Standard Errors by IGFIIE9 Genotypes in the High Line......121

6.4

Least-squares Means and Standard Errors by IGFIIE9 Genotypes in the Low Line......122

6.5

Least-squares Means and Standard Errors for Carcass Traits by IGFIIE9 Genotypes across Lines.........123

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CHAPTER 1 INTRODUCTION

Growth and carcass traits are economically important traits in livestock. In order to obtain animals with desired values for these traits, selection has been done solely on the basis of phenotypic information. This is traditional selection. Traditional selection has been shown to result in slow genetic progress, especially for species with long generation intervals, for example, cattle and sheep. Current technologies enable scientists to improve the accuracy and efficiency of traditional selection by applying genetic markers. This can be done through marker assisted selection (MAS), which combines information on molecular genetic polymorphisms (marker loci) with data on phenotypic variation among individuals (and their relatives). NiemannSorenson and Robertson (1961) were the first to discuss the possible use of molecular marker information directly in a breeding program. It has been estimated that use of genetic markers in beef cattle selection could improve rates of genetic progress by up to 30% and shorten generation interval by 24 mo (Kashi et al., 1990). Therefore, genetic markers that are associated with certain genes and traits of interest could be very useful in MAS. Growth and carcass traits are under the control of multiple genes. One way to identify genetic markers related to these traits is to map quantitative trait loci (QTL). However,

QTL mapping is relatively complicated. Lande and Thompson (1990) pointed out that it is not necessary to map the QTL if the goal is to perform MAS. We only need to identify the markers that are correlated with the traits of interest and base selection on the marker information (Lande and Thompson, 1990). Therefore, genetic markers for candidate genes, which play important roles in growth and development of livestock, can play an important role in MAS. Moreover, analysis of the associations of genetic markers for candidate genes with production traits in animals can also further elucidate the genetic control of the production traits. Genetic polymorphisms in candidate genes that have functionally significant effects on production traits are most useful in association studies. However, the candidate gene approach is limited by the biological information available for the traits of interest. In this study, three genes, the Pit-1 gene, the growth hormone releasing hormone receptor (GHRH-R) gene, and the insulin-like growth factor II (IGF-II) gene, were chosen as candidate genes. All of these genes play crucial roles in animal growth. Therefore, detecting genetic variations in these genes and relating them to growth and carcass traits is important and could be helpful in MAS. Pit-1 is a pituitary specific transcription factor that has been shown to positively regulate the expression of growth hormone (GH), prolactin (PRL) and thyrotrophin subunit (TSH). It also plays a role in pituitary cell differentiation and proliferation ( Andersen and Rosenfeld, 1994). Mutations in the Pit-1 gene lead to the absence of growth hormone and pituitary hypoplasia in mice (Li et al., 1990) and a syndrome of congenital hypothyroidism, dwarfism, and prolactin deficiency in humans (Pfaffle et al., 1992). Association studies have

shown that Pit-1 is associated with growth rate, carcass traits and milk production in domestic animals. Pit-1 was found to be related to birth weight (Yu et al. 1996), weaning weight, average daily gain, and backfat (Yu et al., 1995), as well as lean to fat ratio (Stancekova et al., 1999) in pigs. In cattle, Pit-1 was found to be associated with body weight, and milk, protein and fat yields (Renaville et al., 1997). Therefore, the Pit-1 gene could be a good candidate gene for genetic markers for growth and carcass traits. Growth hormone releasing hormone plays a role in stimulating the secretion of GH (Mayo et al., 1995) and regulating mammalian linear growth (Hammer et al., 1985; Frohman and Jannson, 1986; Gelato and Merriam, 1986). However, no associations of genetic variations in the GHRH gene with a bnormal growth or growth hormone secretion have yet been identified. Therefore, the receptor for GHRH may be a better candidate for genetic markers for growth traits. Mutations in the GHRH-R gene have been found to cause heritable GH deficiency diseases in both mice (Godfrey et al., 1993; Lin et al., 1993) and human (Wajnrajch et al., 1996; Maheshwari et al., 1996). However, no studies have shown significant association of genetic variation in the GHRH-R gene with production traits in domestic animals. One goal of this study was to find genetic markers for this gene in beef cattle. The insulin-like growth factor II gene was another candidate for genetic marker research in this study. IGF-II belongs to a family of structurally related polypeptides, which include IGF-I, insulin and relaxin (Blundell and Humbel, 1980; Dafgard et al., 1985). IGF-II is important in the fetus (Han et al., 1987). It plays a key role in pre-adolescent growth,

influencing fetal cell division and differentiation. IGF-II knockout mice were shown to have significant fetal growth retardation, especially in the early stages of gestation (DeChiara et al., 1990). On the other hand, transgenic mice with overexpression of IGF-II were shown to have organ overgrowth and tumor formation (Ward et al., 1994, Rogler et al., 1994). Therefore, the IGF-II gene may be a good candidate gene for growth. One objective of this study was to detect genetic variations in these three candidate genes in cattle. Association studies were conducted to test the effect of the genetic polymorphisms on growth and carcass traits in beef cattle or on milk production traits in dairy cattle. The final goal was to find genetic markers that have significant effects on economically important traits and thus can be used in development of marker-assisted selection (MAS) programs in cattle breeding.

CHAPTER 2 LITERATURE REVIEW

Pit-1/Growth Hormone Factor I

Pit-1 introduction Pit-1, which is also termed Growth hormone factor 1(GHF1), is a pituitary-specific transcription factor that is responsible for pituitary development and hormone expression in mammals (Cohen et al., 1997). Pit-1 is a member of the POU domain containing proteins, which is a group of transcriptional regulators that have a critical role in differentiation and proliferation of cells (Mangalam et al., 1989). The POU domain was first identified as a novel DNA-binding motif from the cloning of mammalian transcriptional regulators Pit-1, Oct-1, Oct-2, and the Caenorhabditis elegans developmental modulator Unc-86 (Bodner et al., 1988; Clerc et al., 1988; Finney et al., 1988; Herr et al., 1988; Ingraham et al., 1988; Ko et al., 1988). More than 20 other members of the POU family have now been identified in metazoan organisms, ranging from Drosophila to humans (Jacobson et al., 1997) and are grouped into six or seven classes based on the amino acid sequence of their POU domains and conservation of the variable linker region (Wegner et al., 1993; Ryan and Rosenfeld, 1997).

The POU domain is a bipartite DNA-binding domain (Sturm and Herr, 1988; Ingraham et al., 1990; Kristie and Sharp, 1990; Verrijzer et al., 1990). It consists of two highly conserved regions joined by a variable linker (Klemm and Pabo, 1996; Ryan and Rosenfeld, 1997). These two protein domains, POU-specific domain (POU-S), which is unique to these factors, and POU-homeodomain (POU-HD), which is related to that found in the homeobox proteins, are both necessary for DNA binding (Ingraham et al., 1990; Anderson and Rosenfeld, 1994). POU-S is responsible for high affinity DNA binding with site specificity and POU-HD is responsible for low affinity binding with relaxed specificity (Ingraham et al., 1990; Anderson and Rosenfeld, 1994). In vivo, many of the POU proteins have been shown to regulate key developmental processes, which are associated with the nervous system (Ryan and Rosenfeld, 1997; McEvilly and Rosenfeld, 1999). Transcription factor Pit-1 is mainly expressed in the anterior pituitary gland and has been demonstrated to participate in pituitary gland development and gene expression of related hormones.

Biological function of Pit-1

After the genes encoding pituitary hormones, growth hormone (GH), which is required for postnatal growth, and prolactin (PRL), which is required for milk production, were identified, the molecular basis of their regulation at a transcriptional level was investigated and led to the identification of Pit-1 (Andersen and Rosenfeld, 1994). Analysis of the regulatory region upstream of the transcriptional start sites revealed an A/T region common to both GH and

PRL genes, and this A/T region was found to be required for expression of these genes (Nelson et al., 1986; Lira et al., 1988; Crenshaw III et al., 1989). Pit-1 was then cloned in 1988 based on its affinity for these A/T-rich sites and was shown to control transcription of growth hormone and prolactin (Bodner et al., 1988; Ingraham et al., 1988; Nelson et al., 1988; Mangalam et al., 1989). Research further demonstrated that Pit-1 also regulates the transcription of thyroid-stimulation hormone -subunit (TSH) genes (Simmons et al., 1990; Steinfelder et al., 1991; Gordon et al., 1993; Lin et al., 1994), growth hormone releasing hormone receptor (GHRH-R) genes (Lin et al., 1992), and the Pit-1 gene itself (Rhodes et al., 1993). Immunohistological analysis revealed high expression of Pit-1 in three cell types in the anterior pituitary, somatotropes, lactotropes, and thyrotropes (Doll et al., 1990; Simmons et al., 1990), which secrete pituitary hormones GH, PRL, and TSH, respectively. Pit-1 was shown to play a critical role in differentiation and proliferation of these three cell types.

Physiological effect The role of Pit-1 in cell development and hormone secretion has been demonstrated by mutations in the Pit-1 gene of mouse and human. The Snell dwarf mouse (dw) possessed the first genetically transmitted dwarfism observed in mice. This dwarfism was characterized as a single autosomal recessive mutation on the Pit-1 gene (Li et al., 1990). The mutation prevents the development of the pituitary cells that secrete growth hormone, thyrotrophin, and prolactin. It leads to the absence of growth hormone, prolactin, and TSH- gene expression and results in a failure of somatotrope, lactotrope, and thyrotrope proliferation (Bartke, 1964; Sinha et al.,

1975; Cheng et al., 1983). Another dwarf mouse, the Jackson mouse (dwJ), has a gross structural alteration of the Pit-1 gene and no Pit-1 expression (Li et al., 1990). The Jackson mouse also has hypoplastic anterior pituitary gland and combined pituitary hormone deficiency of GH, PRL, and TSH-. Pit-1 carries out similar functions in humans as in rodents. Naturally occurring mutations in the Pit-1 gene have been found to cause combined pituitary hormone deficiency (CPHD) in humans (Cohen et al., 1996). The dimerization interface of the two subdomains turned out to be the mutational hot spot in Pit-1 associated with combined pituitary hormone deficiency (Cohen et al., 1995). Association studies have shown that Pit-1 is associated with growth rate, carcass traits, and milk production in domestic animals. Pit-1 was found to be related to birth weight (Yu et al., 1996), weaning weight, average daily gain and backfat thickness (Yu et al., 1995), as well as lean to fat ratio (Stancekova et al., 1999), in pigs. In cattle, Pit-1 was found to be associated with body weight, and milk, protein and fat yields (Renaville et al., 1997).

DNA binding and target gene activation Pit-1 is characterized by the presence of two conserved regions at the C terminus, POUspecific domain (POU-S) and POU homeodomain (POU-HD), which are responsible for high affinity DNA binding to the GH, PRL and TSH- genes (Laurie et al., 1996). A less conserved domain at the N terminus that is rich in serine and threonine residues

(serine/threonine activation domain, STA) mediates transcriptional activation (Theill et al., 1989; Ingraham et al., 1990). The POU-homeodomain is a 60-amino acid region near the C terminus that has considerable homology to the homeobox, which is a conserved sequence motif identified in genes that regulate developmental processes (Anderson and Rosenfeld, 1994). The homeodomain of Pit-1 is predicted to form a helix-turn-helix motif and is required and sufficient for low affinity DNA binding with relaxed specificity (Lautie et al., 1996). The POU-specific domain is a 75-amino acid region that is 5' to the POU-homeodomain and separated by a nonconserved linker (Anderson and Rosenfeld, 1994). It is necessary for high affinity binding and accurate recognition of Pit-1 response elements (Theill and Castrillo et al., 1989; Ingraham et al., 1990). The POU-specific domain also contributes to DNA-dependent pit-1-pit-1 interaction (Theill and Castrillo et al., 1989; Anderson and Rosenfeld, 1994; Lautie et al., 1996). Analysis of alpha helical domains and conserved structures in Pit-1 suggests that POU domain proteins interact with their DNA recognition sites with both the POU-HD and POU-S domain contacting DNA (Ingraham et al., 1990). Crystallographic studies suggested that POU proteins contain four alpha helices in the POU-S and three alpha helices in the POU-homeodomain (Klemm et al., 1994). The amino alpha helical regions of the POUspecific domain seem necessary for DNA binding and are responsible for mediating proteinprotein interactions, whereas the carboxyl alpha-helical regions seem to be involved in site specificity (Ingraham et al., 1990).

The linker is located between the two subdomains. It can increase the local concentrations of POU-specific and POU homeodomains by tethering these two subdomains, and helps highaffinity, site-specific DNA binding by the two POU subdomains. The length of the linker region, which ranges in size from 15 to 56 amino acids, plays a role in enabling the POU specific domain to have different orientations relative to the POU homeodomain and in determining flexibility with which the POU domain recognizes DNA-binding sites (Ryan and Rosenfeld, 1997). Pit-1 is monomeric in solution but associates as a dimer on most DNA response elements (Ingraham et al., 1990). Some Pit-1 response elements are able to only bind homodimers (such as the distal site in the GH promoter) (Cohen et al., 1996). A high resolution x -ray analysis of Pit-1 POU domain bound to DNA as a homodimer showed that the POU specific domain and the homeodomain bind to the perpendicular face of the DNA and the dimerization interface turns out to be the site of a mutational hot spot in pituitary hormone deficiency (CPHD) in human (Jacobson et al., 1997). The heterodimers of Pit-1 and Oct-1 were found to bind preferentially to the prolactin Prl-1P site (Voss et al., 1991). On certain sites, such as prolactin Prl-1d, Pit-1 preferentially binds as a monomer and with high affinity (Ingraham et al., 1990; Rosenfeld et al., 1991; Holloway et al., 1995; Jacobson et al., 1997). Pit-1 binds to a complex pattern of sites within the promoters and enhancers of the genes it regulates. The sequences of these sites vary widely around a weak consensus sequence (A/T)/(A/T)TATNCAT (Jacobson et al., 1997). This suggests that Pit-1 might be configured differently on the different sites. The expression of the GH gene is controlled by a pituitary-

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specific promoter that contains two binding sites for Pit-1, GH-I (-96 to 70) and GH-II (134 to 106) (Nelson et al., 1988). Both sites are essential for GH promoter activity in vitro and in vivo (Bodner et al., 1988). Transcription of the prolactin gene depends on a distal enhancer segment (-1830 to -1530) containing four Pit-1 binding sites and a proximal promoter region (-422 to -36) containing four Pit-1 binding sites (Nelson et al., 1988). On the prolactin gene, Pit-1 shows a unique promoter spacing requirement for transcription activation. The transcriptional regulation is sensitive to the placement of the most proximal Pit-1 binding site and also the spacing between the TATA box and the proximal binding site on the prolactin gene (Smith et al., 1995). The prolactin gene also has a composite Ets/Pit-1 binding site and Ets protein and Pit-1 functionally cooperate to permit transcriptional regulation (Howard et al., 1995). The 5 flanking region of the TSH- gene (-128 to -92) has a Pit-1 binding site. Three more upstream regions within the 5 flanking region of the TSH- promoter at 274 to 258 (TSH A), at 336 to 326 (TSH B), and at 402 to 385 (TSH C) contain sequences similar to the Pit-1 consensus binding sites and can bind Pit-1 (Mason et al., 1993). The Pit-1 promoter is autoregulated as a consequence of Pit-1 binding to two Pit-1 binding elements, PitB1 and PitB2, with sequence similarity to the consensus sequence of

(T/A)/(T/A)TATNCAT (Chen et al., 1990; McCormick et al., 1990; Cohen et al., 1996). The Pit-1 enhancer contains five binding sites for Pit-1, four of which conform to the TATNCAT/A consensus. Three of the binding sites contribute to enhancer activity (Rhodes et al., 1993; Cohen et al., 1996)

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Phosphorylation of Pit-1 can affect its binding. Results suggest that phosphorylation changes the conformation of Pit-1 on DNA, which could result in different interaction between Pit-1 and its binding site at the target gene (Rosenfeld et al., 1991; Cohen et al., 1996). Pit-1 is phosphorylated at two major sites, serine 115 and threonine 220, and one minor site, threonine 219, through the protein kinase A (PKA) and protein kinase C (PKC) pathways (Cohen et al., 1996)

Pit-1 gene structure Pit-1 cDNA has been cloned in several mammalian species including rat, human, bovine, and swine. It has six exons and most of the distinct functional domains of Pit-1 are encoded by separate exons (Theill et al., 1992). The human Pit-1gene is greater than 14kb in size and is located on chromosome 3p11 (Ohta et al., 1992), whereas the mouse Pit-1 gene is located on chromosome 16 (Li et al., 1990), and the bovine Pit-1 gene is on chromosome 1 (Moody et al., 1995). In mammals, Pit-1 has three different splicing variants, the major type, Pit1, and two other splicing variants, Pit-1 and Pit-1T (Theill et al., 1992; Konzak and Moore, 1992; Morris et al., 1992). All of the splicing variants are biologically active. These Pit-1 variants act differentially on the promoters of target genes. Pit-1 strongly activates Pit-1 and Pit-1 promoters (Voss et al., 1991(2); Tanaka et al., 1999). Pit-1 has a 26-amino acid insert in the transactivation domain because of alternative splicing of the Pit-1 gene transcript at the end of intron one (Mangalam et al., 1989; Konzak et al., 1992; Delhase et al., 1995).

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Consequently it lost the ability to activate the PRL and Pit-1 promoters and preferentially activates the GH gene promoter (Konzak and Moore, 1992; Morris et al., 1992). Pit-1T contains a 14-amino acid insert in the transactivation domain because of an alternate 3 splice acceptor site and was found to be expressed in thyrotroph-derived cells and stimulates only TSH- expression (Haugen et al., 1993; Haugen et al., 1994). In chicken, two pituitary expressed Pit-1 mRNAs encoding cPit-1 and cPit-1, composed of 335 and 327 amino acid residues, respectively, are found. They differ at the N-terminal regions and cPit-1 has higher expression than cPit-1 (Tanaka et al., 1999).

Pit-1 gene expression Research involving the naturally occurring dwarf mouse strains, Snell and Jackson, showed that the lack of functional Pit-1 had no effect on the initial activation of the Pit-1 gene, but did impair sustained expression after ~e18 (embryo day 18). These results indicate that, the initial activation of Pit-1 i not regulated by Pit-1 itself, but Pit-1 does carry out a function of s autoregulating its sustained expression (Rhodes et al., 1993). The initial activation of the Pit-1 gene uses an enhancer located at 3.2 to 5.3kb on the Pit-1 gene; it was found to be stage specific and functioned only in somatotropic progenitor cells (Lew et al., 1993). A pituitary-specific paired-like homeodomain protein, Prophet of Pit1 (Prop-1), is capable of binding to this early enhancer and regulating expression of the Pit-1 gene (DiMattia et al., 1997). A mutation in the Prop-1 gene was found in a dwarf mouse, the Ames mouse. The Ames mouse has similar phenotypes to Snell and Jackson dwarf mice

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(Sornson et al., 1996). This indicates that Prop-1 is necessary for Pit-1 expression. In the mouse pituitary, Pit-1 gene expression is initially detectable at embryonic Day 14 (Crenshaw et al., 1989; Simmons et al., 1990; Dolle et al., 1990; Rhodes et al., 1993). Therefore, activation is likely to occur on or before e13. Once the amount of Pit-1 protein has reached a critical level, Pit-1 transcription is maintained by autoregulation and additional transcriptional regulation. Pit-1 autoregulation is carried out via two Pit-1 response elements. One binding site, PitB1, is located at position -60 to -45 and has a positive effect on transcription. Another Pit-1 recognition element, PitB2, located just downstream of the start site of transcription, has an inhibitory effect. These two elements work together to precisely control the basal level of Pit-1 expression (Chen et al., 1990; McCormick et al., 1990). Pit-1 is regulated by cAMP and two binding sites for cAMPregulatory element binding protein (CREB) are observed in the Pit-1 promoter region around positions 200 and 155. Because CREB does not activate the Pit-1 promoter, the response to CREB requires interaction with pituitary-specific factors (Chen et al., 1990). Pit-1 gene transcription is also regulated by environmental cues that affect the intracellular level of cAMP. This regulation is mediated by the CREB or related family members (McCormick et al., 1990; Theill and Karin, 1993). Activin and inhibin are involved in somatotroph activities and this control is mediated in part by controlling Pit-1 expression/activity. They are capable of restricting the expression of Pit-1 (Theill and Karin, 1993). The activity of Pit-1 is also determined by a regulated balance between a co-repressor complex that contains N-

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CoR/SMRT, mSin3A/B, and histone deacetylases, and a co-activator complex that includes the CREB-binding protein (CBP) and p/CAF (Xu et al., 1998).

