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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 CAROLYN J. MYERS  A THESIS  SUBMITTED IN P A R T I A L F U L F I L M E N T OF  T H E REQUIREMENTS FOR T H E DEGREE OF DOCTOR O F  PHILOSOPHY  in T H E F A C U L T Y O F G R A D U A T E STUDIES Genetics Programme  We accept this thesis as to the  conforming  required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A 23 March  0  1988  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  ^yrTHfctsJ ^  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 equimolar  with  the  insert  accumulate  and  mitochondrial D N A . The  death  occurs when  inserted  form  the  insert  of kalilo  is  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 DNA,  from  intramitochondrial movement  mtFF-kalDNA,  insertion  of  transmitted  kalilo  is  predicted  D N A appears  to  be  to  and an  depend  an  autonomous  intermediate on  the  in  of kalilo  movement.  form  sexually. If a mutagenic insert is transmitted,  form  of  senescence  Novel  mtlS-kalDNA 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 senescence  or less. Transmission of a nonmutagenic  insert delays the  until either a novel insertion or a rearrangement  of the  onset of  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  characteristics behaviour  of  Kauaian of  kalilo  strain  was  identified  senescence.  mtlS-kalDNA  is  erratic  In  this  and  ii  which strain  in , some  shows and  some its  cultures  but  not  derivatives, the  all the  characteristic  mitochondrial biochemical deficiencies, normally accompanying kalilo senescence, not observed. It is suspected  that k a l D N A  is not responsible  are  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  k a l D N A has a viral origin. Only one senescent strain contains detectable  amounts  of  dsRNA  The  survey  identified six nonKauaian strains which contain dsRNA and seven dsRNA  species  were  which  delineated.  senescence,  was  not  homologous  Although the  analysis  of  dsRNA  presence in  a  with  of  a  kalDNA  dsRNA  is  genetically-well  not  probe.  relevant  defined  to  organism  kalilo like  Neurospora may give insight into the significance of dsRNA in fungi in general.  iii  T A B L E OF CONTENTS Abstract  ii  Table of Contents  iv  List of Tables  vi  List of Figures  vii  Acknowledgements  xii  I. Introduction A . Neurospora L I F E C Y C L E .. B. C Y T O P L A S M I C M U T A T I O N S O F F U N G I 1. Petite Mutations of Saccharomyces cerevisiae 2. The ragged Mutation of Aspergillus amstelodami 3. Senescence in Podospora anserina 4. Cytoplasmic Mutations of Neurospora C. P E R S P E C T I V E O N S E N E S C E N C E  1 1 5 6 9 10 21 34  II. Materials and Methods 1. Strains 2. Media and Growth Conditions 3. Nucleic Acid Isolations a. Mitochondrial D N A Isolation b. Nuclear D N A Isolation c. dsRNA Isolation 4. Cytochrome Analysis 5. Restriction Enzyme Digestion and Gel Electrophoresis 6. Labelling of Nucleic Acid a. Nick Translation b. End-labelling of dsRNA 7. Probes 8. Blot Hybridization a. Southern Blot Analysis b. Northern Blot Analysis  39 39 39 40 40 43 44 45 45 46 46 47 48 48 48 53  HI. Chapter 1 A. INTRODUCTION B. RESULTS 1. Transmission of mtlS-kalDNA in Ascospores Initiated from Cross 561-1 X 1766 a. Correlation Between Time of k a l D N A Insertion and Onset of Senescence b. Movement of m t l S - k a l D N A and Identification of a Transient Mitochondrial Autonomous Form of k a l D N A 72 2. Transmission of mtlS-kalDNA in Ascospores Initiated F r o m Other Crosses  54 54 55  iv  58 70 ... 74  a. Ascospore Series from Cross 801-1 X 1836 b. Ascospore Series from Cross 572-5 X 1818 C. D I S C U S S I O N IV. Chapter 2 A. INTRODUCTION B. RESULTS 1. Proposed Genetic Regulation of kalDNA Movement 2. Comparison of Tetrads From Crosses Using a Juvenile Female Parent and a Senescent Female Parent C. D I S C U S S I O N 1. Genetic Regulation of mtlS-kalDNA Movement 2. Comparison of Tetrads Derived From Crosses Using a Juvenile Female Parent and a Senescent Female Parent V . Chapter 3 A. INTRODUCTION B. RESULTS C. D I S C U S S I O N  75 76 131 141 141 143 143 149 182 182 189 192 192 193 227  V I . Chapter 4 A. INTRODUCTION B. RESULTS 1. Identification and Cross Homologies of the dsRNAs 2. Homology with Genomic D N A 3. Hybridizations Using the Pst I-kalDNA Probe C. D I S C U S S I O N  232 232 234 234 238 238 247  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 Locations of the Excised D N A  Showing the 12  Figure 3. M t D N A Restriction Map of Podospora anserina Showing the Locations of the senDNAs Figure 4. The Influence  15  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 Figure 6. M t D N A Restriction Map of Neurospora intermedia Sites of Insertion  25  Showing the  of kalilo D N A  29  Figure 7. Restriction Map of kalilo D N A  33  Materials and Methods  39  Figure 8. Restriction Map the Regions of the Figure 9. Restriction Map Insertions Showing used as probes Chapter 1 Figure 10. Subculture  of the m t D N A of Neurospora intermedia Showing m t D N A Used as Probes of the m t D N A and With two Different k a l D N A the Regions of k a l D N A and Flanking m t D N A  50 52 54  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 m t D N A location of novel insertion in series 4  86  Figure 13. Analysis of Bgl II digested m t D N A s from subcultures of series 7 of cross 561-1X1766  88  Figure 14. Southern analysis showing the m t D N A location of novel insertion in series 7  90  Figure 15. Analysis of Bgl II digested m t D N A s from subcultures of series 8 of cross 561-1X1766  92  vii  Figure 16. Southern analysis showing the m t D N A 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 m t D N A 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 m t D N A 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 m t D N A 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 m t D N A s from series 4 of cross 561-1X1766  '.  112  Figure 26. Southern analysis of mtDNAs from DNase treated mitochondria. Figure 27. Comparison of the Mobilities of A R - k a l D N A and the Autonomous Mitochondrial Form of k a l D N A . Figure 28. Southern analysis of Bgl II digested mtDNAs from the early and late cultures of the three 801 ascospore series ;  ..114  116 118  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 m t D N A 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 Chapter 2  130 141  Figure 35. Lengths of subculture series for tetrads from crosses 561-0 X 605 and 561-5 X 605 Figure 36. Lengths of subculture series for tetrads from crosses 561-0 X 1766 and 561-5 X 1766.  155 157  Figure 3 7A. Analysis of Bgl II digested m t D N A s from the ascospore cultures of ascus 1 from cross 561-0 X 605.  159  Figure 37B. Analysis of Bgl II digested m t D N A from the late cultures of ascus 1 from cross 561-0 X 605  161  Figure 3 8A. Analysis of Bgl II digested m t D N A from the ascospore cultures of ascus 7 from cross 561-0 X 1766 '.,  163  Figure 38B. Analysis of Bgl II digested m t D N A from the late cultures of ascus 7 from cross 561-0 X 1766  165  Figure 39A. Analysis of Bgl U. digested m t D N A 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 m t D N A from the ascospore cultures of ascus 6 from cross 561-5 X 605  171  Figure 40B. Analysis of Bgl II digested m t D N A from the late cultures of ascus 6 from cross 561-5 X 605  173  Figure 41 A . Analysis of Bgl II digested m t D N A from the ascospore cultures of ascus 4 from cross 561-5 X 1766  175  Figure 4 I B . Analysis of B g l II digested m t D N A from the late cultures of ascus 4 from cross 561-5 X 1766  177  Figure 42A. Analysis of Bgl II digested m t D N A from the ascospore cultures of ascus 3 from cross 561-5 X 1766  179  Figure 42B. Analysis of Bgl II digested m t D N A from the late cultures of ascus 3 from cross 561-5 X 1766  181  ix  Chapter 3 Figure 43. Cytochrome  192 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 m t D N A s 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 m t D N A s from subcultures of ascospore 5 from cross 573-1 X 1766  214  Figure 51. Analysis of Bgl Et digested m t D N A 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  242  of dsRNAs 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 thesis.  major  Special  suggestions, committee  asset to my graduate  thanks  and Dr. T.  are  support.  extended I  Grigliatti,  wish  to  to  training and the  Dr.  thank  Dr. B.R. Green,  H . Bertrand the  for  members  Dr. J .  completion of my  of  his my  McPherson,  generosity, supervisory  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 thesis a reality.  xii  help to make the  completion of my  I. INTRODUCTION  A.  NEUROSPORA  LIFE  CYCLE  It was of interest to investigate the transmission of k a l D N A 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  Class  Ascomycete. The  ascomycetes  most  of the  species  which  to the  have  feature of this group is the  Kindom  are  the  been  ascus  Fungi and is a member  largest  widely  class  used  which encloses  of fungi  in genetics.  the  products  and  of the provide  The principal of meiosis,  the  ascopores; there are four or, following an extra mitotic division, eight in number. For  example,  contains  eight  in N . crassa, ascospores.  N . sitophila, and Neurospora  N . intermedia  possesses  a  mycelial  a  mature  form  of  ascus cellular  organization where the mycelium has septa which delineate hyphal  compartments.  Within  central  each  compartment  are  several  nuclei.  The  septa  have  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 propagation  (reviewed by Beadle,  1945). These  1  species  are  asexual  all heterothallic  and  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  On  A. and _a_ and  suitable  crossing  fruiting  bodies,  hyphae  which  become  are  destined  filaments  are  medium,  codominant  either  protoperithecia.  alleles of the  mating  type  is  Protoperithecia,  surrounded  by  to  ascogeneous  become  a  thick  mating type  capable  of  producing  consist  of  coiled  layer  of  hyphae.  hyphae.  gene.  From  female  filaments The  each  of  coiled  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  asexual spores called macroconidia or the less abundant  vegetative  hyphae,  microconidia. It has  or been  proposed that trichogyne growth and localization of the male fertilizing cell is a chemotactic male  response  fertilizing  fertilizing  cell,  initiated by the  cell (Bistis, the  1986). After  nucleus  from  trichogyne into the ascogeneous a  number  of synchronous  ascogenous blackened  the  of a pheromone  released  with  melanin  trichogyne and the  male  transferred  cell  is  through  Nuclear hyphal  fusion  to  maturity,  give a  eventually and  compartment  the  total  of eight  asci elongate  perithecium. The ascospores  male the  small mass of dikaryotic  forms  karyogamy of the  the  mature  eventually  ascogenous  fruiting occurs  hyphae.  and  nuclei which eject  germinate  their under  form  spores  the  through  becomes  body,  between  Immediately  karyogamy, meiosis occurs and the four products of meiosis undergo mitosis  the  hypha. The paternal and maternal nuclei undergo  mitotic divisions to form a  and  by  fusion of the  hyphae. A t the same time the protoperithecium enlarges and  perithecium. penultimate  presence  eight the  high temperatures,  the the after  a round of  ascospores.  At  ostiole  the  of  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).  Introduction / 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 segregation  of  different  inheritance  of  genes.  unordered  phenotypes  in  Furthermore,  the  a  tetrad  can  phenotypic  of ascospores.  detemine  ratios  the  pattern  of  in either  ordered  or  tetrads provide information on various chromosome mutations  nondisjunction,  translocation,  and  inversion, and  gene  The  mutations  such  including  as  gene  conversion. Reciprocal crosses are possible since any strain of Neurospora may be used  as either  ascospores  a male or female parent. Thus, analysis of tetrads or  from  extranuclear  reciprocal crosses  inheritance.  inheritance,  and  therefore  In  aids  in  Neurospora, extranuclear  distinguishing between  the  cytoplasm  inheritance  can  shows be  random  nuclear  strict  and  maternal  distinguished  from  nuclear inheritance based on reciprocal differences in crosses.  B. CYTOPLASMIC  The  topic  MUTATIONS  described  in  this  OF  thesis  FUNGI  is  mitochondrially-based  intermedia. A number of examples of cytoplasmic mutations and  in  this  section  the  most  characterized  examples  senescence have been  described  in  in N . reported  fungi  are  reported  by  presented.  The first observation of non-Mendelian patterns of inheritance  was  Introduction / 6 Correns  (1909)  chloroplast  and  Baur  development  (1909).  in  some  They strains  discovered of  that  flowering  a  factor  plants  (  influencing  Mirabilis  and  Pelargonium) does not follow the normal patterns of Mendelian inheritance. With time many cases of cytoplasmic inheritance were discovered in organisms ranged from  unicelluar algae  plants  as  such  extranuclear  Triticum  (Briggle,  inheritance was  chloroplasts, how they  ( Chlamydomonas, Sager, 1966).  Although  apparent, little was  a  which  1954) to complex higher number  known about  of examples of mitochondria and  interact with nuclear genes, and their importance in the  development of a given organism. The first evidence showing that mitochondria replicate (1964)  and on  information  possess D N A was Neurospora.  Direct  reported evidence  was shown by Diacumakos et  by Luck that  (1963) and  Luck  mitochondria  al (1965). They used  and  contain  Reich genetic  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  Cytoplasm was isolated from Both  the  maternally inherited.  this strain and injected into a wild type recipient.  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.  nonreverting  pleiotropic mutants containing  large deletions of the m t D N A  (p-) or no m t D N A  at all ( p ° ) (Dujon, 1981). In  contrast  which  to  These  strains  filamentous  fungi  are  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 m t D N A  retained  is  variable and usually less than one third of the genome (Dujon, 1981). Since the total  amount  from  which  suggested  of mtDNA it  was  in a  derived,  that the number  p- mutant  is similar  irrespective  of  the  of copies of the  to the  size  retained  of  wild type  the  mtDNA  deletion,  mtDNA  it  was  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 m t D N A mutants (Dujon, 1981). Generally head-to-tail are  observed when the  the  other  the  conserved  other  type  retained  mtDNA  of arrangement, sequence.  p- mutants  are  the  Usually, observed  repeats of the  is less than  repeated  to  contain  conserved sequences  approximately  unit is an  these inverted  sequences in p-  repeats  mixtures  lOOObp. In  inverted duplication of are  of the  not  perfect.  repeats  where  repetition of the conserved sequences may be direct or inverted along the molecule. In addition to these two major rearrangements  may  be  observed  arrangements of the  including  internal  retained  inversions,  Some the same  mtDNA,  deletions  or  illegitimate recombination between different p- mutants (Dujon, 1981).  The degree of suppressiveness, of  p-  mutant  cells,  is  that is the proportion of zygotic clones composed  characteristic  of  a  given  p-  mutant.  (Ephrussi  and  Introduction / 8 Grandchamp, the  1965). Two models were proposed to explain the mode of action of  suppressive  based  on  the  petite m t D N A destructive  in heteroplasmic  recombination  of  crosses.  These  type  mtDNA  wild  include a model 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 the  been suggested  as  a model for suppressiveness,  petite m t D N A in diploid progeny  the  demonstration  from a cross are both physically (Goursot  et al, 1980; Blanc and Dujon, 1980) and genetically (Gingold, those of the  petite parent has  that  focussed  attention  on the  1981) similar to  'out-replication' model.  This model is based on a replicative advantage of petite m t D N A s (Slonimski, al  1968;  Rank,  1970a;  1970b;  Rank  and  Bech  Hansen,  1972;  Carnevali  et and  Leoni, 1981). The 'out-replication' model has received much support from analysis of the  nature of the m t D N A  of very strongly suppressive  petites.  These  petite  m t D N A 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). was  suggested  that  the  high  sequences in highly suppressive the  petite mtDNAs  reiteration  zygotes.  It  zygotes was  crosses  in  further  1986).  medium In  this  containing  (Chambers  and  medium  incorporate  label into their newly synthesized  of  replication  reflects  suggested  the  radioactively only zygotes  to  that p-  lower degree of  analysis of p+  achieved by growing mating mixtures  selective  Gingold,  origin  a replicative advantage  was  degrees of suppressiveness  of  of the origin of replication sequences. Further  heteroplasmic grande  of  petites would confer  in heteroplasmic  mutants showing lesser reiteration  degree  It  and p-  from petite by labeled could grow  uracil and  mtDNA. M t D N A was isolated from  Introduction / 9 the  cultures,  cut  restriction fragments  with  restriction  enzymes,  and  visualized by autoradiography  newly  synthesized  mtDNA  of gels. By focussing on bands  unique to the petite and the grande m t D N A s , the authors were able to ascertain the  relative  determine the  amounts  label  incorporated  their relative level of synthesis  'out-replication'  Hypersuppressive p+  of  mtDNA  hypothesis  does  not  into  in the hold  the  two  mtDNA  species  and  zygote. It was discovered that  true  for  all  suppressive  petites.  petites did exhibit a competitive replication advantage over  molecules, but less suppressive  same pattern. Interestingly,  a  strain  petites do not necessarily  which  was  essentially  show  nonsuppressive  the this gave  an indication of replicative superiority. Thus, the simple 'out-replication' hypothesis does  not  offer  an  explanation  other  than destructive  for  these  inconsistancies.  Clearly,  some  factor(s)  recombination of wild type m t D N A 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 1959; Caten, high frequency  1972). The ragged and 'ragged'  phenotype  arises  appearance (Jinks  spontaneously  at a  1956;  reasonably  mutants characterized by cytochrome deficiencies and  m t D N A rearrangements. Analysis of the m t D N A of 'ragged' mutants revealed the presence  of high  cultures.  A l l the  molecular novel  weight  DNA  D N A which  species  consist  is of  not  observed  tandem  in  repeats  wild that  type are  Introduction / 10 homologous with the m t D N A  (Lazarus et al, 1980). In all but one mutant, the  amplified m t D N A 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.  sequence maps between an unidentified reading frame  This  215bp  (corresponding to U R F 4 of  human mtDNA) and an arginine t R N A gene. It was postulated that the common 215bp  sequence  structure  contains  similar  to  an  yeast  origin and  of replication because  human  mitochondrial  formed (Lazarus et al, 1981). It was suggested origin  of replication sequences could confer  a  hairpin  origin  secondary  sequences  can  be  that these excised and amplified  a replicative advantage to the  high  molecular weight m t D N A species and explain the progressive decline in growth at the  periphery  of  the  mycelial  colony.  The  one  mutant  not  included  possesses a m t D N A sequence excised from the region of the m t D N A  above  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  senescent cultures 1959).  Mycelial  and  from  senescence  juvenile  cultures  with  hyphal fusion experiments is  race  specific  (Rizet,  race A expresses senescence more rapidly than  cytoplasm  (Marcou and  1957;  other  isolated  Marcou,  races.  Schecroun,  1961)  It should be  that two races have been studied in detail, race A and race s.  from  where noted  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 R l ; 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 m t D N A 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 D N A s  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, The  most  frequent  sen  plasmid  is  alpha.  Other  sen  plasmids,  not  1984).  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  that  of  has  shown  no  signs  senescence  even  after  four  years  mutant  of culturing.  Analysis of the m t D N A of this mutant revealed that the sequences of the intron, which normally excise to generate alpha senDNA, are this mutant  absent in the  m t D N A of  (Vierny et al, 1982).  A m t D N A restriction map and the location of four of the sen plasmids is shown in  Figure  cytochrome  3.  Alpha  oxidase  senDNA  subunit  I  consists gene  of  the  intron  (COI gene)  Osiewacz and Esser, 1984). The excision of the the  exon-intron  revealed  that  junction  it belongs  fragments. to  the  Sequence  at  (Cummings  the  5'  and  end  of  Wright,  the  1983;  2.5kb intron occurs precisely at analysis  so called class  of  II introns.  alpha  senDNA  To digress  has  slightly,  introns of fungal m t D N A are classified as class I or class II depending on their overall secondary structure  and the presence  et  mitochondrial  al,  1982).  Class  I  of short conserved sequences (Michel  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  Tetrahymena  thermophila  Dujon,  their  ability  to  nuclear  self-splice  rRNA  intron  as  has  been  (Cech et  al,  shown  for  the  1983; Michel  and  1983; Waring et al, 1983; Garriga and Lambowitz, 1984; V a n der Horst  and Tabak,  1985; Peebles et al, 1986). Class II mitochondrial introns have been  found to contain long open reading frames  (Bonitz et  1984; Osiewacs and Esser, 1984; Michel and Lang, related to reverse transcriptase  al, 1980; Nargang et al,  1985) which encode  proteins  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, A has an extra class II intron referred 1983).  This intron  does not  share  race  to as intron A (Cumrnings and Wright,  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 could  account  races.  for the  This hypothesis  introns  contain  transcriptase  difference was  nucleotide  in the  rates the  of senescence  stimulated  by  finding  sequence  homologies  with  between  these  that these two group the  retroviral  s, two II  reverse  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 sequences  has  been  identified  suggesting  sen plasmid homologous with intron A 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  transcriptase suggest  of the  class  II  intron, evidence  that there is reverse  activity in senescent race A cultures and not in young cultures does  that  alpha  senDNA  may  be  generated  through  an  RNA  intermediate  (Steinhilber and Cummings, 1986).  A more plausible model to explain the amplification of senDNAs is based on the hypothesis mtDNA.  