Mechanism of gene conversion in Ascobolus immersus. II. The relationships between the genetic alterations in b 1 or b 2 mutants and their conversion spectrum

Summary In order to establish a relationship between the type of genetic alteration occurring in the mutant and the conversion spectrum which is associated with it, an attempt was made to characterize the genetic alterations in a sample of five spore colour mutants by specific reversion.All the mutants revert by back-mutation. Reversion of one of them is possible by mutation in external suppressor gene: at least two of the external suppressors behave like super-suppressors. Reversion of two other mutants by intragenic second-site mutation and study of the intragenic suppressors indicate that they are of the frameshift type. One of the frameshift mutants (ICR₁₇₀ induced) reverts by two alkylating agents suggesting that it originates from base(s) addition. The inhability of ICR₁₇₀ to induce reversion of this mutant suggests the preferential occurrence of base addition in ICR₁₇₀ mutagenesis. Conversly the other frameshift mutant (induced by ethyl methanesulfonate) reverts strongly by ICR₁₇₀. Thus it is concluded that it originates from deletion.These two frameshift mutants and all the ICR₁₇₀ induced mutants give no or very few asci with postmeiotic segregation and either an excess of conversion towards the mutant type allele (addition type mutant) or towards the wild type allele (deletion type mutant).Evidence supports the hypothesis that mutants which give many asci with postmeiotic segregation originate from substitution.These data imply differential recognition of non-pairing and mispairing of bases and in the case of non-pairing, that the direction of conversion is determined by the non-pairing itself.     

The intergradation, genetic interchangeability and interpretation of gene conversion spectrum types

In the Pasadena strains of Ascobolus immersus, the gene conversion propperties of 29 induced (nine UV, nine NG, and 11 ICR-170) and nine spontaneous white-ascospore mutations have been studied. Each mutant was crossed to three types of derived wild-type strains; single mutants often gave very different conversion results in the three types of crosses, with any or all of the following changes in: percentage with post-meiotic segregation among aberrant-ratio asci; percentage with conversion to wild type among aberrant-ratio asci; and in total conversion frequency. - These results are compared with those of Leblon (1972 a, b) from Ascobolus immersus and Yu-Sun, Wickramaratne and Whitehouse (1977) from Sordaria brevicollis. It is shown that conversion spectrum types are not necessarily distinct, but can completely intergrade, on the criteria of both post-meiotic segregation frequency and direction of correction. Genetic differences between strains in the present work resulted in much interchangeability of spectrum types for the same mutation in different crosses; e.g., from type C in one cross to type B/D type in another cross, although the mutation is presumably of the same molecular type (addition or deletion frame shift, or base substitution) in each cross. These changes of conversion properties for a given mutation in different crosses mean that previous interpretations of spectrum types in terms of specific conversion properties for various molecular types of mutation are inapplicable, or inadequate on their own, to explain the present data. Other factors, such as heterozygous cryptic mutations or conversion control genes, are probably involved. Because of asymmetric hybrid DNA formation, correction properties may differ from observed conversion properties.     

Genome‐wide survey of artificial mutations induced by ethyl methanesulfonate and gamma rays in tomato

Genome‐wide mutations induced by ethyl methanesulfonate (EMS) and gamma irradiation in the tomato Micro‐Tom genome were identified by a whole‐genome shotgun sequencing analysis to estimate the spectrum and distribution of whole‐genome DNA mutations and the frequency of deleterious mutations. A total of ~370 Gb of paired‐end reads for four EMS‐induced mutants and three gamma‐ray‐irradiated lines as well as a wild‐type line were obtained by next‐generation sequencing technology. Using bioinformatics analyses, we identified 5920 induced single nucleotide variations and insertion/deletion (indel) mutations. The predominant mutations in the EMS mutants were C/G to T/A transitions, while in the gamma‐ray mutants, C/G to T/A transitions, A/T to T/A transversions, A/T to G/C transitions and deletion mutations were equally common. Biases in the base composition flanking mutations differed between the mutagenesis types. Regarding the effects of the mutations on gene function, >90% of the mutations were located in intergenic regions, and only 0.2% were deleterious. In addition, we detected 1 140 687 spontaneous single nucleotide polymorphisms and indel polymorphisms in wild‐type Micro‐Tom lines. We also found copy number variation, deletions and insertions of chromosomal segments in both the mutant and wild‐type lines. The results provide helpful information not only for mutation research, but also for mutant screening methodology with reverse‐genetic approaches.     

