warm. -- TH N EIN- mun: P AND RNA ATIQ INT! EFERE? T . NG CY I D..- of‘Ph’. ‘ egree The WE {for the “D sis UN N STATE {CH 10 HUM MN 1 T. . BY . win->53“ n... u 'II.¢I.’I This is to certify that the thesis entitled RNA AND PROTEIN SYNTHESIS IN FETAL RAT PANCREAS DURING CYTOD IFFERENT IATION presented by JOHN W. BYNUM has been accepted towards fulfillment ' 1 of the requirements for fi‘i/é/Z‘KZv‘n /J Major pro Date December 11, 1972 0-7639 .2 «Mt e-o- _ ' " ".13 CV g swims av ? HMS & SUNS’ 300K BINDERY INC. LIBPARY BINDERS ,mmsvow moment N": 7’: 7" L—J—Z’J I \ RNA AND PROTEIN SYNTHESIS DURING CYTODIFFERENTIATION IN FETAL RAT PANCREAS BY ,3; John W; Bynum AN ABSTRACT OF A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1972 ABSTRACT RNA AND PROTEIN SYNTHESIS DURING CYTODIFFERENTIATION IN FETAL RAT PANCREAS BY John W. Bynum During cytodifferentiation in fetal rat pancreas epithelial cells with very little rough endoplasmic reticulum, a small Golgi apparatus and no zymogen granules differentiate into secretory cells with an extensive endo- plasmic reticulum, an enlarged Grolgi apparatus and numerous zymogen granules. The accumulation of rough endoplasmic reticulum suggested that at some point during this period the fetal pancreas altered the rate at which it metabo- lized RNA, and this alteration resulted in the accumula- tion of ribosomes for the rough endoplasmic reticulum. This cellular transformation occurs between the 14th and 20th day of gestation. An increase rate of rRNA synthesis of a decreased rate of turnover could have caused such an accumulation. This study focuses on changes in the rate of RNA synthesis during cytodifferentiation. Agarose-acrylamide slab gel electrophoresis was used to determine the amount of 288, 188, SS and 4S RNA John W. Bynum at each embryonic age. The method involved homogenization of 100 to 400 ug of tissue in 100 pl of standard RNA extrac- tion buffer (acetate, pH 5.1), 88% phenol and 0.5% SDS, extracting twice with phenol and final aqueous layer, directly on the gel. With this procedure, RNA degradation due to the high ribonuclease content of the pancreas was minimized and a quantity of RNA sufficient to give stained bands on gel was extracted from 19 pg of 15-day fetal pan- creas. Electrophoretic profiles of RNA extracted from pancreatic rudiments of different age, showed no substantial difference in the amount or relative mobility of major RNA species throughout cytodifferentiation. RNA from older, more differentiated rudiments, with a higher content of ribonuclease was generally degraded more during extraction than RNA from younger rudiments. Four modifications were made in developing a satis- factory organ culture system for fetal pancreases; the rudiments were cut into pieces of approximately 5-15 pg of protein to allow labelled precursors and nutrients to penetrate the interior of the tissue; the tissue pieces were attached to a Millipore filter as a substrate and flooded at the liquid gas interphase to facilitate oxygen- carbon dioxide exchange; fetal calf serum and supplemental amino acids were added to Eagle's MEM to provide additional precursors and growth factors; and antibiotics were added to inhibit bacterial contamination. RNA degradation was John W. Bynum used to evaluate tissue necrosis. Using the above modifi- cations distorted RNA bands were resolved from RNA extraced from 17-day pancreas cultured for 24 hours. To provide a control for assessing differentiation and tissue survival, adult pancreas was cultured for 24 hours under the same conditions. RNA from adult pancreas indicated no tissue necrosis and the migration patterns were comparable to 17-day pancreatic RNA. The rates of [3H] leucine incorporation by pancrea- tic rudiments of different ages were determined at pH 6.8 and 7.1. At pH 6.8, the apparent rate of incorporation into TCA precipitable material (dpm/ug protein/hr) de- creased between 14 and 15 days of gestation. The rate increased several fold to a maximum of 200 dpm/ug protein/ hr at 17 days of gestation, then declined to the 15-day level after 20 days of gestation. The rate of protein synthesis in the adult pancreas was comparable to 18 and 19-day rates. At pH 7.1, the apparent rate increased from approxi- mately 150 dpm/ug protein hr at 15 and 16 days of gestation to a maximum of 450 dpm/Lg protein hr, then declined to the 15-day level. The rates were approximately 3-fold greater than the pH 6.8 values at each age. Seventeen—day rudi- ments and adult pancreatic tissue were least affected by the change in pH. John W. Bynum When the apparent rates of protein synthesis were calculated per cell and plotted as a function of embryonic age, there was an apparent transition between 16 and 19 days of gestation at both pH 7.1 and 6.8. At pH 7.1 as well as 6.8, 19-day old tissue incorporated 8-fold more [3H] leucine into protein per cell than did 15 or 16-day tissue. The greatest rate of protein synthesis per cell occurred in adult pancreases which were 2-fold greater than the 19-day rate at pH 7.1. Similar incorporation studies were conducted with [3H] uridine. Changing the pH from 6.8 to 7.1 caused only a slight increase in the apparent rates of RNA synthe- sis, although the apparent rates of protein synthesis were extensively affected. Again, in contrast to protein syn— thesis, the maximal rate in RNA synthesis occurred on day 18, one day before the transition in protein synthesis. From day 15 to day 18, the rate of RNA synthesis increased S—fold at pH 6.8 and 2.5-fold at pH 7.1. Calculating the rates of [3H] uridine incorpora- tion per cell instead of per microgram of protein did not alter the rate curve; maximal rates were attained after day 18 of gestation. Relative to DNA content, the apparent rate of RNA synthesis occurred a day before the maximal rate in protein synthesis; relative to protein content, the apparent maximal rate of synthesis occurred on the same day. These data, therefore, suggest that a transition John W. Bynum in both the apparent rate of RNA synthesis and the apparent rate of protein synthesis occurs in the embryonic rat pan- creas midway through cytodifferentiation. Electrophoredic analysis of [3H] RNA from 14-day pancreatic rudiments indicated that within 4 hours of culture in labelled, peak corresponding to 28S and 188 RNA could be readily distinguished. After 8 hours of con- tinuous labelling there was little variation in the dis- tribution of radioactivity. After 24 hours the percentage of counts in 288 and 18S RNA increased Z-fold. The labell- ing ratio resembled the absorbance profile of total mammalian RNA. [3H] RNA from 17-day pancreas gave radio- active profiles comparable to absorbance scans after 8 hours of culture. Electrophoresis of labelled 19-day pan- creatic RNA gave results similar to 14—day pancreas. For all ages, the percentage of counts in 28S and 18S increased from 4 to 12 hours, while the percentage in SS and 48 decreased from 4 to 24 hours. The 19-day pancreas differed from other ages in that at 4 hours 41% of the label was in low molecular weight RNA. As the labelling time continued, the percentage dropped to 21%. Seventeen-day pancreas also synthesized a high percentage of low molecular weight RNA early and then the percentage of counts in that region gradually declined. RNA AND PROTEIN SYNTHESIS DURING CYTODIFFERENTIATION IN FETAL RAT PANCREAS BY John W. Bynum A THESIS Submitted to Michigan State Univerisity in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1972 To my wife, Brenda, and daughter, Sherrie, whose patience, sacrifice and love made all this possible. ii INVICTUS Out of the night that covers me, Black as the Pit from pole to pole I thank whatever gods may be For my unconquerable soul. In the fell clutch of circumstance I have not winced nor cried aloud. Under the bludgeonings of chance My head is bloody, but unbowed. Beyond this place of wrath and tears Looms but the Horror of the shade, And yet the menace Finds, and shall It matters not how How charged with I am the master of I am the captain of the years find, me unafraid. strait the gate, punishments the scroll my fate; of my soul. --William Ernest Henley iii ACKNOWLEDGMENTS I am grateful to Dr. R. A. Ronzio for providing the materials and guidance for this project. I would like to thank the members of my guidance committee for their helpful evaluation of my educational and professional progress toward the degree--Dr. J. Speck, Dr. D. Bing, Dr. F. Rottman and Dr. J. Boez. Special gratitude is extended to Dr. F. Rottman for providing the RNA standards used in this study. I am deeply indebted to Dr. J. Fairley for being a concerned friend. iv TABLE OF CONTENTS Page PART I INTRODUCTION . . . . . . . . . . . . . . . . . . . l Lymphocytic Series . . . . . . . . . . . . . . l Macrophage . . . . . . . . . . . . . . . . . . 4 Immunogenic RNA . . . . . . . . . . . . . . . . 6 Research Approach . . . . . . . . . . . . . . . 8 METHODS . . . . . . . . . . . . . . . . . . . . . 10 Isolation of Splenocytes . . . . . . . . . . . 10 Isolation of Glass Adhering Cells . . . . . . . 12 Density Separation of Splenocytes . . . . l4 Macrophage Induction in the Peritoneal Cavity . 18 Iodination of GAT . . . . . . . . . . . . 21 [125I] GAT Uptake by Cells from the Peritoneal Cavity . . . . . . . . 24 [1251] GAT Retention by Peritoneal Cells . . . 28 [1251] GAT Stability . . . . . . . . . . . . . 29 RNA Extraction . . . . . . . . . . . . . . . . 29 TEAE Cellulose Chromatography of Low Molecular Weight RNA . . . . . . . . . . 30 Gel Electrophoresis . . . . . . . . . . . . . . 30 Scintillation Counting . . . . . . . . . . . . 32 Photomicroscopy . . . . . . . . . . . . . . . . 33 RESULTS . . . . . . . . . . . . . . . . . . . . . 34 Uptake of [1251] GAT into Peritoneal Cells . . 34 Polymer Stabilit in Cell Culture . . . . . 35 Retention of [112 I] GAT . . . . . . . . 41 Size Distribution of [125I] GAT Released from Prelabelled Cells . . . . . 44 RNA Extraction . . . . . . . . . . . . . 44 TEAE Cellulose Chromatography of Low Molecular Weight Nucleic Acids . . . . . 48 Gel Electrophoresis of Chromatography Fractions . . . . . . . . . . . . . . . . 49 Partial Characterization of Band I . DISCUSSION 0 O O O O O C O O O O O O O O 0 LIST OF REFERENCES 0 O O O O O O O O O C 0 PART II INTRODUCTION 0 O O I O O O C O O O O O C 0 RNA Synthesis in Mammalian Cells . . . Ribosomal RNA . . . . . . . . . . . Transfer RNA . . . . . . . . . . . . Nuclear RNA . . . . . . . . . . . . Control of the Formation of RN Precu RNA Synthesis During Development . . . Differentiation of the Rat Pancreas . . Research Approach . . . . . . . . . . . METHODS O O O I I O O O O O O O O O O O O Rat Breeding . . . . . . . . . . . . . Incubation of Pancreatic Rudiments Uptake Analysis of [3H] Uridine and [3H] Lenoine O O O O O O O O O O O O O C Total Uptake of [3H] Uridine and [3H] Leucine Precursor into Homogenate Incorporation of Labelled Precursors TCA Precipitable Material . . . . Protein Determination . . . . . . . Gel Electrophoresis . . . . . . . . . . Gel Formation . . . . . . . . . . . RNA Extraction . . . . . . . . . . . Electrophoresis Conditions . . . . Determination of Radioactivity in Gel Scintillation Counting . . . . . . . . RESULTS 0 Q I O O O O O I I I I O O O O C Development of RNA Extraction Methods . vi ISOI'S into Page 56 62 66 70 7O 70 71 72 73 74 77 78 80 80 80 81 82 82 83 83 84 84 85 88 88 89 89 Page RNA Extraction from Embryonic and Adult Pancreas . . . . . . . . . . . . 89 Electrophoretic Analysis of RNA from Pancreatic Rudiments During Cytodifferentiation . . . . . . . . 94 Organ Culture of Pancreatic Rudiments . . . . . 101 Short Term Organ Culture . . . . . . . . . . 101 Long Term Organ Culture . . . . . . . . . . . 104 RNA and Protein Synthesis in Fetal Rat Pancreas Cultured at pH 6.8 . . . . . . . . . 104 [ 3H] Leucine Incorporation . . . . . . . . . 104 [3H] Uridine Incorporation . . . . . . . . 107 Electrophoretic Separation of Labelled RNA from Fetal Pancreas . . . . . . . . . 110 Protein and RNA Synthesis by Fetal Rat Pancreas Cultured at pH 7.1 . . . . . . . . . 118 Rates of Protein and RNA Synthesis in 19-Day Pancreas Cultured Under Different 3 Culture Conditions . . . . . . . . . . . . 118 [ H] Leucine Incorporation . . . . . . . . . 125 [3H] Uridine Incorporation . . . . . . . . . 131 Electrophoretic Analysis of Pancreatic RNA from Fetal Rat Pancreas Labelled In vitro at pH 7.1 . . . . . . . . . . . . . . . 141 Analysis of RNA from 14-Day Old Pancreatic Rudiments . . . . . . . . . . . 141 Analysis of RNA from 17-Day Pancreatic Rudiments . . . . . . . . . . . 149 Analysis of RNA from l9-Day Old Pancreatic Rudiments . . . . . . . . . . . 153 Comparison of Distribution of Labelled 288, 188, and SS and 4S RNA I I I I I I I I I I I I I I I I 160 DISCUSSION I I I I I I I I I I I I I I I I I I I I 162 Extraction of RNA from Pancreatic Tissue . . . . 162 Methods of Culturing Pancreatic Rudiment . . . . 164 Rates of Incorporation of [3 H] Leucine into Protein and [3H] Uridine into RNA During Pancreatic Development . . . . . . . . . . . 166 vii Page Electrophoretic Analysis of [3H] RNA . . . . . . 171 Significance I I I I I I I I I I I I I I I I I I 173 LIST OF REFERENCES I I I I I I I I I I I I I I I I 175 viii LIST OF TABLES Table Page PART I 1. Summary of methods of preparing splenocytes . . . 13 2. Summary Of cell induction by pyrogenic SOlutiOn I I I I I I I I I I I I I I I I I I I I 22 3. Summary of [1251] GAT recovery from Bio-Gel P-30 columns . . . . . . . . . . . . . . 27 4. Summary of RNA extracted from different tissues I I I I I I I I I I I I I I I I I I I I I 48 PART II 1. Relative amounts of major RNA species in RNA extracted from pancreatic and liver rUdimentS I I I I I I I I I I I I I I I I I I I I 100 '2. Rates of [3H] uridine and [3H] leucine incroporation into TCA precipitable material . . 124 3. Percent of radioactivity in major RNA species in RNA from pancreatic rudiments labelled EXEC—at pH 7.1 ' 9 ' 9 0 0 O 0 o o o o o o o 152 ix LIST OF FIGURES Figure PART I 1. Density separation of cell populations by sedimentation . . . . . . . . . . . . . . . . 2. Induction of peritoneal cells in rats . . . . 3. Separation of radioiodinated polyGAT from Na 1251 by gel filtration chromatography . . 4. Uptake of [1251] GAT in red blood cells and White cells I I I I I I I I I I I I I I I I I 5. [125I] GAT stability in cell culture medium containing different concentrations of serum 6. Retention of [1251] in cells prelabelled with [1251] GAT I I I I I I I I I I I I I I I I I 7. Molecular weight distribution of [1251] released into the culture medium by pre- labelled cells I I I I I I I I I I I I I I I 8. Separation of lung low molecular weight RNA by TEAE cellulose chromatography . . . . 9. Separation of spleen low molecular weight RNA by TEAE cellulose chromatography . . . . 10. Electrophoretic analysis of lung low molecular weight RNA . . . . . . . . . . . . ll. Electrophoretic analysis of macrophage low molecular weight RNA . . . . . . . . . . . . 12. Partial characterization of band I . . . . . Page 15 19 25 36 38 42 45 50 52 54 57 59 Figure Page 10. ll. 12. 13. PART II Photograph of RNA from varying amounts of lS-day pancreas o o o o o o o o c o o o o o o o 86 Electrophoretic analysis of RNA extracted at different pH's, temperatures and SDS concentrations . . . . . . . . . . . . . . . . 91 A composite photograph of stained gels showing RNA extracted from rat pancreases of different embryonic ages . . . . . . . . . . . . . . . . 