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8. Freinstein, G. (1970) FEBS Lett. 7, 353-355 9. Rick, W. (1965) in Methods of Eitzymatic Analysis (Bergmeyer, H. U., ed.), pp. 811-814, Academic Press Inc., New York and London 10. Dirzynlriewicz, 2. & Arnason, B. G. W. (1974) E-xp. CeM Res. 85, 95-164 11. Quastel, M. R. & KapIan, J. G. (1968) ~Vature219, 198-268 12. Quastel, M. R. Rr. Kaplan, J. G. (1970) Exp. Cell Res. 62,487-420 13. Kast, R. E. (1973) S c a d . J. Immlinol. 4 , 449 14. Vosika, G . & Kennedy, J. B. (1974) Abstracts 9th
134.1
Leucocyte Culture Co~tference,Wil!iamsburg,Virginiadm, p. 241 15. Schnebli, H. P. & Burger, M. M. (1972) Proc. ~Varl. Acad. Sci. U.S.A. 69, 3825-3827 16. Weissman, G., Zurier, R. B. Rr. #offstein, S. (1972) Am. J. Pathd. 68,539-559 17. Havemann, K. R& Schmidt, W. (1972) Proceedings of the 8th Eeucocyte Cukure CmIference (LindahlKiessling, K. R& Ossba, D., eds), pp. 181-189, Academic Press Inc., New York and London 18. Kast, R. E. (1974) Oncology 29, 249-264 19. Saito, M., Yoshizawa, T., Aoyagi, T. & Nagai, Y. (1975) Biochem. Biophys. Res. Commun. 52, 569-575
Hormonal Stimulation of Ribosomal RNA Synthesis in PAULA. HORGEN, ROBINSMITH,JULIEC . SILVER,AND GARYCRAIG Department sf Botany, University o f Toronto, Erindale College, Mississauga, Ontario L5E IC6 Received March 14, 1975' Horgen, P. A., Smith, R., Silver, J. C. & Craig, G. (1975) Hormonal Stimulation of Ribosomal RNA Synthesis in Aclzlya anzbisexualis. Can. J . Biochem. 53, 1341-1345 The experiments reported show that one of the early effects of the steroid sex hormone antheridiol is on the synthesis of rRNA and ribosomes. This is demonstrated in hormone treated cultures of Achlya ambisexualis (strain E87) by an enhancement of the incorporation of [3H]uridine into 26S and 18s rRNA and by an increase in measurable amounts of ribosomes per milligram dry weight of mycelium. Furthermore, since the hormone does not significantly alter the pool size or the specific activity of uridine triphosphate, this effect appears to represent an increased rate of RNA synthesis. Horgen, P. A., Smith, R., Silver, J. C. & Craig, 6 . (1975) Hormonal Stimulation of Ribosomal RNA Synthesis in Achlya ambisexualis. Can. J . Biockenz. 53, 1341-1345 Les expdriences dCcrites montrent qu'en tout premier lieu I'antkCridiol, une hormone sexuelle stCroi'de, agit sur la synthbe du rRNA et des ribosomes. Ainsi, dans des culture dqAchlya ambisexualis (souche E87) traitkes avec cette hormone, l'incorporation de [3H]uridine dans les rRNA 26s et 18S augmente et la quantit6 de ribosomes par milligramme de poids sec de mycklium s'accroit de f a ~ o nmesurable. De plus, comme l'hormone nhlt&re pas de fason significative la grandeur du pool ou lYactivitCspCcifique du triphosphate d'uridine, cet effet impliquerait donc une synth&seaccrue du RNA. [Traduit par le journal]
Introduction The Bsrnycete genus, Aehkya, represents perhaps the most primitive group of organisms which is induced to differentiate by steroid hormones (1). In the presence of antheridiol, a sterol secreted by female strains of Aehkya, male 'Revision received July 9, 1975.
strains are stimulated to produce sex organ initials (antheridiol branches) which eventually differentiate into male sex organs (1). The hormone is active, when assayed with Achlyn ambisexualis (strain E87) at extremely low concentram tiOns (6 X !dml) and is quite specific (1, 2). Because it is extremely fast growing (3), easy to manipulate, and can be induced
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1342
CAN. 9. BKOCHPEM. VOL, 53, 1975
to diRerentiate in a highly uniform manner, Aehka represents a useful 'model system9 to study the effect of steroid horn~oneson macronnolecular synthesis in eukarystes. It has been suggested that a prerequisite for antheridiol stimulation of sex organ initials is a localized softening of the cell wall by the enzynae cellulase (EC 3.2. B .4) (4). Reports of increases in the synthesis and accumulation of this enzyme ( 5 ) and ultrastructural observations of weakened areas of the cell wall (6) tend to strengthen this hypothesis, although no other paranseters have been thoroughly examined. Silver and Horgen (79 reported that antheridiol stimulated the accumulation of a rnRNA-like fraction of RNA with a corresponding enhancement of protein synthesis during the early stages of male sex organ development. The timing of these macromolecular changes corresponds with the morphological appearance of branch initials (7). Furthermore, it was shown that the poly(A)-rich RNA that accumulated was heterogeiaeous ira size indicating that many different gene transcripts were being synthesized. Whether any one gene product is nlsre important than another remains to be seen. Ht is, however, evident that both continued RNA and protein synthesis are required for morphogenesis of sex organ initials in Acbmlya (7, 8). In this study, we have examined the effect of antheridiol on the synthesis of ribosomal RNA and ribosome levels during the early stages of sex-organ differentiation in A . ambisexsrerkis.. We have also exanlined the effects of the hormone on the uridine triphosphate pool size as well as the effect of the hormone on the turnover of ribsomes during differentiation.
