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Effects of 8-Azaguanine Florian, Svatopluk Fred 1957

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THE EFFECTS OF 8-AZAGUANINE ON THE MITOTIC CYCLE AND CELL GROWTH IN VICIA FABA ROOTS by SVATOPLUK FRED FLORIAN  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in the Department of BIOLOGY AND BOTANY  We accept this thesis as Conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA December, 1957  ii ABSTRACT An investigation was made of the effects  of the purine analogs,  8-azaguanine, 8-azaxanthine, and 6-mercaptopurine, and the purines adenine and guanine, on c e l l growth, mitosis, and deso:$rribonucleic acid (DNA) synthesis i n primary roots of Vicia faba seedlings grown i n aerated solutions. Root elongation was used as a measure of c e l l elongation; mitotic frequency was determined i n free c e l l suspensions prepared from 1 mm-long root tips; the relative content of DNA  was  determined microspectrophotometrically by the two-wavelength method. It was shown that the balance between root elongation and mitosis i n the root t i p could be influenced by the amount of aeration and by adenine. Increased aeration stimulated root elongation and depressed mitotic frequency; adenine stimulated mitosis, inhibiting, at the same time, root elongation. 8-azaguanine, i n concentrations of 10 p.p.m. and higher, stopped mitosis within 24 hours and greatly reduced root elongation and the fresh and dry weights of roots within 72 hours.  This inhibitory  effect on both root elongation and mitosis was positively correlated with aeration.  8-azaguanine i n a concentration of 1 p.p.m. significantly  reduced mitotic frequency but slightly stimulated root elongation.  The  inhibition of root elongation could be best, though incompletely, reversed by 40 p.p.m. adenine or guanine. The mitotic inhibition could be partially reversed by 40 p.p.m. guanine; adenine, at a concentration of 80 p.p.m., not only completely relieved mitotic inhibition, but  iii increased the mitotic frequency to a level higher than that of the water controls.  Concentrations of 6-mercaptopurine and 8-azaxanthine comparable  with those of 8-azaguanine had no inhibitory effects. Roots treated with 20 p.p.m. 8-azaguanine for 24 hours and then transferred into 80 p.p.m. adenine showed a higher mitotic frequency than the control within 24 hours after transfer.  Roots transferred from 8-aza-  guanine into water showed some mitosis 48 hours after transfer; i n this case mitosis was restricted to the partial l y differentiated and elongated cells of the provascular bundles. DNA content of interphase nuclei i n the controls showed this distribution:  a sharp 20 peak (about 65 per cent nuclei), a much lower  4C peak (about 20 per cent nuclei), and intermediates (about fifteen per cent).  There were no polyploid nuclei i n the apical meristem of the root.  The DNA content of chromosomal groups i n different mitotic stages demonstrated the accuracy of the two-wavelength method which was used. The DNA content of nuclei i n roots treated with 20 p.p.m. 8-azaguanine was distributed i n a 2C peak (about 80 per cent nuclei) and a 4C peak (about 20 per cent nuclei).  There were no intermediates i n treated roots and  no nuclei contained a higher amount of DNA than 4C. The percentage of 4C nuclei did not increase with time. From the evidence that the mitotic inhibition induced by 8-azaguanine could be completely reversed within 24 hours by subsequent treatment with adenine, and from the findings concerning the distribution of DNA i n inhibited nuclei, i t may be concluded that 8-azaguanine was not  iv  incorporated into DNA.  The possibility that 8-azaguanine exerts i t s  inhibitory effects through interference with ATP metabolism i s discussed.  Wc\z JiStitersttg ai Jirtttsi| Columbia Faculty of Graduate Studies  PROGRAMME OF T H E  FINAL ORAL E X A M I N A T I O N FOR T H E D E G R E E OF  DOCTOR O F PHILOSOPHY of SVATOPLTJK  F R E D  FLORIAN  B.S.A. University of British Columbia M . A . University of British Columbia IN R O O M 33, B I O L O G Y B U I L D I N G M O N D A Y , D E C E M B E R 30th, 1957, at 10:00 a.m.  COMMITTEE IN CHARGE  DEAN G . M . SHRUM, Chairman H. ADASKIN S. H . Z B A R S K Y J . J . R. C A M P B E L L K. I. B E A M I S H  T . M . C. T A Y L O R A.H.HUTCHINSON D . J. W O R T P. F O R D  External Examiner: D r . J. M . N A Y L O R University of Saskatchewan  ABSTRACT A n investigation  was  made  of the effects  of the purine analogs  8 - a z a g u a n i n e „ 8-azaxanthine, and 6-mercaptopurine, and the purines adenine and guanine, on cell growth, mitosis, and desoxyribonucleic acid (DNA)  synthesis  in primary roots of Vicia faba  seedlings  grown in  aerated solutions. Root elongation was used as a measure of cell elongation: mitotic frequency was determined in free cell suspensions prepared from 1 mm-long root tips; the relative content of D N A was determined microspectrophotometricaly by the two-wavelength method. It was shown that the. balance, between* root elongation and mitosis in the root tip could be influenced by the amount of aeration and by, adenine. Increased aeration stimulated root, elongation, and depressed mitotic frequency; adenine- stimulated- mitosis, inhibiting, at the same time, root elongation. . . 8-azaguanine, in. concentrations of. 10, p.p.m., and higher, stopped mitosis within 24" hours and- greatly reduced root elongation and the fresh and dry weights of roots within 72 hours. This inhibitory effect on both root elongation and mitosis was positively correlated with aeration 8-azaguanine in a concentration of 1 p.p.m. significantly reduced mitotic frequency but slightly, stimulated root elongation'. The. inhibition of root elongation could be., best,. though incompletely;, reversed by 40 p.p.m. adenine or guanine. The mitotic, inhibition, could, bgt partially reversed by 40 p.p.m. guanine; adenine, at a concentration of 80 p.p.m., not only completely relieved mitotic inhibition, but increased the mitotic frequency to a level» higher,, than, that' of: the water- controls. Concentrations of 6-mercaptopurine and 8-azaxanthine comparable with those of 8-azaguanine had. no. inhibitory, effects. Roots treated with 20 p.p.m. 8-azaguanine for 24 hours and then transferred into 80 p.pim. adenine, shpw.edj a,, higher mitotic frequency than the control within 2\ hours after transfer. Roots transferred from 8-azaguanine into water showed- some mitosis 48 hours after transfer; in this case. mit.Qsis v^as:i restricted to the partially., differentiated and elongated• cells, ot<the,- proyascular bundles. D N A content of - interphase nuclei in the controls^ showed this distribution: a sharp 2G peak- (about 65 per cent nuclei); a much lower 4C peak (about 20 per cent nuclei), and intermediates (about fifteen per cent). There were no polyploid nuclei in the apical meristem of  the root. The D N A content of chromsomal groups in different mitotic stages demonstrated the accuracy of the two-wavelength method which was used. The D N A content of nuclei in: roots treated with 20 p.p.m. 8-azaguanine was distributed in a 2C peak (about 80 per cent nuclei), and a 4C peak (about 20 per cent nuclei). There were no intermediates in treated roots and no nuclei contained a higher amount of D N A . than 4C. The percentage of 4C nuclei did not increase with time. From  the evidence that the mitotic inhibition induced by 8-aza-  guanine could be completely reversed within 24 hours; by; subsequent treatment  with  adenine,  and from  the findings  concerning the  dis-  tribution of D N A in inhibited nuclei, it may be concluded, that'. 8-azaguanine was  not incorporated into D N A .  guanine; exerjts. its  inhibitory effects  metabolism is discussed.  The possibility that 8-aza-  through interference' with A T P  G R A D U A T E  Field of Study:  S T U D I E S  Botany (Cytology)  Biochemistry  S. H . Zbarsky  Biophysics Cytology Plant Physiology I. Problems in Plant Physiology  Tissue Culture  O. Bluh .  A . H . Hutchinson .  . D . J.  Wort  D . J . Wort  R. Altschul, University of Saskatchewan  Other Studies: Advanced Plant Breeding  V . C. Brink  Biometry  V . C . Brink  Breeding of Horticultural Plants Taxonomy of Higher Plants  _-_C. A . Hornby T . M . C . Taylor  In p r e s e n t i n g the  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  r e q u i r e m e n t s f o r an advanced degree at the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t freely  a v a i l a b l e f o r r e f e r e n c e and  agree t h a t p e r m i s s i o n f o r e x t e n s i v e f o r s c h o l a r l y purposes may  study.  I further  copying of t h i s  be g r a n t e d by the Head o f  Department o r by h i s r e p r e s e n t a t i v e .  be a l l o w e d w i t h o u t my w r i t t e n  Department The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 3, Canada.  my  I t i s understood  that copying or p u b l i c a t i o n of t h i s t h e s i s f o r g a i n s h a l l not  thesis  financial  permission.  TABLE OF CONTENTS  Page Introduction  1  Material and Methods  5  Results:  a. Preliminary experiments  14  b. Effects of purines and purine analogs on root elongation, mitosis, and fresh and dry weight of roots  17  c. Effect of aeration on root elongation and mitosis  28  d. Effect of aeration on the inhibition of elongation and mitosis i n roots treated with 8-azaguanine  • 29  e. Reversal with adenine after inhibition by 8-azaguanine  • 33  f. Spectrophotometry measurements ... 35 Discussion of Methods  43  Discussion of Results  48  Summary  60  Bibliography  63  vi  ACKNOWLEDGMENTS I wish to express my gratitude to Dr. J. Setterfield for suggesting the present study;  to Dr. J.M. Naylor of the University  of Saskatchewan who did not spare time or trouble to supervise the investigation, to align the microspectrophotometer, and to introduce me to the technique of DNA measurementsj  and to Drs. R.W. Begg and  H. Stich of the Department of Cancer Research, University of Saskatchewan, for valuable suggestions. The f a c i l i t i e s to carry out the present investigation were kindly supplied by the Department of Field Husbandry, University of Saskatchewan. The study was carried out under a project supported by a grant-in-aid from the National Cancer Institute of Canada; this financial help i s also greatly  appreciated.  INTRODUCTION A number of analogs of the natural purines, adenine and guanine, * have been shown to suppress growth and c e l l multiplication i n a variety of species.  The hope that such compounds might provide a basis for  practical cancer chemotherapy has l e d to extensive study of their action as antimetabolites. Included i n this group i s the guanine analog, 8-azaguanine. Roblin et al.(1945) reported that this compound acted as a guanine antimetabolite i n Escherichia c o l i .  Kidder et a l . (1949) obtained growth inhibition  in Tetrahymena and an almost complete cessation of growth of adenocarcinoma E 0771 i n mice, though the growth of tumor resumed shortly after the treatment was discontinued.  During these experiments positive results were ob-  tained also with spontaneous mammary cancer and lymphoid leukemia i n mice. However, two years later the same authors (Kidder et al.., 1951) reported that 8-azaguanine did not inhibit some other tumors (sarcoma 37 and melanoma S 91)• Partial reversal of inhibition i n susceptible tumors was produced by guanine. In a series of papers, Matthews (1951, 1952, 1953) reported inhibition of mosaic virus on Nicotiana. leaves, the inhibition being reversible by both guanine and adenine. The results of the studies, only a small part of which i s mentioned above, were summarized by Gellhorn and Hirschberg (1955) as follows: 8-azaguanine was found to be active against 10 experimental tumors out of 15 tested, and interfered with 23 out of 59 non-tumorous systems (such as viruses, bacteria, Drosophilq. and biosynthesis of several organic compounds).  Since their review does not include  2  research which had been done with higher plants, this work will be described here i n more detail. Using very low concentrations of 8-azaguanine (0*1 - 10.0 gamma per liter) de Ropp (l950, 1951) obtained complete or partial growth inhibition in a variety or normal and crown-gall tissues (garden chrysanthemum stem infected with Agrobacterium tumefaciens: bacteriafree crown-gall tissue of sunflower; excised tomato roots; and excised sunflower embryos). The inhibition was not reversed with guanine. On the other hand, the inhibition of virus tumor tissue from Rumex acetosa by concentrations of 8-azaguanine as low as 1 p.p.m. was completely reversed by guanine (Nickell et al., 1950). Fries (1954) noted considerable inhibition of growth of decotylised pea seedlings with 0.10.3 micromolar 8-azaguanine; the inhibition could be partially prevented by guanine, adenine and, to a lesser extent, by high concentrations of yeast nucleic acid. The author also observed a decrease of combined nucleic acids with increasing concentrations of the analog.  