A Transforming Marker That Produces Merodiploids with High Efficiency and Stable Transformants with Low Efficiency in Streptococcus

A mutation (ery-r8) conferring a high level of resistance to erythromycin in the Challis strain of Streptoccus sanguis can be transferred to wild-type erythromycin-sensitive recipients via single molecules of donor DNA. The transformants thus produced are of two types: (1) cells slightly more resistant to erythromycin than wild-type and capable of segregating (at a frequency of 2 X 10(-4)/bacterium/generation) either wild-type or highly-resistant cells like the original donor type; (2) cells phenotypically and genotypically identical to the original donor type. The unstable diploids (ery-r8/+) occur with a frequency equivalent to that obtained with high-efficiency (HE) markers, whereas the stable donor-type (ery-r8) transformants occur with about five hundred times lower frequency. Penetration of the wild-type recipient by more than one molecule of DNA bearing the ery-r8 marker increases by as much as seven times the incidence of stable transformants. UV-irradiation of molecules bearing the ery-r8 marker diminishes their ability to cooperate in producing a stable transformant, although the UV sensitivity of stable transformant production by a single DNA molecule is not different from that of diploid production. Hence, stable transformants do not appear to be produced by a process typical of low efficiency (LE) markers, which are generally highly sensitive to ultraviolet irradiation. Moreover, stable ery-r8 transformants are produced with equally low frequencies in strains of S. pneumoniae that discriminate (hex+) and fail to discriminate (hex--) between HE and LE markers. We postulate that all transformations by the ery-r8 marker result in ery-r8/+ diploids, and that segregation results in the infrequent stable transformants of the original donor type. This hypothesis is supported by the observations that rifampin treatment of ery-r8/+ populations increases the frequency of segregation and similar treatment of wild-type recipients under-going transformation by the ery-r8 marker increases the frequency of stable transformants.--In producing the ery-r8/+ transformant the r8 allele is integrated close to the site of its wild-type homolog, since single molecules of DNA from this transformant can be shown to carry both alleles. Segregation of either the ery-r8 or + allele is not detectably enhanced by acridine orange or thymidine deprivation.--The ery-r8 marker occurs close to a site of mutation (ery-r2) which confers erythromycin resistance upon ribosomes. When the r2 and r8 markers are jointly transferred, ery-r2-r8/+ genomes are produced in which the r2 marker is stably integrated but the r8 marker is unstably adjoined to its wild-type homolog. Thus, the duplicated region can be quite short. When the ery-r8 marker is stably integrated, the region of the marker is refractory to subsequent transformation. Markers with properties like ery-r8 are not particularly rare, being found with a frequency of about 4% among spontaneous mutations to erythromycin resistance.     

A comparison of the genetic and physical size of the streptomycin resistance locus in Pneumococcus.

Summary Polyacrylamide gel electrophoresis of the 30S ribosomal proteins derived from six streptomycin resistant strains indicates that each mutation alters the same ribosomal protein (str-r protein). Preliminary data utilizing SDS gels indicates that thestr-r protein has a molecular weight between 10,000 and 20,000 daltons. No significant differences could be detected between the molecular weight of thestr-r protein when it is derived either from a sensitive or from a resistant strain, including those derived from strains carrying multisite mutations of different genetic size. We have estimated the size of the multisitestr-r mutations to be less than 30 base pairs. Two factor crosses withstr-r markers in the trans position demonstrate recombination frequencies expected of closely linked, intragenic markers although cotransfer frequencies, of these same markers from the cis position, are very low. It is concluded that the cotransfer frequencies represent a marker effect and possible explanations are discussed. A reinterpretation of the genetic map of the pneumococcalstr-r locus is presented.Most of the material is taken from a thesis (ALR) submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Graduate Studies, of the State University of New York, Upstate Medical Center     