Growth Hormone Releasing Hormone Receptor

GHRH and GHRH-R introduction The pituitary polypeptide hormone growth hormone (GH) can regulate linear growth in vertebrate organisms (Mayo et al., 1995; Petersenn et al., 1998). Synthesis and secretion of GH in the somatotroph cells of the anterior pituitary are under multifactoral control from the brain, specifically from the hypothalamus (Martin, 1979; Mayo et al., 1995). The stimulatory signal is from growth hormone releasing hormone and the suppressive signal is from somatostatin (Frohman et al., 1986; Frohman et al., 1992). Growth hormone releasing hormone (GHRH) was initially isolated in 1982 from pancreatic tumors that caused acromegaly (Guillemin et al., 1982; Rivier et al., 1982) and later from the hypothalamus (Spiess et al., 1983; Ling et al., 1984). Substantial information from both the clinical domain and from basic research affirms the primal role of GHRH in the control of growth hormone secretion and in the regulation of linear growth (Hammer et al., 1985; Frohman and Jannson, 1986; Gelato and Merriam, 1986). GHRH is expressed predominantly in the arcuate nuclei of the hypothalamus (Petersenn et al., 1998). It is also expressed in cells and tissues outside the brain such as the placenta (Margioris et al., 1990), ovary (Bagnato et al., 1992), testis (Berry et al., 1992), and lymphocytes (Stephanou et al., 1991), and in the pancreas and gastro-

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intestinal tract (Bosman et al., 1984; Shibasaki et al., 1984), where it may have diverse biological activities unrelated to control of GH secretion (Mayo et al., 1995). Recently, molecular cloning of the growth hormone releasing hormone receptor (GHRH-R) has allowed new insights into the mechanisms of GHRH regulation of growth (Mayo, 1995). GHRH-R complementary DNAs (cDNAs) from rat (Lin et al., 1992; Mayo et al., 1992), mouse (Lin et al., 1992), swine (Hsiung et al., 1993), human (Mayo, 1992; Gaylinn et al., 1993), and bovine (Horikawa et al., 2001) have been identified. The GHRH-R belongs to the family of G-protein-coupled cell surface receptors that activate adenylate cyclase, resulting in increased cAMP levels and activation of protein kinase A (Petersenn et al., 1998). Other members of this family include the receptors for secretin, vasoactive intestinal peptide (VIP), glucagon, GLP-1, PACAP, and gastric inhibitory peptide (Petersenn et al., 1998). The GHRH-receptor transcripts have a highly specific distribution in the anterior pituitary. Also, significant amounts of GHRH-R are found in the hypothalamus, kidney and placenta (Mayo et al., 1995). This further indicates the importance of GHRH-R in GH secretion and somatotroph cell proliferation.

GHRH-R biological function and physiological effect GHRH plays a major role in stimulation of both synthesis and release of GH in the anterior pituitary through its receptor. Its receptor, GHRH-receptor, is able to transduce GHRHdependent increases in intracellular cAMP via Gs activation for stimulating somatotroph proliferation and GH gene expression (Lin et al., 1992; Mayo, 1992; Gaylinn et al., 1993).

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Mutations in the receptor have been found to cause heritable GH deficiency diseases in both mice (Godfrey et al., 1993; Lin et al., 1993) and humans (Maheshwari et al., 1996; Wajnrajch et al., 1996). Lin et al. (1993) demonstrated that one amino acid substitution of the GHRH-R gene in the little mouse, which showed genetically transmitted dwarfism, caused GH deficiency and somatotroph hypoplasia (Lin et al., 1993). This indicates that the integrity of the GHRH signaling pathway is necessary for expansion of the somatotroph cell lineage. In addition, mutations in the GHRH-R gene in humans were demonstrated to cause profound GH deficiency (Wajnrajch et al., 1996; Netchine et al., 1998). Studies of the dwarfism of Sindh, a familial severe dwarfism in Pakistan, shows patients all have a mutation in the GHRH-R gene (Baumann and Maheshwari, 1997; Maheshwari et al., 1998). Mutations in the GHRH-R may also play a role in GH excess and pituitary tumorigenesis (Petersenn et al., 1998). These genetic disorders suggest the physiological significance of GHRH-R in the hypothalamuspituitary GH axis (Iguchi et al., 1999). Also, transcripts for alternatively spliced forms of the GHRH receptor truncated in the third intracellular loop have been found in patients with GHproducing pituitary tumors (Hashimoto et al., 1995; Tang et al., 1995;). In domestic animals, the GHRH-receptor gene has been assigned to bovine chromosome 4 (Connor et al., 1999) and swine chromosome 18 (Sun et al., 1997). An Eco571 PCR-RFLP in intron 6 has been defined on the GHRH-R gene in cattle. Two PCR-RFLP, MseI and TaqI, were identified in pigs (Sun et al., 1997). Thus far, no association has been found between these polymorphisms and growth and carcass traits in domestic animals.

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GHRH-R characterization The amino acid alignment deduced from human cDNA revealed that the GHRH-R consists of 423 amino acids containing seven hydrophobic domains with the potential to serve as membrane-spanning helices (Petersenn et al., 1998). It has been determined, with human and ovine pituitaries, that the GHRH-R is a protein of 55 kd when glycosylated and 45 kd when deglycosylated (Gaylinn et al., 1994(2); Tang et al., 1995). Comparison of predicted primary protein sequences among cloned GHRH-R transcripts from different species demonstrated 78% homology, suggesting a highly conserved function for GHRH-R during evolution. The most conserved part of the receptor is found in the transmembrane domains (89%) and the intracytoplasmic loops (80%), indicating the importance of these parts for ligand binding and/or signal transmission (Tang et al., 1995). Sequence analysis and predicted protein structure indicate that GHRH-R belongs to the superfamily of G protein-coupled cell surface receptors characterized by seven transmembrane helixes joined by three intracytoplasmic and three extracellular loops (Tang et al., 1995). According to peptide similarities, the GHRH-R can be further classified into a subfamily, family B of the G protein-coupled receptor (GPCR) superfamily, consisting of the receptors for vasoactive intestinal peptide (VIP), pituitary adenylyl cyclase activation peptide (PACAP), secretin, calcitonin, parathyroid hormone (PTH), PTH-related peptide, glucagon and glucagon-like peptide 1 (GLP-1) (Segre and Goldring, 1993). Peptide sequence comparison of these receptors reveals several conserved features such as the presence of one or more N-linked glycosylation sites, six highly conserved cysteine residues, and an absolutely

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conserved aspartic acid residue within the relatively large aminoterminal extracellular domain (Segre et al., 1993; Mayo et al., 1995). The integrity of the conserved aspartic acid residue has been shown to be critical for the interaction of the ligands with their respective receptors and is the site of GHRH-R mutation in the little mouse (Godfrey et al., 1993; Gaylinn et al., 1994; Carruthers et al., 1994; Couvineau et al., 1995). Another feature common in the subfamily of receptors is that they all use the G protein as transducer and the adenylate cyclase as effector, and participate in a similar signal transduction mechanism (Tang et al., 1995).

GHRH-R ligand binding and signaling properties High affinity binding of GHRH to its receptors on pituitary somatotrophs results in G protein coupling, adenylate cyclase activation and cAMP production, Ca2+ influx, increased expression of the GH gene, and enhanced GH secretion (Frohman et al., 1986; DeAlmeida and Mayo, 1998). The N-terminal extracellular domain is essential for ligand binding. Studies on the GHRH receptor of the little mouse, which has a mutation at position 60, indicate that the mutation results in loss of the ability of the receptor to bind ligand (Gaylinn et al., 1994) and activate adenylate cyclase (Godfrey et al., 1993; Lin et al., 1993). These studies provided the first indication that the integrity of the N terminus of the GHRH receptor is essential for ligand binding (DeAlmeida and Mayo, 1998). However, neither the N-terminal extracellular domain nor the C-terminal domain, consisting of the transmembrane domains and the associated extracellular loops, can bind ligand in the absence of the other (DeAlmeida and Mayo, 1998).

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A truncated GHRH-R, with part of the first intracellular loop deleted, was found to be insufficient for interaction with GHRH (DeAlmeida and Mayo, 1998). This indicates the transmembrane domains and associated extracellular loop regions can provide critical information necessary for specific interaction with GHRH (DeAlmeida and Mayo, 1998). Studies of chimeric receptors and of GHRH receptor cross-linking sites have shown that the key sites for ligand specificity and signaling are associated with the transmembrane helices and intervening loops (Gaylinn, 1999). Evidence from the ovine GHRH receptor suggests that the C-terminus has an inhibitory function and may be involved in down-regulation via internalization and phosphorylation (Gaylinn, 1999).

GHRH-R gene structure Thus far, the GHRH-R has been cloned in human ( Mayo, 1992; Gaylinn et al., 1993), mouse (Lin et al., 1992), rat (Lin et al., 1992; Mayo et al., 1992), pig (Hsiung et al., 1993), and cattle (Horikawa et al., 2001). In contrast to other G protein-coupled receptors, such as the receptors for somatostatin, the GHRH-R gene has a complex genomic structure including more than 10 exons (Petersenn et al., 1998). It spans more than 8kb and the average size of its exons is approximately 100bp. The h GHRH-R gene was shown to span at least 15kb and to consist of 13 exons (Petersenn and Schulte, 2000). Investigation of the rat GHRH-R gene demonstrated that the coding region contains 14 exons, spanning 15kb of DNA (Miller et al., 1999). The coding sequence for the N-terminal extracellular domain of the rat GHRH receptor is contained within exons 15, whereas exons 514 encode the transmembrane (TM),

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intracellular loop (IL), extracellular loop (EL), and C-terminal domains (Miller et al., 1999). Alignment of the rat GHRH-R gene with the G-protein coupled receptors demonstrated that many of the intron/exon junctions and the lengths of the exons have been conserved, whereas the lengths of the introns are highly variable (Miller et al., 1999). Splicing variants of the GHRH-R gene have been found in the rat (Miller et al., 1999) and humans (Tang et al., 1995; Hashimoto et al., 1995). Three variant forms of the GHRH-R, which originate through differential splicing of intron X located at 1025/1026, have been demonstrated in human (Tang et al., 1995; Petersenn et al., 1998). Complete splicing of this intron generates the regularly spliced transcript; the second transcript contains the entire unspliced intron of 561bp; and the third transcript loses a 123bp intronic sequence at the intron (Petersenn et al., 1998). The functional significance of these transcripts has not been reported. However, the second splicing variant has been observed in some somatotrophic adenomas, which were derived from patients who did not respond to GHRH (Hashimoto et al., 1995). This observation may indicate this variant cannot perform normal functions. The truncated receptor has been shown to be unable to transduce the signal stimulated by GHRH (Hashimoto et al., 1995). The rat GHRH receptor gene is subject to differential splicing of exon 11, which generates two receptor isoforms differing by 41 amino acids within the third intracellular loop (IL) of the protein (Mayo, 1992; Miller et al., 1999). The short isoform of the GHRH receptor is predominant in pituitary cells. The large GHRH-R isoform is much less abundant than the short receptor isoform in normal pituitary; they differ in their ability to stimulate cAMP production in response to GHRH (Miller et al., 1999).

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To gain insight into the developmental and differential regulation of the GHRH-R, characterization of the 5'-promoter elements is essential. The 5-flanking region of the human GHRH-R gene has been cloned. A transient transfection study indicated that the 5-flanking region had pituitary-specific promoter activity and its promoter activity was dependent on Pit1 (Iguchi et al., 1999). RNase protection analysis showed the major transcription start site is 122 base pairs upstream from the translation start site in the normal pituitary gland (Iguchi et al., 1999). Also, some transcripts start from the downstream sites of the major transcript start site, which is often observed in a TATA-less promoter. Typical TATA homologies were not present in the 5-flanking sequence upstream from the transcription start site. Despite the absence of typical TATA homology, several sequences corresponding to the Pit-1 binding consensus (TATNCAT) were identified in the 5-flanking region (Petersenn et al., 1998; Iguchi et al., 1999; Miller et al., 1999). According to deletion mutation analysis, the region 310 to 130 was required for Pit-1 binding promoter activity (Iguchi et al., 1999) and there were two Pit-1 binding elements whose mutation was found to decrease hGHRH-R gene expression in pituitary tumor cells. Another important region for hGHRH-R gene expression was located from 130 to 120 in which one Pit-1 binding site was found. However, mutation of this binding site did not affect GHRH-R gene expression (Iguchi et al., 1999). This region may function in combination with the other two binding elements in region 310 to 130. The 5-flanking region for the rat GHRH-R gene also lacks TATA box motifs and contains two potential Pit-1 binding sites. The 5-flanking region of the rat GHRH-R gene contains three major transcription start sites, whereas only one was found in human (Miller et al., 1999).

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GHRH-R gene expression The GHRH-R transcripts have a highly specific distribution in the anterior pituitary (Mayo et al., 1995). During development of the anterior pituitary gland, GHRH-R transcripts were detected at embryonic day 16 in somatotrophs following the gene activation of Pit-1 (Andersen and Rosenfeld, 1994). Li et al. (1990) and Castrillo et al. (1991) found that GHRH-R was not expressed in the pituitary of Pit-1-defective Snell/Jackson dwarf mice (Li et al., 1990; Castrillo et al., 1991). This result indicates that the pituitary hypoplasia observed in these mice is due to the absence of GHRH-R, which is in turn due to the absence of functional Pit-1. It also showed that the expression of the GHRH-R gene depends on Pit-1 (Lin et al., 1993). The Pit-1 dependent transcription of the GHRH-R gene has also been demonstrated in human (Iguchi et al., 1999) and rat (Miller et al., 1999). Iguchi et al. (1999) found a functional Pit-1 binding site in the human GHRH-R promoter. They showed that the region from 310 to 130 was essential for the Pit-1-dependent expression of the GHRH-R gene. Two putative Pit-1 binding sites, P1 and P2, located from 129 to 123 and from 171 to 160, respectively, were found in this region. Also, mutation of P2 decreased GHRH-R gene expression (Iguchi et al., 1999). In rats, it was found that cotransfection of a Pit-1 expression construct enhances basal activity of the GHRH-R promoter in GH3 cells (Miller et al., 1999). A later study by Nogami et al. (2002) showed a Pit-1 binding sequence at 155 to 146 of the GHRH-R promoter region. Transcription of the GHRH-R gene is enhanced by glucocorticoids, but is inhibited by estrogen. Glucocorticoids were found to potentiate GHRH action and to enhance GH

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secretion in rats (Vale et al., 1983; Stephanou et al., 1992). GHRH-R gene expression was increased by glucocorticoids in rats (Lam et al., 1996). This result suggests that glucocorticoids stimulated GH synthesis and secretion, at least in part, via up-regulation of GHRH-R gene expression (Iguchi et al., 1999). A glucocorticoid-responsive element between 1456 and 1181 was proposed on the human GHRH-R gene. Two functional glucocorticoid response elements were found in the rat GHRH-R gene promoter region (Nogami et al., 2002). On the other hand, estrogen was found to inhibit GHRH-R gene expression (Petersenn et al., 1998; Gaylinn, 1999; Nogami et al., 2002). A recent study observed a significant inhibition of the GHRH-R promoter by -estradiol and a number of putative estrogen receptor response elements were found in the 5-flanking region of the GHRH-R gene (Petersenn et al., 1998). An estrogen-responsive element between 202 and 108 was found on the human GHRH-R gene (Petersenn et al., 1998). Horikawa et al. (1996) found that GHRH may be able to regulate expression of its own receptor. They suggested that GHRH up-regulates GHRH-R expression from the observation of a marked reduction of GHRH-R mRNA in GHRH-deprived neonatal rats.

Insulin-like growth factor II (IGF-II)

IGFs introduction The insulin-like growth factors (IGF-I and IGF-II) are pluripotent factors that regulate growth, differentiation, and the maintenance of differentiated function in numerous tissues and

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in specific cell types (Werner et al., 1994). Both IGFs are produced in largest amount by the liver and are secreted into the circulation, where they function as classical endocrine agents by interacting with specific cell-surface receptors present on target tissues (Werner et al., 1994). In addition to an endocrine effect, the IGFs also employ autocrine and paracrine modes of action (Werner et al., 1994). The IGFs were discovered on the basis of their ability to stimulate cartilage sulphation and to replace the sulphation factor activity of growth hormone both in in vivo and in vitro test systems (Salmon and Daughaday, 1957). Further purification and amino acid sequence determination revealed the existence of two separate molecules, insulin-like growth factor I and II, which were characterized by their high degree of homology with insulin (Rinderknecht and Humbel, 1978a,b). IGF-I and IGF-II belong to a family of structurally related polypeptides, which also includes insulin and relaxin. Members of this hormone family exhibit 40-50% amino acid homology with each other (Blundell et al., 1983; Daughaday and Rotwein, 1989; Sussenbach, 1989; Rechler and Nissley, 1990; Werner et al., 1994). IGF-I and IGF-II display a wider range of developmental and tissue-specific expression than any other known growth factor s(Schofield, 1992; Schofield et al., 1993), and play a pivotal role in promoting embryonic and fetal growth (Engstrm et al., 1998). The IGFs exert most of their biological action via three membrane receptors, the type I and type II receptors, and the insulin receptor (Engstrm and Heath, 1988). The IGFs also associate with six specific IGF-binding proteins (IGFBPs) (McCusker and Clemmons, 1992), some of which are membrane-associated and some of which are soluble (ODell and Day, 1998).

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Biological function of IGF-II High concentrations of IGF-II peptide and mRNA in utero suggest IGF-II is important in the fetus (Han et al., 1987). It plays a key role in mammalian growth, influencing fetal cell division and differentiation and possibly metabolic regulation (ODell and Day, 1998). A restriction fragment length polymorphism in the mouse IGF-II gene was found to be associated with 21-d weight in males and 42-d weight in females. It also indicated an interaction of IGF-II and sex, which would help developing the genetic model for growth (Winkelman and Hodgetts, 1992). IGF-II is also an important regulator of body size. Mice that lack IGF-II are viable dwarfs that are 60% the size of wild type animals. These IGF-II knockouts were shown to have significant fetal growth retardation, especially in the early stages of gestation. The growth rate appeared to be normal after birth (DeChiara et al., 1990). On the other hand, clinical evidence suggests that increased levels of IGF-II have a positive effect on growth and development in vivo (Engstrm et al., 1998). Overexpression of IGF-II can cause rare genetic syndromes, for example, Wiedemann Beckwith syndrome (Engstrm et al., 1988; Nystrm et al., 1992; Ekstrm et al., 1992; Ward, 1997), which leads to overgrowth, as well as growth disturbances, and increased frequencies of neoplasia (Nystrm et al., 1992). Transgenic mice with overexpression of IGF-II were shown to have organ overgrowth and tumor formation (Ward et al., 1994; Rogler et al., 1994; Bates et al., 1995; Rossetti et al., 1996; Vella et al., 2002). IGF-II is involved in other human diseases associated with abnormal cell proliferation, such as cancer and overgrowth syndromes (Toretsky and Helman, 1996; Morison et al., 1996; Eggenschwiler et al., 1997; Sperandeo et al., 2000). IGF II was shown to have a role

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in fetal development in pigs (Daughaday et al., 1986; Hausman et al., 1991). Research also showed a combination effect of IGF-II and IGF-I on post weaning weight gain in pigs (Lamberson et al., 1996). No direct effect of IGF-II on growth and carcass traits for cattle was published. Thus far, polymorphism information for IGF-II gene has not been reported in domestic animals. The goal of this study is to look for genetic polymorphisms in bovine IGF-II gene and analyze the associations of the polymorphisms with growth and carcass traits in beef cattle. Studies of its in vitro role demonstrate that IGF-II exerts a wide range of biological activities in cells in culture. It promotes cell proliferation by acting on the cell division cycle (DNA replication) as well as on the cell growth cycle (Cell enlargement) (Zetterberg et al., 1984; Dafgrd, 1990); it induces differentiation in vitro (Florini et al., 1991); it profoundly affects cellular survival and counteracts apoptosis in some cell systems (Biddle et al., 1988; Granerus et al., 1995; Granerus and Engstrm, 1996); it stimulates hormone synthesis and secretion in ovarian granulos and theca cells (Giudice, 1992); and it can stimulate motility in cultured rhabdomyosarcoma cells (Minniti et al., 1992).