Finally,  Pryzim (1956) reported a complete inhibition in root tips of Vicia faba within 24 hours by concentrations of 10 p.p.m. of 8-azaguanine; the mitotic inhibition was not accompanied by an inhibition of root elongation. The mitotic inhibition was best reversed by adenine and guanine, adenine being superior. While the literature i s voluminous i t does not provide a clear picture of the mechanism of action of 8-azaguanine. These mechanisms have been suggested:  incorporation of the analog into nucleic acids  3  (Bennett et a l ^ , 1950, Parks, 1955, Mandel, 1955, and many others), inhibition of different enzyme systems through interference with ribonucleic acid (Creaser, 1955 &, b), interference with the synthesis of the structural components of nucleic acids (rev. i n Skipper, 1953). The susceptibility of animal tumor tissues probably depends on the concentration of guanase (8-azaguanine deaminase) which converts inhibitory 8-azaguanine into inactive 8-azaxanthine.  Thus Hirschberg  et a l . (1952) found very low concentration of guanase i n 8-azaguaninesusceptible tumors. Similarly, Hirschberg et al.(1953) found that the analog severely damaged a human brain neoplasm, glioblastoma multiforme, which i s devoid of guanase, while the effect on fetal brain tissue containing some guanase was much less severe.  Dietrich and Shapiro  (1953) could potentiate the carcinostatic action of 8-azaguanine with a riboflavin analog, flavotinj the result was interpreted as an indirect inhibition of guanase through product inhibition (xanthine). Finally, Mandel (1955) demonstrated the enhancement of 8-azaguanine toxicity through the inhibition of guanase by  4-amino-5-imidazolecarboxamide.  The history of studies with 6-mercaptopurine i s similar.  After  a demonstration of inhibition of growth i n Lactobacillus casei and of embryogenesis i n Rana niniens. the compound was shown to be inhibitory to mouse sarcoma 180 (Clarke et a l . , 1952); the authors expressed the opinion that the mechanism of action of this analog "may be related to the mercapto group rather than to the purine moiety, i.e., become involved i n sulfhydryl metabolism i n some as yet unknown way".  The  inhibition of mouse sarcoma-180 was confirmed by Biesele (1955)J this author has also demonstrated the prevention of inhibition by very small concentrations of coenzyme A and concluded that the compound might act as an antimetabolite of coenzyme A rather than interfere with nucleic acid metabolism. The review of Gellhorn and Hirschberg (1955) shows that 6-mercaptopurine had been active against 13 tumors out of 15 tested, and interfered with 29 out of 59 non-tumor systems. Very interesting results with this compound have been obtained by Nickell (1955)* while 6-mercaptopurine slightly stimulated the growth of Lemna minor i n aseptic culture, i t strongly inhibited the growth of virus tumor tissue from Rumex acetosa; the results indicated the possibility of different types of metabolism i n these two materials. The present study was undertaken i n order to provide further detailed information on the action of these purine antimetabolites at the c e l l l e v e l , specifically the effects of these compounds on c e l l growth and the mitotic cycle.  With respect to the latter i t was hoped  that the use of cytochemical methods would shot/ whether or not the expected inhibition of c e l l reproduction by 8-azaguanine and 6-mercaptopurine was dependent only on the suppression of mitosis per se, or whether this effect involves the suppression of interphase processes such as DNA reproduction. Furthermore, i t was considered that accurate microspectrophotometric DNA determinations i n single nuclei, i n conjunction with metabolite-antimetabolite studies would provide the technical means of investigating whether these compounds were incorporated into  DNA.  5  MATERIALS AND METHODS  In this investigation the cytological effects of purines and several purine analogs were studied i n roots of v i c i a faba seedlings which had been grown i n aerated aqueous solutions of these substances*  This  method of seedling culture had been previously adapted by Setterfield and Duncan (1955) from the similar technique outlined by Brown and Broadbent (1950) and Brown (1951). Normal, large seeds were selected and soaked i n tap water i n shallow trays for 48 hours, after which the seed coats were peeled off and the naked seeds held at 20+l°C for four days i n f l a t s f i l l e d to a depth of 6" with vermiculite. To diminish bacterial and fungal contamination fresh vermiculite was used for each run and f l a t s were steam-sterilized before use.  Seedlings with healthy, straight primary roots 5-10 cm long  were transferred directly from vermiculite into trays f i l l e d with warm tap water at 20°C for washing and further selection. The seedlings were then separated into groups (usually three) according to the length of the root, and the groups subdivided into replicates (in most cases four replicates of six roots each per group), as uniformly as possible, according to the thickness of the roots.  In most cases an experiment  consisted of six different treatments of twelve roots per treatment. To obtain uniform replicates, i t was necessary to germinate 300-4-00 seeds prior to each run.  Each root was marked at a point 30 mm from  6 the t i p with thick India ink, applied with a fine glass rod. This mark served as a reference point i n measuring root elongation during subsequent treatment. Seedlings with plumule removed were then placed on plastic plates over 1000 ml Pyrex beakers containing the treatment solutions, with roots extending down into the solutions through equally spaced holes i n the plates.  Aeration of the solutions was provided by a small diaphragm  pump which delivered a i r through rubber tubing to a sintered glass diffusion tube i n each beaker.  A i r flow to various beakers was balanced  by means of clamps f i t t e d to the connecting rubber tubing. were carried out at 20*1 °C i n diffuse l i g h t .  The experiments  Relative humidity was close  to 100 per cent. Broad beans of the variety "Windsor" (crops 1955 and 1956) supplied by the Brackman-Kerr Milling Co., New Westminster, B.C., were used i n a l l experiments. The chemicals, 8-azaguanine, 6-mercaptopurine hydrate, 8-azaxanthine, guanine hydrochloride, and adenine, were obtained from the Nutritional Biochemicals Corporation, Cleveland, Ohio. The chemicals were used as supplied, without any further attempt at purification.  The structural formulae of the chemicals are indicated  i n Fig. I* Distilled and subsequently "demineralized" water of less than 1 p.p.m. ion concentration, obtained by letting d i s t i l l e d water run slowly through 111co-Way Research Model De-ionizer, was used throughout the experiments for preparing of solutions and for rinsing of glassware after washing.  7  PLATE I  Figure 1. Structural formulae of purines and purine analogs used i n this investigation.  PLATE I  8-AZAXANTHINE  8 Chemicals were dissolved i n water by heating to less than boiling point directly i n beakers; where necessary, compounds were f i r s t pulverized i n a small porcelain mortar. After cooling the volume of solutions was made up to 1000 ml and the pH adjusted to 6.0*0.1 with dilute HCQ. and K0H.  The pH was measured again at the end of each run.  In a l l experiments, concentrations of chemicals are expressed i n parts per million (pvp.m.). Since the molecular weights of adenine, guanine hydrochloride, and 8-azaguanine differ slightly, a correction was necessary i n order to provide equivalence i n molarity.  For this reason  concentrations of adenine and guanine hydrochloride were adjusted to those of 8-azaguanine, i . e . where 20 p.p.m. of guanine hydrochloride i s indicated, actually 24.66 p.p.m. were administered, and for adenine, 17.76 p.p.m. A l l glassware, except volumetric items, was cleaned by the Calgon-metasilicate method of Scherer (1955)•  Pipette3  and graduate  cylinders were cleaned i n the usual sulphuric acid-potassium dichromate solution, followed • by rinsing i n running, d i s t i l l e d , and f i n a l l y n  de-ionized water. n  The roots were measured to the nearest millimeter at 24, 48, and 72 hours. Where no roots were collected until the end of the run the growth rate curves were constructed on the basis of the root length ( i . e . the distance between the root t i p and the mark less 30 mm) at each time. If some roots were collected during the treatment the growth rate curves were calculated from the average growth increments for each 24  9  hour period.  Most of the runs were started with 12 roots per treatment,  from which three roots were collected at each timej the average increment was then calculated from 12 roots at 24 hours, from 9 roots at 48 hours, and from 6 roots at 72 hours.  If a root had not grown at a l l i t was  discarded and not included i n calculations} this happened occasionally when the root meristem was broken i n measuring. Where the fresh weights were taken, the roots were rinsed i n d i s t i l l e d water and the sections between the mark and the root t i p removed. These portions of the roots were then quickly surface dried with f i l t e r paper, collected i n Cooled, stoppered v i a l s , and weighed immediately i n the vials.  The dry weights were taken after drying to constant weight  i n an oven at 95°C on previously dried and weighed f i l t e r papers. Mitotic frequency determinations were carried out using the method described by Setterfield et al.(1954).  Root tips were fixed i n  freshly prepared ls3 acetic alcohol for 15 min., washed i n running water for 15 min., hydrolyzed 10 min. i n 1 N HC1 at 60°C, washed i n two changes of d i s t i l l e d water, and stained for 1 hour i n leucobasic fuchsin.  The  material was then washed i n three changes of SO2 water of 10 min. each, and rinsed i n d i s t i l l e d water. In a few experiments roots were fixed i n 10 per cent neutral formalin; i n this case the fixing time was extended to six hours and washing to twelve hours. Root caps were removed by gently wiping the roots with f i l t e r paper and one millimeter-long apical segments were removed from the stained roots using a device specially designed to minimuze errors i n cutting.  10 This consisted of a plastic plate provided with a longitudinal groove with a stop at one end and narrow s l i t s transecting the groove at measured intervals.  Roots were placed i n the groove, and 1 mm tips excised  by slicing with a razor blade i n the appropriate s l i t .  The root tips were  then held i n 1 ml aliquots of f i l t e r e d 5 per cent pectinase solution (Nutritional Biochemicals Corporation) at pH 4.0 for 6-24. hours, washed i n two changes of d i s t i l l e d water of 5 min. each, and shaken vigorously i n 1 ml of water i n corked v i a l s .  To obtain free c e l l suspensions from  root tips fixed i n formalin i t was necessary to force the tissue fragments several times through the needle of a hypodermic syringe.  However,  acetic-alcohol fixed root tips disintegrated readily on shaking.  The free  cells were centrifuged down for 5 min. at 1200 r.p.m, and mounted on slides i n a drop of neutral Karo syrup-phosphate buffer mixture. The three roots collected at each time for each treatment were processed together and two slides were prepared as replicates from each sample of cells. Samples of 1700 - 1900 meristematic cells were scored i n random transects of the slide, under high power magnificationi i n order to determine the proportions of cells i n interphase and i n different stages of mitosis. In this scoring prometaphase was included i n prophase, and very early prophases were considered as interphases.  Four samples were counted for  each treatment at each time (one i n the upper and one i n the lower half of each slide), and arithmetic means of the cells i n mitosis were expressed as mitotic frequency.  A l l c e l l types that have been seen to divide  commonly i n controls have been considered as meristematic.  These i n -  cluded, besides isodiametric c e l l s , also partially elongated cells of  11 the central pith.  