95 Representative scans of RNA extracted from rat pancreases at different embryonic ages . . 98 Photograph of RNA from l7uday pancreases ‘cultured for various intervals . . . . . . . . 102 RNA from adult pancreas cultured for various periods at pH 6.8 . . . . . . . . . . . 105 Uptake of [3H] leucine by fetal rat pancreas cultured at pH 6.8 . . . . . . . . . . 108 Uptake of [3H] uridine by fetal rat pancreas cultures at pH 6.8 . . . . . . . . . . 111 Profiles of labelled RNA from 18-day pancreatic rudiments maintained in culture at pH 6.8 for 4 and 24 hours . . . . . . . . . 114 Profiles of labelled RNA from adult pancreas maintained at pH 6.8 for 8 and 12 hours I I I I I I I I I I I I I I I I I 116 Profiles of labelled RNA from 19-day fetal pancreas maintained in culture at pH 7.1 . . . 119 Photograph of RNA from l9-day pancreases cultured at pH 7.1 with and without supplemental amino acids . . . . . . . . . . . 121 Uptake of [3H] leucine by fetal rat pancreases cultured at pH 7.1 . . . . . . . . . . . . . . 12? xi Figure 14I 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. Apparent rates of [3H] leucine incorporation per microgram of protein . . . . . . . . Apparent rates of [3H] leucine incorporation per Cell I I I I I I I I I I I I I I I I Uptake of [3H] uridine by fetal rat pancreases cultured at pH 7.1 . . . . . . . . . . . Apparent rates of [3H] uridine incorporation per microgram of protein . . . . . . . . Apparent rates of [3H] uridine incorporation per Cell I I I I I I I I I I I I I I I I Photograph of RNA from l4-day pancreases at pH 7I1 I I I I I I I I I I I I I I I I Distribution of radioactivity in RNA from l4-day fetal pancreas maintained at pH 7Il I I I I I I I I I I I I I I I I I Percent distribution of radioactivity in 14-day pancreases maintained at pH 7.1 . Percent distribution of radioactivity in 14-day pancreases cultured for 24 hours at pH 7.1 o g o o o o o o o o o o o o o 0 Photograph of RNA extracted from l7-day pancreas cultured at pH 7.1 . . . . . . . Distribution of radioactivity in RNA from 17-day fetal rat pancreas cultured with [3H] uridine at pH 7.1 . . . . . . . . . Distribution of radioactivity in RNA from 19—day fetal rat pancreas cultured with [3H] at pH 701 o o o o a o o o o o o o o xii Page 129 132 134 137 139 142 144 147 150 154 156 158 LIST OF ABBREVIATIONS cpm: counts per minute DNA: deoxyribonucleic acid DPM: disintergrations per minute EBSS: Earle's balanced salt solution EDTA: ethylene diamine tetraacetate GAT: poly-L-glutamine, L-alanine, L-tyrosine (60:30:10) HnRNA: heterogenous nuclear ribonucleic acid MEM: minimum essential medium mRNA: messenger ribonucleic acid PVS: polyvinyl sulfate RNA: ribonucleic acid rRNA: ribosomal ribonucleic acid SAM: S—adenasyl methionine SDS: sodium doderylsulfate Stains-All: l-ethyl-Z-[3-(l—ethylnaptho—[l,2d] thiazolin- 2-y1idene)-2-methylpropenyl]-naptho [1,2d] thiazolium bromide TCA: tricholoracetic acid TEAE cellulose: triethyamino ethyl cellulose tRNA: transfer ribonucleic acid xiii MATERIALS Materials were purchased from the following sources: Agarose - Kinsman Optical Company Ammonium persulfate - Fischer Scientific Company Antibiotics - 10,000 units/ml of penicillin and 10,000 pg/ml of streptomycin - Grand Island Biological Company L-arginine - Sigma Chemical Company Bio-Gel P-30 - Bio Rad Laboratories Bovine serum albumin (35% sterile) - Sigma Chemical Company Bovine serum albumin — Sigma Chemical Company Chick Embryo extract — Grand Island Biological Company Chloramine T — Eastman Organic Chemicals Cyanogum 41 - Fischer Scientific Company 3-dimethylaminopropronitri1e - Eastman Ogranic Chemicals Deoxyribonuclease — Worthington Eagle's minimum essential medium - Grand Island Biological Company Fetal calf serum - Grand Island Biological Company Formamide - Aldrich Chemical Company L-glutamic acid, L-alanine and L-tyrosine polymer (60:30:10) - Pilot Chemical Division of New England Nuclear L-glutamine (200 mM) — Grand Island Biological Company xiv Heparin - Sigma Chemical Company Iodine (1251 ) - New England Nuclear [3H] leucine (25 Ci/mM) - Amersham/Searle Corporation L-methionine — Nutrition Biochemicals Corporation Pancreas ribonuclease (bovine) - Sigma Chemical Company Peptone - Sigma Chemical Company Platicware (tissue culture) - Falcon Plastic Company Polyvinyl-sulfate - General Biochemicals Pronase - Calbiochem Proteose peptone - Difco Laboratories Sprague Dawley rats — Spartan Research Animals Stains-A11 - Eastman Organic Chemicals TEAE cellulose - Sigma Chemical Company [3H] uridine (25 Ci/mM) - New England Nuclear Yeast RNA - Sigma Chemical Company XV PART I INTRODUCTION Current trends in biological research have turned increasingly toward the regulation of cellular differentia— tion in higher organisms. A portion of this interest is due to growing concern over the molecular basis of such" publicized disorders as cancer and organ transplant rejec- tions, and the need to confirm the existence of similar bacterial metabolic phenomena in mammalian systems. How- ever, a substantial number of the myriad laboratories pursuing differentiation are involved in the delineation of the controls and functions of the antibody producing System consisting of the lymphocytic and monocytic series (macrophages). Qymphocytic Series The Lymphocytic series consists of a family of cells having common properties and sharing the function of defending the body against invasion of bacteria and other noxious agents. The induction of this system is initiated by a series of events beginning with the activation of precursor stem cells to differentiate; and terminates with the production of cell capable of removing the extraneous factor by direct cellular contact or the assembly and secretion of antibody. As later references to current publications will show, investigation of this process has been prolific on both the cellular and molecular levels. A large amount of evidence has accumulated suggest- ing that small lymphocytes are the precursors of antibody producing cells (1, 2, 3). Papermaster postulates that small lymphocytes originate in bone marrow from stem cells which are equally capable of becoming erythrocytes, granu- locytes, or lymphocytes. The key factors in determining the developmental path are the cellular environment and the availability of certain hormones. Erythropoietin is indicated as the hormone responsible for committing the stem cell to a pathway of erythropoiesis (4). Failure of thymectomized mice to produce antibody after injections of bone marrow cells denotes the importance of the thymus in developmental lymphopoiesis (3). Further evidence for the function of the thymus has developed around two physiological interactions; one is hormone production and the other is lymphocyte distri- bution. Repeated injections of thymus extract restored the immunological reactivity of thymectomized new-born mice (49.50), but animals so treated remained lymphopenic in certain regions of the lymph nodes and Spleen (51). Consequently, the thymus may have a dual function in that it also seeds lymphocytes to the spleen and lymph nodes. In mice progeny of thymus lymphocytes can be identified by the presence of a specific antigenic site called the "theta" antigen (53). These are referred to as "T" lym- phocytes (54). Studies of lymphocyte function in birds revealed another organ which could compliment or possibly duplicate the activities of the thymus. This organ is the bursa of Fabricus, which lies adjacent to the cloaca (55). Inves- tigations for a site comparable to the bursa in mammals indicates the existence of such tissue, but as yet, its exact location is unknown. Possible sites are the Peyer's patches, bone marrow and the appendix. In rabbits removal of the gut-associated lymphoid organs followed by a lethal dose of radiation resulted in immunological characteristic similar to a bursectomized bird (56). Lymphocytes which do not seem to be thymus dependent but which may be under the influence of a "bursa" are currently called "B" lympho- cytes (66). After the lymphocytes are distributed to the peri- pheral lymphoid organs, further specialization appears to be independent of hormonal control. At this stage the lymphocyte has become sensitive to the physiological sur- roundings, and responds only to certain factors. These include mitogenic agents (phytohemagglutin (5, 6), anti- gens (3, 7, 8), antigen-antibody complexes (9), and macro- phages (10, ll, 12, 13). Within minutes after stimulation with these agents, heterogenous and soluble RNA is synthesized and ribosomal precursor RNA and ribosomal RNA gradually increase (14). The lymphocyte acquires a blast cell morphology resembling the precursor stem cell (15, 16). Through a mitotic transformation, the blast cell divides to form more small lymphocytes capable of further differentiation (17). The labelling patterns shown by small lymphocytes have recently indicated that two classes can result from the transformation of the blast cell. One class of lymphocytes is long-lived; the other is short- lived and proportionately more responsive to induction (18). During subsequent development, the cell is again confronted with what appears to be alternative division paths. One path involves a transformation into plasma cells consisting of enlargement and development of the structures necessary for rapid protein synthesis and secretion, rough endoplasmic reticulum and Golgi apparatus (19). After several divisions and the achievement of maturity, the plasma cell is phagocytized (20). An alter— nate but seemingly unlikely pathway (21) consists of a transformation into macrophages (22). Macrophages The term macrophage is used to define a family of cells which is capable of phagocytosis or engulfment of foreign particulate material. These phagocytes are the reticuloendothelial system. Macrophages have been found in a variety of tissues in a wide physiological distribu- tion: for example, the peritoneal macrophage of the peritoneal cavity (23), the aveolar macrophage of the lung (24), the splenic macrophage of the spleen (25), the Kupffer cell of the liver (31), the microglia cell of the central nervous system (32), the lymphoid macrophage of the thymus and lymph nodes (33), the monocyte of the blood (34). Due to the chemotaxis, macrophages are found in a number of other tissues. However, those listed above appear to be the major foci of macrophage populations. Macrophages differentiate through a series of transitions from precursor cells found in the bone marrow (35, 36, 37). The first differentiated stage of exudate macrophages, which are found in lesions, involves the monocyte which is present in the circulating blood (38). During the course of inflammation the monocyte first leaves the vascular system and migrates into the extra- vascular connective tissue to the site of inflammation. Under normal conditions, this migration is random and usually terminates in the connective tissue. The factors regulating precursor differentiation and monocyte emigra- tion are currently unknown. In the second stage of dif- ferentiation the monocyte in the connective tissue of the inflammatory site matures into a macrophage; the mature macrophages then remain within local sites and engage in phagocytic functions (39). Although the intermediate cell types have not been delineated, macrophages in other loca- tions also have been shown to be derived from cells in the bone marrow (40). "Immunogenic" RNA Macrophages aid significantly in immunity because they phagocytize and process antigen. This processed antigen is apparently coupled to ribonucleic acid (immuno- genic RNA) and transferred to the precursors of antibody forming cells (41, 42, 43, 44, 45). A class of "immuno- genic" RNA composed of the synthetic antigen poly L-gluta- mic acid, L-tyrosine and L—alanine (60:30:10) has been isolated and partially characterized (46). The ribo- nucleoprotein complex was found to band in cesium sulfate at a density of 1.588 grams per cc, to have an 520,w of 1.8 and an approximate molecular weight of 12,000. After removal of the polypeptide, the S was 1.3 and the 20,w guanine cytosine content was 58% (47). After fractiona- tion of the macrophages, the antigen RNA complex was distributed between the supernatant fraction and the ribosomal fraction in a ratio of 2:1. An antigen RNA complex from poly-y-D glutamic acid was stable to sucrose gradient centrifugation, adsorption chromatography, gel electrophoresis and equilibrium sedimentation in cesium sulfate. Furthermore, the labelled polypeptide was not 5 dissociated by 10 fold excesses of unlabelled polypeptide. There was no association between the RNA and polypeptide mixed in_yi£52, but association occurred with a cell-free homogenate (57). A similar complex was obtained with polypeptide mixed with homogenate from HeLa and E. 921i cells. It may be that RNA antigen binding is a nonspecific ionic interaction in that the complex was not found in RNA extracted by the hot phenol method (58). In competition studies, it was found that synthetic polypeptides made from L-amino acids were bound to RNA eight to ten times as well as D-amino acid polymers in yitgg. This effect was attributed to the resistance of D-polypeptides to catabolism (68). Similar studies conducted with synthetic polypeptides of different electrical charges revealed that positively charged L—amino acid polymers were in higher concentration in the RNA fraction than were negatively charged polymers. Neutral polymer were associated to the least extent (69). Immunogenic RNA has also been isolated from liver, spleen and lymph nodes. By assessing the distribution of labelled BSA injected into rabbits, it was found that the antigen BSA was mainly localized in the spleen, lymph nodes and the liver. Of the three tissues, liver retained the highest percentage of antigen for the longest period (59). Cellular studies showed that an antigen RNA complex in liver RNA could induce rapid antibody synthesis in normal spleen cells in vitro. Treatment of the complex with ribonuclease abolished its immunogenicity, while degradation of the protein portion with pronase did not affect the immunogenicity (60). This suggested that immu- nogenicity resides in the RNA and not the protein and that the protein is possibly functioning as a carrier or nuclease inhibitor. In the work of Dray, nonimmune rabbit spleen cells were converted by RNA of lymphoid cells from an immune rabbit to produce antibody of foreign heavy and light chain allotype (61, 62). The results indicated that a component of the RNA extract provided information for synthesis of at least part of the heavy chain as well as the light chain of the IgM and IgG antibody molecule. Johnson demonstrated a regulatory action of poly A:C on the immune response (63, 64). Polynucleotide binding stimulated the production of membrane bound IgG molecules in an anamnestic response (65). Research Approach Increasing evidence points to the existence of a unique species of RNA in macrophages and macrophage con- taining tissues which regulates cellular and humoral immunity. Gottlieb isolated such a component from peri- toneal macrophages using TEAE cellulose chromatography and the synthetic antigen [1251] GAT as a marker (46). I have attempted to utilize his procedures in isolating an antigen-RNA complex from peritoneal exudate cells and other tissues of the reticuloendothelial system. In pur- suit of this