Receptors and binding proteins

The IGFs exert their biological actions by binding to a family of specific membraneassociated glycoprotein receptors that include the insulin receptor, IGF-I receptor (type I receptor), and IGF-II receptor (type II receptor) (Czech, 1989). The type I receptor shares structural similarities and properties with the insulin receptor (Werner et al., 1994). The type II

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receptor is identical to the cation-independent mannose-6-phosphate (M-6-P) receptor (Werner et al., 1994). The affinities and kinetic properties differ between each of the ligandreceptor interactions. The type I receptor has the highest affinity for IGF-I and the type II receptor has the highest affinity for IGF-II. The insulin receptor binds both IGF-I and IGF-II with low affinity (Steele-Perkins et al., 1988; Werner et al., 1992; Engstrm et al., 1998). The type I receptor is a heterodimeric transmembrane protein that consists of two alpha and two beta subunits. Ligand binding induces tyrosine specific autophosphorylation of the receptor, which is followed by different kinds of biological response. The type I receptor is considered to be responsible for nearly all biological effects exerted by the IGFs (de Meyts et al., 1994; Jones and Clemmons, 1995). The type II receptor is a monomeric protein, which consists of a major extracellular portion as well as a single hydrophobic transmembrane helix and a minor cytoplasmic sequence. It is involved in sorting of lysosomal enzymes as well as endocytosis (Engstrm et al., 1998; ODell and Day, 1998). Mouse genetic experiments indicate that the primary functions of the type II receptor are internalization and degradation of IGF-II. Loss or inactivation of the type II receptor gene results in a general overgrowth that is improved in the absence of the IGF-II ligand (Filson et al., 1993; Wang et al., 1994). The IGFs are present in the circulation and in extracellular fluids and tightly bind to a family of specific IGF binding proteins (IGFBP) (McCusker and Clemmons, 1992). Both

membrane-associated and soluble binding proteins exist (ODell and Day, 1998). In human adults, 90% of the circulating IGFs are complexed with IGFBP-3 (ODell and Day, 1998). The roles of the IGFBPs are to prolong the half-life of the IGFs, act as a main transporter,

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inhibit or promote IGF action, and store pre-synthesized IGFs (Jones and Clemmons, 1995). Generally membrane bound forms enhance IGF action by attracting IGFs to the region of the receptor and the soluble forms of IGFBPs are inhibitory (ODell and Day, 1998).

Structure of IGF-II protein and IGF-II gene IGF-II is produced as pre-propeptides that contain a signal peptide of 24 amino acids as well as a carboxy-terminal peptide of 89 amino acids, both of which are cleaved posttranslationally to produce the mature 67 amino acid monomeric plasma protein (ODell and Day, 1998). The mature peptides consist of four distinct domains: A, B, C, and D. The structures of the A and B chains show strong homology with that of pro insulin (Engstrm et al., 1998). The structure of the domains is important in receptor and binding protein recognition (Humbel, 1990). IGF-II cDNA was first cloned in human (Bell et al., 1984) and rat (Dull et al., 1984) in 1984. To date, IGF-II cDNA has been cloned in mouse (Bell et al., 1986), as well as in domestic animals, including sheep (OMahoney and Adams, 1989), pig (Catchpole and Engstrm, 1990), cow (Boulle et al., 1993), mink (Ekstrm et al., 1993), and horse (Otte and Engstrm, 1994; Otte et al., 1996). Comparison of the coding sequences, as well as the primary protein structure, within these species shows a high degree of conservation in the IGFII gene. A maximum of only six out of 67 amino acid differences were shown (Ekstrm et al., 1993).

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The IGF-II gene comprises several exons and multiple promoters and, therefore, has multiple transcripts (Schofield and Tate, 1987; Hedley et al., 1989; Joujou-Sisic et al., 1993; Bcklin et al., 1998). The human IGF-II gene was assigned to the distal end of the short arm of chromosome 11 (Brissenden et al., 1984). It spans 30 kb and is adjacent to the 3 end of the insulin gene (Tricoli et al., 1984). The hIGF-II gene comprises 9 exons and 4 promoters. Exons 7, 8 and 9 encode prepro IGF-II protein, and there is a long 3 untranslated region (UTR) within exon 9 (Ueno et al., 1989; Werner et al., 1994). Exons 1 to 6 are non-coding and form alternative 5-UTRs of different RNA molecules (Dull et al., 1984; Vu and Hoffman, 1994). The four promoters precede some of the leading exons and regulate transcription of the IGF-II gene in a tissue and developmental specific manner (Vu and Hoffman, 1994). The IGF-II gene of mouse, rat, and horse has 6 exons and 3 promoters and is assigned to mouse chromosome 7, rat chromosome 1, and horse chromosome 12, respectively. In these three species, exons 4, 5, and 6 of the IGF-II gene encode the 180bp precursor protein. Exons 1, 2 and 3 are non-coding and are used to form the alternative 5-UTR of different IGF-II transcripts (Engstrm et al., 1998). The Sheep IGF-II gene is on chromosome 21 and consists of 10 exons and 3 promoters. Exons 8, 9 and 10 of the sheep IGF-II gene are the coding region (Engstrm et al., 1998). Thus far, the complete bovine IGF-II gene has not been cloned.

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IGF-II gene expression Transcription of the IGF-II gene is driven by multiple promoters. Each promoter is associated with different 5-untranslated leader exons and thus can generate transcripts of different lengths (Engstrm et al. 1998). In rodents there are three promoters, P1, P2 and P3, which control the transcription of each of the three leader exons (Frunzio et al., 1986; Soares et al., 1986). The human and ovine IGF-II genes have an extra promoter, P1, located 5 to the most upstream leader exon (Schofield and Tate, 1987; Ohlsen et al., 1994; Jin et al., 1995). The P1 promoter contains no TATA or CAAT boxes but includes an SP1 recognition signal and a repeated GC rich motif. It has the capacity to bind the transcription factors CEBP and C-ERB-b, as well as LAP (van Dijk et al., 1992; Sussenbach et al., 1993; Rodenburg et al., 1996). The P1 promoter is a weak promoter in both human and rodent (Ueno et al., 1987; van Dijk et al., 1993) and appears to be silent in some tissues of horse (Otte et al., 1998). The human and rodent P2 promoter contains TATA and CAAT boxes, as well as two SP1 recognition sequences and two Egr 1 binding motifs (Frunzio et al., 1986; Soares et al., 1986; de Pagter-Holthuizen et al., 1987; van Dijk et al., 1993). This promoter can be repressed by binding of the WT1 protein and p53 protein (Drummond et al., 1992; Ward et al., 1995; Zhang et al., 1996). The primary structure of the P2 promoter is strongly conserved in the ovine and bovine IGF-II genes (OMahoney et al., 1991; Boulle et al., 1993). The transcript driven by promoter P3 is predominantly active. It contains a single TATA box and several SP1 recognition sequences (Frunzio et al., 1986; Soares et al., 1986; de Pagter-Holthuizen et al., 1987). The P3 promoter is activated by binding to the AP1

31

complex (Caricasole and Ward, 1993) and repressed by binding to the WT1 protein (Drummond et al., 1994; Ward et al., 1995) and p53 (Zhang et al., 1998). The IGF-II gene is active in nearly all human embryonic and fetal issues. Transcription of the gene declines rapidly after birth in most tissues (Scott et al., 1985). In rodents, IGF-II is expressed at high levels throughout embryogenesis and fetal development in most tissues. All three promoters are downregulated after birth and the transcriptional activity continues only in exchange tissues surrounding the central nervous system (Soares et al., 1985; Brown et al., 1986; Frunzio et al., 1986; Lund et al., 1986; Beck et al., 1987; Gray et al., 1987; Murphy et al., 1987). In human, transcripts derived from P1 are exclusively found in liver and choroids plexusleptomeninges in adults (Teerink et al., 1995; Li et al., 1996). P1 derived transcripts are mostly found in low quantities in fetal liver, whereas P2 and P3 derived transcripts are found in fetal, as well as adult tissues, with the P3 promoter being predominantly active (Nielsen et al., 1990; Ikejiri et al., 1991; de Moor et al., 1994; Newell et al., 1994).

Imprinting of IGF-II gene Genomic imprinting is a phenomenon in which only one of the parental alleles is expressed. The imprint is a mark established during germ-cell development to distinguish between the paternal and maternal copies of the imprinted gene. The imprint is maintained throughout embryo development and erased in the embryonic gonads to prepare for a new imprint (Surani, 1998).

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The IGF-II gene was one of the first genes shown to be imprinted. The paternal IGF-II allele is transcribed, whereas the maternal allele is silent (de Chiara et al., 1991). An exception occurs in the adult l ver, where IGF-II gene expression is biallelic (Vu and Hoffman, 1994). i DNA methylation is essential in the process of parental imprinting (Li et al., 1993). In the mouse IGF-II gene, two differentially methylated regions (DMRs), DMR1 and DMR2, have been mapped. DMR1 is located in the promoter region, and DMR2, an intronic 3 kb CpG island (a region of DNA with a high G +C content), is found in the 3 region of the gene (Birger et al., 1999). Both DMRs are more heavily methylated on the maternal allele that is not transcribed (Sasaki et al., 1992; Feil et al., 1995). These regions are involved in the overall transcriptional regulation of the IGF-II gene (Dell et al., 1997). Studies show that DNA methylation deficiency, or deletion of DMRs, can cause loss of imprinting (LOI) (Elson and Bartolomei, 1997; Wutz et al, 1997; Nicholls et al., 1998), which can cause overexpression of the IGF-II gene. Loss of imprinting and biallelic expression of the IGF-II gene has been reported in Beckwith-Wiedemann syndrome, which is characterized by overgrowth and with risk of developing Wilms kidney tumors (Zhan et al., 1995; Oda et al., 1997; Okamoto et al., 1997; Sohda et al., 1997; Oda et al., 1998). Loss of imprinting in the IGF-II gene has also been suggested in a number of cancers, including Ewing sarcoma and rhabdomyosarcoma (Zhan et al., 1995), Glioma (Uyeno et al., 1996), gynecological tumor (Yaginuma et al., 1997), various testicular tumors (Nonomura et al., 1997), carcinosarcoma of the endometrium (Roy et al., 2000), and epithelial ovarian cancer (Chen et al., 2000). It has been demonstrated that loss of

33

imprinting is a stage specific event during carcinogenesis (Harris et al., 1998). Therefore, loss of imprinting of the IGF-II gene appears to play an important role in tumorigenesis.

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CHAPTER 3 ASSOCIATIONS OF POLYMORPHISMS OF THE PIT-1 GENE WITH GROWTH AND CARCASS TRAITS IN ANGUS BEEF CATTLE

Abstract

Pit-1 is a pituitary specific transcription factor that has been shown to positively regulate the expression of growth hormone, prolactin, and thyrotrophin subunit (TSH). Mutations in the Pit-1 gene can cause growth disorders in human and mice.

Therefore, it may be a good candidate gene for genetic markers for growth and carcass traits. Angus beef cattle, which were divergently selected for high or low blood serum IGF-I concentration, were used for this research. Three polymorphisms, Pit1I3H (Hinf I), Pit1I3NL (Nla III), and Pit1I3N (Nci I), were detected in intron 3 of the Pit-1 gene. One polymorphism, Pit1I4N (BstN I), was found in intron 4 and one single nucleotide polymorphism (SNP) (Pit1I5) was found in intron 5. For the Pit1I3H polymorphism, genotypes CC, CD, and DD were detected with frequencies of .12, .49, and .39, respectively; there was an AAT deletion in allele C. For the Pit1I3NL polymorphism, genotypic frequencies of .12, .47, and .41 were observed for GG, GH, and HH, respectively; a G to C transition in allele G was observed. The Pit1I3N polymorphism was found to have genotypes MM, MN, and NN with frequencies of .12, .51, and .37, respectively. For the Pit1I4N polymorphism in intron 4, genotypes EE, EF, and FF were detected and had frequencies of .12, .47, and .41, respectively; a G to T transition was

35

found in allele E. The genotypic frequencies of the above polymorphisms were observed in 98 Angus beef cattle. The previously reported polymorphism in exon 6, Pit1E6H (Hinf I), was also studied in 416 Angus beef cattle. The frequencies of genotype OO, OP, and PP were .11, .44, and .45, respectively. The intron 5 SNP Pit1I5 was found to have genotypes AB and BB with frequencies of .09 and .91, respectively, among 185 Angus cattle studied. No AA individuals were found. The A allele had a single nucleotide transition from G to T. Associations of the polymorphisms with growth traits, carcass traits, and IGF-I concentration were analyzed using the general linear model procedure in SAS (1989). No significant associations were observed for Pit1I3H, Pit1I3NL, Pit1I3N, Pit1I4N, or Pit1I5. For Pit1E6H, a significant relationship was found with birth weight (P=. 03), where genotype OO had a lower mean than genotypes OP and PP. Genotype OO was found to be superior for preweaning gain (P= .01) and weight gain during the 20-d period between weaning and the beginning of the postweaning test (P= .06). Significant relationships with Pit1E6H genotypes were observed for backfat thickness (P= .01) in the high IGF-I line, and for marbling score (P= .03) in the low line.

Introduction

Growth and carcass traits, which are under the control of multiple genes, are economically important traits in livestock. Selection of animals with higher growth rate and better carcass composition is of great significance. Current technologies enable

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scientists to improve upon the accuracy and efficiency of traditional selection by applying genetic markers. This can be done through marker assisted selection (MAS), which combines information on molecular genetic polymorphisms (marker loci) with data on phenotypic variation among individuals (and their relatives). Therefore, genetic polymorphisms that are significantly associated with certain traits of interest are very useful. Pit-1, which is also termed Growth Hormone Factor 1(GHF1), was used as a candidate gene for genetic markers in this study. Pit-1 is a pituitary-specific transcription factor that is responsible for pituitary development and hormone expression in mammals (Cohen et al., 1997). Pit-1 was first cloned in 1988 and was shown to control transcription of the growth hormone, prolactin (Bodner et al., 1988; Ingraham et al., 1988; Nelson et al., 1988; Mangalam et al., 1989), thyroid-stimulation hormone -subunit (TSH) (Simmons et al., 1990; Steinfelder et al. 1991; Gordon et al., 1993; Lin et al., 1994), and growth hormone releasing hormone receptor (GHRH-R) genes (Lin et al., 1992), and the Pit-1 gene itself (Rhodes et al., 1993). Pit-1 was also shown to play a critical role in differentiation and proliferation of three pituitary cell types, somatotropes, lactotropes and thyrotropes (Doll et al., 1990; Simmons et al., 1990). Mutations in the Pit-1 gene lead to the absence of growth hormone and pituitary hypoplasia in mice (Li et al., 1990), and a syndrome of congenital hypothyroidism, dwarfism, and prolactin deficiency in humans (Pfaffle et al., 1992). Association studies in domestic animals have shown that Pit-1 is associated with growth rate, carcass traits, and milk production. Pit-1 was found to be related to birth weight (Yu et al., 1996),

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weaning weight, and average daily gain in swine (Yu et al., 1995). Also, associations were discovered with backfat, as well as lean to fat ratio in pigs (Yu et al., 1995; Stancekova et al., 1999). In cattle, Pit-1 was found to be associated with body weight, and milk protein and fat yields (Renaville et al., 1997). This study was designed to screen the entire Pit-1 gene for polymorphisms and to analyze the association of these polymorphisms with growth and carcass traits in Angus beef cattle.

Materials and Methods

Animals Angus beef cattle, which were divergently selected for blood serum insulin-like growth factor I (IGF-I) concentration, were used as the experimental animals. Selection began in 1989 at the Eastern Ohio Resource Development Center (EORDC), using 100 spring-calving (50 high line and 50 low line) and in 1990 using 100 fall-calving (50 high line and 50 low line) purebred Angus cows with unknown IGF-I levels. Cows from the base population were randomly assigned to the selection lines. Each year, four bull calves with the highest and four bull calves with the lowest IGF-I concentration were selected for breeding within the selection lines. Approximately eight cows were culled from each selection line each year based on physical unsoundness, failure to conceive in two consecutive years, and oldest age. These cows were replaced with the same number of pregnant heifers having the highest or lowest serum IGF-I concentration (Davis and Simmen, 1997).

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In this study, 98 Angus cattle born in the years of 1995, 1996 and 1997 were genotyped for Pit1I3H, Pit1I3N, Pit1I3NL, and Pit1I4N. The observed genotypes of these four polymorphisms, and the previously reported Pit1E6H (Woollard et al., 1994) indicated the existence of linkage disequilibrium. Therefore, results from the Pit1E6H association analysis could represent Pit1I3H, Pit1I3NL, Pit1I3N and Pit1I4N. Fourhundred-sixteen Angus cattle born in 1995, 1996 and 1997 were genotyped for Pit1E6H. For the intron 5 single nucleotide polymorphism, Pit1I5, 185 Angus cattle born in the years of 1995 and 1996 were genotyped.

Methods

DNA extraction Genomic DNA was isolated from blood of Angus cattle. Blood samples were collected into 15mL tubes containing EDTA during the IGF-I selection experiment at
o EORDC. They were then stored in a 4 C refrigerator until ready for DNA isolation. The

extraction procedure used was: 1. Centrifuge blood sample tubes for 20 min at 2,200 rpm to isolate buffy coats. 2. Pipet 1 to 2 buffy coats into each conical 15mL polypropylene tube. Place no more than two buffy coats into each tube. 3. Mix the remaining EDTA blood. Repeat steps 1 and 2 to ensure the entire buffy coat is removed from the blood sample. 4. Add 4 to 6 mL of .14M NH4 Cl to each polypropylene tube. Invert to mix. 5. Incubate at room temperature for 15 to 20 min.

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6. Centrifuge for 15 min at 2,200 rpm. 7. Discard the supernatant. Add 8 to 10mL of .14M NH4 Cl to each polypropylene tube, vortex to resuspend pellet. 8. Incubate at room temperature for 15 min. 9. Centrifuge for 10 min at 2,200 rpm. 10. Discard supernatant. If pellet is white, continue with step 11. If a large proportion of the pellet is composed of red blood cells, then repeat steps 7 to 9. 11. Carefully rinse the inside of the polypropylene tube with 1X PBS. Be sure not to disturb the pellet. PBS= .15M NaCl .004M NaH2 PO4 .03M Na2 HPO4, pH=7.1

12. Add 3mL of lysis buffer A to each polypropylene tube. Vortex until pellet is in small clumps. Buffer A = .01M Tris-HCl .4M NaCl .002M Na2 EDTA, pH=8.2

13. Add 200L of 10% SDS to each polypropylene tube. Do not vortex. 14. Add 250 L of pre-thawed 5mg/mL proteinase K to each tube without vortexing. 15. Place the tubes on a tube inverter for 10 to 15 min. 16. Incubate in a 37o C water bath overnight.

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17. Add 2mL of saturated NaCl (6M) to each Oakridge centrifuge tube (a leak proof polypropylene copolymer tube, which allows repeatedly autoclave and can be used up to 50,000g in centrifuges). 18. Pour the viscous solution in the 15mL polypropylene tube into the Oakridge tube and shake vigorously for 15 sec or until a white precipitate forms. 19. Centrifuge at 12,000 rpm for 45 min. 20. Prepare glass screw-capped tubes containing 2 volumes of absolute ethanol. 21. Remove the tubes from the centrifuge and pour the supernatant containing the DNA into the glass tubes with 2 volumes of absolute ethanol. Invert the glass tubes several times and recover the DNA (white floculus) with a pipette. 22. Transfer the DNA to a 1.5mL tube containing 50 l of TE. TE= .01M Tris-HCl .0002M Na2 EDTA; pH= 7.5

23. Allow the DNA to resuspend in the TE for at least 30 min or overnight in a 37o C water bath. Shear DNA with pipettes. 24. DNA can be stored indefinitely at 4o C.

