RPD3 (REC3) mutations affect mitotic recombination in Saccharomyces cerevisiae

Prior research identified the recessive rec3-1ts mutation in Saccharomyces cerevisiae which, in homozygous diploid cells, confers a conditional phenotype resulting in reduced levels of spontaneous mitotic recombination and loss of sporulation at the restrictive temperature of 36 °C. We found that a 3.4-kb genomic fragment that complements the rec3-1ts/rec3-1ts mutation and which maps to chromosome XIV, is identical to RPD3, a gene encoding a histone de-acetylase. Sporulation is reduced in homozygous diploid strains containing the rec3-1ts allele at 24 °C, suggesting that this allele of RPD3 encodes a gene product with a reduced function. Sporulation is abolished in diploid strains homozygous for the rpd3Δ or rec3-1ts alleles, as well as in rpd3Δ/rec3-1ts heteroallelic diploids, at the non-permissive temperature. Acid-phosphatase expression has been shown to be RPD3 dependent. We found that acid-phosphatase activity is greater in diploid strains homozygous for the temperature-sensitive rec3-1ts allele than in RPD3/RPD3 strains and increased further when mutant strains are grown at 36 °C. We also tested the rpd3Δ/rpd3Δ strains for their effects on spontaneous mitotic recombination. By assaying a variety of intra- and inter-genic recombination events distributed over three chromosomes, we found that in the majority of cases spontaneous mitotic recombination was reduced in diploid rpd3Δ/rpd3Δ cells (relative to a RPD3/RPD3 control). Finally, although 90% of mitotic recombinant events are initiated in the G₁ phase of the growth cycle (i.e., before DNA synthesis) we show that RPD3 is not regulated in a cell-cycle-dependent manner. These data suggest that mitotic recombination, in addition to gene expression, is affected by changes in chromatin architecture mediated by RPD3. Received: 17 July / 30 November 1998     

The REC46 gene of Saccharomyces cerevisiae controls mitotic chromosomal stability,recombination and sporulation: cell-type and life cycle stage-specific expression of the rec46-1 mutation

Summary The recessive hyperrecombination mutation rec46-1, isolated by ultraviolet light mutagenesis of the MAT n+1 chromosome VII disomic strain LBW (Esposito et al. 1982), enhances the mitotic rates of spontaneous gene conversion, intergenic recombination and restitution of haploidy (due to chromosomal loss or mitotic nondisjunction) in MAT n+1 chromosome VII disomic strains. The rec46-1 mutation does not prevent HO directed homothallic interconversion of mating types. MATaIMaT ree46-1/rec46-1 diploids exhibit the same degree of hyperrecombinational activity as MAT rec46-1 n+1 chromosome VII disomics with respect to gene conversion and intergenic recombination resulting in prototrophy. When compared to MAT rec46-1 n+1 disomics however, MATa/MAT rec46-1/rec46-1 diploids exhibit a ten fold reduced level of hyperrecombinational activity with respect to intergenic recombination and present no evidence of chromosomal loss or nondisjunction resulting in 2n-1 monosomic segregants. MATaIMAT rec46-1/rec46-1 diploids are sporulation-deficient. The results obtained demonstrate that the REC46 gene product modulates mitotic chromosomal stability and recombination and is essential for sporulation (meiosis and ascospore formation).     

Nonrandomly-associated forward mutation and mitotic recombination yield yeast diploids homozygous for recessive mutations

