Photodynamic mutation induction and inactivation in Serratia phages kappa by methylene blue and light

Summary Extracellular treatment with methylene blue and light (MB+Li) caused inactivation and induction of mutations in Serratiaphage . Inactivation is partly due to lesions in the DNA as concluded from marker rescue (CR) experiments where some 67% of UV-and 40% of MB+Li-induced damage could be reactivated. About 32% of the lethal lesions after MB+Li could be reactivated by host cell reactivation (HCR) whereas induced mutations were not influenced. Photoreactivation (PR) of lethal damage and mutations was not detected. Postincubation of MB+Li-inactivated free phage in saline or dye solution at 30°C caused additional inactivation but mutations again were not influenced. The 3 types of plaquetype-mutations scored (clear=c+1, narrow=e, and pale=b) were induced by MB+Li according to concave light-dose curves indicating induction by different average hit numbers (2.3, 1.6, and 3.0). Increase of the fraction of plaquetype-mutants due to selection was excluded. Increase of the mutant types g, however, was due to selection only. The proportion of the 3 mutant-types induced by MB+Li was different (at all light doses) from the ones induced by the guanine-specific mutagens EMS or low pH; thus the MB+Li-induced lesions leading to mutation are due to different changes of guanine moieties or do not have their origin in guanine destruction at all.

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Die Experimente wurden vom ersten Autor als Teil seiner Doktorarbeit bei der Naturwissenschaftlichen Fakultät der Johann Wolfgang Goethe-Universität, Frankfurt a. M. (1966) unter der Leitung des zweiten Autors durchgeführt.     

Toxicity of common biocides used in aquaculture to embryos and larvae of Brachymystax tsinlingensis Li

Brachymystax tsinlingensis Li is a threatened fish species endemic to China. With the problems of environmental factors and seeding breeding diseases, it is important to further improve the efficiency of seeding breeding and the basis of resource protection. This study investigated the acute toxicity of copper, zinc and methylene blue (MB) on hatching, survival, morphology, heart rate (HR) and stress behaviour of B. tsinlingensis. Eggs (diameter: 3.86 ± 0.07 mm, weight: 0.032 ± 0.004 g) of B. tsinlingensis were selected randomly from artificial propagation and developed from eye-pigmentation-stage embryos to yolk-sac stage larvae (length: 12.40 ± 0.02 mm, weight: 0.03 ± 0.001 g) and exposed to different concentrations of Cu, Zn and MB for 144 h in a series of semi-static toxicity tests. The acute toxicity tests indicated that the 96-h median lethal concentration (LC₅₀) values of the embryos and larvae were 1.71 and 0.22 mg l⁻¹ for copper and 2.57 and 2.72 mg l⁻¹ for zinc, respectively, whereas the MB LC₅₀ after 144-h exposure for embryos and larvae were 67.88 and 17.81 mg l⁻¹, respectively. The safe concentrations of copper, zinc and MB were 0.17, 0.77 and 6.79 mg l⁻¹ for embryos and 0.03, 0.03 and 1.78 mg l⁻¹ for larvae, respectively. Copper, zinc and MB treatments with concentrations greater than 1.60, 2.00 and 60.00 mg l⁻¹, respectively, led to a significantly low hatching rate and significantly high embryo mortality (P < 0.05), and copper and MB treatments with concentrations greater than 0.2 and 20 mg l⁻¹ led to significantly high larvae mortality (P < 0.05). Exposure to copper, zinc and MB resulted in developmental defects, including spinal curvature, tail deformity, vascular system anomalies and discolouration. Moreover, copper exposure significantly reduced the HR of larvae (P < 0.05). The embryos exhibited an obvious change in behaviour, converting from the normal behaviour of emerging from the membrane head first to emerging tail first, with probabilities of 34.82%, 14.81% and 49.07% under copper, zinc and MB treatments, respectively. The results demonstrated that the sensitivity of yolk-sac larvae to copper and MB was significantly higher than that of embryos (P < 0.05) and that B. tsinlingensis embryos or larvae might be more resistant to copper, zinc and MB than other members of the Salmonidae family, which benefits their resource protection and restoration.     