that  sen  plasmids  Lazdins and  may  Cummings  show  (1982)  superior showed  replication  that  cloned  relative alpha,  to  the  beta,  and  gamma senDNAs , as well as those young m t D N A 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  the  senDNAs  may  identification  results depend  of only  indicate on  five  the sen  that  the  presence plasmids  amplification  of origin may  define  of  small  circular  of replication sequences the  regions  of the  and  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  m t D N A . It has  that beta senDNA does not contain either spacer  mtDNA  sequences  (Figure  been reported  (Cummings et al, 1987)  intron or exon sequences but  3). For epsilon senDNA,  the  5'  rather  excision site  Introduction / 18 occurs within  the  intron of the  U R F 1 gene  and the  3'  excision  site in the  spacer m t D N A . 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 the  senDNAs  intronic splicing  senDNAs may  are  still  are  not composed solely of intron sequences  apparatus  excised from  be  involved  is probably not the  the  in the  mtDNA.  Indeed  generation  suggests  mechanism by which  the  of these  intronic splicing senDNAs,  but  that these  apparatus  in  an  illicit  manner.  The  onset  number 1980).  of senescence  of nuclear These  in P. anserina  genes  genes are  (Tudzynski  appears  and  pleiotropic because  to depend  Esser,  1979;  on any  Esser  in addition to  and  one of a Tudzynski,  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  showed that senescent ascospores  from  shows  the  longest  lifespan.  Microinjection  experiments  mycelia were not able to infect i viv strains. Moreover,  a cross between  a  senescent  wild  type  female  and  an  i viv  male mutant revealed that i viv progeny did not express senescence whereas wild type progeny were senescent  the  (Tudzynski and Esser, 1979). It was suggested  that the i viv combination counteracts the senDNAs in their active form (Esser and Tudzynski, these  results  1980). M t D N A  indicate that  the  of i viv mutants excision  and/or  has not been investigated but the  probably suppressed by the various nuclear mutations.  amplification  of senDNA  is  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  TO  22  3a  »  / \ w vi«  1  tr _  • 3 3 0 •1250 • 4 0 0 • 5 0 0  n  • 780  >«  1  •0  •650  «r t» *f  90 ••  /  90  ;  CT  ca  •70  •400  •720  70  MS  •0  ?« 1O0  30  96 • 3 0 0 • 110 50-  470 &0  Introduction / 21 4. Cytoplasmic Mutations of Neurospora  Group I Cytoplasmic Mutations of Neurospora crassa  In  1952,  reported  the  first  cytoplasmic respiratory  (Mitchell and Mitchell,  deficient  mutant  1952). This mutant  of N . crassa  was  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 m t D N A and two potential sites of primary defect  being either  Subsequent  small r R N A  gene or  the  S-5  ribosomal protein  gene.  sequence analysis of mi-1 has identified a 4bp deletion in the coding  sequence of the The  the  deletion  mitochondrial small r R N A  results  in  synthesis  of  gene near the  aberrant  small  5' end of the rRNAs  where  exon. 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 r R N A al,  (Collins and Bertrand, 1978; Collins et al, 1979; Lambowitz et  1979). A nuclear suppressor,  referred  to as _f, was discovered (Bertrand and  Introduction / 22 Pittenger, mutants  1972b) yet  additional Bertrand the  which  has  six  no  suppresses  effect  nonallelic  on  the  the  nuclear  initial  growth  lag  cytochrome  content  of these  mutants.  An  isolated  (Kohout  and  suppressors  have  been  of  most  group  1976; Bertrand and Kohout, 1977; Collins, et al 1979) which  initial growth lag and alleviate respiratory  I  suppress  and cytochrome defects  of mi-1  and the other group I mutants.  Group II Cytoplasmic Mutations of Neurospora crassa  Group  II  1972a) rate  mutants  and  exn-5  include mi-3 (Mitchell (Bertrand et  and  are  deficient  completely  by  the  in  nuclear  al,  al, 1976). Both  cytochrome gene  et  su-1  aa . 3  1953; Bertrand  and  have  lag in growth  These  (Bertrand,  an  initial  phenotypes  1971).  The  are  Pittenger,  suppressed  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  no growth. Based on their stop-start 'stoppers'.  Stopper mutants  are  of growth and  growth pattern, these mutants  female  were termed  sterile and deficient in cytochromes  aa  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 m t D N A resulting in heteromorphic m t D N A populations (Bertrand et al, 1980; deVries et al, 1981). In four stoppers analyzed, the sizes of the retained m t D N A 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  contains  a  mtDNA  as  1984)  E35  has  been  studied  extensively.  In  21kb circular molecule consisting of one the^ predominant  (Figure  5).  A  at  frequency  the  of  or  form  (DeVries et  complementary  43kb  circular  near  two  the  directly  depends  on  al,  the  mutant  length of the  1981; Gross et  mtDNA  appears  al,  upon  the  that these circles arise by reciprocal  repeated  their  phase',  third of the  mtDNA  resumption of growth. It was demonstrated recombination  'stop  tRNA(met) sequences  rates  of  replication  from  and  the  origin  of  replication sequences unique to each m t D N A molecule. The 21kb and 43kb circles encompass the entire wild type m t D N A molecule.  In the the  other  majority  stoppers, of the  the  observation  mtDNA  'stop  mutations  phase' that  should  permit  altered  population during the  there is a competition between the  that the  defective  show  strong  resumption  and  molecules  form  phase'  suggested  that  intact m t D N A s  selection  of growth  'stop  mtDNA  and  for visa  such that cells in  nuclear versa  or for  extranuclear 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. forms the and Hind the rRNA  Physical map of the 21kb mitochondrial DNA of mutant E35 which predominant DNA species in 'stop-phase'. Included are Eco R l , Bgl II, III restriction of the region of the mtDNA retained. The bars denote 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.  were identified by their inability to grow the length of a These  variants  deficiencies,  exhibited  abnormal  erratic  stop-start  respiration,  abnormal  growth,  The  serial  subculture  technique  was  aa  mitochondrial ribosome  because  it  and  3  profiles,  b and  A more intense survey  protocol (Griffiths  used  variants  500mm growth tube.  cytochrome  the accumulation of unique m t D N A restriction fragments. was conducted using a serial subculture  These  and Bertrand,  allowed more  1984).  time  asexual propagation and ease of sampling and analyzing mycelium or conidia  for 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  maternally inherited. Proof of the maternal inheritance of senescence  was obtained  from the analysis of ascospores initiated from reciprocal crosses between and  nonsenescent  strains  that juvenile cultures  (Griffiths  of either  and  ascospores  but as a culture goes through the progress  through  the  Bertrand,  or conidia are  senescence  Their  senescent  studies  showed  normal phenotypically,  process, changes  cytoplasm resulting in death.  cytoplasm is rendered heterogeneous  1984).  are  During  originate in and  these  changes,  for the determinative factors, and the  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 m t D N A (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 m t D N A (Bertrand et  al,  1985;  Bertrand,  1987).  Refer  to  the  restriction  map  of N . intermedia  m t D N A (Figure 6) which shows the various locations of mtlS-kalDNA. In juvenile cultures  mtlS-kalDNA  is  present  mtDNA  (Bertrand  al,  1985).  et  show a progressive increase penultimate  subcultures,  The progressive progressive when  low  copy  prepared  number  from  relative  subsequent  is essentially equimolar with  potential of a  stoichiometric  the  molecules suggests that m t D N A  mtDNA.  and  The  the  (Bertrand et rRNA  respiratory  could  result  in  mtDNA. with  the  results  displacement  of  is at the level of mitochondrial division  deficiencies  al, 1986) If this is true,  genes  the  molecules carrying mtlS-kalDNA  pathway of mitochondria becomes disabled as a consequence that  In  death  or at the level of m t D N A replication is not known. Evidence that the  suggested  the  subcultures  molecules corresponds  senescent series  with  are suppressive. Whether suppressiveness  has  to  molecules carrying mtlS-kalDNA.  of normal m t D N A  decline in growth  normal m t D N A  MtDNA  mtlS-kalDNA  is  very  in m t D N A  displacement  mtlS-kalDNA  in  induce  mutations  'renegade'  the  of m t D N A  division  in structural  multiplication of  respiratory mutations  of mitochondria genes, t R N A  mutant  or  mitochondria  during growth displacing normal mitochondria.  Alternatively, efficient  rate  mtDNA of  molecules  replication  hypothesis  could  depend  replication  sequences  on  which  carrying  compared the would  with  presence be  mtlS-kalDNA normal of  associated  extra with  may mtDNA  exhibit  a  more  molecules.  mitochondrial mtlS-kalDNA.  origin  This of  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).  Bgl n[[ psi  r*  -  1 1  EcoRIl  •  1  3 8  |  K>c.11b  •  1 |  3  II  1  1  T . T. Y  I »  2 | 1 4 |  I  10.  |l7fl6|  10b  1  f  *oli-2*  olil co-2  '  ' S-rHNA co-3  *  UMi  Lr  UNA  •  •  I  4  12 14  ( 12a  c  Ha | l 9 (  4 3  "  —' •  H O  o o  Introduction / 30 analysis  has  indicated  that  there  are  no  kalDNA  sequences  homologous  mitochondrial origin of replication sequences (Chan B-S, Doctoral Student, communication). involve two  Alternatively, displacement  between  First, Manella  wild  type  physical markers insertions,  Rl-9,  normal  unidirectional gene conversion. Evidence for  sources:  two  of  are  and  and  mi-1  this  Lambowitz (1979)  mtDNAs  mtDNA  hypothesis  using  personal  molecules  investigated  of N . crassa  with  comes  the  as  may from  interaction  many  as  four  to distinguish the two types of mtDNAs. They discovered that one  of  identified  1200bp  as  sites  in the of  Eco R l - 5 and  high  frequency  the  other  unidirectional  500bp gene  in Eco  conversion  leading to their spread through the m t D N A population in heteroplasmons. Second, some  cultures  rRNA  gene.  of S. cerevisiae possess an This  intron  has  been  optional class  termed  omega.  I intron  of the  It  been  has  large shown  experimentally that omega encodes  a specific transposase which is active in the  gene  and  conversion  process  (Jacquier  Dujon,  activity, omega spreads through the m t D N A genes  that  lack  the  intron  in  zygotes  1985).  Through  this  transposase  population by invading large  containing  both  mtDNA  rRNA  types.  Only  preliminary sequence analysis of kalDNA has been performed (Chan B-S, Doctoral Student, to  decide  personal communication), and consequently whether  there  is  an  open  reading  it is impossible at this time  frame  potentially  encoding  an  endonuclease.  Since  kalDNA  is  unrelated  to  any  Hawaiian  strains  not  homologous  one of  of  the  with  normal  mitochondrial  N . intermedia,  it  was  D N A and  plasmids postulated  was  found  (mtplasmids) that  kalDNA  to  be  found  in  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  k a l D N A was discovered (Bertrand et al, 1986). This element is nucleus associated and called A R - k a l D N A . Analysis of m t D N A tetrad and  of ascospores the  initiated from  nonsenescent  mtlS-kalDNA  are  strain,  maternally  and nuclear D N A (nucDNA) from  a cross between P605,  showed  inherited. It  was  the  senescent  that further  a  strain, P561,  both  AR-kalDNA  and  shown  that A R - k a l D N A  exists in high copy number in both juvenile and senescent cultures and has been proposed as the  precursor  precedes the appearance  of mtlS-kalDNA  because  the  presence  of A R - k a l D N A  of mtlS-kalDNA in subculture series derived from  natural  isolates (Bertrand et al, 1986).  A  restriction  approximately  map  of  kalDNA  is  shown  in  Figure  7.  The  element  9.0kb in length and contains three Eco R l recognition sites,  is one  Hind III site, three Bgl II sites, and no Pst I restriction sites. Sequence analysis of k a l D N A 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  sequences all  strains  junctions  has  revealed  the  presence  which are postulated as recognition sequences analysed,  mtlS-kalDNA  is  flanked  by  long  of  pentanucleotide  for k a l D N A insertion. In inverted  repeats  of  the  in the  mtDNA  m t D N A (Bertrand, 1987).  The  inverted repeats of k a l D N A and flanking repeats generated  as a consequence of insertion of k a l D N A are characteristic of many elements. may  This  suggests that  the  intercompartmental  movement  involve similar mechanisms as for other mobile elements  transposable  of this  element  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  suggests to  the  kalDNA  element  appears  to  move  between  cell  compartments  that in addition to having transposon-like qualities, k a l D N A would have  possess  the  appropriate  signals  for  transport  and  translocation  across  mitochondrial membranes, and insertion into the m t D N A . So far no sequences for any of these functions have been identified (Chan B-S, Doctoral Student, personal communication).  A t 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  consequence,  present information  only  preliminary  on the  results  on  biological significance  kalilo  senescence.  of k a l D N A  its mode of action, and etiology still  remain unknown. From  obvious  complex  that  kalilo  senescence  is  a  phenomenon  the  and  As  a  in senescence, review it is 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. different in  levels of biological organization, this syndrome has different phenotypes  various  organisms,  but  all  metabolism resulting in cellular of age  With  after  the  are  prone  to  irreversible alterations  of  their  death. Experimental interest in senescence  came  development of techniques by which  vertebrate  cells could be  Introduction / 35 cultured 1925).  in vitro (Ebeling, 1913; Carrel and A  number  of provocative  theories  Ebeling,  have  been  1925; Cohn and Murray, put  forth  to explain  progressive decline in growth and the eventual death of higher eukaryotes. are  in  general  programmed (Hayflick hypothesis  two  and  1965;  opposing  aging  is  1972;  (Orgel,  theories:  an  Strehler  1963;  1970;  First,  extension et  al,  the  of  a  1971).  Comfort;  aging  process  normal Second,  1974)  is  which  'error  states  There  genetically  differentiation the  the  process  catastrophe'  that  cells  age  because of the accumulation of mutations. The detrimental mutations would be to functionally  indispensible  replication,  transcription,  cascading senescence  been  agent  in animals due  predicted  exists  Information  and  into catastrophy  proven difficult has  genes,  to  and  to the  serve  as  genes  encoding  translation.  These  errors  ultimately  animal  cell  cell  death.  culture  to  complexity of higher  proteins  would  quickly  Experimental  test  these  eukaryotes  involved in  two  and  explain  aging  compiled on the  (Curtis,  1971;  Comfort,  1974;  three fungi, A . amstelodami,  in a wealth of data on fungal  as model organisms  for the  multiply  research  on  theories  has  furthermore  that it is unlikely that a single principle or one  intermedia, have resulted well  and  such  causative  Hayflick,  P. anserina,  1975). and N .  senescence. They  exploration of senescence at the  it  may  genetic  and biochemical level. These fungi are well suited for studying aging because of their  ease in handling under  accessibility for genetic  laboratory  conditions, but  analysis. Furthermore,  mainly because of their  senescence can be studied  level of the whole organism since fungi are less complex than higher  at  the  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  identified at the onset of this research and  ascospores  available  for  organism  such  aging  and  higher  used  in the  analysis. as  identify  eukaryotes.  In  addition,  intermedia  in  anserina  and A . amstelodami.  of  This  is associated  indicates  that  can  isolates  presented  senescence  cultures  in this  in  a  thesis  were  already  genetically  well  defined  information  potential  on certain  causative  the  molecular  be  distinguished  The latter  with the  the  such that the majority of natural  of the  amplification of specific regions  intermedia  already  should provide  one  develops  and  N.  Analysis  Neurospora  perhaps  were  experiments  of senescent  events  occurring  from  those  senescent systems  of the  mtDNA,  agents  aspects of of aging in  as  senescence  occurring  events  whereas senescence  responsible  for  P.  involve the excision  insertion of a foreign D N A into the  molecular  in  in N . mtDNA.  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. focus  of  this  thesis  is  on  the  characterization  of  the  sexual  and  The  somatic  transmission of mtlS-kalDNA. The main objectives of this study were:  1) To determine arid  if a correlation exists between  longevity. For  genetically  ranges from less than  related  ascospores,  10 to over 20 subcultures  the the  appearance of mtlS-kalDNA time  of growth  cessation  (Griffiths and Bertrand,  1984).  Introduction / 37 It is suspected  that this variability is determined by the behaviour of k a l D N A .  This was investigated by following the senescent  ascospore  series.  The  somatic transmission of mtlS-kalDNA  time  of  appearance  of  mtlS-kalDNA  in 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  mtlS-kalDNA by  performed  to  decide  whether  the  appearance  relies on a particular host nuclear background. This was  comparing  ascospores  was  of  the  derived  somatic  from  crosses  transmission between  patterns  two  of  Kauaian  achieved  mtlS-kalDNA strains  and  of  between ascospores  derived from outcrossing.  3) To determine behaviour  of  subculture  of the  if the  sexual transmission of mtlS-kalDNA  mtlS-kalDNA. female  Ascospores  strain  from  a  P561 generally  cross live  affects  using  longer  a  than  the  somatic  presenescent those derived  from a cross using a senescent subculture of strain P561 (Griffiths and Bertrand, 1984). The sexual using ascospores  and  somatic  transmission  of mtlS-kalDNA  was  characterized  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 was  investigated  to  determine  if  the  sexual  and  somatic  mtlS-kalDNA is similar with other senescent Kauaian strains.  senescence  transmission  of  Introduction / 38 5) A survey for the presence of dsRNA was undertaken to determine if k a l D N A 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  four chapters to follow.  of kalilo senescence is presented in the  II. MATERIALS AND METHODS  1. Strains  Neurospora  intermedia  strains  P561,  collected from Kauai, Hawaii. Strains and  strain  1836 from  Indonesia.  P573,  and  P605  are  natural  isolates  1766 and 1818 were isolated from Taiwan,  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  Kansas City, Kansas. Ascospores described in Chapters  1 and  School,  3 were initiated  from crosses described by Griffiths and Bertrand (1984).  2. Media and Growth Conditions Vegetative  culturing  containing 75mm twice  2%  tubes. a  10  6  glucose Each  week.  cytochrome  was  (Vogel,  series  For  analysis,  performed  the liquid  exclusively  1956).  was  Serial  subcultured  growth  of  Vogel's  subcultures by  mass  mycelium  Vogel's medium  on  for  was  minimal  were  made  conidial  medium in  transfer  10  X  once  or  nucleic  acid  isolation  and  inoculated  with  approximately  conidia/ml and shaken at 200rpm for a minimum of 16 hours. Crosses were  performed on solidified Westergaard's deSerres  (1970). Cross designations  parent is written Mating  type  first  crossing medium as described by Davis and are such that the strain used as the  and followed by the  determination  of  ascospores  strain used  was  performed  Vegetative cultures and crosses were incubated at 25C.  39  as the on  female  conidial parent.  crossing  medium.  Materials and Methods / 40 Subculturing was  performed  subculture  derived  series  as  described  from  natural  by  Griffiths  isolates,  the  and  Bertrand  original  zero, for example 561-0. The same applies for ascospore  culture  (1984). is  In  number  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  was  this  suspension  harvested by suction  Unordered  then  filtration  asci were  added  to  liquid  Vogel's  medium.  Cultures  were  and stored on ice until needed.  collected using the  procedure  of Newcombe and  Griffiths  (1972) slightly modified for use with crosses on solid medium.  All  other  procedures  were  standard  for Neurospora and are  described by Davis  and deSerres (1970).  3. Nucleic Acid Isolations  a. Mitochondrial DNA  Two different  Isolation  protocols were employed for m t D N A  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,  p H 7.6,  then poured through  I m M EDTA),  was  initially  ground in a blender  and  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 l O m M Tris-HCl, p H 7.6 and O . l m M E D T A ) and overlayed with 55% sucrose and then 44% sucrose and  O.lmM  (both consisted of Ultra EDTA)  pure  sucrose  (Lambowitz, 1979). The  in l O m M  step gradients  Tris-HCl,  were  pH  centrifuged  7.6 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  was discarded and the EDTA.  One  tenth  for  15 minutes  pellet suspended  volume of 20%  in an  SS-34 rotor.  SDS was  added  volume of chloroform-amyl alcohol (24:1). Tubes  to  were  lyse  EDTA.  50%  ethanol)  the  and I m M  mitochondria.  phenol and a half  inverted gently  and  then  10,000rpm for 10 minutes in an SS-34 rotor at 25C. The aqueous  phase was removed and dialyzed overnight against ImM  supernatant  in 200mM Tris-HCl, p H 8.0  Extraction involved adding a half volume of Tris-HCl saturated  centrifuged at  The  l O m M Tris-HCl, p H 8.0  and  cesium chloride (0.8gm/ml) and 9ul/ml of bis-benzimide (lmg/ml in were  added  to  the  dialysate.  The  mixture  was  centrifuged  at  53,000rpm for a minimum of 20 hours in an 80Ti rotor. The m t D N A band was illuminated and collected under shortwave diluted mtDNA  three  fold  precipitated  with with  lOmM 2.5  U V light. The m t D N A  Tris-HCl, volumes  of  pH  8.0  ethanol  and  suspension  ImM EDTA  containing  and  was the  0.2M ammonium  Materials and Methods / 42 acetate.  Small scale m t D N A preparations culture. Harvested  mycelium was  (Myers et al, 1988b) required 200mls of liquid ground with acid washed  in isolation buffer. The mitochondrial pellet was suspended pure sucrose  in  lOmM  Tris-HCl,  Ph  44% sucrose.  The flotation gradients  7.6 were  in an SW50.1 rotor. Mitochondria were step gradient two  and  O.lmM  centrifuged  sand  tubes.  