N THE UT} BY MALARTIA PARASHES Thesis for the Degree of Ph.. D. < 'MTGHTGAN STATE UNIVERSITY NORMAN E. KELKER 1967 LlZATTON 0F NUCLETC ACID PRECURSDRS . The: an s This is to certify that the thesis entitled THE UTILIZATION OF NUCLEIC ACID PRECURSORS BY MALARIA PARASITES presented by Norman E. Kelker has been accepted towards fulfillment of the requirements for Ph.D. degree in Microbiology Major professor Date November 17, 1967 0-169 THE UTILIZATION OF NUCLEIC ACID PRECURSORS BY MALARIA PARASITES By .\ Norman E. Kelker A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1967 (3 ,_ a“) o?— 3.) /. -. ABSTRACT THE UTILIZATION OF NUCLEIC ACID PRECURSORS BY MALARIA PARASITES By Norman E. Kelker Studies carried out in this thesis indicate that the erythrocyte nucleus is not utilized by avian malaria parasites for nucleic acid synthesis as previously suggested by some authors (13, 24). Parasites which completed growth and division in avian erythrocytes whose deoxyribonucleic acid was heavily labeled with tritiated thymidine did not become labeled. Further, the DNA content of E. lOphurae-infected blood did not decrease but showed a slight increase during the course of infection. The total nucleic acid content (RNA plus DNA) of heavily infected blood cells was found to be approximately twice that of uninfected blood cells in- dicating that the parasites synthesize nucleic acid precursors and/or utilize a source other than erythrocyte DNA for nucleic acid synthesis. Plasmodium lophurae in ducks and chickens, 2. gallinaceum, in chickens and 2. berghei in mice failed to incorporate intravenously administered tritiated thymidine. Scintillation counting and autoradiography were used to detect in vitro parasite utilization of purine and pyrimidine compounds for nucleic acid synthesis. Intraerythrocytic parasites showed marked incorporation of adenosine and 2'- Norman E. Kelker deoxyadenosine into RNA and DNA and they showed weak incorp- oration of adenine, orotic acid, uridine, cytidine, and 2'- deoxyguanosine into RNA. No incorporation of thymine, thymi- dine, thymidine-S'-monophosphate, cytosine, or uracil was de- tected. Similar results were obtained with parasites isolated free of erythrocytes except that little or no orotic acid in- corporation occurred whereas increased uridine incorporation was observed. Also no incorporation of 2'-deoxyguanosine by free parasites was observed. Tritiated guanosine monophosphate was recovered from RNA of infected erythrocytes labeled with either tritiated adenosine or tritiated Z'adeoxyadenosine indicating that these compounds are converted, presumably by the parasite, to guanosine monophosphate. ACKNOWLEDGEMENTS The author wishes to eXpress his sincere thanks to Dr. David T. Clark for his help and guidance throughout the course of these studies. ii TABLE OF CONTENTS Chapter Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . 2 MATERIALS AND METHODS . . . . . . . . . . . . . . . . 8 RESULTS . . . . . . . . . . . . . . . ... . . . . . . 20 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 30 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . 37 A. Summary . . . . . . . . . . . . . . . . . . . 37 B. Conclusions . . . . . . . . . . . . . . . . . 39 LITERATURE CITED . . . . . . . . . . . . . . . . . . . 40 iii Table LIST OF TABLES Tritiated purine and pyrimidine compounds used in Experiments 4 and 5 . . . . . . . . . . . . . Incorporation of tritiated purine and pyrimidine compounds by intraerythrocytic P. lophurae . . . Incorporation of tritiated purine and pyrimidine compounds by P. lthurae parasites isolated free of host erythrocytes . . . . . . . . . . . . . . Nucleotide analysis of HB-adenosine- and H3- 2'-deoxyadenosine-labeled RNA from Plasmodium lthurae-infected erythrocytes . . . . . . . . . iv Page 16 25 28 29 LIST OF FIGURES Figure Page 1 Photographs showing an autoradiograph of a '2. lo hurae parasite that has undergone growth and nucIear division in an H3-thymidine-labeled duck erythrocyte. . . . .». . . . . . . . . . . . 21 2 Measurements of DNA and RNA in blood cells during the course of Plasmodium lophurae infection in a duck. Measurements made prior to initiation of infection are plotted at zero hours . . . . . . . 