Changes in Isozyme Patterns Between

Transcription

1 JOURNAL OF BACTERIOLOGY, June 1973, p Copyright 1973 American Society for Microbiology Vol. 114, No. 3 Printed in U.S.A. Changes in Isozyme Patterns Between Monokaryons and Dikaryons of a Bipolar Coprinus IAN K. ROSS, ELIZABETH M. MARTINI, AND MARILYN THOMAN Department of Biological Sciences, University of California, Santa Barbara, California 9316 Received for publication 18 January 1973 Proteins and isozymes of several different classes of enzymes in partially purified protein extracts of monokaryons, dikaryons, and monokaryon mixtures of a bipolar Coprinus sp. were separated on polyacrylamide gels by slab electrophoresis. Differences in protein and isozyme spectra were correlated with the operation of the incompatibility factors and with the results of Wang and Raper on Schizophyllum. It was concluded that the shift from monokaryon to dikaryon mediated a major change in the nature, quantity, or distribution of the proteins of this Coprinus sp. The basidiomycetes are eukaryotic organisms characterized by the formation of basidia (often in mushrooms, puffballs, etc.) from a dikaryotic vegetative thallus or mycelium. The dikaryon is generally accepted as the genetic and physiological equivalent of the diploid phase of other organisms and is formed by the union of two compatible monokaryons. In the Coprinus sp. studied here, the monokaryotic strains are distinguishable from the dikaryons only by a more acute branching pattern and the presence of but one nucleus per hyphal segment or cell. Clamp connections, usually associated with dikaryotic hyphae of basidiomycetes, have not been found in this isolate of Coprinus. The factors controlling the mating of two monokaryons are termed incompatibility factors. While many basidiomycetes are tetrapolar and possess two such factors, each with several alleles, the isolate of Coprinus used here is bipolar and possesses only one mating locus, with only two alleles so far isolated, Al and A2 In 197, Wang and Raper (9) reported on isozyme pattern changes and sexual morphogenesis in the tetrapolar basidiomycete Schizophyllum commune. They mated strains that had been rendered isogenic by repeated back-crossing to the same parent and thus differed only at the mating type loci. They expected, therefore, to find differences between monokaryons and dikaryons that were directly related to the modifications necessary to cause the sexually related morphogenetic changes. 183 Since their dikaryons differed from their monokaryons only in having two nuclei per cell rather than one, and in bringing into a common cytoplasm both alleles of each compatibility factor, it was not expected that differences other than those related to the change in morphology would be found. Instead, their results caused them to conclude that practically all of the organisms' arrays of enzymes were functionally altered by events controlled by the four genes of the incompatibility factors. Such an interpretation, if correct, has profound significance conceming the role of the incompatibility factors in regulating overall protein assembly in fungal dikaryons and may even indicate the basis for the fundamental difference between monokaryons and dikaryons-the ability of the latter to undergo differentiation into mature meiosiscompetent mushrooms. Since monokaryons and dikaryons are considered to be functional haploids and diploids, it is possible that such major changes in protein and isozyme patterns occur in other organisms with replicating haploid and diploid phases and that such changes reflect the fundamental differences between the two ploidy states in organisms with alternation of generations. There are several steps that may be taken, two of which are to find whether the protein changes are unique to the tetrapolar S. commune or are common to other dikaryotic basidiomycetes and then to investigate the level at which the change is controlled-deox-