The  tubes  EDTA) at  centrifuged  and  layered with  45,000rpm  collected from the  were  suspended  in 70% sucrose (Ultra  interface  and diluted with 200mM Tris-HCl p H 7.6 and  microcentrifuge  and  for  1 hour  of the  two  I m M E D T A to fill  for  15  minutes  in  microcentrifuge to pellet the mitochondria. The supernatant was discarded and tubes drained. The mitochondria were suspended ImM SDS  a the  in 200mM Tris-HCl, p H 7.6 and  E D T A and pooled into one microcentrifuge tube. One tenth volume of 10% 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  2.5  containing  volumes  of ethanol  and centrifuged  for  0.2M ammonium  15 minutes. Approximately acetate  was  added  aqueous phase to precipitate the m t D N A . The precipitated m t D N A was in  40ul of  lOmM  Tris-HCl,  p H 8.0  and  ImM EDTA,  treated  to  the  suspended  with RNase A  (final concentration of 0.08 ug/ml), and incubated at 55C for 30-60 minutes. The reaction saturated  mixture phenol  was and  extracted one  half  once volume  with  one  half  volume  of  1M  chloroform-amyl alchohol (24:1)  Tris-HCl and  the  aqueous phase precipitated with 2.5 volumes ethanol containing 0.2M ammonium acetate.  Materials and Methods / 43 b. Nuclear DNA  NucDNA  Isolation  preparation  was performed  as described by Collins et al (1981). From  large scale mitochondrial isolations, approximately 1/20 of the harvested mycelium was  set  washed  aside sand  glycerol,  for and  0.25M  nucDNA  preparation.  suspended  EDTA,  in  The  A l buffer  0.5% Triton  mycelium was (1M sorbitol,  ground  with  acid  ficoll  400,  20%  7%  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 l O m M Tris-HCl, p H 7.6, I m M EDTA,  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  minutes  at 25C to precipitate the nucDNA. N u c D N A  for  minutes  10  in  an  SS-34  rotor.  The  pellet  and  let  stand  was pelleted at was  resuspended  for  10  10,000rpm in  lOmM  Tris-HCl, p H 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 10,000rpm for  10 minutes,  and  resuspended  in  lOmM  Tris-HCl  , p H 8.0  at and  ImM  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 l O m M Tris-HCl, p H 8.0 and I m M  Materials and Methods / 44 EDTA  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 S T E (1X= Tris-HCl, of  lOOmM NaCl,  10% SDS, and  tissue.  The  at  was  CF-11  cellulose  ethanol-STE buffer with  shaken  10,000rpm for  was collected and adjusted of  pH7.1) , 0.4ml of B-mercaptoethanol,  30ml of S T E saturated phenol were  mixture  centrifugation  ImM EDTA  vigorously  20 minutes  for  30  added  to the  minutes  at  in a G S A rotor the  10ml  powdered  25C.  After  aqueous phase  to 16% ethanol and poured onto a column of 2.5gm  powder.  The  column  was  washed  with  80ml  of  16%  (STE containing 16% ethanol v/v) and the nucleic acid eluted  15mls of S T E . The eluate was T l RNase digested  and then  50mM  DNase digested  (lU/ml) for 30 minutes  of Cellex N - l was added and shaken  at  (lU/ml) for 30 minutes  37C. Approximately 0.2gm  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 S T E . The dsRNA eluate was precipitated with  1/10 volume of 3 M 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  Tris-HCl  and  the  nucleic  ImM EDTA  acid.  The  and  cesium  nucleic  acid  chloride  was  (0.8  resuspended  gm/ml)  and  in  lOmM  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 l O m M Tris-HCl, p H 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 l O m M Tris-HCl, p H 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 mitochondria buffer  and  spectra set  were  aside  centrifuged  obtained  from at  the  as  described by Nargang et  mtDNA  preparations  were  10,000rpm for  15 minutes  in an  al  (1978). The  diluted in isolation SS-34 rotor.  The  pellet was resuspended in 3ml of 2% (w/v) deoxycholate (in l O m M Tris-HCl , p H 7.2  and  5 m M 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  thiosulphate  were  then  5 minutes added  to  to  remove  reduce  the  debris. A few sample  and  crystals spectra  of sodium 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 m t D N A , 3ug of nucDNA, and lOug of genomic D N A were carried out  Materials and Methods / 46 for  3 hours  at  loading buffer  37C. Digestions were  then  heated  to 60C for  10 minutes  (5% SDS, 25% glycerol, and 0.025% bromophenol blue) was  and added  to make a final volume of 30ul. Samples were loaded into wells of 0.8% agarose gels  and  separated  electrophoresis EDTA,  was  Maniatis et  photographed  by  size  IX  T B E (0.081M Tris  al,  at  1982).  50  volts  Gels were  for  15  base, stained  hours.  The  buffer  0.089M boric with  ethidium  for  gel  acid, 0.002M bromide  and  under shortwave U V illumination.  D s R N A was separated according to size by electrophoresis in 1% agarose minigels run at  40 volts for 2-3 hours. The buffer  (50X T A E =  for gel electrophoresis  was  IX TAE  2 M Tris base, 1 M 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, 7.5,  1975). Typically, 5mM M g C l , 2  dTTP, 0.2mM  20uM 2  labeled in 50ul of 50mM  0.05mg/ml B S A , 10ml 0-mercaptoethanoI,  dATP,  CaCl ,  l u g of D N A was  1.4uM dCTP,  lpg/ul  DNase  I  1.4 and  uCi/ul 0.4U/ul  alpha E.  20uM dGTP,  in  lOmM  EDTA,  pH  20uM  P  dCTP  (3000Ci/mM),  coli  DNA  polymerase  I  90 minutes  at  3  2  (Romberg). The reaction mixture was incubated for approximately 15C. The reaction was terminated  Tris-HCl,  by the addition, of three volumes of 1% SDS  containing 25ug carrier  D N A . Incorporated  labeled  nucleotides  Materials and Methods / 47 were  removed by chromotography  al,  1982).  8.0  and  Labeled D N A was I m M EDTA. use.  b. End-labelling  dsRNA  Purification  of dsRNA  (Maniatis et  al,  The  eluates  dsRNA  phenol  and  volumes  ethanol  labelling were  was  1982).  once  eluated  from the  Labeled D N A was  immediately before  of  on Sephadex  performed  DsRNA  were  with  was  denatured  twice  volume  of  with  to  remove  the  Tris-HCl, p H  dsRNA  troughs  one  chloroform,  lOmM  (Maniatis et  by boiling for  by electroeluting  containing 0.2M ammonium autoclaved  column in  collected from  extracted  one  G-50 spin columns  every  volume and  10  minutes  into  troughs  2-3  minutes.  of S T E saturated  precipitated  with  acetate. A l l solutions used  endogenous ribonucleases.  DsRNA  2.5  for end was  end  labeled according to the method outlined by Maniatis et al (1982) for blunt end or recessed and  boiled for  dsRNA 7.5,  5' termini labelling. D s R N A  was  5-10  mixture adding  3  2  P  was  to  fragment  labeled in 50ul of lOul  O.lM^MgClj,  gamma  minutes  dATP  50mM  from  2ul of 0.5M E D T A  chromotography  on  Sephadex  and  Approximately 0.5ug of I  (0.5M Tris-HCl, p H I m M EDTA)  2 0 U Polynucleotide Kinase. The at  37C. The reaction was  unincorporated  G-50  dsRNA.  I m M spermidine,  30 minutes and  the  in deionized formamide  10X Kinase buffer  dithiothreitol,  (3000Ci/mM),  incubated  was resuspended  spin  labeled  columns.  nucleotides  Labeled  dsRNA  from the column with I X S T E . Labeled dsRNA was denatured minutes immediately before  use.  150uCi reaction  stopped  by  removed  by  was  eluated  by boiling for 10  Materials and Methods / 48 7. Probes  All mtDNA by  and kalDNA clones used as probes were generously given to our lab  Dr. H . Bertrand.  The  location of each  clone in the  mtDNA  is shown in  Figure 8 and clones of k a l D N A shown in Figure 9. Purification of the mtplasmid DNA  of  strain  cesium  chloride  gradients. The plasmid has a different buoyant density than the m t D N A  and can  be isolated free  P561  involved  of m t D N A  isolating  the  plasmid  from  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  nick translation. The glutamate  and  ImM EDTA  dehydrogenase  and  sampled  for labelling by  (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). DNA  separated  NaOH 8.0,  gel electrophoresis  was  denatured  for  30  minutes  in 0.5N  and 1.5M N a C l and then neutralized for 45 minutes in 1M Tris-HCl, p H  and  0.15M  by  3.0M N a C l . D N A was transferred  NaCl,  0.01M sodium citrate,  p H 7.0)  to Genescreen for  24 hours.  with  2 X SSC (1X =  After  transfer,  the  filters were baked at 100C for 3 hours.  DNA  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 DMA >  bglH p»« 1 Hindi*  I  3 ... | 1  EcoRI  6  I We.11b  |  3  4  J10 f  1  1  II  II  1  1  7b olt-2  5  • •  1  6  | 14 | |  1  1 0 . [|17| K 2  b2  |b4|  I  ,4  is  K1  Id  E  • •••  ,  .  Psll-k.l  H I M H I MM • "•  10b  |  4  «•  B  '  —  L-rRNA"  s  3  to rt (T> H H-  ~~~  P> 3 Cu 3 n> rt o CO I  Hlndlll-K1 |  Bgin  EcoRI  ><\  •  B.  Pstl H i n d HI  I  4  * IMHM  S-iHNAco3  oli-l Co-J  |b3  l b .  1  EcoRI-E  i  3 .  J _  8  io  [ Wc.llb  H  | 4  1  1  2  1  3  |  7b "it-2*  |  5 oil" co-2  | 14 |  1 . 1 "  |  ' S-tHNA  H  cTi  10*  lO  1 f|l7|l6  bl  |b3  bZ  |b4|  rNII-tljl K2 |  1  Kl E 1-rRNA"  I  G  I  |  N3  tco R l - B  M,H He  |  1  4  |«.  II. | l 9 3  Materials and Methods / 53 were prehybridized for 24 hours at 55C in a solution containing 40% formamide, 1%  (v/v)  SDS,  IX  polyvinylpyrrolidine,  Denhardt's  solution  2% (w/v) ficoll),  (100X=  1M NaCl,  2%  and  (w/v)  B S A , 2%  0.5mg/ml denatured  herring  sperm D N A (Bertrand et al, 1985). Hybridizations were carried out in the buffer  with the addition of denatured labeled probe to at least  Hybridization for SSC  was for 48 hours at  5 minutes in 2X SSC at at  60C. After  55C. After  25C followed  1 X  (w/v)  same  10 cpm/ml. 6  hybridization, blots were washed  by two 45 minute washes  in 2X  air drying, blots were wrapped in Saran wrap and exposed  to Kodak X-Omat R P film for the appropriate length of time.  b. Northern Blot Analysis  All  buffers  ribonucleases. NaOH minutes  and in  for  Northern blot  DsRNA 1.5M NaCl  separated  analysis  were  autoclaved  to  by gel electrophoresis was  destroy denatured  endogenous in  0.05N  for 30 minutes. The gels were then neutralized for 45  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  described for Southern blots.  were detected  by hybridization to specific probes  as  IH. C H A P T E R 1  A.  INTRODUCTION  This  chapter  mtlS-kalDNA determine  describes during  experiments  vegetative  which  growth.  investigate  These  if a correlation exists between  the  the  experiments  transmission  of  undertaken  to  were  time of insertion of k a l D N A into  the m t D N A and the occurrence of death. This is of interest because it has suggested  that  the  variability in  lifespan  observed  between  different  natural isolates of Neurospora intermedia and between  ascospore  from  of k a l D N A  the  Bertrand,  same cross  may  depend  1984; Bertrand et  al,  on the  behaviour  1985). This suggestion  was  been  senescent  progeny initiated (Griffiths  and  on the  fact  based  that k a l D N A is present only in senescent Kauaian strains (Bertrand et al, 1985).  The prediction that insertion of k a l D N A into the supported  by observations  (Bertrand et  and Bertrand, result  initiates senescence is  that altered m t D N A from insertion of mtplasmid D N A  (Akins et al, 1986), from point mutations deletions  mtDNA  al,  (Bertrand and Pittenger,  1980; Devries et  1972a),  al, 1981), from inversions  1986), and from intramolecular  recombination  (Gross et  from  (Infanger al,  1984)  in irregular growth patterns of Neurospora. Thus, if insertion of k a l D N A  initiates senescence then the time of insertion should correlate with longevity. For example, should  insertion  result  of k a l D N A  in strains  should result in shorter  into  with longer  the  mtDNA  lifespans  later and  in vegetative  insertion  early  lifespans. This model, however, cannot fully  propagation in a  series  explain the  varibility in lifespans of ascospores initiated from the same cross. This is because  54  Chapter  1/55  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  propagation. mtDNA  since  Thus,  variability  molecules  responsible  for  mtlS-kalDNA  carrying  the  is  in lifespan the  initiation  same  present  at  the  onset  of genetically related  mtlS-kalDNA  of senescence  are  indicates  more  of  vegetative  ascospores that  the  complex than  with events  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  mtlS-kalDNA  that  is  accumulation. responsible  for  Interestingly, suppressive  it  is  neutral  the  accumulation, but  variability in lifespan is due to new insertions of k a l D N A different  not  inherited rather  into the  mtDNA  the at  times during growth. The insert transmitted sexually is referred to as because  in none of the  ascospore  series  does  it initiate senescence.  It  was determined that the novel inserts originate from movement of k a l D N A rather than  from  rearrangement  of the  addition, a third form of kalDNA  mtDNA  encompassing the  inherited insert. In  has been identified, denoted as  mtFF-kalDNA,  which may be an intermediate in the movement of k a l D N A .  B.  RESULTS  Subculture  series  shown  Figure  in  were 10.  derived from Ascospore  randomly isolated ascospores  series  which  survived  for  from  more  crosses  than  10  Chapter 1 / 56 subcultures were chosen for analysis. The lengths of the series described in this chapter 10  are  that  shown diagrammatically in Figure 10. It can be seen from Figure  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  mtDNA  was digested with Bgl II and inserts detected by autoradiography using  a  Pst  isolated from  I-kalDNA  restriction  map  probe. of  various subcultures  There  kalDNA,  are  Figure  no  Pst  I  7)  so  the  inserted element together  with the  flanking  Bgl  for  presence  II  digested  because  mtDNA  the  Bgl II digestion of mtlS-kalDNA  fragments  from  each ascospore  sites  in k a l D N A  clone  segments  consisted  series. The  (refer of  to  the  the  entire  of the m t D N A . Analysis of  of k a l D N A  insertion  was  preferred  generates two k a l D N A / m t D N A junction  which give the most information on the relative location of an insert  and  the number of different inserts in the m t D N A of a given strain.  The  strain P605 was used as a nonsenescent  11 digestion of the m t D N A  control in these experiments. Bgl  of this strain gives 15 restriction fragments. This is  typical of all nonsenescent  Kauaian strains. A m t D N A  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,  mtDNA  lane  1 of the  restriction fragments with  the  probe.  autoradiography.  These  are  the  Bgl 11-10 and  which are homologous with the m t D N A  Senescent  Kauaian  strains  are  -12  sequences  distinquished from  associated  nonsenescent  Kauaian strains by the presence of additional Bgl II restriction fragments.  Most  Chapter of these novel bands are  1/57  Bgl II restriction fragments  associated  with  kalDNA.  The k a l D N A restriction map diagrammed in Figure 7 shows that there are three Bgl  II  restriction  unique  Bgl II  labeled b l bl  sites  in k a l D N A  restriction  and  fragments  are  Bgl II  restriction  inserted  created.  and b2 consist of both k a l D N A  and b2 vary depending on where  internal  when  In  Figure  and m t D N A  fragments  are  designated  the 7,  mtDNA the  four  fragments  sequences. The size of  kalDNA is inserted  constant in size in Bgl II digestions. A n example m t D N A prepared  into  into the  b3  and  b4  mtDNA. and  The  remain  of a Bgl II digestion of the  from a senescent strain (P561) is shown in Figure 11, lane 2  of the ethidium bromide stained gel. In addition to the restriction fragments,  15 normal Bgl II m t D N A  seven unique Bgl II restriction fragments  two identified by the  arrows  are  the  are observed. 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). should  be  I-kalDNA fragment 1985). kalDNA which  noticed but  that  only  a  five  total are  of  six  visible  unique  because  bands  the  and the internal k a l D N A Bgl II fragment The  other  five  novel Bgl II  Bgl II  fragment,  b3; and  constitute  junction  fragments  restriction  the of  four two  b l ' and b2'.  normal  with  Bgl 11-12  the  Pst  restriction  b4 comigrate (Bertrand et al,  fragments  higher  include: the  molecular  different  (Bertrand et al, 1985). These junctions fragments  hybridize  It  inserts  weight in  the  internal fragments mtDNA  are designated b l and b2, and  Chapter 1.  Transmission of mtlS-kalDNA  X  1766  1/58  in Ascospores Initiated from  Cross  561-1  The ascospores described in this section were initiated from a cross using strain P561 as the the were  female  parent. A juvenile subculture  nonsenescent strain isolated  and  subjected  beyond 10 subcultures  The  gels  1766  presented  location  above,  in this  observation b2,  strain  of both  to  section  subculturing.  has  is  within  on the  the  two  111-13,18  111-13,18  clone  111-13,18  region  as  a  24,  probe).  of the  fragments  autoradiograph) novel restriction from  as  lived  the  intron  of  the  lane  Refer  mtDNA.  bl  in  well  and  the as  fragments  of both  to  in the  large  mtDNA.  rRNA  The  2 of the  autoradiograph  Figure  for  Hind  111-13,18  the are  b2 fragments female  parent  observation observed  and  that  the  This  (bl and  large  rRNA  using the  Hind  location of the  Hind  probe also hybridizes with fragments.  the  (Figure usually  in Bgl II  The  gene.  intron sequences of the  8  the  parent, P561-1. As  hybridization of all four junction fragments  (Figure  The equimolarity of the b2'  ascospores series  Bgl II profiles  insertions  the normal Bgl II-4, -12, and -14 m t D N A restriction  and  Twenty  of these  senescent female  kalDNA  and b l ' and b2') with a probe of the  gene, Hind  1984).  Seven  include m t D N A  P605, and  P561  inserts  is based  serial  Bertrand,  to  and were chosen for analysis.  nonsenescent control, strain described  (Griffiths and  of this strain was crossed  equimolarity 11,  lane  only the  of the b l ' 2  of  b l ' and  digests of m t D N A  the b2'  prepared  ascospores (details below) indicate that b l and b2 are junction fragments of  Chapter one of the k a l D N A inserts insert.  The  Bgl  11-12  1/59  and b l ' and b2' are junction fragments  and  -14  mtDNA  restriction  fragments  of the  other  constitute  the  majority of the intron of the large r R N A gene (Bertrand et al, 1985) and based on the  sizes of these two restriction fragments  junction fragments  with  Futhermore, junction  junction the  fragments  the  b l and b2, and b l ' and b2' it is suspected  with junction fragments insert  together with  bl  and b2 is within  fragments  different  bl'  and  b2'  levels of intensity  compared  with  the  sizes of the  that the  Bgl 11-12 fragment  within  the  Bgl 11-14  of radioactivity of the  that of the  b l ' and  bl  b2' junction  insert  and  the  fragment. and  b2  fragments  indicates that each insert is in a different m t D N A molecule.  The low copy number of the two inserts relative to the m t D N A (Figure 11, lane 2 of the  ethidium bromide gel) suggests that in addition to m t D N A  molecules  carrying an insert, normal m t D N A molecules are present. This indicates that the m t D N A population of subculture  1 of strain P561 is heterogeneous consisting of  a  mtDNA  minimum  of  three  different  insert with junction fragments with junction fragments  Figures  mtDNA  molecules  carrying  b l and b2; m t D N A molecules carrying the  the  insert  b l ' and b2'; and normal m t D N A molecules.  Mitochondrial D N A prepared lived ascospore-derived  types:  series  from various subcultures for each of the seven long (4,  7, 8,  11, 13, 15, 17, 18, 20, and  12,  13,  14, and  16) are  represented  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  in  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  is  (Figure  11).  This  insert  never  observed  to  accumulate  yet  it  is  maintained in low copy number throughout the entire series. B y subculture 6 two novel Bgl II restriction fragments, (Figure are  showing homology with k a l D N A , are observed  11). These bands are designated by the symbols f l and f2. F l and f2  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  k a l D N A ' . Two more novel Bgl II restriction fragments 12  and are  bands  are  distinquished from  designated  junction fragments novel fragments  bl  fl  and f2 by having a  and b 2 .  1  are detected in subculture  1  It is suspected  slower mobility.  These  that these two bands  of an insert in this ascospore. In order to determine  are  if the  are homologous with k a l D N A , 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 m t D N A 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 k a l D N A sequences of the Pst I-kalDNA probe and constitute junction fragments  of an  insert  located in a  other than in the intron of the large r R N A from  a  rearrangement  of the  region of the  mtDNA  gene. If this insert had originated  inherited insert  then  intron sequences  would  be  associated with the junction fragments of the insertion generated during vegetative  Chapter  1/61  growth.  To  determine  the  cloned regions  relative  of the  mtDNA  junction fragments b l the  large  location  1  as  and b 2  of  this  probes were  II-1  Bgl II restriction fragments  and  -2  restriction  hybridizations  performed.  The  using  various  slow mobility of  indicate that the insert must be within one of  1  of the  with the Hind III- l i restriction fragment. Bgl  insert,  fragments.  m t D N A . The m t D N A  This restriction fragment The  probe  was  probed  spans both the  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  with  the junction the  and -2 fragments have either fragments.  Subclones  of the  been deleted  Hind  site of insertion. The probe Eco R l - 6 i  fragments  (Figure  restriction  fragment  12). The  D N A of this  near  restriction  restriction fragments. delineating  the  kalDNA/mtDNA  the  Ill-li  II-1  and  -2  site  delineating  restriction  The slow mobility and similar sizes of b l II-1 and -2 fragments may be associated of the m t D N A contains a number  used  to  locate  showed homology with both junction clone is located  Bgl II junction fragments  associated  clone were  Insertion into the Bgl II-1 fragment  Bgl  or are  the  and b 2 '  Bgl II-1  Bgl II-1 and  -2  near the restriction site  fragments  which are 1  within the  would  very different  generate  in mobility.  suggest that both the Bgl  with the junction fragments.  This region  of t R N A genes and the small r R N A  gene. It  should be noted that both the  Hind  III-li and Eco R l - 6 i probes hybridize with  lower  The  probes  molecular  fragments  in the  weight  bands.  mtDNA  prepared  from the  hybridize  with  these  same  Bgl II  nonsenescent control indicating that  Chapter 1 / 62 there  is  cross  mtDNA.  The  restriction  homology of Hind  Ill-li  fragment,  and  these  probe both  mtDNA  cross the  probes  with  hybridizes  Hind  Ill-li  other  with and  the Eco  regions  of  Bgl II-4 Rl-6i  the  mtDNA  probes  cross  hybridize with the Bgl II-7 restriction fragment.  