23 INTRODUCTION During its life history the malaria parasite resides in the mosquito vector, in cells of the reticuloendothelial system of the vertebrate host and in erythrocytes or reticula- cytes of the vertebrate host. In all host cells the parasite undergoes rapid growth utilizing host materials and competing with the host for metabolites. The utilization of hemoglobin by intraerythrocytic stages as a source of amino acids for protein synthesis (12) and the depletion of host glucose and glycogen by malaria parasites in monkeys (11) are classical examples. This study was undertaken to determine whether, as suggested by some authors (13, 2h),asexually reproducing stages of avian malaria parasites utilize host erythrocyte deoxyribonucleic acid for nucleic acid synthesis and to determine whether any purine or pyrimidine compounds con- tribute significantly to parasite nucleic acid synthesis. LITERATURE REVIEW Several Species of the genus Plasmodium have been used experimentally to investigate intracellular parasitism. These species produce heavy infections in laboratory animals providing large numbers of intraerythrocytic parasites for study. The intraerythrocytic stages of Plasmodium have been used extensively to investigate the relationship between the parasite and its host cell. These studies include the general physiology and nutrition of intracellular parasitism as well as the resistance and immunity that may result from such infections. Further, the phenomena of susceptibility and resistance to chemotherapeutic agents have been investigated. Of the numerous species of the genus Plasmodium, E. lophurae in ducks,'£.gallinaceum in chickens and P. berghei in rodents have been used most extensively in the numerous laboratory investigations. Their use is directly related to the ease in maintaining the parasite in laboratory animals. Plasmodium lephurae was described from the Fire Back pheasant in Borneo by Coggeshall in 1938 (10). In this species the asexual erythrocytic forms have a generation time of 36 hours and, when maintained under light controlled conditions, good synchrony of growth and division is ob- served (36). The domestic duck has been found to serve as 'mn excellent host. Plasmodium gallinaceum, described by Brumpt (8) from Jungle fowls in 1935, can be easily maintained in Chickens. It has a 36 hour generation time, but it does 2 3 not display a high degree of synchrony. The rodent malaria ‘2. berghei (38) shows a marked preference for reticulocytes and grows asynchronously with a generation time of 24 hours in laboratory mice. Metabolic studies dealing with these three Species, reviewed recently by Moulder (2A), Garnham (13) and Rollo (27), have been concerned largely with the nutrition and carbohydrate metabolism of the parasite and many aspects of parasite physiology have been studied very little. Studies of nucleic acids and nucleic acid metabolism have been especially infrequent. I The literature review which follows includes the infor- mation available and pertinent to nucleic acids in the genus Plasmodium. The review includes those publications which provide a background for the research reported in this thesis. A positive Feulgen reaction is easily demonstrated in erythrocytic schizonte which have compact nuclei (16). Erythrocytic trophozoites have diffuse nuclei and this may explain why Pawan (26) was unable to detect Feulgen positive material in'g. falciparum and‘g.‘gixg§. The cytoplasm of the parasite is rich in RNA. Ribonuclease treatment of smears of infected blood prior to staining completely eliminates the basOphilic prOperties of the parasite cyto- plasm (19). Electron micrographs reveal that the cytOplasm contains large numbers of ribosomes attached to an endo- plasmic reticulum (1, 17, 28). Bahr (A) has carried out a quantitative determination 4 of the nucleic acid contents of‘P. loghurae and P. berghei. Using a cytofluorometric analysis he estimated that a‘g. berghei nucleus contains 0.5 X 10"13 grams of DNA and that the trOphozoite contains roughly twice as much RNA as DNA. A'P. loghurae nucleus was estimated to contain 1.3 X 10"13 gram DNA or approximately 5 percent that of the host erythro- cyte. Plasmodium berghei RNA is rich in purines; the purine to pyrimidine ratio is l.h. Deoxyribonucleic acid, as expected, has a purine to pyrimidine ratio very close to 1.0. Adenine, guanine, cytosine, uracil, and thymine are present in the nucleic acids and no hydroxymethylcytosine has been detected (39). Ball et. al. (5) analyzed.