INVESTIGATION INTO CLASS II P-GLYCOPROTBIN MESSENGER RNA DECAY IN NORMAL LIVER AND LIVER TUMOURS by Janis Alexandra Shandro B.Sc., Lakehead University, 2000 H.B.Sc., Lakehead University, 2001 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in MATHEMATICAL, COMPUTER, AND PHYSICAL SCIENCES (CHEMISTRY) © Janis Alexandra Shandro, 2003 THE UNIVERSITY OF NORTHERN BRITISH COLUMBIA July 2003 All rights reserved. This work may not be Reproduced in whole or in part, by photocopy or other means, without the permission of the author. 1 ^ 1 National Library of C anada Bibliothèque nationale du Canada Acquisitions and Bibliographic Services Acquisitions et services bibliographiques 395 WcWngton Street Ottawa ON K1A0N4 Canada 395, rue Wellington Ottawa ON K1A0N4 Canada Your f it VoOt i*l*rtn e t Our ill NatntéUnnet The author has granted a non­ exclusive licence allowing the National Library of Canada to reproduce, loan, distribute or sell copies of this thesis in microform, paper or electronic formats. L’auteur a accordé une hcence non exclusive permettant à la Bibliothèque nationale du Canada de reproduire, prêter, distribuer ou vendre des copies de cette thèse sous la forme de microfiche/film, de reproduction sur p ^ ier ou sur format électronique. The author retains ownershq) of the co p y ri^ in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author’s permission. L’auteur conserve la propriété du droit d’auteur qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation. 0-612-84626-1 Canada APPROVAL Name: Janis Alexandra Shandro Degree: Master of Science Thesis Title: INVESTIGATION INTO CLASS IIP-GLYCOPROTEIN MESSENGER RNA DECAY IN NORMAL LIVER AND LIVER TUMOURS Examining Committee: Chair: Dr. Robert W. Tait Dean of Graduate Sfddies UNBC Supervisor: Dr. Chow H. Lee Associate P rofessai Chemistry Program UNBC Committee Member: Dr. Brent M urray. Assistant Professor, Ecosystem Science & Management Program UNBC Committee Member: Dr. Kerry Reimer Assistant Professor, Chemistry Program UNBC External Examiner: Dr. Mark Shrimpton Assistant Professor, Ecosystem Science & Management Program UNBC Date Approved: D o \r o b e j/ 3005 To Laila 11 Abstract Class II P-glycoprotein (Pgp2) is a plasma membrane protein associated with multidrug resistance. This protein and its corresponding mRNA are over expressed in many tumours, including liver tumours. Pgp2 has also been found to be overexpressed in all models of rat and mouse liver carcinogenesis. Over expression of Pgp2 in liver tumours is predominantly due to an increase in stability of Pgp2 mRNA. The main objective of this thesis was to compare the degradation pathway of rat Pgp2 mRNA in normal liver and liver tumours, with the goal of understanding why Pgp2 is more stable in liver tumours compared to normal liver. Considerable effort was spent developing the Yeast Poly (A) tailing RTPCR method in the hope of identifying Pgp2 mRNA degradation product(s) in vivo. This study concluded that although in-vitro transcribed RNA can be detected, the Yeast Poly (A) tailing RT-PCR method is not suitable for detecting in vivo mRNA degradation product(s). This study has introduced key issues related to Pgp2 mRNA that were previously unknown. Primer extension identified that endonucleolytic pathway is at least one mechanism for degradation of Pgp2 mRNA in vivo. However, the presence of an endonucleolytic cleaved product in normal liver and liver tumour suggests that endonucleolytic decay is unlikely to contribute to increased stability of Pgp2 mRNA in liver tumours. In summary, the work presented in this thesis does not provide answers as to why Pgp2 mRNA is more stable in liver tumours in comparison with normal liver. It does, however, enhance our knowledge regarding the molecular mechanisms occurring during the processing of Pgp2 mRNA in liver tumours and normal liver. Ill Table of Contents Abstract...............................................................................................................................................iii Table of Contents...............................................................................................................................iv List of Tables..................................................................................................................................... vi List of Figures................................................................................................................................... vii Acknowledgements......................................................................................................................... viii CHAPTER 1- INTRODUCTION 1.0 Multidrug Resistance and P-glycoprotein............................................................................... 1 1.0.1 Overview of Mechanisms related to Multidrug Resistance 1.0.2 ABC Transporters and Multidrug Resistance 1.0.3 P-glycoprotein 1.0.4 Regulation of Pgp Expression I . I Messenger RNA Stability....................................................................................................... II 1.1.1 Messenger RNA Stability in Prokaryotes 1.1.2 Messenger RNA Stability in Lower Eukaryotes (Yeast) 1.2 Messenger RNA decay in Higher Eukaryotes........................................................................ 15 1.2.1 Mode of mRNA decay 1.2.2 cA-Elements in mRNA Decay/Stability 1.2.3 Specific Examples of mRNA Stability/Decay 1.3 Objectives................................................................................................................................... 21 CHAPTER 2-ESTABLlSHlNG AND OPTIMIZING THE YEAST POLY(A) TAILING RTPCR METHOD TO DETECT RNA MOLECULES IN-VITRO 2.0 Methods....................................................................................................................................... 25 2.0.1 In-vitro transcription of pGEM-4Z Pgp2 plasmids 2.0.2 Poly(A)-Tailing, Reverse Transcription of In-vitro transcribed Pgp 2E1 RNA 2.0.3_Dephosphorylation and Phosphorylation of Forward Primers 2.0.4 Amplification of Poly (A) Tailed Pgp2El cDNA through Polymerase Chain Reaction 2.1 Results......................................................................................................................................... 31 2.1.1 Digestion of pGEM4Z-Pgp2 Plasmids 2.1.2 In-vitro transcription of digested pGEM4Z-Pgp2 plasmids 2.1.3 Visualization of unlabeled Poly(A) tailed RT-PCR products on a 1.5% agaorse gel 2.1.4 Visualization of y^^P-ATP labeled Poly(A) tailed RT-PCR products on a 6% denaturing polyacrylamide/urea gel 2.2 Discussion...................................................................................................................................35 IV CHAPTER 3- USE^G YEAST POLY(A)- TAILING METHOD TO SEARCH FOR POTENTIAL RAT PGP2 mRNA DEGRADATION PRODUCTS IN NORMAL LIVER AND LIVER TUMOURS 3.0 M ethods....................................................................................................................................... 38 3.0.1 Poly(A)-Tailing, Reverse Transcription of In-vitro transcribed Pgp2A, Pgp2Bl, Pgp2C, Pgp2D and Pgp2El RNA controls and Total RNA isolated from Normal Liver and Liver Tumour Tissue Samples 3.0.2 Amplification and Purification of Putative Pgp2 mRNA Degradation Products 3.0.3 Preparation of Competent Cells 3.0.4 Ligation of Putative Pgp2 mRNA degradation products 3.0.5 Transformation and Plating 3.0.6 Dideoxy Sequencing of Putative Degradation Product(s) 3.1 Results......................................................................................................................................... 46 3.1.1 Detection of degradation products in Liver and Tumour tissue samples using Yeast Poly(A) Polymerase RT-PCR method 3.1.2 Gel Purification and Secondary PCR of Liver samples with 2L1 Forward Primer 3.1.3 Preparation of pGLM7Zf+ Plasmid Vector for Ligation with Putative Degradation Products 3.1.4. Selection of Bacterial Colonies Positive for Insert and Miniprep of Positive Colonies 3.1.5 Dideoxy Sequencing and Blast Search of Potential Degradation Product(s) 3.2 Discussion...................................................................................................................................64 CHAPTER 4 - USING PRIMER EXTENSION EXPERIMENTS TO DETERMINE THE NATURE OF NUCLEOLYTIC (EXONUCLEOLYTIC OR ENDONUCLEOLYTIC) CLEAVAGE 4.0 Methods....................................................................................................................................... 69 4.0.1 Primer Extension 4.1 Results......................................................................................................................................... 71 4.2 Discussion...................................................................................................................................73 CHAPTER 5 - GENERAL DISCUSSION General Discussion........................................................................................................................... 75 Reference List.................................................................................................................................... 78 List of Tables Table Page 1.1. General mechanisms associated with multidrug resistance........................................ 2 1.2. Summary of human and rodent P-glycoprotein gene homology............................... 7 1.3. P-glycoprotein mRNA half lives...................................................................................11 1.4. A comprehensive list of endonucleases identified and their properties.................. 24 3.1 Forward primer sequences used in PCR reactions.....................................................41 3.2 Sequences of clones identified to contain an insert....................................................62 VI List of Figures Figure Page 1.1. Basic structure of P-glycoprotein...................................................................................... 7 1.2. Deadenylation dependant mRNA decay mechanism in yeast...................................... 14 1.3. General mechanism of endoribonucleic decay in vertebrates...................................... 17 2.1. Pgp2 cDNA constructs...................................................................................................... 26 2.2. pGEM4Z cloning vector....................................................................................................26 2.3. Linearized pGBM4Z Pgp2 plasmids................................................................................31 2.4. In-vitro transcribed RNA.................................................................................................. 32 2.5. Concentrations of in-vitro transcribed Pgp2El RN A ....................................................33 2.6. Concentrations of in-vitro transcribed Pgp2El RN A ....................................................34 3.1. Summary of the Yeast Poly(A) tail RT-PCR method................................................... 39 3.2. Location of forward primers.............................................................................................41 3.3. pGEM7Zf(+) cloning vector.............................................................................................43 3.4. Liver samples analyzed with Pgp2A forward prim er....................................................50 3.5. Tumour samples analyzed with Pgp2A forward prim er............................................... 51 3.6. Liver and tumour samples analyzed with PgpBl forward prim er............................... 52 3.7. Liver samples analyzed with Pgp2C forward prim er.................................................... 53 3.8. Tumour samples analyzed with 2C forward prim er...................................................... 54 3.9. Liver and tumour samples analyzed with Pgp2D forward primer............................... 55 3.10. Liver tissue samples analyzed with Pgp2El forward prim er....................................... 56 3.11. Tumour tissue samples analyzed with Pgp2El forward primer...................................56 3.12. Liver tissue samples analyzed with 2E1 forward primer on a 1.5% agarose g el....57 Vll 3.13. Tumour tissue samples analyzed with 2E1 forward primer on a 1.5% agarose gel.57 3.14. Gel purified, seeondary PCR produets............................................................................58 3.15. pGEM7Zf(+) plasmid........................................................................................................59 3.16. Plasmid clones J1-J5.........................................................................................................61 3.17. Plasmid clones J6-J10.......................................................................................................61 3.18. An example of nucleotide sequences derived from dideoxy sequencing method...63 4.1. Location of Primer R P......................................................................................................69 4.2. Primer extension analysis of liver and tumour tissue samples................................... 72 4.3. Locations of identified endonucleolytic cleavage sites............................................... 