ascospore series 7 from cross 561-1 X 1766  The  mtDNA  insert  from  the  earliest  with junction fragments  parent  (Figure  number  13).  This  relative to the  subculture b l ' and  insert  is  mtDNA.  of this b2'  initially  was  series  transmitted  observed  By subculture  shows  that  from  the  in essentially  4, there is a  only  the  female  equal  copy  reduction in  the  amount of this insert and it is maintained at this level for the remainder of the series.  By  hybridizing  subculture with  the  8,  was  not  unique  Pst I-kalDNA  unique Bgl II fragments 11  two  included  are in  Bgl II  probe  are  the  series  and b l  2  3  and b 2  represented  both  sequences  of the  Pst  (Figure  bl  2  and  b2  2  13). Two more  11 (Figure 24). Subculture in  Figure  13  because  more m t D N A . The bands b l  are probably junction fragments  3  To determine if these novel fragments or  designated  observed  observed in subculture  subculture would not grow enough to prepare b2  bands  of two different  this 2  and  inserts.  are homologous with the kalDNA, m t D N A ,  I-kalDNA  probe,  hybridizations using  the  Hind  111-13,18 and Hind 111-14,15 probes were performed. Together these probes consist of the m t D N A sequences associated with the Pst I-kalDNA probe. The absence of hybridization of either and b 2 with  2  the  Hind  111-13,18 or Hind  111-14,15 probes  bands (Figure 24) 'indicates that these novel fragments  kalDNA  sequences  of  the  Pst  I-kalDNA  probe  and  to the b l  2  are homologous  constitute  junction  Chapter 1 / 63 fragments of a novel insertion into a region of the m t D N A other than the intron of the large r R N A  gene. The sizes of the bands designated b l  hybridization of these bands that  these  originated  bands from  are  a  with the Hind  probably junction  rearrangement  insertion into the  of  and  3  111-13,18 probe (Figure 24) indicate fragments  the  and b 2  3  inherited  intron, the junction fragments  of  an  insert.  insert If  it  which  was  a  has novel  would have been of a smaller  size.  The  location  within Hind  the  of the Hind  novel  III-10b  III-10b m t D N A  probe  fragment  and  junction fragments  2  k a l D N A sequences  junction fragments  restriction fragment  restriction b2  insertion with  of the  is also seen mtDNA.  A  accounts  of the  bl  mtDNA  2  (Figure  to cross hybridize with  l k b difference for  the  of the junction fragments  and  the  in size between  difference  in  size  b2  2  is  14). The Bgl II-4 the b l  between  2  the  implying that the insertion occurred  near the centre of the Hind III-10b fragment, a region containing the majority of the t R N A 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 15).  In subculture  b l ' and b2' was transmitted  1 this insert is essentially equimolar with  copy number is reduced in subcultures by  sexually (Figure the m t D N A .  The  4 and 6 and increases in copy number  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  Figure  24 reveals that accumulation of the  former  mtDNA  b2", To the  preparation.  Two novel  hybridizing with the Pst I-kalDNA determine if these bands Pst  I-kalDNA  probe,  of subculture  prepared  for  inherited insert occurs in only  the  Bgl II  fragments,  10  designated  bl"  and  probe are first detected in subculture 8.  consist of kalDNA, mtDNA,  hybridizations using  the  or both sequences of  Hind  111-13,18  and  Hind  111-14,15 probes was performed. The absence of hybridization of these probes to either  novel fragment  kalDNA  sequences  (Figure 24) indicates that  of the Pst I-kalDNA  they  are  homologous with  probe and constitute junction  the  fragments  of a novel insertion located in a region of the m t D N A 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  (Figure 16). There are no known genes in this region. The Eco R l - 4 i overlaps the  Bgl II-1 and -3 fragments  probe with the m t D N A  Bgl II-1  fragment.  and the hybridization of the Eco R l - 4 i  fragment  in  the  altered. This indicates that insertion occurred in region  overlapping  high  fragment neither  the  Eco  Insertion into this region of the Bgl II-1 fragment  very high molecular weight junction fragment This  when m t D N A  molecular weight junction fragment would  represent  approximately  junction  fragment  is  llkb.  fragment  shows the Bgl II-3 restriction fragment to be intact and  the Bgl II-1 restriction fragment the  mtDNA  of  a  high  together  with  Rl-4i  would generate a  is cut with Bgl II. the  18kb of D N A . Figure molecular  This indicates that about  weight  represent  about  7kb of the  fragment  is not associated with the junction fragments.  restriction  and  other junction 16  shows  together  that they  Bgl II-1 restriction  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 m t D N A 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  (Figure  insert  17).  with junction fragments  This  insert  is  present  in  b l ' and b2' was transmitted sexually very  low copy  number  and  is  never  observed to accumulate. The m t D N A of this series (Figure 17) together with the m t D N A prepared from subculture 11 (Figure 24) reveals a total of six different novel Bgl II fragments subculture  11 was  hybridizing with  not included  the  in the  Pst I-kalDNA  series  (Figure  probe. M t D N A  17) because  the  from culture  would not grow enough to prepare more m t D N A . The bands designated b l b2  and b2  are  5  first observed by subculture 8 and lost by subculture  24). B y subculture are  6  designated  observed bl  7  10, two more novel Bgl II fragments  (Figure  and b 2  7  17).  are  seen  By  subculture  11,  two  5  and  11 (Figures 17 denoted b l  more  (Figures 24). Hybridizations  111-13,18 and Hind 111-14,15 probes were performed to determine  novel  6  and bands  using the  Hind  whether  these  six novel bands are homologous with kalDNA, m t D N A , or both sequences of the Pst  I-kalDNA  bands -12  probe. Hybridization  designated b l  fragments  restriction  7  and b 2  7  of the  the  111-13,18 probe  (Figure 24) and the  with  loss of the  the  novel  Bgl 11-10 and  suggest that an insertion occurred in either the Bgl 11-10 or -12  fragments  and  a  rearrangement  occurred. Furthermore, the sizes of the b l with  Hind  7  including  and b 2  sizes of the Bgl 11-10 and -12 m t D N A  7  both  these  fragments  junction fragments  together  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 , b2 , 5  b l , and  5  b2  6  6  indicates  that  these novel  bands are homologous with the k a l D N A segments of the Pst I-kalDNA probe and constitute junction fragments  of two different novel insertions in regions of the  m t D N A other than the intron of the large r R N A gene.  The  location of the insert with junction fragments  with junction fragments b l  6  and b 2  bl  5  and b 2  and the insert  5  are in the Hind III-14 restriction fragment  6  of the m t D N A (Figure 18). This region contains t R N A genes. This mtDNA probe also cross hybridizes with the m t D N A insert  with junction fragments  rearrangement junction  event  fragments.  of the  bl  first  The third  6  Bgl II-4 restriction fragment.  and  insert  insert,  b2  6  (Figure  The second  17) possibly arose  giving rise to  an  insert  with junction fragments  bl  7  with  by  a  smaller  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 m t D N A 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  This  strain  to  this  ascospore.  insert  is  present  in  low copy  number  relative to the m t D N A  and and is never observed to accumulate. Two novel Bgl  II  and b 2  bands,  first are  denoted  detected  bl  8  in subculture  homologous with  8  hybridizing  with  the  Pst  I-kalDNA  13 (Figure  19). To determine  whether  kalDNA, m t D N A ,  or both sequences  of the  probe these  Pst  are  bands  I-kalDNA  Chapter 1 / 67 probe,  hybridizations using the  performed. fragments  The  absence  Hind  111-13,18 and  of hybridization  of either  Hind probe  111-14,15 probes  were  with  novel  these  two  indicates that they are homologous with the k a l D N A sequences  Pst I-kalDNA probe and are junction fragments  of the  of a novel insertion located in a  region of the m t D N A other than the intron of the large r R N A gene.  It is suspected that the insert is in the Eco R l - 1 1 restriction fragment. The Eco Rl-11  fragment has not been cloned, but a process of elimination, using m t D N A  clones from  the  majority of the  mtDNA,  suggests that k a l D N A is most  likely  inserted into that region. This particular region of the m t D N A 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 k a l D N A is inserted into this region of the The  fast  restriction  mobilities of the junction fragments fragment  suggests  that  some  of  mtDNA.  and the large size of the Bgl II-3 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 m t D N A throughout entire  series.  Two novel  Bgl II  fragments  hybridizing  with  the  Pst  the  I-kalDNA  probe are observed in subculture 6 and maintained through the rest of the series (junction  fragments  bl  9  and  b2 ) 9  (Figure  20). By subculture  10,  four  more  Chapter 1 / 68 unique bands  are  observed and denoted b l  1  and b 2  0  1 0  ,  and b l  and b 2  1 1  1  1  (Figure 24). M t D N A prepared from subculture 10 was not included in the series because  the  determine both  culture  if these  sequences  111-13,18 contain  novel  of  and the  would  Hind mtDNA  not  grow  fragments  the  Pst  enough  are  probes  sequences  prepare  homologous with  I-kalDNA  111-14,15  to  probe, were  associated  mtDNA.  kalDNA,  the  Together,  Pst  To  mtDNA,  hybridizations using  performed. with  more  the  Hind  these  I-kalDNA  probes  probe.  absence of hybridization of either probe to any of the novel fragments  or  The  indicates  that they are homologous with the kalDNA sequences of the Pst I-kalDNA probe and  constitute junction fragments  of three different k a l D N A insertions in regions  of the m t D N A other than the intron of the large r R N A gene.  The  location of the novel insert with junction fragments b l  Hind III-14 restriction fragment are the same as the b l III-14  fragment  in ascospore  and b 2  9  is in the  (Figure 21). The sizes of the junction fragments  and b 2  5  9  5  junction fragments for the insert in the Hind  series  12  (Figure  18).  This  indicates that  both  inserts are located in the same region of the m t D N A . The other two inserts are located  in  the  Eco  RI-6i  restriction  fragment  molecular weight novel bands, designated b l  1 1  (Figure  and b 2  1  1  21).  The  two  , are junction fragments  of one insert, and the two higher molecular weight bands, designated b 1 b2  1  0  ,  are  fragments  junction bl  fragments b l  1 1  1  0  and  fragments b2  and b 2  1  1  1 0  .  of  the  is possibly  other a  1 0  and  The  insert  with junction  deletion of the  insert  with junction  ascospore series 16 from cross 561-1 X 1766  insert.  lowest  Chapter 1 / 69 Analysis of the mtDNA prepared from the first subculture of this series indicates that only the insert with junction fragments the  female  b l ' and b2' was transmitted  (Figure 22). The insert is maintained in low copy number  never observed to accumulate. B y subculture bl  1  and  2  b2  are  1 2  detected  (Figure 22). To determine both  sequences  111-13,18 because  and  of  the  Hind  together  which  whether Pst  these bands  I-kalDNA  111-14,15 probes  they  constitute  probe,  were  the  and is  11 two novel Bgl II bands  hybridize with  the  hybridizations  sequences  These  denoted  I-kalDNA  consist of k a l D N A ,  performed.  mtDNA  Pst  associated  probe  mtDNA  using probes  from  or  the  Hind  were  used  with  the  Pst  I-kalDNA clone. The absence of hybridization of either probe with the novel Bgl II  bands  indicates that  the Pst I-kalDNA  these bands  are  homologous with  probe and are junction fragments  kalDNA  segments of  of a novel insertion located  in a region of the m t D N A 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  insert with junction fragments  cross 561-1 X 1766 inherit only the  b l ' and b2' from  the female parent strain P561.  This insert is located in the intron of the large r R N A  gene. In all series, this  insert does not accumulate and initiate senescence yet is maintained through each series.  During  growth of each  hybridize  with  the  constitute  junction  Pst  series,  I-kalDNA  fragments  novel  probe  of different  Bgl II restriction fragments  are  identified.  kalDNA  inserts  These and  unique the  which bands  absence  of  Chapter 1 / 70 hybridization  of  either  the  Hind  111-13,18  majority of these junction fragments  or  Hind  111-14,15  probes  to  the  indicates that each insert represents a novel  insertion into the m t D N A and not a rearrangement  of the m t D N A encompassing  the transmitted insert with junction fragments b l ' and b2'.  Finally, in Figure 24, note that the m t D N A segments into which k a l D N A inserts are  undetectable  in ascospore  series  4, 8,  Bgl 11-12 or -14 restriction fragments with  junction  segments.  fragments  bl  and  3  Likewise, in ascospore  should not be detectable because  12, 14, and  16. In ascospore  7 the  should not be observed because the insert b2  is  3  14 the  located  in  one  of  these  Bgl I I - l restriction fragment  mtDNA restriction  two insertions are present in this region of the  m t D N A . This indicates that the last subculture from which m t D N A was prepared from  each  ascospore  series  contained  variable  ratios  of  normal  to  abnormal  mtDNAs.  a. Correlation Between Time of kalDNA  The  subculture in which novel inserts  Insertion and Onset of Senescence  are  first  observed varies between  series.  The later in a subculture series that k a l D N A inserts into the m t D N A the longer the  lifespan of that culture. The variables used to determine  whether longevity  correlates with the time of novel insertion of k a l D N A are shown in Table I.  Chapter  T a b l e 1. V A R I A B L E S  USED  1/71  IN THE C O R R E L A T I O N  total ascospore  subcultures  series  preceding  subculture of  kalDNA  insertion  death  ANALYSIS.  subcultures  left  in each series after  kalDNA  insertion  4  26  12  14  7  15  8  7  8  12  8  4  12  13  8  5  13  27  13  14  14  13  6  7  16  19  11  8  Chapter 1 / 72 The  analysis  initiating  revealed  senescence  that  is  the  first  time  detected  during growth is correlated  when  with  the  both  kalDNA  the  insert  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  Autonomous  In  of  Form of  the ascospore  various  times  fragments  mtlS-kalDNA  and . Identification  of  a  Transient  Mitochondrial  kalDNA  series analyzed, k a l D N A  during  formed after  vegetative  inserts initiating senescence  propagation  and  the  sizes  Bgl II digestion in each series are  If these senescence-associated  of  appear  the  at  junction  variable (Figure 24).  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 associated  with  the  junction  fragments  of the large r R N A gene should be  of  these  inserts.  hybridization of the intron probe with most junction fragments senescence-associated than insert.  from  are  absence  two  of  the  exceptions;  mtDNA two  encompassing  of the  of  suggests that the  inserts have originated from the movement of k a l D N A  rearrangement  There  The  the  inherited  four junction  fragments  rather kalDNA of  the  inserts in each of ascospores 7 and 12 hybridize with the intron probe.  In  addition to determining that k a l D N A is capable of assuming new locations, a  Chapter contains  1/73  a mitochondrial plasmid (mtplasmid)  novel bands are  which is labeled in the figure. The  of a higher molecular weight than  using an Eco R l - E probe was performed  the mtplasmid. Hybridization  to determine  whether these two closely  migrating bands show homology with k a l D N A . The Eco R l - E clone is an internal region of the k a l D N A element (see Figure 9 for the location of this clone) and identifies Figure  D N A consisting 25  (Panel  of  kalDNA  B) shows  that  sequences.  both  the  The  uncut  autoradiograph  mtDNA  bands share homology with kalDNA. Homology between mtDNA  is expected  Hybridization  of  because  the  bands must contain whole purified  mtDNA  probe  from  Hybridization  of  with  the  the  nonsenescent  D N A were  sequences  hybridized  probe  presence of the two  novel  the  bands  in the  inserted  into the  novel  E probe and  uncut  form  of kalDNA.  indicates  are  with the  also the  strain  performed  to  associated  with  novel  mtplasmid  bands  with  uncut  P605  and  that  these  determine the  whether  mtDNA  unique  bands.  two  (Figure  25,  mtDNA  radioactively  Panels  C  the  series  of  hybridization to uncut P605 m t D N A indicates that mtplasmids retained  two  inserted  the  in  kalDNA sequences. Hybridizations using radioactively labeled  mtplasmid  mtplasmid  E  of the  and  shown  uncut m t D N A . mtplasmid,  These  neither  labeled or  the  Neither and and  D). no  were probably also  hybridizations indicate that kalDNA is not do the  novel bands contain  any  region of  the mtplasmid.  To  verify  that  extramitochondrial lysis. Lane  the  novel  plasmids  contamination,  1 of Figure  are  mitochondria were  26 is m t D N A  uncut m t D N A from subculture  within  from  the  mitochondrion  rather  than  treated with DNase prior to  a nonsenescent strain,  lane  2 is  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  mitochondria has  treatment  no affect  on the  of  mitochondria novel bands  prior  to  indicating  the  that  lysis the  of  the  autonomous  kalDNA element is within the mitochondrion.  To  determine  AR-kalDNA similar  size  whether  the  mitochondrial  autonomous  form  of  kalDNA  and  (the nucleus-associated linear autonomous form of kalDNA) are of a and  structure,  uncut  nucDNA  and  mtDNA  were  separated  by  electrophoresis and hybridized with the Eco R l - E probe. A R - k a l D N A has a faster mobility relative to the mitochondrial autonomous form  (Figure 27). In addition,  A R - k a l D N A forms a discrete band upon electrophoresis where as two bands, both representing the autonomous mitochondrial element, are observed to migrate from the uncut m t D N A .  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  using strain P561 as a female parent.  initiated  from  crosses  Chapter 1 / 75 a. Ascospore Series from Cross 801-1 X  A  cross  was  nonsenescent  made male  between  strain  the  1836  senescent  (Griffiths  subculture of a series derived from ascospores only  female  and  strain  Bertrand,  P801  1984).  and A  the  juvenile  strain P801 was used in the cross. Twenty  were isolated and subjected to serial subculturing. Of these 20 series  three  survived past  examination. 10.  1836  The lengths  10  subcultures.  of these series  These are  three  series  were  chosen  for  shown diagrammatically in Figure  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.  MtDNA series.  was isolated from The  hybridized  the  autoradiographs with  the  earliest and latest  shown  in Figure  Pst I-kalDNA  (P605) and senescent  probe.  original  predicted because  P801  that  strain  kalDNA  kalDNA  is  28  are  the  of each  mtDNA  The first two lanes  are  ascospore  preparations nonsenescent  (P561-1) controls. A Bgl II digestion of the female  is not included in this section because the  subcultures  could is  not  be  inserted  generally  at the time of the cultured  into  inserted  the into  for  intron this  mtDNA  preparations  mtDNA  preparation.  of  large  the  region  in  parent  It  rRNA  senescent  is  gene  Kauaian  strains (Bertrand et al, 1985; 1986).  KalDNA  sequences  are  present  in  the  mtDNA  prepared  from  the  early  subcultures of each ascospore (Figure 28). The size of the junction fragments  are  the  the  same  ascospore  as  junction  series  fragments  bl'  previously described.  arid In  b2' the  of  the  mtDNA  insert  inherited  prepared  from  in 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  insert within the intron of the large r R N A are similar to junction fragments in  strain  P561.  transmitted  Whether  sexually to  this  these  are junction fragments  of an  gene. The sizes of these two bands  b l and b2 of one of the two inserts described insert  originated  ascospores  from  in very  novel  insertion  low copy number  or  was  cannot  be  determined.  MtDNA  prepared  from  novel  bands  bands  hybridizing are  probe,  the  subculture  hybridizing  Bgl  those  11-10  7 shows no  detectable  probe  (Figure  The  showing homology with the  mtDNA  with  and  26 of ascospore  the  -12  Pst  I-kalDNA  mtDNA  series  restriction  fragments.  28).  only  portions of the To  investigate  whether the nucleus-associated form of k a l D N A is also lost in this series, nuclear DNA  was  prepared  from  subculture  80, run uncut,  and hybridized with the  E  probe. A R - k a l D N A was not detected in the autoradiograph (Figure 29).  b. Ascospore Series from Cross 572-5 X  A  cross  was  nonsenescent  made  between  the  1818  senescent  male strain 1818 (Griffiths  female  strain  P572  and  and Bertrand, 1984). Subculture 5 of a  series derived from strain P572 was used in the cross. Twenty ascospores isolated  and  subjected  to  serial  the  subculturing. The  majority  series exhibited very short lifespans. Only two series one of which showed no signs of senescence  after  were  of ascospore-derived  lived past  10 subcultures,  80 subcultures. The  senescent  Chapter series  died  in  19  subcultures  (Figure  1/77 10). These  two  series  were  chosen  for  analysis.  A  Bgl II digested m t D N A  One  profile of the female parent  is shown in Figure 32.  insert is observed in this culture when hybridized with  probe.  Hybridization  with  the  intron probe  insert is within the intron of the fragments inserts  is apparent  large r R N A  the  in  strain  copy  P561.  number  of  In the  Pst  I-kalDNA  indicates that  this  gene. The sizes of the junction  are comparable with junction fragments  identified  and  the  addition  b l and b2 of one of the two to  Bgl II-5,  this  is  a  in  fragments  and the presence of a new Bgl II restriction fragment (represented by  the  mtDNA  mtDNA  and form  restriction  approximately one quarter  map,  Figure  6).  This  region  -11, and  there  reduction  the arrow). The four Bgl II restriction fragments  -6,  insertion,  represent of the of  -13 restriction  contiguous regions of mtDNA  the  (refer  mtDNA  to  the  contains  the  cytochrome b apoprotein gene and the cytochrome oxidase sub unit 3 gene.  MtDNA  was  prepared  from  various subcultures of each of the  hybridized with the Pst I-kalDNA probe. A description of the  two series and  senescence-associated  insertion events as they relate to each series is presented below.  