Biogenesis of mitochondria. 52

Summary The characteristics of recombination of several petite (rho ^-) mutants of S. cerevisiae that retain the -influenced region of the mitochondrial genome, identified by the markers cap1-r, ery1-r and tsr1, are described. The petites were derived from an ^– grande (rho ⁺) strain and those petites which retain all three markers show recombination properties similar to those of the ^- parental strain. However, other rho ^- mutants that retain the cap1 and ery1 loci but have lost the tsr1 locus, which is located between cap1 and ery1, show markedly different properties of mitochondrial transmission and recombination, consistent with the presence of ⁺ alleles. The association of an internal deletion between the cap1 and ery1 loci with a change in phenotype provides additional evidence for the location of between these two loci.Although the petites deleted for the tsr1 locus exhibited the recombination properties of ⁺ strains, it was not possible to transmit this characteristic to rho ⁺ recombinant cells. Experiments on the kinetics of elimination by ethidium bromide of the cap1 and eryl markers from the petites and measurements of the buoyant densities of their mtDNA species did not indicate major changes (such as selective sequence repetition) in the sequences of the mtDNAs. The possible nature of the changes in the mtDNAs of these petites is discussed in light of recent studies on the physical nature of the alleles.     

Genetic Analysis of Petite Mutants of SACCHAROMYCES CEREVISIAE : Transmissional Types

We have studied a number of petite [rho^- ] mutants of Saccharomyces cerevisiae induced in a wild-type strain of mitochondrial genotype [ome^- CHL ^R ERY^S OLI^S_1,2,3 PAR^S] by Berenil and ethidium bromide, all of which have retained two mitochondrial genetic markers, [CHL^R] and [ERY^S], but have lost all other known markers. Though stable in their ability to retain these markers in their genome, these mutants vary widely among themselves in suppressiveness and in the extent to which the markers are transmitted on crossing to a common wild-type tested strain. In appropriate crosses all of the strains examined in this study demonstrate mitochondrial polarity, and thus have also retained the [ome^-] locus in a functional form; however, five different transmissional types were obtained, several of them quite unusual, particularly among the strains originally induced by Berenil. One of the most interesting types is the one that appears to reverse the parental genotypes with [CHL^R ERY^S] predominating over [CHL^S ERY^R] in the diploid [rho+] progeny, rather than the reverse, which is characteristic of analogous crosses with [rho+] or other petites. Mutants in this class also exhibited low or no suppressiveness. Since all of the petites reported here are derived from the same wild-type parent, and so have the same nuclear background, we have interpreted the transmissional differences as being due to different intramolecular arrangements of largely common retained sequences.     

Biogenesis of mitochondria 49 identification and mapping of a new mitochondrial locus (tsr1) which maps within polar region of yeast mitochondrial genome.

Summary An enrichment procedure which facilitates the isolation of conditional respiratory-deficient mutants of Saccharomyces cerevisiae is reported. Detailed genetic analysis of one mutant which exhibits a respiratory deficient phenotype at low temperature (18°C) is also presented. The phenotype is due to a single lesion at a new locus, tsr1, located on the mitochondrial DNA. By analysis of locus retention patterns in a set of physically characterized petite strains, the tsr1 mutation has been mapped within the segment 0–5 map units on the physical map of the yeast mitochondrial genome. This segment of the mitochondrial DNA also contains the cap1 and ery1 loci and the cistron for the mitochondrial 21S rRNA. Studies of the frequencies of co-retention of markers in petite populations, and of the frequencies of recombination of markers in non-polar crosses (⁺ × ⁺), demonstrate linkage of the tsr1 locus to both the cap1 and ery1 loci. The degree of linkage indicates that tsr1 is closer to the ery1 locus. Comparison of pairwise recombination frequencies for these three markers indicate the order cap1-tsr1-ery1. The tsr1 locus lies within the segment of the mitochondrial genome which is influenced by the polarity locus , and analysis of transmission and recombination frequencies and polarities in a polar (⁺ × ^-) cross show that the behaviour of the tsr1 locus is similar to that of ery1. However striking features of this cross are that the recombination frequency between tsr1 and ery1 is comparable to that observed in non-polar crosses, and that the polarity for recombination between tsr1 and cap1 or ery1 is extremely low.     

