ESQLAflO‘H AND CHARACTERéZAfiGR OF THE DEOXYRIBONUCLE!C ACIDS 0F DROSOPHILA MEMNQGASTER Thesis fix fin 3W“ 3’? Fix. D. MWAK STA“??? QE‘EEVERSITY Chasies {3: Mead fiéé This is to certify that the thesis entitled ISOLATION AND CHARACTERIZATION ". OF THE DEOXYRIBONUCLEIC ACIDS OF DROSOPHILA MELANOGASTER presented by flharles G. Mead has been accepted towards fulfillment of the requirements for 31.11. degreein Genetics M Major professor ) Date MEL—2291950 0-169 LIBRAR Y Michigan State University ISOLATION AND CHARACTERIZATION OF THE DEOXYRIBONUCLEIC ACIDS OF DROSOPHILA MELAHQQAfilER "\ By . ,yr \\ 5 Charles 0‘." Mead ; i AN ABSTRACT Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOC TOR OF PHILOSOPHY Department of Agricultural Chemistry 1960 Approved i (@M 93 - FOX ""I ABSTRACT ISOLATION AND CHARACTERIZATION OF THE DEOXYRIBONUCLEIC ACIDS OF DROSOPHILA_MELANOGASTE_R by Charles C. Mead A procedure is described for the isolation of DNA from Drosophila melanogaster. An enzymatic deproteinization procedure is described which results in a product that is free from RNA and protein and not extensively denatured. Two types of DNA are observed upon precipitation of the isolated product with cold ethanol. One of these is fibrous in nature, typical of most DNA's, whereas the other is Of a flocculent nature. The isolated DNA is found to be relatively non- viscous even in concentrated solutions. Since the DNA is not denatured to any large extent, this observation is interpreted to mean that Drosophila DNA has a relatively low molecular weight. Perchloric acid and formic acid hydrolysates of the DNA contain the purines adenine and guanine, and the pyrimidines thymine, cytosine, and 5-methylcytosine. The 5-methylcytosine is characterized both chromatographically and spectrophotometrically. The exceptional pyrimidine, 5-methylcytosine, is not observed when the DNA is subjected to mild alkaline conditions prior to acid hydrolysis. It is suggested that the amino group at the 6 position of the pyrimidine ring of the 5- methylcytosine nucleoside or nucleotide is alkali labile, and that deamination under mild alkaline conditions results in the corresponding thymine derivative. Charles G. Mead Nucleoside preparations of Drosophila DNA hydrolysed with snake venom and subjected to paper chromatography exhibit two unexpected free pyrimidines. These are identified as uracil and thymine. It is suggested that these two pyrimidines result from the degradation of deoxy-S-methylcytidine under acid conditions. Nucleotide preparations of Drosophila DNA hydrolysed with purified snake venom phosphodiesterase and subjected to paper chroma- tography exhibit the unexpected pyrimidine uracil. This pyrimidine also probably results from the degradation of deoxy-S-methylcytidylic acid under acid conditions. Ion exchange chromatography of snake venom phosphodiesterase digests of Drosophila DNA result in the recovery of five UV. absorbing peaks. The second, third, fourth and fifth peaks eluted are identified as deoxycytidylic, deoxythymidylic, deoxyadenylic and deoxyguanylic acids respectively. The first peak eluted is probably deoxy-S-methyl- cytidylic acid but could not be identified as such by rechromatography on paper due to the small quantities of this compound recovered. A quantitative difference in the molar content of 5-methyl- cytosine is demonstrated between the two types of ethanol-precipitated DNA'S. The flocculent type is demonstrated to contain more 5-methyl- cytosine than the fibrous type. ‘1“ flu l ISOLATION AND CHARACTERIZATION OF THE DEOXYRIBONUCLEIC ACIDS OF QEOSOEHILAMELANOCASTER BY Charles G." Mead A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOC TOR OF PHILOSOPHY Department of Agricultural Chemistry 1960 -7 —.'(_. -.-‘< '-. 1.1 '1 T i.