ascospore series 4 from cross 572-5 X 1818  The  m t D N A prepared from  the early culture of this ascospore series contains at  least six novel Bgl II fragments (Figure 30). A l l six bands  are  which hybridize with the maintained throughout the  Pst I-kalDNA  probe  entire series. Two of  Chapter  1/78  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 and  b2 ' 1  (Figure 30) are of similar size to the junction fragments b l  1  1  fl  and b 2  1  of the insert which accumulates in ascospore 4 from cross 561-1 X 1766 (Figure 12).  This  indicates  that of an  the  bands  insert  also  designated  bl  and  1 f l  junction  fragments  located in the  mtDNA.  The loss of the Bgl II-1 and -2 fragments  b2  1 f t  are  probably  Eco R l - 6 i  region of  in ascospore  4 from  the 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 m t D N A and does not result from a rearrangement. b2  1  5  The novel Bgl II bands denoted b 1  1 5  and  were not characterized.  It is of interest to note that none of the cross  572-5 X 1818 are detected  inserts present in ascospore 4 from  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  with  mtDNA  the  seen  in low copy number  rest of the  restriction fragment  mtDNA  seen  in the  female  parent  in this ascospore. Futhermore, the  in the  mtDNA  prepared  not transmitted to this ascospore (Figure 30).  ascospore series 13 from cross 572-5 X 1818  from  the  are equimolar extra Bgl II  female parent  was  Chapter 1 / 79 The m t D N A 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 fragments  b l bl  1  and  6  b2  and b 2  1  1  are  1 6  a  similar  ascospore  12  fragments b l Figure of  size  as  size  denoted b l  fragments  bl  5  to  the  insert  1  and  and b 2  7  1 7  of an  insert  b2 ,  Figure  5  in ascospore  mtlS-kalDNA  13 are  in common in 18),  and b 2 , Figure 21), and 16 (junction fragments b l  23) from  with junction  cross 561-1 X 1766 (Figure 12).  the junction fragments  (junction 9  similar  of ascospore 4 from  The two unique Bgl II fragments of  of  9  14 1  (junction  and b 2  2  the  1 2  ,  cross 561-1 X 1766. B y subculture 20 no discernible amounts are  seen  in ascospore  series  13. Observation of the  nuclear  D N A prepared from subculture 26 and probed with E reveals that A R - k a l D N A 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  parent. In addition, the Bgl II m t D N A restriction fragments  from  the  female  seen in lower copy  number in the female parent are in normal amounts in the m t D N A 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 cross 561-1 X 1766 and ascospore 4 from  4, 7, 12, 14, and  16, from  cross 572-5 X 1818 are all located  Chapter 1 / 80 in  regions  ascospores  of the 12,  mtDNA  14, and  that  contain  16, from  cross  tRNA  genes.  561-1 X  It  can  1766 and  be  noticed  ascospore  4  that from  cross 572-5 X 1818 all have an insert in the same region of the Hind 111-14 fragment  of the  insertions  are  ascospore  14 from  mtDNA.  also  In  observed  ascospores within  the  7 and intron  12 from of  the  cross large  561-1 X 1766, rRNA  gene.  In  cross 561-1 X 1766, an additional two insertions are located  in the Eco R l - 6 i fragment of the m t D N A . The inserts of ascospores  8 and  13  from cross 561-1 X 1766 are in regions of the m t D N A which contain no known genes. Both these strains carry large deletions encompassing the Bgl II-1 region of the m t D N A .  Chapter  1/81  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.  Chapter  561-1  9  0  X 5  1 / 82  1766 •ubcwttwr« t o  u m b t r 15  20  25  30  •scospore 4 7 0 T2 T3 T4 « 572-5 9 0 • scospore  X 1818CT f  subculture lO  lumber 15  20  4 13  MMMMMMMMMHMMWiMm  801-1  ? X  1836 subculture 10  «aco*pore S 6  7  • •  number 15  20  2 5 -30  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 and b 2 , and f l and f2 are junction fragments of two different inserts of ascospore series 4. The b l ' and b2' bands represent the junction fragments of the inherited insert. The b3 and b4 bands are internal fragments of kalDNA. The b l 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. 1  1  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 and b2 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. 1  1  605 561 561-4-24 605 561 561-4-24 605 561 561-4-24  00  Chapter  1/87  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 and b2 junction fragments of one of the inserts of ascospore series 7. 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 H restriction fragments. The arrows denote the Bgl II fragments of the mtplasmid. 2  2  88  Series 561-7  EtBr  Kal  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 and b2 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 and b 2 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. 2  2  3  3  605 561 561-7-11 605 561 561-7-11  Chapter  1/91  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 b l ' 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.  EtBr  Kal  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.  605 561 561-8-8 605 561 561-8-8  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 and b 2 , and b l and b2 are junction fragments of two different inserts of ascospore series 12. The b l ' 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. 5  5  6  6  Series 561-12  b4/12  EtBr  Kal  Chapter  1/97  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 and b 2 , and b l and b2 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. 5  5  6  6  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 and b2 are junction fragments of the insert of ascospore series 13. The b l ' 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. 8  8  Series m o  561-13  —  S  in 1  7  1 3 15 21 2 3 2 5  Et Br  —  3 5  1  7  1 3 15 21 2 3 2 5  Kal  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 and b2 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. 9  9  \ryz-  Series O  m  561-14 2  4  6  8  2 S  2  4  6  8  I *»  EtBr  Kal  1 0  * - b4/12  Chapter  1/103  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 and b 2 , b l and b 2 , and b l and b2 of the three different inserts in this ascospore 6eries. Junction fragments b l and b 2 , and b l and b2 hybridized with the mtDNA Eco Rl-6i probe. The insert with junction fragments b l and b2 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. 9  1 0  1 1  1  1 0  9  1 0  1  1 0  1 1  1  1  9  9  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 and b 2 are junction fragments of the insert of ascospore series 16. The b l ' 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. 1 2  1 2  Series 561-16 ID  ID  _  S S  1 6  8  EtBr  11 13  15  2  6  Kal  8  11 1 3  15  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 and b 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. 1 2  1 2  605 561 561-16-15 605 561-16-15  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 and b2 are of an insert in ascospore 7. Junction fragments designated b l and b2 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. 3  7  3  7  Kal  H13,18  Chapter  1/111  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 R l - 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 D N A mt plasmid D N A  EtBr  E  mtDNA  mt p l a s m i d D N A  Chapter  1/113  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 R l - E probe (for details on this probe refer to Figure 8).  EtBr  E  Chapter  1/115  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  1/117  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 b l and b2 are the junction fragments of the insert seen in the late cultures of series 5 and 6. The b l ' 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 R l - E probe (for details on this probe refer to Figure 9).  605 801-5-14 801-6-14 801 -7-26  Chapter  1/121  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 * and b 2 * , and b l and b 2 , 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. 1  1 5  1  1 5  V2-2-  Series 5 7 2 - 4  EtBr  Kal  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 and b 2 , and b l and b2 and b l ' 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. 1 6  1 7  1 6  1 7  Et B r  Kal  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 b l and b2 are of an insert observed in the female parent strain. Junction fragments designated b l * and b 2 * 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. 1  1  605 572 572-4-16 572-13-89 605 572 572-4-16 572-13-89 605 572 572-4-16 572-13-89 4^  CO"  (T  Chapter  1/127  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 R l - 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  T  Bgl N Pit 1 Hindis EcoRI  3  | I  8  UM  4  I | Wc.llb  10,11 1,15 • t  T  I  1  |  3  |  II  |  2  1  7b |  •j—m olt-2  5,6,9,12 •  5  |  • •>  «  •  2  • . J_IO_JN2|.4|  | 14 | 1 0 a |  Oti-I c o - 2  3,7,13,14  y  1  ,  , '  .. T  M  1  4 I  |  II« |ig{  l  ••••••••«•  ••• •  S-rNNA co 3  4 3  •  LrRNA  n rf (D  NUMBER  CROSS  1 2 3 4 5 6 7 8 9 10 1 1 12  561 -1  X  13 14  801 -1  15  572-5 X  X  SERIES  1766  1836 •  1818  4 7 7 '8 12 12 12 13 14 14 14 16  JUNCTION FRAGMENTS  H  bl', bl , bl \ b1\ bl , bl«. b l \ b l \ bl \ bl bl ' bl '»  b2« b2 b2 b2' b2 b2« b2 b2« b2» , b2'° , b2" , b2'  5 6  bl bl  , ,  4  b l b l  3  5  1  0  1  1  3  1  J  J  3  5  7  2  b1 b1  , J , J  Chapter C.  1/131  DISCUSSION  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  generally the only insert detected. Because these series are derived from isolates,  it  is  impossible  to  determine  the  history  of  these  and  natural  strains  and  consequently impossible to predict the stage of senescence that these strains were in  when  collected from  nature  and  originally  difficult  to conclude if the inserts detected  inserts  or  ascospore  perhaps  later  inserts  which  subcultured.  Consequently,  in those series are induce  senescence.  it  in fact the  Studying  is first  long-lived  progeny provides a more clearly defined origin to a series, and more  opportunity to chart the course of senescence.  MtDNA  analysis  mtlS-kalDNA designated  by  of  is  the  present  the  early in  cultures  detectable  junction fragments  of  the  amounts. bl'  and  ascospores  In  b2'  all  is  revealed  cultures,  identified. In  the the  that insert 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 prepared  to from  fragments  determine the  the  female  origin  parent.  of this  insert  It is suspected  since  mtDNA  that the  could  not  be  insert with junction  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 k a l D N A characters use  as  the  different  female  inserts.  parent  of this strain such that the culture prepared  and  Alteration  the  of  culture  kalDNA  prepared  for  characters  mtDNA  as  a  for  isolation had  consequence  of  subculturing has also been observed in strain P561 (Bertrand et al, 1985; 1986).  It  should  be  transmitted is  an  noticed  to the  important  that  observation because  Bertrand  referred neutral P561,  et  to as insert a  located Together  al,  neutral  second  the  which  1985;  1986;  of  observations  encodes  the  denoted  intron  intron induce senescence. frame  junction  defective  fragments  1980;  et  al  1982; DeVries  1987).  not initiate senescence. large  large  indicate  rRNA  that  gene  This  b2'  and  et al,  insertion is  As mentioned,  rRNA  by the junction fragments the  and  usually accumulate  al  intron of the  bl'  in the series studied. This  mtDNAs  Lambowitz  since it does  insert,  these  with  molecules (Bertrand et  is located within  within  insert  progeny never initiates senescence  displace normal m t D N A 1981;  the  bl  gene.  strain  and b2, is also  (Bertrand  disruption of certain  In  the  et  al,  regions  1985). of  this  A n important region of this intron is the open reading  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 and  the  neutral  insert  in  that the a  mutagenic  functionally  insert  unimportant  is within region  of  the  S-5 gene  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.  A n important observation is that only the neutral insert (junction fragments  bl'  Chapter II and  b2')  insert  of female  strain  (junction fragments  senescence insert  in the  P561 was  133  transmitted  sexually. In  b l and b2) not transmitted  female  parent  with junction fragments  strain  bl  (Bertrand  and b2  et  strain  561,  the  to its derivatives initiates al,  1985).  Similarly,  the  in the  572  also initiates senescence  female parent strain (unpublished results). This insert was not transmitted to the two 572 ascospore encompassing Bgl  II  the  progeny analyzed. In addition, strain Bgl II-5, -6, -11, and  restriction  fragment  which  572 carried a deletion  -13 restriction fragments  were  not  transmitted  to  and  a novel  either  progeny  analyzed. Together these results indicate there may be a meiotic salvage process which screens defective mitochondria or m t D N A molecules such that they are not transmitted example  sexually. It  the  insert  should  with  be  noted  that  junction fragments  mutagenic  bl  and  kalDNA  b2  in  strain  inserts,  for  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  mtlS-kalDNA are observed. Analysis of the that the  point' in each  subculture  series  801  ascospores,  novel insertions of  561 ascospore-derived series revealed  that  novel inserts  are  detected  and it is usually the last insert observed that initiates senescence equimolar with the m t D N A . The point in each series when the senescence  is  detected  correlation further the  mtDNA  determine  is  if the  is  proportional  to  the  lifespan  of  and  varies becomes  insert inducing  each  strain.  This  validates the idea that some kind of insertion of kalDNA into the  event  same  required  to  initiate  correlation exists for the  senescence.  It  two senescent  is  impossible to  ascospores  from  cross 801 X 1836 since the m t D N A profile of the female strain P801 was not  Chapter II obtainable.  However, observations  accumulated  on the  in these two senescent  that the junction fragments  134 insert  inherited  and  the  series indicates that they are  insert  which  different  of the insert accumulating, in both series, are of the  appropriate size to constitute a novel insertion into the intron of the large gene.  This  suggests  and  that  novel  insertion  probably  occurred  during  rRNA  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  because high molecular weight fragments are  present  determine perhaps these  in the m t D N A whether  were  inserts  transmitted  inserts  presence  these  in  very  prepared  to  low  1818 are  from  the  not  ascospore Ascospore  movement  from  number.  the  during  female  Furthermore,  in  meiosis  parent  to or  carrying  ascospore  4,  the  of mutagenic inserts in the ascospore culture indicates that the lifespan  of the series should be quite short, less than does  early cultures. It is difficult  from  ascospores  copy  of an anomaly  hybridizing with the Pst I-kalDNA probe  originated  the  somewhat  die of  until  subculture  18 from  series  13  cross from  19. It 572-5  this  10 subcultures. In fact this  should be  X  1818  same  cross  that also  noticed that dies  after  shows  a  this a  is the  series only  longer lifespan.  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 m t D N A molecules  and  ascospore-derived anomaly.  ultimately long  series  the from  rate this  of cross  senescence.  Investigation  of  might provide information on  more this  Chapter II  135  The properties of the novel k a l D N A inserts in all series were investigated. The majority  of novel  inserts  observed in the  561  ascospore-derived series  hybridize with the probe of the intron of the large r R N A that  these  novel  insertions originated from  movement  did not  gene. This indicates  of k a l D N A  rather  than  from rearrangement of the m t D N A encompassing the inherited insert, which was located  in the^ intron of the  junction fragments  bl  large  and b 2  3  rRNA  hybridize  fragments  bl  the  and b 2  3  resulted from large r R N A  with  intron  7  rearrangement.  and b 2  probe.  in ascospore  3  The insert  in ascospore 7 from  3  the insert with junction fragments b l cross  gene.  7  Based  series  designated  insert with junction fragments b l  in ascospore 12 from the same on  the  sizes  of  the junction  7, it is predicted that this insert intron of the  would have been smaller in size. The  and b 2  7  the  cross 561-1 X 1766 and  If it were a novel insert into the  gene, the junction fragments  by  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 A R - k a l D N A . In order to deduce the  by  element  which  the  moves,  strains  which  have  only  mtlS-kalDNA  means or  only  A R - k a l D N A are required. Transformation experiments are in progress to construct strains  of  this  mtlS-kalDNA  as  sort.  At  donor  donor D N A is degraded DNA  has  the  present  transformations  D N A have before  opportunity  to  using  either  proven unsuccessful. In  insertion into the established  it  mtDNA as  an  most  AR-kalDNA instances  or before autonomous  or the  the donor element.  Chapter II  136  Incorporation of the Pst I purified k a l D N A fragment Neurospora  transformation  .transformants  are detected  the  (Chueng  nucDNA  have  also  proven  into vectors constructed for  unsuccessful.  In  some  cases,  which carry only vestiges of k a l D N A as insertions in  C . K . , Doctoral Student,  perssonal  communication).  It  is  known that A R - k a l D N A 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  involve  in  vitro  system  which  reassociation  will  of  confer  these  stability to  proteins  with  the  donor  D N A will  AR-kalDNA  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  of  no  homology  with , either  mtDNA  or  mtplasmid  D N A . This  form  k a l D N A is denoted mitochondrial free-form k a l D N A (mtFF-kalDNA).  The observation that m t F F - k a l D N A is transient  and normally seen prior to novel  insertion suggests a role as an intermediate in the movement of k a l D N A . 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;  1983),  bacteriophages  such as P2 (Calender et al, 1977),and lambda (Nash et al, 1977).  The  failure  to  Mossie  observe  et  al,  mtFF-kalDNA  1985),  in  retroviruses  all  of  the  (Varmus,  present  series  may  and  be  Chapter 1 / 137 explained by postulating that if it is an intermediate have  to be present in high copy number  because of the of  heterogeneity  mtFF-kalDNA  mtFF-kalDNA  of m t D N A s  would  not  most  series  in  always  suggests  movement  occurred  was  has  observed  in approximately  been  different Dr.  Kauain  not  sampled  senescent strains  H . Bertrand, personal  be  in movement  for movement  it does not  to occur.  Furthermore,  in this coenocytic fungus,  identification  possible. that  the  Alternatively, the appropriate  for  mtDNA  analysis.  one  quarter  of m t D N A  (Dr. A . J . F .  communication)  Griffiths  absence of  stage  Since  in  which  mtFF-kalDNA  preparations  personal  it is evidently not  from  communication;  an  anomaly  and  deserves to be placed in the general kalilo model.  MtFF-kalDNA  has  not been characterized but the difference  in the  mobilities of  mtFF-kalDNA and AR-kalDNA suggest that mtFF-kalDNA is structurally different from A R - k a l D N A . The structural differences between these two forms of k a l D N A are unknown. One possiblity is that m t F F - k a l D N A is linear. The slower mobility of mtFF-kalDNA, relative to A R - k a l D N A , suggests that if m t F F - k a l D N A is linear, it  consists  kalDNA,  of more generated  mtFF-kalDNA  would  generate  two  the  than  one  after  Bgl  have  to  high  copy II  of k a l D N A . digestion,  consist  molecular  of  differ  tandem  weight  Since  the  by  fragments  approximately  inverted  fragments  end  repeats  (designated  l.Okb,  in order fl  of  and  to f2)  observed after  Bgl II digestion of m t F F - k a l D N A (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  high molecular weight fragment  one may  be  (fl and  explained by postulating that the  inverted repeats is not accurate  as well as  consisting of the ends of kalDNA. The  of two high molecular weight Bgl II fragments than  of k a l D N A  thus forming bands  II digestion. This would also explain the presence  f2; Figure  pairing and  a  presence  11)  rather  union of the  of different  sizes after Bgl  of two bands  migrating from  uncut m t D N A (Figure 27).  Even  though  the  origin of the  novel inserts  and  the  role of m t F F - k a l D N A 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 mtFF-kalDNA  has  only been observed when new locations of k a l D N A  studies, are  seen  and that the mtFF-kalDNA was apparently not required for insertion of k a l D N A 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  regions  regulating  encompassing the  tRNA  these inserts  genes  genes  suggesting  were  will determine  that  destroyed. the  either  the  Sequencing  tRNA of  the  locations of these inserts  gene or regulator}' function has been disrupted.  genes  or  regions  and  what  Chapter II  139  The sites of insertion of k a l D N A in ascospores X  1766, ascopore 4 from  cross  4, 7, and  12 from cross 561-1  572-5 X 1818, and ascospores  cross 801-1 X 1836 are in regions of the m t D N A  5, and 6 from  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 k a l D N A / m t D N A junctions has  identified  pentanucleotide  sequences  which  may be  recognition sites for insertion in the m t D N A (Bertrand, 1987).  The locations of the kalDNA inserts in ascospores 8 and 13 from cross 561-1 X 1766 are in regions of the m t D N A which do not contain any known genes. In both series, deletions are associated with insertion and the regions of the m t D N A deleted  in  include  the  both  strains  subunits  cytochrome  oxidase  1  contain and  gene.  2  functionally of  the  Accumulation  indispensible  ATPase  gene  genes.  and  These  subunit  of mitochondria with  mtDNA  2  genes of  the  molecules  carrying these large deletions would account for the loss of growth potential and eventual death of these cultures.  Ascospore 1836  13 from  appear  mtlS-kalDNA  to  cross 572-5 X have  escaped  were detected  1818 and kalilo  in later  ascospore  senescence.  subcultures from  7 from  Neither either  cross 801-1 X AR-kalDNA  nor  of these series. In  senescent subcultures series, resumption of normal growth in cultures which follow a  senescent  fate  is  not  1984). This phenomenon  a  is referred  1986) which in effect means mtDNA  molecules present  commonly observed to as  the  event  (Griffiths  'Lazarus Effect'  and Bertrand, (Griffiths  et al,  that there is a mixture of normal and abnormal  during the  senescence  process  in transfer  series  and  Chapter sampling  of  cells  in  a  region  1 / 140  containing  only  normal  mitochondria  may  on  occasion result in the resumption of normal growth.  Degenerative among  growth as  fungi.  (reviewed Neurospora  by  In  (Bertrand  still  are  these  unknown.  