We have employed the analysis of spontaneous forward mutations that confer the ability to utilize L--aminoadipate as a nitrogen source (-Aa⁺) to discern the events that contribute to mitotic segregation of spontaneous recessive mutations by diploid cells. -Aa^- diploid cells yield -Aa⁺ mutants at a rate of 7.8±3.6×10^-9. As in haploid strains, approximately 97% (30/31) of -Aa⁺ mutants are spontaneous lys2-x recessive mutations. -Aa⁺ mutants of diploid cells reflect mostly the fate of LYS2/lys2-x heterozygotes that arise by mutation within LYS2/LYS2 populations at a rate of 1.2±0.4×10^-6. Mitotic recombination occurs in nonrandom association with forward mutation of LYS2 at a rate of 1.3±0.6×10^-3. This mitotic recombination rate is tenfold higher than that of a control LYS2/lys2-1 diploid. Mitotic segregation within LYS2/lys2-x subpopulations yields primarily lys2-x/lys2-x diploids and a minority of lys2-x aneuploids. Fifteen percent of lys2-x/lys2-x diploids appear to have arisen by gene conversion of LYS2 to lys2-x; 85% of lys2-x/lys2-x diploids appear to have arisen by mitotic recombination in the CENII-LYS2 interval. lys2-1/lys2-1 mitotic segregants of a control LYS2/lys2-1 diploid consist similarly of 18% of lys2-1/lys2-1 diploids that appear to have arisen by gene conversion of LYS2 to lys2-1 and 82% of lys2-1/lys2-1 diploids that appear to have arisen by mitotic recombination in the CENII-LYS2 interval. The methods described can be used to simultaneously monitor the effects of yeast gene mutations and carcinogens on the principal parameters affecting the genomic stability of diploid mitotic cells: mutation, gene conversion, intergenic recombination, and chromosomal loss or rearrangement.The research of the authors was supported by the Director, Office of Energy Research, Biological Research Division of the U.S. Department of Energy under Contract No. DE-ACO3-76SF00098, grants to M. S. E. and C. V. B. from the National Institutes of Health and the National Aeronautics and Space Administration, and a National Science Foundation postdoctoral research fellowship award to R. M. R.     

Duration of the mitotic cycle of chinese hamster cells cultured at 30–39°C

The duration of the mitotic cycle (T) and of its stages at temperatures of 30, 33, 36, and 39°C was studied by an autoradiographic method in Chinese hamster cells of subline 237. The duration of the mitotic cycle was shortest at 39°C and increased as the cultivation temperature fell. Within the temperature range 33–39°C the increase in duration of the mitotic cycle and its stages was proportional to the temperature studied. The slope of the curve of duration of the mitotic cycle as a function of temperature increased sharply as the temperature changed from 33 to 30°C. The G₁ period was most sensitive and the G₂ period least to a change in the cultivation temperature.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 11, pp. 1365–1367, November, 1976.     

Extragenic revertants of rad50, a yeast mutation causing defects in recombination and repair

Summary The RAD50 gene in yeast is required for recombination-repair (i.e., the double strand break repair pathway) in mitosis, and for meiotic recombination and sporulation. Both of these processes are complex and seem likely to require a relatively large number of gene products. In order to help define other genes required for recombination and repair processes in yeast, we have isolated extragenic revertants of rad50-4 which restore the ability to grow in the presence of MMS. Evidence from segregation indicates the extragenic revertants fall into at least five loci. Two of them reduce sporulation and spore viability at high temperature; another mutation confers a spontaneous hyperrec phenotype on mitotic cells. Thus, at least three revertants are candidates for mutations which affect recombination functions.     

Isolation and characterization of Chlamydomonas temperature-sensitive mutants affecting gametic differentiation under nitrogen-starved conditions

Summary Two conditional mutants of Chlamydomonas reinhardtii, dif-1 and dif-2, affecting gametic differentiation under conditions of nitrogen (N)-starvation, have been isolated. These mutant cells remain vegetative at the restrictive temperature (35°C) in — N medium, as defined by assays of cell body-agglutinin and cell wall lytic enzyme activities in the soluble fractions of cell homogenates. Moreover, the mutants fail to form mating structures at the restrictive temperature, but do so at the permissive temperature (25°C). Temperature-shift experiments show that mutant cells which have differentiated into gametes at 25°C dedifferentiate into vegetative cells under N-starvation conditions after transfer to 35°C, but differentiate again into gametes at 25°C. Genetic analyses indicate that the dif-1 and dif-2 genes are recessive and unlinked to each other or to the matingtype locus; the dif-1 phenotype cosegregates with a conditional flagellales phenotype expressed in both +N and-N medium at the restrictive temperature.     

Site of pheromone action and secretion pathway of a sexual agglutination substance during its induction by pheromone a in α cells of Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae mating-type strains carrying the sec1, sec7 and sec18 genes, pheromone a induces agglutination ability at 24 °C, but not at 36 °C. Even when cells were treated at 36 °C, a biologically active agglutination substance was found in cytoplasm although this activity was not detected in the cell surface fraction. These 36 °C-treated cells became agglutinable with a concomitant appearance of agglutination substance activity in the cell surface fraction. The cells remained agglutinable even when the treatment temperature was dropped to 24 °C under conditions where no de novo synthesis of the agglutination substance occurred. These results indicate that the agglutination substance is transported through the yeast secretory pathway and that pheromone a acts at the step of synthesis of the precursor molecule of the a agglutination substance, similar to the case ofthe -pheromone-induction of the a agglutination substance. Differences in the action of the sex pheromones between agglutination ability induction and induction of G₁ arrest or shmooing is discussed.     

