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Transmission of kalilo DNA in senescent strains of Neurospora intermedia Myers, Carolyn J. 1988

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TRANSMISSION OF KALILO DNA IN SENESCENT STRAINS OF NEUROSPORA INTERMEDIA by C A R O L Y N J . M Y E R S A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF DOCTOR O F P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E STUDIES Genetics Programme We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF BRITISH C O L U M B I A 23 March 1988 0 Carolyn J . Myers, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of y^rTHfctsJ ^   The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ABSTRACT Senescence, the progressive loss of growth potential culminating in death, is common among Kauaian strains of Neurospora intermedia. Senescence is initiated by the insertion of kalilo D N A into the mitochondrial D N A . Mitochondrial D N A molecules carrying the insert accumulate and death occurs when the insert is equimolar with the mitochondrial D N A . The inserted form of kalilo D N A is referred to as mtlS-kalDNA. Studies on the somatic transmission of mtlS-kalDNA in ascospore series have revealed that kalilo D N A is capable of assuming new locations within the mitochondrial D N A . It is proposed that these novel insertions originate from intramitochondrial movement and an autonomous form of kalilo D N A , mtFF-kalDNA, is predicted to be an intermediate in movement. Novel insertion of kalilo D N A appears to depend on the form of mtlS-kalDNA transmitted sexually. If a mutagenic insert is transmitted, senescence is initiated at the onset of vegetative growth of the ascospores and no novel insertions are detected. The lifespans of these ascospores are quite short, death occurring in 10 subcultures or less. Transmission of a nonmutagenic insert delays the onset of senescence until either a novel insertion or a rearrangement of the transmitted insert occurs. The lifespans of these ascospores usually exceed 10 subcultures and are variable. Information obtained from tetrad analysis has revealed that novel insertion of kalilo D N A may also be under the influence of the host genome. A senescent Kauaian strain was identified which shows some but not all characteristics of kalilo senescence. In this strain and its derivatives, the behaviour of mtlS-kalDNA is erratic and in , some cultures the characteristic ii mitochondrial biochemical deficiencies, normally accompanying kalilo senescence, are not observed. It is suspected that ka lDNA is not responsible for senescence in this strain and its derivatives but rather some other unknown factor is affecting the normal growth patterns of these cultures. Kauaian strains were surveyed for the presence of dsRNA to determine whether ka lDNA has a viral origin. Only one senescent strain contains detectable amounts of dsRNA which was not homologous with a kalDNA probe. The survey identified six nonKauaian strains which contain dsRNA and seven dsRNA species were delineated. Although the presence of dsRNA is not relevant to kalilo senescence, analysis of dsRNA in a genetically-well defined organism like Neurospora may give insight into the significance of dsRNA in fungi in general. iii T A B L E O F C O N T E N T S Abstract ii Table of Contents iv List of Tables vi List of Figures vii Acknowledgements xii I. Introduction 1 A . Neurospora L I F E C Y C L E .. 1 B. C Y T O P L A S M I C M U T A T I O N S OF F U N G I 5 1. Petite Mutations of Saccharomyces cerevisiae 6 2. The ragged Mutation of Aspergillus amstelodami 9 3. Senescence in Podospora anserina 10 4. Cytoplasmic Mutations of Neurospora 21 C. P E R S P E C T I V E O N S E N E S C E N C E 34 II. Materials and Methods 39 1. Strains 39 2. Media and Growth Conditions 39 3. Nucleic Acid Isolations 40 a. Mitochondrial D N A Isolation 40 b. Nuclear D N A Isolation 43 c. dsRNA Isolation 44 4. Cytochrome Analysis 45 5. Restriction Enzyme Digestion and Gel Electrophoresis 45 6. Labelling of Nucleic Acid 46 a. Nick Translation 46 b. End-labelling of dsRNA 47 7. Probes 48 8. Blot Hybridization 48 a. Southern Blot Analysis 48 b. Northern Blot Analysis 53 HI. Chapter 1 54 A . INTRODUCTION 54 B . R E S U L T S 55 1. Transmission of mtlS-kalDNA in Ascospores Initiated from Cross 561-1 X 1766 58 a. Correlation Between Time of ka lDNA Insertion and Onset of Senescence 70 b. Movement of mtlS-kalDNA and Identification of a Transient Mitochondrial Autonomous Form of ka lDNA ... 72 2. Transmission of mtlS-kalDNA in Ascospores Initiated From Other Crosses 74 iv a. Ascospore Series from Cross 801-1 X 1836 75 b. Ascospore Series from Cross 572-5 X 1818 76 C. DISCUSSION 131 IV. Chapter 2 141 A . I N T R O D U C T I O N 141 B . R E S U L T S 143 1. Proposed Genetic Regulation of kalDNA Movement 143 2. Comparison of Tetrads From Crosses Using a Juvenile Female Parent and a Senescent Female Parent 149 C. DISCUSSION 182 1. Genetic Regulation of mtlS-kalDNA Movement 182 2. Comparison of Tetrads Derived From Crosses Using a Juvenile Female Parent and a Senescent Female Parent 189 V . Chapter 3 192 A . I N T R O D U C T I O N 192 B . R E S U L T S 193 C. DISCUSSION 227 VI . Chapter 4 232 A . I N T R O D U C T I O N 232 B . R E S U L T S 234 1. Identification and Cross Homologies of the dsRNAs 234 2. Homology with Genomic D N A 238 3. Hybridizations Using the Pst I-kalDNA Probe 238 C. DISCUSSION 247 VII. Conclusion 252 Bibliography 258 v List of Tables Table I. Variables used in the Correlation Analysis 71 Table II. Geographic Origin and Stock Number of Wild Type Isolates of Neurospora crassa and N . intermedia 235 Table 1TL Growth Phenotype, Geographic Origin, and Sizes of dsRNAs of Seven Isolates of Neurospora 238 vi List of Figures Introduction 1 Figure 1. Life Cycle of Neurospora 4 Figure 2. M t D N A Restriction Map of Aspergillus amstelodami Showing the Locations of the Excised D N A 12 Figure 3. M t D N A Restriction Map of Podospora anserina Showing the Locations of the senDNAs 15 Figure 4. The Influence of Seven Nuclear Genes on the Expression of Senescence in Podospora anserina 20 Figure 5. Physical Map of the 21kb Mitochondrial D N A of Mutant E35 25 Figure 6. M t D N A Restriction Map of Neurospora intermedia Showing the Sites of Insertion of kalilo D N A 29 Figure 7. Restriction Map of kalilo D N A 33 Materials and Methods 39 Figure 8. Restriction Map of the mtDNA of Neurospora intermedia Showing the Regions of the mtDNA Used as Probes 50 Figure 9. Restriction Map of the mtDNA and With two Different ka lDNA Insertions Showing the Regions of ka lDNA and Flanking mtDNA used as probes 52 Chapter 1 54 Figure 10. Subculture series for long ascospore series showing growth cessation 82 Figure 11. Analysis of Bgl II digested mtDNAs from subcultures of series 4 of cross 561-1X1766 84 Figure 12. Southern analysis showing the mtDNA location of novel insertion in series 4 86 Figure 13. Analysis of Bgl II digested mtDNAs from subcultures of series 7 of cross 561-1X1766 88 Figure 14. Southern analysis showing the mtDNA location of novel insertion in series 7 90 Figure 15. Analysis of Bgl II digested mtDNAs from subcultures of series 8 of cross 561-1X1766 92 vii Figure 16. Southern analysis showing the mtDNA location of novel insertion in series 8 94 Figure 17. Analysis of Bgl II digested mtDNAs from subcultures of series 12 of cross 561-1X1766 96 Figure 18. Southern analysis showing the mtDNA location of novel insertion in series 12 98 Figure 19. Analysis of Bgl II digested mtDNAs from subcultures of series 13 of cross 561-1X1766 100 Figure 20. Analysis of Bgl II digested mtDNAs from series 14 of cross 561-1X1766 102 Figure 21. Southern analysis showing the mtDNA location of novel insertion in series 14 104 Figure 22. Analysis of Bgl II digested mtDNAs from series 16 of cross 561-1X1766 106 Figure 23. Southern analysis showing the mtDNA location of novel insertion in series 16 108 Figure 24. Southern analysis of Bgl II digested mtDNAs from the late cultures of each ascospore series 110 Figure 25. Southern analysis of uncut mtDNAs from series 4 of cross 561-1X1766 '. 112 Figure 26. Southern analysis of mtDNAs from DNase treated mitochondria. ..114 Figure 27. Comparison of the Mobilities of AR-kalDNA and the Autonomous Mitochondrial Form of ka lDNA ;. 116 Figure 28. Southern analysis of Bgl II digested mtDNAs from the early and late cultures of the three 801 ascospore series 118 Figure 29. Southern analysis of uncut nucDNA from the late cultures of the three 801 ascospore series 120 Figure 30. Analysis of Bgl II digested mtDNAs from ascospore series 4 of cross 572-5X1818 122 Figure 31. Analysis of Bgl II digested mtDNAs from ascospore series 13 of cross 572-5X1818 124 Figure 32. Analysis of Bgl EE digested mtDNA of the late cultures of the two 572 ascospore series and the female parent 126 viii Figure 33. Southern analysis of nucDNAs from the late cultures of the two 572 ascospore series and the female parent 128 Figure 34. Summary of the locations of the novel insertions appearing during growth in all ascospore series analyzed 130 Chapter 2 141 Figure 35. Lengths of subculture series for tetrads from crosses 561-0 X 605 and 561-5 X 605 155 Figure 36. Lengths of subculture series for tetrads from crosses 561-0 X 1766 and 561-5 X 1766. 157 Figure 3 7A. Analysis of Bgl II digested mtDNAs from the ascospore cultures of ascus 1 from cross 561-0 X 605. 159 Figure 37B. Analysis of Bgl II digested mtDNA from the late cultures of ascus 1 from cross 561-0 X 605 161 Figure 3 8A. Analysis of Bgl II digested mtDNA from the ascospore cultures of ascus 7 from cross 561-0 X 1766 '., 163 Figure 38B. Analysis of Bgl II digested mtDNA from the late cultures of ascus 7 from cross 561-0 X 1766 165 Figure 39A. Analysis of Bgl U. digested mtDNA from the ascospore cultures of ascus 5 from cross 561-0 X 1766 167 Figure 39B. Analysis of Bgl II digested mtDNA- from the late cultures of ascus 5 from cross 561-0 X 1766 169 Figure 40A. Analysis of Bgl n digested mtDNA from the ascospore cultures of ascus 6 from cross 561-5 X 605 171 Figure 40B. Analysis of Bgl II digested mtDNA from the late cultures of ascus 6 from cross 561-5 X 605 173 Figure 41 A . Analysis of Bgl II digested mtDNA from the ascospore cultures of ascus 4 from cross 561-5 X 1766 175 Figure 4IB. Analysis of Bgl II digested mtDNA from the late cultures of ascus 4 from cross 561-5 X 1766 177 Figure 42A. Analysis of Bgl II digested mtDNA from the ascospore cultures of ascus 3 from cross 561-5 X 1766 179 Figure 42B. Analysis of Bgl II digested mtDNA from the late cultures of ascus 3 from cross 561-5 X 1766 181 ix Chapter 3 192 Figure 43. Cytochrome spectra of mitochondria from subcultures of series P573. 200 Figure 44. Analysis of Bgl II digested mtDNAs from subcultures of series P573. 202 Figure 45. Cytochrome spectra of mitochondria from subcultures of ascospore 1 from cross 573-1 X 1766 204 Figure 46. Cytochrome spectra of mitochondria from subcultures of ascopore 7 from cross 573-1 X 1766 206 Figure 47. Analysis of Bgl II digested mtDNAs from subcultures of ascospore 1 from cross 573-1 X 1766 208 Figure 48. Analysis of Bgl II digested mtDNAs from subcultures of ascospore 7 from cross 573-1 X 1766 210 Figure 49. Cytochrome spectra of mitochondria from subcultures of ascospore 5 from cross 573-1 X 1766 212 Figure 50. Analysis of Hind i n digested mtDNAs from subcultures of ascospore 5 from cross 573-1 X 1766 214 Figure 51. Analysis of Bgl Et digested mtDNA from subcultures of ascospore 5 from cross 573-1 X 1766 216 Figure 52. Analysis of uncut nucDNAs from the 573 series and three of its derivatives, ascospores 1, 5, and 7 218 Figure 53. Subculture series for long ascospore series from cross 573-1 X 1766 220 Figure 54. Cytochrome spectra of mitochondria from ascospores 2, 4, 15, and 19 from cross 573-1 X 1766 222 Figure 55. Analysis of Bgl II digested mtDNAs from the late cultures of ascospore series from cross 573-1 X 1766 224 Figure 56. Analysis of Eco R l digested nucDNAs from the late cultures of ascospore series from cross 573-1 X 1766 226 Chapter 4 232 Figure 57. Analysis of dsRNAs from seven natural isolates of Neurospora 240 Figure 58. Cross hybridizations of dsRNAs 242 x Figure 59. Hybridization of genomic D N A with the 9.0kb dsRNA species 244 Figure 60. Hybridization of dsRNAs with Pst I-kalDNA 246 Conclusion 252 Figure 61. Summary chart of the molecular events associated with senescence. 257 xi ACKNOWLEDGEMENTS I would like to express appreciation to those who have helped me throughout my graduate career. First, I wish to thank may supervisor Dr. A . J . F . Griffiths for his untiring support, advice, and enthusiasm. His vast knowledge and creativity have been a major asset to my graduate training and the completion of my thesis. Special thanks are extended to Dr. H . Bertrand for his generosity, suggestions, and support. I wish to thank the members of my supervisory committee Dr. T. Grigliatti, Dr. B.R. Green, Dr. J . McPherson, and Dr. H . Brock for their helpful comments and suggestions. I also thank Dr. T. Grigliatti, Dr. B.R. Green, and Dr. J . McPherson for their critical reading of my thesis. Thanks to Dr. B . Martin, L . Piez, and S. Buttrey for teaching me procedures integral to my thesis. I thank all the undergraduate students who help me with my research. I am grateful to all my friends who have made my stay here very enjoyable. Last, I owe a very special thanks to D. Vickery. His unfailing patience, understanding, and support have help to make the completion of my thesis a reality. xii I. INTRODUCTION A. NEUROSPORA LIFE CYCLE It was of interest to investigate the transmission of ka lDNA sexually and during vegetative growth in Kauaian strains of Neurospora intermedia to determine if a correlation exists between the transmission of kalDNA and the expression of senescence. Described in this section is the lifecyle of Neurospora. The genus Neurospora belongs to the Kindom Fungi and is a member of the Class Ascomycete. The ascomycetes are the largest class of fungi and provide most of the species which have been widely used in genetics. The principal feature of this group is the ascus which encloses the products of meiosis, the ascopores; there are four or, following an extra mitotic division, eight in number. For example, in N . crassa, N . sitophila, and N . intermedia a mature ascus contains eight ascospores. Neurospora possesses a mycelial form of cellular organization where the mycelium has septa which delineate hyphal compartments. Within each compartment are several nuclei. The septa have central pores through which the nuclei and cytoplasm can pass thus uniting the various hyphal compartments into a continuous protoplasmic system. This form of cellular organization is referred to as coenocytic. A diagram of the life cycle of Neurospora is shown in Figure 1. The life cyle of N . crassa, N . sitophila, and N . intermedia involves both sexual and asexual propagation (reviewed by Beadle, 1945). These species are all heterothallic and 1 Introduction / 2 consequently require mycelia of opposite mating types to fuse in order to complete the life cycle. Mating type is determined by a mating type gene which is located on Linkage Group 1 (Perkins et al, 1982). The two mating types are designated A. and _a_ and are codominant alleles of the mating type gene. On suitable crossing medium, either mating type is capable of producing female fruiting bodies, protoperithecia. Protoperithecia, consist of coiled filaments of hyphae which become surrounded by a thick layer of hyphae. The coiled filaments are destined to become ascogeneous hyphae. From each filament a sexual hypha, the trichogyne, is produced and fuses with a fertilizing cell of the opposite mating type. The male cells may be either vegetative hyphae, or asexual spores called macroconidia or the less abundant microconidia. It has been proposed that trichogyne growth and localization of the male fertilizing cell is a chemotactic response initiated by the presence of a pheromone released by the male fertilizing cell (Bistis, 1986). After fusion of the trichogyne and the male fertilizing cell, the nucleus from the male cell is transferred through the trichogyne into the ascogeneous hypha. The paternal and maternal nuclei undergo a number of synchronous mitotic divisions to form a small mass of dikaryotic ascogenous hyphae. At the same time the protoperithecium enlarges and becomes blackened with melanin and eventually forms the mature fruiting body, the perithecium. Nuclear fusion and karyogamy eventually occurs between the penultimate hyphal compartment of the ascogenous hyphae. Immediately after karyogamy, meiosis occurs and the four products of meiosis undergo a round of mitosis to give a total of eight nuclei which form the eight ascospores. A t maturity, the asci elongate and eject their spores through the ostiole of the perithecium. The ascospores germinate under high temperatures, 60C, and form Introduction / 3 Figure 1. Life cycle of Neurospora showing both the sexual and asexual cycles. Figure copied from Fincham, et al (1979). I n t r o d u c t i o n / 4 Introduction / 5 mycelia. On vegetative medium aerial mycelia is formed and asexual spores (conidia) are produced through mitosis. The conidia become airborne and give rise to new hyphal colonies which conidiate and continue the asexual life cycle. The advantage of using Neurospora for genetic analysis is the availability of the products of each meiosis which remain together as a tetrad of ascospores. The segregation of different phenotypes in a tetrad can detemine the pattern of inheritance of genes. Furthermore, the phenotypic ratios in either ordered or unordered tetrads provide information on various chromosome mutations such as nondisjunction, translocation, and inversion, and gene mutations including gene conversion. Reciprocal crosses are possible since any strain of Neurospora may be used as either a male or female parent. Thus, analysis of tetrads or random ascospores from reciprocal crosses aids in distinguishing between nuclear and extranuclear inheritance. In Neurospora, the cytoplasm shows strict maternal inheritance, and therefore extranuclear inheritance can be distinguished from nuclear inheritance based on reciprocal differences in crosses. B. CYTOPLASMIC MUTATIONS OF FUNGI The topic described in this thesis is mitochondrially-based senescence in N .  intermedia. A number of examples of cytoplasmic mutations have been reported and in this section the most characterized examples described in fungi are presented. The first observation of non-Mendelian patterns of inheritance was reported by Introduction / 6 Correns (1909) and Baur (1909). They discovered that a factor influencing chloroplast development in some strains of flowering plants ( Mirabilis and Pelargonium) does not follow the normal patterns of Mendelian inheritance. With time many cases of cytoplasmic inheritance were discovered in organisms which ranged from unicelluar algae ( Chlamydomonas, Sager, 1954) to complex higher plants such as Triticum (Briggle, 1966). Although a number of examples of extranuclear inheritance was apparent, little was known about mitochondria and chloroplasts, how they interact with nuclear genes, and their importance in the development of a given organism. The first evidence showing that mitochondria replicate and possess D N A was reported by Luck (1963) and Luck and Reich (1964) on Neurospora. Direct evidence that mitochondria contain genetic information was shown by Diacumakos et al (1965). They used the N . crassa mutant abn-1 in their experiments. This mutant exhibits a slow-growth character and cytochrome a a 3 and b deficiencies, both of which are maternally inherited. Cytoplasm was isolated from this strain and injected into a wild type recipient. Both the slow-growth phenotype and the accompanying cytochrome deficiencies were transmitted. The discovery of D N A within mitochondria set the stage for a cascade of extranuclear inheritance research on many organisms. In this section a review of cytoplasmic mutations in Saccharomyces cerevisiae, Aspergillus  amstelodami, Podospora anserina, N . crassa, and N . intermedia is presented. 1. Petite Mutations of Saccharomyces cerevisiae Mitochondrial genetics of S. cerevisiae was initiated when Ephrussi et al (1949) reported that some respiratory-deficient mutants, obtained after acriflavin induction, Introduction / 7 showed nonMendelian patterns of inheritance. These mutants are called petites or vegetative petites. These strains are nonreverting pleiotropic mutants containing large deletions of the mtDNA (p-) or no mtDNA at all (p°) (Dujon, 1981). In contrast to filamentous fungi which die as a consequence of major mtDNA deletions, yeast is a facultative anaerobe and can survive complete loss or gross alterations of its mtDNA. For p- mutants the fragment of mtDNA retained is variable and usually less than one third of the genome (Dujon, 1981). Since the total amount of mtDNA in a p- mutant is similar to the wild type mtDNA from which it was derived, irrespective of the size of the deletion, it was suggested that the number of copies of the retained mtDNA must be amplified (Hollenberg et al 1972; Nagley and Linnane, 1972; Faye et al, 1973; Fukuhara et al, 1974; Borst et al, 1976). Further investigation revealed that there are two major types of arrangements of the amplified conserved mtDNA sequences in p-mutants (Dujon, 1981). Generally head-to-tail repeats of the conserved sequences are observed when the retained mtDNA is less than approximately lOOObp. In the other type of arrangement, the repeated unit is an inverted duplication of the conserved sequence. Usually, these inverted repeats are not perfect. Some other p- mutants are observed to contain mixtures of the repeats where the repetition of the conserved sequences may be direct or inverted along the same molecule. In addition to these two major arrangements of the retained mtDNA, rearrangements may be observed including internal inversions, deletions or illegitimate recombination between different p- mutants (Dujon, 1981). The degree of suppressiveness, that is the proportion of zygotic clones composed of p- mutant cells, is characteristic of a given p- mutant. (Ephrussi and Introduction / 8 Grandchamp, 1965). Two models were proposed to explain the mode of action of the suppressive petite mtDNA in heteroplasmic crosses. These include a model based on the destructive recombination of wild type mtDNA molecules, thus giving rise to deleted molecules (Coen et al, 1970; Michaelis et al, 1973; Deutsch et al, 1974; Perlman and Birky, 1974; Slonimski and Lazowska, 1977). Although this has been suggested as a model for suppressiveness, the demonstration that the petite mtDNA in diploid progeny from a cross are both physically (Goursot et al, 1980; Blanc and Dujon, 1980) and genetically (Gingold, 1981) similar to those of the petite parent has focussed attention on the 'out-replication' model. This model is based on a replicative advantage of petite mtDNAs (Slonimski, et al 1968; Rank, 1970a; 1970b; Rank and Bech Hansen, 1972; Carnevali and Leoni, 1981). The 'out-replication' model has received much support from analysis of the nature of the mtDNA of very strongly suppressive petites. These petite mtDNA molecules usually consist of short repeats which include one of a small number of closely related sequences thought to represent origins of replication (de Zamaroczy et al, 1979; Bernardi et al, 1980; Blanc and Dujon, 1980; 1981). It was suggested that the high degree of reiteration of origin of replication sequences in highly suppressive petites would confer a replicative advantage to the petite mtDNAs in heteroplasmic zygotes. It was further suggested that p-mutants showing lesser degrees of suppressiveness reflects the lower degree of reiteration of the origin of replication sequences. Further analysis of p+ and p-heteroplasmic zygotes was achieved by growing mating mixtures from petite by grande crosses in selective medium containing radioactively labeled uracil (Chambers and Gingold, 1986). In this medium only zygotes could grow and incorporate label into their newly synthesized mtDNA. M t D N A was isolated from Introduction / 9 the cultures, cut with restriction enzymes, and newly synthesized mtDNA restriction fragments visualized by autoradiography of gels. By focussing on bands unique to the petite and the grande mtDNAs, the authors were able to ascertain the relative amounts of label incorporated into the two mtDNA species and determine their relative level of synthesis in the zygote. It was discovered that the 'out-replication' hypothesis does not hold true for all suppressive petites. Hypersuppressive petites did exhibit a competitive replication advantage over the p+ mtDNA molecules, but less suppressive petites do not necessarily show this same pattern. Interestingly, a strain which was essentially nonsuppressive gave an indication of replicative superiority. Thus, the simple 'out-replication' hypothesis does not offer an explanation for these inconsistancies. Clearly, some factor(s) other than destructive recombination of wild type mtDNA or enhanced replication is involved. 2. The ragged Mutation of Aspergillus amstelodami Jinks (1956) discovered a cytoplasmically inherited mutation of A . amstelodami referrred to as 'ragged'. It was observed that these mutants maintain a state of senescence over long periods of time where vegetative death occurs in the hyphal tips giving the growing front a characteristically ragged appearance (Jinks 1956; 1959; Caten, 1972). The ragged phenotype arises spontaneously at a reasonably high frequency and 'ragged' mutants characterized by cytochrome deficiencies and mtDNA rearrangements. Analysis of the mtDNA of 'ragged' mutants revealed the presence of high molecular weight D N A which is not observed in wild type cultures. A l l the novel D N A species consist of tandem repeats that are Introduction / 10 homologous with the mtDNA (Lazarus et al, 1980). In all but one mutant, the amplified mtDNA region is located between the cytochrome b and ATPase subunit 6 genes. Figure 2 shows the regions of the mtDNA, relative to the r R N A genes, giving rise to the amplified sequences. The length of the excised sequences range from 1.5kb to 2.7kb each having a 215bp sequence in common. This 215bp sequence maps between an unidentified reading frame (corresponding to U R F 4 of human mtDNA) and an arginine tRNA gene. It was postulated that the common 215bp sequence contains an origin of replication because a hairpin secondary structure similar to yeast and human mitochondrial origin sequences can be formed (Lazarus et al, 1981). It was suggested that these excised and amplified origin of replication sequences could confer a replicative advantage to the high molecular weight mtDNA species and explain the progressive decline in growth at the periphery of the mycelial colony. The one mutant not included above possesses a mtDNA sequence excised from the region of the mtDNA downstream of the large subunit of the r R N A gene (Lazarus et al, 1980) (Figure 2). 3. Senescence in Podospora anserina The unavoidable decline in growth potential culminating in vegetative death in P. anserina was discovered to be maternally inherited (Rizet 1953; 1957). This was confirmed by microinjecting juvenile cultures with cytoplasm isolated from senescent cultures and from hyphal fusion experiments (Marcou and Schecroun, 1959). Mycelial senescence is race specific (Rizet, 1957; Marcou, 1961) where race A expresses senescence more rapidly than other races. It should be noted that two races have been studied in detail, race A and race s. Introduction / 11 Figure 2. MtDNA restriction map of Aspergillus amstelodami showing the regions of the mtDNA giving rise to amplified sequences in various 'ragged' mutants. Restriction map outer circle: E=Eco Rl ; inner circle: A = Hpa I, B=Bam HI, P=Pst I, S=Sal I. Restriction map obtained from Lazarus and Kuntzel (1981). I n t r o d u c t i o n / 12 Introduction / 13 It was shown that the 94kb juvenile mtDNA is greatly diminished in senescent cultures and replaced by amplified multimeric sets of small circular D N A (Stahl et al, 1978; Cummings et al, 1979a). These circular DNAs are referred to as sen plasmids and consist of head-to-tail tandem repeats of specific regions of the mtDNA (Stahl et al, 1978; Cummings et al, 1979a; 1979b; Cummings et al, 1980; Jamet-Vierny et al, 1980; Kuck et al, 1981; Osiewacz and Esser, 1984). The most frequent sen plasmid is alpha. Other sen plasmids, not seen as frequently as alpha senDNA, are referred to as beta, epsilon, theta, and gamma (Stahl et al, 1978; Cummings et al, 1980; 1985; Jamet-Vierny et al, 1980; Wright et al, 1982; Osiewacz and Esser, 1984). Confirmation that alpha senDNA is responsible for senescence in some strains came from information on a mutant that has shown no signs of senescence even after four years of culturing. Analysis of the mtDNA of this mutant revealed that the sequences of the intron, which normally excise to generate alpha senDNA, are absent in the mtDNA of this mutant (Vierny et al, 1982). A mtDNA restriction map and the location of four of the sen plasmids is shown in Figure 3. Alpha senDNA consists of the intron at the 5' end of the cytochrome oxidase subunit I gene (COI gene) (Cummings and Wright, 1983; Osiewacz and Esser, 1984). The excision of the 2.5kb intron occurs precisely at the exon-intron junction fragments. Sequence analysis of alpha senDNA has revealed that it belongs to the so called class II introns. To digress slightly, introns of fungal mtDNA are classified as class I or class II depending on their overall secondary structure and the presence of short conserved sequences (Michel et al, 1982). Class I mitochondrial introns are most abundant and are Introduction / 14 Figure 3. Restriction and partial gene map of the mtDNA of Podospora anserina showing the regions of the mtDNA giving rise to amplified senDNAs. A Bgl II, Eco R l , Pst I, and Hae IEt digest are included. The position of the mitochondrial genes are shown. Map obtained from Cumrnings et al (1987). I n t r o d u c t i o n / 15 Introduction / 16 characterized by their ability to self-splice as has been shown for the Tetrahymena thermophila nuclear rRNA intron (Cech et al, 1983; Michel and Dujon, 1983; Waring et al, 1983; Garriga and Lambowitz, 1984; Van der Horst and Tabak, 1985; Peebles et al, 1986). Class II mitochondrial introns have been found to contain long open reading frames (Bonitz et al, 1980; Nargang et al, 1984; Osiewacs and Esser, 1984; Michel and Lang, 1985) which encode proteins related to reverse transcriptase to facilitate their own excision (Michel and Lang, 1985; Steinhilber and Cumrnings, 1987). Senescent cultures of both race A and race s contain alpha senDNA sequences. These two races differ in the number of introns present in the COI gene, race A has an extra class II intron referred to as intron A (Cumrnings and Wright, 1983). This intron does not share overall sequence homology with the intron which excises to generate alpha senDNA. It was proposed that the presence of two class II introns in the COI gene of race A , compared with one in race s, could account for the difference in the rates of senescence between these two races. This hypothesis was stimulated by the finding that these two group II introns contain nucleotide sequence homologies with the retroviral reverse transcriptase of Rous Sarcoma virus and H T L V - 1 viruses (Matsuura et al, 1986). Thus, if reverse transcription of these two class II introns is responsible for the amplification of sen plasmids, then race A would have twice as many copies of sen plasmids as race s. No autonomous sen plasmid homologous with intron A sequences has been identified suggesting this is probably not the mechanism controlling the rate of production of the sen plasmids (Cumrnings et al, 1987). It was suggested that intron A may play a modulating role in the excision of Introduction / 17 alpha senDNA and that it cannot be excised except in the absence of alpha senDNA. Although amplification of alpha senDNA does not appear to depend on the reverse transcription of the class II intron, evidence that there is reverse transcriptase activity in senescent race A cultures and not in young cultures does suggest that alpha senDNA may be generated through an R N A intermediate (Steinhilber and Cummings, 1986). A more plausible model to explain the amplification of senDNAs is based on the hypothesis that sen plasmids may show superior replication relative to the mtDNA. Lazdins and Cummings (1982) showed that cloned alpha, beta, and gamma senDNAs , as well as those young mtDNA sequences which overlap and hybridize with senDNA sequences, confer origin of replication characteristics to the otherwise nonreplicating vector YIp5. It has also been shown that pBR322 containing alpha senDNA is able to replicate in P. anserina (Stahl et al, 1982). Together these results indicate that the amplification of the small circular senDNAs may depend on the presence of origin of replication sequences and identification of only five sen plasmids may define the regions of the mtDNA which contain origin of replication sequences. The other senDNAs do not have sequences homologous with class II introns (Michel and Cummings, 1985), but the presence of direct repeats at both ends of beta senDNA and epsilon senDNA provide a ready mechanism for the excision of these two regions of the mtDNA. It has been reported (Cummings et al, 1987) that beta senDNA does not contain either intron or exon sequences but rather spacer mtDNA sequences (Figure 3). For epsilon senDNA, the 5' excision site Introduction / 18 occurs within the intron of the U R F 1 gene and the 3' excision site in the spacer mtDNA. For theta senDNA, the 3' excision occurs within the 3' exon of the COI gene and the 5' excision site in spacer mtDNA. The fact that epsilon and theta senDNAs are not composed solely of intron sequences suggests that the intronic splicing apparatus is probably not the mechanism by which these senDNAs are excised from the mtDNA. Indeed the intronic splicing apparatus may still be involved in the generation of these senDNAs, but in an illicit manner. The onset of senescence in P. anserina appears to depend on any one of a number of nuclear genes (Tudzynski and Esser, 1979; Esser and Tudzynski, 1980). These genes are pleiotropic because in addition to prolonging life, they alter mycelial morphology. Figure 4 shows the combinations of seven genes and the number of days before death for each single and double mutant. The double mutant ' vivax incoloris shows the longest lifespan. Microinjection experiments showed that senescent mycelia were not able to infect i viv strains. Moreover, ascospores from a cross between a senescent wild type female and an i viv male mutant revealed that i viv progeny did not express senescence whereas the wild type progeny were senescent (Tudzynski and Esser, 1979). It was suggested that the i viv combination counteracts the senDNAs in their active form (Esser and Tudzynski, 1980). M t D N A of i viv mutants has not been investigated but these results indicate that the excision and/or the amplification of senDNA is probably suppressed by the various nuclear mutations. Introduction / 19 Figure 4. The influence of seven morphological genes on the expression of senescence in Podospora anserina. The genes are represented by abbrevations and their relative positions in the genome indicated. The onset of senescence for single mutants (diagonal) and for combinations of double mutants (intersections) are expressed in the figure as number of days. Squares that are crossed out do appear to have an influence on the onset of senescence. Figure obtained from Esser and Tudzynski (1980). I n t r o d u c t i o n / 20 3a 22 TO » 30 / \ ; w 1 tr _ CT ca vi« • 3 3 0 •1250 • 4 0 0 • 5 0 0 • 7 0 • 4 0 0 n • 7 8 0 >« / • 7 2 0 7 0 1 • 0 • 6 5 0 9 0 M S • 0 «r 90 •• ?