§. knowlesi-infected erythro- cytes maintained both in vivo and in vitro and reported large increases in total nucleic acid content during para- site growth. Similar results were obtained by Whitfield (39) who reported that mouse erythrocytes showing a 25 per- cent infection, i.e., 25 parasites/100 erythrocytes, contained about 20 to 25 times as much DNA as uninfected blood cells. Lewert (19) carried out a similar quantitation of RNA and DNA in chicken blood cells infected withlg. gallinaceum. Daily analysis from initiation of infection until death of the chicken revealed on some days an increase and on other days a decrease in the DNA content of infected blood. It appeared that blood predominately infected with actively growing trophozoites had a DNA content less than that of uninfected blood whereas blood predominately infected with 5 multinucleate forms had a DNA content greater than that of uninfected blood. Stained preparations of infected erythro- cytes showed pyknotic nuclei and it appeared that the loss was due to a partial destruction of the red cell nucleus. By the use of a microspectrOphotometer (20) the absorb- ance at 265 millimicrons of individual erythrocyte nuclei of infected and uninfected erythrocytes was measured. Nuclei of infected erythrocytes showed a lower absorbancy than uninfec- ted erythrocytes. It has been suggested that the DNA of the erythrocyte nucleus may serve as a source for the synthesis of nucleic acids by avian plasmodia (13, 24). Whitfield (40) utilized PEZ-phosphate to study the turn- over and synthesis of phosphorous-containing compounds in the parasite. Labeled phosphate injected into'P. berghei- infected mice was rapidly incorporated into parasite DNA and RNA. Analysis of P32-containing parasite fractions at intervals from 2 to A8 hours following administration of the label showed that the DNA contained 3.5 percent of the total P32 present in the parasites at the end of 2 hours and 24 percent at the end of 48 hours. Throughout the 48 hours 10 percent of the P32 was present in the RNA fraction; although the total P32 content of the parasites increased fourfold. Carbon dioxide is rapidly incorporated into DNA and RNA <>f12. lophurae (3A). A sixty minute incubation of para- :aitized erythrocytes in saline or buffer with a 01402 gaseous pfluase results in significant labeling of the nucleic acids. Other areas of malaria research, although not always 6 directly concerned with nucleic acid metabolism, should be considered for their implications in nucleic acid synthesis. Folinic acid (N5—IO methylenylfolate-Hu) has been shown to enhance the growth of P. lophurae cultured free of the duck erythrocyte (35). Folate compounds are synthesized from, among other precursors, para-aminobenzoic acid (22) which is a requirement for growth of malaria parasites cultured with- in erythrocytes (3). It is well known that folinic acid acts as a methyl donor in the synthesis of purine and pyrimidine nucleotides (15), and Siddiqui and Trager (33) have shown that there is a marked increase in the folinic acid content of'E. lophurae parasitized duck erythrocytes correlated with development from the uninucleate to the multinucleate stage. The antimalarial pyrimethamine exerts its effect by inhibiting the activity of dihydrofolate reductase (14) an enzyme necessary for the synthesis of folinic acid (22). This results in a marked inhibition of DNA synthesis but not of RNA synthesis as demonstrated by P32 uptake experiments (30). It appears that folinic acid is necessary for DNA synthesis and presumably for the production of thymidine- 5'-mon0phosphate in the thymidylate synthetase reaction (6). The studies of asexual erythrocytic stages of malaria parasites reported here were carried out in large part using tritiated purine and pyrimidine compounds. The incorporation of these compounds into cellular nucleic acid means not only that the cell possesses the enzymes necessary to utilize the compound but that the cell is permeable to the compound, 7 and that the compound is able to mix successfully with any intracellular pools. The failure of a compound to be utilized does not necessarily mean that the cell lacks the necessary enzyme(s); but nonutilization may be due to im- permeability, a failure to mix with intracellular pools, or repression or other metabolic control. And, as in all studies utilizing radioactive compounds, the assumption must be made that radioisotopic labeling does not alter the biological properties of a compound. MATERIALS AND METHODS The Materials and Methods section of this thesis is presented in two parts. The first describes materials and procedures that were generally and routinely used. The second includes a description of those materials and methods pertinent to each experiment. The strain of 2. berghei used in these studies was KBG 173. It was maintained in laboratory mice by intra- venous or intraperitoneal inoculation of infected blood every 14 to 21 days. Plasmodium lophurae was obtained from Dr. William Trager of Rockefeller University in May, 1965. It was maintained by inoculation of 0.5 ml of heavily infected blood into the jugular vein or