Repair properties in yeast mitochondrial DNA mutators

Summary After ethy1methanesulfonate mutagenesis of the strain Saccharomyces cerevisiae D273-1013, out of 100,000 survivors, 1,000 were selected for their high production of petite mutants at 36 °C. Among these 1,000 mutators, 5 also showed an increased frequency of spontaneous point mutations measured at 25 °C. Further analysis revealed that in all mutators, except 2, petite accumulation proceeded at 25 °C as well as 36 °C. In these 2 mutants, the production of petite mutants was much higher at 36 °C than at 25 °C. In one of them, however, the mutator and the thermosensitive petite phenotypes were due to mutations in two unlinked nuclear genes. In the other mutants, both traits were the result of a mutation in a single nuclear gene. The mutators fell into three complementation groups (tpm1, tpm2, mup1). No complementation was observed between tpm1 mutants and the gam4 mutant previously described by Foury and Goffeau (1979). From the latter and the present works, only four complementation groups (gam1, gam2, gam4 or tpml, mupl) have been identified and it is likely that the number of genes controlling specifically the spontaneous mutability of the mtDNA is low. The mutators exhibited a variety of responses to damaging agents such as UV light and ethidium bromide; especially in a representative mutant from the complementation group tpm1, the induction of ^– mutants was sensitive to UV light and resistant to ethidium bromide. In addition, we found that in the mutants from this complementation group, the synthesis of mtDNA in isolated mitochondria was low; however their mitochondrial DNA polymerase activity was similar to that of the wild type strain. A relationship might exist between the mutator phenotype and the low mtDNA synthesis in the tpm1 mutants.     

Cloning of the Rhizopus niveus pyr4 gene and its use for the transformation of Rhizopus delemar

We have cloned a pyr4 gene encoding orotidine-5-monophosphate decarboxylase of the filamentous fungus Rhizopus niveus. The pyr4 gene of R. nivens has an open reading frame composed of 265 amino-acid residues and has two putative introns. We have also isolated a pyr4 mutant of Rhizopus delemar from 5-fluoroorotic acid-resistant mutants and transformed it with the pyr4 gene of R. niveus as a selectable marker. Introduced DNA was integrated into the chromosome in a multiple tandem array. The mitotic stability of the introduced DNA was increased by a repeated sporulation process. The expression of the Escherichia coli -glucuronidase gene in R. delemar was successfully obtained under the control of the pgk2 gene promoter of R. niveus by co-transformation with the pyr4 gene.     

Effects of the CDC2 gene on adaptive mutation in the yeast Saccharomyces cerevisiae

We have studied the influence of a temperature-sensitive cdc2-1 mutation in DNA polymerase on the selection-induced mutation occurring at the LYS-2 locus in the yeast Saccharomyces cerevisiae. It was found that in cells plated on synthetic complete medium lacking only lysine, the numbers of Lys⁺ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. When cdc2-1 mutant cells, after selective plating, were incubated at the restrictive temperature of 37°C for 5 h daily for 7 days, the frequency of an adaptive reversion of lys ^- Lys⁺ was significantly higher than the frequency in cells incubated only at the permissive temperature, or in wild-type cells incubated either at 23°C or 37°C. Therefore, when the proof-reading activity of DNA polymerase is impaired under restrictive conditions, the frequency of adaptive mutations is markedly enhanced.     

Vergleichende Sauerstoffverbrauchsmessungen bei Kokzidienoocysten der Gattung Eimeria (Protozoa,Sporozoa)

Zusammenfassung Der Sauerstoffverbrauch sporulierender Oocysten von E. falciformis, E. tenella und E. contorta n.sp. Haberkorn, 1970 lag mit 14,9 bis 23,8 l/10⁵ Oocysten/72 Std deutlich unter dem von E. stiedai mit 31,1 l. Der unmittelbar im Anschluß an die Sporulation verminderte Sauerstoffverbrauch sank nach 3- bzw. 10monatiger Lagerung weiter ab. Bis zu einem Temperaturoptimum bei etwa 35°C verbrauchten sporulierte Oocysten von E. stiedai mit zunehmender Temperatur mehr Sauerstoff.

