EFFECYS OF IONIIENG RADIATFON ON THE NUCLEiC ACIDS DURING BARLEY EMBRYONIC DEVELOPMENT Thais m an m at Ph. D. meme»: snuumvsasm Chang Won Chang V 1961 This is to certify that the thesis entitled Effects of ionizing radiation on the nucleic acids during barley embryonic development presented bg Chong Won Chang has been accepted towards fulfillment of the requirements for Ph ,D 3 degree in _9_§_Y__B an /%M I Majof professor Date May 31’ 1961 0-169 LIBRARY Michigan State University ABSTRACT EFFECTS 01' Im mutton ON THE NUCLEIC imammrmoncnsmmr byChongUonChsng me sin of the present study no to investigste radiosensitivity of developing embryos in m in regard to qualitative and quanti- tstive aspects of nucleic scids, end to detersine if any disturbances are induced in the pstterns of nucleic acid netsbolisn during enhryonic develop-mt following treatment with a single dose of l$50 roentgens of l-redietion epplied at specific snbryonio stages. In en attempt to coco-plish the purposes of this study. tun different experieentsl methods were used. its first use s biochelicsl deteninsticn of the purine end pyrinidine been in nature embryos (stsge 6c) utters mg).- dose of #50 r of 1m m epplied st esoh of four different periods in ubryogew. mely. esrly proubryos, lste prosubrycs. lid-differentisting enhryos. end lste differentiating Olin-yes. the other «perinatal nethod involved measuring the relstive haunts of P-32 ineorporstion into the two types of nucleic acid during Olhryonio develop-at. hhryes were irrsdisted (#50 r) st the ssns embryonic stages es shove. Chang Von Chang In the first uperinentel nethod m and mu were extracted with 1 I end 0.5 I perchloric acid, respectively, after excluding alcohol- soluble and alcohol-ether soluble coepounds and acid-soluble carbomdretes. The extracts of m and mm were hydrolyzed to liberate purineendpyrindinebeses. Thenixtureofthe freebaseswere separatedbypaperchroeatographyandthequantityofeachbasewes deterlined by ultraviolet spectroscopy. In the second nethed nicroscope slides were prepared free embryos which were irradiated at various stages of embryo development. These senple-nounted slides were subjected to procedures for differential extraction ofRNAenleiinsuchaweythetthefirstpairofslides (nonel and irradiated) contained an. DNA, and protein: the second pair m and protein: the third pm- protein only. Slides were coated with liquid emulsion, before photographic developing and fixing. then were stained lightly with Delafield's henetcxylin, and eonnted with clarite under a coverslip. Finally. the relative incorporation of P-32 intomendDMwasdetereinedbyavisuelconnting (under oil i-ersien) of the grains (radioactive tracks) of the entire proenbryos and over unit areas of the root. shoot. end scutellua of the differentiated embryos. he naJor findings of the present study were as follows: 1. mu was nore radiosensitive then all during embryogeny. except for theeature embryewheremuwesnore stable than mu. 2. Altered purine-pyridine ratios did not return to the nor-e1 pattern of nucleic acid netabolisa. 3. 4. 5. 6. 7. Chang Won Chang Uracil ofmendtlvnineofDfliappearedtobemorelabile to Lradiation than the other bases. The observed difference in RNA and DNA contents at different stages of embryogeny (with the exception of the DNA in nature embryos) indicated that the younger embryos were more affected. end/or underwent less recovery during the post-irradiation period than the older embryos. em. specificity of radiation effects seemed to be referred more to effects on the DNA rather then the RNA capments of the embryo cells. me order of increasing radiosensitivity to X rays was soutellum, shoot. and root, if radiation was applied during the tine that structural differentiation of the above three regions was occurring. me difference between DNA values in three different parts of an embryo (root. shoot. and scutellun) reflected the degree of tissue heterogeneity, but RNA seemed to be independent of the level of tissue differentiation. EFFECTS OF IOIIZING RADIATION OR THE NUCLEIC ACIDS DURING BIBLE! 313310310 DEVELOPMENT By Chong Won Chang AMSIS Submitted to Michigan State University in partial fulfill-ant of the requirements for the degree of DOCTOR 0! PHILOSOPEI Depart-ant of Botaw and Plant Pathology 1961 /C://) J / a..- // w» ., 1 I s I ’ I ,1 I" U". NW The author wishes to express his sincere gratitude to Dr. L. V. Hericle for his help throughout the course of this study and in preparation of this thesis and to Dr. w. B. Drew, Dr. R. U. Byerrlm, and Dr. I. W. Knobloch for their suggestions. Thanks are also due to Dr. L. G. Wilson, Dr. A. Kivilaan, and Dr. J. L. Fairley for their constructive criticism, and to Mr. R. A. Davis for his unreserved help in preparation of this thesis. The author is also grateful to Dr. U. v. Mostosky for his kindness in irradiating the experimental plants. TABLE OF CONTENTS INTRODUCT ION O O O O O O O O O O O O O O O O O O O O O O 0 O HISNRICAL W O O O O O O O O O O O O O 0 O O O O O O O 0 mm m MODS O O O O O O O O O C O O O O O O O O O 0 Biochemical Analysis of Nucleic Acids in Nature Bnbryos. RailingofPlants................. Preparation of Ehbryo Materials for Tissue Fractional Analysis of Nucleic Acids . . . . . . Tissue Fractionation and Quantitative Determination of Purine and Pyrinidine Bases of RNA and DNA mmtmhbryOSeeeeeeeeeeeee A. Methods of Tissue Fractionation . . . (1) Alcohol-soluble Canpounds . . . (2) Alcohol-ether Soluble Canpounds (3) Acid-soluble Compounds . . . . . (If) wraCtion or RNA e e e e e e e (5) Extraction or DNA e e e e e e e Be Procaduros for MTOMiB e e e e e e 0. Separation of Purines and Pyrimidines by paper Chromatography e e e e e e e e e D. Methods for Determination of Purines and Pyrinidines by Beckmsn Quartz Sectrophotmeter........... (1g Elation Pr00&um8 e e e e e e e e e (2) Ultraviolet Spectroscopy . . Incorporation of P-32 into Nucleic Acids during hbmogw O O O O O O O O O O O O O O O O O O hiSiflgOfPl‘ntSeeeeeeeeeeeeee Procedures for Autoradiographs . . . . . . ‘e Pmparatlon Of Ehbryo Slides e e e B. Methods for Differential Extraction of RNAamDNAOOOOOOOOOOOOOO Ce Application Of Mal-31011 e e e e e e e e e D. Photographic Processing . . . . . . . . . E. Track Counting and Estimation of Relative Amount of P-32 Incorporation . . . . . . iii Page 22 22 EVALUATION OF BIOCHHHCAL METHODS USED WITH EMBRYOS . . . . Determination of Standard Extinction Values of Purine and Pyrimidina Bases e e e e e e e e e e e e e e e RecoveryofFreeBases................ Comparison of Two Types of Hydrolytic Processes in Icaat RNA Sud Salmon DNA e e e e e e e e e e e e e A. Hydrolysis with 12 N Perchloric Acid . . . . B. Hydrolysis with1 N Perchloric Acid . . . . . Recovery of Yeast RNA and Salmon DNA by Processes Applied to EMbnyo SBMPIOB e e e e e e e e e e e e e Survey of Overball Recovery of Purines and Pyrimidines in Yeast RNA and Salmon DNA . . . . . . . . . . . . RESULTS AND DISCUSSION 0 O O O O O O O O O O O O O O O O 0 Biochemical Determination of Nucleic Acid Content in Normal Bud X‘radiated EMbnyOB e e e e e e e e e e e Determination of Purine and Pyrimidine Contents of RNA and DNA in Normal Mature Embryos e e e e e e e e e Determination of Purine and Pyrimidine Contents of RNA and DNA in Nature Einbryos Following X-radiation at Four Differ.nt EIbryoniC Stages e e e e e e e e e e Determination of P-32 Incorporation into Nucleic Acids of Normal and X-radiated Etnbryos during Embryogeny Effect of I—radiation on P-32 Incorporation into Nucleic Acids of Ehibryos as a Whole . . . . . A. Changes in Nucleic Acids during Post- irradiation period Following X—rediation (#50 r) Youngest Proembryo Stages, ape (Group-1) . . . . . . . . (1) Incorporation of P-32 as a Measure or DNA Synthesis e e e e e e e e e (2) Assumptions for Evaluation of R‘su1ts e e e e e e e e e e e ) Comparison of Minimum Values . ) Firat Minimum. e e e e e e e e ) Abortive Recovery . . . . . . ) Second Minilnl e e e e e e e e ; Final Racavery e e e e e e e e e Radiation on Depression of DNA . . (9) Interpretation of the Inter- transformation of RNA and DNA . . B. Changes in Nucleic Acids during Post- irradietion period Following X-radiation (#50 r) of Oldest preembryo Stages, g-1 (Group-2) and at Stage h of Mid-differen- tiating Debryos (Group-3) . . . . . . . C. Changes in Nucleic Acids During Post- Irradiation Period Following I-rediation (#50 r) of Late Differentiating Embryo Stages, L‘to-s (Group-u) e e e e e e e e iv 0 O O O O O O O O O O Theories on the Effects of Ionizing Page 1&6 ’47 50 53 ‘d 59 67 67 78 80 D. Comparisons of To in Four Separate Acids of the of the Embryo O O O O O tal Nucleic Acids Page 81 83 87 89 100 103 116 LIST OF PLATES Plate Page I Photographs of three adjacent sections of an embryo exposed to a single dose of 150 r I rays and treated WithP-BZ...................... 10‘ II Developmental stages of barley embryogeny . . . . . . 102 Figure 1. 2. 3. #. 5. 7. 8. 9. LIST OI‘I FIGURES Comparison of RNA - bases in normal embryos and those X-reyed at four different embryonic stages and analysed at stage 6c, corrected for 21% loss . . . . . . Cuparison of DNA - bases in normal embryos and those Lreyed at four different embryonic stages and analyzed at stage 6c, corrected for 27% loss . . . . . . Comparison of purine and pyrimidine bases of total nucleic acids in normal embryos and those Lirradiated at four different embryonic stages and analyzed at stage 6c, corrected for 21% loss (RNA) and 27% (DNA) . . RNA and DNA content of irradiated embryos, as percent of controls, during 16 days post-irradiation. Embryos were treated with X rays at embryonic stage a-c . . . . RNA and DNA content of irradiated embryos, as percent of controls, during 12 days post-irradiation. Eknbryos were treated with X rays at embryonic stage g . . . . . RNA and DNA content of irradiated embryos, as percent of controls, during 9 days post-irradiation. Embryos were treat“ With X rays at embryonic Stage 3.1+ e e e e e e e RNA and DNA content of irradiated embryos, as percent of controls, during 7 days post-irradiation. thryos were treated with X rays at embryonic stage late-5 . . . Total nucleic acid content of irradiated embryos , as percent of controls, following #50 r I rays. Enbryos were x-rayed at four different stages . . . . . . . . . Nucleic acid content, as percent of controls, of the root, shoot, and scutellum of embryos which were treated with a dose of #50 r X rays at stage a-c and sampled after complete differentiation at stage 6c . . . vii Page 40¢? 10,57 1977/ 108 xfi 11d 0 111 Figure 10. 11. 12. LIST or FIGURES (cont.) Page Nucleic acid content. as percent of controls, of the root, shoot, and scutellum of embryos which were _ treated with a dose of #50 r‘x rays at stage g and fii sampled after complete differentiation at stage 6c . . . 112 Nucleic acid content, as percent of controls, of the root, shoot, and scutellum of embryos which.were treated with a dose of #50 r X rays at stage 3-# and ~9 sampled after complete differentiation at stage 6c . . . 11#’ Nucleic acid content, as percent of controls, of the root, shoot, and scutellum of embryos which were treated with a dose of #50 r X rays at stage late 5 and sampled after complete differentiation at stage 6c . w1/115 Viii Figure 10. 11. 12. LIST or FIGURES (cont.) Page Nucleic acid content, as percent of controls, of the root, shoot, and scutellum of embryos which were treated with a dose of #50 r X rays at stage g and 5-1 sampled after complete differentiation at stage 6c . . . 112 Nucleic acid content, as percent of controls, of the root, shoot, and scutellum of embryos which were treated with a dose of #50 r X rays at stage 3-# and sampled after complete differentiation at stage 6c . . . 11K Nucleic acid content. as percent of controls, of the root, shoot, and scutellum of embryos which were treated with a dose of 450 r X rays at stage late 5 , and sampled after complete differentiation at stage 6c . m /115 V111 Table 1. 2. 3. 7. 8. 9. 10. 11. LIST OF TABLES Page Standard extinction values of purine and pyrimidine bases detemimd from the difference read at the ‘bsorption maximum ‘nd at 290 “P e e e e e e e e e e e 117 Recoveries of five bases after heating at 100°C in 12 N DCTChloric 831d for 1 hour e e e e e e e e e e e e 118 Base recoveries of yeast RNA and salmon DNA hydrolyzed with 12 Nperchloric acid at 100°C . . . . . . . . . . 120 Recoveries of yeast RNA and salmon DNA hydrolysed with 1 N and 0.5 N perchloric acid, respectively, at 10000000000000.000000......0..12a Recoveries of yeast RNA and salmon DNA subjected to the sue procedures as applied to embryo samples . . . 128 Determination of purine and pyrimidine bases of RNA and DNA in normal embryos analyzed at stage 6c according to methOds “35d in Tablo 5 e e e e e e e e e 132 Determination of purine and pyrimidine bases of RNA and DNA in 160.6 mg dry weight of embryos X-irradiated atstageeandanalyzedatstage6c. . . . .. .. . .135 Determination of purine and pyrimidine bases of RNA and DNA in 2110.1 mg dry weight of embryos X-irradiated 3t stage 1 Ind In‘lyzad at Stage 60 e e e e e e e e e e 138 Determination of purine and pyrimidine bases of RNA and DNA in 273.2 mg dry weight of embryos X-irradiated at 3t“. h and ‘nfllyICd .t .tlg. 6c e e e e e e e e e e 141 Determination of purine and pyrimidine bases of RNA and DNA in 203.2 mg dry weight of embryos X-irradiated atstagelateSandanalysedatstage6c .. . . . . . 1114 Quantity in mp moles of purine and pyrimidine bases of RNAandDNApermgdz-yueight ofnornalandxprqed embryos e e e e e e e e e e e e e e e e e e e e e e e e 1“? ix Table 12. 13. 14. 15. 16. 17. 18. 19. LIST OF TABLES (cont.) Page Nucleic acid content, RNA/DNA, and purine/pyrimidine ratios, in normal and Xerayed embryos based on percent or contr013 e e e e e e e e e e e e e e e e e e 148 Purine and pyrimidine bases, as percent of controls, and their ratios in normal and X-rayed embryos . . . . 149 Grain counts per unit area of embryos . . . . . . . . . 150 Summary of Table 14. Nucleic acid content of embryos X-rayed at four different stages and sampled at inter- vals f0110'1ng irradiation e e e e e e e e e e e e e e 161 Relative percent RNA and DNA of the control at each sampling stage during normal barley embryogeny . . . . 162 Average of three determinations of cell number per unit area, except stage d-G of Group-1 . . . . . . . . 163 Summary of Table 14. Nucleic acid content in three different parts of mature embryos X-irradiated at four different embryonic stages . . . . . . . . . . . . 164 Total radioactivity of dissected embryos at each sampling stage except for stages g-1 or younger . . . . 165 C . . \ h ‘ . 7 . l . ‘\ ‘1 V" ‘ n . ‘ d . 01‘. ' 0“ rv ‘3 ' ‘ A l e.- r. . . 'f 1 . . . ‘_ . or INTRODUCTION During the past decade, an increasing amount of work has been directed toward the study of radiation effects on biochemical metabolism in various organisms. Although a complete understanding of the effects of various radiations on biological systems can be determined only by studying all aspects of the diverse changes in the organism that arise as a result of irradiation, a thorough study of nucleic acid metabolism under the influence of radiation is, perhaps, of primary importance since modern concepts link fundamental aspects of growth, develojlent, and protein formation with the synthesis and breakdown of nucleic acids. The first historical observation of the interference of DNA metabolism by X-irradiaticn was discovered by Euler and Hevesy (1944) in their experiments with growing Jensen rat sarcua. Since that time numerous investigators have reported on new aspects of the effects of ionising radiation on nucleic acids such as the mechanisms by which ionising radiation interferes with nucleic acid metabolism, quantitative studies on the effect of X--rays on the amount of mu and DNA, and the effect of Liz-radiation on the mitotic activity of the cell. The majority of these experiments have been carried out on animals and microorganisms with comparatively few dealing with higher plants. There has been some work which considered the effects of Liz-radiation on the mitotic cycle and DNA synthesis in bean roots (Howard and Pele, 1951b,19533 Pele and Howard, 1954, 1955) and his amich dealt with quantitative analysis of nucleic acids in rye and barley seedlings (Sissakian, 1955: Trudova, 1954). Thus far, however, the radiosensitivity of nucleic acid metabolism in developing plant embryos has not been investigated. The objective of this research, therefore, was to determine whether radiosensitive stages occur in nucleic acid metabolism during developmental embryogenv. In order to do this, embryos were irradiated at different stages of development, and quantitative and qualitative studies of the caposition of RNA and DNA were carried out. Barley embryos were chosen as the experimental plant material since they have been shown suitable for research work (Nericle and Nericle, 1957) and X~rm were used as the source of radiation since their dosages could be readily regulated experimentally. HISTORICALREVIEH The primary purpose of the present work was to determine the relationship between the changes in the quantity of nucleic acids, the ages of embryo tissues, and eventual disturbances in the pattern of nucleic acid metabolism after the treatment of Xl-rrays (450 r) at various ubrycnic stages. Host investigations involving the effect of ionising radiation on nucleic acids have been carried out on animals and microorganisms, while higher plants have not been sufficiently studied in this respect. Although embryonic tissues of a higher plant were utilised as the material in the present investi- gation, it was necessary to refer not only to the literature. on plants, but also the "a, dam on mm» The integrity of nucleic acids is of particular importance to the cell. hush data about this subject has stemmed from the earliest experiment of Hahn and Revesy (1940). who studied the incorporation of P—32 into DNA of various organs of rabbits. According to their reports,the turnover of nucleic acids in the liver occurred at a low rate, while all other organic phosphorus compounds intheliverwere foundtobeentirelyrenewedatarspid rate. In the muscle, on the other had, the turnover of the average nucleic acid moleculewasgreaterthaninthelivercrinthetlwms. Inthebrain theturnoverrate of nucleic acidswasfcundtobemarlcedlylcwerthsn that of the phosphatides. Von Euler and Nevesy (1944) observed that in unirradiated Jensen rat sarcoma about twice as much DNA became labeled as could be accounted for by the increase in total DNA, and they concluded that about half of the labeled DNA molecules were l'neu" DNA whereas labeling of the other half was due to renewal of 'old' DNA during the process of synthesis. This conclusion has been supported by Stevens, et al. (1953) and Daeust's, et a1. (1954) work on incorporation of P-32 into DNA in various rat tissues. The first historical observation of interference of DNA meta- bolism due to X-irradiation was discovered by Von Euler and Revesy (1944). They compared the rate of incorporation of labeled phosphate into DNA extracted from irradiated growing Jensen sarsua of rat and of control rat and found that irradiation with an X ray dose of a few hundred roentgens or more reduced the rate of formation of DNA in the sarcoma by about 50$. With similar material Hevesy (1945) studied the uptake of P-32 by DNA after doses of X rays of about 1000 r. The results indicated that isotope administered i-ediately after irradiation was taken up more slowly in the irradiated material than in the non- irradiated control. A large number of papers concerning the effect of ionising radiations on DNA synthesis followed the original discovery of the depression of DNA synthesis by Von Euler and Revesy (1944). New other authors also found that relatively large doses of X rays did not depress DNA synthesis to less than 50$ of the control values (Klein and Perssberg, 1954, mlich ascites tumor; Iorssberg and Klein, 1954, Ehrlich ascites tumor: Lavik and Buckaloo, 1954, chick embryo). In addition, Skipper and Mitchell (1951) repeated Hevesy's experiment (1945), which was concerned with the effect of 950 r radiation on the incorporation of Cut-02 into 6-carbon of DNA purine and of NC‘TOONa into 2-8—carbon of DNA purine of rat intestine. According to their reports, roentgen-ray radiation (950 r) has been shown to inhibit the incorporation of radioactive carbon into the nucleic acids and their purines by about 50$ of the control for a period of six hours. Additional reports concerning the phenomena of initial inhibition of DNA synthesis caused by ionising radiation have been reported by many other investigators, namely Revesy (1948a, rat), Thomsant a1. (1952, rat tlvmus), Vermund, et a1. (1953, mouse mammary carcinoma), Bennett, et a1. (1954, nice), Petersen, et a1. (1955, rat spleen), Harrington, et al. (1955b, rat thymus), 0rd and Stocken (1956, rat), and Sherman and Quastler (1958, mice). The fact that in many cases irradiation depressed DNA synthesis to approximately one-half led Von Euler and Hevesy (1944) to suggest that ionising radiation may interfere with DNA synthesis but not with the high turnover of DNA. This theory of DNA synthesis depression is not accepted by Polo and Howard (1955) on the ground that if Euler and Nevesy's interpretation of the irradiation results was correct, the labeling of single cells after irradiation should be - one-half that of unirradiated ones, which was in no way supported by Polo and Howard's experiment. The strength of the autoradiograph per cell was at least approximately equal for irradiated and unirradiated bean roots. In addition, since Euler and Hevesy's hypothesis was proposed, many investigators have reported results of DNA depression to less than 50% of the control, which is difficult to explain in terms of the 50$ DNA depression theory. For instance, Lutwak - Mann (1951), in his study of the effect of ionizing radiation (500 r as whole body exposure) on the nucleic acids in rat, reported that the greatest depression of DNA content as percent of the control was 9% and 37% in bone marrow and spleen, respectively, after 4 days post-irradiation. In recent work, Kelly, et a1, (1954) found that whole-body irradiation (#50 r to 800 r) of rats after administration of can, depressed DNA synthesis at its minimum to 20% of controls, if animals were irradiated well before the peak, but had no effect if given at the time of maximal synthesis. Polo and Howard (1951*). on the other hand, determined the proportion of cells in the root meristem of 119;; EL); which synthe— sized DNA during a given period of time, as indicated by the incor- poration of P-32 into DNA. According to their reports, the greatest DNA depression fell to 10% of the control at 6 to 8 days post- irradiation. They interpreted this to mean that cells were being prevented from synthesizing DNA if they were in the first part of interphase at the time of irradiation. According to Sissakian's (1955) experiment on the effect of X-irradiation (5 hr) on the nucleic acids of growing rye seedings, the'average DNA depression was recorded as 75% of the control at 3 hours post-irradiation. In a recent work, Nygaard and Potter (1959) investigated the effect of X rays (400 r) on the incorporation of 7 thylidine-Z-Cwintonninratsandreportedfi, 25$, moses depression in flame, spleen, and sell intestine, respectively. within 21+ hours after the treatment of 1100 r. Kore attention has been paid to the mnduental nechani. implicated in the depression of DNA content due to ionising radiation. Howard and Pelc (19519 studied the percent of resting nuclei which showed labeling (P-32) after different periods of growth, their distribution in the bean root cells, and the delay in the appearance in litesis of labeled nuclei. They concluded that P-32 was not incorporated into nucleic acids durim cell divisions, nor during the period inediately preceding it, but during some part of interphase. The incorporation of P-32 took place in cells which sore preparing for division, but not in cells which sill differentiate without further divisions. In addition they reported that the P-32 incorporated mined in the nuclei for considerable periods of time and was translitted to daughter nuclei. 