ACTIVITY IN RELATION TO THE MITOTIC CYCLE

Transcription

1 PERODC NDUCTON OF DEOXYRBONUCLEASE ACTVTY N RELATON TO THE MTOTC CYCLE HERBERT STERN, Ph.D. From the Research Branch, Canada Department of Agricuture, Ottawa. Dr. Stern's present address is Department of Botany, University of inois, Urbana ABSTRACT The behavior of deveoping anthers has been studied with respect to dcoxyribonucease. This enzyme, in contrast to reated hydroytic ones, is sharpy periodic in its activity. Whenever a poo of deoxyribosidcs appears in situ, it is preceded by the appearance of deoxyribonucease. The duration of poo or enzyme does not exceed and is generay ess than 12 hours, even though the ife span of the ces concerned is of the ordcr of 25 to 3 days. The significance of periodic enzyme induction is discussed in reation to ce morphogenesis. NTRODUCTON The requirement of deoxyribosides for deoxyribonuceic acid (DNA) synthesis is patent. n this connection, two probems exist--the mode of dcoxyriboside synthesis and the reguation of such synthesis with respect to time. Much information is aready avaiabe on the first of these questions, and athough the picture is incompete, it wi not be considered here. t is to the probem of reguation that this communication is addressed. The periodic production uf deoxyribosidic compounds associated with chromosome reproduction in iy anthers has been described (1). A simiar periodicity has been observed in synchronousy dividing bacteria (2). The question now posed in whether such puse-ike production of DNA precursors is matched by a corresponding pattern in enzyme activity. To answer the question it has been necessary to seect a pertinent enzyme system, bearing in mind that in this connection its specific cataytic property is of secondary importance to its reguatory behavior. Deoxyribonucease (DNase) was the enzyme chosen as a resut of our observation that microspora DNA synthesis occurred during a pcriod of decine in tota DNA content of the anther and that this period was punctuated with intervas of DNase activity (1). The substance of our resuts is that puses of DNase activity persisting from 4 to 8 hours occur in the course of anther deveopment and that such puses are initiated prior to deoxyriboside formation. We do not know the ocus or nature of the DNase substrate, nor can we account for the tota decine in anther DNA in terms of the puses identified. METODS On the whoe, methods and materias used were simiar to those reported in a preceding pubication (3). Phosphatase was measured by incubating tissue homogenates with appropriate deoxyribonuceotides and separating the reaction products on a butanowater paper chromatogram. Phosphodiesterase was determined by means of the artificia substrates, sodium di-p-nitropheny phosphate synthesized according to Corby et a. (4), or the cacium sat obtained commerciay. The method foowed was that of Sinsheimer and Koerner (5), modified so as to aow for the fact that anther diesterase has a ow ph optimum and does not respond to Mg ++ (1). Thus a.1 u stock soution of dinitropheny phosphate was made up in.1 M acetate buffer (ph 4.7). Before use, a portion of the soution was diuted threefod with water and.75 m. of it pipetted into a tube aong with.25 m. of.5 ~i acetate buffer of desired ph and.1 m. of tissue extract. The mixture was incubated at 25 C. and at, 1, and 2 minute 271

