isozyme analysis in fungal taxonomy and molecular genetics · 2011-08-11 · isozyme analysis in...

In: Arora, Dilip K.; Elander, Richard P.; Mukerji, K.G., eds. Handbook of applied mycology. Vol. 4: Fungal biotechnology. New York: Marcel Dekker, Inc.: 57-79; 1992.

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ISOZYME ANALYSIS IN FUNGAL TAXONOMY AND MOLECULAR GENETICS

J. A. MICALES Forest Service, Forest Products Laboratory, United States Department of Agriculture, Madison, Wisconsin

M. R. BONDE and G. L. PETERSON Agricultural Research Service, United States Department of Agriculture, Frederick, Maryland

I. INTRODUCTION

Isozyme analysis is n powerful technique which has many applications in plant pathology and mycology. For years, its use was restricted to studies in fungal taxonomy, often with ambiguous results [1]. Concurrently, geneticists were using this procedure to examine the population genetics of fish [2-5], mammals [6,7], insects [8,9], nematodes [10], and plants [11]. Isozyme analysis is now used by mycologists to resolve taxonomic disputes, identify unknown fungal taxa, "fingerprint" patentable fungal lines, analyze the amount of genetic variability in a population, trace the origin of pathogens, follow the segregation of loci, and identify ploidy levels throughout the life cycle of an organism. This topic has been previously re-viewed [12,13], but the many advances in recent years necessitate its revision.

Enzymes which are coded by different alleles or separate genetic loci frequently possess different electrophoretic mobilities. Such differences arc due to variations in the amino acid content of the molecule, which in turn is dependent on the sequence of nucleotides in the DNA. Electro-phoretic banding patterns are frequently predictable since they are depend-ent on the genetic and nuclear condition of the organism. Most mycologists and plant pathologists have not taken advantage of this relationship and restrict their interpretation of electrophoretic data to simple "band-counting" techniques [1,14]. A genetic interpretation of the data frequently provides much additional information about the genetics and taxonomy of a group of organisms. Genetic crosses may be necessary to confirm genetic inter-pretations, but certain banding patterns are readily recognizable from comparable studies in human and animal genetics [15].

II. APPLICATIONS OF ISOZYME ANALYSIS

A. Delineation of Fungal Taxa Isozyme analysis is most frequently used to address taxonomic problems, particularly when morphological characteristics overlap or are plastic within

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a genus or species. The application of electrophoretic techniques to syste-matics has been reviewed [16]. A genetic interpretation of banding patterns may not be necessary to allow separation of fungal taxa; genetic similarities and distances may be calculated by simple band-counting procedures. Alter-natively, the interpretation of banding patterns in terms of specific alleles allows the determination of ratios of alleles expressed in common among fungal isolates. These ratios are an excellent means of determining phylo-genetic relationships among organisms. In studies of closely related species, the assumption is made that the same loci are present and are being com-pared among isolates, even though the organisms may be from different species and the number of chromosomes may not be the same.

Extensive electrophoretic studies have been made of fungi pathogenic to insects and mites [17-26], mycorrhizal fungi [27-29], as well as many plant pathogens and saprophytes (as discussed below).

Isozyme analysis has been applied at many different taxonomic levels. It generally is most successful at distinguishing species and has been used to make recommendations on the separation or combination of species. For example, Hodges et al. [30] and Micales at al. [31] used isozymeanalysis to confirm the conspecificity of Endothia eugeniae (Nutman and Roberts) Reid and Booth and Cryphonectria cubensis (Bruner) Hodges, thus supporting studies on cultural characteristics and pathogenicity of these organisms [30]. Also, the close relationship of Peronosclerospora sacchari (T. Miyake) Shirai and Hara and P. philippinensis (Weston) C. G. Shaw, important pathogens of sugarcane and maize in the tropics, has been demonstrated by starch gel electrophoresis [32,33]. Differences in isozyme banding patterns have been used to separate species of Agaricus [34], Aspergillus [35], Botrytis [36], Gaeumannomyces [37], Glomus [27], Penicillium [38], Phytophthora [39,40], Pleurotus [41-43], Puccinia [44-46], Rhizopogon [28], and Scytinostroma [47], and intersterilily groups (bio-logical species) of Heterobasidion annosum (Fr.) Bref. [48] and Armillaria [49, 50].