The kinetics and feedback inhibition of cytidine 5′-triphosphate synthetase in wild-type and mutant Chinese hamster cells

The kinetics and cytidine 5-triphosphate (CTP) feedback inhibition of CTP synthetase in wild-type and four mutants of Chinese hamster V79 cells have been studied. The enzymes of the wild type and three of the four mutants exhibited positive cooperativity with the substrate uridine 5-triphosphate (UTP). Three of the mutants had K _{m app} and S ₅₀ valuves distinctly greater than those of the wild type, while the fourth mutant had values similar to those of the wild type. all four mutants exhibited resistance to CTP feedback inhibition, while the wild type was sensitive to such inhibition. It is postulated that a single mutational event in each mutant had caused a concomitant change of the enzyme in its binding both to the substrate UTP and to the end-product CTP.This work was supported by Grant GM 20608 from the U.S. Public Health Service.     

Caffeine as a comutagen for ethylmethanesulfonate in strains of phage T4

Summary The mutation frequency of DNA polymerase mutants of phage T4 treated with ethyl methanesulfonate (EMS) then incubated in the presence and absence of caffeine was studied using an rII reversion system. The DNA polymerase mutation is shown to be antimutagenic for EMS induction of reversions which occur by a GC to AT transition. Caffeine acts as a comutagen for the induction by EMS of mutant phages and produces a significant increase in the frequency of reversions from rII to r⁺. Caffeine is slightly mutagenic for the phage strain carrying the wild type polymerase and inhibits the action of the 35 exonuclease function of T₄ DNA polymerase as measured in vitro. These findings suggest that caffeine acts by directly influencing nucleotide selection or the editing function of the DNA polymerase.     

A Dual Strategy to Cope with High Light in Chlamydomonas reinhardtii

Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition–deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II.     

Analysis of cytoplasmic membrane from wild type and mutant Paracoccus denitrificans containing excess nitrate reductase protein and cytochrome b

Cytoplasmic membranes were isolated from wild type and mutants strain M-1 of Paracoccus denitrificans grown with low aeration to promote synthesis of nitrate reductase protein and cytochrome b. The presence of 10-100-fold excess of nitrate reductase in the wild type or the corresponding enzymically inactive protein in the mutant did not significantly affect respiratory oxidase activities with NADH, succinate or TMPD-ascorbate as electron donor. A cytochrome b-nitrate reductase complex was resolved by isoelectric focussing of Triton X-100 solubilized membranes from the wild type grown with azide and from the mutant, whereas the enzyme complex from nitrate-grown wild type was not resolved from cytochrome c. Preparations from azideinduced wild type or from the mutant could be a suitable source of the cytochrome b associated with nitrate reductase for more detailed studies.Non standard abbreviations IEF isoelectric focussing - TMPD N, N, N, N-tetramethylphenylenediamine - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

大豆疫霉菌的EMS化学诱变

以甲基磺酸乙酯(ethylmethane sulfonate,EMS)为诱变剂,通过其对大豆疫霉菌Phytophthora sojae休止孢萌发的影响,确定化学诱变条件。通过收集单卵孢子,建立了包含640个单卵孢子系的突变体库,其中约有50%的诱变菌系在培养性状和菌落形态方面发生了明显变化,菌落形态多样,表现出较紧密或松散,近圆形或不规则;气生菌丝减少,生长速度较慢或快;在卵孢子产量方面,8.13%的菌系有增加,20.41%的菌系减少,27.82%的菌系极少或者没有卵孢子产生,43.64%的菌系卵孢子产量类似野生型。以质膜氢离子泵蛋白基因PsPMA1(plasma membrane H+-ATPase1)为对象,通过TILLING技术,从320个大豆疫霉菌突变体中获得9个突变体,进一步确认了EMS对大豆疫霉菌的诱变效果,并且估算EMS对大豆疫霉菌的诱变频率至多每115kb发生一个核苷酸变异。新构建的突变体库为开展大豆疫霉病菌的功能基因组研究奠定了遗传材料基础。     

Molecular analyses of genetically stable mutants of the maize Adh1 gene

Summary 10 genetically stable mutants of the alcohol dehydrogenase-1 gene in maize were examined for ADH1-mRNA size and abundance, large changes in genomic restriction fragments, and intragenic recombination levels. Eight of the mutants were induced with ethyl methanesulfonate (EMS), four of which are CRM^–; the other two mutants followed treatment with ionizing radiation. In general, EMS and ionizing radiation induce point lesions in Adh1 that, with few exceptions, do not alter the abundance or size of ADH1-RNA, or alter the gross genomic restriction map. One EMS mutant produced both normalsized and larger than normal ADH1-mRNA. The larger ADH1-mRNA appears to result from improper termination. Another mutant behaved as expected of an intragenic deletion in recombination tests but we found no abnormalities by restriction site mapping.     