PCR Polymerase chain reaction (PCR) was used to amplify the DNA fragment from genomic DNA. Primers were designed based on either the DNA sequence or the cDNA sequence of the bovine Pit-1 gene from GenBank. Because there is only cDNA information for the bovine Pit-1 gene (except for intron 5), primers were selected on the

41

boundary of each exon in order to cover the whole intron. Exon and intron boundaries were determined by comparing cDNA sequences of bovine with DNA sequences of human. Exons were also covered as much as possible using overlapping primers (forward primer for one PCR reaction overlapped with reverse primer for another PCR reaction, so that the complete exon was covered). PCR amplification was performed using four primers, Pit1I3, Pit1I4, Pit1I53, and Pit1E6 (Table 3.1). Primer Pit1E6 was published by Woollard et al. (1994). The Pit1I3 primer amplified a DNA fragment 3kb in length, which covered all of intron 3 (Genbank accession number: AY183915, and AY183916). The PCR was performed in 30L containing 10pmol of forward primer and the same amount of reverse primer, 200M dNTPs, 1X reaction buffer, which contained 1.5mM MgCl2 , 1 unit of Taq-DNA polymerase and 100ng of genomic DNA as template. Conditions were 97o C for 2 min, followed by 35 cycles of 95o C for 45s, 60o C for 1 min, and 72o C for 90s. After 35 cycles, reactions were finished by an extension of 5 min at 72o C. The Pit1I4 primer amplified a 1kb DNA fragment including all of intron 4 (Genbank accession number: AY183917). The PCR reaction mixture was the same as described above. The conditions were 97o C for 2 min, followed by 35 cycles of 95o C for 45s, 63o C for 1 min, and 70o C for1 min. After 35 cycles, reactions were finished by an extension of 5 min at 72o C. The finished reaction mixtures were stored at 4o C. The Pit1I53 primer amplified a 355bp fragment in intron 5. The PCR conditions were 97o C for 2 min, followed by 35 cycles of 95o C for 45s, 63o C for 1 min, and 72o C for 1 min. After 35 cycles, reactions were finished by an extension of 5 min at 72o C. The finished reaction mixtures were stored at 4o C.

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The Pit1E6 primer amplified a 465bp region in exon 6. Conditions for PCR were described by Woollard et al. (1994).

Detection of Genetic Variation When amplification was achieved, single strand conformation polymorphism (SSCP) was used to screen for mutations within the amplified segment. Since the optimal size of DNA for SSCP is under 400bp, the products of primers Pit1I3 and Pit1I4 were digested with restriction enzymes to reduce the size of the DNA fragments. The reaction mixture, which included 10L of PCR product, 10L of ddH2 O, and 12L of loading dye, was denatured at 95C for 5 min. The samples were placed in ice for 10 min to prevent renaturing of the denatured single strand of DNA. The samples were then loaded on 8-10% polyacrylamide gels. The concentration of the polyacrylamide gel was based on the size of DNA fragment. Adding 10% urea or 10% formamide to the polyacrylamide gels improved the resolution of the DNA bands on the gel. The samples were run in 1x TBE buffer at 200 volts for 16 to 20 h at a constant temperature of 812C or room temperature. The temperature was adjusted to optimize the results. Gels were then stained with .01% Ethidium Bromide for 10 min and viewed under UV light. After a polymorphism was detected by SSCP, and in order to determine the nature of the polymorphism, the PCR products of the two homozygous genotypes (with two copies of same allele) were sent to the Plant-Microbe Genomics Facility at The Ohio State University for sequencing using an ABI 3700 analyzer. Before sending samples for sequencing, PCR products were purified. The DNA fragment, which needed to be sequenced, was cut from the agarose gel and extracted with the Qiagen Gel DNA

43

Extraction kit (Qiagen Inc., Valencia, CA 91355, USA). Sequencing primers were the same as the PCR primers.

Genotyping Sequencing results were analyzed for nucleotide change. If a change was recognized by a restriction enzyme, this corresponding restriction enzyme was used to perform PCR-RFLP (restriction fragment length polymorphism), which was used to genotype the animals. Restriction enzyme HinfI, NlaIII, NciI, BstNI and HinfI were used to genotype polymorphism Pit1I3H, Pit1I3NL, Pit1I3N, Pit1I4N, and Pit1E6H,

respectively. The procedure of PCR-RFLP included amplifying the corresponding DNA segment by PCR; digesting the PCR product with restriction enzymes; and running the digested DNA on an agarose gel containing .003% Ethidium Bromide. The gel was viewed under UV light. Four polymorphisms were detected intron 4. If there was no restriction site for the polymorphism, SSCP was used to genotype the animals. Such was the case for the polymorphism in intron 5 (Pit1I53). in intron 3 and one in

Statistical Analysis

Growth traits and IGF-I concentrations Associations of the animal genotypes with growth traits and IGF-I concentrations were determined by analysis of variance of quantitative traits, which included birth weight, weaning weight, preweaning gain, on-test weight, weight at d 28 and 56 of the

44

140-d postweaning test, off-test weight, weight gain during the 20-d period between weaning and the beginning of the postweaning test (Gain20), postweaning gain, serum IGF-I concentration on d 28, 42, and 56 of the 140-d postweaning test, and mean serum IGF-I concentration, using General Linear Model (GLM) procedures in SAS (1989). Fixed effects of genotypes, year, season of birth (spring vs fall), age of dam, sex (bull vs heifer), and IGF-I selection line (high vs low) were included as independent variables in the linear model. Weaning age of calf was treated as a covariate in the model when analyzing weaning weight, preweaning gain, and Gain20. On-test age of calf was used as a covariate in the model when analyzing on-test weight, weight at d 28 and 56 of the 140-d postweaning test, off-test weight, postweaning gain, serum IGF-I concentration on d 28, 42, and 56, and mean serum IGF-I concentration. Calf age was not included in the analysis of birth weight. Data were also analyzed separately within the high and low IGF-I selection lines using the same model, except that the selection line was omitted from the model.

Carcass traits Carcass traits of approximately 146 Angus bulls that were not saved for breeding were analyzed for Pit1E6H and 71 such Angus bulls for Pit1I5. Age at slaughter ranged from 13 to 15 mo. Bulls born in the years 1995 and 1996 were slaughtered at The Herman Falter Packing Co. in Columbus, OH. Bulls born in 1997 were slaughtered at the Mahan Packing Company in Bristolville, OH. Carcass traits measurements were performed by USDA graders in these two packing companies. Carcass traits analyzed included: Backfat thickness (cm)

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Ribeye area (cm2 ) Kidney, pelvic, and heart fat (KPH) (%) Hot carcass weight (kg) Marbling score (1- devoid, 2- practically devoid, 3- traces, 4- slight, 5- small, 6modest, 7- moderate, 8- slightly abundant, 9- moderately abundant, 10- abundant) Quality grade (6- standard-, 7- standard, 8- standard+, 9- select-, 10- select, 11- select+, 12- choice-, 13- choice, 14- choice+) Yield grade (from 1 to 5)= 2.5+ (2.5 x adjusted fat thickness (inch)) + (.2 x KPH) + (.0038 x hot carcass weight (lb)) (.32 x ribeye area (inch2 )) The model used to analyze carcass traits was the same as the one for growth traits, except that sex was deleted from the model, since only bulls had carcass data, and slaughter age was added in the model as a covariate instead of on-test age.

Results and Discussion

Polymorphisms Three polymorphisms, Pit1I3H (Hinf I), Pit1I3NL (Nla III), and Pit1I3N (Nci I), were detected in intron 3 of the Pit-1 gene. One polymorphism, BstNI, was found in intron 4. Each of these four genetic variations had three genotypes (Table 3.2). For intron 5, one genetic marker (Pit1I5) was found. However, for the 185 animals genotyped for this polymorphism, only genotypes BB and AB were found; no AA individuals were detected (Table 3.2).

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The genotypic frequencies of the intron 3 and 4 polymorphisms were determined in 98 Angus beef cattle (Table 3.3). For the Pit1I3H polymorphism, genotypes CC, CD, and DD were detected with frequencies of .12, .49, and .39, respectively. For the Pit1I3NL polymorphism, genotypic frequencies of .12, .47, and .41 were observed for GG, GH, and HH, respectively. The Pit1I3N polymorphism was found to have genotypes MM, MN, and NN with frequencies of .12, .51, and .37, respectively. For the Pit1I4N polymorphism in intron 4, genotypes EE, EF, and FF were detected and had frequencies of .12, .47, and .47, respectively. The previously reported polymorphism Pit1E6H (Woollard et al., 1994) was also studied in 416 Angus beef cattle. The frequencies of genotypes OO, OP, and PP were .11, .44 and .45, respectively. The observed haplotypes of polymorphisms Pit1I3H, Pit1I3N, Pit1I3NL, and Pit1I4N, and the previously reported polymorphism Pit1E6H indicated the existence of profound linkage disequilibrium in these cattle, with the predominant linkage groups being C-GM-E-O and D-H-N-F-P (Table 3.4). The intron 5 SNP, Pit1I5, was found to have genotypes AB and BB with frequencies of .09 and .91, respectively, among 185 Angus cattle studied. Allele A had a low frequency (frequency of A allele= .048, Table 3.3). Homozygote AA was not observed in these animals. Frequency of AA was estimated based on the frequency of allele A among the 185 animals. The estimated frequency of genotype AA was extremely low (.0025). No significant relationships were found for this polymorphism. Therefore, only 185 Angus cattle were genotyped for this polymorphism. Genotypic frequencies for the various polymorphisms were not found to be significantly different between the high and low IGF-I selection lines (Table 3.3). The

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P-values for the ?2 test were .66, .45, .80, .45, .91, and .89 for polymorphisms Pit1I3H, Pit1I3N, Pit1I3NL, Pit1I4N, Pit1E6H, and Pit1I5, respectively. The characteristics of these polymorphisms were studied using sequencing. The Pit1I3H polymorphism was shown to have an AAT deletion in allele C. The Pit1I3NL polymorphism had a G to C transition in allele G. For the Pit1I4N polymorphism, a G to T transition was observed in allele E. For Pit1I5, the A allele had a single nucleotide transition from G to T (Table 3.2).

Association Analysis No significant associations with growth traits and IGF-I concentration were observed for Pit1I3H (HinfI), Pit1I3NL (NlaIII), Pit1I3N (NciI), or Pit1I4N (BstNI) (Tables 3.5, 3.6, 3.7, and 3.8). These results may be due to the small sample of animals genotyped (98 Angus cattle). However, the observed genotypes of these four polymorphisms, and the previously reported Pit1E6H (HinfI) polymorphism were highly linked. Therefore, results from the Pit1E6H association analysis could represent Pit1I3H, Pit1I3NL, Pit1I3N, and Pit1I4N. The Pit1E6H polymorphism in exon 6 was previously published (Woollard et al., 1994). Our association study showed significant relationships of Pit-1 genotypes with birth weight (P= .03) and preweaning gain (P= .01). Moderate association was also observed with weight gain during the 20-d period between weaning and the beginning the postweaning test (Gain20) (P= .06). Animals with genotype OO had a lower birth

weight than animals with genotypes OP (P= .04) and PP (P= .008). However, animals with genotype OO had higher preweaning gains than animals with genotypes OP (P=

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.06) and PP (P= .004). OO animals also tended to have higher Gain20 than OP and PP animals. When animals in the high and low IGF-I lines were analyzed separately, significant associations were found for preweaning gain in the high line (P= .03) (Table 3.10) and for Gain20 in the low line (P= .04) (Table 3.10). Moderate associations existed between genotypes and birth weight in the high IGF-I line (P= .07) (Table 3.11). These results were consistent with those found when analysis was done including both lines (Tables 3.10, 3.11). These results indicate that genotype OO may be the favorable genotype for preweaning gain and Gain20. Previous studies have shown that Pit-1 was related to birth weight (Yu et al., 1995; Yu et al., 1996), weaning weight, and average daily gain in pigs (Yu et al., 1995). Results from the present study further verify that Pit-1 is related to growth traits in cattle. Although no association between this polymorphism and IGF-I concentration was observed, there was a trend that animals with higher weight tended to have lower IGF-I concentration (Tables 3.9, 3.10 and 3.11). A previous study by Davis and Simmen (1997) showed a negative genetic

correlation of IGF-I concentration with postweaning weights and gains. In this study, we found that most growth traits tended to have lower values when IGF-I concentration was higher. This further supports the results of Davis and Simmen (1997). For the intron 5 polymorphism, Pit1I5, no significant associations between the genotypes and the growth traits were found (Table 3.12). However, when animals in the high and low IGF-I lines were analyzed separately, moderate associations were found between genotypes and Gain20 (P= .07), postweaning gain (P= .08), and IGF-I concentration on d 56 (P= .07) in the high line (Table 3.13). Genotype AB tended to have higher Gain20 than genotype BB. For postweaning gain, animals with genotype

49

AB also had higher means than animals with genotype BB. These results indicate that allele A may be the favorable allele for postweaning gain and Gain20. However, AB animals also had higher IGF-I concentration at d 56 than BB animals. This result is inconsistent with previously published data that showed negative genetic correlations between IGF-I concentration and postweaning weights and gains (Davis and Simmen, 1997). Since the sample size used in analyzing this SNP (185 Angus cattle for growth traits) was small, and since the frequency of the A allele was low, homozygous AA individuals were not observed. Therefore, a larger sample is necessary to further analyze this polymorphism and the effect of allele A. Effects of the Pit1E6H polymorphism on carcass traits were studied. When analysis was done including animals in both high and low IFG-I selection lines, no significant relationships were found. However, a moderate association was observed for backfat thickness (P= .09); genotype OP had a higher value for backfat than genotypes OO and PP. Significant relationships with Pit1E6H genotypes were observed for backfat thickness (P= .01) in the high IGF-I line. Animals with genotype OP had a higher value than animals with genotype PP (P= .005), but genotypes OO and OP were not significantly different. A moderate relationship was observed with quality grade (P= .06) in the high line; animals with genotype OP tended to have a higher grade than animals with genotypes OO and PP. In the low IGF-I line, a significant association was found with marbling score (P= .03); genotype OO had higher scores than genotypes OP (P= .01) and PP (P= .03). A moderate relationship was also observed with quality grade (P= .07) in the high line. Genotype OO tended to have a higher quality grade than genotypes OP and PP.

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Relationships of Pit1I5 genotypes with carcass traits were also analyzed for 71 bulls, but no significant associations were observed (Table 3.18). When data were separated by line, the number of samples was 35 for the high line and 36 for the low line, which are relatively low numbers of observations for a statistically dependable result. Therefore, carcass traits were only analyzed across lines for this polymorphism. The reason for the different association results in the two IGF-I selection lines for Pit1E6H and Pit1I5 is probably because some genes that control IGF-I concentration and growth and carcass traits segregate during divergent selection for IGF-I concentration. These genes may interact with the Pit-1 gene to regulate growth rate and carcass composition.

Conclusions

No significant relationships were found between the five polymorphisms detected in Pit-1 introns, Pit1I3H (HinfI), Pit1I3NL (NlaIII), Pit1I3N (NciI), Pit1I4N (BstNI), and Pit1I5, and growth traits in Angus beef cattle. For Pit1E6H (HinfI), significant associations were found with birth weight (P= .03) and preweaning gain (P=. 01). Animals with genotype OO had a lower mean for birth weight and a higher mean for preweaning gain and Gain20. Significant relationships with Pit1E6H genotypes were observed for backfat thickness (P= .01) in the high line, and for marbling score (P= .03) in the low line. Therefore, Pit1E6 may be useful in marker-assisted selection for growth and carcass traits in Angus beef cattle.

51

References
Bodner, M., J.L. Castrillo, L.E. Theill, T. Deerinck, M.Ellisman, and M. Karin. 1988. The pituitary-specific transcription factor GHF-1 is a homeobox-containing protein. Cell 55:505-518. Cohen, L.E., Wondisford, F.E. and Radovick, S. 1997. Role of pit-1 in the gene expression of growth hormone, prolactin, and thyrotropin. Endocrinology and Metabolism Clinics of North America. 25:523-540. Davis, M.E., and R.C.M. Simmen. 1997. Genetic parameter estimates for serum insulinlike growth factor I concentration and performance traits in Angus beef cattle. J. Anim. Sci. 75:317-324. Doll, P., J.L. Castrillo, L.E. Theill, T. Deerinck, M. Ellisman, and M. Karin. 1990. Expression of GHF-1 protein in mouse pituitaries correlates both temporally and spatially with the onset of growth hormone gene activity. Cell 60:809-820. Gordon, D.F., B.R. Haugen, V.D. Sarapura, A.R. Nelson, W.M. Wood, and E.C. Ridgway. 1993. Analysis of Pit-1 in regulating mouse TSH beta promoter activity in thyrotropes. Molecular and Cellular Endocrinology 96:75-84. Ingraham, H.A., R.P. Chen, H.J. Mangalam, H.P. Elsholtz, S.E. Flynn, C.R. Lin, D.M. Simmons, L. Swanson, and M.G. Rosenfeld. 1988. A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell 55:519-529. Li, S., E.B. Crenshaw III, E.J. Rawson, D.M. Simmons, L.W. Swanson, and M.G. Rosenfeld. 1990. Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene Pit-1. Nature 347:528-533. Lin , C., S.C. Lin, C.P. Chang, and M.G. Rosenfeld. 1992. Pit-1 dependent expression of the receptor for growth hormone releasing factor mediates pituitary cell growth. Nature 360:765-768. Lin, S.C., S. Li, D.W Drolet, and M.G. Rosenfeld. 1994. Pituitary ontogeny of the Snell dwarf mouse reveals Pit-1-independent and Pit-1-dependent origins of the thyrotrope. Development 120: 515-522 Mangalam, H.J., V.R. Albert, H.A. Ingraham, M. Kapiloff, L. Wilson, C. Nelson, H. Elsholtz, and M.G. Rosenfeld. 1989. A pituitary POU-domain protein, Pit-1, activates both growth hormone and prolactin promoters transcriptionally. Genes Dev. 3:946-958.

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Nelson, C., V.R. Albert, H.P. Elsholtz, L.I. Lu, and M.G. Rosenfeld. 1988. Activation of cell-specific expression of rat growth hormone and prolactin gene by a common transcription factor. Science 239:1400-1405. Pfaffle, R.W., G.E. DiMattia, J.S. Parks, M.R. Brown, J. M. Wit, M. Jansen, H. Van der Nat, J. L. Van den Brande, M.G. Rosenfeld, and H.A. Ingraham. 1992. Mutation of the POU-specific domain of Pit-1 and hypopituitarism without pituitary hypoplasia. Science 257:1118-1121. Renaville,R., N.Gengleer, E.Vrech, A. Prandi, S. Massart, C. Corradini, C. Bertozzi, F. Mortiaux, A. Burny, and D. Portetelle. 1997. Pit-1 gene polymorphism, milk yield, and conformation traits for Italian Holstein-Friesian bulls. J. Dairy Science 80:3431-3438. Rhodes, S.J., R. Chen, G.E. DiMattia, K.M. Scully, K.A. Kalla, S.C. Lin, V.C. Yu, and M.G. Rosenfeld. 1993. A tissue-specific enhancer confers Pit-1-dependent morphogen inducibility and autoregulation on the Pit-1 gene. Gene Dev. 7:913-932. SAS Users Guide. 1989.Version 5 Edition. SAS Inst. Inc., Cary, NC. Simmons, D.M., J.W. Voss, H.A. Ingraham, J.M. Holloway, R.S. Broide, M.G. Rosenfeld, and L.W. Swanson. 1990. Pituitary cell phenotypes involve cell-specific Pit1 mRNA translation and synergistic interactions with other classes of transcription factors. Genes Dev. 4:695-711. Stancekova, K., D. Vasicek, D. Peskovicova, J. Bulla, and A. Kubek. 1999. Effect of genetic variability of the porcine pituitary-specific transcription factor (Pit-1) on carcass traits in pigs. Animal Genetics 30:313-315. Steinfelder, H.J., P. Hauser, Y. Nakayama, S. Radovick, J.H. McClaskey, T. Taylor, B.D. Weintraub, and F.E. Wondisford. 1991. Thyrotropin-releasing hormone regulation of human TSH expression: role of a pituitary-specific transcription factor (Pit-1/GHF1) and potential interaction with a thyroid hormone-inhibitory element. Proc. Nat. Acad. Sci. USA 88:3130-3134. Woollard, J., C.B. Schmitz, A.E.Freeman, and C.K. Tuggle. 1994. Rapid communication: Hinf I polymorphism at the bovine Pit-1 locus. J. Anim. Sci. 72:3267. Yu, T.P., C.K. Tuggle, C.B. Schmitz, and M.F. Rothschild. 1995. Association of Pit-1 polymorphism with growth and carcass traits in pigs. J. Anim. Sci. 73:1282-1288. Yu, T.P., M.F. Rothschild, C.K. Tuggle, C. Haley, A. Archibald, L. Marklund, and L. Anderson. 1996. Pit-1 genotypes are associated with birth weight in three unrelated pig resource families. J. Anim. Sci. 74 (Suppl.):22.