al,  different that  fungi. However, In  senescent  of altered  (Borst,  1987),  et  it would appear  disable  consequence  cerevisiae  Cumrnings  altering m t D N A Thus,  S.  a  1972;  A . nidulans  1985; yet  1986),  the  alteration the  N.  molecules  al et  processes  is  P.  anserina  1981)  and  responsible  for  similar in all fungi. the  event  altering the  intermedia,  is common  1980;  events  is very  mtDNA  1973), al,  genetic  result  causative of  et  (Lazarus  of the  strains  Faye  the  end  mtDNA  the  needed  mtDNA  event  to are  initiating  senescence is the insertion of k a l D N A into the m t D N A . Although the etiology of the three forms of k a l D N A 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 A R - k a l D N A gives rise to the first  mtlS-kalDNA,  k a l D N A insertions.  which gives  rise  to  mtFF-kalDNA,  which results  in novel  IV. C H A P T E R 2  A.  In  INTRODUCTION  this  chapter  movement  of kalDNA  initiated  because  parents  showed  same  tetrad  analysis  is influenced by  ascospore that  accumulate  the  performed the  progeny from  mtlS-kalDNA  type of movement  two Kauaian strains  was  can  host  a  to  determine  genotype.  This  whether  the  research  was  cross using geographically unrelated  originate  from  movement  was not observed in ascospores  from  whereas  a cross  this  between  (561-1 X 605). Ascospores initiated from this cross usually  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 this  when  the  hypothesis,  between  the  senescent  tetrads  senescent  female  were female  strain  is outcrossed.  analyzed. The tetrads strain  P561 and  To further  were  isolated  two nonsenescent  investigate  from  crosses  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 was  used  other  as  one of the  marker. From  the  movement is enhanced  markers  2/142  and  the  results presented in series  behaviour of mtlS-kalDNA  it is observed that the  derived from  ascospores  from  as  the  frequency of  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,  normal  to  and it was suggested that the lifespans depend on the ratio of  abnormal  mitochondria transmitted  1984). This ratio should be representative mitochondria abnormal  present  in the  female  mitochondria, the  progressively increase  as  ratio  senescence  presented  indicate  that  and Bertrand,  ratio of normal to abnormal  Since  abnormal  (Griffiths  senescent  to  normal  proceeds such that the  female parent the shorter the average results  of the  parent.  of  sexually  strains  accumulate  mitochondria should more senescent  the  lifespan of its ascospore derivatives. The  mutagenic  kalDNA  inserts  are  transmitted  to  ascospores initiated from crosses using a senescent culture (subculture 5) of strain P561 as the female parent.  Comparison of the m t D N A 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 the onset of growth of the these  ascospores. In contrast,  ascospores would account for the ascospores  initiated  from  from  short lifespans of  crosses using a juvenile  Chapter 2 / 143 culture of strain P561 as the female parent usually only inherit the neutral or nonmutagenic growth  insert, designated  of these  inherited  ascospores,  by the junction fragments  either  insert occur and trigger  novel the  insertions  onset  or  b l ' and b2'.  During  rearrangements  of senescence.  of  The longer  average  lifespans of these ascospores can be accounted for by postulating that the of senescence  is delayed until a mutagenic k a l D N A insert is generated  the  onset  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, m t D N A 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  This  allows for  more  behaviour of mtlS-kalDNA.  One tetrad  from  cross  10  subcultures.  561-0  subjected  to  X  serial  diagrammatically tetrad X  1766  were  examined.  subculturing  and  in Figures 35 and  The the  opportunity  to  study  the  somatic  cross 561-0 X 605 and two ascospores length  36. The mean  of  of each  average  each  tetrad  series lifespans  is for  from were shown each  is included in the Figures. Inspection of the tetrad from the cross 561-0  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  2/144  the ascospores of mating type A_ (Figure 36).  For  each  series,  mtDNA  was  isolated  from  the  subculture from which m t D N A could be prepared. Bgl  II  and  hybridized  with  the  Pst  first  subculture  and  last  The m t D N A was digested with  I-kalDNA  probe.  There  are  no  Pst  I  restriction sites in kalDNA (refer to the restriction map of kalDNA, Figure 7) so the  clone  segments  consisted of  the  of the  entire  mtDNA.  Bgl  k a l D N A / m t D N A junction fragments  inserted  element  II  digestions  are  formed after  together  were  with the  preferred  flanking  because  two  Bgl II digestion which give  more information on the relative location of a novel insertion and the number of different  inserts  present in the m t D N A .  and autoradiograph P561,  are  The first and second lanes of each gel  the nonsenescent control P605 and the  senescent control  respectively (Figure 37A, 37B, 38A, 38B, 39 A , and 39B). As described  in the  previous chapter, the m t D N A  of strain P605 has  with kalDNA and the only Bgl II fragments  no sequence homology  hybridizing with the Pst I-kalDNA  probe are the normal Bgl 11-10 and -12 fragments  (refer  to Figure  11, lane  of the autoradiograph).  In comparison, hybridization of the m t D N A prepared  the  strain  senescent  control  P561  identifies  Figure 11, lane 2 of the autoradiograph). fragments  Bgl II  weight fragments  Bertrand et  al, 1985)  which are junction fragments  strain. The junction fragments intron of the  and the  (refer  to  with the normal Bgl four higher molecular  of two different  inserts  in this  are labeled b l and b2, and b l ' and b2'. Both are  large r R N A gene and each  molecules. Refer to chapter  bands  from  These bands include the internal Bgl II  of k a l D N A , b3 and b4 (note that b4 comigrates  11-12 restriction fragment;  in the  additional  1  inserted  into different  mtDNA  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 m t D N A  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 bl'  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  molecular weight junction fragments. The junction fragments 4,  5, and  7 all hybridize  with  the  intron probe, Hind  inserts  with  of ascospores  higher 2, 3,  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  insert and not from fragments  would  a rearrangement  event of the transmitted  novel insertion. If they were novel insertions, the junction  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 m t D N A 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 the  autoradiograph  this may  was too short to resolve these bands.  be true  comes from  with junction fragments the  2/146  the  senescent  Further evidence that  control, lane  2, where the  insert  b l ' and b2' also fails to show positive hybridization with  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  partially digested with molecular  weight  the  mtDNA  prepared  Bgl II. This may  bands  which  from  this  account for the  hybridize  with  the  ascospore presence  Pst  was  only  of the  high  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  probably not a junction fragment  of a  is  only  one  band  suggests  that  hybridize with  intron  probe.  It  the  Pst  I-kalDNA  is  novel insertion. The significance of this  band is unknown. In ascospore 8, there are two high molecular weight which  it  probe  is possible that these may  and  do not  represent  fragments  hybridize with  the  junction fragments  of a  Analysis of the m t D N A prepared from the early cultures of each ascospore  series  novel insertion.  Ascus 7 from Cross 561-0 X 1766  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  I-kalDNA  weight  probe. Perhaps  the female parent strain.  fragments these are  are  also  seen  to  hybridize  with  the  insertions which were also transmitted  Pst from  Chapter 2 / 147 Analysis of the m t D N A from the late cultures of the ascospores shows that the junction  fragments  of the  different  in size than  inserts  those  accumulating in ascospores  of mating A. are  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 k a l D N A in the m t D N A prepared from 4 but it should be noticed that they are the  same  ascospore  size as junction fragments  bl'  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  proposed to have originated from  2, 4, 5, and a rearrangment  7 of ascus  1, this insert  was  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  weight  of mating type _a_ all accumulate  junction fragments.  It  is  suspected  that  inserts these  with  inserts  movement since the relative sizes of the junction fragments  high molecular originated  from  are similar to those  described in the previous chapter.  Ascus 5 from Cross 561-0 X1766  Analysis of the m t D N A 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  probably originated from rearrangements junction fragments  which  have  of the inherited insert. The sizes of the  of these inserts, with  the  exception of ascospore  the suggestion that these inserts have originated from  rearrangment  3, support events and  are not novel insertions. The m t D N A of ascospore 3 shows junction fragments of the  same  size as  bl  and b2  and  consequently  may  have  arisen  from  novel  insertion or was perhaps inherited in very low copy number.  Only three  of the m t D N A  profiles of ascospores of mating type _a_ are shown.  Ascospores 6 and 8 have inserts with high molecular weight junction fragments. The  mtDNA  fragments.  profile  of  The absence  ascospore  5  shows  of hybridization  an  of the  insert  with  intron probe  smaller junction to  these junction  fragments indicates that they are probably novel insertions.  The  results from ascus  5 and 7 indicate that movement of k a l D N A 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  senescence  crosses  involved  using  isolating  a  female  tetrads  parent  from  strain  crosses  in  different  stages  of  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  were  subjected  to  Taiwanese natural  serial  isolate. The ascospores  subculturing and  the  length  diagrammatically in Figures 35 and 36. Tetrads from  of each  of each series  tetrad  is shown  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 ascospores from  (Figure 35) reveals that the  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 37B,  profiles  40A, and  mtDNA  prepared  of the  early and late  40B. A s from  the  described  in  subcultures are the  early cultures  previous  of the  shown in Figures 3 7A, section,  ascospores  analysis, of  of ascus  1,  the 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  insert  the  (Figure  inherited insert or constitute 37B). In  the  late  cultures  rearrangements of the  of the  ascospores  transmitted  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 bl'  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  rearrangement  Pst  probe  was  proposed to  have  originated from  of the transmitted insert with junction fragments  ascospore 7 of ascus the  1, this- insert  I-kalDNA  6, no novel fragments  probe.  to novel bands  are  b l ' and b2'. In  observed which  In this ascospore, hybridization  constituting junction fragments  hybridize  of the  of an  a  insert  Pst  with  I-kalDNA  should have  been observed since in the early culture of this ascospore series the mutagenic  Chapter 2 1 151 insert with junction fragments does express  bl  and b2 is observed and because  the  culture  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 ascus  3  cross 561-5 X 1766. From cross 561-5 X 1766, the ascospores of  do  show  variability  in  lifespan  subcultures and the other four take from  where  four  series  die  within  10  12 to 20 subcultures to die. In ascus  4 from cross 561-5 X 1766, all ascospores series die within 8 subcultures.  The m t D N A 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  junction fragments  1766, show  or  a  the  neutral  insert  with  b l ' and b2' (Figure 38A and 39A). The late cultures of the  ascospores of mating type A_ from insert  transmission of only  rearrangement  of  these same asci accumulate either the neutral  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  bl  and b2 is transmitted  in higher  Chapter 2 1 152 copy number than the insert with junction fragments and  3 inherit the  number.  insert  with junction fragments  Ascospore 7 inherits both inserts  b l ' and b2'. Ascospores 1  b l ' and  b2'  in higher cop}'  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 bl'  and  inserts  b2' to  to  have  accumulate  accumulated.  The  in  to  addition  insert with junction  mtDNA the  of  ascospore  transmitted  5  fragments  shows  inserts  with  other-  junction  fragments b l and b2, and b l ' and b2'.  Ascus 3 from ascus  4  cross 561-5 X 1766 is somewhat of an anomaly compared  which  was  isolated  from  the  same  cross.  The  transmission  with of  mtlS-kalDNA is reminiscent of ascus 5 and 7 from cross 561-0 X1766; only the insert with junction fragments number  (Figure  b l ' and b2' is transmitted and in very low copy  42A). Analysis  of  the  late  cultures  shows  that  the  accumulating are generally not the transmitted insert (Figure 42B). In 1 and  5, both of mating type  inserts  hybridize with  the  A,  the junction fragments  intron probe.  The  fragments  rearrangements  of the  neutral insert. In ascospore 4, mating type A , two inserts with high  molecular weight junction fragments not  ascospores  accumulating  sizes of these junction  indicate that these insertions probably originated through transmitted  of the  inserts  hybridize strongly  with  these are junction fragments  the  Pst  are  observed. These junction fragments  I-kalDNA  probe  of a novel insert because  but  it  is  suspected  did that  the size of each junction  Chapter 2 1 153 fragment  is  previous  chapter.  ascospore  2,  similar  to The  mating  the  junction fragments  bands  hybridizing  type _A, fail  to  suspected that these junction fragments ascopores  3 and  with  hybridize  of an  insert  the  Pst  I-kalDNA  with  the  intron  in  the  probe  probe.  It  of is  are of a novel insertion. The m t D N A of  8, both of mating type _a_ show the  accumulated. The junction fragments  described  neutral  insert  to  have  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.  Chapter  561-0 ?  605 6  X  subculture  ascus 1  5  0  1A  2 / 155  10  —  i  2a 3A  e i — - • — •  4A  R*—HHi  5A  —  —  6a 7a  H  8a  W  H  K  H  n M  M  —  •  B B  M  9  X  ascus 6  15  -  —  —  —  » w  i  M  average  561-5  number  lifespan=11  605 (J  subculture  number  average lifespan: 7  20 ™  »  Chapter  Figure 561-5 labeled which  2/156  36. Subculture series of tetrads isolated from crosses 561-0 X 1766 and X 1766. The ascsopore culture from which each series were derived is 0. The last number spanned by a horizontal bar indicates the subculture produced no viable conidia. The mating type of each ascospore is shown.  Chapter  2 / 157  »  9  561-0  X 1 7 6 6 Cf  ascus 7  subculture  0  5  1a 2a 3a 4A  number  10  15  20  wmm——mmmmmmmmmmm—mm mmm—mmmmm—mt—^m—mmm wmmmmmmmmmmm—mmmmmmm—mmmmmm wmm^——m—m* 5 A ^m—a^mm^mm* average lifespan=12 6A w—mmmmmmm—mm. 7 a ^——m—mmmmmmm—mmm 8A  MHHHMMMM  ascus 5 1A 2A 3A 4a 5a  m—mmmmmm^mm^mmm—mammm^ 6a wmmmmmm—mm—mmmmmmmm. 7A wmm—mmmmmmmimmmm—m 8a  MHHMMMMHMMMHMMMI  9  561-5  X 1 7 6 6 Cf  ascus 3 0  subculture 5  number 10  1A wmmm—mmmm^m 2A  iBMMHBMa  8a  15  2 0  BMMMMUMMSMMaBMBH  3 a mm—mmm—i 6a 7a  a v e r a g e l i f e s p a n s 15  i  —  a v e r a g e l i f e s p a n = 10 —  MWHM  ascus 4 1a 2A 3A 4A 5a  6 a mmtmmmmmt 7a 8A  average lifespans 5  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 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 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 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).  Kal  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).  Kal  Chapter  2/166  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 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  2/168  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 b l ' 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 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 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 N S 1 2 3 * 4 7 8  A N 5 1  a  2  Kal  3  6  4  7  8  Chapter  2/172  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 b l ' 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).  Kal  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 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 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  5 6 1 - 5 x 1 7 6 6 ascus 4 A N  S  2  3  4  8  3 1  3  7  N  S  A 2  a  3  4  Kal  8  1  5  7  Chapter  2/176  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 b l ' 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 N  S  2  3  4  8  1  a  A 5  6  7  N  S  2  3  4  8  Kal  1  5  6  7  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 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).  Kal  Chapter  2/180  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 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 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  ascospores  of  series  described  isolated from  the  in  cross  the  561-1  previous X  chapter  1766. It  were  was  started  from  observed that  insert inherited did not accumulate and thus initiate senescence;  the  this insert was  termed a neutral insert. A t 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  revealed  that  strain  P605  movement  type _a_ initiated from from  and  to  the  of k a l D N A  cross  strain  occurred primarily  561-0 X  this cross and all but  Taiwanese  1766.  in ascopores  1766. The ascospores  ascospore  1 of ascus  1 from  influenced  of  that  insert.  These  results  by a gene or an allele of a gene  indicate  that  analysis  of mating  of mating type A. cross  accumulate either the inherited insert with junction fragments rearrangement  The  561-0 X 605  b l ' and b2' or a movement  may  be  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  allele or gene  is introduced from  ascospores  mating  of  type  strain  A_ would  and j \ for strain  1766 and linked have  shown  the  1766. If an  to mating type highest  frequency  then of  movement of k a l D N A since mating type A is transmitted from strain 1766. The  Chapter  2/183  fact that ascospores of mating type _a_ of ascus 1766 showed movement indicates that the 1766  probably  proposed  segregates  nuclear  gene  was  independently linked  to  5 and  7 from  cross  561-0 X  allele or gene introduced from of  mating  mating  type,  type. two  In cross  addition, overs  strain if  would  the be  required to obtain the segregation patterns observed in the two tetrads analyzed. This model indicates that in addition to tetrads in which type  &_ show  movement,  tetrads  where  ascospores  of  ascospores of mating  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  the cross 561-0 X 1766 since ascospore  1 of ascus  initiated from  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  ascospores of cross 561-0 X 1766.  in series  derived  from  Chapter 2 / 184 A similar phenomenon has been reported in P. anserina (Vierny et al, 1982). In this  fungus,  generally  mating type  grow  differences  approximately  appear  14cm  to affect  whereas  mt+  longevity. Strains strains  (Vierny et al, 1982). Analysis of the m t D N A prepared different  mating types  revealed  that in mt-  strains,  sequences are present whereas  only the integrated  cultures. This result suggested  that the  juvenile  the  cultures  accounts  for  locus or a  about  from juvenile cultures of  autonomous  alpha  senDNA  state in which alpha senDNA exists in  difference  in longevities  (ie: the  sooner  the  the longevity of that strain).  differ in mating type implies that the  gene closely linked  40cm  sequences are present in mt+  probable appearance of alpha senDNA, the shorter The fact that these strains  grow  of mt-  mating  to mating type might act on, for example,  type the  probability of excision of alpha senDNA from the m t D N A . Although the isolation and characterization of this locus has not been performed the hypothesis that it affects gene  the excision of specific regions of the m t D N A suggests that the affecting  intermedia active  the  may  form  act  of  this  movement  or  movement,  then  Kauaian ascospores  movement  is  genome  in a  of  similar  gene  in  a  protein  the  active  fashion.  It  N . intermedia which  form  since movement  initiated from  mtlS-kalDNA  crosses  may  this  proposed  that  of  N.  that  the  as  a  suppressor  of  movement.  If  a  suppressor  of  gene  two  strains  acts  would  of mtlS-kalDNA between  senescent be  either  facilitates of  in  proposed  be  associated  is generally not  Kauaian  strains.  with  the  observed in  The  nonactive  allele would be introduced through outcrossing and the movement of mtlS-kalDNA observed suppressor  in  those  progeny  which  received  the  nonactive  allele.  How  this  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 mtlS-kalDNA  occurs  junction fragments  in this chapter  only  in  are results revealing that movement of  ascospores  which  inherit  the  neutral  insert  with  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  rearrangement  of  mtlS-kalDNA  of the  neutral  would  be  insert must  observed.  In  reduced strain This  by  50% for  the probability of escape  ascospores  initiated from  in a juvenile stage of senescence reduction  would  be  a  crosses  crossed  consequence  ascospores,  occur to initiate senescence.  would result in those cases where either no rearrangment insert was lost. In contrast,  these  of  Escapees  occurred or the neutral  from senescence using a  would be  Kauaian female  to a nonKauaian male strain.  50% of the  progeny  receiving the  nonactive form of the host suppressor gene. This indicates that the 50% carrying the  nonactive  which  would  form  of the  initiate  safeguard  against  movement  of  the  gene  would  senescence.  Thus,  possible  extinction  mtlS-kalDNA  in  ascospores  strains and the opportunity to escape  An  carry this of from  novel insertions  of  mtlS-kalDNA  model  an  evolutionary  suggests  Kauaian crosses  strains between  by  preventing  two Kauaian  senescence.  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  movement of mtlS-kalDNA generally only occurs in ascospores  genome  initiated from  since the  Chapter outcrossing of a gene  product  examples  senescent female  on  from  the  Kauaian strain.  movement  other  systems  2/186  of  kalDNA  also indicate  is  The  mode  unknown,  of action of this but  a  that functional host  number  nuclear  of  gene(s)  are required for the transposition of a given mobile element.  In the fungus been  P. anserina,  identified  (Tudzynski  which  and  various combinations of recessive nuclear genes have  prevent  Esser,  the  1979;  expression  Esser  and  and/or  propagation  Tudzynski,  pleiotropic because in addition to suppressing  the  1980).  