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Investigation of a kalilo plasmid in Louisiana Neurospora tetrasperma Kuehn, Monica M. 1993

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INVESTIGATION OF A KALILO PLASMIDIN LOUISIANA NEUROSPORA TETRASPERMAbyMONICA MARCINKO KUEHNHonours B.A., McMaster University, 1991A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESBOTANY DEPARTMENTWe accept this thesis as conformingTHE UNIVERSITY OF BRITISH COLUMBIAAugust 1993© MONICA MARCINKO KUEHNIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of ApetiThe University of British ColumbiaVancouver, CanadaDate 421i,r,./Lt A) , /993DE-6 (2/88)ABSTRACTSenescence in Neurospora is caused by the insertion of a linearplasmid, kalilo, into the host mitochondrial genome. Kalilo-related senescence has been studied extensively in Hawaiianfield-isolated strains of Neurospora intermedia. This work,however, investigated the presence of a kalilo-like plasmid,named kalilo', within Louisiana-isolated strains of Neurospora tetrasperma.Comparison of the physical characteristics of the kalilo andkalilo' plasmids revealed that the plasmids have identicalrestriction maps. In addition, digestion of the kalilo' plasmidwith 5' and 3' exonucleases showed a 5' resistance to enzymedigestion and a 3' sensitivity to digestion. This suggested thatkalilo' may have a protein bound to its 5' termini, as is thecase with kalilo.Biological properties of the Louisiana plasmid were alsoexamined. Investigation of inheritance patterns through crossesdemonstrated a maternal inheritance, thereby suggesting thatkalilo' was located within the mitochondria. Additionally,transmission of the plasmid by asexual culturing was relativelystable. In order to determine if the plasmid inserted into thehost mitochondrial genome, DNA from the mitochondria was cut withthe restriction endonuclease Pst I (an enzyme that does not cutthe kalilo plasmid DNA). Unlike the Hawaiian kalilo, kalilo' didiinot appear to insert into the mitochondrial DNA.Finally, given that insertion did not occur, the relationshipbetween kalilo' and senescence was investigated. Various cultureswith and without kalilo' were subcultured (serial vegetativetransfers) and surveyed for senescence. Senescence failed tooccur within the forty subcultures observed. While senescence didoccur within growth tubes, the relationship between senescenceand kalilo' are not tightly correlated.On consideration of these results, several questions arose. Whatwas the evolutionary relationship between kalilo and kalilo'? Ifthe kalilo' plasmid was identical or very similar to kalilo, whydid integration not occur? What was the catalyst for senescencein N. tetrasperma? These matters have been addressed within thetext, but definitive answers are still forthcoming.iiiTABLE OF CONTENTSAbstract^Table of Contents^ ivList of TablesList of Figures^ viAcknowledgements viiI. Introduction^ 1A 1. Life cycle of Neurospora^ 1A 1.1^Heterothallic Neurospora^ 1A 1.2^Neurospora tetrasperma 5A 2. Cytoplasmic senescence in fungi 8A 2.1^Podospora anserina^ 8A 2.2^Aspercallus amstelodami 9A 2.3^Neurospora^ 9A 3. Objectives of this work 11II. Materials and Methods 13B^1. Strains^ 13B 2. Media and growth conditions^ 13B^3. Dot hybridization^ 17B 4. Total DNA isolation 18B 5. Mitochondrial DNA isolation 19B 6. Enzyme digestion and gel electrophoresis^ 20B 7. Labelling of nucleic acids^ 21B 8. Probes^ 21B 9. Southern blot analysis 24III. Results 25C 1. Introduction^ 25C^2. Characterization of a kalilo-like plasmid^ 25C 2.1^Initial dot hybridization^ 25C 2.2^Ascospores from self-fertilization^ 26C 2.3^Restriction map^ 26C 2.4^Confirmation of distinct kalilo' identity  ^27C 2.5^Exonuclease digestion^ 27C 2.6^Examination of unisexual strains^ 28C 2.7^Crosses^ 28C 2.8^Senescence 29C 2.9^Analysis of fortieth subculture^ 30C 2.10^Pst I digestion^ 30IV. Discussion 54V. Bibliography^ 62ivLIST OF TABLESTable 1. List of Louisiana strains of Neurospora^ 14Table 2. Analysis of senescence^ 48vLIST OF FIGURESFigure 1. Life cycle of heterothallic Neurospora^ 3Figure 2. Pseudohomothallic nuclei in N.tetrasperma 6Figure 3. Kalilo plasmid restriction map^ 22Figure 4. Dot blot of Louisiana isolates 32Figure 5. Total DNA from kalilo'-positive isolates 95 and 60...34Figure 6. Dot blot of selfed progeny^ 36Figure 7. Total DNA of selfed progeny 38Figure 8. Restriction mapping of kalilo^ 40Figure 9. Analysis of 5' and 3' exonuclease digestion 44Figure 10. Analysis of unisexual strains^ 44Figure 11. Reciprocal cross^ 46Figure 12. Analysis of 40th subculture^ 50Figure 13. Investigation of kalilo' insertion^ 52viACKNOWLEDGEMENTSI would like to express my appreciation to those who have helpedme throughout my graduate studies. I wish to thank my supervisorDr. A.J.F. Griffiths whose enthusiasm, advice, patience, andexpertise has been most helpful in forming and completing myresearch. Your passion for succinctness is inspiring.I also wish to thank my supervisory committee Dr. N. Louise Glassand Dr. Jim Kronstad for their helpful suggestions and exemplaryresearch practices. Thanks to Xiao Yang and Anne Taylor-Smith fortheir unending patience, advice and moral support.I owe a special thanks to my parents for prompting all theirdaughters to excel in a life of learning, and for believing inme.Finally, I wish to extend my love and gratitude to my husbandMarvin for being an intellectual foil, editor, homemaker, andcontinual source of encouragement when the going got tough.viiINTRODUCTIONA 1. LIFE CYCLE OF NEUROSPORAA 1.1. HETEROTHALLIC NEUROSPORAThe fungi N. crassa, N. intermedia, and N. sitophilia belong tothe Ascomycetes, sub-class Pyrenomycetes, and are usedextensively in genetical studies. The life cycle of Neurosporacan be divided into the vegetative and sexual phases. Thevegetative phase is characterized by hyphal growth. Hyphaeconsist of branched filaments approximately 5 microns in diameter(together forming the mycelium), each hypha being segmented intocompartments by crosswalls (septa). The septa contain a centralpore which allows the movement of cytoplasm between compartments,and thus each hyphal "cell" may have several nuclei, each nucleuscontaining seven chromosomes (the haploid complement). Aerialbranches from the mycelium form and produce the conidiophoreswhere the asexual spores, conidia, are located. Both micro andmacroconidia may be produced, the latter being more frequent.These conidia serve to propagate the fungi. Contact between theconidia and a nutrient medium allows for conidia germination,leading to growth of the mycelium.The sexual cycle of Neurospora (as diagrammed in Fig. 1) requiresthe interaction of mycelia