« 96 t» 1O0 • 3 0 0 • 110 * f 5 0 - 4 7 0 & 0 Introduction / 21 4. Cytoplasmic Mutations of Neurospora Group I Cytoplasmic Mutations of Neurospora crassa In 1952, the first cytoplasmic respiratory deficient mutant of N . crassa was reported (Mitchell and Mitchell, 1952). This mutant was characterized as initially exhibiting slow growth showing a progressive increase in growth rate until a rate of wild type was reached. This mutant was originally designated poky and is now referred to as mi-1 (Mitchell et al, 1953). Young cultures of these mutants are deficient in cytochromes a a 3 and b (Haskins et al, 1953). Rifkin and Luck (1971) and Neupert et al (1971) found that mi-1 mitochondria were deficient in mitochondrial small ribosomal subunits and the small r R N A . It was suspected that the mi-1 mutation resides in the mtDNA and two potential sites of primary defect being either the small rRNA gene or the S-5 ribosomal protein gene. Subsequent sequence analysis of mi-1 has identified a 4bp deletion in the coding sequence of the mitochondrial small r R N A gene near the 5' end of the exon. The deletion results in synthesis of aberrant small rRNAs where 38-40 nucleotides are missing (Akins and Lambowitz, 1984). Bertrand, Pittenger and coworkers (Bertrand and Pittenger, 1972a; 1972b; Bertrand et al, 1976) identified additional mutants ( exn-1, exn-2, exn-3, exn-4, SG-1, SG-3, and stp-B 1) that are phenotypically related to mi-1. Subsequently, six of these mutants were shown to be deficient in the small ribosomal subunits and small rRNA (Collins and Bertrand, 1978; Collins et al, 1979; Lambowitz et al, 1979). A nuclear suppressor, referred to as _f, was discovered (Bertrand and Introduction / 22 Pittenger, 1972b) which suppresses the initial growth lag of most group I mutants yet has no effect on the cytochrome content of these mutants. An additional six nonallelic nuclear suppressors have been isolated (Kohout and Bertrand 1976; Bertrand and Kohout, 1977; Collins, et al 1979) which suppress the initial growth lag and alleviate respiratory and cytochrome defects of mi-1 and the other group I mutants. Group II Cytoplasmic Mutations of Neurospora crassa Group II mutants include mi-3 (Mitchell et al, 1953; Bertrand and Pittenger, 1972a) and exn-5 (Bertrand et al, 1976). Both have an initial lag in growth rate and are deficient in cytochrome a a 3 . These phenotypes are suppressed completely by the nuclear gene su-1 (Bertrand, 1971). The mtDNA defect in these two mutants has not been elucidated. Group HI Cytoplasmic Mutations of Neurospora crassa The group III mutants are characterized by an irregular pattern of growth and no growth. Based on their stop-start growth pattern, these mutants were termed 'stoppers'. Stopper mutants are female sterile and deficient in cytochromes a a 3 and b (Bertrand et al, 1980; DeVries et al, 1981). It has been shown that the generation of the 'stopper' phenotype is correlated with deletions and insertions of the mtDNA resulting in heteromorphic mtDNA populations (Bertrand et al, 1980; deVries et al, 1981). In four stoppers analyzed, the sizes of the retained mtDNA differs but the region maintained in all includes the Eco R l - 1 , -4, and -6 Introduction / 23 fragments. The Eco R l -1 region contains the majority of the tRNA genes and both the large and small r R N A genes. The mutant E35 has been studied extensively. In 'stop phase', the mutant contains a 21kb circular molecule consisting of one third of the length of the mtDNA as the^ predominant mtDNA form (DeVries et al, 1981; Gross et al, 1984) (Figure 5). A complementary 43kb circular mtDNA appears upon the resumption of growth. It was demonstrated that these circles arise by reciprocal recombination at or near the directly repeated tRNA(met) sequences and the frequency of the two depends on their rates of replication from origin of replication sequences unique to each mtDNA molecule. The 21kb and 43kb circles encompass the entire wild type mtDNA molecule. In the other stoppers, the observation that the altered mtDNA molecules form the majority of the mtDNA population during the 'stop phase' suggested that there is a competition between defective and intact mtDNAs such that cells in the 'stop phase' should show strong selection for nuclear or extranuclear mutations that permit resumption of growth and visa versa for cells in the 'growth phase' (Bertrand et al, 1980). This competition is probably dependent on the rates of replication of the mtDNAs as was suggested for mutant E35. Senescence in Neurospora intermedia Five variants having properties similar to the 'stopper' (group III) extranuclear mutants of N . crassa were discovered by Reick et al (1982) in a sample of N . Introduction / 24 Figure 5. Physical map of the 21kb mitochondrial DNA of mutant E35 which forms the predominant DNA species in 'stop-phase'. Included are Eco R l , Bgl II, and Hind III restriction of the region of the mtDNA retained. The bars denote the rRNA genes and the circles the tRNA genes. I n t r o d u c t i o n / 25 Introduction / 26 intermedia strains collected from the Hawaiian island of Kauai. These variants were identified by their inability to grow the length of a 500mm growth tube. These variants exhibited erratic stop-start growth, cytochrome a a 3 and b deficiencies, abnormal respiration, abnormal mitochondrial ribosome profiles, and the accumulation of unique mtDNA restriction fragments. A more intense survey was conducted using a serial subculture protocol (Griffiths and Bertrand, 1984). The serial subculture technique was used because it allowed more time for asexual propagation and ease of sampling and analyzing mycelium or conidia at various times during growth. Using the serial subculture procedure a total of 26 variants were identified. Most variants ceased to grow within 10 subcultures. It was suspected that the cytoplasmic abnormalities of these variants are maternally inherited. Proof of the maternal inheritance of senescence was obtained from the analysis of ascospores initiated from reciprocal crosses between senescent and nonsenescent strains (Griffiths and Bertrand, 1984). Their studies showed that juvenile cultures of either ascospores or conidia are normal phenotypically, but as a culture goes through the senescence process, changes originate in and progress through the cytoplasm resulting in death. During these changes, the cytoplasm is rendered heterogeneous for the determinative factors, and the degree of heterogeneity can be sampled experimentally through ascospores or conidia. Analysis of mtDNA prepared from different subcultures from the prototype senescent strain P561, revealed the presence of extra D N A not homologous with the mtDNA (Bertrand et al, 1985). This D N A has been termed kalilo which is the Hawaiian word for 'at death's door'. Kalilo D N A (kalDNA) is integrated into Introduction / 27 the mtDNA usually within the intron of the large rRNA gene, and termed mtlS-kalDNA in this state. Other locations of kalDNA have been identified which are generally within the Eco R l - 3 , -5, and -6 regions of the mtDNA (Bertrand et al, 1985; Bertrand, 1987). Refer to the restriction map of N . intermedia mtDNA (Figure 6) which shows the various locations of mtlS-kalDNA. In juvenile cultures mtlS-kalDNA is present in very low copy number relative to the mtDNA (Bertrand et al, 1985). M t D N A prepared from subsequent subcultures show a progressive increase in mtDNA molecules carrying mtlS-kalDNA. In the penultimate subcultures, mtlS-kalDNA is essentially equimolar with the mtDNA. The progressive displacement of normal mtDNA molecules corresponds with the progressive decline in growth potential of a senescent series and death results when mtlS-kalDNA is stoichiometric with the mtDNA. The displacement of normal mtDNA molecules suggests that mtDNA molecules carrying mtlS-kalDNA are suppressive. Whether suppressiveness is at the level of mitochondrial division or at the level of mtDNA replication is not known. Evidence that the respiratory pathway of mitochondria becomes disabled as a consequence of mtDNA mutations has suggested that respiratory deficiencies induce the division of mitochondria (Bertrand et al, 1986) If this is true, mutations in structural genes, tRNA or r R N A genes could result in 'renegade' multiplication of mutant mitochondria during growth displacing normal mitochondria. Alternatively, mtDNA molecules carrying mtlS-kalDNA may exhibit a more efficient rate of replication compared with normal mtDNA molecules. This hypothesis could depend on the presence of extra mitochondrial origin of replication sequences which would be associated with mtlS-kalDNA. Sequence Introduction / 28 Figure 6. A linear restriction map of the majority of the mtDNA of Neurospora  intermedia showing the 6ites of insertion of kalDNA in different strains, designated by the arrows. An Eco R l , Hind Ed, Bgl Et, and partial Pst I map are shown. The positions of the mitochondrial genes are presented. The filled in boxes represent the exon regions of each gene and the open boxes the intron regions. The line between the intron and large exon of the large rRNA gene is the S-5 gene. The mitochondrial tRNAs are indicated by dots. Map and location of each insertion obtained from Bertrand (1987). • • T . T . Y • Bgl n[[ - 3 1 1 1 2 I » 12 14 4 I p s i r* I 1 1 8 | K>c.11b | 3 I I 1 | 1 4 | 1 0 . | l 7 f l 6 | 10b ( 12a Ha | l9( 4 EcoRI l f 1 3 * o l i - 2 * o l i l c o - 2 ' ' S - r H N A c o - 3 * UMi L r UNA • " — ' • H O c o o Introduction / 30 analysis has indicated that there are no kalDNA sequences homologous with mitochondrial origin of replication sequences (Chan B-S, Doctoral Student, personal communication). Alternatively, displacement of normal mtDNA molecules may involve unidirectional gene conversion. Evidence for this hypothesis comes from two sources: First, Manella and Lambowitz (1979) investigated the interaction between wild type and mi-1 mtDNAs of N . crassa using as many as four physical markers to distinguish the two types of mtDNAs. They discovered that two insertions, one of 1200bp in the Eco R l - 5 and the other 500bp in Eco R l - 9 , are identified as sites of high frequency unidirectional gene conversion leading to their spread through the mtDNA population in heteroplasmons. Second, some cultures of S. cerevisiae possess an optional class I intron of the large r R N A gene. This intron has been termed omega. It has been shown experimentally that omega encodes a specific transposase which is active in the gene conversion process (Jacquier and Dujon, 1985). Through this transposase activity, omega spreads through the mtDNA population by invading large rRNA genes that lack the intron in zygotes containing both mtDNA types. Only preliminary sequence analysis of kalDNA has been performed (Chan B-S, Doctoral Student, personal communication), and consequently it is impossible at this time to decide whether there is an open reading frame potentially encoding an endonuclease. Since ka lDNA is not homologous with normal D N A and was found to be unrelated to any one of the mitochondrial plasmids (mtplasmids) found in Hawaiian strains of N . intermedia, it was postulated that ka lDNA may be derived from an extramitochondrial element (Bertrand et al, 1985). On looking for Introduction / 31 a precursor of mtlS-kalDNA, an autonomous linear element homologous with ka lDNA was discovered (Bertrand et al, 1986). This element is nucleus associated and called AR-kalDNA. Analysis of mtDNA and nuclear D N A (nucDNA) from a tetrad of ascospores initiated from a cross between the senescent strain, P561, and the nonsenescent strain, P605, showed that both AR-ka lDNA and mtlS-kalDNA are maternally inherited. It was further shown that AR-ka lDNA exists in high copy number in both juvenile and senescent cultures and has been proposed as the precursor of mtlS-kalDNA because the presence of AR-ka lDNA precedes the appearance of mtlS-kalDNA in subculture series derived from natural isolates (Bertrand et al, 1986). A restriction map of ka lDNA is shown in Figure 7. The element is approximately 9.0kb in length and contains three Eco R l recognition sites, one Hind III site, three Bgl II sites, and no Pst I restriction sites. Sequence analysis of ka lDNA has revealed that the ends of the element have inverted long repeats (LTRs) (Bertrand, 1987). The repeats are over 1300bp long. Sequence analysis of the mtDNA/kalDNA junctions has revealed the presence of pentanucleotide sequences which are postulated as recognition sequences for ka lDNA insertion. In all strains analysed, mtlS-kalDNA is flanked by long inverted repeats of the mtDNA (Bertrand, 1987). The inverted repeats of ka lDNA and flanking repeats generated in the mtDNA as a consequence of insertion of ka lDNA are characteristic of many transposable elements. This suggests that the intercompartmental movement of this element may involve similar mechanisms as for other mobile elements characterized. The Introduction / 32 Figure 7. Restriction map for both AR-kalDNA and mtlS-kalDNA. An Eco R l , Hind III, Bgl U , and Pst I map are shown. This map is based on data obtained from Bertrand et al (1985; 1986). The arrows represent the 1300bp inverted repeats. MtDNA sequences which would flank mtlS-kalDNA are represented by dashed lines. I n t r o d u c t i o n / 33 Introduction / 34 fact that the kalDNA element appears to move between cell compartments suggests that in addition to having transposon-like qualities, ka lDNA would have to possess the appropriate signals for transport and translocation across mitochondrial membranes, and insertion into the mtDNA. So far no sequences for any of these functions have been identified (Chan B-S, Doctoral Student, personal communication). At the onset of the work to be presented in this thesis very little information on kalilo senescence was known and from the review presented the majority of molecular work has only been published in the last couple of years. In addition, these studies present only preliminary results on kalilo senescence. As a consequence, information on the biological significance of ka lDNA in senescence, its mode of action, and etiology still remain unknown. From the review it is obvious that kalilo senescence is a complex phenomenon and requires more preliminary experiments to be done before complex questions may be asked. The work presented in this thesis suggests that this system is even more complex than originally predicted and opens up more avenues for potential research. C. PERSPECTIVE ON SENESCENCE Senescence or aging is a syndrome which accompanies most forms of life. With different levels of biological organization, this syndrome has different phenotypes in various organisms, but all are prone to irreversible alterations of their metabolism resulting in cellular death. Experimental interest in senescence came of age after the development of techniques by which vertebrate cells could be Introduction / 35 cultured in vitro (Ebeling, 1913; Carrel and Ebeling, 1925; Cohn and Murray, 1925). A number of provocative theories have been put forth to explain the progressive decline in growth and the eventual death of higher eukaryotes. There are in general two opposing theories: First, the aging process is genetically programmed and aging is an extension of a normal differentiation process (Hayflick 1965; 1972; Strehler et al, 1971). Second, the 'error catastrophe' hypothesis (Orgel, 1963; 1970; Comfort; 1974) which states that cells age because of the accumulation of mutations. The detrimental mutations would be to functionally indispensible genes, such as genes encoding proteins involved in replication, transcription, and translation. These errors would quickly multiply cascading into catastrophy and ultimately cell death. Experimental research on senescence in animals and animal cell culture to test these two theories has proven difficult due to the complexity of higher eukaryotes and furthermore it has been predicted that it is unlikely that a single principle or one causative agent exists to explain aging (Curtis, 1971; Comfort, 1974; Hayflick, 1975). Information compiled on the three fungi, A . amstelodami, P. anserina, and N .  intermedia, have resulted in a wealth of data on fungal senescence. They may well serve as model organisms for the exploration of senescence at the genetic and biochemical level. These fungi are well suited for studying aging because of their ease in handling under laboratory conditions, but mainly because of their accessibility for genetic analysis. Furthermore, senescence can be studied at the level of the whole organism since fungi are less complex than higher eukaryotes. The system chosen for investigation is kalilo senescence of N . intermedia. Of the three fungi, Neurospora, Podospora, and Aspergillus, Neurospora is most amenable Introduction / 36 to genetic manipulation. Second, a number of senescent cultures were already identified at the onset of this research such that the majority of natural isolates and ascospores used in the experiments presented in this thesis were already available for analysis. Analysis of senescence in a genetically well defined organism such as Neurospora should provide information on certain aspects of aging and perhaps identify one of the potential causative agents of aging in higher eukaryotes. In addition, the molecular events occurring as senescence develops in N . intermedia can be distinguished from those occurring in P. anserina and A . amstelodami. The latter senescent systems involve the excision and amplification of specific regions of the mtDNA, whereas senescence in N . intermedia is associated with the insertion of a foreign D N A into the mtDNA. This indicates that the molecular events responsible for senescence in N .  intermedia are unique among the filamentous fungi characterized and will provide information on an alternative mechanism for the induction of senescence in the filamentous fungi in general. Because very little information was available about kalilo senescence at the onset of the research presented in this thesis, a number of different aspects of kalilo senescence were examined to obtain preliminary information on this system. The focus of this thesis is on the characterization of the sexual and somatic transmission of mtlS-kalDNA. The main objectives of this study were: 1) To determine if a correlation exists between the appearance of mtlS-kalDNA arid longevity. For genetically related ascospores, the time of growth cessation ranges from less than 10 to over 20 subcultures (Griffiths and Bertrand, 1984). Introduction / 37 It is suspected that this variability is determined by the behaviour of ka lDNA. This was investigated by following the somatic transmission of mtlS-kalDNA in senescent ascospore series. The time of appearance of mtlS-kalDNA was determined and a correlation analysis performed using the time of appearance of mtlS-kalDNA as one variable and the subcultures remaining in a series after the appearance of mtlS-kalDNA as the other variable. 2) Tetrad analysis was performed to decide whether the appearance of mtlS-kalDNA relies on a particular host nuclear background. This was achieved by comparing of the somatic transmission patterns of mtlS-kalDNA between ascospores derived from crosses between two Kauaian strains and ascospores derived from outcrossing. 3) To determine if the sexual transmission of mtlS-kalDNA affects the somatic behaviour of mtlS-kalDNA. Ascospores from a cross using a presenescent subculture of the female strain P561 generally live longer than those derived from a cross using a senescent subculture of strain P561 (Griffiths and Bertrand, 1984). The sexual and somatic transmission of mtlS-kalDNA was characterized using ascospores initiated from crosses using a female parent sampled in both a presenesent and senescent state. 4) The strain P573 which shows some by not all characteristics kalilo senescence was investigated to determine if the sexual and somatic transmission of mtlS-kalDNA is similar with other senescent Kauaian strains. Introduction / 38 5) A survey for the presence of dsRNA was undertaken to determine if ka lDNA has a viral origin. The survey included a number of Kauaian senescent and nonsenescent strains as well as strains from other geographic locations. Information relating to these five areas of kalilo senescence is presented in the four chapters to follow. II. MATERIALS AND METHODS 1. Strains Neurospora intermedia strains P561, P573, and P605 are natural isolates collected from Kauai, Hawaii. Strains 1766 and 1818 were isolated from Taiwan, and strain 1836 from Indonesia. Other strains used are listed in Table 1 of Chapter 4. A l l strains were collected by Dr. D.D. Perkins. Kauaian strains were obtained directly from Dr. Perkins and all other strains from the Fungal Genetics Stock Centre, Department of Microbiology, University of Kansas Medical School, Kansas City, Kansas. Ascospores described in Chapters 1 and 3 were initiated from crosses described by Griffiths and Bertrand (1984). 2. Media and Growth Conditions Vegetative culturing was performed exclusively on Vogel's minimal medium containing 2% glucose (Vogel, 1956). Serial subcultures were made in 10 X 75mm tubes. Each series was subcultured by mass conidial transfer once or twice a week. For the growth of mycelium for nucleic acid isolation and cytochrome analysis, liquid Vogel's medium was inoculated with approximately 10 6 conidia/ml and shaken at 200rpm for a minimum of 16 hours. Crosses were performed on solidified Westergaard's crossing medium as described by Davis and deSerres (1970). Cross designations are such that the strain used as the female parent is written first and followed by the strain used as the conidial parent. Mating type determination of ascospores was performed on crossing medium. Vegetative cultures and crosses were incubated at 25C. 39 Materials and Methods / 40 Subculturing was performed as described by Griffiths and Bertrand (1984). In subculture series derived from natural isolates, the original culture is number zero, for example 561-0. The same applies for ascospore series except that the ascospore isolation number is used in 'place of a strain designation, for example 4-0. Serial subcultures were then numbered -1, -2, -3,....-n, for example 561-1, 561-2, 561-3, .... 561-n, or 4-1, 4-2, 4-3, .... 4-n. Conidial isolation for inoculation of liquid Vogel's was prepared by pouring conidial suspensions through four layers of cheese cloth. The appropriate volume of this suspension was then added to liquid Vogel's medium. Cultures were harvested by suction filtration and stored on ice until needed. Unordered asci were collected using the procedure of Newcombe and Griffiths (1972) slightly modified for use with crosses on solid medium. A l l other procedures were standard for Neurospora and are described by Davis and deSerres (1970). 3. Nucleic Acid Isolations a. Mitochondrial DNA Isolation Two different protocols were employed for mtDNA preparation. The large scale preparation, described by Bertrand et al (1985), requires a minimum of six litres of liquid culture. The harvested mycelium, in isolation buffer (44mM Sucrose, Materials and Methods / 41 50mM Tris-HCl, pH 7.6, I m M EDTA), was initially ground in a blender and then poured through a mill to completely disrupt the cell walls. The suspension was centrifuged at 3000rpm for 10 minutes in a G S A rotor to pellet cell debris. The supernatant was then centrifuged for 30 minutes at 10,000rpm in an SS-34 rotor to pellet the mitochondria. The isolation buffer was decanted and the small amount of liquid remaining in the tube pipetted out and saved for cytochrome analysis. The pellet was suspended in 60% sucrose (Ultra pure sucrose in lOmM Tris-HCl, pH 7.6 and O. lmM EDTA) and overlayed with 55% sucrose and then 44% sucrose (both consisted of Ultra pure sucrose in lOmM Tris-HCl, pH 7.6 and O . l mM EDTA) (Lambowitz, 1979). The step gradients were centrifuged at 25,000rpm for 2 hours in an SW27 rotor. The mitochondrial band was removed from between the 44% and 55% sucrose layers, diluted with isolation buffer, and centrifuged at 15,000rpm for 15 minutes in an SS-34 rotor. The supernatant was discarded and the pellet suspended in 200mM Tris-HCl, pH 8.0 and I m M E D T A . One tenth volume of 20% SDS was added to lyse the mitochondria. Extraction involved adding a half volume of Tris-HCl saturated phenol and a half volume of chloroform-amyl alcohol (24:1). Tubes were inverted gently and then centrifuged at 10,000rpm for 10 minutes in an SS-34 rotor at 25C. The aqueous phase was removed and dialyzed overnight against lOmM Tris-HCl, pH 8.0 and I m M E D T A . cesium chloride (0.8gm/ml) and 9ul/ml of bis-benzimide (lmg/ml in 50% ethanol) were added to the dialysate. The mixture was centrifuged at 53,000rpm for a minimum of 20 hours in an 80Ti rotor. The mtDNA band was illuminated and collected under shortwave U V light. The mtDNA suspension was diluted three fold with lOmM Tris-HCl, pH 8.0 and I m M E D T A and the mtDNA precipitated with 2.5 volumes of ethanol containing 0.2M ammonium Materials and Methods / 42 acetate. Small scale mtDNA preparations (Myers et al, 1988b) required 200mls of liquid culture. Harvested mycelium was ground with acid washed sand and suspended in isolation buffer. The mitochondrial pellet was suspended in 70% sucrose (Ultra pure sucrose in lOmM Tris-HCl, Ph 7.6 and O. lmM EDTA) and layered with 44% sucrose. The flotation gradients were centrifuged at 45,000rpm for 1 hour in an SW50.1 rotor. Mitochondria were collected from the interface of the two step gradient and diluted with 200mM Tris-HCl pH 7.6 and I m M E D T A to fill two microcentrifuge tubes. The tubes were centrifuged for 15 minutes in a microcentrifuge to pellet the mitochondria. The supernatant was discarded and the tubes drained. The mitochondria were suspended in 200mM Tris-HCl, pH 7.6 and I m M E D T A and pooled into one microcentrifuge tube. One tenth volume of 10% SDS was added to lyse the mitochondria. Extraction involved addition of a half volume of Tris-HCl saturated phenol and a half volume of chloroform-amyl alcohol (24:1). The tubes were inverted and centrifuged for 15 minutes. Approximately 2.5 volumes of ethanol containing 0.2M ammonium acetate was added to the aqueous phase to precipitate the mtDNA. The precipitated mtDNA was suspended in 40ul of lOmM Tris-HCl, pH 8.0 and I m M E D T A , treated with RNase A (final concentration of 0.08 ug/ml), and incubated at 55C for 30-60 minutes. The reaction mixture was extracted once with one half volume of 1M Tris-HCl saturated phenol and one half volume chloroform-amyl alchohol (24:1) and the aqueous phase precipitated with 2.5 volumes ethanol containing 0.2M ammonium acetate. Materials and Methods / 43 b. Nuclear DNA Isolation NucDNA preparation was performed as described by Collins et al (1981). From large scale mitochondrial isolations, approximately 1/20 of the harvested mycelium was set aside for nucDNA preparation. The mycelium was ground with acid washed sand and suspended in A l buffer (1M sorbitol, 7% ficoll 400, 20% glycerol, 0.25M EDTA, 0.5% Triton X-100). The suspension was centrifuged at 500rpm for 5 minutes in an SS-34 rotor to remove cell debris. The supernatant was discarded and the nuclear pellet suspended in lOmM Tris-HCl, pH 7.6, I m M E D T A , and 200mM NaCl . Approximately 25ul/ml of 10% Triton X-100 was added to the nuclear suspension and heated to 60C for 10 minutes. One hundred ul/ml of Proteinase K (4mg/ml) was added and the nuclei incubated at 37C for a minimum of 6 hours. One tenth volume of 20% SDS was added to lyse the nuclei. About 0.8 volumes of isopropanol was added and let stand for 10 minutes at 25C to precipitate the nucDNA. NucDNA was pelleted at 10,000rpm for 10 minutes in an SS-34 rotor. The pellet was resuspended in lOmM Tris-HCl, pH 8.0 and I m M E D T A and extracted with a half volume of Tris-HCl saturated phenol and a half volume of chloroform-amyl alcohol (24:1). Tubes were inverted and centrifuged at 10,000 rpm for 10 minutes in an SS-34 rotor. The aqueous phase was precipitated with 0.8 volumes of isopropanol , centrifuged at 10,000rpm for 10 minutes, and resuspended in lOmM Tris-HCl , pH 8.0 and I m M E D T A . cesium chloride (0.8gm/ml) and 9ul/ml of bis-benzimide (lmg/ml in 50% ethanol) was added. The mixture was centrifuged at 53,000rpm for a minimum of 20 hours in an 80Ti rotor. The nucDNA band was collected under shortwave U V illumination. Three volumes of lOmM Tris-HCl, pH 8.0 and I m M Materials and Methods / 44 E D T A was added and the nucDNA precipitated in 2.5 volumes of ethanol containing 0.2M ammonium acetate. c. dsRNA Isolation Preparations are as described by Myers et al (1988a). Approximately 20gm of mycelium was powdered in liquid Nitrogen. Forty mis of 2X STE (1X= 50mM Tris-HCl, lOOmM NaCl, I m M E D T A pH7.1) , 0.4ml of B-mercaptoethanol, 10ml of 10% SDS, and 30ml of STE saturated phenol were added to the powdered tissue. The mixture was shaken vigorously for 30 minutes at 25C. After centrifugation at 10,000rpm for 20 minutes in a GSA rotor the aqueous phase was collected and adjusted to 16% ethanol and poured onto a column of 2.5gm of CF-11 cellulose powder. The column was washed with 80ml of 16% ethanol-STE buffer (STE containing 16% ethanol v/v) and the nucleic acid eluted with 15mls of STE. The eluate was T l RNase digested (lU/ml) for 30 minutes and then DNase digested (lU/ml) for 30 minutes at 37C. Approximately 0.2gm of Cellex N - l was added and shaken at room temperature for 20 minutes. The mixture was poured onto a column, washed with 20ml of 20% ethanol-STE and the dsRNA eluted with 1.2ml of STE. The dsRNA eluate was precipitated with 1/10 volume of 3M Sodium Acetate and 2.5 volumes of ethanol. The eluate from the 80ml wash of the CF-11 column described in the previous protocol was precipited with 2.5 volumes of ethanol and 0.2M ammonium acetate to precipitate the nucleic acid. The nucleic acid was resuspended in lOmM Tris-HCl and ImM E D T A and cesium chloride (0.8 gm/ml) and approximately Materials and Methods / 45 3ul/ml of ethidium bromide (lmg/ml in distilled water) were added. The mixture was centrifuged at 53,000rpm for a minimum of 20 hours in a 80Ti rotor. Total cell D N A was collected under shortwave U V light, diluted with one volume of lOmM Tris-HCl, pH 8.0 and I m M E D T A , and the ethidium bromide extracted three times with NaCl and water saturated isopropanol. The D N A was then diluted with two more volumes of lOmM Tris-HCl, pH 8.0 and I m M E D T A , and precipitated with 2.5 volumes ethanol containing 0.2M ammonium acetate. 