73 vm Acknowledgements There are many people I would like to thank. First, I would like to thank my supervisor, Dr. Chow Lee, for his support and encouragement during my research and throughout the writing of this thesis. I would also like to thank him for giving me this opportunity to participate in health research. I would like to thank my committee members Dr. Kerry Reimer and Dr. Brent Murray for their involvement in my research and for their guidance. I would like to show appreciation to the Dean of Graduate Studies, Dr. Robert Tait, and the Chair of the Chemistry Department, Dr. Ron Thring, for their kind words of wisdom. I would also like to thank Mrs. Alida Hall for being a very strong mentor during my TAship. Importantly, I would like to acknowledge Vieki Rehaume for her friendship, guidance, and support. I would also like to thank her for at least not openly laughing at the millions of silly questions I have asked her over this two year period. I would also like to thank my family for supporting me in this endeavor; it was not easy being away from you. Last, but by far not least, I would like to thank my husband Joel. Thank you for your undying love, encouragement, friendship, and support. IX Chapter 1 INTRODUCTION I.O Multidrug Resistance and P-glycoprotein 1.0.1 Overview of Mechanisms Related to Multidrug Resistance Chemotherapy is the major form of treatment for many types of human cancers and involves the administration of various anticancer drugs that target various mechanisms related to the growth and development of cancer cells. Alkylating agents are drugs which act to covalently bond to intracellular macromolecules causing cross-linking of DNA strands thereby increasing cytotoxicity in cells. Antimetabolites are drugs that interfere with normal cellular function through inhibition of the formation of normal nucleotides or prevent normal cell division from occurring. Natural products which have been found to interfere with topoisomerase I and topoisomerase II, bind directly with DNA, and interfere with microtubules. Other drugs that are used in chemotherapy include cisplatin and carboplatin, which bind to DNA and produce interstrand crosslinks and adducts; hydroxyurea, which inhibits DNA synthesis; and L-asparaginase, that causes the degradation of the amino acid Lasparagine required for the viability of tumour cells (Tannock and Hill, 1998). The success of these agents is often hindered by the fact that majorities of cancers are resistant to chemotherapy or develop resistance towards the chemotherapeutic drugs during treatment (Chen and Simon, 2000). After exposure to only a few agents cancer cells can become resistant to many drugs of diverse mechanisms and structures. This phenomenon is termed multidrug resistance (MDR) (Chen and Simon, 2000). There are many different mechanisms in which drug resistance can occur (summarized in Table 1.1.). One mechanism involves the impairment of drug influx. Drug uptake can occur through passive diffusion, facilitated diffusion, and active transport. All three mechanisms allow for drug entry into cells down a concentration gradient, and active transport can also lead to transport against a concentration gradient. The development of drug resistance involves impairment of unidirectional drug influx, reduced binding affinity of the transport carrier for drug, reduced number of transport sites, and slower carrier mobility (Tannock and Hill, 1998). Table 1.1. General mechanisms associated with multidrug resistance (modified from Tannock and Hill, 1998) Mechansim Drugs D ecreased Influx Increased efflux by increased expression o f A TP binding cassette transport proteins or decreased expression o f topoisom erase II Increase or decrease in levels o f target enzym es A lterations in target enzym es Inactivation by glutathione Increased D N A repair D eceased ability to undergo apoptosis A lkylating agents, antim etabolites, cisplatin N atural products A ntim etabolites, natural products A ntim etabolites, N atural products A lkylating agents, natural products, cisplatin A lkylating agents, natural products, cisplatin. A lkylating agents, natural products, cisplatin. DNA topoisomerases have also been linked with the development of multidrug resistance. Topoisomerases are enzymes that catalyze topologic changes of DNA structure required for replication and recombination of DNA and for transcription of RNA. These enzymes also play a role in chromosome structure, condensation/decondensation, and segregation. Type I topoisomerases facilitate DNA strand unwinding by passing a single stranded DNA molecule through a strand break in the complementary strand. Type II topoisomerases aid in untwisting of the DNA molecule by catalyzing the passage of double­ stranded DNA molecules through double- strand breaks. Topoisomerases serve as targets for chemotherapeutic agents, which seem to stabilize the DNA-enzyme complex, leading to an increase in DNA strand cleavage, thereby increasing DNA damage and ultimately cell death. Down regulation of both types of topoisomerases, and the production of mutant type I topoisomerase have been reported in drug resistant cells (Tannock and Hill, 1998). Glutathione-s-transferase (GST), has also been reported to play a role in the development of multidrug resistance. GST is a widespread enzyme involved in the detoxification and protection of normal tissue. Reduced forms of glutathione can inactivate peroxides and free radicals produced by some drugs. They can also bind to positively charged electrophilic molecules rendering them less noxious and easily excretable. Drugs conjugated to reduced forms of glutathione are expelled from cells by the GS-X pump, which has a broad specificity for several anticancer drugs. This function is in part reliant on the multidrug resistance protein (MRP) (Borst and Elferink, 2002), therefore drug resistance due to an increase in expression of MRP most likely depends on GSH-mediated conjugation of anticancer drugs (Tannock and Hill, 1998). DNA repair mechanisms also are involved in drug resistance by enhanced removal of DNA adducts and/or crosslinks from resistant strains. Alkylating agents produce DNA lesions that may be repaired by three mechanisms: damage reversal; nucleotide excision repair; and recombination or complementation. 0^-alkylguanine DNA alkyl transferase (AGAT) and 3-methyl-adenine DNA glycosylase are two enzymes involved in DNA repair, and both have been reported to be increased in tumour cells resistant to chemotherapeutic agents (Tannock and Hill, 1998). MDR has also been shown to be associated with an important biological process termed apoptosis. Apoptosis is a process where intracellular signals trigger sequential events leading to cellular death. This complex pathway is regulated by many proteins. Mutation of important genes in tumour cells such as p53, responsible for the stimulation of apoptosis, may inhibit apoptosis. There is also evidence that some tumours exhibit an increase in bcl-2. a gene that inhibits apoptosis, and a decrease in bax, a stimulator of apoptosis. Decreased apoptosis in tumour cells may then lead to drug resistance (Tannoek and Hill, 1998) 1.0.2 ABC Transporters and Multidrug Resistance The best identified actions of multidrug resistance are related to active transport proteins that belong to the ATP-binding cassette (ABC) superfamily (Ruth et al., 2000). This superfamily consists of membrane transporters in yeast, bacteria, parasites, nematodes, plants, and mammals. In mammals, this family includes transporters for bile acids, acylated fatty acids, chloride ions, peptides, and other substrates (Julien and Gros, 2000). ABC transporters play a crueial role in protecting vital bodily structures such as the brain, the testis, the cerebrospinal fluid, and the fetus against the uptake of toxic agents, like food derivatives and drugs into the body (Borst and Elferink, 2002). They have also been associated with many human diseases, ineluding cancer (Gottesman and Ambudkar, 2001). There are four plasma membrane proteins belonging to the ABC superfamily associated with multidrug resistance; multi-drug resistance protein (Cole et ah, 1992), lung-resistance protein (Tannock and Hill, 1998), breast cancer resistanee protein (Doyle et ah, 1998), and Pglycoprotein (Ling, 1974) (to be discussed in detail in section 1.0.3). Multidrug-resistance Protein (MRP) is a glutathione-X conjugate pump and was first diseovered in human lung caneer cells exhibiting multidrug resistance (Cole et ah, 1992). The discovery of MRP has led to the characterization of 6 related proteins, all involved in the transport of anticancer eompounds (Gottesman, 2002). The MRPs identified thus far differ in substrate specifieity, tissue distribution, and intracellular location (Borst and Elferink, 2002). All of the MRPs have been associated with MDR, however, there is yet to be direct evidence implieating MRPs as the root cause of the development of MDR phenotype in human eaneers. A second protein that is associated with resistance to a wide spectrum of drugs is called lung-resistance protein (LRP). This protein is expressed on intraeellular organelles known as vaults, whieh are multisubunit structures involved in nueleocytoplasmie transport. It has been hypothesized that LRP is involved in eausing drug resistanee by pumping the drugs into the vaults, whieh are then exported from the cell (Tannoek and Hill, 1998). Breast cancer resistant protein (BCRP) is a plasma membrane protein located in the apical membranes of placental syneytiotrophoblasts, the epithelial lining of the small intestine and colon, hepatoeytes, and the duets and lobules of the mammary gland (Borst and Elferink, 2002), and has also been associated with multidrug resistanee. The highest concentration of BCRP RNA has been found in the placenta suggesting that this protein has an important defense function by preventing entry of the drug to the fetus. Clinically, BCRP has been identified to be overproduced in MCF7 breast cancer cells and has shown to have high mitoxantrone resistance. Possible inhibitors of BCRP include fumitremorgin C and its analogs (Borst and Elferink, 2002). 1.0.3 P-glycoprotein The first discovered and the only ABC transporter to date shown to be linked with development of clinical MDR is P-glyeoprotein (Juliano and Ling, 1976). P-glycoprotein (Pgp) is synthesized in the endoplasmic reticulum, and modified in the golgi apparatus yielding a glycosylated 170 kDa transmembrane protein. Pgp is found on the luminal surface of epithelial cells in various regions of the body such as the large and small intestine, the biliary canalicular membranes of hepatoeytes, the apical surface of epithelial cells of the proximal cells of the kidney, the blood tissue barriers of the testis and brain, the liver, and the epithelia of the choroid plexus, (Loo and Clarke, 1999). It has been proposed that Pgp functions in several different aspects of normal physiology, such as protection from xenobiotics, resistance to apoptosis, intracellular tracking of sterols, and migration of dendritic cells into lymphatic vessels (Luker et al., 2000). As well as offering protection, Pgp has been associated with the transport of molecules across cell membranes (Brown, Thorgeirsson, and Silverman, 1993). These molecules include chemotherapeutic drugs, calcium channel blockers, steroid hormones, immunosuppressive agents, and peptide antibiotics (Hrycyna et al., 1998). Pgp is a 1280 amino acid polypeptide containing two tandem repeats of 610 amino acids, joined by a linker region of 60 amino acids (Loo and Clarke, 1999). It is comprised of two homologous halves, with each half spanning the plasma membrane bilayer six times (Hrycyna et al., 1998). Each halve of Pgp includes a hydrophobic domain with six transmembrane segments and a nucleotide binding ABC domain (Ruth et al. 2000). This domain arrangement is characteristic of the ABC superfamily (Figure 1.1.) (Loo and Clarke, 1999). Two classes of Pgp genes exist in humans, class I (Pgpl) and class III (Pgp3). Class I (PgpI) is capable of transporting many chemotherapeutic drugs and is associated with multidrug resistance (Lee, Bradley & Ling, 1998). Class III (Pgp3), which is not associated with multidrug resistance, is known to be a phospholipid translocase abundant in the liver (Romsicki and Sharom, 2001). There are three rodent isoforms, class I, class II, and class III. Class I and II are associated with the development of multidrug resistance, and class III, found to be most abundant in the liver is necessary for transport of phospholipids (Lee, Bradley & Ling, 1998). The MDR gene that codes for Pgp in rats is highly conserved with the MDR gene that codes for Pgp in humans (Table 1.1). For example, rat Pgp2 is 79.1 % homologous to human Pgpl, and 71.5% homologous to human Pgp3 (Silverman, et al., 1991). ATP ATP Figure 1.1. Basic structure of P-glycoprotein (modified from Tannock and Hill, 1998). Table 1.2. Summary of human and rodent P-glycoprotein gene homology, (modified from Silverman et al., 1991). Human Pgp3 Genes Human Pgpl Rat Pgp2 Hamster Pgp2 Hamster Pgpl Mouse Pgp3 Mouse Pgp2 Mouse Pgpl % H om ology 79.1 75.7 % H om ology 71.5 47.7 83.5 64.2 71.1 86.1 70.6 71.6 78.7 82.2 Multidmg resistance conferred by overexpression of P-glyeoprotein (Pgp) is one of the best-charaeterized forms of resistanee mediated by transport molecules (Luker et al., 1997). When overexpressed, Pgp funetions to confer multidrug resistance (Loo & Clarke, 1999) by acting as an ATP-dependant efflux pump that transports drugs with cytotoxic activity out of cells before they reaeh intracellular target (Taguchi et al., 1997). These drugs include many natural product derived anticancer agents such as doxorubicin, daunorubicin, vinblastin, vincristine, and taxol (Gottesman, 2002). The binding of these drugs results in activation of one ATP-binding domain. Hydrolysis of ATP results in a conformational change in Pgp, which then releases the drug into the extraeellular spaee. Hydrolysis of a second ATP molecule is required to restore the protein to its original state (Gottesman, 2002). P-glyeoprotein has observed to be overexpressed in many human eaneers, including cancers of the gastrointestinal tract, cancers of the hematopoietie system, cancers of the genitourinary system, and ehildhood cancers (Goldstein et al., 1989). One of the first studies reporting increased Pgp expression in elinical tumour samples involved two women with ovarian cancer being treated by ehemotherapeutic agents (Bell et al., 1985). There is also considerable evidenee of highly expressed Pgp in renal (Fojo et al., 1987) and eolon tumours (Weinstein et al., 1991). In other cases, an inerease in Pgp expression is seen only after exposure to ehemotherapeutie drugs or during relapse (Chan et al., 1996). The majority 8 of clinical efforts to overcome multidrug resistance related to Pgp expression aim to inhibit Pgp’s activity. These clinical trials have had limited success (Gottesman, 2002). Much effort has also been put forth to reverse MDR via inhibition of Pgp expression at the DNA and RNA level (Lee, 2003). For instance, ecteinascidin 743, an anti-tumour agent, has shown promising pre-clinical results in inhibiting Pgp activation (Jin et ah, 2000). Also more recently, transcription has been targeted by modulation of the nuclear receptor SXR (Synold, Dussault, and Forman, 2001). Strategies have also been suggested involving the use of antisense and transcriptional decoy (Marthinet et ah, 2000) and anti-M DRl mRNA hammerhead ribozymes (Wang et ah, 1999). In the treatment of human cancers, Pgp expression has been associated with chemotherapy failure and decreased survival, therefore its regulation is of great clinical importance. 1.0.4 Regulation of Pgp Expression Regulation of Pgp expression is of great importance in cancer research as its overexpression has been implicated as the main reason for the development of multidrug resistance. These observations have provoked profound studies related to the regulation of Pgp expression. It has been shown that anticancer drugs (Fardel et ah, 1997; Schrenk et ah, 1996; Zhou and Kuo, 1998; Furuya et ah, 1997), carcinogens (Fardel et ah, 1996; Nakatsukasa et ah, 1993), UV irradiation (Zhen, Shengkan, and Scotto, 2000; Uchiumi et ah, 1993), extracellular matrix proteins (Tatsuta et ah, 1994), heat shock (Chin et ah, 1990), transcription factors (Thottassery et ah, 1999; Bargou et al, 1997; Chin et ah, 1992), and reactive oxygen species (Ziemann et ah, 1999) participate in overexpression of Pgp in human tissues and animal models. Pgp 1 and Pgp2 have been shown to be overexpressed in all models of rat and mouse liver carcinogenesis (Lee, Bradley, and Ling, 1998; Kren, Trembley and Steer, 1996). An increase in Pgp2 gene expression has been observed during liver regeneration (Nakatsukasa et al., 1993; Teeter et al., 1993; Marino, Gottesman, and Pastan, 1989; Thorgeirsson et al., 1987), during establishment of primary hepatoeytes (Lee, Bradley and Ling, 1995; Hirsch-Ernst et al., 1995; Schuetz et al., 1995), in tissues treated with cyclohexamide (Lee, 2001), and in uteral tissues during pregnancy (Croop et al., 1989; Kuo et al., 1995). Interestingly, mRNA stability has been demonstrated in the regeneration of rat liver (Kren et al., 1996) and in liver tumours (Lee, Bradley, and Ling, 1998). There has also been evidence of post-transcriptional control of Pgp in the uterus (Kuo et al., 1995), intestine (Chianale et al., 1995) and in rat tissues subjected to cyclohexamide (Lee, 2001). It has also been determined that DNA cross linking agents can suppress M DRl gene expression at two independent steps, one at the level of mRNA transcription, and the other at the post transcriptional level (Maitra et al., 2001) More recently, it has been suggested that Pgp expression is regulated at two specific steps in human leukemic cells. These steps include mRNA stabilization and translational initiation (Yague et al., 2003). However, the distinct mechanisms of how regulation occurs at these two steps, in either case, has yet to be determined. Overexpression of Pgp has also been demonstrated in many rat liver carcinogenesis models (Lee et al., 1998; Kuo et al., 1995, Nakatsukasa et al., 1993; Teeter et al., 1993; Teeter et al., 1990). In a transplantable rat liver tumour line. Pgp mRNAs exhibited an increase in stabilization, in comparison to normal liver Pgp mRNA. This has been confirmed through the analysis of mRNA half-lives of the three Pgp. P gpl, Pgp2 and Pgp3 have a half-life of approximately 1-2 hours in normal liver. In liver tumours, all three Pgp mRNA demonstrated a half-life of over 12 hours (Table 1.2) (Lee et al., 1998). This 10 indicates that Pgp mRNA in liver tumours is more stable in comparison to normal liver, which accounts for an increase in Pgp mRNA overexpression in liver tumours. Table 1.3. P-glycoprotein mRNA half lives (t V2) (h) in liver and transplantable liver tumours (modified from Lee et al., 1998) Genes Liver Transplantable Liver Tumours P gpl Pgp2 < 1 < 1 1.8 Pgp3 > 12 > 12 > 12 Mounting evidence suggesting mRNA stability is at least one major mechanism resulting in the overexpression of Pgp (Lee et ah, 1998). This warrents further investigation into the molecular mechanisms responsible for the increase in stability demonstrated. 1.1 Messenger RNA Stability The importance of regulating mRNA stability is shown by the extent of physiological and clinical events that are affected by change in stability (Dodson and Shapiro, 2002). These include development, cell cycle, viral infections, reproduction, and response to cellular environment changes (Dodson and Shapiro, 2002). important in relation to protein expression. Messenger RNA stability is very The length of time mRNA is intact in the cytoplasm directly determines the amount of protein translated (Ross, 1995). Simply, the more stable mRNA transcripts are, the more protein is expressed. In retrospect, if the mRNA transcript is destabilized, less protein production occurs. The molecular mechanisms for activating mRNA degradation or stabilization, particularly in the mammalian system, are not well understood. It also appears that the mechanisms involved and the decay processes that exist in prokaryotic and eukaryotic systems are not conserved, leading to more complication regarding the issue. 11 1.1.1 Messenger RNA Stability in Prokaryotes Prokaryotic mRNA has been observed to be very unstable, and decay rates vary extensively (Coburn and Mackie, 1999). There are many factors and pathways involved in the degradation and stabililty of prokaryote mRNA, however the exact mechanisms are not known, and the main nucleases involved only have loose primary sequenees or secondary structure specificity (Grunberg-Manago, 1999). mRNA stability is also reliant on several external factors such as growth conditions, environmental signals, and efficiency of translation. It is known that decay is always initiated by endoribonucleolytic cleavage by RNase E or RNAse III. RNase E has been found to cleave substrates nonspecifically 5 ’ to AU dinucleotides in single stranded segments (Mackie, 1998). RNase III cleaves double stranded RNA molecules either with a single strand nick or a double strand break. Endonueleolytic cleavage is followed by exonucleolytic decay at the 3’ end. There have been two enzymes responsible for exonucleolytie Pbospborylase (PNPase) and RNase II. decay including Polynucleotide PNPase catalyzes pbospborolysis of mRNA liberating nueleoside diphosphates, while RNase II catalyzes hydrolysis of mRNA releasing 5’ monophosphates (Grunberg-Manago, 1999). A multienzyme complex, eommonly known as the degradosome has also been identified to play a role in the decay process. It appears that the degradosome is eomprised of RNase E, PNPase, a glycolytic enzyme, and different enzymes using ATP as a cofactor. The functional role of the degradosome is unclear, although it is obviously involved in the decay process of bacterial mRNA transcripts (Grunberg-Manago, 1999). The final major enzyme facilitating mRNA decay processes in bacteria is Poly(A) Polymerase. This enzyme adds adenine residues to the 3’ hydroxyl termini of a transcript using ATP as a substrate and releasing pyrophosphate. 12 Unlike in eukaryotic systems where poly (A) tails are used as stability factors for mRNA transcripts, polyadenylation facilitates exonucleolytic decay by providing a single stranded tail for PNPase to attack (Grunberg-Manago, 1999). 1.1.2 Messenger RNA Stability in Lower Eukaryotes (Yeast) The majority of knowledge obtained to date regarding eukaryotic mRNA stability and degradation mechanisms arises from studies in the yeast Saccharomyces cerevisiae. There are three modes of mRNA decay described in yeast; (i) deadenylation dependant decapping, followed by 5’-3’ decay; (ii) deadenylation independent from decapping followed by 3’-5’ decay; and (iii) endonucleolytic cleavage. The major mechanism of mRNA decay is thought to involve the removal of the 3’poly (A) tail, followed by the removal of the 5’ eap. Deadenylation followed by decapping is the mechanism for deadenylation dependent mRNA decay and subjects the mRNA structure to 5’-3’and/or 3’-5’ exonuclease activity (Brewer, 2002; Dodson and Shapiro, 2002; Tucker and Parker, 2000) (summarized in Figure 1.2.). Deadenylation in yeast seems to require the use of two agents with exonuclease activity. The first contributor is composed of two proteins Ccr4P/CaflP, and the second deadenylase is the Pan2p/Pan3p (PAN) exonuclease. The proposed mechanism for deadenylation in yeast involves the PAN subunit that may be responsible for initial trimming of the poly (A) tail, and further deadenylation by the Ccr4P/CaflP complex. This is followed by removal of the 7-methyl guanosine cap. The removal of the 5’ cap in yeast involves the decapping enzyme D cplp, and many other protein factors. Decapping is the key step in yeast mRNA degradation as it precedes and permits the decay of the mRNA transcript body by exposing it to a 5’-3’ exonuclease (Tucker and Parker, 2000). 13 Additionally, yeast transcripts can also be degraded in a 3’-5’ direction following deadenylation. This is catalyzed by a complex of 3 ’-5’ exonucleases termed the exosome. It is apparent that this degradation pathway is slower than 5’-3’ decay, however it is still crucial to development as mutations that inhibit both 5’-3’ decay and 3’-5’ decay are lethal. This suggests that both 5’-3’ and 3’-5’ deadenylation dependant degradation are the two significant pathways for mRNA turnover in yeast (Tucker & Parker, 2000). i i i DEADENYIATTON DBCAPPFSG DEADB\IYl.ASE EXONICLBOLATK: DBCLAY o 3 ' EX O 5 ' EX O Figure 1.2. Deadenylation dependant mRNA decay mechanism in yeast (modified from Wilusz et al., 2001 ) Endonucleolytic cleavage has also been identified to play a role in the stabilization and destabilization in yeast mRNA transcripts (Brewer, 2002; Dodson and Shapiro, 2002). This pathway is reliant on an endoribonuclease to cut the mRNA body internally. This event is followed by exonuclease activity. Endoribonuclease pathway in yeast involves an unfolded protein in the endoplasmic reticulum. The unfolded protein Irel has both protein kinase and 14 endoribonucleic domains. It has been shown to excise introns and degrade RNA in yeast during periods of cell stress (Tirasophon et ah, 2000). Another method of mRNA decay in lower eukaryotes is termed nonsense-mediated decay or RNA surveillance. This process targets mRNA transcripts containing error, and is therefore involved in the regulation of mRNA. Error can include: nonsense mutations or early termination codons and improperly spliced introns. This pathway has been studied extensively in yeast, and occurs through decapping independent of deadenylation (Dodson and Shapiro, 2002), followed by 5’-3’ degradation in the cytoplasm (Brewer, 2002). The same deeapping enzyme used in deadenylation dependant decay, is also used for nonsensemediated decay. Four additional factors have been identified and deemed crucial for nonsense-mediated decay in yeast. These included U pfl, Upf2, Upf3, and H rpl. It is hypothesized that these factors interact to form a ‘surveillance’ complex, and associate with release factors at translation termination. The complex then scans the downstream portion of the transcript for a premature termination signal. H rpl interacts with the premature termination signal and is thought to act as a marker for targeted nonsense mediated decay (Wilusz et al., 2001). 1.3 Messenger RNA decay in Higher Eukaryotes 1.3.1 Mode of mRNA decay The mode of messenger RNA decay in vertebrates is currently an unresolved issue. To date, deadenylation dependant decay has been shown to be an important mode of mRNA decay in mammalian cells. The postulated mechanism for this process involves the enzyme poly (A) ribonuclease (PARN), first purified from the thymus of a calf (Brewer, 2002). The 15 binding of PARN to the 7-methyl guanosine cap activates the processing of the poly(A) tail and suggests a link between decapping and deadenylation. been identified in mammalian systems. A decapping enzyme has also DcpS is an enzyme thought to decap an mRNA transcript that has previously undergone deadenylation and extensive 3’-5’ exonuclease degradation (Brewer, 2002). The exact deadenylation-dependant mRNA decay method has not been fully characterized in vertebrates and research into the exact mechanism is still ongoing (Dodson and Shapiro, 2002). Unlike in lower eukaryotes, there has been no evidence thus far suggesting that 5’-3’ exonuclease decay participates in mRNA regulation in vertebrates. There has been however, substantial evidence indicating that endonucleolytic activity also plays an active role in the decay process of vertebrate mRNA (summarized in Figure 1.3.). mRNA control can be obtained by regulation of endoribonuclease activity, controlling the accessibility of the cleavage site, or regulating both levels of control (Dodson and Shapiro, 2002). Endoribonucleic activity seems to be triggered in response to cellular crisis or infection that is characterized by the presence of double stranded RNA (dsRNA). There are two pathways identified in higher eukaryotes which demonstrate that an endoribonuclease can be activated in response to dsRNA. The first involves the synthesis of a 2 ’-5’ linked oligoadenylate (25A) in response to viral infection. This in turn activates RNAse L that cleaves the RNA strand at UU or UA residues (Starck et ah, 1998). The second mechanism involves interference RNA that targets specific endoribonucleaic cleavage in response to dsRNA. The activated endoribonuclease cleaves the dsRNA into 21-23 nucleotide fragments (Sharp, 2001 ). 16 P ro#ein4ilm di% S ile *Bp * IRecogfudwmby sÊaÈMlizM&r pno#eio fkwÈmnbonwdease r*NDo;;KÛldwMni sile ltgNDO%sfûli 24 nt. 3’-UTR ErEN Not known Nonpolysomal Erythroidenriched aCP Wang et ah, 2000 Undefined' AU-rich, ssRNA RNAse E substrates ARDl 13.3 Polysomes Not identifed Not identifed Wennborg et ah, 1995; ClaverieMartin et ah, 1997 p27kipl UUCGGUUUGUUUU UU, 5 ’-UTR Endonuclease in HUR binding site Not known Not known Not identified HUR Zhao et ah, 2000 Viral RNA AA and AU ssRNA RNase L 4 0 ,8 0 ER INF, 2-5A Not identifed Gallonzi et al., 1998 Irel CUGCAG, AAAACUA, AGUGAA IR El 110 ER Unfolded Protein Not identifed Tirasophon et al., 2000 ’ endogenous substrate unknown ^ isolated from Xenopus laevis Chapter 2 Establishing and Optimizing the Yeast Poly(A) Tailing RT-PCR Method to Detect RNA Molecules In-vitro This chapter describes the establishment and optimization of the Yeast Poly (A) tailing RT-PCR protocol used for detection of putative Pgp2 mRNA degradation intermediates. The sensitivity of the Yeast Poly (A) tailing RT-PCR assay is determined by running Poly(A)-tailed RT-PCR products of in-vitro transcribed RNA, on 6% denaturing polyacrylamide/7M urea gel and on 1.5% agarose gel. Methodology, results and discussion related to these topics are ineluded. 2.0 Methods 2.0.1 In-vitro transcription of pGEM4Z-Pgp2 Plasmids Plasmid constructs designated pGEM4Z-Pgp2A, pGEM4Z-Pgp2B 1, pGEM4Z-Pgp2C, PGEM4Z-Pgp2D, and pGEM 4Z-Pgp2El contain different segments of Pgp2 eDNA were used (Silverman et al., 1991; Fig.2.1.). All plasmids were linearized with the restriction enzyme H indlll to prepare for in-vitro transcription (see Figure 2.2. for detail of pGEM4Z plasmid). Plasmid DNA (15 pg) was added to 10 pL of Hindlll, 3 pL of lOX Reaction buffer, and double distilled water (ddHiO) to make the reaetion mixture up to 30 pL. The mixture was ineubated at 37° C for 3 hours. 2 pL of linearized plasmid was then loaded and run on an ethidium bromide stained 1% agarose gel. The gel was visualized and a photograph was taken using Chemilmager'i'’^ System (Alpha Innoteeh Corporation, San Leandro, CA). Onee linearized 170 pL of ddH20 was added to make the volume up to 200 pL. The standard phenol ehloroform extraetion was performed as follows: 100 pL of phenol and 100 25 [iL of chloroform;isoamylalcohol (CHClsiIAA) (49:1) were added to the linearized plasmid solution. The solution was vortexed and then centrifuged at 12,000 rpm for 5 minutes. The aqueous layer was transferred to a new microcentrifuge tube and one volume of CHCl^iIAA was added to the new tube. The solution was vortexed and centrifuged for an additional five minutes to remove any excess phenol. The A lG W IO j) top layer was llGA(nt3933) ZZZZZHZzlz^^l 3' 4254 nis 1057-IS55 nt pGEVWZfgpZD I&52 2656 nt 12652 3454 nt 13452^229 nt pGE\MZrPg^A Figure 2.1. Pgp2 cDNA constructs used for making in-vitro RNA controls in Yeast PoIy(A) tailing RT-PCR method. (Constructs were designed based on cloning of Pgp2 mRNA accomplished by Silverman et al., 1991) SMI WO II 2 2 6 2 Vector ECOFO SK l K pol A w al XirrM 1 $TTMl B am H 1 XUBi a 'l  cc 1 H f ic 11 S W 'l M n d III 1 S ia n 7 17 23 23 23 25 23 34 40 41 42 SO 56 58 70 T Tt Figure 2.2. pGEM4Z cloning vector used for the construction of Pgp2 cDNA constructs (Promega, Madison, WI). 26 again transferred to a new tube and the Standard Ethanol Precipitation was performed as follows: two volumes of 100% ethanol and I/IO'*’ of the volume of 3 M sodium acetate (NaOAe) pH 5.2 were added and the solution was mixed thoroughly. The mixture was then stored at -20° C for 20 minutes to precipitate the plasmid DNA. The solution was removed and centrifuged for ten minutes at 12,000 rpm. The supernatant was removed and the DNA pellet was washed by gently adding 200 pL of 70% cold ethanol. The sample was then centrifuged for five minutes and the supernatant was aspirated. The DNA pellet was then allowed to air dry for 15 minutes and was resuspended in 10 pL of ddHaO. RNA was synthesized using MEGAscript® SP6 Kit (Ambion, Inc., Austin, Texas). The in- vitro transcribed RNA will be used as a control for verification of the Yeast Poly (A) tailing RT-PCR method. Each in-vitro transcription reaetion entailed: 4 pL of linearized plasmid DNA; 2 uL of 10 X reaction buffer; 2 pL of each 50 mM ATP, CTP, UTP, and OTP; 2 pL of SP6 RNA polymerase; and 4 pL of diethylpyroearbonate (DEPC) H 2 O. The mixture was ineubated at 37° C for 2 hours followed by an addition of 1 pL of DNase I. The reaetion was ineubated again at 37° C for 15 minutes. DEPC H 2 O was then added to make the reaction mixture up to 200 pL and the Standard Phenol/Chloroform Extraetion, Standard Ethanol Precipitation was performed. The pellet was resuspended in 50 pL of DEPC H 2 O, and the solution was added to a ProbeQuant^^ G-50 Micro Column (Amersham Pharmacia Biotech). The column was centrifuged for 2 minutes at 3,000 rpm and the RNA sample was collected in a new microcentrifuge tube. The quality of RNA was checked by running 3 pL of column sample with 3 pL cocktail mix [10 X MOPS (N-morpholino propanesulfonic acid, EDTA, NaOAe, ddH20, DEPC H 2 O), formaldehyde, formamide, ethidium bromide, and 27 DEPC H 2 O and 2[iL loading dye on a 1.3% formaldehyde gel. The gel was visualized and photographed using Chemilmager^M System. 2.0.2 Poly(A)-Tailing, Reverse Transcription of In-vitro transcribed Pgp 2E1 RNA In-vitro transcribed Pgp2El RNA was determined to be of good quality RNA sample (Figure 2.4., lane 5) and therefore was used to test the sensitivity of this assay. Pgp2E 1 RNA (100 ng) was incubated at 65°C for 5 minutes, and then placed on ice. To the sample the following reagents were added: 2 gL of 5X reaction buffer; 1 p,L of 10 mM ATP; 0.5 gL RNasin (40 U/gL) (Promega, Madison, W I ); 0.5 qL 100 mM DL-Dithiothreitol (DTT); 1 gL Yeast Poly(A) Polymerase (600 U/gL) (USB Corp., Cleveland, OH); and 4 gL DEPC HoO. The solution was incubated at 30°C for 10 minutes and then incubated at 65 °C for an additional 10 minutes. The sample then underwent a reverse transcription reaction where the 10 [xL of Pgp2El tailed-RNA was again incubated at 65°C for five minutes and then placed on ice. The following reagents were then added: 2 gL of lOmM deoxyribonucleoside triphosphates (dNTPs); 4 pL of 5X AMV Buffer; 1 pL of OligodTig-Xhol primer 100 mM DTT; 1 pL RNasin (20 U/pL); and 1 pL of AMV reverse transcriptase (5U/pL) (Roche Applied Science, Basel, Switzerland). The solution was incubated at 37°C for 20 minutes, followed by incubation at 50°C for 15 minutes. The enzymes were heat activated by incubation at 65°C for 10 minutes. As a control to test if Poly (A) tailing of Pgp2El invitro transcribed RNA was successful, reverse transcription was carried out on non-tailed RNA samples as well. 28 2.0.3 Déphosphorylation and Phosphorylation of Forward Primers It was necessary to 5’ label forward primers with y^^P-ATP (50pCi) (Amersham Biosciences Corp, Piscataway, NJ) prior to use for PCR of tailed and nontailed RT samples for analysis on 6% polyacrylamide/7 M urea gel. The primers first needed to be dephoshorylated. Each dephosphorylation reaction included: 10 [rg of forward primer, 10 pL of Calf Intestinal Alkaline Phophatase (CIAP) (1 U/pL) (Roche Applied Science, Basel, Switzerland); 10 pL of 10 X dephosphorylation buffer; and ddHaO to make the final reaction volume up to 100 pL. The reaction mixture was incubated at 37 °C for 30 minutes and 100 pL of ddHiO was added. The standard phenol/chloroform extraetion and the standard ethanol precipitation were performed and the DNA pellet was resuspended in 20 pL of ddHzO. The phosphorylation reaction of dephosphorylated primers included the following reagents: 10 pL of dephosphorylated primer; 5pL of 5 X phosphorylation buffer; 5 pL of y32p_ATP; 3 (10 U/pL) of T4 Polynucleotide kinase (New England Biolabs, Beverly, MA); and ddHaO to make the final reaction volume to 25 pL. The reaction mixture was incubated at 37°C for one hour and ddH20 was added to make the volume up to 50 pL. The sample was added to a ProbeQuant^'^ G-50 Micro Column and centrifuged for 2 minutes at 3,000 rpm. The sample was collected in a new microcentrifuge tube and ddHaO was added to make the final volume to 200 pL. The standard phenol/chloroform extraction was performed, followed by the standard ethanol -precipitation. The DNA pellet was resuspended in 200 pL of ddHiO. Ip L of sample was used for scintillation counting. 29 2.0.4 Amplification of Poly (A) Tailed Pgp2El cDNA through Polymerase Chain Reaction In order to test the sensitivity of this technique on both a 6% polyacrylamide/7 M urea gel and a 1.5 % agaorse gel, two different PCR reactions were set up. The reaction intended for polyacrylamide gel analysis included: 2 pL of tailed-RT Pgp2El cDNA template ranging in concentration from 0 ng/pL to 400ng/pL; 3.5 pL of lOX PCR Buffer (New England Biolabs); 3.5 pL of 2.5 mM dNTPs; 1 pL 50 mM MgCl]; 5 pL (100 ng/pL) of y^^P-ATP 5’ labeled EFl forward primer; 1 pL of reverse OligodTig-XhoI (100 ng/pL); 1 pL (5 U/pL) of Taq Polymerase (Invitrogen Life Technologies, Carlsbad, CA); and ddH^O to make the final reaction volume up to 35 pL. The reaction intended for anlaysis on a 1.5% ethidium bromide stained agaorse gel involved the substitution of the labeled EE 1 forward primer for unlabeled EEl forward primer (100 ng/pL). PCR parameters were set at 94°C for 30 sec; 50°C for 30 sec; and 72°C for 45 seconds for 30 cycles on a Minicycler™ Peltier Thermal Cycler (MJ Research, Inc., Reno, NV). Unlabeled products were ran on a 1.5% ethidium bromide stained agarose gel with 0.5 X TBE buffer and were visualized using Chemilmager'i''^ System. Labeled products of the first PCR experiment were run for 3 hours on a 6% polyacrylamide/7M urea gel with I X TBE (tris base, boric acid, EDTA) buffer. The gel was dried and exposed for 24 hours. The image was scanned using Cyclone phosphorimager, and visualized using the OpiQuant software (Hewlett Packard, Palo Alto, CA). 30 2.1 Results 2.1.1 Digestion of pGEM4Z-Pgp2 Plasmids pGEM4Z-Pgp2 plasmids are double stranded eircular DNA moleeules derived from bacteria. The circular structure of the intact plasmid molecule appears as two bands after gel electrophoresis. The major band (approximately 1800 bp) is the plasmid DNA, whereas the top band represents nicked plasmid DNA. All plasmids underwent a restriction digest with H indlll (Figure 2.3.) to prepare for in-vitro transcription. One band is detected in all digested plasmid samples except for pGEM4Z-Pgp2B 1 (lane 5). pGEM4Z-Pgp2B 1 plasmid exhibits two faint bands due to incomplete digestion. Î, I it 1.1 ft "O N « I M a. s I, N « It ft 9 I ft U I I II I! It N « ft 9 ft "c ft 9 I it II ft "0 It ft 9 f t ft, 203616361018- 506- Figure 2.3. pGEM4Z Pgp2 plasmids linearized with restriction enzyme with Hind III in preparation for in-vitro transcription on 1 % agarose gel. Even numbered lanes exhibit intact pGEM4Z Pgp2 plasminds. Odd numbered lanes show plasmid samples that underwent restriction digest with Hind HI. 31 2.1.2 In-vitro transcription of digested pGEM4Z-Pgp2 plasmids A digest (1-3 |iL) sample of each in-vitro transcribed Pgp2 RNA was ran on a 1.3 % formaldehyde gel to visualize the RNA (Figure 2.4.). A major species of in-vitro transcribed RNA was detected for all samples corresponding to the approximate expected length (see Figure 2.2.; most intense band in Figure 2.4.) except for Pgp2A (lane 1). In-vitro transcription of Pgp2A plasmid has resulted in 3 distinct bands. This has been observed in the hands of other experimenters in the lab. In-vitro transcribed Pgp2C, and Pgp2D also exhibit a secondary RNA species (lanes 3 and 4). In-vitro transcribed Pgp2Bl (lane 2) and Pgp2El (lane 5) both exhibit only one species of RNA (lanes 2 and 5). Pgp2El RNA was chosen for further studies as it exhibited one intense distinct major species.(see Methods 2.0.2 and 2.0.4). < '>1 -o 4» e a '< u o. G &t - < < e i fl ^ — t-H ‘2 pa V cu S WD 2 V. T1S) s u 5 AG 6L® K , isS < _ 1 -o aj s a Q ri 523 A G on g A is ■p S C i tH «2 M U M 2S2 cu WD g cu i -1.35 kb- F ig u r e 2 .4 . In -v itr o tr a n sc r ib e d R N A on a 1.3% formaldehyde gel. Linearized plasmid samples were subjected to in-vitro transcription using MEGAscript® SP6 K it (Ambion) 32 2,1,3 Visualization of unlabeled Poly(A) tailed RT-PCR products on a 1,5% agaorse gel Concentrations of poly(A) tailed in-vitro transcribed Pgp2El RNA products from 0 ng/pL to 400 ng/pL were amplified by PCR and were ran on a 1.5% ethidium bromide stained agarose gel to determine the sensitivity of this method (Figure 2.5.). Detection of Poly(A) tailed Pgp2El in-vitro transcribed RNA (most intense band with a size approximate to 1018 bp) was possible from 400 ng/pL to 0.4 ng/pL (lanes 7 to 11). A less distinct band was present at approximately 506 bp and was detected from 400 ng/pL to 1 ng/pL. Two bands shown at the bottom of the gel in Figure 2.4. are primers. I 1 o : 1 s : 1 o o 1 o o 1Tf 1c d 1c Tf 1 ? ÎI Pgp2El RNA 1018- 506396- Primers Figure 2.5, Concentrations of in-vitro transcribed Pgp2El RNA from on a 1.5% agarose gel. Concentrations of poly (A) tailed in-vitro transcribed Pgp2El RNA products from 400 ng/pL to 0 ng/pL were amplified by PCR and visualized to determine tbe sensitivity of tbe Yeast Poly (A) Tailing RT-PCR method. 