Inheritance of citrus nematode resistance and its linkage with molecular markers

Eleven RAPD markers linked to a gene region conferring resistance to citrus nematodes in an intergen-eric backcross family were identified. Two sequence- characterized amplified region markers linked to a citrus tristeza virus resistance gene and one selected resistance gene candidate marker were evaluated for their association with citrus nematode resistance. A nematode-susceptible citrus hybrid, LB6-2 [Clementine mandarin (Citrus reticulata)×Hamlin orange (C. sinensis)], was crossed with the citrus nematode-resistant hybrid Swingle citrumelo (C. paradisi×Poncirus trifoliata) to produce 62 hybrids that were reproduced by rooted cuttings. The plants were grown in a greenhouse and inoculated with nematodes isolated from infected field trees. The hybrids segregated widely for this trait in a continuous distribution, suggesting possible polygenic control of the resistance. Bulked segregant analysis was used to identify markers associated with resistance by bulking DNA samples from individuals at the phenotypic distribution extremes. Linkage relationships were established by the inheritance of the markers in the entire population. A single major gene region that contributes to nematode resistance was identified. The resistance was inherited in this backcross family from the grandparent Poncirus trifoliata as a single dominant gene. QTL analysis revealed that 53.6% of the phenotypic variance was explained by this major gene region. The existence of other resistance-associated loci was suggested by the continuous phenotypic distribution and the fact that some moderately susceptible hybrids possessed the resistance-linked markers. The markers may be useful in citrus rootstock breeding programs if it can be demonstrated that they are valid in other genetic backgrounds. Received: 4 May 1999 / Accepted: 21 September 1999     

Plastid transformants of tomato selected using mutations affecting ribosome structure

Tomato plastid transformants were obtained using two vectors containing cloned plastid DNA of either Nicotiana tabacum or Solanum nigrum and including point mutations conferring resistance to spectinomycin and streptomycin. Transformants were recovered after PEG-mediated direct DNA uptake into protoplasts, followed by selection on spectinomycin-containing medium. Sixteen lines contained the point mutation, as confirmed by mapping restriction enzyme sites. One line obtained with each vector was analysed in more detail, in comparison with a spontaneous spectinomycin-resistant mutant. Integration of the cloned Solanum or Nicotiana plastid DNA, by multiple recombination events, into the tomato plastome was confirmed by sequence analysis of the targeted region of plastid DNA in the inverted repeat region. Maternal inheritance of spectinomycin and streptomycin resistances or sensitivity in seedlings also confirmed the transplastomic status of the two transformants. The results demonstrate the efficacy in tomato of a selection strategy which avoids the integration of a dominant bacterial antibiotic resistance gene.     

Mapping of chloroplast mutations conferring resistance to antibiotics in Chlamydomonas: Evidence for a novel site of streptomycin resistance in the small subunit rRNA

Summary A major obstacle to out understanding of the mechanisms governing the inheritance, recombination and segregation of chloroplast genes in Chlamydomonas is that the majority of antibiotic resistance mutations that have been used to gain insights into such mechanisms have not been physically localized on the chloroplast genome. We report here the physical mapping of two chloroplast antibiotic resistance mutations: one conferring cross-resistance to erythromycin and spiramycin in Chlamydomonas moewusii (er-nM1) and the other conferring resistance to streptomycin in the interfertile species C. eugametos (sr-2). The er-nM1 mutation results from a C to G transversion at a well-known site of macrolide resistance within the peptidyl transferase loop region of the large subunit rRNA gene. This locus, designated rib-2 in yeast mitochondrial DNA, corresponds to residue C-2611 in the 23 S rRNA of Escherichia coli. The sr-2 locus maps within the small subunit (SSU) rRNA gene at a site that has not been described previously. The mutation results from an A to C transversion at a position equivalent to residue A-523 in the E. coli 16 S rRNA. Although this region of the E. coli SSU rRNA has no binding affinity for streptomycin, it binds to ribosomal protein S4, a protein that has long been associated with the response of bacterial cells to this antibiotic. We propose that the sr-2 mutation indirectly affects the nearest streptomycin binding site through an altered interaction between a ribosomal protein and the SSU rRNA.     

Streptomycin and lincomycin resistances are selective plastid markers in cultured Nicotiana cells

Summary Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.     

RAPD markers linked to eastern filbert blight resistance in<Emphasis Type="Italic"> Corylus avellana</Emphasis>

A total of 1,110 decamer primers were screened for RAPD markers linked to a dominant allele in hazelnut (Corylus avellana) that confers resistance to eastern filbert blight caused by Anisogramma anomala. Twenty RAPD markers linked in coupling, and five markers linked in repulsion, were found. A seedling population was used to construct a linkage map of the region flanking the resistance locus. The map spans 46.6 cM, with 14 markers on one side of the resistance locus and eight on the other side. Eleven markers showed less than 3% recombination with resistance, including three that showed no recombination. Seven of these 11 markers are sufficiently robust to allow their use in marker-assisted selection. These include AA12₈₅₀ which shows no recombination, and six markers on one side of the resistance locus: 173₅₀₀, 152₈₀₀, 122₈₂₅, 275₁₁₃₀, H19₆₅₀ and O16₁₂₅₀. Marker 268₅₈₀, which flanks the resistance locus on the other side, is also suitable for use in marker-assisted selection, but shows 5.8% recombination with resistance. Other markers are less suitable for marker-assisted selection because of sensitivity to changes in primer or MgCl₂ concentration, or the long time required for electrophoresis to separate bands of similar size. The 16 markers closest to the resistance locus were cloned and sequenced. The W07₃₆₅ marker, which showed no recombination with the resistance locus but is difficult to score, includes a CT microsatellite repeat. The sequence information will allow the design of SCAR primers and eventual map-based cloning of the resistance allele.Communicated by D.B. Neale     

Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot

Multidrug-resistant strains of Mycobacterium tuberculosis have resulted in several recent outbreaks. Recognition of drug resistance is important both for treatment and to prevent further transmission. Here we use molecular biology techniques to study the basis of streptomycin resistance in single and multi-drug-resistant M. tuberculosis. We demonstrate that streptomycin resistance is associated with mutations implicated in ribosomal resistance. The mutations found either lead to amino acid changes in ribosomal protein SI2 or alter the primary structure of the 16S rRNA. The 16S rRNA region mutated perturbs a pseudoknot structure in a region which has been linked to ribosomal S12 protein.     

Allele-specific CAPS markers based on point mutations in resistance alleles at the pvr1 locus encoding eIF4E in Capsicum

Marker-assisted selection has been widely implemented in crop breeding and can be especially useful in cases where the traits of interest show recessive or polygenic inheritance and/or are difficult or impossible to select directly. Most indirect selection is based on DNA polymorphism linked to the target trait, resulting in error when the polymorphism recombines away from the mutation responsible for the trait and/or when the linkage between the mutation and the polymorphism is not conserved in all relevant genetic backgrounds. In this paper, we report the generation and use of molecular markers that define loci for selection using cleaved amplified polymorphic sequences (CAPS). These CAPS markers are based on nucleotide polymorphisms in the resistance gene that are perfectly correlated with disease resistance, the trait of interest. As a consequence, the possibility that the marker will not be linked to the trait in all backgrounds or that the marker will recombine away from the trait is eliminated. We have generated CAPS markers for three recessive viral resistance alleles used widely in pepper breeding, pvr1, pvr1 ¹, and pvr1 ². These markers are based on single nucleotide polymorphisms (SNPs) within the coding region of the pvr1 locus encoding an eIF4E homolog on chromosome 3. These three markers define a system of indirect selection for potyvirus resistance in Capsicum based on genomic sequence. We demonstrate the utility of this marker system using commercially significant germplasm representing two Capsicum species. Application of these markers to Capsicum improvement is discussed.     

Genetic analysis and molecular cloning of the Escherichia coli ruv gene

Summary The genetic organisation of the ruv gene, a component of the SOS system for DNA repair and recombination in Escherichia coli, was investigated. New point mutations as well as insertions and deletions were generated using transposon Tn10 inserted in eda as a linked marker for site specific mutagenesis, or directly as a mutagen. The mutations were ordered with respect to one another and previously isolated ruv alleles by means of transductional crosses. The direction of chromosome mobilization from ruv:: Mud(Ap^R lac)strains carrying F42lac ⁺ established that ruv is transcribed in a counterclockwise direction. Recombinant phages able to restore UV resistance to ruv mutants were identified, and the ruv ⁺ region was subcloned into a low copy number plasmid. The ruv ⁺ plasmid was able to correct the extreme radiation sensitivity and recombination deficiency of ruv recBC sbcB strains.     

Genetic characterization and mapping of major gene resistance to potato leafroll virus in <Emphasis Type="Italic">Solanum</Emphasis><Emphasis Type="Italic">tuberosum</Emphasis> ssp. <Emphasis Type="Italic">andigena</Emphasis>

Major gene inheritance of resistance to Potato leafroll virus (PLRV) was demonstrated in a parthenogenic population derived from the highly resistant tetraploid andigena landrace, LOP-868. This major gene or chromosome region seems to control a single mechanism for resistance to infection and virus accumulation in this source. About 149 dihaploid lines segregated in a ratio of 107 resistant to 32 susceptible, fitting the expected ratio for inheritance of a duplex gene under random chromatid segregation. A tetraploid AFLP map was constructed using as reference the ultra high density (UHD) map. All AFLP markers associated with PLRV resistance mapped to the same linkage group. Map position was confirmed by analysis of previously-mapped SSR markers. Rl _adg is located on the upper arm of chromosome V, at 1 cM from its most closely linked AFLP marker, E35M48.192. This marker will be used to develop allele-specific primers or a pair of flanking PCR-based markers for their use in marker assisted selection.     