4. Cytochrome Analysis Cytochrome spectra were obtained as described by Nargang et al (1978). The mitochondria set aside from the mtDNA preparations were diluted in isolation buffer and centrifuged at 10,000rpm for 15 minutes in an SS-34 rotor. The pellet was resuspended in 3ml of 2% (w/v) deoxycholate (in lOmM Tris-HCl , pH 7.2 and 5mM EDTA) to clarify the solution. A few crystals of potassium ferricyanide were added to oxidize the sample. The solutions were centrifuged in a microcentrifuge for 5 minutes to remove debris. A few crystals of sodium thiosulphate were then added to reduce the sample and spectra taken. The spectra were run from 610 to 500nm at the appropriate optical density (OD) on a Cary 200 spectrophotometer. 5. Restriction Enzyme Digestion and Gel Electrophoresis Digestion of D N A was standard as described by Boehringer Mannheim. Digestions of 2ug of mtDNA, 3ug of nucDNA, and lOug of genomic D N A were carried out Materials and Methods / 46 for 3 hours at 37C. Digestions were then heated to 60C for 10 minutes and loading buffer (5% SDS, 25% glycerol, and 0.025% bromophenol blue) was added to make a final volume of 30ul. Samples were loaded into wells of 0.8% agarose gels and separated by size at 50 volts for 15 hours. The buffer for gel electrophoresis was I X T B E (0.081M Tris base, 0.089M boric acid, 0.002M E D T A , Maniatis et al, 1982). Gels were stained with ethidium bromide and photographed under shortwave U V illumination. DsRNA was separated according to size by electrophoresis in 1% agarose minigels run at 40 volts for 2-3 hours. The buffer for gel electrophoresis was I X T A E (50X T A E = 2M Tris base, 1M glacial acetic acid, 0.1M EDTA) (Maniatis et al, 1982). Gels were stained and photographed as described above. 6. Labelling of Nucleic Acid a. Nick Translation D N A for use as hybridization probes was labeled by nick translation (Maniatis et al, 1975). Typically, lug of D N A was labeled in 50ul of 50mM Tris-HCl, pH 7.5, 5mM M g C l 2 , 0.05mg/ml BSA, 10ml 0-mercaptoethanoI, 20uM dGTP, 20uM dTTP, 20uM dATP, 1.4uM dCTP, 1.4 uCi/ul alpha 3 2 P dCTP (3000Ci/mM), 0.2mM C a C l 2 , lpg/ul DNase I and 0.4U/ul E . coli D N A polymerase I (Romberg). The reaction mixture was incubated for approximately 90 minutes at 15C. The reaction was terminated by the addition, of three volumes of 1% SDS in lOmM E D T A , containing 25ug carrier D N A . Incorporated labeled nucleotides Materials and Methods / 47 were removed by chromotography on Sephadex G-50 spin columns (Maniatis et al, 1982). Labeled D N A was eluated from the column in l O m M Tris-HCl, pH 8.0 and I m M E D T A . Labeled D N A was denatured by boiling for 10 minutes immediately before use. b. End-labelling of dsRNA Purification of dsRNA was performed by electroeluting the dsRNA into troughs (Maniatis et al, 1982). DsRNA was collected from troughs every 2-3 minutes. The dsRNA eluates were extracted twice with one volume of STE saturated phenol and once with one volume of chloroform, and precipitated with 2.5 volumes ethanol containing 0.2M ammonium acetate. A l l solutions used for end labelling were autoclaved to remove endogenous ribonucleases. DsRNA was end labeled according to the method outlined by Maniatis et al (1982) for blunt end or recessed 5' termini labelling. DsRNA was resuspended in deionized formamide and boiled for 5-10 minutes to fragment the dsRNA. Approximately 0.5ug of dsRNA was labeled in 50ul of lOul 10X Kinase buffer I (0.5M Tris-HCl, pH 7.5, O . l M ^ M g C l j , 50mM dithiothreitol, I m M spermidine, I m M EDTA) 150uCi gamma 3 2 P dATP (3000Ci/mM), and 20U Polynucleotide Kinase. The reaction mixture was incubated from 30 minutes at 37C. The reaction was stopped by adding 2ul of 0.5M E D T A and unincorporated labeled nucleotides removed by chromotography on Sephadex G-50 spin columns. Labeled dsRNA was eluated from the column with I X STE. Labeled dsRNA was denatured by boiling for 10 minutes immediately before use. Materials and Methods / 48 7. Probes A l l mtDNA and kalDNA clones used as probes were generously given to our lab by Dr. H . Bertrand. The location of each clone in the mtDNA is shown in Figure 8 and clones of ka lDNA shown in Figure 9. Purification of the mtplasmid D N A of strain P561 involved isolating the plasmid from cesium chloride gradients. The plasmid has a different buoyant density than the mtDNA and can be isolated free of mtDNA contamination. The mtplasmid D N A was precipitated with 2.5 volumes of ethanol containing 0.2M ammonium acetate. The D N A was resuspended in lOmM Tris-HCl and I m M E D T A and sampled for labelling by nick translation. The glutamate dehydrogenase (am) clone (Kinsey and Rambosek, 1984) of N . crassa was kindly given to our lab by Dr. J . A . Rambosek. 8. Blot Hybridization a. Southern Blot Analysis Southern blot analysis was performed essentially as described by Southern (1975). D N A separated by gel electrophoresis was denatured for 30 minutes in 0.5N N a O H and 1.5M NaCl and then neutralized for 45 minutes in 1M Tris-HCl, pH 8.0, and 3.0M NaCl . D N A was transferred to Genescreen with 2X SSC (1X = 0.15M NaCl , 0.01M sodium citrate, pH 7.0) for 24 hours. After transfer, the filters were baked at 100C for 3 hours. D N A fragments were detected by hybridization to 3 2 P labeled probes. Filters Materials and Methods / 49 Figure 8. A linear restriction map of the majority of the mtDNA from Neurospora intermedia isolated from the island of Kauai. Eco R l , Bgl II, and Hind HI restriction maps as well as a partial Pst I map are shown. The positions of major mitochondrial genes are indicated below the map. The filled in boxes represent the exon regions of each gene and the open boxes the intron regions. The line between the intron and large exon of the large rRNA gene is the S-5 gene. The segments of mtDNA used as probes are shown above the map. M a t e r i a l s and Methods / 50 Materials and Methods / 51 Figure 9. A linear restriction map of the majority of the mtDNA of Neurospora  intermedia isolated from Kauai and map of kalDNA insertion sequences in two regions of the mtDNA. Major mitochondrial genes are shown below the map. The filled in boxes represent the exon regions of each gene and the open boxes the intron regions. The line between the intron and large exon of the large rRNA gene is the S-5 gene. A. KalDNA insertion in the Bgl 11-10 restriction fragment of the mtDNA. Segments of kalDNA and flanking regions of the mtDNA used as probes from this insertion are shown above the map. B. KalDNA insertion in the Bgl 11-12 restriction fragment of the mtDNA. Segments of kalDNA and flanking regions of the mtDNA used as probes from this insertion are shown above the map. A . P i l l - k . I D M A , > E c o R I - E . b g l H 3 I 1 1 1 l b . |b3 | b 4 | b 2 ,4I 4 I p»« 1 . . . 1 P s l l - k . l H i n d i * | 1 6 I We .11b | 3 II 1 | 14 | 1 0 . [ |17| K 2 K 1 is ><\ 10b | « • 4 E c o R I J10 f 4 II 7b 1 5 1 6 | H E I d B 3 o l t -2 • • oli-l Co-J IMHI MM • "• • • • • * I M H M S - i H N A c o 3 ' — L - r R N A " ~~~ • s to rt (T> H H-B. I H l n d l l l - K 1 | | t c o R l - B | B g i n 3 i 1 1 2 | l O b l |b3 | b 4 | b Z M- 4 1 P s t l . 1 r N I I - t l j l , H H i n d HI J _ 8 [ W c . l l b | 3 1 | 14 | 1 0 * f | l 7 | l 6 K 2 | K l H |«. I I . | l 9 E c o R I io H 4 | 7b | 5 1 . 1 H 1 E I G I e 3 " i t - 2 * o i l " c o - 2 " ' S - t H N A cTi P> 3 Cu 3 n> rt o CO N3 1-rRNA" Materials and Methods / 53 were prehybridized for 24 hours at 55C in a solution containing 40% formamide, 1% (v/v) SDS, I X Denhardt's solution (100X= 2% (w/v) BSA, 2% (w/v) polyvinylpyrrolidine, 2% (w/v) ficoll), 1M NaCl , and 0.5mg/ml denatured herring sperm D N A (Bertrand et al, 1985). Hybridizations were carried out in the same buffer with the addition of denatured labeled probe to at least 1 X 10 6cpm/ml. Hybridization was for 48 hours at 55C. After hybridization, blots were washed for 5 minutes in 2X SSC at 25C followed by two 45 minute washes in 2X SSC at 60C. After air drying, blots were wrapped in Saran wrap and exposed to Kodak X-Omat RP film for the appropriate length of time. b. Northern Blot Analysis A l l buffers for Northern blot analysis were autoclaved to destroy endogenous ribonucleases. DsRNA separated by gel electrophoresis was denatured in 0.05N N a O H and 1.5M NaCl for 30 minutes. The gels were then neutralized for 45 minutes in 1M Tris-HCl, pH 7.5 and 3.0M NaCl. R N A was transferred to Genescreen with 10X SSC for 36 hours. After transfer, the filters were baked at 68C for 6 hours. DsRNAs were detected by hybridization to specific probes as described for Southern blots. I H . C H A P T E R 1 A . INTRODUCTION This chapter describes experiments which investigate the transmission of mtlS-kalDNA during vegetative growth. These experiments were undertaken to determine if a correlation exists between the time of insertion of ka lDNA into the mtDNA and the occurrence of death. This is of interest because it has been suggested that the variability in lifespan observed between different senescent natural isolates of Neurospora intermedia and between ascospore progeny initiated from the same cross may depend on the behaviour of kalDNA (Griffiths and Bertrand, 1984; Bertrand et al, 1985). This suggestion was based on the fact that ka lDNA is present only in senescent Kauaian strains (Bertrand et al, 1985). The prediction that insertion of kalDNA into the mtDNA initiates senescence is supported by observations that altered mtDNA from insertion of mtplasmid D N A (Akins et al, 1986), from point mutations (Bertrand and Pittenger, 1972a), from deletions (Bertrand et al, 1980; Devries et al, 1981), from inversions (Infanger and Bertrand, 1986), and from intramolecular recombination (Gross et al, 1984) result in irregular growth patterns of Neurospora. Thus, if insertion of ka lDNA initiates senescence then the time of insertion should correlate with longevity. For example, insertion of ka lDNA into the mtDNA later in vegetative propagation should result in strains with longer lifespans and insertion early in a series should result in shorter lifespans. This model, however, cannot fully explain the varibility in lifespans of ascospores initiated from the same cross. This is because 54 Chapter 1 / 5 5 mtlS-kalDNA is maternally inherited (Bertrand et al, 1986) and ascospores from the same cross usually inherit the same insert in essentially equal copy number (Bertrand et al, 1986). According to the model, the lifespans of these ascospores should be similar since mtlS-kalDNA is present at the onset of vegetative propagation. Thus, variability in lifespan of genetically related ascospores with mtDNA molecules carrying the same mtlS-kalDNA indicates that the events responsible for the initiation of senescence are more complex than the model suggests. This chapter reports that the variability in lifespan between ascospores isolated from the same cross is dependent on the generation of specific defective mtDNAs and their subsequent accumulation. Interestingly, it is not the inherited mtlS-kalDNA that is responsible for suppressive accumulation, but rather the variability in lifespan is due to new insertions of ka lDNA into the mtDNA at different times during growth. The insert transmitted sexually is referred to as neutral because in none of the ascospore series does it initiate senescence. It was determined that the novel inserts originate from movement of ka lDNA rather than from rearrangement of the mtDNA encompassing the inherited insert. In addition, a third form of kalDNA has been identified, denoted as mtFF-kalDNA, which may be an intermediate in the movement of ka lDNA. B. RESULTS Subculture series were derived from randomly isolated ascospores from crosses shown in Figure 10. Ascospore series which survived for more than 10 Chapter 1 / 56 subcultures were chosen for analysis. The lengths of the series described in this chapter are shown diagrammatically in Figure 10. It can be seen from Figure 10 that the time of growth cessation for all the series ranges from 12 subcultures to no expression of senescence even after 80 subcultures. To determine whether longevity correlates with the time of insertion of kalDNA, mtDNA was isolated from various subcultures from each ascospore series. The mtDNA was digested with Bgl II and inserts detected by autoradiography using a Pst I-kalDNA probe. There are no Pst I sites in ka lDNA (refer to the restriction map of ka lDNA, Figure 7) so the clone consisted of the entire inserted element together with the flanking segments of the mtDNA. Analysis of Bgl II digested mtDNA for the presence of ka lDNA insertion was preferred because Bgl II digestion of mtlS-kalDNA generates two ka lDNA/mtDNA junction fragments which give the most information on the relative location of an insert and the number of different inserts in the mtDNA of a given strain. The strain P605 was used as a nonsenescent control in these experiments. Bgl 11 digestion of the mtDNA of this strain gives 15 restriction fragments. This is typical of all nonsenescent Kauaian strains. A mtDNA profile for this strain is shown in Figure 11, lane 1 of the ethidium bromide stained gel. Hybridization using the Pst I-kalDNA probe identifies two Bgl II restriction fragments (Figure 11, lane 1 of the autoradiography. These are the Bgl 11-10 and -12 mtDNA restriction fragments which are homologous with the mtDNA sequences associated with the probe. Senescent Kauaian strains are distinquished from nonsenescent Kauaian strains by the presence of additional Bgl II restriction fragments. Most Chapter 1 / 5 7 of these novel bands are Bgl II restriction fragments associated with kalDNA. The ka lDNA restriction map diagrammed in Figure 7 shows that there are three Bgl II restriction sites in ka lDNA and when inserted into the mtDNA four unique Bgl II restriction fragments are created. In Figure 7, the fragments labeled b l and b2 consist of both ka lDNA and mtDNA sequences. The size of b l and b2 vary depending on where kalDNA is inserted into the mtDNA. The internal Bgl II restriction fragments are designated b3 and b4 and remain constant in size in Bgl II digestions. A n example of a Bgl II digestion of the mtDNA prepared from a senescent strain (P561) is shown in Figure 11, lane 2 of the ethidium bromide stained gel. In addition to the 15 normal Bgl II mtDNA restriction fragments, seven unique Bgl II restriction fragments are observed. The two identified by the arrows are the Bgl II restriction fragments of a plasmid harbored in the mitochondria of strain P561 (Bertrand et al, 1985). Hybridization of the Pst I-kalDNA probe to the mtDNA prepared from strain P561 identifies the five other unique Bgl II bands (Figure 11, lane 2 of the autoradiograph). It should be noticed that a total of six unique bands hybridize with the Pst I-kalDNA but only five are visible because the normal Bgl 11-12 restriction fragment and the internal ka lDNA Bgl II fragment b4 comigrate (Bertrand et al, 1985). The other five novel Bgl II restriction fragments include: the internal ka lDNA Bgl II fragment, b3; and the four higher molecular weight fragments which constitute junction fragments of two different inserts in the mtDNA (Bertrand et al, 1985). These junctions fragments are designated b l and b2, and b l ' and b2'. Chapter 1 / 5 8 1. Transmission of mtlS-kalDNA in Ascospores Initiated from Cross 561-1 X 1766 The ascospores described in this section were initiated from a cross using strain P561 as the female parent. A juvenile subculture of this strain was crossed to the nonsenescent strain 1766 (Griffiths and Bertrand, 1984). Twenty ascospores were isolated and subjected to serial subculturing. Seven of these series lived beyond 10 subcultures and were chosen for analysis. The gels presented in this section include mtDNA Bgl II profiles of both the nonsenescent control, strain P605, and the senescent female parent, P561-1. As described above, strain P561 has two ka lDNA insertions in the mtDNA. The location of both inserts is within the intron of the large r R N A gene. This observation is based on the hybridization of all four junction fragments (bl and b2, and b l ' and b2') with a probe of the intron sequences of the large r R N A gene, Hind 111-13,18 (Figure 24, lane 2 of the autoradiograph using the Hind 111-13,18 clone as a probe). Refer to Figure 8 for the location of the Hind 111-13,18 region of the mtDNA. The Hind 111-13,18 probe also hybridizes with the normal Bgl II-4, -12, and -14 mtDNA restriction fragments. The equimolarity of the b l and b2 fragments and the equimolarity of the b l ' and b2' fragments in the female parent (Figure 11, lane 2 of the autoradiograph) as well as the observation that usually only the b l ' and b2' novel restriction fragments are observed in Bgl II digests of mtDNA prepared from ascospores (details below) indicate that b l and b2 are junction fragments of Chapter 1 / 5 9 one of the ka lDNA inserts and b l ' and b2' are junction fragments of the other insert. The Bgl 11-12 and -14 mtDNA restriction fragments constitute the majority of the intron of the large rRNA gene (Bertrand et al, 1985) and based on the sizes of these two restriction fragments together with the sizes of the junction fragments b l and b2, and b l ' and b2' it is suspected that the insert with junction fragments b l and b2 is within the Bgl 11-12 fragment and the insert with junction fragments b l ' and b2' within the Bgl 11-14 fragment. Futhermore, the different levels of intensity of radioactivity of the b l and b2 junction fragments compared with that of the b l ' and b2' junction fragments indicates that each insert is in a different mtDNA molecule. The low copy number of the two inserts relative to the mtDNA (Figure 11, lane 2 of the ethidium bromide gel) suggests that in addition to mtDNA molecules carrying an insert, normal m t D N A molecules are present. This indicates that the mtDNA population of subculture 1 of strain P561 is heterogeneous consisting of a minimum of three different mtDNA types: mtDNA molecules carrying the insert with junction fragments b l and b2; mtDNA molecules carrying the insert with junction fragments b l ' and b2'; and normal mtDNA molecules. Mitochondrial D N A prepared from various subcultures for each of the seven long lived ascospore-derived series (4, 7, 8, 12, 13, 14, and 16) are represented in Figures 11, 13, 15, 17, 18, 20, and 22, respectively. The sexual and somatic transmission of mtlS-kalDNA as it relates to each series is described below. ascospore series 4 from cross 561-1 X 1766 Chapter 1 / 60 The mtDNA prepared from the first subculture of this series shows that only the insert with junction fragments b l ' and b2' was transmitted from the female parent (Figure 11). This insert is never observed to accumulate yet it is maintained in low copy number throughout the entire series. By subculture 6 two novel Bgl II restriction fragments, showing homology with ka lDNA, are observed (Figure 11). These bands are designated by the symbols f l and f2. F l and f2 are observed through to subculture 12 and are then undetectable. These two unique bands are described in more detail in the section entitled 'Movement of mtlS-kalDNA and Identification of a Transient Mitochondrial Autonomous Form of ka lDNA' . Two more novel Bgl II restriction fragments are detected in subculture 12 and are distinquished from f l and f2 by having a slower mobility. These bands are designated b l 1 and b 2 1 . It is suspected that these two bands are junction fragments of an insert in this ascospore. In order to determine if the novel fragments are homologous with ka lDNA, mtDNA, or both sequences of the Pst I-kalDNA probe, hybridizations using the Hind 111-13,18 and a Hind 111-14,15 probe (see Figure 8 for the location of these regions of the mtDNA) were performed. These probes were chosen for this hybridization because together they constitute the same mtDNA sequences as those associated with the Pst I-kalDNA clone. The absence of hybridization of both the Hind 111-13,18 and Hind 111-14,15 probes with the novel b l 1 and b 2 1 bands (Figure 24) indicates that these novel fragments are homologous with kalDNA sequences of the Pst I-kalDNA probe and constitute junction fragments of an insert located in a region of the mtDNA other than in the intron of the large r R N A gene. If this insert had originated from a rearrangement of the inherited insert then intron sequences would be associated with the junction fragments of the insertion generated during vegetative growth. Chapter 1 / 6 1 To determine the relative location of this insert, hybridizations using various cloned regions of the mtDNA as probes were performed. The slow mobility of junction fragments b l 1 and b2 1 indicate that the insert must be within one of the large Bgl II restriction fragments of the mtDNA. The mtDNA was probed with the Hind III- l i restriction fragment. This restriction fragment spans both the Bgl II-1 and -2 restriction fragments. The probe hybridized with the junction fragments (Figure 12). Absence of hybridization at the position of the normal Bgl II- 1 and -2 restriction fragments indicates that both have been altered. The Hind III- l i probe does not detect any new Bgl II restriction fragments suggesting that the Bgl II-1 and -2 fragments have either been deleted or are associated with the junction fragments. Subclones of the Hind I l l - l i clone were used to locate the site of insertion. The probe Eco R l - 6 i showed homology with both junction fragments (Figure 12). The D N A of this clone is located within the Bgl II-1 restriction fragment near the restriction site delineating the Bgl II-1 and -2 restriction fragments. Insertion into the Bgl II-1 fragment near the restriction site delineating the Bgl II-1 and -2 restriction fragments would generate ka lDNA/mtDNA Bgl II junction fragments which are very different in mobility. The slow mobility and similar sizes of b l 1 and b2 ' suggest that both the Bgl II-1 and -2 fragments may be associated with the junction fragments. This region of the mtDNA contains a number of tRNA genes and the small r R N A gene. It should be noted that both the Hind III- l i and Eco R l - 6 i probes hybridize with lower molecular weight bands. The probes hybridize with these same Bgl II fragments in the mtDNA prepared from the nonsenescent control indicating that Chapter 1 / 62 there is cross homology of these mtDNA probes with other regions of the mtDNA. The Hind I l l - l i probe cross hybridizes with the Bgl II-4 mtDNA restriction fragment, and both the Hind I l l - l i and Eco R l - 6 i probes cross hybridize with the Bgl II-7 restriction fragment. ascospore series 7 from cross 561-1 X 1766 The mtDNA from the earliest subculture of this series shows that only the insert with junction fragments b l ' and b2' was transmitted from the female parent (Figure 13). This insert is initially observed in essentially equal copy number relative to the mtDNA. By subculture 4, there is a reduction in the amount of this insert and it is maintained at this level for the remainder of the series. By subculture 8, two unique Bgl II bands designated b l 2 and b 2 2 hybridizing with the Pst I-kalDNA probe are observed (Figure 13). Two more unique Bgl II fragments are observed in subculture 11 (Figure 24). Subculture 11 was not included in the series represented in Figure 13 because this subculture would not grow enough to prepare more mtDNA. The bands b l 2 and b 2 2 and b l 3 and b 2 3 are probably junction fragments of two different inserts. To determine if these novel fragments are homologous with the kalDNA, mtDNA, or both sequences of the Pst I-kalDNA probe, hybridizations using the Hind 111-13,18 and Hind 111-14,15 probes were performed. Together these probes consist of the mtDNA sequences associated with the Pst I-kalDNA probe. The absence of hybridization of either the Hind 111-13,18 or Hind 111-14,15 probes to the b l 2 and b 2 2 bands (Figure 24) 'indicates that these novel fragments are homologous with kalDNA sequences of the Pst I-kalDNA probe and constitute junction Chapter 1 / 63 fragments of a novel insertion into a region of the mtDNA other than the intron of the large r R N A gene. The sizes of the bands designated b l 3 and b 2 3 and hybridization of these bands with the Hind 111-13,18 probe (Figure 24) indicate that these bands are probably junction fragments of an insert which has originated from a rearrangement of the inherited insert. If it was a novel insertion into the intron, the junction fragments would have been of a smaller size. The location of the novel insertion with junction fragments b l 2 and b 2 2 is within the Hind III-10b restriction fragment of the mtDNA (Figure 14). The Hind III-10b mtDNA probe is also seen to cross hybridize with the Bgl II-4 restriction fragment of the mtDNA. A lkb difference in size between the b l 2 and b 2 2 junction fragments accounts for the difference in size between the ka lDNA sequences of the junction fragments implying that the insertion occurred near the centre of the Hind III-10b fragment, a region containing the majority of the tRNA genes. ascospore series 8 from cross 561-1 X 1766 The mtDNA prepared from the first subculture of this series reveals that only the insert with junction fragments b l ' and b2' was transmitted sexually (Figure 15). In subculture 1 this insert is essentially equimolar with the mtDNA. The copy number is reduced in subcultures 4 and 6 and increases in copy number by subculture 8. The accumulation of this insert is probably not responsible for inducing senescence because comparison of the mtDNA from subculture 10 Chapter 1 / 64 prepared for Figure 15 with the the mtDNA of subculture 10 prepared for Figure 24 reveals that accumulation of the inherited insert occurs in only the former mtDNA preparation. Two novel Bgl II fragments, designated b l " and b2" , hybridizing with the Pst I-kalDNA probe are first detected in subculture 8. To determine if these bands consist of kalDNA, mtDNA, or both sequences of the Pst I-kalDNA probe, hybridizations using the Hind 111-13,18 and Hind 111-14,15 probes was performed. The absence of hybridization of these probes to either novel fragment (Figure 24) indicates that they are homologous with the kalDNA sequences of the Pst I-kalDNA probe and constitute junction fragments of a novel insertion located in a region of the mtDNA other than the intron of the large r R N A gene. The location of the novel insert is within the Eco R l - 4 i region of the mtDNA (Figure 16). There are no known genes in this region. The Eco R l - 4 i fragment overlaps the Bgl II-1 and -3 fragments and the hybridization of the Eco R l - 4 i probe with the mtDNA shows the Bgl II-3 restriction fragment to be intact and the Bgl II-1 restriction fragment altered. This indicates that insertion occurred in the Bgl II-1 fragment in the region overlapping the Eco R l - 4 i restriction fragment. Insertion into this region of the Bgl II-1 fragment would generate a very high molecular weight junction fragment when mtDNA is cut with Bgl II. This high molecular weight junction fragment together with the other junction fragment would represent approximately 18kb of D N A . Figure 16 shows that neither junction fragment is of a high molecular weight and together they represent about l l k b . This indicates that about 7kb of the Bgl II-1 restriction fragment is not associated with the junction fragments. The absence of new Bgl Chapter 1 / 65 II restriction fragments suggests that the 7kb of D N A has been deleted. The exact region deleted was not determined. The Eco R l - 4 i mtDNA probe also cross hybridizes with the Bgl II-1, -6, and -13 restriction fragments of the mtDNA. ascospore series 12 from cross 561-1 X 1766 Only the insert with junction fragments b l ' and b2' was transmitted sexually (Figure 17). This insert is present in very low copy number and is never observed to accumulate. The mtDNA of this series (Figure 17) together with the mtDNA prepared from subculture 11 (Figure 24) reveals a total of six different novel Bgl II fragments hybridizing with the Pst I-kalDNA probe. M t D N A from subculture 11 was not included in the series (Figure 17) because the culture would not grow enough to prepare more mtDNA. The bands designated b l 5 and b 2 5 are first observed by subculture 8 and lost by subculture 11 (Figures 17 and 24). By subculture 10, two more novel Bgl II fragments denoted b l 6 and b 2 6 are observed (Figure 17). By subculture 11, two more novel bands designated b l 7 and b 2 7 are seen (Figures 24). Hybridizations using the Hind 111-13,18 and Hind 111-14,15 probes were performed to determine whether these six novel bands are homologous with kalDNA, mtDNA, or both sequences of the Pst I-kalDNA probe. Hybridization of the Hind 111-13,18 probe with the novel bands designated b l 7 and b 2 7 (Figure 24) and the loss of the Bgl 11-10 and -12 fragments suggest that an insertion occurred in either the Bgl 11-10 or -12 restriction fragments and a rearrangement including both these fragments occurred. Furthermore, the sizes of the b l 7 and b 2 7 junction fragments together with the sizes of the Bgl 11-10 and -12 mtDNA restriction fragments indicate Chapter 1 / 66 that both mtDNA fragments are associated with the junction fragments. The absence of hybridization of either the Hind 111-13,18 or Hind 111-14,15 probes to the fragments designated b l 5 , b 2 5 , b l 6 , and b 2 6 indicates that these novel bands are homologous with the ka lDNA segments of the Pst I-kalDNA probe and constitute junction fragments of two different novel insertions in regions of the mtDNA other than the intron of the large r R N A gene. The location of the insert with junction fragments b l 5 and b 2 5 and the insert with junction fragments b l 6 and b 2 6 are in the Hind III-14 restriction fragment of the mtDNA (Figure 18). This region contains tRNA genes. This mtDNA probe also cross hybridizes with the mtDNA Bgl II-4 restriction fragment. The second insert with junction fragments b l 6 and b 2 6 (Figure 17) possibly arose by a rearrangement event of the first insert giving rise to an insert with smaller junction fragments. The third insert, with junction fragments b l 7 and b 2 7 , is located in the intron of the large r R N A gene (Figure 24). ascospore series 13 from cross 561-1 X 1766 Analysis of the mtDNA from the first subculture of this series shows that onty the insert with junction fragments b l ' and b2' was transmitted from the female parent strain to this ascospore. This insert is present in low copy number relative to the mtDNA and and is never observed to accumulate. Two novel Bgl II bands, denoted b l 8 and b 2 8 hybridizing with the Pst I-kalDNA probe are first detected in subculture 13 (Figure 19). To determine whether these bands are homologous with kalDNA, mtDNA, or both sequences of the Pst I-kalDNA Chapter 1 / 67 probe, hybridizations using the Hind 111-13,18 and Hind 111-14,15 probes were performed. The absence of hybridization of either probe with these two novel fragments indicates that they are homologous with the ka lDNA sequences of the Pst I-kalDNA probe and are junction fragments of a novel insertion located in a region of the mtDNA other than the intron of the large r R N A gene. It is suspected that the insert is in the Eco Rl-11 restriction fragment. The Eco Rl-11 fragment has not been cloned, but a process of elimination, using mtDNA clones from the majority of the mtDNA, suggests that ka lDNA is most likely inserted into that region. This particular region of the mtDNA does not contain any known genes. The region of the mtDNA encompassing the Bgl II-1 restriction fragment has been deleted as shown by the loss of this fragment and the absence of new Bgl II fragments (Figure 19). Alteration of the Bgl II-3 fragment is expected because ka lDNA is inserted into this region of the mtDNA. The fast mobilities of the junction fragments and the large size of the Bgl II-3 restriction fragment suggests that some of the Bgl II-3 fragment has been deleted. ascospore series 14 from cross 561-1 X 1766 Only the insert with junction fragments b l ' and b2' was transmitted sexually (Figure 20). This insert is essentially equimolar with the mtDNA throughout the entire series. Two novel Bgl II fragments hybridizing with the Pst I-kalDNA probe are observed in subculture 6 and maintained through the rest of the series (junction fragments b l 9 and b2 9 ) (Figure 20). By subculture 10, four more Chapter 1 / 68 unique bands are observed and denoted b l 1 0 and b 2 1 0 , and b l 1 1 and b 2 1 1 (Figure 24). M t D N A prepared from subculture 10 was not included in the series because the culture would not grow enough to prepare more mtDNA. To determine if these novel fragments are homologous with kalDNA, mtDNA, or both sequences of the Pst I-kalDNA probe, hybridizations using the Hind 111-13,18 and Hind 111-14,15 probes were performed. Together, these probes contain the mtDNA sequences associated with the Pst I-kalDNA probe. The absence of hybridization of either probe to any of the novel fragments indicates that they are homologous with the kalDNA sequences of the Pst I-kalDNA probe and constitute junction fragments of three different ka lDNA insertions in regions of the mtDNA other than the intron of the large rRNA gene. The location of the novel insert with junction fragments b l 9 and b 2 9 is in the Hind III-14 restriction fragment (Figure 21). The sizes of the junction fragments are the same as the b l 5 and b 2 5 junction fragments for the insert in the Hind III-14 fragment in ascospore series 12 (Figure 18). This indicates that both inserts are located in the same region of the mtDNA. The other two inserts are located in the Eco RI-6i restriction fragment (Figure 21). The two lowest molecular weight novel bands, designated b l 1 1 and b 2 1 1 , are junction fragments of one insert, and the two higher molecular weight bands, designated b 1 1 0 and b 2 1 0 , are junction fragments of the other insert. The insert with junction fragments b l 1 1 and b2 1 1 is possibly a deletion of the insert with junction fragments b l 1 0 and b 2 1 0 . ascospore series 16 from cross 561-1 X 1766 Chapter 1 / 69 Analysis of the mtDNA prepared from the first subculture of this series indicates that only the insert with junction fragments b l ' and b2' was transmitted from the female (Figure 22). The insert is maintained in low copy number and is never observed to accumulate. By subculture 11 two novel Bgl II bands denoted b l 1 2 and b 2 1 2 are detected which hybridize with the Pst I-kalDNA probe (Figure 22). To determine whether these bands consist of ka lDNA, mtDNA or both sequences of the Pst I-kalDNA probe, hybridizations using the Hind 111-13,18 and Hind 111-14,15 probes were performed. These probes were used because together they constitute the mtDNA sequences associated with the Pst I-kalDNA clone. The absence of hybridization of either probe with the novel Bgl II bands indicates that these bands are homologous with ka lDNA segments of the Pst I-kalDNA probe and are junction fragments of a novel insertion located in a region of the mtDNA other than the intron of the large r R N A gene. The location of this insert is within the Hind III-14 restriction fragment (Figure 23). The mobility of the junction fragments is similar to those for the inserts in the Hind 111-14 region in ascospore series 12 and 14. In summary, the ascospores initiated from cross 561-1 X 1766 inherit only the insert with junction fragments b l ' and b2' from the female parent strain P561. This insert is located in the intron of the large rRNA gene. In all series, this insert does not accumulate and initiate senescence yet is maintained through each series. During growth of each series, novel Bgl II restriction fragments which hybridize with the Pst I-kalDNA probe are identified. These unique bands constitute junction fragments of different kalDNA inserts and the absence of Chapter 1 / 70 hybridization of either the Hind 111-13,18 or Hind 111-14,15 probes to the majority of these junction fragments indicates that each insert represents a novel insertion into the mtDNA and not a rearrangement of the mtDNA encompassing the transmitted insert with junction fragments b l ' and b2'. Finally, in Figure 24, note that the mtDNA segments into which ka lDNA inserts are undetectable in ascospore series 4, 8, 12, 14, and 16. In ascospore 7 the Bgl 11-12 or -14 restriction fragments should not be observed because the insert with junction fragments b l 3 and b 2 3 is located in one of these mtDNA segments. Likewise, in ascospore 14 the Bgl I I - l restriction fragment restriction should not be detectable because two insertions are present in this region of the mtDNA. This indicates that the last subculture from which mtDNA was prepared from each ascospore series contained variable ratios of normal to abnormal mtDNAs. a. Correlation Between Time of kalDNA Insertion and Onset of Senescence The subculture in which novel inserts are first observed varies between series. The later in a subculture series that ka lDNA inserts into the mtDNA the longer the lifespan of that culture. The variables used to determine whether longevity correlates with the time of novel insertion of kalDNA are shown in Table I. Chapter 1 / 7 1 T a b l e 1 . V A R I A B L E S U S E D I N T H E C O R R E L A T I O N A N A L Y S I S . t o t a l s u b c u l t u r e s u b c u l t u r e s l e f t a s c o s p o r e s u b c u l t u r e s o f k a l D N A i n e a c h s e r i e s s e r i e s p r e c e d i n g i n s e r t i o n a f t e r k a l D N A d e a t h i n s e r t i o n 4 2 6 12 14 7 1 5 8 7 8 1 2 8 4 12 1 3 8 5 13 2 7 13 14 14 13 6 7 16 19 11 8 Chapter 1 / 72 The analysis revealed that the time during growth when the ka lDNA insert initiating senescence is first detected is correlated with both the length of a subculture series (correlation coefficient of 0.90, using 99% confidence limits) and the number of subcultures remaining in a series after insertion (correlation coefficient of 0.83, using 95% confidence limits). Thus, the variability in lifespan can be predicted for any one series knowing when in a subculture series the insert initiating senescence is first observed. b. Movement of mtlS-kalDNA and . Identification of a Transient Mitochondrial Autonomous Form of kalDNA In the ascospore series analyzed, ka lDNA inserts initiating senescence appear at various times during vegetative propagation and the sizes of the junction fragments formed after Bgl II digestion in each series are variable (Figure 24). If these senescence-associated insertion events originated through a rearrangement encompassing the inherited parental insert (known to be located in the intron of the large r R N A gene) then intron sequences of the large r R N A gene should be associated with the junction fragments of these inserts. The absence of hybridization of the intron probe with most junction fragments suggests that the senescence-associated inserts have originated from the movement of ka lDNA rather than from rearrangement of the mtDNA encompassing the inherited kalDNA insert. There are two exceptions; two of the four junction fragments of the inserts in each of ascospores 7 and 12 hybridize with the intron probe. In addition to determining that ka lDNA is capable of assuming new locations, a Chapter 1 / 7 3 contains a mitochondrial plasmid (mtplasmid) which is labeled in the figure. The novel bands are of a higher molecular weight than the mtplasmid. Hybridization using an Eco R l - E probe was performed to determine whether these two closely migrating bands show homology with kalDNA. The Eco R l - E clone is an internal region of the ka lDNA element (see Figure 9 for the location of this clone) and identifies D N A consisting of ka lDNA sequences. The autoradiograph shown in Figure 25 (Panel B) shows that both the uncut mtDNA and the two novel bands share homology with kalDNA. Homology between the E probe and uncut mtDNA is expected because of the presence of the inserted form of kalDNA. Hybridization of the E probe with the two novel bands indicates that these bands must contain kalDNA sequences. Hybridizations using radioactively labeled whole mtDNA from the nonsenescent strain P605 and radioactively labeled purified mtplasmid D N A were performed to determine whether mtDNA or the mtplasmid sequences are also associated with the two unique bands. Neither probe hybridized with the novel bands (Figure 25, Panels C and D). Hybridization of the mtplasmid with uncut mtDNA of the series and no hybridization to uncut P605 mtDNA indicates that mtplasmids were probably also retained in the uncut mtDNA. These hybridizations indicate that kalDNA is not inserted into the mtplasmid, neither do the novel bands contain any region of the mtplasmid. To verify that the novel plasmids are within the mitochondrion rather than extramitochondrial contamination, mitochondria were treated with DNase prior to lysis. Lane 1 of Figure 26 is mtDNA from a nonsenescent strain, lane 2 is uncut mtDNA from subculture 8 of the ascospore series 4 which was not DNase Chapter 1 / 74 treated, and lane 3 is uncut mtDNA from the same subculture which was DNase treated before lysis of the mitochondria. Hybridization using the E probe revealed that DNase treatment of mitochondria prior to the lysis of the mitochondria has no affect on the novel bands indicating that the autonomous kalDNA element is within the mitochondrion. To determine whether the mitochondrial autonomous form of ka lDNA and AR-kalDNA (the nucleus-associated linear autonomous form of kalDNA) are of a similar size and structure, uncut nucDNA and mtDNA were separated by electrophoresis and hybridized with the Eco R l - E probe. AR-ka lDNA has a faster mobility relative to the mitochondrial autonomous form (Figure 27). In addition, AR-kalDNA forms a discrete band upon electrophoresis where as two bands, both representing the autonomous mitochondrial element, are observed to migrate from the uncut mtDNA. 2. Transmission of mtlS-kalDNA in Ascospores Initiated From Other Crosses It was of interest to determine if the transmission of mtlS-kalDNA is similar in ascospores initiated from crosses using other senescent Kauaian female strains or whether the results presented are exclusive to ascospores initiated from crosses using strain P561 as a female parent. Chapter 1 / 75 a. Ascospore Series from Cross 801-1 X 1836 A cross was made between the senescent female strain P801 and the nonsenescent male strain 1836 (Griffiths and Bertrand, 1984). A juvenile subculture of a series derived from strain P801 was used in the cross. Twenty ascospores were isolated and subjected to serial subculturing. Of these 20 series only three survived past 10 subcultures. These three series were chosen for examination. The lengths of these series are shown diagrammatically in Figure 10. Ascospore series 7 did not show any discernible signs of senescence over a total of 80 subcultures and the other two series died in 26 subcultures. M t D N A was isolated from the earliest and latest subcultures of each ascospore series. The autoradiographs shown in Figure 28 are the mtDNA preparations hybridized with the Pst I-kalDNA probe. The first two lanes are nonsenescent (P605) and senescent (P561-1) controls. A Bgl II digestion of the female parent is not included in this section because at the time of the mtDNA preparations the original P801 strain could not be cultured for mtDNA preparation. It is predicted that kalDNA is inserted into the intron of the large rRNA gene because ka lDNA is generally inserted into this region in senescent Kauaian strains (Bertrand et al, 1985; 1986). K a l D N A sequences are present in the mtDNA prepared from the early subcultures of each ascospore (Figure 28). The size of the junction fragments are the same as junction fragments b l ' arid b2' of the insert inherited in the ascospore series previously described. In the mtDNA prepared from the late Chapter 1 / 76 cultures of ascospore series 5 and 6 two novel bands hybridizing with the Pst I-kalDNA are observed (Figure 28). Hybridization of these bands with the intron probe is apparent and indicates that these bands are junction fragments of an insert within the intron of the large r R N A gene. The sizes of these two bands are similar to junction fragments b l and b2 of one of the two inserts described in strain P561. Whether this insert originated from novel insertion or was transmitted sexually to these ascospores in very low copy number cannot be determined. M t D N A prepared from subculture 26 of ascospore series 7 shows no detectable novel bands hybridizing with the Pst I-kalDNA probe (Figure 28). The only bands hybridizing are those showing homology with the mtDNA portions of the probe, the Bgl 11-10 and -12 mtDNA restriction fragments. To investigate whether the nucleus-associated form of ka lDNA is also lost in this series, nuclear D N A was prepared from subculture 80, run uncut, and hybridized with the E probe. AR-ka lDNA was not detected in the autoradiograph (Figure 29). b. Ascospore Series from Cross 572-5 X 1818 A cross was made between the senescent female strain P572 and the nonsenescent male strain 1818 (Griffiths and Bertrand, 1984). Subculture 5 of a series derived from strain P572 was used in the cross. Twenty ascospores were isolated and subjected to serial subculturing. The majority of ascospore-derived series exhibited very short lifespans. Only two series lived past 10 subcultures, one of which showed no signs of senescence after 80 subcultures. The senescent Chapter 1 / 7 7 series died in 19 subcultures (Figure 10). These two series were chosen for analysis. A Bgl II digested mtDNA profile of the female parent is shown in Figure 32. One insert is observed in this culture when hybridized with the Pst I-kalDNA probe. Hybridization with the intron probe is apparent and indicates that this insert is within the intron of the large r R N A gene. The sizes of the junction fragments are comparable with junction fragments b l and b2 of one of the two inserts identified in strain P561. In addition to this insertion, there is a reduction in the copy number of the Bgl II-5, -6, -11, and -13 restriction fragments and the presence of a new Bgl II restriction fragment (represented by the arrow). The four Bgl II restriction fragments represent contiguous regions of the mtDNA and form approximately one quarter of the mtDNA (refer to the mtDNA restriction map, Figure 6). This region of the mtDNA contains the cytochrome b apoprotein gene and the cytochrome oxidase sub unit 3 gene. M t D N A was prepared from various subcultures of each of the two series and hybridized with the Pst I-kalDNA probe. A description of the senescence-associated insertion events as they relate to each series is presented below. ascospore series 4 from cross 572-5 X 1818 The mtDNA prepared from the early culture of this ascospore series contains at least six novel Bgl II fragments which hybridize with the Pst I-kalDNA probe (Figure 30). A l l six bands are maintained throughout the entire series. Two of Chapter 1 / 7 8 these bands are of the same size as the junction fragments b l ' and b2' of the insert inherited in all the progeny described. The two unique bands denoted b 1 1 fl and b 2 1 ' (Figure 30) are of similar size to the junction fragments b l 1 and b 2 1 of the insert which accumulates in ascospore 4 from cross 561-1 X 1766 (Figure 12). This indicates that the bands designated b l 1 f l and b 2 1 f t are probably junction fragments of an insert also located in the Eco R l - 6 i region of the mtDNA. The loss of the Bgl II-1 and -2 fragments in ascospore 4 from cross 572-5 X 1818 is in accordance with the location of the insert. To determine if this insert is a novel insertion or originated from a rearrangement of the insert in the intron of the large r R N A gene, a hybridization using the Hind 111-13,18 probe was performed. The absence of hybridization (Figure 32) indicates that this insert is probably a novel insertion into the Eco R l - 6 i region of the mtDNA and does not result from a rearrangement. The novel Bgl II bands denoted b 1 1 5 and b 2 1 5 were not characterized. It is of interest to note that none of the inserts present in ascospore 4 from cross 572-5 X 1818 are detected in the female parent upon hybridization with the Pst I-kalDNA probe (Figure 32). Also, the Bgl II-5, -6, -11, and -13 regions of the mtDNA seen in low copy number in the female parent are equimolar with the rest of the mtDNA in this ascospore. Futhermore, the extra Bgl II restriction fragment seen in the mtDNA prepared from the female parent was not transmitted to this ascospore (Figure 30). ascospore series 13 from cross 572-5 X 1818 Chapter 1 / 79 The mtDNA prepared from an early culture of this ascospore series contains six novel Bgl II fragments showing homology with the Pst I-kalDNA probe (Figure 31). Two of these bands probably constitute the b l ' and b2' junction fragments of the insert transmitted to all progeny analyzed thus far. The two novel bands designated b l 1 6 and b 2 1 6 are of similar size to the insert with junction fragments b l 1 and b2 1 of ascospore 4 from cross 561-1 X 1766 (Figure 12). The two unique Bgl II fragments denoted b l 1 7 and b2 1 7 in ascospore 13 are of a similar size as the junction fragments of an insert in common in the ascospore 12 (junction fragments b l 5 and b 2 5 , Figure 18), 14 (junction fragments b l 9 and b 2 9 , Figure 21), and 16 (junction fragments b l 1 2 and b 2 1 2 , Figure 23) from cross 561-1 X 1766. By subculture 20 no discernible amounts of mtlS-kalDNA are seen in ascospore series 13. Observation of the nuclear D N A prepared from subculture 26 and probed with E reveals that AR-kalDNA is also undetectable (Figure 33). As with ascospore 4 from cross 572-5 X 1766 none of the inserts observed in the early cultures of ascospore series 13 were transmitted from the female parent. In addition, the Bgl II mtDNA restriction fragments seen in lower copy number in the female parent are in normal amounts in the mtDNA of ascospore 13. Also, the novel Bgl II band observed in the female parent (designated by the arrow) was not transmitted to this ascospore. Figure 34 summarizes the locations of the inserts which appear during growth of each ascospore. Inserts accumulating in ascospores 4, 7, 12, 14, and 16, from cross 561-1 X 1766 and ascospore 4 from cross 572-5 X 1818 are all located Chapter 1 / 80 in regions of the mtDNA that contain tRNA genes. It can be noticed that ascospores 12, 14, and 16, from cross 561-1 X 1766 and ascospore 4 from cross 572-5 X 1818 all have an insert in the same region of the Hind 111-14 fragment of the mtDNA. In ascospores 7 and 12 from cross 561-1 X 1766, insertions are also observed within the intron of the large r R N A gene. In ascospore 14 from cross 561-1 X 1766, an additional two insertions are located in the Eco R l - 6 i fragment of the mtDNA. The inserts of ascospores 8 and 13 from cross 561-1 X 1766 are in regions of the mtDNA which contain no known genes. Both these strains carry large deletions encompassing the Bgl II-1 region of the mtDNA. Chapter 1 / 8 1 Figure 10. Subculture series for long ascospore series showing growth cessation. The crosses from which the ascospores were initiated are diagrammed. The ascsopore culture from which each series was derived is labeled 0. The last number spanned by a horizontal bar indicates the subculture which produced no viable conidia. Those series showing no signs of senescence even after 80 subcultures are represented by a bar followed by two dots. C h a p t e r 1 / 82 5 6 1 - 1 9 X 1 7 6 6 •ubcwttwr« u m b t r 0 5 t o 15 20 25 30 • s c o s p o r e 4 7 0 T2 T3 T4 « 5 7 2 - 5 9 X 1818CT s u b c u l t u r e l u m b e r 0 f lO 15 20 • s c o s p o r e 4 13 M M M M M M M M M H M M W i M m • • 8 0 1 - 1 ? X 1 8 3 6 s u b c u l t u r e n u m b e r 10 15 20 25 -30 «aco*pore S 6 7 Chapter 1 / 83 Figure 11. Gel electrophoresis analysis of Bgl II digested mtDNAs from various subcultures of a series derived from ascospore 4 from cross 561-1 X 1766. The first and second lanes of the ethidium bromide stained gel and autoradiograph represent the nonsenescent control P605 and the senescent female parent 561-1, respectively. The numbers above the remaining lanes represent the subcultures of the series from which mtDNA was prepared. The autoradiograph shows the bands hybridizing with the Pst I-kalDNA probe (for details on this probe refer to Figure 9). The bands labeled b l 1 and b2 1 , and fl and f2 are junction fragments of two different inserts of ascospore series 4. The bl ' and b2' bands represent the junction fragments of the inherited insert. The b3 and b4 bands are internal fragments of kalDNA. The bl and b2 bands are junction fragments of the other insert presentin the female strain P561 which was not transmitted to its derivatives. The bands designated 10 and 12 are mtDNA Bgl II restriction fragments. The arrows denote the Bgl II fragments of the mtplasmid. Chapter 1 / 85 Figure 12. Southern hybridization analysis showing the location within the mtDNA of the novel insert in series 4. Three autoradiographs are presented. Hybridization using the Pst I-kalDNA probe (for details on this probe refer to Figure 9) shows the relative positions of junction fragments b l 1 and b2 1 of the insert maintained in this ascospore series. Hybridization of the mtDNA Hind EQ-li probe (for the location of this region of the mtDNA refer to Figure 8) with the two junction fragments and the hybridization with a subclone of the Hind EQ-li clone (Eco Rl-6i) localized the insertion to a region of the mtDNA in the proximity of the Bgl U restriction site delineating the Bgl H- l and -2 restriction fragments. Shown diagrammaticaUy in Figure 34 is the location within the mtDNA of this insert. 6 0 5 561 5 6 1 - 4 - 2 4 6 0 5 5 6 1 5 6 1 - 4 - 2 4 6 0 5 5 6 1 5 6 1 - 4 - 2 4 0 0 Chapter 1 / 8 7 Figure 13. Gel electrophoresis analysis of Bgl II digested mtDNAs from various subcultures of a series derived from ascospore 7 from cross 561-1 X 1766. The first and second lanes of the ethidium bromide stained gel and autoradiograph represent the nonsenescent control P605 and the senescent female parent 561-1, respectively. The numbers above the remaining lanes represent the subcultures of the series from which mtDNA was prepared. The autoradiograph shows the bands hybridizing with the Pst I-kalDNA probe (for details on this probe refer to Figure 9). The bands labeled b l 2 and b2 2 junction fragments of one of the inserts of ascospore series 7. The bl ' and b2' bands represent the junction fragments of the inherited insert. The b3 and b4 bands are internal Bgl II fragments of kalDNA. The bands designated 10 and 12 are mtDNA Bgl H restriction fragments. The arrows denote the Bgl II fragments of the mtplasmid. 8 8 S e r i e s 5 6 1 - 7 E t B r K a l Chapter 1 / 89 Figure 14. Southern hybridization analysis showing the location within the mtDNA of one of novel inserts in series 7. Two autoradiographs are presented. Hybridization using the Pst I-kalDNA probe (for details on this probe refer to Figure 9). shows the relative positions of junction fragments b l 2 and b2 2 of the insert maintained in this ascospore series. Both junction fragments hybridized with the Hind III-7b mtDNA probe (for the location of this region of the mtDNA refer to Figure 8). Shown diagrammatically in Figure 34 is the location within the mtDNA of this insert. The junction fragments designated b l 3 and b2 3 of the other insert in this ascospore series are also shown hybridizing with the Pst I-kalDNA probe. The mtDNA restriction fragment designated 4 is seen to cross-hybridize with the Hind HI-7b probe. 6 0 5 561 561 -7 -11 6 0 5 561 561-7-11 Chapter 1 / 9 1 Figure 15. Gel electrophoresis analysis of Bgl II digested mtDNAs from various subcultures of a series derived from ascospore 8 from cross 561-1 X 1766. The first and second lanes of the ethidium bromide stained gel and autoradiograph represent the nonsenescent control P605 and the senescent female parent 561-1, respectively. The numbers above the remaining lanes represent the subcultures of the series from which mtDNA was prepared. The autoradiograph shows the bands hybridizing with the Pst I-kalDNA probe (for details on this probe refer to Figure 9). The bands labeled b l* and b2* are junction fragments of the novel insert of ascospore series 8. The bl' and b2' bands represent the junction fragments of the inherited insert. The b3 and b4 bands are internal fragments of kalDNA. The bands designated 10 and 12 are mtDNA Bgl II restriction fragments. The arrows denote the Bgl H fragments of the mtplasmid. E t B r K a l Chapter 1 / 93 Figure 16. Southern hybridization analysis showing the location within the mtDNA of the novel insert in series 8. Two autoradiography are presented. Hybridization using the Pst I-kalDNA probe (for details on this probe refer to Figure 9) shows the relative positions of junction fragments b l* and b2* of the insert maintained in this ascospore series. Both junction fragments hybridized with the Eco Rl-41 mtDNA probe (for the location of this region of the mtDNA refer to Figure 8). Shown diagrammatically in Figure 34 is the location in the mtDNA of this insert. The mtDNA restriction fragments designated 1 and 6 are seen to cross-hybridize with the Eco Rl-4i mtDNA probe. 6 0 5 561 5 6 1 - 8 - 8 6 0 5 561 5 6 1 - 8 - 8 Chapter 1 / 95 Figure 17. Gel electrophoresis analysis of Bgl II digested mtDNAs from various subcultures of a series derived from ascospore 12 from cross 561-1 X 1766. The first and second lanes of the ethidium bromide stained gel and autoradiograph represent the nonsenescent control P605 and the senescent female parent 561-1, respectively. The numbers above the remaining lanes represent the subcultures of the series from which mtDNA was prepared. The autoradiograph shows the bands hybridizing with the Pst I-kalDNA probe (for details on this probe refer to Figure 9). The bands labeled b l 5 and b2 5 , and b l 6 and b2 6 are junction fragments of two different inserts of ascospore series 12. The bl' and b2' bands represent the junction fragments of the inherited insert. The b3 and b4 bands are internal fragments of kalDNA. The bands designated 10 and 12 are mtDNA Bgl II restriction fragments. The arrows denote the Bgl II fragments of the mtplasmid. S e r i e s 5 6 1 - 1 2 b 4 / 1 2 E t B r K a l Chapter 1 / 9 7 Figure 18. Southern hybridization analysis showing the location within the mtDNA of two of the novel inserts in series 12. Two autoradiographs are shown. Hybridization using the Pst I-kalDNA probe (for details on this probe refer to Figure 9) shows the relative positions of junction fragments b l 5 and b2 5 , and b l 6 and b2 6 of two of the inserts observed in this ascospore series. All four junction fragments hybridized with the mtDNA Hind HI-12 probe (refer to Figure 8 for the location of this region of the mtDNA). The Bgl D-4 mtDNA restriction is seen to cross-hybridize with the Hind HI-12 probe. Shown diagrammatically in Figure 34 is the location within the mtDNA of each insert. Chapter 1 / 99 Figure 19. Gel electrophoresis analysis of Bgl II digested mtDNAs from various subcultures of a series derived from ascospore 13 from cross 561-1 X 1766. The first and second lanes of the ethidium bromide stained gel and autoradiograph represent the nonsenescent control P605 and the senescent female parent 561-1, respectively. The numbers above the remaining lanes represent the subcultures of the series from which mtDNA was prepared. The autoradiograph shows the bands hybridizing with the Pst I-kalDNA probe (for details on this probe refer to Figure 9). The bands labeled b l 8 and b2 8 are junction fragments of the insert of ascospore series 13. The bl' and b2' bands represent the junction fragments of the inherited insert. The b3 and b4 bands are internal fragments of kalDNA. The bands designated 10 and 12 are mtDNA Bgl II restriction fragments. The arrows denote the Bgl K fragments of the mtplasmid. S e r i e s 5 6 1 - 1 3 m — in — o S 1 7 13 15 21 2 3 2 5 3 5 1 7 1 3 15 21 2 3 2 5 E t B r K a l Chapter 1 / 101 Figure 20. Gel electrophoresis analysis of Bgl II digested mtDNAs from various subcultures of a series derived from ascospore 14 from cross 561-1 X 1766. The first and second lanes of the ethidium bromide stained gel and autoradiograph represent the nonsenescent control P605 and the senescent female parent 561-1, respectively. The numbers above the remaining lanes represent the subcultures of the series from which mtDNA was prepared. The autoradiograph shows the bands hybridizing with the Pst I-kalDNA probe (for details on this probe refer to Figure 9). The novel fragments labeled b l 9 and b2 9 are junction fragments of one of the three different inserts of ascospore series 14. The other two inserts in this series are labeled in Figure 21. The b l ' and b2' bands represent the junction fragments of the inherited insert. The b3 and b4 bands are internal Bgl II fragments of kalDNA. The bands designated 10 and 12 are mtDNA Bgl II restriction fragments. The arrows denote the Bgl II fragments of the mtplasmid. \ryz-S e r i e s 5 6 1 - 1 4 O m 2 4 6 8 2 S 2 4 6 8 I 1 0 * » * - b4/12 EtBr K a l Chapter 1 / 1 0 3 Figure 21. Southern hybridization analysis showing the location within the mtDNA of the novel inserts in series 14. Three autoradiographs are presented. Hybridization using the Pst I-kalDNA probe (for details on this probe refer to Figure 9) shows the relative positions of junction fragments b l 9 and b2 9 , b l 1 0 and b 2 1 0 , and b l 1 1 and b2 1 1 of the three different inserts in this ascospore 6eries. Junction fragments b l 1 0 and b 2 1 0 , and b l 1 1 and b2 1 1 hybridized with the mtDNA Eco Rl-6i probe. The insert with junction fragments b l 9 and b2 9 hybridized with the mtDNA Hind HI-12 probe. Shown diagrammatically in Figure 34 is the location within the mtDNA of each of these inserts. The mtDNA Bgl II-7 mtDNA restriction fragment is seen to cross-hybridize with the Eco Rl-6i probe and the Bgl H-4 mtDNA restriction fragment with the Hind HI-12 probe. Chapter 1 / 105 Figure 22. Gel electrophoresis analysis of Bgl II digested mtDNAs from various subcultures of a series derived from ascospore 16 from cross 561-1 X 1766. The first and second lanes of the ethidium bromide stained gel and autoradiograph represent the nonsenescent control P605 and the senescent female parent 561-1, respectively. The numbers above the remaining lanes represent the subcultures of the series from which mtDNA was prepared. The autoradiograph shows the bands hybridizing with the Pst I-kalDNA probe (for details on this probe refer to Figure 9). The bands labeled b l 1 2 and b 2 1 2 are junction fragments of the insert of ascospore series 16. The bl ' and b2' bands represent the junction fragments of the inherited insert. The b3 and b4 bands are internal fragments of kalDNA. The bands designated 10 and 12 are mtDNA Bgl U restriction fragments. The arrows denote the Bgl II fragments of the mtplasmid. S e r i e s 5 6 1 - 1 6 I D _ ID S S 1 6 8 11 13 15 2 EtBr 6 8 11 13 15 K a l Chapter 1 / 107 Figure 23. Southern hybridization analysis showing the location within the mtDNA of the novel insert in series 16. Two autoradiographs are presented. Hybridization using the Pst 1-kalDNA probe (for details on this probe refer to Figure 9) shows the relative positions of junction fragments b l 1 2 and b 2 1 2 of the insert maintained in this ascospore series. The junction fragments of this insert hybridized with the mtDNA Hind EH-12 probe. Shown diagrammatically in Figure 34 is the location within the mtDNA of this insert. The Bgl D-4 mtDNA restriction fragment is seen to cross-hybridize with the Hind HI-12 probe. 