femoral vein of pekin ducklings less than 8 weeks old every 5 to 7 days. Plasmodium gallinaceum was maintained in 6 to 8 week old White Rock chickens and was transferred every 4 to 7 days by intravenous inoculation. Trypanosoma duttoni, used in these studies as an indicator of the presence of tritiated thymidine in the bloodstream of mice, produces mild, non- fatal, and low level infections in laboratory mice. It was maintained in white laboratory mice and was transferred every 2 to 4 weeks by intraperitoneal injection of infected blood. The mice used in these studies were the Swiss-Webster strain of white laboratory mice. They were bred and main- 9 tained at the Department of Microbiology and Public Health, Michigan State University. Domestic pekin ducklings were purchased at the age of one day or two weeks from either the Tulip City Duck Farm, Holland, Michigan or from Ridgeway Hatcheries, La Rue, Ohio. White Rock chickens used to maintain 2. gallinaceum were obtained from the Poultry Science Farms at Michigan State University. One day old White Rock chickens used in experiments were hatched in a 40°C incubator from fertile eggs obtained from the Poultry Science Department Hatchery, Michigan State University. Dirty glassware which accumulated was soaked in tap Water containing detergent (Haemosol). It was then washed and scrubbed in fresh tap water-detergent and rinsed three times in tap water. Any remaining detergent was removed by another rinse in tap water containing approximately 0.1 percent glacial acetic acid. The acetic acid was removed by one rinse in tap water followed by two rinses in distilled Water. Reticulocytes were stained using New Methylene Blue (Biological Research, Inc.). A capillary hematocrit tube was one third filled with a washed suSpension of erythro- cytes and then completely filled with New Methylene Blue Staining Solution. The capillary tube was plugged and placed upright in a clay hematocrit tray so that the blood cells settled through the stain. After 30 minutes the capillary tube was broken and a drop of cell suspension was placed on a slide and smeared. Blood cell counts were made utilizing a Coulter Elec- lO tronic Particle Counter, Model A, with a 100 micron aperture. Cells were suspended in saline and diluted to concentrations within the counting range of the instrument. The nucleic acids from Plasmodium lophurae-infected erythrocytes were prepared for analysis according to the procedure outlined in Merchant et. al. (23) from approximately 108 blood cells. After removal of acid soluble phosphate compounds with cold 0.5 N perchloric acid and removal of lipids with 3:1 ethanol-ether RNA was removed by an 18 hour incubation in 0.3 N KOH. DNA was removed in hot (90°C) perchloric acid. Quantitative determination of DNA was done colori- metrically using a diphenylamine reagent (2). Colorimetric determination of RNA was done using orcinol reagent (23). Optical density measurements were made with a Beckman DU spectrophotometer. The procedure used for autoradiography was derived from the methods described by Leblond et. al. (18). All opera- tions were carried out in a darkroom using a Wratten series 2 safelight. Slides were dipped in Kodak NTB2 emulsion which had been liquefied in a 47°C water bath. The slides were placed upright on a tray of soaked kleenex in an incubator at 28°C. In this way the emulsion did not accumulate at the base of the slide but was absorbed by the kleenex and a coating of more uniform thickness was obtained. The dried slides were removed from the incubator after 30 minutes and placed in slide boxes containing small cheesecloth bags of indicator drierite (CaSOQ). The slide boxes were wrapped in 11 aluminum foil, labeled, and placed in a refrigerator for the designated period of exposure. Photographic processing of the emulsion was completed using a Wratten series 2 safelight and all solutions were used at 17°C. The slides were placed in Kodak Dektol deveIOper for 2 minutes, rinsed in distilled water, fixed in Kodak fixer for 5 minutes, and washed in distilled water for 20 minutes. The slides were air dried and stained with Giemsa stain in the same manner as routine blood smears. Staining could not be done prior to coating of the slides since it was found the Giemsa stain caused considerable chemical fogging of the emulsion. Experiment 1: Test for the incorporation of erythrocytic DNA by Plasmodium lophurae and Plasmodium gallinaceum using tritiated thymidine. The erythrocytic DNA of one day old chickens and a one day old duck was labeled by intraperitoneal injection of tritiated thymidine (Tracerlab, specific activity;6 