The ability of electrophoresis to distinguish fungal species is dependent on the amount of genetic variation within the population. Organisms with high levels of genetic variability will express large levels of intraspecific variation, thus obscuring interspecific differences. The choice of enzyme systems also is important. Johnson [51] proposed, and many studies [52-55] support the concept, that different types of enzymes exhibit different degrees of variation due to the intensity of selection pressure. Regulatory enzymes involved with energy metabolism often display less variation than nonregulatory enzymes, such as esterases and phosphatases. Any study that utilizes only nonregulatory enzymes may detect disproportionately high levels of intraspecific variation arid thus be unable to distinguish species. This problem can be avoided by screening for both regulatory and nonregulatory enzymes.

Electrophoresis also has been used to identify and distinguish sub-specific taxa. Varieties and subspecies of Phytophthora megasperma Drechsler [39] and Leptographium wayeneri (Kendrick) Wingfield [56, 57] have been differentiated. Faris et al. [58] used isozyme patterns to detect two subgroups of Phytophthora megasperma var. medicaginus which also expressed diverse cultural characteristics and differed in pathogenicity. Different researchers were unable to detect subspecific and varietal differ-ences in other fungi including Gaeumannomyces graminis (Sacc. ) Arx and Olivier [37] and Scytinostroma galactinum (Fr.) Donk [47]. Mixed

Isozyme Analysis in Fungal Taxonomy 59

results also have been obtained in differentiating races, formae speciales, and subgroups differing in host preference, pathogenicity and virulence. Such separations have been made for some fungi, including Cryphonectria cubensis [59], Fusarium oxysporum Schlect. emend. Snyd. and Hans. [60], Puccinia graminis Pers. [46], Puccinia sorghi Schwein. [44] and Phyllotopsis nidulans (Pers. ex Fr.) Sing. [ = Pleurotus nebrodensis (Pers. ex Fr. ) Kurrimer] (42). Burdon and Roelfs [61] were able to find such a correlation in asexual populations of P. graminis f. sp. tritici; this asso-ciation did not occur in sexual populations which had a much higher degree of genetic variability. More commonly, host preferences, pathogenicity, and virulence are not correlated with differences in isozyme patterns, e.g., Erysiphe graminis DC ex Murat f. sp. hordei [62], Pleurotus ostreatus (Jacq. ex Fir.) Kummer [41], Rhizopogon [28], and Rhynchosporium secalis (Oud.) Davis [54].

The geographic source of an isolate sometimes can be determined by isozyme analysis. Such information can be used to determine the origin of introduced pathogens and to trace the development of epiphytotics. Local populations of Morchella delictosa Fr., Morchella esculenta (L.) Pers. [63], and Neurospora intermedia Tai [64] were shown to be genetically distinct. Bonde et al. [65] were able to separate isolates of Phakopsora pachyrhizi Sydow, the causal agent of soybean rust, collected from the eastern and western hemispheres. The differences were so great that two species may be causing rust on soybeans. Isozyme patterns have also been used to trace independent introductions of Puccinia recondita Robs. ex Desm. to the United States [66] and P. graminis f. sp. tritici to Australia [67]. Another potential application of isozyme analysis is the tracking of organisms specifically released for biological control of either weed or plant pathogens.

B. Identification of Unknown Fungi The capabitity of using isozyme analysis to distinguish taxa leads to its application in the identification of unknown fungi.

The importance of the correct identification of pathogens cannot be overstated. Regulatory agencies at the federal and state levels in the United States must correctly identify pathogens of regulatory significance. In some cases, only a few spores may be available for examination; isozyme analysis is a tool which can greatly facilitate identification. Scientists at Foreign Disease-Weed Science Research Unit (FD-WSRU) are routinely using isozyme techniques to examine stored grain, storage facilities, and trans-portation vehicles for teliospores of Tilletia indica Mitra, causal agent of Karnal bunt of wheat. This organism has not yet been found established in the United States but has been intercepted in shipments of grain from Mexico. It is easily confused with the endemic pathogen Tilletia barclayana (Bref.) Sacc. and Syd., which causes kernal smut of rice. These two organisms are difficult to differentiate by spore morphology but are easily distinguished by isozyme analysis [69].

Within any species, some genetic loci tend to have little or no variation among individuals. These loci are termed "monomorphic" and are defined as loci in which 99% or more of the individuals express the most common allele. It is, the presence of these monomorphic loci which should be stressed when comparing unknown isolates to previously identified cultures in order to determine identity. The amount of genetic variation within fungi is

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extremely variable [68], but almost all species have some monomorphic loci which can be used for this purpose.

C. Genetic Variation Isozyme patterns can be used as markers to estimate the amount of variation in a fungal population or species. They are more neutral than virulence markers since they are not usually exposed to the strong selection pressures of the host. As genetic markers, isozymes are useful for studying population structure, following epidemics, tracing origins of new pathogenic forms, and analyzing crosses [68].