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Comparative measurements of oxygen consumption in Eimeria oocysts (Protozoa, Sporozoa)

Summary The oxygen consumption of E. falciformis, E. tenella, E. contorta n.sp., and E. stiedai oocysts during sporulation ranged from 14.9–31.1 l/10⁵ oocysts/72 hrs. It decreased to 0.4–1.3 l/24 hrs after a storage period of three months. There was an increase in the oxygen consumption of sporulated E. stiedai oocysts measured from +4°–35°C.

     

Forearm temperature profile during the transient phase of thermal stress

Summary The transient temperature response of the resting human forearm immersed in water at temperatures (T _w) ranging from 15 to 36°C was investigated. Tissue temperature (T _t) was continuously monitored by a calibrated multicouple probe during the 3-h immersions.T _t was measured every 5 mm, from the longitudinal axis of the forearm to the skin surface. Skin temperature, rectal temperature, and blood flow ( ) were also measured during the immersions. The maximum rate of change of the forearm mean tissue temperature ( ) occurred during the first 5 min of the immersion. was linearly dependent onT _w (P<0.001), with mean values (SEM) ranging from –0.8 (0.1) °C · min^–1 at 15°C to 0.2 (0.1) °C · min^–1 at 36°C. The maximum rate of change of compartment mean temperature was dependent (P<0.001) on the radial distance from the longitudinal axis of the forearm. The half-time for thermal steady state of the forearm mean tissue temperature was linearly dependent onT _w between 30 and 36°C (P<0.01), with mean values (SEM) ranging from 15.6 (0.6) min at 30°C to 9.7 (1.2) min at 36°C and not different between 15 and 30°C, averaging 16.2 (0.6) min. There was a significant linear relationship between the half-time for thermal steady-state of the compartment mean temperature and the radial distance from the longitudinal axis of the forearm for each value ofT _w tested (P<0.001). The data of the present study suggest that the forearm is an important determinant of the transient thermal response of the forearm tissue during thermal stress.     

Induced recombination and reversion of theCDC8 gene in relation to the cell cycle of yeast

Summary The induction of mitotic recombination in theCDC8 locus was studied in a diploid strain heteroallelic forcdc8 mutations (cdc8-1/cdc8-3); mitotic reversion was studied in strainscdc8-1/cdc8-1 andcdc8-3/cdc8-3. Conversion and reversion did not occur in those cells blocked at the S stage of the cell cycle by exposure to a nonpermissive temperature. In stationary phase cells irradiated just prior to exposure to temperature stress, the induction of recombinants was rather low and the induction of revertants was minimal. Conversely, a significant induction ofcdc ⁺ occurred in logarithmic phase cells subjected to the same treatment. Irradiation of synchronously dividing cultures revealed that intragenic recombination occurs at all three stages of the cell cycle- G₁, S and G₂. It was also found that UV-induced gene reversion can occur during the S and G₂ stages, but not during the G₁ stage of the cell cycle.     

FLP-FRT mediated intrachromosomal recombination on a tandemly duplicated YEp integrant at the ILV2 locus of chromosome XIII in Saccharomyces cerevisiae

Summary A YEp chimaeric plasmid carrying SMR1 and URA3 genetic markers was integrated into chromosome XIII at the ilv2-1 locus in a [cir°] background. The 1.5 kb BglII deletion of ilv2-1 allowed the clear identification of an integrant structure which consisted of a direct tandem duplication (TD) of the chimaeric plasmid. Within the integrant structure, a single copy of the plasmid sequence was flanked by a direct duplication of the 2m site-specific recombinase (FLP) recognition target (FRT). Isogenic [cir°] and [cir ⁺] diploids formed by crossing the [cir°] TD strain to complementary haploids were analyzed for plasmid marker loss and chromosomal DNA alterations in the presence and absence of selection pressure for the URA3 and SMR1 plasmid borne markers. [cir°] diploids showed no plasmid marker loss and maintained the TD structure. In the absence of selection pressure, the [cir ⁺] diploid underwent FLP-FRT mediated unequal interchromatid recombination, resulting in the breakage-fusion-bridge cycle and homozygotization of chromosome XIII (Rank et al. 1988). Maintenance of selection pressure for the centromere distal plasmid URA3 marker selected against FLP-FRT interchromatid recombinants so that the effects of site specific recombinase on intrachromatid recombination could be evaluated. Intrachromatid recombination at the directly duplicated FRT sites of the TD structure resulted in the loss of a diagnostic internal fragment. These results show that in the presence of FLP, FRT sites separated by up to 13.3 kb of chromosomal DNA function as substrates for intra and interchromatid recombination.     