53

Primer Amplified Name Pit1I3F Pit1I3R Pit1I4F Pit1I4R Pit1I53F Pit1I53R Pit1E6F Pit1E6R

Primer Sequence 5-ATAAGTTTCCTGACCACACG-3 5-TGGGCAGATAGTTGTTTGAC-3 5-AGGATACACCCAGACAAATG-3 5-TACTGATTGTTGTTCTCCGT-3 5-CCTCTGTCCATGGGATTTTC-3 5-AAATGTCCCCCAGAACTCAG-3 5-CCATCATCTCCCTTCTT-3 5-AATGTACAATGTCCTTCTGAG-3

Product Size 3kb 3kb 1kb 1kb 355bp 355bp 451bp 451b Region Intron 3 Intron 3 Intron 4 Intron 4 Intron 5 Intron 5 Exon 6 Exon 6

Table 3.1. Sequences of Primers and Lengths of Their Products

54

Polymorphisms Pit1I3H Pit1I3NL Pit1I4N Pit1I5

Primers Pit1I3 Pit1I3 Pit1I4 Pit1I53

Sequence change AAT deletion in allele C

Restriction Enzymeb HinfI NlaIII BstNI N/Ac

G to C transition in allele G G to T transition in allele E G to T transition in allele A

Table 3.2. Polymorphisms and Their Sequence Characteristics a

a

Pit1I3N was detected using PCR-RFLP. The sequence of this polymorphism was not determined in this study.
b c

Restriction enzyme used to genotype animals.

Pit1I5 did not have a restriction enzyme that recognized the differences between the genotypes. Therefore, SSCP was used to genotype this SNP.

55

Polymorphism Genotype

Across-linea N Frequency .12 .49 .39

High line N 5 23 21 Frequency .10 .47 .43

Low line N 7 25 17 Frequency .14 .51 .34

Pit1I3H

CC CD DD

12 48 38

Pit1I3NL

GG GH HH

12 46 40

.12 .47 .40

5 21 23

.10 .43 .47

7 25 17

.14 .51 .34

Pit1I3N

MM MN NN EE EF FF

12 50 36 12 46 40

.12 .51 .37 .12 .47 .41

5 25 19 5 21 23

.10 .51 .39 .10 .43 .47

7 25 17 7 25 17

.14 .51 .34 .14 .51 .34

Pit1I4N

Pit1I5

AB BB

17 168

.09 .91

9 86

.09 .91

8 82

.09 .91

Pit1E6H

OO OP PP

46 183 187

.11 .44 .45

24 92 98

.11 .43 .46

22 91 89

.11 .45 .44

Table 3.3. Genotypic Frequencies of Polymorphisms by High and Low IGF-I Selection Lines
a

Across line included all animals in both selection lines.

56

Haplotypes CGMEO/DHNFP CGMED/CGMED DHNFP/DHNFP DHMFP/DHNFP CHMEO/DHNFP CGMEP/DHNFP CGMFP/DHNFP CHMFP/DHNFP DHMFP/DHNFP DHNFO/DHNFP CGMEO/DHMFP

Expected Frequency a .0215 .0065 .1023 .0129 .0191 .0191 .0265 .0151 .0204 .0118 .0066

Observed Frequency b .45 .11 .35 .02 .01 .01 .01 .01 .01 .01 .01

Table 3.4. Analysis of Linkage Disequilibrum for the Five Diallelic Loci: Pit1I3H, Pit1I3N, Pit1I3NL, Pit1I4N, and Pit1E6H
a
b

Expected frequency was determined using the Hardy-Weinberg Law. Actual frequency observed. Differences between the actual frequency with and expected frequency indicate disequilibrum.

57

Trait Birth wt (kg) Weaning wt (kg) Prewean. gain (kg) Gain20a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean.gain(kg) D28 IGF-I (ng/mL) D42 IGF-I (ng/mL) D56 IGF-I (ng/mL) Mean IGF-I (ng/mL)

CC 33.7 1.6 199 8 164 9 14.6 3.2 210 9 242 12 281 12 371 13 155 7 275 21 335 27 321 29 311 21

CD 36.1 1.0 204 5 170 6 19.7 2.0 219 6 255 7 291 7 383 8 157 4 273 13 309 17 314 18 299 13

DD 35.4 1.2 201 6 169 7 17.0 2.4 214 7 250 8 286 9 375 10 152 5 280 15 311 20 329 21 306 15

P-value .28 .58 .81 .20 .36 .51 .59 .50 .54 .89 .58 .76 .78

Table 3.5 Least-squares Means and Standard Errors by Pit1I3H Genotype

a

Weight gain during the 20-d period between weaning and the beginning of the postweaning test.

58

Trait Birth wt (kg) Weaning wt (kg) Prewean. gain (kg) Gain20a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean.gain(kg) D28 IGF-I (ng/mL) D42 IGF-I (ng/mL) D56 IGF-I (ng/mL)

GG 33.7 1.6 198 8 164 9 15 3 210 9 241 12 280 12 370 13 157 7 276 21 336 27 321 29

GH 36.1 1.0 205 5.06 171 6 20 2 220 6 256 7 293 7 385 8 157 4 273 13 307 17 314 18 298 13

HH 35.4 1.2 299 6 167 7 17 2 212 7 247 8 283 9 372 10 152 5 280 15 314 20 328 21 307 15

P-value .30 .36 .60 .21 .19 .31 .33 .28 .50 .86 .54 .78 .72

Mean IGF-I (ng/mL) 311 21

Table 3.6 Least-squares Means and Standard Errors by Pit1I3NL Genotype

a

Weight gain during the 20-d period between weaning and the beginning of the postweaning test.

59

Trait Birth wt (kg) Weaning wt (kg) Prewean. gain (kg) Gain20a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean.gain(kg) D28 IGF-I (ng/mL) D42 IGF-I (ng/mL) D56 IGF-I (ng/mL) Mean IGF-I (ng/mL)

MM 33.7 1.6 199 8 164 9 14.6 3.2 210 9 242 11 281 12 371 13 157 1 274 21 334 27 320 29 310 21

MN 36.2 1.0 205 5 172 6 19.9 2.0 220 6 256 7 293 7 385 8 157 4 275 13 313 17 314 18 301 13

NN 35.3 1.2 200 6 167 7 16.8 2.3 213 7 247 8 284 8 373 10 152 5 275 15 303 19 328 21 302 15

P-value .26 .56 .58 .14 .37 .28 .40 .28 .51 .99 .50 .75 .90

Table 3.7 Least-squares Means and Standard Errors by Pit1I3N Genotype

a

Weight gain during the 20-d period between weaning and the beginning of the postweaning test.

60

Trait Birth wt (kg) Weaning wt (kg) Prewean. gain (kg) Gain20a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean.gain(kg) D28 IGF-I (ng/mL) D42 IGF-I (ng/mL) D56 IGF-I (ng/mL) Mean IGF-I (ng/mL)

EE 33.7 1.6 199 8 165 9 14.7 3.3 210 9 243 12 281 12 372 13 157 7 276 21 335 27 321 29 310 21

EF 36.1 1.0 203 5 170 6 19.4 2.0 218 6 254 7 290 7 382 8 156 4 273 13 309 17 315 18 299 13

FF 35.4 .6 202 6 170 7 17.4 2.4 215 7 252 8 288 9 377 10 153 5 280 15 310 20 327 21 306 15

P-value .30 .83 .81 .28 .61 .59 .73 .68 .72 .87 .58 .81 .80

Table 3.8 Least-squares Means and Standard Errors by PitI4N Genotype Weight gain during the 20-d period between weaning and the beginning of the postweaning test.
a

61

Trait Birth wt (kg) Weaning wt (kg) Prewean. gain (kg) Gain20 a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean. Gain (kg) D 28 IGF-I (ng/mL) D 42 IGF-I (ng/mL) D 56 IGF-I (ng/mL) Mean IGF-I (ng/mL)

OO 32.5 .7 162 4 141 5 20.0 1.8 220 5 253 6 290 7 384 7 163 4 242 18 273 20 255 18 257 17

OP 34.0 .4 162 2 127 3 16.4 1.1 218 3 252 4 288 4 386 5 169 3 256 11 286 12 274 11 272 10

PP 34.5 .5 161 3 119 3 15.9 1.1 219 3 252 4 285 4 385 5 166 3 254 11 280 13 281 11 272 10

P-value .03 .72 .01 .07 .90 .98 .73 .91 .24 .36 .80 .33 .65

Table 3.9 Least-squares Means and Standard Errors by Pit1E6H Genotype

a

Weight gain during the 20-d period between weaning and the beginning of the postweaning test.

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Trait

OO

OP

PP

P-value

Birth wt (kg) Weaning wt (kg) Prewean. gain (kg) Gain20 a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean. Gain (kg) D 28 IGF-I (ng/mL) D 42 IGF-I (ng/mL) D 56 IGF-I (ng/mL) Mean IGF-I (ng/mL)

30.3 .9 159 5 145 10 16.3 2.2 210 6 241 7 275 7 367 9 156 5 298 26 331 28 288 24 306 23

32.3 .5 161 2 127 5 15.3 1.2 213 3 246 4 280 4 376 5 164 3 295 14 329 15 311 13 312 12

32.6 .4 156 3 118 5 15.6 1.1 213 3 244 4 278 4 374 4 163 3 298 13 325 14 318 12 314 12

.06 .20 .03 .92 .89 .83 .78 .62 .34 .98 .97 .53 .95

Table 3.10 Least-squares Means and Standard Errors by Pit1E6H Genotype in High IGF-I Line Weight gain during the 20-d period between weaning and the beginning of the postweaning test.
a

63

Trait Birth wt (kg) Weaning wt (kg) Prewean. Gain (kg) Gain20 a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean. Gain (kg) D 28 IGF-I on (ng/mL) D 42 IGF-I on (ng/mL) D 56 IGF-I on (ng/mL) Mean IGF-I (ng/mL)

OO 33.7 1.0 163 5 138 11 22.9 2.6 226 6 260 7 301 10 395 11 170 6 189 19 212 24 219 21 208 19

OP 34.8 .5 161 3 126 6 17.1 1.3 219 3 253 4 290 5 391 6 173 3 222 10 244 12 242 11 236 10

PP 35.7 .6 163 3 121 6 16.0 1.4 221 4 256 5 286 6 390 6 166 3 212 11 234 13 238 12 228 11

P-value .13 .72 .33 .04 .56 .73 .42 .89 .32 .25 .41 .60 .35

Table 3.11 Least-squares Means and Standard Errors by Pit1E6H Genotype in Low IGF-I Line
a

Weight gain during the 20-d period between weaning and the beginning of the postweaning test.

64

Trait Birth wt (kg) Wearing wt (kg) Prewean. Gain (kg) Gain20 a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean. Gain (kg) D 28 IGF-I (ng/mL) D 42 IGF-I (ng/mL) D 56 IGF-I (ng/mL) Mean IGF-I(ng/mL)

AB 33.0 1.2 188 6 158 3 27.5 2.1 214 7 260 8 291 8 390 9 166 5 255 20 241 24 312 24 269 19

BB 34.2 .6 188 3 153 7 25.9 1.1 213 3 252 4 281 4 380 5 162 3 247 10 260 12 285 12 264 10

P-value .30 .97 .43 .42 .94 .27 .19 .28 .33 .67 .42 .25 .77

Table 3.12. Least-squares Means and Standard Errors by Pit1I5 Genotype Weight gain during the 20-d period between weaning and the beginning of the postweaning test.
a

65

Trait Birth wt (kg) Wearing wt (kg) Prewean. Gain (kg) Gain20 a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean. Gain (kg) D 28 IGF-I (ng/mL) D 42 IGF-I (ng/mL) D 56 IGF-I (ng/mL) Mean IGF-I(ng/mL)

AB 33.4 1.9 179 8 151 9 31.0 3.3 207 10 254 11 283 12 391 14 173 8 326 32 357 32 407 34 363 27

BB 33.5 1.2 180 5 149 5 25.9 2.0 205 6 246 6 274 7 373 9 160 5 294 19 344 18 355 20 331 16

P-value .95 .88 .83 .07 .78 .39 .37 .15 .08 .24 .62 .07 16

Table 3.13.Least-squares Means and Standard Errors by Pit1I5 Genotype in High IGF-I Line
a

Weight gain during the 20-d period between weaning and the beginning of the postweaning test.

66

Trait Birth wt (kg) Wearing wt (kg) Prewean. Gain (kg) Gain20 a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean. Gain (kg) D 28 IGF-I (ng/mL) D 42 IGF-I (ng/mL) D 56 IGF-I (ng/mL) Mean IGF-I (ng/mL)

AB 32.8 1.7 194 9 167 11 24.6 2.9 218 10 269 13 302 13 392 14 163 6 200 27 164 32 257 31 207 25

BB 5.4 .8 192 4 154 5 27.2 1.4 219 5 256 6 288 6 388 7 166 3 203 13 172 16 219 15 199 12

P-value .14 .81 .23 .38 .92 .33 .28 .75 .59 .91 .80 .24 .74

Table 3.14. Least-squares Means and Standard Errors by Pit1I5 Genotype in Low IGF-I Line
a

Weight gain during the 20-d period between weaning and the beginning of the postweaning test.

67

Trait Fat (cm) a Ribeye (cm2 ) b KPH (%)c Hotwt (kg) d Marb e Qual f Yield g

AA 9.7 1.0 74.6 2.7 2.4 .2 266.3 8.7 5.1 .3 10.7 .5 2.5 .2

AB 9.8 .5 75.4 1.4 2.2 .1 270.2 4.4 4.9 .1 10.6 .2 2.4 .1

BB 8.3 .5 73.9 1.3 2.0 .1 268.8 4.1 5.0 .1 10.6 .2 2.3 .1

P-value .09 .68 .28 .90 .84 .97 .62

Table 3.15. Least-squares Means and Standard Errors for Carcass Traits by Pit1E6H Genotypes Across Selection Lines
a

Fat: Backfat thickness between 12th and 13th rib (cm) Ribeye: Ribeye area (cm2 ) c KPH: Kidney, pelvic, and heart fat (%) d Hotwt: Hot carcass weight (kg) e Marb: Marbling score (1 to 10, integer only) f Qual: Quality grade (6 to 14, integer only) g Yield: Yield grade (1 to 5)
b

68

Trait Fat (cm) a Ribeye (cm2 ) b KPH (%)c Hotwt (kg) d Marb e Qual f Yield g

AA 9.0 1.2 71.7 3.6 2.2 .3 252.8 13.3 4.1 .4 8.7 .7 2.4 .2

AB 10.6 .6 75.5 1.7 2.3 .2 270.9 6.2 4.7 .2 10.5 .3 2.5 .1

BB 8.3 .5 75.8 1.5 2.1 .1 272.7 5.6 4.8 1.2 10.3 .3 2.3 .1

P-value .01 .52 .71 .35 .33 .06 .24

Table 3.16. Least-squares Means and Standard Errors for Carcass Traits by Pit1E6H Genotypes in High Selection Line
a

Fat: Backfat thickness between 12th and 13th rib (cm) Ribeye: Ribeye area (cm2 ) c KPH: Kidney, pelvic, and heart fat (%) d Hotwt: Hot carcass weight (kg) e Marb: Marbling score (1 to 10, integer only) f Qual: Quality grade (6 to 14, integer only) g Yield: Yield grade (1 to 5)
b

69

Trait Fat (cm) a Ribeye (cm2 ) b KPH (%)c Hotwt (kg) d Marb e Qual f Yield g

AA 12.6 1.9 71.7 4.2 2.9 .4 267.5 13.7 6.3 .5 12.1 .8 3.0 .3

AB 8.1 .9 75.4 2.1 1.6 .2 266.7 7.0 4.7 .3 10.1 .4 2.2 .2

BB 8.4 .9 74.5 2.0 2.0 .2 272.1 6.5 5.0 .2 11.0 .4 2.3 .2

P-value .15 .75 .14 .84 .03 .07 .13

Table 3.17. Least-squares Means and Standard Errors for Carcass Traits by Pit1E6H Genotypes in Low Selection Line
a

Fat: Backfat thickness between 12th and 13th rib (cm) Ribeye: Ribeye area (cm2 ) c KPH: Kidney, pelvic, and heart fat (%) d Hotwt: Hot carcass weight (kg) e Marb: Marbling score (1 to 10, integer only) f Qual: Quality grade (6 to 14, integer only) g Yield: Yield grade (1 to 5)
b

70

Trait Fat (cm) a Ribeye (cm2 ) b KPH (%)c Hotwt (kg) d Marb e Qual f Yield g

AA 9.6 1.9 71.4 4.0 2.7 .3 241.5 15.2 4.9 .2 11.0 .6 2.5 .3

AB 12.3 3.1 70.9 6.5 2.7 .5 239.3 24.7 4.2 .8 10.5 1.0 2.7 .4

P-value .24 .91 .84 .90 .42 .48 .41

Table 3.18. Least-squares Means and Standard Errors for Carcass Traits by Pit1I5 Genotypes Across Line
a

Fat: Backfat thickness between 12th and 13th rib (cm) Ribeye: Ribeye area (cm2 ) c KPH: Kidney, pelvic, and heart fat (%) d Hotwt: Hot carcass weight (kg) e Marb: Marbling score (1 to 10, integer only) f Qual: Quality grade (6 to 14, integer only) g Yield: Yield grade (1 to 5)
b

71

CHAPTER 4 ASSOCIATIONS OF POLYMORPHISMS IN THE GROWTH HORMONE RELEASING HORMONE RECEPTOR GENE WITH GROWTH AND CARCASS TRAITS IN ANGUS BEEF CATTLE

Abstract

Growth hormone releasing hormone (GHRH) plays a major role in stimulation of both synthesis and release of GH in the anterior pituitary through its receptor. Therefore, the GHRH-receptor gene is a candidate gene for growth traits in domestic animals. Genetic marker information on this gene could be used to facilitate selection and breeding through marker assisted selection. One polymorphism, GHRHRE6N (NciI), was detected in exon 6 of the GHRH-receptor gene. Genotypes AA, AB, and BB were observed with frequencies of .80, .19, and .01, respectively, in 455 Angus cattle that were divergently selected for high or low blood serum IGF-I concentration. Genotypic frequencies were not significantly different between high and low IGF-I selection lines. Allele B has a point mutation from A to G in codon 189, which results in an amino acid change from glutamine to arginine. The associations of the polymorphism with growth and carcass traits and with IGF-I concentrations were analyzed using the GLM procedure in SAS. No significant associations of the genotypes with growth, carcass traits, or IGF-I concentrations were observed when data from both selection lines were analyzed together. When animals in the two IGF-I selection lines were analyzed

72

separately, moderate associations were found between genotypes and postweaning gain (P=. 10) in the high IGF-I line.

Introduction

Synthesis and secretion of GH in the somatotroph cells of the anterior pituitary are under multifactoral control from the hypothalamus in the brain (Martin, 1979; Mayo et al., 1995). The stimulatory signal is from growth hormone releasing hormone (GHRH). Substantial information from both the clinical domain and from basic research showed the major role of GHRH in the control of growth hormone secretion and in the regulation of linear growth (Hammer et al., 1985; Frohman and Jannson, 1986; Gelato and Merriam, 1986). Growth hormone response to GHRH has also been shown to be associated with subsequent growth in cattle (Connor et al., 1999a) and carcass traits in sheep (Suttie et al., 1991). Growth hormone releasing hormone extents its influence through its receptor, the GHRH-receptor. The GHRH-R is a member of the G proteincoupled receptor family and is able to transduce GHRH-dependent increases in intracellular cAMP via Gs activation for stimulating somatotroph proliferation and GH gene expression (Mayo, 1992; Lin et al., 1992; Gaylinn et al., 1993). GHRH-R cDNAs from rat (Lin et al., 1992; Mayo et al., 1992), mouse (Lin et al., 1992), swine (Hsiung et al., 1993), human (Mayo, 1992; Gaylinn et al., 1993), and bovine (Horikawa et al., 2001) have been identified. The GHRH-R transcripts have a highly specific distribution in the anterior pituitary. Also, significant numbers of GHRH-R are found in the hypothalamus, kidney and placenta (Mayo et al., 1995). However, the functions of the

73

GHRH-R outside the anterior pituitary and hypothalamus are still unknown. Mutations in the receptor gene have been found to cause heritable GH deficiency diseases in both mice (Godfrey et al., 1993; Lin et al., 1993) and human (Wajnrajch et al., 1996; Maheshwari et al., 1996). Mutations in the GHRH-R gene may also play a role in GH excess and pituitary tumorigenesis (Petersenn et al., 1998). The GHRH-R gene has been assigned to bovine chromosome 4 (Connor et al., 1999b) and swine chromosome 18 (Sun et al., 1997). A Eco571 PCR-RFLP in intron 6 has n

been defined in the GHRH-R gene in cattle (Connor et al., 1999b). Two PCR-RFLP, MseI and TaqI, were identified in pigs (Sun et al., 1997). Thus far, no association has been found between these polymorphisms and growth and carcass traits in domestic animals. In this study, relationships of one newly discovered polymorphism in the GHRH-R gene with growth and carcass traits in Angus beef cattle were analyzed.