The amount of polymorphism detected by isozyme analysis in recent studies of fungal species is presented in Table 1. One factor which must be considered in evaluating any estimate of genetic variability is the quantity of individuals and the number of loci examined. The best estimates of polymorphism are obtained when large numbers of isolates and enzyme systems are utilized. Results may vary among experiments if widely diverse sample sizes and enzyme systems are employed [56, 57].

Levels of genetic variability appear to be quite diverse among fungi. Newton [68] correlated the amount of polymorphism in a species with the physiological specialization of the fungus. Obligate parasites and highly specialized pathogens, which have a relatively uniform substrate and environment, exhibited low levels of genetic variability. Facultative patho-gens and saprophytes, which have a much wider host range and are exposed to more diverse substrates and environments, expressed much greater genetic variation. Recent studies (Table 1) do not entirely support this concept. Uromyces appendiculatus (Pers. ) Unger, the causal agent of bean rust, is an obligate, autoecious, macrocyclic pathogen. yet 67% of its isozyme loci were polymorphic [70]. A worldwide sampling of Puccinia graminis f. sp. tritici revealed that 61% of its loci were polymorphic [71]. In contrast, Fusarium oxysporum, a pathogen with a broad host range, exhibited only 24% polymorphism [60]. The definition of species is essential to evaluations of genetic diversity. Leung and Williams [72] determined that Pyricularia oryzae Cavara collections from rice exhibited limited poly-morphism (11%) compared to isolates from 34 other graminaceous hosts (55%). In such a case, traditional taxonomic criteria may not reflect the true genetic condition of the fungus, and more than one species may actually exist.

Other researchers have suggested that the amount of polymorphism corresponds to the degree of sexual reproduction in a population or species. Burdon et al. [73] determined that there was no genetic variability among Puccinia graminis f. sp. tritici collections in Australia, where the fungus is maintained asexually. Collections from the United States, which under-went sexual reproduction until the 1920s, when the barberry was eradicated, exhibited 38% polymorphism [61]. Cow levels of isozyme diversity also were associated with populations of Phakopsora pachyrhizi [74] and Puccinia striiformis West. [46]; neither pathogen is known to have a functioning sexual cycle. In contrast, sexual populations of Uromyces appendiculatus displayed less genetic diversity than asexual populations [70]. The authors concluded that mutation and selection would lead to greater genetic diver-gence and higher levels of polymorphism in asexual populations since there is no exchange of genes. Lack of genetic variation may indicate that a pathogen has been introduced to an area only recently and that insufficient


TABLE 1. Percentage of Polymorphic Loci in Fungal Populations and Species

Percent Species polymorphism Ref.

Pleurotus ostreatus (Pers. ex Fr.) Sing.

Agaricus campestris Fr. Uromyces appendiculatus (Pers. ) Unger Heterobasidion annosum (Fr. ) Bref., inter-sterility group "spruce" Puccinia graminis Pers. f. sp. tritici (worldwide) Pyricularia oryzae Cavara (graminaceous hosts) Lentinula edodes (Berk.) Pegler Phytophthora infestans (Mont. ) deBary Tillletia indica Mitra Agaricus brunnescens Peck Cryphonectria cubensis (Bruner) Hodges Puccinia graminis f. sp. tritici (United States) Leptographium wageneri (Kendrick) Wingfield

Volvariella volvacea (Bull. ex Fr.) Sing. Fusarium oxysporum Schlect. emend Snyd. and Hans. Peronosclerospora sorghi (Weston and Uppal) C. G. Shaw

Phytophthora cinnamomi Rands Heterobasidion annosum, intersterility group "pine"

Ustilago spinificis Ludw. Pyricularia oryzae (rice isolates) Ustilago bullata Berk. Puccinia recondita Robs. ex Desm. f. sp tritici

Puccinia striiformis West. f. sp. tritici

Puccinia striiformis f. sp. hordei

Puccinia graminis tritici (Australia) Puccinia hordei Otth.

93 132 87 77 67 70

60 48 61 71 55 72 55 88 54 80

44,52 69,133 43 89 38 31 38 61

30,48 56,57 29 83

24 60

23 32 23 75

20 48 12 134 11 72 11 76

9 73

0 46 0 46

0 73

0 46

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time has elapsed to allow for variation [75]. Low levels of variation also may indicate that a pathogen has developed from a limited number of intro-ductions [72]. Clearly, genetic diversity can result from several different factors.