6 0 5 5 6 1 5 6 1 - 1 6 - 1 5 6 0 5 5 6 1 - 1 6 - 1 5 Chapter 1 / 109 Figure 24. Gel electrophoresis analysis of Bgl Ef digested mtDNAs from the late cultures of each ascospore series from cross 561-1 X 1766. The first and second lanes of the ethidium bromide stained gel and autoradiographs represent the nonsenescent control P605 and the senescent female parent 561-1, respectively. The numbers above the remaining lanes represent the ascospore series number followed by the subculture from which mtDNA was prepared. Autoradiographs of the Bgl n digested mtDNA hybridized with the Pst I-kalDNA, Hind 111-13,18, and Hind HI-14,15 are shown (for details on these probes refer to Figures 8 and 9). Bgl H fragments labeled 4, 10, 12, and 14 are mtDNA sequences. Junction fragments designated b l 3 and b2 3 are of an insert in ascospore 7. Junction fragments designated b l 7 and b2 7 are of an insert in ascospore 12. Refer to Figures 12 through 24 for the designations given to the other junction fragments hybridizing with the Pst I-kalDNA probe. The bands labeled b3 and b4 represent the internal Bgl II restriction fragments of kalDNA. K a l H13,18 Chapter 1 / 1 1 1 Figure 25. Gel electrophoresis of uncut mtDNA from the subculture series derived from ascospore 4 showing both the mtplasmid DNA and the autonomous mitochondrial form of kalDNA (mtFF-kalDNA). The first lane of each panel is the nonsenescent control P605 and the second the senescent control P561. A. Ethidium bromide stained gel. B. Hybridization of the uncut mtDNA with the Eco Rl -E probe (Refer to Figure 8 for details on this probe). C. Autoradiograph of uncut mtDNA with radio-actively labeled whole mtDNA prepared from the nonsenescent strain P605. D. Autoradiograph of uncut mtDNA with radioactively labeled mtplasmid DNA isolated from strain P561. Series 561-4 A B C D o w 1 6 8 12 14 16 2Q 22 ° m l 6 8 12 14 16 2022 2 2 1 6 8 12 14 16 20 22 ° S l 6 8 12 1416 2022 I mtDNA mtFF-kal DNA mt plasmid DNA E t B r E m t D N A mt plasmid D N A Chapter 1 / 1 1 3 Figure 26. Gel electrophoresis analysis for the detection of the mitochondrial autonomous form of kalDNA (mtFF-kalDNA) in mtDNA samples isolated from mitochondria which were either DNase treated or not DNase treated prior to lysis. An ethidium bromide stained gel of uncut mtDNA isolated from subculture 8 of ascospore series 4 is presented. Lane 1 of the gel and autoradiograph is of uncut mtDNA isolated from the nonsenescent control P605. The autoradiograph is of uncut mtDNA hybridized with the Eco Rl-E probe (for details on this probe refer to Figure 8). E t B r E Chapter 1 / 1 1 5 Figure 27. Gel electrophoresis analysis of mtDNA and nucDNA prepared from subculture 8 of ascospore series 4 to compare the mobilities of mtFF-kalDNA and AR-kalDNA. An ethidium bromide stained gel of the uncut mtDNA and nucDNA are shown. Lane 1 of both the gel and autoradiograph are of mtDNA prepared from the nonsenescent control P605. The autoradiograph shows the hybridization of the E probe (for details on this probe refer to Figure 8) with mtFF-kalDNA from the mtDNA fraction and with AR-kalDNA from the nucDNA fraction. 605 nucDNA 561-4-8 nucDNA 561-4-8 mtDNA 605 nuc D N A 561-4-8 nucDNA Chapter 1 / 1 1 7 Figure 28. Southern hybridization analysis of Bgl JJ digested mtDNAs from the early and late cultures of ascospores 5, 6, and 7 from cross 801-1 X 1836. The first and second lane of the autoradiograph shown in Panel A are the nonsenescent control P605 and the senescent control P561, respectively (refer to Figure 11 for details on these two controls). In Panel B, only the nonsenescent control P605 is shown (lane 1 of each autoradiograph). The numbers above the remaining lanes represent the ascospore series number followed by the subculture from which mtDNA was prepared. The autoradiographs show the Bgl II fragments hybridizing with the Pst I-kalDNA and Hind EH-13,18 probes (see Figures 8 and 9 for details on these probes). The fragments designated bl and b2 are the junction fragments of the insert seen in the late cultures of series 5 and 6. The bl ' and b2' bands are junction fragments of the inherited insert. The Bgl n 4, 10, 12, and 14 bands are mtDNA restriction fragments. Chapter 1 / 119 Figure 29. Southern hybridization analysis of uncut nucDNA to detect for the presence of AR-kalDNA. The nucDNA was prepared from the late cultures of ascospores 5, 6, and 7 from cross 801-1 X 1836. Above each lane is the ascospore series number followed by the subculture from which nucDNA was isolated. The First lane of the autoradiograph is the nonsenescent control P605. The autoradiograph is of a hybridization using the Eco Rl -E probe (for details on this probe refer to Figure 9). 6 0 5 8 0 1 - 5 - 1 4 8 0 1 - 6 - 1 4 8 0 1 - 7 - 2 6 Chapter 1 / 1 2 1 Figure 30. Gel electrophoresis analysis of Bgl Et digested mtDNAs from various subcultures of a series derived from ascospore 4 from cross 572-5 X 1836. The first and second lanes of the ethidium bromide stained gel and the autoradiograph represent the nonsenescent control P605 and the senescent control P561, respectively. The numbers above the remaining lanes represent the subcultures of the series from which mtDNA was prepared. The autoradiograph shows the bands hybridizing with the Pst I-kalDNA probe (for details on this probe refer to Figure 9). The bands labeled b l 1 * and b2 1 *, and b l 1 5 and b 2 1 5 , and b l ' and b2' are junction fragments of three different inserts of ascospore series 4. The b3 and b4 bands are internal fragments of kalDNA. The bands designated 10 and 12 are mtDNA Bgl II restriction fragments. V2-2-S e r i e s 5 7 2 - 4 E t B r K a l Chapter 1 / 123 Figure 31. Gel electrophoresis analysis of Bgl II digested mtDNAs from various subcultures of a series derived from ascospore 13 from cross 572-5 X 1836. The first and second lanes of the ethidium bromide stained gel and the autoradiograph represent the nonsenescent control P605 and the senescent control P561, respectively. The numbers above the remaining lanes represent the subcultures of the series from which mtDNA was prepared. The autoradiograph shows the bands hybridizing with the Pst I-kalDNA probe (for details on this probe refer to Figure 9). The bands labeled b l 1 6 and b 2 1 6 , and b l 1 7 and b2 1 7 and bl ' and b2' are junction fragments of three different inserts of ascospore series 13. The b3 and b4 bands are internal fragments of kalDNA. The bands designated 10 and 12 are mtDNA Bgl II restriction fragments. Et B r K a l Chapter 1 / 125 Figure 32. Gel electrophoresis analysis of Bgl II digested mtDNAs from the late cultures of two ascospore series from cross 572-5 X 1818. The first and second lanes of the ethidium bromide stained gel and each autoradiograph represent the nonsenescent control P605 and the senescent female parent 572-5, respectively. The numbers above the remaining lanes represent the ascospore series number followed by the subculture from which mtDNA was prepared. Autoradiographs of the Bgl El digested mtDNA hybridized with the Pst I-kalDNA probe (for details on this probe refer to Figure 9) are shown. The junction fragments bl and b2 are of an insert observed in the female parent strain. Junction fragments designated b l 1 * and b2 1 * are of the insert seen in highest copy number in ascospore 4. The bands labeled b3 and b4 represent the internal Bgl II restriction fragments of kalDNA. Bgl II fragments labeled 4, 10, 12, and 14 are mtDNA sequences. 6 0 5 5 7 2 5 7 2 - 4 - 1 6 5 7 2 - 1 3 - 8 9 6 0 5 5 7 2 5 7 2 - 4 - 1 6 5 7 2 - 1 3 - 8 9 6 0 5 5 7 2 5 7 2 - 4 - 1 6 5 7 2 - 1 3 - 8 9 ( T 4^ C O " Chapter 1 / 1 2 7 Figure 33. Southern hybridization analaysis of uncut nucDNA to detect for the presence of AR-kalDNA. NucDNA was prepared from the late cultures of ascospores 4 and 13 of cross 572-5 X 1818. The first lane of the autoradiograph represents nucDNA prepared from the nonsenescent control P605. Hybridization to AR-kalDNA was performed using the Eco Rl-E probe (for details on this probe refer to Figure 9). Chapter 1 / 129 Figure 34. Summary of the locations in the mtDNA of the novel inserts appearing during growth for all the ascospore series analyzed. The restriction map includes Eco R l , Hind HI, and Bgl H digestions and a partial Pst I digestion of the mtDNA. The map was obtained from Bertrand et al (1985; 1986). The numbers above each arrow represent the ascospore series which has an insert in that region of the mtDNA. Below the restriction map is a legend showing the ascospores series which corresponds with each number shown above the restriction map. The junction fragment designations for each insert are shown in the legend. 8 4 10,11 1,15 5 , 6 , 9 , 1 2 3,7,13,14 2 T T • t • • .. T Bgl N 3 I 1 I 2 • . J _ I O _ J N 2 | . 4 | 4 1 P i t 1 y , 1 , ' M | I H i n d i s | I 8 | W c . l l b | 3 | | 1 | 14 | 10a II« |ig{ 4 EcoRI U M II 7b | 5 | « | l 3 •j—m • •> ••••••••«• ••• • olt -2 Oti-I c o - 2 S - rNNA co 3 L r R N A • n rf (D JUNCTION H NUMBER CROSS SERIES FRAGMENTS 1 561 -1 X 1766 4 b l ' , b 2 « 2 7 b l 3 , b 2 J 3 7 b l \ b 2 3 4 '8 b 1 \ b 2 ' 5 12 b l 5 , b 2 5 6 12 b l « . b 2 « 7 12 b l \ b 2 7 8 13 b l \ b 2 « 9 14 b l \ b 2 » 10 14 b l 1 0 , b 2 ' ° 1 1 14 b l ' 1 , b 2 " 12 16 b l ' » , b 2 ' 2 13 801 -1 X 1836 5 b l 1 3 , b 1 , J 14 • 6 bl 1 J , b 1 , J 15 572-5 X 1818 4 b l b l C. DISCUSSION Chapter 1 / 1 3 1 The initial characterization of kalilo senescence was conducted on natural isolates of N . intermedia (Bertrand et al, 1985). In senescent series derived from these natural isolates, the kalDNA insert which initiated senescence was the first and generally the only insert detected. Because these series are derived from natural isolates, it is impossible to determine the history of these strains and consequently impossible to predict the stage of senescence that these strains were in when collected from nature and originally subcultured. Consequently, it is difficult to conclude if the inserts detected in those series are in fact the first inserts or perhaps later inserts which induce senescence. Studying long-lived ascospore progeny provides a more clearly defined origin to a series, and more opportunity to chart the course of senescence. M t D N A analysis of the early cultures of the ascospores revealed that mtlS-kalDNA is present in detectable amounts. In all cultures, the insert designated by the junction fragments b l ' and b2' is identified. In the early cultures of the ascospores from cross 561-1 X 1766, this insert is present in the female parent and it is concluded that this insert is sexually transmitted to the progeny. For the early cultures of the ascospores from cross 801-1 X 1836, it is impossible to determine the origin of this insert since mtDNA could not be prepared from the female parent. It is suspected that the insert with junction fragments b l ' and b2' is present in the 801 female parent and thus transmitted to the progeny. The 572 female parent had no detectable amounts of any of the inserts present in the two progeny analyzed. This may be explained by Chapter 1 / 132 postulating that culturing of the original 572-5 culture for purposes of this work altered the ka lDNA characters of this strain such that the culture prepared for use as the female parent and the culture prepared for mtDNA isolation had different inserts. Alteration of ka lDNA characters as a consequence of subculturing has also been observed in strain P561 (Bertrand et al, 1985; 1986). It should be noticed that the insert with junction fragments b l ' and b2' transmitted to the progeny never initiates senescence in the series studied. This is an important observation because defective mtDNAs usually accumulate and displace normal mtDNA molecules (Bertrand et al 1980; 1982; DeVries et al, 1981; Bertrand et al, 1985; 1986; Lambowitz et al 1987). This insertion is referred to as neutral since it does not initiate senescence. As mentioned, the neutral insert is located within the intron of the large r R N A gene. In strain P561, a second insert, denoted by the junction fragments b l and b2, is also located within the intron of the large r R N A gene (Bertrand et al, 1985). Together these observations indicate that disruption of certain regions of this intron induce senescence. An important region of this intron is the open reading frame which encodes one of the mitochondrial ribosomal proteins, the S-5 gene (Lambowitz, 1979). Although the exact location of each insertion within the intron is unknown, it is postulated that the mutagenic insert is within the S-5 gene and the neutral insert in a functionally unimportant region of this intron. Further studies on these two inserts may give insight into the events required to initiate accumulation of defective mtDNAs and to induce senescence. An important observation is that only the neutral insert (junction fragments b l ' Chapter II 133 and b2') of female strain P561 was transmitted sexually. In strain 561, the insert (junction fragments b l and b2) not transmitted to its derivatives initiates senescence in the female parent strain (Bertrand et al, 1985). Similarly, the insert with junction fragments b l and b2 also initiates senescence in the 572 female parent strain (unpublished results). This insert was not transmitted to the two 572 ascospore progeny analyzed. In addition, strain 572 carried a deletion encompassing the Bgl II-5, -6, -11, and -13 restriction fragments and a novel Bgl II restriction fragment which were not transmitted to either progeny analyzed. Together these results indicate there may be a meiotic salvage process which screens defective mitochondria or mtDNA molecules such that they are not transmitted sexually. It should be noted that mutagenic ka lDNA inserts, for example the insert with junction fragments b l and b2 in strain P561, are usually inherited in ascospores initiated from crosses using a female parent in a later stage of senescence (refer to Chapter 2 for details). During vegetative growth of the 561 and 801 ascospores, novel insertions of mtlS-kalDNA are observed. Analysis of the 561 ascospore-derived series revealed that the point' in each subculture series that novel inserts are detected varies and it is usually the last insert observed that initiates senescence and becomes equimolar with the mtDNA. The point in each series when the insert inducing senescence is detected is proportional to the lifespan of each strain. This correlation further validates the idea that some kind of insertion of kalDNA into the mtDNA is the event required to initiate senescence. It is impossible to determine if the same correlation exists for the two senescent ascospores from cross 801 X 1836 since the mtDNA profile of the female strain P801 was not Chapter II 134 obtainable. However, observations on the insert inherited and the insert which accumulated in these two senescent series indicates that they are different and that the junction fragments of the insert accumulating, in both series, are of the appropriate size to constitute a novel insertion into the intron of the large rRNA gene. This suggests that novel insertion probably occurred during vegetative growth and the time of insertion may be proportional with the longevity of these two series. Ascospores 4 and 13 from cross 572-5 X 1818 are somewhat of an anomaly because high molecular weight fragments hybridizing with the Pst I-kalDNA probe are present in the mtDNA prepared from the early cultures. It is difficult to determine whether these inserts originated from movement during meiosis or perhaps were transmitted to the ascospores from the female parent carrying these inserts in very low copy number. Furthermore, in ascospore 4, the presence of mutagenic inserts in the ascospore culture indicates that the lifespan of the series should be quite short, less than 10 subcultures. In fact this series does not die until subculture 19. It should be noticed that this is the only ascospore of 18 from cross 572-5 X 1818 that dies after a longer lifespan. Ascospore series 13 from this same cross also shows a long lifespan (20 subcultures) before escaping kalilo senescence. Perhaps in these two cultures there is a genetic predisposition for a low rate of accumulation of the defective mtDNA molecules and ultimately the rate of senescence. Investigation of more ascospore-derived long series from this cross might provide information on this anomaly. Chapter II 135 The properties of the novel ka lDNA inserts in all series were investigated. The majority of novel inserts observed in the 561 ascospore-derived series did not hybridize with the probe of the intron of the large rRNA gene. This indicates that these novel insertions originated from movement of ka lDNA rather than from rearrangement of the mtDNA encompassing the inherited insert, which was located in the^ intron of the large r R N A gene. The insert designated by the junction fragments b l 3 and b 2 3 in ascospore 7 from cross 561-1 X 1766 and the insert with junction fragments b l 7 and b 2 7 in ascospore 12 from the same cross hybridize with the intron probe. Based on the sizes of the junction fragments b l 3 and b 2 3 in ascospore series 7, it is predicted that this insert resulted from rearrangement. If it were a novel insert into the intron of the large r R N A gene, the junction fragments would have been smaller in size. The insert with junction fragments b l 7 and b 2 7 in ascospore series 12 is suspected to be a novel insertion into either the Bgl 11-10 or -12 fragment followed by a rearrangement involving these two Bgl II fragments. Although it is known that novel inserts are generated by movement of kalDNA, it is not known whether novel insertion occurs via the inherited mtlS-kalDNA or originates from de novo insertion of AR-ka lDNA. In order to deduce the means by which the element moves, strains which have only mtlS-kalDNA or only AR-kalDNA are required. Transformation experiments are in progress to construct strains of this sort. A t present transformations using either AR-ka lDNA or mtlS-kalDNA as donor D N A have proven unsuccessful. In most instances the donor D N A is degraded before insertion into the mtDNA or before the donor D N A has the opportunity to established it as an autonomous element. Chapter II 136 Incorporation of the Pst I purified ka lDNA fragment into vectors constructed for Neurospora transformation have also proven unsuccessful. In some cases, .transformants are detected which carry only vestiges of ka lDNA as insertions in the nucDNA (Chueng C.K. , Doctoral Student, perssonal communication). It is known that AR-ka lDNA has protein associated with its ends (Chan B-S, Doctoral Student, personal communication) and it is suspected that instability results from the absence of these proteins on the transforming D N A . It is postulated that the only transformation system which will confer stability to the donor D N A will involve in vitro reassociation of these proteins with AR-ka lDNA prior to transformation. A third form of kalDNA has been identified in one series in this chapter. This is the first report of such a form of kalDNA. As shown here, often two sizes are visible. It is located in the mitochondrion and separate from the mtDNA. It shows no homology with , either mtDNA or mtplasmid D N A . This form of ka lDNA is denoted mitochondrial free-form ka lDNA (mtFF-kalDNA). The observation that mtFF-kalDNA is transient and normally seen prior to novel insertion suggests a role as an intermediate in the movement of ka lDNA. This is supported by reports of intermediates involved in the transposition of other mobile elements. For example, the copia element of Drosophila melanogaster, (Flavell and Ish-Horowicz, 1981; Mossie et al, 1985), retroviruses (Varmus, 1983), and bacteriophages such as P2 (Calender et al, 1977),and lambda (Nash et al, 1977). The failure to observe mtFF-kalDNA in all of the present series may be Chapter 1 / 137 explained by postulating that if it is an intermediate in movement it does not have to be present in high copy number for movement to occur. Furthermore, because of the heterogeneity of mtDNAs in this coenocytic fungus, identification of mtFF-kalDNA would not always be possible. Alternatively, the absence of mtFF-kalDNA in most series suggests that the appropriate stage in which movement occurred was not sampled for mtDNA analysis. Since mtFF-kalDNA has been observed in approximately one quarter of mtDNA preparations from different Kauain senescent strains (Dr. A . J . F . Griffiths personal communication; Dr. H . Bertrand, personal communication) it is evidently not an anomaly and deserves to be placed in the general kalilo model. MtFF-ka lDNA has not been characterized but the difference in the mobilities of mtFF-kalDNA and AR-kalDNA suggest that mtFF-kalDNA is structurally different from AR-ka lDNA. The structural differences between these two forms of ka lDNA are unknown. One possiblity is that mtFF-kalDNA is linear. The slower mobility of mtFF-kalDNA, relative to AR-kalDNA, suggests that if mtFF-kalDNA is linear, it consists of more than one copy of ka lDNA. Since the end fragments of ka lDNA, generated after Bgl II digestion, differ by approximately l.Okb, mtFF-kalDNA would have to consist of tandem inverted repeats in order to generate the two high molecular weight fragments (designated f l and f2) observed after Bgl II digestion of mtFF-kalDNA (Figure 11). The presence of two mtFF-kalDNA bands migrating from uncut mtDNA after gel electrophoresis (Figure 27) could be explained by postulating that the difference in sizes depends on the extent of concatamerization. Chapter 1 / 138 An alternative hypothesis is that the element is circular. Circularization would probably involve the pairing of the long inverted repeats. Bgl II digestion of this circle would form the two internal b3 and b4 bands of ka lDNA as well as a high molecular weight fragment consisting of the ends of kalDNA. The presence of two high molecular weight Bgl II fragments (fl and f2; Figure 11) rather than one may be explained by postulating that the pairing and union of the inverted repeats is not accurate thus forming bands of different sizes after Bgl II digestion. This would also explain the presence of two bands migrating from uncut mtDNA (Figure 27). Even though the origin of the novel inserts and the role of mtFF-kalDNA in senescence are unknown, it is proposed that additional mtlS-kalDNA inserts result from movement within mitochondria and that mtFF-kalDNA is an intermediate in movement. This model is based on the observations that in the present studies, mtFF-kalDNA has only been observed when new locations of ka lDNA are seen and that the mtFF-kalDNA was apparently not required for insertion of ka lDNA originating from AR-kalDNA (Bertrand et al, 1986). For ascopores 4, 7, 12, 14, and 16 from cross 561-1 X 1766 and ascospore 4 from cross 572-5 X 1818 the retained novel inserts are located in regions of the mtDNA encompassing tRNA genes suggesting that either the tRNA genes or regions regulating these genes were destroyed. Sequencing of the regions encompassing the inserts will determine the locations of these inserts and what gene or regulator}' function has been disrupted. Chapter II 139 The sites of insertion of ka lDNA in ascospores 4, 7, and 12 from cross 561-1 X 1766, ascopore 4 from cross 572-5 X 1818, and ascospores 5, and 6 from cross 801-1 X 1836 are in regions of the mtDNA where novel insertions have been identified in unpublished work and work described by Bertrand (1987) (see Figure 6). This indicates that insertion may be region specific. Sequence data of ka lDNA/mtDNA junctions has identified pentanucleotide sequences which may be recognition sites for insertion in the mtDNA (Bertrand, 1987). The locations of the kalDNA inserts in ascospores 8 and 13 from cross 561-1 X 1766 are in regions of the mtDNA which do not contain any known genes. In both series, deletions are associated with insertion and the regions of the mtDNA deleted in both strains contain functionally indispensible genes. These genes include the subunits 1 and 2 of the ATPase gene and subunit 2 of the cytochrome oxidase gene. Accumulation of mitochondria with mtDNA molecules carrying these large deletions would account for the loss of growth potential and eventual death of these cultures. Ascospore 13 from cross 572-5 X 1818 and ascospore 7 from cross 801-1 X 1836 appear to have escaped kalilo senescence. Neither AR-kalDNA nor mtlS-kalDNA were detected in later subcultures from either of these series. In senescent subcultures series, resumption of normal growth in cultures which follow a senescent fate is not a commonly observed event (Griffiths and Bertrand, 1984). This phenomenon is referred to as the 'Lazarus Effect' (Griffiths et al, 1986) which in effect means that there is a mixture of normal and abnormal mtDNA molecules present during the senescence process in transfer series and Chapter 1 / 140 sampling of cells in a region containing only normal mitochondria may on occasion result in the resumption of normal growth. Degenerative growth as a consequence of altered mtDNA molecules is common among fungi. In S. cerevisiae (Borst, 1972; Faye et al 1973), P. anserina (reviewed by Cumrnings 1987), A . nidulans (Lazarus et al, 1980; 1981) and Neurospora (Bertrand et al, 1985; 1986), the genetic events responsible for altering mtDNA are different yet the end result is very similar in all fungi. Thus, it would appear that alteration of the mtDNA is the event needed to disable these fungi. However, the causative processes altering the mtDNA are still unknown. In senescent strains of N . intermedia, the event initiating senescence is the insertion of kalDNA into the mtDNA. Although the etiology of the three forms of ka lDNA remain unknown, the information presented here and from Bertrand et al (1986) suggests that the proposed sequence of events ultimately resulting in senescence appear to be that AR-kalDNA gives rise to the first mtlS-kalDNA, which gives rise to mtFF-kalDNA, which results in novel ka lDNA insertions. IV . C H A P T E R 2 A. INTRODUCTION In this chapter tetrad analysis was performed to determine whether the movement of kalDNA is influenced by the host genotype. This research was initiated because ascospore progeny from a cross using geographically unrelated parents showed that mtlS-kalDNA can originate from movement whereas this same type of movement was not observed in ascospores from a cross between two Kauaian strains (561-1 X 605). Ascospores initiated from this cross usually accumulate the sexually transmitted insert or a rearrangment of the inherited insert (Bertrand et al, 1986). In none of the late cultures were novel insertions observed. The series analyzed in the previous chapter were derived from ascospores from the cross 561-1 X 1766, where 1766 is a Taiwanese strain. In all ascospore series studied, novel insertions were apparent. These observations suggest that there may be a host genetic component influencing movement which is seen when the senescent female strain is outcrossed. To further investigate this hypothesis, tetrads were analyzed. The tetrads were isolated from crosses between the senescent female strain P561 and two nonsenescent male strains. The two natural isolates used as the male parents, were strain P605, a Kauaian isolate, and strain 1766, a Taiwanese isolate. If there is a host genetic component influencing movement then segregation of the nuclear gene(s) should be seen in the members of a tetrad. Utilizing tetrads to discern inheritance patterns of nuclear genes is an effective tool because each tetrad is the result of a single meiosis. In the tetrads analyzed, the nuclear gene mating type (alleles _A and _a) 141 Chapter 2 / 1 4 2 was used as one of the markers and the behaviour of mtlS-kalDNA as the other marker. From the results presented it is observed that the frequency of movement is enhanced in series derived from ascospores from cross 561-0 X 1766. It is proposed that movement of mtlS-kalDNA is under the influence of a single gene which is not linked to mating type. In addition, it was of interest to determine whether the sexual and somatic transmission of mtlS-kalDNA is similar in ascospores initiated from crosses where the female parent is in a juvenile state and in a senescent state. It has been observed that the average lifespans of ascospores from crosses using a juvenile female parent generally exceed those of ascospores from crosses using a senescent female parent, and it was suggested that the lifespans depend on the ratio of normal to abnormal mitochondria transmitted sexually (Griffiths and Bertrand, 1984). This ratio should be representative of the ratio of normal to abnormal mitochondria present in the female parent. Since senescent strains accumulate abnormal mitochondria, the ratio of abnormal to normal mitochondria should progressively increase as senescence proceeds such that the more senescent the female parent the shorter the average lifespan of its ascospore derivatives. The results presented indicate that mutagenic ka lDNA inserts are transmitted to ascospores initiated from crosses using a senescent culture (subculture 5) of strain P561 as the female parent. Comparison of the mtDNA from the early and late cultures of these ascospores revealed that the inherited insert(s) accumulated and generally no other inserts were observed. The accumulation of mtlS-kalDNA from the onset of growth of the ascospores would account for the short lifespans of these ascospores. In contrast, ascospores initiated from crosses using a juvenile Chapter 2 / 143 culture of strain P561 as the female parent usually only inherit the neutral or nonmutagenic insert, designated by the junction fragments b l ' and b2'. During growth of these ascospores, either novel insertions or rearrangements of the inherited insert occur and trigger the onset of senescence. The longer average lifespans of these ascospores can be accounted for by postulating that the onset of senescence is delayed until a mutagenic ka lDNA insert is generated sometime during vegetative growth of these ascospores. B. RESULTS 1. Proposed Genetic Regulation of kalDNA Movement To investigate whether the movement of mtlS-kalDNA may be regulated, mtDNA prepared from ascospores of tetrads initiated from crosses 561-0 X 605 and 561-0 X 1766 were compared. A juvenile culture of strain P561 was used as the female parent because the lifespans of its derivatives generally survive for at least 10 subcultures. This allows for more opportunity to study the somatic behaviour of mtlS-kalDNA. One tetrad from cross 561-0 X 605 and two from cross 561-0 X 1766 were examined. The ascospores of each tetrad were subjected to serial subculturing and the length of each series is shown diagrammatically in Figures 35 and 36. The mean average lifespans for each tetrad is included in the Figures. Inspection of the tetrad from the cross 561-0 X 605 reveals that most of the ascospores die within 10 subcultures (Figure 36). Analysis