of  senescence  These  genes  are  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  senDNA is only possible when the wild type gene products In Neurospora, a gene showing similar characteristics senescent strain  to no expression of senescence  in  has been identified in the  addition to  suppressing  as  a  of tetrads exhibiting a 4:4 segregation of (Griffiths,  nuclear gene suppressing the expression of senescence because  are being expressed.  2360 his. Crosses using this senescent auxotrophic strain  female parent results in the ascospores senescence  amplification of  the  expression  unpublished).  The  proposed  is also considered pleiotropic 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 for  movement  supercoiling. recombination  of  mtlS-kalDNA  These events,  genes  have  including  include, been  for  example,  shown  to  transposon-mediated,  host affect  site  genes several  specific  necessary  involved  in  kinds  of  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  For  instances  transposition  example, the  appear  to be  within  a  Fowler  and  affect  the  movement  behaviour of the  subject  plant  or  a  single  developmental maize  tissue  1978).  that  (McClintock,  It  was  suggested  timing  and  the  1983).  In  (Federoff,  is developmentally  Spm and E n mobile elements  to regulation by factors  Peterson,  in  of mobile elements  are  apparent  of Zea maize  differentially distributed  1965; that  regulated.  1971;  similar frequency  Drosophila, P  Peterson, events of  1966;  may  also  Ac and  element  Mp  movement  is  tissue specific; occurring at high frequencies only in the germline tissue (Laski et al,  1986). The P element is regulated  final  at the level of m R N A splicing where  the  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 in  a  particular nuclear background compatible with the  1977;  Levings  Gabay-Laughnan S, T, and  which  1965;  Laughnan  al,  1982;  C, are  genes,  restorer  et  1980;  Laughnan  and  al,  cytoplasm (Pring et al, 1981;  Laughnan  and  1983). The three cytoplasms which cause male sterility,  distinquished on the  restore  et  spontaneously  normal  basis  of nuclear  pollen development  Gabay-Laughnan,  genes appear to affect  1983).  (Duvick At  the  genes,  called  restorer  et  1961;  Duvick,  al,  molecular  level,  the characteristic rearrangements of the  of sterile cytoplasms. S-type cytoplasms have been characterized by the  these mtDNA  presence  Chapter of two linear 1983).  which  (Schardl et contains  plasmids, S I recombine  with  S2 (Pring et  the  mtDNA  to  al, 1985). In addition to the  internally  Reversion  and  2/188  of  background  integrated  S-type  results  Si  and  cytoplasms in  the  recircularization of the  in  loss  mtDNA  with  generate linear  linear m t D N A S2  the  of the  al, 1977; Levings and  sequences  presence linear  Si  only the  mtDNA  molecules  molecules, the (Schardl  of  Sederoff,  the  and  et  mtDNA  al,  1985).  appropriate  S2  plasmids  internally integrated  Si  nuclear and  the  and  S2  sequences remaining (Schardl et al, 1985).  All  these  examples  indicate  that  movement of mobile elements.  host  functions  Although in most  these genes on transposition  is unknown, they  are  important  systems support  in  mediating  the mode of action of the  hypothesis  that  the  active form of the gene identified in N . intermedia may be required to facilitate movement  of  regulating the outbreeding  of  Alternatively,  mtlS-kalDNA. movement  The  of a mutagenic  geographically kalDNA  outbreeding, increases  evolutionary  may  unrelated be  thought  significance  element strains of  the rate of mutation  as  novel  kalDNA  insertions  mutation  would generate new  but,  variants  an  and a  thus  host  promote  mutator  relative to the  explosive  which  a  may be to prevent  rate. In natural populations this would result in decreased carrying  of  factor,  increase  successful speciation.  which  spontaneous fitness  gene  upon  mutation  of the progeny in  the  level  of  would be evolutionarily significant  because genetic variation is necessary for evolutionary change.  Chapter 2. Comparison  of Tetrads Derived  2/189  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 m t D N A prepared from early cultures of ascospores  isolated from crosses using a juvenile female  (561-0), inherited only the neutral insert with junction fragments contrast,  ascospores  isolated from  (561-5)  inherit both  kalDNA  crosses  inserts  involving the  present  in the  parent  b l ' and b2'. In  senescent  female  the  female  parent  parent  strain.  The  different inheritance patterns of mtlS-kalDNA may depend on the ratio of normal to abnormal mitochondria present ratio  of abnormal  to  in the female parent  normal mitochondria, the  more  such that the higher the opportunity  for  abnormal  mitochondria to be transmitted. This does not explain why only the insert junction fragments from  crosses  with  b l ' and b2' is preferentially transmitted to ascospores isolated  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 a juvenile state, the mitochondria  screening process  and mitochondria with  would be  mtDNA  inserts would be exclusively transmitted  where the female is in  very effective  in that normal  molecules carrying neutral  kalDNA  to progeny. The screening process would  not be as efficient in meioses involving a senescent female parent because of the presence  of greater  abnormal hypothesis,  numbers  mitochondria the  would  expression  of  of abnormal mitochondria. Thus, both normal and be  transmitted  senescence  of  to  progeny.  ascospores  According  which  inherit  to  this  abnormal  Chapter 2 / 190 mitochondria  would commence  at  lifespans of these ascospores  the  onset  would be quite short.  observations of the m t D N A prepared ascospore  series from  P561 as the inserts  are  molecules  crosses  begin  Ascus  3  inheritance  patterns  same  from of  to the  ascospores  source  of  mitochondria.  Knowing  at  in  different  culture  This  onset  561-5  (subculture  as  3. This  that  by  5) of strain  that  does  other  defective  growth not  two  mtDNA  and  follow  tetrads  initiates  the  same  isolated  from  Only the neutral insert is transmitted be explained by postulating that  consisted  Neurospora  regions  1766  the  may  transmitted  indicates  of vegetative  X  female parent.  populations are heterogeneous, differ  the  cross  of ascus  mitochondria  This notion is supported  the early and late subcultures of the  insertions.  mtlS-kalDNA  crosses using the senescent  propagation. Thus, the  In these ascospores, the inherited and accumulating  accumulating  senescence.  from  using a senescent  female parent.  generally the  of vegetative  of  is  more  coenocytic  normal and  than  that  the  abnormal  mitochondrial  the ratio of normal to abnormal mitochondria may of  the  cytoplasm  and  the  source  of  cytoplasm  transmitted to these progeny would reflect this.  Analysis from  of the  crosses  mtDNA  prepared  from  the  using a juvenile subculture  late cultures of ascospores initiated  of strain  P561  revealed that either a novel insertion or a rearrangement insert was  required before  senescence  was  as  the  female  parent  of the inherited neutral  induced. This would  account for  the  longer average lifespans of these ascospores.  It  is  of  interest  that  the  accumulate in some ascospore  insert  described  as  being  neutral  is  observed  series. This insert was termed neutral because  to it  Chapter  2/191  was never observed to initiate senescence  in the series described in the previous  chapter. Why it induces senescence  in some but not all ascospore  understood.  of  Perhaps  the  presence  subtle  mutations  carried  series is not on  the  same  m t D N A molecules as the neutral insert were responsible for their accumulation.  In  summary,  the  sexual transmission  of mutagenic  kalDNA  inserts  depend on the age of the strain used as the female parent.  appears  to  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 m t D N A onset  or m t D N A  of senescence  until a  molecules carrying neutral k a l D N A inserts delays the mutagenic  insert  insertion of k a l D N A or through a rearrangment transmitted insert.  is generated  either  through  novel  of the m t D N A encompassing the  V. CHAPTER 3  A.  INTRODUCTION  This chapter  focuses  on a senescent  Kauaian isolate, strain P573, which  some but not all the characteristics of kalilo senescence. of senescence from  (Griffiths  et  a  al,  India  A total of four classes  have been delineated in Neurospora. Two of these four clases were  identified  Aarey,  survey  of natural  1988a).  (Griffiths  nucleus associated  The et  element,  first  al,  isolates class  1988b).  from  was  kalDNA  of and  the  kalilo  marDNA.  element, The  but  second  senescence  is  there  type  are  maternal  geographically unrelated. transmission  of  is suspected  that  senescence  the  results  associated  from  from  with  a  sequence,  is reminiscent of the  sequence  homology  is nonheritable  between and  its  is shown by many natural isolates  class  cytochromes, the lack of m t D N A alterations, It  no  of senescence  This  senescence,  is  locations  population  and a mitochondrial insertion  basis not understood. This type of senescence which  geographic  discovered in a  Here,  AR-marDNA,  different  mtlS-marDNA. The mode of action of these two elements behaviour  shows  is  characterized  presence  of  by  normal  and a unique pattern lethal nuclear  the  lack of  amounts  of  of infertility.  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 m t D N A and are either carried on the mtDNA  molecule or excise and acquire m t D N A  sequences.  disrupting the mtDNA, suppressive  accumulation of the  mtDNA  The  molecules  commences.  192  fourth  case  As a consequence of  altered mtplasmid and/or is  kalilo  senescence.  Chapter  3/193  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 k a l D N A  and  senescence  but  does not  alwaj's  exhibit the  biochemical and molecular events known to accompany kalilo senescence. in this chapter progeny. trace  The  is the analysis  amounts  and  the  senescence  and  Presented  molecular characterization of this strain and some of its reveals  that  behaviour  AR-kalDNA  is present in most  of mtlS-kalDNA  cultures do not exhibit cytochrome a a express  same  eventually  3  is erratic.  In  cultures  addition  in  some  and b deficiencies yet these cultures do  die.  This  strain  is  highly anomalous  when  compared to the prototype of kalilo senescence,  strain P561, and thus provides a  cautionary  cases  note  against  extrapolating  to  all  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  Cytochrome spectra  were  and  Bertrand,  prepared  from  1984;  Bertrand  subcultures  1,  et  al,  1985;  4, 7, 8, and  43) . A normal cytochrome complement is observed for subcultures subculture  detectable.  The progressive loss of cytochromes a a  cytochrome  c  is  crossed  a  female  as  typical  for  hybridized because  with  at the  most  Kauaian  progeny  3  the  strain  aa  3  and b and  3  and b  and b and the  strains.  show  vegetatively. This  as normal kalilo senescence subcultures  senescent  parent  potential when propagated  from  9, no cytochromes  When  same  deviates  9 (Figure  1 and 4. By  7, there is a decline in the amount of cytochromes a a  an increase in cytochrome c. B y subculture  1986).  increase in  this  decline from  are  strain  is  in growth  what  appears  when the m t D N A is examined. M t D N A was isolated  1, 2, 4, 6, and 8. The m t D N A was digested with Bgl II and the  probe  time the  Hind  III-K1.  The  K l clone was  hybridizations were performed  available that consisted of the 9.0kb element). The m t D N A  majority of k a l D N A  sequences  used  this was  sequences  as  the  a  probe  only clone  (ie: 7.0kb of the  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 44) . No novel bands  are detected  in subcultures  in subcultures  1, 4, and 8. In subculture 2,  the sizes of the novel high molecular weight fragments junction fragments  for an insert within the  2 and 6 (Figure  are the same size as the  intron of the large r R N A  gene. B y  subculture 4, this insertion is lost. Reoccurrence of k a l D N A 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  the neutral insert previously described and thus it is postulated  b l ' and b2' of that these two  Chapter bands  are junction fragments  (bl  1 7  3/195  and b 2 ) 1 7  of one  two higher molecular weight bands junction fragments  of the (bl  inserts  and  and b 2 )  1 8  of the  1 8  other insert. Clones encompassing almost the entire m t D N A were used as to determine the location of the insert with junction fragments None  of the  bromide  mtDNA  stained  clones  hybridized with  gel one of the  these  bands.  b l  From  two mtplasmids characteristic  1  8  the  probes  and b 2  the  1 8  .  ethidium  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. B y subculture 8 both inserts  are  lost.  To further investigate this strain, three ascospore-derived series were studied. The ascospores  were isolated from  and the nonsenescent  a cross using culture 573-1 as the female  strain 1766 as the male parent. The ascospores  parent  designated  1 and 7 die within 10 subcultures. Ascospore 5 did not express senescence after  In  80 subcultures.  the  two  senescent  ascospore  series,  subcultures, commencing with subculture are  even  shown  in  Figures  complement  is  observed  45  and  46.  throughout  the  mtDNA  was  isolated  from  alternate  2. The corresponding cytochrome spectra For  ascospore  entire  series  1,  a  (Figure  normal  cytochrome  45). Ascospore 7  shows a normal cytochrome spectra through to subculture 6. B y subculture 8, an abnormal absorbance profile is observed (Figure 46).  The Bgl II digested m t D N A profiles are shown in Figures 47 and 48. Analysis of the  ethidium bromide stained  gels reveals  that the  Bgl II m t D N A  patterns  Chapter are  indistinguishable from  mtDNA  with  either  mtlS-kalDNA  the  the  nonsenescent  E , Hind  in all subcultures,  Hybridizations using the  3/196 control P605. Hybridization  III-K1,  or  even after  B  probes  shows  the  the  absence  two week autoradiograph  intron probe shows no differences  of  between  of  exposures.  strain P605  and the m t D N A s of these two series.  The  cytochrome spectra of subcultures  2, 6, and 76 for ascospore  in Figure 49. No deficiencies in cytochromes a a  5 are shown  or b are observed  3  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 m t D N A 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 k a l D N A 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  111-13,18,  and  constitute  approximately 3.5kb  The  Hind  radioactively-labeled 111-14,15,  which (refer  mtDNA  are to the  clones,  contiguous mtDNA  Hind  within  the  III-10a, mtDNA  restriction map,  that  in addition to  the  deletion  of the  majority  and  Figure 6).  fact that the novel Hind III band hybridizes with all three m t D N A  suggests  Hind  probes  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 A R - k a l D N A is  present  in all subcultures  AR-kalDNA  is barely detectable  Interestingly, weight  the  than  AR-kalDNA ascospore  of the  band  normal. is only  in most  hybridizing In  the  seen  original  in  series  P573 series. Panel B shows  subcultures  subculture derived  in subculture  is detected.  AR-kalDNA  is  It  detected  2  is  in  a  ascospore  of  a  copy  7  series  even after  1 series.  higher molecular  ascospore  6. Panel D is the  should be noticed that  it  is  from  5 which showed no sign of senescence  AR-kalDNA  of the  (Panel derived  far  C), from  80 subcultures. No  in all subcultures  number  that  below  in which  that  generally  encountered in kalilo strains.  Seven the  ascospore-derived series  presence  complements. from  cross  of  AR-kalDNA  As with 573-1  which and  ascospores X  1766.  exhibited long lifespans  mtlS-kalDNA, 1,  5, and  The  and  for  7, these  lengths  of  were  analyzed for  abnormal  ascospores each  series  cytochrome  were initiated are  shown  diagrammatically in Figure 53. The number of subcultures preceding death ranged from  30 subcultures to no signs of senescence  spectra Abnormal The  were  prepared  from  the  late  after  subcultures  spectra were observed for ascospores  2, 4,  80 subcultures. Cytochrome of  each  15, and  other three cultures had normal cytochrome complements.  ascospore  series.  19 (Figure 54).  Chapter MtDNA  3/198  and nucDNA were prepared from the late subcultures of each ascospore  series. The m t D N A was digested with Bgl II and hybridized with Pst I-kalDNA (Figure  55).  fragments  In  all  cultures,  no  novel  Bgl II  fragments  are  detected.  Bgl 11-10 and -12 hybridized with the probe. The nucDNA  Only  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 The  of fragments  of the  restriction fragments  same  mobility  hybridizing  as those of ascospores  in ascospores  4, 9,  and  4 and  19 are  19.  the two  internal fragments of k a l D N A (E and G) and the terminal inverted repeats which co-migrate. These bands is  intact.  Only  one  are  band  of the appropriate sizes indicating that of a  slightly  faster  hybridized in the late cultures of ascospore  mobility  than  the  15 (designated by the  AR-kalDNA fragment  E  arrow). This  indicates that all Eco R l restriction sites are gone. In all the cultures in which AR-kalDNA  is detected,  it is present  encountered in kalilo strains.  in a  copy number  below that generally  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 a a . 3  Chapter 3 /  200  •train 573 I  W A V I I E N O T H (nm)  Chapter  3/201  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 b l and b2 are of an insert detected in the mtDNA prepared from subculture 2. The bands labeled b l and b 2 ,and b l and b2 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. 1 7  1 8  1 7  1 8  2o2_  Series 573 §  1  2  4  6  8  §  1  2  4  6  8  bl b2 8  /  bl bl b2  pi DNA •  7  b2  7  b3  b4 EtBr  Kl  12  8  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 a a . 3  Chapter 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 a a . 3  Chapter 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 R l - 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.  m 2  4  6  8  §  2  4  6  8  12  14 EtBr  E  H13,18  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 R l - 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.  Ascospore  m §  2  4  6  7 8  m 10  §  10  2  4  6  8  10  §  2  4  6  8  IO  S  2  4  6  8  c  12  14  ErBr  H13.18  K l  Chapter  3/211  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 a a . 3  Chapter 3 /  212  Chapter  3/213  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 R l - 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 R l - 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.  Ascospore  5  in  m  m  o  o  o  o  2 76  EtBr  o  2  E  76  o  m  2  H13,18  76  o O  2  m  o  76 O  H14,15  2  76  HlOa  Chapter  3/215  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 R l - 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 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. 2  Ascospore 5 m  2  2  6  m 76 §  2  o  6 76  O £5  " S3  %»#  *  3 2  6  2  6 76  76  - ~-  i EtBr  m  ID  Kl  f E  H13,18  Chapter  3/217  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 R l - E probe (refer to Figure 9 for details on this probe).  218 A 573 2  4  B 6  8  C spore 1 2  4  6  8  Kal  D spore 5  spore 7 2  4  6  8  10  2 6  76  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 10  0  ascospore  2  n  4  •  9  m  15  M  18  H  19  m  20  m  subculture 20  9  number 30  40  50  60  ^ 8  O  Chapter  3/221  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 a a . 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.  Kal  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.  573  spores  m o  in  w  w  in t> jh g J O  Kal  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  characteristics of kalilo strains but the lifespans of ascospore-derived observed  to be quite variable with the  average  lifespan of the  genetic  series  ascospore  were 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 aa  and b and  3  the  both kalilo strains  decrease in growth  and 'stopper'  potential  extranuclear  are  typical  characteristics of  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  from the normal patterns of kalilo senescence  al, 1986). The strain  diverges  when the m t D N A and nucDNA  are  analyzed. This strain shows only trace amounts of A R - k a l D N A , and mtlS-kalDNA is  seen  only  mtlS-kalDNA the  erratically.  to  the  proposed  mechanism  of  is usually observed to accumulate during vegetative  time of death  contrast,  According  be  equimolar  with  the  mtlS-kalDNA does not accumulate  mtDNA  (Bertrand  and in the  senescence,  growth and  et  al,  1985).  at In  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  alternate exceptional  than  subcultures  expected.  of two  in showing a  NucDNA  senescent  normal  and  mtDNA  ascospore-derived  cytochrome  complement  were  series. and  isolated  from  Ascospore  1 is  no  mtlS-kalDNA.  Chapter 3 / 228 This  ascospore  P573  culture.  mobility  series In  relative  shows  traces  subculture to  2  of AR-kalDNA  of  this  normal A R - k a l D N A  series (Figure  as  observed  AR-kalDNA 52)  in the  exhibits  suggesting  that  a  treatment was incomplete. The latter hypothesis is preferred  that  AR-kalDNA  Student,  has  protein  associated  with  its  ends  slower  either  element contains more D N A sequences than normal, is circular, or the K  original  the  proteinase  since it is know  (Chan  B-S, Doctoral  personal communication) and if these proteins are not removed prior to  electrophoresis A R - k a l D N A patterns upon senescence. cytochromes  aa  and  3  shows a slower mobility. Ascospore 7 shows Again, no mtlS-kalDNA is detected,  b become  apparent  as  senescence  different  yet deficiencies in  proceeds.  Interestingly,  A R - k a l D N A is seen in only one subculture of this series. Together these results indicate that neither A R - k a l D N A nor mtlS-kalDNA need be retained in order for senescence ascospore  to  be  In  addition,  normal  1 and deficiencies in cytochromes a a  that senescence inherited.  expressed.  In  may general,  3  cytochrome  and b in ascospore  or may not be mitochondrially-based, yet the  presence  of  normal  complements  cytochrome  by  the  accumulation  cytochromes a a by  postulating  3  of  expression of senescence grossly  defective  mtDNA  complements  subtle  alterations  to  the  is  an  in strain P573  is not always  accompanied  molecules.  loss  and b in the series derived from ascospore that  7 suggest  it is maternally  indicator of wild type m t D N A suggesting that although senescence is maternally inherited, the  in  mtDNA  The  of  7 can be explained  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 m t D N A of N . crassa do result  in their accumulation and the loss of cytochromes a a  3  and b.  