'me discovery of independent inhibition of mitosis and DNA synthesis due to X-irradiation was first made by Howard and Pele (1953) in an experiment with been root unused. It was concluded that the sensitive period for inhibition or delay of DNA synthesis was during the first part of interphase, beginning possibly as early as 2 hours before the previous prophase and ending 2 hours before the beginning of synthesis. Cello which were already synthesising DNA at the time of irradiation were not affected in this respect. In subsequent experiments, Pele and Howard (1951:, 1955) confined the previous results by observing that the number of cells synthesizing DNA (observed by means of autoradiographs) in Eigig gaba root meristem was reduced about 60% by moderate doses of X rays during the subse- quent 12 hours. Reduction was the same after doses of 50 r to 200 r. These results were interpreted to be due to a greater radiosensitivity, resulting in delay or inhibition of DNA synthesis in cells which were in approximately one-third of the cell cycle at the time of irradiation (the first part of interphase). Results obtained from the experiments with bean root.meristem were supported by other authors, namely, Cater, et al. (1956, regenerating liver of the rat), Holmes (1956, rat liver), Kelly, et al. (1955, mouse liver), Kelly, et al. (1957, Ehrlich ascites cells), Kelly, et al. (1957, regenerating rat liver), Lajtha, et al. (1958, human bone marrow). Lajtha, et al. (1958). in their study on the mechanism of radiation effects on DNA synthesis by cell cultures of human bone marrowgig yitgg, concluded that large doses (larger than 500 rads) or X-radiation directly inhibited the process of DNA synthesis in human bone marrow cells, while small doses (smaller than 300 rads) did not inhibit DNA synthesis in cells which already had started DNA synthesis. Low dose radiation during the presynthetic period of the mitotic cycle will produce a 40 to 50% depression of the number of cells which enter the subsequent synthetic period, but will not affect the rate of DNA synthesis in those cells that are already in the synthetic period of the cycle. These results essentially supported the original data obtained by Polo and Howard (1955). As already described above, considerable evidence has been accumulated which has been interpreted as indicating that period preceding DNA synthesis is more sensitive to irradiation than the synthetic period itself. In each case studied, there appears to be a delay of the onset of DNA synthesis by the cells in the presynthetic phase. Howard (1956) and Kelly (1957), however, have critically examined the data and stated that, with the possible exception of the work on regenerating liver by Holmes (1956), the inhibition of DNA syn- thesis could be a secondary one resulting fron mitotic delay, leading to depletion of cells in the presynthetic phase. Evidence contra- dictory to the results of Pelc and Howard (1955) has been presented by Painter and Robertson (1959) and Painter (1960). In their recent experiment the percent of HeLa 53 cells in DNA synthesis at various tines after 500 r of X-irradiation was detemined by means of auto- radiography with H3 - thymine. They found that the percent of DNA synthesis rose during the period of mitotic delay so that at b, - 8 hours after irradiation almost twice the number of cells in the population were synthesising DNA compared to the controls. The interpretation was that many cells synthesising DNA at the time of X-irradiation r-ained in this stage for an abnormally long time and there was no effect on the rate at which cells entered DNA synthesis. Therefore, there was no effect on the cells of the presynthetic phase moving into the synthetic. In other words, the fraction of cells in the synthetic phase, as a result of mitotic delay, increased because of the uninhibited flow of cells from the presynthetic to the synthetic We 10 macro and Guttes (1958). in order to find out the effect of ionising radiation (6000 to 9000 r) on division and on DNA synthesis in slile Iold, irradiated cells at different times from late prophase throughout the early part of the reconstruction period. 'lhey reported that all of the irradiated molds appeared to complete one period of DNA synthesis, even in the cases when normal nuclear division did not occur. In addition, according to their results, irradiation during interphase caused more or less a constant delay of the subsequent division, while Lit-radiation inediately after division or during late prophase gave a greatly increased effect. Gardella and Servelle (1960), on the other hand, studied the labeling of P-32 into DNA in mass synchonous cultures of W‘m exposed to Lirradiation (60 K. rad). They reported that the inhibition of DNA synthesis appeared to be the primary response to irradiation and was not influenced by any particular stage of develop-cat. * Cattaneo, et a1, (1960) conducted an experiment on the effect of rem: (300 red) on mm of hair follicles in mice by the method of labeling with tritiated thymidine and autoradiographv. According to their emperiaental results, the reduction of We incorporation enhanced i-ediately after irradiation and amounted to about 50$ of the control within 30 minutes. This was considered to be an effect on cells in the process of synthesising DNA at the tile of irradiation. For the purpose of determining the pathway of decuposition of nucleic acids in v_i_v_o_ due to l-rays, Scholes, et al. (191:9) performed an 11 experiment in which a 2$ sodium nucleate solution was irradiated with doses of the order et2-tx1o5 r. Theyreportedtbatthechauical changes which occurred under the influence of X-irradiation indicated thataringopeningintbepurineendpyrimidinebaseshadtaloen place. In additien. the results indicated some fission of the glyeosidio linkages with the liberation of purine bases, a declination of the constituent bases, sue breaking of the ester linkages, leading to the formation of inorganic phosphate, and sue splitting of the internucleotide linkages. weiu (1952). citing the results indicated above, theorised the possible biological significance of the action of ionising radiation on nucleic acid in similar ways. Pole and Howard (1952), in their report on chmsme metabolism as determined by autorediographs, theorised that the mechanism of the direct effect of Lrvs on DNA synthesis involved a secondary electron. They said ' that this electron passes through the substrate at one or several steps in the chain of synthesis, releases electrons that can reach the ensyme, and incapacitates it either permanently or t-porarily. In contrast to the direct effect of Lit-radiation, there were a numberefreportswhich indicatedanindirecteffectonDNAdueto X-rsys. According to Von Ahlstra, et al. (19“). the action of Roentgen rays on tumors was an indirect effect which they concluded from the following experiment. Rats were inoculated with two sarcomata. One sarcomawas irradiatedwithuptozooo r, whilethe ether sarcoma was protected by 5- thick lead sheaths. An invesflgaflon 12 An investigation of nucleic acid fonation which took place in both sarcoaata indicated that the shielded sarcoaa exhibited reduced fox-nation of nucleic acid. the cells of a serene protected fra the direct action of Aoentgenrayswere thus actedupononlyif otherparts of thebody becue irradiated. Holnes (19149), in his study of DNA synthesis in irradiated Jensen rat tuner and in other tumors which were themselves not receiving irradiation, reported that on synthesis was reduced to 25 percent of thennornal in the directly irradiated tmor. Kelly and Jones (1950). on the other hand, confined the existence of indirect effects of l-irradiation in their experinents by treating the liver of the aninal directly and the mole indirectly with 11.25 x 105 ergs. DNA was isolated fru the livers of both aninals and the specific activity of P-BZ was neasured. According to their results. the foner showed 66 percent DNA of the control, while the latter 84 percent of the nor-a1 level. It has been quite a canon phenomenon to see overshooting of DNA content. According to the superhent of Willie”. et al. (1955) on D“ synthesis of rat shall bowel epitheliun after I-irradiation (#50 r). the mitotic index was found to be150$ of the control and the adenine and guanine of mu 608$ and “11$ of the control. respectively. is to the interpretation of this overshooting phenmenon of mu, Bennett, et al. (19514) and Howard (1956) explained that periodicity of 1m synthesis recovery night occur due to partial synchronisatim ofDlisynthesiscausedbytheacute celldeath orthedelayofthe 13 DILsynthesisinthesurvivingcelledependingupontheposition of the cell in the mitotic cycle at the tins of irradiation. This partial synchronisation. in turn. could produce an increase of mu content over the control level. Concernim the initial depression and telporary recovery of DNA content due to r-redieticc. Knowlton, et al. (19149 and 1950). in evaluating the results obtained from experiwents involving the effect of trays on the nitotic activity in various nouse tissues. suggested that the tile fru the irradiatim to the ninilun of the witotic index was a measure of duration of nitosis which usually was followed by teIporary return to normal nitotic activity (abortive nitotic recovery). depending upon the doses of I rays, kinds of tissues, and period of post-irradiation. The effects of ionising radiation on the DNA of growing tissues are quite different free, that of differentiating tissues in terns of the synthesis of DEA related to the nitotic phases. However. before surveying the literature cmerned with these tissues, it would be well to conpare then with respect to BIA synthesis. In investigating the relationship between the rate of DNA synthesis and eitetic phases, Pelc and Howard (1952). in their upon-ant on the rate of P-32 incorporation into the on of 3121! m seedlings by use of autoradiographic techniques. reported that a cellwhiehwaspreparingtodivide synthesiseanduringthe first pertoftheinted-phase. inilenomlisynthesieeceurredduring division. In additiai. they suggested that a cell which had capleted 14 its last division and was differentiating did not synthesize DNA and retained the DNA transmitted to it by its parent cell. In contrast to the experiment of Polo and Howard, Hevesy and Ottesen (1945) presented the similar fact that the DNA.of white corpuscles and nucleated erythrocytes. in which mitosis was absent, did not inter- change with P-32 present in the circulation. According to the experiment conducted by Bruce, et al. (19th) on the rate of P-32 incorporation into the DNA of slow growing tissue such as kidney of rat and of partially regenerating liver, only minute amounts of tracer were found in the former, while there was a marked P-32 content in the latter. In addition, they reported that RNA turnover in slow or nongrowing tissue was considerably more active than DNA turnover. Hevesy (1945), on the other hand, studied the uptake of P-32 by the DNA of Jensen rat sarcoma immediately after doses of X rays of about 1000 r and found 60—70% inhibition of DNA renewal compared to the normal. ‘When the administration of labeled phosphorus into the DNA of rat liver was delayed until several days after exposure to the X rays, the difference between the specific activity of the DNA in the control and irradiated tissue was found to be very much less than when the isotope was given immediately after irradiation, while the similar degree of inhibition (60—70% of the control) occurred in the latter case as in sarcoma. He suggested that the rapid diminution in effectiveness of X-rays as a.neans of blocking DNA renewal might well explain the greater sensitivity of growing tissues to irradiation, since, in such tissues 15 the frequency of mitosis and therefore the synthesis of DNA was much greater than in full grown tissues where mitosis was relatively rare. In addition, to explain the phenomenon of recovery of nucleic acid cycle from the effect of X rays on growing and full grown tissues, he hypothesizes (1946) that in the latter case, the average cell was very much further from the mitotic stages than in the growing tissue, and thus had time to recover its normal nucleic acid cycle before any appreciable change in the nuclear structure took place, if enough time was allowed; while in the former, in the absence of a normal nucleic acid cycle, an anomalous nuclear development took place with all its far reaching consequences. In an attempt to visualize the changes of DNA occurring during the post-irradiation period, Quastler and Sherman (1958) studied the recovery of DNA synthesis in the crypts of the small intestine of rats. This was done by injecting tritiated thymidine, sacrificing the rats at various intervals, preparing high resolution autoradiographs, and determining the percent of cells in mitosis which were labeled ‘ after X-irradiation of 800 rad. According to their results, there were two waves of DNA synthesis during post-irradiation. First there was a complete blockage of DNA synthesis that took place in a.half day following irradiation. The second wave of DNA synthesis was characterized‘by a high incorporation rate within 2 days after initial irradiation. Similar multiple effects were observed by other investigators. ‘Willians et al. (1958) conducted an experi- ment on the differential effect of Xpirradiation on the DNA and mitosis of rat small bowel epithelium, and Paigen and Kaufmann.(1953) 16 observed the effect of whole body irradiation (600 r) on the nucleic acids of mouse liver at various times after treatment with X rays. In order to study the recovery after sublethal doses of irradiation (100 and 400 r), Mygaard and Potter (1960a) observed the effect of total body irradiation on DNA synthesis in thymus, spleen, and small intestine of the rat over an extended period by means of thymidine -2 -61” incorporation in DNA. According to their experiments. following an initial period of inhibition the tissues recovered their ability to synthesize DNA. The time for this recovery was found to depend on the tissue and on the radiation dose. In all cases the specific activity exceeded the control value at the time of maximum recovery, usually by a factor of about 2. It was concluded that the effect of radiation on the DNA metabolism of the three tissues were not quantitatively the same, differing only in the degree of acute cell death, in the duration of the delay of DNA synthesis in the surviving cells, and in the rate of recovery resulting from accelerated cell replication during the period of regeneration. Holmes (1947), in his study of the effect of Xeirradiation (ZOOOr) on P-32 incorporation into the nucleic acids of Jensen rat sarcoma, reported that irradiation reduced the rate of P-32 incorporation to one- half that found in the control, while there occurred a lesser degree of inhibition of phosphorus uptake by the RNA of this tissue. In contrast to this result, Abrams (1950), who treated rats with a dose of 500 r, X rays, 1? injected 0“ labeled glycine, fractionated the tissues according to the methods proposed by Schmidt and Thannhauser (1915), and measured the turnover rate of RNA and DNA in terms of tracer incorporation rate into purines of both types of nucleic acids. According to his data, the DNA and RNA were found to be decreased to 89$ and 157$ of the control, respectively, within #8 hours post- irradiation, indicating a more marked depression of RNA than DNA. The recovery percentages of DNA and RNA were h7$ and 23$, respectively, within 96 hours following the treatment of X rays, indicating more DNA recovery than RNA. Lutwak-Hann (1951) also studied the effect of X rays (500 r) on the change in the content of RNA and DNA in the testes of the rats. He indicated more depression. of RNA than DNA either in short or in long periods of post-irradiation tile, and more decrease in DNA than RNA, in bone narrow at four days post- irrsdiatien. These results were supported by Handel, et al. (1951). Using rat spleen and mouse thymus, the same author (Lutwak-Hann, 1952) found a large reduction in the RNA and DNA contents per organ due to x ray treatment (300 - 800 r). This observation was in agreeaent with that of Ueynouth and Kaplan (1952). who treated the nice systemat- ically with It doses of 168 r at 8 day intervals. In an attempt to find the site of action of X rays in a cell, Harriss, et a1. (1952) treated the nucleus and cytoplasm of £2229 m by means of the technique of nuclear transfer, and reported that the cytoplasm and nucleus were damaged independently under the influence of 100,006 to 280,000 r. 18 Payne, et a1. (1952), in a study of the effect of total-body X-irradiation (600 r) on the relative turnover of nucleic acid in mice, observed that the incorporation of P-32 into the cytoplasmic RNA was increased after irradiation, while the uptake of isotope by nuclear RNA and DNA was greatly diminished. Thomson. et al. (1952) treated rat thymus with a dose of 800 r of gamna rays from a source of 60-60 and observed that there occurred a pronounced diminution in P-32 incorporation into DNA and to a lesser extent in RNA in 3 hours post-irradiation. Similar results were observed by Thomson, et al. (1953), who conducted an experiment with rabbit bone marrow exposed to 620 r. In addition Lavik and Buckaloo (195k) allowed chick embryos, X-irradiated with 450 r, to incorporate carbon-11+ labeled formats and cytidine into purines and pyrimidines of nucleic acid. They reported a 50% inhibition of DNA bases and no change in RNA bases. Using barley'seedlings, Trudova (1951+) investigated the effect of X rays on nucleic acid metabolism. He showed that irradiation with a dose of 2 hr completely and irreversibly suppressed the synthesis of DNA in the tips of the roots of barley seedlings, and the synthesis of RNA also was considerably suppressed by 73 percent. In addition, according to his data, within 5 hours after irradiation by a dose of 500 r, DNA and RNA synthesis were suppressed by “4% and 86%, respectively, but within 2h hours the activity of these fractions became equal to the activity in the control. A dose of 65 r was observed to even provoke the synthesis of DNA and RNA. 19 The phenomenon of transformation between RNA and DNA has been reported not only in normal tissues but also in those X-irradiated. According to the theory of nucleic acid starvation proposed by Darlington (19h7), the supply of nucleic acid controls the prophase of mitosis. If the supply of this substance is cut off, the nuclei consume their own cytoplasm and die during mitosis. According to this concept, he concluded that any factors which reduce this substance will induce the interchangeable phenomenon between cytoplasmic RNA and nuclear DNA. This theory was accepted by Davidson and Leslie (1950). The observation that a very large quantity of ribose nucleic acid was localized in the chromosomes during cell division led Caspersson (1940) and Caspersson and Schultz (1938, 1939) to conclude that ribose nucleotides served as the supply of DNA.materials in the nucleus which was the preliminary requirement of’mitosis. Since the action of X rays affected cell division, he added, it might influence the cycle of nucleic acid changes and induce an interchangeable relation between RNA and DNA. The report made by Semenenko (1958) is quite interesting. In his study of summer wheat seeds on quantitative changes of nucleic acids from the time of fertilization to mature seeds, he observed that reciprocal fluctuation existed between RNA and DNA during the first 15 days. In the same experiment, in contradiction to the result above, there was parallel relationship between the contents of RNA and DNA in.maturing pea seeds. On the other hand, Prescot (1960) using hm, labeled DNA with H3. thymidine and RNA with c1 'L adenine 20 and observed that the maximum incorporation of the former tracer into DNA preceded the minimum incorporation of the latter tracer into RNA during a period of one mitotic cycle. The results reported by Caspersson and Schultz (1938, 1939) were demonstrated by Mitchell (1942), who found an accumulation of ultraviolet-absorbing materials which appeared to be of nucleotide nature. He indicated the transformation of nucleic acids from one type to another in irradiated tumor cells. In addition, Nicola (1950), in his experiment with the action of X rays on the metabolism of nucleic acids in proliferative and secretory cells, found that X rays induced an arrest of DNA synthesis and a conversion of DNA into RNA. He indicated some possible counteraction on the reduction process normally taking place in the transformation of RNA to DNA due to the action of substances produced by the interaction of X rays with water*molecules. 