Lethal(1)aberrant immune response mutations leading to melanotic tumor formation in Drosophila melanogaster

Using P element-mediated mutagenesis we have isolated 20 X-linked lethal mutations, representing at least 14 complementation groups, which exhibit melanotic tumor phenotypes. We present the systematic analysis of this interesting group of lethal mutations that were selected for their visible melanotic or immune response. The lethal and melanotic tumor phenotypes of each lethal(1) aberrant immune response (air) mutation are pleiotropic effects of single genetic lesions. Lethality occurs throughout the larval and early pupal periods of development and larval development is extended in some air mutants. The air mutant lethal syndromes include abnormalities associated with the brain, haematopoietic organs, gut, salivary glands, ring glands, and imaginal discs. Additional characterization of the melanotic tumor mutations Tum^l and tu(1)Sz^ts have indicated that the melanotic tumor phenotype is similar to that observed in the air mutants. These studies have led to the proposal that two distinct classes of melanotic tumor mutations exist. Class 1 includes mutants in which melanotic tumors result from “autoimmune responses” or the response of an apparently normal immune system to the presence of abnormal target tissues. The Class 2 mutants display obvious defects in the haematopoietic organs or haemocytes, manifested as overgrowth, and the resulting aberrant immune system behavior may contribute to melanotic tumor formation.     

The dependence of HNO2 mutagenesis in phage T4 on ligase and the lack of dependence of 2AP mutagenesis on repair functions

Summary A temperature sensitive ligase allele of phage T4 reduced or eliminated HNO₂ induced reversion of am mutants. Since at the temperatures used, the ligase mutant is defective in the repair of some types of lethal lesions (i.e., UV, MMS and EMS induced lesions) these results indicate that HNO₂ mutagenesis may occur through a ligase dependent repair pathway. In contrast, 2AP induced mutation was not inhibited by mutants defective in the gene 30 ligase or in genes 32, 39, 41, 44, 45, 46, 47, 49, 52, 56, 58–61 and v. This indicates that 2AP mutagenesis probably does not depend on a repair pathway in phage T4.     

Induction of mutation in phage T4 by extracellular treatment with methylene blue and visible light

Summary Photodynamic inactivation with methylene blue and light (MB+Li) of maximally sensitized phages T4 and T4rII nearly followed single-hit kinetics. Not so mutation induction, which in one phage (T4rII N₁₇) tested showed multi-hit kinetics. This difference and the fact that T4rII phages with a GC base pair at the site of the rII-mutation showed no enhanced backmutation when compared to rII phages with an AT base pair or to sign mutants suggests that there is no guanine specificity for MB+Li-induced mutation in phage T4.     

Mutants with continuous microcycle conidiation in the filamentous fungus Fusarium solani f. sp. pisi

Summary A method of repeated mutation induction and subsequent selection was used to obtain mutants with microcycle conidiation, mc, in Fusarium solani f. sp. pisi. Wild-type, wt, Fusarium has a filamentous (hyphal) growth in a three-stage, conidium—hypha—conidiophore (phialid) vegetative life cycle. The first major mutation induced (by means of the mutagen MNNG) changed the wild-type strain 1–3^- from producing both macro- and microconidia to the mutant strain 19-P2^-, producing only microconidia but preserving the three-stage life cycle. When strain 19-P2^- was exposed to a similar mutagenic treatment mutants without the hyphal stage were obtained. In these mutants the microconidia germinate to form phialids directly, which in turn produce new conidia, thus accomplishing the life cycle as a two-stage, conidium-conidiophore, microcycle. The mc mutants were stabilized after reisolation through back-crossing to the wt of complementary mating type. Mc growth of the mutants is continuous under stable conditions, proceeding uninterrupted to the stationary stage in liquid as well as on solid medium. Mc colonies on solid medium are small and soft, well suited to replication by means of velvet or paper. Large numbers of mature, synchronized conidia are easily obtained from stationary-stage submerged cultures. Sequential mutations are of interest for studies of morphogenetic evolution. The ahyphal mutants make this group of eukaryotes well suited for genetic engineering and biotechnology.     

