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Protein synthesis during fungal spore germination
ARCHIVES
OF
BIOCHEMISTRY
Protein I. Characteristics
AND
BIOPHYSICS
Synthesis
126,
during
13-21 (1968)
Fungal
of an in Vitro Phenylalanine Germinated
of Plant Pathology,
Germination
Incorporating
Spores of Botryodiplodia JAMES
Department
Spore
System Prepared
from
theobromael
L. VAN
ETTES
University
of iVebraska, Lincoln,
Nebraska
Received August 9, 1967 The characteristics of an in vitro amino acid incorporating system prepared from germinated spores of Botryodiplodia theobromae are described. Phenylalanine incorporation was dependent on ribosomes, a 105,OOOgsupernatant fraction, poly I;, sRNA, an ATP generating system, and magnesium concentration. The activity of the system was enhanced by adding ammonium, GTP, and spermine. The incorporation of phenylalanine was inhibited by ribonuclease, puromycin, and actidione, but deoxyribonuclease, chloramphenicol, streptomycin, and neomycin had essentially no effect. Poly A and poly C did not stimulate phenylalanine incorporation. In addition, lysine, isoleucine, and proline incorporation was not stimulated by poly U. A similar system prepared from ungerminated spores was inactive in incorporat.ing phenylalanine. Interchanging ribosomes and the 105,OOOgsupernatant fraction from germinated and ungerminated spores indicated that. the defect in the extract from the ungerminated spores was in the 105,OOOgsupernatant fraction.
Several in vivo studies indicate that there is an extremely rapid synthesis of protein during the germination of many fungal spores (e.g., 4-7). In addibion, both conidiospores and ascosporesof Neurospora cyassa contain only monoribosomes, in contra.st to the presence of polyribosomes after germination (8). However, the sedimentation coefficients of the monoribosomes lated by ATP, GTP, and magnesium, and and the nucleot’ide composition of ribosomal was not inhibited by ribonuclease (1). RNA and sRNA were essentially the same Systems developed from germinated in ascospores, conidiospores, and hyphae spores of t,he obligate parasite Uromyces of N. crassa (9, 10). Therefore, a study of the biological acphaseoli (2) and mycelium of Penicillium cyclopium (3) resemble the systems de- tivities of t,he ribosomes and protein-synveloped from other organisms in that, they thesizing enzymes prepared during various are dependent on an energy source, have a stages of germination may give a better sharp optimum for magnesium, and are understanding of the factors t’hat control sensit,ive to ribonuclease. In the latter two fungal spore germination. The characteriinstances, the studies were conducted with zation of an in vitro amino acid incorporating system developed from germinated poly U as mRNA. spores of the mycelial fungus Botryodiplodia theobromaeis reported here. In addition, the 1 Published with the approval of the Director activities of the ribosomes and proteinas Paper No. 2173, Journal Series, Nebraska Agricultural Experiment Station. synthesizing enzymes from germinat’ed and
Many reports have appeared describing in vitro amino acid incorporating systems of bacterial and mammalian origin, but there are only three reports of in vitro systems from mycelial fungi. One report indicates that the system from Penicillium chrysogenum is quite different from that of other organisms because the system (assayed with endogenous mRKA) was not stimu-
13
14
VAN
ETTEN
ungerminated spores are compared. Botryocliplodia theobromae was chosen for this st’udy because it forms an abundance of spores in culture, and these germinate readily under simple conditions. A brief description of t’he conidia of B. theobromae has been reported (11). MATERIALS
AND
METHODS
Materials used. Tris, pyruvic kinase, PEP,2 GTP, ATP, reduced glutathione, spermine, spermidine, chloramphenicol, puromycin, and streptomycin sulfate were obtained from the Sigma Chemical Co.; synthetic polynucleotides were from the Miles Chemical Co.; yeast sRNA (unstripped) and Escherichia coli K-12 sRNA (stripped) were from the General Biochemical Corp.; actidione (cycloheximide) was from the Nutritional Biochemical Corp.; and uniformly labeled L-phenylalanine-1% (393 ,.&/pmole), Lproline-‘% (204 &/pmole) , L-isoleucine-14C (234 &/Nmole), and L-lysine-14C (237 &/pmole) were from the New England Nuclear Corp. Culturing of Botryodiplodia theobromae. Botryodiplodia theobrmae, obtained from Dr. G. Peterson of this department, was maintained on glucose-Yeast extract agar slants (glucose, 1%; yeast extract, 0.2%; agar, 2%). For the production of spores, the organism was grown at 25” for 4-6 weeks in l-liter Erlenmeyer flasks containing 300 ml of V-8 medium (200 ml of V-8 juice, 3 gm of CaC03, 20 gm of agar, and distilled water to 1 liter). The conidiospores were harvested by flooding the culture surface with sterile distilled water, and the culture was gently scraped with an inoculating needle. Mycelial fragments and spores were separated by filtering the suspension through several layers of cheesecloth and subsequently collecting the spores on filter paper. The spores were washed several times with sterile distilled water and stored at, 4” until use (usually only a few hours). The spores were germinated by adding 100 mg of spores to 100 ml of glucose-yeast extract liquid medium in 500.ml baffled Erlenmeyer flasks. The flasks were shaken on a rotary shaker for 11 hours at 25”, and the germinated spores were harvested by centrifugation and washed three times with sterile distilled water. Preparation of extract. The germinated spores were homogenized at 4” in a prechilled mortar in the presence of twice their weight of sand, and 2 Abbreviations used : mRNA, messenger ribonucleic acid; sRNA, soluble ribonucleic acid; GTP, guanosine t,riphosphate; PEP, phospho(eno1) pyruvic acid; poly U, polyuridylic acid; poly A, polyadenylic acid; poly C, polycytidylic acid.
0
:!
4
6
6
TIME,
IO
0
I2
HRS
FIG. 1. The percentage of germination and the dry weight of the spores per flask of Botryodiplodia theobromae at various periods during incubation.
TABLE
I
CHARACTERISTICS OF PHENYLALANINE-W INCORPORATION RY THE CELL-FREE SYSTEM FROM Botryodiplodia theobromae
Complete system - POlY U - soluble RNA - 105,OOOg supernatant fraction - ribosomes” - ATP, GTP, PEP, pyruvate kinase - GTP + ribonuclease (30 rg) + deoxyribonuclease (30 pg) + puromycin (50 rg) + chloramphenicol (100 CL@;) + streptomycin sulfate (100 fig) + neomycin (100 fig) + actidione (20 rg) + actidione (100 pg)
262.0 5.6 31.3 13.1 6.7 10.9 206.0 12..i 240.0 14.6 266.0 237.0 231.0 108.0 63.4
a Ribosomes were equivalent to 0.300 mg oi RNA, and 105,OOOg supernatant fraction enzymes to 0.354 mg of protein per assay.
PROTEIN
SYNTHESIS
one volume (equal t,o the cells’ weight) of buffer A was slowly added. The pH of the suspension was adjusted to pH 7.8 (when necessary) with 1 M NaOH. Buffer A contained 0.2 M Tris, pH 7.8; 0.4 M sucrose; 0.01 M magnesium acetate; 0.06 M KCl; 0.02 M 2-mercaptoethanol; 0.003 M reduced glutathione; and 40rg spermine/ml. The homogenate was centrifuged at 20,OOOg for 20 minutes, and the residue was discarded. The supernatant, fraction was centrifuged at 30,OOOg for 30 minutes and then at 105,OOOg for 180 minutes. Amino acids present in the 105,000~ supernatant fraction were removed by passing the solution through a column of Sephadex G-25 which was equilibrated with buffer B. Buffer B was similar to buffer A except that sucrose was omitted, 1 X 10W4 M EDTA was added, and the concentration of Tris buffer was reduced to 0.02 M. For some experiments, spermine was omitted from both buffers. The ribosomal pellet from the ultracentrifugation was suspended in buffer B and centrifuged at 10,OOOg for 10 minutes, and this supernatant fraction was finally centrifuged at 105,OOOg for 150 minutes to yield the washed ribosomes. The ratio of protein to RNA in the ribosomes was 56:44. The final ribosome pellet was suspended in a small volume of buffer B, and the enzyme fraction (105,OOOg supernatant fraction) and the ribosome suspension were stored under liquid nitrogen. Sssay for amino acid incorporation. The stand200
180
(I
Oo
I
40
80
120
160
200
ug of Rihomol
240
280
320
3x
RNA
2. Effect of ribosome concentration on the incorporation of phenylalanine-14C by the cellfree system. Each assay tube contained 0.24 mg of the 105,OOOg supernatant fraction protein. The amount of ribosomal RNA was varied as shown. FIG.
IN
0
0
15
FUNGI
50
1x3
150
200
LI g of 105,COOg
250 Supernatont
300
350
400
Protein
FIG. 3. Effect of the concentration of the 105,OOOg supernatant fraction on the incorporation of phenylalanine-14C by the cell-free system. Each assay tube contained 0.22 mg of ribosomal RNA. The amount of the 105,OOOg supernat,ant frart,ion protein was varied as shown.
ard assay contained (in a final volume of 0.5 ml) 50 pmoles Tris, pH 7.8; 5 pmoles magnesium acetate; 25 pmoles of NH&l; 7.5 pmoles 2-mercaptoethanol; 1 kmole of ATP; 1Org of pyruvate kinase; 2.5 pmoles of PEP; 0.1 pmole of GTP; 0.5 pmole of reduced glutathione; 20 pg of spermine; 0.3 pC of phenylalanine-1%; 0.005 pmole each of the remaining nineteen (unlabeled) amino acids; 200 pg of sRNA (yeast); and 60 pg of poly U. The amount of enzyme protein and ribosomal RNA added per assay in a particular experiment is reported in the figures and tables. The reaction mixtures were incubated in conical glass centrifuge tubes at 36” for 60 minutes. Near the conclusion of these experiments, the temperature optimum for the reaction was found to be 20”, and the results reported in Table V were determined at 20”. The reaction was terminated by the addition of 20 pmoles of unlabeled phenylalanine, 500 rg of bovine albumin, and trichloroacetic acid to a final concentration of S70. After being heated in boiling water for 10 minutes, the tubes were kept at 0” for 1 hour, and the precipitates were transferred to cellulose nitrate filters (HA Millipore filter, 25 mm diameter, 0.45 p pore size) and washed three times with 10 ml of 7% trichloroacetic acid. The filters were dried lmder an infrared lamp and
16
VAN
01 0
40
60
I20 yg
160
of Soluble
I 200
Ikin2
RNA
FIG. 4. Effect of yeast sRNA concentration on the incorporation of phenylalanine-1% by the cell-free system. Each assay tube contained 0.17 mg of ribosomal RNA and 0.29 mg of the 105,OOOg supernatant fract,ion protein. The amount of yeast sRNA was varied as shown. placed in counting vials containing 15 ml of counting solution [4 gm of 2,5diphenyloxazole, 50 mg of 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene and toluene to 1 liter]. The samples were counted in a Packard Tri-Carb spectrometer with a counting efficiency of 50%. In the experiments in which proline-l%, isoleucine-14C, and lysine-r4C were employed, the reactions were terminated with a trichloroacet,ic acid-tungstate solution by procedures described previously (12, 13). Other determinations. Protein was determined according to the method of Lowry et al. (14), and bovine albumin was used as a standard. Ribonucleic acid was determined by the orcinol method (15), and yeast, RNA was used as a standard. Each experiment reported was repeated at least once. RESULTS
Under the growth spores began to form hours, and germination of about 90 % after The dry weight of the
conditions used, the germ tubes after 4 reached a maximum 11 hours (Fig. 1). spores remained con-
ETTEN
stant for the first 6 hours of incubation, after which it began to increase until there was approximately a 2-fold increase in dry weight at 11 hours (Fig. 1). If more than 100 mg of spores was used to inoculate one flask, the percentage of germination decreased. This is probably due to the presence of a self-inhibitor in the spores which is common to many fungi (16). Requirement for amino acid incorporation. Table I reports the characteristics of the amino acid incorporating system. Like other in vitro systems, the preparation from germinated spores had an absolute requirement for ribosomes and the 105,OOOg supernatant fraction. Also, there was a strict requirement for poly U. The presence of yeast sRNA increased the incorporation of phenylalanine about g-fold. A small amount of endogenous sRNA is probably present in the 105,OOOg supernatant fraction or on the ribosomes, or both. The activity of the system was strictly dependent on the presence of ATP and of an ATP generating system. Omission of GTP caused a 21% decrease in the activity of the system, a decrease similar to that reported for other systems from plants (e.g., 3, 17). The addition of ribonuclease to the reaction mixture completely inhibited phenylalanine incorporation, but deoxyribonuclease had only a slight effect. The antibiotic puromycin caused 94 % inhibition of the sysbem, and actidione at a concentration of 100 pg per assay inhibited 74 %. Three other antibiotics, chloramphenicol, streptomycin, and neomycin, which commonly inhibit in vitro protein synthesis by bacterial systems, had little or no effect on the B. theobromae system, even at concentrations up to 100 Mg per assay. Increasing the concentration of ribosomes (Fig. 2) produced a proportional increase in phenylalanine incorporation up to about 180 rg of ribosomal RNA per assay, and then the rate began to decrease. Figure 3 shows the effect of various concentrations of the 105,OOOg supernatant fraction. At concentrations above 250 pg of enzyme protein per assay, the reaction rate leveled off. Increasing concentrations of yeast sRNA stimulated Dhenvlalanine incorDoration UD I
PROTEIN
SYNTHESIS
IN
FUNGI
17
Tvere from cell-free extracts prepared in buffers A and B containing 40 pg of spermine per milliliter; in Fig. 6B, spermjne was omitted from t#hesebuffers. The magnesium concentration for optimum activity was slightly higher (13 pmoles) in the complete absence of spermine versus 8 clmolesin the presence of 30 pg of spermine (Fig. GB). The polyamine spermidine has previousl? been reported to produce a similar shift in the magnesium concent,ration necessary for maximum activity for a cell-free preparation from yeast (18). The ammonium concentration in the assay mixture had less effect than that of magnesium since, even in the absence of ammonium, the activity was quite good (Fig. 7). However, the system already contained 15 pmoles of potassium even at zero ammonium concentration since this ion was present in the standard buffers used in the procedure for the preparation of the active fractions. In data not shown,
ug
Of
Pdy uridylic
Acid
FIG. 5. Effect of poly U concentration on the irlcorporation of phenylalanine-14C by the cell-free system. Each assay tube contained 0.19 mg of ribosomal RNA and 0.20 mg of the 105,OOOg supernatant, fraction protein. The amount of poly U was varied as shown. about 200 pg of sRNA (Fig. 4). Soluble RKA prepared from Escherichia coli K-12 was only slightly active in the B. theobromae s-y&em. The incorporation of phenylalanine was stimulated proportionally by increasing concent,rations of poly U up to 50 pg of poly T- per assay (Fig. 5). Phenylalanine incorporation was restricted t,o a narrow range of magnesium concentrations (Fig. 6). The results reported in Fig. 6 are corrected for the amount of magnesium added into the assay mixture from the buffered solutions containing the ribosomes and t’he 105,OOOy supernatant enzymes. The samefigure demonstrates that the addition of spermine to the assay mixture increased activity. The data in Fig. 6A to
FIG. 6. Effect of magnesium acetate in the presence (-) and absence (---) of 30 fig of sperm& on the incorporation of phenylalanine-14C by the cell-free system. A: Cell-free extracts prepared in the presence of spermine as noted in ~~ATERIALS AND METHODS so that all assays contained 10 pg of spermine from the buffered solutions containing the ribosomes and the 105,OOOg fraction. Each assay tube contained 0.20 mg of ribosomal RNA and 0.34 mg of the 105,OOOg supernatant fraction protein. B: Cell-free extracts prepared in the absence of spermine. Each assay tube contained 0.15 mg of ribosomal RNA and 0.29 mg of the 105,OOOg supernatant fraction protein. The amount of magnesium acetate was varied as shown.
18
VAN ETTEN
potassium replaced ammonium and had a optimum concentration similar to that of ammonium. The time course of the reaction (Fig. 8) demonstrates that the reaction reached completion after about 45 minutes. The pH optimum of the reaction was 7.8 (Fig. 9). The incubation temperature for maximum incorporation was 20” (Table II), with the activity decreasing at higher and lower temperatures. Although the system was initially characterized at a temperature of 36’, the effect of the magnesium concentration on activity was rechecked at an incubation temperature of 20”, and the optimum was found to be the same as at 36”. Stability of the system. The ribosomes and
Ok%-
40
60
Incubation
80
I IO0
I 120
time, min.
FIG. 8. Time course of the incorporation of phenylalanine-1°C by the cell-free system. Each assay tube contained 0.21 mg of ribosomal RNA and 0.38 mg of the 105,OOOg supernatant fraction protein.
OoL 120
Ammonium
chloride,
160 200
240
u mdes
FIG. 7. Effect of NH&l on the incorporation of phenylalanine-1°C by the cell-free system. Each assay tube contained 0.17 mg of ribosomal RNA and 0.33 mg of the 105,OOOg supernatant fraction protein. The amount of NH&l was varied as shown.
the 105,OOOgsupernatant enzymes retained complete activity if they were stored in liquid nitrogen for a period of several weeks. However, storage of the ribosomes or enzyme fraction at, 4’ or -20” for a week resulted in almost complete loss of the incorporating activity. E$ect of various polyamines. Table III shows the effect of several concentrations of spermine on phenylalanine incorporation. When the ribosomes were prepared in buffers without spermine, phenylalanine incorporation was stimulated to a greater extent by increasing the spermine concentration in the assay mixture than if the ribosomes were prepared in the presence of spermine. The effect of two other polyamines on the system is reported in Table III. Responseto other synthetic polynudeotides. Phenylalanine incorporation was stimulated only by poly U and not by poly A or poly C (Table IV). Furthermore, the incorpora-
PROTEIN
SYNTHESIS
tions of the amino acids lysine, isoleucine. and proline \vere not st’imulated by poly U.
Activity
o.f wbqerminated spol‘es.Since one -
I
of the obje&ves understand the
of this study leas to t’ry to rapid increase of i?z vivo
IN FUNGI
19 TABLE
EFFECT
OF POLYAMINKS
INCORPOR.\TION FROM
BY
III ON
THK
PHENYLAL.\NINE-14C
CELL-FREE
Botryodiplodia
Polyamine
SYSTEM
theobrmae Phenylalanine-‘4C (~rmoles)/ms
20 pg spermine 50 pg spermine 100 rg spermine 200 pg spermine 20 pg spermidine 20 rg 3,3’-iminobispropylamine None
incorporated ribosomal RNA0
A
B
808 889 748
370 543 684
331 709
236
704
229
649
238
510
a A: Cell-free extract prepared in the presence of spermine as noted in the &~~TERIALS AND RIETHODS. Each assay tube contained 0.24 mg of ribosomal RNA and 0.46 mg of the 105,OOOg supernatant fraction protein. Ten pg of spermine is present in all of the assay tubes in addition to the amount added. B: Cell-free extract prepared in the absence of spermine. Each assay tube contained 0.15 mg of ribosomal RNA and 0.29 mg of the 105,OOOgsupernatant fract,ion protein. TABLE
06,
90
EFFECT
IV
OF POLYNUCLEOTIDES ON PORATION OF PHENYLAL.4NINE-‘4C’
THE
INCOR-
PH
9. Effect of pH on the incorporation of phenylalanine-14C by the cell-free system. Each assay tube contained 0.25 mg of ribosomal RNA and 0.33 mg of the 105,OOOg supernatant fraction protein. The buffer used in the above experiment xas Tris. FIG.