Material and Methods

Animals

Angus beef cattle, which were divergently selected for blood serum insulin-like growth factor I (IGF-I) concentration, were used as the experimental animals. Selection began in 1989 at the Eastern Ohio Resource Development Center (EORDC), using 100 spring-calving (50 high line and 50 low line) and in 1990 using 100 fall-calving (50

74

high line and 50 low line) purebred Angus cows with unknown IGF-I levels. Cows from the base population were randomly assigned to the selection lines. Each year, four bull calves with the highest and f ur bull calves with the lowest IGF-I o concentration were selected for breeding within the selection lines. Approximately eight cows were culled from each selection line each year based on physical unsoundness, failure to conceive in two consecutive years, and oldest age. These cows were replaced with the same number of pregnant heifers having the highest or lowest serum IGF-I concentration (Davis and Simmen, 1997). In this study, 455 animals were genotyped.

Methods

DNA extraction Genomic DNA was isolated from blood samples that were collected during the IGF-I selection experiment at EORDC. The extraction procedures were as described in Chapter 3.

PCR Polymerase chain reaction (PCR) was used to amplify the DNA fragment from genomic DNA. Primers were designed based on the bovine DNA sequence from GenBank. Primer GHRHRE6 (forward 5-CCTGCACTGAGCCCGGTTGTCTTA-3 and reverse 5-CTGGGCTACGGGATGGGAATGAAA-3) was used to amplify exon 6 of the GHRH-R gene.

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The GHRHRE6 primer amplified a 600bp DNA fragment, which covered all of exon 6 and parts of intron 5 and 6 on the boundaries of exon 6. The PCR was performed in a 30L reaction volume containing 10pmol of forward primer and the same amount of reverse primer, 200M dNTPs, and 1x reaction buffer, which contained 1.5mM MgCl2 , 1 unit of Taq-DNA polymerase and 100ng of genomic DNA as template. Conditions were 97o C for 2 min, followed by 35 cycles of 95o C for 45s, 60o C for 1 min, and 72o C for 90s. After 35 cycles, reactions were finished by an extension of 5 min at 72o C, and stored at 4o C.

Detection of Genetic Variation When amplification was achieved, the single strand conformation polymorphism (SSCP) technique was used to screen for mutations within the amplified segment. Since the optimal size of DNA for SSCP is under 400bp, the PCR product was digested with restriction enzyme to reduce the size of DNA fragment. The reaction mixture, which included 10L of PCR product, 10L of ddH2 O and 12L of loading dye, was denatured at 95C for 5 min and put into ice for 10 min to prevent the denatured single stranded DNA from renaturing. The samples were then loaded onto 10%

polyacrylamide gels. Adding 10% urea or 10% formamide to the polyacrylamide gels improved the resolution of the DNA bands on the gel. The samples were run in 1x TBE buffer at 200 volts for 16 to 20 h at a constant temperature of 10C. Gels were then stained with .01% Ethidium Bromide for 10 min and viewed under UV light. After a polymorphism was detected, and in order to determine the nature of the polymorphism, the PCR products of the two homozygous genotypes were sent to the

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Plant-Microbe Genomics Facility at The Ohio State University for sequencing using an ABI 3700 analyzer. Before sending samples for sequencing, PCR products were purified with the Qiagen Gel DNA Extraction kit (Qiagen Inc., Valencia, CA 91355, USA).

Genotyping If there was a restriction site for the polymorphism, corresponding restriction enzymes were chosen and PCR-RFLP (restriction fragment length polymorphism) was used to genotype the animals. For the polymorphism detected in exon 6 of GHRH-R gene, restriction enzyme NciI was used to genotype animals. The PCR-RFLP procedure included amplifying the corresponding DNA segment by PCR; digesting the PCR product with restriction enzymes; and running the digested DNA on an agarose gel containing .003% Ethidium Bromide. The gel was viewed under UV light.

Statistical Analysis

Growth traits and IGF-I concentrations Four-hundred-fifty-five Angus cattle born in the years 1995, 1996, and 1997 were analyzed for growth traits, which included birth weight (kg), weaning weight (kg), preweaning gain (kg), on-test weight (kg), weight (kg) at d 28 and 56 of the 140-d postweaning test, off-test weight (kg), weight gain (kg) during the 20-d period between weaning and the beginning of the postweaning test, and postweaning gain (kg), and serum IGF-I concentration (ng/mL) on d 28, 42, and 56 of the postweaning test and

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mean serum IGF-I concentration (ng/mL). Among these 455 Angus cattle, 234 animals were from the high line and 221 were from the low line.

Carcass traits Approximately 160 Angus bulls born in the years 1995, 1996, and 1997 that were not saved for breeding were analyzed for carcass traits. Ages of bulls at slaughter ranged from 13 to 15 mo. Bulls born in the years 1995 and 1996 were slaughtered at The Herman Falter Packing Co. in Columbus, OH. Bulls born in 1997 were slaughtered at the Mahan Packing Co. in Bristolville, OH. Carcass traits measurements were performed by USDA grader in these two packing companies. Carcass traits analyzed for this polymorphism included: Backfat thickness (cm) Ribeye area (cm2 ) Kidney, pelvic, and heart fat (KPH) (%) Hot carcass weight (kg) Marbling score (1- devoid, 2- practically devoid, 3- traces, 4- slight, 5- small, 6modest, 7- moderate, 8- slightly abundant, 9- moderately abundant, 10- abundant) Quality grade (6- standard-, 7- standard, 8- standard+, 9- select-, 10- select, 11- select+, 12- choice-, 13- choice, 14- choice+) Yield grade (from 1 to 5)= 2.5+ (2.5 x adjusted fat thickness (inch)) + (.2 x KPH) + (.0038 x hot carcass weight (lb)) (.32 x ribeye area (inch2 ))

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Data analysis Associations of the animal genotypes with growth, carcass traits, and IGF-I concentrations were determined by analysis of variance of quantitative traits. General Linear Model (GLM) procedures in SAS (1989) were used to perform the analysis. Fixed effects of genotypes, year, season of birth (spring vs fall), age of dam, sex (bull vs heifer), and IGF-I selection line (high vs low) were included as independent variables in the linear model. Weaning age of calf was treated as a covariate in the model when analyzing weaning weight, preweaning gain, and Gain20. On-test age of calf was treated as a covariate in the model when analyzing on-test weight, weight at d 28 and 56 of the 140-d postweaning test, off-test weight, postweaning gain, serum IGF-I concentration on d 28, 42, and 56, and mean IGF-I concentration. Calf age was not included in the analysis of birth weight. For carcass traits, sex was deleted from the model, since only bulls had carcass data, and slaughter age was added in the model as a covariate. Data were also analyzed separately within the high and low selection lines using the same model, except that selection line was omitted from the model, to test the difference in the association of genotypes with growth and carcass traits and IGF-I concentrations between the high and low selection lines.

Results

Genetic Variation Detected One polymorphism, GHRHRE6N (NciI), was detected in exon 6 of the growth hormone releasing hormone receptor gene. Genotypes AA, AB, and BB were observed

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with frequencies of .80, .19, and .01, respectively, in 455 Angus cattle born in the year of 1995, 1996, and 1997. Genotypic frequencies were not found to be significantly

different between the high and low IGF-I selection lines (Table 4.1). Sequencing revealed that GHRHRE6N has a point mutation from A to G in allele B in codon 189, which changes the amino acid from glutamine to arginine. This polymorphism can be genotyped using restriction enzymes MspIII and NciI. In this study, NciI was used to perform the genotyping.

Association Analysis

No significant associations were observed between genotypes and growth traits when both selection lines were analyzed together (Table 4.2). When animals in the two IGF-I selection lines were analyzed separately, a moderate association was found between genotypes and postweaning gain (P=. 10) in the high IGF-I line (Table 4.3). Animals with genotype AB tended to have higher postweaning gain (169 6 kg) than animals with genotypes AA (162 5 kg) and BB (150 14 kg). For preweaning gain (P= .12), animals with genotype AA tended to have a higher mean (133 9 kg) than animals with genotypes AB (122 11 kg) and BB (92 27 kg). No associations were observed in the low IGF-I line (Table 4.4). Associations of genotypes with carcass traits were also analyzed in approximately 160 Angus bulls across or within selection lines; no significant associations were observed in these animals (Tables 4.5, 4.6 and 4.7).

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Discussion

The role of growth hormone in regulation of growth and metabolism has been well established. As the principal stimulatory factor of GH secretion, GHRH is able to regulate the linear growth of animals (Hammer et al., 1985; Frohman and Jannson, 1986; Gelato and Merriam, 1986). GHRH exerts its influence through its receptor, the GHRH-receptor. In this study, the GHRH-receptor gene was the candidate gene. The GHRH-receptor gene has been assigned to bovine chromosome 4 (Connor et al., 1999b) and encodes a 423-amino acid protein (Petersenn et al., 1998; Connor et al., 2002). It shares 93, 90, 89, 87, and 85% homology with the DNA sequences of ovine, porcine, human, rat, and mouse, respectively (Connor et al., 2002). In human and mouse, mutations have been found to cause abnormal growth. Lin et al. demonstrated that one amino acid substitution of the GHRH-R protein in the little mouse, which showed genetically transmitted dwarfism, caused GH deficiency and somatotroph hypoplasia (1993). In addition, mutations in the GHRH-R gene in humans were demonstrated to cause profound GH deficiency (Wajnrajch et al., 1996; Netchine et al., 1998) and a familial severe dwarfism, Sindh (Baumann and Maheshwari, 1997; Maheshwari et al., 1998). However, thus far, no association has been found between mutations or polymorphisms in the GHRH-R gene and growth or carcass traits in domestic animals. Several polymorphisms have been reported in swine and bovine. Two PCR-RFLP, MseI and TaqI, were identified in pigs (Sun et al., 1997). An Eco571 PCR-RFLP in intron 6 has been defined in the GHRH-R gene in cattle (Connor et al., 1999b). Results of association studies have not been reported.

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In this study, a new RFLP, NciI, was observed in exon 6 of the GHRH-R gene. Exon 6 of the GHRH-R gene encodes part of the transmembrane and associated extracellular loop domain, which can provide critical information for interaction with GHRH (DeAlmeida and Mayo, 1998). However, no significant associations were observed between genotypes and growth traits when high and low IGF-I selection line data were analyzed together (Table 4.2). When animals in the two IGF-I selection lines were analyzed separately, moderate associations were found between genotypes and postweaning gain (P= .10) in the high line, but not in the low line. The reason for the different results in the two lines is p robably the fact that some genes that control IGF-I concentration and growth traits segregate during divergent selection for IGF-I concentration, and these genes may interact with the GHRH-R gene to regulate growth rate. This NciI polymorphism caused an amino acid change from Glutamine to Arginine in the extracellular region of the receptor. Presumably it should change the conformation of the protein and may have an effect on functions of the GHRH-R. However, no evidence showed any effect of this polymorphism on growth or carcass traits in Angus beef cattle. This polymorphism needs to be studied in other populations of cattle to further explore its effect on growth rate and carcass composition.

Conclusions

The GHRHRE6N PCR-RFLP in exon 6 of the GHRH-R gene caused an amino acid change. Therefore, it is assumed that this polymorphism might have an effect on the

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functioning of the GHRH-R. However, no associations of this SNP with growth or carcass traits were identified in a herd of Angus cattle. The polymorphism should be

studied in other populations of cattle in order to better understand its effect.

References
Baumann, G., and H. Maheshwari. 1997. The dwarfism of Sindh: Severe growth hormone (GH) deficiency caused by a mutation in the GH-releasing hormone receptor gene. Acta Paediatr. Suppl. 423:33-38. Connor, E.E., S.M. Barao, L. W. Douglass, S.A. Zinn, and G.E. Dahl. 1999a. Predicting bull growth performance and carcass composition from growth hormone response to growth hormone-releasing hormone. J. Anim. Sci. 77:2736-2741. Connor, E.E., M.S. Ashwell, S.M. Kappes, and G.E. Dahl. 1999b. Rapid communication: Mapping of the bovine growth hormone-releasing hormone receptor (GHRH-R) gene to chromosome 4 by linkage analysis using a novel PCR-RFLP. J. Anim. Sci. 77:2736-2741. Connor, E.E., M.S. Ashwell, and G.E. Dahl. 2002. Characterization and expression of the bovine growth hormone-releasing hormone (GHRH) receptor. Domest. Anim. Endocrinol. 22:189-200. DeAlmeida, V.I., and K.E. Mayo. 1998. Identification of binding domains of the growth hormone-releasing hormone receptor by analysis of mutant and chimeric receptor proteins. Molecular Endocrinology 12:750-765. Frohman, L.A., and J.O. Jansson. 1986. Growth hormone releasing hormone. Endocr. Rev. 7:223-253. Gaylinn, B.D., J.K. Harrison, J.R. Zysk, C.E. Lyons, K.R. Lynch, and M.O. Thorner. 1993. Molecular cloning and expression of a human anterior pituitary receptor for growth hormone-releasing hormone. Mol. Endocrinol. 7:77-84. Gelato, M.C., and C.R. Merriam. 1986. Growth hormone releasing hormone. Annu. Rev. Physiol. 48:569-591. Godfrey, P., J.O. Rahal, W.G. Beamer, N.G. Copeland, N.A. Jenkins, and K.E. Mayo. 1993. GHRH receptor of little mice contains a missense mutation in the extracellular domain that disrupts receptor function. Nat. Genet. 4:227-232.

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Hammer, R.E., R.L. Brinster, M.G. Rosenfeld, R.M. Evans, and K.E. Mayo. 1985. Expression of human growth hormone-releasing factor in transgenic mice results in increased somatic growth. Nature 315:413-416. Horikawa,R., B.D. Gaylinn, C.E. Lyons Jr., and M.O. Thorner. 2001. Molecular cloning of ovine and bovine growth hormone-releasing hormone receptors: the ovine receptor is C-terminally truncated. Endocrinology 142:2660-2668. Hsiung, H.M., D.P. Smith, X.Y. Zhang, T. Bennett, P.R. Rosteck Jr., and M.H. Lai. 1993. Structure and functional expression of a complementary DNA for porcine growth hormone-releasing hormone receptor. Neuropeptides 25:1-10. Lin, C., S.C. Lin, C.P. Chang, and M.G. Rosenfeld. 1992. Pit-1 dependent expression of the receptor for growth hormone releasing factor mediates pituitary cell growth. Nature 360:765-768. Lin, S.C., C.R. Lin, I. Gukovsky, A.J. Lusis, P.E. Sawchenko, and M.G. Rosenfeld. 1993. Molecular basis of the little mouse phenotype and implications for cell typespecific growth. Nature 364:208-213. Maheshwari, I., B.L. Silverman, J. Dupuis, and G. Baumann. 1998. Phenotype and genetic analysis of a syndrome caused by an inactivating mutation in the growth hormone releasing hormone receptor: Dwarfism of Sindh. Journal of Clinical Endocrinology and Metabolism 83:4065-4074. Martin, J.B. 1979. Twenty third annual Bowditch Lecture. Brain mechanisms for integration of growth hormone secretion. Physiologist 22:23-29. Mayo, K.E. 1992. Molecular cloning and expression of a pituitary-specific receptor for growth hormone releasing hormone. Mol. Endocrinol. 6:1734-1744. Mayo, K.E., P.A. Godfrey, S.T. Suhr, D.J. Kulik, and J.O. Rahal. 1995. Growth hormone releasing hormone: Synthesis and signaling. Recent progress in hormone research 50:35-73. Netchine, I., P. Talon, F. Dastot, F. Vitaux, M. Goossens, and S. Amselem. 1998. Extensive phenotypic analysis of a family with growth hormone (GH) deficiency caused by a mutation in the GH-releasing hormone receptor gene. J. Clin. Endocrin. Metabol. 83:432-436. Petersenn, S., A.C. Rasch, M.Heyens, and H.M. Schulte. 1998. Structure and regulation of the human growth hormone releasing hormone receptor gene. Molecular Endocrinology 12:233-247. SAS Users Guide. 1989.Version 5 Edition. SAS Inst. Inc., Cary, NC.

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Sun, H.S., C. Taylor, A. Robic, L. Wang, M.F. Rothschild, and C.K. Tuggle. 1997. Mapping of growth hormone releasing hormone receptor to swine chromosome 18. Anim. Genet. 28:351-353. Suttie, J. M., E.A. Lord, P.D. Gluckman, P.F. Fennessy, and R.P. Littlejohn. 1991. Genetically lean and fat sheep differ in their growth hormone response to growth hormone releasing hormone. Domest. Anim. Endocrinol. 8:323-329. Wajnrajch, M.P., J.M. Gertner, M.D. Harbison, S.C. Chua Jr, and R.L. Leibel. 1996. Nonsense mutation in the human growth hormone-releasing hormone receptor causes growth failure analogous to the little (lit) mouse. Nat. Genet. 12:88-90.

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Genotype AA AB BB

Across-lines .80 .19 .01

High line .78 .21 .01

Low line .83 .16 .01

Table 4.1. Genotypic Frequencies for NciI Polymorphism by IGF-I Selection Line a P-value for the test of difference in genotypic frequency between high and low selection lines was .27.
a

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Trait Birth wt (kg) Weaning wt (kg) Prewean. gain (kg) Gain20a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean.gain(kg) D28 IGF-I (ng/mL) D42 IGF-I (ng/mL) D56 IGF-I (ng/mL) Mean IGF-I (ng/mL)

AA 34 1 163 2 129 4 17 1 219 2 252 3 286 3 385 3 167 2 254 8 282 9 275 9 270 8

AB 34 1 162 3 126 6 17 1 219 4 256 4 291 5 389 6 168 3 264 13 283 15 286 14 277 12

BB 35 1 166 11 115 23 15 6 233 14 271 17 304 20 400 22 160 12 211 53 277 59 248 54 245 49

P-value .93 .87 .76 .86 .56 .39 .46 .64 .83 .51 .99 .61 .71

Table 4.2. Least-squares Means and Standard Errors for Growth Traits by GHRHRE6N Genotype across Lines
a

Weight gain during the 20-d period between weaning and the beginning of the postweaning test.

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Trait Birth wt (kg) Weaning wt (kg) Prewean. gain (kg) Gain20a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean.gain(kg) D28 IGF-I (ng/mL) D42 IGF-I (ng/mL) D56 IGF-I (ng/mL) Mean IGF-I (ng/mL)

AA 33 1 159 4 133 9 19 2 213 6 249 7 282 7 377 9 162 5 301 25 333 27 324 24 319 22

AB 33 1 158 5 122 11 17 3 215 7 257 8 290 9 389 10 169 6 308 29 320 32 323 28 317 26

BB 34 3 156 13 92 27 15 7 230 16 265 20 289 21 385 25 150 14 232 72 285 78 273 70 263 65

P-value .89 .95 .12 .61 .52 .28 .37 .23 .10 .56 .69 .75 .66

Table 4.3. Least-squares Means and Standard Errors for Growth Traits by GHRHRE6N Genotype in High Line
a

Weight gain during the 20-d period between weaning and the beginning of the postweaning test.