'While surveying the literature which deals with the effects of ionizing radiations on nucleic acids, it is readily seen that very few investigators have completely followed all the changes in nucleic acid composition due to X rays. Paigen and Kaufman (1953) observed the effect of whole body irradiation by hard X rays on the nucleic acids of mouse liver. They reported no change in nucleic acid composition when compared to non. irradiated controls. Berenbom and Peters (1956). in contradiction to Paigen and Kaufman's report above, conducted an experiment on changes in nucleic acid composition (rat spleen and thymus treated with 400 r total-body-X-radiation). According to their report, thymus RNA 21 composition did change slightly with a relative loss in adenine and a slight relative increase in cytosine. This change was temporary and the normal pattern r... re-established within 7 days. oh the other*hand, a marked change in splenic DNA composition occurred, resulting in a relative increase in guanine and adenine, and a relative decrease in cytosine and thymine. This increased purine- pyrimidine ratio did not return to the normal pattern within 7 days after irradiation. These results were supported by Harrington and Lavik (1955a) who studied the phosphorus composition and P-32 incorporation in irradiated lymphatic tissue of rats. To determine the relative sensitivity of RNAebases to radiation, Sissakian (1955) treated growing rye seedlings with a dose of 20 hr and analysed the base contents. He found that the effect of radiation clearly manifested itself by the decrease in content of all nitro- genous bases: guanine by 65 percent, adenine by 57 percent, cytosine by 79 percent, and uracil the lowest, by 44 percent, indicating that uraceil was more labile than other bases in this experiment. MATERIALS MWODS Biochemical Analysis of Incleic Acids in Nature hbryos RaisingofPlants Barley, m W I», var. Eannchen, a two-roeed variety, was used for the extraction of m and DNA in nature embryos, and incorporation of P-32 into an and DNA during abzyogem. This variety was chosen as the experimental material because of its unifora crolth due to the availability of long inbred lines, and the fact that it produces a tee-reset! 'flat' head which is particularly sell adapted for X-irrediation since the possibility of partial shielding by other grains is elininated. In addition, this variety of barley produces numerous seeds per head with eabryos in relatively the seas stage of develop-ant, since no lateral florets develop as in a 'six-roeed' variety, and provided that the four basal and the four teninal grains are discarded (Chang. 1957). Plants eere green in the greenhouse shere average temper-stuns ears 75°F during the day and 65°! at night. Optima teaperature differentials for this strain of barley should be 10° - 15°F. It is particularly ilpertant to maintain the loser night temperature to avoid sterility probleas often encountered with higher teaperstunes. 22 o. 23 Since barley is a long day plant, the day length was increased to 20 hours by the use of artificial light in order to hasten flowering, thus permitting emtra I'crops" to be grown during a given period. Preparation of kbryo Materials for Tissue Fractional Analysis of Nucleic Acids he preparation of materials for quantitative deteraination of nucleic acids in normal and Lirradiated nature embryos was conducted as folloss: Patterned after Hericle and hericle (195?), morphological and histological features of barley embryos, from fertilisation time until 'maturity'od'the-bryeasfoundintheseed,mqbedividedinto tee um groups: preembryos and differentiating embryos. The former nay in turn be arbitrarily divided into three subgroups: early (stage a - c), middle (stage d - e), and late preembryos (stage f - g). Early preembryes include developmental stages fra the one-celled mote to the 8-celled stage. middle preembryos from the 16-oelled stage to approximately 72 cells, and late proubryos. Just prior to the initial stages of organogenesis (organ differentiation). Differentiating embryos may be grouped into six stages, namely, early (state 1-2). middle (ates- 3-b). late (stas- 5-6). (plate 2). In addition, stage 6. for convenience, may be divided into 3 subgroups: early (stage 6a), middle (stage 6b), late (stage 6o). with this in nind, approximately 200 embryos at stage 6c eere dissected fra the caryopses of nan-irradiated barley plants, and kept 2h in 70’ ethanol at 0°C for the purpose of fractional analysis of nucleic acids and determination of purine and pyrimidine bases in normal, control embryos. In order to prepare analytical materials for study of the nucleic acids of X-irradiated embryos, a single dose of 450 r of Israys ‘ (at a rate of 65 r/m with a total dose of #50 roentgens) was applied at one of four different periods in embryogeny: namely, stages a-c (Group-i), stage g (Group-B), stages 3-h (Group-C), and stages late-5 (Group-D). "me grains containing irradiated embryos were then allowed to continue \developent in gig; in the greenhouse until reaching stage 6c. Approximately 200 I-irradiated embryos at stage 6c were dissected from each experimental group, and kept in 70% ethanol at 0°C for the purpose of quantitative determination of nucleic acids in I-irrediated embryo materials. A G. E. Maxi-er 250 III therapeutic Lray machine was used as the source of external irradiation. Physical factors to:- application of the doses were as follows: 200 kilo-volts of power, 15 milliamperes of current, inherent filtration of 3 - aluminum, and 0.25 - copper plus I 3 aluminum added filtration. hrther characteristics were: 0.75 - copper half-value-lsyer, 33 cm focal spot distance, and #00 sq. em beam sine. For deteninaticn of embryonic stages at the time of I-irradiation, histological sections were made of embryos fru the middle of each head to be irradiated. Formalin - acetic acid . alcohol, PM (Johansen, 191w), was used for killing and fixing the ovaries. 25 Haterials were dehydrated. embedded in paraffin by standard procedures (Johansen, 19M) and serially sectioned at 12 microns. Staining was carried out by using a combination of safranin and fast green (Johansen, 1940). Overall scheme of embryonic stages irradiated and samples anflyaedchedcallyinthispartoftheexperimcntswasasfollows: Experiment hbryo Stages hbryo Stages lumber Lirradiated Analysed Chemically Group A a - c 6c 01'9“? B 8 6c Group C 3 - lb 6c Group D Late 5 6c Control No Radiation 6c Tissue Fractionation and mantitative Determination of Purine and Pyrimidine Bases of RNA and DHA in Nature hbryos 1. Methods of Tissue Fractionation Host of the recent studies of the nucleic acid content of various animal tissues have been carried out using the tissue fractionation methods developed by Schneider (191's) and by Schmidt and Thannhauser (19%). According to their methods, separation of on tree on is accuplishedbyprolonged contact withalkaliwhichdegradeslillAmore rapidly than DNA, leaving the latter still precipitable with acid. 26 Various difficulties are. however, encountered when one attempts to apply either of these methods to the study of the nucleic acids during the development of plant root tips, pollen cells, or plant embryos. Becauseofthe-allucuntsofmaterialavailable,makingthe successful quantitative precipitation at such levels somewhat questionable, and because of the presence of interfering substances (pentosans and polyuronides) . neither the Schmitt and Thannhauser nor the Schneider procedures were considered adequate for the purposes of the present study. In other words, in their methods all acid- soluble compounds were first extracted with 540$ trichloracetic acid, phospholipids, then with warm ethanolpchlcrofcim (3 x 1), and nucleic acids as sodium nucleate with 10$ Baa. These processes, however. may possibly leave behind one alcoholpsoluble capcunds which are present in most plant materials and which give. an intense purple coloration with diphenylamine. Other methods in current use for tissue fractionation often begin with the extraction of homogenised tissues with cold trichlorc- acetic acid, followed by the hot extraction of phospholipids. Ogur and Boson (1950), however, have modified this approach by beginning with a cold alcohol-extraction to extract as much nonnucleio acid material as possible before separation of Eli and DNA. A modification of their method was found to be most suitable for the present study and is described below. 27 (1) Alcohol-Soluble Compounds Samples, each consisting of approximately 200 embryos, were ' collected and i-ediately after dissection stored in 70$ ethanol at 0°C. Prior to tissue fractionation, both the control and I-irradiated 'maturs' embryos were dried in a dessicator over acetone (B.P. 58° C) under vacuum conditions until constant dry weights were obtained. After dry weights were determined the tissue was homogenised for 10 minutes with a small hand mortar in a cold room (15°C). nie homogenate was washed into a calibrated tube with 70$ ethanol and centrifuged at b0 C. The residue was resuspended in 70$ ethanol containing 0.1‘ perchloric acid and again centrifuged in the cold. The alcohol and acidulated alcohol extracts were cabined. (2) Alcohol-Ether-Soluble Compounds The residue fr:- (1) was suspended in 5 ml of 3:1 ethanol and ethyl ether, a small piece of porous clq plats added, and the mixture boiled gently for 3 minutes in a water bath. this process was repeated end the two extracts then cabined. (3) Acid-Soluble Con-pounds iheresidue from (2) was suspended in‘5mlof cold 0.2)! perchloric acid and centrifuged. This process was repeated and the extracts combined. ibis step was capleted as rapidly as possible, although additional controls in which the residue remained in contact withtheperchlcricacidfor3hoursdiduetloseawmeasurabls amount of in. 28 (a) Extraction of an The residue fru (3) was suspended in 5 nLof 1 N perchloric acid and stored at 149 C for 18 hours. (Roughly over-night contact in the cold is tine enough to split the m frm the tissue residue). The suspension was centrifuged and the residue extracted twice more with 5 :1. portions of cold 1 I perchloric acid and conbined. (5) Extraction of mu he residue fro. (it) was suspended in 5 al. of 0.5 I perchloric acid and heated in a water-bath for 20 ninutes at 70° C. This process was repeated and the extracts cabined. More exhaustive extraction of embryos failed to reveal any additional naterial reacting with diphenylanine. indicating that no D111 remained in the residue. B. Procedures for Hydrolyaing nu and DNA Extracts Various nethods for the hydrolysis of isolated nucleic acids and the separation of the bases have been described (reviews edited by Chargaff and Davidson. 1955: W, 1955) but without aodifi- cation the nethods were not applicable to tissue extracts which contain a variety of interfering substances. A nodificaticn of the perchloric acid asthod (Harshak and Vogel, 1950) described by Wood; (1957) and Taylor (1958) was satisfactory for separating and recovering only uracil. thymine. and guanine. but considerable loss of the base, cystosine. occurs. however. when their nethods are used. Inorderto separateallfourbases chlflandDHAandobtain 800d recovery rates, the writer nodified several steps in these procedures. the nethodsas used in this investigation are described Mae 29 (1) A shall beaker containing 10-15 al. of RNA. or DNA extract. prepared as described above. was placed in a hot water bath at 100° C for 160 minutes to liberate the purine and pyrinidine bases. (2) After coolim, the aixture was diluted to 10 ml. with distilled water. then centrifuged to separate the fluid m. the black particulate residue for-ed. presu-ably fra the ribose or deoxyribose sugar of BIA or DNA, respectively. (3) for the purpose of elininating perchloric acid, the resultim clear hydrolysate was adjusted to a pH of 7.0 with 1 N solution of [2003 (with the aid of a Beck-an pH-neter). (11») The mixture was filtered through matnan No. 1 filter paper. followed by washing with 10 :1. of distilled water and then with so :11. of 801 ethanol. (5) The filtrate was concentrated to a shall volume by the use of a flash evaporator at 58° C. (6) In order to dissolve all purine and pyriaine bases (aostly water-insoluble guanine) which were possibly tied up with the salt of perchlorate, the residue frae (h) was added to 15 al. of 0.1 11 301 and agitated for one hour at 25-30° C by neans of an electric shaker. (7) rhemtnrerree(6)mmtendthmghmmnne.1 filter paper and washed with 15 al. of 0.1 11 1101. (8) Piltrate fra (7) was now cabinet! with that Ira (S) which was alreaw concentrated to a .all volume. and dried by a flash evaporator at 58° C. 30 (9) The dried bases were dissolved in 1 ml. of 0.1 11 301. and diluted to 2 ll. with an additional 1 al. of distilled water for the deter-nation of ”A. For the deteraination of DNA the dried bases were dissolved in 0.5 al. of 0.1 II 301 and diluted to 1 ml. with an additional 0.5 al. of distilled water. The resulting solutions were used for paper chroaotography. c. Separation of Purines and Pyrinidines by Paper Chronatograplw (1) Sheets of Whataan lo. 1 filter paper. 20 on. wide and no on. long. were ruled into five longitudinal sections, each It cm. wide. A transverse line, about 8.5 on. below the top of the sheet. indicated the starting points at which. in the center of each of four lanes. aliquots of solution frau (9) were to be deposited. The fifth section was used as a blank. (2) Prior to spotting and development of the chronatograns. the paper was electrolyaed in the following ways: (a) Sheets were soaked for 5 ninutes in 0.2% EDTA (discdiu salt of ethylene dinitrilotetraacetic acid) solution, which was prepared by dissolving 2 grade of EDTA in 1000 al. of glass- distilled water, then adjusting it to a pH of 8.5 with Bach. (b) The um treated paper sheets were rinsed with distilled water three tines. (o) Sheets were then soaked in 1 I no: for one ainute and rinsed with distilled water until the solution checked neutral. (d) Finally they were washed twice with glass-distilled water and dried at rec tenperature ovemight. 31 (3) The dried papers were now spotted with 10 of base aixture using a aicro pipette and chronategrans were run descendingly at roen tenperature in a solvent containing isoprcpanol, concentrated HCl (sp. gr. 1.19) and water in the proportions 170:“:39 (Wyatt. 1951). All five bases were resolved in the order: guanine, adenine, cytosine. uracil. and twaine. (b) After drying in air, the base resolved chroaatographic ' sheets were placed in a dark room under an ultraviolet leap (253.7 a u) and the bases were located as dark spots against the white paper I then lightly encircled with a pencil. D. Hethods for Deterainaticn of Purine and Pyriaidine Bases by the Bechan mart: Spectrophotoaeter (1) llution For elation, rectangles containing the spots of bases were cut fro- the chraatograns. Rectangles of equal area were cut from the blank at levels corresponding to the position of each base. Each rectangle was cut into shall pieces and placed in an Erlenaeyer flask. To each flask was added 5 ml. of 0.1 N mdrochloric acid. After thorough agitation (by aeans of an electric shaker) at 25-30° C for a mini-an period of 60 ninutes, the eluates were decanted fro. the paper and centrifuged (Marshal: and Vogel, 1950). (2) Ultraviolet Spectroscopy The clarified eluate of each base was read in a Beck-an quarts spectrophotueter in 1 on. cells against the corresponding blank eluate. Acidic extracts of the filter paper itself exhibits a low, 32 but neither constant nor regular. absorption in the ultraviolet region. For this reason. rather than to take the absolute extinction values at the absorption-um (adenine at 262 ‘1. guanine at 2119 m. cytosine at 275 up, uracil at 259 m, and thymine at 2611 up) as the basis of calculation. it was preferable to estiaate the purine and pyriaidins contents of the extracts by using the differences in the extinction values read at the absorption naxiaa and at 290 m. for the standard solution, 10 pg of each of the five bases per one al. of 0.1 II 301, the difference. A , was deteuined as follows: Adenine. D 262.5 = 1.070 E 290 a 0.046 A - 1.02u Guanine, s 2&9 . 0.790 E 290 a 0.299 A a 0.1191 Cytosine, s 275 - 0.960 n 290 a 0.505 “ . 0.155 Uracil, D 259 - 0.7110 s 290 a 0.008 A - 0.732 Tia-ins, l 261} II 0.612 E 290 I 0.076 A . 0e536 33 In order to verify the position of the em. the ultraviolet absorption of the extracts was also deterained at 5 an above and below the characteristic absorption waxim of the purines and pyrinidines in question. i.e. at 267.5 and 257 q: for adenine: 25b andzllllqiforguanine: 280 and2701pfor cytosine: 264and2501p for uracil: 269 and 259 up for thymine. In addition. the extinction oftheeatractsalsowasaeamredatBOOIp. atwhichwavelength the purines and pyrimidines absorb very little. The extinction values found at 300 up should. therefore. be very low. usually between - 0.010 and + 0.0%. Readings outside this range are indicative of contanination. and such extracts should be discarded (Vischer and Chargaff, 19118). Incorporation of P-32 into Nucleic Acids of kbryos During hbryogem' Since developing enbryos. especially in proenbryo stages, are toosnallandnuchtoodifficulttoobtaininadequatembersto perait use of the nethcds of chenical analyses previously described for older. acre nature enbryos. P-32 incorporation has been used as a tracer nethod in order to elucidate changes in nucleic acid content following x-radiation. In any experiaent using a radioactive isotope as a tracer, an obviously inportant requisite for valid results is that the processes studied are not influenced by either the isotopic nature, i.e.. the difference in use number (isotope effect) or the radioactivity 3“ (radiation effect) of the tracer used: if they are. then the results obtained apply only to the presence of the tracer elenent. These are very controversial questions about which nuch has been written. Generally speaking. however. the nagnitude of the isotope effect is .all enough as to be undetected (co-er. 1955) although it might be a vital consideration with elenents of very low atomic nuaber. with regard to potential radiation effects from the use of P-32, papers so far published often report very different results. so that em for liquid cultures it does not appear permissible to set an absolute 'safe' level. Scott. et al. (19119) denonstrated radiation den-age in barley grown for 6 days in nutrient solution containing concentrations of radioactive phosphate as low as 10 no per liter. Hackie. et al. (1952) detected radiation effects in shoot tip cells of barley seedlings grown for 12 days in nutrient culture containing only 11» no per liter at a specific activity of 6.1140 pc P-32/gn P—31; yet 200 pc per liter produced no detectable effects when the specific activity was lower. Levels as high as 1100 no at a specific activity of 9.500 pc/gn resulted in only microscopically detectable effects even in the highly radiosensitive barley proenbryo (nericle and Hericle. 1961 ). In pet experinente. Hendricks and Dean (19118 a, b) reported that radioactive phosphorus in counts as large as 625 pc per kilo- gran of soil did not influence the yield of perennial ryegrass. More recently. Strsenienski (1952) found that P-32 applied in concentrations of fru #0 to 2,560 pc per gran of phosphorus did not influence either 35 dry matter production or distribution in short-rotation rye- grasses. In general. there appears to be a very wide range of |'safe" dose levels for P-32 especially if soil is being used as the 'culture mediul." In the present experiment, 200 no of P-32 was used per kilogrsn sail. a level well below that at which Hendricks and Dean (19118 a, b) found no effects. The validity of using 9-32 incorporation into nucleic acids as a aeasure of nucleic acid content bears Justification at this point. Sons years ago. Sohoenheiaer and his colleagues (19%) ,postu- lated that all constituents of organises. whether functional or structural. were in a state of dynsnio equilibriun. There was said to be a continuous renewal or turnover. in which the synthesis of tissue constituents was exactly balanced by their degradation. In the neantine. it has one to be tacitly assuned that this turnover not only applies to the tissues as a whole but that it occurs as an intracellular event in the various tissues. -Recently. however. the concept of intracellular turnover has been subjected to a critical ennination since studies with bacteria showed that there was probably no renewal of either of the nucleic acids in these cells (Fujisawa, and Sibatani. 1954: Hershey. 19511). The lost convenient and perhaps the only certain nethod of deterrining whether or not. an intracellular constituent is being renewed is to label that constituent with a radioactive isotope and to observe the fate of the incorporated label when the cell is allowed 36 to lultiply in the absence of the extracellular isotope. If turnover of the constituent occurs. its isotope content should decrease in alount. Hershey (1954) applied this method in studying the stability of nucleic acids in I. 99]; and found that P-32 once incorporated into the m and BIA fractions. separated analytically by the Schnidt and Thannhauser nethod, relained there during subsequent cell aultiplications. Recently Silinovitch and Grahll (1956) repeated the eccperilents with bacteria and also showed that RNA and DNA in tissue culture cells behaved in the sane aanner. Pele and Howard (1951+) used autoradiographs to investigate the proportion of cells in the root neristeus of 3121-3 Eh! which synthesised "mu over 10 days, as indicated by the incorporation of P-32 into the nucleic acids. . Taylor and HcHaster (1954). and Moses and ruler (1955) also found that incorporation of v.32 into mm is restricted to a relatively short period of the mitotic cycle which has been shown to coincide with increases in the net anonnt of DNA per nucleus in every instance studied at the cellular level. These data on P-32 incor- poration into nucleic acids at the intracellular level of synthesis will serve as the theoretical basis in this study for applying the sale method to tissues as a whole, In the present study, P-32 was incorporated into embryos at various stages of sabryogeny i-ediately after X-radiation and allowed to continue for extended periods of tine until the last stage of the differentiating e-hryos was reached. During these periods of tine, I II ‘11 il ‘ E1111 37 several eabryo saplings were ads at various representative abryonic stages in order to deteraine relative changes in nucleic acid content as capared with control values. Raising of I’lants light 7-inch clay pots were filled with 2.5 kilograas of haogeneous soil and sown with “i seeds of the barley variety. Blanchen. The plants were later thinned so that seven plants renained in each pet. Tuperatures in the greenhouse where the plants were grownaveraged75°rduringthedayand65°1atnightasprevious1y described. The plants were divided into four different groups. each group consisting of two pots of plants. one control and one x-radiated. . .depeMing upon the initial embryonic stages at the time of the irradiation. The first group was irradiated with a single dose of 450 r at stage a-c, the second group at stage g, the third group at stage 3-10, and the fourth group at stage late 5. 