Proteome analysis on lethal effect of <Emphasis Type="Italic">l</Emphasis><Subscript>2</Subscript> in the sex-linked balanced lethal strains of silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

The sex-linked balanced lethal (SLBL) strains of silkworm serve as an effective system for sex-control in silkworm. To gain comprehensive insight into the effect of one sex-linked balanced lethal gene l ₂, comparative proteomic analysis was carried out between the survival embryos ( W ^{+ l₁} Z^{l₁ + l₂} )\left( {W^{ + l_1 } Z^{l_1 + l_2 } } \right) and lethal embryos ( W ^{+ l₁} Z ^{+ l₁ l₂} )\left( {W^{ + l_1 } Z^{ + l_1 l_2 } } \right) before the lethal stage. The lethal stage of l ₂ was confirmed by observing the typical dead embryo morphology. The two genotype embryos before lethal stage were distinguished using polymorphic simple sequence repeats (SSR) markers closely linked to l ₂ on the sex chromosome. Finally, 11 differentially expressed protein spots were successfully identified by MALDI-TOF/TOF mass spectrometry (MS). Among them, only 1 protein identified as heat shock protein 20.4 (HSP20.4) was up-regulated in the lethal embryos, while the other 10 were down-regulated. The up-regulation of HSP20.4 suggests that there may be abnormal polypeptides produced in the lethal embryos. The gene ontology (GO) annotation indicated those down-regulated proteins are involved in important biological processes including embryo development, nucleoside metabolism, tRNA splicing, translation and protein folding. The biological pathway analysis showed that those down-regulated proteins are mainly involved in spindle assemblage and morphogenesis. Based on our results, we suggest that the l ₂ may be the mutant expressing abnormal polypeptides. Its expression has a negative effect on mitosis and morphogenesis processes. The death of the embryos may be caused by the accumulation of abnormal polypeptides and the handicap of cell proliferation and morphogenesis.     

Alteration of cadmium-induced mutational spectrum by catalase depletion in Chinese hamster ovary-K1 cells

Previously, we have demonstrated that cadmium acetate significantly induces hprt mutation frequency in Chinese hamster ovary (CHO)-K1 and that 3-amino-1,2,4-triazole (3AT), a catalase inhibitor, potentiates the mutagenicity of cadmium [Chem. Res. Toxicol. 9 (1996) 1360–1367]. In this study, we investigate the role of intracellular peroxide in the molecular nature of mutations induced by cadmium. Using 2′,7′-dichlorofluorescin diacetate and fluorescence spectrophotometry, we have shown that cadmium dose-dependently increased the amounts of intracellular peroxide and the levels were significantly enhanced by 3AT. Furthermore, we have characterized and compared the hprt mutation spectra in 6-thioguanine-resistant mutants derived from CHO-K1 cells exposed to 4 μM of cadmium acetate for 4 h in the absence and presence of 3AT. The mutation frequency induced by cadmium and cadmium plus 3AT was 11- and 16-fold higher than that observed in untreated populations (2.2×10⁻⁶), respectively. A total of 40 and 51 independent hprt mutants were isolated from cadmium and cadmium plus 3AT treatments for mRNA-polymerase chain reaction (PCR), genomic DNA-PCR and DNA sequencing analyses. 3AT co-administration significantly enhanced the frequency of deletions induced by cadmium. Cadmium induced more transversions than transitions. In contrast, 3AT co-administration increased the frequency of GC→AT transitions and decreased the frequencies of TA→AT and TA→GC transversions. Together, the results suggest that intracellular catalase is important to prevent the formation of oxidative DNA damage as well as deletions and GC→AT transitions upon cadmium exposure.     