of the two mating types A and a. Onnitrogen-limited medium both mating types form protoperithicia,1the female reproductive structures. The protoperithecia consistof a coiled hypha, the ascogonium, surrounded by a sheath ofhyphae. The ascogonium sends out slender hyphae, the trichogynes,into the air. Fusion of a trichogyne with a male fertilizing cellis aided by the chemotropic response of the trichogyne topheromones released by the male cell. The male cell may be amacroconidium, mycelium, or less frequently a microconidium. Uponfusion of the two fertilizing cells, the opposite mating typenuclei undergo synchronous mitotic division and relocate to thenewly formed ascogonous hyphae. At this time, the mycelial sheatharound the ascogonium darkens with melanin. In the penultimatecells of the croziers, the two nuclei fuse to briefly form adiploid cell. Meiosis then occurs to form four nuclei whichundergo mitosis to give the eight ascospores found within theascus. When ripe, the asci shoot the ascospores through theostiole, and these spores can then be germinated at 60*C. Thegerminated spores produce mycelia and through airborne conidiaare able to disperse to new locations.In certain cases the heterothallic species of Neurospora are ableto form heterokaryons, where different kinds of nuclei exist inthe same cell. The requirements for heterokaryon formation arenot completely understood.2Figure 1. The life cycle of heterothallic Neurospora includingsexual and asexual phases. (Fincham and Day, 1963)34A 1.2. NEUROSPORA TETRASPERMAThe pseudohomothallic nature of N. tetrasperma sets it apart fromthe other Neurospora species. In pseudohomothallism two nuclei ofopposite mating type are included in every spore, and germinatedspores produce mycelia with both A and a nuclei (Alexopoulos andMims, 1979). As such, mating type associated heterokaryonincompatibility genes (tol) are absent in N. tetrasperma (Jacobson, 1992), as are trichogyne structures during the sexualcycle. Since the ascogenous cells already contain nuclei ofopposite mating type, fusion and meiosis proceed without seekingand transporting a fertilizing male cell (Dodge, 1935).Occasionally an ascus will , contain more than four ascospores. Inthis case smaller uninucleate spores are found in addition tothe binucleate spores (Colson, 1934). These uninucleateascospores produce heterothallic strains useful for crossing.Furthermore, the four-spored condition of N. tetrasperma may becontrolled by a single gene, eight spores being dominant to fourspores (Burnett, 1975). It is postulated that pseudohomothallismcould have arisen as a secondary consequence of a mutationaffecting ascus morphology. In this scenario a mutation dictatinga broader and shorter ascus in N. tetrasperma could have lead toa change in spindle orientation, as seen in Fig. 2, andultimately result in the formation of a four-spored ascus.5Figure 2. The formation of pseudohomothallic nuclei in N.tetrasperma. Spindle orientation directs the movement of nucleiduring ascus formation (spindle indicated by a straight, solidline). Nuclei of opposite mating type (represented by shaded andempty circles) are included within the same ascospore. (Burnett,1975)6oA(Ae-^0A 2.CYTOPLASMIC SENESCENCE IN FUNGIA 2.1.PODOSPORA ANSERINAAll natural isolates of P. anserina show senescence patterns thatare repeatable and characteristic for the geographical region(review by Griffiths, 1992). Senescence is characterized byswelling and bursting of hyphal tips, and dark pigmentation. Thesenescence attributes of a particular strain are transmittedmaternally.The elements associated with senescence (senDNA) in P. anserina are a collection of circular DNA molecules derived from themitochondrial DNA (mtDNA). The nature of the senDNA appears to bea set of circular head-to-tail multimers, comprised of a singlemonomeric unit. The monomeric unit varies with individualcultures, and is designated by Greek letters. So for instance,the most common monomer is the alpha-senDNA which has thesequence of the first intron of the gene for subunit 1 ofcytochrome oxidase, COI. During the senescence process, wild-typemtDNA molecules drop to very low levels, and rearranged moleculesare found. These rearrangements are likely the cause of death.Senescence can be temporarily inhibited through chemicaltreatments such as ethidium bromide and inhibitors ofmitochondria' protein synthesis.8A 2.2.ASPERGILLUS AMSTELODAMI The vegetative death mutant in A. amstelodami displays senescentbehaviour, but little work has been done on this strain. Theragged mutant (rqd), on the other hand, is better characterized.It shows stop-start growth patterns similar to senescence andcontains excised mtDNA regions which exist as amplified head-to-tail concatamers (review by Griffiths, 1992). As opposed tosenescent P. anserina, the rgd mutant of A. amstelodami stillcontains detectable levels of wild-type mtDNA.A 2.3.NEUROSPORAPlasmid-induced senescence has become an increasingly well knownphenomenon in the filamentous Neurospora fungus. Uninfectedstrains of Neurospora species are potentially immortal in thatthey are capable of continuous hyphal growth and indefiniteserial transfers. However, some cultures show senescence in lessthan ten subcultures (Griffiths and Bertrand, 1984). The presenceof plasmids such as kalilo (Bertrand, et al., 1985) or maranhar(Court et al., 1991) are known to induce senescence inNeurospora. The best characterized of these plasmids is kalilo.Approximately thirty percent of the natural field-isolates ofNeurospora intermedia from the Hawaiian island of Kauai displaysenescence (Griffiths and Bertrand, 1984). The presence of the9kalilo plasmid is consistently correlated with the onset of theculture's death; consequently, it is concluded that kaliloinduces senescence (Bertrand et al., 1985). Kalilo is a linear,double-stranded 8.6-kb plasmid. The mechanism of senescenceinvolves the integration of kalilo into the host mitochondrialDNA (mtDNA) (Bertrand et al., 1985). During subsequent growth ofthe fungal culture, the inserted form of the plasmid accumulatesin the mtDNA eventually leading to acute biochemical deficienciessuch as decreased levels of cytochromes b and aa3, and subsequentcell death. Kalilo-mediated disruption of the mitochondrialchromosome is often manifest in decreased levels of mitochondrialcytochrome aa3 and b (Bertrand and Griffiths, 1989).Integration of the kalilo plasmid occurs at a number of differentsites, however, at death one insertion site predominates (Myerset al., 1989). The plasmid insertion site is flanked by longinverted repeats of host mtDNA (Dasgupta et al., 1988). Theinsertion mechanism