Changes in messenger RNAs and protein synthesis during germination of Rhizopus stolonifer sporangiospores

EXPERIMENTAL MYCOLOCY 2, 313--325

(1978)

Changes in Messenger RNAs and Protein Synthesis during Germination of Rhizopus stolonifer Sporangiospores~ SHELBY N . FRF-~R AND JAMES L . VAN ETTEN

Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68583 Received August 14, 1978 FREER, S. N., AND VAN ETTEN, J. L. 1978. Changes in messenger RNAs and protein synthesis during germination of Rhizopus stolonifer sporangiospores. Experimental Mycology 2, 313-325. Sporangiospores of Rhizopus stolonifer were pulse-labeled with pS]methionine at various times during germination in order to identify germination-specific proteins, In addition, the in vivo synthesized proteins were compared with in vitro synthesized products encoded by either total messenger RNA or polysomal messenger RNA isolated at comparable time periods to determine if the majority of the proteins synthesized during spore germination were under transcriptional or translational control. We conclude that, with few exceptions, the majority of the proteins synthesized were under transcriptional control. A few proteins were synthesized only at discrete time periods during the germination process and, thus, may be germination-specific proteins. However, at no time during germination did we find a germination-specific protein synthesized which represented a major fraction of the total proteins synthesized. INDEX DESCRIPTORS: Messenger RNA; proteins; translational control; transcriptional control; spores; Rhizopus stolonifer. Fungal spore germination provides a useful experimental system for investigating the concept that cellular development and resumption of growth from a dormant or quiescent state involves unique macromolecular biosynthetic activities. Although protein synthesis is required for germ tube formation in all fungi, essentially nothing is known about the nature and function of the earliest proteins synthesized or about the type of control over protein synthesis operating in the spore (Brambl et al., 1978). Furthermore, there is little informa1 Published with the approval of the Director as Paper No. 56"22, Journal Series, Nebraska Agricultural Experiment Station. The work was conducted under Nebraska Agricultural Experiment Station Project No. 21-17.

tion about the differences in proteins synthesized throughout the germination process (Van Etten et al., 1972; Silverman et al., 1974; Wenzler and Brambl, 1978). Indeed, it is not known whether fungal spores synthesize specific proteins essential for germ tube emergence, or whether the same complement of proteins are synthesized throughout germ tube formation and vegetative growth. In the present paper we examine the spectrum of proteins synthesized in vivo at various times during germination of Rhizopus stolonifer (Ehr. ex Ft.) Lind. sporangiospores in order to determine if germination-specific proteins are synthesized. In addition, the in vivo products are compared with in vitro synthesized products

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014%5975/78/0024-0313502.00/0 Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

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directed by either total messenger (m) RNA or polysomal mRNA isolated at comparable time periods to determine ff the majority of the proteins synthesized during spore germination are under transcriptional or translational control. MATERIALS AND METHODS

Materials. L-[a~S]Methionine (550 Ci/ mmol) and x.-[3H]leueine (55 Ci/mmol) were purchased from New England Nuelear Corp. DNase was purchased from Worthington Biochemical Corp., and protease K was obtained from Miles Laboratories. Techniques for the growth, harvest, and germination of R. stolonifer sporangiospores were described previously (Dunkle and Van Etten, 1979). Pulse-labeling and extraction of total cellular proteins. Fifty-milliliter samples (equivalent to 50 mg of ungerminated spores) were removed from 100-ml cultures at various times, and the spores were harvested by filtration. The spores were resuspended in fresh medium (5 ml) containing L-[asS]methionine (10-25 t~Ci/ml) and incubated with rapid shaking for an additional 15 minutes. Total cellular proteins were extracted by a procedure modified from that of L. C. Lane (personal communication). Pulselabeled spores were combined with 10 g of glass beads (0.5 mm), 9. ml of 80% ( w / w ) phenol, 0.1 M ammonium acetate, 5 mM dithiothreitol, and 2 ml of 0.08 M tris(hydroxymethyl) aminomethane ( Tris ),2 pH 6.8, 0.01 ~ ethylenediaminetetraacetie acid (EDTA), 0.1 M ammonium acetate, and 50 t~g/ml of phenyl methyl sulfonyl fluoride in a 30-ml Corex centrifuge tube, and disrupted by vortexing at maximum speed for 2 minutes (Van Etten and Freer, 1978a). After centrifugation, the phenol 2 Abbreviations used: Tris, tris(hydroxymethyl)aminomethane; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl sulfate; Hepes, N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid.