Isozyme analysis can be used to help interpret the amount of hetero-zygosity in a population. Here the assumption must be made that the isozyme loci tested are representative of the organism's genome; this may be in-correct. Heterozygous banding patterns are often readily recognizable (Section VI). The frequency of heterozygous loci appears to be quite varia-ble in fungi. Bonde et al. [69] estimated 7.3% heterozygosity in Tilleria indica. This level of heterozygosity is comparable to 6.3% reported for humans [15]. Kirby [76] estimated a mean heterozygosity level of 4.8% in populations of Ustilago bullata Berk. in Australia and New Zealand, whereas Burdon et al. [73] obtained estimates of 33 and 15% for populations of P. graminis f. sp. tritici and P. recondita f. sp. tritici, respectively. May and Royse [77] estimated that 28% of the loci were heterozygous for Agaricus campestris Fr. The amount of heterozygosity may be quite different among closely related taxa. No heterozygous loci were detected for the wood decay fungus Scytinostroma galactinum. The heterozygosity of two subspecies of Scytinostroma protrusum (Burt) Nakas, varied from 12.5% (S. protrusum subsp. protrusum Nakas.) to 37.5% (S. protrusum subsp. septentrionale Nakas. ) [47]. Two different intersterility groups of Heterobasidion annosum exhibited heterozygosity levels of 6 and 2% [48]. in most instances, estimates of heterozygosity should be considered conservative since electrophoresis may only detect 2 third of the heterozygosity that actually exists [78]. Likewise, overestimates of heterozygosity can occur for heterokaryotic species since different genetic nuclei may be present [75].

D. Identification of Crosses, Hybrids, and Genetic Lines Isozyme banding patterns have been used as genetic markers to trace crosses and to identify hybrids in the field and the laboratory. Burdon et al. [79] used isozyme analysis to determine that a common Australian race of P. graminis f. sp. tritici originated as a somatic hybrid of other races. An additional study [45] with P. graminis demonstrated that collec-tions made from the Australian grass Agropyron scabrum (Labill. ) Beauv. originated as a somatic hybrid of P. graminis f. sp. tritici and P. graminis f. sp. secalis. Tooley et al. [80] studied the distribution of proteins coded by alleles at two polymorphic loci and determined that mating patterns of Phytophthora infestans (Mont.) deBary were random in Mexico, where the sexual stage of the pathogen exists. Almost every individual in the asexual population, including isolates from the United States, Canada, and Europe, but not Mexico, was heterozygous at these two loci, thus demonstrating the near absence of mating. The lack of recombication of mating types also occurred among Phytophthora cinnamomi Rands isolates in Australia [75, 81]. In the laboratory, isozyme analysis has been used to analyze the progeny of crosses [82-85] and to prove that protoplast fusion has occurred between parental strains [86, 87]. The variability of polymorphic loci can also be used to "fingerprint" patentable lines of edible mushrooms [84,88-90], biological control agents [91,92], and other commer-cially important fungi.


E. Segregation and Linkage of Loci

Careful analysis of the inheritance of isozyme patterns can reveal whether the loci which code for individual enzymes segregate independently or are linked. Genetic maps of the chromosomes also can be constructed. Royse et al. [93] traced the inheritance of six segregating loci in Lentinula edodes (Berk.) Pegler. Random segregation was observed for five of the loci; joint segregation with a putative lethal allele was proposed for the locus coding for aspartate aminotransferase. The loci coding for glucose phosphate isomerase and phosphoglucokinase were shown to be linked. Spear et al. [94] determined that the loci coding for two peptidases in Agaricus brunnescens Peck were linked; the frequency of crossing over was used to estimate the distance of the two loci from the centromere. Similar linkage studies have been conducted with Agaricus campestris [77],Volvarrella volvacea (Bull. ex Fr.) Sing. [83], P. graminis f. sp. tritrici [71], and Ustilago bullata [95].

F. Determination of Ploidy Levels and Tracing Life Cycles The pattern of isozyme bands can sometimes be used to determine the ploidy level of a fungal isolate; studies of the life cycle of the organism can thus be performed. Haploid and homozygous isolates should produce single-isozyme bands. Heterozygous dikaryotes, diploids, and organisms with higher ploidy levels typically produce more complicated banding patterns. Expected banding patterns are discussed in Section VI. Such interpretations should be confirmed by genetic crosses or cytological studies since secondary, artifactual bands can arise for a variety of reasons (Section VI. C), thus invalidating a genetic interpretation.

Atypical meiosis also has been detected by isozyme analysis. Bonde et al. [74] showed that basidiospores arising from germinated teliospores of Tilletia indica did not inherit alleles with equal frequency and that some basidiospores appeared to inherit both alleles. The authors proposed that some basidiospores may receive two haploid nuclei from the promycelium or that the spores are actually aneuploids. This interpretation has been supported by cytological evidence [96]. Atypical meiosis has also been demonstrated in homokaryotic lines of Agaricus brunnescens [94].