SCAR,RAPD and RFLP markers linked to a dominant gene (Are) conferring resistance to anthracnose in common bean

Anthracnose, caused by the fungusColletotrichum lindemuthianum, is a severe disease of common bean (Phaseolus vulgaris L.) controlled, in Europe, by a single dominant gene,Are. Four pairs of near-isogenic lines (NILs) were constructed, in which theAre gene was introgressed into different genetic backgrounds. These pairs of NILs were used to search for DNA markers linked to the resistance gene. Nine molecular markers, five RAPDs and four RFLPs, were found to discriminate between the resistant and the susceptible members of these NILs. A backcross progeny of 120 individuals was analysed to map these markers in relation to theAre locus. Five out of the nine markers were shown to be linked to theAre gene within a distance of 12.0 cM. The most tightly linked, a RAPD marker, was used to generate a pair of primers that specifically amplify this RAPD (sequence characterized amplified region, SCAR).The research was supported by the CNRS and the Ministère Français de l'Education Nationale     

Molecular mechanism of genetic recombination in bacterial transformation

Summary B. subtilis cells auxotrophic for two linked markers (ind-his, ind-tyr, his-tyr) have been transformed by means of DNA preparations obtained by hybridization of wild type DNA with the DNA of a strain auxotrophic for one of the linked markers. It was established that hybridization does not increase the transforming activity of DNA for the heterozygous marker. A genetic analysis of the progeny of cells transformed by hybrid or wild type DNA was performed. On the basis of the data obtained a model of genetic recombination in transformation is proved. According to this model both strands of the donor DNA interact independently with the chromosome, and either strand can be incorporated into the cell genome with equal probability. According to the estimate made on the basis of this hypothesis, the probability of integration of a single DNA strand carrying a particular genetic marker is 8%.With 3 Figures in the Text     

Development of leucine-rich repeat polymorphism, amplified fragment length polymorphism, and sequence characterized amplified region markers to the Cronartium ribicola resistance gene Cr2 in western white pine ( Pinus monticola )

White pine blister rust (WPBR), caused by Cronartium ribicola, is a devastating disease in Pinus monticola and other five-needle pines. Pyramiding a major resistance gene (Cr2) with other resistance genes is an important component of integrated strategies to control WPBR in P. monticola. To facilitate this strategy, the objective of the present study was to identify leucine-rich repeat (LRR) polymorphisms, amplified fragment length polymorphisms (AFLPs), and sequence characterized amplified region (SCAR) markers linked to the western white pine Cr2 (BSA) gene for precise gene mapping. Bulked segregant analysis and haploid segregation analysis allowed the identification of 11 LRR polymorphisms and five AFLP markers in the Cr2 linkage. The closest LRR markers were 0.53 Kosambi cM from Cr2 at either end. After marker cloning and sequencing, AFLP marker EacccMccgat-365 and random polymorphic DNA marker U570–843 were converted successfully into SCAR markers. For a potential application in marker-assisted selection (MAS), these two SCAR markers were verified in two western white pine families. This study represents the first report of LRR-related DNA markers linked to C. ribicola resistance in five-needle pines. These findings may help further candidate gene identification for disease resistance in a conifer species.     

An rpsL Cassette, Janus, for Gene Replacement through Negative Selection in Streptococcus pneumoniae

Natural genetic transformation offers a direct route by which synthetic gene constructs can be placed into the single circular chromosome of Streptococcus pneumoniae. However, the lack of a general negative-selection marker has hampered the introduction of constructs that do not confer a selectable phenotype. A 1.3-kb cassette was constructed comprising a kanamycin (Kn) resistance marker (kan) and a counterselectable rpsL⁺ marker. The cassette conferred dominant streptomycin (Sm) sensitivity in an Sm-resistant background in S. pneumoniae. It was demonstrated that it could be used in a two-step transformation procedure to place DNA of arbitrary sequence at a chosen target site. The first transformation into an Sm-resistant strain used the cassette to tag a target gene on the chromosome by homologous recombination while conferring Kn resistance but Sm sensitivity on the recombinant. Replacement of the cassette by an arbitrary segment of DNA during a second transformation restored Sm resistance (and Kn sensitivity), allowing construction of silent mutations and deletions or other gene replacements which lack a selectable phenotype. It was also shown that gene conversion occurred between the two rpsL alleles in a process that depended on recA and that was susceptible to correction by mismatch repair.     