2 184 ROSS, MARTINI, AND THOMAN J. BACTERIOL. yribonucleic acid, messenger ribonucleic acid (RNA), ribosomal, or later in changes in cellular architecture that may affect the assembly and activity of proteins. Smythe and Anderson (8) have shown that techniques of Wang and Raper can be applied successfully to monokaryons of Coprinus lagopus and that different protein patterns could be obtained from wild type and isogenic strains. They questioned the value of using isogenic strains since variations existed among such laboriously derived strains. This encouraged us to use a recently isolated bipolar Coprinus sp. to see whether the results obtained by Wang and Raper with a tetrapolar form were common to other basidiomycete systems. MATERIALS AND METHODS The Coprinus sp. was isolated as a dikaryotic mycelium from the stalk of a single mushroom and was grown on Emerson's YpSs agar (Difco). Twentyfive basidiospores from the first four mushrooms that developed on this dikaryon were isolated and grown into monokaryotic mycelia. Twenty vigorous monokaryons resulted and were crossed in all combinations of two to determine the incompatibility system. Five clones were selected for study, and dikaryons between four of these were made and maintained in culture (5 x 8, 5 x 9, 16 x 9). Both monokaryons and dikaryons grow well in Emerson's YpSs agar, and mushrooms routinely develop on the dikaryons in 12 to 14 days. We have found that an exposure to light is required to induce mushroom development. This light induction is effective only after the dikaryon is 6 or more days old. Prior to the sixth day of maturation, dikaryons are wholly vegetative and do not respond to light. All cultures used in this study were between 3 and 4 days old to prevent any interfering morphogenetic changes. For acrylamide gel studies, 3- to 4-day-old agargrown mycelia were diced with a sterile spatula and used to inoculate 5 ml of liquid YpSs in 125-ml Belco shake flasks. These were incubated at 22 C in the dark on a New Brunswick gyratory shaker at 125 rpm for 72 h. Mycelia were centrifuged free from medium, washed twice with cold.1 M tris(hydroxymethyl) aminomethane (Tris; Sigma), adjusted to ph 7. with HCI (Tris-hydrochloride), and then lyophilized. Lyophilized material was ground over ice in a mortar and pestle for 3 s with.5 M Tris-glycine (ph 8.5) buffer. The homogenates were centrifuged for 1 h at 37, x g before the supernatant fraction was applied to the gels. Electrophoresis was conducted on acrylamide gel slabs in an E.C. electrophoresis chamber (E.C. Apparatus Corp.). Cyanogum (E.C. Apparatus Corp.) gels of 7% for the running gel and 4% for the spacer gel in.5 M Tris-borate (ph 8.9) were used in the discontinuous gel system. The electrode buffer was.5 M Tris-glycine (ph 8.3). Protein contents of the extracted supernatant fluids were determined by the method of Lowry et al. (7), and samples containing between.5 and 1. mg of protein were mixed with a sample gel and loaded into each slot of the slab. Mixtures of monokaryon samples for the control electrophoresis experiment (see Fig. 13 and 14) were mixed and ground together before centrifugation. Electrophoresis was performed at 4 C at a current of 25 V (8mA) until the sample had passed through the spacer gel and then at 4 V (loma) until the tracking dye had traveled 7 to 9 cm from the origin. Gels were removed and stained with amido blue-black for general protein or incubated with the appropriate substrate systems to reveal specific enzymes. Stains, substrates, incubation times, etc., are listed in Table 1. Densitometer tracings were made on a E.C. densitometer. RESULTS The acrylamide gel pattern of soluble acidic protein of five monokaryons and three dikaryons derived from them is shown in Fig. 1. Even though the slab electrophoresis permits direct comparison of different samples run on the same gel, photographic reproductions rarely record all bands present. Figure 2 is a diagram of the total proteins plotted according to R, values. Densitometer readings for these samples are shown in Fig. 3. All five monokaryons are TABLE 1. Staining procedures Enzyme Incubationa time (h) NADH-dehydrogenaseb 2 NAD-dependent dehydrogenasec Sodium L-malate substrated 2 Monosodium glutamate substrated 6 NADPH-dehydrogenasee 2 NADP-dependent dehydrogenase' Sodium L-malate substrated 8 Glucose-6-phosphate substrated 8 Monosodium glutamate substrated 8 Leucine amino peptidaseg 2 Achromatic bandsh 16 a At 25 C. b NADH-dehydrogenase:.1 M Tris-hydrochloride (ph 8.3) buffer (1 ml), p- nitro blue tetrazolium (p- NBT; 4 mg), and NADH (5 mg). c NAD-dependent dehydrogenase:.1 M Trishydrochloride (ph 8.3) buffer (1 ml), p-nbt (4 mg), NAD (4 mg), and substrate. d A 5-gmol amount. enadph-dehydrogenase:.1 M Tris-hydrochloride (ph 8.3) buffer (1 ml), p-nbt (4 mg), and NADPH (5 mg). ' NADP-dependent dehydrogenase:.1 M Trishydrochloride (ph 8.3) buffer (1 ml), p-nbt (4 mg), NADP (4 mg), and substrate. g Compare with reference 1. h Compare with indole phenol oxidase in reference 1.