- ACKNOW LEDGEMENTS The author expresses his sincere appreciation to Dr. Allen S. Fox for his tireless efforts in guiding the work of this study. The personal interest of Dr. Jean Burnett is also gratefully acknowledged. Acknowledgement is made to the National Institutes of Health for financial support as a Predoctoral Research Fellow. iii»: VII-J ' ‘ -pnkan‘ ._ 9—“, -- In}, It - -,¢ \ C I. II. III. INTRODUCTION ................................... MATERIALS AND METHODS ......................... A. Culture and Collection Methods B. Isolation of Deoxyribonucleic Acid 1. Extraction Procedure 2. Deproteinization and RNA Removal Procedures C. Hydrolysis Methods 1. Chemical Methods 2. Enzymatic Methods D. Chromatographic Methods 1. Column Chromatography 2. Paper Chromatography E. Purification of Nucleic Acid Components with Activated Charcoal 1. Preparation of Charcoal 2. Adsorption and Elution Procedure F. Assay Methods C. Other Materials and Methods RESULTS ......................................... A. Isolation Procedure B. TABLE OF CONTENTS Characterization iii 11 ll 15 .“V .i Purine s and Pyrimidine s 2. Nucleosides 3. Nucleotides IV. DISCUSSION ....................................... V. SUMMARY ......................................... VI. REFERENCES ..................................... 21 24 31 37 39 ‘T able 10. LIST OF TABLES Assay methods ...................................... Composition of product at various stages of the DNA isolation procedure ................................. Recovery of DNA from 1. 0 g. of lyophilized flies by the Duponol extraction procedure ........................ Ultraviolet absorption of DNA preparations ........... RT of purines and pyrimidines from perchloric acid hydrolysates in the isopropanol-HCI solvent .......... RT of purines and pyrimidines from formic acid hydrolysates in the isopropanol-HCI solvent 7 ......... 5- Methylcytosine2thymine ratios in fibrous and flocculent DNA ................................... Chromatographic and spectral constants of compounds isolated from snake venom digests of DNA ........... Molar composition of nucleotides recovered by ion- exchange chromatography from phosphodiesterase digests of Drosophila DNA .......................... Rf1 of nucleotides derived from DNA ................. Page 10 13 14 16 l7 18 2.2 23 29 29 vi LIST OF FIGURES figure Page 1. Ultraviolet absorption spectrum of 5-methylcytosine ..... 19 2. Chromatogram of nucleosides (snake venom hydrolysates). 25 3. Ion-exchange column chromatography of 5'deoxy- {j ribonucleotides ...................................... 27 l C 'JnA:_‘ I‘— :A. a; " I. INTRODUCTION A relationship between the genetic material and nucleic acids has been amply demonstrated in the last twenty years. The relationship of the genetic units of inheritance to the chemical structure of nucleic acids, however, has not been elucidated. The two major difficulties confronting this problem have been the lack of suitable methods for analyzing the structural differences in nucleic acids and the lack of genetically defined nucleic acid. Since suitable techniques are rapidly becoming available, the need for a source of nucleic acid which could be genetically defined with accuracy has become imperative. Of all organisms used in genetic studies, Drosophila melanogaster is the best known genetically. Numerous genetic markers are available which are distributed through its entire genome. Widely varying alterations in chromosomal structure are also available. Genetic backgrounds can be constructed which are identical genetically except for single genes (recons), segments of chromosomes, or whole chromosomes (X, Y, 4). Thus the DNA of Drosophila can be genetically defined with great accuracy and altered genetically with ease. An attempt was therefore undertaken to isolate and characterize the DNA of Drosophila. Very few reports on the nucleic acids of Drosophila have been published. RNA has been isolated and characterIzed for Purines and pyrimidines by Levenbook, Etil' (1958). Recently Kirby 2 (1959) ha. ' 3 Isolated DNA from Drosophila- Guanine, adenine, thymine C t - . . . . and Y 031116 were Identified 1n formic acid hydrolysates of this DNA. addltlon, other components were present in the hydrolysates which In were not identified. I'm-5 11. MATERIALS AND METHODS A. Culture and Collection Methods Stocks of Oregon-R Drosophila melanogaster were grown in half pint milk bottles on standard corn meal-molasseS-agar medium enriched with brewers yeast and seeded with living yeast. Flies were collected after a period of from two to three weeks. The collected flies were frozen, lyophilized, and stored in a freezer at -ZOOC. B. Isolation of Deoxyribonucleic Acid 1. Extraction Procedure Ten grams of lyophilized flies were homogenized with a Davis-Layne Duall tissue grinder in 300 ml. of saline-citrate solution (0. 