Achkya aanbisexunlfs (strain E87) was maintained on Emerson's YPSS (Difco) agar slants. The mycelium was subcultured on PYG ( 1.25 g yeast extract, 1.25 g peptone, 3.0 g glucose per litre deionized H 2 0 ) agar plates for spore preparation according to the method of Griffin and Breuker (9). Spore cysts were then inoculated into PYG broth, grown for 24 h at 24 e 1 "C on a rotary shaker (100 rpm) until finely suspended mycelia had formed. This was used immediately as an inoculum o r stored at 4 2 1 "C for later use. Twenty-five millilitres of the finely suspended mycelia were insculated into 275 ml of mating medium ('9). The cultures were grown at 24 2 I "C for 32-48 Bn. At this time the cultures either received antheridiol (5 X 10-l' giml) o r were left to grow without hormone. Cultures were pulse labelled either with [3H]hlridine
or [l*C]uridine for the times indicated in the presence or absence of hormone. For determination of ribosome levels, cultures of A . ansbisexucrlis were grown and divided into two equal aliquots. One was treated with hormone, the other was left to grow vegetatively. At the designated times, 100 ml samples were taken and collected on a miilipsre filter apparatus. The sample was divided into two equal portions based on wet weight. One portion was used for dry weight measurements; the other used for ribosome isolation. Ribosomes were prepared according to Lin and Key (18). Membrane components were extracted from the ribosome preparations by the procedure of Ingle ( I I ) . WNA was then hydrolyzed in 0.3 IV KQH for 18 h at 37 'C. Nucleotide concentrations were determined as described by Ingle (1 I ) . Data was expressed quantitatively as milligram of ribonucleotides per milligram dry weight of tissue. TO determine whether antheridiol causes the rapid turnover of ribosomes, vegetative cultures were grown in the presence of ['Tjaaridine (0.1 pCi/ml) for 14 h to prelabel ribosomal RNA. Mycelia were then incubated for 30 min in 2 1 of 2.5 mM unlabelled uridine. Cultures were then transferred to fresh mating medium containing [%]uridine (0.5 pCiiml) in the presence or absence sf antheridiol. One-hundred-millilitre samples were collected on a millipore filter and weighed. Ribosomes were prepared according to Lin and Key (10) and the 3H and radioactivity determined. To measure the effect of hormone on incorporation of [3H]uridine into 26s and 18s rRNA cells were grown in the presence of ['W]uridine in the presence or absence of antheridiol. The cultures were harvested by filtration and washed thoroughly with four volumes of deionized water. The mycelia were then suspended in SSC (0.15 A4 NaC1, 0.815 M sodium citrate) containing 1% SSES, (sodium lauryl sulfate), pH 7.6. An equal volume of phenol equilibrated with Tris-HCl buffer pH 7.6, containing 0.1% hydroxyquinoline was added. and the cells were homsgenized for 1.5 min in a Willems Polytron homogenizer (Brinkman Instruments) at the highest setting. %his mixture was centrifuged at 8880 g for 10 min and the aqueous layer extracted several more times with the Tris-equilibrated phenol until the interface between the two layers was clear. The RNA was precipitated overnight from the aqueous phase with 2 volumes s f 95% ethanol at - 15 "C. The mixture was centrifuged at 8080 1: for BO min, the pellet washed with 85% ethanol and dried under vacuum. PoHyacrylamide gel electrophoresis was performed as described by Jaworski and Horgen (12) in 2.4% gels. The gels were scanned in a Gilford Linear Transporter (model 2418-S) attached to a Gilford model 246) spectrophotometer at 268 ram, frozen with dry ice, and sliced in 1-mm slices with a Mickle gel slicer (Brinkman Instruments). The RNA was solubilized from the gels with NCS solubilizer (Amerskana Searle) and counted by liquid scintillation in a toluene based cocktail. The concentration of the RMAs was determined from A r m nm measurements of the gels. Data is expressed as per milligram RNA.