Induction of haploid callus and embryogenesis in in vitro cultured anthers of mulberry (Morus indica)

Anthers of Morus indica L., with microspores at the uninucleate stage were cultured; and the influence of temperature and kinetin pretreatment on induction of androgenic calluses was examined. The effects of various pretreatments revealed that 24 h cold pretreatment increased the percentage of cultures inducing callus. First microspore division was observed after 16 to 20 days of culture. Th anthers split and developed embryogenic calluses on MB medium supplemented with NAA (0.5 mg l^–1 and BA (1.0 mg l^–1)) using 8% sucrose. Rhizogenesis was induced on medium supplemented with NAA and BA (each 0.5 mg l^–1) with reduced myo-inositol (75 mg l^–1). Cytological study of induced roots confirmed the haploid nature of calluses. Different type of embryos were initiated upon transfer of calluses to medium supplemented with NAA, BA (each 0.5 mg l^–1), 2,4-d (1.0 mg l^–1) and PVP (600 mg l^–1). These embryoids further developed roots on removal of 2,4-d from the medium and developed precociously without developing cotyledons and formed elongated shoots.Abbreviations BA 6 benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin: Acetic acid: Alcohol - GA₃ gibberellic acid - IBA indole-3-butyric acid - MB modifed Bourgin (Qian et al., 1982) - NAA 1-naphthalene acetic acid - PVP polyvinylpyrrolidone - RFS-135 rainfed selection 135 - SE standard error     

Cold sensitive mutant of Escherichia coli with altered RNA polymerase

Summary Cold sensitive (cs) mutants of E. coli unable to grow at 26° C were isolated. Three of about 200 mutants tested have RNA polymerase which is about 5 times less active at low temperatures than the wild type RNA polymerase. One of such mutants cs 1 was studied in more detail. The mutation affects core enzyme and is closely linked to rif-r mutations. By transduction analysis the following order of markers was established: argH — rif-r — cs 1.     

Lethal and mutational effects of solar and UV radiation on Staphylococcus aureus

Strains of Staphylococcus aureus, an opportunistic pathogen commonly found on human skin, were exposed to sunlight and UV C radiation, and the lethal and mutational effects measured. Sunlight killed cells with an inactivation constant of 3×10^-5 per joule per square metre; UV C was much more lethal, giving an inactivation constant of approximately 0.1 per joule per square metre. Some strains tested showed a sensitivity to sunlight that was dependent on the growth phase of the cells, exponentially growing cells showing a greater sensitivity. Mutational effects of irradiation were measured by the appearance of mutants sensitive to methicillin following irradiation of a multiresistant strain. Mutants appeared at a frequency of 10^-3; this high frequency of mutation in the region of the mec gene has also been observed when multiresistant strains are subjected to nutritional or thermal stress. Mutants showed the same chromosomal alteration (seen in pulse-field gel electrophoresis of Smal-digested DNA) whether induced by solar or UV C irradiation.     

The proportion of mutants in bacterial cultures

The proportion of mutants in a growing culture of organisms will depend upon (a) the rate at which the wild cells produce them (with or without growth), (b) the back mutation rate, and (c) the growth rates of the wild and mutant cells. If the mutation rate without growth and the back mutation rate are neglected, the growth of a mutant is expressed by See PDF for Equation and the ratio of the mutant to wild by See PDF for Equation in which λ = mutation frequency rate constant, "mutation rate," A = growth rate constant of wild cells W, B = growth rate constant of mutant cells M. If the term [B – (1 – 2λ)A] is positive, the proportion of mutants increases continuously. If it is negative, the proportion of mutants reaches a constant value See PDF for Equation If mutation is assumed to occur without growth at the rate C, then the corresponding equations are (11), (12), and (14). See PDF for Equation If (B + C – A) is negative and t = ∞, See PDF for Equation If C << A, See PDF for Equation     

Altered control of glutamate dehydrogenases in ornithine utilization mutants of Pseudomonas aeruginosa

