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Studies on members of the transferrin family of proteins in humans and mice Hsu, Forrest 1997

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STUDIES ON MEMBERS OF THE TRANSFERRIN FAMILY OF PROTEINS IN HUMANS AND MICE by FORREST HSU B.Sc, Humboldt State University, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology and the Biotechnology Laboratory) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA MARCH 1997 ©FORREST HSU, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. 1 further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Z o ^ k y p)<o^ecli~a\<>^y /-o-bor-^eryr The University of British Columbia Vancouver, Canada Date 9 7 Q S , Z ^ DE-6 (2/88) Abstract Iron is required by all cells. Its chemical properties have been exploited by living organisms to drive a variety of life sustaining metabolic reactions. The demand for iron coupled with its highly reactive nature has forced organisms to evolve specific mechanisms for the adsorption, transport, and storage of this element. The iron binding transferrin family of proteins that includes transferrin (Tf), lactoferrin (LTf), and melanotransferrin (MTf) has been the most well studied class of iron transporters. However, much of the molecular physiology of this family, particularly MTf, is still unclear. There is growing evidence that iron imbalances associated with abnormalities in Tf and MTf expression, localization, and function may be involved in the pathologies of a number of diseases such as cancer and Alzheimer's disease (AD). Mouse as a model system to study Tf and MTf has been poorly characterized. To date, no full length cDNA of mouse Tf has been characterized and no mouse homologue to human MTf has been identified. Possession of full length cDNAs of mouse Tf and MTf will allow us to create a variety of recombinant constructs that will allow us to make transgenic and knockout mice. The characterization of these mice may allow us to determine the importance of Tf and MTf in normal iron transport as well as the role of these molecules in diseases such as AD. In developing the mouse as a small animal model, this thesis describes (1) the cloning of a mouse Tf cDNA from a mouse liver cDNA library, (2) Southern analysis of the genomic organization of human MTf, (3) the identification of a mouse homologue of MTf through Southern analysis, (4) Northern analysis of the distribution of Tf and MTf mRNA expression in normal tissues in humans and mice and (5) identification of a mouse melanoma cell line expressing mouse MTf mRNA. A putative 2.2kb mouse Tf cDNA complete with translation start site, an endoplasmic reticulum (ER) signal peptide, 159 bp of 3' untranslated (UTR) sequence, and a poly adenylation signal was cloned and sequenced. The human MTf gene is approximately 49kb long as compared to the putative mouse MTf gene which spans 38kb. The restriction sites of Bam HI, Eco Rl, and Hind III in the human and mouse MTf genes were mapped. The mRNA expression of Tf and MTf appear to be conserved in both humans and mice. Liver tissue appears to express high amounts of Tf mRNA in relation to other tissues. No detectable MTf mRNA expression was found in normal adult human and mouse tissues. In contrast, MTf mRNA was detected in the human melanoma cell line SKMEL-28, the mouse melanoma cell line JB/MS, and mouse fetal and placental tissues. Finally, cell lines and tissues positive for MTf mRNA expression appear to express multiple forms of the mRNA. In SKMEL-28 and JB/MS cells MTf transcripts of the sizes 3.9kb and 2.6kb were detected. In normal mouse liver tissues a 3.6kb MTf transcript was detected. These results establish the feasibility of using mice as a model to study Tf and MTf and have allowed us to go on to the cDNA cloning of mouse MTf and the construction of a variety of fusion proteins and deletion constructs. iii Table of Contents ABSTRACT 11 TABLE OF CONTENTS I V LIST OF TABLES: V l LIST OF FIGURES: v » ACKNOWLEDGMENTS AND DEDICATION VIII ABBREVIATIONS ' * CHAPTER ONE*. THESIS INTRODUCTION 1 Chemistry of Iron 1 Iron Uptake and Storage 1 Transferrin Famliy of Iron Binding Proteins 2 Transferrin Receptor 4 Iron Transport via Transferrin/Transferrin Receptor 5 Melanotransferrin 6 Iron Transport via Melanotransferrin 6 Iron Response Elements 7 Iron in Disease 10 Iron in Haemochromatosis 10 Iron in Neoplasia 11 Iron in Parkinson's Disease 12 Iron in Alzheimer's Disease 12 Thesis Objectives 13 CHAPTER TWO: MATERIALS AND METHODS. 15 A . Radiolabeling of D N A Probes 15 1. Purification ofcDNA Fragments from Agarose 15 32 2. P Random Primed Labeling of cDNA 15 B. Northern Analysis 15 1. Total RNA Purification from Mouse Tissues and Transformed Cell Lines 15 2. Poly(A)+ RNA Purification from Total RNA 16 3. Northern Blotting 17 4. Northern Blot Hybridization 17 C. cDNA Libray Screening 18 1. Library Titiring and Plating 18 2. Replica Plating •. 18 3. Membrane Hybridization 19 4. Phage Plaque Isolation. 19 5. Polymerase Chain Reaction (PCR) 19 D. Southern Blotting 20 1. Isolation of Genomic DNA 20 2. Restriction Digestion of Genomic DNA 20 3. Genomic Southern Blotting. 21 4. Southern Blot Hybridization 21 E . Phosphorirnaging/Autoradiography 21 F. Cell Methods 22 1. Tissue Culture 22 2. Animals 22 H. Reverse Transcriptase-Polymerase Chain Reaction 23 1. First Strand Synthesis , 23 2. Second Strand Synthesis and PCR Amplification 23 I. Subcloning of PCR Fragments ; 23 1. Fragment Purification 23 iv 2. Ligation ofPCR Products 2 4 3. Electroporation of Bacteria 24 4. Characterization of Transformants 24 J. Sequence Analysis 25 CHAPTER THREE: CHARACTERIZATION OF MOUSE TRANSFERRIN 26 INTRODUCTION 26 Structure of Human Transferrin 26 Structure of Mouse Transferrin 27 Tissue Distribution of Human Transferrin 27 Chapter Three Outline • 27 RESULTS 2 8 mRNA Transferrin Tissue Expression in Humans 28 mRNA Transferrin Tissue Expression in Mice 29 1° cDNA Library Screening 30 2° cDNA Library Screening 30 PCR Analysis of Isolated Clones 31 Subcloning of Putative Mouse Transferrin cDNA 32 Sequence Analysis of cDNA Clones 33 5' R A C E PCR • 34 cDNA Sequence of Mouse Transferrin 36 DISCUSSION 37 Transferrin mRNA Expression in Human and Mouse Tissues 37 1° Structure of Mouse Transferrin 39 CHAPTER FOUR: CHARACTERIZATION OF MOUSE MELANOTRANSFERRIN 42 INTRODUCTION 42 Structure of Human Melanotransferrin 42 Glycosylphosphatidylinositol Anchored Form 43 Protein Maturation and Intracellular Trafficking 44 Soluble Form 44 Tissue Distribution of Human Melanotransferrin 45 Species Homologues of Human Melanotransferrin 45 Chapter Four Outline 45 RESULTS 46 Human Melanotransferrin cDNA 46 Human Genomic Southern Analysis of Melanotransferrin 47 Human Genomic Southern with the 5' and 3' Halves of Melanotransferrin cDNA 48 Genomic Southern Analysis of a Mouse Melanotransferrin Gene 49 Multiple Tissue Northern Analysis of Human Melanotransferrin mRNA Expression 50 Multiple Tissue Northern Analysis of Mouse Melanotransferrin 51 Fetal Tissue and Mouse Melanoma Northern Analysis for Mouse Melanotransferrin 53 DISCUSSION 55 Melanotransferrin Gene in Humans and Mice 55 Melanotransferrin mRNA Expression in Human Tissues 56 Melanotransferrin mRNA Expression in Mouse Tissues 57 CHAPTER FIVE: CONCLUSION 59 Human and Mouse Transferrin 59 Human and Mouse Melanotransferrin 59 Biological Iron Transport Model 60 REFERENCES 62 V List Of Tables: Table 1: Comparison of Transferrin Amino Acid Sequences from Different Species 3 Table 2: Comparison of Amino Acid Sequences of the Human Transferrin Family 3 Table 3: Oligos Used in R A C E PCR Cloning of Mouse Transferrin 34 Table 4: Tissue Distribution of Transferrin mRNA in Humans and Mice 37 Table 5: Structural Features of Human and Mouse Transferrin 39 vi List of Figures: Figure 1: Phylogenetic Tree of the Divergence of Transferrin Molecules 4 Figure 2: Transferrin Receptor 5 Figure 3: Receptor-Mediated Endocytosis of Diferric-Transferrin 5 Figure 4: Proposed Endocytic Pathway of GPI Anchored Melanotransferrin 7 Figure 5: Iron Response Element Consensus Sequence Motif 8 Figure 6: Secondary Structure Model of Human Serotransferrin 26 Figure 7: Northern Analysis of Transferrin mRNA Expression in Human Tissues 28 Figure 8: Northern Analysis of Transferrin mRNA Expression in Mouse Tissues 29 Figure 9: 2° Mouse Transferrin Screen of a tajtlO Mouse Liver cDNA Library 31 Figure 10: PCR Analysis of Putative Mouse Transferrin cDNA Inserts 32 Figure 11: Assembly of Mouse Liver Tf cDNA Fragments 33 Figure 12: Location of RACE Oligos in Mouse Transferrin cDNA 34 Figure 13: Analysis of Mouse Liver Transferrin RT- PCR Products 35 Figure 14: cDNA Sequence of Mouse Transferrin 36 Figure 15: Comparison of Mouse and Human 1° Structures of Transferrin 41 Figure 16: Secondary Structure Model of Human Melanotransferrin 42 Figure 17: General Structure of a GPI Anchor 43 Figure 18: Human Melanotransferin Probe: A3-2 Construct 46 Figure 19: Southern Analysis of Human Melanotransferrin from SKMEL-28.... 47 Figure 20: Southern Analysis of 573' Ends of the Human Melanotransferrin Gene in SKMEL-28 48 Figure 21: Southern Analysis of Mouse Melanotransferrin in JB/MS Cell Line 49 Figure 22: Restriction Maps of the Human and Mouse Melanotransferrin Gene 50 Figure 23: Northern Analysis of Melanotransferrin Expression in Human Tissues 51 Figure 24: Analysis of Melanotransferrin mRNA Expression in Mouse Tissues 52 Figure 25: Northern Analysis of Melanotransferrin mRNA Expression in Mouse Melanoma Cell Lines and Fetal Tissues 53 vi i Acknowledgments and Dedication The completion of this thesis would not have been possible without the enthusiasm and support of many individuals. I would like to thank Dr. Wilfred Jefferies for his supervision of my work and for creating an environment from which I learned many aspects of becoming a good scientist. A special thanks goes to Drs. Reinhard Gabathuler and Malcolm Kennard. Their daily presence and expertise were of invaluable assistance. I would also like to acknowledge Drs. Pauline Johnson and Linda Matsuuchi for their thoughtful comments and advice on my thesis. Much gratitude goes to Drs. Jonas Ekstrand, Sylvia Rothenberger, Catherine Barbey and Jacob Hodgson. Jonas' patience in "showing me the ropes" when I first started in Wilf's lab was much appreciated. Although I did not work with Sylvia that much as she was in the process of leaving as I was starting, her whack of p97 plasmid constructs and meticulous record of her work served as a model for my own work. Cachou, who arrived midway through my M.Sc. training and left last year, provided me with much needed advice and support during a period when I thought I would never finish. And finally Jacob, the Department of Zoology's resident, "Hard-man-of-Science", was always around for consultation in those wee hours of the morning. His zest for and devotion to science has left an indelible impression on me. The people with whom I have had the pleasure to work with on a daily basis also deserve special mention. Daphne Blew, Renee LeNobel, and Alan Giesbrecht for providing crucial technical support. Willem a master at antibody production and transgenic methodologies, introduced me to the complexities and costs of making animal models and reagents. The "Old School", Gregor Reid, Ian Haidl, Kathy Shimizu, Micheal Food, Cyprian Lomas, Roger Lippe and Joseph Yang were senior graduate students when I first arrived in Wilf's Lab. They served as role models as to what a successful graduate student should be. A special thanks to Gregor who showed me how to do my first Northern blots and Mike who passed off much of his expertise on p97 and allowed me to plunder many of his antibodies, cell lines, and journal articles. Many kudos to the "new school", Judie Alimonti, Greg Lizee, Alex Moise, Jacqueline Tiong, and Brandie Thorlaksson for their fellowship and support. Finally, apart from all the technical and academic help I received at school there are a number of individuals whom I would like to acknowledge who have made my life a much richer experience. Edmond Luke, my best friend since grade 8, first introduced me to Wilf and provided me with brotherly advice and support throughout my degree. Mark Trump, a fellow graduate student I met TA'ing Bi 200 in my second year of grad studies, has quickly become one of my closest friends. His rather 'peculiar' life has provided me with much amusement that has kept me in stitches since knowing him. A special thanks to Mark T. and Murray Manson (my TA boss for several semesters) for teaching me basic mountain lore and for including me in many expeditions to Squamish and the Slesse experience last year. To Kevin Brett, Mr. RAP attack, Capoiera acolyte, ski patrol dude, drumming master, and all-around nice-guy for allowing me to live his full life vicariously. Andris Maclnnis, another fellow grad student, for sharing his enthusiasm for science, bikes, and grinding. To the Aquaguard Crew, Terry, Ian, Steve, and Nigel for showing me all those wicked trails on the North Shore, and for allowing me to race with them. Finally to my mother and father-in-law for their parental support and advice. Dedication: To my beloved wife, Neetha and to my parents. viii Abbreviations (3AP b e t a a m y l o i d p r o t e i n A D A l z h e i m e r ' s D i s e a s e A P P b e t a a m y l o i d p r e c u r s o r p r o t e i n b p b a s e p a i r C c a r b o x y t e r m i n u s c D N A c o m p l e m e n t a r y D N A E R e n d o p l a s m i c r e t i c u l u m E t B r E t h i d i u m b r o m i d e G P I g l y c o s y l p h o s p h a t i d y l i n o s i t o l I R E i ron r e s p o n s e e l e m e n t I R P i r on r e s p o n s e p r o t e i n k b k i lo b a s e k D a k i lo d a l t o n L T f l a c t o t r a n s f e r r i n m R N A m e s s e n g e r R N A M T f m e l a n o t r a n s f e r r i n M Y r m i l l i on y e a r s N a m i n o t e r m i n u s P C R p o l y m e r a s e c h a i n r e a c t i o n P I P L - C p h o s p h a t i d y l i n o s i t o l p h o s p h o l i p a s e - C P I P L - D p h o s p h a t i d y l i n o s i t o l p h o s p h o l i p a s e - D R A C E r a p i d a m p l i f i c a t i o n of c D N A e n d s R M E r e c e p t o r m e d i a t e d e n d o c y t o s i s S R P s i g n a l r e c o g n i t i o n p a r t i c l e T f t r a n s f e r r i n T f R t r a n s f e r r i n r e c e p t o r U T R u n t r a n s l a t e d r e g i o n V V o l t s ix Chapter One: Thesis Introduction Chemistry of Iron Iron is required for the growth of all organisms ranging from single celled archebacteria to mammals. Being one of the most abundant reactive elements in the primordial world primitive organisms exploited iron's chemical properties to do a variety of metabolic chores by integrating it into many structural and functional molecules. As a result, free elemental iron went from being one of the most abundantly found elements in the environment to one of the scarcest. The majority of iron above the earth's crust is now sequestered by living organisms or complexed in mineral forms inaccessible to life1"4. This demand for iron has forced living organisms to evolve specialized methods of iron uptake and storage. The necessity of iron in biological systems can largely be attributed to its relative ease in losing or gaining an electron. Under physiological conditions, iron is predominately found complexed within chemically active sites within proteins in the F e 3 7 F e 2 + states. Its energy potential is harnessed by organisms to drive important chemical reactions such as the transfer of electrons to other molecules5, adding or removing hydroxyl moieties in molecules 6, adding or removing dioxygen in molecules 7, or generating or detoxifying oxygen radicals8. Examples of enzymes requiring iron as a cofactor are found in a variety of crucial metabolic pathways such as: (1) A T P generation in the electron transport chain 9 , (2) the synthesis of DNA from RNA precursors 1 0, (3) the detoxification of xenobiotics in the P450 microsomal systems 1 1 , or (4) in free radical generation in immunological and neurological systems 1 2 . Iron Uptake and Storage Although iron is a prerequisite for most living systems, its uptake and storage must be highly regulated because its potential reactivity can lead to toxicity. Free iron in the presence of oxygen, whether it is in the Fe 2 + or F e 3 + state, can easily catalyze the generation of free radicals that are both mutagenic and molecularly destructive, and, if not kept in check, can ultimately destroy the organism. The toxicity of iron has forced organisms to develop systems to regulate the element's uptake and storage8. 