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Uridine triphosphate pool sizes were determined following a modified procedure of Turkington ( 13). The cultures were exposed to mating media containing [3H]uridine (4.0 pCi/m1) in the presence and absence of antheridiol. At designated time intervals 100 ml aliquots of culture were collected on millipore filters and weighed. The mycelia were then suspended in 5 ml of deionized water and homogenized in a Willems Polytron homogenizer at the highest setting for 20 s. The homogenate was made 5% HClOr and centrifuged. The supernatant was neutralized with 0.7 N KOH and centrifuged again. The supernatant was dried down in an Evapo-mix (Bmchler Instruments) and taken up in 500 p1 of water. h e hundred microlitres were streaked along the origin of a polyethyleneimineimpregnated cellulose thin layer plastic plate (Brinkman Instruments). The sample was chromatographed with a UTP standard as described by Turkington ( 13) and the UTP band eluted in 0.6 A4 MgCl,, 4 it4 TrisHC1, pH 7.4 (180: 1 v/v). The eluate was dried down and taken up in water. The residue was chromatographed in two dimensions by the method of Randerath and Randerath (14). The UTP was eluted in 4 A4 NMICPM, assayed spectrophotometricaIly and counted by liquid scintillation. Specific activity was calculated on the basis sf a molar extinction coefficient of 8.1 x ao8 ( 1 5 ) .
Results mcl Discussion In the presence of 5 >( 10-l1g of antheridiol per millilitre, vegetative mycelia of Aehba ambisexualis (strain E87) are induced to undergo sexual morphogenesis resulting in the initiation of branches which differentiate into male sex organs. With the addition of the hormone, there is a dramatic enhancement of the incorporation of [3H]uridine into total cellular RNA when compared to vegetatively growing cells (7). Since the bulk of total cellubar RNA is ribosomal RNA, one might hypothesize that one action of antheridiol is to stimulate the synthesis of rRNA. A valid question to ask is whether the observed incorporation of [3H]uridine into total RNA represents an increase in the total number of ribosomes rather than a rapid turnover of new ribosomes during sexual induction. Ribosomes were quantitatively isolated from identical cultures of A. wmbise.xuali.~in the presence and absence of antheridiol. Lipids were extracted from the ribosome pellets and the amount of rRNA per milligram dry weight of hyphae was determined (Fig. I>. Values from control cultures were unchanged over a period of 7 h. whereas values from hormone-induced diEerentiating cultures indicated an increase in the amount of riboson~esper milligram dry weight of tissue.
Time (hi)
FIG.1. The effect sf antheridiol on ribosome levels in AcBtlya ambisexualis. Ribosomes were isolated from hormone treated and control cultures according to the method of Lin and Key (10). Membrane components were extracted in ethanol-ther-chEorofmm and the W N A was l~ydrolyzed (11). Data is expressed in rPlilligrams of ribonucleotides per miUigams dry weight of tissue as determined by Ingle (1 1).
Possible ribosome turnover was measured by prelabelling Aehlya vegetative ribosomes with [14C]uridine,chasing with unlabelled uridine and then observing the changes in I4C radioactivity in the presence s r absence of hormone. In the absence of antheridiol, cultures showed no loss of radioactivity for at least 4.5 h whereas hormone treated cultures showed a sanall loss sf radioactivity during this time (Fig. 2). This indicates some ribosome turnover in the presence of the hormone. Measurement s f [3H]uridine into RNA of the ribosomes after the addition of the hormone (Fig. 2) follows the pattern previously observed by Silver and Horgen (7). The effect of antheridiol on the incorporatioa~ of [3H]uridine into the 26s and 18s ribosomal RNAs is shown in Fig. 3. In the presence of the hornlone there is a fivefold enhancement of the specific activity of these RNAs at 4 h after the addition of hormone. Not only the specific
CAN. 5. BBQCHEM.
V6L. 53, 1975
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FABLEI. The effect of antheridiol on the uridine triphaphate content and specific activity in A. am bisexual is^
incubation system
Time, h
UITN mmo8/500 mg tissue
Control Hormone Control Hormone Control Hormone
1* 5 1.5 3.0 3 .O 4.5 4.5
115 1211 B 27 116 123 I69
UTP specific activity cpm/500 mg tissue 1246 1211 1141
1643 1082 18364
1[3H]Uridine (4 pCi/ml)was added at time zero in the presence (5 X 10-11 g/ml) or absence of antheridiol. UTP was isolated from mycelial whole cell homogenates as described in Materials and Methods. Spots were eluted and assayed spectrophotometrkally as well as with liquid scintillation. Data is expressed on the basis of 508 rng wet weight of tissue.
1
2 3 Time ah)
5
FIG.2. Determination of ribosome turnover during hormonal stimulation of sexual differentiation in Achlya arnbisex~aiis.Cultures were prelabelled with [lC]uridine (49.1 pCi/ml; specific activity 50 mCi/mmol) for 14 h, chased for 30 min with 2000 ml 2.5 mM unlabelled uridine and suspended in mating media containing [3H]uridine (0.5 yCi/ml; specific activity 28 Ciimmol). One half the culture was treated with antheridiol (5 X 10-llgirnl) the other (control) was left untreated. Samples were taken, fresh weights determined and ribosomes were isolated (10). Radioactivity of WNA in the ribosomes was determined for 14Clabel and 3H label in a Beckman LS- 150 liquid scintillation counter. (- -A--) hormone, (-) control 14C; (-A-) f hormone, (-a-) control, 3H.