38 Overall scheme of eabryonic stages irradiated and saaples analysedbyP-32 grain counting inthispart oftheeacperiaents was organised as follows: kperiaental Inaber Group 1 Group 2 Group 3 Group it Control hbryo stages X-irradiated a- c 8 3-4 Late5 No radiation 1. Preparation of hbryo Slides kbryo stages analysed by P-32 grain countim d - e. g - 1, 1}, 6c 8 - 1e 5. 6c 1*. 6a. 6c 6a, 6b. 6c d-e,g-1.#. 5, 6a, 6b,6c Procedures for Autoradiographs For the first uperiaent (Group-1), involving the youngest proeabryo stages. four to five aain heads free the seven plants in each of two pots were selected as replicates. one pot of plants for the control and one pot to be irradiated. In order to identify the initial eebryonio stages at the tins of irradiation, a single developim oaryopsis was reaoved fru the aiddle of each of the selected heads for histological emination. a single dose of #50 r of trays. with the sane specifications as described earlier. was given to the plants inonepot.whiletheotherpotofplantswasusedaethecontrol. 39 I-ediately following irradiation. 0.5 no of P-32 in 0.01! al. of ROI. diluted to 150 al. with glass-distilled water. was added to each of the two pots. control and treated. nae radioactive phosphorus was obtained fraa the Oak Ridge National Laboratory. Oak Ridge. Tennessee, as a carrier-free separated isotope. the chemical fora of which was H3P04 in weak HCi solution. The first saplings were needs at 2‘» hours post-irradiation at stage dpe; the second saplings at stage g-1; the third at stage 4; andthefourthatstage6c. Ineachsanplingfroatheaainheads of both irradiated and control plants, two caryopses borne oppositely on the rachis were removed from near the top of each spike and proceeding toward the base (except for the four terainal and four basal grains which were discarded). One grain of each sample was used for autoradiographic slides to deteraine P-32 incorporation into the nucleic acids of the eubryos. while the other was used for the neasureaent of total radioactivity. For autoradiographic purposes. the developing caryopses were killed and fixed in fornalin-acetic acidpalcchol. PM (Johansen, 19110). Materials were dehydrated. embedded in paraffin by standard aethods (Johansen, 19M) and serially sectioned at 6 licrons. Three successive near-median dersi-ventral sections of each embryo were selected and separately nounted on each of three slides with Haupt's adhesive. All section-nounted slides were dried in a boo: with a saall dish of 3-“ foraalin for at least two days. |_.I. i {It I! II..II.I!lllallle|lalll.|a|..l||ll‘ #0 Ira the other caryopsis. at each sapling time, the embryo was dissected and its size aeasured under a binocular dissection micro- scope. The eabryc was then squashed between 10 an. squares of alulima foil and saran wrap and placed in the center of a planchet for counting. Total radioactivity of the embryo was calculated by aeans of a Geiger-Muller counter which had been calibrated against a known P-32 simulated standard. The main purpose of the radio- activity aeasur-ents' was to have a better understanding of P-32 absorption fro- the soil into plants, in the event that there night he sue possible significant differences in the snounts and/or rates of P-32 absorption between control and irradiated plants. In the second crperirent (Group-2), the third (Group-3). and the fourth (Group-h), all procedures were carried out in the sane m as in the first experieent, except for different sampling stages. The first saplings in the second superinnt were made after 24 hours when the embryos were in stage g-1: the second at stage 5: the third at stage 6c. In the third experiment. the first saplings were taken after 2" hours) when the embryos had reached stage it: the second sapling-deatstageéa: andthethirdatetageéc. Inthelast superhent. the first saple was taken at 21! hours post-radiation. at stageoax thesecondatstageébzthethirdatstageéc. B. Methods for Differential Extraction of m and on As described in the previous section. the autoradiographic slides were prepared in triplicate. Differential extractions of an and anereaade fro- the sectionsbyprocedures siailartethose which M were applied to the 'aature' embryo tissue described earlier in this work but with minor acdifications (Taylor and Taylor, 1951; Howard and Pele, 1951.;rq1or and HcHaster. 1951+: Taylor. 1958). All sets of slides (three replicates for each radiation level along with their three replicate controls) were grouped in pairs. an irradiated embryo slide back to back with its control. and treated in the following way: 1.’ 2. 3. 4. 5. 6. Paraffin was removed from the sections with ”lens. All slides were passed through an ethanol series, starting with 1001, down through 901, 85¢, to 70$. Preparations were treated in 70$ ethanol containing 0.1:: perchloric acid for 5 ainutes at roo- temperature to raove interfering substances such as pentosans and polyuronides. liter delwdration through another ethanol series, 85$. 90%. and 100$ samle slides were treated with an ether- ethanol (1:3) solution for 5 ainutes at 60° 0 and passed again through an ethanol series. 100%. 90$. 85‘. 70$ dam to water. For the purpose of extraction of acid soluble coapounds. 0.2 I perchloric acid was applied at 2eh° c for 5 minutes. One pair of slides (irradiated and control) of a set of six wee washed in cold water and stored in 70$ alcohol. The remaining four slides were kept in 1 ll perchloric acid at #0 c overnight for the purpose of extraction of RNA. During this period of tine, two changes of solution were aade 7. 1&2 with the sane nomlity of perchloric acid. (has of the pairs of slides (irradiated an! control) was washed in cold water and stored in 701 alcohol. The mining pair of slides was hydrolysed in 0.5 N per- chloric acid at 70° C for 20 ainutes to remove DNA, rinsed in water. and stored in 70% alcohol. All saple preparations stored in 70% alcohol were kept at 1+9 C in a refrigerator until the autoradiographic eanlsion was applied. c. Application of hulsial Liquid mlsion (Type 3TB; No. LSB-3-0) used in these processes was donated to Dr. ‘L. W. Hericle by Eastaan Kodak Capaw. Rochester. new York. Before applying the eamlsion. all slides with embryo sections stored in 70% alcohol were rinsed three tines with glass-distilled water and processed in the following way: 1. 2. 3. Approninately 15 cc. of emulsion was placed in a saall beaker andaslted inawaterbath at 150° Cinthedark. The saple-aounted ends of slides were dipped into the sailsion then inverted in grooved wooden stands so that an excess aelted aalsion flowed evenly downward coating the saples with a very thin lqer of elulsion. After drying sae 20 to 30 minutes. each eaulsion-covered slide was placed in a horisontal position between pieces of lead (75 a. long. 25 ea. wide. and 3 a. thick) alternately inserted into slots of a light-tight slide box. This box ’6 was placed within another larger case and stored for emosure under refrigeration at 1W 0. Slide bones were previously sprqedwith black lacquer to ainiaise possible light leakage. The purpose of the alternate arrangement of lead pieces was to shield each slide to prevent possible fogging frat adjacent slides. In order to be able to rake quantitative caparisons within and between sets of slides. it was aandatory that all slides have identical exposure tines and subsequent photographic processim. The best ensure tine could be estinated fairly closely after several extra slides had been test developed at different tines. on the 21st day after the slides were coated with emulsion. photographic processing was conducted as outlined below. D. Photographic Processing. 1. llon-lmlroquinone (Kodak 13-19) was used as the developer 2. 3. and en acid-hardening-fixing bath (Kodak r-s) as the fixer. These solutions were adjusted and aaintained in a water bath at 20° C. The preparations were developed for 6 ainutes. fixed for 8 ainutes. and washed through three changes of glass-distilled water (each change lasting 15 ainutes). After beiu lightly stained with Delafield's heaatosylin. the slides were dehydrated in an alcohol series. alcohol-xylene. three changes of xylene. and aounted in clarite under a cover slip. M E. Track Counting and Estimation of the Relative Amount of P-32 Incorporation Although most emulsions record only a small part of the complete path of rays or particles. it is possible to correlate the amount of radiation striking an emulsion with the density produced in the emulsion. For visible light. the relation between the intensity of the light and density produced is the well-known Hurter and Driffield curve (Hoes. 1948; Fitzgerald. et al.. 1953). U ll log1o (Io / I) where D density Io = intensity of original beam. and I a intensity of transmitted beam. For beta emitters. similar relationships may be drawn (Marinell and Hill. 19148: Steinberg and Solomon. 19149; Webb. 1951). According to Fitzgerald et a1. (1953). there is a linear relationship between the density of the NTB emulsions and the total number of beta particles per cmz. For this reason. it is possible to measure the relative concen- tration of P-32 striking the emulsion by the number of grains produced. and in turn to estimate the relative P-32 incorporation into DNA and RNA when the geometric relationships between emulsion and object have been held constant. As already described in the previous section. three sets of slides (each comprising two slides. one for control and the other X-irradiated) were differentially treated for extraction of the twa types of nucleic? acids and coated with emulsion for the development of tracks so that #5 the first set of samples nos contained RNA. DNA. and.protein: the second. DNA.and protein: the third. protein only. By visual counting of the grains over a unit area of tissue. estimates of the relative incorporation of phosphorous- 32 into DNA and RNA eere made. The number of grains. in a unit area of #900 sq. p. delimited by a retiele in the eyepiece of the microscope. was separately counted over three different regions or tissues: namely. root. shoot. and scutellum under an oil immersion magnification of 13501. The aserage number of tracks was obtained by counting grains in five to ten.sueh units in each area of tissue. In.these embryos smaller than a single unit of counting area. the relative number of nuclear tracks easldetermined by counting the total grains over'the entire area of therembryos (both control and irradiated) and1mmnnuddng them into units of relative area (obtained.by weighing cut-outs1ot4nnmuuhlucidq drasings of the respective embryos). Corrections for background. which were quite law. were made by counting ten areas outside the tissues on each slide and subtracting the average background count from.the Counts obtained over tissues. By keeping the emulsion refrigerated itciuiruflzseem to seem-ulste.1atent tracks final(“underray'souroes¢hnHUuzIdeeege. the use‘oriuumudlemmlsionibm1mds tantrum-ittmiallmetzhuuhufleuand penmumnn;oontact«ottnounhmmlandcmmfledon.Imnzeaaruramaahmufle in any'muunranuz (Plate 1) EVALUATIOI OF BIOCHDIIGAL METHODS USED m3 31811103 Evaluation of the biochemical procedures for determination of purineamdpyrimidinebases ofnucledc acids applied tofullydif- ferentiated barley embryos (both normal and x-rayed) can best be done by examining the results obtained from using these same pro- cedures on co-ercial samples of free bases. yeast RNA. and salmon mm. and by comparing these results with those from other methods reported by other investigators. Determination of Standard Extinction Values of Purine and Pyrimidine Bases Since the bases from mature embryo samples. after separation by paper chraatography. were eluted with 0.1 N hydrochloric acid. the standard ertinetim values used for detenimtion of unknown base contents in the embryo samples were obtained from spectrophotuetric readings on camercial samples of free bases in 0.1 H hydrochloric acid. For each of the five bases (a grades of guanine. adenine. cytosine. uracil. and thymine. which were obtained from California Corporatim for Biochemical Research. Hedfort Street. Los lsgeles 63. California). 10 mg. samples sere dissolved in 10 m1. of 0.1 l hydro- chloric acid. and an 0.1 ml. aliquot of this solutim was diluted to 10 ml. with 0.1 I hydrochloric acid. Standard extinctim values of 45 47 these bases were determined. as described previously. from the difference read at the absorption maximum for each base. and at 290 mp (Washer and Chargaff. 19%). In Table 1. the averages of each of three determinations for standard extinction coefficients are presented for each of the five bases. Cuparing these values (0.181 mp for guanine. 1.021+ mp for adenine. 0.165 mp for cytosine. 0.732 mp for uracil and 0.536 q: for thymine) with those obtained by Vischer and Chargaff (was). (0.475. 0.900. 0.5115. 0.690. and 0.516. respectively). very close agreement was observed. ihe standard extinction coefficients determined by the writer are. however. the ones used for calculation of unknown base content in embryo materials. Recovery of Free Bases Ml oaplanation has already been made of the procedures for hydrolysis. paper chruatography. and spectrophotcmatry in the previous sections of materials and methods: only minor differences for this part of the experiments will be indicated here. Ten mg. each of guanine. adenine. cytosine. uracil. and thymine were added to1.6ml. of12lperchloric acidandhydrolyaedinasteambathat 100° 0 for one hour. After cooling. the mixture was diluted to 10 ml. with distilled water and the resulting clear. colorless solution was used for paper chromatography. Four replicates of 10 A aliquots of the testing solution were deposited on the paper and four bases were separated with the solvent. isopropanol-hydrochloric acid-water. in the proportions of 170:1":39 respectively (Hyatt. 1951). After 1&8 elution with 0.1 l hydrochloric acid. the clarified eluate of each base was read in a Becltman spectrophotometer and the amount of purines and pyrimidine was calculated accoxding to the standard extinction values shown in the Table 1. As indicated in Table 2. the percent recoveries of the five bases were ranged from 93 to 97. with an average of 96. Approximately 11$ of the bases. therefore. were lost during the processes of hydro- lysis. paper chromatography. eluting. and spectrophotometry. ‘Calparison of ‘hvo ‘fypes of Hydrolytic Processes in Yeast RNA and Salmon DNA Since it was impossible to directly apply previously used techniques to the present work. without modification. it was necessary to cupare the recovery rates obtained by previously used processes to those as modified in the present experiments. A. Recovery Rates After Hydrolysis with 12 N Perchloric Acid Following methods of Marshal: and Vogel (1950). 80 mg. of yeast RNA in 1.6 ml. of 12 N perchloric acid or 20 mg. of salmon DNA (A grades of two commercial samples obtained from California Coupon for Biochemical Research) in 0.1; m1. of 7.5 n perchloric acid was heated in a steam bath at 100° C for #0 minutes. After cooling. the mixture was diluted (10 ml. for REA. and to 5 ml. for DNA) with.distfl.1ed water. and centrifuged to separate the fluid fra the black residue formed (presumably from the sugar moieties). 49 The resulting clear hydrolysate was used for paper chromatography with 10,-»1‘ aliquots being used for each of four replicates (25 A for DNA). Rectangles containing each base separated on the paper were eluted in 5 ml. of 0.1 l hydrochloric acid. The extinction value of each base was determined frcm the difference. A x. read at the absorption maximum and at 290 mp. as explained previously. The quantity of each base eluted in 5 ml. of 0.1 )1 hydrochloric acid was calculated according to the same difference. A . for a standard base solution containing 10 pg of each base per ml. of similar acid solution. (The standard extinction values are listed in Table 1). Fl‘tl the base content in 5 ml. of acid solution. the recovery of each base in )1 moles per 80 mg. yeast RNA (20 mg. for salmon DNA) and mole bases per mole phosphorus were cuputed according to the phosphorus value given by Marshal: and Vogel (1950): a.” P in RNA and 9.371 P in mm; therefore. 23 x 10-5 molesPin80mg. yeastRNAami6x10‘5molesPin20u. salmon DNA. Theactual malyticalvaluesareshovmin‘rable3andthe recoveries were found to be 8% for yeast RNA and 79% for salmon DNA. In order to confirm the reliability of these analytical processes. the present values for DNA-bases. corrected for 1001 recovery. (20 moles for quanine. 27 moles for adenine. 23 moles for cytosine. and 30 moles for thymine per 100 g - atoms of phosphorus) were'cupared with those corresponding values show by Chargaff (1955) (20. 29. 20. and 29 moles. respectively). For the same purpose. the present base recoveries of yeast RNA (0.95 for guanine. 1.05 for adenine. 0.95 for cytosine. and 1.05 for uracil) as molar ratios in a total of 16.00 were compared with 50 corresponding values (1.19. 1.02. 0.83. and 0.96. respectively) from hith and Markham (1950). more was very close agreement observed in both cases of caparisons. B. Recovery Rate after mdrolyeis with 1 N Perchloric Acid The procedures taken in this part of experiment were exactly the same as those in part A. as previously described above. except for using 1 I perchloric acid at 100° c for 160 minutes (with an air stream to hasten evaporation). The main purpose. therefore. was to determine whether an differences in recovery rates existed as a result of the different methods of hydrolysis. As shown in Table I}. the recovery rates in this experiment were 87$ for Rifle-bases and 755 for DNAe-bases. while those corresponding values for the eweriment with 12 N perchloric acid were 84$ and 79$. respectively. Since the results in the foraer were only 3% higher in RNA afi '1" less in DNA than those in the latter. there were no appreciable differences in recoveries observed due to the different treatments in the two experiments. These results are also in agree-e ment with Marshal: and Vogel (1950). who reported that the prolongation of heating time from ‘10 minutes to 160 minutes does not affect recovery. Recovery oerast RlAand SalmonDIAby Processes applied to hbryo Samples he purpose of the previous experiment was to determine the recovery rates spectrophotuetricany i-ediately after hydrolysis following the separation of purine and pyrimidine bases by paper 51 chromatography. This rather simple. stepwise set of procedures. however. could not be amlied to quantitative determination of nucleic acid bases in embryo emaples. because of the various interfering materials in the sample mixtures following hydrolysis. As previously described earlier in the materials and methods. many steps were taken behoen hydrolysis and paper chromatography for the purpose of purification and elimination of a large quantity of salts and various. partially hydrolysed organic substances. The main difficulty encountered" in the processes of purification was the loss of bases due to the differences in the solubility of each base. Arw single solvent always tended to cause loss of certain bases more than others. This obstacle. however. was overcome in the following ways. The solution of bases immediately after hydrolysis was neutralised with potassium carbonate (to eliminate perchloric acid) and the mixture washed with an excess of water and 80$ ethanol. A large quantity of four of the bases. adenine. cytosine. uracil. and thymine. was dissolved in the filtrate. while guanine was always tied up with potassium perchlorate due to its insolubility in water. The greatest quantity of guanine. however. was successfully recovered by dissolving the residue in 0.1 N hydrochloric acid which separated the base from the salt. Recovery of least RNA and Salmon DNA by Processes Applied to hbryo Samples Inordertofindout the recovery rates ofyeastRNAandsalmon DNA by the procedures which were applied to the uhryo samples. the 52 same quantities of yeast RNA (80 mg.) and salmon DNA (20 m.) as those in the preceding experiments were subjected to the embryo sample processes which have been already described in detail in the earlier sections. The calculation of )1 moles per 80 mg. yeast RNA (20 mg. salmon MA) and mole bases per mole phosphorus were con- ducted in the same way as in the previous experiments in this section. As presented in Table 5. 79¢ recovery for yeast RNA-bases and 73$ for salmon DNApbases were observed in this part of experiment. The ranges of recovery shown in a report on animal sources by Chat-gaff and Davidson (1955) are 67 to 99.5 percent. Although the percent recovery in the present data fall within these ranges they do not'reach the higher range. This difference is probably due. in large part. to the different analytic procedures used. since in animal materials. it is usually not necessary to take special pro- cedures to eliminate interfering naterials following hydrolysis. Therefore. after preparing sodium nucleate. the purine and pyrimidine bases can be separated and estimated immediately after hydrolysis by paper chromatography and spectrophotometry. respectively. In contrast. in the present work many prolonged steps have had to be taken to minimise the interfering materials which were the major difficulty encountered in the analysis of nucleic acids of 'embryo samples. Eventually. as a result of these long stepwise processes. the final recovery tended to be somewhat lower due. presumably. to the difference in solubility of each base. These factors are believed enough to explain rather lower recovery rates in this experiment. 