gm: ~. , “ (“tn-w. H ...‘. My”. ,1 ,. ‘ EVIDENCE FOR CYTOPLASMIC EXCHANGE IN MATINGS 0F SCHIZOPHYLLUM COsMaMUNE 'Ehesis for the Degree of Ph. D. MICRIGAN STATE UNWERSITY LEDIA S!CAR1WATRUD 1 972 mare...“ ~—...—.--—. This is to certify that the thesis entitled EVIDENCE FOR CYTOPLASMIC EXCHANGE IN MATINGS OF SCHIZOPHYLLUM COMMUNE. presented by Lidia Sicari Watrud has been accepted towards fulfillment of the requirements for 43h n degreein Botany and Plant Pathology Major professor 1/ Date 31013 A; /‘/ 7X 0-7639 yd?” ‘9 ‘ amomc BY 7 NOAH 8r SUNS' “z 8le BINDERY INC. .‘r‘ARY BINDERS ! .. ~ r. regiment“; l ABSTRACT EVIDENCE FOR CYTOPLASMIC EXCHANGE IN MATINGS OF SCHIZOPHYLLUM COMMUNE BY Lidia Sicari Watrud The objective of the present study was to deter- mine if cytoplasmic exchange occurs in compatible and incompatible matings of the tetrapolar Basidiomycete Schizophyllum commune. Methods were developed to differ- entially label partners in a mating based on the selective uptake of cobalt by mitochondria. Mitochondria so labeled were distinguishable cytologically and ultrastructurally, and could be separated by isopycnic sucrose density grad- ient centrifugation. Matings were analyzed by three methods: 1) direct microscopic examination of individual anastomoses to see if cobalt-stained mitochondria were transferred from the strain grown in the presence of cobalt to the strain grown in the absence of cobalt, 2) microscopic analysis of mycelial plugs of a resident grown in the absence of cobalt to see if cobalt-stained Lidia Sicari Watrud donor mitochondria were present, 3) density gradient anal- ysis of non-labeled residents to determine the presence of mitochondria which were more dense because of cobalt labeling. Mitochondrial transfer was detectable in fully compatible, common-5, and common-gg matings, but not in common-§ matings. Possible inter-relationships between the observed phenomenon of mitochondrial transfer with the time of nuclear migration and mode of action of the incompatibility factors are discussed. EVIDENCE FOR CYTOPLASMIC EXCHANGE IN MATINGS OF SCHIZOPHYLLUM COMMUNE BY Lidia Sicari Watrud A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1972 «q 0&4 Ga DEDICATION To Lee, for his love and understanding To Buck, for being To Mom and Dad, con affetto ACKNOWLEDGEMENTS Special thanks are due to my major professor, Dr. Albert H. Ellingboe and to the members of my guidance committee, Dr. Edward C. Cantino, Dr. Robert P. Scheffer, and Dr. Delbert E. Schoenhard, for meaningful discussions during the progress of this work. The capable technical assistance of Mrs. June Mack in electron microsc0pic procedures is also gratefully acknowledged. iii TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . . . . . . . Vi LIST OF FIGURES O O O O O O O O O O O O O I O O O O Vii INTRODUCTION. . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . 5 MATERIALS AND METHODS . . . . . . . . . . . . . . . 19 Maintenance of Cultures . . . . . . . . . . . . 19 Determination of Mitochondrial Transfer at Points of Anastomosis . . . . . . . . . . . . 20 Determination of Long-Distance Transfer of Mitochondria. . . . . . . . . . . . . . . . . 21 Isolation of Mitochondria . . . . . . . . . . . 22 Succinic Dehydrogenase Assay. . . . . . . . . . 24 Cytochrome Oxidase Assay. . . . . . . . . . . . 25 Electron Microscopy . . . . . . . . . . . . . . 27 Extraction of Mitochondria from Matings . . . . 28 RESULTS . . . . . . . . . . . . . . . . . . . . . . 32 Determination of Tolerance for Cobalt . . . . . 32 Kinetics of Visual Uptake of Cobalt . . . . . . 33 iv TABLE OF CONTENTS (cont.) Page Checks on Cobalt Diffusion on Y-Plates. . . . . 34 Direct Observations of Individual Anastomoses . 37 Determination of Long-Distance Transfer . . . . 41 Characterization of Mitochondria from Mycelia Grown on Co-lOOO ppm and MC Media . . . . . . 43 Electron Microscopy of Mitochondria . . . . . . 53 Analysis of Matings . . . . . . . . . . . . . . 56 DISCUSSION. . . . . . . . . . . . . . . . . . . . . 68 SUMMARY . . . . . . . . . . . . . . . . . . . . . . 78 LITERATURE CITED. . . . . . . . . . . . . . . . . . 79 LI ST OF TABLES Table Page 1. Relationship of class of mating to detection of cobalt-labeled mitochondria on both sides of the anastomosis. . . . . . . . . . 40 2. Determination of long-distance transfer of mitOChondria O O O O O O O O O O O O I O O O 4 2 3. Density gradient analysis of matings. . . . . 67 vi Figure 10. ll. 12. LIST OF FIGURES Methods of differential labeling in matings and determination of tolerance for cobalt. Staining of mitochondria; kinetics of visual uptake of cobalt. . . . . . . . . . Determination of transfer of mitochondria in individual anastomoses. . . . . . . . . Characterization of mitochondria in living cells and in vitro . . . . . . . . . . . . Absorbancy profiles at 254 nm and 620 nm for mitochondria extracted from mycelia grown on MC medium . . . . . . . . . . . . . . . Absorbancy profiles at 254 nm and 620 nm for mitochondria extracted from mycelia grown on Co-lOOO ppm medium. . . . . . . . . . . Cytochrome oxidase profiles for MC and cobalt-labeled mitochondria. . . . . . . . Ultrastructural characterization of mitochondria in situ and in_vitro. . . . . Absorbancy profiles at 254 nm and 620 nm for a common-g mating. . . . . . . . . . . Absorbancy profiles at 254 nm and 620 nm for a common-A mating. . . . . . . . . . . Absorbancy profiles at 254 nm and 620 nm for a common-Ag mating . . . . . . . . . . Absorbancy profiles at 254 nm and 620 nm for a fully compatible mating. . . . . . . vii Page 30 35 38 44 47 49 51 54 58 60 63 65 INTRODUCT ION The objectives of the present study were to deter- mine if cytoplasmic transfer occurs in the various classes of matings of the tetrapolar Basidiomycete Schizophyllum commune, and to assess the role of the cytoplasm with respect to the time and mode of action of the genetic incompatibility factors. Evidence for nuclear transfer and migration can be confirmed morphologically by the development of clamp connections (Buller, 1931), or by the use of appropriately marked biochemical mutants and subsequent tests for complementation on minimal medium (Snider and Raper, 1958; Ellingboe, 1964). It generally has been assumed that transfer of cytoplasm is concommi- tant with nuclear transfer. However no direct test for this has been available. Earlier studies of hyphal anastomosis (Sicari and Ellingboe, 1967), indicate: a) anastomoses occur in all classes of matings, compatible and incompatible; b) there is no immediate incompatibility reaction such as plug formation, protein precipitation or lysis; however within 1 2-8 hours, one of