Chapter  3/229  A n ascospore series which did not die even after for  examination.  spectra. this  MtDNA  series.  present  As  expected,  and  nucDNA  No AR-kalDNA  in  the  mtDNA  cytochrome were  was  analysis  prepared  detected,  preparations.  80 subcultures was also chosen  from  yet  Based  revealed  three of the  about on  normal  absorption  subcultures of  l k b of mtlS-kalDNA  hybridizations using  the  was 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  and includes one of the Hind  1300bp inverted repeats. Absence of hybridization of the  III-K1 probe to novel Bgl II bands  sequences,  element  indicates that in addition to the K l  the other inverted repeat has been deleted. This leaves approximately  300bp of k a l D N A inserted in the m t D N A . This deletion would result in the loss of the three Bgl II and the Hind III restriction sites. Only one novel Hind restriction fragment  III  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 k a l D N A should  include all three Bgl II  fragments been  may  generated  be  explained by  in the  restriction sites.  The  postulating that  a  remaining sequences  presence  Bgl II  of kalDNA.  of two  Bgl II  restriction site  This would account  has for  two novel Bgl II fragments  both hybridizing with the E probe. The hybridization  of the  111-13,18, and Hind  Hind  III-10a, Hind  Hind III band suggests that a rearrangement fragments  the novel  event involving all three restriction  must have occurred. It is difficult to determine the arrangement  altered m t D N A molecules  111-14,15 probes with  and kalDNA, but whatever the arrangement,  should,  defective m t D N A  theoretically,  induce  the  suppressive  molecules and initiate senescence.  the altered  accumulation  of the mtDNA of  Whether this insert and  the the  Chapter  3/230  defects to the m t D N A may be considered neutral as described for the insert with junction  fragments  bl'  and  b2'  in  the  previous  two  chapters  is  undecided.  Sequencing is required to determine the organization of this region of the m t D N A and  to understand  why this m t D N A  alteration does not induce the  suppressive  accumulation of these altered m t D N A molecules.  Observations made from the late subcultures of other ascospore series also show different  combinations  subcultures  of  usual  and  unusual  examined, no mtlS-kalDNA  kalilo  is detected  properties.  and of the  Of  seven  the  late  ascospore,  only three show normal A R - k a l D N A . The cytochrome spectra for only four of the series  showed  between  the  deficiencies in cytochromes presence  of kalDNA  and  aa  and  3  cytochrome  b.  There  is  deficiencies  no correlation 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 k a l D N A from its  genome.  In all series  analyzed, mtlS-kalDNA  yet, in some cases, it was present rather  than  accumulation ma}'  be  observed. P573  and  having a  suppressed  such  Thus, it would its  derivatives  and  remove  the  normal  appear  that not  kalDNA  replication and  that  does  appeared  to accumulate  throughout a series. This may indicate that  predisposition to  of mtlS-kalDNA  never  kalilo the  involve  patterns  mechanism kalDNA,  from  the  retention of  genome,  of A R - k a l D N A  senescence  of senescence but  the  rather  are  not  in strain  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  3/231  somatically transmitted and thus persists in this strain. It has been that  in strain  P573  senescence  indicating  that  senescence  work  the  class  in  nonheritable  of  senescence  is transmissible  cannot  be due  senescent (Griffiths  The  transmission  exclusive to  of  strain  strains, et  defines a fifth class of senescence  to  al,  the  (Griffiths  Bertrand,  1984)  same unknown mechanism  previously  1988).  and  demonstrated  described,  Perhaps  this  at  which  show  Kauaian  strain  in Neurospora.  mtlS-kalDNA  described  P573. This is interesting  strain P573 is the only senescenct  in  this  chapter  because in the  appears  chapter  Kauaian strain to contain detectable  to  to  be  follow, 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 k a l D N A  to give the  results  described.  How these  elements would interact to produce these atypical aging patterns is unknown.  two  VI. C H A P T E R 4  A.  INTRODUCTION  Kalilo D N A shows no homology with either m t D N A or nucDNA indicating that it is foreign in origin (Bertrand et al, 1985; 1986). It was of interest to determine whether  kalDNA  has  a viral  origin. A number  genomes  which  are  double  stranded  et  1984).  intermediates  (Dodds  al,  of fungal viruses contain R N A  or  contain  double  stranded  Thus  double  stranded  RNA  relevant mainly as a indicator of the presence  replicative (dsRNA)  of potential viruses. A survey of  Kauaian as well as nonKauaian natural isolates of Neurospora was undertaken detect for  the  presence  were included in the  of dsRNA.  survey.  A total  is  of nine  Kauaian senescent  to  strains  Of these, only one Kauaian strain, P573, showed  the presence of dsRNA which did not hybridize with the Pst I-kalDNA probe.  Although  it  senescence,  appears Neurospora  viruses. First, the defined  mutant  that  the  is  an  convenient  stocks  presence ideal  et  system al,  interaction of host and viral genomes. been  collected from  around  away from the fungus of fundamental  the  dsRNA  organism  genetic  (Perkins  of  world.  for  is  potential  and the  1982)  would  Second, hundreds These  not  strains  relevant studies  to  kalilo  on  fungal  availability of many facilitate  studies  of natural are  very  on  isolates  few  the have  subcultures  growing in nature so they should reflect well the  genetic elements  well  array  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  232  mitochondrial  introns  (Collins  and  Chapter Lambowitz, kalDNA  4/233  1983; Nargang et al, 1984), and the senescence  and  marDNA  (Griffiths  and  Bertrand,  1984;  determining elements  Bertrand  et  al,  1985;  Griffiths et al, 1986; Bertrand et al, 1986; Griffiths unpublished).  There  are  a  phenotype  few  of the  well host  documented fungus.  cases  in  which  dsRNA  The fungi Saccharomyces  viruses  alter  the  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 Puhalla, Wickner,  strains  of the  1971; Koltin 1981;  same species  and  Bostian,  Agaricus bisporus,  at  1984; least  Peery  five  pathogenic  fungi there is an  is  responsible  (Hankin and  for  chestnut  association between  blight  (Day  et  al,  1987).  In  viruses have  the been  (Tavantzis et al, 1980). In  and a decline in pathogenicity; see for example, the which  et  dsRNA  implicated as the casual agents of L a France disease some  species  and Day, 1975; Rogers and Bevan, 1978; Bussey, 1981;  1983; Tipper  cultivated mushroom  or closely related  the  fungus  al,  presence  of dsRNA  Endothia parasitica  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  Grones,  1983).  (P147).  The  stranded  The  and three  mutants  R N A genomes  Peterson, strains  abnormal-1 (Turna  1972; are  and and  Kuntzel  designated mi-1  Grones,  et  al,  1973;  abnormal-1,  contain  VLPs  1983;  Kuntzel  mi-1, and  which et  Turna  al,  have 1973).  and 2215 single The  Chapter properties  of the  V L P genome  in strain  4/234 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  B.  detected.  RESULTS  1. Identification and Cross Homologies of the  DsRNA  analysis  was  conducted  on  36  wild  dsRNAs  type  geographic origin of each strain is listed in Table II.  strains  of  Neurospora.  The  Chapter  Table  II.  Geooraphic o r i g i n  isolates Strain  of Neurospora  4/235  and s t o c k  crassa*  number o f w i l d  a n d N.  Origin  StocIt Number  Designation  type  intermedia  Australia Tovnsville-1  1833  Townsville,  Continental U.S.A. Labelle-1b Mauricevile-lc  1940 2225*  Labelle, Florida K a u r i c e v i l l e , Texas  India Aarey-le Varkud-lc  2499 1832  Bombay, Varkud,  Indonesia Besakih-1 Besakih-lc Bogor P a s a r Gianjor-lc Jakarta-1 Ratnpong B a b a k a n Tjikini Pasar  1826 1827 2215 1836 1881 2562 2557  Besakih, Bali Besakih, Bali Pasar Bogor, Bogor Gianjor, Bali Jakarta, Java Bandung T j i k i n i Pasar, Jakarta  Tarongong  Queensland  Maharashtra Karnataka  Japan  North  Africa  Pacific Fiji  Fiji  I  Unren,  Japan  430*  Adiopodoume, Ivory Coast  435  Fiji  Islands  N6-6  Hawaiian  Islands P561 P572 P573 P801 P765 P776 P785 2360 2365 3720 3722  Hanalei-1f Lihue-3b Hanapepe Hanalei People's  Beijing  Republic  Harbin Hefei South Monte  P10  America Alegre-1  of  Kauai Kauai Kauai Kauai Oahu Maui Maui Hanalei, Kauai Lihue, Kauai Hanapepe, Kauai Hanalei, Kauai  China 3977 3983 3980  Beijing Harbin, Heilongjiang H e f e i , Anhui  3336  Monte  1766 1767  Taipei Taipei  Taiwan Taipei-lc Taipei-lg  Alegre,  Brazil  Chapter Twenty-four  of the  strains  phenotypes  during  vegetative  4/236  chosen for the  survey exhibited degenerative  propagation.  The  other  12  strains  growth  showed  no  discernible change in phenotype.  DsRNA  was  detected  strain  are  panel  A . The  between  in seven of the  shown in the  the  number  36 strains.  The dsRNA  bands  ethidium bromide-strained gel presented and  mobility  of  the  7 strains.  Listed in Table III  determine  if the  dsRNAs are  the  is  seen  for  each  in Figure 57, to  be  sizes of dsRNAs  variable for  each  dsRNA  from  strain.  In  order  to  9.0kb  dsRNAs  show homology, the  strain P573 was isolated, end-labeled, and used to probe Northern blots of the dsRNAs  from  between  the  that  the  dsRNAs  each 9.0kb  P573  strain. Figure dsRNAs  labeled  58,  present  dsRNA  panel  in the  does  not  A shows  that  three strains. hybridize with  of strain 3336 indicating that there are  It the  there  is homology  should be noticed 2.0kb  or  at least two different  500bp 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  P10 and P573, the probe also hybridized to the the  500bp dsRNA  of strain  1833. Hybridization  9.0kb dsRNAs of strains  18kb dsRNA of strain 435 and to  only  the  500bp  dsRNA  in  strain 1833 and to only the  18kb dsRNA in strain 435 indicates that these two  strains  different  each  carry  two  hybridization between the 9.0kb dsRNA  dsRNA and the  species.  Furthermore,  negative  18kb, 2.0kb and 500bp dsRNAs  Chapter  4/237  (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  exclusively  dsRNA  from  to  3.0kb  the  strain dsRNA  1833 from  was  used  probe  and  hybridized  (Figure  58,  panel  C).  7kb dsRNA in strain 2215 or  the  strain  Hybridization of the dsRNAs with either the  as P776  a  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  T a b l e III. Growth phenotype, dsRNAs o f seven  4/238  g e o g r a p h i c o r i g i n , and s i z e s o f  i s o l a t e s of N e u r o s p o r a  S i z e s of  Growth  Geographic  Strain  dsRNAs  P10  9.0kb  normal  2215  7.0kb  slow  435  18kb  normal  Fiji  Phenotype  Oriain  TJnzen, Japan growth  Java, Indonesia  9.5kb P573  9.0kb  senescent  K a u a i , USA  P776  3.lUb  slow  M a u i , USA  growth,  senescent 1833  3336  3.0*b  slow  growth,  500bp  senescent  Australia  *9.0kb  colonial  Monte A l e g r e ,  2.0kb  growth,  Brazil  500bp  senescent  Queensland,  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  4/241  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.  74 A P10 2215  •  435 P573 P776 1833 3336  CO K" O"  74 A PIO 2215 435 P573 P776 1833 3336  Chapter 4 / 243  Figure 59. Southern hybridization of genomic DNA from various natural isolates hybridized with the 9kb dsRNA of strain P573.  A  A  —10  •  •  Ui O  A  A  -bo* • •  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.  561 PIO 2215 435 P573 P776 1833 3336  Chapter C.  4/247  DISCUSSION  The survey of 36 wild type strains of Neurospora identified seven strains carry  detectable  detected  among  amounts these  of  dsRNA.  A  total  seven  strains.  Figure  of  seven  dsRNA  60,  panel  B  which  species  shows  the  were 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 found  only  include the  in  strain  2215.  18kb dsRNA  The  dsRNA  with  in strain 435, the  cross  homologies  7kb dsRNA  designated  500bp dsRNA in strains  'c'  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, 1980;  1981; 1983; Tipper and Bostian, 1984) and U . maydis ( Koltin et al,  Peery et al, 1982; Dalton et al, 1985; Peery et  several  distinct dsRNA  viruses  consist  encoding  toxin,  of  3  species. to  conferring  capsid. The killer strains  7  The  genome  different  dsRNA  immunity to  the  of the  of S. cerevisiae have  similar products as in U . maydis.  various  segments toxin,  al, 1987) each contain  and  which  types are  of Ustilago involved  production of the  9 distinct dsRNAs  which  in  viral encode  Chapter 4 / 248 Cross hybridization of the  18kb dsRNA of strain 435 with the  strain 3336 and the 500bp dsRNA of strains  3336 and  1833 suggests that the  smaller dsRNAs were derived from deletion of most of the dsRNAs, RNA  generated  preparations  by deletion of the  original  2.0kb dsRNA of  dsRNA,  18kb dsRNA. Cryptic  have  been  observed  in  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 hybridized  with  the  from a number  different  homologous to the dsRNAs. was  the  only dsRNA  dsRNA  of strains  species  to  the  9.0kb  dsRNA  detect  for  The 9.0kb dsRNA in strains  which  hybridized with  genomic  4.5kb and a 2.0kb Eco R l restriction fragment with  included in the  probe.  In  addition,  a  6.5kb  genomic  was  sequences  P10, P573, and  D N A . In  of the  survey  3336  all strains,  a  genomic D N A hybridized Eco RI fragment  in  the  Hawaiian isolates hybridized with the 9.0kb dsRNA probe. It is possible that this additional  region of the  polymorphism. Sequence  D N A originated homology between  from  a duplication and  a 9.0kb dsRNA  restriction  and a 6.5kb  site  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  transcription within the  account  for  a  9.0kb  dsRNA  indicating that  6.5kb D N A may have occurred generating,  aberrant  for example,  Chapter  4/249  an R N A consisting of two copies of the  transcribed  also  probe  hybridizing  and/or  with  the  9.0kb  dsRNA  2.0kb Eco R l fragments  appropriate  sized  transcription  region. Alternatively, bands  may  comigrate  with  the  4.5kb  upon gel electrophoresis  thus accounting for  region  9.0kb  to  generate  a  transcript.  the  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  B T S genome  as  well  as  to  the genomes of other bean cultivars.  No  homology was  1833  and  detected  between  the  genomic D N A implicating a  slow growth and senescent phenotypes  3.0kb viral  dsRNA  origin  for  from  strains  the  3.0kb  senescence  in  been characterized  this  strain  is  and  dsRNA.  The  shared by these two strains suggests that  the 3.0kb dsRNA may be responsible for these altered phenotypes. strain P776 has  P776  at  Senescence in  a molecular level and it is known that  initiated  by  the  insertion  of  kalDNA  into  the  mitochondrial D N A (Griffiths et al, 1988). Senescence of strain 1833 results from a  yet  1988).  unknown phenomenon Thus,  if the  3.0kb  not  related  dsRNA  to kalDNA  does alter  senescence  normal growth  (Griffiths then  et al,  it could  be  responsible for the slow growth phenotype observed in both these strains.  The  7kb  suggesting  dsRNA that  from  this  identified  in this  particles,  and  the  strain  dsRNA  strain  too  2215  does  is  of viral  (Tuveson and  preparation  and  not  hybridize  origin.  Peterson,  Virus  with  genomic D N A  particles  have  been  1972). Isolation of these virus  characterization  of  the  viral  determine if the dsRNA isolated from total nucleic acid preparations  genome  will  constitute  the  Chapter  4/250  viral genome.  The origin of the 2.0kb and 500bp dsRNAs in strain 3336, the  18kb and 9.5kb  dsRNAs  are  Lack also  in strain  435, and the  500bp dsRNA  of homology of these dsRNAs with viral  in origin.  Virus  particles  in strain  1833  not known.  genomic D N A suggests that they  will  have  to be  identified  and  are  isolated  to  discern the origin of these dsRNAs.  The previous chapter described the atypical behaviour of kalDNA in strain P573. In seen  strain  P573, the  observations  that both  only erratically and that this is the  AR-kalDNA  and  mtlS-kalDNA  are  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  geographic  strains locations  in  this  (Table  survey 2).  isolated from Monte Alegre, Unzen,  Japan  generating  the  Alternatively  indicates 9.0kb  if the  dsRNA 9.0kb  The  Brazil,  that  that  presence from the  the may  dsRNA  contain  is  an  be  of the  are  9.0kb  altered  all that  ancestral  from  dsRNA  island of Kauai,  proposed not  dsRNA  in  U S A , and  transcription uncommon  transcript,  different  in  then  strains from  mechanism Neurospora. 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 presence  of the  strain  1833 from Queensland, Australia. The  3.0kb dsRNA in strains collected from different geographic areas  Chapter  4/251  suggests a common ancestor which acquired and transmitted similar hypothesis can be proposed for strains homologous dsRNA  of the  phenotype.  strains, It  is known that  the  al,  1984).  demonstrated  In  vitro  studies  presence  of dsRNA  and/or  with an altered phenotype using  dsRNA  from  showed no altered virus particles  inhibits cell-free  (Burke,  1977),  and  is  translation  (Burke,  involved in the  1977),  regulation  induces  in  in the host (Dodds  various  the involvement of dsRNA in some cellular processes.  dsRNA  dsRNA  1833 and 3336.  2215 and 435, found to contain dsRNA  fungi is not necessarily associated et  435, 1833, and 3336 which carry  sequences except that deletion of the majority of the  occurred in the ancestor of strains  Two  the 3.0kb dsRNA. A  systems  has  For example,  interferon  of gene expression  production (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. the  Cross hybridizations reveal seven different  dsRNA  species  among  seven strains. A t present, we do not know the biological significance of the  dsRNAs.  VII.  CONCLUSION  Mitochondrial aging in a number of organisms has received much attention. This is  primarily  because  mitochondria are  and changes  at  biochemical or genetic  aerobes.  fungi  In  the  such  as  considered the level  N . intermedia,  'power  are  N.  house'  detrimental  crassa,  P.  of the  to all obligate  anserina,  amstelodami, alterations of the m t D N A have proven to be the events for causing degenerative  growth, sometimes resulting in death,  cell  and  A.  responsible  and mitochondrial  biochemical deficiencies. Although m t D N A damage causing altered growth patterns is relatively well understood  in these fungi,  not yet  been  validated as models for aging in higher eukaryotes. Nonetheless, there are  some  striking similarities between higher  organisms  genetically longevity  (Finch  programmed and  verification  the  of  the and  this  mitochondrial functions Hayflick,  1977)  instability of the  occurrence  of  hypothesis  fungal systems  mtDNA  senescence  would  and  be  in  associated fungi  may  to  with  aging of  suggesting  exist  higher  difficult  have  which  organisms. attain  and  produce  a  mitochondrial  aging may  consequently  go  characterization  great be  associated  undetected.  of m t D N A  diversity with  Although  defects  as  this they  of  mtDNA  At  because  only  a  particular  hypothesis relate  defects.  to the  is  present aging in different  Alternatively,  tissue difficult  type  and  to  test,  growth of fungi  suggest another mechanism by which aging may occur in higher organisms.  252  a  determines  different tissues and cell lineages in higher eukaryotes might proceed at rates  that  does  Conclusion / 253 The work reported fungus  in this  thesis  N . intermedia. The major  was  on mitochondrial-based senescence  of the  findings reported in this thesis are summarized  below:  1) K a l D N A is capable of assuming new locations within the m t D N A . •  In  all series  insertion  were  analyzed, novel insertion of k a l D N A generally  within  functionally  was  important  apparent. regions  of  The  sites 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 k a l D N A  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 k a l D N A has been identified which is a free element in the mitochondria.  This form of k a l D N A 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  The  inserted  form  of  senescence.  kalDNA,  mtlS-kalDNA,  was  seen  only  A R - k a l D N A seen often but in low copy number. It is suspected  erratically  and  that k a l D N A is  not responsible for the fate of strain P573 and its derivatives but rather  some  other unknown factor is responsible.  5) D s R N A has been detected in seven strains of Neurospora.  Among  seven  geographically  dsRNA  were delineated. Although the presence  kalilo senescence,  distincy  strains  of  Neurospora,  of dsRNA  seven  species  is not associated  Neurospora is an ideal organism to determine  the  of with  significance  of the presence of dsRNA in fungi in general.  The  information  senescence  where  presented  in  this  thesis  reveals  the  it is characterized by a mobile element  complexity  of  which resides  kalilo 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 in  the  initiation  senescence  and propagation of senescence  differs from senescence  are  involved  shown in Figure 61. Kalilo  of P. anserina and A . amstelodami in that it  involves the insertion of foreign D N A into the m t D N A rather  than the excision  of specific regions of the m t D N A . 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 k a l D N A have been demonstrated. general, the overall molecular events resulting in senescence determined. are  In  order  to  answer  these questions,  required. Initial transformation experiments  kalDNA  is  conjunction  the  cause  of  senescence.  with in vitro mutagenesis  Once  have not been  transformations  will  determine  demonstrated,  will allow one to follow  using  In  fully  kalDNA  unequivocally that transformations  in  the behaviour of  k a l D N A during vegetative and sexual growth and thus determine the sequence of molecular intermedia.  events  responsible  for  the  senescent  fates  of Kauaian strains  of N .  Conclusion / 256  Figure 61. A flow chart summarizing the senescence of Kauaian strains of N. nucleus-associated linear plasmid. MtlS-kalDNA of kalDNA. MtFF-kalDNA is the mitochondrial  molecular events associated with intermedia. AR-kalDNA is the is the mitochondrial inserted form free form of kalDNA.  mtFF-kalDNA  n o. 3 o i— c  \  1  AR-kalDNA"  CYTOSOL  mtlS-kalDNA  accummulation of m t D N A w i t h mtlS-kalDNA  MITOCHONDRIA  loss of "•respiratory potential  CO HO 3  ••DEATH  BIBLIOGRAPHY  Akins,  R . 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