1 Bacteria and plants are believed to take up iron via a system of siderophores and gated membrane channel receptors, where the bacteria or root cell synthesize and excrete siderophores into its surrounding medium. The siderophores scavenge displaced ferric (Fe3 +) iron from the medium and are then captured and transported into the cell via gated receptors on the cell surface 1 3. Yeast appear to have a slightly more sophisticated system involving a number of membrane bound proteins (fre, fet, and ftr) that can dislodge iron sequestered by foreign molecules, scavenge it and transport it into the cell 1 4 . It is also known that yeast can exploit bacterial siderophores in iron uptake1 5. Multicellular organisms such as mammals require another level of sophistication in iron uptake and storage. Mammals need to obtain iron from their environment by ingesting it. Then the iron must be delivered in the appropriate amount and correct form to the various tissues. The mechanism by which mammals take up iron from the environment, through their digestive tracts is still unclear, but it is believed that the system may be similar to the fre, fet, ftr system found in yeast based on the similarity of structure and function of cerruloplasmin in mammals to the fet protein in yeast 1 5. The mechanism by which iron is absorbed into and transported between cells is still unclear despite the large number of studies on this subject. However, it is generally believed that cellular iron adsorption and transport occurs predominately through a receptor mediated endocytosis (RME) pathway involving Tf and TfR 1 , 2 , 1 3 . Transferrin Famliy of Iron Binding Proteins Tf, LTf, and MTf are all members of the iron binding Tf family of proteins based on their structural and functional similarities. All three genes are highly conserved between species and possess about a 60% overall cDNA sequence homology to one another. The 3° structure of all three proteins may be separated into N and C terminal domains. Phylogenetic analysis of the Tf family predicts that the internal homology between the N and C terminal domains arose through a gene duplication event that occurred before the divergence of the mammalian and insect lines some 700 million years (Myr) ago 1 6 . Using this phylogenetic model, the divergence of 2 various Tfs from human serum Tf can be estimated. A table comparing the amino acid identities of Tfs from various species is shown in Table 1. The same model can be used Table 1: Comparison of Transferrin Amino Acid Sequences from Different Species Tfs amino acid sequences were divided into their N and C-terminal iron binding lobes and compared using BLAST algorithmn at the National Center of Biological Information (NCBI). Species % Identity to Human Tf N C Human N C 49 Pig N 72 C 45 71 Horse N 76 45 C 45 71 Rabbit N 82 49 C 44 76 Rat C 39 67 Chicken N 54 46 C 46 54 Frog N 4 3 40 C 42 51 Hornworm N 28 30 C 31 33 to compare Tf-related sequences such as LTf and MTf. An amino acid comparison between members of the Tf family is shown in Table 2. Using these protein identity comparisons, the evolutionary distance of a Tf family molecule from human Tf may then be calculated based upon the percentage difference at a cDNA level between two molecules. The divergence of various Tfs from human serum Tf can then be plotted on a phylogenetic tree that illustrates the ancestral relationship of human Tf. Tables 1 and 2 illustrate that the 700+ MYr old gene duplication event that created the N and C terminal lobes of Tf remains remarkably conserved Table 2: Comparison of Amino Acid Sequences of the Human Transferrin Family 1 6 Tf % Identity to Human Tf Sequence N C Human Tf N 49 C ^ 3 4 ^ ^ Human LTf N 63 C 46 62 human MTf N 46 47 C 39 45 3 among all animals compared and among all members of the Tf family 1 6 1 7 . The same model predicts the appearance of MTf occurring between 650-390 Myr, suggesting that there may be animal homologues to MTf reaching as far down the evolutionary hierarchy as fish and perhaps even some classes of invertebrates. The recent cDNA cloning of a putative chicken homologue to human MTf supports this prediction18. A more detailed treatment of the primary structure and tissue distribution of Tf and MTf can be found in the introductions to Chapters 3 and 4 respectively. Figure 1: Phylogenetic Tree of the Divergence of Transferrin Molecules Transferrin Receptor The TfR is a transmembrane protein composed of two identical subunits joined together by disulfide bridges 1 9 (see figure 2, pg 5). Each subunit may bind Tf preferring the halo- form (iron saturated Tf) as opposed to the apo-form(iron free Tf) and is involved in the receptor mediated endocytic pathway of iron transport into cells 2 1" 2 3. Preliminary evidence suggests that the TfR may also be able to bind MTf but the location and number of MTf's that bind is unknown 2 4. TfRs are found expressed on the surfaces of a variety of cell types. Its expression is a function of a cell or tissue's iron requirements and state of proliferation25 ,26. There have been a number of studies that have demonstrated that the numbers of TfR's expressed on the cell surface is directly proportional to the rate of cell proliferation leading some to propose that the interaction of Tf with its receptor triggers a cell proliferative response that is independent of its iron transport function 2 7 2 8. However, recent evidence indicates that iron is 4 C Y T O P L A S M I C E X T R A C E L L U L A R 65 AMINO ACIDS M E M B R A N E G 7 I A M , N O A C L D S Q <^  ( ^ T r a n s f e r r i n g ) ( NH2 i4 : ^yp:^^^^^ COOH |SS i s s NH2 l ^ ^ i ^ ^ COOH P H ° " ( [ T r a n s f e r r i n ^ ) ^ Figure 2: Transferrin Receptor20 linked with DNA synthesis leading to cell proliferation and that the observation of high numbers of TfR's expressed on the surfaces of proliferative cells could be a product of these cells acquiring iron through the Tf/TfR pathway20. The specific function of iron in DNA synthesis is unclear. Iron Transport via Transferrin/Transferrin Receptor Once iron is absorbed into the blood plasma from the digestive tract, its transport to other sites in the body is believed to be mediated by Tf /TfR 2 9 , 3 0 . Tf, primarily synthesized in the liver, is secreted into the blood stream where it picks up two atoms of ferric iron. Once loaded Clathrin Figure 3: Receptor-Mediated Endocytosis of Diferric-Transferrin30 5 with iron, Tf binds with the TfR and may be sequestered into the cell through a receptor mediated endocytic pathway (RME) summarized in Figure 3. Briefly, the pathway involves (1) the binding of two atoms of ferric iron to apo-Tf at a pH of >7. (2) Iron laden holo Tf binds to the TfR on the outer surface of cell membranes. (3) Tf/TfR complexes cluster in clathrin coated pits and are internalized into endosomes. (4) The pH in endosomes falls to 5.5 causing iron to be released from the complex. (5) Released iron is then transported across the endosome membrane through an unknown pathway. (6) Cytosolic iron is stored complexed to the iron storage protein, ferritin. (7) The apoTf remains bound to the TfR within the endosome where it is sorted back (8) to the cell surface. The increase in pH in the extracellular space causes apoTf to dissociate from the TfR where it may bind iron and begin the cycle anew 1 2 , 2 9 , 3 0 . Melanotransferrin The evolutionary distance of MTf from serum Tf has prompted the question of whether MTf is a trivial relic of T fs structural and functional evolution; or a significant molecule having a unique role in the metabolism of iron in animals. It is structurally related to Tf, sharing a 55% amino acid homology 3 1, contains apparantly one functional iron binding site at its N-terminal lobe 3 2 4 8 , and unlike Tf, it may exist as both a soluble protein and glycosylphosphatidylinositol (GPI) anchored cell membrane protein3 3 3 4 . MTf has been studied in in vitro systems where its molecular structure and some of its metal binding properties have been investigated 3 5 ' 4 8. However, the physiological role of MTf in iron transport is still poorly understood and is a field of study that could be aided by the development of a small animal model. A more detailed description of MTf's structure and tissue distribution may be found in Chapter 4 page 42. Iron Transport via Melanotransferrin MTf appears to be capable of binding one molecule of iron in its N-terminal lobe at a pH>6 and releases it over a pH range of 6-5 under in vitro conditions 3 2 , 4 8. The significance of MTf in iron uptake and transport is poorly understood. It has been hypothesized that iron may 6 Figure 4: Proposed Endocytic Pathway of GPI Anchored Melanotransferrin30 be transported into the cell through MTf via two pathways. The first pathway, shown in Figure 4, involves (1) GPI anchored MTf binding a Fe 3 + atom from the extracellular medium at the cell surface. (2) Iron loaded MTf can then enter an endocytic pathway. (3) MTf's iron load is then released in the acidic endosomes and (4) transported into the cytosol by an unknown process. (5) The destination of this released iron in the cytosol is unknown. (6) Once MTf releases its iron, it is free to cycle back to the cell membrane where it may repeat the process anew 3 0. The second model currently being studied may involve iron bound soluble MTf binding to the TfR at the cell surface and being internalized via RME (similar to Figure 3). The MTf-TfR complexes may aggregate in clathrin coated pits before being endocytosed into the cell; where the complex releases iron into the acidic endosome. The MTf-TfR complex is then free to cycle back to the cell surface where the MTf/ TfR complex dissociates and the process is renewed. Iron Response Elements Expressional control of proteins involved in iron transport and storage through the Tf/TfR R M E pathway are believed to be controlled by intracellular concentrations of iron through iron response elements(IREs). IREs are RNA sequences that form 2° structural motifs (Figure 5) 7 that are recognized and bound by iron regulatory proteins (IRP's). Binding of IRP's to IRE's on mRNA transcripts leads to one of two fates: 1. repression of translation by physically blocking the progress of the ribosomal translational machinery along the mRNA transcript or 2. protection from RNase degradation4 9. The IREs currently characterized are found in the untranslated regions (UTR) of mRNA transcripts and location of the IREs appears to determine the fate of the transcript when IRP's bind to the motif. An IRE found in the 5' UTR of mRNA transcripts results in repression of translation and IREs found in the 3'UTR of mRNA transcripts results in protection from RNase degradation. The binding of IRPs to IREs is regulated by the availability of iron. When there is an abundance of iron, iron binds to IRPs in iron-sulfur clusters containing 4 atoms of iron and 2 atoms of sulfur. Iron bound IRPs cannot bind to IREs 4 9 ' 5 1 . (1) G U / A A G C H (2) 0-0 (3) 0-0 0-0 0-0 0*0 (4) C 0»0 OO (5) 0*0 0*0 0«0 0*0 0«0 0»0 5'-NNNN NNNN-3' F i g u r e 5: I ron R e s p o n s e E l e m e n t C o n s e n s u s S e q u e n c e Mo t i f 5 0 IRE structural requirements are as follows: (1) nulceotides in single letter code are mandatory (2) H can not be G (3) 1st 4 base pairs must pair in top helix (0-0) (4) bulge C is mandatory (5) 2nd helix base pairing is optional (0»0), total pairing must meet a minimum stability score. In ferritin one IRE is found in it's 5' UTR, in the TfR five IREs are found in it's 3'UTR. Cells starved of iron causes high affinity binding of IRP's to IRE's leading to (1) the repression of translation of ferritin mRNA and (2) protection of TfR mRNA from RNase degradation 4 9. 8 Recently, a putative IRE has been identified in the 5' UTRs of human Tf mRNA 5 1 . Using ferritin as a model, binding of an IRP to an IRE in the 5' UTR will result in the repression of translation of the mRNA. However, in the case of human Tf, when IRP binds to its IRE, translation of Tf mRNA appears to increase, suggesting that location of the IRE may not be the only determinant of the fate of the transcript when IRP binds to the structural motif51. Thus under iron scarce conditions cells inhibit iron storage in ferritin and increase the rate of iron uptake through the Tf/TfR R M E pathway by effectively decreasing the number of ferritins and increasing the number of Tfs and TfRs translated 4 9 , 5 1. It is important to note that most cells do not express both Tf and TfR. Mammals as an example, synthesize the bulk of their serum in liver cells and TfR expression on cells is generally a function of local intracellular iron concentrations. The length of human MTf's 5' and 3' UTR suggests that MTf expression is also subject to a level of post-transcriptional control. Compared to Tf, MTf has a 5' UTR that is 321 bp longer and a 3'UTR that is 1157bp longer than their counterparts in Tf. The role of these UTRs in MTf in transcriptional regulation is unknown. A search of human MTf cDNA using the established base pairing rules for IRE motifs found no plausible IREs. Thus if MTf is indeed regulated post transcriptionally through its UTRs, then it does not occur by the known IRP-IRE systems currently characterized. The IRP/IRE system of post transcriptional control of expression appears relatively simple when considered in the context of regulating the concentrations of ferritins and TfRs in the iron uptake and storage pathway. However, when considering the importance of iron in many biological systems the IRP-IRE regulation of expression adds another degree of complexity to the regulatory mechanisms of many metabolic pathways. IRP-IRE regulation may even extend to some pathologies. In Alzheimer's disease (AD) the integral amyloid precursor protein(APP) is abnormally cleaved producing p -amyloid (PAP), the principal component of senile plaques. APP mRNA transcripts contain one putative IRE within the 3' end of the mRNA transcript. The APP IRE is unique from previously characterized IREs in that it is located within the transmembrane coding region of the mRNA 5 2 . The 9 significance of the location of this IRE and how it is involved in the regulation of A P P by IRP and iron concentrations is unknown. Iron in Disease A normal human body contains about 4 to 5 grams of iron largely complexed to either hemoglobin or ferritin53. Daily internal turnover of iron stores from erythrocyte destruction and replacement provide a pool of "free" iron that may be redistributed to iron requiring sites throughout the body in order to maintain homeostasis. Approximately 0.05% of the total body iron stores are lost each day through sloughing off of skin, intestinal mucosa, and bleeding. This loss is involuntary as there is no known metabolically active process of iron excretion 5 4. Therefore, the burden of homeostatic regulation of body iron stores is thought to be borne by the physiological mechanisms of iron adsorption and distribution. Only 1 to 2 mg of iron must be adsorbed each day to maintain iron balance and this requirement is generally met by the diet which usually contains far more than this5 5. Abnormally high levels of iron either in the body in general or in specific tissue compartments or cell types have been implicated in a number of diseases such as haemochromatosis, cancer; and more recently, Parkinson's and AD. The major biochemical threat of iron overload is its ability to generate free radicals that can react with and alter proteins, lipids, and nucleic acids thereby compromising the integrity of the cell and ultimately altering the function and structure of tissues. Iron in Haemochromatosis People afflicted with haemochromatosis suffer from having too much iron accumulating in such tissue compartments as the pancreas, liver and heart. Iron overloads in these compartments lead to diabetes, cirrhosis, liver cancer, and cardiac dysfunction. The defect in the regulation of iron adsorption is unknown and study in this field is made even more difficult by the fact that the mechanisms for iron adsorption are not understood5 6. 