appears to involve pairing of the last 20-bpof both plasmid ends with 5-bp of homologous mtDNA sequences(Bertrand and Griffiths, 1989).The kalilo plasmid contains 1361-bp terminal repeats, with aprotein covalently bound at the 5' termini (Vierula et al.,1990). Two open reading frames exist within the plasmid,potentially encoding a DNA polymerase and a RNA polymerase (Chanet al., 1991). The kalilo sequence contains no homology with the1 0host mitochondrial or genomic DNA.Suppression of senescence in kalilo strains may occur by means ofnuclear genome variant hosts (Griffiths et al., 1992).Suppression is by two mechanisms. First, the level of free andinserted kalilo forms are reduced to very low levels, or second,plasmid levels remain high but the host becomes tolerant to theplasmid. Either mechanism allows a kalilo-infected strain to berestored to immortality.Another senescence-inducing plasmid in Neurospora is the maranharplasmid: a 7-kb plasmid found in natural isolates of Neurospora crassa from India (Court et al., 1991). It shares many of theproperties seen in kalilo,_for instance linearity, two openreading frames, 5' protein bound to the terminal repeat, theabsence of homology with its host, insertion into mtDNA, as wellas the correlation between insertion and senescence. However,there is no hybridization of kalilo and maranhar (Bertrand andGriffiths, 1989).A 3. OBJECTIVES OF THIS WORKThe focus of this work has been an investigation of a kalilo-related plasmid within Neurospora tetrasperma. The unique hostand geographical location for the plasmid were the impetus ofthis study. To date, kalilo has only been isolated from Hawaiian1 1Neurospora intermedia strains, whereas this kalilo-like plasmidwas isolated from Louisiana strains of Neurospora tetrasperma.Fifty isolates were collected from Louisiana; none of the 40 N.crassa strains contained kalilo, but DNA from 2 of the 10 N.tetrasperma strains showed hybridization with the kalilo plasmid.Incentives for this investigation were three-fold. First, thedegree of plasmid structure similarity was of interest. Didkalilo' have the characteristics of the Hawaiian kalilo plasmid:5' terminal proteins, restriction sites, and location within themitochondria?Second, plasmid inheritance patterns in N. tetrasperma were ofinterest. Was the plasmid transmitted maternally, and was thepseudohomothallic nature of the host a factor in plasmiddistribution to progeny? Asexual plasmid transmission was alsoobserved.Third, senescence in the Louisiana isolates was observed. Waskalilo' presence correlated to culture senescence? Did plasmidintegration occur, and if so, how was integration correlated tosenescence? These matters were investigated to increase theunderstanding of plasmid function and plasmid-host interactions.12MATERIALS AND METHODSB 1. STRAINSNeurospora intermedia strains P561, P605 are natural isolatescollected from Kauai, Hawaii (Griffiths and Bertrand, 1984).Ascospore derivative C4 is from the cross P561 x 1766 describedby Griffiths and Bertrand (1984).Neurospora crassa and Neurospora tetrasperma strains 48 through101 are natural isolates collected from soil samples of one fieldin Louisiana, U.S.A. (Table 1). They were obtained from Dr. D.Jacobson, Michigan State University.Strains 1-1 to 1-10 and 2-1 to 2-24 are progeny from crosses ofthe above N. tetrasperma isolates and were obtained from Dr. D.Jacobson, Michigan State University (Table 1). The unisexualstrains 95A, 95a, 60A, 60a were also received from Dr. D.Jacobson, Michigan State University (Table 1). Strains 95-1 to95-10 and 60-1 to 60-17 are single ascospore cultures from theself-fertilization of strains 95 and 60 respectively.B 2. MEDIA AND GROWTH CONDITIONSAll strains were grown on Vogel's minimal medium (Vogel, 1956)for vegetative culturing. Sexual crosses were performed on13Westergaard's medium (Westergaard and Mitchell, 1947).Serial subculturing was performed on Vogel's minimal medium in 10mm x 75 mm tubes. Mycelial samples were transferred three times aweek. Growth tubes also contained Vogel's minimal medium;mycelial growth was measured daily, and a 5 mm x 5 mm section ofrecent growth was used for transfers to new growth tubes.Random ascospores were isolated by suspending the ascospores fromthe petri plate lid in water. Alliquots of the suspension wereplaced on a piece of agar and individual spores were picked undera dissection microscope. Ascospores were placed separately intotubes containing Vogel's minimal medium and heat shocked at 60 Cfor 0.5-1.0 hour, and incubated at 25 C for three days.The mating type of each ascospore was determined. Petri plates ofcrossing medium were inoculated with a unisexual N. tetrasperma strain of known mating type--this was performed for each matingtype. When protoperithia formed, a drop of conidial suspensionfrom the strain in question was placed on the two types of petriplates. If the conidia crossed with the plated culture, as seenby the release of black ascospores, then the strain in questioncontained the mating type opposite to that of the plated culture.14Table 1. A list of Louisiana strains of Neurospora, includingspecies and mating type. Strains were obtained from Dr. D.Jacobson, Michigan State University.15al0 cti al U) ni CO 0 co ai ai co (LI ai aiGOCO0coU)RScoU) SI04-)coco U)a$U)U)aiU)U)RS0144-)cocoCAci)aScoRScc11,4U)U)ai1-I4-)coalcoaS roU)coaiN 1-1 14 1-1 W W 54 $4 1-1 140 4-) 0 4-)r) 4-) c..) 4-) 0 0 0z • z z z z •U) coco U)nso rtooE E Ew^W0 0E Ew W irERRRRRa) a) a) a) a) a) a) a) 0 0s REERw w (1)^(1)CO CA co 0) co co o) CO CO CO CO GO CO IA CO CO CO CA CA CO CO CO CO GO CO CO CA CO CO CO CO CO GO GO GO CO CO co0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 cc1 cti 0 0 0 Id al 0 of 0 0 0 Id 0 0 0 0 0 0 0 cc)14 14 14 11 14 14 11 14 14 1-I 1-1 11 14 14 14 14 $4 14 14 14 1i 14 11 14 14 14 14 14 1.1 14 1i 14 14 1 1 1i 11 14 1-14-) 4.) 4-) 4-) 4-) 4-) 4-) 4.) 4-) 4-3 4-) 4-)^4-) 4-) 4-)^4-) 4) 41 4-) 4-) 4-) 4-) 4)^4-) 4-) 4.3 4-) J.) 4-) 4.3 4-3^.1.J ▪ 4.1(1) W^a) Q.) a) (ll W (1) cll (1) w^(1)• (1)• CI) W^(1) WWWW^(1)^WWWWWWW4-) 4-) 4-) 4-) 4-) 4.) 4-1 4.) 4.3 4-1^▪^4-) 4-) 4..) 4-) 4-) 4-) 4-)^4-) 4-) 4-) 4-) 4-) 4-) 4.) 4-) 4-) 4-) 4-) 4-) 4.) 4-)4J4)4)4) -)Z ZZZZZ ZZ► ZZZ ZZZZ ZZZZZ ZZZ► ZZ zZZZZ ZZZZ ZZZ0^ ri CV rl .41 111 VD^co al CD ri CV 01 dig4^4^CV el VI LC) VD r, oo on r4 rq CV 01 -41 Ul VD r, co CA rA^rq ri rA rA r4 rA rA r4 CV CV CI CV CVin Li) 0011111111111111111111111111111111110) 0) t.0^H r-I r-I c-I c4r--le-11-1T-IHNOINC•INCNCNINOINCSICNOINCNNCICINOINC\10101nj ni njcr) Cr) WRS ai11 1-40U)U)1-10•njU)1-1Uai njU)U)ni ai0aiU)U)nifA U)co cotd ai1i 1.4•ci)cdU)U)aiU)coofniNU)Ri•U)U)1-1coccl0U)U)alz z z z ZZZ Z z. z zai 0 01.4• Wco co cnal 0 air)• 1-1 1-1.4) 4) 4) alWWk4-) 4) 4)• aSEI Ea)co coO 01-I4-) 4-)W4.) 