53 Sirvey of Over-All Recovery Rates of Purines and Pyrimidines in Yeast RNA and Salmon DNA From the results obtained in the three previous experiments (summarised in Table 2. 3. and 5). an average of 96% recovery was observed in the experiment utilising free bases (Table 2). This indicates that bases once liberated fro! the polymerized nucleic acids will not be recovered more than 96% by the analytical processes used in the present study and that the net loss is mainly due to the steps taken during paper chromatography. eluting. and spectrophoto- ‘ metry. As shown in Table 3. the base recovery of yeast RNA and that of salmon DNA. following hydrolysis with 12 N perchloric acid. were 84$ all! 80$. respectively. These data indicate that up to the process of hydrolysis (which was not followed by the steps taken for purification) 16% of yeast RNA and 20% of salmon mu were lost. Since 15$ loss was caused by the manipulation of paper chromatography. eluting. and spectrophotometry. as already explained above. approxi- mately 12$ yeast an and 16% salmon on was estimated to be lost. due mainly to incomplete liberation of bases (the processes involved in filtering the black pentose and decrypentose residues from the mixture of bases. and probably also. in part. to possible commercial Linearity). Observing the data presented in Table 5 in which processes appliedtoembryosampleswereusedonyeastRNAandsalmonDNA. 79$ recovery is shown for yeast an and 73$ for salmon DNA. col-perm 54 the recovery results (814 for am and 80$ for mu) indicated in Table 3 with those obtained frm the experiment shown in Table 5 above. 5‘yeutRlAand 7% salmonDNA were lost. duemainlyto the different solubility of each base and to other long manipulations of the purification processes. It is quite useful in the evaluation of recovery rates of an experiment and for caparing the data from one experiment with others. to consider awe of the factors which affect the loss of saple materials during analytical processes. First. the different solubility of each base greatly affects the. recovery rate. Reviewing the properties of each base: of the purines. guanine is water- insoluble. very slightly soluble in alcohol and ethyl ether. and soluble in potassium hydroxide. while adenine is soluble in cold water (0.09 grus per 100 ml.). slightly soluble in alcohol. insoluble in ethyl ether or chloroform. and soluble in hot a-oniu hydroxide: of the pyrimidines. cytosine is water soluble (1.0 gram per 130 ml.). slightly soluble in alcohol. insoluble in ethyl ether. and forms salts with acid. while uracil. on the other hand. is very slightly soluble in cold water. insoluble in alcohol. and soluble in ether and min hydroxide. The last pyrimidine base of DNA. thymine. is water-soluble (0.71» grams per 100 al.). slightly soluble in alcohol. very slightly soluble in ether. and soluble in alkali and sulfuric acid. As described above. there are no omen properties of RNA and DNA base solubilities. Any single solvent. therefore . will not be 55 satisfactory for the recovery of all the bases during the analytical process. unless more than one kind of solvent is applied at the same time or separately at the different steps conducted during purifi- cation in this experiment. ' The second factor. which has by far the most effect on recovery rates. is the degree or base liberation from polymerized nucleic acid during hydrolysis. The possible hydrolytic products of both RNA and DNA include purine and pyrimidine bases. pentose and pentose phosphate. nucleosides. nucleotides. and oligonucleotides. It is characteristic of RNA and DNA. however. that the purine riboside or dewriboside linkage is unusually labile to acid hydrolysis. while the pyrimidine ribose or deoayriboside linkage is relatively resistant. The free purine bases. adenine and guanine. are readily toned during acid hydrolysis of RNA and DNA. whereas the pyrimidine bases remain for the most part as mononucleotides. in the case of RNA. or as nucleoside diphosphates. in the case of DNA. As the purine bases are cleaved. reducing groups from the N-riboside or N-deoayribo- side linkages are liberated and. depending on the conditions used. free ribose or deoxyribose may be formed. As to the degree of purine liberation after acid hydrolysis of RNA. apparent discrepancies in results have been reported. According to indirect evidence frtm the recovery of adenine (as adewlic acid) and of guanine from sodium guanylate. approximatcly 95‘ and 100$. respectively. were reported by Vischer. and Chargaff (19118) after Indrolysis in 1 N hydrochloric acid at 100° c for 1 hour. Other It"|ll|l'll ll 56 experiments by Abrams (1951). in which an isotope dilution method was used to estimate the purine concentration. have indicated that as much “7.87éorbothsdenineshdgushineusyhedestroyedhy1Nhydro- chloric acid at 100° 0. Although both experiments above were operated with the sane normality of acid and at the same temperature. some differences in results were reported. Therefore. it is especially difficult to compare analytical data on purines of any two experiments when different processes are undertaken under different conditions and with different materials. As to the liberation of pyrimidine bases of RNA. Chargaff and Davidson (1955) reported that hydrolysis of yeast mm with 12 N perchloric acid for 1 hour at 100° 0 causes no appreciable destruction of either adenine. guanine. cytosine. uracil. and thymine. However. the recovery of total pyrimidine base; indicates an incomplete liberation fro! nucleic acid when this procedure was applied to yeast RNA (0.37 moles per mole P and 0.12 moles per mole P in the present experi. ment as shown in Table 3). At present. therefore. under the conditions studied. neither 20% N01. concentrated formic acid. nor 12 N perchloric acid can be said to lead to the complete liberation of the pyrimidine components of RNA in the form of free bases. The data analysed on base components differ greatly depending upon the quantitative distribution of purines and pyrimidines in nucleic acids of any species. When the compositions of many specimens of DNA from different cellular sources are compared. a very striking feature emerges. as was pointed out some years ago: two principal. 57 groups can be distinguished. namely. the "AT type. " in which adenine and thymine predominate. and the "ac type." in which guanine and cytosine are the major constituents. In addition. an intermediate group was discovered in 3,. ml; which is characterised by the presence of almost equimolar proportions of all four components. Total DNA preparations from animal sources described so far belong to the AT type while the GC type has been encountered in several microorganisms and in some insect viruses. The molar ratio of guanine and cytosine to adenine and thymine will usually be less than 1 in the nucleic acids of the AT type but greater than 1 in those of the 00 type. In other words. the analysis of purified DNA preparations isolated from a great variety of cells has led to the conclusion that the DNAs of different species of organisms have different nucleotide compositions and those of different organs of the same species have identical compositions. Because of the different distribution of purines and pyrimidines in each different species of materials. it is very difficult to compare the quantity and compositions of bases recovered from one material with that from another kind of ample. Finally. the recovery rates may vary depending upon the different techniques utilised by different workers. In regard to this factor. Chargaff and Davidson (1955) have stated that 'ths comparison of analytical results obtained by different workers using procedures that are almost never identical and often radically different suffers from a great deal of uncertainty. " Eggs AID DISCUSSION Determination of Purine and Pyrimidine Contents ofmanleAinlonalHaturehbryos he purpose of this section of the work was to determine the purine and pyrimidine contents of RNA and DNA of normal. mature embryos so that these date night be used as control values for the results to be obtained fr. Lraying embryos at four different developmental stages. Four replicate embryo samples. each consisting of 138.5 rg dry weight were subjected to the analytical processes which have been described previously in Hatorials and Hethods. The data on the electrophotuetrie estimation of purine and pyrimidine bases. following paper chromatography. with their quantities expressed in a p moles per lg dry weight of embryos are presented in Table 6. As shown in the salary of Table 6 and 12. the ratio of RNA to on sea found to be 5.9 (calculated by dividing the total quantity of RNA bases: [mine 37. adenine 28. cytosine 32. and uracil 26 my soles per mg dry weight of eabryos by the total content of their DNA- bases: guanine #J. adenine 5.0. cytosine ht. and thy-ins 7.2 m ; soles per lg dry weight). In comparison to this value. Car and Boson (1959) reported BIA to DNA ratios of 5.7 in corn root tips and 1.9 in 58 59 rabbit liver (determined by direct ultra-violet absorption of polymerisod nucleic acids isolated with 1 l and 0.5 N perchloric acid). Thus the ratio of RNA to DNA in both corn root tips and mature barley enbryos are in close agreement. Reference to the same tables shows that the ratio of purines to pyrimidines in RNA was found to be 1.1. According to Chargaff and Davidson (1955). the range for this ratio is fr. 0.98 to 1.69: pregnant rabbit liver 0.98, rabbit liver 1.08. chicken liver 1.12. and calf pancreas 1.69 (deterninod spectrophoto- metrically after separation by paper chromatography). It will be noted that the present data fall within this range. Purine and Pyrimidine Contents of RNA and DNA in nature hbryos X-irradiatod at Four Different Mryonic Stages Four replicate embryo samples. each consisting of 160.6 n dry weight which were Lirradiated at the youngest preembryonic stages s-c (Group-A) were subjected to the same analytical procedures as the control samples. after reaching stage 6c in embmgem. In the same way. four replicate embryo samples. each consisting of $0.1 mg dry weight for Group-B. 273.2 mg for Group-O. and 203.2 mg for Group-D, Lradiated at stage g. 3-4. and late 5. respectively. were subjected to the same analytical methods as the control samples when these embryos in the three above groups reached stage 6c. The values for extinction at wave lengths in m n for the spectrophetuetrio estimation of each base. and the base content in an moles per mg dry weight of embryo samples are shown in Table 7 (Group-A), m1. 8 (amp—a), “\ 60 Table 9 (Group-C). and Table 10 (Group—D). Those analytical data obtained from the four experimental groups and control are summarised in Table 11 . This table presents the analytical purine and pyrimi- dine amounts of the four different irradiated groups of embryos cupared with the control in m )1 moles per mg dry weight. Table 12 indicates the percent nucleic acids of the control. the cmparison of the ratios of mom and purine-pyrimidine ratios of the four different experimental groups as compared with the control. while Table 13 shows the percent base content of each experimental group cupared with that of the control. The effects on the total amount of RNA-bases following a #50 r dose of I-radiation given at four different stages of embryogew may be more easily visualised by referring to Figure 1 (based on Table 11). As shown in this histo- gram. thoro was considerable depression of RNA. #8 to #2 m )1 moles per mg dry weight in the I-rayed embryo groups. from the cmtrol value of 123 m ’1 moles. Differences within the Lirradiated groups were small. the maximum difference being only 6 m p moles between Group—A (which showed the greatest depression) and Group-C and -D (which had the same depression). The depression occurring in Group-B was found to be intermediate between those two extremes. In general. the degree of depression of RNA. as observed at stage 6c. was greatest the earlier the stage of embryegev at the time of irradiation. while irradiation at either stages 3-!» or late 5 of the differentiating embryo produced the same degree of RNA depression. The amounts of DNA-bases in Lrayod and control groups of embryos (Figure 2) showed depression; of fra 6.8 to 1.4 m p moles for 61 the irradiated groups of embryos as a whole as cmpared with the control value. Comparing differences in DNA content among irradiated groups. there occurred a maximum of 5‘} m )1 moles difference between Group-C and -D. and a minimum of difference (1.3 m )1 moles) between Group-B and -C. The degree of depression of IDEA content was greatest with Group-D. followed by lessening degrees of depression in Group-A. -B, and -C. respectively. If the RRA-base and DNA-base contents are summed to give total nucleic acid values. the control shows 1mm m p moles per mg dry weight. while in the Lirradiated groups. Group-C with the least depression had 100.6 m p moles. Group-D 95.2, Group-B 914.3. and Group-A. the greatest depression with a value of 91.9 (Figure 3). In order to more easily assess the degree of relative differences in RNA. DNA. and total nucleic acid content in these four irradiated groups of embryos. it seems reasonable to express these analytical data on a percent basis with the control expressed as 100 percent (Table 12). The total nucleic acid content of the experimental groups showed rather large depressions of from 31$ to 36$ below that of the A control. yet only a maximum of Si difference among the irradiated groups. The RNA content of the experimental groups behaved much the same as the total nucleic acid. largely because the RNA makes up the major part of the total nucleic acid (so 435%). In terms of m content. among the x-rayed embryo groups. Group-C and -D showed the highest values of 66$. Group-B 62$. and Group-A the lowest value of 615. Thus, while there was observed a 33-39% depression of RNA in the X-rayod groups as a whole capared with the control values. only a maximum 62 of 5p difference occurred among the irradiated groups. use as shown in Table 12. the highest value of DRA (as percent of control) was 93$ in Group-C. 871 in Group-B. so; in Group-A. and the lowest 68$ in Group-D. It would seem that there is a gradual tendency toward a decrease of BIA as younger stages are irradiated. except for Group-D. It seems important. therefore. to capers the relative changes of RNA and DRA within the tissues of barley embryos in several different ways. he points should be brought out for the purposes of these considerations. First. the Group-A experiment underwent the longest postirradiation time. 16 days. between I-ray treatment and sampling time. Group-B 12 days. Group-c 9 days. and Group-D the shortest time. 7 days. If one considers only postirradiation time and neglects the physiological differences of the embryos at the time of irradiation in each experimental group. one might expect the percent of RNA to be the highest in Group-A. second highest in Group-B. third highest in Group-C. and lowest in Group—D. The actual order of recovery was found to be exactly reversed. Group-A lowest. Group-B next. and Group-c and -D highest. A A similar situation is found for DNA in Group-A through -C, but Group -D instead of having a DEA content equal to that of Group -C (as was the case with BIA). has instead the lowest amount. With the exception of the MA value of‘ Group -D. these results demonstrate that the differences in post-irradiation time cannot in themselves account for the order of the observed radiation induced differences in RIA and DNA. me only other way in which those groups differed experimentally from one another was in the embryonic stage of development at the 63 time of radiation. The order of the observed differences in all and mm (with the exception of the an value in Group -9) certainly implies that the younger embryos were more affected and/or underwent less recovery during the post-irradiation time until sampling than the older ubryos. In other words. the results obtained from these experiments are in agreement with the general statement (Borgonie and Tribondeau. 1906) that the younger the tissue. the more the effect due to ionising radiation. The divergent results observed in Group -D. however. would appear to contradict the above general statement. since the percent DNA in this Group is seen to he the lowest among the four experiments in spite of the fact that it was the oldest tissue (stage late 5) at the time of irradiation. This could be explained by comparing the metabolic activity in the nucleus. which is the site of mu synthesis. with that in the cytoplasm where the greatest quantity of RNA is synthesised. It is generally agreed that in more mature tissue (such as embryos in stage 6) the physiological activity ofz’ncuclous is lower. while that of the cytoplasm is higher (Ha—arms and Hovesy. 19%). In addition. the time spent in interphase by cells caprisiag mature tissue is much longer than that in cells of actively growing tissues. due to the lower mitotic activity of the former. If more mature tissue. therefore. is once affected by ionising radiation. mu recovery is more difficult than that of Eli because of the lower metabolic activity of the nucleus as. compared to the cytoplasm. In addition. it also requires a much longer time for mature tissue to recover following irradiation due to its longer time spent in interphase. which usually is the only period 61; in the mitotic cycle during which DNA synthesis occurs (HOV-m 191‘s). Secondly. in cupsring the differences in recovery of either RNA or DNA (as percent of control) it can be seen (Table 12) that the DNA of the four experimental groups shows wider variation (6 - 2 5%) than does the m (o - 5%). This was in spite of the fact that in all cases the percent of RNA depression exceeded that for DNA. These results lead to the important suggestion that the stage specificity of radiation effects observed by others (Hericle and Hericle. 1957: 1961) may be referred more to effects on the DNA rather than the RNA components of the embryo cells. From Table 12 it can be seen that the ratios of RNA to DNA were the same for Group-A. -B. and -C and the values were lower than that of the control. The ratio in Group -D. however. was comparable to the control. In general. it is very difficult to detenine the relative sensitivity of RNA and DNA to ionizing radiation since the ratio of RNA-DNA varies depending upon the period of post-irradiation. No conclusion may be drawn as to their comparative sensitivity to Lirradiation at any particular sampling period. As shown in Table 13. the content of DNA-bases was lower than the control for all experimental groups. The purine-pyrimidine ratios in Group -A. -C. and -D correspond to that of the control. but the ratio in Group -B was increased by 2013 as cupared with the control. “This increased ratio of purine-pyrimidine in this experiment did not return to the normal pattern of nucleic acid metabolism during the 12-day post-irradiation period. Analysis of the DNA compositions of 65 Group -B showed a considerable decrease in thymine content (6 5% of the control value). while the contents of guanine. adenine. and cytosine were comparable to the control. On the other hand the content of RNA-bases was also lower than the control for all experimental groups. The purine-pyrimidine ratios in Group -A. -B. and -D were comparable to the control. but the Group -C ratio was increased by 20$. This ratio did not return to the normal pattern of nucleic acid metabolism during 9 days post- irradiation. Analysis of the RNA compositions of Group-C showed a considerable decrease in uracil content (5% of the control) which was the lowest for the four ' experimental groups. The contents of guanine. adenine, and cytosine. in Group -C were cmparable to the other treatments. The results obtained from the experiments of Group -B and -C indicate that uracil of m and thymine of mm W bo’ more labile to X-irradiation than other bases. Comparing the present data with those by previous investigators. Boronbom and ;‘Peters (1956). in their study of nucleic acid changes in rats after a total-body X-irradiation (M0 roentgens as a single dose). reported that spleen DNA and thymus RNA showed evidence of ‘ altered composition. The increase in the purine-pyrimidine ratio of spleen DNA was accompanied by a substantial reduction in relative content of cytosine and mmine. resulting in a disproportionate - increase in the guanine and adenine content. The ratio in this case did not return to the normal pattern. This irreversible alteration of purine-pyrimidine ratio was observed in the present experiments. Borenbom and Peters (1956) also noted sue modification in thymus RNA 66 composition whereby the purine-pyrimidine ratio increased more than the control value. In their material. however. this alteration appeared to be temporary. since an essentially normal pattern was observed in 7 days after irradiation. In an attempt to determine the effect of X rays on nucleic acid purine and pyrimidine bases of rye seedlings. Sissakiln (1955) reported that uracil was most labile due to I-irradiaticn (20 Kr). He concluded that under the influence of X rays nucleic acid synthesis became disturbed. This Lability of uracil seems to be in agreement with the results observed in the present work. although different materials and different doses of radiation were used. Effect of I-irradiation on P-32 Incorporation Into Nucleic Acids of hbryos as a hole A. Changes in Nucleic Acids during the Period Following X-radiation (b50r) at Youngest Proenbrye Stage a-c (Group-1 ). The main purpose of this experiment was to determine the contents of RNA and DNA as percent of the control values during developental abryogeny. General methods have been already explained in Naterials and Methods. Minor differences in procedures for this experiment will be explained prior to the discussion of results. After I-irradiation and treating of the plants with P-32. samplings (control and X-rayed) were made as sunarised on page 39. Table 1h shows the P-32 tracks counted for RNA and DNA. These were computed according to the methods which have been described in the section of Materials and Nethods. The percent of the two types of nucleic acids as cupared to the control values are recorded in Figure 4. The content of DNA fell to about 35$ of the normal level in 2b hours (at stage d-Le) and slightly recovered to #55 of the control in three days (at stage g-1) post-irradiation. The quantity of DNA then decreased to 28$ of the normal value in 7 days (stage 1+) while the final recovery value (83$ of the control) was reached in 16 days pest-treatment. It will be noted that there were two minima. the first 35$ of the control at stage d-e and the second 28$ of the normal at stage it 67 68 Observing the percent content of RNA compared with usual values. the first sample showed 681 at stage d-e: the second 52$ at stage g-1: the third 67‘ at stage It; the fourth 58$ of the normal level at stage 6c. As shown in Figure fl. there existed an interrelationship of reciprocal fluctuation between RNA and DNA throughout the 16-day pest-irradiation period. The content of DNA was much lower than RNA at 21‘ hours post-irradiation and recovered more than RNA at the final sample stage (Table 1*). Cuparing the difference between minimum and maximum values of RNA with those of DNA during the 16-day post- irradiation period. an was found to be 161. while DNA was 55% of the control. This indicates the relatively higher radiosensitivity of DNA as compared to RNA. Since the results obtained in this experiment (Group-1) seemed to be most representative of the patterns of response of embryo tissue to ionising radiation. the discussion will be focused on this experiment. Results of other experiments will be discussed wherever relevant. (1) Incorporation of P-32 as a measure of DNA snythesis. In these and other studies (Hershey. 