Biogenesis of mitochondria: XLI. Physical mapping of mitochondrial genetic markers in yeast

This paper describes the physical mapping of five antibiotic resistance markers on the mitochondrial genome of Saccharomyces cerevisiae. The physical separations between markers were derived from studies involving a series of stable spontaneous petite strains which were isolated and characterized for the loss or retention of combinations of the five resistance markers. DNA-DNA hybridization using ³²P-labelled grande mitochondrial DNA was employed to determine the fraction of grande mitochondrial DNA sequences retained by each of the defined petite strains.One petite clone retaining four of the markers in a segment comprising 36% of the grande genome was then chosen as a reference petite. The sequence homology between the mitochondrial DNA of this petite and that of the other petites was measured by DNA-DNA hybridization. For each petite, the total length of its genome derived by hybridization with grande mitochondrial DNA and the fraction of the grande genome retained in common with the reference petite, together with the genetic markers retained in common, were used to position the DNA segment of each petite relative to the reference petite genome. At the same time the relative physical location of the five markers on a circular genome was established. On the basis of the grande mitochondrial genome being defined as 100 units of DNA, the positions of the markers were determined to bo as follows, measuring from one end of the reference petite genome. chloramphenicol (cap1) ~ 0 units erythromycin (ery1) 0 to 15 units oligomycin (oli1) 18 to 19 units mikamycin (mik1) 22 to 25 units paromomycin (par1) 61 to 73 unitsThe general problems of mapping mitochondrial genetic markers by hybridizations involving petite mitochondrial DNA are discussed. Two very important features of petite genomes which could invalidate the interpretation of DNA-DNA hybridization experiments between petite mitochondrial DNAs are the possible presence in the reference petite of differentially amplified DNA sequences, and/or “new” sequences which are not present in the parent grande genome. A general procedure, which overcomes errors of interpretation arising from these two features is described.     

A PIF-dependent recombinogenic signal in the mitochondrial DNA of yeast

From their recombination properties, tandem rho^- mutants of the mitochondrial genome of Saccharomyces cerevisiae were divided into two categories. In crosses between PIF-independent rho^- and rho⁺ strains, the recombination frequency is low and similar in PIF/pif and pif/pif diploids. In crosses between PIF-dependent rho^- and rho⁺ strains, the recombination frequency is stimulated 10-50 times in PIF/pif diploids and is drastically decreased in pif/pif diploids. These results suggest that a recombinogenic signal is present in the mitochondrial (mt) DNA of PIF-dependent rho^- clones. This signal is not recognized in pif mutants. Sequence analysis of a series of small (<300 bp) overlapping tandem rho^- genomes located in the ery region of the 21S rRNA gene led us to identify an essential element of this signal within a 41-bp A+T sequence exhibiting over 26 bp a perfect dyad symmetry. However the recombinogenic signal is not sequence-specific since the sequence described above does not characterize PIF-dependent rho^- clones located in the oli1 region. Our results rather suggest that the recombinogenic signal is related to the topology of rho^- DNA. Denaturated sites in the double helix or cruciform structures elicited by local negative supercoiling might be preferred sites of the initiation of recombination.     

Mitochondrial resistance to miconazole in Saccharomyces cerevisiae

Summary One mutant of mitochondrial origin resistant to miconazole has been isolated and characterized in S. cerevisiae. The mutation is linked to the locus oli1, the structural gene for subunit 9 of ATPase on mitochondrial DNA. Miconazole inhibited the mitochondrial ATPase of the wild type while the enzyme of the resistant mutant was insensitive to this effect. Levels of ATP decreased to one-third of the control in the wild type in the presence of miconazole, while they were unaffected in the mutant.Abbreviations MNNG N-methyl-N-nitrosoguanidine - Mic^s/Mic^r phenotypic sensitivity/resistance to miconazole - M ₁ ^R mitochondrial locus conferring miconazole resistance - rho⁺/rho^- grand/cytoplasmic petite - rho^o cytoplasmic petite deleted of all mitochondrial DNA - w⁺ mitochondrial locus conferring polarity of recombination     

Comparative studies of the effects of acridines and other petite inducing drugs on the mitochondrial genome of Saccharomyces cerevisiae.