Time (Osl FIG.3. The eflect of antheridiol on the synthesis of 26s and 18s rRNA. Cultures were continuously labelled with 10 pCi [3M]uridine per millilitre (specific activity 28 Ci/mmol). RNA was extracted and. electrophoresd as described in Materials and Methods. The concentration of the 26S and 18s RNAs was determined from the A260nm measurement of the gels. The gels were sliced and radioactivity determined by liquid scintillation. The data is expressed as specific activity of the RNA taken at various time points. ( a ) 24s RNA from cultures induced with 5 X 10-llgiml antheridiol; (u) 18s RNA from 26s RNA from control hormone-treated cultures; (6) cultures (no hormone); (8) 18S RNA from control cultures.
activity but also the total amount of rRNA is enhanced by the hormone. Antheridiol appears to affect the synthesis of both 26S and B8S rRNA in a similar manner (Fig. 3). No measurements have yet been made spa the hormonal effect on 5S rRNA metabolism. To determine whether the antheridiol was
actually affecting the synthesis of WNA or perhaps simply affecting the nucleotide precursors within the cells, specific activity of UTP in the cells was determined. Table 1 shows that at given time points, there were only minor differences in the specific activ-
-a-
+
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ity of UTP in hormone and non-hormone treated cells. Furthermore, the UTB pool size did not seem to alter significantly with time from the control. These results suggest that the increase in incorporation of labelled precursors into rRNA in the presence of antheridiol indicates a true stimulation of rRNA synthesis. Associated with antheridiol stimulation of the morphogenesis of sex organ initials, there is a concomitant enhancement of the synthesis of RNA and of protein (7, 8). The hormone initially stimulates total RNA synthesis (within 30 min) and after a lag period (3 h), selectively stimulates poly(A)-rich RNA and protein accumulation (7). Thus, the hormone elicits an apparent gene activation step followed by a period where gene products accumulate. Horgen and Ball (16) have shown that before the time of poly(A)-rich RNA accumulation there is a chemical change in the structure of the chromosomal proteins. This change involved acetylation of specific histonelike, basic nuclear proteins (16). In this study we have shown that before the steroid enhancement of mRNA production and protein synthesis, there is an increase in the apparent number of ribosomes in the hyphae. This is manifested by an increase in the total number of ribosomes extracted per milligram dry weight of tissue in the presence of antheridiol and by the small amount of ribosome turnover. Furthermore, the total incorporation of [3W]uridineinto both 26s and 18s ribosomal RNA increases dramatically in the presence of the hormone. This incsrporation is not due to changes in the UTP cellular pools. These results are very similar to those reported for the effects of hormones on ribosome levels and rRNA synthesis in both higher animal cells (17) and higher plant cells (18). Since the response by Achlya to hormones parallels that of
1345
other eukaryotic systems (16-19), we feel that Achlyn provides an unique and valuable model system for the study of hormonal effects on differentiation. Supported by grants from National Research Council of Canada and Brown-Hazen to P. A. H. We thank Dr. A. W. Barksdale and Dr. T. C. MeMorris for the generous gifts of antheridiol. The technical assistance of E. Thompson is gratefully acknowledged. 1. brksdale, A. W. (1969) Science 166,831-837 2. Barksdale, A., McMsrris, T., Seshadri, R., Arunachalarn, T., Edwards, J., leundeen, J. & Green, D. (1974) J. Gert. ~Wicrobiol.82, 295-299 3. Griffin, D. H., Timberlake, W. E. & Cheney, J. C . (1974) J. Gen. A4icrobiol. 80, 381-388 4. Thomas, D. & Mullins, J. T. (1965) Science 158,84-85 5. Thomas, B. & Mullins, J. T. (1969) Plrysiol. Plant. 22, 347-3 53 6. Nolan, R. A. & Bal, A. K. (1974) J. Bmteriol. 117, 840-4343 7. Silver, J. C. & Horgen, P. A. (1974) Nature (Lorzdon) 249,252-254 8. Kane, B. E., Reiskind, J. B. & Mullins, J. T. (1973) Scierzce 180, 1192-1 193 9. Griffin, D. H. & Breuker, C. (1969) J. Bacterial. 88, 689-696 10. Lin, C. Y. & Key, J. L. (1967) J. ~Wol.Biol. 26, 237247 11. Ingle, J. (1963) Phytochernistry 2, 353-370 12. Jaworski, A. J. & Horgen, P. A. (1973) Arch. Biochern. Bioplrys. 157, 260-267 13. Turkington, R. W. (1970) J. Blol. Cizern. 245, 66% 6697 14. Randerath, K. & Randerath, E. (1967) Methods Enzyrnol. 12, 232-347 15. Bucher, N. L. R. & Swaffield, Ma N. (1966) Biochim. Biophys. Acta 129, 445-459 , A. & Ball, S. F. (1974) Cytobios 10, 18116. ~ o i g e n P. 185 17. Tata, J. R. (1968) Nature (London) 219, 331-337 18. Key, J. L. (1969) Amnu. Rev. Plmit Physiol. 20, 449474 19. B'Malley, B. & Means, A. (1974) Science 183, 610-620