Two classes of ornithine-nonutilizing (oru) mutants of Pseudomonas aeruginosa PAO were investigated. Strains carrying the oru-310 mutation were entirely unable to grow on l-ornithine as the only carbon and nitrogen source and were affected in the assimilation of a variety of nitrogen sources (e.g., amino acids, nitrate). The oru-310 mutation caused changes in the regulation of the catabolic NAD-dependent glutamate dehydrogenase; this enzyme was no longer inducible by glutamate but instead could be induced by ammonia. The oru-310 locus was cotransducible with car-9 and tolA in the 10 min region of the chromosome. An oru-314 mutant was severely handicapped in ornithine medium but could grow when a good carbon source was added; the mutant also showed pleiotropic growth effects related to nitrogen metabolism. The oru-314 mutation affected the regulation of the anabolic NADP-dependent glutamate dehydrogenase, which was no longer repressed by glutamate but showed normal derepression in the presence of ammonia. The oru-314 locus was mapped by transduction near met-9011 at 55 min. Both oru mutants could grow on l-glutamate, l-proline, or l-ornithine amended with 2-oxoglutarate, albeit slowly. We speculate that insufficient 2-oxoglutarate concentrations might account, at least in part, for the Oru^- phenotype of the mutants.     

Mutations affecting the mitochondrial genes encoding the cytochrome oxidase subunit I and apocytochrome b of Chlamydomonas reinhardtii

Mitochondrial mutants of the green alga Chlamydomonas reinhardtii that are inactivated in the cytochrome pathway of respiration have previously been isolated. Despite the fact that the alternative oxidase pathway is still active the mutants have lost the capacity to grow heterotrophically (dark + acetate) and display reduced growth under mixotrophic conditions (light + acetate). In crosses between wild-type and mutant cells, the meiotic progeny only inherit the character transmitted by the mt ^– parent, which indicates that the mutations are located in the 15.8 kb linear mitochondrial genome. Two new mutants (dum-18 and dum-19) have now been isolated and characterized genetically, biochemically and at the molecular level. In addition, two previously isolated mutants (dum-11 and dum-15) were characterized in more detail. dum-11 contains two types of deleted mitochondrial DNA molecules: 15.1 kb monomers lacking the subterminal part of the genome, downstream of codon 147 of the apocytochrome b (COB) gene, and dimers resulting from head-to-head fusion of asymmetrically deleted monomers (15.1 and 9.5 kb DNA molecules, respectively). As in the wild type, the three other mutants contain only 15.8 kb mitochondrial DNA molecules. dum-15 is mutated at codon 140 of the COB gene, a serine (TCT) being changed into a tyrosine (TAC). dum-18 and dum-19 both inactivate cytochrome c oxidase, as a result of frameshift mutations (addition or deletion of 1 bp) at codons 145 and 152, respectively, of the COX1 gene encoding subunit I of cytochrome c oxidase. In a total of ten respiratory deficient mitochondrial mutants characterized thus far, only mutations located in COB or COXI have been isolated. The possibility that the inactivation of the other mitochondrial genes is lethal for the cells is discussed.     

Toxicity and mutagenicity of X-rays and [I]dUrd or [H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

The reactivity of cytochrome c with soft ligands

The spectral changes caused by binding soft ligands to the cytochrome c iron and their correlation to ligand affinities support the hypothesis that the iron—methionine sulfur bond of this heme protein is enhanced by delocalization of the metal l₂, electrons into the empty 3d orbitals of the ligand atom. These findings also explain the unique spectrum of cytochrome c in the far red.     