Morphology and ultrastructure of 11 barley shrunken endosperm mutants

Summary Eleven Na-azide induced barley shrunken endosperm mutants expressing xenia (sex) were characterized genetically and histologically. All mutants have reduced kernel size with kernel weights ranging from 11 to 57% of the wild type. With one exception, the mutant phenotypes are ascribable to single recessive mutant alleles, giving rise to a ratio of 31 of normal and shrunken kernels on heterozygous plants. One mutant (B10), also monofactorially inherited, shows a gene dosage dependent pattern of expression in the endosperm. Among the 8 mutants tested for allelism, no allelic mutant genes were discovered. By means of translocation mapping, the mutant gene of B10 was localized to the short arm of chromosome 7, and that of B9 to the short arm of chromosome 1. Based on microscopy studies, the mutant kernel phenotypes fall into three classes, viz. mutants with both endosperm and embryo affected and with a non-viable embryo, mutants with both endosperm and embryo affected and with a viable embryo giving rise to plants with a clearly mutant phenotype, and finally mutants with only the endosperm affected and with a normal embryo giving rise to plants with normal phenotype. The mutant collection covers mutations in genes participating in all of the developmental phases of the endosperm, i.e. the passage from syncytial to the cellular endosperm, total lack of aleurone cell formation and disturbance in the pattern of aleurone cell formation. In the starchy endosperm, varying degrees of cell differentiation occur, ranging from slight deviations from wild type to complete loss of starchy endosperm traits. In the embryo, blocks in the major developmental phases are represented in the mutant collection, including arrest at the proembryo stage, continued cell divisions but no differentiation, and embryos deviating only slightly from the wild type.     

An efficient plating system for rapid isolation of mutants from plant cell suspensions

Summary A plating system for cell suspensions of soybean, SB-1, (Glycine max L. cv. Mandarin) and Datura innoxia D.I. (Mill) was developed using feeder cells. The characteristics of the system are: a) the efficiency of plating (EOP) is high (0.5–0.6), b) over a range of 10–300 plated clumps the EOP is constant, c) the growth rate of plated cells resembles that of suspension cultures (generation time 24 hr.). Clumps with few or with many cells have similar plating efficiencies.Employing the plating system, a mutant resistant to 8 azaguanine (8AG) was isolated from SB-1 in 7 days and purified and tested within an additional 3 weeks. Feeder plates were used to selectively re-isolate 8 AG resistant and maltose utilizing mutants from a 1000-fold excess of wild type cells.The plating technique also can be utilized to isolate auxotrophic mutants since free amino acids are not produced by the feeder suspension. Other applications of this plating technique are discussed.Abbreviations 8AG 8 Azaguanine - 6TG 6 Thioguanine - EMS Ethyl methanesulfonate - EOP Efficiency of plating - HGPRT Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8)     

Non-Locus-Specific Polygenes Giving Responses to Selection for Gene Conversion Frequencies in Ascobolus Immersus

Selection for higher and lower meiotic conversion frequencies was investigated in the fungus Ascobolus immersus. Strains carrying the same known gene conversion control factors, which have major effects on conversion frequencies at their specific target locus, sometimes gave significant differences in conversion frequency. Selection for high or low conversion frequencies at the w1-78 site was practiced for five generations, giving significant responses in both directions. These responses were due to polygenes, or genes of minor effect, not to new conversion control factors of major effect. Crosses of selected strains to strains with other mutations showed that the genes' effects were not specific to w1-78, but could affect conversion frequencies of another mutation, w1-3C1, at that locus and of two other loci, w-BHj and w9, which are unlinked to w1 or to each other. The proportional changes in gene conversion frequency due to selection varied according to the locus and site involved and according to the conversion control factor alleles present. There were differences of >/=277% in conversion frequency between ``high' and ``low' strains. Selection for conversion frequency had little effect on other features of conversion, such as the frequency of postmeiotic segregation or the relative frequencies of conversion to mutant or wild type.     