10 Iron In Neoplasia Tumor growth is enhanced by iron; and attempts to divert or chelate iron from tumors is a common strategy used by hosts to inhibit tumor growth57. Tumor cells appear to utilize more than half of their iron pool incorporated in metabolically active membrane fractions as opposed to normal cells who store it in the iron storage protein ferritin58. This difference in the intracellular distribution of iron pools suggests that tumor cells are metabolically more active than normal cells and may have an increased iron requirement. Tumor cells whose iron uptake mechanisms have been studied appear to be able to adsorb iron through a variety of pathways that are generally deregulated. In malignant B lymphocytes TfR expression is constitutive compared to the tight regulation of expression observed in normal B lymphocytes 5 9. Cultured clonal leukemic cell populations appear to express TfR whereas their normal pluripotent haematopoietic progenitor counterparts do not 6 0. Melanoma and some types of hepatoma cells express MTf in addition to TfR suggesting that these cells can take up iron through an alternate pathway other than the Tf/TfR R M E pathway 4 3 , 4 8. Finally, small lung carcinoma cells can synthesize their own Tf suggesting that these cells are able to take up iron more efficiently by increasing the local Tf concentration surrounding the cell 6 1. The various methods used by tumorgenic cells to acquire iron causes harm to the host on two levels. First, the tumor cells are able to satisfy their increased iron requirements and are able to metabolically function and grow uninhibited by this factor. Secondly, increased iron pools in these cells may increase the number of mutated genes through DNA damage caused by free radicals generated from free iron. A common strategy used by the host immune response to tumors is to withhold or scavenge iron from tumor cells. Hepatocytes and macrophages appear to respond to tumors by increasing their capacity to adsorb and store iron by expressing more TfR and ferritin57. Macrophages activated against tumor cells will also actively scavenge iron from these cells as well as synthesize and secrete reactive nitrogen intermediates such as nitric oxide, nitrites and nitrates, to inhibit the flux of iron in tumor cells 6 2. The net effect of these host defense mechanisms is to block or inhibit tumor cell function and growth by reducing the local iron concentrations surrounding these cells. Tumor cells with 11 disrupted iron metabolisms do not undergo cell division and are often forced to use less efficient metabolic pathways to generate cellular energy6 3"6 5. Iron in Parkinson's Disease Parkinson's disease involves the destruction of substantia nigra neuronal cells involved in the control of coordinated motion. The pathology of the disease is reviewed in refrence 66. Briefly, the disease is characterized by episodes of involuntary jerky movements that are progressively more frequent and intense. The disease culminates in paralysis and eventual death due to loss of organ muscle function. Substantia nigra neurons produce dopamine, a neurotransmitter characterized as responsible for the "smooth" functioning of muscles. Symptoms of the disease occur when approximately 70% of the dopamine producing neurons are destroyed. The normal aging process destroys about 4% of these neurons for every ten years of life. The accelerated destruction of these neurons in people afflicted with Parkinson's is believed to occur through abnormal levels of free radicals in this area of the brain. The origins of these free radicals are unclear. One hypothesis involves the role of activated microglia cells against dopamine producing neurons. Like macrophages activated against tumor cells, activated microglia cells associated with dopamine producing neurons of the substantia nigra secrete nitric oxide and superoxide radicals. These highly reactive compounds not only produce different species of radicals through interactions with cellular molecules, but may release intracellular iron stores 6 7. The presence of free iron increases the levels of destructive free radicals within the neuron. The levels of these reactive compounds generated by this pathway is believed to overwhelm the neurons antioxidative defense mechanisms. Damage caused by these molecules ultimately cause the death of the neuron and the reduction of dopamine synthesized and excreted from the substantia nigra. How the microglia cells become activated against the dopamine producing neurons of the substantia nigra is unknown. Iron in Alzheimer's Disease AD is a neurodegenerative disease that results in the loss of memory. Two theories of how damage occurs in AD are based on the two histological landmarks of AD brains, neurofibrillary tangles and senile plaques 6 8. The molecular pathology of AD is reviewed in reference 68. Briefly, tangles arise from tau protein, normally involved in the stabilization of a 12 neuronal cell's microtubules, clustering together forming helical paired filaments. The inability of tau protein to interact with the neuron's microtubule assembly may disrupt cellular structure causing dysfunction and eventual death. Senile plaques are primarily composed of insoluble aggregates of (3AP, a normally soluble cleavage product of the integral transmembrane protein A P P . The function of A P P and PAP is unclear, although the highly conserved nature of the protein, its presence in a variety of tissues outside of the brain, and its apparent necessity in growth in cultured fibroblasts69, suggests that the protein is important to a number of mammalian species and that it may be involved in growth. There is increasing evidence that iron and other metals such as zinc and aluminum may play a role in AD pathology. Increased levels of iron laden ferritin were found in AD brains as opposed to normal, particularly in microglia associated with senile plaques 7 0 7 1 . Elevated levels of iron in plaques and tangles have also been observed 7 2 . In vitro studies have shown that low levels of free iron, zinc and aluminum can increase the rate of PAP precipitation73. The details of how iron is involved in creating tangles and plaques is still unclear. The ability of tau protein to bind iron have led some to speculate that iron binding may cause a disruption of tau binding to microtubules74. Evidence of the ability of iron to modulate the processing of A P P at the level of oc-secretase, an enzyme involved in the cleaving of membrane embedded A P P into soluble products, suggests that the enzyme may use iron as a co-factor or allosteric modifier65. The rate at which A P P is processed into its soluble form and PAP may be invovled in the rate of formation of pAP plaque formation. The identification of a putative IRE in A P P mRNA and a mutated, non-functional IRE in APP mRNA's of AD patients suggests that iron may be involved in the post transcriptional control of A P P expression 5 2. Finally, the recent identification of elevated cerebral spinal fluid and blood serum levels of the soluble form of the iron binding protein MTf suggests that AD brains may have a higher iron requirement than normal brains 7 6. Thesis Objectives In order to establish the physiological mechanisms involved in iron transport in normal and disease states, it is important to have a physiological model with which many of the necessary biochemical and molecular experiments may be conducted with relative ease and economy. Much of the work done to characterize Tf/TfR was carried out in rats, rabbits and 1 3 humans. MTf has only been studied in invitro studies with much of the metal binding assays done in cultured human melanoma and MTf transfected hamster cell systems. This thesis will attempt to further characterize the use of mice as a small animal model to study Tf and MTf biology. Mice as a physiological model system for iron biology is currently being developed. A hypotransferrinanemic mouse has been made and the mouse TfR, LTf, and ferritin have been cloned and characterized. However mouse Tf has only been partially cloned from various tissues and a mouse homologue of MTf has not yet been identified. Having full length cDNA's of both mouse Tf and MTf would allow for the expression and purification of both of these molecules for use in controlled functional studies, generation of mono and poly clonal antibodies, and the creation of novel transgenic hybrids and knockout mice. In order to further characterize the mouse as a small animal model for studying the role of MTf and Tf in iron biology we need to carry out the following: • compare the mRNA expression and tissue distribution patterns of Tf in humans and mice • determine the full length cDNA sequence of mouse Tf • determine the size of the human MTf gene • identifiy a putative mouse homologue to human MTf • compare the mRNA expression distribution patterns of MTf in various tissues and cell lines in humans and mice. Expression studies were undertaken to gain more evidence for the validity of using mice as a physiological model for humans as well as to identify a Tf mRNA tissue source. This was done by first determining the cross hybridization activity between human Tf and MTf cDNA and then probing Northern blots containing mRNA from a variety of tissues from humans and mice. The cDNA cloning of mouse Tf will allow us to study its physiological activity at a genetic level and was undertaken by screening a mouse liver cDNA library and completed using Rapid Amplification of cDNA Ends (RACE) methodology. Finally, the identification of a putative mouse MTf gene through Southern analysis will suggest that a mouse homologue of human MTf exists. 14 Chapter Two: Materials and Methods A. Radio-labeling of DNA Probes 1. Purification of cDNA Fragments from Agarose 10 u.g of an expression vector containing a cDNA fragment of interest was digested to completion over 4 hours at 37°C with selected restriction enzyme systems in the presence of 1mM spermidine. Digests were then loaded onto a 1% agarose Tris-Acetate EDTA(TAE) 100ng/mL EtBr gel and electrophoresed for 4 hours at 40 Volts. Separated cDNA fragments were excised from the gel and purified using Qiagen's Qiaex 11 kit. Purified cDNA was eluted from Qiaex II glass beads with 2x25uL of 50°C warmed 1 mM NaOH and spectrophotometrically quantified at 260nM. 32 2. P Random Primed Labeling of cDNA 25ng of purified cDNA was denatured at 100°C for 5 minutes, snap cooled on ice for 1 min, and radiolabeled for 4 hours at 37°C using Boehringer Mannheim's Random 32 Primed DNA Labeling Kit and 50u.Ci redivue [a P]dCTP from Amersham. Radiolabeled cDNA was purifed from unincorporated nucleotides by passing the reaction mix through a d d H 2 0 equilibrated Pharmacia Nick Spin column at 2300rpm for 5 minutes. The purified cDNA probe was denatured at 100°C for 5 minutes and snap cooled on ice for 1 minute prior to addition to hybridization solutions. Efficiency of labeling was monitored by aliquoting 1u.L of hybridization solution into 1mL of 32 Scintillation fluid and assayed using a P calibrated Beckman Scintillation counter. B. Northern Analysis 1. Total RNA Purification from Mouse Tissues and Transformed Cell Lines Organs and tissues were immediately extracted and cryofrozen from sacraficed C57BI/6 mice. Frozen tissue samples were then bathed in liquid nitrogen and pulverized with a -80°C cooled morter and pestle. Approximately 1g of powdered tissue was lysed in 6mL of GITC (4M Guanidineisothiocyanate, 25mM NaCitrate pH 7.0, 0.8% v/v 2-Mercaptoethanol) and layered onto a 4mL CsCI cushion (5.7M CsCI, 25mM NaAc pH 15 5.0, 0.1 mM EDTA) in SW41 tubes (344059 Beckman). The samples were spun at 32000 rpm at 23°C for 16 hours. The pellet was redissolved in 200uL of d d H 2 0 with 0.2u/mL RNasin and1.07mM DTT. The RNA was ethanol precipitated, pelleted and redissolved in 100-200uL of RNasin/DTT solution. The total RNA prep was quantified by spectrophotometry at 260nM. Samples were stored at -80°C before use. Total RNA from transformed tissue culture cell lines was purified in the same way except for the harvesting of material. 2x107 adherent cells were collected from 2x 800mL Nunc flasks with 0.0125% trypsin. The cell suspension was pelleted at 1200rpm and washed with PBS before lysis with GITC and ultracentrifugation on a CsCI cushion. Poly(A)+ RNA Purification from Total RNA A silanized 1 mL column (BioRad) was rinsed with 2mL of 5M NaOH and rinsed with ddH20. A suspension of 0.5g of dry oligo dT cellulose powder (Pharmacia) in 1 mL of 0.1 M NaOH was prepared. Depending on the amount of total RNA to be processed, various amounts of the oligo dT slurry was loaded onto the column; keeping in mind that a 100uL packed volume of oligo dT is capable of processing 1 mg of total RNA. Oligo dT columns were rinsed with 5mL of ddH20 and equlibrated to ph 7.5 with 5mL of loading buffer (0.5M LiCI, 10mM Tris-CI pH 7.5, 1mM EDTA, 0.1% SDS). Total RNA was denatured at 70°C for 10 minutes and snap cooled on ice for 1 minute. 10M LiCI was added to a final concentration of 0.5M. The RNA solution was loaded onto the column and washed with 1 mL of loading buffer. The effluent was collected and reloaded onto the column twice. The column was then washed with 2mL wash buffer (0.15M LiCI, 10mM Tri-CI pH 7.5,1mM EDTA, 0.1% SDS) and the poly(A)+ RNA eluted with 2mL of 2mM EDTA/0.1% SDS. The Eluted RNA was ethanol precipitated for 30 minutes at -20°C and centrifuged at 10,000xg for 20 minutes at 4 °C . Resulting pellet was redissolved in ddH 2 0 and quantified by Spectrophotometry at 260nM. Quality of RNA was checked by denaturing a 1ug sample of purified Poly(A)+ RNA at 70°C for 16 5 minutes and analyzed on a 1% agarose gel (1x T A E , EtBr). 3. Northern Blotting RNA samples were thawed on ice and denatured at 70°C for 5 minutes and snap cooled on ice for 1 minute. An appropriate amount of 10x loading buffer (50%v/v glycerol, 0.25% wA/ bromphenol blue, 0.25% (w/v) xylene cyanol, 1mM EDTA pH 8.0) was added to make up a final 1x loading solution that did not exceed the comb well volume. The RNA solution was loaded onto a 50ml_ 1% agarose formaldehyde gel (2.2 M Formaldehyde, 40mM M O P S pH 7.0, 10 mM NaAcetate, 1mM E D T A pH 8.0). Electrophoretic Tank, combs, and gel casting trays were treated with 10% H 2 0 2 for 1 hour to minimize the presence of RNase prior to electrophoresis. Gel was electrophoresed for 5 hours at 40v. The gel was then soaked and rinsed in d d H 2 0 whilst a downward capillary blot pyramid was assembled. The separated RNA was blotted onto Nylon N membrane (Amersham) for 16 hours using 20x S S C (3M NaCI, 0.3M NaCitrate) as transfer solution. Membrane was rinsed once in 5x S S C (0.75M NaCI, 0.06M NaCitrate) and the RNA was fixed to the membrane by baking at 80°C for 2 hours. 