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A Comparison of in Vitro Nucleic Acid Metabolism of Normal Bovine Red Blood Cells and Red Blood Cells from Calves Infected with Anaplasma Marginale

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A Comparison of in Vitro Nucleic Acid Metabolism of Normal Bovine Red Blood Cells and Red Blood Cells from Calves Infected with Anaplasma Marginale Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1973 A Comparison of in Vitro Nucleic Acid Metabolism of Normal Bovine Red Blood Cells and Red Blood Cells From Calves Infected With Anaplasma Marginale. Michael Edward Billups Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Billups, Michael Edward, "A Comparison of in Vitro Nucleic Acid Metabolism of Normal Bovine Red Blood Cells and Red Blood Cells From Calves Infected With Anaplasma Marginale." (1973). LSU Historical Dissertations and Theses. 2516. https://digitalcommons.lsu.edu/gradschool_disstheses/2516 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. INFORMATION TO USERS This material was produced from a microfilm copy of the original document. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the original submitted. The following explanation of techniques is provided to help you understand markings or patterns which may appear on this reproduction. 1.The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting thru an image and duplicating adjacent pages to insure you complete continuity. 2. When an image on the film is obliterated with a large round black mark, it is an indication that the photographer suspected that the copy may have moved during exposure and thus cause a blurred image. You will find a good image of the page in the adjacent frame. 3. When a map, drawing or chart, etc., was part of the material being photographed the photographer followed a definite method in "sectioning" the material. It is customary to begin photoing at the upper left hand corner of a large sheet and to continue photoing from left to right in equal sections with a small overlap. If necessary, sectioning is continued again — beginning below the first row and continuing on until complete. 4. The majority of users indicate that the textual content is of greatest value, however, a somewhat higher quality reproduction could be made from "photographs" if essential to the understanding of the dissertation. Silver prints of "photographs" may be ordered at additional charge by writing the Order Department, giving the catalog number, title, author and specific pages you wish reproduced. 5. PLEASE NOTE: Some pages may have indistinct print. Filmed as received. Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 74-18,312 BILLUPS, Michael Edward, 1944- A COMPARISON OF IN VITRO NUCLEIC ACID METABOLISM OF NORMAL BOVINE RBC AND RBC FROM CALVES INFECTED WITH ANAPLASMA MARGINALE. The Louisiana State University and Agricultural and Mechanical College, Ph.D., 1973 Microbiology University Microfilms, A XERQ\ Company , Ann Arbor, Michigan THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. A COMPARISON OF IN VITRO NUCLEIC ACID METABOLISM OF NORMAL B O W n E M C AND RBC FROM CALVES INFECTED WITH ANAPLASMA MARGINALE A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Microbiology by Michael Edward Billups B.S., McNeese State College, 1968 M.S., McNeese State University, 1970 December, 1973 Acknowledgment The author wishes to express his sincere apprecia­ tion to Dr. George T. Dimopoullos for his suggestions and encouragement during the experimental work and in prepara­ tion of this dissertation. Acknowledgment is made to Dr. L. T. Hart for his guidance and assistance. The assistance of fellow students and student workers in handling experimental calves and procurement of blood samples is appreciated. Appreciation is also extended to those secretaries and technicians who aided in the preparation of this dissertation. Acknowledgment is made to the Department of Veterinary Science and the Agricultural Experiment Station of Louisi­ ana State University and to the National Institutes of Health for the funds which supported this study. Table of Contents Chapter Page Acknowledgment .............................. ii Table of Contents...............................iii List of Tables .............................. v List of Figures.............................. vi Abstract ..................................... x I Introduction ................................. 1 II Selected Literature.......................... 2 Anaplasmosis.............................. 2 Nucleic Acids ............................ 6 DNA isolation.......................... 6 RNA isolation......................... 8 Base composition analysis............ 10 Hydrolysis.......................... 10 Separation and analysis ......... n Enzymatic assays ..................... 12 Electron microscopy................... 13 Radiotracer methodology ................ 14 III Materials and Methods....................... 16 Experimental animals..................... 16 Hemato logy................................. 16 Preparation of materials................ 17 RBC and plasma ....................... 17 Marginal bodies ....................... 18 Chemically-induced anemia............ 18 Incubation of materials ................ 21 TCA fractionation of RBC................ 21 Hydrolysis and separation of bases. 22 Assay of radioactivity................... 23 Enzyme assay.............................. 24 DNA isolation and sucrose gradient centrifugation.......................... 25 iii Chapter Page RNA isolation and sucrose gradient centrifugation......................... 2 7 Electron microscopy.......................... 30 IV Results and Discussion........................... 32 Introduction .............................. 32 Incubation of materials..................... 32 TCA fractionation and assay of radioactivity .......................... 33 Hydrolysis and assay of radioactivity. 50 Enzyme assay .............................. 59 DNA isolation and sucrose density gradient centrifugation .............. 62 RNA isolation and sucrose density gradient centrifugation .............. 64 Electron microscopy.......................... 69 V Summary and Conclusions ........................ 72 In vitro uptake of labeled precursors. 72 In vitro incorporation of label into individual nitrogenous bases in RBC from normal and infected calves......................................73 In vitro enzyme assay........................74 DNA and RNA isolation........................75 Electron microscopy.......................... 75 VI Literature Cited ..................................77 VII V i t a ................................................85 iv List of Tables Table Page 1 Dosage, injection time and hema­ tologic values, for a calf with anemia induced with phenyl- hydrazine HC1 ............................... 20 2 OMP pyrophosphorylase activity of partially-purified Anaplasma marginale bodies .......................... 6 1 v List of Figures Figure Page 1 Diagrammatic scheme showing pro­ cedure utilizing sucrose density gradients and ultra­ centrifugation to purify Anaplasma bodies ......................... 19 2 Modified scheme for phenolic extraction of DNA from A. m a r g i n a l e .............................. 26 3 Scheme for hot phenol RNA e x t r a c t i o n ................................ 29 14 4 Disappearance of C-hypoxanthme from normal plasma incubated with normal RBC and from normal plasma incubated with RBC from Anaplasma marginale-infected calves................ 34 5 Radioactivity ( C-hypoxanthine) in the cold TCA soluble fraction of RBC from normal and Anaplasma marginale-infected calves. I ............ 36 6 Disappearance of -^C-sodium formate from normal plasma incubated with normal RBC and from normal plasma incubated with RBC from Anaplasma marginale infected calves................ 3 Q 7 Disappearance of ^C-orotic acid from normal plasma incubated with normal RBC and from normal plasma incubated with RBC from Anaplasma marginale- infected calves............................ 3 9 8 Radioactivity (l^C-orotic acid) in the cold TCA soluble fraction of RBC from normal and Anaplasma marginale- infected calves. I 7 ..................... 40 vi Radioactivity (14C-orotic acid) in the cold TCA soluble fraction of RBC from normal and Anaplasma marginale-infected calves. 7~. Disappearance of ^H-uridine from normal plasma incubated with normal RBC, from normal plasma incubated with RBC from Anaplasma marginale- infected calves, and from plasma of infected calves with RBC from normal calves......................... Radioactivity (^H-uridine) in the cold TCA soluble fraction of RBC from normal, and Anaplasma marginale- infected calves, which were suspended in normal plasma; and from normal RBC suspended in plasma from infected calves....................... Radioactivity (^H-uridine) in the cold TCA soluble fraction of RBC from normal and Anaplasma marginale- infected calves which were suspended in normal plasma; and from normal RBC suspended in plasma from infected calves......................
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