Summary The effects of the acridines euflavine and proflavine on mitochondrial DNA (mtDNA) replication and mutation inSaccharomyces cerevisiae have been compared. In contrast to previous results we found that under our conditions proflavine can indeed induce high levels (>80%) of petite mutants, although six times less efficiently than euflavine. The parameters measured for mutagenesis of the mitochondrial genome and inhibition of mtDNA replication in whole cells suggest that the modes of action of euflavine and proflavine are very similar. After extended (18h) treatment of growing cells with each drug the percentage loss of mtDNA or genetic loci was almost coincidental with the extent of petite induction.It was found that proflavine is equally as effective as euflavine in inhibiting mtDNA replication in isolated mitochondria in contrast to the differential between the drugs observed in vivo. However, proflavine and euflavine inhibit cellular growth at almost the same concentrations. It is therefore proposed that there is some intracellular permeability barrier which impedes proflavine access to the mitochondrial DNA replicating system.The petites induced by euflavine (and proflavine) are characterized by there being a preferential induction ofrho ⁰ petites lacking mtDNA as opposed torho ^- petites retaining mtDNA. This is in contrast to the relative proportions of such petites induced by ethidium bromide or berenil. A scheme for the production of petites by euflavine is presented, in which euflavine inhibits the replication of mtDNA, but does not cause direct fragmentation of mtDNA (unlike ethidium bromide and berenil). The proposed scheme explains the production of the high frequency ofrho ^o cells, as well as therho ^- cells induced by euflavine. The scheme also accounts for previous observations that euflavine only mutants growing cultures, and that the buds, but not mother cells, become petite.     

Temperature-sensitive respiratory-deficient mitochondrial mutations: Isolation and genetic mapping

Summary In order to find new genetic loci and functions on the yeast mitochondrial DNA, especially mutations affecting the mitochondrial protein synthesis apparatus, temperature sensitive mutants have been isolated after MnCl₂ mutagenesis and mitochondrial and nuclear mutants classified according to their pattern of recombination with three rho^- tester strains.Eighteen cold- and heat-sensitive respiratory deficient mitochondrial mutants have been isolated and localized on the mitochondrial genome by deletion mapping using 113 rho^- strains. Eight of them appear to represent new loci, among which some are probably mutations of the tRNA and rRNA genes.     

Maintenance and Integrity of the Mitochondrial Genome: a Plethora of Nuclear Genes in the Budding Yeast

Instability of the mitochondrial genome (mtDNA) is a general problem from yeasts to humans. However, its genetic control is not well documented except in the yeast Saccharomyces cerevisiae. From the discovery, 50 years ago, of the petite mutants by Ephrussi and his coworkers, it has been shown that more than 100 nuclear genes directly or indirectly influence the fate of the rho⁺ mtDNA. It is not surprising that mutations in genes involved in mtDNA metabolism (replication, repair, and recombination) can cause a complete loss of mtDNA (rho⁰ petites) and/or lead to truncated forms (rho⁻) of this genome. However, most loss-of-function mutations which increase yeast mtDNA instability act indirectly: they lie in genes controlling functions as diverse as mitochondrial translation, ATP synthase, iron homeostasis, fatty acid metabolism, mitochondrial morphology, and so on. In a few cases it has been shown that gene overexpression increases the levels of petite mutants. Mutations in other genes are lethal in the absence of a functional mtDNA and thus convert this petite-positive yeast into a petite-negative form: petite cells cannot be recovered in these genetic contexts. Most of the data are explained if one assumes that the maintenance of the rho⁺ genome depends on a centromere-like structure dispensable for the maintenance of rho⁻ mtDNA and/or the function of mitochondrially encoded ATP synthase subunits, especially ATP6. In fact, the real challenge for the next 50 years will be to assemble the pieces of this puzzle by using yeast and to use complementary models, especially in strict aerobes.     

The genetic map of the mitochondrial genome in yeast

Summary An approach for the screening of mit ^- mutants, the isolation and preliminary classification of a series of such mutants is reported. Loss and retention of 8 mit ^- and 6 drug ^r markers in mitDNA was analyzed in populations of rho^- clones derived from four yeast strains. The populations studied constitute a representative fraction of the rho^- petites formed during growth at 35° C under the influence of mutation tsp-25 which is in common to the four strains. The majority of the rho^- clones retained several of the markers studied. Depending on the marker regarded retention frequencies between 15% (oxi3) and 45% (oli1, cob) were observed. Loss of one and retention of the other of a pair of markers was determined in all rho^- clones of the four populations. The frequencies of marker separation by rho^- deletion thus obtained are assumed to reflect the distance between markers on the mitochondrial genome: the higher the frequency of separation the longer the distance between two markers. Based on these frequencies a unique order of markers on a circular map was determined. Positions of markers on a scale from 0 to 100 were found to be: cap/ery (0) — olil (16) — cob1-1354 (21) — ana101 (22) — cob2-1625 (24) — oli2 (35) — pho1 (40) — oxi3-2501 (44) — oxi3-3771 (47) — par (65) — oxi2 (79) — oxil (87) tms8 (93) —cap (100). The relevance of this map as to the faithful representation of the topology of gene loci on mitDNA is discussed. Correlation of retention frequencies of markers to their map positions reveals a pronounced polarity: mitDNA segments carrying the cob-oli1 segment prevail whereas segments retaining oxi3 are the least frequent.     