III. SELECTION OF FUNGAL TISSUES ANDSAMPLEPREPARATION

Electrophoresis can be performed successfully with a wide variety of fungal tissues, including vegetative mycelia, spores, basidiocarps, and sclerotia. Care must be taken in the selection of fungal tissues since inconsistent results may be obtained when a mixture of different tissues is used. Such variation may be due to differential expression of alleles at different stages of development or from variations in the nuclear condition, as in the ex-pression of monokaryotic and dikaryotic stages of an organism. Variations in catalase banding patterns have been reported for Fusarium solani (Mart. Sacc. [97]. Similar differences in isozyme banding patterns have been observed among different portions of the basidiocarp in Lentinula edodes [98], Coprinus cinerens (Schaeff. : Fr.) [99], and Agaricus brunnescens [100]. Variations in isozyme patterns reported for Schizophyllum commune Fr. at different stages of the life cycle [101] have been disputed by other researchers [102,103].

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Proteins can be extracted from spores, mycelia, or fruiting bodies which have been grown in culture or collected in the greenhouse or field. Uniform conditions of light, temperature, and nutrition should be used when sample material is grown in culture in order to minimize the chance for the differential expression of induced enzymes. Ideally, samples should be collected at a specific stage of the growth curve: this is usually diffi-cult to coordinate, and most researchers harvest their material after a specified period of growth. Initial studies should be conducted to determine the amount of electrophoretic variation detected at different stages of the growth cycle. The amount of protein in the mycelium can also vary with the stage of growth; protein assays [104,105] should be conducted to determine the time of maximum protein production. Culture media should be used that allow rapid growth of the fungus; slowly growing cultures often possess low enzyme activity.

Proteins can be extracted from fungal tissues by a number of different procedures. Thin-walled structures, such as certain conidia and vegetative tissues, can be crushed with a glass rod after the sample has been frozen in liquid nitrogen. Lyophilization is frequently used with vegetative tissues and large fruiting bodies; this process removes the water from the sample and allows it to be crushed readily. The preparation of acetone powders can be used in lieu of lyophilization although we have obtained lesser quantities of enzymes with this technique. Large fungal structures, such as basidiocarps, ascocarps, or mycelial mats, can be disrupted with a variety of commercial homogenizers or simply crushed in a chilled mortar and pestle. Sample buffer should be added to frozen and freeze-dried samples before they thaw or rehydrate; nonfrozen samples should be dis-rupted in sample buffer. The amount of buffer which should be added will vary with the nature and size of the sample and is best determined empirically. There is a thin line between the addition of enough buffer to generate adequate sample size and diluting the sample below the level of detection. Proteins in a sample may precipitate or aggregate if they are in low concentrations, if insufficient sample buffer is added, or if the sample buffer has the wrong pH or ionic strength.

The denaturation of enzymes must be prevented during and after homogenization. Samples should be kept cold (below 4°C) during preparation and storage. Agents which inhibit proteases and stabilize enzymes such as phenylmethylsulfonyl fluoride (PMSF) and polyvinylpyrrolidone (PVP), can be added to the sample buffer to maintain enzyme activity. Sample preparation is the most critical step in isozyme analysis and other electro-phoretic techniques. Poor resolution, faint staining of bands, and irregular banding patterns are frequently caused by the incorrect choice of sample buffer or improper extraction techniques. Troubleshooting techniques associated with extraction procedures have been described [13].

IV. ELECTROPHORETIC TECHNIQUES

A variety of electrophoretic procedures, including starch gel electrophore-sis, polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, and two-dimensional electrophoresis, have been used to separate isozymes. Each technique has advantages and disadvantages. The goal of this chapter is to acquaint the reader with the different procedures rather than to provide detailed protocols. References will be provided for more elaborate treatments of each technique.


A. Starch Gel Electrophoresis Starch gel electrophoresis is one of the oldest electrophoretic procedures, and it is still one of the best for isozyme analysis. Most of the major studies in fungal genetics and taxonomy have been conducted with this technique. It is inexpensive, uses nontoxic chemicals, and can be used to screen rapidly for many different enzyme activities. The preparation of starch gels is something of an art and requires some practice in order to obtain reproducible electrophoretic patterns. Many different systems are available commercially, but most researchers build their own equipment quite inex-pensively. Highly detailed descriptions of equipment and electrophoretic procedures are available [13, 106-110]. A list of troubleshooting tips was assembled by Micales et al. [13].