curies/ millimole). Four chicks and one duckling received 150 micro- curies each. Twenty four hours later two of the chicks and the duck were infected with.2. lophurae and two chicks were infected with‘P.gallinaceum. Blood smears were taken at approximately 12 hour intervals until the death of the host or, in the case of the chicks which recovered fromlg. lo hurae, until the parasitemia dropped to less than one parasite per 100 erythrocytes. The slides were coated with photographic emulsion and exposed for one week at 4°C. 12 Experiment 2: Measurement of RNA and DNA in blood cells in- fected with Plasmodium lophurae. A four week old duck was infected with washed erythro- cytes prepared from 1.5 ml of heavily infected blood and kept in a room with the lights on for 13 hours and off for 11 hours each day in order to obtain synchrony of parasite growth (36). Blood samples were taken 24 hours and one hour prior to initiation of infection and daily thereafter until a level of 21 parasites per 100 erythrocytes was reached. Samples were then taken at approximately 8 hour intervals until the death of the bird. Immediately after each sample was taken the blood cells were washed 3 times in saline and resuspended in 5 m1 of saline. Two 0.1 ml aliquots were removed and placed in the deep freeze. The cells were counted with a Coulter Counter and preparations were made for microscopic examina- tion to determine growth and percent reticulocytes and leucocytes. Experiment 3: Test for the incorporation of tritiated thymidine by Plasmodium lophurae, P. gallinaceum, P. ber hei, and Trypanosoma duttoni in vivo. Two mice were infected with‘P. berghei and when the parasitemia reached a level of approximately 20 parasites per 100 erythrocytes, tritiated thymidine (Volk Biochemical Com- pany, specific activity 3.5 curies/millimole, in 10 percent 2-pr0panol) mixed with phosphate buffered saline at pH 6.8 was inoculated intraperitoneally at a dose of 5 microcuries per gm. body weight. One of the mice received additional 13 injections of thymidine at 12, 24, and 36 hours following the first. Blood smears were taken at 12 hour intervals for 6 days. The smears were coated with photographic emulsion and exposed for l, 2, and 3 weeks. A similar procedure was followed with P. lthurae-in- fected ducks. Two one day old ducks were infected and kept under light controlled conditions to obtain synchronous parasite growth. When the majority of the parasites were commencing nuclear division and when the parasitemia was approximately 50 parasites per 100 erythrocytes, tritiated thymidine (Tracerlab, specific acitivity 6.0 curies/milli- mole) was administered intravenously at a dose of 5 micro- curies per gm. body weight. Blood smears were taken one hour later and at 12 hour intervals until the death of the birds. The smears were coated with photographic emulsion and exposed for 7 and 14 days at 4°C. A one day old chicken was infected with P. gallinaceum and when the parasitemia reached 50 parasites per 100 red blood cells tritiated thymidine (Tracerlab, Specific activity 6.0 curies/millimole) was prepared in phosphate buffered saline (pH 6.8) and administered intravenously at a dose of 5 microcuries per gm. body weight. Blood smears were taken at 12 hour intervals thereafter. The slides were coated with photographic emulsion and exposed for 7 and 14 days. In order to determine the presence of tritiated thymi- dine in the bloodstream of mice, a mouse was infected with Trypanosoma duttoni and when the parasitemia reached a level of approximately one parasite per 100 red blood cells 14 5 microcuries per gm body weight tritiated thymidine was inoculated intraperitoneally. Blood smears taken at 12 hour intervals thereafter were coated with emulsion and eXposed for one week. Experiment 4: Incorporation of tritiated purine and pyrimi- dine compounds by 3. lophurae incubated within erythrocytes. The medium used in this eXperiment is a modification of Trager's buffer (31). It was prepared immediately prior to use and sterilized by filtration through a Seitz sterilizing filter. The final pH was 7.4. Its components are: Mg50437H20 .370 gm/liter MgClZ-6H20 .400 gm/liter NaCl 3.3 gm/liter KCl 4.4 gm/liter NaBHPo4 2.9 gm/liter KHZPo,+ .670 gm/liter NaHCO3 .330 gm/liter D-glucose 2.5 gm/liter penicillin 50,000 units streptomycin 50,000 units phenol red indicator. A four week old duck was infected with P. lophurae and maintained under light controlled conditions (36) to obtain synchrony of parasite growth. When the parasitemia exceeded 100 parasites per 100 erythrocytes and when the majority of the parasites were commencing