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8. Freinstein, G. (1970) FEBS Lett. 7, 353-355 9. Rick, W. (1965) in Methods of Eitzymatic Analysis (Bergmeyer, H. U., ed.), pp. 811-814, Academic Press Inc., New York and London 10. Dirzynlriewicz, 2. & Arnason, B. G. W. (1974) E-xp. CeM Res. 85, 95-164 11. Quastel, M. R. & KapIan, J. G. (1968) ~Vature219, 198-268 12. Quastel, M. R. Rr. Kaplan, J. G. (1970) Exp. Cell Res. 62,487-420 13. Kast, R. E. (1973) S c a d . J. Immlinol. 4 , 449 14. Vosika, G . & Kennedy, J. B. (1974) Abstracts 9th
134.1
Leucocyte Culture Co~tference,Wil!iamsburg,Virginiadm, p. 241 15. Schnebli, H. P. & Burger, M. M. (1972) Proc. ~Varl. Acad. Sci. U.S.A. 69, 3825-3827 16. Weissman, G., Zurier, R. B. Rr. #offstein, S. (1972) Am. J. Pathd. 68,539-559 17. Havemann, K. R& Schmidt, W. (1972) Proceedings of the 8th Eeucocyte Cukure CmIference (LindahlKiessling, K. R& Ossba, D., eds), pp. 181-189, Academic Press Inc., New York and London 18. Kast, R. E. (1974) Oncology 29, 249-264 19. Saito, M., Yoshizawa, T., Aoyagi, T. & Nagai, Y. (1975) Biochem. Biophys. Res. Commun. 52, 569-575
Hormonal Stimulation of Ribosomal RNA Synthesis in PAULA. HORGEN, ROBINSMITH,JULIEC . SILVER,AND GARYCRAIG Department sf Botany, University o f Toronto, Erindale College, Mississauga, Ontario L5E IC6 Received March 14, 1975' Horgen, P. A., Smith, R., Silver, J. C. & Craig, G. (1975) Hormonal Stimulation of Ribosomal RNA Synthesis in Aclzlya anzbisexualis. Can. J . Biochem. 53, 1341-1345 The experiments reported show that one of the early effects of the steroid sex hormone antheridiol is on the synthesis of rRNA and ribosomes. This is demonstrated in hormone treated cultures of Achlya ambisexualis (strain E87) by an enhancement of the incorporation of [3H]uridine into 26S and 18s rRNA and by an increase in measurable amounts of ribosomes per milligram dry weight of mycelium. Furthermore, since the hormone does not significantly alter the pool size or the specific activity of uridine triphosphate, this effect appears to represent an increased rate of RNA synthesis. Horgen, P. A., Smith, R., Silver, J. C. & Craig, 6 . (1975) Hormonal Stimulation of Ribosomal RNA Synthesis in Achlya ambisexualis. Can. J . Biockenz. 53, 1341-1345 Les expdriences dCcrites montrent qu'en tout premier lieu I'antkCridiol, une hormone sexuelle stCroi'de, agit sur la synthbe du rRNA et des ribosomes. Ainsi, dans des culture dqAchlya ambisexualis (souche E87) traitkes avec cette hormone, l'incorporation de [3H]uridine dans les rRNA 26s et 18S augmente et la quantit6 de ribosomes par milligramme de poids sec de mycklium s'accroit de f a ~ o nmesurable. De plus, comme l'hormone nhlt&re pas de fason significative la grandeur du pool ou lYactivitCspCcifique du triphosphate d'uridine, cet effet impliquerait donc une synth&seaccrue du RNA. [Traduit par le journal]
Introduction The Bsrnycete genus, Aehkya, represents perhaps the most primitive group of organisms which is induced to differentiate by steroid hormones (1). In the presence of antheridiol, a sterol secreted by female strains of Aehkya, male 'Revision received July 9, 1975.
strains are stimulated to produce sex organ initials (antheridiol branches) which eventually differentiate into male sex organs (1). The hormone is active, when assayed with Achlyn ambisexualis (strain E87) at extremely low concentram tiOns (6 X !dml) and is quite specific (1, 2). Because it is extremely fast growing (3), easy to manipulate, and can be induced
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1342
CAN. 9. BKOCHPEM. VOL, 53, 1975
to diRerentiate in a highly uniform manner, Aehka represents a useful 'model system9 to study the effect of steroid horn~oneson macronnolecular synthesis in eukarystes. It has been suggested that a prerequisite for antheridiol stimulation of sex organ initials is a localized softening of the cell wall by the enzynae cellulase (EC 3.2. B .4) (4). Reports of increases in the synthesis and accumulation of this enzyme ( 5 ) and ultrastructural observations of weakened areas of the cell wall (6) tend to strengthen this hypothesis, although no other paranseters have been thoroughly examined. Silver and Horgen (79 reported that antheridiol stimulated the accumulation of a rnRNA-like fraction of RNA with a corresponding enhancement of protein synthesis during the early stages of male sex organ development. The timing of these macromolecular changes corresponds with the morphological appearance of branch initials (7). Furthermore, it was shown that the poly(A)-rich RNA that accumulated was heterogeiaeous ira size indicating that many different gene transcripts were being synthesized. Whether any one gene product is nlsre important than another remains to be seen. Ht is, however, evident that both continued RNA and protein synthesis are required for morphogenesis of sex organ initials in Acbmlya (7, 8). In this study, we have examined the effect of antheridiol on the synthesis of ribosomal RNA and ribosome levels during the early stages of sex-organ differentiation in A . ambisexsrerkis.. We have also exanlined the effects of the hormone on the uridine triphosphate pool size as well as the effect of the hormone on the turnover of ribsomes during differentiation.