Use of reporter genes for the isolation and characterisation of different classes of sporulation mutants in the yeast Saccharomyces cerevisiae

Reporter genes consisting of sporulation-specific promoters fused to lacZ were used as markers to monitor the sporulation pathway of the yeast Saccharomyces cerevisiae. Strains transformed with these lacZ gene fusions expressed -galactosidase (assayable on plates using the substrate 5-bromo-4-chloro-3-indolyl--D-galactopyranoside, X-gal) in a sporulation-dependent manner. Mutagenesis experiments performed on transformed strains resulted in the recovery of a number of novel sporulation mutants. Three classes of mutants were obtained: those which overexpressed the reporter gene under sporulation conditions, those which did not express the gene under any conditions, and those which expressed the gene in vegetative cells not undergoing sporulation. On the basis of the blue colony-colour produced in the presence of X-gal these have been described as superblue, white, and blue vegetative mutants, respectively. These were further characterised using earlier reporter genes and other marker systems. This study established that the multicopy reporter plasmids chosen do not interfere with sporulation; they are valid tools for monitoring the pathway and they provide a way to isolate mutations not readily selected by other markers.     

Cloning and mapping of CDC40, a Saccharomyces cerevisiae gene with a role in DNA repair

Summary The cdc40 mutation has been previously shown to be a heat-sensitive cell-division-cycle mutation. At the restrictive temperature, cdc40 cells arrest at the end of DNA replication, but retain sensitivity to hydroxyurea (Kassir and Simchen 1978). The mutation has also been shown to affect commitment to meiotic recombination and its realization. Here we show that mutant cells are extremely sensitive to Methyl-Methane Sulfonate (MMS) when the treatment is carried out at restrictive temperature. Incubation at 37 °C prior to, or after MMS treatment at 23 °C, does not result in lower survival. It is concluded that the CDC40 gene product has a role in DNA repair, possibly holding together or protecting the DNA during the early stages of repair.The CDC40 gene was cloned on a 2.65 kb DNA fragment. A 2 plasmid carrying the gene was integrated and mapped to chromosome IV, between trp4 and ade8, by the method of marker loss. Conventional tetrad analysis has shown cdc40 to map 1.7 cM from trp4.     

The influence of environmental conditions and parasite-intermediate host-related factors on the transmission ofEchinostoma liei

Moderate freezing, desiccation, and pH levels of 5 and 6 killed theEchinostoma liei egg immediately or after only partial development, and increasing salinity levels above 3.79 and temperatures of 33°C and 35°C reduced the developmental potential. Temperatures of 6°C and 8°C and maintenance of eggs in mouse faeces arrested the development of the egg, and increasing temperatures decreased the time of development from 40 days at 18°C to nine days at 35°C. Maintenance of unembryonated eggs for 14 weeks in faeces at 12°C and 22°C and for 20 weeks in filtered pond water at 4°C allowed a subsequent normal development, while embryonated eggs maintained at 4°C only retained an unchanged hatchability for three weeks.A miracidium/snail density of 1010 in 18 litres of water gave rise to an average level of parasitisation of 21%. Increasing miracidium/snail densities gave rise to increasing levels of parasitisation but to a level less than expected.The first intermediate host spectrum ofE. liei was shown to be restricted to the genusBiomphalaria, but species variability in susceptibility within the genus and differences in survival of infected susceptible species was also demonstrated to exist.B. glabrata (Puerto Rico and St. Lucia),B. alexandrina (Qalyub), andB. pfeifferi (Malumfashi) were found to be susceptible, whileB. camerunensis (Kinshasa) was almost refractory. Increasing size ofB. glabrata (Puerto Rico) resulted in increasing daily cercarial production, but resistance to infection with increasing snail size was also demonstrated to exist.E. liei cercarial infectivity to the second intermediate host snail and the subsequent metacercarial infectivity to the mouse was found to be independent of the species of the first intermediate host and of the age of the infection inB. glabrata (Puerto Rico) for up to at least 6 1/2 weeks after the end of the prepatent period. No obvious peak in cercarial shedding from the host snail occurred during the day.Five species of the genusBulinus andPhysa acuta (Egypt) were found to be highly susceptible as second intermediate host snails, whereas three species of the genusBiomphalaria, Planorbarius corneus (Rabat),Lymnaea natalensis (Egypt), andHelisoma duryi (Florida) all had a lower degree of susceptibility. On the other hand,E. liei metacercarial infectivity to the mouse was independent of the species and size of the second intermediate host snail. Also, the infectivity of metacercariae encysted on snail mucus, in snails harbouring patent redial infections, and in clean second intermediate host snails was comparable.Metacercarial infectivity remained unchanged for at least 12 and 18 weeks, respectively, when encysted in livingB. glabrata (Puerto Rico) or when maintained in freshwater at 4°C. Metacercariae in dead decayingB. glabrata remained unharmed for one week, followed by a loss of infectivity after 4 1/2 weeks, while the infectivity of metacercariae maintained in freshwater with snail debris at 22°C declined for four weeks, followed by a loss in infectivity after five weeks.     