problem, I have investigated cell induction in the peritoneal cavity of rats and evaluated antigen uptake and retention by these cells. Using chromatography and polyacrylamide gel electophoresis, I have found no evidence to support the existence of a unique low molecu- lar weight RNA species in these tissues. METHODS Isolation of Splenocytes Dissociation of rat spleen gave a suspension rich in cells from the lymphocytic and monocytic series with minor contamination by the other component of peripheral blood. However, in the isolation of spleen cells, the nature of the disruption produced contaminants such as connective tissue and cell aggregates which prevented pre— paration of pure macrophages. To alleviate this problem, techniques were sought which would more thoroughly disso- ciate the cells and more effectively remove the stroma. All manipulations were performed under sterile conditions. The number of cells was approximated with a hemacytometer. Splenocytes were isolated from tissue treated by three different procedures: mincing, mincing and filtra- tion, and mincing and gentle homogenization. Mincing Sprague Dawley of various ages and rats were de— capitated and the spleen aseptically transferred to a beaker containing 5 ml of sterile Earle's balance salt solution (EBSS). The cells were dissociated by mincing the tissue with scissors until a paste—like suspension 10 11 was formed. Large tissue aggregates were removed by trans- ferring the suspension to a conical centrifuge tube and allowing them to sediment for five minutes. Smaller cell aggregates were removed by transferring the upper 7/8 (vol.) of the suspension to a conical centrifuge tube and allowing them to sediment for 10 minutes. Cells were collected by transferring the upper 7/8 (vol.) of the sus- pension to a conical centrifuge tube and pelleting in a clinical centrifuge. Mincingyand Filtration The tissue was treated as in mincing but instead of sedimenting, the suspension was forced through a Swinney adaptor (Millipore Corp.) containing a 4000 mesh nylon screen to remove small cell aggregates. Mincing and Homogenization The tissue was treated as in mincing. To further dissociate the cell aggregates, the suspension was homo- genized by ten strokes of a modified, large clearance, hand homogenizer. A cell suspension in that the 15 ml Ten Broeck tissue grinder completely disrupted the tissue to subcellular fragments. A modified homogenizing vessel with a clearance sufficient for cell dissociation was made from a 65 mm polypropylene powder funnel with a 25 mm stem bored to accommodate a 150 x 16 mm test tube. 12 The test tube was shortened by 15 mm to allow the pestle from the 15 ml Ten Broeck grinder to reach the bottom. The funnel provided a basin for the test tube. Spleens were excised and dissociated by mincing, mincing and filtration, and mincing and gentle homogeniza- tion. Mincing yielded 2.2 x 107 cells per spleen with substantial contamination by cell aggregates and connec— tive tissue (Table l). Mincing and filtration yielded 2.2 x 107 cells per spleen but with very little contmina- tion. Mincing and gentle homogenization yielded 4.2 x 107 cells per spleen with a large number of cell clumps and pieces of stroma. Consequently, a procedure involving mincing, followed by gentle homogenization and then fil- tration of the resulting cell suspension was found to be the most desirable cell isolation procedure. Isolation of Glass Adhering Cells Splenocytes were cultured for 24 hours in complete medium: 89% (v/v) Eagle's minimum essential medium (MEM) containing Hank's balanced salt solution, 10% (v/V) fetal calf serum, 100 units/ml of penicillin and 100 ug/ml of 7 cells were added to 100 x 20 mm streptomycin. 1.7 x 10 plastic dishes containing 10 ml of medium and incubated in a moist atmosphere of 5% carbon dioxide and 95% air (standard incubation conditions). Unattached cells were removed after 1 hour incubation with a pipette and the 13 mannmo HMHSHHmonsw mammflu m>Huowcaoo coflumNflcmmoEon .mmesao Hamu boa x m.v cam mcfiocflz mwunmo x . cowbmuuaflm umHSHHmonnm woa N N van masocflz mwunmp umHsHHOOQSm .mnmmflu m>Huowccoo .mmasau Hamo hoe x m.~ mceocflz doflumceamudoo cmmamm\oamew demo cored: .mmoomonowe an ommmmmmm wumB mmuhooamamm mo camflm cam muflusm tam moonpme ucmnmmmac ha pmpMHOOmmHU mm3 somamm pom .mmsmoosmamm mzwummwnm mo moonume mo humfifism .H magma 14 attached cells washed with EBSS and evaluated. Similar procedures were followed using cover slips inserted in Leighton tubes. Splenocytes were cultured for 24 hours to allow macrophages and monocytes to attach to the plastic sur- face. The attached cells had the morphology of macrophage, but the yield of cells was low as compared to other methods. Density Separation of Splenocytes The density separation procedure used was a modification of the technique of Bennett and Cohn (26). A 35% sterile solution of bovine serum albumin (BSA) was diluted with EBSS to make a discontinuous gradient. Splenocytes at a concentration of 2 x 107 cells/ml in a volume of 1 ml were layered over a discontinuous gradient of 1 ml of 19% BSA, 1 ml of 23% BSA and 2 ml of 27% BSA. The gradient was centrifuged at 2,357 x g for 20 minutes in a Sorvall refrigerated centrifuge with a swinging bucket rotor (HS-4). Cells of macrophage morphology banded at the interphase of 23% and 27% BSA. Similar gradients were utilized with brain, liver and lung cell suspensions. The suspension of splenocytes layered over the discontinuous BSA gradient and sedimented was separated into three distinct bands and a pellet (Figure 1A). Band I, at the interphase of the layering Figure l. 15 Density separation of cell populations by sedi- mentation. Suspensions of various tessues were layered over a discontinuous gradient of BSA and sedimented 2,357 x gravity for 20 minutes as described in METHODS. Spleen is A, brain is B, lung is C and liver is D. BSA concentra- tions used were 27%, 23% and 19%. 16 DENSITY SEPARATION OF CELLS V/A cells % 2357X920min. E -1 w - |9°/oBSA *m. _ ll -23°oBSA _‘ / ------- -m -27°/oBSA I U W -Pellel l , A l @‘I E - I __ m-fi zzzzzz-[fl U \Vzpj-Pellel B w C -II m :zzzz w- Pel let D 17 solution and 19% BSA and composed mainly of cellular debris and particles the size of mitochandria. Band II was located at the interphase of 19% and 23% BSA and contained clusters of cells and undissociated tissue. Band III, located at the interphase of 23% and 27% BSA, contained cells of macrophage morphology, red blood cells and lympho- cytes. The pellet contained mostly lymphocytes and red blood cells with some macrophages. The cells in Band III were sedimented and counted. The yield was 5.97% of the sedimented cells: 90% were macrophages. After 8 days in culture, splenocytes gave only a pellet upon sedimenting. Macrophages and lymphocytes were the major cell types in the pellet. To determine whether the liver, lung and brain contained cells of similar density, suspensions of these tissues were prepared and sedimented using the procedure for spleen. Brain tissue gave a single band at the inter- phase of the layering solution and 19% BSA (Figure 1B). Lung tissue sedimented into three bands and a pellet (Figure 1C). Band I contained membranes and organelles, Band II was composed of small lymphocytes, connective tissue and septal cells, and Band III was mostly debris with a few lymphocytes. In the pellet red blood cells were the predominant type. The liver suspension sedimented into two bands and a pellet (Figure 1D). Bands I and II were cellular debris, and the pellet contained tissue clumps 18 and red blood cells. This indicated that the monocyte from spleen is the only phagocyte which sediments at a BSA density comparable to blood monocytes (26). Macrophage Induction in the Peritoneal CaVity Several investigators reported that relatively pure populations of peritoneal macrophages could be induced in four days with pyrogenic solutions of protein or mineral oil. To confirm these reports, the following experiments were conducted to find a pyrogenic solution and a time after pyrogen injection which induced the greatest number of macrophages in the peritoneal cavity of rats. In an attempt to determine the time necessary for the maximum induction of cells in the peritoneal cavity, rats were injected with 10 ml of 50% mineral oil in 10% peptone and the accumulation of cells in the cavity was evaluated. The number of cells in the cavity reached a maximum on the third day with a gradual decline and level- ing off by the sixth day (Figure 2). Macrophages were more prominent on the fourth, fifth and sixth day as judged by the number of cells with the characteristic morphology. Consequently, the fourth day was selected as the appropriate time to harvest the optimum number of cells having the greatest percentage of macrophages. Figure 2. 19 Induction of peritoneal cells in rats. Rats were injected intraperitoneally with 10 ml of a solution of 50% mineral oil and the accumula- tion of cells in the peritoneal cavity was evaluated at various times after the injection. 20 _ _ _ Wu 5 .6 838 do Emsaz :05 DAYS 21 Rats were injected intraperitoneally with 10 ml of various pyrogenic solutions to determine which gave optimal cell induction: 10% peptone in EBSS, 50% mineral oil in EBSS, 50% mineral oil in 10% peptone and 50% mineral oil in proteose peptone. Four days after the injections the rate were sacrificed. Thirty milliliters of EBSS containing 5 units/ml of heparin were injected into the peritoneal cavity and the resulting exudate aspirated. The cells were counted and compared to control injections of EBSS. All mineral oil solutions were soni- cated to make an emulsion. Recovery of exudate from the cavity was 67% to 83% of the 30 ml injected (Table 2). Peptone and 50% mineral oil gave the highest cell yield. Rats injected with proteose peptone and mineral oil were dead by the third day, half of the rats died on the second day after the injection. The symptoms displayed before death suggested anaphylaxis. Iodination of GAT A random synthetic polymer of L—glutamic acid, L—alanine and L—tyrosine (GAT) with a molecular weight of approximately 55,000 was radioiodinated according to a modified procedure of Greenwood, gt_al. (27). The reac- tion mixture contained 50 pl of carrier free Nalzsl, 30 pg of GAT (1 mg/ml), 25 pl of 0.5 M sodium phosphate (pH 7.5) and 15 pl of the catalyst 7 mM choloramine T in 0.05 M 22 m m e m on mOH x o vOH x N HE HN mOH x N.v H mmmm OH x mo.H 0.0m OH x m.h OH x vm.N HE dN OH x mo.m N HHO HMMOCHE h w m m wom paw m.mm N.OH x v.H mOH x h.v HE ON mOH x mv.m H mcoummm NOH mOH x v.v m.mm mOH x m.v mOH x vm.H HE mN moH x om.m N o.wo mOH x N.v mOH x mv.H HE ON mOH x mm.H H HHO HmumcHz 00H x hv.m w.mm N.OH x m.H mOH x mH.m HE ON wOH x MN.H N o.m> mOH x mv.m vOH x mH.m HE MN mOH x wN.h H mcoummm NOH Ga ”wwwwumo >Hm>oomm huH>mo CH wumwsxm mo Um>oEmm Um>OEmm .mwmxm coHusHom .dz mmmnm>d unmoumm mHHmo Hmuoe Ha\mHHmo mssao> mHHmo Heads .02 doeeomHaH .Umucsoo mums wuH>mo HmmcouHHom on» CH mHHmo map can .coHpmuHmmomp can COHDMNqumnumwcm an UmoHMHHomm mnt mum“ .m»dp Hsom Mmumfl .mGOHusHOm oHcmmoumm mDOHum> mo HE 0H nqu wuH>mo mecouHumm 0:» GH omuommsH mnm3 mumm .mcoHunHOm UHCOmoumm ND COHuosch HHmo mo wnmEEsm .N mHQmB 23 sodium phosphate (pH 7.5). The reaction time was varied according to the concentration of GAT approximately three minutes per 10 pg of polymer. The reaction was stopped by adding 50 ml of 12.6 mM sodium metabisulfite in 0.05 M sodijm phosphate buffer, pH 7.1. A solution of 0.047 M sucrose, 0.023 M potassium iodide and 0.1 mg/ml of bromphenol blue was added to the reaction mixture and the resulting solution transferred to a l x 32 cm Bio Gel P-30 column equilibrated with 0.005 M sodium phosphate buffer, pH 7.1. The reaction vessel was washed with 100 pl of a rinsing solution similar to the transfer solution containing only 0.023 M sucrose. The polymer was eluted with 0.05 M sodium phosphate buffer in 50 drop fractions (approximately 1 ml). Aliquots were counted in a Packard scintillation counter and the absorbance at 230 nm in each fraction was measured to determine the elution profile of labelled polypeptide. Several reaction times and polymer concentrations were employed in an effort to incorporate more 1251 into GAT. The time and concentration chosen had to be suffi- cient to allow maximum iodination of the tyrosine residues without deaminating the amino acids in the polymer. Reaction times of 8 and 10 minutes with 20 and 30 pg of polymer, respectively, were found to incorporate over 50% of the label into GAT. 24 The labelled polymer was separated from free NA125I on a Bio Gel P-30 column having an exclusion limit of 30,000. The radioactivity elution profile indicated that no labelled components of a molecular weight between those of the exclusion limit and the iodide salt (Figure 3). Based upon absorbance at 230 nm four peaks were eluted most of Peak I eluted with the radioactivity peak of GAT. Peak III contained the bromphenol blue marker in addition to the radioactive iodide salt. Peak II eluted near the GAT peak and may have been fragment of polymer lacking tyrosine. Peak I did not completely coincide with the [125n GAT which could have been due to a contaminant in the presence of polymer containing little tyrosine. The recovery of label added to the Bio Gel column was evaluated by adding a known amount of [1251] GAT to reaction mixtures having varying quantities of unlabelled polymer. Using three separate columns, the recovery was consistently 71% (Table 4). 125I] GAT Uptake by Cells from the Peritoneal Cavity [ Phagocytic cells were induced in the peritoneal cavities of rats by intraperitoneal injections of 5.to 10 ml of 50% mineral oil in 10% peptone sonicated to make an emulsion. Three to four days later the rats were sacri- ficed and the cells harvested. Rats were sacrificed by“ Figure 3. 25 Separation of radioiodinated poly GAT from Na 1251 by gel filtration chromatography. Labelled iodine (Na 1251) was reacted with 30 pg of GAT for 8 minutes and the reaction mix- ture was fractionated on a l x 32 cm Bio Gel P-30 column equilibrated with 0.05 M sodium phosphate buffer (pH 7.5). The absorbance at 230 nm was determined in a Gilford spectro- photometer and 10 ml aliquots were evaluated for radioactivity by scintillation counting. Fractions were collected in 50 drop aliquots (approximately 1 ml). A maximum absorbance in Peak III, bromphenol blue, was reached at fraction 10. h 30 mix- Bel 3 at .ed gl ts it _ 0.6 a 0.4 26 0———0COUNTS/I‘4INUTE 'AODzso IODINATION A"-.. El ' ‘ —- a .- — ‘- — "———- ’- .— -‘ ~u.- -“- I ——~_-_-h.---~‘ ------——- —.._. ----~----_-- . o-— -- ‘-——- -— -“ --.- --.-.--.d 'O I ' l9 I8 9 IO II I2 I3 l4 I5 I6 I7 FRACTIONS 8 l2l—o IO_ «3 \1‘ (..0Ix) Binwlw (\l 83d SINOOD 27 .cEsHoo 30mm ou poopm wm3 Emu mnm.on* Hm Hmm.mo om Hm mmH.mm oN Ha mem.om OH onoutz >Hm>oomm w Umum>oomm .CHE\mpcsoO COHpommm CH ado m0 m: I I|I|‘ «.UmsHEumumo mm3 sEsHoo msu Eoum mnm>oomn map can sEsHoo omlm How on Eo Nm x H m CH poopm mnm3 HwEMHom pmumcHGOH mo Emu vOH x H.h mchHmucoo mmusuxHE coHuommu mchoH .mGEdHoo omlm Hmo OHm Eoum mnm>oomu Eda HHmNHH mo mumEEsm .m mHQme 28 a) cervical dislocation and decapitation, b) ether asphyxi- ation, and c) anesthethization and decapitation to deter- mine which method gave the lowest contamination by red blood cells. Anesthetization and decapitation gave the least red blood cell contamination. The cells were washed several times in EBSS to remove residual mineral oil and resuspended in sterile MEM. The cell yield was approxi- mately 5 x 105 white cells per rat with 80 to 95% of the cells having macrophage marphology. Aliquots of cells were mixed with complete medium containing 0, 5 and 10% fetal calf serum and dispensed in 1 m1 volumes in 12 x 75 mm sterile plastic centrifuge tubes or 10 x 30 mm plastic dishes. After various periods of incubation under standard conditions, the cells were sedimented, washed with MEM and cell number and the con- tent radioactivity was determined. Similar procedures were followed with red blood cells isolated from peripheral blood. 