4.)zl zf ZZ • •Z Zof nt ai rt:1^at^ daicel^ni ni^ (Ti^ni^ai+ + + + ^+ + ai4 (Ti 0 0 0 4 (Ti (Ti 0 4 4 4 4 4 4 0444  (Ti 0 (Ti 0 444  0 4 4 04 0 4 0 4 4 4 4 f4C 0 4 0 4 (Ti 4 4 (Ti 0^0 r-1CO ON CD ri CI 01 di 0 VD C, CO CD ri CV 01 V U1 UD IN OD al CD r-1 CV 01 di In VO C.- CO CA CD rl CV 01 V 0 VD r, CO CA CD ri CV el .4 0 VD r■ CO cn CD CD• .41^Ul Ul^0^Ul(nlnVD VD V, VD V) VD VD VD VO VD C.- C,^r■ r■ C, r, r, r, r■ OD CO oa CO OD CO CO CO CO CO CA cn CA CA CA Ch CA CA CA CA ri rlGrowth of mycelia for dot blots and total DNA preparations wasperformed by inoculating 1 ml of liquid Vogel's medium andincubating at 25 C for a maximum of one week (C. Myers, labmanual).Growth of mycelia for mitochondrial DNA preparations used a fourday old 88 mm x 88 mm culture tube to inoculate 125 ml liquidVogel's medium, and was then shaken at 200 rpm for three days.Another 125 ml of liquid Vogel's medium was added to thissuspension and shaken for three days (A.J.F. Griffiths, labmanual). Mycelia were harvested by suction filtration throughWhatmann #1 filters in Buchner funnels.B 3. DOT HYBRIDIZATIONCrude nucleic acid isolation was performed as follows. Themycelial mats were taken from the liquid medium and the excessliquid was removed. The mats were ground at 4 C with acid-washedsand, and were suspended in 600 ul LETS buffer (0.1 M LiC1, 10 mMEDTA, 10 mM Tris, 0.5% SDS, ph 8.0). The suspension was placed ina microfuge tube and spun at 15 krpm for 10 minutes. Thesupernatant was removed and loaded into a dot hybridizationapparatus containing Hybond filter. Suction was allowed toproceed overnight. The filter was removed from the apparatus andwas placed face up on Whatmann paper soaked with alkalinesolution (1.5 M NaC1, 0.5 M NaOH) for 5 minutes, and was then17transferred to Whatmann paper soaked with neutralizing solution(3 M NaC1, 0.5 M Tris-HC1, pH 8.0) for 5 minutes. The filter wasbaked in 80 C oven for 2 hours. (C. Myers, lab manual)B 4. TOTAL DNA ISOLATIONAll steps were performed at 4 C unless stated otherwise. Themycelial mats were transferred from liquid medium and the excessliquid was removed. Using acid-washed sand, the mats were groundand suspended in 0.7 ml LETS buffer. The suspension wastransferred into a microfuge tube and spun 1 minute at 15 krpm(revolutions per minute x 1000). The supernatant was removed, and40 ul 20% SDS was added to lyse organelles. 10 ul 2% proteinase Kwas added to digest proteins, and then the solution was invertedto mix and incubated at 37 C overnight. Proteins were extractedby adding half a volume each of Tris-HC1 saturated phenol, andchloroform-isoamyl alcohol (24:1). The solution was inverted tomix, and spun at 15 krpm for 3 minutes. Using the top aqueousphase, this previous step was repeated one time. 0.5 volume of7.5 M ammonium acetate and 2 volumes 100% ethanol were added andthe solution was placed at -20 C overnight to precipitate theDNA. The solution was centrifuged at 4 C for 10 minutes and thesupernatant was discarded. The pellet was rinsed with 70%ethanol, and then dried at room temperature. The pellet wasdissolved in approximately 100 ul T10E1 (10 mM Tris-HC1, 1 mMEDTA, pH 8.0), and the RNA was digested by adding 1 ul 20 mg/ml18RNase and incubating for 1 hour at 55 C. (Myers, Ph.D. thesis1988)B 5. MITOCHONDRIAL DNA ISOLATIONAll steps were performed at 4 C unless stated otherwise. Mycelialmats were homogenized by the bead-beater procedure (Turner,1971). The mats were placed into a 300 ml beater flask half fullwith glass beads. The flask was filled with DNA isolation buffer(44 mM Sucrose, 50 mM Tris-HC1, pH 8.0, 1 mM EDTA) so that whensealed no air bubbles remained. For every 2.5g of mycelia thebeater was allowed to proceed for 1 minute. Flask contents werestrained through cheesecloth, and centrifuged at 2 krpm for 5minutes in a SS-34 rotor to pellet debris. The supernatant wascollected and spun at 15 krpm in a SS-34 rotor for 15 minutes topellet the mitochondria. The pellet was resuspended in 3 ml 70%sucrose, and 1 ml 44% sucrose was layered on top. This tube wascentrifuged at 45 krpm for 2 hours in a SW50.1 rotor. Theinterface band, containing the mitochondria, was removed into amicrofuge tube. The tube was filled with T200E1 (200 mM Tris-HC1,1 mM EDTA, pH 8.0), mixed by inversion, and spun at 15 krpm for15 minutes to pellet the mitochondria. The pellets were pooledand resuspended in 0.25 ml T200E1. 40 ul 20% SDS was added tolyse the mitochondria, and 10 ul 2% proteinase K was added todigest the proteins. This solution was incubated overnight at 37C. Proteins were extracted with half a volume each of Tris-HC119saturated phenol, and chloroform-isoamyl alcohol (24:1). Usingthe top aqueous layer, the extraction was repeated. MitochondrialDNA was precipitated by adding 0.5 volume 7.5 M ammonium acetateand 2 volumes 100% ethanol to supernatant. This solution wasplaced at -20 C overnight. It was then centrifuged at 15 krpm for15 minutes, and the supernatant was discarded. The mtDNA pelletwas rinsed with 1 ml 70% Ethanol and air dried. The pellet wasresuspended in approximately 100 ul T10E1, 1 ul 20 mg/ml RNasewas added, and was incubated at 55 C for 1 hour to digest RNA.The concentration of DNA was measured using UV absorption.(Myers, Ph.D. thesis 1988)B 6. ENZYME DIGESTION AND GEL ELECTROPHORESISRestriction enzyme digestion of nucleic acids was carried out asdescribed by Bethesda Research Laboratories. 10 ug mtDNA wasdigested for 2 hours at 37 C. When using lambda exonuclease,mtDNA was digested for 2 hours at 37 C in the buffer (67 mMglycine KOH, pH 9.4, 2.5 mM MgCl2, 50 ug/ml BSA) using a finalconcentration of 10 U/ul enzyme. When using exonuclease III,mtDNA was digested for 2 hours at 37 C in the buffer (50 mM Tris-HC1, pH 7.5, 5 mM MgC12, 5 mM DTT, 50 ug/ml BSA) using a finalconcentration of 50 U/ul enzyme.5 ul of 6X loading buffer (5% SDS, 50% glycerol, 0.025%bromophenol blue) was added to the digestion mixture and samples20were loaded into wells of 0.8% agarose gels. Gels were run at 40volts for 16 hours in a 1X TAE buffer (40 mM Tris acetate, pH7.6, 2 mM EDTA) to separate DNA fragments by size. Gels werestained using ethidium bromide and photographed under short waveUV illumination.B 7. LABELLING OF NUCLEIC ACIDSOligolabelling was performed as described by Sambrook et al. (1989). 50 ng of probe DNA was denatured by placing in 100*Cwater bath for 5 minutes. 