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3 intervas.25 m. was pipetted into a cuvette containing 1 m. of.2 N NaOH. The absorption was immediatey read at 44 m#. DNase was measured either by foowing the formation of acid-soube products from poymerized caf-thymus DNA (6) or by assaying microbioogicay the production of deoxyribosides from endogenous substrate (7). RESULTS Deveoping anthers are the seat of DNase, phosphodiesterase, and monoesterase activities, the sum of which woud account for the production of deoxyribosides from some form of poydeoxynuceotide (1). The set of measurements which therefore engages our attention is the variation in activities during anther deveopment. Phosphomonoesterase was not studied systematicay, but scattered observations indicate its pattern of behavior to be simiar to that of diesterase. There is, however, a striking difference in the respective deveopmenta patterns of diesterase and DNase (Fig. 1). The one increases continuousy; the other fuctuates widey. Mature anthers are a rich source of diesterase. Dr. Y. Hotta has made some initia purifications of the enzyme and found it to be separabe from the monoesterase present. The partiay purified enzyme, ike the crude extract, is competey inhibited in phosphate buffer. The curve for DNase is a pot of the percentage of anthers in a given interva of bud ength having an activity above an arbitrary estabished minimum. This arbitrariness does itte to bias the picture as the active group breaks down 4 to 8 #g. DNA-P/hour, whereas the inactive one varies within the range of 5 to 15 #g. (Tabe ). ntermediate vaues are found but they are infrequent. t wi be observed that among the 8 buds thus tested here, those beow 43 mm. and above 15 ram. had no appreciabe DNase activity in their anthers. The interva spanning these two engths is marked by arge fuctuations. At some points, no DNase activity was observed; at others, a variabe percentage of the anthers were active. Athough the fuctuations correspond roughy to intervas of deoxyriboside production, such correspondence is ceary insufficient to provide for a definitive reationship. The spread of the fuctuations can be attributed in part to the arge changes in greenhouse temperature over the period studied (1), but the ony concusion which can be reasonaby drawn is that at certain defined intervas of growth DNase may or may not be present in an DNase TABLE and Diesterase Activities in Pre-Mitotic Anthers Bud ength DNase Diestcrase DNase activities are expressed as #g. DNA-P/ anther X 1 hour. Diesterase activities are expressed as /x O.D. 44mJ1 em. minutes for a soution containing the extract of one anther per m. Assays were run at 25 C. The imited interva of bud ength here chosen is meant to iustrate the difference in behavior of the two enzymes. anther. Obviousy, the question to be answered is why a-or-none fuctuations occur in DNase but not in reated hydroytic enzymes. Our first effort was to test for the in situ presence of deoxyribosides simutaneousy with and 18 hours after DNase assay. We supposed that if DNase were tied to deoxyriboside production in the iving anther, deoxyribosides shoud be found either whie the enzyme is active or afterwards. The idea was tested by removing one anther from a bud and assaying it for DNase, removing a second for measurement of the interna deoxyriboside poo, and cuturing the remainder, which had been excised with fiaments intact, in a drop of buffer overnight at 15 or 25 C. Dupicate sets of the cutured anthers were anayzed in the same way as the initia ones. The resuts provided itte enightenment. Anthers with initiay high DNase activity had much the same activity after 18 or even 4 hours in cuture, making it appear unikey that the variabe percentages shown in Fig. were due to brief periods of enzyme activity. Moreover, the presence or absence ofdeoxyribosides appeared unreated to the presence or absence of DNase either at the time of its measurement or 18 hours ater. There were two shortcomings, however, to the above experiments. First, we coud not be sure that the cutured anther woud behave ike the in situ one. Second, we had no reason to presuppose that 18 hours was a meaningfu interva. t coud aso be argued that anthers of a singe bud were not necessariy at simiar stages of de- H. STERN Deoxyribonucease Activity and the Mitotic Cyce 273