4. Northern Blot Hybridization Fixed membranes were prehybridized in a shaking water bath at 37°C or 42°C for 4 hours in 50% deionized formamide, 5x S S P E (0.75M NaCI, 0.05 M N a H 2 P 0 4 , 0.005M Na 2 EDTA, pH 7.4), 10x Denhardt's (0.2% (w/v) Ficoll 400, 0.2% (w/v) polyvinylpyrrolidone, 0.2% (w/v) BSA), 0.1% SDS, and 0.1mg/mL sheared herring sperm DNA. Before addition of radiolabeled probe the pre hybridization solution was replaced with fresh hybridization solution without herring sperm DNA. Blots were hybridized to radiolabeled probes for 16 hours at 37°C/42°C. Hybridization solution was stored for decay and the blots were washed 3 times for 20 minutes at 42°C. Wash stringency was controlled by varying the salt content of the wash buffers. Low stringency wash buffers were composed of 2X S S C ( 0.3M NaCI, 0.03M NaCitrate, pH 7.4), 0.1% SDS; Moderate 1 7 stringent wash buffers were composed of 1X S S C , 0.1% SDS; and high stringency wash buffers were made of 0.1 X S S C and 0.1% SDS. Washed membranes were wrapped in saran wrap before phosphor imaging or autoradiography. C. cDNA Libray Screening 1. Library Titiring and Plating A A.gt10 phage cDNA library was titered and plated using the E. coli strain c600hfl as the bacterial host. A frozen C600hfl stock was streaked onto an LB agar (1.5% (w/v) agar, 1% (w/v) bacto-tryptone, 0.5% (w/v) bacto-yeast extract, 0.5% (w/v) NaCI) plate and incubated at 37°C for 12 hours. An isolated colony was picked and cultured in a 37°C shaker for 16 hours in 5mL of LB Broth (10mM MgSC-4, 1% (w/v) bacto-tryptone, 0.5% (w/v) bacto-yeast extract, 0.5% (w/v) NaCI, 0.2% maltose). Titering of the library was acheived by preparing two serial dilutions,1:500, and 1:250,000, of the library stock and mixing 0, 2, 5, 10 uL of the last dilution with 100uL 1x Lamda dilution buffer (0.1M NaCI, 0.01 M MgS04 , 0.1 M Tris-CI pH 7.5) and 200uL of C600 overnight culture. Prepared tubes were incubated in a 37°C shaker for 15 minutes before addition of 3mL of melted LB soft top agar (0.75% (w/v) agar, 1% (w/v) bacto-tryptone, 0.5% (w/v) bacto-yeast extract, 0.5% (w/v) NaCI, 10 mM MgS04). Mixtures of phage and top agar were mixed well before pouring onto a warmed 37°C LB agar plate. Plates were swirled quickly and allowed to set at roomtemperature before incubation at 37°C for 12 hours. Library titre was determined using the formula: plaque forming units (pfu)/mL = number of plaques/uL x dilution factor x 1 0 3 u.L/mL. 2. Replica Plating Based on the library titre, large 150mm diameter LB-agar plates were constructed carrying 5,000 pfu/plate. Round 130mm Nylon N+ (Amersham) membranes were numbered and placed onto the plates using sterile forceps. The membrane was asymmetrically marked using an 18G needle while on the plate. The membrane was allowed to blot the plaques for 1 minute before placing on top (plaque side up) of a 18 Whatman 3mm filter paper soaked with denaturing solution (1.5M NaCI, 0.5M NaOH) for 1 minute followed by a 5 minute incubation on top of filter paper soaked with neutralizing solution (1.5M NaCI, 0.5M Tris-CI pH 8.0). The membrane was then rinsed in 5xSSC and the cDNA fixed to the filters by baking at 80°C for 2 hours. A duplicate membrane of each plate was also made in the same way except for a lengthened 5 minute incubation on top of plates before processing. 3. Membrane Hybridization Fixed membranes were prehybridized for 4 hours in a shaking water bath at 6 0 7 6 5 ° C in 50% (v/v) deionized formamide, 5xSSPE, 5x Denhardt's, 0.1% S D S and 0.1 mg/mL herring sperm DNA. Before addition of radiolabeled probe, membranes were immersed in fresh hybridization solution without herring sperm DNA. Membranes were hybridized for 8-12 hours at 60°C before subjecting them to three 20 minute washes in either low (2xSSC, 0.1% SDS), moderate (1x S S C , 0.1% SDS), or high (O.lxSSC, 0.1% SDS) at 60°C. Membranes were wrapped in Saran wrap before autoradiography. 4. Phage Plaque Isolation Positive signals seen on the autoradiographs of screened membranes were mapped to the original plate. Isolated plaques were cored using sterile 200u,L pipetman tips. The agar plugs were soaked in 200 uL 1xLDB and a drop of chloroform at 4°C for 16 hours. The suspension was microcentrifuged at 10,000 rpm for 5 minutes and the supernatant transferred to fresh eppendorfs before titering. Phage eluates were stored at 4 °C for short term use. 5. Polymerase Chain Reaction (PCR) cDNA inserts from isolated phage DNA were amplified using 5' forward and 3' backward primers surrounding the .^gt10 cloning site. Phage DNA were prepared by cycling 10u.L of eluate 3 times through 100-0°c for 3-1 minutes. 1oL of this phage DNA prep was used as template in PCR. Reactions were carried out in 50p± volumes containing 0.2mM dNTP's, 0.3uM each of forward and reverse primers, 2.6units of High Fidelity 19 PCR enzyme mix, and 1X Expand High Fidelity buffer with 1.5mM MgCl2- Reaction was cycled in Perkin Elmer thin wall PCR tubes in a Perkin Elmer GenAmp 9600 thermocycler using a cycling strategy of 1 cycle at 94°C for 2 minutes, 30 cycles of 94-50-72°C for 20-30-120 seconds, followed by 1 cycle of 72°C for 10 minutes and soaked at 4°C. 5uL of the reaction was taken for analysis on a 1 % agarose T A E , EtBr gel. D. Southern Blotting 1. Isolation of Genomic D N A Genomic DNA was isolated from tissue cultured cells using Qiagen's Genomic DNA kit. 2 x 10 7 cells were harvested with 0.0125% trypsin and washed in cold phosphate buffered saline (PBS). Cells were resuspended in 2ml_ of PBS prior to addition of 2mls of lysis buffer C1 (320mM sucrose, 5mM MgCI 2, 10mM Tris, 1% (v/v) Triton X-100, pH 7.5). Sample was mixed by inversion and incubated on ice for 10 minutes before centrifugation at 4°C for 15 minutes at 1300xg. The pellet was washed a second time with buffer G2 and water before votexing 10-30s in 5ml_ of nuclei lysis buffer G2 (800mM GuHCI, 30mM EDTA, 30mM Tris, 5% (v/v) Tween 20, 0.5% (v/v) Triton X-100,1mg/ml_ protease). G2 suspension was incubated at 50°C for 30-60minutes before vortexing and loading on to a tip 100 equilibrated with buffer QBT (750mM NaCI, 50mM M O P S pH 7, 15% (v/v) ethanol 0.15% Triton X-100). Loaded tip 100's were washed several times with buffer Q C (1.0M NaCI, 50mM MOPS pH 7, 15% (v/v) ethanol) and eluted with buffer Q F (1.25MNaCI, 50mM MOPS, 15% (v/v) ethanol, pH 8.5). Column flow rates were typically 12 drops/minute. Genomic DNA eluate was ethanol precipitated and redissolved overnight on rollers at room temperature in 0.1 -2mL 1mM NaOH. Genomic DNA prep was spectrophotometrically quantified at 260nM and analyzed on a 1% agarose gel (1x T A E , EtBr). 2. Restriction Digestion of Genomic D N A 20u.g of Qiagen purifed genomic DNA was digested for 4 hours at 37°C with a selected Boehringer-Mannheim restriction enzyme system in the presence of 1 mM spermidine in a total volume of 50uL. Digests were monitored to completion by 2 0 electrophoretically analyzing a 100ng sample on a 1% agarose gel ("IX T A E , 0.1 u.g/mL EtBr). Completely digested samples were prepared for loading onto a 1% agarose gel by adding 5uL of 10x DNA loading buffer, incubating at 65°C for 5 minutes and snap cooling on ice for 1 min prior to loading. Loaded gels were electrophoresed for 16 hours at 1.75v/cm length of gel. 3. Genomic Southern Blotting Electrophoresed gels were treated in 0.25 M HCI for 15 minutes or until the bromphenol blue marker turned yellow. The gel was then rinsed several times with d d H 2 0 before soaking in 0.4M NaOH while a downward capillary blot pyramid was constructed. DNA was blotted onto Nylon N+ membranes (Amersham) using 0.4M NaOH as transfer buffer for 4 hours. The blotted membrane was rinsed in 5X S S C before placing in pre-hybridization solution. 4. Southern Blot Hybridization Fixed membranes were pre-hybridized/hybridized in a shaking water bath at 60 or 65 °C for 4 hours in Amershams recommended solution containing 5x S S P E , 5x Denhardt's, 0.5% SDS, and 100uxj/mL sheared herring sperm DNA. Prior to radiolabeled probe addition, blots were pre-hybridized at 65°C or 60°C for 4 hours in a shaking water bath. Hybridization was carried out for 16 hours under the same conditions before subjecting the blots to 3 high stringent washes using 0.1XSSC and 0.1% SDS. "Hot" hybridization solution was held for decay. Blots were wrapped in saran wrap before phosphorimaging or autoradiography. E. Phosphorimaging/A utoradiography 1. Phosphorimaging was performed using a Molecular Dynamics 7200 phosphorimaging cassette and MD 7200 imager. Images were scanned in at a resolution of 200u,m and enhanced using the auto level curve function in Adobe Photoshop. 2. Kodak XAR film and cassettes with intensifying screens were used in autoradiography. 21 Blots were typically autoradiographed at -80°C for 1 or 3 days before developing. F. Cell Methods 1. Tissue Culture Adherent melanoma cell lines from humans and mice were cultured in Nunc flasks in a C O 2 , 37°C incubator. Cells were grown in DMEM supplemented with 10% fetal bovine serum, 10mM L-glutamine, 10mM H E P E S , and 10mM non-essential amino acids. The melanoma cell lines used were human SKMEL-28 , mouse S91 (ATCC); mouse B16F10, JB/MS, and JB/RH (Vincent J Hearing at NIH). Cells were grown to 80-90% confluency before detaching with 0.0125% (v/v) trypsin in phosphate buffered saline (PBS) + 1mM EDTA and diluting into fresh complete media in NUNC tissue culture flasks. Cells were incubated in a 37°C, C O 2 controlled incubator. Typical generation to confluency times from a 1/10 dilution in a 250ml_ flask were 2 days for SKMEL-28 and JB/MS; 3 days for B16F12, 5 days for JB/RH, and 7 days for S91. 2. Animals All animals used were cared for in accordance with the Canadian Council on Animal Care guidelines. Animals were housed at the South Campus Animal Care Facility at UBC under normal conditions. Food and water were provided ad libitum. C57 B6 male mice from 5 to 20 weeks old were sacraficed either by C O 2 or fracturing of the neck. Tissues were immediately excised and cryo-frozen in liquid nitrogen. Mouse placentas and fetuses were taken from pregnant mice in their last week of gestation. Mothers were sacraficed by fracturing of the neck and placentas and pups were immediatly removed and cryo-frozen in liquid nitrogen. Tissues were stored at -80°C before use. 2 2 H. Reverse Transcriptase-Polymerase Chain Reaction I. First Strand Synthesis First strand synthesis was performed using Boehringer Mannheim's EXPAND First Strand Synthesis Kit. 100pmols of primer was added to 1u.g of Total RNA and dentaured at 65°C for 10 minutes. Mixture was snap cooled on ice for 1 minute and the first strand synthesis reaction carried out in 1x Expand reverse transcriptase buffer (50mM Tris-CI, 40mM KCI, 5mM MgCl2, 0.5% (v/v) tween 20, pH 8.3), 10mM dithiothreitol, 1mM dNTPs, 20 units of RNase inhibitor, and 50 units of reverse transcriptase. Total volume was 20uL and the reaction was carried out at 42°C for 1-2 hours. 2. Second Strand Synthesis and PCR Amplification Second strand cDNA synthesis and amplification of the resulting cDNA was carried out immediatly after first strand synthesis using the polymerase chain reaction. 5uL of first strand synthesis reaction was used as a source for template cDNA. PCR was carried out in a 50u.L reaction volume containing 0.2mM dNTP's, 0.3ulv1 each of forward and reverse primers, 2.6units of High Fidelity PCR enzyme mix, and 1x Expand High Fidelity buffer with 1.5mM MgCI 2. Reaction was cycled in thin wall PCR tubes in a Perkin Elmer GenAmp 9600 thermocycler using a cycling strategy of 1 cycle at 94°C for 2 minutes, 25 cycles of 94-42-72°C for 15-30-120 seconds, followed by 1 cycle of 72°C for 10 minutes and soaked at 4°C. 5uL of the reaction was taken for analysis on a 1 % agarose TAE-EtBr gel. /. Subcloning of PCR Fragments 1. Fragment Purifcation Desired PCR amplified DNA fragments were separated by electrophoresis on 1% agarose T A E gels, excised and purified using Qiagen's Qiaex II kit. Purified cDNA was eluted from Qiaex II glass beads with 2x25uL of 50°C warmed 1 mM NaOH and 2 3 spectrophotometrically quantified at 260nM. 2. Ligation of PCR Products 3' dA overhangs in purified PCR products were polished and the resulting blunt ended product was ligated into 5° dephosphorylated SMA I linearized pBluescript in a 20u.L reaction containing 1x reaction buffer (50mM Tris-HCI, 5mM MgCl2,1mM ATP, 1mM DTT, 0.1 uM dNTPs, 5% (w/v) polyethylene glycol 8000), 2 units of T4 ligase, and 1 unit of T4 DNA polymerase. 2:1 insert to vector ratios were commonly used. Ligation reactions were incubated in a 16°C water bath for 16 hours. DNA was n-butanol precipitated from ligation reactions and redissolved in 10u.L 1mM NaOH before electrotransformation into either DH5a or JM110 bacteria. 3. Electroporation of Bacteria 10u,L of ligated DNA was mixed with 40uL of electrocompetent DH5a or JM110 and allowed to sit on ice for 10 minutes. Samples were the loaded onto chilled 0.2cm cuvettes and shocked using a Biorad electroporator set to 200Q resistance, 2.5u.F capacitance, and 1.8kv voltage. No arcing was observed and time constants were in the range of 3.8 to 4.6 seconds. Electroporated samples were immediatly transferred to 450(iL S O C media and incubated in a shaking 37°C incubator for 1 hour. A maximum of 200u.L of broth per plate was plated onto lOOuq/mL Ampicillin and 40u,g/ml IPTG/X-Gal LB plates and incubated at 37°C for 12 hours. 4. Characterization of Transformants White colonies were picked and characterized either by miniprep and restriction digest analysis or by PCR using T7 and T3 primers. Minipreps were completed using Promega's Wizard Miniprep system or Qiagen Tip 20 Miniprep kit. Plasmid DNA for PCR analysis was prepared by innoculating 5uL of 10xTNT buffer and freeze thawing at -80°C. PCR reactions were performed in a 50 u.L reaction volume containing 1x TNT, 1.5% (v/v) DMSO, 0.2mM dNTP, 2mM MgCI 2, 0.2mM each of T7 and T3 primers, 2.5 2 4 units of Taq DNA polymerase. They were cycled in thin wall PCR tubes in a Perkin Elmer GenAmp 9600 thermocycler using a cycling strategy of 1 cycle at 94°C for 2 minutes, 30 cycles of 94-50-72°C for 15-30-120 seconds, followed by 1 cycle of 72°C for 10 minutes and soaked at 4°C. 5ul_ of the reaction was taken for analysis on a 1% agarose TAE-EtBr gel. Sequence Analysis 500ng of purified cDNA clones, 3.2pmols of T3 or T7 primers, and 8uL of Taq FS/fluorescineated dideoxynuclotide terminator mix were mixed and cycled 25 times at 95°C for 15s, 50°C for 30s, 68°C for 4 min. Taq FS PCR reaction was then purified using Centrisep columns and sent to UBC Biotechnology Laboratory NAPS unit for sequencing. 2 5 Chapter Three: Characterization of Mouse Transferrin Introduction Structure of Human Transferrin Serum Tfs are glycoproteins with a molecular weight of approximately 80kDa. They are single polypeptide proteins that range in length from 680 to 700 amino acids in various species 7 7 . Human Tf mRNA is 2076 bp long flanked by 54bp of 5' and 170 bp of 3' untranslated sequence 7 8" 8 2. The translated polypeptide, shown in Figure 6 8 0, is 680 amino acids in length and can be divided into an N and C terminal lobe which share a 40% amino acid homology to each other. The protein contains 19 disulfide bridges and two potential glycosylation sites 8 1 , 8 2 . The Tf gene in humans is 33.5kbp long, containing 17 exons; and is localized to chromosome 3q2 1 8 3 , 8 4 . HtSD-umanaEflS V P D K T V R M C A V 8 E H E A T K C O S F R0HHK8 HA A I A R . 