In some treatments the ratio of prophases: telophases  was calculated. The DNA content was measured i n Feulgen stained nuclei. In preparing material, the staining procedure was identical with that described above except that the staining time was extended to 90 min. and the temperatures and time of fixing, washing, hydrolyzing, staining, and washing i n S62 water were carefully standardized.  Single lots of 1 N  HC1, leucobasic fuchsin, and of the stock solution of potassium metabisulfite were used for preparation of a l l the material used for measurements. The control and treated root tips i n each experiment were carried through the various steps i n the procedure i n the same v i a l .  Each root  t i p was then placed on a slide i n a drop of 45 per cent glacial acetic acid, the root cap removed and the epidermis peeled off under a dissecting microscope.  An apical portion of the root 0.2 mm i n length was then cut  off, squashed, and the coverslip floated off i n a Petri plate f i l l e d with 45 per cent acetic acid.  The time was controlled so that the cells  were treated with the acetic acid for a total time of 90 min. (this Included dissecting, squashing, and floating off the coverglasses).  The slides  were then passed through two changes of five min. each, of 70 per cent, 95 per cent, and absolute ethanol, xylene, and toluene, and were f i n a l l y mounted i n Shillaber's immersion o i l of N  n  = 1.5150. Prepared slides  were stored i n darkness i n a refrigerator. The Feulgen dye content of individual nuclei was measured by the tiro-wavelength method of Patau (Patau, 1952, Patau and Swift, 1953)  12 using a microspectrophotometer of the type described by Swift (1950 a, b). The instrument included the following main components: a ribbon-filament (tungsten) light source, a Beckman DU spectrophotometer which isolated essentially monochromatic light ( s l i t width 0.1 mm), a Leitz binocular microscope with Aristophot stand, a 90 X apochromatic o i l immersion objective (NA 1.32) and 10 x periplanatic ocular, an I P 21 electron multiplier phototube, a Farrand Type B control unit, and a Rubicon galvanometer.  Details of the assembly and alignment of the optical  components, as well as a thorough discussion of procedures used i n operating and testing the apparatus are given by Swift and Rasch (195&). The choice of the two wavelengths used i n these measurements was made i n the usual way, by f i r s t obtaining the absorption curve of the central portion of a homogeneous nucleus, and from this selecting two wavelengths ( >)± and ^2^> '^ sao  t h a  *  E  *^  1=  V  2  S^z.  On this basis  wavelengths 490 millimicrons (^^) and 511 millimicrons ( h^) were chosen and used throughout these experiments. The following procedure was followed i n measuring individual nuclei:  the image of the nucleus was centered i n the aperture of an i r i s  diaphragm located immediately beneath the photocell and the diaphragm stopped down to a diameter slightly greater than that of the nuclear image. At each wavelength the intensity of light passing through this aperture to the photocell was measured both with the nucleus i n position ( l ) and through a blank area on the slide ( l ) . n  Q  •  E = extinction.  Transmission (T) i s  !3  given by the ratio I n / l *  •^  0  his  ra  ^ i  0  ^  a  determined twice by replicate  measurements, yielding an average value of T at each wavelength (T ^, and T  M. The relative dye content within the measured area A i s given  by M = L^Dc^, where L ] _ = l - T / \ , c i s the diameter of A, and D =  i  —  2 - LaAa  T  a-4,—  n  ~  T  . The value D i s a correction factor for  1  inhomogeneity of dye distribution within A.  This correction i s based  on the fact that inhomogeneity increases T to a proportionally greater extent at ^2 than a t ^ j .  The ratio Ig/lQ. =  — , which i s the 1 •* T A1 l  variable i n the calculation of D, therefore decreases with decreasing homogeneity. Convenient tables of D prepared by Swift and Rasch (1956) were used i n calculations. A l l measurements were carried out with microscope condenser stopped down to a numerical aperture of approximately 0.4, and with the monochromater s l i t width of 0.1 mm.  Where frequency distributions of DNA  per nucleus were sought, nuclei were measured at random i n several fields on the slide.  These areas were mapped and the nuclei numbered so that  unintentional duplications of measurements of individual nuclei could be avoided and, where necessary, nuclei could be remeasured.  14  RESULTS  About one quarter of the results reported here have been obtained i n runs under high aeration. After i t had been realized that high aeration, besides stimulating root elongation (Pig. 14), considerably depresses mitosis (Pig. 15) and thus renders data on mitotic frequencies less suitable for expressing differences due to treatments, the reit of the runs have been done under medium aeration. The average a i r flow has been measured with a "flowrator" tube (Fischer and Porter) and gave these values:  high aeration - cca 0.0070 CFM (cubic feet per minute) per  beaker; medium aeration - cca 0.0035 CFM per beaker.  Results presented  without a note have been obtained under medium aeration. A note about pH has been made only where f i n a l value deviated from the original pH 6.0+0.1 by more than 0.2.  a.  Preliminary experiments:  In preliminary experiments an investigation was made of the effects on root elongation of seed size, presence of the plumule, and degree of aeration of water (used later as control).  An examination  was also made of the effect of fixatives on mitotic scoring, and of the effects of washing during preparation of roots on mitotic frequency and the prophase: telophase ratio.  15 Concerning the f i r s t three preliminary experiments, only a short summary i s reported here. A small but noticeably greater rate of root elongation was observed i n the roots of seedlings grown from relatively small seeds.  However, these roots were also much thinner, provided  smaller amounts of tissue per unit root length for the determination of fresh and dry weight and for mitotic counts, and thus were less satisfactory. Increased a i r flow through the culture beakers stimulated root elongation, though when the rate of aeration reached the point where gas bubbles settled on the roots growth declined sharply.  The presence of the plumule  slightly stimulated root elongation but the difference was very small. TABLE I Effect of fixative on scoring of mitotic frequency i n root t i p s . Roots were collected directly from vermiculite. Three root tips from each treatment were processed together and from the mixture of cells two slides per treatment were prepared.  Treatment  Slide  Aceticalcohol fixation  Oa/la 0a/2a Oa/lb 0a/2b  formalin fixation  Ob/la 0b/2a Ob/lb 0b/2b  Number of cells Total Pro Meta Ana Telo mitosis 106 103 101  115  34 29 21 20  425  104  125  24 19 19 21 83  106 111 132  474  Inter- Grand phases total  Per cent Mitosis (+S.E.) 10.68 10.37 9.65 9.89  740  1,815 1,821 1,823 1.829 6,548 7,288  52 56 .48 38  206 187 184 197  1,624 1,626 1,664 1.680  11.25 10.31 9.95 10.49  23 194  774  1,830 1,813 1,848 l 877 6,594 7,368  37 35 40 33  194 189 176 181  66 145  17 22 14  5 6 6 6  1,621 1,632 1,647 1.648  f  10.15 + .23  i°:19  16 Table I i s included to illustrate i n detail the technique of mitotic frequency scoring.  While the difference between mitotic  frequencies of the two samples i s not s t a t i s t i c a l l y significant (difference - .35 per cent, t calc. - .97, t at.05 - 2.45), the difference between the average number of anaphases per count i s s t a t i s t i c a l l y highly significant (fixed i n acetic alcohol - 16.50+2.02, fixed i n formalin 5.75+.25, difference - 10.75, t calc. - 5.29, t at .01 - 3.71).* The nuclei and chromosomes treated with acetic-alcohol appeared larger, swollen, while those from formalin were more compact and more densely stained.  The difference i n average number of anaphases per count was  obviously due to the fact that a large number of anaphases with contracted and shrunken arms due to formalin fixation were counted as telophases. Since i t was much more d i f f i c u l t to prepare c e l l suspensions from formalinfixed root tips i t was decided to use acetic alcohol fixative i n a l l subsequent runs. The prophase:telophase ratio for root tips fixed i n acetic alcohol was 2.93. TABLE II Effect of preparatory washing and handling of roots, on mitotic frequency and the prophase:telophase ratio. Unwashed roots collected directly from vermiculitej washing and handling lasted for approximately two hours. The t test was used for estimation of significance. Mitotic frequencies are means of four counts as shown in Table I. Treatment Run  Mitotic frequency {%) + S.E. Unwashed Washed  IS/57 20/57  11.17+ .16 12.59+ .27  Prophase:Telophase ratio  2.82  9.21+ .45 11.18+ .12  Difference (%) 1.96** 1.41*  2.23  * Statistical methods and values of t from Goulden (1953)  17 During the preparatory washing and handling of roots preceding fixation the mitotic frequency i n the root tips decreased.  The decrease  was highly significant i n the f i r s t run and significant i n the second run. The decrease i n mitotic frequency was accompanied by a decrease i n the prophase:telophase ratio.  The significance of the changes i n prophase:  telophase ratio i s considered i n the discussion.  b.  Effects of purines and purine analogs on root elongation, mitosis, and fresh and dry weight of roots.  Responses of root elongation to various concentrations of 8-azaguanine are represented graphically i n Figs. 2 and 4» Low concentrations (up to 1.0 p.p.m.) consistently gave a small stimulation which increased with concentration (Fig. 4)» Even the stimulation with 1.0 p.p.m. was not statistically significant: gave these results:  the analysis of variance  F value for treatment = 1.98, F at .05 = 4«60.  Within the range (5^40 p.p.m.) the effect was reversed - inhibition was increased with concentration (Fig. 2). The inhibition by 80 p.p.m. of 8-azaguanine (not shown i n Fig. 2) was slightly greater than that by 40 p.p.m. However, this result was unreliable since the chemical tended i n some runs to precipitate i n this highest concentration. The growth curves of roots inhibited by 8-azaguanine showed a typical levelling off with time:  for example the total elongation of roots treated with  20 p.p.m. 8-azaguanine was 77.6, 56.2, and 44.7 per cent of that of control at 24, 48, and 72 hours respectively, or, expressed i n another way, the average consecutive daily increments of roots treated with  18 PLATE II  Figure 2.  Inhibition of elongation of Vicia faba roots by "high" concentrations of 8-azaguanine. The concentrations are expressed as parts per million (p.p.m.). Values i n the figure represent means of following number of runs of 12 roots each: control - 8, 5 p.p.m* - 4 , 10 p.p.m. - 7, 20 p.p.m. - 6, 40 p.pim. - 3. Growth of treated roots was calculated as per cent of control i n each run, and the individual controls adjusted to the mean of a l l the controls.*  Figure 3.  Effects of "high" concentrations of 8-azaguanine on mitosis i n root tips. In each run four separate scorings of 1,700 - 1,900 meristematic cells were carried out for each concentration at each time. Presented values are means of following number of runs: control - 4> 5. 10, and 40 p.p.m. 8-azaguanine - 1, 20 p.p.m. 8-azaguanine - 4« Mitotic rates i n treated roots were calculated as per cent of control and the means adjusted to the average control.*  *  Methods described for Figs. 2 and 3 were used i n a l l subsequent figures showing root elongation and mitotic frequencies. A l l experiments were caried out on primary roots of Viciq faba.  PLATE I I  l0  £  Time (hours)  19  PLATE I I I  Figure 4-. Effect of "low" concentrations of 8-azaguanine on root elongation. Presented values are means of following number of runs: control - A> 0*1 p.p.m. 1, 0.5 p.p.m. - 3, 1.0 p.p.m. - 2.  Figure 5. Effect of "low" concentrations of 8-azaguanine on mitosis i n root tips. Presented values are means of following number of runs: control - 3, 0.5 p.p.m. 2, 1.0 p.p.m. - 1.  PLATE I I I  -J  24  I  48  Time (hours)  L_  72  20 20 p.p.m. 8-azaguanine were 19.2, 4»9> and 1.5 mm while those of control were 24.8, 18.2, and 14.3 mm. The effects of 8-azaguanine on mitotic frequency i n the root meristem are shown i n Figs. 3 and 5» Concentrations of 10-80 p.p.m. caused a complete cessation of mitosis within 24 hours while with the concentration of 5 p.p.m. the mitotic rate declined to about 1 per cent within 24 hours and ceased within 48 hours (Fig. 3). In 8-azaguanineinhibited root tips, about 5 - 1 0 per cent of nuclei had the appearance of very early prophases. These "pseudo-prophases" (term used by Firket, 1957) are larger than interphase nuclei, show some condensation of chromosomes and very large nucleoli with diffused borders as compared with smaller, sharply delineated nucleoli i n normal interphasic nuclei. A concentration of 0.5 p.p.m. had practically no effect on mitotic ratej a concentration of 1.0 p.p.m. lowered mitotic frequency to about one half of that of control (Fig. 5). The observed inhibitory effects of 8-azaguanine at rather low concentrations might be interpreted on the basis of the concept that this compound interfered with purine metabolism.  