10 ul Reagent mix (random primers-Pharmacia), 3 ul 32-P labelled dCTP, and 1 ul Klenow enzyme wereadded to a total volume of 50 ul, and incubated at roomtemperature overnight. A G-50 Sephadex spin column was used toremove unincorporated nucleotides.B 8. PROBESThe ka1DNA X3 fragment (as seen in Fig. 3) cloned in pUC18 wasprovided by D.J. Vickery, and the whole kalDNA (obtained by Pst Iremoval of the integrated form of kalilo) cloned in pUC18 wasprovided by X. Yang. The whole ka1DNA was used for all probesunless stated otherwise.21Figure 3. The kalilo plasmid restriction map. Fragments createdby the restriction of kalilo DNA with the enzymes: Xba I, Eco RI,Eco RV, Bgl II, Hind III, and Pst I are shown. Restrictionfragments are labelled and are referred to by these designationsin the text. Two open reading frames exist within the plasmid, asindicated by the dashed boxes, and 1361-bp inverted terminalrepeats are illustrated by the arrows.22ORF 1 893 aa^ ORF 2 811 aaIMO WWI J^L^JX3 I^X1 J X2b^I X2a IC I G I E I CEl I^E3 I E2B2 I B4 I B3 I B1kl I^k2Xba IEcoR IEcoR VBgl HHind IIIPst I1 kbB 9. SOUTHERN BLOT ANALYSISSouthern blots were performed following the procedure outlined bySambrook, et al. (1989). The gel was placed in alkaline solution(1.5 M NaC1, 0.5 M NaOH) and shaken for 30-45 minutes to denatureDNA. The gel was rinsed, placed in neutralizing solution (3 MNaC1, 0.5 M Tris-HC1, pH 8.0) and shaken for 45 minutes.Capillary transfer was set up using 2X SSC (0.3 M NaC1, 30 mMSodium citrate, pH 7.0) as the buffer, 3MM Whatmann paper as thewick, Hybond for the filter, and paper towels topped with a lightweight to aid buffer movement. Transfer was allowed to proceedovernight. Filters were baked for 2 hours in an 80 C oven.Baked filters were hybridized with 32-P labelled probes. Filterswere prehybridized for 2 hours at 42 C in 40% deionizedformamide, 1% SDS, 1X Denhardt's solution (100x = 2% BSA, 2% PVP,2% ficoll), 1 M NaC1, and 0.4 mg/ml denatured herring sperm DNA.Labelled probe (10 6 cpm/ml) was added and filters were hybridizedovernight at 42 C. Filters were washed in 2X SSC at roomtemperature for 5 minutes. This step was repeated once. Filterswere washed in a 65 C solution of 0.5% SDS and 2.0x SSC for 1hour. Once air dried, filters were wrapped in Saran Wrap, andexposed to Kodak (X-Omat RP) film for the appropriate length oftime as determined by band strength on the developed film.24RESULTSC 1. INTRODUCTIONTo date, the kalilo plasmid has been isolated only fromNeurospora intermedia strains of Kauai, Hawaii. In a routine scanof some Neurospora isolates from Louisiana I discovered that twoof the fungal cultures contained DNA homologous to kalilo. Thiswas the first discovery of a kalilo plasmid outside of Hawaii.This discovery prompted many questions. How similar were thekalilo DNA (kalDNA) sequences? Was the plasmid transmittedmaternally? Did senescence occur? Did the plasmid integrate intothe mtDNA? This work aimed to resolve these questions.C 2. CHARACTERIZATION OF A KALILO-LIKE PLASMID IN N. TETRASPERMAC 2.1 INITIAL HYBRIDIZATIONSDot blot hybridization using a kalilo probe was performed withthe 50 Neurospora Louisiana isolates (Table 1). Figure 4 shows aselection of these strains; only the two N. tetrasperma strains,60 and 95, hybridized with the probe. Isolation of the total DNAof strains 60 and 95 allowed the kalDNA to be examined further.Analysis of the kalilo-probed filter in Figure 5 revealed thatthe kalilo-like DNA (designated "kalilo'") was the same size asthe original kalilo plasmid. Figure 5 also suggested that pieces25of DNA larger or smaller than the original plasmid, referred toas kalilo' "derivatives", existed within these two strains.C 2.2 ASCOSPORES FROM SELF-FERTILIZATIONTo investigate the sexual transmission of kalilo', thepseudohomothallic cultures 60 (A + a) and 95 (A + a) werepermitted to self-fertilize. Twenty-five random ascospores wereisolated from each selfed culture: 10 ascospores from strain 95,and 17 ascospores from strain 60 germinated successfully. A dotblot of these new cultures (Fig. 6) revealed that two of the 27cultures, 95-4 and 95-10, did not contain the kalilo' DNA.Cultures 95-2, 95-3, 95-6, 95-7, and 95-8 contained low levels ofkalilo' (Fig. 7, total DNA). None of the ascospore progenycontained kalilo' derivatives.C 2.3 RESTRICTION MAPRestriction analysis of kalilo' was performed with DNA isolatedfrom the mitochondria of strains 95-2 and 60-13. Restrictionenzymes Xba I, Eco RI, Eco RV, Bgl II, Hind III, and Pst I wereutilized. Bands seen in the autoradiographs (Fig. 8) representfragments of DNA that hybridize to the kalilo probe, and thusindicate homology of the fragment DNA to kalilo DNA.Autoradiographs (Fig. 8) showed no discernable restriction26variation between the two N. tetrasperma plasmids and the kaliloplasmid in N. intermedia. Thus, the restriction map of kalilo(Fig. 3) for these particular enzymes is also applicable to thekalilo' plasmid.C 2.4 CONFIRMATION OF DISTINCT KALILO' IDENTITYThe presence of kalDNA in strains other than N. intermedia raisedthe possibility of contamination. In order to discount thispossibility several DNA preparations of strains 95 and 60 wereperformed using different culture tubes, and particularly, usingnew cultures derived from silica gel stocks. Since the silica gelstocks were a recent acquisition from another lab, in whichresearch is unrelated to kalilo, contamination of these stocks isunlikely. All DNA preparations confirmed the original results.C 2.5 EXONUCLEASE DIGESTIONDNA from the mitochondria of strains 95 and 60 was digested withlambda exonuclease and exonuclease III to test for the presenceof 5' and 3' terminally bound proteins associated with kalilo'.Note that 1 kb ladder DNA was digested with lambda exonucleaseand exonuclease III (data not shown) as a control of exonucleaseefficacy; complete digestion of ladder DNA was observed in bothcases. Results indicated that kalilo' was sensitive to 3'degradation, but resistant to 5' degradation (Fig. 9,27autoradiograph). The presence of a 5' terminal protein is likely.Note the presence of kalilo and kalilo' plasmid derivatives inlanes C4, 95 and 60.C 2.6 EXAMINATION OF UNISEXUAL STRAINSTo determine if the unisexual cultures of the pseudohomothallicisolates retained the kalilo' plasmid, DNA from the mitochondriaof cultures 95A, 95a, 60A and 60a were examined by means ofkalilo-probed Southern filters. All strains were positive for theplasmid (Fig. 10), and plasmid size remained constant between thebisexual and unisexual N. tetrasperma strains.C 2.7 CROSSESCrosses were performed as follows (females listed first):Cross 1-^60a (kalilo'+) x 66A (kalilo'-)Cross 2 -^66A (kalilo' - ) x 60a (kalilo'+)Note: "+" = present; "-" = absentIndividual random ascospore progeny were collected and labelled1-1, 1-2,...1-10 for cross 1 and labelled 2-1, 2-2,...2-24 forcross 2. Since all of the cross 1 progeny contained kalilo', andnone of the cross 2 progeny contained kalilo' (Fig. 11), kalilo'inheritance is maternal.The weak autoradiograph signal in lanes 1-1 to 1-10 may be due to28decreased plasmid copy number as a culture initiates mycelialgrowth from a germinated ascospore.C 2.8 SENESCENCEThe relationship between the presence of the kalilo' plasmid andsenescence was studied using both growth/race tubes and serialvegetative subculturing (Table 2). The strains analyzed in thisfashion included kalilo'+ and kalilo'- pseudohomothallicLouisiana isolates, ascospore cultures from self-fertilizedstrains 60 and 95, unisexual cultures of 60 and 95, as well asthe ascospore cultures from the reciprocal cross mentioned above.Mycelial growth in race tubes was analyzed for a minimum of 40days, and serial subcultures were performed 40 times. The N.tetrasperma growth tube results are summarized in the chartbelow:SENESCENT^NOT SENESCENTKALILO PRESENT^14^21^/35KALILO ABSENT^1 9 /10Note that strain 66 did not contain kalilo', but senesced by 36days in the growth tube (Table 2). The data presented in Table 2suggests that a general correlation exists between senescence andthe presence of kalilo'.29C 2.9 ANALYSIS OF FORTIETH SUBCULTUREIn order to account for the low frequency of senescence inkalilo'-containing strains, 40th subcultures of ascospore progenyfrom self-fertilization (strains 60-1 to 60-17, and 95-1 to 95-10) were examined for kalilo' content. Analysis of the total DNAcontent (Fig. 12) revealed that N. tetrasperma strains 95-6, 95-8, and 60-12 did not contain kalilo' at the 40th subculture. Notethat the 40th subcultures of strains 60-3, 60-7, 60-8, and 60-15contain low levels of kalilo', and that strain 95-10 did notoriginally contain the kalilo' plasmid. Thus plasmid loss is notsufficient to explain the long life spans observed duringsubculturing of kalilo'-positive strains.C 2.10 PST I DIGESTIONSenescence of Hawaiian N. intermedia is caused by the insertionof kalilo into the host mtDNA. To determine if this processoccurred in N. tetrasperma, kalilo' insertion was investigatedusing Pst I digestion. Since the kalilo' plasmid does not containPst I restriction sites, inserted plasmid (IS-ka1DNA) would beexcised at the adjacent host mtDNA Pst I sites, thereby forming afragment larger in size than the free cytoplasmic form of kalilo'(AR-kalDNA).Strains 95A, 95a, 60A, and 60a were used to test for insertion.30The DNA of unisexual strains 95 and 60 was isolated atsubcultures 0, 33 and 40, and was digested with the restrictionenzyme Pst I. Using autoradiograph band patterns as an indicationof insertion, none of the subcultures subjected to Pst 1digestion had kalilo' insertions (Fig. 13).31Figure 4. A dot blot of selected Louisiana isolates probed withkalilo DNA (X3 fragment--refer to Fig. 3). Two isolates, 95 and60, contain DNA homologous to kalilo. Strain "+P561" is apositive N. intermedia control for kalilo, and strain "-P605" isa negative N. intermedia control for kalilo.32+P561 -P605 95 9780 60 73 883 3Figure 5. Autoradiograph of the total DNA from kalilo-positiveLouisiana isolates 95 and 60. Both isolates are N. tetrasperma.N. intermedia strains P561 and P605 are positive and negativecontrols. The X3 kalilo fragment (refer to Fig. 3) was used as aprobe.340S609S09dT9SdFigure 6. A dot blot of random individual ascospores from theselfed cultures of N. tetrasperma 95 and 60 probed with the X3fragment of kalilo (refer to Fig. 3). In naming these spores thefirst two digits indicate the parent strain and the number afterthe hyphen identifies the particular ascospore. Only ascosporecultures 95-4 and 95-10 lack kalilo. N. intermedia strains P561and P605 are positive and negative controls. N. tetrasperma strain 82 is a negative control, and N. tetrasperma strains 95and 60 are positive controls.3695-1 95-2 95-3 95-495-5 95-6 95-7 95-895-9 95-10 60-1 60-260-3 60-4 60-5 60-660-7 60-8 60-9 60-1060-11 60-12 60-13 60-1460-15 60-16 60-17 9560 82 P605 P56137Figure 7. Kalilo content of the ascospore cultures from self-fertilization of strains 95 and 60. Plasmid size is apparent inthis autoradiograph of total DNA. N. intermedia strains C4 andP605 are positive and negative controls. The Pst I-isolatedkalilo DNA was used as the probe (refer to Fig. 3).38i-03 9Figure 8. Restriction analysis of kalilo'. Cultures C4 (Kalilo N.intermedia), 95-2 and 60-13 are restricted with six enzymes andcompared with respect to their restriction fragment patterns.Both 95-2 and 60-13 display the same pattern as C4, and thus, themap of kalilo' corresponds to the one constructed for kalilo (seeFig. 3 for restriction map and fragment designations). The Pst I-isolated kalilo DNA was used as a probe (refer to Fig. 3).40C4P60595-260-13C4+XbaI95-2+28.160-13+XbaIC4+EcollI95-2+1col160-13+1con1C4+icolV95-2+HcoRV60-13+1ScoRVC4P60595-260-13C4+Xba/95-2+18.160-13+XbaIC4+8coRI95 -2+ItcoRI60-13+6coR124+BcoRVP5 -2+EcoR•50 -13+8coRVto00•60-13+PStI95-2+PstIC4+PstI60-13+HindIII95-2+HindIIIC4+HindIII60-13+1911I95-2+89111C4+141IIP605C460-13+PstI95-2+PstIC4+PstI60-13+HindIII95-2+HindIIIC4+HindIII60-13+8911195-2+89111C4+891IIP605C44 1Figure 9. The 3' and 5' exonuclease digestion of DNA isolatedfrom the mitochondria of strains 95 and 60. Resistance to 5'exonuclease activity as seen on the autoradiograph suggests thepresence of a protein on the 5' termini of the kalilo' plasmid.N. intermedia strain C4 is a positive control. Pst I-isolatedkalilo DNA (refer to Fig. 3) was used as the probe.42EtBr kalilo-wholeOko0o411.1in+0 0W %godi inO crt-kaliloFigure 10. Analysis of unisexual strains 95A, 95a, 60A and 60afor kalilo' content. The N. tetrasperma strains 95 and 60 arepositive controls. The N. intermedia strains . C4 and P605 arepositive and negative controls for kalilo. Pst I-isolated kaliloDNA (refer to Fig. 3) was used as the probe.444 5Figure 11. Reciprocal crosses. Random ascospore progeny areexamined from the following crosses (female parent listed first):Cross 1: 60a x 66A and Cross 2: 66A x 60a. Progeny are identifiedby the parental cross (first number), and by the assignedascospore number (written after the hyphen). Autoradiographs showtotal DNA. The lane locations for the autoradiograph containingsamples 2-1 to 2-12 are indicated with pen marks. N. intermedia strains P561 and P605 are the positive and negative controls. PstI-isolated kalilo DNA (refer to Fig. 3) was used as a probe.464 7Table 2. Analysis of Senescence. Various cultures are examinedfor senescence during serial vegetative subcultures, and growthtube passage. The strains under examination include:pseudohomothallic