3 VOL. 114, 1973 ISOZYME PATTERNS IN COPRINUS 185 ent monokaryon patterns, all different from individually different dikaryon patterns; (ii) individually different monokaryons different from a uniform dikaryon pattern; and (iii) a uniform monokaryon pattern different from a uniform dikaryon pattern. The single exception in this work showed the same pattern for all - =mycelia. The patterns shown in Fig. 4 and 5 of nicotinamide adenine dinucleotide, reduced form, (NADH) and NADH phosphate (NADPH) dehydrogenase activity show differences in all six samples. Both patterns show characteristics that might be typical of a dikaryon pattern. NADPH dehydrogenase activity, for example, consistently appears as two sets of bands in the monokaryons and as three sets of bands in the dikaryons. In the case of NADH dehydrogenase, there is a fast component moving just behind the tracking dye front in all dikaryons. Figures 7, 8, and 12 represent patterns in XB 5X9 16X9 which nonuniform monokaryon banding I I - I--- changes to a uniform pattern in the dikaryon. M*ONOKARYONS MONOKARYONSIIDIKARYONSI DIKRONS NAD-dependent dehydrogenases with sodium FIG. 1. Total soluble protein from.5 M Tris-gly- gamatepande sodi maenasses ndi eartdo o7 glutamate and sodium malate as cine (ph 8.5) extracts separated on a 4 to {V ND-eedn eydoeaesubstrates ihgu and cine~~~~~~~~~ (p_.)etat discontinuous gel slab. NADP-dependent dehydrogenases with glu X8 5X9 16X9 MONOKARYONSRYN FIG. 2. Diagram of gel slab shown in Fig. 1, plotted ',9' by Rf values. c Z w. distinct and different, whereas, with the exception of one band, all three dikaryons are virtu- / ally identical. Isozyme patterns for several classes of enzymes have been similarly plotted in Fig l With one exception, these pattems fall into FIG. 3. Densitometric tracings of gel slab shown in three groups: (i) those with individually differ- Fig X 9

4 186 ROSS, MARTINI, AND THOMAN J. BACTERIOL..5 ityfrom.5 M Tris-glycin(pH8.5)extractssep.6 ratedon a 4 to7% disconinuousgeslabwitspecifi X8 5X9 16X9 Rf FIG. 4. Diagram of NADPH-dehydrogenase activity from.5 M Tris-glycine (ph 8.5) extracts separated on a 4 to 7%1 discontinuous gel slab with specific activity determined in.1 M Tris-hydrochloride (ph 8.3) with p-nbt m - ~~~~~~~~ I ~ X8 5X9 16X9 Rf FIG. 5. Diagram of NADH-dehydrogenase activity from.5 M Tris-glycine (ph 8.5) extracts separated on a 4 to 7%o discontinuous gel slab with specific activity determined in.1 M Tris-hydrochloride (ph 8.3) with p-nbt. 1IT cose-6-phosphate as substrate show many differences among the monokaryons but a remarkable imposition of similarity in the dikaryons. Figures 1 and 11, both of NADP-dependent dehydrogenases, show a uniform monokaryon pattem that changes to a different, but still uniform, dikaryon pattern. In Fig. 6 there are slight differences in both monokaryon and dikaryon groupings, but the appearance of a fast-moving band appears characteristic of the dikaryon. This particular enzyme prevents the darkening of the stain in the presence of light and shows up as colorless bands in a dark purple gel. The exact identity of the enzymes is not known, but Brewer (1) indicates that it may be an indole phenol oxidase. The activity of leucine amino peptidase (Fig. 9) was the only one found in this study to remain the same. A control electrophoretic experiment was performed with mixtures of pairs of monokaryon I4-.7 U X8 5X9 16X9 Rf FIG. 6. Diagram of achromatic activity from.5 M Tris-glycine (ph 8.5) extracts separated on a 4 to 7% discontinuous gel slab with specific activity determined in.3 M Tris-hydrochloride (ph 8.) with phenazine methosulfate and 3(4,5-dimethyl thiazolyl- 2)-2,5 diphenyl tetrazolium bromide. -.4 ; ~~~~~~~~~~~ X8 5X9 16X9 Rf FIG. 7. Diagram of NAD-dependent glutamate dehydrogenase activity from.5 M Tris-glycine (ph 8.5) extracts separated on a 4 to 7%o discontinuous gel slab with specific activity determined with monosodium glutamate as substrate in.1 M This-hydrochloride (ph 8.3) with p-nbt and NAD.