12 [ SI] GAT Retention by Peritoneal Cells 125 The retention of I by peritoneal cells in vitro was measured by adding 3 ml of cells in MEM (8.7 x 106 cells/ml. 20% white cells) to 2 ml of [125 I] GAT (2.5 Ug/ml at 8.9 x 104 cpm/pg) in 2.5% BSA and 0.05 M phosphate buffered physiological saline (pH 7.5) and incubating 29 under standard conditions for 12 hours. The cells were harvested and washed and the radioactivity in the cells and incubation medium determined. 1 [ 25:] GAT Stability The extent of degradation of [1251] GAT during cell culture as well as after storage at -20°C was evaluated by gel filtration on chromatography on a 1 x 32 cm Bio Gel P-30 column equilibrated with 0.05 M phosphate buffer 9pH 7.5). One ml samples were applied to the column and 50-drop fractions were collected at room temperature. RNA Extraction Rats were sacrificed and the liver, brain, lung, spleen and pancreas were excised and frozen on dry ice. The frozen tissue was placed in a chilled semimicro blender container with 25 ml of acetate EDTA buffer containing 0.01 M sodium ethylenediamine tetraacetate (EDTA), 0.01 M sodium acetate buffer (pH 5.1) and 5 pg/ml of polyvinyl sulfate (PVS), and 30 ml of redistilled phenol. Sodium dodecyl sulfate (SDS) was added to a final concentration of 1%. The tissue was homogenized for four 30-second intervals with interim 60-second periods of chilling in a 0°C ice bath. The phases were separated by centrifugation and the aqueous phase re-extracted twice with an equal volume of phenol. Residual phenol was removed by ether 30 extraction and the ether evaporated by bubbling the aqueous phase with nitrogen. The RNA was precipitated twice in 0.1 volume of l M NaCl and 2 volumes of ethanol at -20°C for 4 hours. All glassware was acid or base washed to reduce ribonuclease cOntamination. TEAE Cellulose Chromatography of Low MbIecular Weight RNA Low molecular weight RNA was separated on a l x 24 cm TEAE-cellulose column equilibrated with 0.02 M Tris-Cl (pH 8.2) and eluted with a linear gradient of 0.1 to 0.75 M sodium chloride in Tris buffer. The column eluent was monitored with an ISCO ultraviolet analyzer and recorder. The concentration of sodium chloride was monitored with a conductivity meter (Radiometer Copenhagen) and the absorb- ance at 280 nm was determined with a Model 240 Gilford spectrophotometer. Gel Electrophoresis Gel electrophoresis was conducted in the vertical analytical cell of Raymond, E-C470 according to the method of Peacock (29, 29, 30). Gel Buffer A stock solution of Tris EDTA borate (10X) buffer (pH 8.3) containing 0.89 M Tris, 0.89 M boric acid and 0.025 .M disodium EDTA was prepared and diluted as needed. 31 Slab Gels The gels used were either agarose acrylamide com- posite gels or straight acrylamide gels. Agarose acryla- mide composite gels were 2% acrylamide and N,N‘- Methylenebisacrylamide (bis), and 0.5% agarose and were used to analyze total or high molecular weight RNA. The acrylamide and his used were purchased as Cyanogum 41 in a ratio of 19:1. Gels were prepared from 160 ml of gel solution contained 0.8 g of agarose, 109 m1 of water, 16 ml of 20% Cyanogum 41, 10 m1 of 6.4% 3—dimethylaminopro- pronitrile, 16 ml of 10 x Tris EDTA borate buffer and 5 m1 of 1.6% ammonium persulfate. The acrylamide gels used were either 5% or 7.5% acrylamide-bis. The gel solution varied in that it con- tained 69 ml of water and 60 ml of Cyanogum 41 for 7.5% gels, and 89 ml of water and 40 m1 of Cyanogum 41 for 5% gels. Acrylamide gels did not contain agarose. Cylindrical Gels Cylindrical gels were prepared according to Loening (48) with recrystallized bis and acrylamide. A gel solution of 7.5% acrylamide (w/v) and 0.38% bis (w/v) was prepared. To polymerize the gels, 33 pl NNN/N/— Tetramethylethylene diamine and 2.5 ammonium persulfate (w/w) were added per gram of acrylamide. Gels were poly- merized in 0.5 x 12 cm glass tubes for 30 minutes at room 32 temperature and pre-run at 5 mA per gel for 1 hour. Sam- ples were mixed with an equal volume of 40% sucrose and 0.01% bromphenol blue and layered on the gel. Electro- phoresis was conducted at 5 mA/gel for 30 minutes. The final buffer concentration was: Tris, 0.04 M; sodium acetate, 0.02 M; EDTA, 2 mM; acetic acid was used to adjust the pH to 7.8. Staining A stock staining solution of 0.1% Stains-All in 100% formamide was diluted to 0.01% with 100% formamide and then further diluted to 0.005% with water to give a working solution having 50% formamide. The gels were stained overnight in the dark at room temperature and destained in the dark in running water for 4 hours. Because of the light sensitivity of stains all, all solu- tions were stored in the dark. Scintillation Counting Radioactivity was quantitated in a Packard Tri-Carb spectrometer. Samples of 1251 in 1 m1 of solution were added to 10 ml of scintillation fluid containing 66.7% of toluene (w/v), 33.3% of Triton X-100 (w/v), 0.01% 1,4—bis [2-(4-methyl-S-phenyloxazolyl)] benzene (dimthyl POPOP) (w/v) and 0.55% 2,5 diphenyloxazole (PPO) (w/v), and counted with a gain of 40% and a window of 50 to 1000 with an efficiency of approximately 80%. 33 Photo Microscopy Photomicroscopy was performed with an A. 0. Spencer phase microscope equiped with a Kodak Colorsnap 35 camera. The photomicrographs were taken with Kodachrome II profes- sional color film at shutter speeds of 6 to 12 seconds with normal viewing illumination. RESULTS 125 Uptake of‘[ I] GAT into Peritoneal Cells The peritoneal cells employed in the first experi- ment contained 80-90% red blood cells. A preliminary experiment was performed to assess the contribution of red blood cells to the uptake of [125 I] GAT by white cells. Peritoneal cells in culture were monitored for 18 hours. During this period the number of red blood cells gradually declined with only a 10% decrease in the white cell popula- tion. Cells from the peritoneal cavity enriched in macro- phages were incubated in the presence of 0.15 pg [1251] GAT/ml with a specific activity of 6.6 x 105 cpm/pg in different concentrations of fetal calf serum to determine if the serum had an effect on polymer uptake. Each cul- 5 ture tube initially contained approximately 10 cells. 4 cells were white and 90% of those Approximately 1.5 x 10 were macrophages. Red blood cells constituted 85% of the cells. To determine whether contaminating red blood cells incorporated a significant amount of polymer, parallel sets of cultures were prepared containing greater than 99% 6 red blood cells (1.5 x 10 cells/ml). The red blood cells 34 35 were cultured with medium containing 10% (v/v) fetal calf serum or no fetal calf serum. At the time of maximum uptake, the enriched white cell population in control cultures (no serum) contained 0.5% of the label; those in 5% serum, 2.85%; and those 10% serum, 2.92%. Red blood cells in control cultures took up 0.57% of the polymer; those in 10% serum, 1.41% (Figure 4). All of the culture reached a maximum rate in about 6 hours. While the cells in 10% serum leveled off after 6 hours, those in 5% serum and no serum continued to increase for 24 hours. By evaluating red blood cell contamination of macrophages, the contribution to the maximum uptake was estimated to be 1.5%. Fetal calf serum stimulated the uptake of [1251] GAT by both red blood cells and white cells. Polymer Stability in Cell Culture To determine whether fetal calf serum had an effect on [1251] GAT stability in culture, the incubation media from uptake experiments described above was frac- tionated on a Bio Gel P-30 column. The tissue culture media contained phenol red which eluted in Fractions 9 to 12 (Figure 5) and was used as a low molecular weight marker. Degradation was assessed by the percentage of 1251 eluted in fractions 9 to 14. Sixteen percent of the Figure 4. 36 Uptake of [lZSI] GAT in red blood cells and white cells. Peritoneal cells containing 1.5 x 10 white cells/ml and purified red blood cells containing 1.5 x 106 cells/ml were incubated under standard conditions in the presence of [125I] GAT at a concentration of 4.6 x 104 cpm/ml with a specific activity of 6.6 x 10 cpmAJg for polymer uptake at various serum concentrations. (----) red blood cells, ( ) white blood cells. and I 1.5 X I cells [ted e of 105 CPM (x 10‘?) I6- I2— IO— 37 ---- Red blood cells - White cells I IO % l ' 4. i I/4 50/0 8 I2 I6 20 24 INCUBATION TIME (Hours) Figure 5. 38 [1251] GAT stability in cell culture medium containing different concentrations of serum. The sterile conditioned culture media containing no serum4 or 5 or 10% serum were incubated with 3.6 x 10 cpm/ml of [1251] GAT with a specific activity of 6.6 x 105 cpm/pg. After 4 hours (A), 12 hours (B), and 24 hours (C), the media were fractionated on a l x 32 cm Bio Gel P-30 columns and collected in SO—drop fractions. 39 mum—2:2 ZO_._.Uta<40243< I 00 . . QOoIo m0 . Zm m IE m Figure 10. 54 Electrophoretic analysis of lung low molecular weight RNA. Electrophoresis was performed on a 7.5% acrylamide slab gel for 3 hours at 4°C and 200 v with TEAE cellulose fractions of lung RNA described in Figure 10. Slot 1 con- tained fraction 76; slot 2, fraction 73; slot 3, fraction 70; slot 4, fraction 67; slot 5, fraction 64; slot 6, fraction 61; slot 7, fraction 58; and slot 8, 0.4 OD260 units of E. coli tRNA. 55 ’VHH HIGHS :1 56 distinct bands. The first five of the six bands comigrated with bands in the E. 221i tRNA marker. The elution sequence was bands II, III, IV and V first, band VI second and band I third. Bands VI and I were present in all fractions analyzed. Macrophage low molecular weight RNA resolved on a 7.5% disc gel gave only bands I and VI (Figure 11). This suggested that macrophages contain no unique species of low molecular weight RNA. Partial Characterization of Band I Spleen low molecular weight RNA was resolved on a 5% acrylamide gel from TEAE cellulose fractions 55 through 90 of Figure 11. Again, the initial fractions contained tRNA but band I was proportionately greater. In the latter fractions band I was the sole component. To determine the nature of band I, fractions 71 to 81 were electrophoretically analyzed in the presence of pronase, ribonuclease and deoxyribonuclease on an acrylamide agarose composite gel (Figure 12). Pronase was used to identify protein and also to remove any protein which might be associated with RNA and preventing nuclease degradation. Each fraction was ethanol precipitated, dried and redis- solved in 100 m1 of water. Five pg of the appropriate enzyme was added and the solution was incubated at 0°C for 10 minutes before applying the sample on the gel. Band I was conspicuously absent in slots containing Figure 11. 57 Electrophoretic analysis of macrophage low molecular weight RNA. Electrophoresis was performed on a 7.5% cylindrical gel for 30 minutes at 5 mA/gel as described in METHODS. Fractions 44, 45 and 46 were on gel (A); fractions 49, 50 and 51 were on (B); 0.5 OD260 units of E. coli: tRNA were on gel (C). Figure 12. 59 Partial characterization of band I. Electro- phoresis was performed on an acrylamide agarose composite gel for 2.5 hours at 4°C and 200 v with spleen TEAE cellulose fractions and various degradative enzymes. Slot 1 con- tained 0.6 OD260 units of total spleen RNA and ribonuclease; slot 2, 0.6 OD260 units of total spleen RNA and deoxyribonuclease; slot 3, fraction 81 and deoxyribonuclease; slot4, fraction 79 and ribonuclease; slot 5 fraction 77, pronase and deoxyribonuclease; slot 6 and slot 8, 0.6 OD260 units of total spleen RNA. 60 QMHDZOM 83H 61 deoxyribonuclease and pronace and deoxyribonuclease alone. Degradation was also evident in slots having whole spleen RNA preparation and deoxyribonuclease. This established Band I to be low molecular weight DNA. Such a species of DNA could have resulted from fragmentation of larger mole- cules during extraction oriizcould be an endogenous species of satellite DNA or mitochondrial DNA. DISCUSSION Many of the characteristics essential to a cellu— 1ar defense system were inherent in splenocytes maintained ig_zi§gg. Such a system would have to be rugged enough to survive the onslaught of microbial invasion. The cells of this system would have to accommodate environmental modula- tions with a minimum loss of efficiency. Metabolic activities would have to be modulated with the external milieu and the nature of the invader. Lymphocytes and macrophages were cultured for periods up to two weeks with no visible evidence of necrotic cells, whereas the £2.2iEEQ environment destroyed other white cells in a day or two. During incubation, monocytes formed macrophages and giant cells and small lymphocytes enlarged. These transformations also occur i2 yizp with a corresponding increase in the ability of lymphocytes to produce antibody and monocytes to phagocytize particles. Macrophage—like cells from rat spleen sedimented at the interphase of 27% BSA. After incubation, these cells did not band at this interphase. However, many of the cultured splenocytes had the morphology of macrophages. This suggests that the cells banding in BSA were monocytes and not the larger and more active macrophage of the next 62 63 differentiated state in agreement with Bennett and Cohn (26). The failure to detect cells of similar density in brain, liver, and lung is evidence against the existence of monocytes in these tissues. However, this does not negate the possibility that a pre or post monocyte cell type exists in these tiSsue and that these cells differentiate into the Kupffer cells of liver, the microglial cell of brain and the alveolar macrophage of lung. The lung pos— sesses septal cells, which banded a 23% BSA in association with lymphocytes. This is significant because some histo- logists believe that septal cells can differentiate into alveolar macrophage. The white cells induced in the peritoneal cavity were consistently greater than 90% macrophage. In medium containing 10% serum, [1251] GAT uptake by red blood cells was only 50% of the maximum uptake achieved by macrophages (4.2 x 10"4 cpm/cell). Macrophages took up 9 x 10"3 cpm/cell or 5.77 x lo'3 ng/cell, a ZOO-fold increase over the red blood cell uptake. Since the reliability of the cell count is 90%, a 1% or 2% contamination of red blood cells by macrophages would not have been detected. There- fore, the contribution to uptake by red blood cells is negligible. Fetal calf serum stimulated [1251] GAT uptake in cultured macrophages. The addition of 10% fetal calf serum caused a 5-fold increase in the uptake of polymer by 64 macrophages. Macrophages cultured in 5% serum required a longer incubation to achieve a maximum uptake and were incapable of sustaining such a level once achieved. This effect can be partially attributed to protection of the labelled polymer from protease degradation. However, limited [1251] GAT degradation is