phase was extracted twice with an equal volume of the aqueous buffer. The proteins were precipitated from the phenol phase with 5 vol of methanol which contained 0.1 ~ ammonium acetate and stored at -20°C. The precipitate was collected, washed twice with methanol, dried under vacuum, and resuspended in an appropriate volume of 80 mM Tris, pH 8.8, 10 mM EDTA, 5 m ~ dithiothreitol, 270 ( w / v ) sodium dodecyl sulfate (SDS), and 0.02570 ( w / v ) mTstal violet just prior to eleetrophoresis. Extraction of total R N A and polysomal RNA. The procedures used to isolate total BNA from the cells were identical to those described previously (Van Etten and Freer, 1978b). The ethanol-precipitated nucleic acids were resuspended in 2.0 ml of 0.01 M Tris, p H 7.4, 0.01 ~ KCI, and 0.01 M MgC12, and treated for 20 minutes at room temperature with DNase (50/~g/ml). Two milliliters of 0.01 M Tris, pH 7.4, 0.01 EDTA, 17o ( w / v ) SDS were added, and the samples were incubated with protease K (20 ~g/ml) for 20 minutes at room temperature. An equal volume of phenol:chloroform :isoamyl alcohol (50:48:2) was added, and the nucleie acids were precipitated from the aqueous phase with ethanol. The RNA was dissolved in a small volume of 0.075 M NaC1 and 0.0075 M sodium citrate, pH 7.0, (0.5 x SSC), and desalted on a 2.5 × 20-cm column of Sephadex G-25 equilibrated in 0.5 × SSC. The RNA was reprecipitated with ethanol, dissolved in sterile, deionized water, and stored at -80°C. Polysomes were isolated by a procedure modified from that of Freer et al. (1977). Spores (50-100 mg) were combined with 10 g of glass beads (0.5 mm) in a 30-ml Corex centrifuge tube and vortexed (15 s, maximum speed) (Van Etten and Freer, 1978a) in 5 ml of 0.02 ~ N-2-hydroxyethylpiperazine - N' - 2 - ethanesulfonic acid (Hepes), pH 7.9, containing 0.06 ~ KC1,

PROTEINS SYNTHESIZED DURING SPORE GERMINATION 0.03 M MgC12, and 0.25 ~ sucrose. The homogenates were centrifuged at 12,000g for 15 minutes to remove cell debris. A polysomal pellet was obtained by layering the supernatant over 1 ml of 600 mg/ml of sucrose in the same buffer and centrifuging (8 hr, 4°C, 45,000 rpm) in a Spinco SW-50.1 rotor. The pellets were suspended in 0.05 M Tris, pH 9.0, 0.2 ~ NaC1, 10-4 EDTA, 1% ( w / v ) SDS; and deproteinated with an equal volume of 80% phenol containing 0.01% ( w / w ) 8-hydroxyquinoline. The phases were separated, and the phenol phase was extracted with an equal volume of aqueous buffer. After centrifugation, the aqueous phases were combined and extracted with an equal volume of phenol: chloroform : isoamyl alcohol (50:48 : 2), and the RNA was precipitated with ethanol. The samples were suspended in 0.5 × SSC, reprecipitated, dissolved in sterile deionized water, and stored at - 8 0 ° C . Preparation of the cell-free extract and translation of exogenous RNA. In vitro translation of fungal RNA was carried out with the wheat germ cell-free system as described ,by Roberts and Paterson (1973) using a reaction mixture identical to that described in the preceding paper (Van Etten and Freer, 1978b). Each wheat germ extract was optimized with respect to K + and Mg ~+ concentrations to give products of maximum molecular weight. The largest polypeptides were not necessarily formed under the same conditions which gave the maximum incorporation of isotope into hot trichloroacetic acid-insoluble material. The final concentrations of KC1 and magnesium acetate were about 100 and 3.0 mM, respectively. The in vitro synthesized translational products were treated as described previously (Van Etten and Freer, 1978b). All samples, including the in vivo products, were heated at 100°C for 2 minutes just prior to electrophoretic analysis. Electrophoresis of samples and interpre-

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tation of gels. The labeled peptides were electrophoresed on polyacrylamide gradient slab gels and examined by fluorography as described in the preceding paper (Van Etten and Freer, 1978b). The procedures used only enabled us to detect proteins which contain methionine. Since the blackening of the film is proportional to the amount of radioactivity and the exposure time (Laskey and Mills, 1975), the number of methionine residues in a protein influences the estimation of its relative abundance. In all experiments, an equal amount of radioactivity from each sample was placed on the gels. Thus, the degree of blackening of the film measures the rate of synthesis of a peptide relative to the total cellular proteins or total cell-free translational products. Therefore, any increases or decreases in band intensities between samples indicate that the rate of synthesis of that particular peptide has increased or decreased relative to the total proteins in that particular sample. It does not mean that the absolute rate of synthesis of that peptide has increased or decreased. The protein bands from the fluorograms in Figs. 1, 4, and 5 are represented schematically in Fig. 2; Fig._ 2 was drawn after examining several films exposed to the same gel for various lengths of time. The fluorograms shown in Figs. 1, 4, and 5 only represent one exposure time. Comparisons between different gels were made visually by superimposing small areas of two fluorograms. In this way, it was possible to match equivalent polypeptide bands between gels. Other procedures, t/NA was always heated to 65°C for 5 minutes before in vitro translation in order to dissociate preformed aggregates. RNA concentrations were determined by assuming i mg of RNA/ml had an A26o~m of 24 after correcting for light scattering (Bonhoeffer and Schachman, 1960). all-Labeled proteins (phosphorylase A,

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PROTEINS SYNTHESIZED DURING SPORE GERMINATION bovine serum albumin, alcohol dehydrogenase, chymotrypsinogin A, myosin, eytochrome c) were prepared by the method of Montelaro and Reuckert (1975) and run on each gel as molecular weight standards. All experiments described in this report were performed two or more times, and the examples cited represent typical results. RESULTS Polyacrylamide gel electrophoresis of proteins synthesized in vivo during various 15-minute pulse-labeling periods with L[8~S]methionine is shown in Fig. 1. Eighty distinct polypeptide bands were identified on the fluorogram. Alterations in the relative rate of synthesis of 52 of these bands could be detected during germination. The polypeptides on this fluorogram are numbered consecutively from the highest molecular weight to the lowest. The bands, which change in intensity relative to the other bands within that sample, are marked on the edge of the fluorogram and are represented schematically in Fig. 2A. The diffuse manner in which the 0- to 15-minute sample migrated is due to an extraction problem characteristic of dormant spores. Several other extraction procedures were tried with limited success. The reproducibility of any given extraction procedure was quite variable when dormant spores were used. The procedure finally chosen gave the most reproducible results of the methods tested. Changes in the in vivo protein patterns. The majority of the differences between the polypeptide patterns of spores pulse-

317

labeled from 0 to 15 and 45 to 60 minutes involved increases in the relative rate of synthesis of 20 polypeptide bands (e.g., bands 24, 42, and 60) (Figs. 1 and 2A). The initial synthesis of 11 polypeptides (e.g., 1, 38, and 64) was detected for the first time in the 45- to 60-minute sample. Four polypeptides (2, 39, 54, and 69) were synthesized in the first 15 minutes of germination but were not detected in tile 45to 60-minute sample. By 2 h, approximately 25% of the spores had swollen. At this time, the relative rate of synthesis of four polypeptides (34, 41, 43, and 63) increased. The synthesis of band 23 was first detected at 105 to 120 minutes, while the synthesis of band 26 ceased at this time. Three hours after the initiation of germination, approximately 75% of the spores had swollen, yet none had germ tubes. The synthesis of five new polypeptides appeared at this time (12, 30, 39, 46, and 57), and one polypeptide (11), which was visible in the 2-h sample, disappeared. Changes in the relative rate of synthesis of three polypeptides (9, 19, and 23) were also detected at this time. Approximately 5 to 10% of the spores had germ tubes by 4 h. Two polypeptides were detected for the first time (37 and 53) one polypeptide disappeared (21), and changes in the relative rate of synthesis occurred for 10 polypeptides (e.g., 13, 24, and 30). After 5 h, about 60% of the spores had germ tubes. The synthesis of one peptide (69), which was only detectable in the 0-

