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SPRAGUE-DISSERTATION.Pdf (6.772Mb) i STRUCTURE AND FUNCTION OF THE DELETED IN AZOOSPERMIA GENE A Dissertation by DAVID CHASE CAMERON SPRAGUE Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY December 2006 Major Subject: Medical Sciences ii STRUCTURE AND FUNCTION OF THE DELETED IN AZOOSPERMIA GENE A Dissertation by DAVID CHASE CAMERON SPRAGUE Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Co-Chairs of Committee, Thomas J. Kuehl Allison C. Rice-Ficht Committee Members, Fuller W. Bazer Steve A. Maxwell Head of Department, J. Martin Scholtz December 2006 Major Subject: Medical Sciences iii ABSTRACT Structure and Function of the Deleted in Azoospermia Gene. (December 2006) David Chase Cameron Sprague, B.S., Saint Edward’s University Co-Chairs of Advisory Committee: Dr. Thomas J. Kuehl Dr. Allison C. Rice-Ficht A number of genes have been associated with variation in human spermatogenesis related to fertility. One of these, the Deleted in Azoospermia (DAZ) gene, exists as copies on two chromosomes, 3 and Y. The autosomal copy, DAZ-like (DAZL), has one RNA recognition motif (RRM) and is homologous to the DAZL gene found throughout the vertebrate lineage. There are four copies of DAZ on the Y chromosome with a pair at each of two sites. One pair contains a single RRM and the other has three RRMs. Human DAZ is homologous to genes in old world primates and ape Y chromosomes. Both DAZ and DAZL bind messenger RNAs at U-rich sequences near the poly-A tail in a manner that facilitates translation. Both are expressed in spermatogonia during the transition from mitotic cellular expansion through meiotic chromosomal reduction and during spermiogenesis. This study examined genomic variation in DAZ and DAZL, including deletion of DAZ from individuals with various levels of sperm cell production and mutations of DAZL in male partners of infertile couples. Deletions in DAZ are not as common in azoospermic men from central Texas as compared to other reports. Single nucleotide polymorphisms (SNPs) were identified in anonymous infertility patients, but were not located in the exons of the RRM. Proteins produced from transcripts encoded by genes from human DAZL, DAZL with iv SNPs within and outside the RRM, and a DAZ with single RRM were identified. Binding activity of DAZL to mRNA was confirmed using a microarray method, and mRNA from human testes was screened to identify at least 1,313 mRNA potential targets for DAZL. These targets were involved in ribosome construction, pyruvate metabolism, cell cycle control, and proteasome function. Variations in binding of protein to a high and a low bound target mRNA were demonstrated between protein constructs of DAZL, DAZL with mutations, and DAZ. Binding of DAZL to mRNA was also confirmed using electrophoretic mobility shift assays. With materials and procedures developed during this study, comparisons of genetic variants of DAZ and DAZL can be performed to identify mechanisms responsible for structural and functional differences in control of spermatogenesis. v DEDICATION To my Wife, Dawn. There is no other way to say it without being trite, but I couldn’t have done it without you. You have been with me for the whole way. You gave me encouragement when I needed it most and comforting when I could not go on any further. Mostly, you reminded me to forgive when I got lost in myself. Thank you and I love you very much. …and this is for you too- William, and Maren. vi ACKNOWLEDGEMENTS I would like to thank both of my co-principal investigators, Dr. Thomas J. Kuehl and Allison C. Rice-Ficht, for their continual support of my project, my professional growth, and my personal development. I have learned much in addition to the book work and lab work. Additionally, Dr. Bazer and Dr. Maxwell used their valuable time to review my work over many iterations and for that I am grateful. I also want to thank the faculty in the Department of Molecular & Cellular Medicine for guidance. There also have been numerous staff at Scott & White over the years who have contributed greatly to this project and include, Monica Brown, Patrick Conley, Janet Dye, and Rob LeFever. My peers during my tenure at Texas A&M, who supported me include Kenny Carson, Brian O’Shea, Angela Arenas, Barbara Ruef, Mike Remedi, Jason Schmittschmitt, and Sean Conlan. Finally, I thank the support staff in my department- Lydia Mousner, Chantel Plaag, Janis Chmiel, Rebecca Hogard, and J.D. Luza. I also received a great amount of support from my family. Thank you Da, Cha, D, Quin, Momma, Jim, and Nancy. Thank you for your patience with me! Especially, I would like to honor my older brother Quin whose lifelong interest in the sciences served as a starting point for my own scientific endeavors. You all are number one in my book. vii TABLE OF CONTENTS Page ABSTRACT.......................................................................................................................iii DEDICATION .................................................................................................................... v ACKNOWLEDGEMENTS ...............................................................................................vi TABLE OF CONTENTS..................................................................................................vii LIST OF FIGURES............................................................................................................. x LIST OF TABLES ...........................................................................................................xiii CHAPTER I INTRODUCTION: INFERTILITY AND THE DAZ FAMILY OF GENES ............................................................................................ 1 Infertility.......................................................................................... 1 Structure of Genomic DNA on the Y Chromosome ....................... 3 DAZ Family of Proteins.................................................................. 5 Deletions, Inversions, and Reordering of Genomic DNA in the AZF Region................................................................................... 12 Putative Role for DAZL................................................................ 14 II GENOMIC ANALYSIS OF DAZ ........................................................... 17 Introduction ................................................................................... 17 Materials and Methods.................................................................. 22 PCR ................................................................................... 22 sY254 Screening............................................................... 22 SNP Analysis..................................................................... 25 Results ...........................................................................................26 DAZ Deletion Screening................................................... 26 Are There Point Mutations in the DAZL RRM?............... 33 Discussion ..................................................................................... 38 III ISOLATION, CLONING, AND VERIFICATION OF DAZL PROTEINS AND VARIANTS..................................................... 42 Introduction ................................................................................... 42 viii CHAPTER Page Materials and Methods.................................................................. 43 PCR from Phage Library to Amplify DAZL .................... 43 Cloning of PCR Products.................................................. 44 Streak Plates...................................................................... 45 Midipreps .......................................................................... 45 Digests of pMAL and DAZL............................................ 47 Ligation Reactions............................................................. 48 Test Expression of Clones................................................. 48 Freezer Stocks of Clones................................................... 48 Electroporation .................................................................. 49 Assessing Purity of DNA Samples.................................... 49 Sequencing Verification.................................................... 49 Site-Directed Mutagenesis................................................ 50 Expression and Purification of DAZL Proteins ................ 51 Cloning of DAZ+TEV from Clontech Phage Library ...... 52 Creation of RNA Probe for DAZL Binding...................... 53 Specific Activity Calculations........................................... 56 RNase Detection................................................................ 59 Gel Shift with CDC25C.................................................... 60 Nitrocellulose Filter Binding............................................. 60 Gradient Centrifugation..................................................... 61 Affinity Chromatography Binding Experiments............... 61 Results ...........................................................................................63 Discussion ..................................................................................... 85 IV FUNCTION OF DAZL............................................................................. 89 Introduction ..................................................................................
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