15 M NaCl and 0.015 M Na3C6HSO7' ZHZO) at 0°C. The resulting homogenate was centrifuged at 10, 000 x g. for 20 minutes at 40C. The supernate was discarded and the residue re-homogenized in 300 ml. of saline—citrate solution. This process of homogenization and centri- fugation was repeated four more times (a total of six homogenizations and centrifugations). After the final centrifugation the residue was homogenized in 300 ml. of 0. 15 M NaCl at 00C. Twenty-seven m1. of 5% Duponol C (sodium lauryl sulfate, E. I. Du Pont de Nemours and Co.) in 45% ethanol was added and the mixture stirred with a magnetic stirrer for 3 hours at room temperature. The mixture was then made 1 M with respect to NaCl, stirred an additional 10 minutes and :9. I i C 4 Cfintrifu ed - 0 . g at 10, 000 x g. for 20 minutes at 4 C. The resulting super- gate was treated with an equal volume of cold 95% ethanol and placed in the cold for 1 hour to assure complete precipitation. The precipitate was collected by centrifugation at 10, 000 x g. for 20 minutes at 40C. and was then ready for deproteinization. 2, Deproteinization and RNA Removal Procedures The deproteinization methods of Sevag Sta—1. (1938) using amyl alcohol and chloroform and of Kirby (1957) using methoxyethanol were utilized and found to be effective. Due to the extreme lability of Drosophila DNA constituents, the following enzymatic deproteinization and RNA removal procedure was elaborated. The ethanol-precipitated DNA from the isolation procedure was dissolved in 150 m1. of O. 15 M NaCl without mechanical agitation. 3. 5 m1. of 5% Duponol C in 45% ethanol was added and the mixture stirred with a magnetic stirrer for 1 hour at room temperature. The mixture was made 1 M with NaCl, stirred 10 minutes, centrifuged at 10, 000 x g. for 2.0 minutes at 4°C. and the supernate precipitated with an equal volume of cold 95% ethanol. The precipitate was collected by centrifugation at 10, 000 x g. for 20 minutes at 40C. and the whole procedure repeated once more. The resulting precipitate was then dissolved in 50 m1. of 0. 15 M NaCl to which was added 10 mg. of trypsin (Worthington, 2X crystalline) in 1. 0 m1. of water. The mixture was incubated at 370C. for 3 hours, made 1 M with NaCl, centrifuged at 0 0 I 00 x g. 1 r, fiqual VOlume of cold 95% ethanol. The precipitate was collected by a ffi'frlfilgation and redissolved in 50 m1. of O. 15 M NaCl to which was c6 added 10 mg. of chymotrypsin (Worthington, 3X crystalline) in l. 0 m1. of Water. The mixture was incubated for 3 hours at room temperature after which it was made 1 M with NaCl, centrifuged at 10, 000 x g. for 20 minutes at 40C. and the resulting supernate precipitated with an equal volume of cold 95% ethanol. The precipitate was collected by centrifugation and dissolved in 50 ml. of O. 1 M ammonium acetate- ammonium hydroxide buffer at pH 5. 0 (0. l M with respect to acetate). 10 mg. of ribonuclease (Worthington, 4X crystalline) in 1. 0 m1. of water was added and the mixture incubated at 370C for 3 hours. The mixture was then made 1 M with NaCl, centrifuged at 10, 000 x g. for 20 minutes at 40C. and the supernate precipitated with an equal volume of cold 95% ethanol. The precipitate was collected by centrifugation and dissolved in an appropriate solvent. C. @drolysi 5 Methods 1. Chemical Methods a. Perchloric acid: DNA was hydrolysed in O. 1 m1. of 70% perchloric acid at 100°C. for 1 hour (Marshak and Vogel, 1951). The hydrolysate was neutralized with KOH, centrifuged in the cold, and the supernate chromatographed. . 0 . . . for 20 minutes at 4 C. , and the supernate preCIpltated With 5 . -- _ '.“,77. ‘V._4 t “L'W‘FFu v i"?! s n' 3",th b. Formic acid: DNA was hydrolysed in 2. 5 m1. of 90% £ofn'lic. acid at 1750C. for 30 minutes (Wyatt and Cohen, 1953) in a 19,58 lined steel bomb. The contents were then evaporated to dryness, g. xaKen Up in 0. 1 m1. of 0. 1 N HCl and chromatographed. 2” Enzymatic Methods a. Deoxyribonuclease: DNA was dissolved in 0. 