nuclear division 10 ml of blood was removed in a heparinized syringe. Five m1 of blood 15 was removed from an uninfected duck of the same age. Both samples were washed three times in buffer at 40°C. Following centrifugation associated with each washing the buffy coat was discarded. After washing and removal of DNA and RNA synthesizing leucocytes, the red cells were resuspended in buffer, counted, and distributed in 0.5 ml quantities to sterile 25 ml Erlenmeyer flasks containing 0.5 ml buffer and 20 microcuries of a labeled compound. The tritiated com- pounds used are listed in Table 1. The flasks were placed on a shaker which traversed a linear 1.5 inch path at the rate of 75 oscillations per minute for 5 hours at 40°C. Following incubation the cells in each flask were washed four times in 6 ml quantities of 0.9 percent saline. Smears of the washed cells were made and saved for auto- radiography. DNA and RNA were extracted from the remaining cells as previously described (23). The radioactivity in the DNA and RNA samples was deter- mined with a Packard Tri Carb liquid scintillation counter which counts tritium with 40 percent efficiency. One-half m1 of each sample was placed in a glass counting vial (Packard tm solubilizer (Nuclear Instrument Corporation). Two ml of NOS Chicago Corporation), a quaternary ammonium base which re- duces quenching in aqueous samples, was added. Fifteen m1 of scintillation fluid composed of 0.4 percent 2,5- diphenyloxazole (PPO, Packard Instrument Corporation) and 0.01 percent 1,4-di (2-(5-diphenyloxazole))benzene (POPOP, Packard Instrument Corporation) was added and the samples were counted. l6 TABLE 1. Tritiated purine and pyrimidine compoundsa used in EXperiments 4 and 5 1 Position of Specific tritium in activity the purine Radio- in curies/ or pyrimi- chemical Compound Jmillimole dine ring purity Source Adenine . . . . T 2.1 2,8 99% Tracerlah' Cytosine . . . 1 2.38 4,5 98% Tracerlab Uracil. . . . . A 5.6 4,5 -- Tracerlab Thymine . . . . . 7.6 5-methy1 -- Tracerlah Orotic Acid . . I 7.0 5 -- Nuclear Chicago Adenosine . . . g 6.0 --b 99% Schwarz Cytidine. . . . J 6.0 -- 99% Schwarz Uridine . . . . 1 5.0 -- 99% Tracerlab 2'-deoxyadenosina 3.5 -- 99% Schwarz 2'-deoxycytidinej 5.0 -- 99% Schwarz 2'-deoxyguanosine 4.8 8 98% Schwarz Thymidine . . . 4 6.0 5-methyl -- Tracerlab Thymidine-5'- monOphOSphate . 4 5.2 5-methyl 96% Schwarz aThese compounds were supplied in sterile aqueous solutions and stored in a refrigerator. bInformation was not given by the supplier. 17 Prior to autoradiography the slides were fixed in absolute methanol for one minute. For each experimental condition 3 slides were prepared. One was treated with deoxyribonuclease (Calbiochem, B grade, 38,250 dornase units per mg) solution containing 500 microgram/ml DNAase, 0.05M M5012, and .0025M CaCl2 for 1 hour at room temperature and another was treated with a solution containing 333 micro- gram/ml ribonuclease (crystalline, Nutritional Biochemicals Corporation) for one hour at room temperature. The enzyme treated Slides and the remaining untreated slide were soaked in 0.5N HClO at 4°C for 30 minutes (32) to remove soluble 4 nucleotides. After 3 rinses in distilled water the slides were dried at room temperature, coated with emulsion, and exposed for three weeks. ExperimentiS: Incorporation of tritiated purine and pyrimi- dine compounds by Plasmodium 10phurae isolated free of the host erythrocyte. Parasites were isolated free of their host erythrocytes according to the method of Sherman and Hull (31). A four week old duck was infected with P. lthurae and when the parasitemia reached approximately 120 parasites per 100 erythrocytes and when most of the parasites were commencing nuclear division 40 m1 of blood was removed. The blood cells were washed three times in buffer at 40°C and resuSpended in 4 ml buffer containing 0.15 percent saponin in a 100 m1 Erlenmeyer flask. The flask was incubated on a shaker moving at a speed of 75 linear oscillations per minute in a 1.5 18 inch path for 30 minutes. The cells were transferred to a 12 ml conical polyethylene tube and centrifuged at full speed on a clinical centrifuge (Clay Adams Safety Head) for 15 minutes. The dark brown top layer containing the para- sites, most of which were attached to erythrocyte nuclei, was removed and resuspended in 10 ml buffer. Separation of the parasite from its host cell nucleus was accomplished by adding 2 mg deoxyribonuclease (Nutritional Biochemicals Corporation, 38,250 dornase units/mg) to the suspension and incubating it at 40°C for 15 minutes. The suspension was washed 3 times in buffer and with