33 2.1.4 Visualization of y^^P-ATP labeled Poly(A) tailed RT-PCR products on a 6% denaturing polyacrylamide/urea gel. I eg ÎÎ I I I I W O o II ? I Pgp2El RNA 622- 439430- 370- Primers 8 1 10 Figure 2.6. Concentrations of in-vitro transcribed Pgp2El RNA on a 6% denaturing poiyacrylamide/7M urea gel. Concentrations of poly (A) tailed in-vitro transcribed Pgp2El RNA products from 400 ng/pL to 0 ng/pL were amplifîed by PCR and visualized to determine the sensitivity of the Yeast Poly (A) Tailing RT-PCR method. The marker used is plasmid pBR322 cut with restriction enzyme H aelll. To determine whether the Yeast Poly(A) tailing RT-PCR method would be more sensitive, experiments were also carried out using 5’ y^^P-ATP labeled Pgp2El forward primer. Concentrations of poly(A) tailed in-vitro transcribed Pgp2El RNA products from 34 400 ng/fxL to 0 ng/gL were amplified by PCR and were run on a 6 % denaturing poIyacrylamide/7M urea gel to determine the sensitivity of this method (Figure 2.6.). Detection of Poly (A) tailed Pgp2El in-vitro transcribed RNA (most intense band with a size approximate to 1048 bp) was possible from 400 ng/^iL to 0.001 ng/fxL (lanes 3-10). Several less distinct bands were also detected at approximately 600 bp and 400 bp in the same samples. Large smears present at the bottom of the gel are primers. 2.2 Discussion All pGEM4Z-Pgp2 plasmids were successfully linearized after the restriction digest with Hind III except for pGEM-Pgp2B 1, as only one distinct band was exhibited when a small amount of sample was run on a 1% agarose gel (Figure 2.3.). pGEM-Pgp2B 1 exhibited partial digestion as one faint band was apparent. In other experiments, complete linearization of pGEM-Pgp2B 1 has been observed. This sample was still used for in-vitro transcription as some linearized plasmid sample is present. In-vitro transcription was performed on all pGEM4Z-Pgp2 plasmids. Small samples of the newly synthesized RNA were loaded and run on a 1.3% formaldehyde gel to confirm successful transcription (Figure 2.4.). Ideally in-vitro transcribed RNA should only exhibit one band as seen in Pgp2El and Pgp2Bl RNA (Figure 2.4.). In-vitro transcribed Pgp2C, and Pgp2D RNA display two bands. In-vitro transcribed Pgp2A RNA exhibits 3 bands. It is not clear why in-vitro transcribed RNA from these plasmids exhibits more than one species. One reason could be related to the presence of a premature termination sequence. If a premature termination sequence is present in the sequence being transcribed, which is recognizable by SP6 RNA polymerase, early termination of transcription would occur. This would explain why two or three different samples of in-vitro transcribed RNA are present. However, there 35 is no premature stop signal present in any of the plasmid sequences. Therefore, it is possible that the structure of the plasmid at certain points may promote dropping off of the SP6 RNA polymerase, yielding the possibility of an incomplete transcript being present. A more likely explanation involves transcription of incomplete digested plasmid which was not observable on an agarose gel. This would lead to the presence of a larger species of RNA, which is evident in this case. In any case, this has not hindered analysis of liver and tumour samples for Pgp2 mRNA degradation products, as the in-vitro transcribed RNA is only acting as a control to test if the Yeast Poly (A) RT PCR method is valid, and does not participate in the actual detection of putative decay products. The Yeast Poly(A) tailing RT-PCR method was able to amplify concentrations of invitro transcribed Pgp2El RNA from 400 ng/pL to 0.4 ng/pL using unlabelled Pgp2El forward primer (Figure 2.5.). The sensitivity of this method was improved when using 5’ y^^P-ATP labeled Pgp2El forward primer and run on a 6% denaturing polyacrylamide/urea gel (Figure 2.6.). In this case, in-vitro transcribed Pgp2El mRNA could be detected down to 0.001 ng/pL. These results indicate that the Yeast Poly (A) tailing RT-PCR method is highly sensitive in detecting low concentrations of RNA in-vitro. It is imperative to bear in mind that the concentration of putative Pgp2 mRNA degradation products in vivo is not known, therefore, it is not possible to determine if this method is suitable for in vivo applications. One can speculate however, that there is potential for this method to be successful as 0.4 ng/pL and 0.001 ng/pL are very low concentrations. Upon visualization of the 1.5% agarose gel (Figure 2.5.), it was noticed that a less distinct band was visible with a size of approximately 506 bp. Several less distinct bands were also present on the 6% polyacrylamide/7M urea gel (Figure 2.6.) with sizes approximately 500 bp and 400 bp. It is 36 possible that these bands were amplified and represent regions of in-vitro transcribed Pgp2 mRNA rich in adenine residues where the Oligo dTis-XhoI primer could nonspecifically hybridize. It is possible that such nonspecific nature of the Oligo dTig-XhoI primer could pose a problem for in vivo applications (Chapter 3); however it will depend on the concentration of tailed Pgp2 mRNA degradation products. If the concentration of successfully tailed Pgp2 mRNA degradation products is high enough, they should be successfully amplified. Due to the sensitivity of this method, observed here in-vitro it was determined that this method will be used to detect putative Pgp2 mRNA degradation products in normal liver and liver tumour tissue samples. 37 Chapter 3 Using Yeast PoIy(A) Tailing Method to Search for Putative Rat Pgp2 mRNA Degradation Products in Normal Liver and Liver Tumours This chapter describes experiments performed to detect putative Pgp2 mRNA degradation intermediates in total RNA from normal liver and liver tumours from three different animals using the Yeast Poly (A) tailing RT-PCR method. All experimental procedures, results, and a discussion related to the use of Yeast Poly (A) tailing RT-PCR method are included. This chapter assesses the efficiency of the Yeast Poly (A) tailing RTPCR method in detecting Pgp2 mRNA degradation intermediates. This could potentially have implications for detecting any mRNA degradation intermediates in cells or tissues. 3.0 Methods 3.0.1 Poly(A)-TaiIing, Reverse Transcription of In-vitro transcribed Pgp2A, Pgp2Bl, Pgp2C, Pgp2D and Pgp2El RNA controls and Total RNA isolated from Normal Liver and Liver Tumour Tissue Samples All tissue samples previously collected from animals, were subjected to in vivo transcriptional inhibition, and were used in experiments described in this thesis (Lee et al., 1998). Briefly, tissue samples of liver and tumours were collected from rats, which had received an intravenous bolus of 50 pg per 100 g body weight of a-amanitin and an intraperitoneal dose of 150 pg per 100 g body weight of aetinomyein D. Aetinomyein D and a-amanitin are transcriptional inhibitors, therefore halting the process in which mRNA is synthesized from a DNA template. Tissue samples obtained from three different animals at time points 0, 1, 3, 6, and 12 hours post-injection with transcriptional inhibitors were subjected to total RNA isolation (Lee et al., 1998). Total RNA samples at 0 time point were analyzed for the possible presence of Pgp2 mRNA degradation products. Figure 3.1. is a diagrammatic scheme of the Yeast Poly(A) tailing RT-PCR method. 38 These samples were named Liver 0.1, 0.2, 0.3 and Tumour 0.1, 0.2, 0.3. In-vitro transcribed Pgp2 RNA (100 ng) and 10 [xg of total RNA from each tissue sample was used for the Poly(A) Tailing method (see Methods 2.0.2). All samples then underwent a reverse transcription reaction (see Methods 2.0.2) As a control to test if Poly(A) tailing was successful, reverse transcription was carried out on non-tailed in-vitro transcribed RNA samples. To test for the presence of possible degradation products, reverse transcription was also carried out on nontailed total RNA isolated from all six tissue samples. degraded RNA AAAAAAAAAAAA Poly (A) Tail AAAAAAAAAAA reverse transcribe ^ OligodTlS-XhoI primer * PCR with labeled mRNA-specific primer Figure 3.1. Summary of the Yeast Poly(A) tail RT-PCR method used to search for putative Pgp2 mRNA degradation intermediates. 39 3.0.2 Amplifîcation and Purification of Putative Pgp2 mRNA Degradation Products PCR was used to amplify possible degradation products of Pgp2 mRNA in normal liver and liver tumour. It was initially thought that identification of possible degradation products would only be possible through analysis of 5’ labeled y^^P-ATP PCR products on a 6% polyacrylamide/7M urea gel. However, due to the apparent sensitivity of the technique (see Discussion 2.2), some analysis on cold PCR products were carried out on a 1.5% ethidium bromide stained agarose gel. Five general PCR reactions were set up to analyze the five segments of Pgp2 mRNA represented by pGEM4Z plasmid constructs (Figure 3.2.). Each general reaction included tailed and nontailed samples of the in-vitro transcribed RNA sample corresponding to the region of study (i.e. Pgp2El), normal liver (0.1, 0.2, 0.3), and liver tumour (0.1, 0.2, 0.3). To each individual reaction the following was added: 3.5 [xL of lOX PCR Buffer; 3.5 fxL of 2.5 mM dNTPs; 1 piL 50 mM MgCli; 1 tiL of reverse Oligo dTig-XhoI primer (100 ng/pL); 1 pL of Taq Polymerase (5 U/pL); and ddH20 to make the final reaction volume up to 35 pL. For PCR reactions involving Pgp2A, Pgp2B 1, and Pgp2El as the region of study, 5 pL of y^^P-ATP 5’ labeled 2A, 2B1 and 2E1 forward primer respectively, were added to each individual reaction. For PCR reactions involving Pgp2C and Pgp2D as the region of study, 5 pL (lOOng/pL) of unlabeled 2C and 2D Forward Primers respectively were added to each individual reaction (see Table 3.1 for primer sequences). PCR parameters entailed: 94°C for 30 seconds; 50°C for 30 seconds; and 72°C for 45 seconds for 30 cycles on a Minicyeler™ Peltier Thermal Cycler. The PCR products utilizing the labeled primers (2A, 2B1, and 2E1) were loaded and ran for 3 hours on a 6% polyacrylamide/7M urea gel with 1 X TBE buffer. The gel was dried and exposed for 24 hours. Autoradiography was carried 40 out using Cyclone phosphorimager using the OpiQuant software. PCR products utilizing unlabeled forward primers (2D and 2C) were ran on a 1.5% ethidium bromide stained agarose gel with 0.5 X TBE buffer and were visualized using C h e m i l m a g e r ^ M System. A lC (oH 03) 5 '[ _L ly _J____ o n 52 o f - 72 nf DF1A52 HI 1071 BFI 2652 nt- 2671 nt AF3452 nt-3471 nt Figure 3.2. Location of forward primers used in the polymerase chain reaction to amplify possible Pgp2 mRNA degradation products. Primer Sequence (5’ - 3’) * 2A forw ard prim er CTCGGATCCAGGAGCCCATCCTGTTTGAC 2 B 1 forw ard prim er CTCGGATCCCTTAGTCTATGGCTGGCAGC 2C forw ard prim er CTCGGATCCATAGCTCACCGCTTGTCTAC 2D forw ard prim er CTCGGATCCCCTCCTTGGTCCTCTAAAT 2E1 forw ard prim er CTCGGATCCACATTCTTGGCGGACTTCGCGA Bold print represents B am H I restriction site. 41 Tissues exhibiting differences between tailed and nontailed samples, indicating potential Pgp2 mRNA degradation product(s) underwent another PCR reaction with unlabeled forward primer and were ran on a 1.5% ethidium bromide stained agarose gel with 0.5 X TBE buffer. The gel was visualized using Chemilmager™ System. The potential product(s) was then excised from the gel using a sharp scalpel under UV light, and gel purified using QIAEX II Gel Extraction Kit (QIAGEN, Mississauga, ON). Two pL of the purified sample then was used for 6 PCR reactions and included the following reagents: 3.5 pL of lOX PCR Buffer; 3.5 pL of 2.5 mM dNTPs; 1 pL 50 mM MgCla; 5 pL (100 ng/pL) of EEl forward primer; 1 pL of reverse Oligo dTig-XhoI (100 ng/pL) primer ; I pL of Taq Polymerase (5 U/pL); and ddHzO to make the final reaction volume up to 35 pL. The PCR products were pooled together totaling a final volume of 210 pL. Two volumes of 100% ethanol and I/IO'*’ the volume of 3 M NaOAc pH 5.2 was added and the solution was mixed thoroughly and precipitated at -20°C for 24 hr s. The solution was removed and centrifuged for ten minutes at 12,000 rpm. The supernatant was removed and the DNA pellet was washed by gently adding 200 pL of 70% cold ethanol. The sample was then centrifuged for 5 minutes and the supernatant was aspirated. The DNA pellet was resuspended in 7 pL of ddHaO and was loaded on a 1.5% ethidium bromide stained agarose gel. Again the potential product(s) were excised from the gel and purified. The purified sample was then used to perform ligation and transformation experiments in an attempt to sequence the potential degradation product(s). 3.0.3 Preparation of Competent Cells DH5-a competent cells required for transformation, were then prepared via the 42 following methodology: 10 ^iL of frozen stock DH5-a cells were added to 3 mL Luria- Bertani (LB) broth in a sterile 15 mL tube and incubated at 37°C overnight with agitation. Ten |iL of this culture was transferred to a flask containing 50 mL LB broth and was again incubated at 37°C with agitation until the OD reached 0.3 (2 hours) at 600 nm. The culture was then centrifuged for 10 minutes at 2,500 rpm and the supernatant was removed. The pellet was resuspended in 3.5 mL of cold 50 mM CaCl 2 and the volume was increased to 25 mL. The cells were then placed on ice for 30 minutes and then centrifuged at 2,500 rpm for 10 minutes. The supernatant was again removed and the competent cells were resuspended in 3.5 pL of cold 50 mM CaClz. 3.0.4 Ligation of Putative Pgp2 mRNA degradation products. To prepare for the ligation of the potential product(s) it was necessary to perform a restriction digest on both the purified product and pGEM7Zf(+) cloning vector (Fig. 3.1.). All of the gel purified product (20 pL) was added to the restriction digest, which included the following reagents: 6 pL of BamRV, 6 pL of Xhol; 3.6 pL of lOX double digest buffer; and 4 pL of dd H 20. The solution was incubated at 37°C for 3 hours. ddHzO was added to make a final colume of 100 pL, and standard ethanol precipitation was performed. The pellet was resuspended in 10.5 pL of ddHiO. The resuspended product was added to 1 pL of digested pGEM7Zf(+) vector (Figure 3.3.) and the following reagents were added: 3 pL of T4 DNA Ligase (400 LF/pL) (New England Biolabs); and 1.45 pL of 10 X Ligase Buffer (New 43 England Biolabs). The reaction mixture was T7 i incubated at 4°C for 24 hours. 