Two types of experiments are usually conducted with starch gel electro-phoresis. A "screening" run should be conducted early in a study with a limited number of isolates [8-10] and a large number of stains and buffer systems. This experiment will determine which enzymes are active in the taxon and which buffers provide optimal resolution and activity of each enzyme. Many different buffer systems have been used for starch gel electrophoresis. The taxonomy and genetics of large numbers of isolates can then be studied in subsequent "analytical" runs, using the enzyme systems determined to be best in the screening run. The arrangement of samples on the gel depends on the type of experiment. In screening runs at FD-CVDRU, eight to 10 samples are repeated four times across the width of the gel. Dye markers are placed between each group to iden-tify places where the gel will be cut following electrophoresis. A larger number of samples are used for analytical experiments. The sequence of samples should be repeated at least once in an analytical run if sufficient space is available; variations in migration in different portions of the gel can then be detected. This greatly facilitates interpretation of the results. It is advisable to repeat one or two samples at intervals on the gel in order to provide a standard for comparison. If specific comparisons are desired among isolates, those samples should be placed in close proximity to ensure accurate analysis.

Starch gels can be quite thick and must be adequately cooled. Special-ized cooling plates are commercially available, and ice packs can also be used. This thickness dramatically increases the number of enzymes which can be analyzed at any one time. The gel can be sliced vertically between each group of samples, and thin horizontal slices can also be made with a commercial gel slicer or with nylon thread and Plexiglas supports. Slicing techniques are illustrated in Micales et al. [13]. More than twenty different pieces can be easily obtained from a single gel. Each slice is then placed in a staining tray. Staining procedures are described in Section V.

B. Polyacrylamide Gel Electrophoresis PAGE also is commonly used to separate proteins. It usually provides much better resolution than starch gel electrophoresis, and more complex banding patterns are usually encountered. There is a greater chance that secondary, "artifactual" bands will be detected due to the increased resolution. Un-fortunately, fewer staining procedures are available for PAGE and only a small number of enzymes can be studied at one time. Detailed procedures for PAGE are presented by Hames [111] and Andrews [112]; the former contains a useful list of troubleshooting procedures.

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C. isoelectric Focusing Isoelectric focusing has also been used to study the distribution of isozymes, primarily in taxonomic studies [37, 40, 49, 50, 113-117]. In isoelectric focusing, a pH gradient is established across the gel due to the inclusion of highly charged moieties, termed "ampholytes," in the gel. Proteins migrate to their isoelectric point, where they have a net charge of zero. Excellent resolution is obtained with isoelectric focusing, and more is learned about the physical properties of the enzymes with this technique. Unfortunately, proteins often precipitate at their isoelectric point and may lose activity. Isoelectric focusing cannot be used for the genetic interpretations in Section VI, but simple band-counting techniques can be substituted. Detailed protocols are presented by Andrews [112], An der Lan and Chram-bach [118], and Righetti and Drysdale [119].

D. Two-Dimensional Electrophoresis This technique has only been used rarely in taxonomic studies. Torp and Andersen [62] separated 174 proteins from Erysiphe graminis f. sp. hordei, the causal agent of barley powdery mildew. Howes et al. [120] used two-dimensional electrophoresis to separate races of P. graminis f. sp. tritici; they detected 290 separate polypeptides with this technique. As demon-strated by these two examples, two-dimensional electrophoresis has tremen-dous resolution, and quantification of the numerous protein spots can be a major problem. In two-dimensional electrophoresis, proteins are separated by two different parameters, typically isoelectric focusing in the first dimension and PAGE or SDS-PAGE in the second dimension. Proteins are usually inactivated by this procedure and thus are detected by general protein stains. Detailed descriptions on the theory and practice of two-dimensional gel electrophoresis are presented by Sinclair and Rickwood [121] and Andrews [112].

V. STAINING PROCEDURES

Specific activity stains are used to detect the location of specific enzymes after completion of electrophoresis. Detection of specific enzymes is possible because the appropriate substrates and cofactors required for activity are provided in the staining solution. The enzymatic reaction then forms a colored product, either through direct activity with a dye or by involving other enzymes in a series of reactions with the generation of a colored product as a final result. Fluorescent products can be detected with ultra-violet light ; alternatively, nonfluorescent products can also be visualized as a negative stain by reacting the starch with a fluorescent compound. The biochemistry of such staining reactions has been discussed by Gabriel [122] and Vallejos [123].

