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

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Change in Chromosome Number Associated with a Double Deletion in the Neurospora Crussa Mitochondrial Chromosome Copyright 0 1989 by the Genetics Society of America 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, sponta- neously 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 particular interest 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 gener- ated 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 be derived 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""se- quences 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.
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