of the tetrads from cross 561-0 X 1766 show the average lifespan of the ascospores of mating type a to be 16.5 subcultures and 11 subcultures for Chapter 2 / 1 4 4 the ascospores of mating type A_ (Figure 36). For each series, mtDNA was isolated from the first subculture and last subculture from which mtDNA could be prepared. The mtDNA was digested with Bgl II and hybridized with the Pst I-kalDNA probe. There are no Pst I restriction sites in kalDNA (refer to the restriction map of kalDNA, Figure 7) so the clone consisted of the entire inserted element together with the flanking segments of the mtDNA. Bgl II digestions were preferred because two ka lDNA/mtDNA junction fragments are formed after Bgl II digestion which give more information on the relative location of a novel insertion and the number of different inserts present in the mtDNA. The first and second lanes of each gel and autoradiograph are the nonsenescent control P605 and the senescent control P561, respectively (Figure 37A, 37B, 38A, 38B, 39 A , and 39B). As described in the previous chapter, the mtDNA of strain P605 has no sequence homology with kalDNA and the only Bgl II fragments hybridizing with the Pst I-kalDNA probe are the normal Bgl 11-10 and -12 fragments (refer to Figure 11, lane 1 of the autoradiograph). In comparison, hybridization of the mtDNA prepared from the senescent control strain P561 identifies additional Bgl II bands (refer to Figure 11, lane 2 of the autoradiograph). These bands include the internal Bgl II fragments of ka lDNA, b3 and b4 (note that b4 comigrates with the normal Bgl 11-12 restriction fragment; Bertrand et al, 1985) and the four higher molecular weight fragments which are junction fragments of two different inserts in this strain. The junction fragments are labeled b l and b2, and b l ' and b2'. Both are in the intron of the large r R N A gene and each inserted into different mtDNA molecules. Refer to chapter 1 for a more detailed description of these inserts. Chapter 2 1 145 A description of the sexual and somatic transmission of mtlS-kalDNA as it relates to each tetrad is presented below. Ascus 1 from Cross 561-0 X 605 In the mtDNA prepared from the early cultures of each ascospore series, only the insert with junction fragments b l ' and b2' was transmitted from the female parent (Figure 37A). In the late cultures of ascospores 1, 2, 3, 4, 5, and 7, the the junction fragments of accumulating inserts are of a different size than junction fragments b l ' and b2' of the inherited insert (Figure 37B). In the late cultures of ascospores 2, 4, 5, and 7, the junction fragments of the inserts accumulating are of the same size. Ascospores 1 and 3 each accumulate inserts with higher molecular weight junction fragments. The junction fragments of ascospores 2, 3, 4, 5, and 7 all hybridize with the intron probe, Hind 111-13,18 (Figure 37B). The sizes of the junction fragments in ascospores 2, 3, 4, 5, and 7 indicate that each insert has probably arisen from a rearrangement event of the transmitted insert and not from novel insertion. If they were novel insertions, the junction fragments would have been of a different size. The junction fragments of ascospore 1 do not hybridize with the intron probe. It is possible that this insert may represent a novel insertion in a region of the mtDNA other than the intron of the large r R N A gene. It is suspected that the two lower molecular fragments in each of ascospores 6 and 8 should hybridize with the intron probe because of the similar sizes to the b l ' and b2' junction fragments. Perhaps the exposure of Chapter 2 / 1 4 6 the autoradiograph was too short to resolve these bands. Further evidence that this may be true comes from the senescent control, lane 2, where the insert with junction fragments b l ' and b2' also fails to show positive hybridization with the intron probe. In ascospore 3, high molecular weight fragments are observed which hybridize with the Pst I-kalDNA probe. Inspection of the ethidium bromide stained gel suggests that the mtDNA prepared from this ascospore was only partially digested with Bgl II. This may account for the presence of the high molecular weight bands which hybridize with the Pst I-kalDNA probe. In ascospore 6, a high molecular weight band is seen which hybridizes with the Pst I-kalDNA probe. The fact that there is only one band suggests that it is probably not a junction fragment of a novel insertion. The significance of this band is unknown. In ascospore 8, there are two high molecular weight fragments which hybridize with the Pst I-kalDNA probe and do not hybridize with the intron probe. It is possible that these may represent junction fragments of a novel insertion. Ascus 7 from Cross 561-0 X 1766 Analysis of the mtDNA prepared from the early cultures of each ascospore series reveals that the insert with the junction fragments b l ' and b2' was transmitted sexually (Figure 38A). In the early cultures of the ascospores of mating type A., high molecular weight fragments are also seen to hybridize with the Pst I-kalDNA probe. Perhaps these are insertions which were also transmitted from the female parent strain. Chapter 2 / 147 Analysis of the mtDNA from the late cultures of the ascospores shows that the junction fragments of the inserts accumulating in ascospores of mating A. are different in size than those of ascospores of mating type _a_ (Figure 38B). The insert accumulating in ascospores 5 and 6, both of mating type A , is the insert with junction fragments b l ' and b2' (Figure 38B). Unfortunately, it is difficult to detect the junction fragments of ka lDNA in the mtDNA prepared from ascospore 4 but it should be noticed that they are the same size as junction fragments b l ' and b2'. In ascospore 8, also of mating type A , the junction fragments of the insert accumulating are the same size as the junction fragments of the insert accumulating in ascospores 2, 4, 5, and 7 of ascus 1 from cross 561-0 X 605 (Figure 37B). In ascospores 2, 4, 5, and 7 of ascus 1, this insert was proposed to have originated from a rearrangment of the transmitted insert with junction fragments b l ' and b2'. In ascus 7, the high molecular weight fragments which hybridized with the Pst I-kalDNA probe in the early cultures of the ascospores of mating type _A are not observed in the late cultures. The ascospores of mating type _a_ all accumulate inserts with high molecular weight junction fragments. It is suspected that these inserts originated from movement since the relative sizes of the junction fragments are similar to those described in the previous chapter. Ascus 5 from Cross 561-0 X1766 Analysis of the mtDNA prepared from the early cultures of each ascospore from ascus 5 indicates that only the insert with junction fragments b l ' and b2' was Chapter 2 / 148 transmitted from the female parent (Figure 39A). In the late cultures, the inserts accumulating in ascospores of mating type _A are not the inherited insert (Figure 39B) however, hybridization of the intron probe with these junction fragments indicates that they belong to inserts which have probably originated from rearrangements of the inherited insert. The sizes of the junction fragments of these inserts, with the exception of ascospore 3, support the suggestion that these inserts have originated from rearrangment events and are not novel insertions. The mtDNA of ascospore 3 shows junction fragments of the same size as b l and b2 and consequently may have arisen from novel insertion or was perhaps inherited in very low copy number. Only three of the mtDNA profiles of ascospores of mating type _a_ are shown. Ascospores 6 and 8 have inserts with high molecular weight junction fragments. The mtDNA profile of ascospore 5 shows an insert with smaller junction fragments. The absence of hybridization of the intron probe to these junction fragments indicates that they are probably novel insertions. The results from ascus 5 and 7 indicate that movement of ka lDNA appears to occur almost exclusively in ascosopores of mating type a. This same segregation is not observed in the ascospores of ascus 1 from cross 561-0 X 605 indicating that outcrossing of a Kauaian senescent strain introduces a gene or an allele of a gene which influences movement. Chapter 2 / 149 2. Comparison of Tetrads From Crosses Using a Juvenile Female Parent and a Senescent Female Parent Comparison of the sexual and somatic transmission of mtlS-kalDNA in ascospores initiated from crosses using a female parent strain in different stages of senescence involved isolating tetrads from crosses 561-0 X 605, 561-5 X 605, 561-0 X 1766, and 561-5 X 1766. One ascus was isolated from each of crosses 561-0 X 605 and 561-5 X 605 and two asci from each of crosses 561-0 X 1766 and 561-5 X 1766. The first subculture (o) of series 561 was used as the juvenile female parent and subculture five of the same series as the senescent female parent. Strain P605 is a nonsenescent Kauaian natural isolate and strain 1766 a nonsenescent Taiwanese natural isolate. The ascospores of each tetrad were subjected to serial subculturing and the length of each series is shown diagrammatically in Figures 35 and 36. Tetrads from crosses 561-0 X 605 and 561-0 X 1766 were described in the previous section of this chapter. Comparison of Tetrads from Crosses 561-0 X 605 and 561-5 X 605 Comparison of the average lifespans of each tetrad (Figure 35) reveals that the ascospores from cross 561-0 X 605 have an average lifespan of 11 subcultures compared to an average of 7 for the ascospores from cross 561-5 X 605. The mtDNA profiles of the early and late subcultures are shown in Figures 3 7A, 37B, 40A, and 40B. As described in the previous section, analysis, of the mtDNA prepared from the early cultures of the ascospores of ascus 1, from cross 561-0 X 605, showed that only the insert with junction fragments b l ' and Chapter 2 1 150 b2' was transmitted sexually. In the early cultures of the ascospores of ascus 6, from cross 561-5 X 605, both inserts present in the female parent strain are inherited (Figure 40A). The majority of the ascospores of this ascus inherit the insert with junction fragments b l and b2 in higher copy number than the insert with junction fragments b l ' and b2'. Ascospore 2 shows the insert with junction fragments b l ' and b2' to be in very high copy number relative to the insert with junction fragments b l and b2. Ascospores 4 and 8, both of mating type a, inherit the insert with junction fragments b l ' and b2' in higher copy number indicating that they are probably spore pairs. As previously described, the inserts accumulating in the ascospores from ascus 1 are either the inherited insert or constitute rearrangements of the transmitted insert (Figure 37B). In the late cultures of the ascospores of ascus 6, the inserts which accumulate are the transmitted inserts (Figure 40B). In ascospores 2, 3, 6, and 8 of ascus 6, the insert with junction fragments b l and b2 is seen in highest copy number. In ascospore 4, the insert with junction fragments b l ' and b2' is observed to accumulate. Ascospore 1 has junction fragments of the same size as the junction fragments of the insert accumulating in ascospores 2, 4, 5, and 7 of ascus 1 from cross 561-0 X 605 (Figure 37B). In ascospores 2, 4, 5, 7 of ascus 1, this- insert was proposed to have originated from a rearrangement of the transmitted insert with junction fragments b l ' and b2'. In ascospore 7 of ascus 6, no novel fragments are observed which hybridize with the Pst I-kalDNA probe. In this ascospore, hybridization of the Pst I-kalDNA probe to novel bands constituting junction fragments of an insert should have been observed since in the early culture of this ascospore series the mutagenic Chapter 2 1 151 insert with junction fragments b l and b2 is observed and because the culture does express senescence. Comparison of Tetrads from Crosses 561-0 X 1766 and 561-5 X 1766 Two tetrads from each cross were analyzed. The lengths of each ascospore series are shown in Figure 36. The average lifespan of the tetrads from cross 561-0 X 1766 is 16.5 subcultures compared to an average of 8.5 subcultures for the two tetrads from cross 561-5 X 1766. From cross 561-5 X 1766, the ascospores of ascus 3 do show variability in lifespan where four series die within 10 subcultures and the other four take from 12 to 20 subcultures to die. In ascus 4 from cross 561-5 X 1766, all ascospores series die within 8 subcultures. The mtDNA profiles for the early and late cultures of each series are shown in Figures 38A, 38B, 39A, 39B, 41A, 4 IB, 42A, and 42B. As previously described, the autoradiograph of the early cultures of each ascospore from ascus 5 and 7, from cross 561-0 X 1766, show transmission of only the neutral insert with junction fragments b l ' and b2' (Figure 38A and 39A). The late cultures of the ascospores of mating type A_ from these same asci accumulate either the neutral insert or a rearrangement of the neutral insert, whereas the ascospores of mating type _a_ accumulate novel insertions (Figure 38B and 39B). In ascus 4 from cross 561-5 X 1766 transmission of both inserts present in the female parent is apparent (Figure 41 A). In the early cultures of ascospores 2, 4, and 5, the insert with junction fragments b l and b2 is transmitted in higher Chapter 2 1 152 copy number than the insert with junction fragments b l ' and b2'. Ascospores 1 and 3 inherit the insert with junction fragments b l ' and b2' in higher cop}' number. Ascospore 7 inherits both inserts in essentially equal copy number. In ascospore 8, novel bands other than junction fragments b l and b2, and b l ' and b2' are observed to hybridize with the Pst I-kalDNA probe. Analysis of the late cultures reveals that in all cases the transmitted inserts accumulate (Figure 4 IB). Ascospores 1, 2, 3, 4, 6, and 7 show the insert with junction fragments b l and b2 to have accumulated. Ascospore 8 shows the insert with junction fragments b l ' and b2' to have accumulated. The mtDNA of ascospore 5 shows other-inserts to accumulate in addition to the transmitted inserts with junction fragments b l and b2, and b l ' and b2'. Ascus 3 from cross 561-5 X 1766 is somewhat of an anomaly compared with ascus 4 which was isolated from the same cross. The transmission of mtlS-kalDNA is reminiscent of ascus 5 and 7 from cross 561-0 X1766; only the insert with junction fragments b l ' and b2' is transmitted and in very low copy number (Figure 42A). Analysis of the late cultures shows that the inserts accumulating are generally not the transmitted insert (Figure 42B). In ascospores 1 and 5, both of mating type A, the junction fragments of the accumulating inserts hybridize with the intron probe. The sizes of these junction fragments indicate that these insertions probably originated through rearrangements of the transmitted neutral insert. In ascospore 4, mating type A , two inserts with high molecular weight junction fragments are observed. These junction fragments did not hybridize strongly with the Pst I-kalDNA probe but it is suspected that these are junction fragments of a novel insert because the size of each junction Chapter 2 1 153 fragment is similar to the junction fragments of an insert described in the previous chapter. The bands hybridizing with the Pst I-kalDNA probe of ascospore 2, mating type _A, fail to hybridize with the intron probe. It is suspected that these junction fragments are of a novel insertion. The mtDNA of ascopores 3 and 8, both of mating type _a_ show the neutral insert to have accumulated. The junction fragments of the inserts observed in the late cultures of ascospores 6 and 7, both of mating type _a_, fail to hybridize with the intron. In addition, the sizes of the junction fragments are similar to junction fragments of inserts described in the previous chapter indicating that the inserts in each of these two ascospores originated from movement. In this tetrad novel insertions are associated with two ascospores of mating type A_ and two of mating type a. Chapter 2 1 154 Figure 35. Subculture series of tetrads isolated from crosses 561-0 X 605 and 561-5 X 605. The ascsopore culture from which each series was derived is labeled 0. The last number spanned by a horizontal bar indicates the subculture which produced no viable conidia. The mating type of each ascospore is shown. C h a p t e r 2 / 155 5 6 1 - 0 ? X 6 0 5 6 subcul ture number ascus 1 0 5 10 15 2 0 1A — i - — ™ » 2 a e i — - • — 3 A • — — — 4 A R*—HHi 5 A — n — » 6 a • 7 a H H K M B M w i 8 a W H M B M a v e r a g e l i f e s p a n = 1 1 5 6 1 - 5 9 X 6 0 5 ( J subculture number ascus 6 a v e r a g e l i f e s p a n : 7 Chapter 2 / 1 5 6 Figure 36. Subculture series of tetrads isolated from crosses 561-0 X 1766 and 561-5 X 1766. The ascsopore culture from which each series were derived is labeled 0. The last number spanned by a horizontal bar indicates the subculture which produced no viable conidia. The mating type of each ascospore is shown. C h a p t e r 2 / 157 » 5 6 1 - 0 9 X 1 7 6 6 Cf a s c u s 7 s u b c u l t u r e n u m b e r 0 5 1 0 1 5 2 0 1a wmm——mmmmmmmmmmm—mm 2 a mmm—mmmmm—mt—^m—mmm 3 a wmmmmmmmmmmm—mmmmmmm—mmmmmm 4 A wmm^——m—m* 5A ^m—a^mm^mm* a v e r a g e l i f e s p a n = 1 2 6A w—mmmmmmm—mm. 7 a ^——m—mmmmmmm—mmm 8A MHHHMMMM a s c u s 5 1A 2 A 3A 4 a 5 a m—mmmmmm^mm^mmm—mammm^ a v e r a g e l i f e s p a n s 15 6a wmmmmmm—mm—mmmmmmmm. 7A wmm—mmmmmmmimmmm—m 8 a MHHMMMMHMMMHMMMI 5 6 1 - 5 9 X 1766 Cf s u b c u l t u r e number a s c u s 3 0 5 10 15 2 0 1A wmmm—mmmm^m 2A B M M M M U M M S M M a B M B H 3 a mm—mmm—i i B M M H B M a a v e r a g e l i f e s p a n = 10 6 a 7a i — — 8 a MWHM a s c u s 4 1a 2A 3A 4 A 5 a a v e r a g e l i f e s p a n s 5 6 a mmtmmmmmt 7 a 8 A Chapter 2 / 158 Figure 3 7A. Gel electrophoresis of Bgl II digested mtDNA prepared from the early cultures of the ascospores from ascus 1 from cross 561-0 X 605. The mating type (alleles A_ or _a) of each ascospore is shown. The first and second lanes of the ethidium bromide stained gel and the autoradiograph are the nonsenescent control P605 and the senescent control P561, respectively. The numbers above the remaining lanes represent the ascospore isolation number from the ascus. The autoradiograph represents a hybridization using the Pst I-kalDNA probe (for details on this probe refer to Figure 9). The bands labeled b l and b2, and b l ' and b2' are junction fragments of two inserts present in the female parent strain P561. B3 and b4 are internal Bgl II fragments of kalDNA. The bands denoted 10 and 12 are mtDNA Bgl II fragments (note that the Bgl 11-12 restriction fragment comigrates with the b4 fragment). Chapter 2 / 160 Figure 37B. Gel electrophoresis of Bgl II digested mtDNA from the late cultures of the ascospores from ascus 1 from cross 561-0 X 605. The mating type (alleles A_ or _a) of each ascospore is shown. The first and second lanes of the ethidium bromide stained gel and each autoradiograph are the nonsenescent control P605 and the senescent control P561, respectively. The numbers above the remaining lanes represent the ascospore isolation number from the ascus. The autoradiographs represent hybridizations using the Pst I-kalDNA and Hind HI-13,18 probes (for details on these probes refer to Figures 8 and 9). The bands labeled bl and b2, and bl ' and b2' are junction fragments of two inserts present in the female parent strain P561. B3 and b4 are internal Bgl H fragments of kalDNA. The bands denoted 4, 10, and 12 are mtDNA Bgl H fragments (note that the Bgl H-12 restriction fragment comigrates with the b4 fragment). Chapter 2 / 1 6 2 Figure 3 8A. Gel electrophoresis of Bgl II digested mtDNA prepared from the early cultures of the ascospores from ascus 7 from cross 561-0 X 1766. The mating type (alleles or _a) of each ascospore is shown. The first and second lanes of the ethidium bromide stained gel and the autoradiograph are the nonsenescent control P605 and the senescent control P561, respectively. The numbers above the remaining lanes represent the ascospore isolation number from the ascus. The autoradiograph represents a hybridization using the Pst I-kalDNA probe (for details on this probe refer to Figure 9). The bands labeled b l and b2, and bl ' and b2' are junction fragments of two inserts present in the female parent strain P561. B3 and b4 are internal Bgl II fragments of kalDNA. The bands denoted 10 and 12 are mtDNA Bgl II fragments (note that the Bgl 11-12 restriction fragment comigrates with the b4 fragment). K a l Chapter 2 / 164 Figure 38B. Gel electrophoresis of Bgl II digested mtDNA prepared from the late cultures of the ascospores from ascus 7 from cross 561-0 X 1766. The mating type (alleles A^  or _a) of each ascospore is shown. The first and second lanes of the ethidium bromide stained gel and the autoradiograph are the nonsenescent control P605 and the senescent control P561, respectively. The numbers above the remaining lanes represent the ascospore isolation number from the ascus. The autoradiograph represents a hybridization using the Pst I-kalDNA probe (for details on this probe refer to Figure 9). The bands labeled b l and b2, and bl* and b2' are junction fragments of two inserts present in the female parent strain P561. B3 and b4 are internal Bgl II fragments of kalDNA. The bands denoted 10 and 12 are mtDNA Bgl II fragments (note that the Bgl 11-12 restriction fragment comigrates with the b4 fragment). K a l Chapter 2 / 1 6 6 Figure 3 9A. Gel electrophoresis of Bgl II digested mtDNA prepared from the early cultures of the ascospores from ascus 5 from cross 561-0 X 1766. The mating type (alleles A. or a) of each ascospore is shown. The first and second lanes of the ethidium bromide stained gel and the autoradiograph are the nonsenescent control P605 and the senescent control P561, respectively. The numbers above the remaining lanes represent the ascospore isolation number from the ascus. The autoradiograph represents a hybridization using the Pst I-kalDNA probe (for details on this probe refer to Figure 9). The bands labeled b l and b2, and bl' and b2' are junction fragments of two inserts present in the female parent strain P561. B3 and b4 are internal Bgl II fragments of kalDNA. The bands denoted 10 and 12 are mtDNA Bgl II fragments (note that the Bgl 11-12 restriction fragment comigrates with the b4 fragment). Chapter 2 / 1 6 8 Figure 39B. Gel electrophoresis of Bgl Et digested mtDNA prepared from the late cultures of the ascospores from ascus 5 from cross 561-0 X 1766. The mating type (alleles A_ or _a) of each ascospore is shown. The first and second lanes of the ethidium bromide stained gel and each autoradiograph are the nonsenescent control P605 and the senescent control P561, respectively. The numbers above the remaining lanes represent the ascospore isolation number from the ascus. The autoradiographs represent hybridizations using the Pst I-kalDNA and Hind EQ-13,18 probes (for details on these probes refer to Figures 8 and 9). The bands labeled b l and b2, and bl ' and b2' are junction fragments of two inserts present in the female parent strain P561. B3 and b4 are internal Bgl H fragments of kalDNA. The bands denoted 4, 10, and 12 are mtDNA Bgl II fragments (note that the Bgl El-12 restriction fragment comigrates with the b4 fragment). Chapter 2 / 1 7 0 Figure 40A. Gel electrophoresis of Bgl II digested mtDNA prepared from the early cultures of the ascospores from ascus 6 from cross 561-5 X 605. The mating type (alleles or _a) of each ascospore is shown. The first and second lanes of the ethidium bromide stained gel and the autoradiograph are the nonsenescent control P605 and the senescent control P561, respectively. The numbers above the remaining lanes represent the ascospore isolation number from the ascus. The autoradiograph represents a hybridization using the Pst I-fcalDNA probe (for details on this probe refer to Figure 9). The bands labeled bl and b2, and bl' and b2' are junction fragments of two inserts present in the female parent strain P561. B3 and b4 are internal Bgl II fragments of kalDNA. The bands denoted 10 and 12 are mtDNA Bgl H fragments (note that the Bgl II-12 restriction fragment comigrates with the b4 fragment). I 5 6 1 - 5 x 6 0 5 ascus 6 A a A a N S 1 2 3 * 4 7 8 N 5 1 2 3 6 4 7 8 K a l Chapter 2 / 1 7 2 Figure 40B. Gel electrophoresis of Bgl II digested mtDNA prepared from the late cultures of the ascospores from ascus 6 from cross 561-5 X 605. The mating type (alleles A_ or _a) of each ascospore is shown. The first and second lanes of the ethidium bromide stained gel and each autoradiograph are the nonsenescent control P605 and the senescent control P561, respectively. The numbers above the remaining lanes represent the ascospore isolation number from the ascus. The autoradiograph represents a hybridization using the Pst I-kalDNA probe (for details on these probes refer to Figures 8 and 9). The bands labeled b l and b2, and bl ' and b2' are junction fragments of two inserts present in the female parent strain P561. B3 and b4 are internal Bgl H fragments of kalDNA. The bands denoted 10 and 12 are mtDNA Bgl U fragments (note that the Bgl Et-12 restriction fragment comigrates with the b4 fragment). K a l Chapter 2 / 1 7 4 Figure 41 A . . Gel electrophoresis of Bgl Et digested mtDNA prepared from the early cultures of the ascospores from ascus 4 from cross 561-5 X 1766. The mating type (alleles A_ or _a) of each ascospore is shown. The first and second lanes of the ethidium bromide stained gel and the autoradiograph are the nonsenescent control P605 and the senescent control P561, respectively. The numbers above the remaining lanes represent the ascospore isolation number from the ascus. The autoradiograph represents a hybridization using the Pst I-kalDNA probe (for details on this probe refer to Figure 9). The bands labeled bl and b2, and bl ' and b2' are junction fragments of two inserts present in the female parent strain P561. B3 and b4 are internal Bgl Et fragments of kalDNA. The bands denoted 10 and 12 are mtDNA Bgl Et fragments (note that the Bgl Et-12 restriction fragment comigrates with the b4 fragment). \1S 561-5x1766 ascus 4 A 3 A a  N S 2 3 4 8 1 3 7 N S 2 3 4 8 1 5 7 K a l Chapter 2 / 1 7 6 Figure 4 IB. Gel electrophoresis of Bgl II digested mtDNA prepared from the late cultures of the ascospores from ascus 4 from cross 561-5 X 1766. The mating type (alleles A^  or _a) of each ascospore is shown. The first and second lanes of the ethidium bromide stained gel and the autoradiograph are the nonsenescent control P605 and the senescent control P561, respectively. The numbers above the remaining lanes represent the ascospore isolation number from the ascus. The autoradiography represent hybridizations using the Pst I-kalDNA and Hind HI-13,18 probes (for details on these probes refer to Figures 8 and 9). The bands labeled b l and b2, and bl ' and t>2' are junction fragments of two inserts present in the female parent strain P561. B3 and b4 are internal Bgl E fragments of kalDNA. The bands denoted 4, 10, and 12 are mtDNA Bgl U fragments (note that the Bgl D-12 restriction fragment comigrates with the b4 fragment). rn 5 6 1 - 5 x 1 7 6 6 ascus 4 A a A a N S 2 3 4 8 1 5 6 7 N S 2 3 4 8 1 5 6 7 K a l Chapter 2 / 1 7 8 Figure 42A. Gel electrophoresis of Bgl II digested mtDNA prepared from the early cultures of the ascospores from ascus 3 from cross 561-5 X 1766. The mating type (alleles _A or _a) of each ascospore is shown. The first and second lanes of the ethidium bromide stained gel and the autoradiograph are the nonsenescent control P605 and the senescent control P561, respectively. The numbers above the remaining lanes represent the ascospore isolation number from the ascus. The autoradiograph represents a hybridization using the Pst I-kalDNA probe (for details on this probe refer to Figure 9). The bands labeled bl and b2, and b l ' and b2' are junction fragments of two inserts present in the female parent strain P561. B3 and b4 are internal Bgl II fragments of kalDNA. The bands denoted 10 and 12 are mtDNA Bgl II fragments (note that the Bgl 11-12 restriction fragment comigrates with the b4 fragment). K a l Chapter 2 / 1 8 0 Figure 42B. Gel electrophoresis of Bgl Et digested mtDNA prepared from the late cultures of the ascospores from ascus 3 from cross 561-5 X 1766. The mating type (alleles A_ or _a) of each ascospore is shown. The first and second lanes of the ethidium bromide stained gel and each autoradiograph are the nonsenescent control P605 and the senescent control P561, respectively. The numbers above the remaining lanes represent the ascospore isolation number from the ascus. The autoradiographs represent hybridizations using the Pst I-kalDNA and Hind EQ-13,18 probes (for details on these probes refer to Figures 8 and 9). The bands labeled bl and b2, and bl' and b2' are junction fragments of two inserts present in the female parent strain P561. B3 and b4 are internal Bgl Et fragments of kalDNA. The bands denoted 4, 10, and 12 are mtDNA Bgl Et fragments (note that the Bgl E-12 restriction fragment comigrates with the b4 fragment). Chapter 2 / 182 C. DISCUSSION 1. Genetic Regulation of mtlS-kalDNA Movement The majority of series described in the previous chapter were started from ascospores isolated from the cross 561-1 X 1766. It was observed that the insert inherited did not accumulate and thus initiate senescence; this insert was termed a neutral insert. At some point in each subculture series novel insertions were detected which initiated senescence. In all cases, these inserts were proposed to have originated from movement of the neutral insert with junction fragments b l ' and b2'. To investigate whether movement of mtlS-kalDNA may be influenced by the host genome, tetrads were isolated from crosses of a juvenile female parent (561-0) to the Kauaian strain P605 and to the Taiwanese strain 1766. The analysis revealed that movement of ka lDNA occurred primarily in ascopores of mating type _a_ initiated from cross 561-0 X 1766. The ascospores of mating type A. from this cross and all but ascospore 1 of ascus 1 from cross 561-0 X 605 accumulate either the inherited insert with junction fragments b l ' and b2' or a rearrangement of that insert. These results indicate that movement may be influenced by a gene or an allele of a gene introduced upon outcrossing. It is suspected that this gene or allele is unlinked from mating type for the following reasons. The mating type of strain P561 is and j \ for strain 1766. If an allele or gene is introduced from strain 1766 and linked to mating type then ascospores of mating type A_ would have shown the highest frequency of movement of ka lDNA since mating type A is transmitted from strain 1766. The Chapter 2 / 1 8 3 fact that ascospores of mating type _a_ of ascus 5 and 7 from cross 561-0 X 1766 showed movement indicates that the allele or gene introduced from strain 1766 probably segregates independently of mating type. In addition, if the proposed nuclear gene was linked to mating type, two cross overs would be required to obtain the segregation patterns observed in the two tetrads analyzed. This model indicates that in addition to tetrads in which ascospores of mating type &_ show movement, tetrads where ascospores of mating type A_ show movement should be observed equally frequently. Tetrads of the later type were probably not observed because only two tetrads from cross 561-0 X 1766 were isolated and examined. It should be noticed that, unexpectedly, ascus 3 from cross 561-5 X 1766 also showed a 4:4 segregation for the movement of mtlS-kalDNA. In this case, two of the ascospores were of mating type _A and two of mating type _a. If there is a gene influencing movement and unlinked to mating type, then it is suspected that a crossover may have occurred either between this locus and its centromere or between mating type and its centromere. Movement does not appear to be entirely confined to ascospores initiated from the cross 561-0 X 1766 since ascospore 1 of ascus 1 from cross 561-0 X 605 may have accumulated a novel insert. Although movement of mtlS-kalDNA may have occurred in this ascospore, where it was not expected, it can be proposed that intramitochondrial movement occurs most frequently in series derived from ascospores of cross 561-0 X 1766. Chapter 2 / 184 A similar phenomenon has been reported in P. anserina (Vierny et al, 1982). In this fungus, mating type differences appear to affect longevity. Strains of mt-generally grow approximately 14cm whereas mt+ strains grow about 40cm (Vierny et al, 1982). Analysis of the mtDNA prepared from juvenile cultures of different mating types revealed that in mt- strains, autonomous alpha senDNA sequences are present whereas only the integrated sequences are present in mt+ cultures. This result suggested that the state in which alpha senDNA exists in juvenile cultures accounts for the difference in longevities (ie: the sooner the probable appearance of alpha senDNA, the shorter the longevity of that strain). The fact that these strains differ in mating type implies that the mating type locus or a gene closely linked to mating type might act on, for example, the probability of excision of alpha senDNA from the mtDNA. Although the isolation and characterization of this locus has not been performed the hypothesis that it affects the excision of specific regions of the mtDNA suggests that the proposed gene affecting the movement of mtlS-kalDNA in senescent strains of N .  