1 C O L Y S A K K V C A V 8 P C 0 3P t O A V T L O A C L Y Y D A Y L A P N N L K P V V A E F C 2 > G 8 K E O P 0 «8C L C T&C 8 K K C R L Q N M O 8 A 8 O K K V V A V A Y<2F M C C A F K C L K D 6 A C O V A F V K H 8 T I F E N L A N K A O R D O Y E L L C L D N T A l ^ M & K E Q A O N L L O H t L 0 E K C C H 6 ® A V V T 6 Q P Y O A L H C O K Y E O V C->sk. P R K P L E K A V A N F F 8 H* P y R \ A T V r E Y C t Y n i C A O M n P P V K L F C K M C A L a t j H E R L K C O E M T V .C Ntl I K A I C O E T TEA S V CE t ICC V 8 i - L * 8 T 8 C K R L N C V A K V Y E E C L Y K E Y T N F i g u r e 6: S e c o n d a r y S t r u c t u r e M o d e l of H u m a n S e r u m T r a n s f e r r i n ,80 Disulfide bridges between cysteine residues are denoted by a line. Amino acid residues circled are predicted to compose the iron binding site of Tf. 2 6 Structure of Mouse Transferrin The complete primary structure of mouse Tf is still unknown. Only fragments from mouse placenta and mammary gland tissues have been c loned 8 5 , 8 6 . Sequence homology analysis between these fragments and human Tf show a 55-86% homology between the two species suggesting the conservation of structure between the two species. Tissue Distribution of Human Transferrin Histochemical analysis of the tissue distribution of Tf in humans has identified liver, blood, and fetal tissues as having the highest concentrations of serum Tf 8 7 ' 9 4. Other tissues that have relatively low amounts in comparison are submaxillary gland, ovary, spleen, brain, lung, testis, bone marrow, lymph nodes, thymus, placenta, and cerebral spinal fluid91"94. Chapter Three Outline In order to determine the entire cDNA sequence of mouse Tf and to obtain a full length cDNA clone, Northern analysis of a variety of mouse tissues was performed to determine the best tissue source from which mouse Tf mRNA could be isolated. A Xgt10 cDNA library constructed from mouse liver was then screened using 3 2 P radiolabeled human Tf cDNA as probe. Overlapping positive clones were then purified and sequenced and R A C E PCR was used to determine the sequence of the 5' and 3' ends of the full length cDNA. Sequence analysis using the BLAST algorithm was used to identify the cDNA as mouse Tf and the sequence was analyzed for conserved structural features as well as the presence of possible iron response elements. Possession of a full length cDNA clone of mouse Tf will aid in the construction of a mouse model to study the physiology of iron uptake and storage. 2 7 Results mRNA Transferrin Tissue Expression in Humans A human multiple tissue Northern blot purchased from Clontech was hybridized with 3 2 P random primed labelled probes generated from the 2.2kb human Tf cDNA insert isolated from the pBSTRANS construct. The pBSTRANS construct is a pBluescript SK- vector containing the complete 2.2kb cDNA of human Tf cloned into the XBA l/SMA I cloning site. The vector was a gift from Bee and Dr. Ross MacGillvary at the Department of Biochemistry in the University of British Columbia. Each lane was loaded with 5u.g of poly(A+) purified RNA. Blot was pre-hybridized for 4 hours and hybridized with human Tf probes for 16 hours at 4 2 ° C in 6mL of 50% formamide/SSPE buffer. Hybridized blot was washed 3 X 20 minutes in 50ml_ of 0 .1XSSC. The Tf cDNA probes detected one species of mRNA in liver tissues at about 2.4kb A c >> 8 i CQ CL -1 _J S 3 V DL F i g u r e 7: N o r t h e r n A n a l y s i s o f T r a n s f e r r i n m R N A E x p r e s s i o n in H u m a n T i s s u e s A: Multiple Tissue Blot hybridized with 3 2 P random primed probes generated from human Tf cDNA. Probe activity was 1.7x106cpm/ml_. Autoradiography at - 8 0 ° C for 24 hours. B: Multiple Tissue Blot hybridized with 3 2 P random primed probes generated from human B-Actin cDNA. Probe activity was 1.1 x 106 cpm/mL. Autoradiography at - 8 0 ° C for 12 hours. 28 and a weaker signal of the same size in pancreas. Longer exposures of the Northern blot revealed a similar size signal present in brain, spleen, lung, and placenta. This mRNA agrees with the expected size of 2.2kb of human Tf mRNA. Human Tf mRNA is reported to be expressed in high amounts in liver and relatively low amounts in brain, spleen, lung, ovary, thymus, lymph nodes, bone marrow, and placenta. Our results appear to agree with published findings85"94. m R N A T r a n s f e r r i n T i s s u e E x p r e s s i o n i n M i c e The hybridization and wash conditions used to study Tf expression in human tissues was also used to study the expression pattern of Tf in mouse tissues. A mouse multiple tissue blot purchased from Clontech was hybridized with 3 2 P random primed labelled probes generated S u § .2 I I I " s l s ls I C Q w -J J « 2 * F Figure 8: Northern Analysis of Transferrin mRNA Expression in Mouse Tissues A: Multiple Tissue Blot hybridized with 3 2 P random primed probes generated from human Tf cDNA. Probe activity was 1.7x106cpm/mL. Autoradiography at -80°C for 24 hours. B: Multiple Tissue Blot hybridized with 3 2 P random priemd probes generated from human B-Actin cDNA. Probe activity was 1.1 x 1x06 cpm/mL. Autoradiography at -80°C for 12 hours. 29 from the 2.2kb human Tf cDNA insert isolated from the pBSTRANS construct. Each lane was loaded with 5|xg of poly(A+) purified RNA. Blot was pre-hybridized for 4 hours and hybridized with human Tf probes for 16 hours at 42°C in 6mL of 50% formamide/SSPE buffer. Hybridized blot was washed 3 X 20 minutes in 50ml_ of 0.1XSSC. The pBSTRANS probe detected a single species of mRNA at approximatley 2.2kb in liver and weaker signals of the same size in brain, spleen and lung. A non-specific signal centered at approximately 4.4kb was also detected in heart tissue. The predicted size of mouse transferin mRNA is thought to be about 2.3-2.4kb in length. Our results suggest the actual size of message may be smaller. The expression pattern found in the various tissues coincides with published expression studies of Tf in human tissues. 1° cDNA Library Screening tajtlO phage library constructed from C57B6 mouse liver cDNA was titred and plated at a density of 10,000 pfu per 150mm plate. The plate was incubated to near confluency. The resulting plaques were replica transferred to duplicate nylon membranes and hybridized using radiolabeled human Tf cDNA, pBS TRANS, as probe. Plaques that were positive on both the original and replica filters were cored from the master plate and eluted in 1x X phage dilution buffer at 4°C for eight hours. A total of 38 plaques were cored from five of the 1 ° plates. 2° cDNA Library Screening Phage eluted from cored samples isolated from the 1° screen were pooled by plate, titred and plated at a density of 1,000 pfu per 150mm plate. Phage eluates were pooled by plate to minimize the number of 2° membranes that needed to be made. The low density plating resulted in large well defined plaques that were generally well isolated from each other. These plaques were lifted and screened as in the 1° screening. Plaques positive on both the original and replica filters were again cored from the master plate and eluted in 1x lambda phage dilution buffer at 4°C for eight hours. A total of 27 plaques were isolated from four 2° plates. 3 0 Figure 9: 2° Mouse Transferrin Screen of a TigtlO Mouse Liver cDNA Library Plaques were blotted to Amersham Nylon N+ membranes and fixed by baking for 2 hours at 80°C. 5 filters and their duplicates were pre-hybridized/hybridized in 100ml_of 50% formamide/ 5X SSPE hybridization solution at 37°C for 16 hours . 3 2 P random primed Tf cDNA was used as probe. Activity of the hybridization solution was measured to be 2.5 x 106 cpm/mL. Membranes were washed 3 x 40 minutes at 37°C in 200mls 1xSSC before autoradiography at -80°C on Kodak X-AR film for 1 day. PCR Analysis of Isolated Clones Phage eluted from the 2° cDNA library screen were analyzed for their purity using PCR. Eluted phages were treated with three cycles of 95°C for 5min, ice for 1min to release the DNA contained in the phage heads. A 2u.L aliquot of this processed mix was used as template in a PCR reaction using the 5' and 3' cDNA library insert screening oligos recommended by Clontech. A 5u.L aliquot of the resulting PCR amplified products were analyzed by agarose gel electrophoresis. Product length was estimated by constructing a power function relating the distance travelled by the A-hindlll markers. Results from this analysis showed that some cored plugs contained a mixture of phages containing different cDNA inserts. The clones choosen for subcloning into pBluescript and subsequent sequence analysis were chosen based upon two criteria: i) the clone must come from a phage eluate containing only one species of cDNA insert and ii) clones of different lengths. Only one of the cDNA inserts from different phage preps that 31 F i g u r e 10: P C R A n a l y s i s o f Pu ta t i ve M o u s e T r a n s f e r r i n c D N A Inser ts PCR analysis was performed on Tf positive phage eluates prepared from a 2° screen. Mouse insert cDNA's were amplified from phage vectors using 0.2mM 5° forward and 3° backward primers, 0.2mM dNTP, 2mM MgCI2, "IxTNT, 1.5%(v/v) DMSO, and 2.5u Taq polymerase. Reactions were denatured at 94°C for 2 minutes before cycled 30 times using a cycling strategy of 94-50-72°C for 20-30-60 seconds. 5uL of PCR reaction was analyzed on a 1% agaorse gel and electrophoresed for 4 hours at 100v. Gel was illuminated with a 216nmUV transilluminator and photographed using Polaroid 667 film. had similar estimated lengths were chosen for further analysis as it was assumed that these were probably the same clone. Two similar sized clones isolated from different plates, 1.6 and 4.1, were subcloned and analyzed to test this prediction. Subcloning of Putative Mouse Transferrin cDNA The remaining PCR reaction generated from the analysis of selected clones was loaded onto a 1% agarose gel and separated for 1 hour at 70v. The desired cDNA fragments were excised from the gel and purified using Qiagen's Qiaex II kit. The purified PCR amplified cDNA fragments had their 3° dA overhangs blunted with T4 DNA polymerase and the blunted molecule ligated into 5° dephosphorylated SMA linearized pBluescript (pBS) all in one tube at 16°C for 16 hours. Ligation mixes were n-butanol precipitated and the pellets redissolved in 32 ddH 2 0 before transformation into the E. coli strain JM110. Transformed bacteria were selected using ampicillan and blue white selection on IPTG/X-GAL plates. Typically 15-30 white colonies were isolated from each transformation experiment and these were analyzed for cDNA inserts by P C R . Transformants containing desired cDNA inserts were minipreped using Qiagen's Miniprep tip 20 kit and sequenced. Sequence Analysis of cDNA Clones Putative mouse Tf pBluescript subclones were sequenced using Taq FS technology and analyzed by U B C Biotechnology Laboratory's NAPS unit. The identities of the resultant sequences were determined using the BLAST algorithmn at NCBI. From the 28 cDNA clones isolated from the mouse liver cDNA library only 10 were selected for subcloning and sequencing; and from those 10 only 8 resulted in identities ranging from 68-86% to human serum Tf. Assembly of these fragments allowed the construction of a 1670 base pair mouse Tf contiguous sequence (contig) that includes 53bp of 3' untranslated sequence and a poly A signal. The 5' end of the contig is not complete as there are no identifiable translation start sites or ER targeting signal sequences. A homology comparison of this assembled full length human serum Tf indicates that 500bp of 5'sequence is still missing if the full length size of mouse Tf mRNA is conserved between humans and mice. 0 250 500 750 1000 1250 1500, 2£ tin ."11111111111111111111 IIIIIHII " " " " ' Clone 1.2 • iii-xjiw^Mff^Tcione 3.12 4 " K . ^ . - : . ^ . * . - ^ - . ^ Clone 1.6 4 fiMtviWt<k-z$™\ Clone 4.1 < Clone 3.10 • ''"'r !5^i^.^.<-^r f'.^.^V.1 Clone 1.1 • ^^^l^U.^*.^ Clone 1.5 • Figure 11: Assembly of Mouse Liver Tf cDNA Fragments Sequenced cDNA fragments were sent for BLAST analysis at NCBI. Sequences identified as Tf were loaded into Kodak's MacVector and Assemblylign sequence analysis program and assembled into the figure as shown. Resulting consensus sequence has a 65% identity to human seroTf and 68% identity to rat seroTf. 3 3 5'RACE PCR To determine the sequence of the 5' end of mouse Tf, three internal oligos derived from the mouse Tf contig sequence, one degenerate oligo designed from cDNA homology alignments between 5'ends of human and rat Tf cDNA, and one poly dT oligo were constructed. The degenerate oligo targets the sequence encoding the translation start site, Table 3: Oligos Used in RACE PCR Cloning of Mouse Transferrin Oligo Length Tm Sequence bp r-c MTD 27 81.3 A T G A G G Y T C G C Y G T G G G W G C C C T G C T G MT1 22 62.0 A A G C C A G T G A A T C A G T A T G A G G MT2 22 65.7 T G G T T C T C T C C A G G T G A C T C A G MT3 18 57.2 A A T T C C A C C C T C T G T G A C dT 18 42.0 I I I I I I I I I I I I I I I I I I I I methionine, in human Tf mRNA and the poly dT oligo targets the poly A tail at the 3' end of human Tf mRNA. The degenerate oligo MTD (see Table 3), was designed with as low a complexity as possible to minimize non-specific priming. MTD has only 8 possible permutations. First strand cDNA synthesis was performed using 1u,g of total C57B6 mouse RNA, oligo dT, and Boehringer Mannheim's Expand RT Kit. Second strand synthesis and subsequent PCR amplification was performed using different combinations of oligos and MTD» MT1 * MT3i 5" 500bp 1670 bp Mouse Transferrin Contig -MT2 Figure 12: Location of RACE Oligos in Mouse Transferrin cDNA Oligonucleotides were synthesized by Genosys Biotechnologies Inc. Oligos were provided deprotected, desalted and dried. Purity was assayed on a polyacrylamide gel. Oligos were resuspended in 1mM NaOH and stored at -20°C before use. 3 4 Boehringer Mannheim's Expand High Fidelity Kit. To check the efficiency of the reverse transcriptase-PCR reaction, internal oligos MT1 and MT2 were used as primers in one of the reactions. A single PCR product approximately 360 base pairs was generated using the MT1/MT2 oligo pair which matched the predicted size. All of these products were also subjected to hybridization screening using radiolabeled random primed human Tf cDNA using stringent wash conditions. F i g u r e 13 : A n a l y s i s of M o u s e L i v e r T r a n s f e r r i n R T - P C R P r o d u c t s A: 5 u.L of PCR reaction was analyzed on a 1% agarose gel for 1 hour at 70v. B: Gel from A was blotted to a Nylon N+ membrane and pre-hybridized for 4 hours and hybridized for 8 hours with random primed human Tf cDNA probes at 65°C in 5 mL of 5 X S S P E buffer. Activity of the hybridization buffer was 1.8x106 cpm/mL. Hybridized blot was washed 3x30 minutes in 50 mL of O. lxSSC. Blot was autoradiographed for 12 hours at -80°C on Amersham Hyperfilm-MP. The MT1/dT, MT1/MT2, and MTD/MT2 PCR reactions contained predicted sized fragments that strongly annealed to the Tf probe. From the remainder of the PCR reaction, the 1112 base pair product generated using the MTD/MT2 oligos was electrophoretically separated on a 1% agarose gel, isolated, purified, quantified, and subcloned into pBluescript. The resulting 3 5 subclones were insert-size characterized using PCR and pBluescript oligos T3 and T7 before sequencing. cDNA Sequence of Mouse Transferrin Sequence from the 1112 base pair PCR product generated using oligos MTD/MT2 was analyzed using the BLAST algorithmn at NCBI and assembled into the 1670 bp Tf contig to yield the complete translated cDNA sequence of mouse Tf. Sequence from the 5' end primed by the MTD oligo revealed the presence of an ATG start site followed by 54 bp coding for an ER signal sequence. The entire cDNA sequence of mouse Tf is shown in Figure 14. F i g u r e 14: c D N A S e q u e n c e of M o u s e T r a n s f e r r i n Sequence formatted using Kodak Eastman's Mac Vector 4.1. Total Length of Mouse cDNA sequence is 2130bp. ER signal sequence is underlined, S T O P codon is bold and underlined, and poly A signal is marked in bold. A T G A G G C A G C C C G T G G G T G C C C T G C T G G C C T G C G C T G C C C T G G G G C T G T G T C T G G C T G T C C C T G A C A A A A C G G T C A A A T G G T G C G C A C T G T C N G A G C A C G A G A A T A C C A A G T G C A T C A G C T T C C G T G A C C A C A T G A A G A C C G T C C T T C C G C C T G A T G G C C C C C G G C T T G C C T G T G T G A A T A A A A C C T C C C A T C C G G A T T G C A T C A R G G C C A T T K C T G C A A R T G A A S C C G A T G C T A T G A C C T T G G A T G G G G G K T K G G T G T A C G A T G C C G G C C T G A C T C C N A A C A A C C T G A A K C C C G T G G C S G Y G G A G T T T T A T G G A T C A G T G G A A C A T C C A C A G A C C T C C T A C T C C G C T G T G G C T G T G G T A A A G A A G G A A C G A G A C T T C C A G C T G A A C C A G C T N G A