TABLE EFFECT OF TEMPER.~TURE INCORPORATION BY FROM
Temperature
Boturodiplodia (“C)
II
ON PHENYL.4L.4NINI+‘4C THE CELL-FREE SYSTEM
Phenylalanine-1°C Incorporated (mpmoles)/mg ribosomal RNAa
0.08 1.93
20
2.40 2.03 1.7.5
30 36
None Poly u Poly A Poly c
Phenylalanine-W (wmoles)/mg
incorporated ribosomal RNAa
13.7 598.0 12.6 12.3
(1Each assay tube contained 0.207 mg of ribosomal RNA and 0.260 mg of the 105,OOOg supernatant, fraction protein.
theobromae
0 16 24
Addition
0.92
(LRibosomes were equivalent to 0.173 mg of RNA, and the 105,OOOg supernatant fraction cont.ained 0.38 mg of protein per assay.
protein synthesis during spore germination, ribosomes and the 105,OOOgsupernatant fraction \\-ere also prepared from ungermi-
nated spores. The complete system from the ungerminated sporeswas inactive (Table V). The interchange of the ribosomes and the lOZ,OOOg supernatant fraction from germinated and ungerminated spores indicated that the defect in the ungermina,ted spores was in the 105,000g supernatant fraction since the ribosomes from the ungerminated spores had some activity with
20
VAN
ETTEN
antibiotic known to promote the release of the unfinished peptide chains from ribosomes. In addition, the system is partially inhibited by the ant’ibiotic actidione, which inhibits in vitro amino acid incorporation by yeast but not bacPhenylalanineW incorterial systems (19). Ribonuclease com105,000g Supernatant Ribosomes porated fraction b.wmoles)/ pletely inhibited the system, but deoxyriboaSSaya nuclease had essentially no effect on amino I I acid incorporation. Germinated 2.1 It is interesting that the enzyme fraction Germinated 0.3 from the ungerminated spores is inactive, Germinated Germinated 344.0 while the ribosomes from the spores have Germinated Ungerminated 4.0 fair activity when assayed with the enzyme Germinated Germinated + 258.0 ungerminated fraction from the germinated spores. ExUngerminated 3.9 periments in progress indicate that at least Ungerminated 1.9 part of the inactivity of the enzyme fracUngerminated Ungerminated 5.0 tion from the ungerminated spores is due Ungerminated Germinated 81.2 to t’he extremely low activity of phenylalanyl-sRNA synthetase. The inhibitory n When added, ribosomes from both germinated effect of the enzyme fraction from the unand ungerminated spores were equivalent to 0.102 germinated spores on the complete system mg of RNA/assay; the 105,OOOg supernatant fracfrom the germinated spores is being intion enzymes from both germinated and ungerminated spores contained 0.190 mg of protein/ vestigated also. assay. The assay mixture was incubated at 20”. It may be characteristic of microbial spores that the mechanism for protein synthesis is inhibited during unfavorable the 105,OOOgsupernatant fraction from the conditions and that the inhibition is regerminated spores. The 105,OOOg supermoved during or preceding germination. natant fraction from the ungerminated Staples et al. (2) have reported that ribospores slightly inhibited the complete somes prepared from both germinated and system from the germinated spores (Table ungerminated uredospores of the obligate VI* parasite Uromyces phaseoli have about the same activity when assayed with an enzyme DISCUSSION fraction prepared from rice seedlings, but The amino acid incorporating activity an enzyme fraction prepared from the unof the system prepared from germinated germinated uredospores completely inspores of B. theobromaehas properties and hibits this system. More recenbly, however, requirements similar to those of other systems developed from plants, and micro- Staples and Bedigian have obtained an active enzyme fraction from ungerminated organisms. The incorporation of phenyluredospores (20). Another recent report of alanine by the cell-free preparation is not an in vitro amino acid incorporating due to bacterial contamination since plate system developed during various stages of counts performed on the assay mixture showed that the bacterial population was germination of Bacillus cereus spores indicates that both the ribosomes and transfer negligible (e.g., less than 500 bacteria per enzymes from the dormant’ spores are assay mixture). Furthermore, the antidefective (21). bacterial antibiotics, streptomycin, chloramphenicol, and neomycin, had essentially ACKNOWLEDGMENTS no effect on the amount of phenylalanine The author is indebted for technical help reincorporated. Amino acid incorporation by the cell- ceived at various stages of this work from R. free system is inhibited by puromycin, an Brambl. TABLE
V
PHENYLALANINE-W INCORPORATION BY CELLFREE SYSTEMS PREPARED FROM GERMINATED AND UNGERMINATED SPORES OF Botryodiplodia theobrmae
PROTEIN
SYNTHESIS
REFERENCES W., KORNFELD, J.M., ANDKNIGHT S. G., Arch. Mikrobiol. 63, 41 (1966). 2. STBPLES, R.C.. APP, A. A,, MCCARTHY, W.J., .IND GEROSA, M. M., Contrib. Boyce Thompson Inst. 23, 159 (1966). 3. VAN ETTEN,J.L.,PrZRISI,B.,SND CIFERRI,O., Nature 212, 932 (1966). 4. STAPLES, R. c., SYAMANANDA, R., KAO, V., AND BLOCIC, R. J., Contrib. Boyce Thompson Inst. 21, 345 (1962). 5. GOTTLIEB, D., AND CALTRIDER, P. G., Nature 197, 916 (1963). 6. BaRaSH, I., CONWAY, M. L., AND HOW:~RD, D. H., J. Bacterial. 93, 656 (1967). 7. SHEPHERD, C. J.,J. Gen. Microbial. 16, i (1957). 8. HENNEY, H. R., AND STORCK, R., Proc. Natl. Acad. Sci. U.S. 61. 1050 (1964). 9. HENNEY, H. R., AND STORCK, R.,Science142, 1675 (1963). 10. HENNEY, H., AND STORCK, R., J. Bacterial. 86, 822 (1963). 11. PETERSON, G. W., Phytopathology 67, 825 (1967). 1. H~IDLE,C.
IN
21
FUNGI
12. So, A. G., AND DAVIE, E. W., Biochemistry 3, 1165 (1964). 13. DOWNEY, K.M.,So, A. G., AXD DAVIE, E.W., Biochemistry 4, 1702 (1965). 0. H., ROSEBROUGH,X. J., FARR, A. 14. LOWRY, L., AND RANDALL, R. J., J. Biol. Chem 193, 265 (1951). 15. SCHNEIDER, W. C., Methods in Enzymol. 3. 680 (1957). 16. SUSSMAN, A. S., in “The Fungi” (G. C. Ainsworth and A. S. Sussman, eds.), Vol. 2, p. 733. Academic Press, New York (1966). 17. PARISI, B., AND CIFERRI, 1638 (1966). R. K.,MARCUS, 18. BRETTHAUER, J., HALVORSON, H. O.,
O.,
AND
Biochemistry
6,
L., CHALOUPKA, BOCK, R. M.,
Biochemistry 2, 1079 (1963). M. R., AND SISLER, H. D., Biochim. 19. SIEGEL, Biophys. Acta 103, 558 (1965). R. C., AND BEDIGIbN, D., Contrib. 20. STAPLES, Boyce Thompson Inst. 23, 345 (1967). 21. KOBAYASHI,~., AND HALVORSON, H.O., Bacteriol. Proc. 1967, 22 (1967).
OF
BIOCHEMISTRY
Protein I. Characteristics
AND
BIOPHYSICS
Synthesis
126,
during
13-21 (1968)
Fungal
of an in Vitro Phenylalanine Germinated
of Plant Pathology,
Germination
Incorporating
Spores of Botryodiplodia JAMES
Department
Spore
System Prepared
from
theobromael
L. VAN
ETTES
University
of iVebraska, Lincoln,
Nebraska
Received August 9, 1967 The characteristics of an in vitro amino acid incorporating system prepared from germinated spores of Botryodiplodia theobromae are described. Phenylalanine incorporation was dependent on ribosomes, a 105,OOOgsupernatant fraction, poly I;, sRNA, an ATP generating system, and magnesium concentration. The activity of the system was enhanced by adding ammonium, GTP, and spermine. The incorporation of phenylalanine was inhibited by ribonuclease, puromycin, and actidione, but deoxyribonuclease, chloramphenicol, streptomycin, and neomycin had essentially no effect. Poly A and poly C did not stimulate phenylalanine incorporation. In addition, lysine, isoleucine, and proline incorporation was not stimulated by poly U. A similar system prepared from ungerminated spores was inactive in incorporat.ing phenylalanine. Interchanging ribosomes and the 105,OOOgsupernatant fraction from germinated and ungerminated spores indicated that. the defect in the extract from the ungerminated spores was in the 105,OOOgsupernatant fraction.
Several in vivo studies indicate that there is an extremely rapid synthesis of protein during the germination of many fungal spores (e.g., 4-7). In addibion, both conidiospores and ascosporesof Neurospora cyassa contain only monoribosomes, in contra.st to the presence of polyribosomes after germination (8). However, the sedimentation coefficients of the monoribosomes lated by ATP, GTP, and magnesium, and and the nucleot’ide composition of ribosomal was not inhibited by ribonuclease (1). RNA and sRNA were essentially the same Systems developed from germinated in ascospores, conidiospores, and hyphae spores of t,he obligate parasite Uromyces of N. crassa (9, 10). Therefore, a study of the biological acphaseoli (2) and mycelium of Penicillium cyclopium (3) resemble the systems de- tivities of t,he ribosomes and protein-synveloped from other organisms in that, they thesizing enzymes prepared during various are dependent on an energy source, have a stages of germination may give a better sharp optimum for magnesium, and are understanding of the factors t’hat control sensit,ive to ribonuclease. In the latter two fungal spore germination. The characteriinstances, the studies were conducted with zation of an in vitro amino acid incorporating system developed from germinated poly U as mRNA. spores of the mycelial fungus Botryodiplodia theobromaeis reported here. In addition, the 1 Published with the approval of the Director activities of the ribosomes and proteinas Paper No. 2173, Journal Series, Nebraska Agricultural Experiment Station. synthesizing enzymes from germinat’ed and
Many reports have appeared describing in vitro amino acid incorporating systems of bacterial and mammalian origin, but there are only three reports of in vitro systems from mycelial fungi. One report indicates that the system from Penicillium chrysogenum is quite different from that of other organisms because the system (assayed with endogenous mRKA) was not stimu-
13
14
VAN
ETTEN
ungerminated spores are compared. Botryocliplodia theobromae was chosen for this st’udy because it forms an abundance of spores in culture, and these germinate readily under simple conditions. A brief description of t’he conidia of B. theobromae has been reported (11). MATERIALS
AND
METHODS
Materials used. Tris, pyruvic kinase, PEP,2 GTP, ATP, reduced glutathione, spermine, spermidine, chloramphenicol, puromycin, and streptomycin sulfate were obtained from the Sigma Chemical Co.; synthetic polynucleotides were from the Miles Chemical Co.; yeast sRNA (unstripped) and Escherichia coli K-12 sRNA (stripped) were from the General Biochemical Corp.; actidione (cycloheximide) was from the Nutritional Biochemical Corp.; and uniformly labeled L-phenylalanine-1% (393 ,.&/pmole), Lproline-‘% (204 &/pmole) , L-isoleucine-14C (234 &/Nmole), and L-lysine-14C (237 &/pmole) were from the New England Nuclear Corp. Culturing of Botryodiplodia theobromae. Botryodiplodia theobrmae, obtained from Dr. G. Peterson of this department, was maintained on glucose-Yeast extract agar slants (glucose, 1%; yeast extract, 0.2%; agar, 2%). For the production of spores, the organism was grown at 25” for 4-6 weeks in l-liter Erlenmeyer flasks containing 300 ml of V-8 medium (200 ml of V-8 juice, 3 gm of CaC03, 20 gm of agar, and distilled water to 1 liter). The conidiospores were harvested by flooding the culture surface with sterile distilled water, and the culture was gently scraped with an inoculating needle. Mycelial fragments and spores were separated by filtering the suspension through several layers of cheesecloth and subsequently collecting the spores on filter paper. The spores were washed several times with sterile distilled water and stored at, 4” until use (usually only a few hours). The spores were germinated by adding 100 mg of spores to 100 ml of glucose-yeast extract liquid medium in 500.ml baffled Erlenmeyer flasks. The flasks were shaken on a rotary shaker for 11 hours at 25”, and the germinated spores were harvested by centrifugation and washed three times with sterile distilled water. Preparation of extract. The germinated spores were homogenized at 4” in a prechilled mortar in the presence of twice their weight of sand, and 2 Abbreviations used : mRNA, messenger ribonucleic acid; sRNA, soluble ribonucleic acid; GTP, guanosine t,riphosphate; PEP, phospho(eno1) pyruvic acid; poly U, polyuridylic acid; poly A, polyadenylic acid; poly C, polycytidylic acid.
0
:!
4
6
6
TIME,
IO
0
I2
HRS
FIG. 1. The percentage of germination and the dry weight of the spores per flask of Botryodiplodia theobromae at various periods during incubation.
TABLE
I
CHARACTERISTICS OF PHENYLALANINE-W INCORPORATION RY THE CELL-FREE SYSTEM FROM Botryodiplodia theobromae
Complete system - POlY U - soluble RNA - 105,OOOg supernatant fraction - ribosomes” - ATP, GTP, PEP, pyruvate kinase - GTP + ribonuclease (30 rg) + deoxyribonuclease (30 pg) + puromycin (50 rg) + chloramphenicol (100 CL@;) + streptomycin sulfate (100 fig) + neomycin (100 fig) + actidione (20 rg) + actidione (100 pg)
262.0 5.6 31.3 13.1 6.7 10.9 206.0 12..i 240.0 14.6 266.0 237.0 231.0 108.0 63.4
a Ribosomes were equivalent to 0.300 mg oi RNA, and 105,OOOg supernatant fraction enzymes to 0.354 mg of protein per assay.
PROTEIN
SYNTHESIS
one volume (equal t,o the cells’ weight) of buffer A was slowly added. The pH of the suspension was adjusted to pH 7.8 (when necessary) with 1 M NaOH. Buffer A contained 0.2 M Tris, pH 7.8; 0.4 M sucrose; 0.01 M magnesium acetate; 0.06 M KCl; 0.02 M 2-mercaptoethanol; 0.003 M reduced glutathione; and 40rg spermine/ml. The homogenate was centrifuged at 20,OOOg for 20 minutes, and the residue was discarded. The supernatant, fraction was centrifuged at 30,OOOg for 30 minutes and then at 105,OOOg for 180 minutes. Amino acids present in the 105,000~ supernatant fraction were removed by passing the solution through a column of Sephadex G-25 which was equilibrated with buffer B. Buffer B was similar to buffer A except that sucrose was omitted, 1 X 10W4 M EDTA was added, and the concentration of Tris buffer was reduced to 0.02 M. For some experiments, spermine was omitted from both buffers. The ribosomal pellet from the ultracentrifugation was suspended in buffer B and centrifuged at 10,OOOg for 10 minutes, and this supernatant fraction was finally centrifuged at 105,OOOg for 150 minutes to yield the washed ribosomes. The ratio of protein to RNA in the ribosomes was 56:44. The final ribosome pellet was suspended in a small volume of buffer B, and the enzyme fraction (105,OOOg supernatant fraction) and the ribosome suspension were stored under liquid nitrogen. Sssay for amino acid incorporation. The stand200
180
(I
Oo
I
40
80
120
160
200
ug of Rihomol
240
280
320
3x
RNA
2. Effect of ribosome concentration on the incorporation of phenylalanine-14C by the cellfree system. Each assay tube contained 0.24 mg of the 105,OOOg supernatant fraction protein. The amount of ribosomal RNA was varied as shown. FIG.
IN
0
0
15
FUNGI
50
1x3
150
200
LI g of 105,COOg
250 Supernatont
300
350
400
Protein
FIG. 3. Effect of the concentration of the 105,OOOg supernatant fraction on the incorporation of phenylalanine-14C by the cell-free system. Each assay tube contained 0.22 mg of ribosomal RNA. The amount of the 105,OOOg supernat,ant frart,ion protein was varied as shown.
ard assay contained (in a final volume of 0.5 ml) 50 pmoles Tris, pH 7.8; 5 pmoles magnesium acetate; 25 pmoles of NH&l; 7.5 pmoles 2-mercaptoethanol; 1 kmole of ATP; 1Org of pyruvate kinase; 2.5 pmoles of PEP; 0.1 pmole of GTP; 0.5 pmole of reduced glutathione; 20 pg of spermine; 0.3 pC of phenylalanine-1%; 0.005 pmole each of the remaining nineteen (unlabeled) amino acids; 200 pg of sRNA (yeast); and 60 pg of poly U. The amount of enzyme protein and ribosomal RNA added per assay in a particular experiment is reported in the figures and tables. The reaction mixtures were incubated in conical glass centrifuge tubes at 36” for 60 minutes. Near the conclusion of these experiments, the temperature optimum for the reaction was found to be 20”, and the results reported in Table V were determined at 20”. The reaction was terminated by the addition of 20 pmoles of unlabeled phenylalanine, 500 rg of bovine albumin, and trichloroacetic acid to a final concentration of S70. After being heated in boiling water for 10 minutes, the tubes were kept at 0” for 1 hour, and the precipitates were transferred to cellulose nitrate filters (HA Millipore filter, 25 mm diameter, 0.45 p pore size) and washed three times with 10 ml of 7% trichloroacetic acid. The filters were dried lmder an infrared lamp and
16
VAN
01 0
40
60
I20 yg
160
of Soluble
I 200
Ikin2
RNA
FIG. 4. Effect of yeast sRNA concentration on the incorporation of phenylalanine-1% by the cell-free system. Each assay tube contained 0.17 mg of ribosomal RNA and 0.29 mg of the 105,OOOg supernatant fract,ion protein. The amount of yeast sRNA was varied as shown. placed in counting vials containing 15 ml of counting solution [4 gm of 2,5diphenyloxazole, 50 mg of 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene and toluene to 1 liter]. The samples were counted in a Packard Tri-Carb spectrometer with a counting efficiency of 50%. In the experiments in which proline-l%, isoleucine-14C, and lysine-r4C were employed, the reactions were terminated with a trichloroacet,ic acid-tungstate solution by procedures described previously (12, 13). Other determinations. Protein was determined according to the method of Lowry et al. (14), and bovine albumin was used as a standard. Ribonucleic acid was determined by the orcinol method (15), and yeast, RNA was used as a standard. Each experiment reported was repeated at least once. RESULTS
Under the growth spores began to form hours, and germination of about 90 % after The dry weight of the
conditions used, the germ tubes after 4 reached a maximum 11 hours (Fig. 1). spores remained con-
ETTEN
stant for the first 6 hours of incubation, after which it began to increase until there was approximately a 2-fold increase in dry weight at 11 hours (Fig. 1). If more than 100 mg of spores was used to inoculate one flask, the percentage of germination decreased. This is probably due to the presence of a self-inhibitor in the spores which is common to many fungi (16). Requirement for amino acid incorporation. Table I reports the characteristics of the amino acid incorporating system. Like other in vitro systems, the preparation from germinated spores had an absolute requirement for ribosomes and the 105,OOOg supernatant fraction. Also, there was a strict requirement for poly U. The presence of yeast sRNA increased the incorporation of phenylalanine about g-fold. A small amount of endogenous sRNA is probably present in the 105,OOOg supernatant fraction or on the ribosomes, or both. The activity of the system was strictly dependent on the presence of ATP and of an ATP generating system. Omission of GTP caused a 21% decrease in the activity of the system, a decrease similar to that reported for other systems from plants (e.g., 3, 17). The addition of ribonuclease to the reaction mixture completely inhibited phenylalanine incorporation, but deoxyribonuclease had only a slight effect. The antibiotic puromycin caused 94 % inhibition of the sysbem, and actidione at a concentration of 100 pg per assay inhibited 74 %. Three other antibiotics, chloramphenicol, streptomycin, and neomycin, which commonly inhibit in vitro protein synthesis by bacterial systems, had little or no effect on the B. theobromae system, even at concentrations up to 100 Mg per assay. Increasing the concentration of ribosomes (Fig. 2) produced a proportional increase in phenylalanine incorporation up to about 180 rg of ribosomal RNA per assay, and then the rate began to decrease. Figure 3 shows the effect of various concentrations of the 105,OOOg supernatant fraction. At concentrations above 250 pg of enzyme protein per assay, the reaction rate leveled off. Increasing concentrations of yeast sRNA stimulated Dhenvlalanine incorDoration UD I
PROTEIN
SYNTHESIS
IN
FUNGI
17
Tvere from cell-free extracts prepared in buffers A and B containing 40 pg of spermine per milliliter; in Fig. 6B, spermjne was omitted from t#hesebuffers. The magnesium concentration for optimum activity was slightly higher (13 pmoles) in the complete absence of spermine versus 8 clmolesin the presence of 30 pg of spermine (Fig. GB). The polyamine spermidine has previousl? been reported to produce a similar shift in the magnesium concent,ration necessary for maximum activity for a cell-free preparation from yeast (18). The ammonium concentration in the assay mixture had less effect than that of magnesium since, even in the absence of ammonium, the activity was quite good (Fig. 7). However, the system already contained 15 pmoles of potassium even at zero ammonium concentration since this ion was present in the standard buffers used in the procedure for the preparation of the active fractions. In data not shown,
ug
Of
Pdy uridylic
Acid
FIG. 5. Effect of poly U concentration on the irlcorporation of phenylalanine-14C by the cell-free system. Each assay tube contained 0.19 mg of ribosomal RNA and 0.20 mg of the 105,OOOg supernatant, fraction protein. The amount of poly U was varied as shown. about 200 pg of sRNA (Fig. 4). Soluble RKA prepared from Escherichia coli K-12 was only slightly active in the B. theobromae s-y&em. The incorporation of phenylalanine was stimulated proportionally by increasing concent,rations of poly U up to 50 pg of poly T- per assay (Fig. 5). Phenylalanine incorporation was restricted t,o a narrow range of magnesium concentrations (Fig. 6). The results reported in Fig. 6 are corrected for the amount of magnesium added into the assay mixture from the buffered solutions containing the ribosomes and t’he 105,OOOy supernatant enzymes. The samefigure demonstrates that the addition of spermine to the assay mixture increased activity. The data in Fig. 6A to
FIG. 6. Effect of magnesium acetate in the presence (-) and absence (---) of 30 fig of sperm& on the incorporation of phenylalanine-14C by the cell-free system. A: Cell-free extracts prepared in the presence of spermine as noted in ~~ATERIALS AND METHODS so that all assays contained 10 pg of spermine from the buffered solutions containing the ribosomes and the 105,OOOg fraction. Each assay tube contained 0.20 mg of ribosomal RNA and 0.34 mg of the 105,OOOg supernatant fraction protein. B: Cell-free extracts prepared in the absence of spermine. Each assay tube contained 0.15 mg of ribosomal RNA and 0.29 mg of the 105,OOOg supernatant fraction protein. The amount of magnesium acetate was varied as shown.
18
VAN ETTEN
potassium replaced ammonium and had a optimum concentration similar to that of ammonium. The time course of the reaction (Fig. 8) demonstrates that the reaction reached completion after about 45 minutes. The pH optimum of the reaction was 7.8 (Fig. 9). The incubation temperature for maximum incorporation was 20” (Table II), with the activity decreasing at higher and lower temperatures. Although the system was initially characterized at a temperature of 36’, the effect of the magnesium concentration on activity was rechecked at an incubation temperature of 20”, and the optimum was found to be the same as at 36”. Stability of the system. The ribosomes and
Ok%-
40
60
Incubation
80
I IO0
I 120
time, min.
FIG. 8. Time course of the incorporation of phenylalanine-1°C by the cell-free system. Each assay tube contained 0.21 mg of ribosomal RNA and 0.38 mg of the 105,OOOg supernatant fraction protein.
OoL 120
Ammonium
chloride,
160 200
240
u mdes
FIG. 7. Effect of NH&l on the incorporation of phenylalanine-1°C by the cell-free system. Each assay tube contained 0.17 mg of ribosomal RNA and 0.33 mg of the 105,OOOg supernatant fraction protein. The amount of NH&l was varied as shown.
the 105,OOOgsupernatant enzymes retained complete activity if they were stored in liquid nitrogen for a period of several weeks. However, storage of the ribosomes or enzyme fraction at, 4’ or -20” for a week resulted in almost complete loss of the incorporating activity. E$ect of various polyamines. Table III shows the effect of several concentrations of spermine on phenylalanine incorporation. When the ribosomes were prepared in buffers without spermine, phenylalanine incorporation was stimulated to a greater extent by increasing the spermine concentration in the assay mixture than if the ribosomes were prepared in the presence of spermine. The effect of two other polyamines on the system is reported in Table III. Responseto other synthetic polynudeotides. Phenylalanine incorporation was stimulated only by poly U and not by poly A or poly C (Table IV). Furthermore, the incorpora-
PROTEIN
SYNTHESIS
tions of the amino acids lysine, isoleucine. and proline \vere not st’imulated by poly U.
Activity
o.f wbqerminated spol‘es.Since one -
I
of the obje&ves understand the
of this study leas to t’ry to rapid increase of i?z vivo
IN FUNGI
19 TABLE
EFFECT
OF POLYAMINKS
INCORPOR.\TION FROM
BY
III ON
THK
PHENYLAL.\NINE-14C
CELL-FREE
Botryodiplodia
Polyamine
SYSTEM
theobrmae Phenylalanine-‘4C (~rmoles)/ms
20 pg spermine 50 pg spermine 100 rg spermine 200 pg spermine 20 pg spermidine 20 rg 3,3’-iminobispropylamine None
incorporated ribosomal RNA0
A
B
808 889 748
370 543 684
331 709
236
704
229
649
238
510
a A: Cell-free extract prepared in the presence of spermine as noted in the &~~TERIALS AND RIETHODS. Each assay tube contained 0.24 mg of ribosomal RNA and 0.46 mg of the 105,OOOg supernatant fraction protein. Ten pg of spermine is present in all of the assay tubes in addition to the amount added. B: Cell-free extract prepared in the absence of spermine. Each assay tube contained 0.15 mg of ribosomal RNA and 0.29 mg of the 105,OOOgsupernatant fract,ion protein. TABLE
06,
90
EFFECT
IV
OF POLYNUCLEOTIDES ON PORATION OF PHENYLAL.4NINE-‘4C’
THE
INCOR-
PH
9. Effect of pH on the incorporation of phenylalanine-14C by the cell-free system. Each assay tube contained 0.25 mg of ribosomal RNA and 0.33 mg of the 105,OOOg supernatant fraction protein. The buffer used in the above experiment xas Tris. FIG.
TABLE EFFECT OF TEMPER.~TURE INCORPORATION BY FROM
Temperature
Boturodiplodia (“C)
II
ON PHENYL.4L.4NINI+‘4C THE CELL-FREE SYSTEM
Phenylalanine-1°C Incorporated (mpmoles)/mg ribosomal RNAa
0.08 1.93
20
2.40 2.03 1.7.5
30 36
None Poly u Poly A Poly c
Phenylalanine-W (wmoles)/mg
incorporated ribosomal RNAa
13.7 598.0 12.6 12.3
(1Each assay tube contained 0.207 mg of ribosomal RNA and 0.260 mg of the 105,OOOg supernatant, fraction protein.
theobromae
0 16 24
Addition
0.92
(LRibosomes were equivalent to 0.173 mg of RNA, and the 105,OOOg supernatant fraction cont.ained 0.38 mg of protein per assay.
protein synthesis during spore germination, ribosomes and the 105,OOOgsupernatant fraction \\-ere also prepared from ungermi-
nated spores. The complete system from the ungerminated sporeswas inactive (Table V). The interchange of the ribosomes and the lOZ,OOOg supernatant fraction from germinated and ungerminated spores indicated that the defect in the ungermina,ted spores was in the 105,000g supernatant fraction since the ribosomes from the ungerminated spores had some activity with
20
VAN
ETTEN
antibiotic known to promote the release of the unfinished peptide chains from ribosomes. In addition, the system is partially inhibited by the ant’ibiotic actidione, which inhibits in vitro amino acid incorporation by yeast but not bacPhenylalanineW incorterial systems (19). Ribonuclease com105,000g Supernatant Ribosomes porated fraction b.wmoles)/ pletely inhibited the system, but deoxyriboaSSaya nuclease had essentially no effect on amino I I acid incorporation. Germinated 2.1 It is interesting that the enzyme fraction Germinated 0.3 from the ungerminated spores is inactive, Germinated Germinated 344.0 while the ribosomes from the spores have Germinated Ungerminated 4.0 fair activity when assayed with the enzyme Germinated Germinated + 258.0 ungerminated fraction from the germinated spores. ExUngerminated 3.9 periments in progress indicate that at least Ungerminated 1.9 part of the inactivity of the enzyme fracUngerminated Ungerminated 5.0 tion from the ungerminated spores is due Ungerminated Germinated 81.2 to t’he extremely low activity of phenylalanyl-sRNA synthetase. The inhibitory n When added, ribosomes from both germinated effect of the enzyme fraction from the unand ungerminated spores were equivalent to 0.102 germinated spores on the complete system mg of RNA/assay; the 105,OOOg supernatant fracfrom the germinated spores is being intion enzymes from both germinated and ungerminated spores contained 0.190 mg of protein/ vestigated also. assay. The assay mixture was incubated at 20”. It may be characteristic of microbial spores that the mechanism for protein synthesis is inhibited during unfavorable the 105,OOOgsupernatant fraction from the conditions and that the inhibition is regerminated spores. The 105,OOOg supermoved during or preceding germination. natant fraction from the ungerminated Staples et al. (2) have reported that ribospores slightly inhibited the complete somes prepared from both germinated and system from the germinated spores (Table ungerminated uredospores of the obligate VI* parasite Uromyces phaseoli have about the same activity when assayed with an enzyme DISCUSSION fraction prepared from rice seedlings, but The amino acid incorporating activity an enzyme fraction prepared from the unof the system prepared from germinated germinated uredospores completely inspores of B. theobromaehas properties and hibits this system. More recenbly, however, requirements similar to those of other systems developed from plants, and micro- Staples and Bedigian have obtained an active enzyme fraction from ungerminated organisms. The incorporation of phenyluredospores (20). Another recent report of alanine by the cell-free preparation is not an in vitro amino acid incorporating due to bacterial contamination since plate system developed during various stages of counts performed on the assay mixture showed that the bacterial population was germination of Bacillus cereus spores indicates that both the ribosomes and transfer negligible (e.g., less than 500 bacteria per enzymes from the dormant’ spores are assay mixture). Furthermore, the antidefective (21). bacterial antibiotics, streptomycin, chloramphenicol, and neomycin, had essentially ACKNOWLEDGMENTS no effect on the amount of phenylalanine The author is indebted for technical help reincorporated. Amino acid incorporation by the cell- ceived at various stages of this work from R. free system is inhibited by puromycin, an Brambl. TABLE
V
PHENYLALANINE-W INCORPORATION BY CELLFREE SYSTEMS PREPARED FROM GERMINATED AND UNGERMINATED SPORES OF Botryodiplodia theobrmae
PROTEIN
SYNTHESIS
REFERENCES W., KORNFELD, J.M., ANDKNIGHT S. G., Arch. Mikrobiol. 63, 41 (1966). 2. STBPLES, R.C.. APP, A. A,, MCCARTHY, W.J., .IND GEROSA, M. M., Contrib. Boyce Thompson Inst. 23, 159 (1966). 3. VAN ETTEN,J.L.,PrZRISI,B.,SND CIFERRI,O., Nature 212, 932 (1966). 4. STAPLES, R. c., SYAMANANDA, R., KAO, V., AND BLOCIC, R. J., Contrib. Boyce Thompson Inst. 21, 345 (1962). 5. GOTTLIEB, D., AND CALTRIDER, P. G., Nature 197, 916 (1963). 6. BaRaSH, I., CONWAY, M. L., AND HOW:~RD, D. H., J. Bacterial. 93, 656 (1967). 7. SHEPHERD, C. J.,J. Gen. Microbial. 16, i (1957). 8. HENNEY, H. R., AND STORCK, R., Proc. Natl. Acad. Sci. U.S. 61. 1050 (1964). 9. HENNEY, H. R., AND STORCK, R.,Science142, 1675 (1963). 10. HENNEY, H., AND STORCK, R., J. Bacterial. 86, 822 (1963). 11. PETERSON, G. W., Phytopathology 67, 825 (1967). 1. H~IDLE,C.
IN
21
FUNGI
12. So, A. G., AND DAVIE, E. W., Biochemistry 3, 1165 (1964). 13. DOWNEY, K.M.,So, A. G., AXD DAVIE, E.W., Biochemistry 4, 1702 (1965). 0. H., ROSEBROUGH,X. J., FARR, A. 14. LOWRY, L., AND RANDALL, R. J., J. Biol. Chem 193, 265 (1951). 15. SCHNEIDER, W. C., Methods in Enzymol. 3. 680 (1957). 16. SUSSMAN, A. S., in “The Fungi” (G. C. Ainsworth and A. S. Sussman, eds.), Vol. 2, p. 733. Academic Press, New York (1966). 17. PARISI, B., AND CIFERRI, 1638 (1966). R. K.,MARCUS, 18. BRETTHAUER, J., HALVORSON, H. O.,
O.,
AND
Biochemistry
6,
L., CHALOUPKA, BOCK, R. M.,
Biochemistry 2, 1079 (1963). M. R., AND SISLER, H. D., Biochim. 19. SIEGEL, Biophys. Acta 103, 558 (1965). R. C., AND BEDIGIbN, D., Contrib. 20. STAPLES, Boyce Thompson Inst. 23, 345 (1967). 21. KOBAYASHI,~., AND HALVORSON, H.O., Bacteriol. Proc. 1967, 22 (1967).