195k: Pole and Howard. 1951;) the incorporation of P-32 into DNA has been used as a measure of DNA snythesis. Nhen expressed in terms of number of nuclear tracks per cell. the incorporation data acquire a meaning analogous to the mitotic index. Nhereas the mitotic index indicates the relative number of cells undergoing mitosis at a given moment. the number of grains or nuclear tracks due to DNA per cell is proportional to the 69 relative number of cells engaged in DNA synthesis during the period of incorporation. This phenomenon is demonstrated by data obtained 'for the first control samples of Group-3 and -1. (Tables 114 and 17) where 0.77 and 0.37 tracks per cell. respectively. were observed. Since those samples were taken at the same number of hours after irradiation (2h hours) the data show. as expected. that DNA synthesis is more rapid in the younger embryo tissue (Group-3) than in the older tissue (Group-h) where differentiation is more advanced. in spite of a larger reservoir of P-32 in the latter (Table 19). As shown in Table 19. the range of total radioactivity (in counts per minute) per mg dry weight of X-rayed embryos was found to be 8‘} to 126 percent of the controls. These differences in the amounts of P-32 between I-rayed and control plants. however. should not significantly affect the amount of nucleic acid synthesis in embryos. since the penetration of inorganic phosphorus into nuclei is so slow (Hahn and Hevesy. 19110) that a large reservoir of intracellular and extracellular inorganic phosphorus is built up. (2) Evaluation of the Results The interpretation of data on the labeling of DNA in a tissue is complicated by the fact that few tissues consist of a single population of cells. When a tissue contains cell populations of different mitotic activities. the contents of the isolated DNA will depend on the relative abundance of the different cell types and on their mitotic index. In studying the effect of irradiation on the tissue as a whole, we obtain the summation value for the effect on the various cell types. 70 Evaluation can be simplified if the assumption is made that the relatively abundant types of cells exhibit a greater rate of repro- duction than the others. therefore the observed changes of P-32 incorporation following irradiation will essentially reflect the changes in these normally more active cells. (3) Ccmparison of minimum values The maximum depression of RNA and DNA in the various tissues of the rat treated with 500 r of I-ray. according to Lutwak-Hann (1951). was found to be 91 and 28% of the control in DNA and RNA of bone marrow. respectively: this maximum depression occurred four days after X-irradiation. According to Sissakian's (1955) recent report on the nucleic acid content in rye. seedlings treated with a dose of 5 Kr. x-radiation. the maximum depression of P-32 incorporation into DNA was found to be 251 of the control value. The relation between the time period of post-irradiation and the minimum of DNA synthesis was shown by Pole and Howard (1954). The first depression of DNA in £23 £513; I-irradiated with mo roentgens was down to 10% of tho control value at 6 to 8 days. post-irradiation. Berenbom and Peters (1956) reported a 75% decrease of DNA at 2 days. post-irradiation. in an experiment with thymus of male rats treated with #00 r total-body X-irradiation. The maximum depression of DNA in the present study was down to 35% of the control for the first minimum and 28$ for the second minimum at 214 hours and 7 days post-irradiation. respectively. This fell in the range reported by other workers even though materials were different from those of other investigators. 71 (It) The First Minimum Knowlton and co-workers (19b9 and 1950) have concluded that the time fra irradiation to the initial minimum for the mitotic index is a measure of the duration of mitosis. Analogous to this. the time frn irradiation to the minimum for the incorporation of tracer into DNA might indicate the rough duration of DNA synthesis in the more active groups of cells in the tissue. The initial decrease in P-32 labeling of DNA can then be explained as a reduction in the number of cells entering the period of DNA synthesis. This may in part be due to a delay in the initiation of DNA synthesis (Howard and Polo. 1953) by temporarily or irreversibly affecting the presynthetic phase in some groups of cells. In part it may be due to a direct. irreversible inhibition of DNA synthesis which in turn may be due to an interference with existing DNA in such a way that the presumed Self-duplication is made impossible (Nygaard and Potter. 1959). Although many other factors may be involved in the decrease of DNA due to X-irradiation. it was found that part of the decrease in DNA was due to the fact that there were fewer cells counted per unit area in the tissue of X-rayod embryos. The significance of this will be discussed in the section of "Theories on the Effects of Ionising Radiation on Depression of DNA. " (5) Abortive Recovery As mentioned previously. after the first sampling the content of DNA had recovered to 455 of the control within 2 days post-irradiation. This "abortive" recovery of DNA may have been caused by cells that 72 were not inhibited and would have entered DNA synthesis at the time of irradiation. and also by the recovery of cells that were originally delayed by the irradiation. (6) The Second Minimum After this temporary recovery. the percent DNA content fell to 285 of the normal value. This second minimlm in mitotic activity no he a result of the failure of MA synthesis brought on by the previous abortive mitosis which occurred in predominant groups of active cells withimthetissue. ThiscouldbepossibleduetethefaotthatDNA synthesis and mitosis do not occur simultaneously in any single cell. not within the more active groups of cells in on cell population. In other words. the replication of DNA is a necessary prerequisite for mitotic division (Howard and Polo. 1953). 6 (7) The Final Recovery The final recovery rate my depend in part on the relative number of cells which were “released" from previous suppression of DNA synthesis. and in part on the'degroe of metabolic disturbances which occur within individual cells as a result of the direct effects of ionisiu radiation. On the other hand. the final recovery rate may be governed by the period of post-irradiation and the plusiological nature of the tissue itself. According to hgaard and Potter (1959). on relative irregular shapes of the curves. in general. may indicate the relative cell ‘ death caused by radiation. Since in Group-1 no samplings were made between the embryonic stages.‘b and 6c. it was not possible to estimate ifcelldeathoccurredinthisexperiment. . w A! . .. I .o 5“ a. r! ' w . . s n . w , . . _ m . ‘3 . l n) . ‘. . I ' 1 I .7 r. , . . o J t 1 ... l '4 x l' .' . . ; .- v x } f 1 1 . h e . . . .‘ .. “ ”r. , e | e \ DU.) I. . . . . ’ O . o .7 ‘ _ . .7 . e I " - a .. . u N 'a - ... \. ' ‘\ ‘ .s ( V m . J . .' I v 4.! '5; a 1‘, .L\’ ., o ~_ 1 e '. .\ As O ‘.)-..'.. O "I , - r 1 I~ ' "'C .l J v ‘.'. 11 J I O .0( VA 0 ' l- ., I 1‘ 1 e . . I ,‘ t . O.- , - . .- . ,' ery~ o . o e ‘ -, a" * ‘ , - s .. - '1 V‘ ~ ") -.o . . I' ' o P. . _ 4 o ' e' U .w e m I ' ‘.' '. )’ \- ' t.‘ . t m I o. - ‘ ' e- e . . - . _' o g o -‘ ,H , . . . I‘ A . e . > . . _ ~ t " ‘ h r O .o . o. . . t e w s , v . - ' r s o e .- ". .s' .- 1. I ' A , A \. . ‘9 l ‘1'. ' . 1 ‘sq r. u . 1 e . . e e —.A- s ‘ e v r ‘- A A . ,‘ ' I a .\ e f ‘0 y m . I . _' ". O "r ,. «w A o ~ I l l e o f' '."~ "" - ‘0! e’ y ' ~ A .5 'r. _ , - .i g, a.) ‘ I ~. ‘6’ l" ‘ . . . . . . . . 9 e .o . - J ,. . o- l .r ' r r -. r .. v- . , a. . . ‘ . r A ’ Q Q - ' . 4-. . o . e , . o‘ n/ - . ' - " c ‘ v o , o - < J r . 1 , - 1 ' e ‘ r T . a ' '3 ‘ ' 'u 0 ‘I ' " ‘ o e r a \ .'. . ’ t [e -' l . 4' I D .. . . t / o: I“ e e‘ ‘ l‘ o . ' l l ‘u'x a i-e.. ’ or ‘ I. m '7 Q -o e. . I o ‘ov‘.’ ' _ ' _ . . 1 ,_ ., , i a O ‘ .. . . . ' . a. - I i _ .o t ,‘. .9. ' ,1 ‘1 ‘ I» ' . 5 ' I "4', e I" m 0‘ e c r- s h 3 ' ‘ .4 , . ' V I . 1 1 , 1 a m . s- r4 r 1 .(< ' I . I ‘4 4 a. e .‘ .J. 73 (8) Theories of the Effect of Ionizing Radiation On the Depression of DNA Many papers in the past have cited the blocking effects of ionising radiation on mitosis as the main factor for the depression of In. This explanation. however. seems not to be the primary answer to this question. Since DNA synthesis precedes nitosis. major emphasis must be placed first of all on changes of the metabolic processes due to I-irradiation. In the preliminary stages of mitosis. the transfer of nucleotides fromthscytoplasmtothenucloustakesplace. andthis resultsintheir conversion of ribose nucleic acid to desoxy ribose nucleic acid through the reduction process. This conclusion is based upon the very large quantities of RNA localised within the chromosaes during cell division. (Casporsson and Schults. 1938). As to the effects of ionising radiation upon RNA and DNA. Mitchell (19'02) found an increase in the absorption of ultraviolet radiation (wave-length. 2537 2) within tho cytoplasm of proliferating and differentiating cells following therapeutic doses of roentgen radiation. Re interpreted this increase to be due to the accumulation of ribossnu- cleotidesintheoytoplasmandasaresultofdisturbancesinthe normal metabolic processes. either by an increased rate of formation. or a decreased rate of removal. It is a widely accepted theory that oxidising substances are produced as a result of radiatimi interaction with water molecules. These oxidising reagents could counteract the reduction process of RNA to DNA and thereby sense an interference with the formation of DNA. with a oomccmitant increased accumlation of RNA in the cytoplau. \ . ‘ . . I l T‘ I v I ‘ . 7-7 I, _ . . *v ' ‘ ~ 1. . , . . I . ,. _ ' ,, o, | i O ‘ a O a V 6 t 4 r\" fi 5 . e I s u " I _x \ . i . as ~ I' . . ‘ . ,_ - . . . . . . 's- . « V -s ' I _ ‘ ' a, . e g. - . t + . - '. A --' ' , '4 . P ‘ I a .A , . \ - - _ c , ‘ ' -'\ '.\ - I ‘;- ‘ .‘I _,,.,..u I. .- ,. .— > ‘ ‘ ‘ \ ‘ v- . ' l . i '0 .1 I ‘ e . . e O r ' I e ‘( '| t ‘ " ‘0 '5.) .. ‘ ‘ I "e' ‘: u-I . _, - . m 3‘ _ 0 ,i .l .."-" _"') fle,. 0“" - -.. ‘ea c» 4'- etc ‘0' ‘ ‘ I I A ., .-- . . ' v T" ‘ we 0 «C . ' o - . ’- . e , ' . 5 i - 2:» ‘ a l ' . I ID - l’ < - ‘ Q - . . .‘ . . H". l . /‘ C‘s Os 1» -.-' . q J . .—. . ..( ‘ ‘ r .‘ ~ I‘ .‘ n n .- ~ ' . o'W .‘ . ’ ' . ‘ ' . 'I ‘ ‘ a ' -« - 1" .l ' t - .i ' -I {.x e '- ‘ ~‘u _ , ‘ . . . e . t “ g s‘ ‘ . . . . ‘. -J .. . . w . . _ . . r I. ‘i... ‘ v we ‘ *‘ ‘ \ ' ' ‘ - ' . ' “ . . '- s I , L ' ‘. . l s-r- _e- , ‘ .‘ ‘ .. _ t .. a. ‘ . . . .., _ I _- , i v . . e ' fl >I‘.’ 0“ ‘ . ' . l , ‘ .. ' ’ . 1' ‘ , i “ , n I 'I . .. - . . , . . i. ‘ u' .1 I '1}. f} .' .’I' r . . ,. s s ' . . . I‘ ' '.. .. .,. hm ‘ .' 0 '1‘: , _’ . . ‘3 ‘ _ _.- ..- .0 v I ‘ . v - V“ -"'-'_4' ’.r. v. 0 .‘h ' I . . e ., ' t- , 3,- p - .3 , .. , i. . I l' . l . ‘ e- " - I ‘ " n ‘ ‘ - . , s cg . n g’ i e \i‘ \ A a t . . r‘ — . 74 According to the results reported in new papers. even relatively large doses of X rays do not depress DEA synthesis to less than about 50$ of the control. therefore Von Euler and Hevesy (19%) suggested that ionisim radiation lay interfere with mu synthesis but not with the high turnover of DIA which occurs at the tins of synthesis of new DEA. In an attupt to solve the nechanisn iapiioatao in the initial drop of DIA after aoderate doses of laws (50r to 200r). Polo and Howard (1955) reported that the nuaber of cells synthesising DllA (observed by aesns of autoradiographs as uptalne of 9.32 into mu in a fora not'renoved by acid Imirolysis) in bean root'aeristens is reduced to about 60$ of the control value during the subsequent 12 hours. Those cells already undergoing synthesis (or shortly before it) when irradiated. are unaffected in this respect even if subjected to 200r. This result is interpreted as being due to a greater radiosensitivity to delay or inhibition of BIA synthesis on the part of cells which are in approailately the first one—third of the cell aitotic cycle at the ties of irradiation (the first part of interphase) than cells at other stages of the cycle. This contradicts Euler and Bevesy's lupcthssis on the ground that. if their interpretation of the irradiation results is correct. the labeliu of single cells after irradiation should be one-half that of unirradiated ones. which is in no way supported by Pele and Howard 's experimnt. The main difference existing between the two views is that the latter (Pele and Board) attributes the depression of on to the nusber of cells delayed or inhibited in their DIA synthesis while those cells 75 entering DNA synthesis. even if irradiated. are unaffected by x-irradiation. The for-er (Euler and Reveey) . however. insist that radiation induced decrease of DNA free: the control is due to the labeling of each cell in the irradiated tissue being one—half that of unirradiatod tissue. Pelo and Howard's theory say very well apply to experiments conducted in such a way that the dose of irradiation is low enough not to affect the synthetic phase of the mitotic cycle. Bit since the tissue as a whole is composed of cells having different radiosensitivities. it is very difficult to say whether any dose of radiation has affected the pre- synthetic phase only. On the other hand. if one follows Euler and Hevesy's theory as to the fundanental biochenical motions inplicated in the depression of DNA. it is very difficult to explain values below 50%. which have been reported by new investigators. and which occur in the present experiments. It is quite interesting to note that the views of sons other investigators are a conpronise between the above two interpretations. Kelly. et a1. (1955). Holmes (1956). and Cater. at al. (1956). working with regenerating rat liver. found that once DNA synthesis starts large doses of radiation are necessary to produce a partial inhibition while snall doses delivered before co-encenent of DNA synthesis produce a delay in onset of DNA synthesis. although not necessarily affecting its rate. Lajtha. et al. (1958). irradiating (with absorbed doses of 300-1100 reds) cell suspensions of huaan bone narrow (labeling DNA by the incorporation of formate-Cm). reported that large doses of Lirradiation directly inhibited the process of D)“ synthesis. 76 and that this effect appeared to be dose-dependent. Small doses did not inhibit DNA synthesis in cells which already had started the synthesis of DNA. In cells. however. which were in the presynthetio period of mitosis at the time of radiation. small doses produced a l+0-50$ depression on the number of cells entering the subsequent synthetic period. without affecting the rate of subsequent DNA synthesis. It is quite difficult to define "large or small” doses of X-irradiation. since the response of any tissue to a single dose of radiation may vary depending upon the kinds of tissues and the relative number of cell types within the tissues. It seems logical to asstime. however. that within the tissue of an entire barley embryo as was used in this experiment. a dose of #50 r of X-radiation will affect not only the presynthetic mitotic phases of a certain number of cells reversibly or irreversibly. but also may directly affect the DNA synthetic phases as well. An important factor in determining DNA depression by track counting is the number of cells counted per unit area in a tissue. As shown in Table 17. fewer numbers of cells were counted per unit area in the first. second. and third samples of Group-1: the first and second samples of Group-2; and in the third sample of Group-3. These observations show that while the growth of the embryo in size was retarded due to X-radiation. the individual cell size in the I-rayed embryo increased from 110% to 120% of the normal cell size. Thus. DNA values shown in Table 15 would be even somewhat higher if recal- culated on this basis of difference in cell size. 77 (9) The Interpretation on Interchangeable nu and mi According to the theory of nucleic acid starvation proposed by Darlington (19”). an adequate supply of nucleic acid is essential for mitosis. Aw factor which reduces the supply of this substance will result in the 'consumption' of cytoplasmic nucleotides by the nuclei. It is wall known“ that ionising radiation decreases the amount of on either In direct effect on tho on synthetic phase or by blocking the radiosensitive presynthetic stage of the mitotic cycle. This depression of on will in turn lead to two possible phenomena: one is an effect on normal cell division and the formation of anaelous mitotic products in the irradiated tissue. since the doubling of mu synthesis is a prerequisite for mitosis: the other is an interchangeable relationship between BIA an! DIA due the consumption of cytoplasmic nucleotides by starved nuclei. Ira the observation that very large quantities of ribose nucleic acids are localised in the chromosues during cell division. Gaspersson and Schulta (1938, 1939). conclude that the cytoplasmic nucleotides serve as a source of nucleic acids from which the chrome- saes derive their supply. The preliminary stages of mitosis involve the transfer of nucleotides fru the cytoplasm to the chumosomes. their conversion flu ribose into dewribose nucleotides. and their polgmerisation into long chains. Since ionisiu radiation blocks cell division. it will influence this cycle and thus reciprocal inter- ehamgebetweenRIAanleAoculdocour. Thisenpectationhasalready been denonstrated by Mitchell (19112) who used microspectrophctuetric techniquesinhis etudycnirrediatedandnon-irradiatedtnmortissue. 78 Hefoundintheoytoplasmcftheirradiatedtumoroellsan accumulation of ultraviolet- absorbing material which appeared to be nucleotide in nature. indicating the transformation of nucleic acids fru one type to another. Consider-in Caspersson's conclusion (19%) that MA is formed fr. BIA and with experimental demonstration that accumulation of RNA in the cytopla- results after I-irradiation (llitchell. 1913). it secs reasonable that radiation mu counteract the reduction process taking place normally in the transformation of BIA to DEA by means of the action of oxidising substances which are produced as a result of tho interaction of ionising radiation and water. This interpretation is supported by experiments conducted by licola (1950). In studying the metabolism of nucleic acids in proliferative and secretory cells he found. that l-rays caused a stoppage of DIA synthesis and a conversion of mu into m. 8. Changes of holeic Acids Dorm the Period Following I-radiation (1150 r) of Oldest Pro-bryo Stages. g-1 (Group-2) and at Stage It of Hid-differentiating hbryos (Group-3) Tracks freer-32 counted formenleAinGroup-Z ale‘listedin Table Mandgraphedinl‘igures. Itwillbenotedthstatstegeg-l the DIA content fell to 81 percent of the contlel in 2b hours post- irradiation. This DIA continually decreased until stage 5 was reached, ‘5 due poet-irradiation,mlere the lowest value was only 23 percent of thenormallevel. Betweenstage5and6cthequantityofDNAhad recovered to 97 percent of the control. On the other hand. at stage g-1’2b hours post-irradiation. BIA content was 70 percent of the 79 control. RNA recovered up to 80 percent of the normal value at stage 5 and fell to 57 percent at the final stage. As seen in Figure 5. reciprocal fluctuations betwem RNA and DNA occurredas in the previous (Group-1) experiment. Cuparing the rate of changes in the content of DNA with that of RNA. the difference between the minimum and the final recovery value of DNA was found to be 7h percent of the control. while the corresponding value of RNA was 23 percent. men compared with Group-1. the content of DNA seems to be more variable than RNA during 12 due post-irradiation. DatafortheemperimentofGroup-BareshowninTable Mend Figure 6. It will be noted at 211 hours post-irradiation (at stage 11) that the content of DNA increased to more than the control value (110 percent of tho control). Following this 'over-shooting' behavior theDNAcurve suddenlydroppedtozzpercent ofthenonal at stage 6ainhdaysbutrecoveredupto81$bythefinalelbryonic stage (9 days post-irradiation). when cupared with DNA. the RNA cmtent was found to be 68% of the control yet recovered slightly. up to 78%. at stage 6a (b days post-irradiation). After this. the RNA gradually droppedto'lbfiofthenolmalvalueuponreachingthefinalstage. As. in the previous experiments (Group-1 and -Z) DNA recovery was greater than RNA during the 9 days post-irradiation. he difference betweenmaximumandminiwumvslueswas foundtobe88$inDNA. while in RNA only 10$. indicating that DNA was also much more variable than RNA in this experiment. As found in the previous two experiments. the changes in contents of RNA and DNA were reciprocal. Comparing the curves of Group-2 (Figure 5) with. those of Group-3 (Figure 6). tho 80 shape in both experiments was essentially the same except for the ”overshooting' DNA content at the first sample stage in the latter. As already explained in the Group-1 experiment. there are two possible phenaena which could have occurred during the first period of post-irradiation: one being the continuous mitotic activity of one groups of cells which were not inhibited by the action of bras and would have entered DNA synthesis at the time of irradiation: the other. the recovery of those cells that were originally delayed by the ionising radiation. Furthermore. a summation of DNA synthesis in‘the above two groups of cells. which. conceivably. could be caused by a synchronising effect of Lirradiaticn. would explain the "over- shoot" incorporation seen at 215 hours post- irradiation in the Group-3 experiment. C. Changes in Nucleic Acids During the Period Following x-radiation (use r) of Late Differentiating hbryo Stages. Late-5 (Group-b) The experimental data for this group appears in Table 1t and Figure 7. Results obtained from this experiment were found to be quite different from those of the previous experiments. Group-1. -2. and .3. First. tho mu content fell to only 95$ of tho control in the first sample (zu hours posts-irradiation). yet kept more or less constant (96$) during tho next 3 duo. after which it decreased to 72$. lacking the ability to recover during tho entire 7-dav post-irradiation period. The content of RNA. on the other hand. dropped to 86$ of the control atthezll-hour: initial sample. continudtodecreasetoMofthe normallevelinlidayspoet-irradiation. thenrecoveredupto58$of 81 the control value. The conspicuous feature here is the. phenonenon of non-recovery in mu but recovery in RNA. If one compares the relative changes of these two types of nucleic acids. the difference between mummiormndtobozafimnmmszstnm. indicating that DNA is Inch lore stable than an. is seen in the previous uperinents. there occurred a conpetitive relation between an and mu for estabolic substrates. The divergent results observed in Group-bperhaps can be explained‘by caparing the netabolic activity of the nucleus. Ihich is the site of 1m synthesis. with that of the cytoplasa. where the greatest quantity of an is synthesised. The netsbolic activity of the nucleus is lower than that of cytopla- in lore nature tissue (Ea-euten and Hevesy. 