Change in Chromosome Number Associated with a Double Deletion in the Neurospora Crussa Mitochondrial Chromosome

Change in Chromosome Number Associated With a Double Deletion in the Neurospora crussa Mitochondrial Chromosome

Samson R. Gross, Ann Mary and Pearl H. Levine

Department of Biochemistry, Division of Genetics, Duke University, Durham, North Carolina 27710 Manuscript received October 27, 1988 Accepted for publication December 19, 1988

ABSTRACT The mitochondrial genome of Neurospora is usually found in a single covalently closed circular 62-kbp DNA molecule. We report here that the mitochondrial genome of a phenotypic revertant of a stopper mutant (stp-ruv) is contained primarily in two separate, nonoverlapping, autonomously replicating circular chromosomes. The circles, one about 21 kbp and the other somewhat less than 36 kbp are derived from the most frequent classes of recombinant chromosomes (21 and 41 kbp) in the chromosomal population of mitochondria in the original stopper mutant. The new, more stable chromosomal configuration, is associated with the deletion of two sequences (1 kbp and 4 kbp) at the splice junctions of the two circles. The data suggest that both deletions are likely to have originated from a single recombinational event involved in generating the 36-kbp circle. Secondary, spontaneously arising derivatives of stp-ruv have been found to yield, at high copy number, shortsections of the 21-kbp circle in covalently closed supercoiled circles varying from unit length to very high multimers. The amplified segments span a common segment likely to contain the replication origin of the 2 1-kbp chromosome. -

NTRACHROMOSOMAL recombination is a fre- and LEVINE1984). As indicated in the following sec- I quent event during the normal growth and repli- tions, the stability of the n = 2 chromosomal comple- cation of mitochondria in many different plant and ment is associated with the loss of two extended se- fungal species (CUMMINGS,BELCOUR and GRAND- quences of the single chromosome of normal mito- CHAMP 1979; PALMERand SHIELDS1984; GROSS, chondria. One of these deletions includes the coding HSIEHand LEVINE1984). In Neurospora, intramolec- sequences forthe NADH dehydrogenasesubunits ular recombination appears to generate a variety of found deficient in the original st@ mutant. The defi- circular derivatives of the normal 62-kbp mitochon- ciency of the subunits cannot be crucial for growth, a drialchromosome (ALMASANand MISHRA 1988; fact already pointed out by DEVRIES,DEJONGE and DEVRIESet al. 1986; GROSS,HSIEH and LEVINE1984). SCHRAGE(1985) with respect to the deletion of the These, usually smaller,circular forms are found same region in anotherstopper mutant (E35). We among a heterogeneousclass of molecules making up show here, and describe more fully in the accompa- only a small fraction of the total DNA of normal nying paper (GROSSet al. 1989) that thestp-ruv strain mitochondria; however, some of the derivatives are appears to behave like some derivatives of the senes- the most frequent molecules found in mitochondria cent strains of Podospora anserina which have been from stopper(stp) mutants (GROSS,HSIEH and LEVINE found by TURKER,DOMENICO and CUMMINGS(1987) 1984). The characteristic stop-start mode of growth to amplify short sections of mitochondrial DNA. of at least one of thesemutants was shown tobe correlated with severe reversible fluctuations in the MATERIALS ANDMETHODS frequency of different classes of recombinant mole- The origin of theparental stp mutant, IAr155(11)2,107A, cules which result in a deficiency of a region shown as well as a description of its nutritional requirements and likely to code for subunits of NADH dehydrogenase mode of growth are described in GROSS,HSIEH and LEVINE (GROSS,HSIEH and LEVINE1984; DEVRIESet al. 1986). (1984). Methods employed for the isolation of mitochon- Selection for continuously growing derivatives of dria, the separation of linear and nicked mt DNA from supercoiled, covalently closed molecules by CsCI-ethidium the original stp mutant yielded a wide variety of rea- bromide density gradientcentrifugation, electrophoretic sonably stable mitochondrialgenotypes. One of them, analysis, as well as the procedures employed in isolating and stp-run, was of particularinterest because its mito- cloning Hind111 fragments of mt DNA in pSCC31 (CHENG chondrial chromosomalcomposition seemed to consist and MODRICH1983) are described in the same paper. In several instances DNA hybridization was determined by the primarily of the two most frequent recombinant chro- biotinylation method of WAHL, STERNand STARK(1 979) as mosomes foundto fluctuate as afunction of the described in the kit supplied by Bethesda Research Labo- growth state of the original stp mutant (GROSS,HSIEH ratories. Otherwise, the standard SOUTHERN(1975) hybrid-