each centrifugation the tap brown parasite layer was removed and transferred to another tube. Giemsa stained smears showed that there were approximately 12 erythrocyte nuclei per 100 parasites re- maining in the final preparation. The free parasites (a volume of approximately 0.25 ml) were divided among eleven 25 m1 Erlenmeyer flasks, each containing 1 ml of buffer. .The tritium labeled compounds (described in Exp 4) were added at a concentration of 20 microcuries per ml and the flasks were incubated on a shaker at 40°C for 5 hours as previously described. The parasites were washed 4 times in saline, smears were made, coated with emulsion and exposed for 3 weeks at 4°C. Experiment 6: Nucleotide analysis of HB-adenosine- and H3-2'-deoxyadenosine-labeled RNA from Plasmodium lophurae- infected erythrocytes. 19 The 3'-(2')-ribonucleotides obtained from KOH hydro- lysis of HB-adenosine- and H3-2'-deoxyadenosine-labeled erythrocytes in Experiment 4 were adsorbed to charcoal, washed free of salts, separated by two dimensional chromatography and counted in a scintillation counter. The above was done in the following manner. The hydrolysates were adjusted to pH 6.0 with phOSphate buffer and acidified charcoal was added. After five washes in 5 ml of glass distilled water the nucleotides were re— covered from the charcoal by a wash in 80 percent ethanol- one percent NH#0H and two additional washes in 50 percent ethanol-one percent NHAOH. The supernates were combined, evaporated to dryness and the nucleotides were dissolved in 0.5 ml of a carrier solution containing 150 micrograms/0.05 m1 of a mixture of equimolar amounts of 3i-(2')-ribonucleo- tides. Fifty lambda was Spotted on Whatman No. 1 paper and two dimensional chromatography was done in isoprOpanol:HCl: H20 (195:50355) and g-propanol:ammonia:H20 (60:30:10). Spots were detected with a Mineralight UV lamp and identi- fied by comparison with standards. Nucleotides were re- covered from the paper by two overnight elutions in 0.1N HCl. The eluates were evaporated to dryness, 0.5 m1 H20 was added and the radioactivity of each sample was deter- mined as previously described. RESULTS Experiment 1: Test for the incorporation of erythrocytic DNA by Plasmodium gallinaceum and Plasmodium lophurae using tritiated thymidine. Injection of tritiated thymidine into the day old duck and into day old chickens at 24 hours prior to initiation of infection resulted in strong labeling of erythrocyte nuclei. Labeled erythrocytes became numerous in the blood when the parasitemias reached a level of approximately 50 parasites per 100 erythrocytes. No incorporation of radio- activity could be demonstrated in various stages of E. lophurae present in labeled duck or chicken erythrocytes. Exhaustive examination of autoradiographs revealed that in every case the radioactivity remained confined to the erythro- cyte nucleus (Figure 1). Similar results were obtained with .2. gallinaceum in tritium labeled chicken erythrocytes. 20 21 Figure l. The upper photo shows an autoradiograph of a P. 10 hurae parasite that has undergone growth and nuclear div—‘I—_is on 1n an H3-thymidine-labeled duck erythrocyte. The lower photograph Shows the same parasite. The area of the erythrocyte cytoplasm occupied by the parasite is enclosed within the broken line. The dark areas enclosed within the broken line are parasite nuclei and parasite pigment granules. The darkened silver grains are confined to the area above the erythrocyte nucleus. 22 Experiment 2: Measurement of RNA and DNA in blood cells infected with Plasmodium lophurae. Good synchrony of parasite growth was obtained as in- dicated by the nature of the growth curve (Figure 2) thus providing a good pOpulation for evaluating DNA and RNA synthesis. The DNA level does not fall significantly below that observed in uninfected erythrocytes. The RNA concen- tration is observed to increase in relation to increasing numbers of parasites. The total nucleic acid content (RNA plus DNA) of the cells at the final reading (139 hours post infection) is approximately twice that of uninfected erythro- cytes. Reticulocytes and leucocytes constituted less than one percent of the blood cells throughout the experiment. Experiment_3: Test for the incorporation of tritiated thymidine by Plasmodium lophurae, P. gallinaceum, P. ber hei, and Trypanosoma duttoni in vivo. Although leucocytes of all hosts and erythrocytes of infected ducks and chickens showed marked incorporation of tritiated thymidine, no radioactivity could be detected in any of the three Species of Plasmodium studied. 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