A G G C A A G A A G T C N T G C C A C A C A G G C C T G G G A A G G T C T G C A G G C T G G G T C A T C C C C A T T G G N T T G T T A T T C T G T A A G C T G T C G G A G C C C C G C A G T C C T C T T G A G A A A G C T G T G T C C A G T T T C T T C T C G G G C A G T T G T G T C C C C T G T G C A G A T C C A G T G G C C T T C C C C A A A C T G T G T C A A C T G T G C C C A G G C T G T G G C T G C T R C T C C A C T C A A C C A T T C T T T G G C T A C G G G G C A T T C A A G T G T C T G A A A G A T G G C G G T G G G G A T G T G G C C T T T K T C A A G C A M C A C A A C C A T T G G G A G T C T T G G C C G G A G A A G G C T G A C A G G G A C C A A T A T G A A C T G T T G T G C C T T G A A A A T A C C C G C A A G C C A G T G G A T C A G T A T G A G G A T T G C T A C C T G G C T C G G A T C C C C T C T C A T G C T G T T G T G G C T C G R A A A A A C A A T G G C A A G G A A G A C T T G A T C T G G G A G A T T C T C A A A G T G G C A C A G G A A C A C T T T G G C A A A G G C A A A T C A A A A G A C T T C C A A C T G T T C A G C T C Y C C T C T T G G G A A A G A C C T G C T G T T T A A A G A T T C T G C C T T T G G G C T G T T A A G G G T C C C C C C A A G G A T G G A C Y A C A G G C T G T A C C T T G G C C A W A A C T A T G T C A C T G C C A T T C G G A A T C A G C A G G A A G G C G T G T G C C C G G A G G G C T C G A T C G A C A A C T C G C C A G T G A A G T G G T G T G C A C T G A G T C A C C T G G A G A G A A C C A A G T G T G A C G A G T G G A G C A T C A T C A G T G A G G G A A A G A T A G A G T G T G A G T C A G C A G A G A C C A C T G A G G A C T G C A T C G A G A A G A T T G T S A A C G G A G A A G C G G A C G C C A T G A T T T T G G A T G G A G G A C A T G C Y T A C A T T G C A G G C C A G T G T G G T C T A G T G C C T G T C A T G G C A G A G T A C T A C G A G A G C T C T A A T T G T G C C A T C C C A T C A C C C G A A G G T G G G T A T T A T G C C G T G G C T G T G G T G A A G G C A T C G G A C A C T A G C A T C A C C T G G A A C A A C C T G A A A G G C A A G A A G T C C T G C C A C A C T G G G G T A G A C A G A A C C G C T G G T T G G A A C A T C C C T A T G G G C A T G C T G T A C A A C A G G A T C A A C C A C T G C A A A T T C G A T G A A T T T T T C A G T C A A G G C T G C G C T C C C G G G T A T G A G A A G G A T T C C A C C C T C T G T G A C C T G T G T A T T G G C C A C T C A T G T G A T C C G A A C A A C A A A G A G G A A T A T A A T G G T T A C A C A G G G G C T T T C A G G T G T C T C G T T G A G A A A G G A G A T G T G G C C T T T G T G A A A C A C C A G A C T G T C C T G G A T A A C A C C G A A G G A A A G A A C C C T G C C G A A T G G G C T A A G A A T C T G A A G C A G G A A G A C T T C G A G T T G C T C T G C C C T G A T G G C A C C A G G A A G C C T G T G A A A G A T T T T G C C A G N T G C C A C C T G G C C C A A G C T C C A A A C C A T G T T G T G G T G T C A C G A A A A G A G A A G G C A G C C A A G G C T G T A C T G A C T A G C C A G G A G A C T T T A T T T G G G G G A A G T G A C T G C A C C G G C A A T T T C T G T T T G T T C A A G T C T A C C A C C A A G G A C C T T C T G T T C A G G G A T G A C A C C A A A T G T T T C G T T A A A C T T C C A G A G G G T A C C A C A C C T G A A A A G T A C T T A G G A G C M G A G T A C T T G C A A G C T G T T G G A A A C A T A A G G A A G T G T T C A A C C T C A C G A C T C C T A G A A G C C T G C A C T T T C C A C A A A A G T T A A A A T C C A A G A G G T G G G T G C C A C T G T G G T G G A G G A G G A T G C C C C C G T G A T C C A T G G G C T T C T C C T G G C C T C C A T G C C C T G A G C G G C T G G G G C T A A C T G T G T C C G T C T T C A C T G C T G T G T G T T A C C A C A T A C A C A G A G C A C A A A A T A A A A A A T G A C T G T T G A C T T T A 3 6 Discussion T r a n s f e r r i n m R N A E x p r e s s i o n i n H u m a n a n d M o u s e T i s s u e s The distribution of mRNA transcripts in human and mouse tissues was studied by Northern Analysis using human Tf cDNA as a probe. The expression patterns were found to be similar between the two species. The tissue distribution observed generally agrees with the Table 4: Tissue Distribution of Transferrin mRNA in Humans and Mice Tissue Humans Mice Brain + + Heart - smear Kidney - -Liver +++++ +++++ Lung + + Pancreas ++ NA Placenta + +++ Spleen + + Skeletal Muscle - -Testis NA -+++++ high expression + low expression no expression NA not analyzed established physiological role of Tf as an iron binding and transport protein. The current model involves free serum Tf scavenging either free iron or displacing complexed iron from other molecules and then delivering it to sites requiring iron through TfRs expressed at the cell surface. Hepatocytes in liver tissues are responsible for producing a large amount of seceretory proteins that make up blood plasma, one of which is Tf 8 7 , 8 e . Thus the observed high Tf mRNA expression in liver tissue agrees with this blood serum circulatory model and with Tf protein expression studies. The peripheral expression of Tf in other tissues may be explained in two ways. The first possibility is the need for Tf to overcome local barriers such as the blood brain barrier in brain tissue; the blood testis barrier in testis tissue, or the maternal/fetal blood placenta barrier in placental t issue 9 0 . These tissues highly regulate substances that travel through the circulatory system through a layer of endothelial cells surrounding vessels perfusing the tissue. The barrier 3 7 model proposes that the endothelial cells play a role in iron transport into these tissue by not only controlling its TfR expression but the local Tf concentration as well. Immunohistochemical and in situ hybridization studies have identified endothelial cells in the choroid plexus and oligodendrocytes in brain 9 2 , 9 3 , testis endothelial cel ls 1 , 8 8 , 9 0 , and placenta tissue 9 4 that synthesize and express Tf protein. The second explanation of Tf expression in peripheral tissues is based on the idea that increased iron is necessary for proliferation or differentiation in cells and that Tf/TfR is the primary iron acquiring system used by the cel l 2 7 , 9 5 . Active cells unable to meet their iron requirements solely through the Tf concentrations in blood plasma may synthesize their own Tf thereby increasing the local concentration of Tf, and thus increasing transport. Cells such as those found in the fetus and placenta; epithelial cells in lung tissue, and lymphoid cells in the thymus and spleen may enhance their iron uptake in this manner 9 1 , 9 4. A related explanation of Tf expression in peripheral tissues is based on a zinc requirement by active cells. Zinc is required in DNA synthesis and transcription96"98. A common motif in DNA binding proteins are "zinc" finger motifs where zinc is complexed with a charged pocket of amino acids that make up the DNA binding site. Actively dividing cells will have a greater zinc requirement than dormant cells as DNA synthesis and metabolic activity is occurring at a higher rate. The primary zinc binding and transport protein is plasma serum albumin although Tf is also able to play a minor role in zinc binding and transport when cells require increased levels of zinc. Thus the presence of Tf in peripheral tissues may play a role in modulating the cells zinc requirements. The last possibility of peripheral Tf synthesis is based on its ability to bind other metals and complex compounds that may be toxic to the ce l l 8 , 1 7 , 1 8 , 2 9 . Cytotoxic effects of reactive compounds make a significant impact on unique tissues or tissues that are actively dividing. Neuronal cells involved in memory processes may be extremely sensitive to toxic compounds because once damaged, it is believed that the memory information contained in these cells is damaged or lost. On the other hand, actively dividing cells are sensitive to toxic compounds because of the deleterious mutational effects that may be caused by such compounds. Thus, 3 8 tissues such as the brain, placenta, fetal, liver, spleen, lung will synthesize a basal level of Tf to act as a guard against potentially threatening compounds. 1 ° Structure of Mouse Transferrin Using a combination of library screening methods and reverse transcriptase PCR techniques, the cDNA sequence of mouse liver Tf was determined and compared to human Tf. Some of the features of mouse Tf in relation to human Tf are summarized in Table 5. The 5' end of the mouse Tf mRNA transcript remains to be determined. A 5' UTR approximately 30-60 nucleotides in length should be present based on complete Tf cDNA sequences determined from other species 1 , 1 1 , 8 1 . The size of the mouse Tf mRNA transcript appears to be conserved with the human Tf mRNA transcript. Northern analysis of Tf from human and mouse tissues suggested that the human Tf transcript is approximately 100 nucleotides longer. The complete cDNA length of human Tf is 2294bp where a 2070bp coding region is flanked by a 58bp 5'UTR and a 170bp 3 'UTR 8 1 8 2 . Mouse Tf cDNA determined so far, contains a 2070 bp of coding region flanked by an unknown length of 5' UTR and 90bp 3'UTR. The 100 nucleotide difference between human and mouse Tf detected from Northern analysis may largely be accounted for by the 80 nucleotide shorter mouse Tf 3'UTR. If this is true then mouse Tf may either have a very short 5' UTR or none at all. Feature Number of Nucleotides Composing 5 ' UTR Number of Nucleotides Composing Coding Region Number of Nucleotides Composing 3 ' UTR Number of Nucleotides of entire mRNA transcript Number of Polyadenylation Signal Motifs Number of Potential IRE Motifs (location) Number of Amino Acid (A.A.) Residues ER signal Peptide? (A.A. residue length) Number of Cysteine Residues Number of Potential Disulfide Bridges Number of Potential Iron Binding Residues Number of Potential N-Glycosylation Sites Number of Potential O-Glycosylation Sites Overall cDNA Homology Overall A.A. Homology N-terminal Lobe A.A. Homology C-terminal Lobe A.A. Homology Overall A.A. Homology to Human MTf Humans Mice 58 ? 2070 2070 170 60 2298 ? 1 i 1 (5'UTR) 0 690 690 yes (19) yes (19) 40 38 20 19 14 14 2 1 71 76 40% 78% 71% 73% 69% _ L _ 38% Table 5 : Structural Features of Human and Mouse Transferrin 3 9 Mouse Tf mRNA also differs from human Tf mRNA in that there are no apparent IRE motifs. Recently, an IRE capable of binding IRP was identified in the 5' UTR of human Tf. Binding of IRP to this IRE caused an increase in translation of human Tf 5 1. An analysis of the mouse Tf cDNA consensus sequence for IRE motifs found no matches. However, this analysis is not yet complete since the 5'UTR of mouse Tf, if it exists, has yet to be characterized. The primary structure of mouse Tf aligned with human Tf is shown in Figure 15. Translation of the coding sequence of mouse Tf predicts that the protein is 690 residues long with a 19 residue ER signal peptide at the N-terminal end of the protein suggesting that the size of mouse Tf is conserved with human Tf. Furthermore, all the residues thought to form the iron binding site in the N- and C- terminal lobes of the Tf protein in humans remain conserved in mice, and the 71% overall amino acid homology between human and mouse Tf suggests that mouse Tf structure and function is similar to that of human Tf. 4 0 Mus T f Hu T f MR 3PUGflLLfi hR-ri»GfiLLU CflfeLGLCLRU P O K T U K E C R L gEHETffKCll IS FRDHMKIllOlJP Cfl|j|_GLCLRU PPKTURHCRp (SEHEpJTKCpjS F R P H I 1 K f l 4 | P Mus T f Hu T f F tJGPPURCUN k m s HFCCI bp \m\ yRJjfr LDpp[^'VDR[t' ^ ppHML[<|PUp Mus T f Hu T f XEFVGS flEFVGS if QTEttaRURUU KKERC-Q.HQ DP QT^rWURUU K K 3 S G - Q 1 H Q L E G K K S C H T G L G R S R G U J H P L ^ G K K S C H T G L G R S R G U ^ I P Mus T f Hu T f I G L L T C IGLL«IC BE P R 3 P L E K R U B S F F S G S C J P C flDPURFPkLC QLCPGCGC<S = E PR < PLEKRUR N F F S G S C = f C flDpTDJFPpLc Q L C P G C G C 3 T Mus T f Hu T f T Q P F - G Y - G R F K C L K D G 3 GD UAF < i <HMHU L N Q V - G V 3 G R F K C L K D G =l GD U R F J U S T I F EWPEkflDRD Q V E L L C L a H T NLRNKRDRD Q V E L L C L p N T Mus T f Hu T f PSHAW URRKNNGIED LIUEl K P U D B V k p C rf-flpUfSHnp URRBMGpKED L I W E l X N o A Q E H F G K p K S K E ILKURQ EHFGKpKSRp Mus T f Hu T f F Q L F S S P U G FQLFSSpLJG 3K D L L F K D S R : G 3K D L L F K D S R H G |_L. ^ JPPRMD < R L Y L G O W U T R I F N 3 Q E G C <LiPPRMD=l K M Y L G / E W T fl IRN _F EG T C Mus T f Hu T f ^Ebsi ih-sp U K U C R L S H L E - R l r k c o E w s p i I : € p P T p ^ C K J P UKUCRLSHhte R | - K C D E W S | J N IS! I I feSGKI ECBSR ETTEDC IEKI LlGK I ECUSfl ETTEDC IRK I Mus T f Hu T f UNGERDRMIlC MpGERDRMpf-DGG DGG HRVIRGR CGLUPUHf lEk ' FMVIRG K C G L U P U L p E N mm SCRIPS P E G G V i H U R U T'hKGDNCEDT P E * 5 V : R U R U Mus T f Hu T f UK LIKl PP DTSimT kfeRSDLnrw H'HLKGKKSCH T3U]RTflGUN IPMGUrTHI NHC<FDEFFS ]NLKGKKSCH TRU3RTFM3UN IPMG._VN<I NHC1FDEFFS Mus T f Hu T f qGCRPGI/Ef^sTll-Cbm RL S EpPNNK EEpfCVTGRF RCLUEKGDUR E)GCRPGp:K|<D S ^ - C ^ c f y p GLNI_|CJ£NNKE 3 f < 3VTGRF RCLUEKGDUR Mus T f Hu T f FUKHQTU FUKHQTU TEGKIHPl T3GKNPD fl KNL <QEpF EL L C ' D G T R K P U R KNLHEKEWEL L C . D G T R K P U K D F K E H L R 3 I C H L F H Mus T f Hu T f PPNHJUUE=IK pPNH^UrRK ERn^-KRUClTJ ](d^huHKl|__RC F G 3 - -SpCrpNFCL F<S1"TKDLLF FGpN UTpCEJGNFCL F 1 S E T K D L L F Mus T f Hu T f F D D T k t R D D T L C UKL^ L f l k L HDRi €GTffr 1NJJ EKVL G EKVL G VLORUGN IRKCS.TSRLL ERCTF VUKpUGM LpKCSTSELL EflCTFRRP HKS Figure 15: Comparison of Mouse and Human 1 ° Structures of Transferrin" Mus Tf: Mouse Tf Hu Tf : Human Tf bar line: ER Signal Peptide • : Amino Acid residues involved in iron binding 41 Chapter Four: Characterization of Mouse Melanotransferrin Introduction Structure of Human Melanotransferrin MTf is a glycoprotein with an approximate weight of 97 kDA. It is a single polypeptide protein that is translated into a 738 amino acid precursor form and processed to a mature 694 residue form 3 8 ' 4 2 . Human MTf is translated from an mRNA transcript 3916bp long where 2214 bp of coding sequence is flanked by 375bp of 5' and 1327 bp of 3' untranslated sequence. The translated polypeptide can be divided into an N and C terminal lobe which share a 36% amino CMC VflMCA T S O P E O H K C C MMSE AF n O A A ILftVcMOABTCftVCUtlPd I CA OA tTLOCGA! YEACKEMCl.PCPVVCEV«DO£V ? K 8 N 1 C T Q C S K V C K L T 0 I T V H S S R R V V A V A Y& B C A F R c C A E C A G O V A F V K H f T V L E N T D C K T L P 6 H C Q A L L 6 Q D F E L L C R D C S R A a Q B M « F L H 0 C E N « _ L « F 1 U G C 0 T 0 A 8 V V V A < 3 » P V R A I . H C 0 R W 6 T V 8 F O M F a S E A Y C Q K O L L F K Q ' L P S K O C V C E C 8 8 6 Y S E S U C R l - C R C 0 L T E C A C P V C , P f M e C O V L K A v a O Y F C C T A ^ R H P O t L U C K M A H U Y E H C L H A E Y T 0 L R H C Y t _ 8 T P E f O K C C D H A V A F R . C A O t A E M C M O P S K A S V C O 1 E P K L R O * * 0 VOAVTU . 9 C E I>t Y T ACKKYCLVP A A C E K S K P E O S . o A r $ C F C ^ 4 J C s n K C n i . E o i . T F A M 8 0 o n n v v A V V Y < Z a N i (e n e A F n 2 u v C N A C r > V A F Y n t l T T Y F O N T N O H N f l E P M A A C k - n S E O Y E L U C P N C A n A » <p ^ K M I I O O C r i _ O 0 A K O L l . C V V ? f I N T O f ^ p V H V A O P P I 0 A L N C A A F 0 8 V £ \ „ ° F K M F 0 8 8 M Y H C O O L L F K O . A K r L O S N C V C K N R C O S Y O Y P 9 S I . C A l . C V C 0 * C _ N K P N N V P V C K P N N V P V C ? R P K O C O V t . r A V 9 E F F N A E C s i 0 0 S 8 M C E L A A V Y O L Q L H C R Y T 7 K G P I S i g n a l P e p t i d e F i g u r e 16: S e c o n d a r y S t r u c t u r e M o d e l o f H u m a n M e l a n o t r a n s f e r r i n Disulfide bridges between cysteine residues are denoted by a line. Amino acid residues circled are predicted to compose the iron binding site of MTf and residues thought to contain the GPI anchor attachment signal are indicated. 