Generics 121: 685-691 (April, 1989) 686 S. R. Gross. A. Mary and P. H. Levine

ization procedure was employed using "P-labelednick trans- the stp parent. The loss of one of the tRNA"" se- lated DNA. In each case restriction fragments were generated according to instructions supplied by the vendor of the quences in stp-ruv, along with the deletion of a seg- enzyme. mentof the chromosome common to EcoRI, 6, The stp-ruu mutant was obtained by selecting for a con- BamHI, 3, and HincII, 8, was established previously tinuously growing derivative of the original stp after irradi- by hybridization analysiswith the homologous ation of stp conidia with ultraviolet light in the following tRNA'"" sequence of HindIII, 9 (GROSS,HSIEH and way: About 1 X IO7, 7-day-old conidia were irradiated with ultraviolet light to about 10% survival,then used as an LEVINE 1984). Confinementof the deleted sequences inoculum for growth tubes prepared as described by RYAN, to the region of the 41-kbp circle is reflected further BEADLEand TATUM(1 943). A derivative which grew con- by retention of the 7.2-kbp HincII junction fragment tinuously was obtained duringthe first growth tube passage. of the 21-kbp circle, which joins HincII, 8 to 9, and Upon subsequent growth tube passages and purification by the replacement of the 1.1-kbp HincII junction frag- conidial reisolation the growth of the isolated strain, stp-run, though continuous, was at a highly variable rate, averaging ment of the stp's 41-kbp circle with a 2.9-kbp frag- about half that of wild type. Derivatives of stp-ruv which ment, joining a segment of HincII, 8, within EcoRI, yield amplified small circular DNA molecules arose sponta- 6, tosome point within HincII, 6, and HindIII,3. The neously upon standing at room temperature. In addition to deleted segment, indicated in Figure 1, which extends a requirement for methionine, stp-ruu requires inositol for froma point in HindIII, 9, beyond the tRNA""' growth. As a consequence, it does not survive when the concentration of available inositol is low (LESTERand GROSS sequence, to a point about 4-5 kbp away in HindIII, 1959). All cultures were routinely started from conidia 3, is included in the5-kbp deletion of stp E35 of stored in silica gel at -20". DEVRIESet al. (1986) which contains two open reading frames specifying two peptides resembling subunitsof RESULTS NADHdehydrogenase (CHOMYN et al. 1985). The electrophoretic patterns obtained after subjecting co- The organization of the mitochondrial genome of valently closed circular DNAs to SalI hydrolysis illus- a derivative of the original stopper (stp) mutant, trate the relative mobility and yield of the uncut 41- IAr155(11)2,107A capable of continuous growth, stp- and 36-kbp circular forms and the linearized deriva- run, was found to be quite different from that of its tives if the 2 l-kbp circle and the 62-kbp wild-type parent which, in the growth phase, consisted largely chromosome. Particularly heavily loaded samples of 2 1- and 41 -kbp circular molecules. These chromo- were run so that the minor, linearized molecules in somes were shown to bederived from the normal the 5- 17-kbp range could be seen. These are partic- single 62-kbp chromosome by intramolecular recom- ularly obvious in the stp-ruv lanes and might represent bination within the directly repeated tRNA""sequences illustrated in Figure 1. The entire nucleotide linear derivatives of small circular molecules whose sequenceappeared represented in the two circular replication were initiated at an ori sequence not too forms which were capable of reversion to something distant from the unique Sal1 site of the mitochondrial resembling wild type, albeit at very low frequency chromosome (see below). The relative intensity of the (GROSS,HSIEH and LEVINE 1984). Figure 1, which is staining of the Sal1 bands indicate that the 21- and based on the electrophoretograms of Figure 2, illus- 36-kbp circles of stp-ruv are notpresent in equal trates that the genome of the stp-ruv contains two amounts. This is indicated clearly by the 1.5-2.0-fold largedeletions, one a 4-5-kbp segmentextending excess of the 7.5-kbp HindIII junction fragment of from apoint within the tRNA""' sequence of HindIII, the 2 1 -kbpcircle over the 1 .O-kbp1 junction fragment 9, to some point in HindIII, 3, and another of about of the 36-kbp circle in DNA derived from circular as 1 kbp extendingfrom the tRNA"" sequence of well as linear molecules. HindIII, 1,to some point in EcoRI, 6. The breakpoints Two subclones of the stp-ruv mutant strain were of the two deletions appearto form the splice junctions found to produce, athigh copy number, small circular of the two derivative circular molecules. The salient derivatives of a short regionof the 2 l-kbp circle. The feature of the stp-ruv mitochondrial genome is that it first of these produced circular multimers of a 1.9- consists primarily of two independently replicating kbp segment including some or all of HindIII, 19a, nonhomologouschromosomes along with recombi- and extending into HindIII, As14. indicated in Figure nant derivatives of the two at low frequency. 3, the segment includes the chromosome's single Sal1 The data of Figure 2 are consistent with the notion site. It yields a 1.9-kbp linear molecule after cutting that the smaller of the two chromosomes of stp-ruv is with SalI that hybridizes with HindIII, 15 and 19a. identical to the 21-kbp circular chromosome of its Unfortunately, this stp-ruv derivative died before the parent. They arealso consistent with the idea that the analysis could be extended much further. A search deletions arose as a consequence of a single intramo- among isolates fromthe original strain, however, lecular recombination event either within a 41-kbp yielded one which produced, at high copy number, circular chromosome or some larger component of circular derivatives of two short sequences, 286 and the chromosomal population in the mitochondria of 291 bp (GROSSet al. 1989), which hybridized with Mitochondrial ChromosomeMitochondrial Number 687