intermedia may act in a similar fashion. It may be proposed that that the active form of this gene in N . intermedia either acts as a suppressor of movement or is a protein which facilitates movement. If a suppressor of movement, then the active form of this gene would be associated with the Kauaian genome since movement of mtlS-kalDNA is generally not observed in ascospores initiated from crosses between two Kauaian strains. The nonactive allele would be introduced through outcrossing and the movement of mtlS-kalDNA observed in those progeny which received the nonactive allele. How this suppressor would affect movement is unknown, but one possibility is that it may act as a repressor of the expression of kalDNA-specific genes which are required Chapter 2 / 185 for movement. Presented in this chapter are results revealing that movement of mtlS-kalDNA occurs only in ascospores which inherit the neutral insert with junction fragments b l ' and b2'. This suggests that the evolutionary significance of a host allele which suppresses movement may allow for more opportunity for a culture carrying only a neutral insert to escape senescence. Thus, if the proposed nuclear gene is a. suppressor of movement then all progeny from a cross between two Kauaian strains would receive the active form of the host gene and no movement of mtlS-kalDNA would be observed. In these ascospores, rearrangement of the neutral insert must occur to initiate senescence. Escapees would result in those cases where either no rearrangment occurred or the neutral insert was lost. In contrast, the probability of escape from senescence would be reduced by 50% for ascospores initiated from crosses using a Kauaian female strain in a juvenile stage of senescence crossed to a nonKauaian male strain. This reduction would be a consequence of 50% of the progeny receiving the nonactive form of the host suppressor gene. This indicates that the 50% carrying the nonactive form of the gene would carry novel insertions of mtlS-kalDNA which would initiate senescence. Thus, this model suggests an evolutionary safeguard against the possible extinction of Kauaian strains by preventing movement of mtlS-kalDNA in ascospores from crosses between two Kauaian strains and the opportunity to escape senescence. An alternative hypothesis suggests that the active form of the proposed nuclear gene promotes movement of mtlS-kalDNA. According to this hypothesis, the active form of this gene would be associated with the nonKauaian genome since movement of mtlS-kalDNA generally only occurs in ascospores initiated from the Chapter 2 / 1 8 6 outcrossing of a senescent female Kauaian strain. The mode of action of this gene product on the movement of ka lDNA is unknown, but a number of examples from other systems also indicate that functional host nuclear gene(s) are required for the transposition of a given mobile element. In the fungus P. anserina, various combinations of recessive nuclear genes have been identified which prevent the expression and/or propagation of senescence (Tudzynski and Esser, 1979; Esser and Tudzynski, 1980). These genes are pleiotropic because in addition to suppressing the expression of senescence, they alter the morphology of the mycelium. M t D N A from these mutants has not been investigated but these results indicate that the excision and/or amplification of senDNA is only possible when the wild type gene products are being expressed. In Neurospora, a gene showing similar characteristics has been identified in the senescent strain 2360 his. Crosses using this senescent auxotrophic strain as a female parent results in the ascospores of tetrads exhibiting a 4:4 segregation of senescence to no expression of senescence (Griffiths, unpublished). The proposed nuclear gene suppressing the expression of senescence is also considered pleiotropic because in addition to suppressing the expression of senescence, this gene is thought to be responsible for the reduced development of aerial mycelium. Other examples supporting the hypothesis that a gene product may be necessary for movement of mtlS-kalDNA include, for example, host genes involved in supercoiling. These genes have been shown to affect several kinds of recombination events, including transposon-mediated, site specific recombination (Reid, 1981), bacteriophage lambda integration (Nash et al, 1980), Tn3 movement Chapter 2 1 187 (Heffron, 1983), mating-type switching in S. cerevisiae (Nasmyth, 1982), and movement of the Ty element (Roeder and Fink, 1983). In some instances transposition of mobile elements is developmentally regulated. For example, the behaviour of the Spm and En mobile elements of Zea maize appear to be subject to regulation by factors that are differentially distributed within a plant or a single tissue (McClintock, 1965; 1971; Peterson, 1966; Fowler and Peterson, 1978). It was suggested that similar events may also affect the developmental timing and the apparent frequency of Ac and Mp movement in maize (Federoff, 1983). In Drosophila, P element movement is tissue specific; occurring at high frequencies only in the germline tissue (Laski et al, 1986). The P element is regulated at the level of mRNA splicing where the final intron is removed only in the germline. This indicates that some host gene product, expressed only in the germline, regulates the processing and ultimately the movement of this element. In maize, reversion of male cytoplasmic sterility to fertility occurs spontaneously in a particular nuclear background compatible with the cytoplasm (Pring et al, 1977; Levings et al, 1980; Laughnan et al, 1981; Laughnan and Gabay-Laughnan 1982; 1983). The three cytoplasms which cause male sterility, S, T, and C, are distinquished on the basis of nuclear genes, called restorer genes, which restore normal pollen development (Duvick et al, 1961; Duvick, 1965; Laughnan and Gabay-Laughnan, 1983). At the molecular level, these restorer genes appear to affect the characteristic rearrangements of the mtDNA of sterile cytoplasms. S-type cytoplasms have been characterized by the presence Chapter 2 / 1 8 8 of two linear plasmids, S I and S2 (Pring et al, 1977; Levings and Sederoff, 1983). which recombine with the mtDNA to generate linear mtDNA molecules (Schardl et al, 1985). In addition to the linear mtDNA molecules, the mtDNA contains internally integrated S i and S2 sequences (Schardl et al, 1985). Reversion of S-type cytoplasms in the presence of the appropriate nuclear background results in the loss of the linear S i and S2 plasmids and the recircularization of the mtDNA with only the internally integrated S i and S2 sequences remaining (Schardl et al, 1985). A l l these examples indicate that host functions are important in mediating movement of mobile elements. Although in most systems the mode of action of these genes on transposition is unknown, they support the hypothesis that the active form of the gene identified in N . intermedia may be required to facilitate movement of mtlS-kalDNA. The evolutionary significance of a host gene regulating the movement of a mutagenic element may be to prevent successful outbreeding of geographically unrelated strains and thus promote speciation. Alternatively, kalDNA may be thought of as a mutator factor, which upon outbreeding, increases the rate of mutation relative to the spontaneous mutation rate. In natural populations this would result in decreased fitness of the progeny carrying novel kalDNA insertions but, an explosive increase in the level of mutation would generate new variants which would be evolutionarily significant because genetic variation is necessary for evolutionary change. Chapter 2 / 1 8 9 2. Comparison of Tetrads Derived From Crosses Using a Juvenile Female Parent and a Senescent Female Parent Analysis of ascospores from crosses utilizing juvenile and senescent female parents validates the supposition that the form of mtlS-kalDNA transmitted sexually plays a major role in the expression of senescence. In mtDNA prepared from the early cultures of ascospores isolated from crosses using a juvenile female parent (561-0), inherited only the neutral insert with junction fragments b l ' and b2'. In contrast, ascospores isolated from crosses involving the senescent female parent (561-5) inherit both ka lDNA inserts present in the female parent strain. The different inheritance patterns of mtlS-kalDNA may depend on the ratio of normal to abnormal mitochondria present in the female parent such that the higher the ratio of abnormal to normal mitochondria, the more opportunity for abnormal mitochondria to be transmitted. This does not explain why only the insert with junction fragments b l ' and b2' is preferentially transmitted to ascospores isolated from crosses using the juvenile female parent. As mentioned in the previous chapter there may be a meiotic process which screens mitochondria and prevents the transmission of abnormal mitochondria. In ascospores where the female is in a juvenile state, the screening process would be very effective in that normal mitochondria and mitochondria with mtDNA molecules carrying neutral ka lDNA inserts would be exclusively transmitted to progeny. The screening process would not be as efficient in meioses involving a senescent female parent because of the presence of greater numbers of abnormal mitochondria. Thus, both normal and abnormal mitochondria would be transmitted to progeny. According to this hypothesis, the expression of senescence of ascospores which inherit abnormal Chapter 2 / 190 mitochondria would commence at the onset of vegetative propagation. Thus, the lifespans of these ascospores would be quite short. This notion is supported by observations of the mtDNA prepared from the early and late subcultures of the ascospore series from crosses using a senescent culture (subculture 5) of strain P561 as the female parent. In these ascospores, the inherited and accumulating inserts are generally the same insertions. This indicates that defective mtDNA molecules begin accumulating at the onset of vegetative growth and initiates senescence. Ascus 3 from cross 561-5 X 1766 does not follow the same inheritance patterns of mtlS-kalDNA as the other two tetrads isolated from crosses using the senescent female parent. Only the neutral insert is transmitted to the ascospores of ascus 3. This may be explained by postulating that the source of mitochondria transmitted consisted of more normal than abnormal mitochondria. Knowing that Neurospora is coenocytic and that mitochondrial populations are heterogeneous, the ratio of normal to abnormal mitochondria may differ in different regions of the cytoplasm and the source of cytoplasm transmitted to these progeny would reflect this. Analysis of the mtDNA prepared from the late cultures of ascospores initiated from crosses using a juvenile subculture of strain P561 as the female parent revealed that either a novel insertion or a rearrangement of the inherited neutral insert was required before senescence was induced. This would account for the longer average lifespans of these ascospores. It is of interest that the insert described as being neutral is observed to accumulate in some ascospore series. This insert was termed neutral because it Chapter 2 / 1 9 1 was never observed to initiate senescence in the series described in the previous chapter. Why it induces senescence in some but not all ascospore series is not understood. Perhaps the presence of subtle mutations carried on the same mtDNA molecules as the neutral insert were responsible for their accumulation. In summary, the sexual transmission of mutagenic ka lDNA inserts appears to depend on the age of the strain used as the female parent. Transmission of a mutagenic insert generally results in its accumulation and initiation of senescence at the onset of vegetative growth of the ascospores. Contrastingly, transmission of normal mtDNA or mtDNA molecules carrying neutral ka lDNA inserts delays the onset of senescence until a mutagenic insert is generated either through novel insertion of ka lDNA or through a rearrangment of the mtDNA encompassing the transmitted insert. V . C H A P T E R 3 A. INTRODUCTION This chapter focuses on a senescent Kauaian isolate, strain P573, which shows some but not all the characteristics of kalilo senescence. A total of four classes of senescence have been delineated in Neurospora. Two of these four clases were identified from a survey of natural isolates from different geographic locations (Griffiths et al, 1988a). The first class was discovered in a population from Aarey, India (Griffiths et al, 1988b). Here, senescence is associated with a nucleus associated element, AR-marDNA, and a mitochondrial insertion sequence, mtlS-marDNA. The mode of action of these two elements is reminiscent of the behaviour of the kalilo element, but there is no sequence homology between ka lDNA and marDNA. The second type of senescence is nonheritable and its basis not understood. This type of senescence is shown by many natural isolates which are geographically unrelated. This class is characterized by the lack of maternal transmission of senescence, the presence of normal amounts of cytochromes, the lack of mtDNA alterations, and a unique pattern of infertility. It is suspected that senescence results from lethal nuclear mutations that are suppressive in the vegetative cultures (Griffiths et al, 1988a). The third class of senescence, discovered by Akins et al (1986), is mitochondrial-based. In this class, mitochondrial plasmids recombine with the mtDNA and are either carried on the mtDNA molecule or excise and acquire mtDNA sequences. As a consequence of disrupting the mtDNA, suppressive accumulation of the altered mtplasmid and/or mtDNA molecules commences. The fourth case is kalilo senescence. 192 Chapter 3 / 1 9 3 Characterization has been extended to more cases from Kauai and to cases from other Hawaiian islands (Griffiths et al, 1988a). Reported here is work on a variant Kauaian strain, P573, which shows maternal transmission of ka lDNA and senescence but does not alwaj's exhibit the same biochemical and molecular events known to accompany kalilo senescence. Presented in this chapter is the molecular characterization of this strain and some of its progeny. The analysis reveals that AR-ka lDNA is present in most cultures in trace amounts and the behaviour of mtlS-kalDNA is erratic. In addition some cultures do not exhibit cytochrome a a 3 and b deficiencies yet these cultures do express senescence and eventually die. This strain is highly anomalous when compared to the prototype of kalilo senescence, strain P561, and thus provides a cautionary note against extrapolating to all cases of senescence in Kauaian strains. B. RESULTS Strain P573 was chosen for investigation because the lifespans of series derived from its progeny generally exceed 20 subcultures and are quite variable. This is of interest because the number of subcultures preceding death is usually less then 20 subcultures for series derived from ascospores isolated from crosses using other Kauaian senescent strains as female parents (Griffiths and Bertrand, 1984). Strain P573 was subjected to serial subculturing. Vegetative death occurred in the tenth subculture. The progressive decline in growth potential is as expected for Chapter 3 / 194 kalilo senescence (Griffiths and Bertrand, 1984; Bertrand et al, 1985; 1986). Cytochrome spectra were prepared from subcultures 1, 4, 7, 8, and 9 (Figure 43) . A normal cytochrome complement is observed for subcultures 1 and 4. By subculture 7, there is a decline in the amount of cytochromes a a 3 and b and an increase in cytochrome c. By subculture 9, no cytochromes a a 3 and b are detectable. The progressive loss of cytochromes a a 3 and b and the increase in cytochrome c is typical for senescent Kauaian strains. When this strain is crossed as a female parent most progeny show the same decline in growth potential when propagated vegetatively. This strain deviates from what appears as normal kalilo senescence when the mtDNA is examined. M t D N A was isolated from subcultures 1, 2, 4, 6, and 8. The mtDNA was digested with Bgl II and hybridized with the probe Hind III-K1. The K l clone was used as a probe because at the time the hybridizations were performed this was the only clone available that consisted of the majority of ka lDNA sequences (ie: 7.0kb of the 9.0kb element). The mtDNA sequences of the Bgl 11-12 restriction fragment are associated with this clone (see Figure 9 for the location of this clone). The Hind III-K1 probe hybridizes with novel Bgl II bands in subcultures 2 and 6 (Figure 44) . No novel bands are detected in subcultures 1, 4, and 8. In subculture 2, the sizes of the novel high molecular weight fragments are the same size as the junction fragments for an insert within the intron of the large r R N A gene. By subculture 4, this insertion is lost. Reoccurrence of ka lDNA is seen in subculture 6. In this subculture, two inserts are present based on the hybridization of K l with the four high molecular weight novel fragments. The sizes of the two lower molecular weight bands are similar with the junction fragments b l ' and b2' of the neutral insert previously described and thus it is postulated that these two Chapter 3 / 1 9 5 bands are junction fragments ( b l 1 7 and b 2 1 7 ) of one of the inserts and the two higher molecular weight bands junction fragments ( b l 1 8 and b 2 1 8 ) of the other insert. Clones encompassing almost the entire mtDNA were used as probes to determine the location of the insert with junction fragments b l 1 8 and b 2 1 8 . None of the mtDNA clones hybridized with these bands. From the ethidium bromide stained gel one of the two mtplasmids characteristic of this strain is absent in subculture 6 (Figure 44). It is postulated that the junction fragments belong to an insert carried on this mtplasmid. By subculture 8 both inserts are lost. To further investigate this strain, three ascospore-derived series were studied. The ascospores were isolated from a cross using culture 573-1 as the female parent and the nonsenescent strain 1766 as the male parent. The ascospores designated 1 and 7 die within 10 subcultures. Ascospore 5 did not express senescence even after 80 subcultures. In the two senescent ascospore series, mtDNA was isolated from alternate subcultures, commencing with subculture 2. The corresponding cytochrome spectra are shown in Figures 45 and 46. For ascospore 1, a normal cytochrome complement is observed throughout the entire series (Figure 45). Ascospore 7 shows a normal cytochrome spectra through to subculture 6. By subculture 8, an abnormal absorbance profile is observed (Figure 46). The Bgl II digested mtDNA profiles are shown in Figures 47 and 48. Analysis of the ethidium bromide stained gels reveals that the Bgl II mtDNA patterns Chapter 3 / 1 9 6 are indistinguishable from the nonsenescent control P605. Hybridization of the mtDNA with either the E, Hind III-K1, or B probes shows the absence of mtlS-kalDNA in all subcultures, even after two week autoradiograph exposures. Hybridizations using the intron probe shows no differences between strain P605 and the mtDNAs of these two series. The cytochrome spectra of subcultures 2, 6, and 76 for ascospore 5 are shown in Figure 49. No deficiencies in cytochromes a a 3 or b are observed throughout the series. M t D N A was isolated from these same subcultures. Figures 50 and 51 show Hind III and Bgl II digestions of the mtDNA respectively. These gels were hybridized with the E probe. One Hind III band and two Bgl II bands hybridized with the E probe. The Hind III-K1 probe was hybridized with the Bgl II digested mtDNA. The only fragment hybridizing is the Bgl 11-12 fragment (Figure 51). This hybridization indicates that the K l region of ka lDNA has been deleted. The presence of only one novel Hind III band (Figure 50) implies that the Hind III restriction site delineating K l and K 2 is included in the deletion. In addition, the terminal inverted repeat sequences of K 2 must be deleted otherwise the K l probe, carrying the other terminal inverted repeat, would have hybridized with one of the novel Bgl II fragments (Figure 51). It is difficult to discern the location of the insertion in this series because the novel Hind III band hybridizes with three different radioactively-labeled mtDNA clones, Hind III-10a, Hind 111-13,18, and Hind 111-14,15, which are contiguous within the mtDNA and constitute approximately 3.5kb (refer to the mtDNA restriction map, Figure 6). The fact that the novel Hind III band hybridizes with all three mtDNA probes suggests that in addition to the deletion of the majority of mtlS-kalDNA, a Chapter 3 / 197 rearrangement event has occurred. Southern analysis of uncut nucDNA from the original P573 series and its three ascospore derivatives is shown in Figure 52. A l l panels represent two week autoradiograph exposures using E as the probe. Panel A shows that AR-ka lDNA is present in all subcultures of the original P573 series. Panel B shows that AR-kalDNA is barely detectable in most subcultures of the ascospore 1 series. Interestingly, the band hybridizing in subculture 2 is of a higher molecular weight than normal. In the series derived from ascospore 7 (Panel C), AR-kalDNA is only seen in subculture 6. Panel D is the series derived from ascospore 5 which showed no sign of senescence even after 80 subcultures. No AR-kalDNA is detected. It should be noticed that in all subcultures in which AR-kalDNA is detected it is in a copy number far below that generally encountered in kalilo strains. Seven ascospore-derived series which exhibited long lifespans were analyzed for the presence of AR-kalDNA and mtlS-kalDNA, and for abnormal cytochrome complements. As with ascospores 1, 5, and 7, these ascospores were initiated from cross 573-1 X 1766. The lengths of each series are shown diagrammatically in Figure 53. The number of subcultures preceding death ranged from 30 subcultures to no signs of senescence after 80 subcultures. Cytochrome spectra were prepared from the late subcultures of each ascospore series. Abnormal spectra were observed for ascospores 2, 4, 15, and 19 (Figure 54). The other three cultures had normal cytochrome complements. Chapter 3 / 1 9 8 M t D N A and nucDNA were prepared from the late subcultures of each ascospore series. The mtDNA was digested with Bgl II and hybridized with Pst I-kalDNA (Figure 55). In all cultures, no novel Bgl II fragments are detected. Only fragments Bgl 11-10 and -12 hybridized with the probe. The nucDNA from the late cultures was cut with Eco R l and hybridized with the Pst I-kalDNA probe (Figure 56). The late cultures of ascospores 4, 15, and 19 showed detectable amounts of fragments hybridizing with the probe. Ascospore 9 also showed trace amounts of fragments of the same mobility as those of ascospores 4 and 19. The restriction fragments hybridizing in ascospores 4, 9, and 19 are the two internal fragments of ka lDNA (E and G) and the terminal inverted repeats which co-migrate. These bands are of the appropriate sizes indicating that AR-kalDNA is intact. Only one band of a slightly faster mobility than the fragment E hybridized in the late cultures of ascospore 15 (designated by the arrow). This indicates that all Eco R l restriction sites are gone. In all the cultures in which AR-ka lDNA is detected, it is present in a copy number below that generally encountered in kalilo strains. Chapter 3 / 199 Figure 43. Cytochrome spectra from mitochondria isolated from various subcultures of a series derived from the natural isolate P573. The number of the serial transfer is given above the corresponding spectrum. The cytochromes are identified as c, b, and aa 3 . C h a p t e r 3 / 200 •train 573 I W A V I I E N O T H (nm) Chapter 3 / 2 0 1 Figure 44. Gel electrophoresis of Bgl Et digested mtDNA from various subcultures of a series derived from the natural isolate P573. The first lane of the ethidium bromide stained gel and the autoradiograph represents the nonsenescent control P605. The numbers above the remaining lanes are the subcultures of the series from which mtDNA was prepared. The autoradiograph is a hybridization using the Hind DI-K1 probe (for details on this probe refer to Figure 9). The fragments designated bl and b2 are of an insert detected in the mtDNA prepared from subculture 2. The bands labeled b l 1 7 and b 2 1 7 ,and b l 1 8 and b 2 1 8 are junction fragments of two different inserts detected in the mtDNA prepared from subculture 6. Bands b3 and b4 are internal Bgl H restriction fragments of kalDNA. The fragment denoted 12 is a mtDNA Bgl Et restriction fragment The designation, plDNA, refers to the plasmid harbored in the mitochondria of strain P573. 2o2_ Ser ies 573 § 1 2 4 6 8 § 1 2 4 6 8 b l / 8 b 2 8 p i D N A • b l b l 7 b 2 b 2 7 b 3 b4 12 E t B r K l Chapter 3 / 203 Figure 45. Cytochrome spectra from mitochondria isolated from various subcultures of a series derived from ascospore 1 from cross 573-1 X 1766. The number of the serial transfer is given above the corresponding spectrum. The cytochromes are identified as c, b, and aa 3 . C h a p t e r 3 / 204 •Kotpor* 1 WAVELENGTH (nm) Chapter 3 / 205 Figure 46. Cytochrome spectra from mitochondria isolated from various subcultures of a series derived from ascospore 7 from cross 573-1 X 1766. The number of the serial transfer is given above the corresponding spectrum. The cytochromes are identified as c, b, and aa 3 . C h a p t e r 3 / 206 WAVELENGTH (nm) Chapter 3 / 207 Figure 47. Gel electrophoresis of Bgl Et digested mtDNA from various subcultures of a series derived from ascospore 1 from cross 573-1 X 1766. The first lane of the ethidium bromide stained gel and each autoradiograph represents the nonsenescent control P605. The numbers above the remaining lanes are the subcultures of the series from which mtDNA was prepared. Three autoradiographs are presented. The probes Eco Rl-E and Eco Rl-B were used to detect for the presence of kalDNA sequences. For details on these probes refer to Figure 9. In addition the mtDNA was hybridized with the Hind HI-13,18 probe (refer to Figure 8 for the location of this region of the mtDNA). The fragments designated 4, 12, and 14 are mtDNA Bgl Et restriction fragments. The mtplasmid is identified by the arrow. E t B r E m 2 4 6 8 § 2 4 6 8 12 14 H 1 3 , 1 8 B Chapter 3 / 209 Figure 48. Gel electrophoresis of Bgl II digested mtDNA from various subcultures of a series derived from ascospore 7 from cross 573-1 X 1766. The first lane of the ethidium " bromide stained gel and each autoradiograph represents the nonsenescent control P605. The numbers above the remaining lanes are the subcultures of the series from which mtDNA was prepared. Three autoradiographs are presented. The probes Eco Rl-E and Hind HI-K1 were used to detect for the presence of kalDNA sequences. For details on these probes refer to Figure 9. In addition the mtDNA of this was hybridized with the Hind HI-13,18 probe (refer to Figure 8 for the location of this region of the mtDNA). The fragments designated 4, 12, and 14 are mtDNA Bgl II restriction fragments. The mtplasmid is identified by the arrow. A s c o s p o r e 7 m m § 2 4 6 8 1 0 § 2 4 6 8 1 0 E r B r 10 § 2 4 6 8 I O S 2 4 6 8 c 12 1 4 H13.18 K l Chapter 3 / 2 1 1 Figure 49. Cytochrome spectra from mitochondria isolated from various subcultures of a series derived from the ascospore 5 from cross 573-1 X 1766. The number of the serial transfer is given above the corresponding spectrum. The cytochromes are identified as c, b, and aa 3 . C h a p t e r 3 / 212 Chapter 3 / 2 1 3 Figure 50. Gel electrophoresis of Hind HI digested mtDNA from subcultures 2 and 76 of a series derived from ascospore 5 from cross 573-1 X 1766. The first lane of the ethidium bromide stained gel and each autoradiograph represents the nonsenescent control P605. Four autoradiographs are presented. The Eco Rl-E probe was used to detect for the presence of kalDNA sequences. For details on these probes refer to Figure 9. The band identified by the arrow was observed to hybridize with the Eco Rl-E probe. To locate this band within the mtDNA hybridizations using the Hind HI-13,18, Hind EQ-14,15, and Hind HI-10a probes was performed. Refer to Figure 8 for the location of these regions of the mtDNA. The fragments designated 12, 15, 16, and 20 are mtDNA Bgl H restriction fragments. A s c o s p o r e 5 in m m m m o o o o o o 2 76 o 2 76 o 2 76 O 2 76 O 2 76 E t B r E H 1 3 , 1 8 H 1 4 , 1 5 H l O a Chapter 3 / 2 1 5 Figure 51. Gel electrophoresis of Bgl II digested mtDNA from various subcultures of a series derived from ascospore 5 from cross 573-1 X 1766. The first lane of the ethidium bromide stained gel and each autoradiograph represents the nonsenescent control P605. The numbers above the remaining lanes are the subcultures of the series from which mtDNA was prepared. Four autoradiographs are presented. The Eco Rl-E and Hind EQ-K1 probes were used to detect for the presence of kalDNA sequences. For details on these probes refer to Figure 9. The bands identified as b l , and b2 2 hybridized with these two probes. In addition the mtDNA was hybridized with the Hind HI-13,18 probe (refer to Figure 8 for the location of this region of the mtDNA). The fragments designated 4, 12, and 14 are mtDNA Bgl H restriction fragments. Ascospore 5 m 2 2 6 76 m § 2 6 76 ID o O 2 6 76 m 3 2 6 76 £5 " S 3 % » # * - ~ -f i E t B r K l E H13,18 Chapter 3 / 2 1 7 Figure 52. Southern hybridization analysis of uncut nucDNA to detect for the presence of AR-kalDNA. NucDNA was prepared from various subcultures of the three ascopore series, 1, 7, and 5, and the natural isolate P573. Hybridization to AR-kalDNA was performed using the Eco Rl-E probe (refer to Figure 9 for details on this probe). 218 A B C D 573 spo re 1 spore 7 s p o r e 5 2 4 6 8 2 4 6 8 2 4 6 8 10 2 6 76 K a l Chapter 3 / 2 1 9 Figure 53. Subculture series for long The ascospores are from cross 573-1 each series were derived is labeled 0. bar indicates the subculture which showing no signs of senescence even bar followed by two dots. ascospore series showing growth cessation. X 1766. The ascsopore culture from which The last number spanned by a horizontal produced no viable conidia. Those series after 80 subcultures are represented by a 573-1? X 1766 9 s u b c u l t u r e number ^ 0 10 2 0 3 0 4 0 5 0 6 0 8 a s c o s p o r e 2 n 4 • 9 m 15 M 18 H 19 m 2 0 m O Chapter 3 / 2 2 1 Figure 54. Cytochrome spectra from mitochondria isolated from the late cultures of a series derived from ascospores 2, 4, 15, and 19 from cross 573-1 X 1766. The number of the serial transfer is given above the corresponding spectrum. The cytochromes are identified as c, b, and aa 3 . Chapter 3 / 222 WAVELENGTH Chapter 3 / 223 Figure 55. Southern hybridizatin analysis of Bgl II digested mtDNA from the late cultures of series derived from ascospores 2, 4, 9, 15, 18, 19, and 20 from cross 573-1 X 1766. The first lane of the autoradiograph represents the nonsenescent control P605. The numbers above the remaining lanes are the ascospore series numbers followed by the subculture from which mtDNA was prepared. The autoradiograph is a hybridization to the mtDNA using the Pst I-kalDNA probe (refer to Figure 9 for details on this probe). The fragments designated 10 and 12 are mtDNA Bgl II restriction fragments. K a l Chapter 3 / 225 Figure 56. Southern hybridization analysis of Eco R l digested nucDNA to detect for the presence of AR-kalDNA. NucDNA was isolated from the late cultures of series derived from ascospores 2, 4, 9, 15, 18, 19, and 20 from cross 573-1 X 1766. The first lane of the autoradiograph represents the nonsenescent control P605. The numbers above the remaining lanes are ascospore series numbers followed by the subculture from which mtDNA was prepared. The autoradiograph is a hybridization using the Pst I-kalDNA probe. The bands referred to as E and G are internal Eco R l restriction fragments of kalDNA. The designation LTR corresponds to the long terminal repeats generated after Eco R l digestion of AR-kalDNA. The arrow identifies a deleted form of AR-kalDNA in ascospore 15. 