Fia. 1. Polyacrylamidegel eleetrophoresis of proteins synthesized at various times throughout the germination of R. stoloni[er spores. Spores at 10 mg/ml were pulse-labeled for 15minute periods with 10 to 25 /zCi/ml of r~-[~S]methionine. Proteins were extracted and prepared for electrophoresis as described in Materials and Methods. Slot 0 refers to spores pulse-labeled from 0 to 15 minutes, slot 1 from 45 to 60 minutes, slot 2 from 105 to 120 minutes, slot 3 from 165 to 180 minutes, slot 4 from 225 to 240 minutes, slot 5 from 285 to 300 minutes, and slot 6 from 345 to 360 minutes. Each slot contained 8 × 10~ cpm, and the film was exposed to the gel for 16 h. Standard 8H-labeled proteins were co-electrophoresed on the gel, and their molecular weights are indicated on the right.

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PROTEINS SYNTHESIZED DURING SPORE GERMINATION to 15-minute sample reappeared. The relative rate of synthesis of five polypeptides (6, 56, 60, 62, and 63) decreased, while the rate of synthesis of polypeptides 37 and 55 increased. Six hours after the initiation of germination, 9 5 ~ of the spores had germ tubes. No new polypeptides were synthesized, nor did the synthesis of any of the proteins which were previously being synthesized cease. However, decreases in the relative rate of synthesis of seven polypeptides (6, 7, 9, 17, 20, 56, and 70) occurred. Translation of total BNA. In order to understand the regulation of a particular gene, one must be able to identify the gene product and quantitate the amount of mRNA encoded by that gene. It is possible to estimate the amount of a specific mRNA by translating it in a cell-free translational system. For example, Alton and Lodish (1977a) demonstrated that the amount of actin produced in the wheat germ cell-free protein-synthesizing system was directly proportional to the amount of translatable actin mRNA present in Dictyostelium discoideum cells.

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The translation of mRNAs in the wheat germ system and the subsequent identification of the products by polyaerylamide gel electrophoresis are subject to several complications. One problem is that proteins which undergo post-translational modification in vivo probably will not undergo similar modifications in the cell-free system. For example, some proteins are synthesized as larger precursors and then processed to the mature protein (preproalbumin to proalbumin to albumin; Strauss e~ al., 1977). In addition, many proteins are phosphorylated, acetylated, glycosylated, or covalently bound to lipids after translation. Such proteins would probably not be detected as translation products in the wheat germ system. Another inherent problem with the cell-free wheat germ system is that the translation of some mRNAs is prematurelly terminated. Premature termination can be partially overcome by altering the K ~ and Mg 2+ concentrations; however, it cannot be completely eliminated. If one uses the in vitro wheat germ system to quantitate the amount of a mRNA present in a cell, the system must meet

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certain criteria. The first of these requirements is that the amount of protein synthesized in the wheat germ system should be a linear function of the amount of exogenous mRNA present. Figure 3 shows that

the amount of L-[3H]leueine incorporated into hot triehloroaeetic acid-preeipitable material was a linear function of the amount of exogenous RNA. This was true of all the I1NAs extracted, both total IINA

PROTEINS SYNTHESIZED DURING SPORE GERMINATION and polysomal RNA, at various times throughout germination. However, the RNAs extracted from spores at different times during germination did not stimulate the system to the same degree. Total RNA isolated from dormant and 1-h spores was only about 25 to 50% and 75% as eflqcient, respectively, in serving as mRNA as the RNAs isolated at later times. Also, if too much spore RNA was added, the in vitro protein synthesizing system was inhibited (Fig. 3A); this did not occur with t/NA isolated from germinated spores (Fig. 3B). The nature of the inhibition by the spore RNA is not known. Another criterion is that the endogenous background of the wheat germ system must be low enough that it does not interfere with the identification of products. As can be seen in Fig. 4 (slot E), the endogenous protein products were barely visible and did not interfere with the identification of products directed by the exogenous RNA. Figure 4 also indicates that total RNA isolated from spores at various times throughout germination encodes for many polypeptides which migrate at the same rate as authentic R. stolonifer polypeptides. However, not all of the polypeptides synthesized in rive were identifiable among the in vitro translation products. The largest peptide (19) which was detected when fungal RNA was translated in the wheat germ system had a molecular weight slightly greater than 100,000. Differences in the mRNA population were detected within 15 minutes after the initiation of germination (Fig. 4 and schematically in Fig. 2B ). The cell-free translation products of tlNA isolated after 15 minutes include bands 19 and 70. These polypeptides were not visible in the cellfree translation products directed by dormant spore RNA. The relative rate of synthesis of many of the products increased in the 15-minute sample (e.g., 32, 46, 64,