FIGURE1 .-A composite restric- tionmap ofthe Neurospora niito- chondrial chromosome showing the deletionsand major circularchro- mosomalderivatives present in the stp-ruu mutant strain. The map of type I1 mitochondrialDNA is derived largely from those of TAYLOR and SMOLICH (1985). COLLINS (1986). DEVRIFSet al. (1986) and NELSON and MACINO (1987). Ribo- somal RNA sequences are noted by open boxes and the intronwithin the 25s rRNA region by a jagged line. The two tRNA"" sequencesare noted by boxes with arrows indicat- ing sequence orientation. The deletions of stp-ruu are cross hatched and the junctionsof the 2 1- and <36-kbp circular derivatives are notedby lines drawn across the circle. The 286-hp and 1.9-kbp circular derivatives are drawn to includethe Hindlll. 7a- 1%. junction.This region has not beenshown definitively to be included within the 1.9-kbp circle. It is left unshaded.

HindIII, 19a and 7a (Figure 4). The electrophoreto- recombinantchromosomes to a genomeconsisting gramsand hybridizations of Figure 4 (usingDNA primarily of two complementary, separately replicat- from the cloned 286-bp sequence) indicate that only ing chromosomes, and (2) a proclivity to amplify de- a very small fraction of the minicircles are in mono- rivatives of a short section of one of thechromosomes. meric form. The extent andcomplexity of the ladder A discussion of therelation of base sequenceto mech- of the two circular derivatives suggest the involvement anisms of recombination and replication of the nested of a large number of polymeric forms, some of them, set of plasmid-like elements derived from the 2 1-kbp perhaps, unresolved knottedcircles. Of special note is the curious 5.3-kbp HindIII fragment that hybridizes chromosome can be found in GROSSet al. (1 989). We strongly with the 286-bp sequence but appears to be reserve the discussion here to the overall change in only a minor constituent as judged by ethidium bro- the chondriotype from the n = 1 of the wild type to mide staining. Since it has only a single HindIII site n = 2 of stp-ruv and its relationto replication initiation and is very much larger than either HindIII, 7a or from a replication origin within the 21-kbp chromo- 19a, it is likely to contain a highly iterated sequence some. derived from HindIII,19a. The 5.3-kbpHindIII frag- The genotypic analysis of the chromosomal com- ment was transient, appearing only in DNA from a position of mitochondria in the original stopper mu- few subcultures ofstp-ruv 286129 1. tant,IArl55(11)2,107A and normal mitochondria The base sequences of the 286 and 291 subunits from its inositol requiringparent, 89601, revealed are presented in GROSSet al. (1989) along with se- thatintramolecular and intermolecular recombina- quencesof other plasmids derivedfrom the same tion were normal events during the life history of a strain and ananalysis of the recombination andrepli- mitochondrialchromosomal population. However, cationmechanisms involved in generating the plasmids ofstp-ruv. the maindifference between normal and stopper chromosomal populationswas found confined largely DISCUSSION to frequency rather than kind of chromosomes Two properties ofstp-ruv distinguish it from its stp present. We were able to show that the 2 1- and 4 1- parent: (1) a switch from a fluctuating population of kbpcircular chromosomes, the major fluctuating 688 S. R. Gross, A. Mary and P. H. Levine

FIGURE2.-Restriction fragment patterns of mitochondria stp-ruv and wild-type II DNA. The electrophoretic patterns of BamHI, EcoRI. Hincll, Hind111 and Sal1 restriction patterns of covalently closed circular DNA from sgruu mf (mu)are compared with the corresponding patterns obtained from total wild-type II mf DNA. Restriction patterns of linear and nicked stp-ruu DNA were found. in each case, to be similar to those obtained for covalently closed molecules. This is illustrated by the patterns obtained after cutting covalently closed circular (H) and nicked circular plus linear (L) DNA molecules with Hindlll. The fragments are identified by the numbers listed to the right of the type I1 channels, and the sizes of each of the junction fragments, in kbp, are listed to the left of the stp-ruu channels. The threeSal1 patterns on the right are presented to illustrate the classes of DNA molecules, found at low frequency, in preparations of closed circular sfp-ruu DNA. Nothing was detectable on these gels beyond the smallest listed fragment. Uncut covalently closed circular (c) DNA is at the top of the gel and the remaining bands are derived from circular forms linearized by Sal1 (A). Electrophoresis of uncut samples (not presented) yield only broad bands near the topof the gel. classes of the stp mutant, were present, albeit at low ates the kinds of plasmid-like objects produced by stp- frequency, in normalmitochondria (GROSS,HSIEH ruu. Furthermore, several continuous growing deriv- and LEVINE1984) andthat the two chromosomes atives of the stp mutant havebeen obtained in addition were generated by homologous, reciprocal recombi- to stp-ruu. The most pertinent of these has a chondri- nation within the directly repeated tRNA"" sequences otype consisting of a single chromosome with a defi- located about 20 kbp apart on theEcoRI, 1 fragment ciency in the same region and about the same size as (Figure 1). No specific explanation for the difference that in E35 (ourunpublished observation). This strain between the chondriotypes of the normal or stp mu- does not generate plasmids. In fact, the generationof tants was obtained, but because reversion to wild type polymeric derivatives of shortsections of the HindIII, was very rare it was assumed that a reasonably stable, 7a and 19a, region have only been found under two rarely reversible sequence alteration was responsible circumstances, growth of mutant and normal strains for thestp phenotype. It was clear from the studies of on inhibiting levels of acriflavin (our unpublished E35 mitochondria (DEVRIES,DEJONCE and SCHRAGE observation) and spontaneously, during thegrowth of 1985), that the region of the mitochondrial chromo- stp-ruu. some containing nd2 and nd3, subunits of NADH There are two major manifestations of the differ- dehydrogenase, that was poorlyrepresented in stp ences between the chondriotype of stp-ruu and the mutantmitochondria was dispensable since it was strain fromwhich it was derived, both perhapsdirectly deleted from the E35 genome. (It has been shown relatedto the short (about 1kbp) deletion of the recently that stp-ruu, which also is missing the two nd EcoRI, 1-6, junction (Figure 1). The first of these is coding units respires by a CN insensitive pathway (J. the fact that onlya single tRNA""is present per SIEDOW,personal communication). genome. Its absence from the 36-kbp circle may ac- While E35 andstp grow and support thereplication count for failure to generate a single large, 57-kbp of recombinant chromosomes, neither strain gener- chromosome. Such a role for the tRNA sequence is MitochondrialNumber Chromosome 689 Hind Hindrn Su/I Uncut 15 19a 15 19a 15 15 15 19a 19a 15 15 WtII ruvl9K WtII ruv 1.9K ruv 1.9K ruv 1.9K

1.9"- -15 15-

14- I, "19a

FIGURE 3.-Electrophoretic and hybridi7ation patterns of sgruv mt DNA containing 1.9-kbp circular derivatives. Biotinvlated, cloned Hindlll, 15 and I%, fragments were hybridized withHind111 and Hincll cut total DNA as well as Sal1 cut and uncut covalently closed circular DNA from stp-ruv 1.9-kbp mitochondria. The linearized 1.9-kbp fragment (marked with arrow) is clearly visible after cutting with Sall. It, as well as the ladder of multimers derived from it, hybridizes with HindIII, 15. To help visualize these very light blots each of the polymeric forms is marked by a line beside each channel. The size of the monomeric unit, 1.9 kbp, is no shorter:I after cutting with HincII than Sall. Hence the unit does not extend significantly beyond the HincII, 14 interval. The restriction fragments containing homologous sequences to each of the probes is identified in each case either by number or size. implicit in the reversible, reciprocal recombination sive independent replication. It is of some interest to mechanism proposed for generating the population note that the EcoRI, 4-6, region has been proposed of chromosomes of the original stp mutant (GROSS, previously to be involved in some replication function HSIEHand LEVINE1984). The second and somewhat because of thegeneration of tandemreiterations surprising consequenceis that thesingle chromosome within the region (MANNELLA,GOEWERT and LAM- configuration has not been found to be regenerated BOWITZ 1979).However, the essential point to be by recombination at the many other highly repeated made from the existence of the two independently sequences distributed throughout the chromosome. replicating chromosomes of stp-ruu is that the Neu- These repeated sequences havebeen found to be sites rospora mitochondrial chromosome has, at the very forthe legitimate and illegitimate recombination least, two replication initiation sites and if yeast and events involved in the generation of the multimeric Podospora are indicative models, the true number of plasmid subunits and theirresolution after replication ori sequences in Neurospora is likely to greater than (YIN et al. 1982; DEVRIFSet al. 1986; GROSSet al. two. 1989). Repeated sequences are not confined to the At this point, too much evolutionary significance 21-kbp regionspanned by the smaller of the two should not beattributed to the changein chromosome circular chromosomes but many, associated with tan- number in the Neurospora mitochondrion. It is not dem PstI site repeats, are distributed widely in the 36- known how the Neurospora mitochondrial chromo- kbp chromosome as well (NELSONand MACINO 1987; some was derived; whether it is a recombinant com- ALMASANand MISHRA 1988). It seems a possibility, at posite of several chromosomes or whether multiple least, that recombinationdoes occur butthat the origins arose by duplication over time. What is clear replication of composite chromosomes is faulty. This is that most fungal and mammalian mitochondrial implies that there is some kind of replicative function chromosomes have multiple origins and except for for the region near EcoRI, 1-6, which, when missing, the two ori sequences in mammals, one foreach of the may occasionally cause premature termination of rep- DNA strands (CREWSet al. 1979), the function and lication from an ori sequence within the HindIII, 7a- regulation of the multiple origins are, for the most 19a region. Sequences containing an origin of repli- part, unknown. The information currently available cation, once synthesized, might then undergo exten- about mitochondria in Podospora,Neurospora and 690 S. R.and Gross,Mary A. P. H. Levine Hinc II Uncut Hind III wtII ruv286 1291 ruv286 1291 wtII ruv286/291 ruv2861291 286286 286 286286 15 190

2- +5.3

-70

15-

"190

,286 ,286 291

FIGURE4.-Electrophoretic and hybridization patterns of stp-ruv 286/291 DNA. '*Pnick translated cloned 286-bp sequence and biotinylated cloned HindlII. 15 and 19a, DNA were hybridized with HincII, and Hindlll, cut and uncut covalently closed DNA from stp-mv (ruv) mitochondria containing 286- and 291-bp subunit circles. The hybridbation ladders were matched to the gel photographs as well as practicable in view of the compression distortions resulting from the blotting procedure itself. Note that very few of the circular forms migrate as monomers and noteespecially the highly reactive circular form which hybridizes with a very faint 5.3-kbp Hind111 band. Although not cut by Hincll, the migration of the circular forms is altered after, exposure to the enzyme. The hybridized restriction fragments are marked by number and the novel linear derivatives of the circles by size (arrow). yeast, outlines situations where differentori sequences 1979 Nucleotide sequence of a region of human mitochon- are exploited in the amplification of specific sections drial DNA containing the precisely identified origin of replication. Nature 277: 192-198. of the mitochondrialchromosome. It is yet to be CUMMINCS,D. J., L. BELCOUR and C. GRANDCHAMP, shown whether this amplification process is physiolog- 1979 Mitochondrial DNA from Podospora anserina. Mol. ically significant. Gen. Genet. 171: 239-250. DEVRIES,H., J. C. DEJONGEand C. SCHRAGE,1985 The Neuro- This work was supported by a research grant from the U.S. spora mitochondrial stopper mutant, [E35], lacks two protein National Institutes of Health. BRSG SO7 RR 05405. genes indispensable for the formation of complexes I. 111, and IV, pp. 285-292 in Achievements and Perspectives of Mitochon- drial ResearchVolume 11: Biogenesis, edited byE. QUAGLI- LITERATURECITED ARIELLO. Elsevier Science Publishers, New York. ALMASAN,A., and N. C. MISHRA,1988 Molecular characteri~ation DEVRIES,H., B. ALZNER-DEWEERD,C. A. BREITENBERCER,D. D. of the mitochondrial DNA of a new stopper mutant ER-3 of CHANG,J. C. DEJONGEand U. L. RAJBHANDARY,1986 The Neurospora crassa. Genetics 120 935-945. E35 stopper mutant of Neurospora crassa: precise locali7ation CHENG,S.-C., and P. MODRICH,1983 Positive-selection cloning of deletion endpoints in mitochondrial DNA and evidence that vehicle useful for overproduction of hybrid proteins. J. Bacte- the deleted DNA codes for a subunit of NADH dehydrogenase. riol. 154: 1005-1008. EMBO J. 5: 779-785. CHOMYN,A., P. MARIOTINI. M. W. J. CLEETER,C. I. RAGAN,A. GROSS,S. R., T. HSIEHand P. H. LEVINE,1984 Intramolecular MATSUNO-YAGI,Y. HATEFI.R. F. DOOLITTLEand G.ATTARDI, recombination as a source of mitochondrial chromosome het- 1985 Six unidentified reading frames of human mitochon- eromorphism in Neurospora. Cell 38: 233-239. drial DNA encode componentsof the respiratory chain NADH GROSS, S. R.,P. H. LEVINE,S. METZGER and G. GLASER, dehydrogenase. Nature 314: 592-597. 1989 Recombination and replication of plasmid-like deriva- COLLINS,R. A., 1986 Neurosporacrassa laboratory strain 74- tives of a short section of the mitochondrial chromosome of OR23-1A: mitochondrial genes, pp. 322-324 in Genetic Maps Neurospora crassa. Genetics 121: 693-701. 1987, edited by S. J. O'BRIEN.Cold Spring Harbor Laboratory, LESTER,H., and S. R. GROSS,1959 Efficient method for selection Cold Spring Harbor, N.Y. of auxotrophic mutants of Neurospora. Science 129 572. CREWS,S., D. OHALA,J. POSAKONY,J. NrsHIGuCHIand G.ATTARDI, MANNELLA, A.,C.R. GOEWERTand A. M. LAMBOWITZ, M itochondrial Chromosome NumberChromosome Mitochondrial 69 1

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