the hyphal tips involved in the anasto- mosis or a cell adjacent to the anastomosed cell may de- velop a lysed appearance; and c) the frequency of such lysed hyphal tips can generally be correlated with the degree of incompatibility of the mating; i.e., the lowest frequency is found in fully compatible matings. The suggestion was made that the action of the incompatibility factors was of an inducible rather than constitutive nature. Modifier mutations of S. commune which do not directly affect the incompatibility factors, but disrupt the morphogenetic sequence associated with each, are believed to act via or in the cytoplasm (Raper, 1966). Hyphal tips isolated from doubly modified heterokaryons in which each mate carries a modifier mutation retain the ability to produce pseudoclamps, a property which neither parent has, upon hundreds of subsequent isolations (Raper and Raper, 1964). Much earlier (Harder, 1927), it was reported that subcultures of uninucleate subterminal cells from dikaryotic hyphae of g, commune isolated by micrur- gical procedures had the ability to produce pseudoclamps, again suggesting the activation of normally suppressed morphogenetic sequences, or perhaps the activation of a self-replicating cytoplasmic factor (Raper, 1966). The behavior of unilateral maters has also been suggested to be related to the state of the cytoplasm (Papazian, 1956). Other instances of persistent cytoplasmic information can be found in the transfer of senescence factors in Podospora anserina (Jinks, 1964), in the spread of vegetative death in Aspergillus glaucus (Jinks, 1959), in the apparent "transformation" of wild type mitochondria to abnormal ones in Neurospora crassa (Diacumakos, et al., 1965), and most strikingly in the alga Acetabularia, where informa- tion for specific cap type formation persists for many days after enucleation of the young plant (Hammerling, 1953; Harris, 1968). Nucleo-cytoplasmic interactions have also been studied in animal and bacterial systems, where the analogues to fungal heterokaryons Would be cultures of somatic cell hybrids (Harris and Watkins, 1965), and heterogenotes (Morse, et al., 1956). Thus it can be seen that the role of the cytoplasm in cellular control mechan- isms and morphogenesis is of basic biological significance. The immediate objective of the present study was to determine if cytOplasmic transfer occurs following hyphal fusion in the various classes of matings. A specific organelle of the cytoplasm, the mitochondrion, was chosen to be monitored, since it had been reported that cobalt uptake resulted in its in give staining (Lindegren and BeMiller, 1969). Further development of the technique to differentially label mates in a diffusion- controlled system could then be used to visually determine, by direct phase contrast examination, if mitochondria were transferred at individual sites of anastomosis. In addi- tion, if it could be shown that such vitally stained mito- chondria differed in density from non—stained mitochondria, then analyses based on the detection of donor density- labeled mitochondria in non—labeled residents could be used. Information on whether or not transfer occurs in the various classes of matings would help to assess the role of the cytoplasm, and perhaps that of one of its components, on the times and mode of action of the incom- patibility factors. LITERATURE REVIEW The wood rotting Basidiomycete Schizophyllum commune, variously placed in the Agaricales and Poly- porales, is characterized morphologically by a fan shaped basidiocarp having split gills on the underside (Buller, 1941; Raper, 1953, 1966). Haploid basidiospores germi- nate to produce elongate cylindrical hyphae having simple septa perforated by circular openings, and/or dolipore septa characterized by flange shaped domes which project into the cytoplasm of adjoining cells (Moore and McAlear, 1962; Giesy and Day, 1965). The term tetrapolar Basidiomycete applied to S. commune follows from the independent segregation of the A and g factors at meiosis, which results in production of basidiospores of four different mating types (Kniep, 1918, 1920). Thus if we term the mating type factors in one strain élEl and those in its fully compatible mate gagg, we would expect to find, following completion of the sexual cycle and basidiospore production, the two parental types A131 and A2B2, and the recombinant types A132 and 5 gag; in l:1:1:1 frequencies, if there were no linkage be- tween the A and g factors. The total number of different A factors is esti- mated at 450, the total number of different g factors at 93 (Raper and Raper, 1968). The A factor is made up of two linked subunits, perhaps representing cistrons, termed the alpha and beta (Raper et al., 1958; Raper et al., 1960). The E factor likewise is made up of two and pos- sibly three such subunits (Koltin et al., 1967). A dif— ference at only one of the subunits is sufficient to confer compatibility with regard to a single mating type factor. If hyphae which have germinated from A121 and §3§£ basidiospores come into contact and anastomose, effecting a fully compatible mating, we would expect reciprocal ex- change and migration of nuclei to occur, followed by the association of nuclei in pairs. Conjugate, synchronized nuclear divisions of the two paired, unfused nuclei occurs in the dikaryon (Kniep, 1918, 1920; Bensaude, 1918). The dikaryon, with its buckle-like clamp connections at the septa, is the form one is most likely to encounter in nature. During fructification two nuclei in a dikaryotic tip cell fuse to form a short-lived diploid nucleus which migrates into the base of the developing basidium. Subse- quent meiosis produces four haploid nuclei which migrate into the developing basidiospores. Thus the sexual cycle is completed. One may speak also of common-1, common-S and common-SS matings in which, respectively, the mates share in common the 1 factor, i.e., A1S1 x S1S1; the S factor, §1S1 x 51S1; and both factors, §1S1_x S1S1. In S. cgmmune these different types of matings can be distinguished morphologically (Papazian, 1950a, 1950b). The common—S mating results in the formation of a heterokaryon charac- terized by a "Flat," or appressed appearance; the common-S interaction by a region of low hyphal density between the mates (Barrage phenomenon), and by the presence of pseudo- clamps, i.e., clamps that have failed to fuse with the adjacent cell; and the common-AS interaction in growth essentially like that of the unmated homokaryons. The phenomenon of nuclear migration (Buller, 1931) can be quantitatively estimated and shown to be greater than the rate of intrusive hyphal growth by the use of appropriate morphological mutants, or by tests for com- plementation between auxotrophic mutants (Snider, 1963; Snider and Raper, 1958; Ellingboe, 1964). Nuclear migration is generally associated with fully compatible and common-S matings; it is limited in common-S and common-1S interactions. Thus control of nuclear migration is inferred to reside in paired unlike S factors. Control of the subsequent process leading to formation of clamp connections requires the presence of paired unlike 5 factors (Raper, 1966). Mutations at the incompatibility factor loci lead to loss of discrimination normally due to those factors (Parag, 1962; Raper and Raper, 1964; Raper, et al., 1965). Thus a strain with a mutated A mimics a common-S hetero- karyon; one with a mutated S mimics a common-A heterokaryon; and an A mut S mut homokaryon mimics a dikaryon. Modifier mutations which do not affect the incompatibility factors directly or their primary regulatory functions, affect all stages subsequent to nuclear migration (Raper and Raper, 1966, 1968). Such mutations are not normally expressed phenotypically in the homokaryon and are believed to be involved in sequential stages of dikaryosis, following their release or induction by paired or mutated incompat— ibility factors (Raper, 1966). They are viewed as muta- tions of structural genes that are under the direct or indirect control of the incompatibility factors. A dominant gene, Q1EE, has been identified (Koltin and Raper, 1968) that is required for stable and persistent dikaryosis; the presence of S1§+ is required in one or both partners; in the presence of S1k the nuclei fuse to form stable diploid nuclei. Normally, the diploid phase is restricted to a single nuclear generation in the basidia. Various models have been proposed to explain com- patibility in the tetrapolar Basidiomycetes, among them differences in charge or complementation between the mat- ing type factors. The former model has been rejected since the number of incompatibility factor alleles is greatly in excess of two, the latter also since alleles at each locus are numerous and functionally equivalent (Prévost, 1962). Two models presented as alternatives by Prévost would require anti-repressor substances to release suppression of metabolic processes; in one case these would be induced, in the other, he constitutive. In a complementary'mode of action to explain ster- ility in Angiosperms, growth of the pollen tube can be attributed to recognition of unlike S alleles to produce a substance required for pollen tube growth. In an oppo- sitional model interaction of like S alleles would inhibit 10 growth of the pollen tube (Lewis, 1954). Each of these modes, complementary and oppositional, is rejected for Schizophyllum. The complementary model is rejected since disomic S and disomic S strains are completely compatible with normal homokaryons carrying a factor in common with the disomic. The oppositional model is rejected, since individual alleles at each locus are known to be distinct but equivalent in function and no overlapping specifi- cities occur (Raper, 1966). Serological evidence has been presented for spe- cific pollen, style, and pollen-style proteins (Lewis, 1960, 1964). A unique type of RNA is found in styles pollinated with incompatible pollen; its production is thought to be induced, and its function to code for in— hibition of growth of the pollen tube. A dimer made up of two polypeptide chains, alpha and beta, is proposed to be coded for by S and 1 genes, specific dimers then re- sulting from the interaction of specific S and S alleles (Lewis, 1964). In Schizophyllum, differences between protein spectra in acrylamide gels and serological dif- ferences between homokaryons and dikaryons have been reported (Dick, 1965; Wang and Raper, 1970; Esser and Raper, 1961); however, these are not interpreted to be 11 the primary products of the incompatibility factors. With regard to postulated control mechanisms in bacteria (Monod and Jacob, 1961), a model has been proposed (Dick, 1965; Raper, 1966), in which the S_and S factors are considered "master switches," with the subunits of each, alpha and beta, considered as dual regulator genes. It has been suggested that the products of such subunits may be pro- tein dimers which act to suppress the dikaryotic sequence. In compatible matings, the combination of two such dimers would produce a tetramer ineffective as a repressor, allow- ing the dikaryotic morphogenetic sequence to proceed. Cell fusion appears to be indiscriminate in S, commune and Coprinus lagopus (Sicari and Ellingboe, 1967), and is not accompanied by an immediate incompatibility reaction. In Neurosppra crassa, however, there is super- :5 imposed upon the bipolar mating system a cytoplasmic com- patibility controlled by the C, D, and E loci (Wilson and Garnjobst, 1966). Fusion of cells having only one gene difference, e.g., CDE x CDe, results in rapid death of the fusion cell. Artificial mixing of cytoplasms by micrurgical techniques, although admittedly drastic, also results in plug formation in septal pores (Wilson, 1961; Wilson, et al., 1961). These results suggest a 12 constitutive nature to cytoplasmic incompatibility. By contrast, in cultures of interspecific somatic hybrids of various vertebrates, there appear to be no intracellular mechanisms for recognition and destruction of tissue, as one might normally expect in tissue grafts (Harris, 1968). Cultures of heterokaryotic vertebrate cells produced with a killed virus pretreatment can transcribe from both genomes, and may even form common mitotic spindles (Harris and Watkins, 1965). In the slime mold Physarum, in which four loci controlling fusion have been identified, strains which do not have identical alleles at all four loci will not fuse, but only collide (Collins and Haskins, 1972). Extreme incompatibility reactions may be seen in the proto- zoan Paramecium, where extended conjugation occurs between kappa-containing and sensitive non-kappa—containing mates (Sonneborn, 1943, 1947). In higher animals we see mani- festations of incompatibility in clumping or 1ytic reac- tions of red blood cells, based on the production of spe- cific antibodies to specific antigens present on the mem- brane of the erythrocytes (Race and Sanger, 1968). Thus it becomes apparent that whether incompati- bility is expressed immediately or its expression is de- layed, the roles of both nucleus and cytoplasm should be 13 considered. In the present study an organelle of the cytoplasm, the mitochondrion, was chosen for "tagging" to determine if cytoplasm was transferred following anasto- mosis. A brief review of mitochondrial structure and re- lated functions will follow. The term mitochondrion, coined by Benda, refers to the thread-like and grain-like appearances these organelles can assume (in Lehninger, 1964). Composed primarily of phospholipid and protein, mitochondria house the enzymes of the Krebs cycle for cellular aerobic res— piration of pyruvate to CO2 and H20, with the concommitant production of energy in the form of ATP by interactions with the electron transport system, and also enzymes for the oxidation of fatty acids (Lehninger, 1964). Mitochon- dria vary greatly in size, principally in the dimension of the long axis, and occasional reports have been made of granular forms coalescing to form the filamentous form and vice-versa (Chévremont, 1963). The number of mito- chondria per cell may be numerous, or just one, as has been reported for the water mold Blastocladiella emersonii, in which a single large mitochondrion is found in close proximity to the flagellum of the motile spore (Cantino, et al., 1963). Histochemically, mitochondria are 14 distinguished by their affinity for Janus Green B, a dye which can serve as an oxidation-reduction indicator. Bio- chemically, cytochrome oxidase and succinic dehydrogenase activity can be used as criteria for mitochondrial ac- tivity. On the ultrastructural level, they are distin- guished by double unit measures (Robertson, 1958), the inner one possessing characteristic projections called the cristae (Palade, 1952b). The unit membrane (Davson and Danielli, 1952) is conceived as a protein lipid bi- layer sandwich. Protein is on the outside, and polar groups of the fatty acids are oriented toward the protein coats; the hydrocarbon chains are oriented inwards.. The mitochondrial membrane, especially the inner membrane, is thought to have repeating tripartite units having a base, stalk and knob-like head (Fernandez—Moran, et al., 1964). There may be a functional division of labor between these sections; i.e., with respect to oxidation and phosphory- lation mechanisms, energy transfer, and movement of ions. Exact mechanisms and locations for these vital processes are far from settled. Some workers suggest that reactions of the electron transport system occur in the basepiece (Green and Young, 1971), and that ATP synthesis occurs in the head piece (Kagawa and Racker, 1966), the stalk serving 15 as a link between the two systems (MacLennan and Asai, 1968). Postulated conformational changes of the tripar- tite repeating units may be related to trapping of free energy released by the electron transfer process (Green and Young, 1971). Conformational energy would then drive ATP synthesis or active transport. Some ultrastructural support for this model is visualized in the energized, non-energized and energy-twisted forms of mitochondria (Green, et al., 1968). Mitochondria have been demonstrated to exhibit changes of volume (Lehninger, 1964); this has been related to uptake of certain anions, especially phos- phate. A contractile protein in the mitochondrial mem- brane exhibits characteristics quite similar to myosin, the contractile protein of muscle tissue (King, et al., 1965). The concept of a "proton pump" has been advanced, in which two protons would traverse the mitochondrial membrane for two electrons going the opposite way; this method of uptake would be directly related to the electron transport system (Mitchell and Moyle, 1965). It has been shown that mitochondria accumulate Ca++, Mg++, and Mn++ by active transport mechanisms, and that an anion requirement for this exists (Lehninger, 1964; Chance, 1967). During 1S vitro Ca++ uptake in the 16 presence of phosphate, it is believed that an insoluble phosphate, Ca-hydroxyapatite, precipitates in the intra- mitochondrial space (in Lehninger, 1964). Mitochondria so loaded with Ca++ show an increased density when pur- ified by sucrose density gradient centrifugation (Lehnin- ger, 1964). Differences in the bouyant densities of mitochondria isolated from a choline mutant of Neurospora crassa grown for different periods of time in choline have also been reported (Luck, 1965). It has been reported that a slow 1S vivo uptake of + . Co + for 72 hours by Saccharomyces from a growth medium containing CoC12-6H20 results in a visible darkening of the mitochondria as viewed by phase contrast microscopy (Lindegren and BeMiller, 1969). Cobalt, atomic number 27, atomic weight 58.94, is a first transition series element, as are its neighbors iron and nickel, and differs in the number of electrons in the 3 d shell (Hodgman, et al., 1961). Solutions of cobalt chloride hexahydrate are pink at room temperature, but become blue when treated with concentrated sulfuric acid or dehydrated with heat, as when it is used for invisible ink (Piatnitski, 1969; Hodgman, et al., 1961). Changes of color by cobalt solutions may be due to l7 solvation or dehydration of the cobalt ions, the state of coordination of cobalt atoms, the presence of complexes, or simply electron transfers between quantum levels (Young, 1960). The tolerance for cobalt by different organisms varies widely, and may vary between parts of the same organism. Thus the concentration of cobalt in root nodules of legumes may greatly exceed concentrations in leaves (Bertrand and DeWolf, 1955). Cobalt concentrations in stigma, style and pollen may greatly exceed those found in petals (Yamada, 1958). Microorganisms generally appear to have a greater tolerance for cobalt than do higher plants. Actinomycetes, Saccharomyces, Aspergillus, barley, and wheat may tolerate, respectively, 10,000, 750, 1500, 29.5, and 7 ppm (Young, 1960; Perlman and O'Brien, 1954). It is interesting to note that much of the cobalt appears to be chemically’cOmbined, not merely adsorbed to protein, and that the protein content of organisms grown in the presence of cobalt may increase (Young, 1960; Ballentine and Stephens, 1951). Cobalt has been reported to stimulate certain en- zymes, e.g., pyruvate decarboxylase, malate dehydrogenase, peptidases, and to inhibit esterase activity (Dixon and 18 Webb, 1964). It forms an essential part of vitamin B12 (cobalmin), and carbamide enzymes; it is chelated in vitamin 312 by a porphyrin system, much as is iron in hemoproteins and magnesium in cholorophyll (Dwyer and Mellor, 1964). MATERIALS AND METHODS Maintenance of Cultures All strains of Schizophyllum commune were highly isogenic except for mating type (Ellingboe and Raper, 1962). Cultures were maintained on 2% agar Migration Complete medium (Snider and Raper, 1958), or on Migration Complete medium (MC) modified by the addition of disodium ethylene diamine tetraacetic acid (EDTA) and cobalt chloride hexahydrate in equimolar quantities, prior to addition of other components of the medium. At high con- centrations of cobalt, it was necessary to increase the agar to 3.5%. In matings made on Y—plates as described below, the concentrations of EDTA and CoC12°6H20 used were 0.39 and 0.25 g/liter, respectively. This medium will henceforth be referred to as Co—250 ppm. In the analysis of matings based on the detection of density differences of mitochondria, the concentrations of EDTA and cobalt chloride were 1.56 and 1.0 g/liter, respectively. This medium will henceforth be referred to as Co-1000 ppm. 19 20 Determination of Mitochondrial Transfer at Points of Anastomosis One well of a Petri dish containing three compart- ments separated by plastic divisions (Falcon Plastics, Y- plate), was filled with approximately six ml of Co-250 ppm medium, the remaining two with MC medium. The volume was such as to ensure that the plastic dividing walls were plainly visible above the levels of the agar media. Strips of dialysis membrane 5 cm x 2.5 cm, previously sterilized in distilled water, were freed of obvious moisture by blotting with sterile filter paper and trans- ferred with sterile forceps to the Y-plate such that each of two strips straddled the divisions between Co-250 ppm and MC media. Inocula consisted of mycelia grown on Co-250 ppm or MC agar media for 72-96 hours. Blocks of agar (approximately 4 cm x 0.5 cm) containing mycelia of the appropriate mating type were positioned parallel to each other and to the division between them. Each inoc- ulum block was approximately 1.5 cm distant from the appropriate divider. The supporting medium in the well of the Petri plate corresponded to the medium used for the inoculum block. Thus two sets of matings of strains 21 maintained on Co-ZSO ppm medium x strains maintained on MC medium could be done per Y—plate (Figure 1a). After 48 hours incubation at 32 C, the hyphae had generally grown out far enough from the inocula to establish anas- tomoses at points of contact in the vicinity of the plas- tic divider. This central area of the dialysis membrane, not in direct contact with either supporting medium, was cut out, mounted in water and examined by phase contrast microscopy (Zeiss Standard WL), using a green filter. A scan at low power was performed to find points of anasto- mosis, followed by examination at 800 X, to determine if dark staining cobalt mitochondria could be seen on only one or both sides of the anastomosis. Determination of Long— Distance Transfer of Mitochondria A resident mycelium (Snider and Raper, 1958) was prepared by pour-plating molten MC agar with two m1 of hyphal fragments in suspension. The latter was prepared by macerating a colony approximately 5 cm in diameter with 50 ml of MC broth for 30 seconds in a Waring Blendor. The resident was incubated 72 hours at 32 C prior to 22 application of a cobalt donor mycelium. A modification of the "racetrack" technique (Ellingboe, 1964) was used, in which transfer and migration are allowed to proceed essentially unidirectionally by cutting out all agar of the resident plate except for a narrow 3 cm strip down the center of the plate (Figure 1b). Donor mycelium was applied without any adhering cobalt agar. This was accomplished by growing macerated hyphal fragments on sterile squares of dialysis membrane on plates of Co-250 ppm medium. A narrow strip (3 cm x 1 cm) of dialysis membrane with mycelium growing on it could then be cut with a scalpel, peeled off with forceps, and be applied mycelial side down at one end of the resident mycelium. After 48 hours at 32 C, small plugs 2 mm in diameter (Figure 1b) were cut out from the resident with a metal tube. The mycelium in the upper part of the plug was examined by phase contrast microscopy to see if any cobalt—stained mitochondria were present. Isolation of Mitochondria Methods used for the isolation and purification of mitochondria were similar to those employed for 23 Neurospora crassa (Luck, 1963a, b; Kuntzel and Noll, 1967), and differed primarily in methods of growing and harvesting mycelium. Cultures were grown on squares of dialysis mem— brane on either MC or Co-1000 ppm media. Inoculum con- sisted of approximately six m1 of a suspension hyphal fragments prepared by macerating a 5 cm diameter colony on agar medium with 50 m1 of the appropriate broth for 30 seconds. The plates were incubated at 32 C for 72-96 hours prior to harvesting mycelium. Dialysis membranes with adherent mycelia from a total of six plates were peeled off and macerated 20 seconds in cold 0.25 M sucrose containing 0. 01 M Tris—HCl and 0.001 M EDTA, buffered at pH 7.0, in prechilled Waring Blendor cups. The homogenate was centrifuged at 4 C at 3,000 x g for 20 minutes to pellet nuclei, cell walls, and fragments of dialysis mem— brane. The supernatant was filtered through 400 mesh nylon prior to a second centrifugation at 4 C for 20 minutes at 10,000 x g to obtain the crude mitochondrial pellet. The pellet so obtained was resuspended in 1.0 m1 of 0.25 M sucrose buffer described above, using a glass rod in a test tube or several strokes in a Toenbroeck tissue homogenizer. It was then carefully pipetted onto a previously chilled linear sucrose gradient ranging in 24 molarity from 0.9 M to 2.0 M, buffered at pH 7.0 (0.001 M Tris-HCl), and containing EDTA (0.001 M). The gradient was prepared according to the method of Britten and Roberts (1960). One ml of sample was layered on top of the gradient and centrifuged at 35,000 rpm (SW 50L rotor in Beckman L2-65 B Ultracentrifuge) at 4 C for 4-5 hours. The gradient was pumped through a Uvicord II flowcell to obtain the profile of absorbency at 254 nm. Fractions were collected with an LKB Ultrorac 7000. Five drOp fractions were collected starting from the top of the tube by pumping a 2.0 M sucrose solution containing neutral red dye through a bottom puncture of the tube at constant rate. Addition of the dye served to indicate the beginning and end of the sample. Fractions so collected could then be tested for enzymatic activity. Succinic Dehydrogenase Assay A histochemical method (Gomori, 1957) was modified for routine and rapid estimation of localization of ac- tivity in the sucrose gradients following centrifugation and banding of mitochondria. The reaction mixture con— sisted of 0.25 M sodium succinate, 0.1 M phosphate buffer 25 pH 7.0, 0.1% w/v nitro blue tetrazolium chloride, and 0.1 M NaCN in a ratio of 3:3:3:1. Three-tenths ml was added to 0.1 m1 samples of each fraction collected and the mixture was incubated at 32 C for one hour. One ml of water was added to each sample and the absorbancy at 620 nm was determined with a Beckman DU spectrophotometer. A profile of succinic dehydrogenase activity was thus obtained for each sucrose gradient. The control mixture contained the same components minus the succinate sub- strate. Cytochrome Oxidase Assay Cytochrome oxidase activity was monitored by fol- lowing the rate of decrease in absorbancy at 550 nm of reduced cytochrome c (Nielsen and Lehninger, 1955; Smith, 1955). The cytochrome c had been reduced with sodium borohydride and dialyzed overnight at 4 C against 0.1 M phosphate buffer (pH 7.4) containing EDTA, to remove the excess reducing agent (Martin, et al., 1957). This assay is based on the oxidation of iron in the ferrous state to the ferric state, thus one had to be certain that a sig- nificant portion of the iron in the cytochrome c was 26 indeed in the reduced state prior to running the assay. The cytochrome c reduced as above was checked for its absorbancy at 550 nm and 565 nm. If the ratio was greater than six, then the cytochrome was considered to be in a form suitable for the assay (Smith, 1955). Change in absorbancy at 550 nm with time following addition of the mitochondrial suspension was then monitored for each fraction to determine which one had the greatest activity. The reaction mixture in a 3.0 m1 cuvette consisted of 2.4 ml of phosphate buffer (0.1 M, pH 7.4), 0.5 m1 of 2.0 x 10-4M reduced cytochrome c, and at zero time, 0.1 m1 of the fraction being tested. The change in absorbancy at 550 nm was monitored with a Beckman DU spectrophoto- meter connected to a recorder with a chart speed of one inch per minute. The reaction was followed 2-3 minutes at room temperature, and the oxidation of ferrocytochrome c brought to completion by the addition of 0.04 ml of satur- ated potassium ferricyanide. The rate of reaction could be estimated by determining the rate of change of absor- bancy at 550 nmkwith time (i.e., the slope). The reaction is considered to be first order for the time interval studied (Smith, 1955). 27 Electron Microscopy Inocula were grown on squares of dialysis membrane on MC or Co-1000 ppm medium for 96 hours. Small pieces of dialysis membrane covered with mycelium were prefixed overnight at room temperature in 2.5% glutaraldehyde (Sabatini, et al., 1963), and rinsed three times with 0.1 M phosphate buffer pH 7.4 at 20 minute intervals prior to fixation in buffered (pH 7.4, 0.1 M P04) 2% 0304 for 1-1/2 hours (Palade, 1952a). Dehydration was in ethanol (25, 50, 70, 95%, 10 minutes each; absolute ethanol, two 15 minute rinses). The samples were then placed in two changes of propylene oxide for 30 minutes each, followed by infiltration with a 1:1 mixture of Epon 812 (7A:3B) and propylene oxide. Final embedding was in Epon complete mix (Luft, 1961), in a 00 gelatin capsule at 60 C. The embedded samples were sectioned on a Blum Ultramicrotome with a diamond knife. Sections were floated onto 400 mesh Copper grids and stained 30 minutes with a saturated solu- tion of uranyl acetate. This was followed by a water rinse, and a 5 minute staining in saturated lead citrate; a rinse in 0.02 M NaOH preceded a final water rinse prior 28 to viewing in a Philips 300 electron microscope. All electron micrographs were made at 32,000 X magnification. Mitochondria rich fractions were pooled, prefixed in 2-1/2% glutaraldehyde, and pelleted by centrifugation at 10,000 x g. Three rinses with phosphate buffer as described above preceded fixation in 050 subsequent 4; steps were as described for mycelia above. Fixation of mitochondrial fractions with 2% aqueous KMnO was preceded by an overnight pre-fixation 4 in glutaraldehyde, pelleting at 10,000 x g, and phosphate buffer rinses as above. Fixation was in 2% KMnO4 for 24 hours at room temperature followed by several rinses in phosphate buffer and dehydration in ethanol. Subsequent steps for embedding and sectioning were as described in the preceding section with the elimination of uranyl acetate and lead citrate stains. Extraction of Mitochondria from Matings The MC resident mycelium was prepared by pouring a suspension of hyphal fragments onto sterile squares of dialysis membrane on MC plates. The suspension was pre- pared by macerating a colony 5 cm in diameter with 50 m1 29 of MC broth for 30 seconds in a Waring Blendor. Approx- imately 6 ml were poured on each plate. The plates were incubated 72-96 hours at 32 C. The cobalt-labeled donor to be used for the mating was similarly prepared using Co—1000 ppm broth and plates. Strips of dialysis membrane (2.5 x 8 cm), containing cobalt-labeled donor mycelium, free of supporting medium, were cut, removed from the Co-1000 ppm medium with forceps, and applied mycelial side down onto the MC resident. Two such strips were placed on a plate for a mating (Figure 1c). After incu- bation for 24 hours at 32 C, the entire area covered by the cobalt-labeled implant, as well as the implant, was discarded. Mycelium on the dialysis membrane which pre- viously surrounded the implants was then extracted as described above for isolation and purification of mito- chondria by isoPycnic sucrose density gradient centrifu- gation. Analysis of absorbancy profiles at 254 nm, along with profiles of succinic dehyrogenase activity was then made to determine if denser cobalt—labeled mitochondria had been transferred in the various classes of matings. A minimum of four replicate experiments was performed for each class of matings. This included the use of at least two different combinations of strains and/or the alterna- tion of the mate which was labeled with cobalt. matings la. 1b. 1c. 1d. 1e. 1f. 30 Fig. l.--Methods of differential labeling in and determination of tolerance for cobalt. Triplate mating. Dialysis membrane strips are straddled between the MC and Co-250 ppm wells. Dialysis membrane in the region of the plastic divider was later cut out for microscopic examination of individual anastomoses. Racetrack method for determination of long distance transfer of mitochondria. Donor (d) placed at one end of resident (r) prior to removal of plugs for microscopic examination 48 hours later. Agar-free cobalt donor implants (d) on MC resident (r) plates. Both donor and resident mycelium are contained on dialysis membranes. Mitochondria from resident areas surrounding the donor implants were extracted and puri- fied; donor and resident immediately below it were discarded. Tests for mating type on Co-1000 ppm medium. Clockwise, C: fully compatible mating, ring of fruit body initials is visible. B: common-S mating; Barrage area between the mates is evident. A: common-S mating demon- strates "Flat," appressed mycelium. AB: common-SS_mating; mates remain essentially as homokaryons. Tolerance test for cobalt; no inhibition apparent to 900 ppm Co++. Tolerance test for cobalt; complete inhibition at 2500 ppm Co . 31 Figure 1 RESULTS Determination of Tolerance for Cobalt Plugs of mycelium grown on MC medium were cut out with a sterile cork borer at equal distances from the initial inoculum on plates which had been previously incu- bated for 96 hours at 32 C. The plugs were then trans- ferred to Petri plates containing MC broth supplemented with cobalt chloride hexahydrate and disodium EDTA in a range of equimolar amounts. As can be seen in Figure 1e, up to 900 ppm cobalt does not appear to inhibit growth; growth was possibly somewhat enhanced. Examination of Figure 1f indicates a slight inhibition of growth at 1000 ppm, definite inhibition at 1500 ppm, and complete inhibition at 2500 ppm of cobalt added. Mating type tests performed on the cobalt media proceeded normally as could be determined by gross macro- scopic as well as microscopic morphology (Figure 1d). In the fully compatible matings, both partners formed clamp connections, and normal looking fruiting bodies were 32 33 formed. Common-§_matings showed the characteristic "Flat" morphology. Common-S matings developed the expected "Barrage" reaction. The common-SS interaction also ap- peared to be as normal; i.e., the homokaryons retained the morphology of homokaryons. Kinetics of Visual Uptake of Cobalt Small plugs of mycelium grown on MC agar were placed in Co-ZSO ppm broth and samples of the mycelia were removed after 24, 48, and 72 hours at 32 C and examined by phase contrast microscopy. At zero time, hyphae from MC media have indistinct mitochondria and walls that are thick (Figure 2b). A histochemical stain for mitochondria based on cytochrome oxidase activity (Gomori, 1957) showed that mitochondria were numerous in the cells. However, the procedure results in an apparent swelling of both hyphae and mitochondria (Figure 2a). Mycelia 24 hours after transfer to Co-250 ppm broth had cell walls that were obviously darkened, but the mitochondria were normal in appearance (Figure 2c). By 48 hours, darkening was not particularly apparent in the walls, but a general darken- ing of the cytOplasm was visible (Figure 2e). After 34 72 hours in Co-250 ppm broth, mycelia had mitochondria that were darker and somewhat smaller in size (Figure 29). Normal appearing mitochondria were sometimes also present. In Co-1000 ppm broth, the walls were also visibly darker by 24 hours (Figure 2d); by 48 hours a few mitochondria occasionally were seen that were darker (Figure 2f), and by 72 hours virtually all the mitochondria were darker and somewhat smaller (Figure 2h). Checks on Cobalt Diffusion on Y-Plates Cobalt-5000 ppm water-agar blocks were positioned as if for inoculum on dialysis membranes straddling the divisions between wells of cobalt and MC media in Y-plates. There was no visual or spectrophotometric (510 nm absorp- tion peak for aqueous cobalt chloride) evidence of diffu- sion on the dialysis membrane in the vicinity of the divider after 48 hours incubation at 32 C. Some diffusion to the supporting medium below the cobalt block was evi- dent visually however. 2a. 2b. 2c. 2d. 2e. 2f. Zg. 2h. 35 Fig. 2.--Staining of mitochondria; kinetics of visual uptake of cobalt. Histochemical test for cytochrome oxidase; mitochondria stained dark bluish-purple, some are quite swollen in appearance. Normal appearing hyphae and on MC medium. Mitochondria distinct; several seen near Hyphae maintained in Co-250 hours; the walls are darker controls. mitochondria grown are relatively in- arrow along wall. ppm medium for 24 than those of the Hyphae maintained in Co-lOOO ppm medium for 24 hours; the walls are much darker than those of the controls. Hyphae maintained in Co-250 ppm medium for 48 hours; neither the walls nor the mitochondria are particularly dark. Hyphae maintained in Co-lOOO ppm medium for 48 hours; a few mitochondria may be somewhat darker. Hyphae maintained in Co-250 ppm medium for 72 hours; mitochondria are darker. Hyphae maintained in Co-1000 ppm medium for 72 hours; mitochondria appear darker and also smaller in size than those in controls. 36 Figure 2 37 Direct Observations of Individual Anastomoses A11 observations were made on anastomoses already established at the time of microscopic examination. Only those in which both partners could be clearly traced back to their respective origins on opposite sides of the slide were scored for transfer of mitochondria. If the darker (bluish) mitochondria were present on both sides of the anastomosis, it was scored as positive evidence for transfer. If the dark mitochondria were visible on only one side of the anastomosis, it was scored as having no transfer of mitochondria. All anastomoses examined in common-S (Table 1, Figure 3a), and common-SS (Table 1, Figure 3b) matings showed positive evidence for transfer for at least two and often to four cells beyond the point of anastomosis. It was difficult to trace hyphae involved further than that since the hyphal density became too great to trace single hyphae by microscopic examination. In common-S matings, on the other hand, there was no evi- dence for transfer in any of the more than 120 anastomoses observed (Figures 3c and 3d, Table 1). In fully compat- ible matings, either of haploid x haploid, or haploid x diploid, a11 anastomoses showed evidence for mitochondrial 38 Fig. 3.--Determination of transfer of mito- chondria in individual anastomoses. 3a. 3b. 3c. 3d. 3e. 3f. Common-S_mating; dark mitochondria are visible (arrows) on both sides of the anastomosis. Common-SS mating; dark mitochondria are vis- ible in both mates. Common-S_mating; dark mitochondria are visible in only one of the mates; nucleus (n) is vis— ible near the anastomosis. Common-S_mating; dark mitochondria are visible in only one mate; possible developing pseudo- clamp (pc) visible; one of mates has branch with lysed appearance to cytoplasm (1t). Fully compatible mating; dark mitochondria are visible in each mate; dense cytoplasm is present in the anastomosis. A clamp connec— tion is visible in one of the mates. Same anastomosis as in Figure 3e photographed two minutes later; a mass movement of cyto- plasm out of area of anastomosis has occurred, leaving a clear zone in the region of the anastomosis. Figure 3 40 TABLE 1.--Relationship of class of mating to detection of cobalt- 1abe1ed mitochondria on both sides of the anastomosis. Mating Mating Number Having Number of . . Class of Type Type Anastomoses Dark Mitochondria Mating Cobalt Unlabeled on both sides of . . Scored _ Strain Strain the AnastomOSis Fully A41 B41 A42 B42 42 42 Compatible A42 B42 A41 B41 24 24 n x n A42 B41 A41 B42 29 29 A41 B42 A42 B41 _S_ ._S Class totals 98 98 Fully A41 B42 ---——— A42 B41 60 60 Compatible A41 B42 2n x n A41 B41 ---- A 2 5 A41 B41 4 B42 -£L- —2§- Class totals 155 155 Common-A A42 B42 A42 B41 22 22 A42 B41 A42 B42 2 2 A41 B41 A41 B42 12_ '11 Class totals 41 41 Common-B A42 B42 A41 B42 19 0 A41 B42 A42 B42 25 0 A41 B41 A42 B41 74 0 A42 B41 A41 B41 7 _11 Class totals 125 0 Common-AB A41 B41 A41 B41 22 22 A42 B42 A42 B42 10 10 A41 B42 A41 B42 23 23 A42 B41 A42 B41 44 44 Class totals 99 99 41 transfer (Figures 3e and 3f, Table 1). That the anasto- moses were indeed functional (i.e., nuclei had been trans- ferred), was evidenced by the presence of clamp connec- tions in one or both mates in fully compatible matings, and by the production of pseudoclamps in the growing hy- phal tips of common-S matings. Thus in all classes of matings examined except the common-S, there was direct visual evidence for transfer of mitochondria via func- tional anastomoses. Determination of Long- Distance Transfer Resident racetracks (Figure 1b), and cobalt- 1abe1ed donor mycelia were prepared as described in the materials and methods and incubated 48 hours at 32 C. Plugs 2 mm in diameter were removed from the resident for microscopic examination to determine if the darker stain- ing cobalt-labeled mitochondria could be detected when .examined at high magnification (800 X) with the phase contrast microscope. Plugs from up to 1.2 cm from the cobalt-labeled donor were examined. The darker mitochon- dria were detected in plugs up to 6 mm from the cobalt- labeled donor. As in the direct observation of individual 42 anastomoses, there was evidence for transfer in fully com- patible, common-S, and common-SS_matings, but none in the common-S matings (Table 2). No attempt was made to count the number or proportion of darker mitochondria in these samples, only the presence or absence of darkly stained mitochondria was scored. TABLE 2.—-Determination of long-distance transfer of mitochondria. Mating Type Mating Type Number Number Plugs Class of Meeting Cobalt-labeled Unlabeled Plugs Having Dark strain strain Scored Mitochondria Common-A A41 B41 A41 B42 48 26 A42 B41 A42 B42 96 SS_ Class totals 144 72 Common-B A41 B42 A42 B42 32 0 A41 B41 A42 B41 48 0 A42 B41 A41 B41 16 _S_ Class totals 96 0 Common-AB A41 B42 A41 B42 32 14 A42 B42 A42 B42 48 .SS Class totals 80 40 Fully compatible A42 B41 A41 B42 32 24 A41 B41 A42 B42 32 20 Class totals 64 44 43 Characterization of Mitochondria from Mycelia Grown on Co-1000 ppm and MC Media Mitochondria purified by isopycnic sucrose density gradient centrifugation banded at characteristic regions of the gradient after 5 hours at 35,000 rpm. Macroscopic observations of the mitochondrial bands indicate that mitochondria isolated from mycelium grown on Co-1000 ppm medium are denser, i.e., band is lower in the tube, than mitochondria isolated from mycelium grown on MC medium (Figure 4e). A size difference between MC and Co mito- chondria is apparent in living cells, as examination of Figures 4a and 4b and Figures 2b and 2h indicates. Samples taken from the respective fractions of greatest absorbancy at 254 nm show a difference in size and color of isolated mitochondria (Figures 4c and 4d). The cobalt- 1abe1ed mitochondria banded in a lower position in the centrifuge tube (Figure 4e) as viewed macroscopically. The absorbancy at 254 nm for each centrifuge tube was obtained by passage of the gradient through a Uvicord II flowcell as described in the materials and methods. The maximum absorbance at 254 nm for purified mitochondria extracted from mycelium maintained on MC medium was in a 44 Fig. 4.--Characterization of mitochondria in living cells and 1S_vitro. 4a. Hypha grown on Co-1000 ppm medium; mitochon- dria are darker and smaller than MC counter- parts. Several are visible near the arrow tip. 4b. Hypha grown on MC medium; mitochondria are relatively indistinct. Several are visible near the wall in the region indicated by the arrow. 4c. Mitochondria extracted from mycelium grown on Co-lOOO ppm medium and purified by isopycnic sucrose density gradient centrifugation; sample taken from fraction having maximal absorbancy at 254 mu (see Figure 6). Size and color of mitochondria comparable to those seen 15 situ in Figure 4a. 4d. Mitochondria extracted from mycelium grown on MC medium and purified by isopycnic sucrose density gradient centrifugation; sample taken from fraction having maximal absorbancy at 254 mu (see Figure 5). Mitochondria are not as dark as those labeled by cobalt. 4e. Comparison of banding patterns of non-labeled and cobalt-labeled mitochondria. Cobalt- labeled mitochondria sediment lower in tube. 4f. Mitochondria extracted from Common-S (Figure 9), and common-SS (Figure 11), matings; band of cobalt-dense mitochondria is evident in latter, 'as well as the band of unlabeled mitochondria. 45 Figure 4 46 different position in the centrifuge tube than for mito- chondria similarly extracted from mycelium maintained on Co-1000 ppm medium (Figures 5 and 6). This difference in banding positions was consistent in five replicate exper- iments. Analysis of fractions for succinic dehydrogenase activity (absorbancy at 620 nm) corresponded with the profiles of absorbancy at 254 nm (Figures 5 and 6). Occasionally, succinic dehydrogenase activity appeared at the top of the tube. A failure of material to sediment at the meniscus perhaps suggests the presence of broken mitochondrial fragments bearing the enzyme. The activity of cytochrome oxidase was also determined as another means to monitor mitochondria in density gradients. As is evi— dent in Figure 7, the mitochondria grown on MC media show the greatest activity at fraction 8, which corresponds well with the maximum absorbancy at 254 nm (Figure 5). The cobalt-labeled mitochondria have maximal absorbancy at 254 nm and maximum cytochrome oxidase activity at fraction 10 (Figures 6, 7). 47 Fig. 5.--Absorbancy profiles at 254 nm and 620 nm for mitochondria extracted from mycelia grown on MC medium: Absorbance at 254 nm (-—-) represents a constant monitoring of effluent from the top of the gradient tube. Maximal absorbance at 254 nm for unlabeled purified mitochondria occurs in the region of fraction 8. Measurement of succinic de- hydrogenase activity in aliquots from five drop fractions collected starting from the top of the tube was made by determining absorbancy at 620 nm (o----o) of reduced nitro blue tetrazolium chloride. The maximum activity occurs at fraction 8. 48 HmHHU< H>HHHHU< M>HHHHO< H>HHHHU< H>HHHHU< H>HBHHO< H>HH