Biogenesis of mitochondria 47

Summary This paper consolidates and refines the physical map of genetic loci previously established in our laboratory, by molecular analysis of seven genetically characterized new petites (deletion mutants of mtDNA). A modified DNA-DNA hybridization procedure employing filters simultaneously bound with mtDNA from two different petites has been used to measure the overlaps in mtDNA sequences between the different petite mutants.Thus, by analysis of three new petites carrying the antibiotic-resistance loci, ery1, cap1 and par1 on their mitochondrial genomes, it has now been possible to improve our estimation of the maximum distance between the cap1 and ery1 loci. The cap1, ery1 loci, and the 21S ribosomal RNA gene have now been mapped within 5 units in the same region (map position 0 to 5 units). Similarly, by analysis of four new petites carrying the O _II and/or par1 loci on their mtDNAs, the map position of the O _II locus is also more accurately determined within 2 units in a region (map position 34 to 36 units) between the par1 and ana1 loci. The positions of other loci including par1, the 15S ribosomal RNA gene, and some mit ^- loci are also discussed.We have thus extended our library of genetically and molecularly defined petite mutants, resulting in a set of petites having overlapping regions distributed throughout the entire wild-type mitochondrial genome, consistent with the idea that yeast mtDNA is physically circular.     

A genetic and biochemical analysis of petite mutations in yeast

A series of spontaneous cytoplasmic petite mutants was isolated from a grande strain of Saccharomyces cerevisiae doubly marked with the cytoplasmically inherited determinants to erythromycin and oligomycin resistance. The petites were characterized with regard to the genetic stability of these antibiotic resistance markers and to their degree of suppressivity. No relation was found between the genetic instability of a petite mutant and the degree of suppressivity exhibited by that mutant. Three petites of 19.4%, 57.4% and 90.4% suppressivity were selected and their mitochondrial DNA characterized with regard to molecular weight, buoyant density in analytical cesium chloride density gradients, and the percentage of the total cellular DNA represented by the mitochondrial DNA. From these results it appears that the molecular weight of the mitochondrial DNA of the petite strains examined is the same as that shown by the parental grande strain, regardless of the degree of suppressivity exhibited.     

Biogenesis of mitochondria 44

Summary A comparative study of eight independently isolated mitochondrial oligomycin resistant mutants obtained from three laboratories show a variety of phenotypes based on cross resistance to venturicidin and sensitivity to low temperature. Analysis of recombination between pairs of markers indicate the existence of at least three genetic classes; class A, cross resistant to venturicidin and including the mutations O III, [oli1-r], [OLG1-R], [tso-r]; class B, mutations O _I, [oli17-r], [OLG2-R]; and class C, the mutation O _II. The recombination data is consistent with mutations of each class residing in three separate genes, although mutations of class A and B show very close linkage.Recombination in non-polar crosses has demonstrated that markers of all three classes are linked to the mik1 locus in the configuration (AB)-mik1-C. The mapping of this segment with respect to other markers of the mitochondrial genome and the order of classes A and B was established by analyses of co-retention frequencies of markers in primary petite isolates as well as by analysis of marker overlap of genetically and physically defined petite genomes. The unambiguous order ery1-A-B-mik1-C-par was obtained. DNA-DNA hybridization studies using mtDNA isolated from selected petites confirms this map and estimates the physical separation of markers. A reasonable correlation exists in this region of the genome between distances estimated physically by hybridization and genetically by frequency of recombination in non-polar crosses.It is postulated that the oligomycin-mikamycin linkage group represents a cluster of genes involved in determining a number of mitochondrial membrane proteins associated with the mitochondrial ATPase and respiratory complex III.This work was supported by the Australian Research Grants Committee, Project D65/15930     

Cytoduction as a new tool in studying the cytoplasmic heredity in yeast

Summary When crossing the haploid cells of genetically marked yeast strains we observed the appearance of both normal diploid zygotes and haploid nuclear cytoplasmic hybrids. The latter had the nuclear markers of one and the cytoplasmic marker (rho⁺) of the other parent. The autonomous cytoplasmic factor transfer was termed as cytoduction. Cytoduction is supposed to be the abortive form of yeast cell mating. Only about 1% of cytoductants is usually observed.Cytoduction can be used as a simple test on cytoplasmic determination of some characters. We observed the transfer into cytoductant cells of not only rho⁺ marker but of resistance factors to antibiotics (erythromycin, neomycin) and killer factor as well. Cytoduction can be applied towards constructing strains having the identical nucleus genotype with mitochondria and other cytoplasmic factors of different origin.In crossing strains with doubly marked mitochondria recombination of mitochondrial markers in cytoductant haploid cells was observed, the pattern of which was similar to that of mitochondrial recombination in normal zygotes.     

Mitochondrial and nuclear myxothiazol resistance in Saccharomyces cerevisiae

Summary Mitochondrial and nuclear mutants resistant to myxothiazol were isolated and characterized. The mitochondrial mutants could be assigned to two loci, myx1 and myx2, by allelism tests. The two loci map in the box region, the split gene coding for apocytochrome b. Locus myx1 maps in the first exon (box4/5) whereas myx2 maps in the last exon (box6). The nuclear mutants could be divided into three groups: two groups of recessive mutations and one of dominant mutations. Respiration of isolated mitochondria from mitochondrial mutants is resistant to myxothiazol. These studies support the conclusion that myxothiazol is an inhibitor of the respiratory chain of yeast mitochondria. The site of action of myxothiazol is mitochondrial cytochrome b.Abbreviations box mosaic gene coding for apocytochrome b - cyt b cytochrome b - MIC minimum inhibitory concentration - MNNG N-methyl-N'-nitro-N-nitrosoguanidine - Myx ^R/Myx ^S allelte forms of a locus conferring myxothiazol resistance - myx1, myx2 mitochondrial loci conferring myxothiazol resistance - rho ⁺/rho ^– grande/cytoplasmic petite - rho ⁰ cytoplasmic petite that is deleted of all mitochondrial DNA     

Restriction enzyme analysis of mitochondrial DNAs of petite mutants of yeast: Classification of petites, and deletion mapping of mitochondrial genes

Summary We have analyzed the restriction digest patterns of the mitochondrial DNA from 41 cytoplasmic petite strains of Saccharomyces cerevisiae, that have been extensively characterized with respect to genetic markers. Each mitochondrial DNA was digested with seven restriction endonucleases (EcoRI, HpaI, HindIII, BamHI, HhaI, SalI, and PstI) which together make 41 cuts in grande mitochondrial DNA and for which we have derived fragment maps. The petite mitochondrial DNAs were also analyzed with HpaII, HaeIII, and AluI, each of which makes more than 80 cleavages in grande mitochondrial DNA. On the basis of the restriction patterns observed (i.e., only one fragment migrating differently from grande for a single deletion, and more than one for multiple deletions) and by comparing petite and grande mitochondrial DNA restriction maps, the petite clones could be classified into two main groups: (1) petites representing a single deletion of grande mitochondrial DNA and (2) petites containing multiple deletions of the grande mitochondrial DNA resulting in rearranged sequences. Single deletion petites may retain a large portion of the grande mitochondrial genome or may be of low kinetic cimplexity. Many petites which are scored as single continuous deletions by genetic criteria were later demonstrated to be internally deleted by restriction endonuclease analysis. Heterogeneous sequences, manifested by the presence of sub-stoichiometric amounts of some restriction fragments, may accompany the single or multiple deletions. Single deletions with heterogeneous sequences remain useful for mapping if the low concentration sequences represent a subset of the stoichiometric bands. Using a group of petites which retain single continuous regions of the grande mitochondrial DNA, we have physically mapped antibiotic resistance and mit^- markers to regions of the grande restriction map as follows: C (99.3-1.4 map units)-OXI-1 (2.5-15.7)-OXI-2 (18.5-25)-P (28.1-34.2)-OXI-3 (32.2-61.2)-O_II (60-62)-COB (64.6-80.8)-O_I (80.4-85.7)-E (95-98.9).Supported by USPHS Training Grant 5-T01-GM-00090-19.Supported by USPHS Training Grant T32-GM-07197.The Franklin McLean Memorial Research Institute is operated by the University of Chicago for the U.S. Energy Research and Development Administration under Contract EY-76-C-02-0069.     