I f this was the case,  i t might be possible to reverse this inhibition by supplying the natural purine metabolites - guanine and adenine - to the roots. The effects of guanine alone and guanine i n combination with 20 p.p.m. of 8-azaguanine on root elongation and mitosis are represented i n Figs. 6 and 7. Guanine alone slightly stimulated both root elongation and mitosis.  Best but not complete reversal of the inhibition of root elong-  21  PLATE IV  Figure 6 . Effects of guanine along and guanine i n combination with 2 0 p.p.m. 8-azaguanine on root elongation. Concentrations of guanine-HCl are equimolar with the concentrations of 8-azaguanine. Presented values are means of following number of runs: control -At 2 0 8-azaguanine - At 2 0 guanine H C 1 - 1 , 4-0 guanine H C 1 - 1 , 2 0 8-azaguanine plus 2 0 guanine H C 1 - 2 , 2 0 8-azaguanine plus 4-0 guanine H C 1 - A* A l l concentrations i n p.p.m.; high aeration.  Figure 7 . Effects of guanine alone and guanine i n combination with 2 0 p.p.m. 8-azaguanine on mitosis i n root t i p s . Presented values are means of following number of runs: control, 2 0 8-azaguanine, and 2 0 8-azaguanine plus AO guanine H C 1 - 3 each, 4-0 guanine H C 1 - 1 . A l l concentrations i n p.p.m.; guanine equimolar; high aeration.  PLATE IV  24  48  72  Time (hours) 0^  "^o 40 ppm gua - ~o control * L40ppm quo. —S— ZOppm 8-aza. 72  22 ation by 20 p.p.m. 8-azaguanine was achieved with 4-0 p.p.m. of guanine hydrochloridej mitotic inhibition was relieved effectively at 24 hours only, while later mitotic rates sank to negligible levels.  It was not  possible to carry out reversal experiments with higher concentrations of guanine because of precipitation. Adenine alone inhibits root elongation, the inhibition being correlated with the concentration (Fig. 8). High concentrations of adenine depressed root elongation considerably but the growth curves did not level off more than did those of the control (e.g. 160 p.p.m. of adenine reduced root elongation to 37.1, 39.6, and 38.1 per cent of control at 24, 48, and 72 hours, respectively).  The highest concentrations  of adenine produced swellings located about 3 to 10 mm from the root tips, and completely depressed the development of secondary roots that normally appeared at the end of runs i n controls.  The mitotic rates i n  root tips treated with adenine increased with the dosage (Fig. 9) though the effect on root elongation was i n the opposite direction.  From data  showing the effect of adenine when applied i n combination with 20 p.p.m. of 8-azaguanine, i t i s evident that best reversal of inhibition of root elongation  was obtained with 40 p.p.m. adenine (Fig. 10).  The growth  curves obtained with 20 p.p.m. 8-azaguanine combined with high concentrations of adenine (80, 160, and 320 p.p.m.) closely resemble the growth curves for adenine alone and show no inhibitory effect of 8-azaguanine. The growth curve for 20 p.p.m. 8-azaguanine with 20 p.p.m. adenine levels off after 24 hours, that for 20 p.p.m. 8-azaguanine with 40 p.p.m. adenine after 48 hours.  The mitotic frequency for 20 p.p.m.  23  PLATE V  Figure 8. Effect of adenine on root elongation. Concentrations of adenine are equimolar with those of 8-azaguanine. Presented values are means of following number of runs: control - 6 , 20 adenine - 1, 4-0 adenine - 2 , 80 adenine 3, 160 adenine - 1. Concentrations i n p.p.m.  Figure 9 .  Effect of adenine on mitosis i n root t i p s . Presented values are means of following number of runs: control 2, 80 p.p.m. adenine - 1, 160 p.p.m. adenine - 1. Concentrations of adenine are equimolar with the concentrations of 8-azaguanine.  PLATE V  _l  24  I  1—  48  72  Time (hours)  PLATE VI  Figure 10, Effects of adenine i n combination with 20 p.p.m. 8-azaguanine on root elongation. Presented values are means of following number of runs: control - 6, 20 8-azaguanine - 6, 20 8-azaguanine plus 20 adenine 3, 20 8-azaguanine plus 40 adenine - 2, 20 8-azaguanine plus 80 adenine - 4, 20 8-azaguanine plxis 160 adenine 2, 20 8-azaguanine plus 320 adenine - 1. Concentrations i n p.p.m.j concentrations of adenine are equimolar with concentrations of 8-azaguanine.  Figure 11. Effect of 80 p.p.m. adenine i n combination with 20 p.p. 8-azaguanine on mitosis i n root t i p s . Presented values are means of 1 run per treatment.  PLATE VI  •  control  •  20ppm 8-ozo + 4 0 ppm ode 80 20  160  320  24  48  Time (hours) 20  ppm  8 0 ppm  o  Time (hours)  72  azo.t  ade.  conlrol  20ppm  48  8  8aza.  25  8-azaguanine with 80 p.p.m. adenine was stimulated to about the same level (Fig. 11) as i n treatment with 80 p.p.m. adenine alone (Fig. 9 ) . The preceding experiments have shown that complete inhibition of mitosis was accomplished at 24 hours, using 8-azaguanine i n concentrations exceeding 5 p.p.m. The rate of descent of the mitotic rate within the f i r s t 24 hours of treatment with 20 p.p.m. of the compound was investigated i n the three experiments represented i n Fig. 12. I t i s indicated that the onset of inhibition occurred after eight hours, with the mitotic rate declining to nearly zero after 14 hours. The decrease i n mitotic frequencies was accompanied by a simultaneous drop i n the prophase:telophase ratio^  These were the values of the ratio:  0 time - 2.83, 2 hours - 3.43, 4 hours - 2.48, 6 hours - 4.24, 8 hours 3.80, 10 hours - 1.79, 12 hours - 1.53, 14 hours - . 7 5 , 16 hours - .40, 18 hours - . 2 5 . The effects of 8-azaguanine on fresh and dry weight during a treatment period of 72 hours i s shown i n Fig. 13. Concentrations of 10 p.p.m. and 80 p.p.m. reduced the fresh weight to 69.06 per cent and 61.70 per cent of that of the control; dry weight was reduced to 71.26 per cent and 66.13 per cent, respectively.  These data were calculated  from total weights of the roots to the mark; i f increments only had been considered (i.e. f i n a l weight less original weight) the relative differences between treatments would be greater.  The main point of  interest i n the results of the two experiments i s that dry weight as well as fresh weight decreased with increasing dosage.  This shows that  PLATE VII  Figure 12.  Time effect of 20 p.p.m. 8-azaguanine on mitosis i n root t i p s . Three different runs; mitotic frequencies were calculated from four repeated counts at each time on material prepared from two root tips each.  Figure 13. Effects of 8-azaguanine on fresh and dry weights of roots. Treatments lasted for 72 hours. Run I represents 12, run II 24 roots per treatment. Seeds from crop 1955 were used i n run I, seeds from crop 1956 i n run I I . In run II the average fre3h weight was .0965 gm, the average dry weight .0054 gm per root at 0 time i n 30 mm long sections of roots.  PLATE VII  I controi  10  PPM  20  8-azaguanine  40  27 the inhibition of root elongation by this compound i s accompanied by a reduction i n dry matter synthesis and i s , therefore, not principally dependent on an induced reduction i n water content.  TABLE III Effect of 6-mercaptopurine on root elongation. The results are an average of three runs for the control, 20, and 160 p.p.m., and of two runs for the other concentrations. High aeration. Concentration 10 20 40 80 160  Elongation as per cent of control 24 hours 48 hours 72 hours  p.p.m. p.p.m. p.p.m. p.p.m. p.p.m.  107.1 105.5 103.2 97.3 109.6  104.3 99.2 94.6 95.4 105.2  107.2 98.4 90.6 95.6 100.6  TABLE IV Effect of 6-mercaptopurine on mitosis i n root tips after 72 hours. Presented data are means of four different counts of 1,700 - 1,900 meristematic cells each. Mitotic frequency at 0 time was 7.57+ .33 per cent. High aeration. Treatment  Control  10  20  40  80  160 p.p.m.  Mitotic frequency  2.00  1.91  1.87 2.06  2.11  1.11  Standard Error  +.21  +•15  +.12  +.35  +.08  +.17  28 TABLE V Effect of 8-azaxanthine on root elongation. The presented data are an average of two runs for the control, 20, and 160 p.p.m., and of 1 run for the other concentrations. High aeration. Concentration  Elongation as per cent of control 24 hours 48 hours 72 hours  20 p.p.m.  103.5  40  85.4 96.6 101.00  100.7  87.0  88.1  "  80  160 320  •  " n  102.3  104.8 87.5 91.0  85.5  94.7  106.0 92.4  The effects of two other purine analogs, 6-mercaptopurine and 8-azaxanthine on root elongation (both analogs) and mitosis (6-mercaptopurine only) are given i n Tables I I I , IV and V.  The former  compound had no apparent effect on root elongation at any of the concentrations tested, and gave only partial inhibition of mitosis at the highest dosage (160 p.p.m.). Treatments with 8-azaxanthine had no marked effect on root elongation;  the apparent slight depression at  40, 80, and 320 p.p.m. may have resulted from chance variation and from differences i n aeration between beakers i n the single experiment i n which these three concentrations were tested.  c.  Effect of aeration on root elongation and mitosis.  The preliminary experiments have shown that within a considerable range increased a i r flow stimulated root elongation.  But i t became  apparent that high rates of aeration resulted, at the same time, i n a progressive decrease i n mitotic rates i n the controls during the  29 incubation period (72 hours).  This drop i n mitotic rate was not observed  i n similar experiments reported by Setterfield and Duncan (1955)•  There-  fore, experiments were undertaken i n order to clarify the responses of root elongation and mitosis to the rate of aeration, the results of which are plotted i n Figs, L4 and 15.  The rates of aeration had opposite  effects on mitosis and root elongation, i . e . increased aeration depressed the mitotic rate and stimulated elongation; the converse was true for no aeration.  The pH of non-aerated water decreased during the runs from the  original 6.0 to about 5.5; this change i n pH i s too small to account alone for any of the effects reported above.  d.  Effect of aeration on the inhibition of elongation and mitosis i n roots treated with 8-azaguanine.  The demonstration that rate of aeration profoundly influences both root elongation and mitotic activity suggested that aeration might also influence the degree of effect of exogenous purines and their analogs on the two aspects of root growth. This possibility was tested i n experiments i n which 8-azaguanine was administered at different rates of air-flow. The results are plotted Figs. 16, 17, 18, and 19.  In  agreement with the earlier experiments control roots elongated more rapidly at the relatively higher rate of aeration (cf. Figs. 16, 17, and 18).  Moreover the degree of inhibition of root elongation at both  concentrations (20 and 80 p.p.m.) of 8-azaguanine increased with the rate of a i r flow (cf. Figs. 16, 17, and 18).  Similarly, 8-azaguanine  inhibited mitosis more effectively i n aerated solutions: while the  PLATE VIII  Figure 14. Effect of aeration on root elongation. Presented values are means of following number of runs: control (medium aeration) - 5, high aeration - 3, no aeration - 5 .  Figure 15. Effect of aeration on mitosis i n root tips. Presented values are means of following number of runs: control (medium aeration) - 5, high aeration - 3, no aeration - 5.  PLATE VIII  Time (hours)  _L_  24  I  48  .  Time (hours)  i  72  PLATE IX  Figure 16. Effect of high aeration on inhibition of root elongation by 20 and 80 p.p.m. 8-azaguanine. Presented values are means of 1 run (12 roots) per treatment.  Figure 17.  Effect of medium aeration on inhibition of root elongation by 20 and 80 p.p.m. 8-azaguanine. Presented values are means of 1 run (12 roots) per treatment.  PLATE IX  •  control  •  20 ppm  E 60  £ O 40  a>  320  35.5%  ^80  o  24  48  Time(hours)  44.1%  • •  48  Time (hours)  m  72  •  24  P P  control  20 ppm 80 ppm  PLATE X  Figure 18, Effect of lack of aeration on inhibition of root elongation by 20 p.p.m. 8-azaguanine. Presented values are means of two runs per treatment.  Figure 19. Effect of aeration on mitosis i n root tips treated with 20 p.p.m. 8-azaguanine. Presented values are means of two runs per treatment.  