Achkya aanbisexunlfs (strain E87) was maintained on Emerson's YPSS (Difco) agar slants. The mycelium was subcultured on PYG ( 1.25 g yeast extract, 1.25 g peptone, 3.0 g glucose per litre deionized H 2 0 ) agar plates for spore preparation according to the method of Griffin and Breuker (9). Spore cysts were then inoculated into PYG broth, grown for 24 h at 24 e 1 "C on a rotary shaker (100 rpm) until finely suspended mycelia had formed. This was used immediately as an inoculum o r stored at 4 2 1 "C for later use. Twenty-five millilitres of the finely suspended mycelia were insculated into 275 ml of mating medium ('9). The cultures were grown at 24 2 I "C for 32-48 Bn. At this time the cultures either received antheridiol (5 X 10-l' giml) o r were left to grow without hormone. Cultures were pulse labelled either with [3H]hlridine
or [l*C]uridine for the times indicated in the presence or absence of hormone. For determination of ribosome levels, cultures of A . ansbisexucrlis were grown and divided into two equal aliquots. One was treated with hormone, the other was left to grow vegetatively. At the designated times, 100 ml samples were taken and collected on a miilipsre filter apparatus. The sample was divided into two equal portions based on wet weight. One portion was used for dry weight measurements; the other used for ribosome isolation. Ribosomes were prepared according to Lin and Key (18). Membrane components were extracted from the ribosome preparations by the procedure of Ingle ( I I ) . WNA was then hydrolyzed in 0.3 IV KQH for 18 h at 37 'C. Nucleotide concentrations were determined as described by Ingle (1 I ) . Data was expressed quantitatively as milligram of ribonucleotides per milligram dry weight of tissue. TO determine whether antheridiol causes the rapid turnover of ribosomes, vegetative cultures were grown in the presence of ['Tjaaridine (0.1 pCi/ml) for 14 h to prelabel ribosomal RNA. Mycelia were then incubated for 30 min in 2 1 of 2.5 mM unlabelled uridine. Cultures were then transferred to fresh mating medium containing [%]uridine (0.5 pCiiml) in the presence or absence sf antheridiol. One-hundred-millilitre samples were collected on a millipore filter and weighed. Ribosomes were prepared according to Lin and Key (10) and the 3H and radioactivity determined. To measure the effect of hormone on incorporation of [3H]uridine into 26s and 18s rRNA cells were grown in the presence of ['W]uridine in the presence or absence of antheridiol. The cultures were harvested by filtration and washed thoroughly with four volumes of deionized water. The mycelia were then suspended in SSC (0.15 A4 NaC1, 0.815 M sodium citrate) containing 1% SSES, (sodium lauryl sulfate), pH 7.6. An equal volume of phenol equilibrated with Tris-HCl buffer pH 7.6, containing 0.1% hydroxyquinoline was added. and the cells were homsgenized for 1.5 min in a Willems Polytron homogenizer (Brinkman Instruments) at the highest setting. %his mixture was centrifuged at 8880 g for 10 min and the aqueous layer extracted several more times with the Tris-equilibrated phenol until the interface between the two layers was clear. The RNA was precipitated overnight from the aqueous phase with 2 volumes s f 95% ethanol at - 15 "C. The mixture was centrifuged at 8080 1: for BO min, the pellet washed with 85% ethanol and dried under vacuum. PoHyacrylamide gel electrophoresis was performed as described by Jaworski and Horgen (12) in 2.4% gels. The gels were scanned in a Gilford Linear Transporter (model 2418-S) attached to a Gilford model 246) spectrophotometer at 268 ram, frozen with dry ice, and sliced in 1-mm slices with a Mickle gel slicer (Brinkman Instruments). The RNA was solubilized from the gels with NCS solubilizer (Amerskana Searle) and counted by liquid scintillation in a toluene based cocktail. The concentration of the RMAs was determined from A r m nm measurements of the gels. Data is expressed as per milligram RNA.