Staining recipes for many different enzymes have been described [13, 15, 107, 108, 110, 122-124]. At FD-WSRU, stain components are weighed into prelabeled styrofoam cups during an electrophoretic run. This can be done quite rapidly if stains with similar reagents are grouped together. One or two key enzymes and cofactors should be left out initially to prevent premature reactions. They can be added just before the staining solution is poured over the gel. Many stains are light-sensitive. so that develop-ment of the gels should be done in the dark. Reactions occur more rapidly


if the staining reactions are performed in a 37°C incubator. Agar overlays can be used for enzymes with low activity or soluble products. This is done by incorporating the staining reagents and buffer into 6-8% molten agar and immediately pouring the mixture over the gel. A more detailed description is provided by Vallejos [123].

VI. GENETIC INTERPRETATION OF BANDINGPATTERNS

Genetic interpretations of electrophoretic banding patterns can result in a better understanding of the nuclear condition and genetic makeup of an organism. A brief summary of the principles of genetic interpretation is provided; the reader is also referred to more detailed accounts [15, 124].

Isozymes are defined as multiple molecular forms of a given enzyme. These forms usually have similar, if not identical, enzymatic properties. Isozymes can occur within a single individual or among different individuals of the same species or taxa. Isozymes also can be localized in specific tissues of an organism or be compartmentalized within different areas of the cell. Only those isozymes with amino acid compositions of different net charge, or those which result in large differences in the shape of the enzyme, will be differentiated by electrophoresis. This represents only one-third of all possible isozymes which may be present within a genetic system [78].

Detectable isozymes can arise from three different genetic and bio-chemical phenomena: (a) multiple allelism at a single locus; (b) multiple loci coding for a single enzyme; and (c) posttranslational processing and the formation of secondary isozymes [15]. All of these possibilities must be considered when trying to interpret electrophoretic data.

A. Multiple Alleles at a Single Locus Variations in banding patterns frequently are caused by the expression of multiple alleles at a single locus, each allele coding for a structurally distinct polypeptide chain. Isozymes which are coded by alleles at a single locus are termed "allozymes." The actual electrophoretic banding pattern is dependent on the number of alleles present at the locus, which in turn is dependent on the nuclear condition (monokaryotic, dikaryotic), ploidy number (haploid, diploid, polyploid), and genetic makeup (homozygous, heterozygous) of the organism. Individuals which are haploid or homozygous will produce simple banding patterns due to the expression of a single allele. Organisms which are dikaryotic or diploid and heterozygous will produce more complex banding patterns due to the expression of two sepa-rate alleles.

Banding patterns are also dependent on the quaternary structure of the enzyme. Enzymes which are monomeric are composed of a single poly-peptide chain; monomeric isomers of a heterozygote will appear as a mixture of the allozymes produced by the two corresponding homozygotes (Fig. 1). In contrast, multimeric enzymes consist of two or more polypeptide chains; hetcromeric (hybrid) bands will be produced by heterozygous individuals as the result of the random combination of polypeptide chains which produce hybrid enzyme molecules. Heteromeric bands cannot occur unless different alleles (coding for the different polypeptide chains) are present in the

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FIGURE 1. Predicted banding patterns for one locus with two alleles (A and A') and two segregating loci which share the same two alleles for monomeric, dimeric, and tetrameric enzymes of a diploid organism. Geno-types are listed beneath each banding pattern; alleles are designated by capital letters. The subunit composition of each protein band is shown on the right; lower case letters refer to subunit designations. The expected ratios of banding intensity for each phenotype is presented beneath the genotype. (Originally adapted from Ref. 3 and reproduced from Ref. 13.)

same individual. The expected banding patterns for monomeric, dimeric, and tetrameric enzymes coded by two alleles at a single locus are presented in Fig. 1. The frequency of occurrence of the different allozyme molecules, assuming random combinations of polypeptide chains, should follow Mendelian ratios. The ratio of isomers formed in heterozygotes will be 1:1 for mono-mers, 1:2:1 for dimers, 1:3:3:1 for trimers, and 1:4:6:4:1 for tetramers. In some cases, a particular polypeptide will not contribute equally to the activity of the enzyme due to a slow rate of synthesis, low stability, or a tendency to break down before it can be assembled into the final enzyme. A particular polypeptide chain can also reduce the effectiveness of an enzyme by decreasing its stability or reducing its catalytic ability, such enzymes would not be detected in the expected ratios of intensity on the electrophoretic gel. A complex series of banding patterns is produced when three or more alleles arc present in a population [15]. Further examples of banding patterns of dimeric and tetrameric enzymes are dia-grammed in Fig. 2.