PLATE X  control (med. aeration) non aeraled control 20ppm 8-aza. non aerated  24  48  Time (hours)  72  33 mitotic rate reached zero at 24 hours i n both high and medium aerated runs (and  also with 10 p.p.m. S-azaguanine - Fig. 3) i t stayed above two per cent  i n non-aerated solution of 20 p.p.m. 8-azaguanine at 24 hours (Fig. 19).  e.  Reversal with adenine after inhibition by 8-azaguanine.  The preceding experiments have shown that the inhibition of root elongation and mitosis by 8-azaguanine could be reversed by either adenine or guanine when both the analog and the metabolite were administered together.  While this suggested that the two compounds acted competitively  i n one or more c r i t i c a l metabolic processes leading to c e l l growth and nuclear division, the possibility could not be excluded that the reversal resulted merely from an interaction of the two substances i n the culture solution.  This possibility was tested i n an experiment i n which roots  were held i n 20 p.p.m. of 8-azaguanine for 24 hours, and then placed i n water or i n a solution of 80 p.p.m. adenine for 48 hours.  I f reversal by  adenine were dependent principally on interaction with 8-azaguanine i n solution, reversal should not occur i n this experiment.  The results  plotted i n Fig. 20 show that water slightly relieved the inhibition of root elongation, while the growth curve for roots transferred into adenine somewhat resembled those i n Figs. 8 and 10 for roots treated with adenine alone, or with 8-azaguanine plus adenine:  a depressed but  steadily ascending curve. However, within the period of reversal i n this experiment, adenine had a rather small effect on root elongation. Interestingly, the reversal of mitosis by adenine i n this experiment was very marked (Fig. 21). Partial recovery (0.27 per cent mitosis) was  PLATE XI  Figure 20. Reversal of the inhibition of root elongation by 80 p.p.m. adenine (equimolar) after the roots had been inhibited by 20 p.p.m. 8-azaguanine. The values are means of following number of runs: control - 2, 20 p.p.m. 8-azaguanine - 1, 20 p.p.m. 8-azaguanine then 80 p.p.m. adenine 3, 20 p.p.m. 8-azaguanine then water - 2. The values at 24- hours i n different treatments with 20 p.p.m. 8-azaguanine (range 17.2 - 19.8 mm) were adjusted to the value of the control treatment with 20 p.p.m. 8-azaguanine (19.8 mm) and the differences were added up at 4-8 and 72 hours.  Figure 21. Reversal of mitotic inhibition i n roots treated for 24- hours with 20 p.p.m. 8-azaguanine by transferring into solution with 80 p.p.m. (equimolar) adenine. Presented values are means of following number of runs: control 2, 20 p.p.m. 8-azaguanine - 1, 20 p.p.m. 8-azaguanine then 80 p.p.m. adenine - 2, 20 p.p.m. 8-azaguanine then water - 1.  PLATE XI  35  f i r s t observed 12 hours after the transfer from 8-azaguanine to adenine; mitotic figures observed at this time were a l l at prophase.  Complete  recovery had been established a few hours later as indicated by the rapidly rising curve. The rate of mitosis levelled off after i t s ascent (at 24 hours after transfer), but remained well above the control until the termination of the experiment.  In roots transferred from 8-azaguanine  into water some recovery of mitosis was observed only 4-8 hours after transfer.  In these roots mitotic figures were restricted to the  elongated provascular c e l l s .  The ratio of prophasesHelophases i n controls  at a l l times and i n roots transferred from 8-azaguanine into adenine at 24 and 48 hours after transfer was between 2.5 and 3.5; the ratio i n roots transferred from 8-azaguanine into water was 5*62 at 48 hours after transfer, indicating a sharply rising mitotic frequency at the end of the experiment.  f.  Spectrophotometric measurements.  This aspect of the work was undertaken primarily to determine whether or not DNA synthesis occurred i n cells i n which mitosis was blocked by 8-azaguanine.  In principle, frequency distributions of  DNA content per nucleus were determined i n both treated and control roots after various periods of incubation. The results of microspectrophotometric measurements i n control roots fixed at 24 and 72 hours are shown i n Figs. 22 and 25, respectively.  The essential characteristics of these curves are very  36 similar:  i n each case there are two well marked peaks representing  DNA values i n a ratio of approximately 1:2, and there are a few intermediate values lying between the peaks.  It w i l l be noted that the lower  peak for interphasic distribution coincides with the modal DNA value for telophasic and anaphasic nuclei.  In common usage (e.g. Swift, 1950a, b,  Deeley et aL., 1957) this amount i s designated as ™2C , and the amount n  characteristic of prophasic and metaphasic nuclei, "40".  There were  no values exceeding 4-C level indicating that there were no polyploid nuclei i n the root t i p . The curves representing treated roots fixed at 24, 48, and 72 hours are plotted i n Figs. 23, 24, and 26, respectively.  These  differ from those of controls chiefly i n one respect, namely that intermediate values are rare or absent.  It i s apparent that DNA syn-  thesis decreases prior to 24 hours and reaches zero before 48 hours. This finding i s also supported by the fact that the proportion of nuclei containing 4C level of DNA remained constant (l6 per cent at 24, 16 per cent at 48, and 18 per cent at 72 hours) throughout the treatment while no mitoses were observed.  TABLE VI DNA values of nuclei i n different mitotic stages i n control at 24 hours. DNA i n arbitrary units; values presented here are plotted i n Fig. 22 Mitotic stage Prophase Metaphase Anaphase Telophase  Number measured  13 4 6 21  Ranee  DNA content Mean  28.23-31.43 28.69-31.37  14.37-16.52  13.24-16.23  29.95 29.63 15.51 15.05  S.E. +.29  +.59 +.35 +.14  PLATE XII  Figure 22.  DM content of the nuclei i n apical portion of the root i n control at 24 hours. Mitotic frequency 7.44 per cent. Number measured: interphases - 100, prophases - 13, metaphases - 4 , anaphases - 6, telophases - 21.  Figure 23.  DNA content of the nuclei i n apical portion of the root treated with 20 p.p.m. 8-azaguanine for 24 hours; 100 nuclei measured.  PLATE XII  •—• interphases x—x ana- and telophases o—o pro- and metaphases  ^  •  •  20 DNA  in  I  •  24  II  28  L-ii  .  32  arbitrary units  • 12  16 DNA  20  in arbitrary  24 units  28  32  36  38  PLATE XIII  Figure 24-.  DNA content of the nuclei i n apical portion of the root treated with 20 p.p.m. 8-azaguanine for 4-8 hours and DNA content of telophases from control at 4-8 hoursj 51 treated nuclei and 4- telophases (control) were measured.  plate x i i i  DNA  in arbitrary  units  plate xiv  Figure 25. DM content of the nuclei i n apical portion of the root i n control at 72 hours. Mitotic frequency 6.87 per cent. Number measured: interphases - 100, prophases - 5, anaphases - 3 , telophases - 6.  Figure 26.  DNA content of the nuclei i n apical portion of the root treated with 20. p.p.m. 8-azaguanine for 72 hours 100 nuclei measured.  PLATE XIV  DNA in arbitrary  12  16  20  units  24  DNA in arbitrary units  28  32  40  TABLE VII DNA values of four telophases i n control at 4-8 hours. DNA i n arbitrary units; values presented here are plotted i n Fig. 24. Mitotic stage Telophase  Number measured 4  DM  content Mean  14.21 - 15.65  15.05  Range  . S.E. 1*30  TABLE VIII DNA values of nuclei i n different mitotic stages i n control at 72 hours. DNA i n arbitrary units; values presented here are plotted i n Fig. 25. Mitotic stage Prophase Anaphase Telophase  Number measured 5 3 6  ' Range  DNA content Mean  27.67 - 33.35 14.13 - 16.90 13.20 - 16.23  30.19 15.12 14.38  S.E. +1.04 + .88 + .54  Tables VI, VII, and VIII contain the DNA values for 2C (anaphase and telophase) and 4C (prophase and metaphase) nuclei i n control at the three different times (24,48, and 72 hours). These data show that the internuclear variation i n each class i s rather small and that the ratio of DNA content between 2C and 40 nuclei closely approximates to the theoretical ratio of 1:2.  The average values of  2C and 40 groups i n treated nuclei agree well with the values obtained by measuring mitotic groups of chromosomes i n controls; for example the mean of the 2C group i n roots treated with 8-azaguanine for 48 hours equals 15.15+.13, C.V.»= 5.83 (43 nuclei), and that of 4C group 29.85+.35, G.V. = 3.31 (8 nuclei). These values differ very slightly #  G.V. = coefficient of variability.  41  from the values obtained at different mitotic stages of controls, e.g. telophase at 24 hours where mean = 15•05+ .14, C.V. = 4*26 (21 telophase groups).  The close agreement between the theoretical and obtained  ratios and also between the DNA values obtained at different times shown i n the three tables indicates the accuracy of the method as well as a satisfactory standardization of fixation, hydrolysis, and staining procedures. The data presented i n Figs. 23, 24, and 26 were obtained i n random measurements and for this reason the possibility could not be excluded that occasional nuclei i n treated roots contained amounts of DNA substantially greater than 40 l e v e l .  This might be considered to  occur as a consequence of the direct suppression of the onset of prophase by  8-azaguanine, provided that DNA synthesis was not also entirely  suppressed.  To test this possibility slides prepared from treated root  tips at different times were searched for large, deeply stained nuclei which might contain unusually large amounts of DNA.  These were the  "pseudo-prophases" described previously.  TABLE IX DNA values of selected "pseudo-prophases" i n roots treated with 20 p.p.m. 8-azaguanine. DNA i n arbitrary units. Time of 8-aza treatment 24 hours 48 hours 72 hours Total  Number measured  Ranee  DNA content Mean  3 4 9  29.35 - 33.52 28.38 - 32.37 27.45 - 30.53  31.70 30.88 28.99  +1.23 + .87 + .34  16  27.45 - 33.52  29.97  1 .44  S.E.  42 The results given i n Table IX show that this variation does not occur, since the mean DNA content agrees closely with that of the unselected populations (Figs. 22-26, Tables VI and VIII).  A3  DISCUSSION OF METHODS The Calgon-metasilicate method of washing glassware i s now being used generally i n animal tissue culture. The method has the advantage over the usual acid cleaning that i t avoids the risk of residual chromium ions l e f t on the glassware,  unfortunately i t cannot  be used for the cleaning of volumetric glassware. In preparing seeds for germination, removal of the seed coats after preliminary soaking results i n more uniform germination and reduces the contamination of the seedlings by molds. While the seeds could be treated with antibiotics, this might introduce the possibility of effects of these substances on growth and mitosis, as well as interaction with the tested chemicals.  Though some fungal growth was often  found on the naked cotyledons toward the end of an experiment, this did not seem to affect either growth of the roots or mitosis. Since the diffusion of oxygen into solution depends not only on the air flow but also on the size of a i r bubbles released by the sintered glass tubes, i t was necessary to select tubes of uniform performance. A voltage stabilizer was introduced to eliminate variation i n the speed of the pump due to changes i n the line current. It was stated i n Materials and Methods that growth rate curves i n the case3 where some roots were collected during the treatment, were  44  constructed on the basis of daily average increments rather than from an average total length of 24, 48, and 72 hours.  This was done i n order  to eliminate a distortion of the curves introduced by withdrawing roots for mitotic counts during the experiment.  I f , for example, the three  roots collected at 48 hours were, by chance, considerably longer than the remainder, the growth curve constructed from the average total root length would be depressed i n the 48-72 hour interval and might even appear to reflect a negative growth rate. This i s particularly important in treatments ;where the growth rate declines sharply toward the end of the experiment (e.g. i n 8-azaguanine treatments).  This problem i s  avoided when the growth curves are obtained by summation of daily average increments. Under the given experimental conditions the period of 72 hours appeared to be the maximal time span for which the root elongation and mitotic counts i n the root meristem could give reliable results; after 72 hours considerable development of secondary roots usually appeared on the upper portion of the roots (the development of secondary roots was sometimes observed as early as at 48 hours) and roots i n some treatment solutions became necrotic. An i n i t i a l drop i n mitotic rate during the f i r s t 24 hour period was generally observed i n both treated and control roots.  