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Trait Birth wt (kg) Weaning wt (kg) Prewean. gain (kg) Gain20a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean.gain(kg) D28 IGF-I (ng/mL) D42 IGF-I (ng/mL) D56 IGF-I (ng/mL) Mean IGF-I (ng/mL)

AA 35 0 163 2 126 5 18 1 221 3 255 3 289 4 392 4 171 2 211 8 230 10 233 9 224 8

AB 35 1 161 4 134 9 18 2 220 5 254 7 290 8 386 8 164 5 223 15 245 18 252 17 240 15

BB 36 4 177 23 186 48 18 12 226 28 288 37 335 44 438 46 191 26 249 84 334 102 270 94 284 82

P-value .74 .77 .33 .91 .96 .67 .58 .46 .26 .67 .45 .49 .46

Table 4.4. Least-squares Means and Standard Errors for Growth Traits by GHRHRE6N Genotype in Low Line Weight gain during the 20-d period between weaning and the beginning of the postweaning test.
a

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Trait

AA

AB

BB

P-value

Fat (cm)a Ribeye (cm2 )b KPH (%)c Hotwt (kg) d Marbe Qualf Yieldg

10.3 1.7 71.7 4.4 2.7 .3 261.0 14.5 5.0 .1 11.4 .7 2.7 .3

10.1 1.9 68.9 4.9 2.7 .4 265.4 16.0 4.9 .2 11.8 .7 2.8 .3

9.7 3.6 69.8 9.5 1.4 .7 244.7 31.0 4.9 .2 10.7 1.5 2.3 .62

.93 .74 .14 .69 .98 .57 .57

Table 4.5. Least-squares Means and Standard Errors for Carcass Traits by GHRHRE6N Genotype across Selection Lines
a

Fat: Backfat thickness between 12th and 13th rib (cm) Ribeye area: Ribeye area (cm2 ) c KPH: Kidney, pelvic, and heart fat (%) d Hotwt: Hot carcass weight (kg) e Marb: Marbling score (1 to 10, integer only) f Qual: Quality grade (6 to 14, integer only) g Yield: Yield grade (1 to 5)
b

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Trait Fat (cm) a Ribeye (cm2 ) b KPH (%)c Hotwt (kg) d Marb e Qual f Yield g

AA 10.5 1.9 70.9 4.6 2.8 .4 264.3 17.3 4.7 .1 11.3 .9 2.8 .3

AB 10.4 2.0 71.8 4.9 2.7 .5 275.0 18.4 4.7 .3 11.2 .9 2.8 .4

BB 9.9 3.5 68.1 8.5 1.3 .8 240.2 32.1 4.9 .8 10.4 1.7 2.4 .6

P-value .97 .85 .12 .33 .96 .79 .73

Table 4.6. Least-squares Means and Standard Errors for Carcass Traits by GHRHRE6N Genotype in High Selection Line
a

Fat: Backfat thickness between 12th and 13th rib (cm) Ribeye: Ribeye area (cm2 ) c KPH: Kidney, pelvic, and heart fat (%) d Hotwt: Hot carcass weight (kg) e Marb: Marbling score (1 to 10, integer only) f Qual: Quality grade (6 to 14, integer only) g Yield: Yield grade (1 to 5)
b

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Trait Fat (cm) a Ribeye (cm2 ) b KPH (%)c Hotwt (kg) d Marb e Qual f Yield g

AA 9.1 .6 75.0 1.4 2.1 .1 271.2 3.9 5.1 .2 10.7 .2 2.4 .1

AB 9.3 1.8 73.2 4.5 2.5 .4 266.2 12.5 5.4 .4 11.9 .8 2.5 .4

BBh

P-value .91 .73 .45 .72 .59 .16 .75

Table 4.7. Least-squares Means and Standard Errors for Carcass Traits by GHRHRE6N Genotype in Low Selection Line
a

Fat: Backfat thickness between 12th and 13th rib (cm) Ribeye: Ribeye area (cm2 ) c KPH: Kidney, pelvic, and heart fat (%) d Hotwt: Hot carcass weight (kg) e Marb: Marbling score (1 to 10, integer only) f Qual: Quality grade (6 to 14, integer only) g Yield: Yield grade (1 to 5) h: No BB genotypes were detected in the 77 low line Angus bulls.
b

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CHAPTER 5 ASSOCIATIONS OF POLYMORPHISMS IN THE PROMOTER REGION OF THE GROWTH HORMONE RELEASING HORMONE RECEPTOR GENE WITH MILK PRODUCTION IN JERSEY CATTLE

Abstract
Milk production is an economically important trait for dairy cattle. Selection on milk merit can be improved by applying genetic markers on candidate genes. In this study, the growth hormone releasing hormone receptor gene was screened for useful gene markers. One polymorphism was detected in the promoter region of the gene. Two

alleles, A and B, were found in 30 Jersey cows. Genotypes AA, AB, and BB were observed with frequencies of .44, .53, and .03, respectively. This polymorphism was due to a C to T mutation in allele B. Restriction enzyme AciI can be used to genotype this SNP. The associations of this polymorphism with milk merit were analyzed using the GLM procedure in SAS. Fixed effects of genotype and lactation number were included in the model. Significant relationships were observed between genotypes of this polymorphism and relative value of milk production (P= .05). In addition, moderate associations were found for 305-d milk production (P= .08). For both traits, animals with genotype AB tended to have a higher mean. Therefore, it is assumed that this polymorphism had an overdominant effect on milk production.

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Introduction

The Jersey is an efficient dairy producer which produces high quality milk for human food. Milk production is thus an economically important trait for the Jersey cow. However, milk production traits can only be evaluated after the first calving, which is a major obstacle to dairy cattle selection. Current technologies enable scientists to improve the accuracy and efficiency of traditional selection by applying genetic markers. This can be done through marker-assisted selection (MAS), which combines information on molecular genetic polymorphisms (marker loci) with data on phenotypic variation among individuals (and their relatives). Therefore, identification of genetic markers for candidate genes associated with milk production could lead to a more efficient selection program. Growth hormone (GH) secretion is associated with milk yield and lactation performance in cattle (Reinecke et al., 1993; Parchury et al., 1993; Woolliams et al., 1993). Exogenous administration of GH induces an increase in milk yield of 10 to 40% (Barber et al., 1992). Beef cows divergently selected for milk production have different GH responses to a challenge dose of growth hormone releasing hormone (GHRH) (Auchtung et al., 2001). Synthesis and secretion of GH in the somatotroph cells of the anterior pituitary are under multifactoral control from the hypothalamus in the brain (Martin, 1979; Mayo et al., 1995). The stimulatory signal is from GHRH. However, no associations of genetic variations in the GHRH gene with abnormal growth or growth hormone secretion have

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yet been identified. Therefore, the receptor for GHRH may be a better candidate for genetic markers. The GHRH-R is a member of the G protein-coupled receptor family and is able to transduce GHRH-dependent increases in intracellular cAMP via Gs activation and stimulate somatotroph proliferation and GH gene expression (Mayo, 1992; Lin et al., 1992; Gaylinn et al., 1993). GHRH-R cDNAs from rat (Lin et al., 1992; Mayo et al., 1992), mouse (Lin et al., 1992), swine (Hsiung et al., 1993), human (Mayo, 1992; Gaylinn et al., 1993), and bovine (Horikawa et al., 2001) have been identified. The GHRH-R gene transcripts have a highly specific distribution in the anterior pituitary. Mutations in the receptor have been found to cause heritable GH deficiency diseases in both mice (Godfrey et al., 1993; Lin et al., 1993) and human (Wajnrajch et al., 1996; Maheshwari et al., 1996). Mutations in the GHRH-R gene may also play a role in GH excess and pituitary tumorigenesis (Petersenn et al., 1998). The GHRH-R gene has been assigned to bovine chromosome 4 (Connor et al., 1999) and swine chromosome 18 (Sun et al., 1997). An Eco571 PCR-RFLP in intron 6 has been identified in the GHRH-R gene in cattle (Connor et al., 1999). Two PCR-RFLP, MseI and TaqI, were identified in pigs (Sun et al., 1997). Thus far, no association has been found between these polymorphisms and growth and milk merit in domestic animals. In this study we further explore the effect of GHRH-R gene polymorphism on milk merit.

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Material and Methods

Animals Thirty Jersey cows from the Waterman Dairy farm at The Ohio State University were genotyped. Cows were born from 1983 to 1991 and their lactation number ranged from 2 to 10. DNA samples stored in the Animal Genetics Laboratory of The Ohio State University were used as template DNA. Milk production traits were measured for these 30 Jersey cows during their stay at the Waterman Dairy farm.

Methods

PCR Polymerase chain reaction (PCR) was used to amplify the GHRH-R DNA fragment. Primers were designed based on the bovine DNA sequence of the GHRH-R promoter from GenBank. Primer GHRHRP (forward 5-CCCTTCAGACTTCACCTCGCCAT CC- 3 and reverse 5-CTTCCACTGTCCCCTGTTGTTCACC- 3) was used to amplify a 624bp region of the promoter of the GHRH-R gene. The PCR was performed in a 30L reaction volume containing 10pmol of forward primer and the same amount of reverse primer, 200M dNTPs, 1x reaction buffer, which contained 1.5mM MgCl2 , and 1 unit of Taq-DNA polymerase, and 100ng of genomic DNA as template. Conditions were 97o C for 2 min, followed by 35 cycles of 95o C for 45s, 63o C for 1 min, and 70o C for 90s. After 35 cycles, reactions were finished

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o by an extension of 5 min at 72o C, and samples were finally bulked at 4 C in a DNA

Thermal Cycler.

Detection of Genetic Variation When amplification was achieved, single strand conformation polymorphism (SSCP) analysis was used to screen for mutations within the amplified segment. Since the optimal size of DNA for SSCP is under 400bp, the PCR products were digested with restriction enzyme PvuII to reduce the 624bp DNA fragment to two fragments of approximately 200 and 400bp. The reaction mixture, which included 10L of PCR product, 10L of ddH2 O and 12L of loading dye, was denatured at 95C for 5 min. The samples were placed in into ice for 10 min to prevent the denatured single-stranded DNA from renaturing. The samples were then loaded on 10% polyacrylamide gels. Ten percent formamide was added to the polyacrylamide gels to improve the resolution of the DNA bands on the gel. The samples were run in 1x TBE buffer at 200 volts for 16 to 20 h at a constant temperature of 12C. Gels were then stained by .01% Ethidium Bromide for 10 min and viewed under UV light. After a polymorphism was detected, and in order to determine the nature of the polymorphism, the PCR products of the two homozygous genotypes were sent to the Plant-Microbe Genomic Facility at The Ohio State University for sequencing using an ABI 3700 analyzer. Before sending samples for sequencing, PCR products were purified with the Qiagen Gel DNA Extraction kit.

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Genotyping Rrestriction enzyme AciI was able to detect the three genotypes. Therefore, AciI was used to genotype the cows. The PCR-restriction fragment length polymorphism (RFLP) procedure included amplifying the corresponding DNA segment by PCR; digesting the PCR product with restriction enzymes; and running the digested DNA on an agarose gel containing .003% ethidium bromide. The gel was viewed under UV light.

Statistical Analysis Associations of the animal genotypes with milk production characteristics were determined by analysis of variance of quantitative traits. General Linear Model (GLM) procedures in SAS were used to perform the analysis. Fixed effects of genotypes and lactation number were included as independent variables in the linear model. Least squares means and standard errors were determined for all measurements. The measurements of milk production included Average milk production per day (kg), Average fat production in milk (kg), Average protein production in milk (kg), 305-d milk production (kg), 305-d fat production (kg), 305-d protein production (kg), Relative value of milk production (kg) compared to mean milk production of herd mates (cows with similar age and calving at same season), Relative value of fat production (kg), and Relative value of protein production (kg).

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Results and Discussion

The promoter region of the GHRH-R gene was screened for polymorphism using a breed panel that consisted of 12 Angus beef cattle, 6 Jersey dairy cattle, and 6 Holstein dairy cattle. No polymorphism was found in the Angus or Holstein breeds within the breed panel and among 40 more Angus beef cattle analyzed. However, one single nucleotide polymorphism (SNP), GHRHRP, was detected in the promoter region in Jersey. Two alleles, A and B, were found in 30 Jersey cows, which included the 6 Jersey cows in the breed panel. Genotypes AA, AB, and BB were observed with frequencies of .44, .53, and .03, respectively. This polymorphism had a C to T mutation in allele B. Restriction enzyme AciI was used to genotype this polymorphism. Association of this polymorphism with milk production was analyzed in 30 Jersey cows. Significant relationships were observed between genotypes of this polymorphism and relative value of milk production (P= .05). In addition, moderate associations were found for 305-d milk production (P= .08). For the relative value of milk production, animals with genotype AB had a higher mean (402 256kg) than animals with genotypes AA (-392 200kg) and BB (-475 762). For the 305-d milk production, AB animals also tended to have higher mean than the other two genotypes (Table 5.1). It seems this polymorphism may have an overdominant effect on milk production. Direct result of relationships of GHRH-R and milk production has not been published yet. However, GH has been shown to be associated with milk yield and lactation performance in cattle (Parchury et al., 1993; Reinecke et al., 1993; Woolliams et al., 1993). Therefore, it was assumed that the receptor of the stimulator of GH, GHRH-R,

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could have an indirect effect on milk production in cattle. Our discovery supported this assumption by showing that this polymorphism in the promoter of GHRH-R gene was associated with both relative value of milk production and the 305-d total milk production. However, 30 Jersey cows is a relatively small sample. In order to confirm our assumption and findings in this study, more Jersey cows need to be studied.

Conclusion

Milk production is an economically important trait and genetic markers that are significantly associated with this trait will be useful to improve selection for milk production. The GHRHRP polymorphism was examined in 30 Jersey cattle and was found to be significantly related to 305-d milk, fat, and protein yield. Therefore, it might be a useful marker for milk production in Jersey cattle. This observation must be confirmed in a larger number of animals. Also, other breeds of cattle should be studied for this polymorphism to determine if it has an effect on growth and carcass traits.

References
Auchtung, T.L., D.S. Buchanan, C. A. Lents, S. M. Barao, and G. E. Dahl. 2001. Growth hormone response to growth hormone-releasing hormone in beef cows divergently selected for milk production. J. Anim. Sci. 79:1295-1300. Barber, M.C., R.A. Clegg, E. Finley, R.G. Vernon, and D.J. Flint. 1992. The role of growth hormone, prolactin, and insulin-like growth factor in the regulation of rat mammary gland and adipose tissue metabolism during lactation. J. Endocrinol. 135:195-202.

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Baumann, G., and H. Maheshwari. 1997. The dwarfism of Sindh: Severe growth hormone (GH) deficiency caused by a mutation in the GH-releasing hormone receptor gene. Acta Paediatr Suppl. 423:33-38. Connor, E.E., M.S. Ashwell, S.M. Kappes, and G.E. Dahl. 1999. Rapid communication: Mapping of the bovine growth hormone-releasing hormone receptor (GHRH-R) gene to chromosome 4 by linkage analysis using a novel PCR-RFLP. J. Anim. Sci. 77:27362741. Gaylinn, B.D., J.K. Harrison, J.R. Zysk, C.E. Lyons, K.R. Lynch, and M.O. Thorner. 1993. Molecular cloning and expression of a human anterior pituitary receptor for growth hormone-releasing hormone. Mol. Endocrinol. 7:77-84. Godfrey, P., J.O. Rahal, W.G. Beamer, N.G. Copeland, N.A. Jenkins, and K.E. Mayo. 1993. GHRH receptor of little mice contains a missense mutation in the extracellular domain that disrupts receptor function. Nat. Genet. 4:227-232. Horikawa,R., B.D. Gaylinn, C.E. Lyons Jr., and M.O. Thorner. 2001. Molecular cloning of ovine and bovine growth hormone-releasing hormone receptors: the ovine receptor is C-terminally truncated. Endocrinology 142:2660-2668. Hsiung, H.M., D.P. Smith, X.Y. Zhang, T. Bennett, P.R. Rosteck Jr., and M.H. Lai. 1993. Structure and functional expression of a complementary DNA for porcine growth hormone-releasing hormone receptor. Neuropeptides 25:1-10. Lin, C., S.C. Lin, C.P. Chang, and M.G. Rosenfeld. 1992. Pit-1 dependent expression of the receptor for growth hormone releasing factor mediates pituitary cell growth. Nature 360:765-768. Lin, S.C., C.R. Lin, I. Gukovsky, A.J. Lusis, P.E. Sawchenko, and M.G. Rosenfeld. 1993. Molecular basis of the little mouse phenotype and implications for cell typespecific growth. Nature 364:208-213. Maheshwari, I., B.L. Silverman, J. Dupuis, and G. Baumann. 1998. Phenotype and genetic analysis of a syndrome caused by an inactivating mutation in the growth hormone releasing hormone receptor: Dwarfism of Sindh. Journal of Clinical Endocrinology and Metabolism 83:4065-4074. Martin, J.B. 1979. Twenty third annual Bowditch Lecture. Brain mechanisms for integration of growth hormone secretion. Physiologist 22:23-29. Mayo, K.E. 1992. Molecular cloning and expression of a pituitary-specific receptor for growth hormone releasing hormone. Mol. Endocrinol. 6:1734-1744.

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Mayo, K.E., P.A. Godfrey, S.T. Suhr, D.J. Kulik, and J.O. Rahal. 1995. Growth hormone releasing hormone: Synthesis and signaling. Recent progress in hormone research 50:35-73. Parchury, N., J. Chester-Jones, K.J. Loseth, J.E. Wheaton, L.B. Hansen, D.M. Ziegler, and B.G. Crabo. 1993. Somatotropin concentrations in plasma and scrotal circumference in bulls calves with different dairy merit. J. Dairy Sci. 76:445-452. Petersenn, S., A.C. Rasch, M.Heyens, and H.M. Schulte. 1998. Structure and regulation of the human growth hormone releasing hormone receptor gene. Molecular Endocrinology 12:233-247. Reinecke, R.L., M.A. Barnes, R.M. Akers, and R. E. Pearson. 1993. Effect of the selection for milk yield on lactation performance and plasma growth hormone, insulin and IGF-I in first lactation Holstein cows. J. Dairy Sci. 76 (suppl. 1):286 Sun, H.S., C. Taylor, A. Robic, L. Wang, M.F. Rothschild, and C.K. Tuggle. 1997. Mapping of growth hormone releasing hormone receptor to swine chromosome 18. Anim. Genet. 28:351-353. Wajnrajch, M.P., J.M. Gertner, M.D. Harbison, S.C. Chua Jr, and R.L. Leibel. 1996. Nonsense mutation in the human growth hormone-releasing hormone receptor causes growth failure analogous to the little (lit) mouse. Nat. Genet. 12:88-90. Woolliams, J.A., K.D. Angus, and S.B. Wilson. 1993. Endogenous pulsing and simulated release of growth hormone in dairy calves of high and low genetic merit. Anim. Prod. 56:1-8.

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Trait

AA

AB

BB

P-value

avemilk a avefat b avepr c milk d fat e ps f rvmilk g rvfat h rvps I

14.9 .7 .74 .04 .56 .03 5998 213 296 12 220 11 -392 200 -8.1 11.2 -16.3 11.0

16.1 .9 .79 .05 .59 .04 6781 272 329 16 249 15 402 256 27.0 14.2 13.6 15.3

17.2 2.7 .84 .15 .65 .12 6151 811 319 47 229 39 -475 762 -20.3 42.5 -5.3 40.0

.47 .64 .66 .08 .24 .26 .05 .11 .26

Table 5.1. Least-squares Means and Standard Errors for Milk Merit by Genotypes of the GHRHRP Polymorphism
a

avemilk: Average milk production per day (kg), avefat: Average fat production in milk(kg), c aveps: Average protein production in milk (kg), d milk: 305-d milk production (kg), e fat: 305-d fat production (kg), f ps: 305-d protein production (kg), g rvmilk: Relative value of milk production (kg) compared to mean milk production over all cows, h rvfat: Relative value of fat production (kg), I rvps: Relative value of protein production (kg).
b

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CHAPTER 6 ASSOCIATIONS OF POLYMORPHISMS IN THE INSULIN-LIKE GROWTH FACTOR II GENE WITH GROWTH AND CARCASS TRAITS IN ANGUS BEEF CATTLE

Abstract

Insulin-like growth factor II (IGF-II) plays a key role in pre-adolescent growth, influencing fetal cell division and differentiation. Therefore, it is a candidate gene for genetic markers for growth and carcass traits. Genetic markers could be used to facilitate genetic improvement by marker-assisted selection. An AciI polymorphism was observed in intron 8 of the IGF-II gene. Two alleles, A and B, and three genotypes, AA, AB, and BB, were observed in Angus cattle. Genotypic frequencies of AA, AB, and BB were .15, .43, and .42, respectively. Sequencing results demonstrated a transition from T to G in allele A. Restriction enzyme AciI was used to genotype this SNP. Relationships of genotypes with growth and carcass traits and with IGF-I concentrations were studied using the GLM procedure in SAS. Significant associations were found between genotypes and weight gain during the 20-d period between weaning and the beginning of the postweaning test (P= .05) and IGF-I concentration at d 28 of the 140-d postweaning test (P= .02). In addition, moderate relationships between the genotypes and preweaning gain, on-test weight, off-test weight, and weight at d 28 and 56 of the

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postweaning test were observed. For all of these traits, genotype AB had a significantly higher mean than genotype AA. Significant relationships were also observed for ribeye area (P= .01) and yield grade (P= .03). Animals with genotype AB had a higher mean for ribeye area, and a lower mean for yield grade.