FIGURE 2. Predicted banding patterns of dimeric (A) and tetrameric (B) enzymes coded by a single locus and three electrophoretically distinct alleles (A. A', and A") of a diploid organism. Genotypes are listed below each banding pattern. The subunit composition of each protein band is shown on the right; lower case letters refer to subunit designations. (Originally adapted from Ref. 3 and reproduced from Ref. 13.)

B. Multiple Loci Multiple loci also can code for a structurally distinct series of isozymes: up to four different loci have been reported for some enzymes. Multiple loci are thought to originate from gene duplications and subsequent diver-gence by point mutations; they are frequently expressed in different tissues of an organism or compartmentalized in different areas of the cell. The distribution of loci is usually constant within a given species [15]. Isozymes coded by different loci are frequently detected in separate regions of the gel due to their differences in charge and conformation from that usually associated with multiple alleles at a single locus. Heteromeric bands can be formed by polypeptides coded by different loci, and banding patterns can thus become quite complex when a number of different loci and alleles are involved (Fig. 3). For this reason, it is often difficult to interpret banding patterns of enzymes which are not substrate-specific (such as esterases and peroxidases).

C. Secondary Isozymes Banding sequences may not appear to follow expected genetic patterns due to posttranslational processing and other events which form secondary

70 Micales et al.

FIGURE 3. Predicted banding patterns of a dimeric enzyme coded by two loci which share the same three electrophoretically distinct alleles (A, A', and A"). Genotypes are listed below each banding pattern. The subunit composition of each protein band is shown on the right; lower case letters refer to subunit designations. (Originally adapted from Ref. 3 and reproduced from Ref. 13. )

isozymes. Common modifications include the deamidation of glutamine and asparagine residues, acetylations, oxidation of sulfhydryl groups, additions and removals of carbohydrate and phosphate moieties, cleavage of the enzyme by proteases, and aggregation or polymerization of the protein. The formation of such secondary isozymes is usually quite uniform within a species or group and can be recognized by the production of a series of bands for each allele [15].

Another process responsible for the generation of secondary isozymes is conformational isomerism. Some enzymes may have several stable con-figurations which vary in tertiary or quaternary structure ; these will frequently have different electrophoretic mobilities. All configurations are usually isolated from a single preparation, and a series of electrophoretic bands will be observed for a single allele.

Enzymes which require cofactors, such as NAD and NADH, may vary in their electrophoretic mobility depending on the degree of saturation of the enzyme with the cofactor. The amount of cofactor in the staining solution must not be limiting or inconsistent results are likely to occur. It may be necessary to add the cofactor to the sample buffer and/or the gel buffer in order to maintain the stability of the enzyme during electro-phoresis [15].

Artifact bands can also arise from the proteolysis of the sample during extraction and storage. Sample material can be preserved at -80°C or below, but it should not be kept for more than a year as activity will generally decrease even at ultracold temperatures. Some enzymes, such as those located in membranes, may be bound to other cell constituents and will not migrate freely in an electrophoretic field. Alternate methods of sample


extraction and storage may be necessary. Stabilizing agents can be added to the sample to protect the enzymes from other cell constituents [15].

VII. GENIC NOMENCLATURE AND PRESENTATION OF RESULTS

Taxonomic and genetic information can be summarized by several different methods. The technique of May et al. [126], as further described by Bonde et al. [32], allows for direct comparison among experiments regardless of absolute migration distances as long as standard alleles are included in each test. Alleles at a single locus are described by the relative anodal or cathodal movement from the origin as compared to the movement of the homomeric protein products coded by the most frequently observed allele (which is designated 100). A dimeric heterozygote (or heterokaryon), which produces three bands on the electrophoretic gel, would be designated by two different alleles (e. g., 100/130). A diploid homozygous individual would be described by two identical alleles (e. g., 100/100 or 130/130). The frequency of the shared alleles can be used to calculate the coefficients of similarity or distance among organisms. Several different formulas can be used to determine these quantities [125, 127, 128] ; commercial computer programs are available to do these calculations [129]. The final information can be summarized in the form of a dendrogram or by cluster analysis. This type of diagram can be constructed with a variety of clustering pro-cedures [125, 130, 131] using commercial computer programs.

VIII. SUMMARY

Isozyme analysis is not a new technique, but only recently has it been used to help address important problems of fungal taxonomy and genetics. A virtual explosion of publications is anticipated in this area within the next few years. Objections raised by "classical" geneticists are subsiding as genetic interpretations of banding patterns are being confirmed by crossing experiments. Isozyme analysis will soon become a standard tool for plant pathologists and mycologists, and will help resolve many mysteries of the fungal kingdom.

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