This was probably due to the change i n  the environment (transfer of seedlings from vermiculite into water) and due to handling of the seedlings at the beginning of the experiments. This effect would have been eliminated by allowing the roots a 24 hour recovery period i n water prior to the treatment period.  However,  45 i n this case i t would have been necessary to shorten the duration of treatment for the reasons given above. .Apical, 1 mm segments of root tips were used i n mitotic frequency determinations, though Setterfield and Duncan (1955), Pryzina (1956), and others have used 2 mm portions for this purpose. Jensen (1955) has shown that c e l l elongation for the majority of cells begins at about 1.7  mm.  Errors i n cutting of the order of about 0.2 mm are to be reckoned with since the Feulgen-stained roots are soft and easily deformed i n handling. If 1 mm sections are cut the variation i n actual length of the cut portions i s restricted to an area of more or less homogeneous, dividing cells while the variation at 2 mm distance w i l l add to the sample greater or smaller amount of non-dividing cells and thus increase the error i n counting of mitotic frequencies. Fresh roots could be cut more accurately but short sections are d i f f i c u l t to handle during staining procedures. The apical 1 mm segment i n the Vicia faba, root t i p does not consist entirely of meristematic cells, though several authors (Holmes et a l . . 1955, Deeley et a l . , 1957) considered even 2 mm sections to be completely meristematic.  As noticed by Jensen (1955), the elongation and differentiation  of some central provascular cells starts as close to the t i p as 0.8  mm.  Partially elongated, non-vacuolated cells which have commonly been seen to divide on controls, together with isodiametric c e l l s , were scored as "meristematic".  Greatly elongated c e l l s , occasional root cap c e l l s , and  epidermal cells were omitted i n the counting.  For microspectrophotometric measurements i t i s necessary to standardize carefully the times and temperatures of fixing, hydrolysis,  46 and s t a i n i n g , since these factors influence the density of developed Feulgen dye and, consequently, the amount of measured DNA Swift, 1955)*  (Lessler, 1953,  Mounting i n o i l of r e f r a c t i v e index matching that of the  cytoplasm was recommended by P o l l i s t e r and Ornstein (1955) t o diminish the non-specific l i g h t l o s s i n the specimen.  In material prepared by  t h i s method the cytoplasm was nearly i n v i s i b l e and i t s absorption n e g l i g i b l e (less than 2 per cent of the s p e c i f i c absorption of the nucleus).  The two-wavelength method of microspectrophotometric measurement of DNA  (Patau, 1952, Patau and Swift, 1953, Swift and Rasch, 1956)  was used i n these experiments.  While the procedure i s somewhat more  tedious than the conventional one-wavelength method, i t has several important t e c h n i c a l advantages.  Accuracy of measurement i s not dependent  on the d i s t r i b u t i o n of the absorbing material, hence interphase n u c l e i of a l l shapes can be included because s i z e , number, and p o s i t i o n of n u c l e o l i does not matter.  Moreover, chromosomal groups of a l l m i t o t i c  stages can be measured accurately by the two-wavelength but not by the one-wavelength method.  The superior accuracy of the method was considered  to be p a r t i c u l a r l y important i n t h i s type of study, since i t was necessary to detect changes of a few per cent i n DNA content.  The discussion of photometric errors as f a r as instrumentation i s concerned i s omitted, because, f i r s t l y , these have been discussed many times (e.g. Ris and Mirsky, 1949, Patau, 1952, Grundraann and Marquardtj 1953b, P o l l i s t e r and Ornstein, 1955, Swift and Rasch, 1956) and, secondly, the r e s u l t s obtained here indicate that these errors  47 had a very minor effect.  It should be mentioned only that the alignment  of the apparatus i s c r i t i c a l .  The remark of Swift and Rasch (1956) that  "the alignment of microphotometers i s simple i n principle but i n practice takes patience and empirical manipulation" i s rather an understatement. Suitable tests for checking the alignment are given i n the above-mentioned excellent discussion.  The f i n a l check on the performance of the instrument  consisted of a series of measurements on nuclei of known relative content.  DM  Thus i n diploid dividing cells of a growing tissue the Feulgen  dye uptake, and DNA content, of telophases and prophases i s i n the ratio of 1:2 (review i n Swift, 1953, Vendrely and Vendrely, 1956)'^ Preliminary measurements of nuclei i n these two stages i n several Vicia root tips gave a very satisfactory f i t to this ratio.  Furthermore, the results  were not influenced by dye distribution, since variation i n the size of the measured area surrounding the nuclei had no effect on the results.  48  DISCUSSION OF RESULTS  Patau and PatH (1951) proposed that the relative number of cells i n prophase and telophase could be used as a sensitive indicator of changes i n the rate of nuclear division.  When the mitotic rate i s  steady the ratio i s constant because equal number of nuclei w i l l be entering prophase and telophase per time unit and the ratio w i l l be given by the relative duration of each stage.  I f the mitotic rate  increases the change w i l l f i r s t appear as an increase i n relative number of prophases, and, consequently, an increased prophase:telophase ratio; and when the mitotic rate decreases the ratio w i l l be depressed as well. By comparing the ratio i n treated nuclei with that of control, where the mitosis proceeds at a more or less steady rate, i t can be determined, i n a single count, whether at the time of collection the mitotic rate was increasing, steady, or decreasing i n the treated material.  Data  presented here are i n agreement with the above considerations; the ratio was increased at the beginning of periods of increasing mitotic rate, and was correspondingly depressed when the rate was decreasing  (Table I I ,  results of Figs. 12 and 21). Mitosis vra.3 more sensitive than root elongation to inhibition by 8-azaguanine. With the concentrations as low as 10 p.p.m. i n aerated solutions, mitosis was completely inhibited within 24 hours while root elongation was decreased i n the f i r s t 24 hour period by only about 10 per  49 cent and some elongation was s t i l l evident at 72 hours.  This result  was the f i r s t indication that mitosis and root elongation were not necessarily correlated. S t i l l more striking were the results obtained with the concentration of 1.0 p.p.m. 8-azaguanine: mitotic frequency was inhibited to about 50 per cent of control, though root elongation was slightly stimulated.  The finding that small amounts of anti-  metabolites stimulate growth i s quite common. For example Goldacre (1957) found that very low concentration (3 X 10"%)  of 2,6-diaminopurine  stimulated the growth of both isolated root tips and roots of intact seedlings of Subterranean clover while higher concentrations (1.5 X 10~%) were inhibitory.  The treatment i n these experiments lasted for 15 days  and root elongation only, but not mitosis, was investigated. Similarly, Hohn (1955) noticed growth stipulation of cress roots by very low concentrations of thiouracil.  Taking into consideration the demonstrated  greater sensitivity of mitosis than of root elongation to inhibition by 8-azaguanine, the results can be interpreted i n the following way: substrates and/or energy that cannot be u t i l i z e d for c e l l multiplication can s t i l l be utilized for increase i n c e l l size.  It should be stressed  that stimulation of growth occurs only when very low concentrations of antimetabolites are usedj when the concentrations are increased both mitosis and growth are inhibited. The responses of root elongation and mitosis to aeration observed here are interesting i n the light of previous experiments on the respiratory capacity of different root tissues. It had been found repeatedly that the respiration rate i s higher i n elongating cells  50 adjacent to the meristem of roots than i n meristematic cells of the root t i p i f oxygen consumption i s calculated per unit weight of protoplasm (or N content). Thus Kopp (194-8) reported a nearly threefold O2 consumption by cells of zone of elongation as compared to that i n meristematic cells i n primary roots of wheat seedlings. Brown and Robinson (1955) agreed with these findings and also showed that the activity of several enzymes (invertase, dipeptidase, phosphatase, glycine oxidase, and proteinase) i s low i n the meristematic region of bean root. Similar results i n respect of respiration were obtained by Betz (1955) i n experiments with primary roots of corn and peas; the author also demonstrated the presence of fermentation i n root meristems.  Jensen (1955) found very low O2 uptake  i n root meristem of Vicia faba and suggested that "the low oxygen consumption could indicate that the energy required for the synthetic activities i s derived either from glycolysis or the u t i l i z a t i o n of high energy compounds produced by other c e l l s " .  There are several papers  reporting opposite results but i n a l l these cases O2 consumption was calculated on the basis of fresh weight or volume unit of tissuej this would bias the results substantially, since differentiated plant cells contain relatively much more water (central vacuole) than meristematic cells.  Lettre (1951) suggested that while aerobic respiration i s not  necessary for c e l l division, glycolysis* i s sufficient to supply the energy, and i s probably indispensable. This author also demonstrated the decrease of glycolytic processes i f respiration i s increased, and suggested that the reaction of ribose with l a c t i c acid to produce  * The terms glycolysis and fermentation are used interchangeably i n accordance with the terminology of the original authors.  51 desoxyribose, pyruvic acid, and water, i s a part of the glycolytic process.  The results presented here may indicate that c e l l elongation  i s principally dependent on aerobic respiration, and that the energy of glycolysis can be more readily utilized i n c e l l division.  Stimulation  of mitosis by lack of oxygen has been reported by Wagner (1957) i n many animal tissues; mitosis i n at least some embryonic tissues i n animals also i s independent of aerobic respiration (rev. in Ris, 1955).  On the  other hand, aerobic respiration i s necessary for mitosis i n adult mammalian epidermis (Bullough, 1952), and i n the cleavage divisions i n echinoderm eggs (Drahl, 1950). While similar detailed information on the metabolic characteristics of dividing plant cells i s not available, the dat^i presented here suggest that the former mechanism, namely stimulation of mitosis by lowered tension of oxygen, i s operating i n roots of Vicia faba. The observed relationship between rate of aeration and mitosis might also be explained i n the following way:  the increased root elonga-  tion i s probably, at least partially, due to the increased uptake of water dependent on enhanced respiration (Rosene and Bartlett, 1950); Swann (1957) suggested that ascertain concentration rather than an absolute amount of some unknown substance i s necessary for triggering of mitosis.  In cells that take up greater amount of water the  establishment  of the c r i t i c a l concentration, and consequently, the mitosis i n individual cells would be delayed, and the mitotic rate i n the tissue would be decreased. The rate of aeration also exerted a considerable influence on the degree of inhibition obtained with a given dose of 8-azaguanine  52  (Figs. 16 - 19). high aeration.  This agent has.been relatively more inhibitory under This was true for both root elongation and mitosis.  With  high or medium aeration the time required for complete mitotic inhibition with 20 p.p.m. 8-azaguanine was less than 24- hours, though only about 70 per cent inhibition had been established with this concentration at 24 hours i n unaerated cultures. Though the physiological basis of this relationship i s not clear, the increased inhibition probably reflects an enhanced uptake of 8-azaguanine i n aerated solutions, i . e . an "active" uptake correlated with the magnitude of respiration. This sensitivity of inhibition emphasizes the importance of reproducibility of experimental conditions i n experiments where the effects of different compounds at different concentrations are compared. The effects of adenine alone on mitosis and root elongation were similar to those produced by low aeration, i . e . mitosis was  stimulated  whereas root elongation was depressed. Setterfield and Duncan (1955) also reported a slight stimulation of mitosis with adenine but not with adenosine, and suggested that this effect resulted from a specific requirement of free adenine for nuclear division.  