not sufficient to explain the differences which occurred at zero time. Although at this time the concentration of intact polymer was approximately the same in all cultures, uptake was greatest in cultures containing 10% serum. Even though the polymer in both 5% and 10% serum was protected to the same extent, cells in 10% serum still took up polymer at a much faster rate. Stimulation of uptake can best be explained by the presence of a single serum factor or a series of serum factors which enhances pinocytosis. Such a factor could originate from epigenetic regulation in the developing calf or could merely be metabolic cofactor such as vitamins or coenzymes. The retention of such a high percentage of polymer in macrophages in the presence of 10% serum implies a specific role for the GAT taken up. Cells involved in nonspecific pinocytosis and general metabolism would rapidly equilibrate with the protein in the external environment. Macrophages could contain an enzyme system capable of utilizing iodinated tyrosine and free iodine as in the pituitary gland. Conceivably, the polymer retained 65 by the cells is in a processed form capable of invoking an antibody response from lymphocytes. A processed anti- gen could exist as a free protein or in conjunction with RNA. Regardless of the function, labelled iodine is released into the medium in a form having a molecular weight of at least 30,000 daltons. Gottlieb stated that immunogenic RNA constituted 4% of total macrophage RNA and that it eluted from a TEAE cellulose cOlumn with 0.32 M sodium chloride (46). Since examination of those fractions by gel electrophoresis revealed no unique species of RNA in spleen or macrophages, the antigen-RNA complex either comprises less than 4% of macrophage RNA or it elutes at a different salt molarity. Characterization of slower eluting nucleic acid suggests that the antigen—RNA complex must have a greater affinity for TEAE cellulose than tRNA because the elution profiles had no major ultraviolet radiation absorbing component in the region between nucleotides and the major peak. How— ever, it is possible that immunogenic RNA migrates with tRNA and constitutes a major portion of the difference in the percentage of low molecular weight nucleic acid observed in macrophage and lung total RNA. In this case, identification can only be accomplished by using a radio- active antigen marker. Once the labelled complex is identi- fied, further characterization could be accomplished by degrading the RNA or protein and investigating the nature v of the linkage. LI ST OF REFERENCES 10. 11. 12. 13. 14. 15. LIST OF REFERENCES Mc Gregor, D., et all, Proc. Royl. Soc. B. 168, 229 (1967). Gowans, J. L., J. W. Uhr., J. Exptl. Med., 124, 1017 (1966). Papermaster, B. 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PART I I INTRODUCTION RNA Synthesis in Mammalian Cells Ribosomal RNA Ribosomal RNA (rRNA) is mainly composed of three RNA species and comprises over 80% of total cellular RNA. The three rRNA species are classified according to Svedberg units (S) and are designated 288, 188 and SS. The initial recognizable precursor of the 28S and 188 components, 458, was first observed during studies of rapidly sedimenting nucleolar RNA (8, 9, 10). Confirmation of these species was made using polyacrylamide gel electrophoresis (11, 12). Because of the slow equilibration of nucleoside pools (66, 67) the typical "pulse chase" experiments were ineffective and precursor studies were conducted using actinomycin D. The inhibitor was administered after brief exposure to labelled uridine to inhibit further isotope incorporation and to allow the processing of precursors to be traced. Labelling kinetics in rat liver suggested a half-life of 6 to 8 minutes for the 458 precursor (9). After a partial hepatectomy total RNA synthesis in regene- rating rat livers increased 5-fold (13) and the half-life 70 71 of 458 rRNA was reduced to 3.7 minutes (14). The next probable step in the synthesis of 288 and 188 rRNA is the cleavage of 458 RNA to 418 and then 368 RNA (11). The next major precursor is a 328 molecule which is converted to the 288 species found in cytoplasmic ribosomes. This path is 458 -+ 418 -+ 368 -+ 328 -* 288. Eighteen—S rRNA is also synthesized from the 458 presursor via a 208 or 238 intermediate (11, 15): 458 + 238 + 188. Only 50 to 60% of the 458 molecule is used to makelHfiSand 288 RNA (16, 17). The only acceptor of methyl groups is the 458 rRNA precursor, and all methyl groups are conserved in further processing (11, 18). The 58 molecule is found in the nucleolar particle with the 458 species (19), but the 58 species does not hybri— dize with other rRNA molecules (20) and is apparently made from different precursors. In addition, the 58 molecule must pass through a 58 pool before entering the ribosomes (21). Transfer RNA Transfer RNA (tRNA) is the next most abundant RNA species in mammalian tissue. In polyacrylamide gels, tRNA migrates more rapidly than ribosomal 58 RNA. Recent .investigations have established the existence of a tRNA Precursor. After a 30 minute labelling period in HeLa cells, a radioactive peak was observed in the region of 72 tRNA and 58 rRNA distinct from tRNA and 58 RNA (22, 23). At later times the peak gradually disappears and a new radioactive peak comigrates with the 48 peak of tRNA. 2p_yi£pp conversion of earlier labelled species to tRNA has been accomplished (24). The slower migration of the pre-tRNA molecule is due to a longer nucleotide sequence and not merely a conformational structure different from tRNA (24). Pre~tRNA is deficient in methyl groups and has a lower content of pseudouridine (25). Nuclear RNA Harris noted that the amount of label appearing in the cytoplasm of macrophages and connective tissue was insufficient to account for all the RNA synthesized in the nucleus (26, 27). Short term labelling experiments indi- cate that the nucleus contains rapidly labelled RNA which sediments throughout a 5% to 20% sucrose gradient and is almost totally degraded without ever entering the cyto- plasm (28). Sedimentation values for this heterogenous nuclear RNA (HnRNA) range from 2 to 1008. Although the very large HnRNA molecules have a low guanine and cytosine content as in DNA (1, 29, 30), total nuclear RNA is diverse in base composition (31, 32). The DNA-like base <:omposition of the larger molecules is partially attributed to the presence of polyadenylate segments on the 3' terminus of messenger RNA which is also found in HnRNA 73 (33, 34, 35, 36). It was postulated that the polyadenylate attachment may be a necessary processing step which stabili- zes the messenger RNA and facilitates transport into the cytoplasm (37), but the conspicuous absence of the poly- adenylate segment on histone messenger RNA makes such an encompassing hypothesis unlikely (38). This concept is further abated by the occurrence of messenger RNA in pro- tein complexes which are also postulated as the form of the processed messenger (39, 40, 41, 42). Messenger RNA sediments at approximately 108 (43, 44) and can be extracted from the nucleus or removed from cytoplasmic polysomes. Control of the Formation of RNA Precursors RNA precursor formation and cleavage in mammalian cells is a process which normally occurs in minutes and occupies a small fraction of the cellular generation time. By utilizing suboptimal cell culture conditions and inhibi- tory drugs, precursor processing can be manipulated to allow each cleavage step to be thoroughly evaluated. Cycloheximide at a concentration which inhibits 99% of protein synthesis reduces RNA synthesis and causes the accu- mulation of rRNA precursors. But the drug fails to block precursor processing and ribosome assembly completely (45). Puromycin also reduces RNA synthesis while allowing 458 precursor and methylation to occur, but unlike cyclohexi- mide, ribosome formation is completely blocked in that 288 74 rRNA is made and 188 rRNA is not. Once the drug is removed normal processing continues. Puromycin and cycloheximide together allow reduced ribosome formation (46). Methionine deprivation to an extent which inhibits 70 to 80% of pro- tein synthesis allows precursor formation but inhibits 288 and 18S rRNA formation by reducing methylation of4SSqu1by 80% and 328 by 60%; valine deprivation to the same extent permits reduces synthesis of 288 and 188 RNA molecules (47). Incubation of HeLa cells at reduced temperatures stops rRNA processing. Below 20°C,no 458 RNA is formed; at 15°C conversion of 458 to 328 is completely inhibited and at 25°C,328 cleavage to 288 is stopped (48). Prolonged incubation at low temperatures causes the transient 418 intermediate to accumulate. Transfer RNA precursor can be isolated as a distinct peak in cells that have been methionine starved (49) or maintained at a suboptimal temperature (50). RNA Synthesis During Development By far the most intensive studies of RNA synthesis during development system have been the investigation of oogenesis in echinoderms and amphibians. In both systems the small immature oocytes which occur before yolk disposi- tion or during the previtellogenesis stage are active in RNA synthesis and are responsible for RNA accumulation; the late mature oocytes are relatively quiescent (51, 52). 75 In Xenopus laevis oocytes at all stages of development can be found maturing simultaneously in the ovary. Duryee classified the maturing oocytes into distinct numerical stages with each having its own chromosomal physiology and nuclear structure (53). Stage 3 constitutes the early lampbrush stage which marks the appearance of lampbrush chromosomes. Stage 4 is the maximum lampbrush period. According to the results obtained by Davidson (54), the stage 3 oocyte possesses about 20% of the RNA contained in the mature stage 6 oocyte. The total RNA increases to the stage 6 value by the end of the maximum lampbrush stage. In Xenopus oocytes at either stage 4 or 6, 95% of the RNA is ribosomal and therefore measurement of total RNA content is essentially a measure of rRNA content. Labelled RNA extracted from lampbrush stage oocytes after a 30 minute exposure to labelled precursor has the base composition and sedimentation characteristics of rRNA. The same 1&5 true of RNA extracted from oocytes labelled several hundred times longer. In contrast, a large pro- portion of the high molecular weight RNA synthesized per unit of time in HeLa or liver cells is nonribosomal (54). .After stage 4 no more rRNA is made until the nucleoli appear in the fertilized egg during gastrulation. In the chick embryo new rRNA begins to be made at the mid- cleavage stage (55); mouse embryos, however, are able to synthesize new rRNA soon after fertilization (56). 76 Brown and Littna noted a sudden burst of informa- tional RNA (mRNA) synthesis in Xenopus toward the end of oogenesis (57, 58). Using its DNA-like base composition as a means of characterization, they estimated that the unfertilized ovulated egg contains at least one nanogram of new mRNA. The total new mRNA in the early Xenopus gastrula containing thousands of active nuclei is only about lOng. Consequently, the synthesis of one nanogram of mRNA within the 12 hours of the ovulation period con- stitutes a tremendous increase in mRNA synthesis. Hor— mones which induce ovulation are believed to be the cause of the sudden burst of mRNA synthesis. In sea urchin, mRNA synthesis appears to occur at least a month before maturation of the egg. Female sea urchins incubated with labelled RNA precursor three months before analysis has labelled mRNA still present in mature oocytes (59). Synthesis of mRNA continues under stimulatory conditions up to a week before shedding (60). The fact that in both species enucleated and actinomycin D treated fertilized eggs are able to develop to the gastrula stage without synthesizing new RNA indicates the present of maternal "programmed" or “masked" messenger RNA which is somehow activated by fertilization to direct early embryogenesis (61). 77 Differentiation of the Rat Pancreas Past studies of the embryonic rat and mouse pan— creas have partially defined the transitory stages in the differentiation of the anlage to the mature organ (1, 2, 3, 4, 5). Most of the investigations were concerned with the enzymatic and morphological differences which accompany phenotypic maturation. Therefore, considerable data are available on the synthesis of secretory proteins and orga- nelles and the effects of various metabolites and drugs on their 22.22529 occurrence (6). Currently, Parsa, Marsh and Fitzgerald have observed the methionine dependent differentiation of rat pancreas ip_yipgo, Day 13 anlages maintained in methionine deficient medium for nine days were able to grow, but there was only limited morphological differentiation and little increase in the enzymatic activity of amylase, lipase or chymotrypsin, proteins produced specifically by the mature tissue in large amounts. Addition of methionine or S-adenosyl methionine (SAM) resulted in considerable morphological differentia- tion and increased level of these proteins within 24 hours (62). Acinar cell proliferation required 30 mg/l of methionine and differentiation required 50 mg/l. Because SAM or choline (methyl donors) could substitute for methionine and homocysteine (demethylated methionine) could not, it was concluded that the concentration of 78 available methyl groups influences the differentiation of pancreas ip|zippo (63). Because of previous studies of methionine deprivation in HeLa cells (47), the most obvious place to investigate an effect is in RNA process— ing. Unfortunately, few laboratories have attempted to elucidate the role of RNA in the differentiation of the pancreas and those that have were unable to detect any diversity in the RNA synthesized at the different fetal ages (7). Mainly this can be attributed to the small amount of tissue obtainable from rat and mouse embryos; the prodigious concentration of ribonuclease in the more matured pancreatic rudiments; the lack of analytical methods capable of resolving the possible differences which may occur. Research Approach As a standard technique for analyzing RNA, poly- acrylamide gel electrophoresis is replacing sucrose zonal sedimentation and because of its superior speed and resolu- tion, it removes several of the obstacles confronting pancreatic RNA studies. The technique permits analysis of small samples and provides rapid separation of RNA from the contaminating residual ribonuclease which survives phenol extraction. Polyacrylamide gel electrophoresis has been instrumental in elucidating the discrete steps in the processing of RNA by mammalian tissues. 