5 VOL. 114, 1973 ISOZYME PATTERNS IN COPRINUS 187 extracts to determine whether the isozyme pattern of such a mixture was merely the sum of the two individual patterns or a pattern different from either. NADP-dependent glucose- 6-phosphate and the achromatic band enzyme were chosen because they could both be detected on the same gel without interference and both had a multiplicity of bands. In Fig. 13 and 14 the mixtures of 5 + 8, 5 + 9, and represent mixtures of different mating types, whereas and are mixtures of the same mating types. The results shown in Fig. 13 and 14 indicate that mixed monokaryon samples are similar to the addition of the bands of individual monokaryons and are not the same as true dikaryon patterns. There are a few minor band changes, such as (Fig. 13) the loss of the slowest band of the 5 and 9 monokaryons on mixing and a broad general band in the mixture rather than three separate bands, but x8 5X9 16X9 RF FIG. 8. Diagram of NAD-dependent malate dehydrogenase, determined as described in legend of Fig. 7 with sodium L-malate as substrate XB 5X9 16X9 Rf FIG. 9. Diagram of leucine amino peptidase activity, from.5 M Tris-glycine extracts separated on a 4 to 7% discontinuous gel slab with specific activity determined with L-leucyl-f3-naphthylamide HCI as substrate in.2 M Tris-malic acid (ph 5.2) with Fast Black K. w -~~~~~~~~~~.3 4~ ~ ~ ~ ~~~~~~ KXS 5X9 16X9 Rf FIG. 1. Diagram of NADP-dependent glutamate dehydrogenase activity from.5 M Tris-glycine (ph 8.5) extracts separated on a 4 to 7% discontinuous gel slab with specific activity determined with monosodium glutamate as substrate in.1 M Tris-hydrochloride (ph 8.3) with p-nbt and NADP. w I- ~~~~~~~~~~.3 z X8 5X9 16X9 R, FIG. 11. Diagram of NADP-dependent malate dehydrogenase activity, determined as described in legend of Fig. 1, with sodium L-malate as substrate. none of the mixtures shows the appearance of the new bands characteristic of the dikaryons. DISCUSSION From the evidence presented here, it appears that total soluble protein spectra and seven out of nine enzymes studied show changes from monokaryon types to patterns that could be termed characteristically dikaryotic, while five of those seven show patterns that are uniform in the dikaryon. The patterns found in the dikaryons are not due merely to the chemical addition of two monokaryon patterns, but are an integral function of the intact dikaryon (Fig. 13 and 14). The use of extracts of soluble proteins (obtained by cold-temperature grinding followed by a single rate of centrifugation) automatically discards several major protein populations, particularly insoluble and membrane- and organelle-bound proteins. For this reason, the

6 188 ROSS, MARTINI, AND THOMAN J. BACTERIOL X8 5X9 16X9 RF FIG. 12. Diagram of NADP-dependent glucose- 6-phosphate dehydrogenase, determined as described in legend of Fig. 1, with glucose-6-phosphate as substrate. 5 a 9 IS SXS 55X ESX S +S Rf mow""ousaoi OISAYowS MIXTURE FIG. 13. Diagram of NADP-dependent glucose- 6-phosphate dehydrogenase, determined as described in the legend of Fig. 1. The 12-slot gel slab was subjected to electrophoresis with four monokaryons, three dikaryons and five mixtures of monokaryons (+). protein spectra examined here represent but a small fraction of the total protein content of this Coprinus, but the consistency of the different pattems obtained is, we believe, significant and indicative of possible general effects on most of the proteins of the organism. General protein pattems may be affected by various parameters, and variation in such patterns is often the norm rather than the exception. However, if consistent patterns are found to be associated with particular genetic arrangements, it is not unreasonable to assume that such patterns may be the result, however indirectly, of such arrangements. This is further borne out by the changes in the pattems obtained when the gels were examined for specific enzymes. The actual nature of the multiple staining bands on acrylamide gels stained for specific enzyme activity is not unequivocally understood (1). Multiple bands may be the result of degradation during extraction and processing of a single molecular species or they may be artificial aggregates of protein-subunits released during extraction, or they may represent truly different molecular species having the same enzyme function, and hence justifiably termed isozymes. In the context of this paper, exactly which of these or other explanations is valid is not directly pertinent to the central theme of a change between the monokaryon and the dikaryon states. If the bands are indeed degradation products or artificial aggregates, then the evidence indicates that degradation-aggregation patterns for monokaryons are different from those of dikaryons. This could result from either the protein molecules being different so that the same extraction procedure affects them differently, or from the cell environments being different in ways that result in different degradation-aggregation extractions of the same molecules. If the former, there is a molecular difference possibly resulting from transcriptional or translational differences; if the latter, a physiological or possibly architectural difference: either way, a change resulting from the monokaryon-dikaryon shift. If the multiple bands from any one monokaryon represent true isozymes, i.e., different molecular species with the same enzymatic activity, then the monokaryon-dikaryon shift would be the result of the appearance of different proteins in the dikaryon. Fawole and Casselton (5) have recently examined the specific activities of NAD-dependent and NADP-dependent glutamate dehydrogenases extracted from a monokaryon and a dikaryon of C. lagopus. They found that the specific activities of the two enzymes changed when either the monokaryon 5 a 9 Is '9 S*S a MOO A DISMYO MIXTURES FIG. 14. Diagram of achromatic band activity, determined as described in the legend of Fig. 6. The 12-slot gel slab was subjected to electrophoresis like that described in the legend of Fig. 13.