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Uridine triphosphate pool sizes were determined following a modified procedure of Turkington ( 13). The cultures were exposed to mating media containing [3H]uridine (4.0 pCi/m1) in the presence and absence of antheridiol. At designated time intervals 100 ml aliquots of culture were collected on millipore filters and weighed. The mycelia were then suspended in 5 ml of deionized water and homogenized in a Willems Polytron homogenizer at the highest setting for 20 s. The homogenate was made 5% HClOr and centrifuged. The supernatant was neutralized with 0.7 N KOH and centrifuged again. The supernatant was dried down in an Evapo-mix (Bmchler Instruments) and taken up in 500 p1 of water. h e hundred microlitres were streaked along the origin of a polyethyleneimineimpregnated cellulose thin layer plastic plate (Brinkman Instruments). The sample was chromatographed with a UTP standard as described by Turkington ( 13) and the UTP band eluted in 0.6 A4 MgCl,, 4 it4 TrisHC1, pH 7.4 (180: 1 v/v). The eluate was dried down and taken up in water. The residue was chromatographed in two dimensions by the method of Randerath and Randerath (14). The UTP was eluted in 4 A4 NMICPM, assayed spectrophotometricaIly and counted by liquid scintillation. Specific activity was calculated on the basis sf a molar extinction coefficient of 8.1 x ao8 ( 1 5 ) .
Results mcl Discussion In the presence of 5 >( 10-l1g of antheridiol per millilitre, vegetative mycelia of Aehba ambisexualis (strain E87) are induced to undergo sexual morphogenesis resulting in the initiation of branches which differentiate into male sex organs. With the addition of the hormone, there is a dramatic enhancement of the incorporation of [3H]uridine into total cellular RNA when compared to vegetatively growing cells (7). Since the bulk of total cellubar RNA is ribosomal RNA, one might hypothesize that one action of antheridiol is to stimulate the synthesis of rRNA. A valid question to ask is whether the observed incorporation of [3H]uridine into total RNA represents an increase in the total number of ribosomes rather than a rapid turnover of new ribosomes during sexual induction. Ribosomes were quantitatively isolated from identical cultures of A. wmbise.xuali.~in the presence and absence of antheridiol. Lipids were extracted from the ribosome pellets and the amount of rRNA per milligram dry weight of hyphae was determined (Fig. I>. Values from control cultures were unchanged over a period of 7 h. whereas values from hormone-induced diEerentiating cultures indicated an increase in the amount of riboson~esper milligram dry weight of tissue.
Time (hi)
FIG.1. The effect sf antheridiol on ribosome levels in AcBtlya ambisexualis. Ribosomes were isolated from hormone treated and control cultures according to the method of Lin and Key (10). Membrane components were extracted in ethanol-ther-chEorofmm and the W N A was l~ydrolyzed (11). Data is expressed in rPlilligrams of ribonucleotides per miUigams dry weight of tissue as determined by Ingle (1 1).
Possible ribosome turnover was measured by prelabelling Aehlya vegetative ribosomes with [14C]uridine,chasing with unlabelled uridine and then observing the changes in I4C radioactivity in the presence s r absence of hormone. In the absence of antheridiol, cultures showed no loss of radioactivity for at least 4.5 h whereas hormone treated cultures showed a sanall loss sf radioactivity during this time (Fig. 2). This indicates some ribosome turnover in the presence of the hormone. Measurement s f [3H]uridine into RNA of the ribosomes after the addition of the hormone (Fig. 2) follows the pattern previously observed by Silver and Horgen (7). The effect of antheridiol on the incorporatioa~ of [3H]uridine into the 26s and 18s ribosomal RNAs is shown in Fig. 3. In the presence of the hornlone there is a fivefold enhancement of the specific activity of these RNAs at 4 h after the addition of hormone. Not only the specific
CAN. 5. BBQCHEM.
V6L. 53, 1975
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FABLEI. The effect of antheridiol on the uridine triphaphate content and specific activity in A. am bisexual is^
incubation system
Time, h
UITN mmo8/500 mg tissue
Control Hormone Control Hormone Control Hormone
1* 5 1.5 3.0 3 .O 4.5 4.5
115 1211 B 27 116 123 I69
UTP specific activity cpm/500 mg tissue 1246 1211 1141
1643 1082 18364
1[3H]Uridine (4 pCi/ml)was added at time zero in the presence (5 X 10-11 g/ml) or absence of antheridiol. UTP was isolated from mycelial whole cell homogenates as described in Materials and Methods. Spots were eluted and assayed spectrophotometrkally as well as with liquid scintillation. Data is expressed on the basis of 508 rng wet weight of tissue.
1
2 3 Time ah)
5
FIG.2. Determination of ribosome turnover during hormonal stimulation of sexual differentiation in Achlya arnbisex~aiis.Cultures were prelabelled with [lC]uridine (49.1 pCi/ml; specific activity 50 mCi/mmol) for 14 h, chased for 30 min with 2000 ml 2.5 mM unlabelled uridine and suspended in mating media containing [3H]uridine (0.5 yCi/ml; specific activity 28 Ciimmol). One half the culture was treated with antheridiol (5 X 10-llgirnl) the other (control) was left untreated. Samples were taken, fresh weights determined and ribosomes were isolated (10). Radioactivity of WNA in the ribosomes was determined for 14Clabel and 3H label in a Beckman LS- 150 liquid scintillation counter. (- -A--) hormone, (-) control 14C; (-A-) f hormone, (-a-) control, 3H.