79 Using polyacrylamide gel electrophoresis, I have attempted to determine if ribosomal RNA synthesis in the differentiating rat pancreas occurs differentially as in amphibian oogenesis or continuously as in other mammalian systems. By resolving RNA made at a suboptimal pH, I have obtained contrasting profiles of RNA made under conditions of normal and abnormal synthesis. I have devised methods for preparing undegraded RNA from rat pancreas suitable for electrophoretic analysis. The sensitivity of existing methods for gel electrophoresis has been increased. I have defined culture conditions for [3H] uridine incor- poration into RNA and evaluated changes in the labelled population of RNA during cytodifferentiation. This study clearly establishes the feasibility of polyacrylamide gel electrophoresis as an analytical tool in the study of RNA in embryonic systems. METHODS Rat Breeding Male and female Sprague Dawley rats were paired in breeding cages for 12 hours. Dropped vaginal plugs marked conception. Approximately 70% of the plugged females were pregnant and litters routinely exceeded 12 rats. Incubation of Pancreatic Rudiments Pregnant female rats were sacrificed by cervical dislocation and decapitation. Placentas were aseptically removed and placed in Earle's balanced salt solution. Fetal dissections were performed in Earle's balanced salt solution (EBSS) under a Nikon dissecting microscope. Embryonic pancreases excised and placed in Eagle's mini- mum essential medium (MEM) containing Hank's balanced salt solution. The incubation conditions were similar to those described by Shaffer (67). The pancreases were then cut into pieces of 5—15 pg and placed on sterile Millipore filters (pore size 0.22p, 13mm). When the excess media was removed, the tissue became attached to the filter. To study the kinetics of RNA and protein synthesis, six filters each containing approximately 20 pg of tissue were 80 81 floated (tissue down) in a 100 x 20 mm Falcon dish contain- ing 7 ml of complete MEM having 5pCi/ml of [3H] uridine (25.7 Ci/mM) or [3H] leucine (23 Ci/mM). For electro- phoretic analysis of RNA, four filters each containing approximately a total of 50 to 150 pg of tissue were floated on 7 m1 of complete MEM having 5pCi/m1 of [3H] uridine. Complete culture medium contained the following components: 87% (V/V) Eagle's MEM, 10% (V/V) fetal calf serum, 100 unit/ml of penicillin and 100 pg/ml of strepto- mycin, an amino acid supplement was added which increased the final concentration of L-methionine from 0.1 mM to 0.3 mM of L-argonine from 0.6 mM to 1.8 mM, of L-glutamine from 2 mM to 4 mM. The tissue was incubated at 37°C under a humidified atmosphere of 95% air and 5% C02. Uptake AnaIysis of [3H] Uridine and [3H] Leucine After various incubation periods, the filters were removed, washed with 1 ml of Eagle's MEM at 4°C and the tissue was scraped off. The pieces of pancreas were placed in a 1 ml tissue grinder (Duall) with 1 m1 of acetate EDTA buffer containing 10 mM sodium ethylenedia- mine tetraacetate (EDTA), 10 mM sodium acetate buffer (pH 5.1) and 5 pg/ml of polyvinyl sulfate (PVS); homogeniza- tion was performed by 20 strokes of a teflon motorized pestle at 0°C. 82 Total Uptake of [3H] Uridine and [3H] Leucine Precursor lnto Homogenates To evaluate total uptake of label, 0.2 ml aliquots of homogenate were added to scintillation vials, and solu- bilized in 0.5 ml of 1 M KOH at 90°C for 1 hour. Ten m1 of triton-toluene scintillation fluid (METHODS, part I) were added and the radioactivity was counted in a scintillation counter. Incorporation of Labelled grecursors into TCA Precipitable Material 0.2m1 aliquots of homogenate were precipitated at 0°C for 15 minutes with 10% TCA and 100pg of BSA as a car- rier for [3H] leucine labelled protein or 100pg of yeast RNA as carrier for [3H] uridine labelled RNA. The precipi-‘ tate was collected on a 2.4 cm glass fiber filter (GF/C). Alkaline resistant uridine counts were assessed by hydroly- zing 0.2 ml of homogenate with 0.5 m1 of l M KOH at 60°C and precipitating in the presence of 100pg of BSA with 10% TCA. The precipitate was collected on filters, and the filters were incubated at 90°C for 1 hour in 0.5 ml of l M KOH and counted. Soluble counts were determined by subtracting the TCA precipitable counts from the counts in an equal volume of homogenate. Because TCA causes quenching in our scintillation system, the soluble counts could not be measured directly. 83 Protein Determination Two 0.1 m1 aliquots of a homogenate were placed in 0.4 m1 plastic microfuge tubes (Beckman Instrument Co.) with 0.2 ml of 10% TCA and precipitated at 4°C for 4 hours. The solution was centrifuged for 5 minutes in a Microfuge (Beckman), the supernatant removed and the pellet dried. Protein was estimated according to the method of Lowry (64) as modified by Rutter (65). Gel Electrophoresis Slab gel electrophoresis was conducted in a cus- tomized Raymond vertical analytical cell according to the methods of Peacock (see Gel Electrophoresis, Part I). The apparatus was made to accommodate gels of 1/16 inch and 1/8 inch thickness with slots for thirteen samples. Because of the limited amount of tissue and the ribonu— clease susceptibility of the pancreatic RNA, a method was sought which allowed sensitive analysis of crude RNA extrac- tions. Agarose acrylamide gels of 1/8 inch thickness did not give the sensitivity of the 1/16 inch gel. The 1/16 inch gel was used in later analysis, but because of its fragility, the thinner gel had to be handled with great care. 84 Gel Formation The gels used were agarose acrylamide composites with 2% acrylamide and N,N'-ethylenebisacry1amide (bis) and 0.5% agarose. The acrylamide and his were purchased as Cyanogum 41 in a ratio of 19:1. Two gels 1/16 inch thick and 12 cm long were formed from an 80 ml gel solu- tion of 0.4 9 agarose, 54.5 ml of water, 8 m1 of 20% Cyanogum 41, 5 ml of 6.4% 3-dimentylaminopropionitrile, 8 ml of 10x Tris EDTA borate buffer and 2.4 ml of 1.6% ammonium persulfate. Gels were polymerized for 1 hour at 25°C and pre-run for 1 hour at 4°C 100v. Extra gels were stored horizontally under buffer at 4°C up to 4 days with no visible distortion. RNA Extraction After evaluating various extraction procedures, the following standard procedure was employed. Washed labelled tissue was homogenized with 20 strokes of a 1 ml teflon tissue grinder (Duall) at 0°C in the presence of 47.5 1 of standard RNA extraction buffer (10 mM sodium acetate, pH 5.1, 10 mM sodium EDTA and 5 pg/ml PVS), 2.5 p1 of 10% SDS and 50 pl of 88% redistilled phenol. The homogenate was placed in a microfuge tube and chilled for 30 seconds in an ice bath. The aqueous and phenol phases were separated by centrifuging the mixture for 2 minutes in a Beckman Microfuge at 4°C. The aqueous layer was removed and 85 re-extracted in 25 pl of redistilled phenol by mixing with a Micromixer (Beckman) at 4°C. The mixture was chilled and centrifuged, and the aqueous phase was removed and frozen at -20°C. After two phenol extractions the final volume of aqueous solution ranged from 5 to 20 pl and this was suf- ficient for direct gel application without further adjust- ment of the RNA concentration. With this procedure the RNA from 19 pg of lS—day fetal pancreas was sufficient for electrophoretic analysis (Figure l). The optimal amount of RNA for electrophoretic analysis was 0.1 to 0.5 OD260 units. Electrophoresis Conditions After the last phenol extraction, between 5 and 15 p1 of aqueous phase were rapidly mixed with 5 p1 of layering solution at 0°C. The layering solution was 40% sucrose, 0.1 mM EDTA and 0.01% bromphenol blue. The sample was then layered in the gel slots. Gels were run at 4°C and 50 v for 30 minutes to allow the sample to enter the gel and then at 100vfor 2 hours. Staining was conducted overnight in a Stains—All working solution. The gels were scanned in a model 240 Gilford equipped with a Gilford linear scanner and a Sargent recorder. Figure l. 86 Photograph of RNA from varying amounts of 15- day pancreas. RNA was extracted in 100 pl total volume as described in METHODS from 15- day pancreas cultured for 6 hours. Slot 1 contained RNA extracted from approximately 19 pg of tissue; slot 2, RNA from approxi— mately 57 pg of tissue; slot 3, RNA from approximately 95 pg of pancreas; slot 4, RNA from approximately 190 pg of tissue; slot 5, RNA from 15-day liver cultured for 6 hours, and alot 6, 0.1 OD260 units of hepatoma rRNA. 87 E8 woz<._.m_o O 4 8 H 88 Determination of Radioactivity ln Gels After destaining the gel slots were cut out in strips and sliced into approximately 1 mm pieces with the razor blades spaced 1 mm apart. Each slice was hydrolyzed in 0.5 m1 of 1 M KOH at 90°C for 1 hour and analyzed for radioactivity. Scintillation Counting Radioactivity was quantitated in a Packard Tri- Carb spectrometer. Alkaline treated samples were brought to 1 ml with distilled water and added to 10 ml of scin— tillation fluid containing 66.7% of toluene (v/v), 33.3% of Triton X-100 (v/v), 0.01% 1,4-bis [2-(4-methyl—5- phenyloxazoly)] benzene (dimethyl POPOP) (w/v) and 0.55% 2,5-diphenyloxazone (PPO) (w/v). Counting was conducted with an efficiency of 20% at a gain of 60% and a window of 50 to 1000. RESULTS Development of RNA Extraction Methods RNA Extraction from Embryonic and Adult Pancreas Because of the production and secretion of ribo- nuclease by rat pancreas, undegraded RNA is very difficult to extract from this tissue. RNA isolation usually entails homogenization in pH 9.0 to 5.1 buffer containing 0.5 to 1% SDS followed by phenol extraction. However, none of the major RNA species in RNA from liver (control) could be detected in electropherogram of RNA prepared according to METHODS, Part I from adult pancreas. To determine exactly where degradation occurred in the isolation process, elec- trophoresis was conducted on the extraction solution after every manipulation. Previously, control experiments showed that PVS, SDS and phenol had no effect on the migration of hepatoma rRNA on agarose acrylamide gels. Complete degra- dation of 28S and 188 RNA was evident after the first phenol extraction. This indicated that degradiation had occurred between disruption of the tissue and addition of the phenol and SDS. To shorten this interval, the pancreas was homogenized in a solution of extraction buffer as 89 90 above together with an equal volume of 88% phenol. Elec- trophoretic profiles of this pancreatic RNA were comparable to migration patterns of RNA from liver, lung and spleen. The pH and extraction buffer composition were varied to determine whether different RNA species could be selectively extracted and if degradation could be reduced. Brawerman, g£_32. (69) reported that sequential extraction with Tris-C1 at pH 7.6 and 9.0 resulted in extraction of rRNA and selective extraction of polyadenosine enriched RNA. RNA from 17-day fetal rat pancreas extracted in 0.1 M Tris-C1 at pH 7.6 and pH 9.0 and resolved by gel elec- trophoresis after each extraction yielded only decreasing quantities of rRNA and tRNA. To further define optimal conditions for RNA extrac- tion, buffer composition, pH, temperature and SDS concentra- tion were varied. Pancreatic rudiment from 17-day fetal rat pancreas was extracted under different conditions and subjec- ted to electrophoresis (Figure 2). Extractions at pH 5.1 in RNA buffer, pH 7.6 in Tris-Cl buffer and pH 9.0 in Tris-Cl yielded increased quantities of DNA and increased RNA degra- dation. Increasing the SDS concentration to 1% from 0.5% in RNA buffer, pH 5.1, resulted in extraction of more DNA. Re- ducing the SDS concentration to 0.25% in Tris—Cl, pH 9.0 resulted in extraction of less DNA than in the same buffer with 0.5% SDS. RNA extracted in pH 5.1 RNA extraction buf— fer and in pH 7.6 Tris-C1 yielded comparable RNA extractions Figure 2. 91 Electrophoretic analysis of RNA extracted at different pH's, temperatures and SDS concen- trations from approximately 360 pg of l7-day pancreas. Slot 1 contained RNA extracted at pH 5.1 in standard RNA extraction buffer (acetate buffer) with 0.5% SDS at 4°C. Slot 2 contained RNA extracted at pH 7.6 in 0.1 M Tris-C1 buffer with 0.5% SDS and 5 pg/ml of PVS at 4°C. Slot 3 contained at pH 9.0 in 0.1 M Tris-Cl with 0.5% SDS and 5 pg/ml of PVS at 4°C. Slot 4 contained RNA extracted by shaking on a micromixer (Beckman) for 30 seconds and incubating at 60°C for 30 seconds over a 4 minute period. The extraction buffer was standard RNA extraction buffer containing 2% SDS and 30 pg/ml of PVS. Slot 5 contained RNA extracted at pH 9.0 in 0.1 M Tris-C1 having a reduced SDS concentration of 0.25%. Slot 6 contained RNA extracted in standard RNA extrac- tion buffer having an increased SDS concentra- tion of 1% SDS. Slot 7 contained 0.1 OD260 units of hepatoma rRNA (marker). 92 HEoH muz<._.m_n_ 4 H 93 with slightly more degradation occurring at pH 7.6. RNA extracted at pH 9 in 0.1 M Tris-C1 was more degraded and contained considerably more DNA than RNA extracted at pH 7.6. Elevating the extraction temperature from 4°C to 60°C in pH 5.1 RNA extraction buffer and 1% SDS resulted in extraction of DNA and degraded RNA. More DNA was extracted by increasing the SDS concentration or increasing the pH of the extraction buffer. It was concluded that intact RNA was optimally extracted in pH 5.1 standard RNA extraction buffer and 0.5% SDS at 4°C. These conditions were employed for all subsequent experiments. To evaluate the efficiency of the micro RNA extrac- tion, approximately 200 pg of l7-day pancreas was labelled 23 12322 with [3H] uridine at a concentration of 5 pCi/ml for 24 hours as described in METHODS. The tissue was washed and the RNA extracted. Samples were counted after each step in the extraction. The homogenizer and vessel used to disrupt the tissue was washed three times with buffer and the wash solution counted. The pooled solutions accounted for 35.3% of the total [3H] uridine counts. The phenolic phase and the interphase of denatured protein from the phenol extraction contained 23.2% for the first extrac- tion and 7.9% for the second. 33.5% of the total [3H] uridine was in the final RNA solution (9 pl) which would normally have been applied to a gel. Similar percentages Were obtained with l4-day and 19-day pancreas. TCA 94 precipitation of phenol extracted counts yielded the same percentage of counts as was found in the tissue homogenate. Homogenization of the tissue in the 1 ml homogenizer caused the small volume of rather viscous homogenate to be distributed over a large area of the homogenization vessel. Consequently, a large percentage of uridine counts could not be removed for extraction without using a centrifuge. Centrifugation would have resulted in a longer extraction time and more degradation. So, an increase in extracted RNA counts was comprised for minimum degradation. Electrophoretic Anal sis of RNA from Rancreatfc Rudiments Durlng_§ytodifferentIaEion RNA was extracted from fetal rat pancreas of dif— ferent embryonic ages to determine whether any major dif- ferences in extracted RNA could be detected during differen- tiation. Figure 3 demonstrates that gels of RNA prepared from 15, l6, l7 and 20-day pancreases gave similar band patterns. The major bands corresponded to 288 and 188 rRNA in hepatoma rRNA; 58 and 4S RNA were detected, but were not well resolved on these gels. This suggested that no difference in the percentage and relative mobility of major RNA species, extracted under standard conditions, occur during pancreatic cytodifferentiation. An inherent problem in this study was that older rudiments contain more ribonuclease than younger less Figure 3. 95 A composite photograph of stained gels showing RNA extracted from rat pancreases of different embryonic ages after various periods in vitro RNA was extracted in volumes from loo‘Eo 400 U1 having 49.5% standard RNA extraction buffer, 0.5% SDS and 50% phenol (88%) and sub- jected to electropharesis as described in METHODS. The total extraction volume was varied to accommodate the limited amount of tissue obtained from younger embryos. Slot 1 contained RNA from lS-day pancreas cultured for 4 hours; slot 2, RNA from lS-day pancreas cultured for 24 hours; slot 3, RNA from 16- day pancreas cultured for 6 hours; slot 4, RNA from l7-day pancreas cultured for 12 hours, and slot 5, RNA from 20-day pancreas cultured for 6 hours. 