4 TABLE Endogenous Production of Deoxyribosides by Anther Homogenates Micrograms thymidine eequivaents per anther Bud ength 52. mm mm mm mm. Time, hrs. }4 }4 1 }4 1 }4 1 Treatment : Acetate aone Acetate, then sucrose Sucrose aone Sucrose, then acetate Conditions for assay described in text. -- indicates a vaue of ess than.2 #g. per anther, veopment, but our experience in measuring deoxyriboside poos of individua anthers ed us to beieve that this coud not be a significant factor in the experiments. To avoid the above shortcomings a fap was made in each bud by two ongitudina sits and the anthers removed for anaysis at 2 to 3 hour intervas. During interim periods the buds were encosed in poyethyene bags and the pants kept in the greenhouse. To faciitate enzymatic measurements, it was decided to use the endogenous DNA of the anther as substrate. A number of tests had to be carried out, however, in order to estabish the reiabiity of the method. Essentiay, the method was to measure the amount of deoxyriboside reeased by homogenized anthers over a given period of time. The effects of tonicity and ph of the medium were first tested. Anthers of a singe bud were sit ongitudinay and corresponding haves were pooed in two groups. One group was homogenized in,1 M acetate buffer (ph 5.25), the other in.35 M sucrose. Enough soid sucrose was added to a portion of the acetate homogenate to make a concentration of.35 u, and enough 1 M acetate buffer added to a portion of the sucrose homosenate to make it.1 M with respect to acetate. The four suspensions were incubated at 25 C. for different intervas of time and the reaction stopped by addition of trichoroacetic acid to a fina concentration of.36 M or of absoute methano to a fina concentration of 7 per cent. The supernatant fuids obtained upon centrifugation were assayed microbioogicay for deoxyriboside content. Resuts of the experiments are summarized in Tabe. t is apparent that the amounts of deoxyribosides reeased by anthers of the same bud are simiar. t is easy to distinguish between those which reease appreciabe amounts of deoxyriboside and those which do not by any of the four procedures. n view of this, it seems unikey that the appearance of DNase is due to osmotic shock of subceuar partices. This is in ine with the observation that fuctuations in DNase activity (as measured by diphenyamine anaysis) occur even though the anther homogenates were diuted fifteenfod with water for measurement. The.35 M sucrose was chosen as the simpest test medium. A comparison was next made between the reease of deoxyribosides in a sucrose suspension and the capacity of suitaby modified portions of these suspensions to degrade poymerized cafthymus DNA. The resuts are reported in Tabe. The coincidence of the two methods is cear. Athough far more deoxyribosidic materia is reeased in the presence of added substrate (to compare the two coumns, thymidine equivaents shoud be divided by 7.8), the differences between active and inactive buds are adequatey defined by either procedure. t might be added that treating the endogenousy derived soube products with diesterase does not appreciaby ater their microbioogica activity, a resut which woud be expected in view of the continuous presence of diesterase in anther extracts. n our first set of experiments, two anthers were removed initiay, then grouped as described. One group was assayed for DNase, the other was homogenized in 7 per cent methano and assayed 274 TnE JOURNAL OF BOPHYSCAL AND BOCHEMCAL CYTOLOGY VOLUME 9, 1961

5 TABLE Endogenous Deoxyviboside Production and DNase Activity of Anther Homogenates Deoxyri- Deoxyri- Bud ength bosides produced DNase Bud ength bosides produced DNase Deoxyriboside vaues are for micrograms thymidine equivaents reeased per anther in onehaf hour at 25 C. DNase vaues are for micrograms DNA-P reeased from poymerized caf-thymus DNA under simiar conditions. -- indicates ess than 3.1 #g. thymidine equivaents or 5/zg. DNA-P. for deoxyriboside content. Singe anthers were removed at subsequent intervas and ony their deoxyriboside content measured. 12 of the 16 buds thus tested showed no DNase initiay, and no significant content of deoxyribosides over a period of 9 hours; a few showed an increase 24 hours ater. The course of the deoxyriboside poo in the few buds with initiay high DNase activity is shown in Fig. 2. Deoxyribosides appear in a cases but they may ag in appearance and do not persist for periods onger than 12 hours. The resuts made it apparent why measurements of deoxyriboside content simutaneousy with or 18 hours after that of DNase do not yied meaningfu reationships. n our second set of experiments we sought to characterize the cyces of both DNase activity and deoxyriboside production. To do this, anthers were removed successivey and spit ongitudinay, one-haf anther being used for enzyme assay, the other for deoxyriboside content. Three typica exampes have been chosen for iustration in Fig. 3, among those buds in which deoxyribosides were found ony after the beginning of the experiment. Apart from those which showed neither DNase activity nor deoxyriboside formation throughout the course of the experiment, there were, of course a variety of situations which may be found by picking any point aong the time axes in Fig. 3. The rue which has thus far proved to be invariant is that the appearance of deoxyribosides is preceded by DNase activity. A few situations have 1 59MM. 57MM. r onoso 2o -,.)N s. ssmm FGURE f "--7,-~ HOURS Reationship of DNase to cycic production of deoxyribosides by in situ anthers. Buds were attached to pant during experiment (see text). Vertica ines represent initia DNase activity expresscd as micrograms thymidine equivaents produced per homogenized anther in one-haf hour at 25 C. Dots represent deoxyriboside content of in situ anthcrs as measured in 7 per cent methanoic extracts. "59 MM.," etc., is bud ength in ram. H. STERN Deoxyribonucease Activity and the Mitotic Cyce 275