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and 73), while the relative rate of synthesis of one protein (54) decreased. At 1 h, three new polypeptides were detected (40, 57, and 66) while the synthesis of polypeptide at band 54 disappeared. Also, there were changes in the relative rate of synthesis of several other polypeptides (e.g., 31, 41, and 57). The synthesis of polypeptide at band 33 was first detected at 2 h. At this time, the relative rate of synthesis of one polypeptide decreased (63), while four others increased (34, 38, 40, and 57). The only changes observed at 3 h into germination were the initiation of synthesis of polypeptides 39 and 55 and the accelerated relative rate of synthesis of polypeptides 44, 57, and 66. Four hours after the initiation of germination, band 53 appeared. The only other observable change in the protein patterns was a decrease in the relative rate of synthesis of polypeptide at band 44. At 6 h, five polypeptides showed a decrease in their relative rates of synthesis (20, 24, 34, 43, and 67). The majority of the changes in the patterns of the translatable mRNAs, as detected by their in vitro translation products, indicates that the mRNA encoding a particular protein was detected in the cell slightly before the appearance of the protein in vivo (e.g., 19, 25, 38, 41, 43, and 63). Also, the relative rate of synthesis of several proteins labeled in vivo and the abundance of their mRNAs showed parallel changes (e.g., 24, 27, 28, 29, and 31). Translation of polysomal mRNA. In order to measure qualitatively as well as quantitatively the "active" mRNA population at discrete times during germination, polyseines were purified, and the polysomal mRNAs were translated in the wheat germ system. The results of these experiments are reported in Fig. 5 and schematically in Fig. 2C. Fewer differences in the translatable polysomal mRNA population were observed during germination than in the

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PROTEINS SYNTHESIZED DURING SPORE GERMINATION total mRNA population. The only differences between the translation products directed by dormant spore mtlNA and 15minute tlNA were changes in the relative rates of synthesis of several peptides (e.g., 20, 31, 42, and 60). The synthesis of polypeptides 54 and 69 ceased at 1 and 2 h, respeetively. The relative rate of synthesis of several polypeptides changed during the remainder of the germination period while the synthesis of polypeptide at band 56 could not be detected at 6 h. In general, these results indicate that many of the mtlNAs encoding a particular protein (41, 43, 63, 69, and 70) were associated with polysomes slightly before the deteetion of the produet in vivo. Also, many parallel changes in the amounts of "active" mRNA and the relative rate of synthesis of a protein labeled in vivo oecurred (e.g., 28, 29, 31, 42, 54, 60, and 61). Although the majority of the changes in the in vivo protein patterns and the amount of mtlNA present correlated well, exeeptions were observed. For example, the synthesis of polypeptide 57 was detected easily after 3 h in in vivo labeled spores. The mBNA was detected after 2 h in total RNA extraets; however, the mtlNA was not detected in the polysomal t/NA extraets. Conversely, protein 55 was just detectable in in vieo labeled spores. The mBNA was detectable in low quantities after 3 h in total t/NA extracts, but the polysomal fractions were enriched in this mRNA. DISCUSSION Gradient slab gel electrophoresis was used to analyze the protein patterns pro-

323

dueed by germinating R. stolonifer sporangiospores. The total mRNA and polysomal mRNA populations were analyzed by isolating either total cellular RNA or polysomal RNA at various times throughout germination and translating this RNA in the wheat germ cell-free system. In this report we have assumed that polypeptides which migrate at the same rate on polyacrylamide gels (both in comparisons on the same gel and between gels) are identical. However, this is an oversimplification; what can be said is that the polypeptides have identical molecular weights. Thus, the interpretation of the results in this paper are subject to this qualification. The protein patterns from in vivo labeled spores revealed that several proteins were synthesized at specific stages of germination. The majority of these changes occurred immediately after the initiation of germination or just about the time of germ tube emergence (3-4 h). When the protein patterns from spores pulse-labeled from 0 to 15 and 45 to 60 minutes were compared, 11 proteins were identified in the later sample which were not detected in the early sample. In addition to these changes, the relative rates of synthesis of 20 of the proteins were altered. At 2 h, the major changes were in the relative rates of synthesis of several proteins. Either the initiation or cessation of synthesis of six proteins could be detected at 3 h and of three proteins at 4 h into germination. The majority of the changes that occurred in the 5- and 6-h samples were the result of a change in the relative rates of synthesis. At no time did we find a major germination-specific protein during germination;

Fro. 5. Polyacrylamidegel electrophoresis of proteins synthesized by the wheat germ cellfree system programmed with polysomal RNA from R. stolonifer isolated from dormant spores (slot 0), 15 minutes (slot 15'), 1 h (slot 1), 2 h (slot 2), 3 h (slot 3), 4 h (slot 4), 5 h (slot 5), and 6 h (slot 6) into germination. Each slot contained 3.5 X 10~ epm (from L-[3~S]methionine), and the film was exposed to tile gel for 17 (bottom of the figure) or 40 h (top part of the figure). ~H-Labeled protein standards were co-eleetrophoresed, and their molecular weights are given on the right.

324

FREER AND VAN ETTEN

i.e., one peptide that represented a major fraction of the total protein; however, a few proteins specific to various times during germination were identified. In general, the majority of the changes which oeeurred with germination were changes in the relative rates of synthesis. Several investigators ( Staples et al., 1968; Brambl and Van Etten, 1970; Brambl, 1975; Mirkes, 1974) have demonstrated that dormant fungal spores contain polysomes and, thus, a priori mRNA. However, except for the recent report of Wenzler and Brambl (1978), no one has shown that the produets translated from spore mtlNA resemble authentic proteins. Figure 4 demonstrates that RNA isolated from dormant spores is translated in vitro into proteins which migrate at the same rate as R. stolonifer proteins synthesized in vivo. Thus, dormant spores of R. stolonifer contain preformed functional mRNAs. During germination, the majority of the changes in the mRNA population occur just prior to or coincidental with changes in the proteins labeled in vivo. Thus, the majority of the proteins synthesized during R. stoloriifer spore germination appear to be regulated at the transcriptional level. However, this is not the only type of regulation. Several of the mt/NAs appear to be masked (30, 37, 46, 57, and 69), since the mRNAs for these proteins can be detected in vitro several hours before the synthesis of these proteins can be detected in vivo. Alton and Lodish (1977b) described another type of regulation in D. discoideum cells in which specific proteins were synthesized ir~ vivo early in differentiation; however, the mRNA remained in a translatable form in the cells for several hours. From our results, it appears that polypeptide 26 exhibits this type of control. In vivo, this polypeptide cannot be detected after 2 h, but the mtlNA coding for this protein is present throughout germination.

Polysomal BNA directs the synthesis of many, but not all, of the R. stolonifer proteins synthesized in vivo. In general, the presence of polysomal mBNAs occurs slightly before or parallel with the appearanee of the in vivo labeled peptides (41, 54, and 70). This provides additional support for the proposition that the synthesis of the majority of the proteins is controlled by the amount of mRNA present in the cell; i.e., they are under transcriptional control. However, this control mechanism does not explain all of our results. For example, band 57 is easily detected in the in vivo sample, and the mRNA is easily detected in total RNA. However, this mlqNA was not detected in the polysomal RNA. Conversely, polypeptide at band 55 is synthesized sparingly in the in vivo samples, and its mRNA cannot be detected until 3 h in total RNA extracts. However, this mRNA is present in very high amounts in the polysomal RNA extracts. Thus, it appears that certain mRNAs may have different affinities for the ribosomes. In summary, we show that the preformed mRNA in the dormant spore codes for the synthesis of many proteins and that the protein patterns vary during germination. There are a few specific proteins which are synthesized only at discrete times during germination and thus may be germination-specific; however, at present, nothing is known about the function of any of these proteins. ACKNOWLEDGMENTS We are indebted to Les Lane for his helpful advice and to Bryan MeCune for his technical assistance. This investigation was supported in part by Public Health Service Grant AI-08057 from the National Institute of Allergy and Infectious Diseases. REFERENCES ALTON,T. H., A~D Lonis~, H. F. 1977a. Developmental changes in messenger RNAs and protein synthesis in Dieti]ostelium discoideum. Develop. Biol. 60: 180-206,