Elevated Levels of Petite Formation in Strains of SACCHAROMYCES CEREVISIAE Restored to Respiratory Competence. II. Organization of Mitochondrial Genomes in Strains Having High and Moderate Frequencies of Petite Mutant Formation

Restriction enzyme analysis of aberrant mtDNA molecules in restored strains of Saccharomyces cerevisiae that displays an elevated level of petite formation has shown the occurrence of novel junction fragments and nonstoichiometric amounts for some unaltered bands. Five aberrant mitochondrial genomes from high-frequency petite-forming (hfp) strains (greater than 60% petites per generation) contain like-oriented duplications and single copy regions. High-frequency petite formation is postulated to arise from increased intramolecular recombination between duplicated segments. Mitochondrial DNA structures in two other hfp strains cannot be easily interpreted and might arise from intramolecular recombination. Mitochondria DNA from moderate-frequency petite-forming (mfp) strains (5-16% petites per generation) contains inverted duplications in two cases. The elevated petite formation is postulated to arise from homologous recombination between directly repeated sequences. In mtDNA from one mfp strain, deletion end-points have been shown to overlap. Such deletion endpoint overlap is postulated to be required for the maintenance of the tandem duplication in hfp strains. Two regions of the wild-type mtDNA (between cyb and oli2 and between SrRNA and oxi2) appear to be dispensable for mitochondrial function.     

Elevated Levels of Petite Formation in Strains of SACCHAROMYCES CEREVISIAE Restored to Respiratory Competence. I. Association of Both High and Moderate Frequencies of Petite Mutant Formation with the Presence of Aberrant Mitochondrial DNA

When recently arisen spontaneous petite mutants of Saccharomyces cerevisiae are crossed, respiratory competent diploids can be recovered. Such restored strains can be divided into two groups having sectored or unsectored colony morphology, the former being due to an elevated level of spontaneous petite mutation. On the basis of petite frequency, the sectored strains can be subdivided into those with a moderate frequency (5–16%) and those with a high frequency (>60%) of petite formation. Each of the three categories of restored strains can be found on crossing two petites, suggesting either that the parental mutants contain a heterogeneous population of deleted mtDNAs at the time of mating or that different interactions can occur between the defective molecules. Restriction endonuclease analysis of mtDNA from restored strains that have a wild-type petite frequency showed that they had recovered a wild-type mtDNA fragmentation pattern. Conversely, all examined cultures from both categories of sectored strains contained aberrant mitochondrial genomes that were perpetuated without change over at least 200 generations. In addition, sectored colony siblings can have different aberrant mtDNAs. The finding that two sectored, restored strains from different crosses have identical but aberrant mtDNAs provides evidence for preferred deletion sites from the mitochondrial genome. Although it appears that mtDNAs from sectored strains invariably contain duplications, there is no apparent correlation between the size of the duplication and spontaneous petite frequency.     

Biogenesis of mitochondria. 30

Summary Mitchondrial gene recombination in S. cerevisiae was investigated using four combinations of mitochondrial markers: [oli1-r ery1-r], [oli1-r spi2-r], [oli1-r spi3-r] and [oli1-r spi4-r] in cis bifactorial crosses to [oli-s ery-s spi-s] strains. A number of sensitive strains including representatives of both mating types and of diverse origin were used. The crosses were analysed for frequency and polarity of mitochondrial gene recombination as well as the frequency of transmission into the diploid progeny of individual mitochondrial determinants.The results show that the polarity of recombination varied markedly in crosses between a single pair of mitochondrial markers and many unrelated sensitive strains. For example, one series of crosses included polarity values of 1.7,0.34,0.081, and 0.021. Furthermore, there was also considerable variability in frequency of recombination and frequency of transmission of individual markers and these frequencies were not correlated in many cases with polarity values. However, in certain other crosses involving different marker combinations there was a correlation between extreme polarity, high recombination frequency and high transmission frequency of one marker. The results are not compatible with polarity being determined by a simple mitochondrial sex factor and suggest that several different interactions are operating which might include nuclear phenomena.