1916). In addition. the tine spent in interphase by cells caprising nature tissue is such longer than that in cells of actively growing tissues. because of the lover nitotic activity of the former. when sore nature tissue. therefore. is once affected by ionising radiation. mu recovery is sore difficult than that of mu because of the lover neta- bolicactivityofthenucleusascuparedtothecytoplau. In addition. it also requires a mob longer tile for nature tissue to recover following irradiation since a longer period of tine is spent in interphase during inch mu synthesis occurs (Bevesy. 1945). D. Couparison of ‘l'otal Nucleic Acids in Group-1. -2. -3. and J Itwasaotedinriguresb. 5. 6. and7thatthechangesoftotal nucleic acids always followed the sale direction as that of mm in all experilents during the entire post-irradiation period with the 82 exception of Group-4. As shown in Figure 7. the changes of total nucleic acids of Group-4 were comparable to those of RNA. they being opposite to that of the previous experiments. Figure 8 indicates the percent total nucleic acids obtained from all four experiments. It will be seen that the total nucleic acids. as percent of the controls. was found to be 50. 72, 85. and 89 in Group-1. -2. -3. and -h. respectively. at 2h hours post-irradiation. The minhnwn values in these four experiments were found to be hhfi at 7 days post-irradiation (Group-1). 59% at 4 days (Group-2). 5h$ at 4 days (Group-3). and 5#% at h days post-irradiation (Group-4). Following these depressions. the total nucleic acid of Group-1 had recovered up to 64% of the control at 16 days post-irradiation. 69% at 12 days (Group-2). 79% at 9 days (Group-3). and 66% at 7 days (Group-4). These results indicate that the younger the embryonic stages at the time of X-irradiation. the greater the depression of total nucleic acid at 2h hours post-irradiation. In addition. this indicates the greater radiosensitivity of'younger embryonic tissues to X rays during 2h hours post-irradiation. The pattern of susceptibility was the same for all groups except for Group-h at the final sampling stage. Only minor differences were found in the minimum values for all four experiments. 83 Effect of Lit-radiation on P-32 Incorporation Into lucleic Acids of Different Parts (Root. Shoot. and Scutellun) of an hbryo The-ainpurpose ofthispartofthe uperiaentswastodetenine the influence of bradiation on the nucleic acids in three different parts of an ubryo. Table 18 indicates percent nucleic acids in three different parts (root. shoot. and scutellun) of 'Iature' esbryoe which had been previously treated with doses of #50 r at four different enbryonic stages. lhe results obtained free Group-i are shown in Table 18 and W9. theDMcontentrangedfroIMto83$inthreedifferent parts oftheenbryo. while therange ormmronno tobs52$to 58$ of the control. As shownin‘rable 18andll'igurs 10. Group-ZhadaDncontentin root. shoot. and scutellun of 98$. 95%. and 1025, respectively. while the m of these tissues were deterlined to be 18$. 88$ and 77$ of the none]. level. The results found for Group-3 are recorded in fable18 and Figure 11. Inthis casethepercentmuofroot. shoot. andscutelluawas 70$. 77$. and 91‘. respectively. while the nu values in these tissues were 67$. 581. and 81$ of the control. Finally Figure 12 (fron the data indicated in Table'18) shows the results of the last eaperhent of the stuflv (Group-IO). nu values are noted as 7% 72¢. and 1011 of the norsal level in root. shoot. and scutellue. respectively. while the an of uses parts is observed to be M. 66$. and 66;“: the control, respectively. 84 After evaluating the data shown in Table 18. it was found that the difference between maximal and sinilun m values in the three differentparts ofthe enbrycwas6$ofthe controlintheGroup-1 experiment. 7‘ in Group-2. 21$ in Group-3. and 29$ in Group-h. rho differences reflect the degree of tissue heterogeneity within the embryo at the time of x-rediation. is shown here. the differences between mu values in three different parts of the embryo in Group-1 and Group-2 were similar to each other and lower than those in other experimental groups. Both tho embryos of Group-1 and -2 at the tins of Lradiaticn (ot stage a-c and g) were couposed of the undifferentiated proenbryos. Since the three parts of a 'aature' embryo (at stage 6c) were derived fru those morphologically hoaogenous groups of cells. the swell differences (6% in Group-1 and 75 in Group-2) between mu contents in'the three parts of the mature embryos say be due to the hoaogenous effect of Ins-aye on the enbryos at the tine of Lirradiaticn. Free Figure 11. it can be seen that the D“ content of Group-3 was 701 in tho root. 77$ in shoot. and 91$ in tho contouuo. This indicates that the order of increasing susceptibility to irradiation is (1) scutellun. (2) shoot. and (3) root. According to Hericle one Hericle (1957). tho norphological ~meiooono1t1v1ty of three regions (root. shoot. and scutellue) of an enbryo varies with the stage of enbryogew at the tins of irradiation. It was reported that the root region showed greater radio sensitivity than did the shoot region if radiation was applied during the time that structural differentiatim of these regions was occurring. may explained this 85 increased radiosensitivity of the root region as a reflection of the fact that. since structural differentiation occurs first in the shoot region. the root region is. therefore. 'ontogenetically younger.” This explanation of the morphological radiosensitivity of two parts (root and shoot) of an embryo could account for the different effects of X rays on the DNA of two parts of an embryo. In comparison with the root and shoot regions. the region of the scutellum is composed 'of a relatively small amount of’meristematic tissue and a large quantity of differentiated tissue. Since more differentiated tissue is less radiosensitive than younger tissue (Bergonie and Tribondeau. 1906) the higher DNA value in this region could be explained in this fashion. The results of Group-h appear in Figure 12. The percent DNA content of the root and shoot were found to be 7h¢ and 72%. respectively. indicating similar responses for these two tissues. while that of the scutellum.(101%) indicates the highest resistance to ionizing radiation. The explanation of the results in Group-3 could be applied to those of Group-4. The largest differences between the content of RNA in the three embryo parts are 23% in Group-1 experiment. 16% in Group-2. 23% in Group-3. and 26% in Group-h. As shown here. the response of RNA content to X rays seemed to be quite independent of degree of tissue differen- tiation. This confirms previous results obtained in the experiments dealing with the biochemical analysis of purines and pyrimidines in normal and X-rayed nature embryos. As seen here. the largest variation in RNA among the three tissues was in Group-2. This could be explained by pointing out the relationship between protein synthesis and RNA 86 content. Stage g in Group-2 is the stage Just prior to morphological differentiation. The embryos undergoing differentiation are the site of marked protein synthesis. which in turn requires large quantities of RDA. Since morphological differentiation of the parts (root. shoot. and scuteJluI) of an embryo does not occur simultaneously. the spacial distribution of the RNA required for protein synthesis may be markedly different within an abryo at stage g. SUMMARY The purpose of the study was to investigate the effect of a single dose (#50 r) of ionising radiation (X rays) on the RNA and DNA content of barley embryos developing in 1119. First,the purines and pyrimidines of RNA and DNA of mature embryos were separated and detereined spectrophotometrically after irradiation at different stages of embryogerw. In the second technique ,tracer levels of P32 phosphate were supplied to the roots inediately after irradiation when the embryos were at the different stages of embryogeny and histological sections of the embryo tissue were prepared and analysed for P32 incorporation into am one mu at subsequent stages of enbryo development. The conclusions reached during the course of the observations were as follows: 1. DNA was more radiosensitive than RNA during embryogerw. except for the mature embryo where DNA was more stable than RNA. ‘ 2. Altered purine-pyrinidine ratiogdid not return to the normal pattern of nucleic acid metabolism. 7 3. Uracil ofRNAandthymineofDNAappearedtobemorelabile to X-radiation than other bases. 4. The observed differences in RNA and DNA contents at different stages of embryogew (with the exception of the DNA in nature 87 5. 7o 8. 88 enbryos) indicated that the younger embryos were more affected and/or underwent less recovery during the post- irradiation period than the older embryos. Stage specificity of radiation effects seemed to be referred more to effects on the DNA rather than the RNA components of the enbryo cells. If irradiation occurred prior to structural differentiation. root. shoot. and scutellum regions show similar levels of radiosensitivity with regard to the content of DNA. If radiation was applied during the time that structural differentiation of the above three regions was occurring. the order of increasing radiosensitiv'ity to X rays was scutellum. shoot. and root. as based upon DNA content. 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Effect of I-radiation on DNA notebolisn in various tissue of the rat. II. Recovery after sublethal doses of irradiation. Radiation Research 12: 120-130. s- / 95 Nygaard. 0. F. .' and R. L. Potter. 1960.. Effect of X-radiation on DNA metabolism in various tissues of the rat. III. Retention of labeled DNA in nonal and irradiated annals. Radiation Research 12 : 131-116». Ogur. H.. and G. Bosch. 1950. The nucleic acids of plant tissues. I. The extraction and estiaation of deosypentose nucleic acid and pentose nucleic acid. Arch. Biochen. 25: 262-276. 0rd. 14. G.. and L. A. Stocken. 1956. The effects of I-and l' radiation on nucleic acid metabolisn in the rat in vivo m 1.2 “tree Bloch“. Je 638 3-8e c/ Paigen. K. . and B. N. Kaufman. 1953. Effect of X-irradiation on amount and couposition of nucleic acid in liver. J. Cellular Coup. Pigsiol. #2: 163-178. Painter. R. B. 1960. Nucleic acid metabolism in Hela S3 cells after X-ray-induced mitotic delay. Radiation Research 13 3 726-735 e Painur, Re Be, 8111 Je Se Romnaone 1959e Eff.” of much and theory of role of mitotic delay on the tine course of labeling of Hela 33 cells with tritieted tIUIidine. Radiation Research 11: 206-216. Payne. A. H.. L. S. Kelly. and C. Ezrtemnan. 1952. Effect of total-body X-irradiation on relative turnover of nucleic acid phosphorus. Proc. Soc. Exper. Biol. and Med. 813 698-701e Pelc. S. H.. and A. Howard. 1952. Chromosome metabolism as shown by autoradiograls. Exptl. Cell Research (mppld 2: 269.279] Pole. 5. R.. and A. Howard. 1955. Effect of irradiation on DNA synthesis in Vicia as sheen by autoradiographs. Acta Radiol. (W116: 699. Pelc. S. R.. and A. Howard. 1955. Effect of various doses of X rays on the nuaber of cells synthesizing DNA. Radiation Research 3: 135-lh2. Pmraen, De Fe. Fe U. Fitch, w I. P. Wile 1955e 810°11‘10“ change- in spleens of rats after localised X-irradiation. Proc. Soc. Exptl. Biol. Ned. 88: 39'4-397. 96 Prescot, D. H. 1960. Relation between cell growth and cell division. IV. The synthesis of DNA, RNA, and protein from division to division in Tetanus. Exptl. Cell Research 19: 228-239. Quastler, H.. and F. G. Sherman. 1958. The recovery of DNA synthesis after irradiation. Radiation Research 9: 169. Schmidt, G., and S. J. Thannhauser. 191-6. A nethod for the determination of DNA and RNA, and phosphoprotein in 8111”]. 111881198. J e B101. Chane 1613 83.89e Schneider, W. C. 19’45. Phosphorus compounds in animal tissues. I. Extraction and estimation of DNA and RNA. J. Biol. Chem. 161: 293-303. Schoenheiler, R. 19%. The dynamic state of body constituents. Harvard Univ. Press. Cambridge, Massachusetts. Scholes, G., G. Stein, and J. Weiss. 191:9. Action of X rays on nucleic acids. Nature 16“: 709-710. Scott, R. R., and R. P. Martin. 1949. Use of radioactive phosphorus in plant nutritional studies. Nature 163: 717’719 e Semenenko, G. I. 1958. The transformation of nucleic acids in gernimting and maturing seeds. Plant Physiol. 4: 320-325 ( Trans. from Russian ). Sherman, F. G. , and H. Quastler. 1958. The nature of radiation- induced inhibition of DNA synthesis. Radiation Research 9: 182 (suppl.). Sininovitch, L., and A. F. Graham. 1956. Significance of ribonucleic acid and deozwribonucleic acid turnover studies. Jour. Histcchem. and Cytochem. 1?: 503-515. Sissald.an, N. H. 1955. On the nature of changes in metabolism under irradiation effects. International Conference on peaceful uses of Atomic Energy 11: 248-255. 97 Skipper, H. E., and J. H. Mitchell, Jr. 1951. Effect of roentgen- ray radiation on the biosynthesis of nucleic acids and nucleic acid purines. Cancer 4: 363-366. Smith, J. D.. R. Markham. 1950. Chromatographic studies on nucleic acids. II. The quantitative analysis of ribonucleic acid. Biochem. J. 46: 509-513. Stem“, De ’ 8111 Ae Ke salmon. 1919 e The detOCtion of Ca-J45, I-13l, P-32, and Zn-65 by photographic film. Rev. Scientific Instruments 20: 655-659. SW, Ce Ee Re, Re Daoust, and C. P. LOblOrde 1953e Rate of synthesis of DNA and mitotic rate in liver and intestine. J. Biol. Chem. 202: 177-186. Strzemienski, K. 1952. Radiation injury from radioactive phosphorus in tracer experiments with plants. New Zealand J. Sci. Technol. 34: 313-319. Taylor, J. H. 1958. Incorporation of phosphorus-32 into nucleic acids and proteins during microgametogenesis of Tulbaghia. Amere Joure BOte “58 123-1314“ Taylor, J. H.. and R. McMaster. 1954. Anteradiographic and nicrophotometric studies of DNA during microgameto- genesis in Lilium long-form. Chromosoma 6: 489-521. Taylor, J. H.. and s. H. Taylor. 1951. The autoradiograph- A tool for cytogeneticists. J. Heredity 44: 128-132. Thomson, J. F., W. W. Tourtellotte, and M. S. Carttar. 1952. Some observation on the effect of gamma radiation of the biochemistry of the rat tl'wmus. Proc. Soc. Exptl. Biol. and Med. 80: 268-272. Thomson, J. F., W. W. Tourtelotte, M. S. Cartter, and J. B. Storer. 1953. Some observation on the effect of gamma irradiation on the biochemistry of rabbit bone marrow. Arch. Biochen. Bioplws. 42: 185-196. 98 Trudova, R. G. 1954. The effect of X rays on nucleic acid metabolisn of barley seedlings. Plant P133101. 1: 146-149. ( Trans. from Russian ). vow, He, Ce P. km, R. A. Huseby, 8111 K. we Stemtm 1953e The effect of roentgen radiation on the incorporation of radiophosphorus into nucleic acids and other constituents of mouse mamary carcinoma. Cancer Research 13: 633-638. Vischer, E., and E. Chargaff. 1948. The composition of the pentose nucleic acids of yeast and pancreas. J. Biol. Chen. 176 : 715-743- Von Ahlstrom, L. , H. Euler, and G. Hevesy. 1944. Action of X rays on nucleic acid metabolism in rat organs. Arkiv. Kemi, Mineral. Geol. 19A, No. 9: 1-16 (1944) ( Quoted by G. Hevesy, Radioactive Indicators, Interscience Publisher, New York and London, 1948 ). Von Euler, H. , and G. Hevesy. 191m. Wirkung Der R'6ntgenstrahlen auf den Umsatz der Nuklein-S'éure in Jensen-Sarkoma. Det Kgl. Danslce Videnskabernes Selskab, Biol. Med. 17: N0. 8, 3.38e Webb, J. M. 1951. The reaction of ionizing particles with the photographic emulsion to produce the latent image. Manual for Autoradiographic Course, Oak Ridge Institute of Nuclear Studies. Oak Ridge, Tenn. Weiss, J. 1952. Possible biological significance of the action of ionizing radiation on the nucleic acids. Nature 169 : 460-461. Weynouth, P. P., and H. S. Kaplan. 1952. Effect of irradiation on lymphloid tissuetnucleic acids in C57BL mice. Cancer Research 12: 680. Williams, R. 13., J. N. Teal, and J. 0. Reid. 1958. Coordinated morphological and biochemical studies of the effect of total-bow x-radiation upon cell population in interphase in the rat small bowel epithelium. Radiation Research (suppl.) 9: 204. 99 “m, Re Be, Je White, Je Ne T031, am Je Ce Reid. 1955e Relationship between mitotic activity, nucleic acids, and protein synthesis in the rat small bowel epithelium after X-radiation. Radiation Research (eupp1.) 3: 358. Woods, P. S. 1957. A chromatographic study of mdrolysis in the Feulgen nuclear reaction. J our. Biochem. Biophya Cytol. 33 71-88e Wyatt, G. R. 1951. The purine and pyrimidine composition of DNA. Bloch“. J e “83 581-58l’e APPENDIIA Plate is Photographs ( x 1350 under oil immersion ) of three adjacent sections (at 6 p) of an embryo exposed to a single dose of 450 r X rays and treated with P-32 (200 p c/kg of soil). Top tissue, processed with 0.2 N perchloric acid at room temp. for 5 min. so that neither RNA nor DNA is removed. Middle tissue, processed with 1 N perchloric acid at 4'0 for 18 hours so that RNA is removed, but DNA is not. 9 Bottom tissue, processed with 0. 5 N perchloric acid at 7° C for20min.sothatbothRNAandDNAareruoved. Time for exposure, 21 days after the slides were 60““ with emulsion. Plato 2e Developmental stages of barley ubryogew Stages a, b, and c-d are early preembryos. Stage e-f is a hid-proenbryo. Stage g - 1 is a stage intermediate between a late preembryo and an early differentiating eebryo. Stage 3 - 4 is a mid-differentiating embryo. Stages 5 and 6 are late differentiating embryos. 102 s-a 5 6 APPENDIIB Fig. 1. Comparison of RNA - bases (- ,1 moles per mg dry night) in nomal embryos and those erd at four different embryonic stages and analyzed at stage 6c, corrected for 21% loss ”2:. “Ir ..'I" mt 103 A 160 h b F 150a m o E . E o g, 110 . o 12’ . to 90. E” 'U r--n E? ' ' 56 Q 70. n o m V m ,o -< a 50' 0) m H a g x 50. E 10 , Irradiation a - c g 5-4 late 5 control stages Final sample 6 - c 6 - c 6 -c 6 - c 6 - c stages Fig. 2. Comparison of DNA- mu(npno1uporlzdry weight) in normal embryos and thou I-royod at four different embryonic stages and analyzed at etage 6c, corrected for 27% lose In.gxxnoles DNA bases per mg dry weight of embryos 104 150 . 110 70 10 b O Irradiation stages Final sample stages a .. c g 5.. 14 late 5 control 6 - c 6 - c 6 ' C 6 ’ c 6 - c Fig. 3. Comparison ofpurinsandpyrhidinabasasoftotal nucleic acids ( a p nolaa per as dry night) in normal embryos and those Liz-radiated at four different embryonic stages and analysed at stage 6c, corrected for 21% loss (am) and 27$ (mu) 105 170 . DNA bases 150 RKA bases U) o E; _ C aw’1350_ c... O E D to ‘8 mg >3 , A . 'U 2° 3 90, Q. U) m m b a ,Q :g 70. 0 a O . -H ,3 8 50» $3 (D o H D 0 E r 50> E 10 . O Irradiation a- c g 5 - h late 5 control stages Final sample 6 - c 6 -c 6 - c 6 - c 6 - c stages Fig. lb. RNA and DNA content of irradiated ubryos, as percent of controls. during 16 days post-irradiation. hbryos were treated with x rays (#50 r) at subryonic stage a-c (Group - 1) 106 perm: 525:6 'r“ 100. 90.. U] '3 4’3 80. ADI-IA c: o o ('6' 70. is? o 60L 3-. a) Q. 53 5o. 2 a) *5 140. o o :3 33°- 0 ‘8‘ r: 20 . o :5 z 10. 0 L4 1 1 A L A A a-b c d-e g-l 1} 5 6a 6b 6c Embryonic stages of samples Fig. 5. RNA and DNA content of irradiated embryos. as percent of controls, during 12 days post-irradiation. hbryoe were treated with X rays (#50 r) at eebryonic stage 3 (Group-2) Nucleic acid content as percent of controls 107 100 . 80 . 7O . o 0 RNA + DNA 60 b ‘ . ‘ RNA 20. 10. 