42 acid homology to one another. The mature protein contains 14 disulfide bridges and three potential glycosylation sites 3 1. The MTf gene in humans localizes to 3q21, the same region were Tf resides 1 0 0" 1 0 2. The size and intron/exon organization of the gene has not been published. Glycosylphosphatidylinositol Anchored Form MTf was first characterized as a membrane protein tethered to the cell membrane through a short 24 residue hydrophobic region in its C-terminus. This hydrophobic region has been recently shown to be bound to the cell membrane through a GPI anchor. The GPI signal peptide and the peptide cleavage and anchor attachment site in human MTf has not been identified. However, peptide sequence analysis of the 24 residue hydophobic C-terminus of the 738 amino acid precursor reveals possible sequences that conform to GPI signal peptide motifs that have been characterized in other proteins 3 3 , 3 4. The general structure of a GPI anchor is shown in Figure 17. Briefly, the anchor is composed of 1 to 3 ethanolamine residues NH Protein Ethanolamine 0 = C - N H C H , C H , |Nitrous A c i d | Membrane (CH 2) n ( C H ^ F i g u r e 17 : G e n e r a l S t r u c t u r e of a G P I A n c h o r .24,103 4 3 each bound through a phosphodiester bond to a glycan composed of a variety of sugars built around a core of mannose and glucosamine residues. The glycan is in turn bound to a phosphatidyinositol fatty acid embedded within the phospholipid membrane in the ER. The anchor is susceptible to cleavage by phosphotidylinositol phospholipase-C and D (PIPL-C,-D) and by nitrous acid. Cleavage by these agents results in the release of the membrane anchored protein into the extracellular medium 1 0 3" 1 0 7. Protein Maturation and Intracellular Trafficking The intracellular trafficking of MTf is believed to follow the classical pathway of integral membrane proteins. Briefly, translation of the protein begins in the cytosol. Once the ER signal sequence is translated, and the signal recognition particle (SRP) binds to it, translation is halted until the whole complex docks with the SRP receptor on the ER membrane. Once docked, the complex resumes translation with the nascent protein being extruded into the cytosol of the ER until the GPI signal peptide is translated and enters the ER phospholipid membrane. The hydrophobic nature of this peptide is believed to contain a halt signal that stops the peptide from being extruded into the hydrophillic ER cytosol or cell cytoplasm. The protein remains anchored to the ER membrane through this peptide until the GPI cleavage and attachment machinery processes the protein. Studies have shown that GPI signal peptide cleavage and attachment of preassembled GPI anchors occurs within 1 minute of translation 1 0 5 , 1 0 8. Soluble Form A soluble form of MTf has been identified in the supernatants of tissue cultured human melanomas and in the blood serum of humans. The origin and identity of the soluble form is not well understood. The soluble form may originate as a result of a default mechanism where pre-assembled GPI anchors are not attached to MTf's; endogenous PIPL-D cleavage of the GPI anchored form of MTf at the cell surface; it may originate from an alternatively spliced mRNA transcript lacking a GPI signal coding sequence; or it may come from all three sources 3 3 , 3 4 . The soluble form of MTf can bind iron like its membrane bound form; and it is suspected that MTf may be capable of binding and internalizing with TfR. 4 4 Tissue Distribution of Human Melanotransferrin GPI anchored MTf was first characterized on the surfaces of a variety of melanoma and hepatocarcinoma cell lines. Histochemcial analysis of the distribution of MTf in normal human tissues identified comparable levels expressed in fetal intestine and liver1 0 9. Other tissues that express relatively low amounts in comparison are liver sinusoidal cel ls 1 1 0 , brain endothelial cel ls 1 1 1 , brain microglia activated against P-amyloid plaques in Alzheimer's d isease 1 1 2 , and a soluble form of MTf found in blood and cerebral spinal fluid 7 6. Species Homologues of Human Melanotransferrin Two putative homologues of human MTf have been discovered in chicken eosinophil cells 1 8 and pig intestinal cells 1 1 3 . The chicken eosinophil cells appear to synthesize two forms of MTf, a GPI-anchored form and a secreted form. Each form arises from a 3860bp mRNA transcript that may be alternatively spliced to transcripts giving rise to a GPI-anchored protein or a secreted protein. The distribution of chicken MTf in normal tissues by Northern and histochemcial analysis identified that most tissues endogenously expressing both the secreted and GPI anchored form with the secreted form predominating. Chicken MTf shares a 68% cDNA homology to human MTf; is 698 amino acids long, has three potential glycosylation sites and has all 14 potential disulfide bridges found in human MTf conserved in its polypeptide sequence 1 8 . Much less is known about the pig homologue other than the characterization of a GPI anchored molecule found in intestinal epithelial cells that appears to bind and internalize iron. The 1' structure of pig MTf is unknown and no soluble form has been detected 1 1 3. Chapter Four Outline The existence of a mouse homologue to human MTf has been determined by Southern analysis of human and mouse genomic DNA using 3 2 P radiolabeled human MTf as probe. The tissue distribution of MTf in human and mouse tissues was studied by Northern analysis of a variety of tissues and melanoma cell lines in order to determine a tissue source from which mouse MTf mRNA could be isolated. These studies were undertaken in order to obtain a full length cDNA clone of the mouse homologue to human MTf. Having the cDNA's of mouse Tf, TfR and MTf will enable one to create a variety of in vitro and in vivo physiological models to study iron transport and the interaction of these three molecules with one another. 45 Results Human Melanotransferrin cDNA Human MTf cDNA was isolated from the pSV2p97a construct, a gift from Joseph Brown at Oncogene, using Hind III. The Hind III cDNA fragmets was subcloned into p G E M 3Zf+ by Sylvia Rothenberger, amplified, characterized, and named A3-2. All hybridization experiments using a MTf cDNA as a template to generate 3 2 P labeled random primed probes refer to the Hind III cDNA fragment, or Hind Ill/Sal I cDNA fragments cut from the A3-2 construct shown below. Figure 18: Human Melanotransferin Probe: A3-2 Construct Size: 6772bp Restriction Fragments Isolated from A3-2 5'-3' MTf cDNA 343 Hindlll to 3916bp Hind 5'-MTf cDNA 343bp Hindlll to1616 Sal I 3'-MTf cDNA 1617 Sal I to 3916 Hind III 3573bp 1212bp 2300bp pGEM3Zf(+) Hindlll to Hind 3260bp A3-2 Construct Hind III 343bp Sal I 1616bp Hind III 3916bp • Human MTf cDNA coding region • 5' and 3' Untranslated regions — pGEM3Zf(+) Complete human MTf cDNA ~3.9kb 46 Human Genomic Southern Analysis of Melanotransferrin A total of 2.2 mg of Genomic DNA from 1 x 106 SKMEL-28 human melanoma cells was isolated and purified using Qiagen's genomic DNA extraction kit. Purified DNA was spectrophotemetrically quantified at 260nm and its quality checked by gel electrophoresis before cutting with restriction enzymes. 20|xg of genomic DNA was subjected to cutting by Bam HI, Eco Rl, Hind III, Bam HI and Eco Rl, Bam HI and Hind III, and Eco Rl and Hind III for 4 hours in the presence of 1mM spermidine and the appropriate restriction enzyme buffer. Digestion was monitored to completion by loading 1 txg of digested DNA onto a 1% gel and electrophoresing at 70 v for 1 hour. Complete digestion was indicated by a smear of relatively Figure 19: Southern Analysis of Human Melanotransferrin from SKMEL-28 Genomic DNA isolated from the human melanoma cell line SKMEL-28 was digested with restriction enzymes, separated on a 1% agarose gel and blotted to a nylon N+ membrane. Blot was hybridized overnight with random primed 3 2 P labeled probes generated from human MTf cDNA at 65°C and washed in 0.1 X S S C three times at 65°C. Probe activity was 1.8x106 cpm/mL. Blot was autoradiographed at -80°C for 1 day. uniform intensity, extending from the lane wells to the top of the gel. 10 \ig of completely digested DNA reactions were loaded onto a new 1% agarose gel and electrophoresed in a clean tank with fresh running buffer for 16 hours at 20v. The gel was photographed and prepared for blotting by treating in HCI and NaOH. Gel was rapid alkaline blotted to Amersham 47 Nylon N+ membranes for 2 hours before prehybridizing for 4 hours at 65°C. Blot was then hybridized with 3 2 P random primed cDNA for 16 hours before washing the blot in three 20 min cycles in an excess of 0 .1XSSC at 65°C. Blot was then phosphorimaged for 1 hour and autoradiographed for 1 day. Human Genomic Southern with the 5' and 3' Halves of Melanotransferrin cDNA The results from probing the genomic Southern blot with the entire MTf cDNA yielded a complex pattern that was difficult to analyze (Figure 19). In order to identify the fragments that represented the 5' ends of the gene, the Hind III cDNA fragment which was used as template to generate random primed probes, was cleaved into two halves using Sal I. The two halves were electrophoretically separated and purified from a 1% agarose gel. The digest resulted in a 5' cDNA half that included 32 base pairs of 5' untranslated sequence and 1241 base pairs of coding sequence; and a 3' cDNA that included 973 base pairs of coding sequence and 1327 Figure 20: Southern Analysis of 573' Ends of the Human Melanotransferrin Gene in SKMEL-28 A: Genomic DNA blot fromthe human melanoma SKMEL-28 cell line was hybridized with 3 2 P random primed probes generated from the 5' Hind Ill/Sal I human MTf cDNA fragment. B: Genomic DNA blot in figure A was stripped and rehybridized with probes generated from the 3' Hind Ill/Sal I human MTf fragment. 48 base pairs of 3' untranslated sequence. The two MTf cDNA fragments represent the two halves of the protein where the 5' cDNA corresponds to the first 406 amino acids of the protein (the N terminal lobe) and the 3' cDNA corresponds to the last 332 amino acids (the C terminal lobe). Each of the fragments was used as a template to generate 3 2 P labeled random primed probes that were used to probe the genomic Southern blot created in Figure 20. Briefly, the blot was stripped with 0.1% boiling SDS, pre hybridized and hybridized with probes generated from either the 5' or 3' half of the cDNA. The blot was subjected to the same stringent wash conditions and phosphorimaged and autoradiographed for 1 and 24 hours. The size of the genomic fragments hybridizing with the MTf probes were estimated using a power function curve generated by plotting the distance the Hind III fragments traveled to their respective sizes. The fragments were size catalogued and the restriction map for the gene determined (Figure 22). Genomic Southern Analysis of a Mouse Melanotransferrin Gene 70 ug of mouse genomic DNA was purifed from 1 x10 6 mouse melanoma JB/MS cells using Qiagen's Genomic DNA extraction kit. Purified DNA preps were spectrophotometrically F i g u r e 21: S o u t h e r n A n a l y s i s of M o u s e M e l a n o t r a n s f e r r i n in J B / M S C e l l L i n e Genomic DNA, isolated from the mouse melanoma cell line JB/MS, was digested with restriction enzymes, separated, and blotted to a Nylon N+ membrane. Blot was hybridized overnight with random primed ^ P labeled probes generated from human MTf cDNA and washed three times in 0.1XSSC at 60°C. Blot was phosphorimaged for 24 hours at room temperature. 49 quantified at 260nm and its quality checked by gel electrophoresis. 20u.g of genomic DNA was subjected to restriction digestion by the same enzymes used in the Southern analysis of the human MTf gene. The pre-hybridization/hybridization, wash conditions, phosphorimaging and autoradiography times from the analysis of the human melanotrasferrin gene were repeated except that the temperature of hybridization and washing was lowered to 60°C . The entire human MTf cDNA was used as a template to generate random primed 3 2 P probes. The size of the genomic fragements hybridizing with the MTf probes were estimated using a power function curve generated by plotting the distance the Hind III fragments travelled to their respective sizes. The fragments were size catalogued and the restriction map (figure 22) for the gene determined and compared to the human MTf gene. B E B H H H E B H H E I I I I I I I I I I I Human MTf Gene " 4 9 k b H E B B E E B E H B I I I I I I I I I I Mouse M T f Gen* ~38kb Figure 22: Restriction Maps of the Human and Mouse Melanotransferrin Gene Restriction site markers are abberviated as follows B : Bam HI, E: Eco Rl, H: Hind III Multiple Tissue Northern Analysis of Human Melanotransferrin mRNA Expression A human multiple tissue Northern blot was purchased from clontech and probed with 3 2 P labeled random primed probes generated from the Hind III cDNA fragment of human MTf. The blot was loaded with 5uq of poly(A+) purfied mRNA from each tissue represented. Pre-hybridization/hybridization was carried out in 50% formamide/5XSSPE buffer,and the blot washed 3 times at 0.1XSSC all at 42°C. The resulting blot shows no predominant bands after 24 hours of autoradiography at -80°C. A transcript approximately 4.4kb in length began to appear in all tissues after 3 days of autoradiography at -80°C. The signals corresponding to 50 A kb 9.5 • 7.5 • 4.4 • 2.4 • 1 . 35» B 1 . 35» Figure 23: Northern Analysis of Melanotransferrin Expression in Human T issues A: Multiple tissue blot screened with 3 2 P random primed probes generatd from human MTf cDNA. Probe activity was 1 x 10 s cpm/ml. Autoradiography for 36 hours at -80°C. B: Multiple tissue blot screened with 3 2 P Random Primed probes generated from human B-Actin cDNA. Probe activity was 1.5 x 106 cpm/ml. Autoradiography at -80°C for 45 minutes. these transcripts became more intense when the hybridization was repeated using low stringent washes with 2xSSC (data not shown). Under stringent hybridization conditions, transcripts of MTf were not detected in normal human tissues. Decreasing the stringency resulted in the MTf cDNA probes annealing to a 4.4kb transcript appearing in all tissues as well as an approximate 2.3kb transcript found in liver and placenta tissues. Multiple Tissue Northern Analysis of Mouse Melanotransferrin Poly(A+) RNA was purified from tissue pooled from five C57B6 mice. The quality of RNA preparations were electrophoretically anlayzed on a 1% agarose T A E gel. Good preps were delineated as having minimal low molecular weight smearing. 28 and 18s ribsomal subunits were barely visible. 10|ig of RNA was electrophoretically separated on a 1% formaldehyde M O P S gel and blotted to Amersham Nyon N membranes for 16 hours using a high salt 20xssc buffer. The blot was then pre hybridized and hybridized in 50% formamide/5xSSPE buffer at 37°C and washed in 0 .1XSSC wash buffer three times at 37C 51 before phosphorimaging for 1 hour and autoradiography for 1 and 3 days. After 1 hour of phosphorimaging and 24 hours of autoraidography only the 3.9 kb transcript in SKMEL-28, corresponding to melanotransferin was detected. After 3 days of autoradiography at -80°C, a weak signal corresponding to a transcript of approximately 3.6kb in length was detected in A c a i . « 5 •a c » = 8 ca _J _ i s*: c <u c <u K -J « 5 . F i g u r e 24 : A n a l y s i s o f M e l a n o t r a n s f e r r i n m R N A E x p r e s s i o n in M o u s e T i s s u e s A: Multiple Tissue blot screened with 3 2 P random primed probes generated from human MTf cDNA. Poly (A) purified RNA from the MTf expressing human melanoma cell line SKMEL-28 was loaded as a positive control. Probe activity was 2x106 cpm/mL. Autoradiogrphy at -80°C for 72 hours. B: Multiple Tissue blot screened with 3 2 P random primed probes generated from mouse p-Actin cDNA. Probe activity was 5 x 10 6 cpm/mL. Phosphorimaged at room temperature for 1 hour. mouse liver tissue. No other signals were apparant in other tissues. The possibility that the detected transcript in mouse liver was mouse Tf was investigated. The blot was stripped and reprobed with human Tf cDNA using the same hybridization and wash conditions. A 2.2 kb transcript corresponding to mouse Tf was detected in large amounts mouse liver tissues after autoradiography for 24 hours at -80°C. The 3.6 kb transcript detected with human MTf cDNA probes in mouse livers disappeared (data not shown). 