In a l l these studies  aerated solutions were used. Woll (1953), using non-aerated solutions, reported inhibition of both root elongation and mitosis with comparable concentrations of adenine i n roots of Vicia faba:  i n addition, nearly  a l l dividing cells i n these adenine-inhibited roots were i n prophase. Under the conditions prevailing i n the present experiments, mitotic stimulation i s proportional to dose over a rather wide range of adenine concentrations, as indicated by results of experiments i n which 80 and  53  160 p.p.m. of adenine were administered  (Figs. 8 and 9). Interestingly,  where either adenine concentration or the level of aeration were changed, the rates of mitosis and root growth were both influenced, but i n opposite directions (Figs. 8, 9, 14, and 15).  This may reflect a  physiological balance between the rates of c e l l growth and mitosis which can be disturbed by several means, with the result that the specific stimulation of one of the processes ( i . e . c e l l growth or mitosis) results i n the suppression of the other, and vice versa. While both 8-azaguanine and adenine inhibited root elongation with increasing concentrations (Figs. 2 and 14), the growth curves for these two treatments are basically different.  The depressed growth rate  obtained with adenine was approximately constant throughout the 72 hour period of incubation.  On the other hand, inhibition by 8-azaguanine  increased with time, indicating that the inhibition i n this case was cumulative.  This would be expected on the assumption that this antimeta-  bolite progressively displaces natural purines from active sites i n the cell.  The curves show a modification of the metabolic processes i n the  former, and a progressive disruption of these processes i n the latter case. Inactivity of 6-mercaptopurine i n concentrations equivalent to highly inhibitory levels of 8-azaguanine possibly indicates that the mechanism of action of these txro purine analogs i s different.* Biesele (1955) reported prevention of mitotic inhibition of sarcoma 180 cells caused by 1.0 mM 6-mercaptopurine by as l i t t l e as 0.02 *  mH  It i s not possible to compare the concentrations of analogs used i n work with animals with those used here since the concentrations i n former studies are usually expressed as mg/day/ kg weight; besides diffusion factors are quite different.  54 coenzyme A, and suggested that 6-mercaptopurine might act as an antimetabolite of coenzyme A, interfering with the introduction of two-carbon units into the respiratory cycle.  Accordingly, the concentration of  coenzyme A i n roots can be considered as being high enough to prevent the inhibition.  The differences i n concentration of endogenous coenzyme  A might be also responsible for different effects of this analog on duckweed and virus tumor tissue reported by Nickell (1955). The absence of inhibition of root elongation by 8-azaxanthine agrees with the findings of Hirschberg et a l . (1952) that this compound had no carcinostatic potency. The results are also compatible with the demonstrated deamination of 8-azaguanine to 8-azaxanthine carried out by cells resistant to this analog (Hirschberg et al,., 1953, Mandel, 1955).  Since the meristematic cells were not resistant to 8-azaguanine,  i t may be suggested that root tips of Vicia faba are low i n 8-azaguanine deaminase (guanase) content. The demonstration that the inhibition of mitosis by 8-azaguanine can be reversed by subsequent treatment with adenine (Fig. 21) i s of some significance i n that i t eliminates the possibility that the reversal i s dependent on a non-metabolic interaction between the two compounds i n culture solution.  It should ne noted that complete mitotic  inhibition and i t s complete reversal each took about the same period of time, somewhat less than 24 hours. Since the average duration of one complete mitotic cycle i n root tips of Vicia faba, was reported to be 25.9 hours by Setterfield and Duncan (1955) and about 30 hours by Pelc and Howard (1955), complete mitotic inhibition and also complete reversal of the inhibition was accomplished i n a time period shorter  55 than the duration of one complete mitotic cycle.  In roots transferred  from 8-azaguanine into water, mitosis f i r s t appeared 4-8 hours after transfer but only i n the partially elongated provascular c e l l s .  This  suggests that endogenous substances capable of reversing the inhibition of mitosis by 8-azaguanine were carried downwards to the root tips through the vascular tissue. The results of spectrophotometric measurements of controls are i n excellent agreement with the recently published data of Deeley et a l . (1957) who measured the DNA content of root t i p nuclei of Vicia faba, using the technique of photoelectric scanning of crushed c e l l s .  There  i s also a good general agreement with the results of Jensen (1956) obtained by microchemical analysis.  On the other hand, there i s a  discrepancy with the results presented by Grundmann and Marquardt (1953 a, b). These authors used the one-wavelength method, and concluded that DNA synthesis started immediately after telophase and was followed, again immediately upon the completion of 4-C amount of DNA, by mitosis. The curves presented here for controls have to be interpreted i n the following way:  there i s a rather long lag period at the beginning of  interphase before DNA synthesis starts and another, shorter, lag period before beginning of the prophase, after completion of DNA synthesis. The microspectrophotometric measurements yielded the expected frequency distribution of DNA per nucleus at 24, and 72 hours i n the control roots.  This bimodal distribution, i n which the DNA values  representing the frequency maxima are i n the ratio 1:2 (2C, 4C) has been found typical of dividing tissues where the chromosome number per  56  c e l l i s constant, (for review see Vendrely and Vendrely, 1956, 1953)•  Swift,  The high reproducibility of these measurements i s indicated i n  the small error calculated for nuclei i n known states of division (Tables VI to VIII). Comparison of the distribution of DM  values for treated and  control roots indicates two main effects of 8-azaguanine on the mitotic cycle.  The virtual absence of intermediate values after 4-8 hours shows  that this agent blocks synthesis of at least one chromosomal constituent DNA.  Moreover, i n spite of the fact that "new"  4-C nuclei were not  produced after 4-8 hours, the proportion of the nuclei i n this class did not decline during the 4-8 - 72 hour interval (Figs. 24. and 26). This shows that the entrance into prophase from 4-C was also prevented. Perhaps the best evidence that no DNA  synthesis occurs i n  inhibited interphasic nuclei i s found i n the close agreement between the DNA values of inhibited 2C nuclei and the telophases and anaphases of the controls.  The means are almost identical and the coefficient of  variation (standard deviation i n per cent of the mean) i n each group i s less than 6 per cent.  Errors i n measurement can be expected to  provide an observed variation of at least 3 per cent (Swift and Rasch, 1956). Therefore, the actual internuclear variation i s no greater than 3 per cent i n the inhibited nuclei. Since the mean DNA values for the inhibited nuclei and control telophases are i n close agreement, i t i s evident that l i t t l e , i f any, DNA  synthesis occurred i n the former.  Pryzina (1956) also investigated  57 the i n f l u e n c e of 8-azaguanine i n V i c i a faba roots using the s i n g l e wavelength method of microspectrophotometric  measurement.  Though he  found complete m i t o t i c i n h i b i t i o n at 24 hours using 10 p.p.m. of the a n t i metabolite, the observed DNA values of the interphase n u c l e i formed a continuous d i s t r i b u t i o n from 2C t o 40.  These data do not exclude the  p o s s i b i l i t y that 8-azaguanine may block DNA values.  synthesis at  intermediate  However, the magnitude of e r r o r s i n measurement cannot be  c a l c u l a t e d from the presented data, and consequently t h i s r e s u l t cannot be  evaluated. The present r e s u l t s permit of the f o l l o w i n g comment on the  p h y s i o l o g i c a l b a s i s of 8-azaguanine-induced i n h i b i t i o n of the m i t o t i c cycle.  Two l i n e s of evidence i n d i c a t e that i n h i b i t i o n i s not accomplished  through i n c o r p o r a t i o n of t h i s compound i n t o DNA. i n DNA measurable by the microspectrophotometric i n i n h i b i t e d interphases.  F i r s t l y , no  increase  method could be shown  Secondly, i n view of considerable evidence  that DNA i s m e t a b o l i c a l l y s t a b l e , i t would not be expected that an incorporated purine antimetabolite could be r a p i d l y replaced by n a t u r a l purines.  Therefore, the f i n d i n g that i n h i b i t i o n e s t a b l i s h e d by 8-aza-  guanine could be completely reversed w i t h i n 24 hours by subsequent t r e a t ment with adenine provides strong a d d i t i o n a l evidence that the i n h i b i t i o n d i d not depend on i n c o r p o r a t i o n of t h i s compound i n t o DNA.  I t should be  pointed out that the biochemical studies on 8-azaguanine i n c o r p o r a t i o n i n t o the n u c l e i c acids of various animal t i s s u e s have not y i e l d e d consistent r e s u l t s . For example, the i n c o r p o r a t i o n of l a b e l l e d 8-azaguanine i n t o RNA of both rumor and normal t i s s u e s has been reported  58 (rev. i n Parks, 1955), the incorporation being sometimes higher i n the latter.  Using a similar technique Parks (1955) found incorporation of  the antimetabolite into RNA, but not into DNA, i n the protozoa Tetrahymena gelei.  On the other hand, Mandel (1955), after injecting  labelled 8-azaguanine into mice, found that this substance could be located i n the l i v e r nucleic acid hydrolysate, mainly i n RNA, but also i n DNA.  However, there appears to be no conclusive evidence that the  carcinostatic effect of this compound depends chiefly on the incorporation into either of the nucleic acids or free nucleotides.  The present  experiments and those of Parks (ibid.) provide two examples where the inhibition of c e l l reproduction i s not dependent on the incorporation of the antimetabolite into  DNA.  Though the incorporation of 8-azaguanine into RNA cannot be eliminated on the basis of the present results, other possible mechanisms, such as interference with adenine-containing  coenzymes and especially  interference with the synthesis or action of ATP, should be considered. It i s known that interconversion of guanine into adenine occurs (Mandel, 1955)5 since the same enzyme, guanase (8-azaguanine deaminase), deaminates both guanine and 8-azaguanine, i t i s possible that conversion of 8-azaguanine into 8-azaadenine also occurs.  Adenine was found to be  superior to guanine i n reversal of inhibition by 8-azaguanine (Pryzina, 1956).  This might mean that 8-azaguanine interferes with the metabolism  of adenine or adenine-containing  compounds. It i s now generally accepted  that ATP or a similar high-energy compound has to be accumulated before prophase can begin, i.e. until the amount of energy i s sufficient to  59 support complete mitosis (Bullough, 1952, Swann, 1957). The results obtained here are compatible with this hypothesis because even with the highest concentration of 8-azaguanine (80 p.p.m.) mitosis was completed and the mitotic process was interrupted i n interphase.  The presence of  a similar mechanism which would accumulate energy before DNA synthesis can start might be indicated by the lag period of nuclei containing 2G amount of DNA.  A definite correlation between the utilization of ATP  and the synthesis of DNA has been reported recently (Kanazir and Errera, 1956). I f the energy supply were suppressed, either by interference with the synthesis of high energy compounds (presumably ATP) or by producing "fraudulent" high energy compounds, the mitotic process would be interrupted only i n the two c r i t i c a l stages when the energy i s being accumulated. This was exactly the situation found i n nuclei inhibited with 8-azaguanine.  