28$— 96 97 differentiated rudiments (2). Even mild degradation could decrease the amount of RNA in the major classes and pro— duce species of intermediate size. To evaluate the degree of degradation more accurately, similar stained gels of l6, l7, l9 and 20—day pancreatic RNA were scanned. As shown by Figure 4, the major RNA species—-288, 188, 58 and 4S--were present at each day, and there was little varia- tion in relative mobilities. The amount of DNA extracted was variable. In addition, occassionally an RNA band was detected between the 188 and 58 regions. This was probably an artifact of extraction. By measuring the area under the major peaks in gel scans of RNA from fetal pancreas at different ages, the extent of degradation between tissues containing different quantities of ribonuclease could be assessed (Table 1). All ages had essentially the same relative amounts of each major RNA species. These were also comparable to RNA extracted from fetal rat liver which was used as a low ribonuclease control. The ratio of 288 to 188 RNA was approximately 2:1 for pancreas of all ambryonic ages in Table 1 except 15 and 20—day. The variation in the ratio of 288 and 188 RNA in lS—day pancreas was due to the small amount of RNA applied to the gel. This caused the background absorbance to be higher and created uncertainty in approximating the peak area. The same situation holds for 20—day pancreatic RNA. Figure 4. 98 Representative scans of RNA extracted from rat pancreases at different embryonic ages. RNA was extracted and resolved by gel electro- phoresis. Gels were stained with Stains-A11 and scanned at 570 nm as described in METHODS. Gel migration is from left to right; major peaks were identified by the migration of standard hepatoma rRNA. ABSORBANCE,570nnI ZO— 288 IGDAjt I.5~ IO— 48 I88 0.5— w 58 0 IS— 285 I7DAI IO- DNA I88 0.5— 53 4S 0 11 L0— 288 ISDAY 05” l88 43 W 58 O 288 go I ZODAY l5- IIBS IO— 05 )J W O_ . o ' '2 ‘ 4 ‘ é ‘ é DISTANCE (cm) 100 .COHHMH>mp CmoE H EC ohm um OOCMQHOQO mo uCoOHmQ mm Co>Hm mum mmCHm> .xmmm oCu CH conHHomCH mHmCMHHu m mo moum esp mCHumEonummm an UmumEHumm oHOB mCmom Hom CH mmHoomm sz we UCm mm .mmH .mmN mo mmoudm m.o H m.oH H.e H m.o o.m H N.o~ H.o H as m HH oH a em mm H mH HMHHH NH o.o em oo H om e.o H m.om o.H H o.m m.o H m.Hm o.m H He m aH m.m H H.HH N.N H o.m o.~ H m.om m.o H m.om oH HH mH a mm Hm H oH 4H s om mo H mH mmmmmmmm mCoHHMCHE Awe me Awe mm Ase mmH Hoe mom Iwoooo cod Ho .oz m.mpCoEHtoH Ho>HH oCm OHHMOHOCMQ Eoum Umuomnuxm «2m CH mmHommm fizm HonmE mo muCCOEm m>HHMHmm .H mHQmB 101 Organ Culture of Pancreatic Rudiments Short Term Organ Culture Seventeen-day pancreatic rudiments were aseptially removed and cultured intact in 1 ml of Eagle's MEM alone in a 12 x 75 mm sterile disposable centrifuge tube, for intervals up to 8 hours. Under these conditions, the tissue was completely submerged and the total rudiment mass varied from age to age. RNA degradation was used to evaluate tissue necrosis. Seventeen-day pancreases cultured for 15 minutes to 4 hours gave identical RNA patterns (Figure 5). Uptake of [3H] uridine by 14, 17, and l9-day pancreas was linear for 4 hours with no indication of saturation kinetics. Electrophoretic analysis of phenol extractable counts revealed that none of the major RNA species were labelled by 4 hours. Most of the label was in fast migrating RNA with the remainder being dispersed throughout the gel. It was apparent that the labelling time was insufficient to permit labelling of rRNA. Using the same procedure, l7-day rudiments were cultured for 24 hours. At 12 hours in culture all the RNA extracted was completely degraded. This indicated that necrosis had occurred. Four modifications were made in developing a satisfactory long term organ culture system; the rudiments were cut into pieces of approximately 5-15 pg of protein Figure 5. 102 Photograph of RNA from l7-day pancreases cul- tured for various intervals. RNA was extracted from 360 pg of tissue in 100 p1 total volume as described in METHODS. Slot 1 contained RNA from l7-day liver; slot 2, RNA from l7-day pancreas cultured for 15 minutes; slot 3, RNA from l7-day pancreas cultured for 30 minutes; slot 4, RNA from l7-day pancreas cultured for 1 hour; slot 5, RNA from l7—day pancreas cul- tured for 2 hours; slot 6, RNA from l7-day pancreas cultured for 3 hours, and slot 7, RNA from l7-day pancreas cultured for 4 hours. 103 (“19) EONVISICI 104 to allow labelled precursors and nutrients to penetrate the interior of the tissue; the tissue pieces were attached to a Millipore filter as a substrate and floated at the liquid gas interphase to facilitate oxygen-carbon dioxide exchange; fetal calf serum and supplemental amino acids were added to Eagle's MEM to provide additional precursors and growth factors; and antibiotics were added to inhibit bacterial contamination. These modifications are evaluated in a subsequent section. Long Term Organ Culture Using the above modifications distinct RNA bands were resolved from RNA extracted from l7-day pancreas cultured for 24 hours. To provide a control for assessing differentiation and tissue survival, adult pancreas was cultured under the same condition. Before this study, it was believed that adult rat pancreas could be successfully cultured only for short intervals. RNA from adult pancreas indicated no tissue necrosis. _, The migration patterns were comparable to l7-day pancreatic RNA (Figure 6). RNA and Proteip_8ynthesis in Fetal Rat Pancreas Cultured’atng .8 [3H] Leucine Incorporation When the air—CO2 mixture was bubbled through water in the bottom of the 37°C incubator, it was observed that the equilibration pH of the tissue culture medium dropped Figure 6. 105 RNA from adult pancreas cultured for various periods at pH 6.8. RNA was extracted from approximately 400 pg of tissue under standard conditions. Slot 1 contained RNA from adult pancreas cultured for 4 hours; slot 2, 8 hours; slot 3, 12 hours; slot 4, 24 hours, and slot 5, 0.1 OD260 units of hepatoma rRNA. 106 HEoH mozHPmB 107 from 7.1 to 6.8. This unusual occurrence was probably due to a faulty gas meter which measured CO2 incorrectly. This pH change upon equilibration was used to adjust the pH of the medium to 6.8. Labelling studies were conducted using 5 pCi/ml of [3H] leucine in standard medium which contained 0.4 mM leucine. The added labelled leucine did not alter the total leucine concentration in the medium. Fourteen, 17, 18, 19, 20-day old pancreatic rudiments took up leucine into the soluble pool and incorporated it into TCA precipitable products at approximately the same rate (Figure 7). Leucine entered the soluble and protein pools at approximately the same rate at each age. Fifteen-day pancreas incorporated leucine linearly for 24 hours with no indication of pool equilibration or steady state kinetics. At this age less than 30% of the label taken up by the tissue was incorporated into TCA precipitable material. Adult pancreas incorporated leucine into the soluble pool for 12 hours and then the rate started to level off. Leucine was incorporated into protein at a linear rate for 24 hours and approximately the same rate at which it went into the soluble pool. This indicated that fetal and adult pancreas synthesized protein in culture. [3H] Uridine Incorporation Uridine uptake analysis was conducted at 5 poi/ml of [3H] uridine in complete medium. The Eagle '8 MEM contained no Figure 7. 108 Uptake of [3H] leucine by fetal rat pancreas cultured at pH 6.8. Fetal rat pancreases of different embryonic ages were cultured at pH 6.8 on Millipore filters in 5 pCi/ml of [3H] leucine as described in METHODS. (H) TCA soluble counts, (o——o) TCA precipitable counts. LEUCINE-‘H DPM/pg PROTEIN (x I03) 109 6" IflDAI H TCA SOLUBLE COUNTS ~ ° ° H TCA PRECIPITABLE COUNTS 4_ I 0 2H - . soar 7? I O l g I I I 4*— LQDAI o 3.. 3— 2... . 2~ l~ e I o 0 o I’- O l l l l I JIDAI o 9 I L i 3— 2” e I- I O I I l I 1 181241 4.. 3H 2_ 2- o '30 I” O 8 O 1 l l l I O 1 l I 1 l 24 8 I2 24 24 8 I2 24 INCUBATION TIME (HOURS) INCUBATION TIME (HOURS) 110 uridine or uracil other than labelled uridine and that in fetal calf serum. Fourteen-day pancreas incorporated uridine into the soluble pool at a linear rate for 4 hours (Figure 8). Incorporation into TCA precipitable material increased for 24 hours. During the first 8 hours 75% more uridine was incorporated into the soluble pool than in the insoluble material. By 24 hours the percentage of soluble counts was reduced to 50%. Seventy-five per- cent of the counts were soluble in.151and 19-day pancreases cultured for 24 hours. At both ages, incorporation was linear for 24 hours. Seventeen and 18-day pancreases incorporated uridine linearly into both pools with 50% of the counts in the soluble pool. Adult and 20-day pancreases had a 24 hour linear uptake with a percentage in soluble counts of approximately 60%. Alkaline resistant TCA percipitable counts were taken as a measure of [3H] label incorporated into DNA. In uridine studies at pH 7.1 and 6.8, the percentage of alkaline resistant counts did not exceed 10% in rudiments labelled for 24 hours. This indicated that very little 3H was incorporated into protein and DNA during the study. Electrophoretic Sgparation of Qabelled RNA from Fetal Pancreas RNA extracted from 18-day pancreas labelled with [3H] for 4 and 24 hours displayed a very low number of Figure 8. 111 Uptake of [3H] uridine by fetal rat pancreas cultured at pH 6.8. Fetal rat pancreases of different embryonic ages were cultured at pH 6.8 on Millipore filters in 5 pCi/ml of [3H] uridine as described in METHODS. URIDINE-BH DPM/pg PROTEIN (x IO'3) 112 H TCA SOLUBLE COUNTS 0—0 TCA PRECIPITABLE COUNTS O l 2 4 8 I2 INCUBATION TIME O l I O l 24 24 8 l2 24 (HOURS) INCUBATION TIME (HOURS) 1 113 counts (Figure 9). After 4 hours of labelling, none of the major RNA species were distinctly labelled. The [3H] uridine appeared as a broad peak in the region of 288 and 188 RNA and as a double peak in the 48 region. After incu- bating 24 hours in the presence of [3H] uridine, 288 and 188 labelled peaks could be distinguished. A major low molecular peak migrated in the region of 58 RNA. Adult pancreatic RNA labelled for 8 hours gave two main peaks after electrophoresis (Figure 10). One migrated with a molecular weight greater than 288 and the other migrated around 108. In 12 hours the high molecular weight peak migrated closer to 288, a small percentage of label appeared in a 288 peak and a double peak migrate near 58 RNA. The amount of [3H] uridine incorporated into rRNA was low. Comparable results were obtained with 17 and 20-day pancreases. None of the rRNA peaks were labelled until 12 hours of incubation , and a number of unusual peaks were detected. The results suggested that at pH 6.8 rRNA synthesis in fetal and adult pancreases is diminished. Furthermore, much of the radioactivity was incorporated into RNA species which normally constitute a small percent of total RNA. Figure 9. 114 Profiles of labelled RNA from 18-day pancreatic rudiments maintained in culture at pH 6.8 for 4 and 24 hours. Approximately 50-100 pg of tissue was incubated with 5 pCi/ml of [ H] uridine in complete medium containing Eagle's MEM; 10% fetal calf serum, antibiotics and supplemental animo acids at pH 6.8 as described in METHODS. After incubation at 37°C for 4 and 24 hours, RNA was extracted and subjected to electrophoresis and the gels were stained and scanned. Distribution of radioactivity was determined as described in METHODS. CPM (—*—) 115 IOO—I 60* 4O— 20 l IGO I I40— I20 T IOOI- 20— 5 I I0 28S I I I I5 2025 ‘ v ISS II 1L1 L 1 E1 The... 30354045505560657075 GEL NUMBER Figure 10. 116 Profiles of labelled RNA from adult pancreas maintained at pH 6.8 for 8 and 12 hours. Adult pancreas was labelled and the RNA extrac- ted and subjected to electrophoresis as described inMETHODS.(o-—o) 8 hours, (o—o) 12 hours, ( ----- ) absorbance. mum—>52 mofiw w 2-3.8, mm 8 9. . .-¢I.--U-HI.-II‘..--.(M.\.£- ._.§u.u....z. Ct.(.- ..... 11:11.11 0 ‘3‘ \\\\\ :2), A ...... (-I, I ‘ I-) \ \\\\\ ”p \s . . II \\’/ ._ lllllll 1“!!! \a.\ ow . . . u . u l w . .ra : % i u l n H .. u _. u . I : _ _ I N _ _ 7 j... _ 1‘ __ loo. 1 C.- _ _ K 1. i u no _. U . u n m me. : looN " x _ : _ ___ (I a -omm _ x E wdo 118 Protein and RNA_§ynthesis by Fetal Rat Pancreas Cultured at pH 7.1 Rates of Protein and RNA Synthesis z§_T§:55§—Pancreas Cultured Under Different Culture Conditions By adjusting the air-CO2 mixture flow, the pH was stabilized at pH 7.1. A series of experiments was carried out to determine the effects of culture conditions on appa- rent rates of [3H] uridine incorporation into RNA and [3H] leucine incorporation into protein. Nineteen-day pancreatic rudiments were cultured for 24 hours in medium without supplemental amino acids and com- pared with rudiments cultured with complete MEM. L-glutamine in liquid medium is unstable and tend to break down upon storage. To alleviate this problem, this amino acid was added in the supplemental amino acids. Some investigators believe that L-arginine plays a paramount role in the sur- vival of tissue in culture (70). The special function of the amino acid is undefined. Some believe it is involved in histone control of DNA transcription; others believe it is no more important than other amino acids. It was assumed that arginine was essential and it was added in the supple- mental amino acids. Because Parsa 22.2i' demonstrated a requirement for methionine in cultured fetal pancreas, methionine was included in the supplemental amino acids. Electrophoretic analysis of 19-day pancreatic RNA labelled without supplemental amino acids gave profiles com- parable to l9-day pancreas cultured with supplemental amino Figure 11. 119 Profiles of labelled RNA from l9-day fetal pan- creas maintained in Culture at pH 7.1 for 4 (A) and 12 (B) hours in the absence of supplemental amino acids. Nineteen-day pancreas (150-200 pg) was incubated on Millipore filters in Eagle's MEM-10% fetal calf serum with and without supplemental amino acids: 0.12 mM L-arginine, 0.2 mM l-methionine and 2 mM L-glutamine. After incubating t 37°C for 4 and 12 hours in 5 uCi/ml of [ H] uridine, the tissues were rinsed and the labelled RNA was extracted and resolved by electrophoresis as described in Figure 4. The gel was stained and scanned as described in METHODS. The distribution of radioactivity was then deter- mined. (-—-—) RNA from rudiments labelled in medium without supplemental amino acids. (O——o) RNA from rudiments labelled in medium with supplemental amino acids. ' 120 mmmzaz moflm mNONmmowmmommvovmmommNONQ O. m __qa —_ _ _ fi . _ _ _ .. -- - fl _ _ _ _ {1-6.1 ll--- \\ OI \\0 I \ ” '-' "."I“.. 0 -0 < mo_o< OZ_S_< .SOICB I mead. 02.53 1.2273} (3.0: X) was N—OCD Figure 12. 121 Photography of RNA from 19-day pancreases cultured at pH 7.1 for various intervals with and without supplemental amino acids. Tissues (150—200 g) were incubated and RNA was extracted in 100 pl total volume as describediJIMETHODS. Slotl contained 0.1 OD250 units of hepatoma rRNA. Slots 2 through 6 contained RNA from 19-day pancreas cultured without additional amino acids in a standard concentration of MEM and fetal calf serum: slot 2, cultured for 4 hours; slot 3, cultured for 12 hours; slot 4, cultured for 15 minutes; slot 5, cultured for 30 minutes, and slot 6, cultured for 1 hour. Slots having RNA from l9-day pancreas cultured with supplemental amino acids (standard cul- ture conditions) were: slot 7, cultured for 4 hours; slot 8, cultured for 8 hours; slot 9, cultured for 12 hours; slot 10, cultured for 24 hours, and slot 11, cultured for 36 hours. 