Multiple biochemical effects of a series of X-ray induced mutations at the albino locus in the mouse

Further studies of the four radiation-induced lethal albino mutations causing glucose 6-phosphatase deficiency in the mouse have shown that there is also a deficiency of tyrosine aminotransferase in the newborn albino mutants. These two enzymes can be induced in fetal and newborn heterozygous or homozygous normal littermate controls by injection of glucagon or dibutyryl cyclic AMP, but not in the albino mutants. Microsomal NADH-cytochrome c reductase was found to be increased in the albino mutants. The multiple biochemical abnormalities in the albino mutants, in addition to the lack of gene dosage effect in the heterozygotes, suggest the involvement of genes other than the structural genes for particular enzymes. When a new radiation-induced lethal albino mutation was tested against the four original alleles, complementation resulted, and double heterozygotes were found to be viable, with normal enzyme levels.Supported by grants from the National Institutes of Health, 5 R01 AM 11448-05, 5R01 HD-00193-17, 5T01 GM00110-09, GM19100, and by a grant from the American Cancer Society, VC-64N.     

Characterisation of new symbiotic Medicago truncatula (Gaertn.) mutants, and phenotypic or genotypic complementary information on previously described mutants

From a pool of Medicago truncatula mutants—obtained by gamma-irradiation or ethyl methanesulfonate mutagenesis—impaired in symbiosis with the N-fixing bacterium Sinorhizobium meliloti, new mutants are described and genetically analysed, and for already reported mutants, complementary data are given on their phenotypic and genetic analysis. Phenotypic data relate to nodulation and mycorrhizal phenotypes. Among the five new mutants, three were classified as [Nod⁺ Fix^– Myc⁺] and the mutations were ascribed to two loci, Mtsym20 (TRV43, TRV54) and Mtsym21 (TRV49). For the two other new mutants, one was classified as [Nod^–/+ Myc⁺] with a mutation ascribed to gene Mtsym15 (TRV48), and the other as [Nod^– Myc^-/+] with a mutation ascribed to gene Mtsym16 (TRV58). Genetic analysis of three previously described mutants has shown that [Nod^–/+ Myc⁺] TR74 mutant can be ascribed to gene Mtsym14, and that [Nod^–/+ Myc^–/+] TR89 and TRV9 mutants are ascribed to gene Mtsym2 (dmi2). Using a detailed analysis of mycorrhizal phenotype, we have observed a delayed typical arbuscular mycorrhizal formation on some mutants that present thick lens-shaped appressoria. This phenotype was called [Myc^–/+] and mutants TR25, TR26, TR89, TRV9, P1 and Y6 were reclassified as [Myc^–/+]. Mutant P1 was reclassified as [Nod^–/+] because of a late nodulation observed on roots of this mutant.     

Biochemical,genetic and molecular characterization of new respiratory-deficient mutants inChlamydomonas reinhardtii

Eight respiratory-deficient mutants ofChlamydomonas reinhardtii have been isolated after mutagenic treatment with acriflavine or ethidium bromide. They are characterized by their inability to grow or their very reduced growth under heterotrophic conditions. One mutation (Class III) is of nuclear origin whereas the seven remaining mutants (Classes I and II) display a predominantly paternalmt ^- inheritance, typical of mutations residing in the mitochondrial DNA. Biochemical analysis has shown that all mutants are deficient in the cyanide-sensitive cytochrome pathway of the respiration whereas the alternative pathway is still functional. Measurements of complexes II + III (antimycin-sensitive succinate-cytochromec oxido-reductase) and complex IV (cytochromec oxidase) activities allowed to conclude that six mutations have to be localized in the mitochondrial apocytochromeb (COB) gene, one in the mitochondrial cytochrome oxidase subunit I (COI) gene and one in a nuclear gene encoding a component of the cytochrome oxidase complex. By using specific probes, we have moreover demonstrated that five mutants (Class II mutants) contain mitochondrial DNA molecules deleted in the terminal end containing the COB gene and the telomeric region; they also possess dimeric molecules resulting from end-to-end junctions of deleted monomers. The two other mitochondrial mutants (Class I) have no detectable gross alteration. Class I and Class II mutants can also be distinguished by the pattern of transmission of the mutation in crosses.Anin vivo staining test has been developed to identify rapidly the mutants impaired in cyanide-sensitive respiration.     

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.