PROTEINS SYNTHESIZED DURING SPORE GERMINATION

ALTON, T. H., AND LODISH, H. F. 1977b. Translational control of protein synthesis during the early stages of differentiation of the slime mold Dictyostelium discoideum. Ceil 12: 301-310. BONHOEFFER, F., AND SCttACHMAN, H. K. 1960. Studies on the organization of nucleic acids within nucleoproteins. Biochem. Biophys. Res. Commun. 2: 366-371. BRAMBL, R. 1975. Presence of polyribosomes in conidiospores of Botryodiplodia theobromae harvested with nonaqueous solvents. J. Bacteriol. 122: 1394-1395. BBAMBL, R., DUNKLE, L. D., AND VAN ETTEN, J. L. 1978. Nucleic acid and protein synthesis during fungal spore germination. In The Filamentous Fungi (J. E. Smith and D. R. Berry, Eds.), Vol. 3, pp. 94-118. Edward Arnold (Publishers) Limited, London. BRAMBL, R. M., AND VAN ETTEN, J. L. 1970. Protein synthesis during funga] spore germination. V. Evidence that the nngerminated conidiospores of Botryodiplodia theobromae contain messenger ribonucleic acid. Arch. Biochem. Biophys. 137: 442-452. DUNKLE, L. D., ANn VAN ETTEN, J. L. 1972. Characteristics and synthesis of deoxyribonucleic acid during fungal spore germination. In Spores V (H. O. Halvorson, R. Hanson, and L. L. Campbell, Eds.), pp. 283-289. American Society of Microbiology, Washington, D.C. FREER, S. N., MAYAMA, M., AND VAN ETTEN, J. L. 1977. Synthesis of polyadenylate-containing RNA during the germination of Bhizopus stolonifer sporangiospores. Exp. Mycol. 1: 116127. LASKEY, R. A., AND MILLS, A. D. 1975. Quantitative film detection of *H and 14C in polyacrylamide gels by fluorography. Eur. ]. Bioehem. 56: 335-341. MIRKES, P. E. 1974. Polysomes, ribonucleic acid, and protein synthesis during germination of

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Neurospora crassa conidia. J. Baeteriol. 117: 196-202. MONTELARO, R. C., AXD RUEC~ERT, R. R. 1975. Radiolabeling of proteins and viruses in vitro by acetylation with radioactive acetic anhydride. J. Biol. Chem. 250: 1413-1421. ROBERTS, B. E., AND PATERSON, B. M. 1973. Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell-free system from commercial wheat germ. Pro& Nat. Acad. Sci. USA 70: 2330-2334. SILVERMAN, P. M., HuH, M. M. O., AND SUN, L. 1974. Protein synthesis during zoospore germination in the aquatic phycomycete Blastocladiella emersonii. Develop. Biol. 40: 59-70. STAPLES, R. C., BEDIGIAN, D., AND WILLIAMS, P. H. 1968. Evidence for polysomes in extracts of bean rust uredospores. Phytopathology 58: 151-154. STRAUSS, A. W., DONOHUE, A. M., BENNETT, C. D., RODKEy, J. A., AND ALBERTS, A. W. 1977. Rat liver preproalbumin: in vitro synthesis and partial amino acid sequence. Proc. Nat. Acad. Sci. USA 74: 1358-1362. VAN ETTEN, J. L., AND FREER, S. N. 1978a. Simple procedure for disruption of fungal spores. Appl. Environ. Microbiol. 35: 622-623. VAN ETTEN, J. L., AND FREER, S. N. 1978b. Potyadenylate-containing RNA in dormant and germinating sporangiospores of Rhizopus stolonifer. Exp. Mycol. 2: 301-312. VAN ETTEN, J. L., ROKER, H. R., AND DAVIES, E. 1972. Protein synthesis during fungal spore germination: Differential protein synthesis during germination of Botryodiplodia theobromae spores. 1. Bacteriol. 112: 1029-1031. WENZLER, H., AND BRAMBL, R. 1978. In vitro translation of polyadenylate-containing RNAs from dormant and germinating spores of the fungus Botryodiplodia theobromae. J. Bacteriol. 135: 1-9.