5 7 3 spores o m in w w in t> jh g J O K a l Chapter 3 / 227 C. DISCUSSION The prototype case of senescence in N . intermedia was provided by previous studies on a small sample of isolates from the island of Kauai (Bertrand et al, 1985). Among the isolates initially characterized, strain P573 showed genetic characteristics of kalilo strains but the lifespans of ascospore-derived series were observed to be quite variable with the average lifespan of the ascospore series being greater than 20 subcultures (Griffiths and Bertrand, 1984). Further analysis of the P573 natural isolate has revealed that the progressive loss of cytochromes a a 3 and b and the decrease in growth potential are typical characteristics of both kalilo strains and 'stopper' extranuclear mutants of N . crassa (Bertrand et al, 1976; Bertrand et al, 1980; DeVries et al, 1981; Reick et al, 1982; Gross et al, 1984; Bertrand et al 1985; Bertrand et al, 1986). The strain diverges from the normal patterns of kalilo senescence when the mtDNA and nucDNA are analyzed. This strain shows only trace amounts of AR-kalDNA, and mtlS-kalDNA is seen only erratically. According to the proposed mechanism of senescence, mtlS-kalDNA is usually observed to accumulate during vegetative growth and at the time of death be equimolar with the mtDNA (Bertrand et al, 1985). In contrast, mtlS-kalDNA does not accumulate and in the late subculture of series 573 no mtlS-kalDNA is detected, even after long autoradiograph exposures. Analysis of ascospores from the cross 573-1 X 1766 show this strain to be even more anomalous than expected. NucDNA and mtDNA were isolated from alternate subcultures of two senescent ascospore-derived series. Ascospore 1 is exceptional in showing a normal cytochrome complement and no mtlS-kalDNA. Chapter 3 / 228 This ascospore series shows traces of AR-kalDNA as observed in the original P573 culture. In subculture 2 of this series AR-ka lDNA exhibits a slower mobility relative to normal AR-ka lDNA (Figure 52) suggesting that either the element contains more D N A sequences than normal, is circular, or the proteinase K treatment was incomplete. The latter hypothesis is preferred since it is know that AR-kalDNA has protein associated with its ends (Chan B-S, Doctoral Student, personal communication) and if these proteins are not removed prior to electrophoresis AR-ka lDNA shows a slower mobility. Ascospore 7 shows different patterns upon senescence. Again, no mtlS-kalDNA is detected, yet deficiencies in cytochromes a a 3 and b become apparent as senescence proceeds. Interestingly, AR-kalDNA is seen in only one subculture of this series. Together these results indicate that neither AR-ka lDNA nor mtlS-kalDNA need be retained in order for senescence to be expressed. In addition, normal cytochrome complements in ascospore 1 and deficiencies in cytochromes a a 3 and b in ascospore 7 suggest that senescence may or may not be mitochondrially-based, yet it is maternally inherited. In general, the presence of normal cytochrome complements is an indicator of wild type mtDNA suggesting that although senescence in strain P573 is maternally inherited, the expression of senescence is not always accompanied by the accumulation of grossly defective mtDNA molecules. The loss of cytochromes a a 3 and b in the series derived from ascospore 7 can be explained by postulating that subtle alterations to the mtDNA occurred which went undetected in Eco R l , Bgl II, and Hind III digests. It has been shown (Bertrand and Pittenger, 1972) that point mutations to the mtDNA of N . crassa do result in their accumulation and the loss of cytochromes a a 3 and b. Chapter 3 / 2 2 9 A n ascospore series which did not die even after 80 subcultures was also chosen for examination. As expected, cytochrome analysis revealed normal absorption spectra. M t D N A and nucDNA were prepared from three of the subcultures of this series. No AR-kalDNA was detected, yet about lkb of mtlS-kalDNA was present in the mtDNA preparations. Based on hybridizations using the Hind III-K1 probe, it is concluded that the majority of mtlS-kalDNA has been deleted. The Hind I1I-K1 probe consists of approximately 7.0kb of the kalDNA element and includes one of the 1300bp inverted repeats. Absence of hybridization of the Hind III-K1 probe to novel Bgl II bands indicates that in addition to the K l sequences, the other inverted repeat has been deleted. This leaves approximately 300bp of ka lDNA inserted in the mtDNA. This deletion would result in the loss of the three Bgl II and the Hind III restriction sites. Only one novel Hind III restriction fragment is observed which corresponds with the loss of the Hind III restriction site in kalDNA. The presence of two Bgl II novel bands hybridizing with the E probe is an anomaly because the deletion of the majority of ka lDNA should include all three Bgl II restriction sites. The presence of two Bgl II fragments may be explained by postulating that a Bgl II restriction site has been generated in the remaining sequences of kalDNA. This would account for two novel Bgl II fragments both hybridizing with the E probe. The hybridization of the Hind III-10a, Hind 111-13,18, and Hind 111-14,15 probes with the novel Hind III band suggests that a rearrangement event involving all three restriction fragments must have occurred. It is difficult to determine the arrangement of the altered mtDNA and kalDNA, but whatever the arrangement, the altered mtDNA molecules should, theoretically, induce the suppressive accumulation of the defective mtDNA molecules and initiate senescence. Whether this insert and the Chapter 3 / 2 3 0 defects to the mtDNA may be considered neutral as described for the insert with junction fragments b l ' and b2' in the previous two chapters is undecided. Sequencing is required to determine the organization of this region of the mtDNA and to understand why this mtDNA alteration does not induce the suppressive accumulation of these altered mtDNA molecules. Observations made from the late subcultures of other ascospore series also show different combinations of usual and unusual kalilo properties. Of the late subcultures examined, no mtlS-kalDNA is detected and of the seven ascospore, only three show normal AR-kalDNA. The cytochrome spectra for only four of the series showed deficiencies in cytochromes a a 3 and b. There is no correlation between the presence of kalDNA and cytochrome deficiencies in these seven ascospore series. The observation that the presence of both AR-kalDNA and mtlS-kalDNA is erratic suggests that strain P573 may be predisposed to eliminating kalDNA from its genome. In all series analyzed, mtlS-kalDNA never appeared to accumulate yet, in some cases, it was present throughout a series. This may indicate that rather than having a predisposition to remove kalDNA from the genome, the accumulation of mtlS-kalDNA and the replication and retention of AR-kalDNA ma}' be suppressed such that normal kalilo patterns of senescence are not observed. Thus, it would appear that the mechanism of senescence in strain P573 and its derivatives does not involve kalDNA, but rather some other mechanism not presently understood. It should be noted that although the causal agent of senescence does not appear to be kalDNA, kalDNA is sexually and Chapter 3 / 2 3 1 somatically transmitted and thus persists in this strain. It has been demonstrated that in strain P573 senescence is transmissible (Griffiths and Bertrand, 1984) indicating that senescence cannot be due to the same unknown mechanism at work in the class of senescent strains, previously described, which show nonheritable senescence (Griffiths et al, 1988). Perhaps this Kauaian strain defines a fifth class of senescence in Neurospora. The transmission of mtlS-kalDNA described in this chapter appears to be exclusive to strain P573. This is interesting because in the chapter to follow, strain P573 is the only senescenct Kauaian strain to contain detectable amounts of double stranded R N A (dsRNA). Although this dsRNA shows no homology with the Pst I-kalDNA probe (refer to Chapter 4), it is possible that this dsRNA may affect the behaviour of ka lDNA to give the results described. How these two elements would interact to produce these atypical aging patterns is unknown. V I . C H A P T E R 4 A . INTRODUCTION Kalilo D N A shows no homology with either mtDNA or nucDNA indicating that it is foreign in origin (Bertrand et al, 1985; 1986). It was of interest to determine whether ka lDNA has a viral origin. A number of fungal viruses contain R N A genomes which are double stranded or contain double stranded replicative intermediates (Dodds et al, 1984). Thus double stranded R N A (dsRNA) is relevant mainly as a indicator of the presence of potential viruses. A survey of Kauaian as well as nonKauaian natural isolates of Neurospora was undertaken to detect for the presence of dsRNA. A total of nine Kauaian senescent strains were included in the survey. Of these, only one Kauaian strain, P573, showed the presence of dsRNA which did not hybridize with the Pst I-kalDNA probe. Although it appears that the presence of dsRNA is not relevant to kalilo senescence, Neurospora is an ideal organism for potential studies on fungal viruses. First, the convenient genetic system and the availability of many well defined mutant stocks (Perkins et al, 1982) would facilitate studies on the interaction of host and viral genomes. Second, hundreds of natural isolates have been collected from around the world. These strains are very few subcultures away from the fungus growing in nature so they should reflect well the array of fundamental genetic elements that abound in natural populations. Examples of elements discovered by such surveys are the mitochondrial plasmids (Collins et al, 1981; Nargang et al, 1983; Lambowitz et al, 1985; Akins et al, 1986; Nargang, 1986; Lambowitz et al; 1987), optional mitochondrial introns (Collins and 232 Chapter 4 / 2 3 3 Lambowitz, 1983; Nargang et al, 1984), and the senescence determining elements ka lDNA and marDNA (Griffiths and Bertrand, 1984; Bertrand et al, 1985; Griffiths et al, 1986; Bertrand et al, 1986; Griffiths unpublished). There are a few well documented cases in which dsRNA viruses alter the phenotype of the host fungus. The fungi Saccharomyces cerevisiae and Ustilago  maydis contain a number of dsRNA segments which are responsible for the killer phenotype of certain strains. The viruses encode and secrete toxic proteins lethal to sensitive strains of the same species or closely related species (Hankin and Puhalla, 1971; Koltin and Day, 1975; Rogers and Bevan, 1978; Bussey, 1981; Wickner, 1981; 1983; Tipper and Bostian, 1984; Peery et al, 1987). In the cultivated mushroom Agaricus bisporus, at least five dsRNA viruses have been implicated as the casual agents of L a France disease (Tavantzis et al, 1980). In some pathogenic fungi there is an association between the presence of dsRNA and a decline in pathogenicity; see for example, the fungus Endothia parasitica which is responsible for chestnut blight (Day et al, 1977; Van Alfen, 1982; Fulbright, 1984; Elliston, 1985; L'Hostis et al, 1985) and the fungus Rhizoctonia  solani which is a pathogen of many chlorophyllous plants (Castanho et al, 1978; Zanzinger et al, 1984). Virus-like particles (VLPs) have been identified in three slow growing strains of Neurospora (Tuveson and Peterson, 1972; Kuntzel et al, 1973; Turna and Grones, 1983). The three strains are designated abnormal-1, mi-1, and 2215 (P147). The mutants abnormal-1 and mi-1 contain V L P s which have single stranded R N A genomes (Turna and Grones, 1983; Kuntzel et al, 1973). The Chapter 4 / 2 3 4 properties of the V L P genome in strain 2215 have not been determined and consequently this strain is included in the present survey. The survey revealed dsRNA of various sizes in seven strains of Neurospora. Seven distinct dsRNAs were detected which show patterns of homology with each other. Homology of genomic D N A with one of the dsRNA species was detected. B. RESULTS 1. Identification and Cross Homologies of the dsRNAs D s R N A analysis was conducted on 36 wild type strains of Neurospora. The geographic origin of each strain is listed in Table II. Chapter 4 / 2 3 5 T a b l e II. G e o o r a p h i c o r i g i n and s t o c k number of w i l d t y p e  i s o l a t e s of Neurospora c r a s s a * and N. i n t e r m e d i a S t r a i n D e s i g n a t i o n A u s t r a l i a T o v n s v i l l e - 1 C o n t i n e n t a l U . S . A . L a b e l l e - 1 b M a u r i c e v i l e - l c StocIt Number 1833 1940 2225* I n d i a A a r e y - l e V a r k u d - l c I n d o n e s i a B e s a k i h - 1 B e s a k i h - l c Bogor P a s a r G i a n j o r - l c J a k a r t a - 1 Ratnpong Babakan T a r o n g o n g T j i k i n i P a s a r J a p a n N o r t h A f r i c a I P a c i f i c I s l a n d s F i j i F i j i N 6 - 6 H a w a i i a n I s l a n d s H a n a l e i - 1 f L i h u e - 3 b Hanapepe H a n a l e i P e o p l e ' s R e p u b l i c o f C h i n a Beijing H a r b i n H e f e i S o u t h A m e r i c a Monte A l e g r e - 1 T a i w a n T a i p e i - l c T a i p e i - l g 2499 1832 1826 1827 2215 1836 1881 2562 2557 P10 430* 435 P561 P572 P573 P801 P765 P776 P785 2360 2365 3720 3722 3977 3983 3980 3336 1766 1767 O r i g i n T o w n s v i l l e , Q u e e n s l a n d L a b e l l e , F l o r i d a K a u r i c e v i l l e , T e x a s Bombay, M a h a r a s h t r a V a r k u d , K a r n a t a k a B e s a k i h , B a l i B e s a k i h , B a l i P a s a r B o g o r , B o g o r G i a n j o r , B a l i J a k a r t a , J a v a Bandung T j i k i n i P a s a r , J a k a r t a U n r e n , J a p a n A d i o p o d o u m e , I v o r y C o a s t F i j i K a u a i K a u a i K a u a i K a u a i Oahu M a u i M a u i H a n a l e i , K a u a i L i h u e , K a u a i H a n a p e p e , K a u a i H a n a l e i , K a u a i B e i j i n g H a r b i n , H e i l o n g j i a n g H e f e i , A n h u i M o n t e A l e g r e , B r a z i l T a i p e i T a i p e i Chapter 4 / 2 3 6 Twenty-four of the strains chosen for the survey exhibited degenerative growth phenotypes during vegetative propagation. The other 12 strains showed no discernible change in phenotype. DsRNA was detected in seven of the 36 strains. The dsRNA bands for each strain are shown in the ethidium bromide-strained gel presented in Figure 57, panel A . The number and mobility of the dsRNAs is seen to be variable between the 7 strains. Listed in Table III are the sizes of dsRNAs for each strain. In order to determine if the 9.0kb dsRNAs show homology, the dsRNA from strain P573 was isolated, end-labeled, and used to probe Northern blots of the dsRNAs from each strain. Figure 58, panel A shows that there is homology between the 9.0kb dsRNAs present in the three strains. It should be noticed that the P573 labeled dsRNA does not hybridize with the 2.0kb or 500bp dsRNAs of strain 3336 indicating that there are at least two different dsRNA, species in this strain. The three dsRNAs from strain 3336 were pooled, end-labeled, and used to probe the dsRNAs (Figure 58, panel B). In addition to the 9.0kb dsRNAs of strains P10 and P573, the probe also hybridized to the 18kb dsRNA of strain 435 and the 500bp dsRNA of strain 1833. Hybridization to only the 500bp dsRNA in strain 1833 and to only the 18kb dsRNA in strain 435 indicates that these two strains each carry two different dsRNA species. Furthermore, negative hybridization between the 9.0kb dsRNA and the 18kb, 2.0kb and 500bp dsRNAs Chapter 4 / 2 3 7 (Figure 58, panel A) indicates that it is the 2.0kb and 500bp dsRNAs of strain 3336 that are homologous with the 500bp dsRNA of strain 1833 and the 18kb dsRNA of strain 435. The 3.0kb dsRNA from strain 1833 was used as a probe and hybridized exclusively to the 3.0kb dsRNA from strain P776 (Figure 58, panel C). Hybridization of the dsRNAs with either the 7kb dsRNA in strain 2215 or the 9.5 kb dsRNA in strain 435 was not performed. It is suspected that these two dsRNAs are not homologous such that seven distinct dsRNAs are delineated. The cross homologies of the dsRNAs are represented by the letters 'a' through V in Figure 57, panel B. Chapter 4 / 2 3 8 Table III. Growth phenotype, geographic o r i g i n , and s i z e s of dsRNAs of seven i s o l a t e s of Neurospora S t r a i n S i z e s of dsRNAs Growth Phenotype Geographic O r i a i n P10 2215 435 P573 P776 1833 3336 9.0kb 7.0kb 18kb 9.5kb 9.0kb 3.lUb 3.0*b 500bp *9.0kb 2.0kb 500bp normal slow growth normal senescent slow growth, senescent slow growth, senescent c o l o n i a l growth, senescent TJnzen, Japan J a v a , Indonesia F i j i K a u a i , USA Maui, USA Queensland, A u s t r a l i a Monte A l e g r e , B r a z i l Chapter 4 / 239 Figure 57. A. Gel electrophoresis of dsRNA preparations from seven natural isolates of Neurospora. B. A diagrammatic view of the cross homologies of the seven distinct dsRNAs, represented by the letters 'a' through V . A A A A N|<CO oow £ crcrrj of?z Chapter 4 / 2 4 1 Figure 58. Cross hybridizations of the dsRNAs. A. The dsRNA of strain P573 used as a probe. B. The pooled dsRNAs of strain 3336 used as a probe. C. The 3.0kb dsRNA of strain 1833 used as a probe. 7 4 A P10 2 2 1 5 4 3 5 P 5 7 3 P 7 7 6 1 8 3 3 3 3 3 6 • CO K" O" 7 4 A P I O 2 2 1 5 4 3 5 P 5 7 3 P 7 7 6 1 8 3 3 3 3 3 6 Chapter 4 / 243 Figure 59. Southern hybridization of genomic DNA from various natural isolates hybridized with the 9kb dsRNA of strain P573. A A A A — 1 0 -bo* • • • • Ui O in in 7r cr o- o-o-Chapter 4 / 245 Figure 60. Northern hybridization of the dsRNAs with Pst I-kalDNA probe. Lane 1 contains mtDNA prepared from the senescent strain P561 used as a positive control. 5 6 1 P I O 2 2 1 5 4 3 5 P 5 7 3 P 7 7 6 1 8 3 3 3 3 3 6 C. DISCUSSION Chapter 4 / 2 4 7 The survey of 36 wild type strains of Neurospora identified seven strains which carry detectable amounts of dsRNA. A total of seven dsRNA species were detected among these seven strains. Figure 60, panel B shows the seven different dsRNA species and their cross homologies. The 9.0kb dsRNA species is present in strains P10, P573, and 3336. The second species is the 7kb dsRNA found only in strain 2215. The dsRNA with cross homologies designated 'c' include the 18kb dsRNA in strain 435, the 500bp dsRNA in strains 1833 and 3336, and the 2.0kb dsRNA in strain 3336. The variable sizes of these dsRNAs account for three of the dsRNA species identified. The 9.5kb dsRNA found only in strain 435 is the sixth dsRNA species discovered. The seventh dsRNA species is the 3.0kb dsRNA common in strains P776 and 1833. From Figure 60B, it is evident that strains 435, 1833, and 3336 each carry two different species of dsRNA. The presence of different dsRNA species in the same strain has been observed in other systems. The killer strains of S. cerevisiae (reviewed by Bussey, 1981; Wickner, 1981; 1983; Tipper and Bostian, 1984) and U . maydis ( Koltin et al, 1980; Peery et al, 1982; Dalton et al, 1985; Peery et al, 1987) each contain several distinct dsRNA species. The genome of the various types of Ustilago viruses consist of 3 to 7 different dsRNA segments which are involved in encoding toxin, conferring immunity to the toxin, and production of the viral capsid. The killer strains of S. cerevisiae have 9 distinct dsRNAs which encode similar products as in U . maydis. Chapter 4 / 248 Cross hybridization of the 18kb dsRNA of strain 435 with the 2.0kb dsRNA of strain 3336 and the 500bp dsRNA of strains 3336 and 1833 suggests that the smaller dsRNAs were derived from deletion of most of the 18kb dsRNA. Cryptic dsRNAs, generated by deletion of the original dsRNA, have been observed in R N A preparations from Endothia parasitica (Tartaglia et al, 1986), from wound tumour virus (Nuss and Summer, 1984), and from S. cerevisiae (Fried and Fink, 1978; Bruenn and Brennan, 1980; Thiele et al, 1984; Lee et al, 1986). In all these examples, the cryptic dsRNAs consist of the termini of the original dsRNA. We have not determined what region of the 18kb dsRNA from strain 435 is retained in the 2.0kb and 500bp dsRNAs. Genomic D N A prepared from a number of strains included in the survey was hybridized with the different dsRNA species to detect for genomic sequences homologous to the dsRNAs. The 9.0kb dsRNA in strains P10, P573, and 3336 was the only dsRNA which hybridized with genomic D N A . In all strains, a 4.5kb and a 2.0kb Eco R l restriction fragment of the genomic D N A hybridized with the 9.0kb dsRNA probe. In addition, a 6.5kb Eco RI fragment in the Hawaiian isolates hybridized with the 9.0kb dsRNA probe. It is possible that this additional region of the D N A originated from a duplication and restriction site polymorphism. Sequence homology between a 9.0kb dsRNA and a 6.5kb stretch of genomic D N A prepared from all strains tested suggests that the 9.0kb dsRNA may have originated through the transcription of the 6.5kb region of the genomic D N A in the distant past. If presently transcribed, then the normal transcription of 6.5kb of D N A cannot account for a 9.0kb dsRNA indicating that aberrant transcription within the 6.5kb D N A may have occurred generating, for example, Chapter 4 / 2 4 9 an R N A consisting of two copies of the transcribed region. Alternatively, bands also hybridizing with the 9.0kb dsRNA probe may comigrate with the 4.5kb and/or 2.0kb Eco R l fragments upon gel electrophoresis thus accounting for the appropriate sized transcription region to generate a 9.0kb transcript. Cross hybridization of genomic D N A and dsRNA has been reported by Wakarchuk and Hamilton (1985). They showed that a high molecular weight dsRNA in Phaseolus  vulgarus L . 'Black Turtle' (BTS) hybridizes to the BTS genome as well as to the genomes of other bean cultivars. No homology was detected between the 3.0kb dsRNA from strains P776 and 1833 and genomic D N A implicating a viral origin for the 3.0kb dsRNA. The slow growth and senescent phenotypes shared by these two strains suggests that the 3.0kb dsRNA may be responsible for these altered phenotypes. Senescence in strain P776 has been characterized at a molecular level and it is known that senescence in this strain is initiated by the insertion of ka lDNA into the mitochondrial D N A (Griffiths et al, 1988). Senescence of strain 1833 results from a yet unknown phenomenon not related to kalDNA senescence (Griffiths et al, 1988). Thus, if the 3.0kb dsRNA does alter normal growth then it could be responsible for the slow growth phenotype observed in both these strains. The 7kb dsRNA from strain 2215 does not hybridize with genomic D N A suggesting that this dsRNA too is of viral origin. Virus particles have been identified in this strain (Tuveson and Peterson, 1972). Isolation of these virus particles, and the preparation and characterization of the viral genome will determine if the dsRNA isolated from total nucleic acid preparations constitute the Chapter 4 / 2 5 0 viral genome. The origin of the 2.0kb and 500bp dsRNAs in strain 3336, the 18kb and 9.5kb dsRNAs in strain 435, and the 500bp dsRNA in strain 1833 are not known. Lack of homology of these dsRNAs with genomic D N A suggests that they are also viral in origin. Virus particles will have to be identified and isolated to discern the origin of these dsRNAs. The previous chapter described the atypical behaviour of kalDNA in strain P573. In strain P573, the observations that both AR-ka lDNA and mtlS-kalDNA are seen only erratically and that this is the only senescent Kauaian strain tested which contains dsRNA implies that these two elements may somehow interact to give the atypical aging patterns described in the previous chapter. Exactly how this interaction would be conducted is unknown. The seven strains in this survey that contain dsRNA are from different geographic locations (Table 2). The presence of the 9.0kb dsRNA in strains isolated from Monte Alegre, Brazil, from the island of Kauai, U S A , and from Unzen, Japan indicates that the proposed altered transcription mechanism generating the 9.0kb dsRNA may not be all that uncommon in Neurospora. Alternatively if the 9.0kb dsRNA is an ancestral transcript, then these three strains probably had a common ancestor. The 3.0kb dsRNA is present in strains also collected from different geographical locations. Strain P776 was isolated from the island of Maui, U S A and the strain 1833 from Queensland, Australia. The presence of the 3.0kb dsRNA in strains collected from different geographic areas Chapter 4 / 2 5 1 suggests a common ancestor which acquired and transmitted the 3.0kb dsRNA. A similar hypothesis can be proposed for strains 435, 1833, and 3336 which carry homologous dsRNA sequences except that deletion of the majority of the dsRNA occurred in the ancestor of strains 1833 and 3336. Two of the strains, 2215 and 435, found to contain dsRNA showed no altered phenotype. It is known that the presence of dsRNA and/or virus particles in fungi is not necessarily associated with an altered phenotype in the host (Dodds et al, 1984). In vitro studies using dsRNA from various systems has demonstrated the involvement of dsRNA in some cellular processes. For example, dsRNA inhibits cell-free translation (Burke, 1977), induces interferon production (Burke, 1977), and is involved in the regulation of gene expression (Travers, 1984). Perhaps the dsRNAs in phenotypically normal fungi play a similar role. From a geographic survey, seven strains of Neurospora have been identified that contain dsRNA. Cross hybridizations reveal seven different dsRNA species among the seven strains. A t present, we do not know the biological significance of the dsRNAs. VII . C O N C L U S I O N Mitochondrial aging in a number of organisms has received much attention. This is primarily because mitochondria are considered the 'power house' of the cell and changes at the biochemical or genetic level are detrimental to all obligate aerobes. In fungi such as N . intermedia, N . crassa, P. anserina, and A .  amstelodami, alterations of the mtDNA have proven to be the events responsible for causing degenerative growth, sometimes resulting in death, and mitochondrial biochemical deficiencies. Although mtDNA damage causing altered growth patterns is relatively well understood in these fungi, fungal systems have not yet been validated as models for aging in higher eukaryotes. Nonetheless, there are some striking similarities between the mitochondrial functions associated with aging of higher organisms (Finch and Hayflick, 1977) and fungi suggesting that a genetically programmed instability of the mtDNA may exist which determines longevity and the occurrence of senescence in higher organisms. A t present verification of this hypothesis would be difficult to attain because aging in different tissues and cell lineages in higher eukaryotes might proceed at different rates and produce a great diversity of mtDNA defects. Alternatively, mitochondrial aging may be associated with only a particular tissue type and consequently go undetected. Although this hypothesis is difficult to test, characterization of mtDNA defects as they relate to the growth of fungi does suggest another mechanism by which aging may occur in higher organisms. 252 Conclusion / 253 The work reported in this thesis was on mitochondrial-based senescence of the fungus N . intermedia. The major findings reported in this thesis are summarized below: 1) K a l D N A is capable of assuming new locations within the mtDNA. • In all series analyzed, novel insertion of ka lDNA was apparent. The sites of insertion were generally within functionally important regions of the mtDNA. Some clustering of integration sites was observed. 2) The appearance of novel insertions is strongly correlated with longevity. This correlation supports the notion that insertion of ka lDNA is the ultimate cause of death. Furthermore, the point in a subculture series when an insert is observed determines the length of a given series. 3) A third form of ka lDNA has been identified which is a free element in the mitochondria. This form of ka lDNA is termed mtFF-kalDNA. It is suspected that mtFF-kalDNA is an intermediate in novel insertion. Conclusion / 254 4) Novel insertion appears to depend on two criteria: a) the age of the female parent; only when a presenescent subculture is used were novel insertions detected b) the host nuclear genotype; outcrossing resulted in an increased appearance of novel insertions 5) The senescent Kauaian strain P573 and its derivatives show some but not all characteristics of kalilo senescence. The inserted form of ka lDNA, mtlS-kalDNA, was seen only erratically and AR-ka lDNA seen often but in low copy number. It is suspected that ka lDNA is not responsible for the fate of strain P573 and its derivatives but rather some other unknown factor is responsible. 5) DsRNA has been detected in seven strains of Neurospora. Among seven geographically distincy strains of Neurospora, seven species of dsRNA were delineated. Although the presence of dsRNA is not associated with kalilo senescence, Neurospora is an ideal organism to determine the significance of the presence of dsRNA in fungi in general. The information presented in this thesis reveals the complexity of kalilo senescence where it is characterized by a mobile element which resides in two Conclusion / 255 different compartments within the cell which apparently exhibits both inter- and intra-compartmental movement. A general model of the molecular events involved in the initiation and propagation of senescence are shown in Figure 61. Kalilo senescence differs from senescence of P. anserina and A. amstelodami in that it involves the insertion of foreign D N A into the mtDNA rather than the excision of specific regions of the mtDNA. Together, these observations indicate that kalilo senescence involves a unique sequence of events not observed in other organisms and suggests an alternative model for aging in filamentous fungi and perhaps an example of one mechanism of aging in higher eukaryotes. Knowledge on kalilo senescence is increasing, but as of yet neither de novo insertion nor intramitochondrial movement of ka lDNA have been demonstrated. In general, the overall molecular events resulting in senescence have not been fully determined. In order to answer these questions, transformations using ka lDNA are required. Initial transformation experiments will determine unequivocally that kalDNA is the cause of senescence. Once demonstrated, transformations in conjunction with in vitro mutagenesis will allow one to follow the behaviour of ka lDNA during vegetative and sexual growth and thus determine the sequence of molecular events responsible for the senescent fates of Kauaian strains of N . intermedia. Conclusion / 256 Figure 61. A flow chart summarizing the molecular events associated with senescence of Kauaian strains of N. intermedia. AR-kalDNA is the nucleus-associated linear plasmid. MtlS-kalDNA is the mitochondrial inserted form of kalDNA. MtFF-kalDNA is the mitochondrial free form of kalDNA. A R - k a l D N A " C Y T O S O L m t F F - k a l D N A \ m t l S - k a l D N A accummulation of m t D N A w i t h m t l S - k a l D N A loss of " • r e s p i r a t o r y po ten t ia l M I T O C H O N D R I A • • D E A T H n o. 3 o i—1 c CO H-O 3 B I B L I O G R A P H Y Akins, R .A. , and Lambowitz, A . 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