FLP recombinase induction of the breakage-fusion-bridge cycle and gene conversion in Saccharomyces cerevisiae

Summary A YEp chimaeric plasmid containing URA3 and SMR1 [sulfometuron methyl resistant (SM^R) allele of ILV2] as selectable markers, and the 2 m site-specific recombination FLP recognition target (FRT), was integrated at the ilv2-1 site in chromosome XIII in a cir°] haploid. Southern analysis defined two integrant structures. Structure I had URA3 distal and SMR1 proximal to FRT whereas in structure II both markers were distal to FRT. Selectable markers were stably inherited in [cir°] haploids and [cir°] diploids heterozygous for the integrant and ILV2. Approximately 14% of heterozygous [cir ⁺] diploid cells exhibited homozygotization for the distal (500 kb) ade4 marker in trans. In [cir ⁺] diploids FLP-FRT recombination resulted in the simultaneous loss of both structure II markers, whereas the structure I distal URA3 marker loss always preceded the variable loss of the proximal SMR1 marker. URA^– cells continued to segregate for loss of SMR1 until stable URA^– SM^R or URA^–SM^S cells were produced. Gene conversion was identified in stable URA^–SM^R cells that were homozygous SMR1/SMR1 but contained wild type ILV2 restriction endonuclease sites. These observations support a model based on concerted FLP-FRT action resulting from the secondary integration of native 2 m DNA followed by unequal sister chromatid exchange (USCE) within inverted FRTs. The resultant chromatid bridge resulted in a double-stand break. Fusion of the broken ends of sister chromatids generated a breakage-fusion-bridge cycle (BFBC). Repeated rounds of the BFBC resulted in proximal marker loss and the generation of additional double-strand breaks. Recombinogenic properties of the double-strand break initiated events leading to homozygotization and gene conversion.     

Cloning and integrative deletion of the RAD6 gene of Saccharomyces cerevisiae

Summary Three overlapping plasmids were isolated from a YEp24 library, which restore Rad+ functions to rad6-1 and rad6-3 mutants. Different subclones were made and shown to integrate by homologous recombination at the RAD6 site on chromosome VII, thus verifying the cloned DNA segments to be the RAD6 gene and not a suppressor. The gene resides in a 1.15 kb fragment, which restores Rad⁺ levels of resistance to U.V., MMS and -rays to both rad6-1 and rad6-3 strains. It also restores sporulation ability to rad6-1 diploids.Integrative deletion of the RAD6 gene was shown not to be completely lethal to the yeast. Our results suggest that the RAD6 gene has some cell cycle-specific function(s), probably during late S phase.     

Biased inheritance of optional insertions following mitochondrial genome recombination in the basidiomycete fungus Coprinus cinereuss

Summary Two mitochondrial genomes of Coprinus cinereus, H and J, were found to have alternative 1.23 kb insertions. Using the Neurospora crassa cytochrome oxidase-1 (co-1) gene as a probe, the J insertion site was shown to be located within the Coprinus co-1 gene, whereas the H insertion was some 2 kb distant. The insertions showed biased inheritance following mitochondrial genome recombination. Recombination between H and J genomes was detected using the mitochondrial gene mutations acu-10, which causes a cytochrome oxidase defect, and cap-1, which confers chloramphenicol resistance. Fourteen of fifteen independently derived recombinants for these two genes were shown to have both DNA insertions. In a second series of H x J crosses, intragenic recombination between different cap-1 alleles was detected. These mutations are assumed to be in the large ribosomal RNA gene some 6 kb distant from the nearest insertion site. Each of eight independently derived cap-1 ⁺ recombinants had both DNA insertions. Despite their similar size and similar behaviour following recombination the insertions do not share extensive sequence homology.