1 Kbifl As*II 20 ^11 26 XWI 31 37 X/N)l tooHI 43 Kpr;l 63 56 Sffa I C ^ l 61 Oil I 67 Wfid III 73 |M97hn) 76 91 & k 'l 8^X1 100 TSPG 123 109 Figure 3.3. pGEM7Zf(+) cloning vector used in ligation reaction (Promega, Madison, WI). 3.0.5 Transformation and Plating In a prechilled sterile tube all of the ligation mix was added to 200 pL of DH5-a competent cells. The mixture was stored on ice for 30 minutes and then heat shocked at 42°C for 2 minutes. One pL of LB Broth was added and the reaction was incubated at 37°C for 30 minutes with agitation. 2pL of isopropyl b-D-thiogalactoside (IPTG) and 50 pL of 5bromo-4-chloro-3- indolyl- beta- D- galactopyranoside (X-GAL) were added and the mixture was poured on a pre-warmed agar plate. The plate was incubated at 37 °C with the lid partially off in order for the solution to dry for 20 minutes and then inverted. After 24 hours 10 white colonies were picked with a sterile pipette tip, and then placed in a sterile tube containing 3 mL LB broth and 1.5 pL 50 mg/mL ampicilin. The cultures were grown overnight at 37°C with agitation. To determine if any of the white bacterial colonies positively contained an insert that might be a possible degradation product it was necessary to purify the plasmid DNA from the rest of the culture. The QIAprep Spin Miniprep Kit (QIAGEN) was used for this procedure and yielded a 50 pL sample 44 of purified plasmid DNA. 3 jxL of purified plasmid sample then underwent a restriction digest with 1 pL of Xhol, IpL of BamHI, 1 pL of lOX Double Digest Buffer, and 4 pL ddH20. The reaction mixture was incubated for 1 hour at 37°C and then ran on a 1.5% ethidium bromide stained agarose gel. The gel was visualized using ChemilmagerTM System. 3.0.6 Dideoxy Sequencing of Putative Degradation Product(s) Sequenase Version 2.0 DNA Sequencing Kit (United States Biochemical, Cleveland, OH) was used to sequence purified plasmid samples that contain an insert. The protocol is as follows: 5 pL of purified plasmid sample for clones J l, J2, J7, and JIG was added to 15 pL of ddHaO. 2 pL of 2 M NaOH, 2 mM EDTA mix was added and the solution was incubated at 80°C for 5 minutes in order to hydrolyze any RNA that may be present. ddH20 was added to increase the volume of the solution to 100 pL. Standard ethanol precipitation was performed and the pellet was resuspended in 7 pL of ddH20. sequencing buffer and 10 ng of 7Z-T7 primer was added. To the DNA, 2 pL of The solution was mixed and incubated at 37°C for 15 minutes. The following was then added to the DNA solution: IpL of 100 mM DTT; 2 pL of diluted dGTP labeling mix (1:20 with ddH20); 1 pL of 1 mM MnCU; 1 pL ^^S-dATP (lOpCi/pL) (Amersham Biosciences Corp.), and 2 pL Sequenase™ (1:10 dilution with TE). The mixture was left at room temperature for 3 minutes. 3.5 pL of labeling reaction mix was then added to four separate microcentrifuge tubes, each containing 2.5 pL of a different dideoxy triphosphate. The solutions were incubated at 37°C for 5 minutes and 4 pL of Stop Solution was added. The sequencing reaction for Clones J6 and J9 required the use of all purified plasmid sample (45 pL) as the concentration of these plasmid samples were lower than the others. The amount of reagents used for these two samples doubled in all respects. 45 Prior to loading on an 8% polyacrylamide/ 7 M urea sequencing gel, the solutions were incubated at 80°C for 2 minutes, and then quickly placed on ice. The sequencing gel was also pre-loaded with Formamide Loading Solution to ensure the lanes were not leaking. The gel was ran for 30 minutes at 60 W constant current to pre-warm it. After the samples were loaded, the gel was ran at 60 W constant current until the blue dye of the stop solution reached the very bottom of the gel (approximately 1.5 hours). The gel was then dried and exposed for 48 hours. The image was scanned using Cyclone phosphorimager and visualized using OpiQuant software. Results 3.1 3.1.4 Detection of degradation products in Liver and Tumour tissue samples using Yeast Poly (A) Polymerase RT-PCR method. The detection of putative Pgp2 mRNA degradation intermediates in total RNA from normal liver and liver tumour from three different animals involved using the Yeast Poly (A) tailing RT-PCR method. In order to detect potential degradation products it was necessary to include nontailed-RT-PCR products, as presence of bands in tailed samples that were absent in nontailed samples would indicate the existence of potential decay products. The analysis of normal liver and liver tumour tissue samples for putative Pgp2 mRNA decay products involved the use of five different forward primers (see Figure 3.2.) ensuring that the entire Pgp2 mRNA sequence was analyzed. Samples of tailed and nontailed in-vitro transcribed RNA corresponding to the region under study were also included as verification that the Yeast Poly (A) tailing RT-PCR method was employable. Analysis of liver tissue samples with forward primer 2A (Figure 3.4) was carried out 46 on an ethidium bromide stained 1.5% agarose gel. Several bands are noticed in both the tailed and nontailed samples (lanes 5, 7, 8, and 9), however, there is no significant differences present between tailed and nontailed samples. There were PCR products present/detected in tailed samples that correspond to in-vitro transcribed Pgp2A. This sample exhibits two distinct bands, one of approximately 1 kb, while Pgp2A nontailed sample exhibits several. The 1 kg fragment likely corresponds to the Pgp2A RNA size. Differences are noted between tailed and nontailed in-vitro transcribed Pgp2A RNA, indicating that the Yeast Poly (A) tailing RT-PCR method was successfully applied. Analysis of tumour samples with forward primer 2A was carried out on a 6% denaturing polyacrylamide/urea gel (Figure 3.5.). Again, there are no differences between tailed and nontailed tumour samples detected. Similar results were obtained with in-vitro transcribed Pgp2A RNA. The tailed sample exhibits two distinct bands (lane 2), while Pgp2A nontailed sample exhibits several (lane 3). As with normal liver samples (Figure 3.4.), no distinct PCR products were seen in tailed tumour samples versus nontailed samples (Figure 3.5., lanes 4-9). Analysis of liver and tumour samples with forward primer 2B 1 was carried out on a 6% denaturing polyacrylamide/urea gel and results are shown in Figure 3.6. As seen in Figure 3.6. there are no differences between tailed and nontailed normal liver and liver tumour tissue samples present (lanes 4-15). In-vitro transcribed Pgp2B 1 tailed sample exhibits one band of approximately 1 kb, while Pgp2B 1 nontailed sample exhibits several. The presence of tailed in-vitro transcribed PCR products (lane 2) indicate that the Yeast Poly (A) tailing RT-PCR method was successful. 47 Analysis of liver samples (Figure 3.7.) and tumour samples (Figure 3.8.) with forward primer 2C were carried out on ethidium bromide stained 1.5% agarose gels. There are no significant differences between tailed and nontailed tissue samples in both normal liver (see Figure 3.7., lanes 4-9) and liver tumour (see Figure 3.8., lanes 4-9). In tailed samples of both normal liver (lanes 4, 6, and 8) and liver tumour (lanes 4, 6, and 8), a small smear was observed near the well. Curiously, this smear was also present in tailed in-vitro transcribed RNA (lane 2). Due to its presence in all tailed samples, this smear was not further investigated as a putative degradation product. In-vitro transcribed Pgp2C tailed sample exhibits one distinct band approximately 1 kb in size, while Pgp2C nontailed sample exhibits several (Figure 3.7. and 3.8., lanes 2 and 3), again indicating that the Yeast Poly (A) tailing RT-PCR method was again successful in detecting in-vitro transcribed RNA. Analysis of liver and tumour samples with forward primer 2D was also carried out on a 1.5% ethidium bromide stained agarose gel (Figure 3.9.). There were no differences between tailed and nontailed tissue samples present (lanes). In-vitro transcribed Pgp2D tailed sample exhibits one distinct band (lane), while Pgp2D nontailed sample exhibits several (lane). Once again, the differences present in tailed and nontailed in-vitro transcribed Pgp2D RNA samples indicate that the Yeast Poly (A) tailing RT-PCR method was successfully employed. PCR products of normal liver and liver tumour samples with 2E1 forward primer were both analyzed on a 6% denaturing polyacrylamide/urea gel (Figure 3.10. and 3.11. respectively). Distinct differences are present between the tailed samples of both tissue samples (Figure 3.10. and 3.11., lanes 4-9). In tailed liver samples (Figure 3.10., lanes 4, 6, and 8) a smear is present from approximately 500 bp to approximately 181 bp in all three 48 tissue samples. This smear is not present in nontailed samples (lanes 5, 7, and 9). In tailed liver tumour samples a very intense smear is present from approxiamtely 439 bp to approximately 181 bp in two tissue samples (Figure 3.11., lanes 6 and 8). In-vitro transcribed PgpEl tailed sample exhibits one very intense distinct band approximately 1 kb in size (lane 2), and four faint bands, while Pgp2El nontailed sample exhibits several bands (lane 3). The distinct band of approximately 1 kb likely corresponds to in-vitro transcribed RNA indicating that the Yeast Poly(A)tailing method was used successfully in this experiment. Liver and tumour samples with 2E1 forward primer were then ran on an ethidium bromide stained 1.5% agarose gel (Figure 3.12. and 3.13. respectively) to determine if further analysis of these potential Pgp2 mRNA degradation products could proceed without the use of radioactive material. Consistent with analysis on the 6% denaturing polyacrylamide/urea gel (Figure 3.10.), tailed normal liver samples exhibit a smear around the 500 bp size in all three tissue samples (lanes 4, 6, and 8). Nontailed samples do not have a smear present, but one distinct band at approximately 1000 bp (lanes 5, 7, and 9). Tailed tumour samples also exhibit the same smear (lanes 6 and 8) when ran on a 1.5 % agarose gel (Figure 3.13.) which is consistant with results obtained from 6% denaturing polyacrylamide/urea gel analysis (Figure 3.11.). Nontailed samples have 2 bands at approximately 1000 and 506 bp that are present in Tumour 0.1 and Tumour 0.2 (lanes 5 and 7). 49 1636- 1018- 506- 298- 1 2 3 4 5 6 7 8 9 Figure 3.4. Liver samples analyzed with Pgp2A forward primer on a 1.5% agarose gel. Prior to amplification with Pgp2A forward primer, liver samples were either tailed and reverse transcribed (even lanes), or reverse transcribed (odd lanes) to search for putative Pgp2 mRNA degradation intermediates. Pgp2A cDNA control is 799 base pairs (bp) long. 50 •i # 620- 439430- 370- 1 8 Figure 3.5. Tumour samples analyzed with Pgp2A forward primer on a denaturing 6% polyacrylamde/7M urea gel. Prior to amplification with Pgp2A forward primer, liver samples were either tailed and reverse transcribed (even lanes), or reverse transcribed (odd lanes) to search for putative Pgp2 mRNA degradation intermediates. Pgp2A cDNA control is 799 bp long. The marker used was pBR322 cut with the restriction enzyme Hae II. 51 ■g I! I I I ■s I I O o 1I z 2 o I i I1 0 1 0 1 1I z 2 I 2 o )j 622- 439- 370- 181- Figure 3.6. Liver and tumour samples analyzed with Pgp2Bl forward primer on a 6% denaturing polyacrylamde/7M urea gel. Prior to amplification with Pgp2Bl forward primer, samples were either tailed and reverse transcribed (even lanes), or reverse transcribed (odd lanes) to search for putative Pgp2 mRNA degradation Intermediates. Pgp2Bl cDNA control Is 800 hp long. The marker used was pBR322 cut with the restriction enzyme Hae II. 52 1 I I I I I I s 3 ■g I I a u I O Ï o Ï I z 2 Ï S I 1018- 506396298- 220- Figure 3.7. Liver samples analyzed with Pgp2C forward primer on a 1.5 % agarose gel. Prior to amplification with Pgp2C forward primer, samples were either tailed and reverse transcribed (even lanes), or reverse transcribed (odd lanes) to search for putative Pgp2 mRNA degradation intermediates. Pgp2C cDNA control is 800 hp long. 53 ■2 I I I ! 21 2 I I Jj ! 1 j I I 1 I e c o N O z © u 1018- 506298220 - 1 8 Figure 3.8. Tumour samples analyzed with 2C forward primer on a 1.5% agarose gel. Prior to amplifîcation with Pgp2C forward primer, samples were either tailed and reverse transcribed (even lanes), or reverse transcribed (odd lanes) to search for putative Pgp2 mRNA degradation intermediates. Pgp2C cDNA control is 800 bp long. 54 1a a 1s ra Z H a Q 'â M v-4 & Z Q 'n, 0* CU 1018- 506298- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Figure 3.9. Liver and tumour samples analyzed with Pgp2D forward primer on a 1.5% agarose gel. Prior to amplification with Pgp2D forward primer, samples were either tailed and reverse transcribed (even lanes), or reverse transcribed (odd lanes) to search for putative Pgp2 mRNA degradation intermediates. Pgp2D control is 800 bp long. 55 i I I J II I I III 1 H 1 I - 1048- - 1048- 622622 439 370370 181 181- t 1 2 3 4 5 6 7 8 9 1 Figure 3.10. Liver tissue samples analyzed with Pgp2El forward primer on a 6% denaturing polyacrylamide gel. Prior to amplification with Pgp2El forward primer, samples were either tailed and reverse transcribed (even lanes), or reverse transcribed (odd lanes) to search for putative Pgp2 mRNA degradation intermediates.Pgp2El control is 1048 hp long. The marker used was pBR322 cut with restriction enzyme Hae II. 56 2 3 4 5 6 7 8 9 Figure 3.11. Tumour tissue samples analyzed with Pgp2El forward primer on a 6% denaturing polyacrylamide gel. Prior to amplification with Pgp2El forward primer, samples were either tailed and reverse transcribed (even lanes), or reverse transcribed (odd lanes) to search for putative Pgp2 mRNA degradation intermediates.Pgp2El control is 1048 bp long. The marker used was pBR322 cut with restriction enzyme Hae 11. ■2 ■s II i a I H I h J 1III1 Ï Ï I - ) h J DC h J DC ^ 1048- 1048- 1 2 3 4 1 5 6 7 8 Figure 3.12. Liver tissue samples analyzed with 2E1 forward primer on a 1.5% agarose gel. The same Poly (A) tailed and nontailed samples from Figure 3.10. were analyzed to determine if further analysis could he accomplished without the use of radioactive material. 