Introduction

Growth and carcass traits are economically important traits in livestock. It has been shown that these traits are under the control of multiple genes. Genetic marker information on related genes can be used to facilitate selection and breeding through marker assisted selection (MAS) in domestic animals. Insulin-like growth factor II (IGF-II) belongs to a family of structurally related polypeptides, which include IGF-I, insulin, and relaxin (Blundell and Humbel, 1980; Dafgard et al., 1985). IGF-II is important in the fetus (Han et al., 1987). It plays a key role in pre-adolescent growth, influencing fetal cell division and differentiation. IGF-II knockouts in mice were shown to have significant fetal growth retardation, especially in the early stages of gestation (DeChiara et al., 1990). On the other hand, clinical evidence suggested that increased levels of IGF-II had an effect on growth and development in vivo (Engstrm et al., 1998). Overexpression of IGF-II can cause rare genetic syndromes, such as Wiedemann Beckwith syndrome, which leads to overgrowth, as well as growth disturbances, and increased frequencies of neoplasia (Engstrm et al., 1988; Nystrm et al., 1992; Ekstrm et al., 1992; Ward, 1997). T ransgenic mice with overexpression of IGF-II were

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shown to have organ overgrowth and tumor formation (Ward et al., 1994; Rogler et al., 1994). IGF II was shown to have a role in fetal development in pigs (Daughaday et al., 1986; Hausman et al., 1991). Research also showed a combinational effect of IGF-II and IGF-I on postweaning weight gain in pigs (Lamberson et al., 1996). No direct effect of IGF-II on growth and carcass traits of cattle has been published. Thus far, polymorphism information for the IGF-II gene has not been reported in domestic animals. IGF-II cDNA has been cloned in human (Bell et al., 1984), rat (Dull et al., 1984), and mouse (Bell et al., 1986), as well as in domestic animals, including sheep (OMahoney and Adams, 1989), pig (Catchpole and Engstrm, 1990), mink (Ekstrm et al., 1993), and horse (Otte and Engstrm, 1994; Otte et al., 1996). Bovine cDNA has not been completely cloned; therefore, the ovine IGF-II cDNA sequence from Genbank was used to design primers in this study. The sheep IGF-II gene is on chromosome 21 and consists of 10 exons and 3 promoters. Exons 8, 9 and 10 of the sheep IGF-II gene are the coding region (Engstrm et al., 1998). In this study, the IGF-II gene was examined as a candidate gene for growth and carcass traits. Detecting genetic variations in the IGF-II gene and relating them to growth rate and carcass traits could be helpful in development of marker-assisted selection (MAS) programs in animal breeding. This study was designed to examine the IGF-II gene for possible polymorphism and to test the association of the polymorphism with growth and carcass traits in Angus beef cattle.

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Materials and Methods

Animals

Angus beef cattle, which were divergently selected for blood serum insulin-like growth factor I (IGF-I) concentration, were used as the experimental animals. Selection began in 1989 at the Eastern Ohio Resource Development Center (EORDC), using 100 spring-calving (50 high line and 50 low line) and in 1990 using 100 fall-calving (50 high line and 50 low line) purebred Angus cows with unknown IGF-I levels. Cows from the base population were randomly assigned to the selection lines. Each year, four bull calves with the highest and four bull calves with the lowest IGF-I concentration were selected for breeding within the selection lines. Approximately eight cows were culled from each selection line each year based on physical unsoundness, failure to conceive in two consecutive years, and oldest age. These cows were replaced with approximately the same number of pregnant heifers having the highest or lowest serum IGF-I concentration (Davis and Simmen, 1997). In this study, 371 animals born in the years of 1995, 1996, and 1997 were genotyped.

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Methods

DNA extraction Genomic DNA was isolated from blood samples that were collected during the IGF-I selection experiment at EORDC. The extraction procedures were as described in Chapter 3.

PCR Polymerase chain reaction (PCR) was used to amplify the DNA fragments from genomic DNA. Primers were designed based on the ovine DNA sequence from GenBank. Primer IGFIII8 (forward 5-GGGCCCGCCTCTCGCTTCCTTCT-3 and reverse 5-CGCGGGGCTGCAGAGGGAGAGA-3) was used to amplify the region from intron 8 to intron 9. The IGFIII8 primer amplified a 385bp DNA fragment, which covered all of exon 9 and the boundaries of intron 8 and intron 9. The PCR was performed in a 30L reaction volume containing 10pmol of forward primer and the same amount of reverse primer, 200M dNTPs, 1x reaction buffer, which contained 1.5mM MgCl2 , 1 unit of Taq-DNA polymerase, and 100ng of genomic DNA as template. Conditions were 97o C for 2 min, followed by 35 cycles of 95o C for 1 min, 67o C for 1 min, and 72o C for 90s. After 50 cycles, reactions were finished by an extension of 5 min at 72o C.

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Detection of Genetic Variation When amplification was achieved, single strand conformation polymorphism (SSCP) procedures were used to screen for mutations within the amplified segment. The reaction mixture, which included 10L of PCR product, 10L of ddH2 O and 12L of loading dye, was denatured at 95C for 5 min, and then placed on ice for 10 min to prevent the denatured single stranded DNA from renaturing. The samples were then loaded on 10% polyacrylamide gels. Ten percent urea or 10% formamide were added to the polyacrylamide gels in order to improve the resolution of the DNA bands. The samples were run in 1x TBE buffer at 200 volts for 16 to 20 h at a constant temperature of 10C. Gels were then stained using .01% ethidium bromide for 10 min and viewed under UV light. After a polymorphism was detected, and in order to determine the nature of the polymorphism, the PCR products of the two homozygous genotypes were sent to the Plant-Microbe Genomics Facility at The Ohio State University for sequencing using an ABI 3700 analyzer. Before sending samples for sequencing, the PCR products were purified with the Qiagen Gel DNA Extraction Kit.

Genotyping PCR-RFLP (restriction fragment length polymorphism) using AciI was used to genotype the cattle. The PCR-RFLP procedure included amplifying the corresponding DNA segment by PCR; digesting the PCR product with AciI; and running the digested DNA on an agarose gel containing .003% ethidium bromide. The gel was then viewed under UV light.

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Statistical Analysis

Growth traits and IGF-I concentrations Three-hundred-seventy-one Angus cattle were analyzed for growth traits, which included birth weight (kg), weaning weight (kg), preweaning gain (kg), on-test weight (kg), weight (kg) at d 28 and 56 of the 140-d postweaning test, off-test weight (kg), weight gain (kg) during the 20-d period between weaning and the beginning of the postweaning test, postweaning gain (kg), serum IGF-I concentration (ng/mL) on d 28, 42, and 56, and mean serum IGF-I concentration (ng/mL).

Carcass traits Approximately 127 Angus bulls that were not saved for breeding were analyzed for carcass traits. Ages of these bulls at slaughter ranged from 300 to 500 d. Bulls born in 1995 and 1996 were slaughtered at The Herman Falter Packing Co. in Columbus, OH. Bulls born in 1997 were slaughtered at the Mahan Packing Co. in Bristolville, OH. Carcass traits analyzed for this polymorphism included: Backfat thickness (cm) Ribeye area (cm2 ) Kidney, pelvic, and heart fat (KPH) (%) Hot carcass weight (kg) Marbling score (1- devoid, 2- practically devoid, 3- traces, 4- slight, 5- small, 6modest, 7- moderate, 8- slightly abundant, 9- moderately abundant, 10- abundant)

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Quality grade (6- standard-, 7- standard, 8- standard+, 9- select-, 10- select, 11- select+, 12- choice-, 13- choice, 14- choice+) Yield grade (from 1 to 5)= 2.5+ (2.5 x adjusted fat thickness (inch)) + (.2 x KPH) + (.0038 x hot carcass weight (lb)) (.32 x ribeye area (inch2 ))

Data analysis Associations of the animal genotypes with growth and carcass traits and with IGF-I concentrations were determined by analysis of variance of quantitative traits. General Linear Model (GLM) procedures in SAS were used to perform the analysis. Fixed effects of genotypes, year, season of birth (spring vs fall), age of dam, sex, and IGF-I selection line (high vs low) were included as independent variables in the linear model. Weaning age of calf was treated as a covariate in the model when analyzing weaning weight, preweaning gain, and weight gain during the 20-d period between weaning and the beginning of the postweaning test (Gain20). On-test age of calf was treated as a covariate in the model when analyzing on-test weight, weight at d 28 and 56 of the 140d postweaning test, off-test weight, postweaning gain, serum IGF-I concentration on d 28, 42, and 56, and mean IGF-I concentration. Calf age w not included in the analysis as of birth weight. For carcass traits, sex was deleted from the model since only bulls had carcass data, and slaughter age was added in the model as a covariate. Data were also analyzed separately within the high and low IGF-I selection lines using the same model, except that line was omitted from the model, to test the difference in association of genotypes with growth and carcass traits between the selection lines. Multiple comparisons of production traits were performed among three genotypes.

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Results

One polymorphism, IGFIIE9 (AciI) was detected in intron 8 of the bovine IGF-II gene. Two alleles, A and B, were observed. Three genotypes, AA, AB, and BB, were identified with frequencies of .15, .43, and .42, respectively. Genotypic frequencies were not significantly different between the high and low IGF-I lines (Table 6.1). Sequence results demonstrated a transition from T to G in allele A, which can be recognized by digestion with restriction enzyme AciI. Significant associations were observed between the genotypes and Gain20 (P= .05) and IGF-I concentration at d 28 of the 140-d postweaning test (P= .02) (Table 6.2). Genotype AB had a higher mean for Gain20 than genotype AA (P= .02), but not significantly higher than genotype BB. For IGF-I concentration at d 28, AB animals had a higher mean than BB animals (P= .007), but not significantly higher than AA animals. Moderate associations were observed between the genotypes and preweaning gain (P= .07), on-test weight (P= .10), off-test weight (P= .07), weight at d 28 (P= .10), and weight at d 56 (P= .08). For all of these traits, the heterozygotes tended to have a higher means than the homozygotes. For most of these traits, animals with genotype AA had the lowest mean (Table 6.2). When data from the two IGF-I selection lines were analyzed separately, significant effects of this polymorphism were observed on preweaning gain (P= .03) in the high line (Table 6.3), and on off-test weight (P= .03) and postweaning gain (P= .02) in the low line (Table 6.4). Genotype AB was superior for all of these traits. In the high line, AB animals also tended to have a higher mean for Gain20 (P= .09). In the low line,

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animals with genotype AB tended to have a higher mean for weight at d 56 than animals with AA and BB genotypes (P= .06). For all these traits, genotype AA had the lowest mean. One-hundred-twenty-seven bulls among the 371 Angus cattle had carcass data. When data were separated by line, the number of samples in each line was only approximately 50 to 60, which is a relatively low number for a statistically dependable result. Therefore, carcass data were only analyzed across lines. Significant relationships with genotypes were observed for ribeye area (P= .01) and yield grade (P= .03). For ribeye area, genotype AB had a higher mean than genotypes AA (P= .04) and BB (P= .007). For yield grade, genotype AB had a lower mean than genotypes AA (P= .05) and BB (P= .01) (Table 6.5).

Discussion

IGF-II is able to regulate growth, especially fetal growth, body size, and tumor formation (DeChiara et al., 1990; Rogler et al., 1994; Ward et al., 1994; Bates et al., 1995; Engstrm et al., 1998; ODell and Day, 1998). Therefore, the coding region of the IGF-II gene was studied for genetic markers. In sheep, exons 8, 9 and 10 of the IGF-II gene are the coding region (Engstrm et al., 1998). The complete bovine IGF-II gene has not been cloned thus far. Because of the similarity of sheep and cattle, it was assumed that exons 8, 9, and 10 w ould be the coding region for cattle as well. The DNA segment from intron 8 to intron 9, including exon 9, was amplified and examined for

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polymorphism. No polymorphism was detected in exon 9; one AciI PCR-RFLP was observed in intron 8. In this study, significant associations were observed between an AciI polymorphism in the IGF-II gene and growth and carcass traits, including weight gain during the 20-d period between weaning and the beginning of the postweaning test (P= .05) (Table 6.2), ribeye area (P= .01), and yield grade (P= .03) (Table 6.5). Previous studies have shown that IGF-II is an important regulator of body size. An ApaI polymorphism in the 3UTR of the IGF-II gene was reported to be associated with body weight in middle-aged men (ODell et al., 1997). Mice that lack IGF-II are viable dwarfs 60% the size of wild type animals. These IGF-II knockouts were shown to have significant fetal growth retardation, especially in the early stages of gestation (DeChiara et al., 1990). Results of this study demonstrate that the IGF-II gene affects growth rate and body composition in cattle. Although the main function of IGF-II is to control fetal growth, studies show that it also has an effect on adult growth. In human, an ApaI polymorphism was associated with body weight in middle-aged men (ODell et al., 1997). In addition, the results from a transgenic mouse model showed IGF-II regulated postnatal growth (Wolf et al., 1998). The current study also demonstrated the effect of IGF-II on postnatal growth and carcass traits. Animals with genotype AB for this AciI polymorphism were heavier in general and had higher means for traits that had significant relationships with this polymorphism. However, although genotype AB had significantly higher means for growth traits than genotype AA, it did not show a significant difference with genotype BB. Therefore, allele B could be the dominant allele and could be associated with

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higher growth rate. When the high and low lines were analyzed separately for growth traits, significant effects of this polymorphism were observed on preweaning gain (P= .03) in the high line (Table 6.3) and on off-test weight (P= .03) and postweaning gain (P= .02) in the low line (Table 6.4). The B allele was again associated with higher growth rate. For ribeye area, genotype AB had a significantly higher mean than the homozygotes. Therefore, this polymorphism may have an overdominant effect on ribeye area. For yield grade, genotype AB had the lowest mean. Lower yield grades are desirable, because they are indicative of higher yield of lean meat. Therefore, genotype AB may be the favorable genotype for ribeye area and yield grade. The IGF-II gene has been shown to be imprinted in rodents (de Chiara et al., 1991) and human (Giannoukakis et al., 1993), but thus far not in domestic animals. The AciI polymorphism detected in intron 8 of the IGF-II gene could be used to analyze the possible imprinting pattern in cattle. Sires and dams of the 371 Angus beef cattle used in this study need to be genotyped in order to study possible associations of offspring growth and carcass traits, which showed significant relationships with genotypes of the AciI polymorphism, with sire or dam production traits.

Conclusions

Significant associations of an AciI polymorphism in intron 8 of the IGF-II gene with Gain20 and with serum IGF-I concentration at d 28 of the postweaning test were observed. In addition, moderate relationships between the genotypes and preweaning

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gain, on-test weight, off-test weight, and weight at d 28 and 56 of the 140-d postweaning test were found. Among the carcass traits, significant relationships were observed for ribeye area and yield grade. This polymorphism had a dominant effect on growth traits; allele B was the favorable allele. Genotype AB was the favorable genotype for ribeye area and yield grade. More breeds of cattle should be tested for this polymorphism to study its effect in other populations.

References
Bates, P., R. Fisher, A. Ward, L. Richardson, D.J. Hill, and C.F. Graham. 1995. Mammary cancer in transgenic mice expressing insulin like growth factor II. Br. J. Cancer 72:1189-1193. Bell, G.I., J.P. Merryweather, R. Sanchex-Pescador, M.M. Stempien, L. Priestley, J. Scott, and L.B. Rall. 1984. Sequence of a cDNA clone encoding human preproinsulinlike growth factor II. Nature 310:775-777. Bell, G.I., M.M. Stempien, N. M.Fong, and L.B. Rall. 1986. Sequences of liver cDNAs encoding two different mouse insulin like growth factor I precursors. Nucl. Acid Res. 14:7873-7882. Blundell, T.L., S. Bedarkar, and R.E. Humbel. 1983. Tertiary structures, receptor binding, and antigenicity of insulin-like growth factors. Fed. Proc. 42:2592-2597. Catchpole, I., and W. Engstrm. 1990. Nucleotide sequence of a porcine insulin like growth factor II cDNA. Nucl. Acids Res. 18:6430. Dafgard, E., M. Bajaj, A.M. Honegger, J. Pitts, S. Wood, and T. Blundell. 1985. The conformation of insulin-like growth factors: relationships with insulins. J. Cell Sci. Sppl. 3:53-64. Daughaday, W.H, C.E. Yanow, and M. Kapadia. 1986. Insulin-like growth factors I and II in maternal and fetal guinea pig serum. Endocrinology 119:490-494. Davis, M.E., and R.C.M. Simmen. 1997. Genetic parameter estimates for serum insulinlike growth factor I concentration and performance traits in Angus beef cattle. J. Anim. Sci. 75:317-324.

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Genotype

Across-lines

High line

Low line

AA AB BB

.15 .43 .42

.16 .41 .43

.14 .46 .40

Table 6.1. Genotypic Frequencies for IGFIIE9 (AciI) Polymorphism by IGF-I Selection Linea P-value for test of difference in genotypic frequency between high and low selection lines was .47.
a

119

Trait Birth wt (kg) Weaning wt (kg) Prewean. gain (kg) Gain20a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean.gain(kg) D28 IGF-I (ng/mL) D42 IGF-I (ng/mL) D56 IGF-I (ng/mL) Mean IGF-I (ng/mL)

AA 33 1 157 3 122 5 15 2 216 4 250 5 283 5 379 6 162 4 283 13 307 15 305 14 298 12

AB 34 0 162 2 134 3 20 1 222 3 258 3 294 3 393 4 168 3 297 10 333 10 312 10 314 9

BB 34 0 161 2 129 3 17 1 216 3 252 3 289 3 386 4 168 3 269 9 315 10 306 10 296 8

P-value .63 .39 .07 .05 .10 .10 .08 .07 .33 .02 .11 .82 .14

Table 6.2. Least-squares Means and Standard Errors by IGFIIE9 Genotypes across the Two Selection Lines Weight gain during the 20-d period between weaning and the beginning of the postweaning test.
a

120

Trait Birth wt (kg) Weaning wt (kg) Prewean. gain (kg) Gain20a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean.gain(kg) D28 IGF-I (ng/mL) D42 IGF-I (ng/mL) D56 IGF-I (ng/mL) Mean IGF-I (ng/mL)

AA 34 1 153 5 126 11 15 3 212 5 250 7 282 7 378 9 162 6 323 23 345 24 344 23 337 20

AB 33 1 157 4 150 9 20 2 216 4 254 5 287 6 382 7 160 5 344 19 376 20 336 19 352 16

BB 33 1 157 3 139 8 18 2 212 4 250 5 284 5 380 7 165 4 318 18 371 18 350 17 346 15

P-value .83 .52 .03 .09 .49 .53 .69 .82 .48 .24 .32 .65 .70

Table 6.3. Least-Squares Means and Standard Errors by IGFIIE9 Genotypes in the High Line Weight gain during the 20-d period between weaning and the beginning of the postweaning test.
a

121

Trait Birth wt (kg) Weaning wt (kg) Prewean. gain (kg) Gain20a (kg) On-test wt (kg) D 28 wt (kg) D 56 wt (kg) Off-test wt (kg) Postwean.gain(kg) D28 IGF-I (ng/mL) D42 IGF-I (ng/mL) D56 IGF-I (ng/mL) Mean IGF-I (ng/mL)

AA 34 1 156 5 139 11 16 3 215 6 245 7 278 8 373 10 157 6 227 17 256 20 276 20 253 16

AB 34 1 162 3 144 7 19 2 223 4 260 5 297 5 400 6 175 4 246 11 281 13 289 13 271 10

BB 35 1 162 3 142 7 17 2 218 4 256 5 294 5 392 6 171 4 222 10 257 12 267 12 248 10

P-value .89 .59 .91 .41 .26 .16 .06 .03 .02 .13 .17 .31 .13

Table 6.4. Least-squares Means and Standard Errors by IGFIIE9 Genotypes in the Low Line Weight gain during the 20-d period between weaning and the beginning of the postweaning test.
a

122

Trait Fat (cm) a Ribeye (cm2 ) b KPH (%)c Hotwt (kg) d Marb e Qual f Yield g

AA 10.6 1.5 69.9 4.5 2.6 .3 259.3 14.8 5.1 .3 11.3 .7 2.8 .3

AB 10.9 1.6 75.6 4.7 2.7 .3 261.6 15.5 4.8 .2 11.0 .7 2.6 .3

BB 12.4 1.7 69.2 4.9 2.7 .4 251.7 16.3 5.0 .3 11.4 .7 2.9 .3

P-value .09 .01 .98 .41 .58 .48 .03

Table 6.5. Least-squares Means and Standard Errors for Carcass Traits by IGFIIE9 Genotypes Across Lines
a

Fat: Backfat thickness between 12th and 13th rib (cm), b Ribeye: Ribeye area (cm2 ), c KPH: Kidney, pelvic, and heart fat (%), d Hotwt: Hot carcass weight (kg), e Marb: Marbling score (1 to 10, integer only), f Qual: quality grade (6 to 14, integer only), g Yield: Yield grade (1 to 5).

123

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