strains 95 and 60, the ascospore progeny fromself-fertilization of strains 95 and 60, unisexual N. tetraspermastrains, progeny from unisexual crosses, negative N. tetrasperma controls, and positive and negative N. intermedia controls (P561and P605). ">>" refers to unarrested growth.48r.rain no.,mating typeSpecies Strain no.,mating typeSpecies48A N. crassa 95A N. tetrasperma49a N. crassa 95a N. tetrasperma50a N. crassa 60A N. tetrasperma51a N. crassa 60a N. tetrasperma52a N. crassa 1-1 N. tetrasperma53A N. crassa 1-2 N. tetrasperma54a N. crassa 1-3 N. tetrasperma55a N. crassa 1-4 N. tetrasperma56a N. crassa 1-5 N. tetrasperma57a N. crassa 1-6 N. tetrasperma58A+a N. tetrasperma 1-7 N. tetrasperma60A+a N. tetrasperma 1-8 N. tetrasperma61A+a N. tetrasperma 1-9 N. tetrasperma62A+a N. tetrasperma 1-10 N. tetrasperma63A N. crassa 2-1 N. tetrasperma64A N. crassa 2-2 N. tetrasperma65a N. crassa 2-3 N. tetrasperma66A+a N. tetrasperma 2-4 N. tetrasperma67A N. crassa 2-5 N. tetrasperma68A N. crassa 2-6 N. tetrasperma69a N. crassa 2-7 N. tetrasperma70a N. crassa 2-8 N. tetrasperma71a N. crassa 2-9 N. tetrasperma72a N. crassa 2-10 N. tetrasperma73A+a N. tetrasperma 2-11 N. tetrasperma74A+a N. tetrasperma 2-12 N. tetrasperma75A+a N. tetrasperma 2-13 N. tetrasperma76a N. crassa 2-14 N. tetrasperma77A+a N. tetrasperma 2-15 N. tetrasperma78A+a N. tetrasperma 2-16 N. tetrasperma79a N. crassa 2-17 N. tetrasperma80A N. crassa 2-18 N. tetrasperma81a N. crassa 2-19 N. tetrasperma82A+A N. tetrasperma 2-20 N. tetrasperma83a N. crassa 2-21 N. tetrasperma84A N. crassa 2-22 N. tetrasperma85A N. crassa 2-23 N. tetrasperma86A87A88A+a89a90A91a92A+a93a94A95A+a96a97a98a99a100a101aN. crassa 2-24 N. tetraspermaN. crassaN. tetraspermaN. crassaN. crassaN. crassaN. tetraspermaN. crassaN. crassaN. tetraspermaN. crassaN. crassaN. crassaN. crassaN. crassaN. crassa49Figure 12. Total DNA from ascospore progeny of self-fertilizationare tested for kalilo' content at the fortieth subculture. N.intermedia strains C4 and P605 are positive and negativecontrols. Pst I-isolated kalilo DNA (refer to Fig. 3) was used asthe probe.5051Figure 13. Investigation of kalilo' insertion. Pst I digestion oftotal DNA from the original, 33rd, and 40th subcultures ofunisexual strains 95 and 60 was performed. N. intermedia strainsC4 and P605 are positive and negative controls. Pst I-isolatedkalilo DNA (refer to Fig. 3) was used as the probe.52to1.-1-I-05 3DISCUSSIONUpon examination of Louisiana natural isolates of Neurospora, twocultures of N. tetrasperma were found to contain DNA homologousto the Hawaiian kalilo plasmid of N. intermedia; this new plasmidwas named "kalilo'". Many similarities existed between kalilo andkalilo'. Plasmid size was identical, the restriction enzyme mapwas identical on the basis of fragment patterns from 6 enzymes,and both plasmids were resistant to 5' exonuclease digestion.This identity with kalilo was remarkable in several respects. Itwas the first time a kalilo plasmid had been observed outside ofHawaii, and it was the first time that kalilo had been discoveredin a fungal strain other than N. intermedia. This was no trivialfinding considering the fact that hundreds of Neurospora isolatesfrom around the world have been examined for kalilo content (Yangand Griffiths, 1993).Furthermore, it was the first time that kalilo had been isolatedfrom a pseudohomothallic fungus. Neurospora tetrasperma usuallyundergoes self-fertilization, and thus, does not utilize thetrichogyne crossing mechanisms. As a result, it was intriguing toobserve the effects of forced outbreeding in pseudohomothallic N.tetrasperma strains. The outcome was suggestive of heterothallicmechanisms: trichogyne-mediated fertilization and maternalmitochondrial inheritance patterns.54The kalilo' plasmid displayed relatively stable asexualtransmission through vegetative transfers. In most cases theplasmid persisted through extensive subculturing. However, givenenough time plasmid levels fluctuated in some strains. Ascosporeprogeny from self-fertilized strains 95 and 60 were analyzed forkalilo content at the 40th subculture (Fig. 12). In the initialsubcultures (Fig. 7) plasmid levels were fairly constant whereasin Fig. 12 there was considerable variation. (Note that theamount of DNA loaded in the lanes within each figure were similarby visual inspection of the EtBr-stained gels--data not shown).In particular, strains 95-6, 95-8, and 60-12 lost the plasmid,and strain 60-3 had faint traces of the plasmid. Other cases ofplasmid loss during subculturing have been reported for N. crassa (May and Taylor, 1989; Yang, M.Sc. thesis 1991). Plasmid loss,however, did not sufficiently account for the long life spansobserved during subculturing of kalilo'-positive strains.On the other hand, some mode of suppression (Griffiths et al.,1992) could have been responsible for plasmid loss. I did not seea Mendelian ratio characteristic of a single gene suppressor(Table 2), but a greater data set or an ordered tetrad analysiswould provide more conclusive results to discern betweensuppression versus random plasmid loss.Upon examination of the kalilo' plasmid in random ascospore-derived cultures (Fig. 7) the size of the plasmid remained55constant; only lane 60-1 showed a slightly smaller autoradiographband. Since standard plasmid size was seen for strain 60-1 inother experiments (data not shown), it is unlikely that 60-1contains a smaller plasmid. Rather, fragment mobility wasprobably affected by altered buffer salt concentrations.The appearance of "kalilo-derivative" DNA was also unexpected.Some derivatives were smaller than kalilo (undigested lanes ofC4, 95 and 60 in Fig. 9), and some were larger (lanes 95 and 60,Fig. 5). These bands did not appear in the lanes digested with 5'or 3' exonuclease even though the same DNA samples were used.While the appearance of plasmid derivatives is not a newphenomenon (Yang and Griffiths, 1993) their sensitivity to 5'exonuclease was of interest because it was known that the kalilo'plasmid was resistant to 5' exonuclease digestion. The nature ofthis resistance is unknown--perhaps a 5' protein or a 5' hairpinstructure exists. Whatever the cause of 5' exonuclease resistancein kalilo', this element appears to be absent in the plasmidderivatives. The exact DNA composition of these derivatives wasnot investigated.The general rate of senescence of N. tetrasperma strains appearedto be slower than in N. intermedia (approximately 40 subcultureswere required for senescence to occur compared to less than 20subcultures). This delayed senescence may have been due to thesubculturing technique itself. Vegetative subculturing performed56on N. intermedia