2 M ammonium acetate-ammonium hydroxide buffer, pH 7. 0,and assayed for total phosphorus (Table 1, No. 14). Magnesium ion was added so that the ratio of Mg++ to phosphorus was 3:1. Deoxyribonuclease (Worthington, 1X crystalline) was then added dissolved in water. The mixture was incubated at 37°C. until digestion was complete as judged by UV. absorption or titration with alkali (Table 1, No. 4 and 5). b. Snake Venom Phosphodiesterase: 500 mg. of lyophilized Crotalus atrox venom (Ross Allen‘s Reptile Institute) was processed by the Razzel—Khorana (1959) modification Of the Koerner-Sinsheimer (1957) method. The resulting preparation was Chromatographed on DEAE cellulose by the method Of Boman and Kaletta (1957). The phosphodiesterase was recovered in two peaks followed by a third peak of S'nucleotidase. Only the first phosphodiesterase peak was used for enzymatic hydrolysis Of DNA since the second peak was slightly con- taminated with 5'nucleotidase. A deoxyribonuclease limit digest was adjusted to pH 8. 9 by the addition of O. 2 M ammonium acetate-ammonium hydroxide 7 ifer pH - - ' ‘}. ’ 8. 9. Phosphodlesterase was added and the mixture 1ncubated o 97 C- Until digestion was complete as judged by UV. absorption. The at eSt was then either applied directly to an ion-exchange column or 3.1 adéorbed to charcoal and eluted for paper chromatography. c. Whole Snake Venom: Whole venom was used to degrade DNase limit digests to free nucleosides. The digestion procedure used was identical to that used with the purified snake venom phosphodiesterase. D. CHROMATOGRAPHIC METHODS 1. Column Chromatography The method of Sinsheimer and Koerner (1951) was modified by using a gradient elution system (Bock and Ling, 1954) rather than the original stepwise elution system. 2. Paper Chromatography Descending chromatography using Whatman No. 1 filter paper was used exclusively. Papers were routinely washed with 0. l N HCl for 36 hours followed by distilled water for 2.4 hours and dried in air at room temperature. The following solvents were used: a. n-butanol Saturated with Water in an Ammonia Atmosphere (Hotchkiss, 1948): n-butanol and distilled water in the ratio of 2:1 by volume were shaken in a separatory funnel for 10 minutes, and the two phases were allowed to separate during a period of 1 hour. The upper butanol phase was placed in the chromatographic trough after equilibration 8 h p e chamber. n-Butanol, distilled water, and 28% ammonium hydroxide O . bafibEr and the system equilibrated for 1 hour before introduction of c the golvent. b. 65% Isopropanol Made 2N with HCl (Wyatt, 1951): 650 m1. of isopropanol and 167 m1. of concentrated HCl were mixed and water added to make 1 1. A portion of the solvent was placed in the bottom of the chamber and the system equilibrated for 1 hour, after which a second portion of the solvent was added to the chromatographic troughs. , c. Isobutyric Acid-Ammonia (Magasanik e_t _a__l_. , 1950): Isobutyric acid, distilled water, and concentrated ammonium hydroxide were mixed in the ratio of 66:33:1 by volume. A portion of the mixture was placed in the bottom of the chamber and the system equilibrated for 1 hour, after which the solvent was added to the chromatographic troughs. ‘ E. Purification of Nucleic Acid Components with Activated Charcoal 1. Preparation of Charcoal Commercial activated charcoal (Norit A) was further purified by the method of Lipkin e_t a}; (1954). 2. Adsorption and Elution Procedure 0. 5 g. of purified charcoal was placed in a sintered glass funnel, washed with 50 m1. of distilled water, 50 m1. of eluting solvent \2 07 7 0 ethanol, 24% water, 3% concentrated ammonium hydroxide; gf’nett e_t 8:1: , 1955), and again with 50 m1. of distilled water. The 1/1 of the mixture of purines, pyrimidines, nucleosides, or nucleotides ‘0 be adsorbed was adjusted to 5. 