4 o A A a a a a a a-b c d-e g-l ll 5 6a 6b 6c Drbryonic stages of samples Fig. 6. RNA and DNA content of irradiated eebryos. as percent of controls. during 9 days poet-irradiation. hbryos sere treated with X rays (#50 r) at embryonic stage 3-!» (Group " 3)e 108 pens: :' fins W. 3-1 6". b b D D ' ’ m m m ,w w. o m m m o a-b c mHonocoo mo pnoonom mo pcopcoo Ufloo owoaod: Dnbryonic stages of s "Iples Fig. 7. RNA and DNA content of irradiated embryos, as percent of controls. during 7 ms post-irradiation. hbryos vere treated with X rays (450 r) at ubryonic stage late-5 (Group - h) 109 I J»; 5.39761}: 9 . m m m + Mn. N O D h b b F D D P m, w N ,w W m m/ % mHofiEoo mo pcoouom mm. pcopcoo ounce 9.8352 10» 6b 6c 69. a-b c d-Q g—l Elnbryonic stages of samples Fig. 8. Total nucleic acid content of irradiated .bryos, as percent of controls, following #50 r X rays. hbryos were I—rayed at four different stages (Group 1«atlnagec, Group 2 at stage g, Group 3 at stage 3-1}. and Group it at stage late 5) 73:. a 1km ‘ I mi; ' Total nucleic acid as percent of controls 100 70 2O 10 O A 110 ‘ L a-b C d-e g-l 4 5 Embryonic stages of 6a samples 6b 6c Fig. 9. Nucleic acid content. as percent of controls, of the root, shoot, and scutellua of embryos which were treated withadose of450rxraysatstagea-cands-pled after complete differentiation at stage 60 (Group - 1) 111 //4 ./ /////7////, //////7//////////////////////, 7/477///////////////////51/554 fix? “a wm + H m m u.“ . mm m .6,////////////////////////////// ////// ////x7///////z&/////l////////.. 2 a R \n F a22222433223222222222272222232223322;. b h F F b b b F b O O. O O m“ mp s. 7: Ac mw mw mfl Mw mm nu mHonpcoo.mo pcooaog mm pcopcoo neon ofioaosz Shoot Scutellum Root Fig. 10. Nucleic acid content. as percent of controls. of the root, shoot. and scutellma of elbryos which were treatedwithadose of1+50 rXraysatstagegendsaned after complete differentiation at stage 6c (Group - 2) "2 '1 33:3 '03 gm RNA 4' DN -m I: .‘.\\\' A DI IA V I a I‘ U. . _! 1C.» ‘ I m .////////////////////////////////////%//////////////////////Z//////////A////IZ//////%I/é/Ag/él/Ié 7//////,,///////////////////////%7//////////////V///,7///////////V/A * P ’ b mwmw mu, m 2 1 maoppcoo mo pcooaoa we psopcoo ofloo oHoHozz m/ 0 O 0‘ Shoot Scutellum Root Fig. 11. Nucleic acid content. as percent of controls. of the root. shoot. and scutellun of .bryos which were treated withadose ofbjo r1 rays at stage 3-handsaapled after cauplete differentiation at stage 6c (Group - 3) 113 RNA + DNA -m: 1:: DNA 100 e 7///////////////fl/’7////////////lgg/A/IZV////////607/A//éél7/47/1////////////////////4 F 7///////////4¢//////////////////////////////é///////////////////////////////////////////é .///////////////////////////////////////////7/////////////////////////////////////////. D b D D # l- m m n m w m. w m m o naoupcoo mo punched no panacea ounce ofieaosz Scutellum Fig. 12. Nucleic acid content, as percent of controls, of the root. shoot, and scutellua of embryos which were treated with a dose of 450 r X rays at stage late 5 and s-pled after complete differentiation at stage 60 (Gromp-u) 1111/1»- A Y I RIIA + DNA - m1 C3 DL'A 100 De '6 :h,d‘ 1 I 7////////////////////////////////////////4///////////////A///47/4 Z7////////////fl//fi/47/////é///////////V/AI///////////fl////l//Z 7//////////////////////////////////////////////f////////4 D D D by ’ L mmwwm/mmwmmo machpcoo mo pcoouog mo pcoucoo cave oaoaosz Shoot Scutellum Root APPENDIX C 11? Table 1. Standard extinction values of purine and pyrimidine bases* (iopgpornioro. inayerochioric acid ) determined from the difference (A) read at the absorption maxim ( guanine at 209 up, adenine 262.5 m, cytosine 275 mp, arac11259np, andthyninezéhm)aniat290np Guanine Repli- Extinction at wave a in Average sates A 1 . 1 . . . 1 0.088; 0.030 0.299 0.721 0.790 0.721 0.091 0.091 2 0.02? 0.298 0.218 0.792 0. 71? 0.494 ‘Ererage A 1 5 1.05 2 0.005 0.006 1.020 1.071 1.020 1.025 1.020 2 0.005 0.006 1.018 1.071 1.015 1.025 Cytosine Repli- ' Average A ' .5 ' 0.357 2 0.028 0.500 0.919 0.960 0.895 0.456 0.055 _3 0.030 0 .506 0.918 0.958 0.890 0.052 ' The commercial samples? five bases ( A grades ) were omined from California Corporation for mechanical Research, Medfozd St... Los Angeles 63, Calif. 2 0.002 0.008 0.700 0.700 0:690 0:732 0.732 _3 0.____003 0.010 0.700 0.702 0.6_91 0.222 . __ Iver-age A 6.535 2 0. 003 0. 078 03598 0. 616 0.565 0.538 _3 0.003 0.080 0.597 0.612 0.26: 0.222 0.236 ,l, -eI>s , .. ,. -a ‘ ‘ ’ _ . . -‘ O o e .. ,1 a .‘ i r _ . v ' - O O O o‘ - ’ . _ . ,.. .- . - a ‘. . O . c . O _. .e "" '6 ‘7- . ' ' . . . I n ‘. _ ...--"' .. . . ‘ 4 ‘ . t ‘ “’"" - -.e-“'.--I . . I . I . C a. . . a e‘-"'" . _....— .- ~ ‘,_... ..,.n~" . ‘ .‘l .. . v»' f. .> 118 Table 2. Recoveries of five bases‘ after heating at 100°C in 12 n perchloric acid for 1 hour Guaninein W11“ at wave 1 ' flowery cat0- mm 2’“ in"2 A" “ 00.1 0.0 0. "0 0.1“; 0. " 0.0“ 0 “0 "‘ 2 0.005 0.037 0.127 0.130 0.127 0.090 9.55 96 3 0.002 0.033 0.123 0.126 0.123 0.093 9.55 96 0 0.005 0.03; 0.121 0.1%)I 0.127 0.090 . 96 A I . Ax- erence naflm inétion 0 ' " 0, z; . i. 0 2 0.003 0.011 0.197 0.201 0.189 0.190 9.30 93 3 0.001 0. 007 0.191 0.195 0.183 0.188 9.20 92 10 0.008 0.010 0.199 0.20L 0.190 0.19j5_ 9.50 9L I a I. osinein Motion at wave ¢r . fifiicovery cates mm A“ u 0. 0 0 ."0 0. .2 0. . 0.0‘; . °. 2 0.015 0.099 0.180 0.191 0.180 0.092 10.10 101 3 - - - - - 10 0.012 0. 0.179 0.188 0.176 0.089 94; 96 2 ' 0.033 * Sane as 1555510 1 Uracilin Minotfon at wave-T A 1360317 cm: W: x . . .. 0.0"0 0.0Y0- 0. 0 . 0.l .. . 2 0.006 0.007 0.133 0.150 0.131 0.1103 9.75 97 3 0. 008 0.015 0.150 0.155 0.130 0.100 9.55 96 '0 0. 0010 0.010 9229. 0.1108 0.129 0.12 9.'!§_____9j__ A ' . 2 Teaser-y Ax ee 1 U. e O 2 0. 002 0. 00 0.096 0.100 0.093 0.093 8.75 3 0.0010 0.01; 0.122 0.126 0.116 0.107 10.10 1:: '0 0. 007 0.021 0.125 0.129 0.121 0.108 10.15 1 __ "‘:T'1:1::Ei§ ” '3 ”Ax/A :5 Mar 119 Salary of Table 2 M adenine Wine W Iverage "emf! 97 93 97 96 96 * Four replicates 120 Table 3. hse recoveries of yeast 2 ll 8* and niece D l At ludrolyued with 12 ll perchloric acid at 100°C YeastRll Guanine a. wt. 1.1 ) .- en a wave in A move cates 2 2 .1 pm 1.... . 1 p 7- “ 80.8111 oranleP 1.0 0.00. 0. 0.0‘ 0.00 . W 0.0‘ 2 0.013 0.009 0.116 0.123 0.108 0. 070 7.50 09 0.220 3 0.012 0.005 0.108 0.112 0.100 0.067 6.80 05 0.196 as 0 0.010 0.052 0.112 0.116 0.103 0.060 6.60 0.190 I . 1 Ax= saleas a e P 8 8.9 f by Marshal: and Vogel, 1950 Adflninfl . ,. . . .. . .. - . ’l - O O ‘Q ‘ . . . . . _ t . e s e .. ' . . . , t. O . i . I‘ e I . . I ‘ .l . . . ~ -. 1110 Smary of Table 8 R NA npnofes para; gait. Kean I 8 rd deviaTion 03a... M 1.75 Adenine 18 1 0.605 1.21 Cytosine 20 1 1.005 2.01 Uracil 15 1 0.150 0.30 D NA 7131.. It. 1 findard deviation 03mins . + . w- Adenine 0.6 1 0.160 0.331 0:06.111. 0.5 1 0.200 (MOO Thur-1m 0.7 1 0.160 0.282 1101 “hi- 9. Determination of purine and pyrimidine 0.... of m and mm in273.2lgdryreightofembryosX-irradiated(lb50r)at stagehvandanalysedatstage6c RleIX-rayedenbryos muction a‘F wave T%t_l;in 1211 ng bises up noles cates 2 2 in total per A‘ * ' 'It. ‘1 ‘ 1112. 0.01 0.0 " 0.0 0.0-0 00 4. 1.170 .0 0 .°. 2 . 2 0.010 0. 023 0 .058 0.060 0.057 0. 037 3.75 1.184 28.6 2 0.009 0. 023 0.011»? 0.051 0.0116 0.028 2.85 0.886 21.5 4 and A: 1' sane as previoflable Adenine RepE- Emotion at wave 1%! in :01 lg bases In noles cates 3 2 . 22 intotal per 4“ e . wt. .; wt. 1.0 0 .01 1.0.‘0 0.20. 0.0 9 0.0V"? 2.0 0.": 0.~ 2 _0.008 0.012 0.059 0.061 0.058 0.009 2.110 0.758 20.5 a 0.012 0.019 0.066 0.069 0.066 0.050 2.115 0.7711 20.9 0.008 0.010 0.03% 0.061 0.0% 0.051 2.50 0.189 21.10 Tend Axtsaneasp ous a e 1 sine ROPE- Emotion at wave %% in am lg bases up noIes cates 2 2 in total per 01 ‘ ° ' Ute ‘ “e 0. 0'0'0 0. 0 ; 0. 0 0.07.1.01 0.1 2 2.1" '. "e; 2 -0.002 0. 015 0.032 0 0.35 0.032 0.020 2.20 0.695 22.9 3 0.000 0.012 0.028 0.029 0.026 0.017 1.85 0.580 19.3 ‘1' «0.002 0. 016 0 .020 0.01% 0.028 0.018 2.00 0.622 20.8 la? Ax-saneasprevious a e Uracil W In... .01.. cates W mg total r? per A; ’ "Ht w... 1 0.0'0" 0.0 - 0. 0 0 .0 0.0 0 0.“ ." "e” 2 0.008 0.012 0.030 0.032 0.029 0.020 1.35 0.026 13.9 3 0.005 0.010 0. 028 0 .030 0.027 0.020 1.35 0.026 13.9 b 0.010 0.016 0.032 0.03; 0.032 0.019 1.3 0.011 12.10 JanIAIBsaneasp ousae * 1012/21 2 5 Mg 1102 lel. 9 contixmed D N A of X-reyed embryos Guanine Mp - mcfloniinnfgfiggmg ngBes nap-31's? cetee 2 2 2 4‘ . , in total per 2 0.003 0.009 0.027 0.028 0.026 0.017 1:75 0.1.82 0:3 3 0.006 0.0110 0.025 0.028 0.023 0.0110 1.115 0.151 3.6 0 0.012 0.022 0.035 0.032 .034 0.016 1.65 0.192 10.2 41nd szsmaaprevioua'ra e 1 Adenine Mafia. 0.? wavrf%g_h 1n ¥ 1:; Ba... I91 non-a eaten 2 .5 2 2 25 A in total per " "' wt. wt. 1 .007 . . 7 . . 32 . .1 . 2 0.007 0.017 0.051 0:053 0.051 0.036 1.75 0.182 10.9 2 0.008 0.010 0.042 0.0104 0.0111 0.0310 1.65 0.172 “.7 0.008 0.01L 0.007 0.0109 0.0106 0.022 1.52 0.161 10.10 Tend ix =- 38!» as previous sine EGPE- Elncflon at wave $2852 in 032 lug bases mp 100013;- catea 0 275 2 A in tote]. per 1 . ° ' “to '1 ' ' it. 0.00 0.0 000.2 00.2 00.2".1 1.". '015 ‘0. 2 0.002 0.013 0.023 0.025 ‘ 0.023 0.012 1.30 0.135 10.5 130 0.015 0.028 0.036 0.039 0.035 0.011 1.20 0.125 10.2 [ma 1:8 use as previous“. T e Wctm .0. wave $100 2 me bases 9 331-07 cute: 2 2 2 9 in total per 0,. 0 . 1 wt. ..._ - - wt. 1 0.0V“: 0.01 0.0, 0.01; 0.0 0.0 .° ”.2 e 2 0.007 0.013 0.035 0.038 0.030 0.025 2.30 0.239 6.9 3 0.0010 0.022 0.0005 0.006 0.0003 0.0210 2.25 0.234 6.7 10 0.0016 0.028 0.008 0.020 0.0107_0.922__g:_1i_ 0.22“ 6-:___. Aflxxemeeprevioue e ‘ ’0 lat/.0 xi ,0! 103 801-817 of Table 9 a 111 nEnolee are; ggz‘ifi i on Emma. W “07858-— Adenine 21 3 0.2310 0.1169 0:06.111. 21 1 0.510 1.020 0:00.11 11 3 0.883 1.766 D 001 We: r 00%. Mean 1 Mn! Emu» Wain. . 1 . W— Adenine 0.6 1 0.1100 0.221 Cytoeme 0.5 1 0.1520 0.361 mun- 6.5 3; 0.2397 0.079 1411 Table 10. Deterumtion of purine and pyrimidine bases of RNA and DNA in 203.2 :3 dry weight of embryos Liz-radiated at stage lute 5 and analyzed at stage 60 R l A of. Land embryos Guanine Won .76 wave 1 in “Times up .61.. 2 onto: 2 A. in total per "' flit. 5932101.. 1 a n g - - u c - e. 2 0.015 0.028 0.056 0.060 0.057 0.031 3.20 1 0.695 7 22.6 130 0.009 0.025 0.059 0.060 0.059 0.035 3.55 0.772 25.1 0.012 0.028 0.061 0.062 0.061 0.02; 2.55 0.750 210.10 Fenddx=eaneaee in p ouaee Adenine W11“ at man%% in g I; Tine: up noIee cute: 2 . 2. 2 A in total per ‘ . ' “te ' - ' "to 0.2070 0.001.; 0.0 . 0.0 90.0 . 0.0 1 2.0 0.*4"0 . 2 0.007 0.005 0.050 0.052 0.0108 0.047 2.30 0.500 18.2 3 0.012 0.015 0.067 0.069 0.066 0.0510 2.65 0.576 21.0 ‘0 0.008 0.011 0.061 0.062 0.066 0.021 2. 20 OJAO 19.7 Ianqux I came as shown in ihe previous 2 1$3311“ ROPE- Extinction at wave a? in F 7 lg bases I}: leIee catee 2 2 in total per 4-1 0 . ~ 001:. -’ . 1 001:. 0.00 00.2 0.0 2 0.0 0.0 ; 0.200 2.20 0.: ; 2.2 2 0.010 0.027 0.01010 0.0105 0.0101 0.018 1.95 0.420 18.8 3 0.007 0.026 0.0105 0.047 0.0103 0.021 2.30 0.500 22.1 ‘0 0.009 0.028 0.010 0.0108 0.010 0.020 2.20 0.1078 21.2 4 A: =- em as a in p on: able Uracil Mon at waveT in I; bases 90 soles cute: 2 259 2 A in total per ‘ . ' ' "to ’1 ' ' it. 00 0.01 0.0‘ 0.0‘” 0.0‘610.0 . .;’0 0.1“ . 2 0.005 0.007 0.029 0.030 0.029 0.023 1.55 0.337 110.8 3 0.007 0.010 0.032 0.035 0.032 0.025 1.70 0.370 16.2 llv 0.011 0.015 0.0102 0.0102 0.0100 0.027 1.85 0.1002 17.7 A 8 dx 3 an as e p one a e * Iota: Id 00 5 #9 115 I‘m. 10. mm D II A of Land embryos Guanine Minuet: often 1% in # mg been up nolee oetee 2 2 u in total per 1 * QEZVUt. !£;g£z:flt. 2 0.000 0.009 0.0210 0.027 0.021 0.018 1.85 0.079 2.6 3 0.003 0.010 0.023 0.027 0.020 0.017 1.70 0.072 2.0 4 0.02% 0.009 0.021 0.026 0.019 0.017 1.70 0.072 2.“ X Tend I sane as shown in the previoname M01111). 3.. Wadi” at wave £252 in ? Ifbaeee m nolee cetee 2 . 2 1 intotel per " 0 . 000-. --. - wt. 1 'e‘ He1 .e.'(; 'e'{. .0.” .e' 2 2e e .Y. 2 0.007 0.008 0.056 0.060 0.052 0.052 2.55 0.109 3:9 2 0.002 0.006 0.051 0.053 0.035 0.01-P7 2.30 0.098 3.5 0.006 0.007 0.051 0.052 0.0 0. 2.20 0.090 2.11 lend Ax 1- sale n 3110000111 the 00.0 e e osine EEK-Emma .0 an. e h in lg 13.... a, $13.? .00.. 355 $5 585 " "275 77'; in total! per 4‘ . ' -'t0 ‘2 ' "to 1 0..V.1. ”.22 0.70' 0.0 n 0.0 0.0 2 -0.002 0.0110 0.029 0.031 0.027 0.017 1.85 0.079_ 3.5 3 .0.002 0.015 0.028 0.031 0.027 0.017 1.85 0.079 3.5 '0 0.000 0.012 0.028 0.020 0.026 0.015 1.65 0.020 2.1 70nd IIBeeneaeehounin epnevioue'ra e 1' 1 V T" - 300W “um ”I” ” “to. 4,. 1. in total per .0, . . O “:5 2 0.01 0.024 0.052 0.053 0.008 0.029 2.70 0.115 3 0000; 0.016 0.01010 0.008 0.0106 0.032 3.00 0.123 2.: ‘0 0.010 0.018 0.0106 0.009 0.9503 0.021 2.20 0.123 . 10nd 7x=aeneueho00nintbepre01ou8hble 0 [0941/le M5 0.0 0.0) 0.0 0.0 0.0-.- 'o' 1106 Sunny of Table 10 10101 We: r 1ft. Kean 1 0.1180181501301611 Gian. W 1.03f 10.1113. 20 1 0.550 1.10 Cyboaine 21 1 0.650 1.30 Uracil 16 -_0-_ 0.605 1.21 on "'1 ”W Kean i on 60310111. . 1 . 15:100-— Adenine 3.6 1 0.132 0.2610 Cytoeine 3.3 i 0.100 0.200 Thu-1m 0.8 1 0.150 0.310 1w Tablo 11. mantity in up :01» of purine and pymidino bases of m and DNA por la dry might or nond Ind X-rayod ( 1150 r ) .10on RNA DNA _ Emorinonul stucco of days Groups embryo: post- : p :0qu per 1 p .0103 por X—mod radio lg dry it. I; dry wt. -tion G A C U T A C '1' Control - - 37 28 32 26 11.11 5.0 11.11 7.2 Group-A 0.0 16 21 20 18 16 3.“ 11.3 3.11 5.8 Group-B g 12 23 18 20 15 11.5 11.6 16.5 11.7 Group-C 3-10 9 25 21 21 111 11.0 11.6 1+.5 6.5 G I Gumbo. A - Adonino, C I Cytooino, U - Uracil, ‘l' =- Thynino ”to 1118 Table 12. nucleic acid content. REA I DNA. and purine / pyrimidine retloo. in nor-:1 and Iéreycd elbryc: be:ed on percent of control: Exporilontal :tege: dry: lucleic Acid: R N A, D N A group: of after fi—' fi' 4" elbryo: irradi- f of ‘53; f or f of irradiated tion control DNA control control Control - - 100 5. 9 100 1 .1 100 0.81 Group-A one 16 6b “.0 61 1.1 80 0.8h Group-B g 12 65 0.2 62 1.2 87 0.98 Group-C 3-h 9 69 0.1 66 1.3 93 0.78 Group-D late-5 7 66 5.7 66 1.2 68 0.75 1&9 Table 13. Purine tnd pan-Incline boo», a: percent of control: in nor-:1 and brand embryo: *4 'iTWHTK *fiiiif7i Elporilontnl . G A c U G A c 'r W Control 100 100 100 100 100 100 100 100 Group—A 57 71 56 58 77 . 86 77 81 Group-B 62 63 63 57 102 92 102 65 Group-C 68 75 66 5‘1 91 92 102 90 GronpdD 65 71 66 62 57 72 75 6? 150 Table 1h. Grain count: ( corrected for background ) per unit area (#900‘n2 ) of eibryo: ( or part: of embryo: ) Group-1 1st :alpling: Type: of nucleic Total acid: R N A D N A nucleic acid: libryo‘ Control 22 26—* " #8 X-reyed 15 9 Zh $ of control 68 35 5° ‘ Average of three determination: of an entire embryo 2nd sampling: Type: of nucleic Total acid: R N A D N A nucleic acid: nan-yo" Control 56 712 58 X-rayed 29 19 ‘18 f of control 52 45 49 * Awerege of three determination: of an entire olbrro . . ‘u ‘ _ , ' .o 1 _ , . - v ' ‘ ' . 7 - ._.... , -_ . -. - - .. . u. -. V . .. ‘ 1 _. -. , ..r~. . . .or'.‘ a- ' . .o. , , . 1 1 ‘ . e ,, _ . . . . . ., c ' ‘ r -u “ .n . _, ,_ ‘ _- _ -‘ .. .7 . ‘ r 0 ' .1, . . .7 .... . . -_. , . l7 V a... --‘ 3- ' ' ‘ I ' . . ‘ ‘ ‘ ' : 151 Table 111- Gronp-1 continued 3rd :anpling: Type: of nucleic Tom—— acid: R N A D N A nucleic acid: of Three Average Three Average Three Average Control 2% 75 259 86 1181+ 161 Boot 1.1-mo ' 86 29 31 27 20:1 68 11 or control 36— 31 112 Control 239 . 80 2 59 86 £197 166 Shoot Lrayed 176 59 61 20 222 711 i of control 711 211 1.5 Control 130 113 211-6 82 3110 113 acutellun X-rayed 133 M1 119 16 150 51 5 of control 102 20 £15 Control 593 66 6‘18 72 1311 1'16 Total Lrayed 395 114 183 20 578 611 f of control 67 28 W 152 Table 11+ MID-1 contimed 11th :anpling: Type: of. nucleic Total acid: R N A D N A nucleic acid: Part: counted of Three Average Three Average Three Average embryo Control 1 7911 359 1160 92 21 511 14.31 Root X-rayed 1039 208 382 76 11111 282 f of control 58 83 66 Control 1807 361 610 122 21117 1183 Shoot X-rayed 953 191 519 1011 11172 290 $ of control 52 85 61 Control 567 1 13 359 72 926 186 Scutellun X-rayed 1128 56 286 57 7111 1113 i of control 75 79 77 Control 11088 273 11119 95 5517 368 Total X-rq'ed 21107 160 1178 79 3585 239 1. of control 58 83 611 -0:- 1 53 Table 111 continued Group - 2 1:t coupling: Type: of nucleic Total * acid: R N A D N A nucleic acid: hbryo Control 87 21 108 Iprayed 61 17 78 1 of control 70 81 72 It Average of three determination: of an entire elbryo 2nd :anpling: “Type: of nucleic Total acid: N N A D N A nucleic acid: Three Average Three Average Three Average “m“ 76 25 37 13 113 38 Root erd 37 1; g g 111 1.1L 119 16 38 A Control 57 19 29 10 86 29 Shoot I-rayed 5Q #_ 12 9 J 59 29 __ as 31 a: Control '32 11 29 1o 61 20 scutellum x-rqod 1.5 15 7 _3 42 ‘2 9342.293129]. ”9 2“ 8L— Control 155 55 95 32 260 87 Total X-rayed 1 3; 114 g 7 1 5!! 51 1mm 8° 23 59 Table 111 Group-2 continued 3rd :alpling: 151+ Type: of nucleic Total acid: 8 N A D N A nucleic acid: Part: 0 Ten Average Ten Average Ten Average Olbm Control 749 75 2M 24 979 93 Root X-rayed 320 32 237 23 558 56 f of control “3 98 57 Control 328 33 117 12 l+115 “6 Shoot 1.1-mo 289 29 111 11 1101 110 f of control 88 95 90 Control 117 16 58 19 10“ 35 Scutellun I-rayed 36 12 59 20 96 32 f 0: 3011131301 "’7‘? 102 92 1 Control 1120 1+9 1116 18 1538 67 Total X-rayed 6115 28 1107 17 1055 ‘16 i or control 57 97 v 69 155 Table 111» continued Group - 3 1:t :npling: Type: of nucleic Total R N A D N A nucleic acid: Two Average ‘hro Average Two 1 Average Part: of embryo Control 1 59 80 92 116 751 376 Root X-rayed 98 119 88 M 186 93 i of control — 61 96 ‘ 7“ Control 72 36 88 M 160 80 Shoot X-rayed 61 31 1011 52 165 83 7” °f °°ntr°1 85 118 101 Control 95 I18 63 32 158 79 Scutcllun X-rayed 63 32 75 33 135 69 fl of control 66 103 — 37 Control 326 54 2113 M 569 95 Total I-rayed 222 37 267 I115 1189 82 f of control W 68 110 v 35 Table 14 Group-3 continued 2nd earpling: 156 Type: of nucleic acid: Total D NAA nucleic acid: Three Average Three Average Three Average Control 520 173 325 108 845 282 Root X-rayed #371, 129 .35 fi_, 12 £96 135 $ or control 71 11 #8 Control 273 91 186 62 1159 1 53 Sheet l-rayed 185 62 56 19, 25] 83 f of control 68 30 53 Control 169 55 170 58 338 113 Scutellun. X-rayed 197 1 2 1 8 f of control 120 37 77 Control I-rayed f of control Total _753 251 151 JEL 908 957 319 685 228 16112 5117 2392.. 78 22 5“ Table 111 Group-3 continued 3111 :alpling: 157 Type: of nuclei? Total R N A D N A nucleic acid: Five Average Five Average Five Average Control 79 16 11-09 82 1487 9? Root X-raved 53 11 "387 57 359 Q 5 or control 67 70 67 Control 137 27 363 73 500 100 Sheet X-rayed m _ ._J.§..__.E9.L__§5____3.ZQ____Z‘L_ i of control 58 77 7‘1 Control 289 58 381 76 670 1311 Scutellun X-raved $31 ‘17 3119 ML 112 i of control 81 91 87 Control 505 101 11 52 230 1657 331 Total X-raved 327 75 1 1 2 1 5 or control 74 81 79 . 4-... A '.. 158 Table 11+ continued Group - l1 1 :t :anpling: Type: of nucleic Total R N A D N A nucleic acid: Five Average Five Average Five Average embryo Control 72 15 13 3 89 17 Root X-raved 5g 1g R g 29 111 f of control 81 99 83 Control 36 7 16 11 52 11 Shoot X-rayed 33 6 W 15 w 3 47 9 f of control 88 93 90 Control 6 - 111 - 20 - Scutellun X-rayed it .- # 13 _- 17 - 9 of control 67 92 85 Control 109 22 112 9 156 32 Total X-rayed 9L L 9 AL 8 139 28 f of control 86 95 39 Table 111 Group-h continued 2nd :anpling: 159 Type: of nucleic Total acid: R N A D N A nucleic acid: nit area: # counted Five Average Five Average Five Average Part: of embryo Control 111-0 28 152 31 292 58 Root X-rayed 121 2 1 2 22 46 9 or control 86 70 78 Control 232 1+6 85 18 317 611 Sheet X-rayed 75 15 WM 157 3_2___ i of control 33 96 50 Control 350 70 108 22 1158 92 Scutellun X-rayed __5g_ 13 1 2 8 2 95 or control 15 130 112 Control 722 1115 3115 69 1067 2111 Total I-rayed .212 50 W 5 of control 34 96 51“ 160 Table 1b Group-h continued 3rd :alpling: Type: of nucleic Total acid: R N A D N A nucleic acid: Five Average Five Average Five Average embryo Control 394» 69 150 30 494' 99 Root X—rqyed 138 29 112 2} 350 50 5 of control #0 74 51 Control 369 73 76 1h #30 86 Shoot X-rayed guz 59 55 11 79397 59 f of control 66 72 69 Control 209 50 85 17 339 67 Scutellun. X-rayed 153_ .31 99 12 239 __99 5 of control 66 101 72 Control 9“? 189 351 70 1298 260 Total X-rayed 533 192......253 51 29L _157 % of control 58 72 66 ’ P 161 Table 15. Sunnary'of Table 1“. Nucleic acid content ( a: percent of control: ) of enbryo: Xprayed at four different :tage: and :anpled at interval: following irradiation. d - e g - 1 h 5 6 - a 6 - b 6 - c 50 1+9 14 - - - a» 68 52 67 - - - 58 DNA 35 us 28 - - - 83 n+0" - 72 - 59 - - 69 Group 2 RNA - 70 - 80 - - 57 DNA - 81 - 23 - - 97 R+D"' -_ - 85 - 54 - 79 Group 3 RNA - — 68 - 78 - 74 DNA - - 110 - 22 - 81 R + D * - - - - 89 5“ 65 Group 9 RNA - - - v 86 3“ 58 DNA - - - - 95 96 72 *R+0-mu+mu 162 Table 16. Relative percent RNA and DNA of the control at each :anpling stage during normal barley elbrygeny Stage: R N A D N A ealpled d - e number of 22 26 track: 1‘ 115 511 g - 1 number of 56 “2 track: 1% 57 “2 9 number of 67 28 track: 9 71 29 6c number of 53 83 track: 7; 11 58 163 Table 17. Average of three determination: or cell number’per unit area ( 11900912 ). except :tage d-fi of Group-1 ( unit area . 1100 ’12 ) number of cell: counted m. Stages ~— w group: d - 8 g - 1 l1 5 6a 6b 60 Control l~12 311 5 5 - .. .. 59 Group 1 X-reyed 37 30 116 - - - 58 5 or control 89 89 80 - - - 100 Control - 36 - 1.18 .. .. 59 Group 2 X-rayed - 32 - 111 - - 53 91 of control - 90 - 87 - - 100 Control - - 53 - 57 - 52 Group 3 X-rayed - - 1+9 - 118 - 50 $ of control - - 93 - 35 - 96 Control - - - - 60 58 56 Group 1+ Lrayed - - - - 56 56 55 5 of control - - - - 93 98 99 164 Table 18. Summary of Table 1“. Nucleic acid content (a: percent of controls) in three different part: (root. :hoot, and scutellum) of mature embryo: X-irradiated at four different embryonic :tage: Group 1 Group 2 Group 3 Group 9 a - c 8 3 - 4 18t° 5 6c 60 60 6c 66 57 67 51 Root RNA 58 #3 67 40 DNA 83 98 70 7‘1 R + D " 60 90 71+ 69 Shoot RNA 52 88 58 66 DNA 85 95 77 72 a + D 3 77 92 87 72 Scutellum RNA 75 77 81 66 DNA 79 102 9‘ ‘0‘ n + D * 61+ 69 79 63 Embryo RNA 58 57 7“ 58 *R-l-DBRNA'FDNA 165 465:8 no 828 .u .35! coho: .n 493:8 .a .Qcofiuxoon .8 8366.88 v oboe. no .533 be as non .38? non 8.58 5 .hafifioocg .o o 4:86.988 now 86.8280 v ch33 choc. «6 3852 can 388 5 .398888 .m . .651. no .332. can u: .4 o 8 on 83 n can «9. 68.0 nmcn «rm 82° 38 A..: one... a 8.68.8 8n 8: 38¢ coon «nu Res 38 n9 mac... .— «3 8“ man u Rm 3.. to... «and on Rod «no 3 m3... N n13 an nor 80.0 can“ on 036 can 3 .86 a no“ «3 e m? non 86 SR 3 ~86 a «185.5 «or own 0&6 moon Q. macs 3 m3 8 w 8.. 8n c8... «“8 «9 32¢ a 7888 o m 4 u m 4 o m 4 o m 4 o m 4 8 8 8 n r . 4 anemones no «1» none»- .uou .388: one». no.3”? node a: eoheale nevuenodv No 138.308? gen. .9 93:9 ROOM USE cm a? fix a s r *9 “ ‘ 9. \,2€-,’E‘:'é. UH}? 0;:va - .m ‘0» q "‘ 3%! IL VWWVN Y LIB MITITIGWIWIWIW“WWW film“ 3 12 9 3 0 3 0 4 6 1838