2 3 4 5 6 7 8 Figure 3.13. Tumour tissue samples analyzed with 2E1 forward primer on a 1.5% agarose gel. The same Poly (A) tailed and nontailed samples from Figure 3.10. were analyzed to determine if further analysis could he accomplished without the use of radioactive material. 57 3.1.5 Gel Purification and Secondary PCR of Liver samples with 2E1 Forward Primer. To confirm specificity of the smear observed in normal liver and liver tumour tailed samples, the smear observed in liver samples analyzed with Pgp2El forward primer were excised for the gel and purified (see Methods 3.0.2). Secondary PCR was also performed using the purified products as a template. The secondary PCR products were ran on a 1.5% agarose gel to check if the putative degradation products were successfully purified and amplified (Figure 3.14.). All purified secondary PCR liver tissue products exhibit a smear from approximately 600 bp to 250 bp, suggesting that the smear is specific. Putative degradation product(s) present in Liver 0.1 were used in further experiments to determine if the PCR product(s) belong to Pgp2 mRNA. 1018506- 298- Figure 3.14. Gel purifîed, secondary PCR products from tailed liver samples on a 1.5% agarose gel. Smears identified in Figure 3.12. were excised and purified. Purified samples underwent secondary PCR with the same primers to determine the specificity of the smears. 58 3.1.6 Preparation of pGEM7Zf(+) Plasmid Vector for Ligation with Putative Degradation Products. Plasmid vector pGEM7Zf(+) underwent a linearization reaction with restriction enzymes BamYll and Xhol (Figure 3. 15.). The circular structure of the intact plasmid molecule appears as two bands after gel electrophoresis. The major band (approximately 2036 bp) is the plasmid DNA, whereas the top band represents residual chromosomal and nicked plasmid DNA. Complete digestion of the pGEM7Zf(+) plasmid is noted as only 1 band is present in the sample that was digested with BamHI and Xhol. T 203616361018- 506298- Figure 3.15. pGEM7Zf(+) plasmid linearized with restriction enzymes BamHI and X h o\ in preparation for ligation reaction on a 1.5% agarose gel. Lane 2 exhibits undigested plasmid, and lane 3 shows digested plasmid sample. 59 PCR products (see Results section 3.1.2) which are putative Pgp2 mRNA degradation intermediates were purified and digested with BamYU. and Xhol, and then ligated into linearized pGEM7Zf(+) plasmid (see Method 3.0.4). The ligated reaction mixture was then transformed into DH5-a competent cells (See Method 3.0.5). 3.1.4. Selection of Bacterial Colonies Positive for Insert and Miniprep of Positive Colonies. White and blue colonies were present on agar plates plated with transformed DH5-a competent cells. Colonies that are positive for the inserted putative degradation product(s) are coloured white. Colonies that do not contain an insert are coloured blue. Ten white colonies were picked and grown in liquid medium overnight (See Methods 3.0.5). The ten plasmid DNA samples were then isolated and subjected to a restriction digest with BamHI and Xhol. Digested plasmid samples were ran on an ethidium bromide stained 1.5% agarose gel (Figures 3.16. and 3.17.) to check for the presence of an insert (See Methods 3.0.5). Figure 3.16. shows results of elones J l to J5 after restriction digest with BamHI and Xhol. Clone J l exhibits 2 distinct bands after digestion with BamHI and Xhol (lane 2). The top band represents linearized plasmid DNA, and the second band is approximately 500 bp in size. Clone J2 exhibits 3 bands after digestion (lane 3); the top band again representing plasmid DNA, and two additional bands approximately 506 bp and 220 bp in length. Clones J3, J4, and J5 exhibit only plasmid DNA after the restriction digest (lanes 4, 5, and 6). Figure 3.17. exhibits clones J6 to JIO after a restriction digest with BamHI and Xhol. Clones J6, J7, J9, and JIO all exhibit two bands (lanes 2, 3, 5, and 6), the top band representing linearized plasmid DNA, and the second band approximately 506 bp in size . Clone J8 exhibits plasmid DNA only. Clones J l, J2, J6, J7, J9, and JIO, deemed to contain 60 inserts were subjected to sequence analysis (see Methods 3.0.5) Samples containing only plasmid DNA after digestion were disregarded and not used in further experiments. Figure 3.16. Plasmid clones J1-J5 on a 1.5% agarose gel. Clones (lanes 2-6) underwent restriction digest with enzymes BamHI and Xhol to check for the presence of an insert. 1018- 1 2 3 4 5 6 Figure 3.17. Plasmid clones J6-J10 on a 1.5% agarose gel. Clones (lanes 2-6) underwent restriction digest with enzymes BamHI and Xhol to check for the presence of an insert. 61 3.1.5 Dideoxy Sequencing and Blast Search of Potential Degradation Product(s) Clones J l, J2, J6, J7, 19, and JIO were manually sequenced (see Methods 3.0.4) to determine if they contained an insert that was a possible Pgp2 mRNA degradation product (See Figure 3.18.). All clones contained the multiple cloning site sequence from the vector pGEM7Zf(+). Sequences for clones J l, J6, J9, and JIO revealed an identical sequence that was different from the vector sequence. This sequence was subjected to a nucleotide BLAST search on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/) to determine if they were Pgp2 mRNA degradation products. The nucleotide search provided no significant matches. The sequence determined for clone J2 also contained vector sequence and a potential inserted sequence that was unique. This sequence was also subjected to a nucleotide BLAST search that yielded no significant results. This was also the case for clone J7 that again contained the vector sequence plus a potential insert. In conclusion, no Pgp2 mRNA degradation products were detected. Table 3.2 Sequences of clones identified to contain an insert C lone S eq u en ce * Jl 5 ’... G C C G G G G T C G C G A A G T C C G T T A A G A T G G G A T C C A A G C T T A T C G A T T T C G A A C C C G G G G T A C C G A A T T C C T C G A G C .. .3 ’ J2 5 '... T C A G C A T G C T C C T C T A G A C T C G A G C C G G G G T A T A T A G C C T T T G A A C A A C G G T A G C TAGTTTCTAGGCATTCTTTGTCCCTTCCTAACCTCTTGTGCTCGCTGAGACAATTGCCAG TG TC G C A G TC TA A G G ,.. 3 ’ J6 5 ’G C A T G C T C C T C T A G A C T C G A G C C G G G G T C G C G A A G T C C G C C A A G A T G G G A T C C A A G C T T A T C G A T T T C G A A C C C G G G G T A C C G A A T T C C T C G A G G A T T ... A G A G C T C C C A A C G C G T T G G A T G C A T A G C T T G A G T A .. .3 ’ J7 5 ’C C T C T A G A C T C G A G G A A T T C G G T T G A C C C C G G G T T C G A A A A T C C G A T A A G C T T G G A T C A A G C T T A T C G A T T C G A C C .. ..3 ’ J9 5 ’GC A T G C T C C T C TA G A C T C G A G C C G G G G T C G C G A A G T C C G C C A A G A T G G G A T C C A A G C T T A T C G A T T T C G A A C C C G G G G T A C C G A A T T C C T C G A G G A T T .. .A G A G C T C C C A A C G C G T T G G A T G C A T A G C T T G A G T A .. .3 ’ JIO 5 ’C C T C T A G A C T C G A G G A A T T C G G T T G A C C C C G G G T T C G A A A A T C C G A T A A G C T T G G A T C A A G C T T A T C G A T T C G A C C .. ..3 ’ ' X hol restriction site 62 T G C A II Figure 3.18. An example of nucleotide sequences derived from dideoxy sequencing method (see Methods 3.0.5). The Nucleotide sequence of clones J2 (left) and J l (right) on an 8% denaturing polyacrylamide/urea gel. 63 Discussion 3.2 The main objective of this chapter was to use the Yeast Poly (A) tailing RT-PCR method to detect Pgp2 mRNA degradation products in vivo. Results using in-vitro trasnscribed RNA (Pgp2A-2El) have shown that RNA can be tailed and amplified using this method. Analysis of tailed and nontailed normal liver and liver tumour samples amplified using five specific forward primers that covered the entire length of Pgp2 mRNA was carried out on both denaturing 6% polyacrylamide gels and 1.5% agarose gels. Analysis using forward primers 2A, 2B1, 2C, and 2D yielded no detection of putative degradation intermediates. This is consistent with data retrieved using ligation-mediated PCR (Lee et ah, unpublished results) Analysis of normal liver and liver tumour tissue samples with 2E1 forward primer revealed a large smear present in tailed samples, that was absent in nontailed samples (Figure 3.10. and figure 3.11.). This also was consistent with results obtained from ligand-mediated PCR (Lee et ah, unpublisbed data). The presence of the smear indicated the presence of putative Pgp2 mRNA decay intermediate(s), and required further investigation to confirm this. In summary, although it appeared it appeared that the Yeast Poly (A) tailing RTPCR method was successful in detecting in-vitro RNA, detection of specific tailed RNA in vivo posed a problem. To recap, a smear was present in tailed samples and absent in nontailed samples. This was detected in both normal liver and liver tumours analyzed with E F l forward primer only. These results were reproducible and PCR products could be reamplified using the same primers (Figure 3.14.). It was also possible to clone amplified fragments into pGEM7Zf+ vector (Figure 3.16. and figure 3.17.). This strongly suggested that the insert contained both BamHI and Xhol restriction sites, consistent with a putative 64 Pgp2 mRNA decay product amplified with 2E1 forward primer containing a BamHI site, and an Oligo dTis-XhoI reverse primer containing an Xhol site. Surprisingly, sequence analysis revealed that inserts in all positive clones were not that of Pgp2 mRNA. To understand this phenomenon, a few points must be considered. Firstly, the specificity of the Oligo dTigXhol primer must be taken under consideration. This primer is nonspecific as it consists of a stretch of T bases. Due to its nonspecific nature, the OligodTig-XhoI primer can hybridize to any stretch of A bases. A second point that needs consideration relates to the samples used. The samples used to search for Pgp2 mRNA decay intermediates were total RNA samples isolated from liver and tumour. This means that not only did the samples contain possible degraded Pgp2 mRNA, but also various other RNAs. One would think that forward primer 2E1 would be specific for Pgp2 mRNA. However, based on the amplification and sequencing of products that are not Pgp2 mRNA, it is possible that the Pgp2El primer is not specific for only Pgp2 mRNA. A nucleotide BLAST search was performed on the 2E1 forward primer sequence, however no significant matches were made other than with Pgp2 mRNA. This result is not surprising though, as the sequences determined contained no significant match after a nucleotide BLAST search either. The sequence of 2E1 forward primer was not detected in sequence analysis, however, manual sequencing only enables reading of the sequence up to a certain point before it becomes illegible. The specificity of 2E1 forward primer is questionable, and can not definitively be ruled out as a possible explanation as to why this method failed in detecting Pgp2 mRNA decay intermediates in vivo. The success of this experiment was also concentration dependant. stated, the concentration of Pgp2 mRNA decay products is unknown. 65 As previously It is possible that putative Pgp2 mRNA decay products were successfully poly (A) tailed and amplified, however, the concentration of successfully amplified decay products had to have been lower than the fragments that were detected. It is also possible that decay product(s) were not poly (A) tailed due to low abundance, however this is unlikely as a decay intermediate was identified using ligation-mediated PCR, which is less sensitive than the Yeast Poly (A) Tailing RT-PCR method in detecting in-vitro RNA (Lee et al., unpublished results). It is likely that the nonspecific nature of the Oligo dTig-XhoI primer, coupled with probable low concentration of decay intermediates and a possible conserved Pgp2El forward primer region within another gene were the root causes as to why Pgp2 mRNA decay intermediates were not detected. As previously stated, all determined sequences did not have a significant match when a nucleotide BLAST search was performed. All one can say regarding the fragments cloned, is that they are located in similar tissue samples as Pgp2 mRNA. Another interesting feature pertaining to the sequencing results, is that one would expect to have a string of T bases at the 5’ end of the sequence. This result however, is not present. It is likely that an Xhol restriction site was present upstream from the region where the Oligo dTig-XhoI primer hybridized to. This would explain the presence of the Xhol restriction site in all sequences determined, and a lack of T bases (See Table 3.2). Detection of decay intermediates in vivo has previously been reported. Albumin mRNA degradation products have been identified in vivo using ligation-mediated PCR (Hanson and Schoenberg, 2001). This method involves the annealing of a specific primer to the hydroxyl terminus of decay products, followed by amplification using two specific primers. This method has also been used to successfully identify a Pgp2 mRNA decay 66 intermediate (Lee et al., unpublished results; See Chapter 4 and 5 for discussion). Taken together, it is concluded that primer specificity is very critical for specific amplification of degradation products. This study failed to detect Pgp2 mRNA decay products in vivo. Chapter 4 Using Primer Extension Method to determine the Nature of Nucleolytic (Exonucleolytic or Endonucleolytic) Cleavage 67 As previously mentioned (Chapter 3, Discussion 3.2) an mRNA degradation product belonging to Pgp2 was identified using ligation-mediated PCR. This method was first described in the identification of albumin mRNA decay intermediates in vivo (Hanson and