7 VOL. 114, 1973 ISOZYME PATTERNS IN COPRINUS or the dikaryon was transferred to media differing in nitrogen sources and catabolites, but they also found that the enzymes of the monokaryon responded differently from those of the dikaryon. They did not offer any explanation as to why the enzymes should respond differently in the monokaryons and the dikaryon, but concluded, "Clearly the results can be interpreted as regulation of the enzymes either by different mechanisms, or equally well, by the same mechanism which is responding differently for physiological reasons which may or may not be connected with the different nuclear states." One such regulating mechanism could be the synthesis of different molecules in the dikaryon from those of the monokaryon. Wang and Raper (9) made the tentative suggestion that the products of the incompatibility loci may resemble the "activator-rnabattery-of-producer-genes" model of Britten and Davidson (2) and concluded that the four genes of the A and B factors are capable of controlling the activity of most or all of the enzymes of S. commune. The protein-subunit aggregation model for self-incompatibility in higher fungi (6) offers a mechanism whereby the products of the incompatibility loci may indeed act as general repressors or derepressors, or possibly interact with the molecular architecture of the cell. It is not without reason, we believe, to suggest that a similar effect takes place under the influence of the two genes of this bipolar Coprinus sp. incompatibility system. This is not the place, and the evidence does not warrant any attempt at this time, to determine the nature of this influence, whether transcriptional, by the derepression of a partial or complete set of "dikaryon" genes, or translational by the reading of hitherto masked mrnas, or differential "dikaryon" reading of existing "monokaryon" RNAs, or a function of a changing cell architecture with different binding and activation sites so that monokaryon molecules are differentially extracted from dikaryons. The essential point is to establish the existence of the change and its presence in species other than S. commune. Even though the strains we used were not isogenic, a requirement debated by Smythe and Anderson (8), and this Coprinus is a bipolar form, the results obtained are quite similar to those of Wang and Raper (9) in reference to the. major changes that occur in the transition from monokaryon to dikaryon. Since all the strains used did come originally from a single dikaryon, they are all products of different meioses of a 189 single pair of nuclear types and may be expected to have a certain degree of natural isogenicity. Such a condition is possibly indicated by the similarity of pattern of several enzymes and the presence of several major protein bands in common. However, the lack of isogenicity and the variability among the monokaryons makes it the more remarkable that there is so much uniformity imposed on the final behavior of molecules composing the isozyme and protein spectra so that distinctive dikaryon patterns may be recognized. We think that the imposition of uniformity and the development of a dikaryon pattern of proteins is controlled by the incompatibility loci in basidiomycetes and that this represents an interesting method of protein regulation. The discovery by Casselton (3) that diploid strains of C. lagopus could be derived and that these were quite stable (4) suggests that in Coprinus we have an organism in which it may be possible to compare monokaryons to dikaryons and both to true diploids. We think that the Coprinus system with a single locus of control offers an excellent opportunity to investigate the actual level at which the influence that regulates the change in protein spectra is exerted. ACKNOWLEDGMENTS We would like to thank John R. Raper for kindly reading the manuscript before it was submitted for publication and for the many suggestions he offered, and Marvin Cassman for his review of the electrophoretic techniques. This work was partially supported by National Science Foundation grant GB-8537 to the senior author. LITERATURE CITED 1. Brewer, G. J An introduction to isozyme techniques. Academic Press Inc., New York. 2. Britten, R. J., and E. H. Davidson Gene regulation for higher cells: a theory. Science 165: Casselton, L. A The production and behavior of diploids of Coprinus lagopus. Genet. Res. 6: Casselton, L. A., and D. Lewis Compatibility and stability of diploids in Coprinus lagopus. Genet. Res. 8: Fawole, M. O., and P. J. Casselton Observations on the regulation of glutamate dehydrogenase activity in Coprinus kagopus. J. Exp. Bot. 23: Kuhn, J., and Y. Parag Protein-subunit aggregation model for self-incompatibility in higher fungi. J. Theor. Biol. 35: Lowry,. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein assessment with the folin phenol reagent. J. Biol. Chem. 193: Smythe, R., and G. E. Anderson Electrophoretic protein spectra of wild-type and isogenic monokaryons of Coprinus lagopus. J. Gen. Microbiol. 66: Wang, C., and J. R. Raper Isozyme patterns and sexual morphogenesis in Schizophyllum. Proc. Nat. Acad. Sci. U.S.A. 66: