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Characterization of several mitochondrial variants of natural isolates of Neurospora intermedia Rieck, Anne Carolyn 1981

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CHARACTERIZATION OF SEVERAL MITOCHONDRIAL VARIANTS OF NATURAL ISOLATES OF NEUROSPORA INTERMEDIA by ANNE CAROLYN RIECK B.Sc, The University of Vermont, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Botany Department, University of B r i t i s h Columbia) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1981 (c) Anne Carolyn Rieck, 1981 In presenting th i s thesis in par t i a l fu l f i lment of the requirements for an advanced degree at the Univers ity of B r i t i s h Columbia, I agree that the Library shal l make i t f ree ly avai lable for reference and study. I further agree that permission for extensive copying of th i s thesis for scholar ly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publ icat ion of th i s thesis for f inanc ia l gain shal l not be allowed without my written permission. The Univers ity of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 D E - 6 B P 75-51 I E i i ABSTRACT A survey of natural i s o l a t e s of N. intermedia revealed f i v e variants with respect to growth phenotype. These variants showed stop-start behavior i n growth tubes, sometimes never a t t a i n i n g the f u l l tube length. These s t r a i n s are mainly.female s t e r i l e . The stop-start phenotype was not transmitted v i a the male parent i n any cross. In two crosses, maternal transmission was demonstrated. One s t r a i n was investigated for the presence of v i r u s - l i k e p a r t i c l e s , but none were found. Cytochrome spectra show a deficiency of cyto-chromes a. and/or b_ r e l a t i v e to the amount of c.. In r e s p i r a t i o n studies, the one s t r a i n tested proved to be cyanide r e s i s t a n t and s a l i c y l hydroxamic acid s e n s i t i v e . These c h a r a c t e r i s t i c s are also found i n extranuclear mutants of N. ovassa. However, the analogy no longer holds at the mitochondrial ribosome l e v e l . An analysis of four of the s t r a i n s disclosed that three are large subunit d e f i -c i e n t . Only small subunit d e f i c i e n t s t r a i n s have been found among the cytoplasmic mutants of N. ovassa. R e s t r i c t i o n enzyme analysis was also c a r r i e d out on the v a r i a n t s . I t was found that they possess a d d i t i o n a l mitochondrial DNA compared to normal N. intermedia. One Eco RI fragment of M.W. 2.4 x 10 was found to be common to a l l variants tested, but i s not found i n the normal s t r a i n s . There were other DNA differences which were unique to each abnormal s t r a i n . These findings suggest that the basis f o r the abnormal phenotypes .is some h e r i t a b l e f a c t o r associated with the mitochondria. Dedicated to the memory of my father. His enthusiasm f or science and devotion to h i s work have been a true i n s p i r a t i o n . i v TABLE OF CONTENTS Abstract i i L i s t of Tables v i L i s t of Figures v i i Acknowledgements i x Section I. Genetic Analysis 1 Introduction 2 Materials and Methods 16 Strains 16 Media and Growth Conditions 16 Methods • • -20 Growth Rate Tests 20 Heterokaryon Tests .21 Virus Search 22 Results 24 Growth Characteristics of Original Strains ..24 Colony Isolates ..24 Reciprocal Crosses 33 Growth Characteristics of Progeny 40 Growth on Complete Media 40 Heterokaryon Formation 40 Virus Search 43 Discussion 48 Section I I . Biochemical Analysis 54 Introduction 55 Materials and Methods 71 V Strains '. 71 Media and Growth Conditions 71 Methods 71 Cytochrome Spectra 71 Respiration 72 Mitochondrial Isolation 72 Preparation and Analysis of Mitochondrial Ribosomes 73 rRNA Extraction 74 mt DNA Preparation and Digestion 75 Results 78 Cytochrome Spectra 78 Oxygen Uptake Studies 78 Ribosome P r o f i l e s 81 Mitochondrial Ribosomal RNA Analysis 81 DNA Re s t r i c t i o n Enzyme Patterns 83 Discussion 102 Bibliography 112 v i LIST OF TABLES Page Table I. Neurospora intermedia Isolates Obtained from the FGSC 17 Table I I . Neurospora intermedia Isolates Obtained from D.D. Perkins 18 Table I I I . Neurospora orassa and Neurospora intermedia Auxotrophic Strains Used for Heterokaryon Testing 19 Table IV. Summary of Results of Conidial Isolation for Neurospora intermedia Strains P594, P608, and P804 38 Table V. Summary of Results of Reciprocal Crosses of the Normal and Abnormal Isolates of N. inter-media 39 Table VI. Results of Heterokaryon Tests Between N. inter-media Strain 2360his and N. orassa Auxotrophs 44 Table VII. Results of Heterokaryon Tests Between N. inter-media Strain 2360his and N. intermedia Auxo-trophs 45 Table VIII. Summary of the Results of Heterokaryon Tests Between 2360his and Progeny of the Crosses: 3401 x 2360 and 3386 x 2360... 46 Table IX. Ratios of Large to Small Subunits for the Various N. intermedia Strains 86 Table X. Molecular Weights ( x 10 ) of N. intermedia mt DNA Eco RI Fragments 91 Table XI. A Summary of the Results of the Characteri-zation of the N. intermedia Isolates I l l v i i LIST OF FIGURES Page Figure 1. Genetic and Physical Map of Yeast mt DNA 4 Figure 2. Growth Curves of Poky vs. Not Poky 7 Figure 3. Heterokaryon Formation 12 Figure 4. Growth Curves of the Five Anomalous N. inter-media Strains (2360, 2363, P804, P608, P594) and one Normal Strain(2361) 25 Figure 5. Two Growth Curves of N. intermedia Strain 2360... 27 Figure 6. Growth Curves of Conidial Isolates of N. inter-media Strains 2360 and 2363 29 Figure 7. Conidial Isolation of N. intermedia Strain P608.. 31 Figure 8. Conidial Isolation of N. intermedia Strain P594.. 34 Figure 9. Conidial Isolation of N. intermedia Strain P804.. 36 Figure 10. Growth Curves on Complete Media .41 Figure 11. The Electron Transport Chain 56 Figure 12. Cytochrome Spectra of Two N. orassa Strains 58 Figure 13. Branched Electron Transport 61 Figure 14. The Eco RI Re s t r i c t i o n Enzyme Map of Wild Type N. orassa mt DNA..... 67 Figure 15. Cytochrome Spectra of N. intermedia Strains 79 Figure 16. Oxygen Uptake of Intact C e l l s 82 Figure 17. Ribosome P r o f i l e s of N. intermedia Strains....... 84 Figure 18. Ribosomal RNA Analysis 88 Figure 19. Eco RI Re s t r i c t i o n Enzyme Digestion of N. orassa and N. intermedia Strains 90 Figure 20. Eco RI Res t r i c t i o n Enzyme Digest of N. inter-media Strains 92 v i i i Figure 21. Kpn Rest r i c t i o n Enzyme Digest of N. intermedia Strains . ... ; 94 Figure 22. Cla Rest r i c t i o n Enzyme Digest of N. intermedia Strains.. 97 Figure 23. Uncut DNA of the N. intermedia Strains 100 XX ACKNOWLEDGEMENTS I wish to express appreciation to those who have been h e l p f u l throughout the work of t h i s t h e s i s . I am indebted to Dr. A.J.F. G r i f f i t h s f o r h i s advice, patience and encouragement. Thanks are also due to Dr. B.R. Green and Dr. R.C. M i l l e r f o r t h e i r suggestions on procedures and the time spent reviewing the th e s i s . 1 would l i k e to thank a l l my friends f o r t h e i r support and understanding. Very s p e c i a l thanks go to Dr. H. Bertrand f o r the long hours spent teaching me procedures, for h i s enthusiasm, and for the motivation he gave me. 1 SECTION I Genetic Analysis 2 INTRODUCTION In 1901 C. Correns and E. Baur (Correns, 1909; Baur, 1909) noted non-Mendelian patterns of inheritance for a factor influencing chloroplast development i n some strains of flowering plants. Work-ing with MivabiUs, Correns found maternal inheritance governed the presence of mutant white sectors of leaf tissue. Baur also found non-Mendelian ratios for the same t r a i t i n the genus Pelargonium. While they attributed the phenomenon to extrachromosomal heredity, l i t t l e interest was aroused i n the s c i e n t i f i c community since work on chromosomal genetics was progressing so rapidly (Sager, 1972). However, as more cases of cytoplasmic genes were discovered, their significance was soon recognized. A vast array of organisms was shown to possess extranuclear genes. These ranged from u n i c e l l -ular algae (Chlamydomonas, Sager, 1954) to complex higher plants (such as Tvitioum, Briggle, 1966). An example of the p r a c t i c a l importance of extranuclear inheritance was found i n Zea mays (Rhoades, 1933). A cytoplasmic gene causes male s t e r i l i t y and i s consequently used i n commercial corn hybridization to eliminate the cost of manual detassling. Also, i n man, two congenital abnormalities, anencephaly and spina b i f -ida, are suspected to be the result of cytoplasmic genes (Nance, 1969). Another case of extrachromosomal genes which i s of human interest i s seen i n mosquitoes (Lavens, 1967). These insects carry a cytoplasmic incompatibility between different populations. When incompatible males are introduced the females w i l l randomly mate with them reducing the number of the t o t a l population. This procedure was experimentally carried out i n Burma when a mosquito population was abolished i n a two 3 month period. Further examples of cytoplasmic inheritance are continuously being discovered. Yet, l i t t l e i s known about these organelle genes: how they interact with nuclear genes, their importance i n development of the organism and their evolution. Therefore, research i n this area i s becoming widespread. The most studied organisms are the lower eukaryotes. One, the green alga Chlamydomonas> shows maternal inheritance for resistance to certain a n t i b i o t i c s , as w e l l as temperature s e n s i t i v i t y , slow growth and photosynthetic mutations (Sager, 1972). Sager (1954) found that both parents contribute their cytoplasmic genes to the progeny. How-ever, the DNA from the two parents are treated d i f f e r e n t l y i n the zygote so that i t s phenotype resembles the female parent (Sager and Lane, 1969). Another well studied organism i s Saacharornyaes oerevisiae. A class of mutants of this yeast, referred to as cytoplasmic "petites" (Ephrussi and Hottinguer, 1951), shows non-Mendelian inheritance. These mutants are characterized by a small colony size and a deficiency i n cytochrome a c t i v i t y . Some petites have also been shown to retain only about 0.1% of their mitochondrial DNA and others have an altered base r a t i o so that the G + C content i s only 18% (Borst and G r i v e l l , 1978). In the past decade, the yeast mitochondrial genome has been extensively mapped and much has been learned regarding which genes are located i n the mitochondria and which are nuclear genes (Borst and G r i v e l l , 1978). The genetic and physical map of yeast mtDNA i s given i n Figure 1. The fungi, Podospora and Aspergillus also show cytoplasmic i n -heritance. The phenomenon of senescence i n Podospora (Tudzynski and 4 Figure 1. Genetic and Physical Map of Yeast mt DNA. The inner ring shows the location of various markers ( i e . erythromycin resistance). The black bars i n the inner ring represent insertions present i n the mt DNA of S. cevevisiae and absent i n S. carlsbergensis. The outer ring gives the positions of recognition s i t e s for endonucleases Hind I I + I I I and Eco RI and the pos-i t i o n - of 4S RNA genes. The open c i r c l e s are tRNA m e t genes. The approximate positions of other transcripts are given outside the outer r i n g , the bars indicating uncertainty i n the exact positions. The open part of the 21S rRNA represents the intervening sequence. Sal and Pst indicate the single recognition s i t e s for r e s t r i c t i o n endonuclease Sal I and Pst I, respectively. The figure and legend are taken from Borst and G r i v e l l , 1978. 6 Esser, 1979), and sexual d i f f e r e n t i a t i o n i n Aspergillus (Mahoney and Wilkie, 1958), are controlled by extranuclear genes. The fungus Neurospora orassa has been the subject of much research. This species i s an i d e a l genetic t o o l . The ease with which i t i s cultured and man-i pulated renders i t invaluable. The f i r s t case of unusual growth pat-terns i n Neurospora was found by M i t c h e l l and M i t c h e l l (1952). Their characterization of the s t r a i n [poky] (la t e r referred to as [mi-1] by M i t c h e l l ert a l . , 1953) showed a greatly reduced growth rate with respect to normal strains (Figure 2). U t i l i z i n g genetic analysis they showed that this growth habit could only be inherited through the maternal parent (with rare exceptions given i n the Discussion). They proposed a cytoplasmic factor as the cause, since the female presumably contrib-utes the bulk of the cytoplasm i n a cross. I t has also been found that [poky] lacks cytochromes a. and b_ and has an excess of c^  (Haskins et a l . , 1954). This fact, along with other mitochondrial abnormalities to be discussed i n Section I I , indicates that the unusual growth phenotype of [poky] i s associated with some heritable factor within the mitochon-dri a . Subsequently, several other maternally inherited growth mutants have been found i n N. orassa: maternally inherited-3 ([mi-3]), which shows slow growth and abnormal cytochromes (Mitchell e_t a l . , 1953) ; slow growth mutants ([SG]), these also show maternal inheritance, slow growth, and abnormal cytochromes (Srb, 1958); abnormals ([abn]), these strains show slow and abnormal growth, they are female s t e r i l e , so that cytoplasmic inheritance i s indicated by non-Mendelian ratios and transmission of the irre g u l a r growth through heterokaryons; the [abn]'s also have a defective cytochrome system (Garnjobst et a l . , 1965); 7 Figure 2. Growth Curves of Poky vs. Not Poky. Taken from M i t c h e l l and M i t c h e l l , 1952. 8 cn 120-i CD 0 S vHo 3o 35o 4l)0 H O U R S 9 stoppers ([stp]) are characterized by irregular growth and abnormal cytochromespectra, they, too are female s t e r i l e (McDougall and P i t -tenger, 1966); and extranuclear mutants ([exn]), these have a pronounced "lag" phase (very slow growth at f i r s t , followed by much faster growth), an abnormal cytochrome system, and are female f e r t i l e (Bertrand and Pittenger, 1972). A l l of the N. orassa growth mutants mentioned are laboratory derived s t r a i n s . They either arose i n laboratory stocks or were induced through mutation. Consequently, l i t t l e i s known regarding the adaptive significance, i f any, of the unusual growth rates. I f this behavior could be shown to exist i n nature, i t would provide an opportunity to explore i t s r o l e , and, perhaps to understand how such an extraordinary growth habit could survive and compete i n nature. Genetic and bio-chemical work on natural isolates possessing abnormal growth may add insight to the relationship between nuclear and cytoplasmic genes. Thus, i t was indeed of interest when a population study, undertaken to invest-igate v a r i a t i o n i n growth rates among natural populations (carried out by Dr. A.J.F. G r i f f i t h s ) , of N. intermedia i s o l a t e s , disclosed two with a stop-start growth pattern. The strains used for G r i f f i t h s ' study were collected by D.D. Perkins from locations around the world. No abnormalities were noted when the strains were f i r s t isolated by Perkins (the strains are l i s t e d i n Materials and Methods). Once these two ex-ceptional isolates were recognized, a search was undertaken for additional strains with a similar growth phenotype. The strains used for this were also collected by Perkins and were from Kauai, Hawaii. The research described i n this thesis includes the i d e n t i f i c a t i o n of several strains among these natural isolates showing abnormal growth 10 rates. The objectives were not only to discover these st r a i n s , but to characterize them genetically and biochemically. I f these strains could be shown to possess mitochondrial abnormalities, as do the N. orassa s t r a i n s , they would provide a new source of mutants with which to probe the mitochondrial genome. Also, information would be gained as to the ecological role of such strains and the mechanism of the mutation. The f i r s t question which arises i s whether or not the growth habit of the N. intermedia isolates shows cytoplasmic inheritance. Several methods can be used i n Neurospora to fi n d the mode of i n h e r i t -ance of this type of t r a i t . Two of these are: 1) reciprocal crosses and, 2) heterokaryon testing. In reciprocal crosses, only the female parent should pass on i t s phenotype, i f the t r a i t i s extranuclear. Thus, when an abnormal i s o l a t e i s used as the female, the progeny should show the abnormal phenotype. When the normal s t r a i n i s used as the female, the progeny should be normal. I t has been found i n N. orassa that the anomalous growth habit can be passed through a heterokaryon (Garnjobst et. a l . , 1965). A het-erokaryon consists of genetically d i s t i n c t nuclei within a. common cyto-plasm. Heterokaryons are formed frequently i n fungi and arise through hyphal anastomosis. A heterokaryon can be forced to occur i f two compat-i b l e strains possessing different auxotrophic markers are inoculated onto minimal media together. Both cultures i n i t i a t e l imited growth. The hyphae fuse and the nuclei migrate through the cytoplasm enabling the culture to continue growing. I f however, the strains are incompatible, there i s anastomosis, but the single fusion c e l l i s blocked off form the rest of the hyphae 11 (Burnett, 1975). Nuclei can not migrate so no heterokaryon re s u l t s . Both strains w i l l cease growth due to the lack of a nutrient, the one required by the auxotrophic marker. Heterokaryon compatibility i n the species N. orassa requires that the two strains be of the same mating type. In other words, two strains which w i l l form a sexual cross w i l l not produce vegetative heterokaryons. However, not only must they be of l i k e mating type, but they must also be homozygous at three other known l o c i : C^, D, E_ (Garnjobst, 1953; Mylyk, 1975). An example of compatible strains would be two A mating type cultures both carrying the three recessive a l l e l e s : c_, d., and e^ . Only when a l l these requirements have been met are the strains compatible and a successful heterokaryon formed. The conidia which are produced by a heterokaryon w i l l consist of some which contain the nucleus of one s t r a i n with the cytoplasmic factors of the other (Figure 3). Thus, a heterokaryon test can be used to show a cytoplasmic factor as the cause of a t r a i t . When a normally growing s t r a i n and an abnormal growth phenotype s t r a i n are forced into a heterokaryon, some resulting conidia should possess the cytoplasm from the abnormal s t r a i n and nuclear markers of the normal s t r a i n . The N. orassa cytoplasmic growth mutants which have been shown to pass the i r phenotype through a heterokaryon i n this way are: [abn-1] and[abn-2] (Garnjobst et a l . , 1965), [mi-l] and[mi-4] (Pittenger, 1956), and [exn-l] and [exn-4] (Bertrand and Pittenger, 1968). There were some d i f f i c u l t i e s encountered with [abn-l]and[abn-2]: the growth rate of the heterokaryons varied and many cultures did not survive s e r i a l trans-fer; however, a few cultures were isolated which possessed nuclear markers of the wi l d type s t r a i n and an abnormal growth phenotype (Garn-12 Figure 3. Heterokaryon Formation. A represents a slow growth mutant with abnormal cytoplasmic factors (A) and unmarked nuclei ( O ) . JB represents a s t r a i n with a normal growth phen-otype. I t has normal cytoplasmic factors (A) and marked nuclei (•) . C_ i s the heterokaryon with developing conidia. JJ i s part of the mycelium resulting from one of the conidia. I t contains the abnormal cytoplasmic factors with marked nucl e i . Adapted from Suzuki and G r i f f i t h s , 1976. 13 c 14 jobst e_t a l . , 1965) . Some of these cytoplasmic growth mutants of N. orassa are female s t e r i l e ([abn-1] and [abn-2]). They are thought to be cytoplasmic mutants because, when they are used as the male parent their pheno-type i s not passed on as would be expected of a nuclear mutation. Also, i n these cases heterokaryotic transfer i s often used as an a l t -ernative indication of an extranuclear cause of e r r a t i c growth. When heterokaryons were obtained involving cytoplasmic growth mutants of N. orassa, the strains used were known to be compatible with respect to vegetative heterokaryosis. In the case of N. inter-media, however, the heterokaryon incompatibility system has not yet been researched. Consequently, strains have not been i d e n t i f i e d as possessing certain incompatibility genes ( i f they do e x i s t ) . Once extranuclear inheritance i s determined, there are several possible causes. F i r s t , the t r a i t could be carried i n the genetic information of an organelle such as the mitochondrion or, i n the case of green plants, the chloroplast. This p o s s i b i l i t y i s discussed i n Section I I . Second, the unusual phenotype could be the result of a v i r a l i n f e c t i o n . A disease (known as "die-back") of the common cultivated mush-room and characterized by slow, abnormal growth, i s caused by a virus (Rollings e_t a l . , 1963). More s i g n i f i c a n t l y , v i r u s - l i k e p a r t i c l e s (VLP) have been found associated with three slow growing strains of N. orassa, one of which was a natural i s o l a t e (Tuveson and Peterson, 1972). These VLP are polyhedral p a r t i c l e s with a diameter of 170 nm. Tuveson and Peterson examined [poky],[abn-l], and another variant s t r a i n designated P147, as well as a wild type s t r a i n . D.D. Perkins 15 had collected s t r a i n P147 i n Indonesia and noted that i t grew slowly even upon the i n i t i a l i s o l a t i o n . A l l three slow growing strains had VLP associated with them, the wild type* did not. The researchers did not claim that these VLP necessarily caused the abnormal growth phenotype, since they did not show i n f e c t i v i t y of the p a r t i c l e s . Such p a r t i c l e s would, however, be expected to show cytoplasmic inheritance, just as the unusual growth habit does. To explore the inheritance and cause of the e r r a t i c growth of the N. -intermedia i s o l a t e s , they were: 1) put through reciprocal crosses with normal i s o l a t e s , 2) tested for heterokaryotic transfer, 3) exam-ined for the presence of v i r u s - l i k e p a r t i c l e s , and 4): characterized with respect to several biochemical t r a i t s (Section I I ) . * Strain P147 can be considered a w i l d type s t r a i n since i t i s a natural i s o l a t e . However, any such s t r a i n ( i e . a natural i s o l a t e which shows abnormal growth behavior) w i l l be referred to as aberrant or variant. Wild type w i l l only be used to describe strains which do not show any unusual growth characteristics. 16 MATERIALS AND METHODS Strains Neurospora intermedia strains 2360, 2361, 2363, and 2366 are natural isolates o r i g i n a l l y collected from Kauai, Hawaii, by D.D. Perkins. They were obtained from the Fungal Genetics Stock Center (FGSC), Areata, C a l i f o r n i a , and are l i s t e d i n Table I along with other N. intermedia strains tested. An additional 83 isolates also from Kauai, were donated by Perkins and are l i s t e d i n Table I I . Neurospora intermedia FGSC auxotrophic strains 3386 and 3401 were used for some crosses. A h i s t i d i n e requiring mutation was induced into s t r a i n 2360 by D.L. Robbins using u l t r a v i o l e t l i g h t . This s t r a i n i s abbreviated 2360his. Other strains used for the heterokaryon tests included N. orassa and N. intermedia auxotrophs obtained from the FGSC and l i s t e d i n Table I I I . The only s t r a i n examined for v i r u s - l i k e p a r t i c l e s was FGSC s t r a i n 2360. Media and Growth Conditions Minimal Vogel's, N u t r i t i o n a l Testing, Pl a t i n g , and Crossing media are a l l described by Davis and deSerres (1970). Complete medium (containing 2% Vogel's solution, 0.005% tryptophane, 0.5% casein hydroly-sate, 0.5% yeast extract, 1% dextrose, 1% vitamin solution, and 2% agar) was used for some tests. Supplementation consisted of 0.25 mg/ml for h i s t i d i n e , methionine, leucine, and cysteine; and 0.05 mg/ml for panto-thenic acid, i n o s i t o l , and r i b o f l a v i n . Water and acetone washed agar was used for heterokaryon tests to assure the absence of extraneous nutrients. 17 Table I. Neurospora intermedia isolates obtained from the FGSC. FGSC Strain Mating Collection FGSC Strain Mating Collection Number Type Site Number Type Site 2316 A Florida 1826 A Indonesia 1940 a Flo r i d a 1827 a Indonesia 2236 A Florida 1836 A Indonesia 2237 a Flo r i d a 1837 a Indonesia 2360 A Hawaii 1820 A India 2361 a Hawaii 1821 a India 2363 a Hawaii 1803 A India 2365 a Hawaii 2496 a India 2366 A Hawaii 2367 a Hawaii 1768 A Japan 1766 A Taiwan 1767 a Taiwan 1818 A Taiwan 1819 a Taiwan 1762 A P h i l l i p i n e s 1763 a P h i l l i p i n e s 1797 A Malay Pen. 1798 a Malay Pen. 1799 A Malay Pen. 1800 a Malay Pen. 1784 A New Guinea Pen. = Peninsula 1785 a New Guinea 1937 A New Guinea 1938 a New Guinea 1830 A Aus t r a l i a 1831 a Au s t r a l i a 1792 A Indonesia 1793 a Indonesia 1796 A Indonesia 1795 a Indonesia 18 Table I I . Neurospora intermedia isolates obtained from D.D. Perkins, ( a l l from Kauai, Hawaii). Perkins Perkins Stock Mating Stock Mating Number Type. Number Type P 558 a P 608 a P 561 a P 609 A P 562 A P 612 A P 563 a P 613 a P 564 A P 614 a P 565 A P 615 A P 566 A P 616 a P 567 A P 617 A P 568 a P 618 a P 570 a P 619 A P 572 A P 620 A P 573 a P 621 A P 574 a P 622 A P 576 A P 623 A P 577 A P 624 A P 587 A P 625 A P 591 A P 626 A and P 592 a P 628 a P 593 A P 630 a P 594 A P 631 A and P 595 a P 632 A P 596 A P 633 a P 597 a P 634 a P 598 A P 635 a P 599 a P 636 A and P 602 a P 638 a P 603 a P 639 a. P 604 A P 640 A and P 605 A P 641 a P 606 A P 643 A and P 607 A and a P 644 A and Perkins Stock Mating Number Type p 645 A p 646 A p 789 a p 790 A p 791 A p 792 A p 793 A p 794 a p 795 a p 796 a p 797 a p 798 a p 799 A p :800 A p 801 a p 802 a p 803 A p 804 a p 805 A p 806 A p 809 a mixed cultures, A and a, are probably N. in-termedia but were not seperated for d e f i n i t e testing by Perkins. Note that none were of interest to this study. 19 Table I I I . N. orassa and N. intermedia auxotrophic strains used for Heterokaryon Testing. N. orassa auxotrophic s t r a i n s * FGSC het auxotrophic number genes marker 1423 CDE pan 1424 CdE pan 1425 cDE pan 1426 cdE pan 1454 CDe inos 1453 Cde inos 1455 cDe inos 1422 cde inos 478 CDE rib-2 476 . cDE inos 538 CdE inos 474 cdE inos * a l l strains are mating A Auxotrophic requirement abbreviations: pan-pantothenic acid i n o s - i n o s i t o l r i b - r i b o f l a v i n arg-arginine cys-cysteine h i s - h i s t i d i n e N. intermedia auxotrophic s t r a i n s * FGSC auxotrophic number marker 3370 arg 3378 arg 3389 cys 3395 u- A his 3397 his 3399 u- A his 3393 asn 3391 his AThis his locus i s a different one from the 2360his locus ( i e . at least one of these strains would possess a different his locus from that of 2360his). 20 Strain 2360 was grown i n l i q u i d media consisting of minimal Vogel's medium without agar, i n preparation for electron microscopy. The sucrose buffer used for harvesting the mycelia was 0.44M sucrose, lOmM Tris-HCl pH 7.2, and 5mM EDTA (ethylenediamine-tetraacetic acid disodium s a l t ) . Growth rate tests were carried out i n 50 cm. growth tubes contain-ing 30 mis. of Vogel's medium, at room temperature. A l l other c u l -ture and crosses were grown at 25°C. Any s t r a i n that showed a stop-s t a r t growth pattern was also grown i n the 25°C incubator to confirm i t s e r r a t i c pattern. Methods A. Growth Rate Tests Growth rate measurements involved inoculating a culture at one end of a growth tube, and marking i t s d aily progress u n t i l i t reached the opposite tube end. Variant strains that never attained this goal were allowed a maximum of 39 days to ensure, within reason, that they would not resume growth. Once strains possessing an abnormal growth phenotype had been i d e n t i f i e d , conidia suspended i n 0.1% agar were plated on p e t r i dishes using an overlayer technique (Newmeyer, 1954). For each s t r a i n a minimum of t h i r t y - f i v e colonies, presumably a r i s i n g from a single conidium, were isolated i n an attempt to obtain more homogeneous cultures. These were put i n growth tubes, and one i s o l a t e showing an e r r a t i c pattern was selected for each s t r a i n , to be used for genetic analysis and biochemical studies (Section I I ) . A l l the, anomalous strains were also tested on complete media i n growth tubes. The abnormal strains were put through reciprocal crosses to either normal s t r a i n 2361 (mating type a.) or 2366 (mating type A) . These two strains are also from Kauai. Each cross was simultane-ously replicated 20 times. Reciprocal crosses are unusually straightforward i n Neurospora, since either mating type (A or a.) can be used as the maternal or paternal parent. The maternal par-ent i s that which i s i n i t i a l l y inoculated onto crossing medium. These are f e r t i l i z e d 7-10 days l a t e r by a co n i d i a l solution of the s t r a i n acting as the paternal parent. After 21 days either random spores or tetrads are isol a t e d , heat shocked for 30 minutes at 60°C to i n i t i a t e germination, and grown at 25°C. B. Heterokaryon Tests The i n i t i a l step i n forcing heterokaryons i s to grow the strains on appropriately supplemented vegetative media. After 4-5 days growth, a con i d i a l suspension i s prepared containing approximately 4 10 conidia per ml. of d i s t i l l e d water. A single drop of the suspen-sion i s placed into test tubes of minimal media. Added to this i s a drop of the suspension from another s t r a i n with a different auxo-trophic marker. Each s t r a i n i s also inoculated singly to test for "leakiness" (growth of the supposed auxotrophic s t r a i n on minimal media). Each test i s repeated i n f i v e tubes. After 3, 4, and 5 days the results are recorded. A l l N. orassa auxotrophic strains which showed a degree of l e a k i -ness, were replaced by a s t r a i n carrying the same het (heterokaryon incompatibility genes such as C_, JJ, and E as previously described) genes, but a different auxotrophic mutation. Heterokaryons f a i l e d to form between 2360his and the N. orassa auxotrophs. At about the same time as these experiments ended, auxotrophic strains of N. intermedia became available through the FGSC. A l l these strains were tested. 22 When these also f a i l e d to form heterokaryons, a new avenue was explored. I f s t r a i n 2360 was used as the male parent i n a cross to a N. intermedia auxotroph, some progeny would be expected to have nuclear genes of 2360 within a normal (with respect to growth phenotype) cytoplasm. These new strains could thus possess the het genes of 2360, be of mating type A (as i s 2360), and s t i l l show a different auxotrophic marker than s t r a i n 2360his. They would then be expected to form heterokaryons with 2360his, i f the het genes were the same. However, this depends on whether or not the heterokaryon incompatibility system i n N. intermedia i s si m i l a r to N. orassa. In other words, i f the incompatibility a l l e l e s must be homogenic for a heterokaryon to form. Although nothing i s known about the incompat-i b i l i t y system of N. intermedia t h i s approach was s t i l l worthy of t r i a l . So, crosses were made using strains 2360 as the male and 3401 and 3386 as the female. 200 progeny were isolated per cross. 104 progeny grew from the cross 3401 x 2360 and 121 grew from the cross 3386 x 2360. This t o t a l of 225 isolates was tested for n u t r i t i o n a l requirements, by spot testing on supplemented media as well as minimal media. Mating type tests were performed as described by Davis and deSerres (1970). The 43 auxotrophic mating type A isolates of the desired type were put through heterokaryon tests with 2360his. C. Virus Search 10^ conidia per ml. of s t r a i n 2360 was inoculated into 300 mis. of l i q u i d media and grown at 25°C i n a shaker incubator. The 24 hour old culture was harvested by suction f i l t r a t i o n , washed with a sucrose buffer, and ground with acid washed sand i n a mortar and pestle. The 23 preparation was centrifuged twice at 3,000 rpm to remove sand and large clumps of mycelium. The remainder of the procedure consisted of standard preparation f or electron microscopy (Dawes 1971) and was c a r r i e d out by Dr. Stace-Smith at A g r i c u l t u r e Canada, University of B r i t i s h Columbia. 24 RESULTS Growth Characteristics of the Original Strains Most isolates studied showed linea r growth rates and attained the 50 cm. tube length i n 5-8 days. Two out of 39 N. intermedia isolates obtained from the FGSC, 2360 and 2363, revealed a stop-s t a r t growth pattern. Of 83 additional strains donated by D.D. Per-kins three, P804, P608, and P594, proved to be variant cultures. Thus, there was a t o t a l of f i v e abnormal strains (plus one auxotroph, s t r a i n 2360his). The growth curves of these are shown i n Figure 4. As can be seen, the cultures may stop and star t growth several times, with no consistency i n the duration of either the growth or stop phases. Normal growth rates are between 3.5 mm/hr and 5.3 mm/hr. The rates of abnormal strains vary, on the average, from 0 mm/hr to 5.3 mm/hr. Colony Isolates I t was also noted that the same i s o l a t e , simultaneously put into two growth tubes, by mass transfer, w i l l give different patterns (Fig-ure 5). This created suspicion about the homogeneity of the culture. Therefore, the abnormal isolates were put through a p u r i f i c a t i o n pro-cedure by thi n l y plating c o n i d i a l suspensions, and picking the colonies which arise from a single conidium. Each colony was then examined with respect to i t s growth pattern. A l l 52 isolates recovered for s t r a i n 2360, and a l l 37 recovered for 2363, showed e r r a t i c growth of which representative patterns are shown i n Figure 6. Out of 43 colonies of P608(Figure 7), 28 came to a complete stop at least once, 10 slowed growth down to 1.6 mm/hr and 25 Figure 4. Growth Curves of the Five Anomalous N. inter-media Strains (2360, 2363, P804, P608, P594) and One Normal Strain (2361). A l l were grown i n 50 cm. growth tubes on minimal medium at 25°C. i i i i i l i i i i I I 1 I 1 l — l — l — l — I — i — l — l — I — I — i — i — i — i — r 0 4 8 12 16 20 24 28 T I M E ( d a y s ) 27 Figure 5. Two Growth Curves of N. intermedia Strain 2360. The inoculum was from the same culture tube and produces two unique growth curves when put into two growth tubes simultaneously. 28 2360-1 2360-2 \ i rn i i i i i i rn m i i i i i — i — i — r n — i — m — m n 0 4 8 12 16 20 24 28 T I M E ( d a y s ) 29 Figure 6. Growth Curves of Conidial Isolates of N. intermedia Strains 2360 and 2363. Three representative curves are shown for each. 30 0 4 8 12 16 20 24 28 T I M E ( d a y s ) 31 Figure 7. C o n i d i a l Isolates of N. intermedia S t r a i n P608. Four representative growth curves of c o n i d i a l i s o l a t e s show the heterogeneity of t h i s s t r a i n . 32 33 eventually resumed more normal speeds (approximately 2.3 mm/hr), but 5 grew without slowing or stopping. However, none of the isolates grew the tube length i n less than 9 days. The colonies isolated for strains P594 and P804 gave similar results as P608: some stopping, some only slowing down, and others neither slowed not stopped i n the growth tube tests. Representative growth curves are given i n Figures 8 and 9. A summary of a l l these results i s given i n Table IV. I t was also noted for s t r a i n P594 that i f some of the nonstopping cultures were repeatedly subcultured they would eventually y i e l d a stopping s t r a i n . These stop-start strains never resumed normal growth. They did, however, become more s i c k l y and often died. Reciprocal Grosses For each s t r a i n , one of the above colonies that consistently showed the stop-start pattern, was chosen to be analyzed genetically. Each abnormal i s o l a t e was crossed to a normal one (see Methods) and 100 random progeny analyzed for growth rates. Two st r a i n s , 2360 and P608, appear to be female s t e r i l e , only rarely producing perithecia ( i e . i n only 1 cross i n 10). As either the male or female parent strains P594, P804, and P608 y i e l d approximately 1/3 to 2/3 hyalin-colored spores, as opposed to the t y p i c a l black spores. The hyalin spores never germinated, and thus appear to be aborted. Table V summarizes the results of a l l crosses. Note: 1) that transmission of the abnormal growth behavior i s very i n e f f i c i e n t , 2) s t r a i n 2360 as male or female yields some progeny that show "slow" growth, that i s , they never stop, but maintain a slow speed ( the highest speed i s approximately 1.8 mm/hr) u n t i l reaching the f u l l length of the growth tube, 3) strains 2360 and P594 do show a degree 34 Figure 8. Conidial isolates of N. intermedia Strain P594. Four representative growth curves of conidial isolates show the heterogeneity of the strain. 35 T IME ( d a y s ) 36 Figure 9. Conidial Isolates of N. intermedia Strain P804. Four representative growth curves of conidial isolates show the heterogeneity of this s t r a i n . 37 6 31 0 4 8 12 16 20 24 28 T l M E ( d a y s ) 38 Table IV. Summary of Results of Conidial Isolation for N. intermedia Strains P594, P608, and P804. P608 Slowest growth approximately 5 2.3 mm/hr Slowest growth approximately 5 0.8 mm/hr Slowest growth approximately 5 0.4 mm/hr umber of Isolates of Strain: P594 P804 24 13 11 4 Slowest growth O.Omm/hr 28 8 10 39 Table V. Summary of Results of Reciprocal Crosses of the Normal and Abnormal Isolates of N. intermedia. Number of progeny germinated per 100 Number of Maternal Slow Cross isolated spores stoppers Inheritance growers 2361 X 2360 72 0 - 5 2360 x 2361 79 2 yes 3 2361 X 2363 86 1 - 0 2363 X 2361 53 2 0 2366 X P594 12 A 0 - 0 P594 X 2366 72 4 yes 0 2361 X P608 31 0 - 0 P608 X 2361 71 0 * 0 2361 X P804 87 2 - 0 P804 X 2361 58 2 * 0 AOut of 200 isolated spores *These strains do not show maternal inheritance i n these crosses. How-ever that does not rule out cytoplasmic inheritance. The maternal parent i s written as the f i r s t s t r a i n i n a cross ( i e . 2361 x 2360: 2361 i s the female and 2360 i s the male). Slow growth i s defined a r b i t r a r i l y as not reaching the tube end i n less than 12 days. 40 of maternal inheritance, 4) strains 2363 and P804 transmit the abnormal phenotype through either the male or female parent (but only rarely i n both cases), 5) s t r a i n P608 never yields abnormal progeny, 6) out of 200 spores from the cross involving s t r a i n P594 as the male, only 12 germinated, this may be due to an incompatibility between the normal strain's cytoplasm and nuclear genes of s t r a i n P594 since this low v i a b i l i t y was only noted when P594 was the male parent. Hence, i t appears that this s t r a i n shows cytoplasmic inheritance. However, too few progeny were obtained when i t i s used as the male parent, to make a conclusive statement on the patterns of inheritance of this s t r a i n . This was not studied more closely. Growth Characteristics of the Progeny A majority of the progeny showed normal growth rates. Those pro-geny labelled stoppers i n Table IV possess phenotypes reminiscent of their parents: they stop and start i n no discernible patterns. The only progeny that do not f i t into one of these two categories are the 8 offspring of s t r a i n 2360 which show slow growth as previously mentioned. Growth on Complete Media To rule out the p o s s i b i l i t y that the abnormal phenotype i s caused by a n u t r i t i o n a l requirement, a l l the o r i g i n a l strains were grown on complete media. The growth curves are given i n Figure 10. These are the same as those using minimal media. Heterokaryon Formation The f i r s t attempts at heterokaryon formation involving the stop-41 Figure 10. Growth Curves on Complete Media. The f i v e stop-start strains of N. intermedia (2360, 2363, P594, P608, P804) and one normal s t r a i n (2361) have been grown on complete media i n growth tubes. Representative growth curves are given. 42 43 start s t r a i n 2360his, used N. orassa auxotrophic strains possessing the eight different combinations of heterokaryon incompatibility genes (i e . CDE, CDe, Cde, etc.)- Every attempt at forming heterokaryons between 2360his and the N. orassa auxotrophs was unsuccessful. The results are l i s t e d i n Table VI. The only tubes showing growth are those which contain a "leaky" s t r a i n . Each test was carried out sim-ultaneously i n 5 tubes, and the whole procedure was performed 3 times. Thus, each s t r a i n was tested with 2360his a t o t a l of 15 times. Two compatible N. orassa strains (1-37-21 and 1453) were tested to assure r e l i a b i l i t y of the procedure. Five out of f i v e tubes formed heterokaryons and neither s t r a i n revealed i t s e l f as leaky. The N. -intermedia auxotrophic strains tested also did not form heterokaryons with s t r a i n 2360his (Table V I I ) . The f i n a l attempts at forcing heterokaryons with 2360his involved progeny from crosses of 2360 as the male parent and N. intermedia strains 3401 and 3386. Out of the 225 progeny iso l a t e d , a t o t a l of 43 were mating type A and auxotrophic mutants for either methionine or leucine. Again, a l l attempts at heterokaryon formation f a i l e d . The results are summarized i n Table VIII. Virus Search Strain 2360 was examined for v i r u s - l i k e p a r t i c l e s but no evidence of them was found. Tuveson and Peterson (1972) examined three slow grow-ing strains of N. orassa and, on the f i r s t attempt, found VLP associated with each. However, they found only a few p a r t i c l e s i n [poky]. The choice of the word "few" implies a scarcity of such p a r t i c l e s . Thus, i t i s possible that VLP exist i n s t r a i n 2360 and are too scarce to have been 44 Table VI. Results of Heterokaryon Tests Between N. intermedia Strain 2360his and N. orassa Auxotrophs. Heterokaryon Test 1423 + 2360his 1424 + 2360his 1425 + 2360his 1426 + 2360his 1454 + 2360his 1455 + 2360his 1422 + 2360his 1453 + 2360his 478 + 2360his 538 + 2360his 474 + 2360his Result + + + + Control 1423 1424 1425 1426 1454 1455 1422 1453 478 528 474 2360his Result + + + + + indicates growth - indicates absence of growth * the N. orassa strains involved i n these tests are leaky auxotrophs Table VII. Results of Heterokaryon Tests Between N. intermedia Strain 2360his and N. intermedia Auxotrophs. Heterokaryon Test 3370 + 2360his 3378 + 2360his 3389 + 2360his 3395 + 2360his 3397 + 2360his 3399 + 2360his 3393 + 2360his 3391 + 2360his Result Control 3370 3378 3389 3395 3397 3399 3393 3391 Result * very s l i g h t growth occurred i n 3/15 tubes, but not enough for transfer. The cultures soon died. + indicates growth - indicates absence of growth 46 Table VIII. Summary of the Results of Heterokaryon Tests Between 2360hls and Progeny of the Crosses: 3401 x 2360 and 3386 x 2360. Number of Pro-geny of A mating Number of Pro- Number of Pro- type with a Cross geny isolated geny germinated marker  3401 x 2360 200 104 19 3386 x 2360 200 121 24 NONE OF THE PROGENY OF THESE CROSSES FORMED HETEROKARYONS WITH 2360his. found i n the one attempt. 48 DISCUSSION Five i s o l a t e s of N. intermedia (strains 2360, 2363, P594, P608, P804) were found to have s i g n i f i c a n t l y d i f f e r e n t growth curves from those of the majority of s t r a i n s . Two of these, 2360 and 2363, are homogeneous cultures as indicated by a l l the c o n i d i a l i s o l a t e s possess-ing the stop-start phenotype, However, the other three abnormal s t r a i n s , P594, P608, and P804, did not show such homogeneity. Not only did some i s o l a t e s not stop, but these same i s o l a t e s when tested again could produce a d i f f e r e n t r e s u l t i n the growth rate test (ie. the stop-s t a r t character). This suggests both a heterogeneity of the conidia of each s t r a i n , and also a heterogeneity within a single conidium. The f a c t that subculturing eventually y i e l d s a homogeneous stop-start s t r a i n (noted i n s t r a i n P594) may i n d i c a t e some advantage of t h i s phenotype over the normal growth behavior; or an advantage of the mutant fa c t o r over the normal fa c t o r (such as r e p l i c a t i v e advantage of the mutant DNA over normal DNA). However, i t i s not possible at t h i s point to specu-l a t e what the nature of t h i s advantage may be. This suppressiveness of the anomalous phenotype exists when hetero-karyons between N. orassa [abn] and normal s t r a i n s are made (Garnjobst et a l . , 1965). Conidia a r i s i n g from these heterokaryons showed various growth rates. However, even the normal cultures eventually became ab-normal or died. Thus, the factor(s) responsible apparently became phenotypically suppressive. Why the abnormal phenotype becomes mani-fested i s not known, but i t appears that t h i s i s also happening with the N. intermedia i s o l a t e s of heterogeneous o r i g i n . I t i s unfortunate that two s t r a i n s (P608 and 2360) tend to be 49 largely female s t e r i l e . The occassional successful cross i s therefore rather suspect, since i t takes an unusually long time to produce p e r i -thecia. This indicates that the anomalous s t r a i n allows the normal parent to contribute protoperithecia (theoretically rendering this normal s t r a i n as the actual female parent). When M i t c h e l l and M i t c h e l l (1952) performed crosses using [poky] as the female culture they, too, noted that protoperthecia did not form within the expected time (about f i v e days for normal cultures). However, i f they waited long enough, [poky] did produce proto perithecia. Crosses i n which the [poky] s t r a i n was f e r t i l i z e d before protoperithecia had formed, did not pass on the [poky] character. This may explain the lack of progeny with the aberrant phenotype i n crosses involving s t r a i n P608. I f cytoplasmic inheritance i s involved, and this s t r a i n never poses as the actual f e-male, i t would not pass on the stop-start phenotype. Strain 2360, on the other hand, does rarely (2 out of 79) pass on i t s phenotype when used as the female parent. The assumption then i s that i t can occassion-a l l y produce protoperithecia. However, i t i s possible that determinants from the normal male parent are being passed on to these rare abnormal progeny. This female s t e r i l i t y i s also seen i n the N. orassa cytoplasmic mutants [abn-l] and[abn-2] (Garnjobst et a l . , 1965), [mi-4] (Pittenger, 1956), and [stp] (Srb, 1963). In crosses of a l l these female s t e r i l e s t r a i n s , including the two N. intermedia i s o l a t e s , the aberrant pheno-type has never been transmitted when the abnormal s t r a i n poses as the male parent. The fact that, even i n the female f e r t i l e s t r a i n s , the abnormal growth phenotype i s rarely passed on to progeny, i s i n contrast to the 50 results found with [poky]. M i t c h e l l and M i t c h e l l (1952) noted that i n most crosses involving [poky] as the female parent, a l l progeny were [poky]. The reason that the female f e r t i l e N. intermedia strains only rarely pass on thei r abnormal growth phenotype i s not clear. There are two li n e s of evidence that imply that a cytoplasmic factor i s associated with the abnormal growth phenotype of the N. in-termedia i s o l a t e s . F i r s t l y , as mentioned, this growth behavior i s not commonly transmitted when the abnormal s t r a i n poses as the male parent. However, strains 2363 and P804 did transmit the stop-start phenotype when they were used as either the male or female parent (although i n both cases very r a r e l y ) . There i s no explanation for this i f maternal inheritance i s involved since no anomalous progeny should be produced when the abnormal s t r a i n i s the male parent. Sec-ondly, a nuclear gene explanation i s not suitable since none of the data show a simple Mendelian segregation r a t i o as expected for a single nuclear gene. Neither does a polygenic nuclear explanation appear as a l i k e l y candidate, since the progeny do not show a wide range of phenotypes. The only nonparental phenotype produced i s that of 8 progeny from crosses involving s t r a i n 2360. These isolates never stop i n the growth tube, but do take an unusually long time to reach the tube end (up to 13 days). Since a range of phenotypes i s not observed i n the progeny, a polygenic explanation would have to include a thres-hold effect. M i t c h e l l and M i t c h e l l (1952) noted that i n some crosses involving [poky]mutants a number of progeny grew too slowly to be called wild type but not as slowly as [poky]. They offer no explanation for t h i s . This result was unexpected not only because this was a nonparent-a l phenotype, but also because i n some cases [poky]had served as the 51 f e r t i l i z i n g parent, so that growth was expected to be normal. Even with these unusual re s u l t s , extranuclear inheritance i s the most l i k e l y p o s s i b i l i t y , since there i s no simple nuclear gene explanation. In other cases, such as [abn-l] and [abn-2] (Garnjobst e_t a l . , 1965), where the t r a i t i s not passed on at a l l due to female s t e r i l i t y (also,[mi-4] Pittenger, 1956, and [stp] Srb, 1963), cyto-plasmic inheritance has been demonstrated by eliminating the poss-i b l i t y of nuclear inheritance. This can also be done for the N. inter-media strains. If the abnormality was the result of a single gene, half the progeny would be normal and half abnormal, regardless of how the cross was made. No such segregation i s ever found. On the other hand, i f the abnormality was the result of several nuclear genes, the progeny should show an entire range of various phenotypes. Again, this was not observed. Further evidence of cytoplasmic inheritance for the female s t e r i l e strains [abn-l] and [abn-2], i s that i n heterokaryons with wild type the abnormal phenotype eventually becomes suppressive (Garnjobst et a l . , 1965). Also, when the cytoplasm of the [abn-2] s t r a i n was microinjected into w i l d type strains the resulting culture developed the abnormal character (Garnjobst et al., 1965). No microinjection studies were performed on the N. intermedia strains due to technical d i f f i c u l t y i n the procedure. Therefore, to strengthen the evidence for cytoplasmic inheritance i n the N. intermedia i s o l a t e s , attempts were made at forcing heterokaryon formation with normal strains . Unfortunately, no hetero-karyons were formed. Heterokaryon incompatibility systems vary among species. In N. sitophila heterokaryosis depends on only 1 pair of a l l e l e s , h e t + and 52 het . Two h e t + strains w i l l form a heterokaryon, regardless of mating type. N. tetrasperma has absolutely no r e s t r i c t i o n s on het-erokaryon formation and even depends on vegetative heterokaryons to complete i t s l i f e cycle (Fincham ^ t a l . , 1979). However, the system i n N. orassa i s more stringent. There have been more than 10 incompat-i b i l i t y l o c i i d e n t i f i e d i n the N. orassa genome (Mylyk, 1975). Studies on N. orassa populations revealed a great deal of v a r i a b i l i t y i n the heterokaryon genotypes of strains within a population, as we l l as from one population to another (Mylyk, 1976). Consequently, i t i s not very suprising that heterokaryons f a i l e d to form i n the N. intermedia strains tested. They are a l l natural i s o l a t e s , not laboratory derived s t r a i n s , and thus most probably rep-resent a vast array of genotypes a l l lumped under the heading of "wild type". I t was, therefore, reasonable to cross the het genes of the abnormal s t r a i n , 2360, into a normal cytoplasm, although the pro-geny of this type also f a i l e d to form heterokaryons with 2360his. Per-haps further backcrossing of these progeny to 2360 would have yielded strains with even more 2360 het genes. The number of backcrosses would depend on the number of het genes involved. However, the N. inter-media incompatibility system may not depend on homogeneity of these het genes and then these crosses would be f u t i l e . I t i s l i k e l y that the incompatibility system i s more complex than i n N. sitophila and N. tetrasperma, since such a large number of N. in-termedia auxotrophic strains was tested. No successful N. intermedia heterokaryons have ever been reported i n the l i t e r a t u r e . I t would cl e a r l y require further tests of a l l combinations of the N. intermedia auxotrophs available to unravel the genetics of incompatibility i n this 53 species. Regardless of the f a i l u r e of heterokaryosis, the evidence for extranuclear inheritance was strong enough that various cytoplasmic causes were explored. The theory that a v i r a l i nfection was causing the stop-start phen-otype i s by no means unfounded. A l l f i v e N. intermedia strains were isolated from the same Hawwaiian Island, and could consequently have been exposed to the same source of v i r a l i n f e c t i o n . Also, as mentioned v i r u s - l i k e p a r t i c l e s had previously been found associated with slow growing strains of N. orassa '('Tuveson and Peterson, 1972). Since only one s t r a i n , 2360, was examined for VLP, i t i s possible that some or a l l of the other abnormal strains do possess VLP. However, the sim-i l a r i t y of the aberrant phenotype suggests a common cause i n a l l f i v e s t r a i n s . As mentioned, i t i s also possible that VLP are associated with 2360 but were not found. Other possible cytoplasmic causes are discussed i n Section I I . SECTION II Biochemical Analysis 55 INTRODUCTION The biochemical characterization of the N. intermedia isolates involved three aspects: 1) respiration and cytochrome studies, 2) an investigation of mitochondrial ribosomes, and 3) mitochondrial DNA r e s t r i c t i o n enzyme analyses. The respiratory system of wi l d type N. orassa mitochondria consists, i n part, of cytochromes a., b_, and c_, and i s cyanide, azide, and antimycin sensitive (Diacumakos ej: a l . , 1965; Haskins et a l . , 1954). The diagram of the electron transport chain (Figure 11) shows the different points at which i t i s inhi b i t e d by the various toxins (Lehninger, 1975). In this species of Neurospora there are several viable respira-tory-deficient mutants. The most extensively studied of these i s the renowned [poky] mutation. This s t r a i n possesses an excess of cyto-chrome _c up to 16 f o l d that of wild type (Haskins et a l . , 1954) . A spectrum of the[poky] cytochromes i s shown i n Figure 12, along with the wi l d type. The cytochrome system requires the presence of cyto-chromes a. and lb. The lack of a detectable amount of these cytochromes suggests that respiration i n [poky] does not t o t a l l y depend on the cytochrome system. Further evidence of this was found when studies on the effects of known respiratory i n h i b i t o r s showed that [poky] i s mainly resistant to cyanide, azide and antimycin, while wild type i s not (Tissieres et a l . , 1953; Lambowitz et a l . , 1972). For example, the addition of cyanide to wi l d type blocks the electron transport from cytochrome a_ to oxygen, yielding completely reduced cytochrome a. (Lambowitz et a l . , 1972). [poky] i s not cyanide sensitive. This points to an alternate oxidase system shunting the electrons from the 56 Figure 11. The Electron Transport Chain. A diagrammatic representation of the electron transport chain. The various i n h i b i t o r s are shown, as we l l as the probable sites of ATP production. FP designates flavoproteins. Taken from: Lehninger, 1975. Rotenone, Amytal NAD — > FP Succinate ^ FP antimycin cyanide >^cyt b ; > cyt c > cyt aa^ . > 0^ * ATP production sites. 58 Figure 12. Cytochrome Spectra of Two N. orassa Strains. A) wild type, B) poky mutation. Both are room temperature spectra taken during expo-nential growth. Taken from: Bertrand and Kohout, 1977. 59 60 substrate to oxygen. Both wi l d type and [poky] appear to have this alternate oxidase pathway. However, i n wi l d type i t accounts for less than 10% of t o t a l r espiration, unless the fungus i s grown i n the presence of antimycin A, cyanide, or chloramphenicol. In this case the alternate oxidase a c t i v -i t y can be increased 20-fold (Lambowitz and Slayman, 1971). In [poky], both systems are present, the alternate oxidase being two to three times as active as the cytochrome system (Lambowitz,et a l . , 1972). This alternate oxidase system i s blocked by s a l i c y l hydroxamic acid (SHAM) while the cytochrome system i s not (Lambowitz and Slayman, 1971). Further work with various i n h i b i t o r s , and experiments i n which electrons are donated d i r e c t l y to cytochrome _c, have shown that there i s no connection between the cytochromes and alternate oxidase. The evidence indicates a model of a branched electron transport system as shown i n Figure 13. As can be seen the cytochromes and alternate oxidase share dehydrogenases and flavoproteins (Lambowitz et a l . , 1972). Although much work has been carried out on the alternate oxidase system of many organisms, the components of this pathway are s t i l l un-known (Vanderleyden ej; air-,- 1980) . However, evidence has accumulated that points to the involvement of quinones i n this system (Moore and Rupp, 1978). Cyanide-resistant respiration occurs i n some higher plants (Arum James and Beevers, 1950; Symploaarpus foetidus Hackett, 1957), whose respiratory system also involves an alternate oxidase sensitive to hydroxamic acids. The model proposed by Storey and Bahr (1969) for Symploaarpus i s that of a branched electron transport system similar to the one proposed for N. orassa. Several species of algae also have 61 Figure 13. Branched Electron Transport. The model for a branched electron transport system in[poky] mitochondria i s shown. X depicts the cyanide-resistant oxidase and Y an unspecified compo-nent which can transfer electrons from the f l a v i n step to the cyanide resistant oxidase or to the b_ type cytochromes. Taken from: Lambowitz et a l . , 1972. 62 63 cyanide-insensitive respiration (Chlamydomonas reinhardi Hammersand and Thimann, 1965; Euglena graoilus Sharpies and Butow, 1970; Atasia klebsii van Dach, 1942), as we l l as some protozoa (Mayorella palen-stinensis Reich, 1955; Trypanosoma vivax Ryley, 1956; Paramecium caudatum Clark, 1945), yeast {Rhodotorula glutinus Matsunaka at a l . , 1966) and other fungi (Myrotheoium verruaaris Kidder and Goddard, 1965). Other cytoplasmic growth mutants of N. orassa also show cytochrome spectra lacking cytochromes a_ and b_, but with an excess of c^  ([exn] , [stp], Bertrand and Pittenger, 1972; [abn], Diacumakos et a l . , 1965). The abnormal N. intermedia strains resemble these mutants i n growth phenotype, as we l l as i n showing maternal inheritance. Thus, i t was of interest to study the cytochrome system of the N. intermedia i s o -lates . The second phase of the biochemical investigation involved the mitochondrial ribosomes. Mitochondrial ribosomes have been isolated from a variety of organisms including Neurospora, Aspergillus, yeast, ra t , mouse, hamster, man, locust, Tetrahymena, Xenopus, and Euglena (Borst and G r i v e l l , 1971). The sedimentation coefficients of these ribosomes range from 55-60S for animal c e l l s to 70-80S for microorg-anisms and higher plants (Lambowitz, 1979). They are unlike cytosol ribosomes, but similar to prokaryotic ribosomes i n that they are chlor-amphenicol sensitive and cyclohexamide resistant (Borst and G r i v e l l , 1971; Lamb et a l . , 1968). Mainly through work on the [poky] mutant, i t has been found that mitochondrial ribosome assembly i n N. orassa depends on nuclear as we l l as mitochondrial genes (LaPolla and Lambowitz, 1977). Hybridization studies show that the mitochondrial (mt) ribosomal RNA's (rRNA) are 64 transcribed from mt DNA while most of the mt ribosomal proteins are coded for by nuclear DNA, synthesized in the cytosol, and transported into the mitochondria (Schatz and Mason, 1974). There i s , however, at least one exception. The protein designated S-5 which is associated with the mt small ribosomal subunit i s synthesized within the mitochon-dria (Lambowitz et a l . , 1976). N. orassa's mt ribosomes are 73S with subunits of 50S and 37S (Kuntzel and Noll, 1967; Kuntzel, 1969). The 50S subunit is composed of 25S rRNA and various proteins. The 37S subunit consists of 19S rRNA and proteins. At one point, low molecular weight RNA (ie. 5S) was considered a universal component of ribosomes, but i t has been found that there is no low molecular weight RNA (excluding tRNA) in N. orassa mitochondria (Lizardi and Luck, 1971). This is also known to be true for mitochondria of other fungi, as well as animal cells (Chua and Luck, 1974). During the investigation of [poky], the mitochondrial ribosomes were found to be defective. They are deficient in small subunits (30S) when compared to wild type N. orassa (Rifkin and Luck, 1971). This deficiency of small subunits is accompanied by a deficiency of 19S • rRNA. There i s , however, no alteration of the structure of the 19S rRNA that is present (Lambowitz and Luck, 1976) . LaPolla and Lambowitz (1977) proposed that the 19S rRNA is transcribed in wild type amounts and would be functional, except that i t i s rapidly degraded when not integrated into ribosomes. Studies involving the effect of chloram-phenicol on mt ribosome assembly show that protein S-5 may be required for maturation of small subunits (LaPolla and Lambowitz, 1977). The [poky] mutant is deficient in several small subunit proteins, possibly 65 including S-5, and this may be the basis of the unusual phenotype since S-5 i s intramitochondrially synthesized (Lambowitz et a l . , 1976). The N. intermedia stop-start strains have been characterized with respect to growth and inheritance patterns, as w e l l as mitochondrial res p i r a t i o n (see Section 1 and Results i n Section I I ) . However, none of these t r a i t s reveals information as to the basis of the unusual phenotype, but instead may only be manifestations. Consequently, f o l -lowing the history of the investigation of the [poky] mutation, research on the N. intermedia strains turned to analysis of mt ribosomes. Mito-chondrial ribosome p r o f i l e s were carried out to determine whether or not there were subunit deficiencies. F i n a l l y , the t h i r d aspect studied was mt DNA. The f i r s t evidence for mt DNA was found by Nass and Nass (1962). The same material was also being found i n other organisms (Amoeba, Pappas and Brandt, 1959; mouse oocyte, Parsons, 1961) and was eventually recognized as DNA. The mt DNA of animal c e l l s i s c i r c u l a r with a molecular weight of 10^ daltons, while that of higher plants i s generally larger (up to about 7 x 10^ daltons, Quagliarello, .1976). mt DNA of the yeast Saccharomyees aerevisiae has been shown to code for a number of mitochondrial transfer RNA's, ribosomal RNA's, and messenger RNA's. These mitochondria possess the a b i l i t y to transcribe this DNA and translate the RNA into proteins (Borst, 1971). The gene products of this DNA i n yeast include three of the seven subunits of cytochrome j : oxidase, one of the seven subunits of the cytochrome bc^ complex, and three of the ten subunits of mitochondrial ATPase (Borst and G r i v e l l , 1978). The mitochondrial DNA of yeast has been thoroughly studied and several noteworthy discoveries have been made. This DNA 66 can possess deletions that remove 20-99% of i t s sequence. The re-maining DNA of these "p e t i t e " mutants i s amplified by tandem duplication so that the amount of mt DNA i s the same as i n the w i l d type (Borst and G r i v e l l , 1978). In addition to this interesting phenomenon, the gene for large rRNA i n yeast mitochondria i s s p l i t by an intervening sequence (Borst et a l . , 1977). A map of the yeast mitochondrial genome i s given i n Figure 1. The same si t u a t i o n has recently been found i n N. orassa (Mannella et a l . , 1979). mt DNA of the N. orassa cytoplasmic growth mutants has also been well studied (Bertrand e_t a l . , 1980) . Current mitochondrial DNA research includes mapping techniques involving the use of r e s t r i c t i o n enzymes. These enzymes cleave the mitochondrial DNA at s p e c i f i c s i t e s and thus y i e l d fragments of vary-ing sizes which can be seperated by electrophoresis. Restriction enzymes have been used to map mt DNA of S. oerevisiae, as well as N. orassa (Borst and G r i v e l l , 1978; Terpstra et_ a l . , 1976). In N. orassa this technique i s not only being used to map mitochondrial genes, but also to compare mutant and w i l d type strain s . A map of the mt DNA of a wild type s t r a i n i s given i n Figure 14. Four stopper mutants have been shown to possess insertions or deletions i n their mt DNA (Bertrand ^ t a l . , 1980). Restriction enzyme analysis of the mt DNA of the [poky] mutant shows that most [poky] strains possess a wild type fragment pattern. Many, however, do show an addition (Man-n e l l a and Lambowitz, 1978) and one shows a deletion (Mannella et a l . , 1979). These alterations, therefore, are not the cause of the [poky] phenotype. Work i s currently being carried out on a l l of the N. orassa cytoplasmic growth mutants i n several laboratories, so that more inform-ation on the precise mechanism of the mutations may soon be available. 67 Figure 14. The Eco RI Restriction Enzyme Map of Wild Type N. orassa mt DNA. The positions of the eleven r e s t r i c t i o n fragments are given. A) the 25S RNA gene with an intervening sequence, B) the 19S RNA gene. Taken from: Bertrand et^ a l . , 1980. 69 No evidence has been found i n any N. orassa s t r a i n that would indicate a heterogeneity of mt DNA ( i e . l i n e a r and c i r c u l a r etc.)- The r e s t r i c tion maps support a c i r c u l a r genome (Bertrand, personal communication) The relationship between the nuclear and mitochondrial genomes re mains unclear. For example, the role of organelle genes i s not neces-s a r i l y obvious. In f a c t , mt DNA of yeast contributes only about 5% of the t o t a l mitochondrial protein (Borst and G r i v e l l , 1978). However, a mutant completely lacking mt DNA i s unable to form a functional i n -ner mitochondrial membrane. Yet, the advantage for a c e l l to have two seperate genetic systems i s unknown. Since there are s t i l l so many unknowns regarding mt DNA, i t s function, i t s evolution, i t s i n t e r -relationship with nuclear DNA, work i n this f i e l d i s becoming more extensive. R e s t r i c t i o n enzymes have also been used i n population analyses. Avise and coworkers (Avise e_t a l . , 1979) compared enzyme fragment patterns of mt DNA from natural populations of the mouse Peromysous. They showed that the heterogeneity i n mt DNA sequences can be used to estimate relatedness between individuals and populations. Clearly, this unique application of r e s t r i c t i o n enzyme analysis w i l l be of considerable value to population b i o l o g i s t s . Since the abnormal N. intermedia strains are analogous to the stoppers of N. orassa i n growth phenotype, i t was of interest to ana-lyze their cytochromes, mt ribosomal subunits, and mt DNA. The goal of these analyses would be to gain insight into 1) the mechanism of natural variants ( i e . how they arise and function), 2) the o r i g i n of the "slow" progeny that were found i n some of the reciprocal crosses (Section I ) , 3) the nature of the mitochondrial genome through dissec-tion of these new mutants, and 4) even to gain insight into the evolu-70 tionary advantage of variant mt DNA. Perhaps, t h i s could be ac-complished through a thorough characterization of the factors which are known to be involved i n the [poky] phenotype of N. orassa. MATERIALS AND METHODS Strains Eight Neurospora intermedia strains were used. Three possess normal growth phenotypes: FGSC strains 2361, 2365, and 1940. The f i v e abnormal strains were 2360, 2363, P608, and P804, and the auxo-trophic s t r a i n 2360his. One co n i d i a l i s o l a t e (obtained as described i n Section I ) , which consistently showed stop-start growth was select-ed for the remaining analyses of each s t r a i n . An attempt was made to use s t r a i n P594 i n the remaining studies, however, this s t r a i n did not grow upon subsequent transfers, rendering i t no longer usable for research. N. orassa wild type s t r a i n 74A obtained from Dr. H. Bertrand was studied for comparison of some char a c t e r i s t i c s . Media and Growth Conditions A l l cultures were grown at 25°C i n either l i q u i d or s o l i d Vogel's medium (Davis and deSerres, 1970). Liquid cultures of 10 conidia per ml. ( f i n a l concentration) were grown i n 800 mis. of media, i n a 2 l i t e r f l a s k , and kept i n shaker incubators at 25°C. The normal strains were harvested after 12-14 hrs. growth, while the slow growing strains were harvested after 24-48 hrs. Eco RI, Cla, and Kpn r e s t r i c t i o n en-zymes were obtained from Miles Research Laboratories. Methods A. Cytochrome Spectra The l i q u i d cultures were harvested by f i l t r a t i o n and washed with i s o l a t i o n buffer (0.44 M sucrose, 10 mM Tris-HCl pH. 7.2, 5 mM EDTA). 72 The mycelia was ground with acid-washed sand, resuspended i n the i s o l a t i o n buffer, and centrifuged at 3,000 rpm for 10 minutes i n a Sorvall centrifuge at 4°C. The supernatant was centrifuged at 13,000 rpm for 30 minutes. The resulting p e l l e t was resuspended with 2.5% deoxycholate (in 10 mM Tris-HCl pH. 7.2, 5 mM EDTA, bringing i t to a concentration of 1%) to c l a r i f y the solution. If the suspension was s t i l l unclear, i t was sonicated with a sonic d i s -membrator for approximately 20 seconds. I t was then centrifuged at 10,000 rpm for 10 minutes. 1 ml of the supernatant was diluted with spectra buffer (l-O.'mM Tris-HCl pH. 7.2, 5 mM EDTA), and put into each of two spectrophotometer c e l l s . A spectrum was obtained by running oxidized vs. oxidized, then oxidized vs. reduced. A few crystals of potassium ferricyanide were used to oxidize the solutions and a few crystals of sodium d i t h i o n i t e were used to reduce them. Spectra were run from 650-500 nm. at the appropriate O.D. on a Beckman Acta V spectophotometer. B. Respiration 3 mis. of Vogel's l i q u i d medium i n which 10 conidia per ml. had been growing for 12-24 hrs (depending on growth phenotype) was used as respiration media. Oxygen uptake was measured with a Clark oxygen electrode at 25°C. The respiration media was saturated with a i r (240 uM O2)• S a l i c y l hydroxamic acid was dissolved i n absolute etha-nol, while potassium cyanide was dissolved i n d i s t i l l e d water. C. Mitochondrial Isolation Mitochondria free of c y t o s t o l i c ribosome contamination can be obtained by f l o t a t i o n gradient centrifugation, a l l steps being carried 73 out at 4°C. The suction f i l t e r e d cultures were washed with cold i s o l a t i o n buffer (0.44 M sucrose, 10 mM Tris-HCl pH 7.6, 0.1 mM EDTA), ground with acid washed sand and resuspended with i s o l a t i o n buffer. The suspension was centrifuged at 4,000 rpm for 10 min-utes to p e l l e t out debris, and the supernatant was centrifuged at 13,000 rpm for 30 mins. Both spins were carried out i n a Sor-v a l l SS-34 rotor. However, i f the sample was large a Sorvall GSA rotor was used and the sample was centrifuged twice at 4,500 rpm for 10 mins. and then at 13,000 rpm for 1 hr. In either case, care was taken to remove a l l buffer from the r e s u l t i n g mitochondrial p e l -l e t which was then resuspended i n 60% sucrose ( u l t r a pure sucrose i n 10 mM Tris-HCl pH. 7.6 and 0.1 mM EDTA was used) and brought to a t o t a l of 7 mis. i n an ultracentrifuge tube. This was overlayed with 2-3 mis of 55% sucrose and then 2-3 mis of 44% sucrose (both were ultrapure sucrose i n 10 mM Tris-HCl pH. 7.6 and 0.1 mM EDTA). These gradients were centrifuged at 39,000 rpm for 1% hours during which time the mitochondria form a tight band between the 44% and 55% sucrose layers. Exclusion of EDTA or the addition of Mg during this i s o l a t i o n leads to contamination by cytosolic ribosomes (Lambowitz, 1979). D. Preparation and Analysis of Mitochondrial Ribosomes The band of mitochondria was pipetted from the gradient to a Sorvall SS-34 centrifuge tube and diluted 1:3 with HKCTD (500 mM KC1, 50-.mM CaCl 2, 25 mM Tris-HCl pH. 7.6, 5 mM d i t h i o t h r e i t o l ) . I t has I | been shown that Ca suppresses nuclease a c t i v i t y i n Neurospora (Linn and Lehman, 1966), and thus i t i s included i n the buffer at this point. The suspension was centrifuged at 12,000 rpm for 20 mins. 74 The p e l l e t s were resuspended with enough HKCTD to y i e l d approxi-mately 12 mg. of mitochondrial protein per ml. The protein e s t i -mation was carried out by p r e c i p i t a t i n g a 50 u l aliquot with 500 u l of 10% TCA. I f the protein concentration was correct, the solution immediately became cloudy and after 1 min. had a l i g h t precipitate. More HKCTD was added as needed. Once the d i l u t i o n was correct, puromycin (20 mM i n 100 mM T r i s -HC1 brought to pH 7.6 with KOH and stored at -20°C, then heated for 10 mins. at 37°C before use) was added to 250 y l of mitochondria to a f i n a l concentration of 1 mM puromycin. The puromycin dissociates the ribosome monomers into subunits. This was incubated at room temperature for 10 mins. Trit6n-X-100 at a f i n a l concentration of 1% lyses the mitochondrial membrane. The sample was then overlayed onto sucrose gradients of ultrapure 5%-20% sucrose i n HKMT (500 mM KC1, 25 mM MgCl 2, 25 mM Tris-HCl pH 7.6). Mg"1"1" i s now substituted I | for Ca on the assumption that nucleases are no longer active, and I | since the effects Ca may have on the a c t i v i t y of puromycin i s un-known. These gradients were centrifuged at 39,000 rpm for 3% hrs. i n a Beckman SW 40 rotor (or i t s equivalent), then analyzed with a density gradient fractionator at E. rRNA Extraction rRNA was extracted from whole mitochondria obtained from the f l o t a t i o n gradient described above. The pelleted mitochondria were resuspended i n 2 mis of HKCTD. In a glass homogenizer 50 mM T r i s -HCl pH 7.6, 5 mM MgCl 2, 1% SDS (sodium dodecyl sulfate) and 0.15 ml diethylpyrocarbonate (a nuclease i n h i b i t o r ) were mixed for 2 minutes at 4°C. The mitochondria were added and homogenized for 2-3 mins. at 75 4°C. The homogenate was then incubated at 37°C for 5 mins. and centrifuged at 9,000 rpm for 10 mins. at 24°C. 0.5 g NaCl was added to the supernatant and the solution was kept on ice for 10 mins. I t was then centrifuged at 10,000 rpm for 30 mins. at 4°C, to remove the SDS-protein precipitate. Two volumes of ice cold ethanol were added to the supernatant and i t was stored overnight at -20°C to precipitate the RNA. The sample was spun for 45 mins. at approximately 1,000 rpm i n a c l i n i c a l centrifuge. The RNA was dissolved i n a buffer of 0.04M Tris (ultapure), 0.3 mM EDTA, 22% sucrose, 0.1% SDS, 0.42% NaH^O^O for electrophoresis through an agarose-acrylamide composite gel (Pea-cock and Dingman, 1968). F. mt DNA Preparation and Digestion Mitochondria were isolated by sucrose f l o t a t i o n gradients as de-scribed, except that a l l glassware was acid cleaned for this procedure. The mitochondrial band was removed from between the 44% and 55% sucrose layers, diluted 1:3 with HKCTD and spun for 30 mins. at 18,000 rpm. The supernatant was discarded, and the p e l l e t was resuspended i n 3 volumes of HKCTD with 2% ( f i n a l concentration) Nonidet added to lyse the mitochondria. The suspension was centrifuged at 55,000 rpm for 16 hrs. on a 1.85 M sucrose (in HKCTD) cushion. The resulting r i b o -nucleoprotein p e l l e t s were caref u l l y washed with cold d i s t i l l e d water. At this point the samples could be frozen at -20°C u n t i l ready for use. The p e l l e t s were resuspended i n 2 mis. N-SET (100 mM NaCl, 100 mM Tris-HCl pH 8.2, 2 mM EDTA, 1% SDS). Then, 2 mis. of phenol buffer (10 volumes d i s t i l l e d phenol to 3.5 volumes H-NET; H-NET contains 150 mM NaCl, 100 mM T r i s - HC1 pH 8.2, 1 mM EDTA) were added and the samples were l e f t at room temperature for 10 mins. They were then cen-76 trifuged at 10,000 rpm for 10 mins. i n a warm centrifuge. An ad-d i t i o n a l 2 mis. of phenol buffer was added to the upper aqueous phase and the centrifugation was repeated. The upper phase was dialyzed overnight against 4 l i t e r s of H-NET. 50 y l RNAse solution (2 mg/ml RNAse A, 25 yl/ml RNAse i n H-NET, preincubated at 80°C for 10 mins) was added to the dialysate and incubated at 37°C for 30 mins,. 200 y l of protease solution (2 mg/ml Sigma protease VI i n H-NET, preincubated for 1 hour at 35°C) was added and the solution was incubated for 40 mins at 37°C. At the end of a l l incubations 2 mis of phenol buffer were added, the samples were centrifuged, and the upper phase was dialyzed overnight against 4 l i t e r s of F i n a l buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.1, 0.1 mM EDTA). This dialysate was put on ice with 5 mis ice-cold ethanol and stored at -20°C for at least 24 hrs. The samples were centrifuged at 40,000 rpm for 1 hr and a l l the ethanol was removed. The p e l l e t s were resuspended i n 50 y l L-NET (10 mM NaCl, 10 mM Tris-HCl pH 7.5, 0.1 mM EDTA) and O.D. readings were obtained to estimate 1 ygm of DNA to be loaded onto an agarose gel. To carry out digestion of the DNA a reaction mixture was used as described by the suppliers of the re-s t r i c t i o n enzyme. For Eco RI the mixture was: 2.5 y l 1 M Tris-HCl pH 7.5, 2.5 y l 0.1 M MgCl'2, 2.5 y l 0.5 M NaCl, 15.5 y l L- NET, 1.0 y l Eco RI. For Kpn the reaction mixture consisted of: 2.5 y l mercapto-ethanol ( 0.42% i n d i s t i l l e d water and made fresh for each use), 5yl L-NET, 8 y l Kpn r e s t r i c t i o n enzyme. The Cla digest was carried out i n 2.5 y l Cla mix (100 mM Tris-HCl pH 8.0 and 100 mM MgCl 2), 16.5 y l d i s t i l l e d water, 4 y l Cla enzyme. The samples were digested for 4 hrs at 37°C. They were then electrophoresed on a 0.8% agarose gel at 50 v o l t s f o r 15 hrs. The gel was stained with ethidium bromide (0.5 ug/ml of d i s t i l l e d water) f o r one hour. 78 RESULTS Cytochrome Spectra TheN. •intermedia strains which have a normal growth phenotype (FGSC 2361, 2365, and 1940) a l l possess cytochrome spectra similar to that of N. orassa wild types. The a peaks for cytochromes b_ and c: are at 550 nm and 561 nm, respectively, i n both species. However, the a peak of cytochrome a_ i s at 608 nm in N. intermedia rather than at 601 nm as i t i s i n N. orassa (Lambowitz e_t a l . , 1972). The spectrum of s t r a i n 2361 i s given i n Figure 15 and i s also representative of spectra for 2365 and 1940. The spectra of strains 2360 and the related auxotrophic s t r a i n 2360his show l i t t l e or no cytochrome b_ r e l a t i v e to cytochrome c^ , and compared to the normally growing strains . Strains 2363 and P804 have a very small amount of cytochrome a. and no b_, again r e l a t i v e to the amount of cytochrome c_. The abnormal s t r a i n P608 appears to have a "wild type" spectrum. A l l of these spectra are shown i n Figure 15. The cytochrome peaks are consistently located at the same position ( i f present) i n a l l spectra (cytochrome a_ at 608 nm, b_ at 561 nm, and c_ at 550 nm) . Oxygen Uptake Studies Strains 2361 and 2360his were studied with respect to their sen-s i t i v i t y to'respiratory i n h i b i t o r s . Strain 2361 proved to be 92% cya-nide sensitive, while s t r a i n 2360his was only 34% sensitive; both of these experiments were carried out only once. However, s t r a i n 2360his was 68% sensitive to SHAM (this i s the average of two runs: 64% and 79 Figure 15. Cytochrome Spectra of N. vntevmedia Strains. A) a spectrum representative of 2363 and P804, B) s t r a i n P608, C) a spectrum representative of 2360 and 2360his, and D) a spectrum representa-tive of strains 2361, 2365 and 1940. 81 72% s e n s i t i v i t y ) . These results are shown i n Figure 16. Ribosome P r o f i l e s A l l abnormal, and one normal s t r a i n , of N. intermedia were ana-lyzed for mitochondrial ribosomal subunit deficiencies. Figure 17 shows the results of sucrose gradient analyses, and Table IX l i s t s the r a t i o s of large to small subunits. The values for ratios are means and are based on the area under the curve; units are a r b i -trary. The normal s t r a i n , 2361, shows both subunits and i n a r a t i o of 2.2 : 1, large to small. This i s comparable to that found i n N. orassa wild type 74A which shows a r a t i o of 2.4 : 1 (Collins et a l . , 1979) . The stop-start s t r a i n , 2363, has a deficiency of small subunits so that the r a t i o of large to small i s 7.3 : 1. A l l of the other stop-sta r t strains show a deficiency of large subunits. The ratios vary from 1 : 2.0 to 1.1 : 1. Note that the ribosome p r o f i l e for s t r a i n 2360his shows two peaks, but i n different locations than the other p r o f i l e s . I t i s probable that the gradient was inaccurate, thus changing the location of the large and small subunit peaks. Another point to note, i s seen i n the p r o f i l e of s t r a i n 2360. This shows a small unlabelled peak which sediments more slowly than the small sub-units. A s i m i l a r peak found i n some mutants of N. orassa i s thought to be incomplete mt small subunits (Collins ejt a l . , 1979) . Mitochondrial Ribosomal RNA Analysis Three strains were analyzed by gel electrophoresis to see i f a deficiency of rRNA could be detected i n 2360his. One s t r a i n was 82 Figure 16. Oxygen Uptake of Intact C e l l s . The arrows indicate the time when either KCN or SHAM was added. The l i n e labelled "a" shows normal respiration and i s taken to be 100%. The l i n e labelled "b" i s i n -hibite d respiration. The percent i n h i b i -t i o n i s calculated from the slope of the l i n e . 1) s t r a i n 2360his, 2) s t r a i n 2360his 3) s t r a i n 2361. 84 Figure 17. Ribosome Profiles of N. intermedia Strains. A) strain 2361, B) 2363, C) P804, D) P608, E) 2360his, F) 2360. Peak 1 is the small subunit peak, and peak 2 is the large subunit peak. • 1 T I T 1 0 3 6 mis 0 3 6 mis Sedimentation-* Sedimentation-* 86 Table IX. Ratios of large to small subunits for the various N. intermedia s t r a i n s . Values are obtained from areas under the appropriate peaks. Strains Ratio of Large : Small subunits 2361 2.2 : 1 (5) 2363 7.3 : 1 (3) 2360 1 : 1.3 (3) 2360his 1 : 2.0 (1) P608 1.1 : 1 (3) P804 1 : 1.8 (2) These are average values. The number of runs for each s t r a i n i s given i n ( ). 87 N. orassa wild type 74A, which showed bands corresponding to 25S rRNA and 19S rRNA (Figure 18). N. intermedia s t r a i n 2361 also showed two clear bands corresponding to 25S and 19S rRNA. Strain 2360his, the only stop-start s t r a i n analyzed, shows almost no band i n the expected location of 25S rRNA, but a heavy band for 19S rRNA. This confirms the above observation that there i s a deficiency of large ribosomal subunits i n this s t r a i n as mentioned above. DNA R e s t r i c t i o n Enzyme.Patterns Figure 19 shows the Eco RI r e s t r i c t i o n enzyme patterns of N. orassa wild type s t r a i n 74A and .N. intermedia s t r a i n 2361 (a normal phenotype i s o l a t e ) . Both show eleven mt DNA fragments, seven of which comigrate. Not suprisingly, since they are unique species, some fragments are seen i n d ifferent locations on the gel. The molecular weights of the frag-ments for a l l strains analyzed are given i n Table X, and show that 2361 has a t o t a l molecular weight of approximately 41.5 x 10 . This i s com-parable to the molecular weight of 74A, 40.9 x 10^. Figure 20 shows the Eco RI digest of strains 2363, P804, 2360, 2361, and 2365. The two strains with a normal phenotype (2361, 2365) show precise comigration of fragments. The only exception i s that s t r a i n 2365 possesses an additional band. This band w i l l be referred to as band E, and i s not seen i n 2361. Since s t r a i n 2361 shows Eco RI fragments 1-10 as does E. orassa (although not the same molecular weights), the DNA of this s t r a i n w i l l be referred to as "standard" DNA. The additional DNA seen i n the Eco RI digest of 2365, i s not seen i n the Kpn digest (Figure 21). There are no additional bands and a l l four bands present comigrate with those of 2361. This disappearance of the 88 Figure 18. Ribosomal RNA Analysis. A). N. orassa-w i l d type 74A, B). N. intermedia s t r a i n 2361, C). N. intermedia s t r a i n 2360his, D). N. orassa s t r a i n 74A. A l l three strains show a f a i n t band of mt DNA. Note that s t r a i n 2360his shows l i t t l e 25S rRNA. 89 A B C D m t D N A 2 5 S 19S W W 90 Figure 19. Eco RI R e s t r i c t i o n Enzyme Digestion of N. orassa and N. intermedia Strains. A) . N. orassa w i l d type s t r a i n 74A, B) . N. intermedia s t r a i n 2360his, C). N. intermedia s t r a i n 2361. The numbers 1-10 correspond to the fragments of s t r a i n 74A. Table X. Molecular weights ( x 10 ) of N. intermedia mt DNA Eco RI fragments. The M.W. was obtained by p l o t t i n g the log of the molecular weight vs. the distance from the well for s t r a i n 74A (molecular weights ob-tained from Terpstra et a l . , 1976) and then extrapo-l a t i n g the weights for the N. intermedia s t r a i n s . STRAINS Eco RI fragment number 2361 2365 2363 2360 P804 1 12.7 12.7 12.7 12.7 12.7 2 7.1 7.1 7.1 7.1 7.1 3 6.2 6.2 6.2 6.2 A 4.3 B 4v05 4 3.6 3.6 3.6 3.6 3.6 C 3.15 5 2.9 2.9 2.9 2.9 2.9 D 2.4 2.4 2.4 E ' 2.1 2.1 6 1.8 1.8 1.8 1.8 1.8 * 7a 1.71 1.71 1.71 1.71 1.71 * 7b 1.71 1.71 1.71 1.71 1.71 8 1.6 1.6 1.6 1.6 1.6 9 1.2 1.2 1.2 1.2 1.2 10 1.0 1.0 1.0 1.0 1.0 Total 41.52 43.62 46.92 50.32 41.27 The values for fragments 7a and 7b are approximate since they migrate so close together. 1 2 3 4 5 6 7a 7b 8 9 10 92 Figure 20. Eco RI Re s t r i c t i o n Enzyme Digest of N. intermedia Strains. A). P804, B). 2361, C). 2363, D). 2361, E). 2365, F). 2363, G). 2360, H). 2361. The num-bers 1-10 correspond to the fragments of s t r a i n 2361. A-E to the right and l e f t of the gels are the extra bands present i n the various strain s . 93 94 Figure 21. Kpn Re s t r i c t i o n Enzyme Digest of N. inter-media Strains. A). 2360, B). 2363, C). 2361, D). 2365, E). P804, F). 2361, G). 2363. The numbers 1-4 correspond to the fragments of s t r a i n 2361. 96 extra DNA may indicate that the DNA of band E comigrates with an-other Kpn band. I t i s also possible that this DNA i s shredded by Kpn, and hence i s not v i s i b l e . The Eco RI patterns of stop-start s t r a i n 2363 show "standard" DNA, as we l l as two additional bands. These bands are labelled B and D. There i s no band E present. The Kpn digest shows that this s t r a i n may have an extra Kpn r e s t r i c t i o n s i t e i n band 2. One of the resulting fragments would be the second band that appears i n this digest, and the other fragment would be too small to be seen. The Kpn digest shows another band not seen i n s t r a i n 2361. This may correspond to the extra DNA seen i n the Eco RI digest. The same situ a t i o n i s seen i n the Cla digest of this s t r a i n (Figure 22). The fragment pattern shows additional DNA with respect to the "standard" DNA. The Eco RI digest pattern of s t r a i n P804 shows several obvious variations from the pattern of standard DNA. Band D i s present i n this s t r a i n , as well as a band unique to this s t r a i n , band C. The Kpn digest also shows a very f a i n t band which comigrates with one of the extra Kpn bands of s t r a i n 2363. Eco RI band 3 i s not seen i n s t r a i n P804 and may represent a deletion or may be migrating close to Eco RI band 2 (there i s a f a i n t band between Eco RI-1 and-2). Also, a band between Eco RI fragments 4 and 5 (band C) i s highly ampli-f i e d i n this s t r a i n with respect to i t s other bands and may represent a plasmid (see Discussion). The DNA of this band i s also probably shredded i n the Kpn digest, since i t i s not seen. However, i t may be showing up i n the Cla digest as band 6 which appears somewhat amplified. 97 Figure 22. Cla Restriction Enzyme Digest of N. inter-media Strains. A). P804, B). 2361, C) . 2363. The numbers 1-7 correspond to the fragments of s t r a i n 2361. 98 99 Abnormal s t r a i n 2360 has "standard" DNA, as we l l as the addi-t i o n a l bands A, D, and E. This extra DNA i s seen i n the Kpn digest as w e l l . Band E i s the same band seen i n s t r a i n 2365. The uncut DNA,shows only one band (with some smearing) i n each s t r a i n (Figure 23). Thus, a l l three abnormal strains (2363, P804, and 2360) show additional DNA with respect to the "standard". Of this additional DNA, the abnormal strains a l l possess a common frag-ment, band D of M.W. 2.4 x 10 , i n their Eco RI digestion patterns that i s not seen i n either of the normal strains tested. 100 Figure 23. Uncut DNA of the N. -intermedia Strains. The uncut DNA of each strain (2361, 2363, P804, 2365, 2360) is shown in slots be-tween the Eco RI digested DNA. There are no differences seen in the migration of uncut DNA and only one band is seen for each strain. 102 DISCUSSION As would be expected i f there i s a common cause of unusual growth phenotypes for both species, most of the abnormal N. •inter-media strains (2363, 2360, 2360his, and P804) possess cytochrome spectra reminiscent of the cytoplasmic mutants of N. orassa ([poky], [exn], [stp], Bertrand and Pittenger, 1972; [abn], Diacumakos et a l . , 1965). There i s l i t t l e or no cytochrome a or b present, and an excess of cytochrome c_ r e l a t i v e to the amounts of a. and b_. However, the cytochrome spectrum of s t r a i n P608 showed a l l cytochromes present and i n proportions similar to the normally growing strains 2361, 2365, and 1940. In Section I i t was noted that P608 never passes i t s abnormal growth phenotype on to i t s progeny. This was actually expected, though, i f cytoplasmic inheritance i s involved since the s t r a i n i s female s t e r i l e . The suprising r e s u l t was that i t shows a normal cytochrome spectrum. This indicates a difference between P608 and the other abnormal strains which do show a lack of cytochromes a. and b_. However, P608 does show a deficiency of large mt ribosome subunits as do most of the other stop-start strai n s . Abnormal cytochrome spectra may strengthen the evidence for cyto-plasmic inheritance since the mitochondrion i s known to code for sub-units of cytochrome oxidase and may code for other proteins involved i n the cytochrome system. However, a cytochrome spectrum cannot prove or disprove maternal inheritance. There are nuclear mutants of N. orassa which show a lack of certain cytochromes (strains C-115 and C-117, Mi t c h e l l ejt a l . , 1953). These two strains also show a growth phenotype 103 s i m i l a r to [poky] . The same i s t rue i n yea s t ; there are both n u -c l e a r and cy top la smic cy tochrome^def i c ient mutants ( Ephrus s i , 1953). Thus, the cytochrome spec t r a of the abnormal N. intermedia s t r a i n s demonstrates two p o i n t s : 1) that most of the e r r a t i c growth h a b i t s t r a i n s (2363, 2360, 2360his and P804) resemble the [poky] mutant of N. orassa i n one more c h a r a c t e r i s t i c , and 2) that the abnor-mal N. intermedia s t r a i n s are not a l l i d e n t i c a l . R e s p i r a t i o n s t ud i e s a l s o show a s t r i k i n g s i m i l a r i t y between s t r a i n 2360his and [poky]. Both s t r a i n s r e s p i r e , a t l e a s t i n p a r t , by a cyanide r e s i s t a n t pathway. This i n s e n s i t i v i t y to cyan ide, and the l a c k of cytochromes a. and ]D, p o i n t to the involvement of an a l t e r n a t e ox idase i n the r e s p i r a t o r y cha in of s t r a i n 2360his. Whi le f u r t h e r work i s needed to e l u c i d a t e the nature of the a l t e r n a t e ox idase , i f i t does e x i s t , i t i s probable that i t w i l l resemble that which i s found i n [poky]. The o r i g i n a l work w i t h [poky] supported the theory that f l a v o p r o t e i n s ac t as a l t e r n a t e ox idases . There i s tw ice as much f l a v i n i n [poky] as i n w i l d type and n e a r l y a l l i s found as FAD ( T i s s i e r e s e t a l . , 1953). However, f u r t h e r work then i n d i c a t e d an i r o n - s u l p h u r spec ies as the a l t e r n a t e ox idase (Benda l l and Bonner, 1971), and f i n a l l y the cu r ren t hypothes i s i s tha t a quinone ac t s as the a l t e r n a t e ox idase (Moore and Rupp, 1978). Whi le the work on the N. intermedia slow growing s t r a i n s does not add i n f o rma t i on on the p r o p e r t i e s of the a l t e r n a t e ox idase , i t does i nc rea se the number of known r e s p i r a t o r y v a r i a n t s . I t a l s o shows that they can e x i s t i n v. natu re , a t l e a s t i n Neurospora. The r e s u l t s i n d i c a t e that the aberrant growth phenotype of• s t r a i n s 2360, 2360his, 2363, P804 and P608 i s r e l a t e d to a d e f i c i e n c y of one 104 of the mitochondrial ribosomal subunits. The large subunit d e f i -cient strains show that a large subunit defect can have a normal cytochrome spectrum (as i n s t r a i n P608) or an abnormal spectrum (as seen i n strains 2360his, 2360 and P804). While the nature of the p a r t i c u l a r defect of the mt ribosomes remains unclear, i t i s expected to show maternal inheritance i f i t i s coded for by mito-chondrial genes. So far two defects of the stop-start strains have been associated with mitochondria: the abnormal cytochrome system and the deficiencies of mt ribosomal subunits. I f the de-fect were within the genetic material of the mitochondria, i t would show cytoplasmic inheritance. Since i t i s known that i n N. orassa genes for mt rRNA are located within the mitochondrial genome, these are l i k e l y suspects for the defect. An abnormality i n the rRNA would probably give r i s e to a defect i n the ribosomal subunits. However, electrophoretic studies on [poky] showed that, although there was a deficiency of 19S rRNA (corresponding to the 37S subunit deficiency), there was no difference i n the stoichiometry of 19S rRNA of wild type and [poky] (Lambowitz and Luck, 1976). Thus, a mi.tochondrially coded protein, such as S-5, i s more l i k e l y to be the basis of subunit deficiency, and therefore of the unusual pheno-type i n the N. orassa cytoplasmic mutants. Only one N. intermedia s t r a i n , 2363, shows a deficiency of small subunits. In this respect i t i s similar to [poky]. Several other cytoplasmic mutants of N. orassa have also been analyzed and a l l show poky-like characters: a small subunit deficiency with 19S rRNA being rapidly degraded (Collins e_t al.,1979). Further work on s t r a i n 2363 Is necessary to see i f i t , too, shows 19S rRNA degradation. 105 The remaining stop-start strains a l l show a deficiency of large subunits, although to varying degrees (see Results). No other maternally inherited mutations i n Neurospora have been shown to possess large subunit deficiencies. However, three nuclear mutants of N. orassa which possess abnormal cytochrome spectra are large subunit deficient. They also have a decreased r a t i o of 25S to 19S RNA when compared to wi l d type (Collins et_ a l . , 1979). The rRNA analysis of N. intermedia large subunit deficient s t r a i n 2360his shows very l i t t l e 25S rRNA. I t i s probable that this s i t u a t i o n i s analogous to [poky], i n that the 25S rRNA i s rap-i d l y degraded (as i s the 19S rRNA i n [poky]) when mature large sub- . units are not formed. I f this i s the case, i t would suggest another mitochondrial ribosomal protein i s coded for by the mitochondrial DNA. However, other p o s s i b i l i t i e s s t i l l e x i s t , such as a defect i n the 25S rRNA i t s e l f , that cannot be ruled out. Clearly, a l l of these large subunit deficient strains are worthy of continued research. The results of these ribosome analyses have shown s t r a i n P608 to be similar to the majority of stop-start strains (2360, 2360his, and P804). The reason P608 shows a s i m i l a r i t y to these other abnormal strains i n i t s ribosome p r o f i l e and not i n i t s cytochrome spectrum i s unclear. I t i s also interesting that s t r a i n 2363 shows the t y p i c a l cytochrome spectrum for these strains but shows a different ribosome p r o f i l e . Thus, the abnormal s t r a i n s , at this point can be put into three groups with respect to cytochrome spectra and ribosome p r o f i l e s . Group I includes s t r a i n 2363 which lacks cytochromes a. and b_, and has a deficiency of small mt ribosome subunits. Group I I includes P608 which shows a normal cytochrome spectrum but a deficiency of large mt 106 ribosome subunits. Group I I I consists of the remaining abnormal strains: 2360, 2360his and P804. These lack cytochromes a_ and b_ and large mt ribosome subunits. Only Group I (2363) i s tr u l y simi-l a r to [poky] . The analysis of mitochondrial ribosomes reinforced the evidence for cytoplasmic inheritance i n the stop-start strains of N. inter-media. I t also added to the l i s t of s i m i l a r i t i e s between these strains and [poky] ( i e . they a l l have a ribosomal subunit deficiency). However, i t also revealed a difference not only between some of the strains and [poky], but also between the strains themselves. Thus, two characteristics show differences among the abnormal strains ( i e . cytochrome spectra and ribosome p r o f i l e s ) . A l l of these N. inter-media strains were collected from one Hawaiian Island, and a l l possess a sim i l a r growth phenotype. Yet, the genetic cause of their pheno-type may not be i d e n t i c a l . Therefore, the remainder of this work was devoted to investigating the mitochondrial DNA. Any major alterations of the DNA of the anomalous strains should be i d e n t i f i a b l e by r e s t r i c -tion enzyme analysis. Also, studies of this type have been carried out on the N. orassa growth mutants so that they were available for comparison. The present theory regarding the growth phenotype of the N. orassa stopper mutants requires the existence of two species of mt DNA (Ber-trand et al., 1980). One species would be mutant DNA which, when accumulated i n the hyphal t i p , would cause cessation of growth. The other nonmutant DNA species would eventually take over and growth would resume. This theory c a l l s for some form of r e p l i c a t i v e advan-tage for the mutant DNA, but the details of this are speculative> Re-107 striction enzyme analysis added credit to this theory by showing that two types of mt DNA may exist in these mutants. Certain wild type bands are amplified with respect to the other bands on the gels. This indicates that these fragments are present in both the mutant DNA and the more intact DNA. In other words, two types of mt DNA are' present in a c e l l . The mutant type contains only certain wild type bands (the amplified ones, amplified because ,they are present in both types of DNA) and is missing others (the nonamplified ones). The other type of mt DNA contains a l l (or most) of the wild type bands. Thus, when the mutant DNA accumulates the hyphae stops growing. When the intact DNA takes over growth would resume. The phenotype of the N. intermedia isolates so closely resembles that of the N. orassa [stp] mutants, that i t seemed probable they would have an analogous cause. Thus, major additions to, and deletions of, the mt DNA were looked for in the stop-start strains. In a re-striction enzyme pattern a deletion of mt DNA is expected to shift a band to a new position further down the gel. An addition, on the other hand, would increase the molecular weight of the fragment and thus keep the band from migrating as far. It is not expected that an entire band w i l l either be lost or added without affecting some other band. For this to happen the deletion would have to be the precise size of the fragment. Also, an addition would have to occur exactly between two other fragments without disturbing the restriction enzyme recognition sites. These types of deletions and additions are highly unlikely. Two of the ,N. intermedia abnormal isolates (2360 and 2363) appear not to have any mt DNA deletions. The "standard" DNA df strain 2361 108 i s present i n both strains as seen i n the Eco RI digest patterns. The only s t r a i n which does have a band missing i s P804 ( i e . Eco RI-3). However, there are no new bands seen that add up to the molecular weight of the missing fragment. Also, there i s no evidence for a deletion i n P804 i n the Kpn digest. Therefore this Eco RI-3 frag-ment may have a s l i g h t addition causing i t to migrate between Eco RI -1 and -2. There i s also no evidence of additions to the mt DNA of the ab-normal strains (except as mentioned i n s t r a i n P804). While there i s d e f i n i t e l y more DNA i n these st r a i n s , i t does not appear to be i n s e r t -ed into the mt DNA genome since none of the standard bands have been altered. Therefore, i t i s more probably "extrachromosomal" ( i e . plas-mid-like DNA);. Plasmid-like DNA exists i n mitochondria of cytoplasmic male-sterile maize (Kim et a l . , 1979), as w e l l as i n wi l d type strains of N. orassa (Collins e^ t a i l . , i n press). Strain P804 shows a very amplified Eco RI band which i s not pres-ent i n "standard" DNA. This DNA most l i k e l y exists as several tandem repeats giving the band i t s int e n s i t y . I t i s not clear why this extra DNA of P804 and the extra fragments of strains 2360, 2363.and P804 do not show up as separate bands i n the undigested DNA. I t i s assumed that they are too large to be separated from the rest of the mt DNA since this i s the case i n N. orassa: plasmids occur as large pieces with tandem repeats and are not separated from the rest of the mt DNA when the undigested DNA i s electrophoresed (Bertrand, personal com-munication) . Band E cannot be associated with the abnormal growth habit of s t r a i n 2360 since i t i s also found i n the normal s t r a i n , 2365. How-109 ever, band D i s found i n a l l of the stop-start s t r a i n s , and not i n either of the normal strains . I t i s possible that this DNA i s related to the abnormal phenotype. The stopper hypothesis for N. orassa requires the existence of two species of mt DNA. The N. intermedia isolates did not show additions or deletions (with the exception, possibly, of s t r a i n P804) within the i r mt DNA (although they do possess extra DNA intra-mitochondrially). However, this i s not necessarily inconsistent with the N. orassa hypo-thesis. I t i s possible for even a point mutation to cause stopping. This would not be detectable through r e s t r i c t i o n enzyme analysis. Thus, i t cannot be concluded that the extra DNA causes the e r r a t i c growth patterns of the N. intermedia s t r a i n s . Yet, i t i s interesting that a l l the stop-start strains possess band D i n their Eco RI digests. Further work on the genetic function of this DNA i s sure to be p r o f i t -able. Another area which would be interesting for further studies i s the difference between two normal strains . Strain 2365 contains an addition-a l Eco RI band which i s obviously nonessential for normal growth since 2361 does not possess i t . Strains 2361 and 2365 are not true controls for the r e s t r i c t i o n enzyme patterns of the anomalous strains . Since a l l the N. intermedia strains were obtained from nature there are no true controls available. This would require normal strains from which the anomalous isolates were derived. Strain 2361, does however, give an indication of what DNA i s required for normal growth. The r e s t r i c t i o n enzyme patterns suggest the p o s s i b i l i t y of plasmid-l i k e DNA within the mitochondria of the stop-start s t r a i n s . This leaves open to investigation the genetic function of this DNA. 110 These s t r a i n s of N. intermedia are f a s c i n a t i n g tools of re-search. They show many c h a r a c t e r i s t i c s s i m i l a r to the laboratory derived mutations' i n the c l o s e l y r e l a t e d species N. orassa. They possess an abnormal growth phenotype which appears to be cytoplasmic-a l l y i n h e r i t e d . They show an excess of cytochrome c_ and lack of cytochromes a. and/or b_. At l e a s t one i s cyanide r e s i s t a n t . They show defects i n t h e i r mt ribosomal subunits ( i n one the small sub-unit, i n the others the large subunit). They also have a d d i t i o n a l DNA independent of "standard" mt DNA. A summary of the characters studied and the r e s u l t s obtained i s given i n Table XI. This char-a c t e r i z a t i o n has revealed much information about these s t r a i n s . Yet, there i s considerable room for more work. The two areas which would be most i n t e r e s t i n g and probably the most p r o f i t a b l e f o r further stud-ies are the mt ribosomes and mt DNA. Perhaps a protein analogous to S-5, which i s associated with small mt ribosomal subunits i n N. orassa and i s intramitochondrially synthesized, could be found f o r the large subunits. Studies on the mt DNA w i l l indeed be informative. There i s much to be learned about the mechanisms behind t h i s unusual growth pheno-type. These s t r a i n s are unique i n that they are natural i s o l a t e s . I t w i l l be i n t e r e s t i n g to be able to hypothesize how these i s o l a t e s arose and have survived while i n competition with normally growing i s o l a t e s . I l l Table X I . A Summary of the Re su l t s of the C h a r a c t e r i z a t i o n of the N. intermedia i s o l a t e s . Transmiss ion of growth phenotype to progeny when male when female produces v a r i - cytochromes S t r a i n parent parent ab le c o n i d i a present 2360 no * yes no _c 2363 * yes * yes no c P594 no * yes yes P608 no no yes a.,b_,_c P804 yes yes yes c^  * i n a l l these cases, on ly a few abnormal progeny were produced. mt r ibosomal subuni t d e f i c i e n c y l a r ge sma l l rRNA mt DNA S t r a i n subuni t subun i t a n a l y s i s a n a l y s i s 2360 yes no 25S d e f i c i e n t band D present 2363 no yes band D p resent P594 — P608 yes no P804 yes no band D present S t r a i n Remarks 2360 possesses e x t r a mt DNA which may be i n the form of a p l a sm id . 2363 possesses e x t r a mt DNA which may be i n the form of a p la smid . P594 does show materna l i n h e r i t a n c e of growth phenotype. P608 has normal spectrum, yet shows abnormal growth and a d e f i c i e n c y of l a r ge mt r ibosomal subun i t s . P804 possesses e x t r a mt DNA which may be i n the form of a p lasmid w i t h a tandem repeat . 112 BIBLIOGRAPHY Avise, J.C., P.A. Lansman and R.O. Shade. 1979. The Use of Restric-tion Endonuclease to Measure Mitochondrial DNA Sequence Relatedness i n Natural Populations I. Population Structure and Evolution i n the Genus Peromysous. Genetics. 92:279-295. Baur, E. 1909. Das Wesen und die Erblichkeitsverhaltnisse der "Var-ietates albumarginatae hort" von Pelargonium sonale. Z. Vererbungs-lehre. 1:330-352. Bendall, D.S. and W.D. Bonner. 1971. Cyanide-insensitive Respiration i n Plant Mitochondria. Plant Physiol. 47:236-245. Bertrand, H., R.A. C o l l i n s , L.L. Stohl, R.R. Goewert and A.M. Lambowitz. 1980. Deletion Mutants of Neurospora orassa Mitochondrial DNA and Their Relation to the "Stop-Start" Growth Phenotype. Proc. Nat. Acad. Sc i . 77:6032-6036. 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