456789|0 122 (”13) 33NV1SICI 123 acids (Figure 11). The RNA patterns suggest no differences in the migration of the RNA extracted from tissue cultured under the two conditions (Figure 12). The pattern shown in slot 8 depicts degradation. This could have occurred dur- ing tissue necrosis or RNA extraction. Because the RNA ex- tracted from tissue cultured for longer periods was unde- graded, the degradation probably occurred during extraction. The rate of RNA synthesis did not vary under the two condi- tions (Table 2), but the rate of protein synthesis was increased 2-fold in supplemental amino acids. Therefore, we included them in our standard culture medium. The removal of antibiotics from the culture medium had no effect on the initial rate of RNA and protein syn- thesis, but after 4 hours a drastic reduction in the uptake of both [3H] uridine and [3H] leucine was observed. The pH of the medium dropped and in 24 hours the trubidity of bac— terial contamination was evident. The drop in label uptake could have been caused by cellular necrosis due to the acid pH or depletion of the label in the medium by rapidly pro- liferating bacteria. It was thought that antibiotics were not necessary for 24 hour cultures. This study indicated that antibiotics were needed to prevent bacterial contami- nation of growing cultures. Chick embryo extract is often added to culture media to provide additional growth factors. The extract is usually the supernatant fraction from a sedimented 124 .omumowpca mmfi3um£uo mmmacd mc0fluflpcoo .mmon ESEmeE cam EDEAGHE on» no mmmnm>m on» mo coauma>mp coma u + Q UHMUGMUm .HmUGD UmHDMHSO 0Hm3 mmflummflfim mm mm 5 mm mm om mm on ma +I44 +I +l +| +I44 +I +I mad mom mm mom mam 0mm m.~m.u m.m~H m.~m_H m.mmH m.¢H H m.~v ~.m H m.m~ m.-_H ~.mm ¢.o~.H m.mm mm H .ou m.m H o.>m ma ma mm mm om mm mm vm mm ov +I +I +l-+l +l +I-+l+l +| +I mmH mmH mh moa omm mhm mad mmH mam oam h.mv v.m m.HH m.mH h.mm m.m +l+4 +I +I +I-+I +I +I +| .oom AH.» may pasc< m.mm Am.m may pano¢ Am.m may Enumm oz . m o.ov Am m m V Esfipme Gunpcmum m.maa womuuxm omnnfim wm+ m.HoH moflpowawucm oz . mpflom OGHEM m we Hmwcmamammsm oz Assflpme cum o.mm upcmumv cowuwn Inocflmum H50: vm . Awnsuaso m baa mumoflamscv m.¢HH Edwpme pumpcmum mmmuocmm mmolma .us\cflmuoum mn\zmo Amoacua\aamo\zmo .Hn\cflmuoum ma\zmo Amoavun\aamo\zmo mcflcofluflwcoo musuasu a mcfloflua mama Q mcflosmq hmmg . ' CH mmcmno m.mmsmmflu owummuocmm omusuaso ca ammumume manmuamwomum «UH oucfl coaumuomnoocw mcflosma mama can mcwwwus Emma mo mmumm .N manna 125 homogenate of chicken embryos and may contain a variety of factors ranging from growth promoters to essential vita- mins. Addition of 3% embryo extract had no effect on the rate of RNA and protein synthesis (Table 3). This suggested that either the necessary growth factors for pancreas cul- tured for 24 hours are provided in the other components of the medium, or that they are not needed by the tissue at the ages studied. Cultures incubated more than 2 days may require embryo extract. Nineteen—day pancreases were cul- tured for 24 hours in the absence of [3H] uridine and [3H] leucine and then transferred to labelled medium for 24 hours. The rates obtained from the uptake curves were similar to control values (Table 2). This showed that the studies conducted in 24 hours were not performed at the limit of in zitgg survival of the tissue. Fetal calf serum was added to tissue culture medium to provide physiological hormones and factors. Addition of 10% fetal calf serum to cultures resulted in a 0.3-fold increase in protein synthesis and a 3-fold increase in RNA synthesis (Table 2). Fetal calf serum is heterogenous in composition, and therefore, the stimulation could have been caused by one or more factors. [3H] Leucine Incorporation Kinetic studies of [3H] leucine incorporation described in the preceding section were repeated. Equilibration of the 126 soluble pools was generally apparent within 4 to 12 hours (Figure 13). This is in contrast with the results obtained at suboptimal pH. The incorporation of counts into TCA insoluble material increased linearly for approximately 24 hours. The soluble pool accounted for 30-50% of the total counts in the pancreatic homogenates after 12 hours. The uncertainty in the estimates of radioactivity in the soluble pool, obtained as the difference of two relatively large numbers, is rather high. This may account for the scatter in these points as observed, for example, in the l9-day tissues. The apparent rate of [3H] leucine incorporation into TCA precipitable material varies throughout differen- tiation of the pancreas as shown by Figure 14. At pH 6.8, the apparent rate of incorporation (dpm/ug protein/hr) decreased between 14 and 15 days of gestation. The rate increased several fold to a maximum of 200 dpm/ug protein/ hr at 17 days of gestation, then declined to the lS-day level after 20 days of gestation. The rate of protein synthesis in the adult pancreas was comparable to 18 and 19-day rates. At pH 7.1, the apparent rate increased from approxi- mately 150 dpm/ug protein/hr at 15 and 16 days of gesta- tion to a maximum of 450 dpm/ug protein/hr then declined to the lS-day level. The rates were approximately 3-fold greater than the pH 6.8 values at each age. Seventeen—day Figure 13. 127 Uptake of [3H] leucine by fetal rat pancreases cultured at pH 7.1. Fetal rat pancreases of different embryonic ages were cultured at3pH 7.1 on Millipore filters in 5 uCi/ml of [ H] leucine as described in METHODS. (o——o) TCA soluble counts, (o-—o) TCA precipitable counts. crease es of l 2 8 H TCA SOLUBLE COUNTS 0—0 TCA PRECIPITABLE COUNTS m. L l I J ()1 J l I l I LEUCINE-sH DPM/ug PROTEIN (x 163) O O INCUBATION TIME I I ‘ 1 24 e3 2 é4 (HOURS) 24 £3 2 24 INCUBATION TIME (HOURS) Figure 14. 129 Apparent rates of [3H] leucine incorporation per microgram of protein. The average of the maximum and minimum slopes were taken for each incorporation curve for TCA precipitable counts at pH 6.8 (Figure 6) and pH 7.1 (Figure 11), and plotted as a function of embryonic age. (o———o) pH 7.1, (o---o) pH 6.8. The bars indicate the maximum and minimum slope. [3H] LEUCINE DPM mg protein / hr (I02) 130 U" l «b. 1' N1 j r \ l I L I I I l4 l5 l6 I7 l8 I9 20 Embryonic Age ADULT 131 rudiments and adult pancreatic tissue were least affected by the change in pH. As the pancreas differentiates, the ratio of pro- tein to DNA content in the tissue increases. The cells become larger and more specialized, as indicated by the formation of new membrane structures and the accumulation of tissue specific products such as amylase. The rates of incorporation per microgram of protein were converted to rates per cell by using the quantity of protein per cell calculated for each embryonic age by Rutter EE.2£° (unpub- lished data). When the apparent rates of protein synthe- sis per cell are plotted as a function of embryonic age, there is an apparent transition between 16 and 19 days of gestation at both pH 7.1 and 6.8 (Figure 15). At pH 7.1 as well as 6.8, l9-day old tissues incorporated 8-fold more [3H] leucine into protein per cell than did 15 or 16— day tissue. The greatest rate of protein synthesis per cell occurred in adult pancreases which ‘was 2—fold greater than the 19—day rate at pH 7.1. [3H] Uridine Incorporation Fifteen-day pancreatic rudiments incorporated [3H] uridine into TCA insoluble material at a linear rate for 12 to 24 hours during the developmental transition (Figure 16). The amount of label in the TCA soluble pool increased at a linear rate up to 24 hours for several sets of tissue, e.g., rudiments from 15 and l7-day old embryos. In other 132 Figure 15. Apparent rates of [3H] leucine incorporation per cell. The apparent rates in Figure 14 were converted to rates per cell by using the protein per cell values calculated by Rutter, et al. (unpublished data). (o——o) pH 7.1, (o---o) pH 6.8. The bars indicate the maximmn and minimum slopes. [3H] LEUCINE DPM/CELL/hr.(|02) 5 CD 03 I4 133 /%’+,/:\} \ / \i/ I I I I I I l5 l6 l7 l8 I9 20 EMBRYONIC AGE 134 Figure 16. Uptake of [3H] uridine by fetal rat pancreases cultured at pH 7.1. Fetal rat pancreases of different embryonic ages were cultured on Millipore filters in 5 uCi/ml of [3H] uridine as described in METHODS. 135 H TCA SOLUBLE COUNTS 0—0 TCA PRECIPITABLE mUNTS reases s of URIDINE-SH 0PM mg PROTEIN (XIO'3) I? 5—Y ISDAY O 6“ ' 8’ o . _ O 4’- 4~, . - O b O o . . I . 2” O I I I I I 3,. 2.0.09.1 2’. O I” o O O I l 1 1 I 3.. ADULT 2- II. 0 O l I 24 8 I2 24 INCUBATION TIME (HOURS) I2- 0 24 é I2 “ INCUBATION TIME 24 (HOURS) 136 cases, the soluble pool apparently equilibrated in 4 to 12 hours, e.g., 18 and l9-day old rudiments. After 12 hours incubation, approximately 30% of the [3H] uridine in the homogenate was TCA precipitable. The apparent rates of [3H] uridine incorporation (dpm/mg protein/hr) were plotted as a function of age and the data are summarized in Figure 17. Changing the pH from 6.8 to 7.1 caused only a slight increase in the apparent rates of RNA synthesis, although the apparent rates of protein synthesis were extensively affected (Figure 13). Again, in contrast to protein synthesis, the maximal rate in RNA synthesis occurred on day 18, one day before the transition in protein synthesis. From day 15 to day 18, the rate of RNA synthesis increased 5-fold at pH 6.8 and 2.5-fold at pH 7.1. Calculating the rates of [3H] uridine incorporation per cell instead of per microgram of protein did not alter the rate curve; maximal rates were attained after day 18 of gestation (Figure 18) . Relative to DNA content, the maximal apparent rate of RNA synthesis occurred a day before the maximal rate in protein synthesis; relative to protein content, the apparent maximal rate of synthesis occurred on the same day. These data, therefore, suggest that a transition in both the apparent rate of RNA synthesis and the apparent rate of protein synthesis occurs in the embryonic rat pancreas midway through cytodifferentiation. Figure 17. 137 Apparent rates of [3H] uridine incorporation per microgram of protein. The average of a maximum and minimum slope was taken for each incorporation curve for TCA precipitable counts at pH 6.8 (Figure 7) and pH 7.1 (Figure 14), and plotted as a function of embryonic age. (o——-c) pH 7.1, (o---o) pH 6.8. The bars indicate the maximum and minimum slopes. 138 \ \ \ ‘\ \ \ _ . _ 1. I. \- 6 . 5 4 3 2 149 v 2.: 5208 03.2% ”125.5 21.1 ADULT I6 I? I8 IS 20 EMBRYONIC AGE I5 l4 Figure 18. 139 Apparent rates of [3H] uridine incorporation per cell. The apparent rates in Figure 17 were converted to rates per cell by using the protein per cell values calculated by Rutter, et al. (unpublished data). (o———o) pH 7.1, (o———o) pH 6.8. The bars indicate the maximum and minimum slopes. 140 _ _ _ ._ nlu 8 6 .4 .2 IZ— 1.035348an NEE: EL l5 I6 I? I8 I9 20 EMBRYONIC AGE I4 141 An increased rate of synthesis per cell precede an increased rate of protein synthesis per cell. Electro horetic Analysis of_Pancreatic RNA from Fetal Rat Pancreas Labelled In Vitro at pH 7.1 Anal sis of RNArfrom l4-Day 01d Pancreatic Rudiments RNA was extracted from l4-day labelled with [3H] uridine in_yi3£g at pH 7.1 and electrophoretically analyzed on an agarase-acrylamide gel. This experiment is summarized in Figure 19. The photograph of the gel indicates that each of the major rRNA species, as well as 48 RNA, are dis- tinct bands. There is little evidence for degradation, even after 24 hours in culture. After the gel was photographed, it was cut into 5 mm strips, each of which corresponded with a sample slot. Representative electropherograms were scanned to determine the position of 288 and 18S rRNA as described in METHODS. The strips were cut into sections of approximately 1 mm and the distribution of radioactivity was determined. These data are summarized by Figure 20. Within 4 hours of culture in labelled medium, peaks corresponding to 288 and 183 RNA could be readily distinguished. After 8 hours of continuous labelling there was little variation in the distribution of radioactivity. Throughout the labelling period, a large number of counts migrated with an apparent Figure 19. 142 Photograph of RNA from 14-day pancreases cultured for yarious intervals at pH 7.1 in 5 pCl/ml of [ H] uridine. RHA was extracted from 150-200 pg of tissue as described in METHODS. Slot 1 contained RNA from adult liver (marker); slot 2, RNA from l4-day pan- creas cultured for 24 hours; slot 3, l4-day pancreas cultured for 12 hours; slot 4, RNA from l4-day pancreas cultured for 8 hours with adult liver RNA as carrier; slot 5, RNA from 14-day pancreas cultured for 4 hours and adult liver RNA. 143 (”13) BONVISIO O V (I) I I I In .Il:-° , ”I'M 3 '5. 58— 48-- I I I < m a) z co co C) N - Figure 20. 144 Distribution of radioactivity in RNA from 14- day fetal pancreas maintained at pH 7.1. The gels shown in Figure 19 were cut into 5 mm wide strips, cut into 1 mm slices and counted for radioactivity. Representative gels were scanned to indicate position of 28S and 188 RNA. Labelling times were: A, 4 hours; B, 8 hours; C, 12 hours, and D, 24 hours. (o———o) counts per minute, (----) absorbance. 1 ..... V 5:on . mozqmmommq 9 3 b a mu 145 AAA 1 A IA- *1 l5202530354045505560657075 IO 5 AI:~.o_ xv .28 S B IIIII lat II I. I. a A m ... IN; I. A IIIIIIIIIIIIIIIIIIIIIII I I W «.IHHIIIIIIIHHH IIIIIIIIIIIIIIIIII Ill, IL I, m... p I _ - A L _ AI» A _ _ u _ I O 3 2 I 4 4 0 6 nI/I. 8 4. 2 2 I SLICE NUMBER 146 molecular weight greater than 288. Because the division time for mammalian cells in culture is 12 to 24 hours, it is unlikely that the counts observed in this high molecular weight region of the gel at 4 and 8 hours are in DNA. This suggested that the counts migrating with an apparent molecular weight greater than 288 at 4 and 8 hours are probably in RNA, at the later times, they are probably in DNA. During the extraction of labelled RNA, some of the denatured protein at the interphase of the phenolic and aqueous phases is removed with the small volume of aqueous phase. This insoluble contaminate contains trapped [3H] uridine and represents the high number of counts at the origin of the gels. Green, gt_§l. (71) noted that the amount of labelled RNA varied by more than Z-fold for a given set of mammary gland explants undergoing hormone induced differentiation. To compare radioactive profiles of labelled RNA for dif- ferent sets of tissue, they expressed their results as percent of total counts per minute. To provide a compar- able analysis, the data from Figure 20 was treated simi- larly and are summarized in Figure 21. Figure 22 compares the percent of counts per minute to the absorbance of stained gels. A relatively large percentage of count migrates with a molecular weight greater than 288. As mentioned earlier, the counts are assumed to be in RNA because of the early labelling. However, the percentage I” it...” . II. I . -. '. l. Figure 21. 147 Percent distribution of radioactivity in 14- day pancreases labelled with [ H] uridine for 4 hours. The data from the 4 hour profile of Figure 20 were converted to percentages and plotted with an absorbance scan from that stained gel. 148 lurid-turgil t... 1.3-15: Rm . moz