Gene-directed mutagenesis on the chromosome of Bacillus subtilis 168

Summary We have devised a method whereby any mutagenized cloned DNA from Bacillus subtilis can be reinserted at the original site on the B. subtilis chromosome. The procedure depends on the accuracy and high frequency of homologous recombination between the B. subtilis chromosome and the DNA taken up by the cell. The method makes use of two drug resistance selection markers (the chloramphenicol resistance gene and the neomycin resistance gene) and a marker gene which functions as a catalyst. The utility of the method has been demonstrated using leuB and pro of B. subtilis as target gene and catalyst, respectively, and mutations such as leuB: : cat, leuB ^–, and pro: : neo constructed in vitro on the cloned DNA fragments. Transformation in sequential steps as (leuB ⁺ pro⁺)(leuB: : cat pro ⁺) (leuB ^– pro: : neo)(leuB ^– pro ⁺) resulted in a leuB ^– single mutant without affecting other regions of the B. subtilis chromosome (gene-directed mutagenesis). We also demonstrate that other single mutations such as metD ^– and pro ^–, as well as the double mutation leuB ^– pro ^– can be introduced by the same procedure. In principle, true isogenies with multiple mutations can be constructed by the method described in this paper. Furthermore, the procedure should be generally applicable to any organisms in which homologous recombination is proficient.     

Competence Mutant of Haemophilus influenzae with Abnormal Ratios of Marker Efficiencies in Transformation

In studies of competence-deficient mutants of Haemophilus influenzae which absorb deoxyribonucleic acid (DNA) but fail to produce transformants, it was observed that in some mutants the residual transforming activity for different markers varied widely, i.e., produced a ratio effect. One of these mutants, com⁻⁵⁶, was studied intensively to determine the cause of the residual efficiency of transformation and the reason for the ratio effect. The residual frequency of transformation was higher for markers considered single-site mutations (like naladixic acid resistance), whereas the least efficient markers tested were those conferring resistance to high levels of streptomycin or novobiocin which are more complex than single-site mutations. Measurement of frequencies of cotransformation indicated that overall genetic linkage was reduced. Transfection was fairly efficient with phage S2 DNA, but not prophage DNA. Donor marker activity could be detected in transformed cell lysates, but not linked to recipient markers in recombinant molecules. Sucrose gradient analysis of such lysates revealed that donor material was associated with recipient DNA in at least normal quantities, but lacked detectable genetic activity. Material from donor DNA labeled with heavy isotopes was incorporated into recipient chromosomal fragments having a density indistinguishable from normal density, unlike the hybrid density recombinant material found in normal cells. No excessive solubilization or nicking of unincorporated donor was detected. It is postulated that this strain contains a hyperactive nuclease, which reduces the effective size of the input DNA during the integration process.     

A detailed linkage map around an apple scab resistance gene demonstrates that two disease resistance classes both carry the V f gene

A detailed genetic map has been constructed in apple (Malus x domestica Borkh.) in the region of the v _f gene. This gene confers resistance to the apple scab fungus Venturia inaequalis (Cooke) Wint. Linkage data on four RAPD (random amplified polymorphic DNA) markers and the isoenzyme marker PGM-1, previously reported to be linked to the v _f gene, are integrated using two populations segregating for resistance to apple scab. Two new RAPD markers linked to v _f (identified by bulked segregant analysis) and a third marker previously reported as being present in several cultivars containing v _f are also placed on the map. The map around v _f now contains eight genetic markers spread over approximately 28 cM, with markers on both sides of the resistance gene. The study indicates that RAPD markers in the region of crab apple DNA introgressed with resistance are often transportable between apple clones carrying resistance from the same source. Analysis of co-segregation of the resistance classes 3A (weakly resistant) and 3B (weakly susceptible) with the linked set of genetic markers demonstrates that progeny of both classes carry the resistance gene.This work was supported in part by grants from the New Zealand Foundation for Research Science and Technology (FoRST) Programme 94-HRT-07-366 and ENZA New Zealand (International)     

Next generation synthetic vectors for transformation of the plastid genome of higher plants

Plastid transformation vectors are E. coli plasmids carrying a plastid marker gene for selection, adjacent cloning sites and flanking plastid DNA to target insertions in the plastid genome by homologous recombination. We report here on a family of next generation plastid vectors carrying synthetic DNA vector arms targeting insertions in the rbcL-accD intergenic region of the tobacco (Nicotiana tabacum) plastid genome. The pSS22 plasmid carries only synthetic vector arms from which the undesirable restriction sites have been removed by point mutations. The pSS24 vector carries a c-Myc tagged spectinomycin resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. The synthetic vectors will enable direct manipulation of passenger genes in the transformation vector targeting insertions in the rbcL-accD intergenic region that contains many commonly used restriction sites. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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