6 -- Z b,,- "a- W \ 53.MM. 14 \ DNase 1 - \ i/ MM. uj 12 Z )- "1" -- ~.8 2- " J ~X. 8.q 56.MM. 1 ;, Fmu~ 3 ~ 2 -/]~ ~\%_ o 2 4 HOURS Periodicity in DNase activity and deoxyriboside appearance. "n situ" refers to deoxyribosides. Other representations as in Fig. 2. been encountered at bud engths of 8 to 9 mm. in which DNase activity rose during the course of the experiment but no deoxyribosidic poo coud be detected. At these bud engths too, however, if a poo did appear, it was preceded by DNase. The point of greatest interest to these studies was the cycica nature of DNase activity. ts duration, under conditions of growth as cose to normaity as possibe, is of the order of 6 hours. This interva is brief compared with the 25 days covering the growth of microspores from the tetrad stage to postmitotic DNA synthesis, and even briefer compared with the ife span of the wa ces. Athough we have no knowedge of the moecuar basis for the fuctuations in enzyme activity, it woud be difficut to cassify them other than as inductions of enzymatic activity. We have mixed suspensions of active and inactive anthers, but found the effects to be additive. Thus no evidence has been found for the existence of an inhibitory agent. Whether such induction represents a biosynthesis at the moecuar eve or transformation at a more compex one is incidenta to the point that one of the ways in which ce deveopment is governed is by a comparativey brief cycica activation of enzymes. We have strong reason to suppose that DNase is not aone in this category. Our current studies on phosphoryation of the deoxyriboside poo (Hotta and Stern) indicate a simiar pattern in phosphoryation. Evidence suggestive of periodic enzyme activity has aso been found by Lark (2) in his studies of syncht onousy growing bacteria. 276 THE JOURNAL OF BOPtYSCAL AND BOCHEMCAL CYTOLOGY VOLUME 9, 1961

7 The ocus and nature of the substrate which yieds deoxyriboside remains an open question. Thus far we have been unabe to identify the poydeoxyribonuceotide materia which woud appear to be the source of deoxyribosides. Superficiay the DNA of the degenerating tapetum woud appear to be the substrate but recent radioautographic studies of S. Takats (persona communication) indicate that abeed tapeta DNA does not transfer its abe to the microspore chromosomes. ndeed, over the 8 to 12 hour period studied it has been found virtuay impossibe to draw up any equation between the appearance of soube deoxyriboside and disappearance of DNA in the anther. The tota deoxyriboside appearing in situ is not much greater in amount than the variations in DNA content between individua anthers. CONCLUSONS The principa concusion to be drawn from these studies is that periodic induction of enzyme activity over a brief interva in the ife span of a ce is a mechanism for its morphogenetic deveopment. The particuar enzyme or process here studied is probaby beside the point. The enzymatic mechanisms supporting the process may vary and the BBLOGRAPHY 1. STERN, H., Tr. New YorkAcad. Sc., 196, inprcss. 2. LARK, K. G., Biochim. et biophysica, acta, 196, in press. 3. HOTTA, Y., and STERN, H., J. Biophysic. and Biochem. Cyto., 1961, 9, CORBY, N. S., KeNNEK, H. W., and TODD, A. R.. J. Chem. Soc., 1952, Part, process itsef may be continuous or discrete depending upon the ce type concerned. But insofar as sharpy defined events occur during ce deveopment, mitosis furnishing a striking exampe, then such events may we foow equay defined transformations in metaboic machinery. The microspore, because of its reativey sow deveopment, probaby makes possibe a refined study of its tempora properties which woud be difficut in other ces. Even so, we have aready pointed out (3) that broad cyces of metaboic change occur in the anther which bear ony a secondary reationship to the briefy enduring structura changes which the ces undergo. The contrast is not that of reative importance but that of immediacy of effect. Much has been written about comparative increases and decreases in enzymatic activities of proiferating ces in norma and maignant tissues (1), but from our studies of anther deveopment, it may we be that such changes are of the secondary type. What agent reguates the sharp cycica enzymatic changes woud appear to be a most significant question. Contribution No. 118, Pant Research nstitute, Ottawa. Received for pubication, September 4, , SNSHEMER, R. L., and KOERNER, J. F., J. Bio. Chem., 1952, 198, ALLFREY, V. G., and MmsKv, A. E., J. Gen. P~ysio., 1952, 36, HOFF-JCRoENSEN, E., Biochem. J., 1951, 5, 4. H. STERN Deoxyribonucease Activity and the Mitotic Cyce 277