52 Fetal Tissue and Mouse Melanoma Northern Analysis for Mouse Melanotransferrin Poly(A+) purified RNA from mouse fetal tissue and placentas were isolated from pregnant mice in their last week of gestation. Poly(A+) purified RNA was also isolated from a panel of mouse melanoma cell lines obtained from A T C C and from Dr. Vincent Hearing at NIH, A k b 3.9 3.5 2.6 Figure 25: Northern Analysis of Melanotransferrin mRNA Expression in Mouse Melanoma Cell Lines and Fetal Tissues A: Multiple Melanoma/Fetal tissue blot hybridized with 3 2 P random primed probes generated from human MTf cDNA. Probe activity was 1.6 x 10 cpm/mL. Autoradiogrpahy at -80°C for 3 days B: Multiple Melanoma/Fetal tissue blot hybridized with 3 2 P randomprimed probes generated from mouse (3-actin cDNA. Probe activity was 5 x 10 5 cpm/mL. Phosphorimaged at room temperature for 1 hour. Bethesda, Maryland. 10uq of RNA was separated and blotted onto Amersham Nylon N membranes. Membranes were hybridized using the same conditions that were used to analyze the mouse multiple tissue blot using random primed 3 2 P labeled probes generated from the Hind III fragment of human MTf cDNA. Poly (A+) RNA from the human melanoma cell line SKMEL-28 was also included on the blot as a positive control. After 6 hours of autoradiography at -80°C 53 a 3.9 and 2.6 kb transcript were detected in the SKMEL-28 cell line. After a 24 hour exposure similar sized transcripts appeared in the JB/MS cell line. A 3.6kb transcript was also detected in normal mouse placenta and fetal tissue. A longer autoradiography of 3 days at -80°C improved the signals found in the mouse tissues. However, the transcripts corresponding to human MTf in the SKMEL-28 cell line swamped out. 54 Discussion Melanotransferrin Gene in Humans and Mice Analysis of the results from Southern analysis of human and mouse genomic DNA using human MTf must be approached carefully keeping in mind that the cDNA probe will only identify genomic fragments containing exons. The restriction enzymes Bam HI, Eco Rl, Hind III were selected on their ability to cut human MTf cDNA. Bam HI cleaves twice, once in the 5' end and once in the 3' end of the cDNA; Eco Rl does not cut the cDNA at all, and Hind III cuts once in the 3' end of the cDNA. The low number and location of cut sites helped in orientating the fragments and estimating probable size of the gene. The restriction maps determined for the human and mouse genes contain some inconsistancies between fragments due to these problems. The total size of the MTf gene in humans is predicted to be about 49 kb long and in mice to be about 38kb long. The exon/intron organization and size of Tf, LTf, and TfR are conserved between species studied. Since the MTf gene resides in the same genomic region as the other members of the Tf family and considering its similarity to Tf and lactoTf, it is likely that the size and organization of the MTf gene is also conserved between species. The extra 9 kb in the human MTf gene may be accounted for by the 19kb 5' end Bam HI fragment having only a small portion of its 3' end containing exons. If we use the 5' end Eco Rl restriction site as a more suitable marker that delineates the start of the gene, the human MTf gene reduces to a length of about 37 kb thus bringing the sizes of the human and mouse genes more in line with each other. An alternative explanation of the size discrepency between the human and mouse MTf genes may arise from the possiblity that MTf is polymorphic 1 1 4. Tf, LTf, and TfR all have restriction fragment length polymorphisms making it highly probable that MTf is also polymorphic. Fragments found in some of the digests, such as the 28kb and 13kb fragments (produced from the Eco Rl digest that anneal to both the 5' and 3' human MTf cDNA probes) suggested that SKMEL-28 cells may carry a heterozygotic Eco Rl restriction site phenotype. This lends weight to the idea that MTf is polymorphic. Assuming that the 28 kb and 13 kb 55 fragments contain the same or similar exon organization, which is likely since no smaller fragments annealing to the 5' or 3' MTf cDNA probes were detected, then the polymorphic Eco Rl restriction site cuts away a 15kb intron fragment that is not detectable by MTf cDNA probes. This reduces the total size of the gene from 49 kb to either a 36.5 or 20.5 kb allele of MTf. The exon/intron arrangement of human and mouse MTf could not be determined using the full length human MTf cDNA as probe. In the Southern analysis of human genomic DNA where the 5' and 3' ends were resolved by probing in succession with the 5' and 3' halves of MTf cDNA, only the orientation of fragments could be crudely resolved. To map the exon/intron arrangement of the gene it would be best to subclone the genomic fragments hybridizing to MTf cDNA probes, sequencing, and then analyzing the sequence for open reading frames and splice sites. Melanotransferrin mRNA Expression in Human Tissues The distribution of mRNA transcripts in human tissues was analyzed by Northern analysis using human MTf cDNA as probe. Using stringent wash conditions and short exposure times no transcripts were detected in any of the human tissues. Longer exposures detected a 3.8 and 2.6 kb transcripts in placenta tissue and moderate wash conditions with long exposure times detected a 4.4 kb fragment in all human tissues. The 3.8 kb transcript detected on the Northerns for placental tissue agreed with the published size of MTf isolated from the human melanoma cell line SKMEL-28. The 2.6kb transcript found under the same wash and exposure conditions in placenta can also be detected in some preparations of RNA isolated from SKMEL-28. The recent cloning of the chicken homologue of human MTf has identified a 3.5 and 2.8 kb sized transcript of chicken MTf 1 8. Analysis of their cDNA sequences reveal that the only difference between the two transcripts occurs in their 3' untranslated regions and that the longer transcript has a GPI signal motif in the 3' end of the coding region, whereas the shorter transcript lacks this motif. It is proposed that the GPI-anchored and soluble forms of chicken MTf may arise not only from a post transcriptional RNA splicing level where the alternative splicing determines the type of MTf the transcript is destined to encode, but from a cleavage of 56 the GPI anchor by phospholipases which generates soluble forms of MTf. A similar explanation may be used to explain the two different sized transcripts found in human placenta, where the 3.8 and 2.6kb forms correspond to mRNA transcripts encoding the GPI-anchored and soluble forms. The 4.4 kb transcript found in relatively low levels in all tissues does not agree with the reported tissue distribution of MTf in normal t issues 1 0 3 , 1 0 4 . In humans, MTf is found in low levels in brain, liver and placenta tissues, relative to human melanomas. Fetal tissue appears to express MTf at levels similar to those found in human melanomas. Other known human tissue that express MTf are derived from transformed cells such as melanomas and hepatomas. Furthermore, the need to lower the wash stringency by 20 times also argues against the idea that this 4.4 kb transcript represents some alternatively spliced form of MTf. The signal probably represents weak homology with an unrelated protein. Melanotransferrin mRNA Expression in Mouse Tissues Under high stringency wash conditions and short exposure times, mouse MTf mRNA transcripts were only detected in liver, placenta, fetus, and certain mouse melanoma tissues. 3.8 and 2.6kb sized transcripts were detected in the mouse melanoma cell line JB /MS expressed in amounts rivaling those found in the human melanoma cell line SKMEL-28. In normal mouse tissues only fetal tissues appeared to express large amounts of MTf. Two forms were also detected in fetal tissues, a 3.5kb and 2.6kb form. The longer of the two transcripts differed from the longer form found in mouse and human melanomas. Other tissues that expressed low levels of MTf in comparison were liver and placental tissue only the shorter, 3.6kb form was detected. Like human and chicken MTf, the different sized mouse MTf transcripts may be explained by alternative splicing where the 3.8kb and 3.5kb forms are GPI-anchored variants and the 2.6kb transcript corresponds to the soluble form of mouse MTf. The presence of two different sized GPI-anchored forms may be explained by the tissues form which the transcripts originate. The 3.8kb form is found in the mouse melanoma JB/MS. The 3.5kb form is found in mouse fetal tissues and placenta. The difference may arise from a mutation or a splice 57 variation in the MTf gene's 5' or 3' untranslated regions altering its efficiency of expression. Untranslated regions are believed to be involved in the transcriptional and translational regulation of expression of the protein by either stablizing or destabilizing the transcript from degradation by RNases. Control of protein expression at this level has been well characterized for ferritin and TfR; where both of these transcripts are either stabilzed or destabilized by binding of iron response proteins to iron response elements located in their 5' and 3' untranslated regions. MTf is not expressed in normal tissues with the exception of liver and brain endothelial cells and placenta and fetal tissues. In fetal tissues, expression is similar to that found in melanomas. However, once the organism is fully developed, the expression is turned off and the presence of MTf quickly disappears. It has been speculated that transformed cells have a much higher iron requirement than "normal" proliferating cells and have evolved the use of MTf to increase their iron uptake. It is possible that the 3.6 kb form in placenta and fetal tissue represents the properly regulated transcript of MTf and the 3.8kb form represents the tumorigenic form of MTf. 58 Chapter Five: Conclusion Human and Mouse Transferrin Northern analysis of Tf mRNA transcripts in a variety of human and mouse tissues confirmed previous findings that the predominant tissue expressing Tf is liver. The complete translated cDNA sequence of mouse Tf was successfully cloned from mouse livers using a combination of RT PCR techniques and conventional cDNA library screening methods. The size of the mouse transcript is 2235 bp in length and has a 68% cDNA homology to human Tf. All the amino acids involved in iron binding are conserved between humans and mice. A full length cDNA clone of mouse Tf containing the entire 5' untranslated sequence can now be isolated and the resulting cDNA subcloned into an appropriate expression vector and transfected into cell lines not expressing Tf. The protein can then be characterized using mono and polyclonal antibodies against mouse Tf. Human and Mouse Melanotransferrin The size of the human MTf gene is predicted to be about 49 kb long. Evidence for a mouse homologue to human MTf was found via Southern analysis of the mouse genome using human MTf cDNA as a probe. The putative mouse MTf gene is predicted to be about 38 kb in length. Northern analysis of MTf mRNA transcripts in humans and mice have shown that MTf is either not expressed or expressed in very low amounts in normal adult human and mouse tissues. Human fetal tissues, placentas, and certain melanoma cell lines express high levels of MTf mRNA in comparison. The same expression patterns were found in mice with the exception of a 3.6kb transcript identified in normal mouse livers. A mouse melanoma cell line JB/MS, was identified to express amounts of a putative mouse MTf mRNA transcript in amounts comparable to the characterized human MTf expressing melanoma cell line SKMEL-28 . Recently, a 500 bp fragment with 83% cDNA homology to human MTf was cloned by the IMAGE consortium from a mouse fetal tissue cDNA library. An R T - P C R and cDNA library screening strategy is currently being pursued to isolate a full length cDNA clone of mouse MTf from mouse livers, fetuses, and the melanoma cell line JB/MS. Isolation of a full length cDNA clone will be followed by characterization of the protein in transfected tissue culture cell systems with mono and polyclonal antibodies raised against the recombinant protein. 59 Biological Iron Transport Model When creating an animal model to study the molecular physiology of a known human protein it is important to determine whether the animal expresses a homologous protein and whether the expression of this protein occurs in the amounts and tissue locations similar to humans. In creating a mouse model in which to study the physiology of Tf and MTf, the work thus far has established the following. 1. Distribution and relative activity of normal tissue expression of Tf in humans and mice are conserved. 2. 1° structure of mouse Tf is highly conserved to human Tf suggesting that 3° structure and function are conserved. 3. Determination of the existence of a mouse homologue to human MTf. 4. Distribution and relative activity of normal tissue expression of MTf in humans and mice are conserved. Knowing that the tissue distribution of Tf and MTf is similar in mice and humans provides initial evidence that the physiology of Tf and MTf may be similar in humans and mice and thus the feasibility and utility of using mice as a model to study Tf and MTf. Further work must be carried out to determine the distribution of mRNA expression of Tf and MTf within tissue compartments. Possession of a full length cDNA clone of mouse Tf and MTf will allow for a number of physiological studies to be carried out at a genetic level. Some of these studies include: 1. Identification and analysis of a putative IRE in the 5' UTR of Mouse Tf. 2. Creation of a recombinant Tf carrying the MTf GPI anchor. 3. Creation of an MTf knockout mouse. 4. Creation of a hyper-expressed MTf transgenic mouse. 5. Analysis of the regulation of expression of GPI anchored and soluble forms of MTf. Although the 1° structure of mouse Tf was determined, no 5'UTR has yet been isolated. Should mouse Tf be conserved in structure and function with human Tf, then mice should possess a 5'UTR containing an IRE. Tf and MTf share a 40% amino acid homology to each other and can both bind and transport iron into cells in tissue culture systems. The major 60 structural feature that separates the two other than primary structure is that Tf is a secreted soluble protein whereas MTf has two forms, a GPI-anchored membrane form, and a secreted soluble form. Characterization of the phenotype of a transgenic mouse expressing a recombinant form of Tf tethered to the cell surface by a GPI-anchor will attempt to address the importance and function of a GPI-anchored iron binding molecule. The biological importance of MTf may be addressed through the characterization of the phenotypes of MTf knockout and a hyper-expressed MTf transgenic mice. Comparison of the phenotypes of these mice to Tf knockout (Hp) mice and Tf hyper-expressed mice may enable us to determine the specific physiological role Tf and MTf play in iron transport and storage. A study of the tissue distribution of the GPI anchored and soluble forms of MTf and how the two are regulated may yield more information on the physiological function of MTf. Finally, creation of a mouse model will allow us to test a number of diagnostic and therapeutic strategies developed around MTf for the diagnosis and treatment of a variety of neurological disorders such as AD. The work listed above is intimidating in both breadth and depth; however, with the increasing scientific and therapeutic interest in the role of iron in pathologies affecting the aged more attention will be focused on the field of iron biology, particularly on the molecular physiology of Tf and MTf. 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