60  SUMMAHT An investigation was made of the effects of the purine analogs, 8-azaguanine, 8-azaxanthine, and 6-mercaptopurine, and the purines adenine and guanine, on c e l l growth, mitosis, and desoxyribonucleic acid (DNA) synthesis i n primary roots of Vivia faba seedlings grown i n aerated solutions.  Root elongation was used as a measure of c e l l elongation;  mitotic frequency was determined i n free c e l l suspensions prepared from 1 mm-long root tips; the relative content of DNA was determined microspectrophotometrically by the two-wavelength method. It was shown that the balance between root elongation and mitosis i n the root t i p could be influenced by the amount of aeration and by adenine.  Increased aeration stimulated root elongation and  depressed mitotic frequency; adenine stimulated mitosis, inhibiting, at the same time, root elongation. 8-azaguanine, i n concentrations of 10 p.p.m. and higher, stopped mitosis within 24- hours and greatly reduced root elongation and the fresh and dry weights of roots within 72 hours.  This inhibitory  effect on both root elongation and mitosis was positively correlated with aeration.  8-azaguanine i n a concentration of 1 p.p.m. significantly  reduced mitotic frequency but slightly stimulated root elongation. The inhibition of root elongation could be best, though incompletely, reversed by 4-0 p.p.m. adenine or guanine.  The mitotic inhibition could  be partially reversed by 4-0 p.p.m. guanine; adenine, at a concentration of 80 p.p.m., not only completely relieved mitotic inhibition, but  61  increased the m i t o t i c frequency t o a l e v e l higher than that o f the water c o n t r o l s .  Concentrations o f 6-mercaptopurine and 8-azaxanthine  comparable w i t h those o f 8-azaguanine had no i n h i b i t o r y e f f e c t s . Roots t r e a t e d w i t h 20 p.p.m. 8-azaguanine f o r 24 hours and then t r a n s f e r r e d i n t o 80 p.p.m. adenine showed a higher m i t o t i c frequency than the c o n t r o l w i t h i n 24 hours a f t e r t r a n s f e r .  Roots t r a n s f e r r e d from  8-azaguanine i n t o water showed some m i t o s i s 48 hours a f t e r t r a n s f e r ; i n t h i s case m i t o s i s was r e s t r i c t e d t o the p a r t i a l l y d i f f e r e n t i a t e d and elongated c e l l s o f the provascular bundles. DNA content o f interphase n u c l e i i n the c o n t r o l s showed t h i s distribution:  a sharp 2C peak (about 65 per cent n u c l e i ) , a much lower  4C peak (about 20 per cent n u c l e i ) , and intermediates (about f i f t e e n per c e n t ) .  There were no p o l y p l o i d n u c l e i i n the a p i c a l meristem o f  the r o o t .  The DNA content o f chromosomal groups i n d i f f e r e n t m i t o t i c  states demonstrated the accuracy o f the two-wavelength method which was used.  The DNA content o f n u c l e i i n roots t r e a t e d w i t h 20 p.p.m.  8-azaguanine was d i s t r i b u t e d i n a 2C peak (about 80 per cent m i c l e i ) and a 4C peak (about 20 per cent n u c l e i ) .  There were no intermediates  i n t r e a t e d roots and no n u c l e i contained a higher amount o f DNA than 4C.  The percentage o f 4C n u c l e i d i d not increase w i t h time. From the evidence t h a t the m i t o t i c i n h i b i t i o n induced by  8-azaguanine could be completely reversed w i t h i n 24 hours by subsequent treatment w i t h adenine, and from the f i n d i n g s concerning the d i s t r i b u t i o n of DNA i n i n h i b i t e d n u c l e i , i t may be concluded that 8-azaguanine  w a  ?  62  not incorporated into DNA.  The possibility that 8-azaguanine exerts  i t s inhibitory effects through interference with ATP metabolism i s discussed.  63  BIBLIOGRAPHY  Biesele, J.J., 1955• Antagonistic effects of 6-mercaptopurine and coenzyme A on mitochondria and mitosis i n tissue culture. J. Biophys. and Biochem. Cyt. 1:119-126. Bennett, L.L., H.E. Skipper, J.H. Mitchell, and K. Sugiura, 1950. Studies on the distribution of radioactive 8-azaguanine (guanozolo) i n mice with E 0771 tumors. Cancer Research  10:644-649.  Betz, A., 1955. Zur Atmung wachsender Wurzelspitzen.  Planta 46:381-402.  Brown, R., 1951. The effects of temperature on the durations of the different stages of c e l l division i n the root t i p . J . Exptl.  Bot. 8:96-110.  and D. Broadbent, 1950. The development of cells i n the growing zones of the root. J. Exptl. Bot. 1:249-263. and E. Robinson, 1955. Cellular differentiation and the development of enzyme proteins. In "Biological Specificity and Growth". Ed. by E.G. Buttler. Princeton Un. Press, 1955. Bullough, W.S., 1952. The energy relations of mitotic activity. B i o l . Rev. (Cambridge) 27:133-168. Clarke, D.A., F.A. Philips, S.S. Sternberg, C.C. Stock, G.B. Elion, and G.H. Hitchings, 1953. 6-Mercaptopurine: effects i n mouse sarcoma 180 and i n normal animals. Cancer Research 13s593-604. Creaser, E.H., 1955. a. Inhibition of induced enzyme formation by purine analogues. Nature 175*8991955 b. Effect of 8-azaguanine on enzyme formation. Nature 176:556-557. Deeley, E.M., H.G. Davies, and J. Chayen, 1957. The DNA content of cells i n the root of Vicia faba. Exptl. Cell Res. 12:582-591. Dietrich, L.S., and D.M. Shapiro, 1953. Combination chemotherapy of cancer: Potentiation of carcinostatic action of 8-azaguanine by a riboflavin analog. Cancer Research 13:699-702  64 Flrket, H., 1957. Effet du chauffage de cultures de tissue a des temperatures supranormales sur l a synthese de I'acide desoxyribonucleique. C.R. Soc. Biol. (Paris) 151:188-190. Fries, N., 1954* Chemical factors controlling the growth of decotylised pea seedlings. Symb. Bot. Upsal. 13:1-83. Gellhorn, A., and E. Hirschberg, Ed., 1955. Investigation of diverse Systems for. Cancer Chemotherapy Screening. I. Summary of results and general correlations. Cancer Research, Suppl. 3:1-13. Goldacre, P.L., and H. Unt, 1957. Effects of 2:6-diaminopurine on the growth of roots of Subterranean clover. Nature 179:877-878. Goulden, C.H. Methods of Statistical Analysis. Second edition. Wiley and Sons, Inc., New York, 1952.  John  Grundmann, E., and H. Marquardt, 1953 a. Die DNS.-Synthese im Wurzelmeristem von Vicia faba,. Naturwiss. 40:557-558. 1953'1>. Untersuchungen an Interphasekernen des Wurzelmeristem von Vicia faba,. Chromosoma 6:115-134. Hirschberg, E., J . Kream and A. Gellhorn, 1952. Enzymatic deamination of 8-azaguanine i n normal and neoplastic tissues. Cancer Research 12:524-528. Hirschberg, E., M.R. Murray, E.R. Peterson, J . Kream, R. Schafranek, and J.L. Pool, 1953. Enzymatic deamination of 8-azaguanine i n normal human brain and i n glioblastoma multiforme. Cancer Research 13:153-157. Hoehn, K., 1955. Zur frage der Bedeutung der Nucleinsaeuren fuer die Wachstumsvorgaenge der Pflanze. Beitraege zur Biol, der Pflanzen 31:261-292. Holmes, B.E., L.K. Mee, S. Hernsey, and L.H. Gray, 1955* The nucleic acid content of cells i n the meristematic, elongating, and elongated segments of roots of Vicia faba,. Exptl. Cell Res. 8:101-113. Jensen, W.A., 1955* A morphological and biochemical analysis of the early phases of cellular growth i n the root t i p of Vicia, faba. Exptl. Cell Res. 8:506-522. 1956. On the distribution of nucleic acids i n the root t i p of Vicia faba,. Exptl. Cell Res. 10:222-224.  ?  6  Kanazir, D., and M. Errera, 1956. Alterations of intercellular deoxyribonucleic acid and their biological consequence. In Cold Spring Harbor Symposia on Quant. Biol. XXI. Genetic Mechanisms: Structure and Function, p. 19-29. Kidder, G.W., V.C. Dewey, R.E. Parks, Jr., and G.L. Woodside, 1949. Purine metabolism i n Tetrahymena and i t s relation to malignant cells i n mice. Science 109:511-514. , 1951. Further evidence on the mode of action of 8-azaguanine (guanozolo) i n tumor inhibition. Cancer Research 11:204-211. Kopp, Maja, 1948. Ueber das Sauerstoffbeduerfnis wachsender Pflanzenzellen. Ber. der Schweiz. Bot. Ges. 58:283-318. Krahl, M.E., 1950. Metabolic activities and cleavage of eggs of the sea-urchin, Arbacia punctulatet. Biol. Bull., 98:175-217. Lessler, M.A., 1953. The nature and specificity of the Feulgen nucleal reaction. Int. Rev. Cyt. 2:231-247. Lettre, H., 1951. Zellstoffwechsel und Zellteilung. 38:490-496.  Naturwiss.  Mandel, H.G., 1955. Some aspects of metabolism of 8-azaguanine. In "Antimetabolites and Cancer", p. 199-218. Ed. by C P . Rhoads. A.A.A.3., 1955. Matthews, R.E.F., 1951. Effect of some substituted purines on the development of plant virus infections. Nature 167, 892. 1952. Effect of purine on the multiplication of plant viruses. Nature 169:500. 1953. Chemotherapy of plant viruses. 8:277-288.  J . Gen. Microbiol.  Nickell, L.G., P. Greenfield, and P.R. Burkholder, 1950. Atypical growth i n plants. III. Growth responses of virus tumors of Rumex to certain nucleic acid components and related compounds. Bot. Gazette 112:42-52. .......... 1955* Effects of antigrowth substances i n normal and atypical plant growth. In "Antimetabolites and Cancer", p. 129-151. Ed. by CP. Rhoads. A.A.A.S., Washington, D.C, 1955. Parks, R.E., Jr., 1955. Antimetabolite studies i n Tetrahymena and tumors. In "Antimetabolites and Cancer". Ed. by C P . Rhoads. A.A.A.S., Washington, D.C, 1955.  66 Patau, K., 1952. Absorption microphotometry of irregular-shaped objects. Chromosoma 5 s341-362. and R.P. P a t i l , 1951. Mitotic effects of sodium nucleate i n root tips of Rhep discolor. Chromosoma 4-:4-39-4-58. .......... and H. Swift, 1953. The DNA-content (Feulgen) of nuclei during mitosis i n the root t i p of onion. Chromosoma 6:14-9-169. Pelc, S.R., and A. Howard, 1955. Effect of various doses of x-rays on the number of cells synthesizing deoxyribonucleic acid. Radiation Research 3:135-14-2. Pollister, A.W., and L. Ornstein, 1955* Cytophotometric analysis i n the visible spectrum. In "Analytical Cytology", p. 1-71. Ed. by R.C. Mellors, Blakiston Division, McGraw-Hill, 1955. Pryzina, E.A., 1956. Cytological effects of 8-azaguanine i n terms of mitotic inhibition, i t s prevention, and i t s relation to DNA synthesis i n the root meristem of Vivia faba. Ph.D. Thesis, University of Wisconsin. Ris, H., 1955. Cell Division. In "Analysis of Development", p. 91-125. Ed. by B.H. I&llier, P.A. Ifeiss, and V. Hamburger, W.B. Saunders Co., 1955. • and A.E. Mirsky, 194-9. Quantitative cytochemical determination of desoxyribonucleic acid with the Feulgen nucleal reaction. J. Gen. Phys. 33:125-146. Roblin, R.O., Jr., J.O. Lampen, J.P. English, Q.P. Cole, and J.R. Vaugham, Jr., 1945. Studies i n chemotherapy. VIII. Methione and purine antagonists and their relation to sulfonamides. J. Am. Chem. Soc. 67:290-294. de Ropp, R.S., 1950. Some new Plant-growth inhibitors.  Science 112:500-501.  1951. The action of some chemical growth inhibitors on healthy and tumor tissue of plants. Cancer Research 11:663-668. Rosene, Hilda F., and L.E. Bartlett, 1950. Effect of anoxia on water influx of individual radish root hair c e l l s . J. C e l l , and Comp. Physiol. 36:83-96. Scherer, W.F., 1955. Glassware and i t s cleaning. In "An Introduction to Cell and Tissue Culture" by the Staff of the Tissue Culture Course, Cooperstown, N.Y., 1949-1953. Burgess Publishing Co., Minneapolis, Minn., 1955.  67  Setterfield, G., and R.E. Duncan, 1955* Gytological studies on the antimetabolite action of 2,6-diaminopurine i n Vicia faba roots, J. Biophys. and Biochem. Cyt. 1:399-419. .......... R. Schreiber, and J. Woodard, 1954* Mitotic frequency determinations and microphotometric Feulgen dye measurements i n root t i p s . Stain Technol. 29:113-120. 7  Skipper, H.E., 1953. A review: On the mechanism of action of certain temporary anticancer agents. Cancer Research 13*545-551. Swann, M.M., 1957. The control of c e l l division: a review. mechanisms. Cancer Research 17:727-757.  I. General  Swift, H., 1950 a. The desoxyribose nucleic acid content of animal . nuclei. Physiol. Zool. 23:169-198. 1950 b. The constancy of desoxyribose nucleic acid i n plant nuclei. Proc. Nat. Acad. Sci., U.S. 36:643-654. 1953. Quantitative aspects of nuclear nucleoproteins. Int. Rev. Cytology 2:1-76. 1955. Cytochemical techniques for nucleic acids. In "The Nucleic Acids", II, P. 51-92. Ed. by E. Chargatt and Y.N. Davidson, Acad. Press, New York, 1955. and Ellen Rasch, 1956. Microphotometry with visible light. In "Physical Techniques i n Biological Research", Vol. I l l : 353-400. Ed. by G. Oster and A.W. Pollister. Acad. Press, New York, 1956. Vendrely, R., and C. Vendrely, 1956. The results of cytophotometry i n the study of the deoxyribonucleic acid (DNA) content of the nucleus. Int. Rev. Cytology 5:171-197. Wagner, R., 1957. Biologische Regelung und Gewebsbildung.  44: 97-107.  Naturwiss.  Woll, E., 1953. Einwirkung von Nucleinsauren und ihren Baustoffen auf die Wurzelspitzenmitose. Chromosoma 5:391-427.  

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