Time (Osl FIG.3. The eflect of antheridiol on the synthesis of 26s and 18s rRNA. Cultures were continuously labelled with 10 pCi [3M]uridine per millilitre (specific activity 28 Ci/mmol). RNA was extracted and. electrophoresd as described in Materials and Methods. The concentration of the 26S and 18s RNAs was determined from the A260nm measurement of the gels. The gels were sliced and radioactivity determined by liquid scintillation. The data is expressed as specific activity of the RNA taken at various time points. ( a ) 24s RNA from cultures induced with 5 X 10-llgiml antheridiol; (u) 18s RNA from 26s RNA from control hormone-treated cultures; (6) cultures (no hormone); (8) 18S RNA from control cultures.
activity but also the total amount of rRNA is enhanced by the hormone. Antheridiol appears to affect the synthesis of both 26S and B8S rRNA in a similar manner (Fig. 3). No measurements have yet been made spa the hormonal effect on 5S rRNA metabolism. To determine whether the antheridiol was
actually affecting the synthesis of WNA or perhaps simply affecting the nucleotide precursors within the cells, specific activity of UTP in the cells was determined. Table 1 shows that at given time points, there were only minor differences in the specific activ-
-a-
+
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ity of UTP in hormone and non-hormone treated cells. Furthermore, the UTB pool size did not seem to alter significantly with time from the control. These results suggest that the increase in incorporation of labelled precursors into rRNA in the presence of antheridiol indicates a true stimulation of rRNA synthesis. Associated with antheridiol stimulation of the morphogenesis of sex organ initials, there is a concomitant enhancement of the synthesis of RNA and of protein (7, 8). The hormone initially stimulates total RNA synthesis (within 30 min) and after a lag period (3 h), selectively stimulates poly(A)-rich RNA and protein accumulation (7). Thus, the hormone elicits an apparent gene activation step followed by a period where gene products accumulate. Horgen and Ball (16) have shown that before the time of poly(A)-rich RNA accumulation there is a chemical change in the structure of the chromosomal proteins. This change involved acetylation of specific histonelike, basic nuclear proteins (16). In this study we have shown that before the steroid enhancement of mRNA production and protein synthesis, there is an increase in the apparent number of ribosomes in the hyphae. This is manifested by an increase in the total number of ribosomes extracted per milligram dry weight of tissue in the presence of antheridiol and by the small amount of ribosome turnover. Furthermore, the total incorporation of [3W]uridineinto both 26s and 18s ribosomal RNA increases dramatically in the presence of the hormone. This incsrporation is not due to changes in the UTP cellular pools. These results are very similar to those reported for the effects of hormones on ribosome levels and rRNA synthesis in both higher animal cells (17) and higher plant cells (18). Since the response by Achlya to hormones parallels that of
1345
other eukaryotic systems (16-19), we feel that Achlyn provides an unique and valuable model system for the study of hormonal effects on differentiation. Supported by grants from National Research Council of Canada and Brown-Hazen to P. A. H. We thank Dr. A. W. Barksdale and Dr. T. C. MeMorris for the generous gifts of antheridiol. The technical assistance of E. Thompson is gratefully acknowledged. 1. brksdale, A. W. (1969) Science 166,831-837 2. Barksdale, A., McMsrris, T., Seshadri, R., Arunachalarn, T., Edwards, J., leundeen, J. & Green, D. (1974) J. Gert. ~Wicrobiol.82, 295-299 3. Griffin, D. H., Timberlake, W. E. & Cheney, J. C . (1974) J. Gen. A4icrobiol. 80, 381-388 4. Thomas, D. & Mullins, J. T. (1965) Science 158,84-85 5. Thomas, B. & Mullins, J. T. (1969) Plrysiol. Plant. 22, 347-3 53 6. Nolan, R. A. & Bal, A. K. (1974) J. Bmteriol. 117, 840-4343 7. Silver, J. C. & Horgen, P. A. (1974) Nature (Lorzdon) 249,252-254 8. Kane, B. E., Reiskind, J. B. & Mullins, J. T. (1973) Scierzce 180, 1192-1 193 9. Griffin, D. H. & Breuker, C. (1969) J. Bacterial. 88, 689-696 10. Lin, C. Y. & Key, J. L. (1967) J. ~Wol.Biol. 26, 237247 11. Ingle, J. (1963) Phytochernistry 2, 353-370 12. Jaworski, A. J. & Horgen, P. A. (1973) Arch. Biochern. Bioplrys. 157, 260-267 13. Turkington, R. W. (1970) J. Blol. Cizern. 245, 66% 6697 14. Randerath, K. & Randerath, E. (1967) Methods Enzyrnol. 12, 232-347 15. Bucher, N. L. R. & Swaffield, Ma N. (1966) Biochim. Biophys. Acta 129, 445-459 , A. & Ball, S. F. (1974) Cytobios 10, 18116. ~ o i g e n P. 185 17. Tata, J. R. (1968) Nature (London) 219, 331-337 18. Key, J. L. (1969) Amnu. Rev. Plmit Physiol. 20, 449474 19. B'Malley, B. & Means, A. (1974) Science 183, 610-620