involved the serial transfer of conidia.However, N. tetrasperma produces few conidiophores, and thereforepieces of hyphal growth were used for transfers instead.This change in protocol may also explain the inconsistentsenescence patterns. The four strains (95-3, 95-5, 95-7, 95-9)that senesced during subculturing did not senesce when theexperiment was repeated. These results were contrary to therepeatable nature of conidial subculture life-spans in N.intermedia.While there was no direct causal link between kalilo' presenceand senescence, there was a significant correlation. Out of 35kalilo'-positive strains, 14 senesced (growth tube analysis,Table 2), and given more time a larger proportion of thesestrains would have likely senesced.Kalilo' independent senescence was seen in the kalilo'-negativestrain 66 which senesced at day 36 (growth tube analysis, Table2). In regard to strain 66, note that the reciprocal crossesinvolved strains 66 (kalilo'-negative, senescent) and 60(kalilo'-positive, not senescent). Maternal inheritance of thekalilo' plasmid from strain 60 was seen, and no senescence wasobserved in these progeny. Furthermore, if there was a"senescence factor" in strain 66, it was apparently notconsistently expressed or inherited; none of the progeny showed57signs of senescence that could be correlated with this"senescence factor".The lack of kalilo' insertion also argued against kalilo'-inducedsenescence. It is believed that senescence is induced by theinsertion of kalDNA into the mitochondrial genome, therebydisrupting normal host functions (Bertrand et al., 1985). YetFig. 13 showed no Pst I digestion of the plasmid and, therefore,no insertion. If kalilo' did not insert then either it was notresponsible for the senescence observed during subculturing andgrowth tube analysis, or kalilo' induced senescence by analternate mechanism.At this point two questions arose. First, why did kalilo' fail tointegrate? The exact method of plasmid integration is not known,but the terminal inverted repeats (TIR) and perhaps the 5'terminal protein are thought to be important in this process.While kalilo' had a 5' terminal protein (Fig. 9), it wasuncertain if kalilo' had the same TIR's as kalilo. Thus, kalilo'may have been defective at the termini (either the TIR's or theprotein), or perhaps Neurospora tetrasperma was an unsuitablehost for the plasmid. The easiest approach to distinguish betweenthese two hypotheses would involve transferring the kaliloplasmid from N. intermedia into N. tetrasperma by means ofprotoplast fusion, and analyzing the transformed strains for58senescence.Second, what was the cause of senescence? It is possible that inthese N. tetrasperma isolates senescence was not the result of aplasmid, but was caused by a single or multiple nuclear mutation.Nuclear mutation induced senescence has also been postulated byGriffiths and Yang (1993, in press) to explain the senescenceobserved in natural populations of N. intermedia. In the afore-mentioned study, the senescent N. intermedia strains displayheterogeneous life-spans and senescence is generally lesspredictable during culturing and crossing than kalilo-inducedsenescence. This variable expression of senescence is analogousto the N. tetrasperma strains in this work.Alternatively, the spontaneous alteration of apparently "benign"plasmids to produce "host unfriendly" plasmids could haveresulted in senescence. Benign plasmids "gone bad" have beenreported by Akins et al. (1986). The authors state thatoccasionally variants of the Mauriceville and Varkundmitochondrial plasmids in N. crassa and N. intermedia are foundto be suppressive to the host mtDNA--the mechanism of this newtype of senescence does not necessarily involve plasmidintegration. Similar plasmids may exist in N. tetrasperma. Thevariable expression of senescence in sexual crossing andunrepeatable life -spans may have been due to the unintendedselection of diverse forms of these plasmids during culturing.59A very similar case of non-kalilo senescence was observed by C.Myers (Ph.D. thesis, 1988) in the N. intermedia strain P573.According to Myers, ascospore progeny of strain P573 exhibitvariable life-spans; senescence occurs after 20 subcultures.Senescent P573-derived strains contain small amounts of AR-kalDNA(cytoplasmic kalilo plasmid). Furthermore, the appearance of IS-kalDNA (inserted kalilo plasmid) is erratic and IS-ka1DNA isgenerally absent in late senescent subcultures. It appears thatalthough senescence is transmitted to progeny, kalilo is not thesource of senescence in strain 573.The discovery of the kalilo' plasmid provoked some thought. Themovement of kalilo was of interest: did kalilo' arrive throughhorizontal transfer or was_the kalilo plasmid present beforespecies divergence and conserved through evolution? Inconsidering these two options, May and Taylor (1989) support theidea of horizontal transfer of plasmids in N. crassa based on twolines of evidence: "the distribution of plasmid DNAs in[different species and among mitochondrial types] in naturalpopulations and the experimental transfer of plasmid independentof the host mitochondrion at an evolutionarily significant rate."(p. 322) Independent transfer of plasmids in Neurospora may beachieved sexually (May and Taylor, 1989) or asexually (Collinsand Saville, 1990).Allowing that independent transfer of Neurospora plasmids can60occur, the geographic relocation of the plasmid may have occurredthrough the movement of sugar cane between Hawaii (EncyclopediaAmericana, 1979) and Louisiana. Sugar cane has been a leadingcrop in Hawaii and Louisiana for over a century. Sugar canefields are routinely burned before harvesting to remove leavesand to seal the juice in the stalks. Neurospora ascospores areactivated by burning, and sugar cane provides an ideal substratefor growth. In fact, natural isolates are often isolated fromburnt sugar cane fields, and so relocation of Neurospora by thisroute appears plausible.61BIBLIOGRAPHYAkins, R.A., Kelley, R.L., and Lambowitz, A.M. (1986).Mitochondrial plasmids of Neurospora: Integration intomitochondrial DNA and evidence for reverse transcription inmitochondria. Cell 47: 505-516.Alexopoulos, C.J., and Mims, C.W. (1979). Introductory Mycology,third ed. (New York, John Wiley & Sons).Bertrand, H., Chan, B.S.-S., and Griffiths, A.J.F. (1985).Insertion of a foreign nucleotide sequence intomitochondrial DNA causes senescence in Neurospora intermedia. Cell 41: 877-884.Bertrand, H., and Griffiths, A.J.F. (1989). Linear plasmidsthat integrate into mitochondrial DNA in Neurospora. Genome31: 155-159.Burnett, J.H. (1975). 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