Schoenberg, 2001). The ligation-mediated PCR protocol is useful in detection of mRNA decay intermediates that possess 3’ hydroxyl termini. A DNA ligation primer that has a 5’phosphate and a 3’- amino group is ligated to the decay intermediate using RNA ligase. Reverse transcription is performed with a complementary primer, and the cDNA is amplified using specific primers (Hanson and Schoenberg, 2001). A Pgp2 mRNA decay intermediate was identified using this method and amplified with Pgp2El forward primer. This product was confirmed to be a Pgp2 mRNA decay product through sequence analysis (Lee et al., unpublished results). It was then necessary to determine if the product was the result of endonucleolytic cleavage. Primer extension is an accurate assay used to map in vivo endonuclease cleavage sites. This experiment is able to identify unique cleavage sites that will appear as bands that correlate with the exact position where the polymerase runs off the template. Cleavage sites are distinct from polymerase pausing sites, which are identified in the primer extension performed on an in vitro control transcript. A DNA sequencing ladder is also included in the analysis to enable precise identification of the cleavage sites. The methodology for this experiment was derived from a detailed review of experiments related to the characterization of mRNA endoribonucleases (Schoenberg & Cunningham, 1999). This chapter describes the use of Primer Extension method to determine the nature of cleavage of putative Pgp2 mRNA degradation product(s). Results and discussion related to the nature of decay of 5 ’ end Pgp2 mRNA degradation products in normal liver and liver 68 tumour are included. A discussion regarding possible mechanisms for increased Pgp2 mRNA stability is also included. Methods 4.0 4.0.1 Primer Extension Based on results from ligation-mediated 5’CGTGAATGACGCTGGGGAGCTCAACACC3’ was PCR, designed primer 50 RP nucleotides downstream from the detected degradation product by ligation-mediated PCR (Figure 4.1). AllGntlOJ llQ \(nt3933) ■S' I I , Pri mer RP (nt 591 - nt é l 9) V2 eiem%esiie idbntified % LM fCR {see Discussion Figure 4.1 Location of Primer RP in relation to previously identified Pgp2 degradation product (Lee et al., unpublished data) for use in primer extension experiment. V2 cleavage site is located at 541 nt. RP primer was 5’ labeled with y^^P-ATP and then used for PCR reaction described below (see Methods 2.0.3). Standard ethanol precipitation (see Methods 2.0.1) was performed on 3 pL of 5’ y^^P-ATP labeled primer with 10 pg of total RNA samples extracted from liver 0.1, 0.2 and tumour 0.1, 0.2. As a control, the same amount of 5’ labeled primer with 20 ng of in-vitro transcribed Pgp2El RNA also underwent standard ethanol precipitation. The RNA pellets were resuspended in 10 pL of annealing buffer (50 mM TrisHCl; pH 8.7; 0.54 M KCl; and 1 mM EDTA), and incubated at 65°C for 10 minutes, 54°C for 90 minutes and slowly cooled to room temperature. 69 30 pL of Reverse Transcription mixture (10 mM dATP, dCTP, dGTP, dTTP; 0.25 M Tris-HCl, pH 8.7; 65 mM MgCb; 0.1 M DTT; MMLV reverse transcriptase; ddHaO) was added to each sample and the mixture was incubated for 1.5 hours at 42°C. The reaction was stopped by an addition of 260 pL of NET buffer (0.3 M NaAc; 10 mM Tris-HCl, pH 8.0; 1 mM EDTA) and 600 pL of cold 100% ethanol. The samples were precipitated at -20°C for 30 minutes and centrifuged for 10 minutes at 12,000 rpm. The pellets were washed with 200 pL of 70% cold ethanol and centrifuged for an additional 5 minutes. The supernatant was removed and the pellets were allowed to air dry for 15 minutes. The pellets were resuspended in 4 pL of formamide loading buffer, boiled for 5 minutes and placed on ice prior to loading on an 8% polyacrylamide/7 M urea sequencing gel. Dideoxy Sequencing (see Methods 3.0.4) was carried out on pGEM4Z-Pgp2El plasmid with y^^P-ATP 5’ labeled primer extension primer with the following changes: After standard ethanol precipitation the pellet was resuspended in 5 pL of ddH 20 and ^^S-dATP was not used as a labeled primer was utilized. Samples from the Primer Extension and Dideoxy Sequencing experiments were ran at 60 W constant current on a pre-ran 8% polyacrylamide/7 M urea sequencing gel for approximately 1.5 hours. The gel was dried and exposed as previously described (see Methods 3.0.4). Results 4.1 Primer extension is an accurate assay for mapping endonucleolytic cleavage sites. The presence of bands potentially indicates cleavage sites where the polymerase runs off the 70 template. Cleavage sites are distinet from polymerase pausing sites, which are identified in the primer extension performed on in vitro transcribed Pgp2El RNA transcript. The cleavage site positions were determined relative to a DNA sequencing ladder prepared from pGEM4Z-Pgp2El plasmid. This experiment was performed based on results from section 3.1.4, and due to the successful detection and sequencing of a Pgp2 mRNA degradation product present in both liver and tumour tissue samples using ligation-mediated PCR with Pgp2El forward primer (Lee et al., unpublished data). The primer extension experiment revealed 5 cleavage sites, one of them consistent with the ligation-mediated PCR detected degradation product (See Figure 4.2. and 4.3. lanes 5, 6, 7, and 8). For simplicity, the cleavage site related to the detected decay intermediate identified by ligand-mediated PCR, is termed V2. All cleavage sites detected were present in both liver and tumour tissue samples (see Figure 4.2., lanes 5, 6, 7, and 8). TGCA I Hf Ji lJ lft, H 71 o T G C A S ites Site V2 Site 4 Site 3 Site 2 S ite l 12 12 34 5 6 7 8 3 4 5 6 7 8 9 9 Figure 4.2. Primer extension analysis of liver and tumour tissue samples. Left picture details entire length of experiment. Right picture details important information pertaining to endonucleolytic cleavage sites present in hoth liver and tumour. 72 Site 1 'i ' Site 2 Site3 '1 ' '1 ' 5’ TGGAAAAAC TTGCCTAA CTGTGTATT7TTGTGTTCCAG Site 4 V2 Site CCAGCTGCCAGGCACCA AA GTGAAACCTGG ATGTGGC Site 5 AA CAATAGCCCGCACCAATCCGGTT 3’ Figure 4.3. Locations of identified cleavage sites in Pgp2 mRNA sequence from primer extension analysis (see figure 4.2.). Discussion 4.2 Based on findings from primer extension analysis it can be concluded that the Pgp2 mRNA degradation product previously identified by ligation-mediated PCR is the result of endonucleolytic cleavage. Aside from the V2 cleavage site, five major cleavage sites were also identified and were present in both liver and tumour. These sites appear very intense in comparison to the V2 cleavage site, indicating that the V2 cleavage site is a minor site. Why then was it not possible to detect degradation products that parallel these major sites? A possible explanation as to why this was not possible requires investigation into the general process of mRNA decay and into the process of how ligation-mediated PCR detects decay products. mRNA degradation products can be of two natures in regards to their 3’ end; the 3’ terminus can either have a phosphate or hydroxyl group. As previously described, ligation-mediated PCR employs the use of a specific primer, which is ligated to hydroxyl 73 termini of decay intermediates (Hanson and Schoenberg, 2001). Therefore, this method, as well as the Yeast Poly(A) tailing RT-PCR method can only detect decay intermediates with hydroxyl termini. It is possible that the major cleavage sites demonstrated in primer extension analysis represent products with phosphate termini, which could not be detected using ligation-mediated PCR or Poly (A) tailing method. Presently, there is no method available to detect mRNA decay products with 3’ phosphate termini in vivo. However, such possibility would require further investigation. Identification of the decay intermediate by ligation-mediated PCR in both liver and tumour, and the identification of the exact same cleavage sites by primer extension analysis indicate that the decay pathway of Pgp2 mRNA is the same in hoth liver and tumour. This result is significant as it demonstrates that increased mRNA stability exhibited in tumours (Lee et ah, 1998) is not likely related to endonucleolytic decay of Pgp2 mRNA between normal liver and liver tumour. This suggests that one should examine possible differences in deadenylation and 3 ’-5’ decay. In conclusion, this experiment identified that endonucleolytic cleavage plays a role in the decay process of Pgp2 mRNA in vivo. The mode whereby Pgp2 mRNA is degraded has not been previously described. Identification of endonucleolytic cleavage in Pgp2 mRNA adds to the list of mammalian mRNAs that are degraded by endonucleolytic pathway. Based on the current findings, it is not possible to determine if the cleavage sites are the result of one or more enzymes. Future research related to this finding can pertain to purification and characterization of the endoribonuclease(s) responsible for cleavage of Pgp2 mRNA. 74 Chapter 5 General Discussion This study aimed at comparing the in vivo degradation pathway of Pgp2 mRNA, through identification of decay intermediates in liver tumour and normal liver. The rational behind the study also relates to the lack of knowledge surrounding decay mechanisms associated with Pgp mRNA. On a global scale, it is also related to the lack of understanding of mRNA decay processes within mammalian systems, especially in an animal model (Ross, 1995). This study attempted to detect Pgp2 mRNA degradation products using the Yeast Poly (A) Tailing RT-PCR method. At first this method was determined to be suitable due to its apparent sensitivity and specificity demonstrated in in-vitro assays (see Chapter 2, Discussion 2.2). It became apparent however, that this method was not suitable for use in detection of mRNA decay intermediates in vivo because it lacked specificity (See Chapter 3, Discussion 3.2). A Pgp2 mRNA degradation product was identified through an alternative method (ligation-mediated PCR; Lee, et al. unpublished results) in both normal liver and liver tumours. Primer extension analysis provided insight into the decay pathway by confirming that the decay product identified was the result of endonucleolytic cleavage, and by revealing four major cleavage sites within the 2E1 portion of Pgp2 mRNA. These cleavage sites were identical in both normal liver and liver tumour (See Chapter 4). Therefore, even though this study failed to detect Pgp2 mRNA decay products using the Yeast Poly (A) Tailing RT-PCR method, the main objective has been accomplished. Based on the results of the primer extension analysis, there is no indication of differential endonucleolytic decay pathways present between normal liver and liver tumour. The increased Pgp2 mRNA stability demonstrated in liver tumours in comparison to normal liver 75 (Lee et al., 1998) could be related to some other decay mechanism. Observations made here suggest that future studies should be directed towards examining possible differences in deadenylation and 3’-5’ decay. As discussed previously (see Chapter 1), there are few explanations and mechanisms related to mRNA stabiliy in P-glycoprotein. It is the objective of this chapter to identify possible areas of research that could potentially shed more light on the issue. Many recent observations suggest that Pgp2 mRNA is post-transcriptionally regulated. It was recently demonstrated that the application of cyclohexamide (a protein synthesis inhibitor) significantly increased Pgp2 mRNA in all normal rat tissues. The increase in mRNA transcripts was mainly through post-transcriptional control as there was no indication of a parallel increase in transcriptional activity (Lee, 2001). It has been suggested that this increase in Pgp2 mRNA transcript levels is due to mRNA stability (Lee, 2001). It has also been suggested that trans-acting factors are most likely involved in Pgp2 mRNA stability (Lee, 2001). There are a few explanations for this suggestion. First, even though a change in mRNA stability has been noticed between normal liver and liver tumour, there has been no obvious change in Pgp2 mRNA structure (Kren et al., 1996; Lee et al., 1998). Also, an AU-rich element at the 3’UTR of Pgp mRNA failed to exhibit destabilizing capability demonstrated by other AU-rich elements (Prokipcak et al., 1999). Therefore, investigation into the presence or absence of RNA binding proteins in normal liver and liver tumour would be appropriate. The involvement of mRNA decay during aging and development has not been widely explored. It has been recognized that specific changes in gene expression can occur during aging and development (Brewer, 2002). It is widely accepted that mRNA decay processes 76 contribute to the control of gene expression. In some cases, mRNA stability has been linked to development. For instance, it has been demonstrated in mouse albumin mRNA, that stability is partially dependant on development and aging. This conclusion was based on the identification of significant levels of albumin mRNA in fetal mouse liver and undetectable levels in adult liver. This suggests that mRNA turnover rate is higher in fetal liver and decreases during development (Tharun and Sirdeshmukh, 1995). Opposite to the results determined for albumin mRNA, granulocyte/macrophage colony-stimulating factor (GMCSF) mRNA levels are four fold lower in activated neonatal mononuclear cells from umbilical cord blood, as compared to adult peripheral blood (Brewer, 2002). It has also been proposed that in young cells there is an equal balance of mRNA degradation and stabilization factors present, where as in older cells degradation factors are dominant (Brewer, 2002). While many details related to age-dependant changes in mRNA are currently unknown, it would be interesting to determine if endonucleolytic decay of Pgp2 mRNA in normal liver changed during development. 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