0 and the mixture applied to the char- coal. The charcoal was washed with water after which the adsorbed Compounds were eluted with the eluting solvent. Elution was continued until no more UV. absorbing compounds were eluted. If the substances eluted were to be chromatographed, the eluate was evaporated to dryness on a rotary evaporator, the residue taken up in 15 ml. of distilled water, lyophilized, and redissolved in a small volume of an appropriate solvent. F. Assay Methods Assay methods used are contained in Table 1, page 10. C. Other Materials and Methods Nativeness of DNA was determined by the method of Hotchkiss (1957). This method consists of a standard procedure for the alkaline denaturation of DNA. Degree of denaturation produced by the alkali treatment is expressed as percent increase in absorption at 260 mu. All authentic compounds were Obtained from the California Corporation for Biochemical Research. All spectra were obtained on a Beckman DKZ spectro- photometer. {kBLE l.- Assay methodS. 10 /¢\]me or substance Procedure no. 6" Reference “if \I‘P sin Chymotr ypsin Ribonuclease Deoxyribonuclea s e Phosphodie sterase 5'nuc1eotidase Protein Pentose Deoxypentose Phosphorus 10 ll 12 13 14 15 Schwert and Takenaka (1955) Schwert and Takenaka (1955) Kunitz (1946) Kunitz (1950) Titration with NaOH Koerner and Sinsheimer (1957) Sinsheimer and Koerner (1951) Gorna11_e_t_a_1. (1949) Lowry e_t 31. (1951) Dische (1955) Dische (1955) Stumpf (1947) Buchanan (1951) Allen (1940) Bandurski and Axelrod (1951) 11 III. RESULTS A. Isolation Procedure Preliminary analysis of lyophilized Drosophila by the SC‘hrrlldt-Thannhauser-Schneider procedure (Schneider, 1946) revealed that the ratio of RNA to DNA in Drosophila was 3 or 4 to 1 and that lyophilized flies contained from 1 to 3 mg. Of DNA per gram dry weight. In early attempts to isolate DNA from Drosophila, several unsuccessful methods were used. The weak salt method of Crampton Etfil.‘ (1950), the strong salt method of Signer and Schwander (1949, 1950), and the phenol method of Braun et__a_l. (1957) all proved unsuccessful. In light Of the experience of Daly e_t_a_l. (1950) and Chargaff 3331: (1952) where NaCl solutions of high molarity were necessary to extract spermatozoa nucleoprotein, it was thought that increasing the NaCl molarity of the extracting solvent might meet with success. The NaCl molarity was increased stepwise to 5 M without successfully extracting DNA from Drosophila. Due to the large content of RNA in extracts of Drosophila it was thought possible that the DNA was not being detected. A saline extract was fractionated into 8 fractions by precipitating the nucleic acids with from 1 to 6 volumes of cold ethanol. All fractions were found to contain RNA and none of the fractions contained DNA. The isolation procedure which finally proved successful was modified from the method of Kay, Simmons and Dounce (1952). The — a 1" : is 1.: *“n’f‘z'lq-w I , . ._,_.~’ ‘x': 12 '01- . . . . . . K73 modifications conSISted of more extenSlve washing of the saline- , cfate: homogenized residue, in the avoidance of dehydration of the 0" '15 Olated DNA-protein, and in maintenance of the salt content at or above Q }6 M NaCl at all times. The latter two modifications were introduced to afissure an undenatured product. An example of the effectiveness of the isolation procedure in obtaining a purified product of DNA is given in Table 2. About 90% of the RNA is removed in the first six saline-citrate extractions leaving a residue which is highly enriched in DNA. Upon Duponol extraction, r.".‘~.’.4‘u pt. -- . 1 the DNA is apparently recovered quantitatively (Table 3). The amyl alcohol-chloroform, methoxyethanol, and the enzymatic deproteinization procedures all resulted in a product which is free of RNA as determined by the orcinol method (Table 1, No. 10) and free of protein as determined by the biuret method (Table 1, No. 8). When the first Duponol extract containing DNA was pre- cipitated with cold ethanol or ethoxyethanol, a flocculent precipitate was formed. This contrasts with the fibrous precipitates formed upon similar precipitation of DNA's from most other sources. When this flocculent precipitate was redissolved in saline, even in highly concen- trated solutions, it was far less viscous than comparable solutions of salmon sperm DNA. Upon deproteinization of this crude DNA preparation two types of ethanol-precipitable DNA'S were observed. L. . It] filliulFllll. :r I! 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