Combining next‐generation sequencing and progeny testing for rapid identification of induced recessive and dominant mutations in maize M2 individuals

Molecular identification of mutant alleles responsible for certain phenotypic alterations is a central goal of genetic analyses. In this study we describe a rapid procedure suitable for the identification of induced recessive and dominant mutations applied to two Zea mays mutants expressing a dwarf and a pale green phenotype, respectively, which were obtained through pollen ethyl methanesulfonate (EMS) mutagenesis. First, without prior backcrossing, induced mutations (single nucleotide polymorphisms, SNPs) segregating in a (M₂) family derived from a heterozygous (M₁) parent were identified using whole‐genome shotgun (WGS) sequencing of a small number of (M₂) individuals with mutant and wild‐type phenotypes. Second, the state of zygosity of the mutation causing the phenotype was determined for each sequenced individual by phenotypic segregation analysis of the self‐pollinated (M₃) offspring. Finally, we filtered for segregating EMS‐induced SNPs whose state of zygosity matched the determined state of zygosity of the mutant locus in each sequenced (M₂) individuals. Through this procedure, combining sequencing of individuals and Mendelian inheritance, three and four SNPs in linkage passed our zygosity filter for the homozygous dwarf and heterozygous pale green mutation, respectively. The dwarf mutation was found to be allelic to the an1 locus and caused by an insertion in the largest exon of the AN1 gene. The pale green mutation affected the nuclear W2 gene and was caused by a non‐synonymous amino acid exchange in encoded chloroplast DNA polymerase with a predicted deleterious effect. This coincided with lower cpDNA levels in pale green plants.     

Mutagen specificity in the induction of karyotic versus cytoplasmic respiratory deficient mutants in yeast by nitrous acid and alkylating nitrosamides

Summary In the haploid eukaryotic organism Saccharomyces cerevisiae the induction of cytoplasmic and genic (karyotic) RD mutants was studied, using nitrous acid, nitrosomethylurethane (NMU) and nitrosoimidazolidone (NIL).The cytoplasmic or genic origin of the induced RD mutants was determined by prescreening in complementation tests with ^– and wild type tester strains. Among the mutants of all three agents we could thus score the incidence of three RD mutant types: genic, suppressive and cytoplasmic (both primary and secondary). The final identification of the cytoplasmic type was only possible through tetrad analysis, performed in the cases of HNO₂ and NMU.A distinct difference in cytoplasmic versus genic mutagen specificity was observed between HNO₂ and NMU. HNO₂ was unable to induce cytoplasmic RD mutants but it proved to be highly efficient in the induction of genic RD mutants. In contrast, NMU induced more cytoplasmic effects was it possible to detect mutagenic specificities which, solely on the basis of karyotic action, were not detectable.     

Enhanced ferredoxin-dependent cyclic electron flow around photosystem I and α-tocopherol quinone accumulation in water-stressed <Emphasis Type="Italic">ndhB</Emphasis>-inactivated tobacco mutants

Dissipation mechanisms of excess photon energy under water stress were studied in ndhB-inactivated tobacco (Nicotiana tabacum cv. Xanthi) mutants, which are impaired in NAD(P)H dehydrogenase-dependent cyclic electron flow around PSI. Relative leaf water content and net CO₂ assimilation decreased to 30% and almost zero, respectively, after 11-day water stress in the mutant and wild type plants. Similar reductions in PSII activity (by ca. 75%), and increases in malondialdehyde (by ca. 45%), an indicator of lipid peroxidation, were observed in both the plant groups when subjected to water stress. The stressed mutant and wild type plants showed similar P700 redox kinetics, but only the stressed mutant demonstrated an enhanced operation of the antimycin A-sensitive, ferredoxin-dependent cyclic electron flow around PSI, as indicated by a transient increase in chlorophyll fluorescence after turning off of actinic light. Further, the stressed mutant showed higher oxidation of -tocopherol to -tocopherol quinone, as compared with that in the stressed wild type. Thus, a deficiency in NAD(P)H dehydrogenase-dependent cyclic electron flow around PSI does not lead to oxidative damage because the mutant compensates for this deficiency by activating alternative dissipating routes of excess photon energy, such as up-regulation of ferredoxin-dependent cyclic electron flow around PSI and increased accumulation of -tocopherol quinone.     

Xanthommatin biosynthesis in wild-type and mutant strains of the Australian sheep blowfly Lucilia cuprina

The synthesis of eye pigments has been studied in the seven eye color mutants of the Australian sheep blowfly, Lucilia cuprina. Six appear to be affected primarily in the synthesis of xanthommatin. In wild type, the onset of xanthommatin biosynthesis occurs midway through metamorphosis. Developmental patterns of accumulation of the xanthommatin precursors tryptophan, kynurenine, and 3-hydroxykynurenine have also been established for wild type. By determining the levels of these precursors in late pupae of the mutants, it has been shown that the mutant yellowish accumulates excess tryptophan and the mutant yellow accumulates excess kynurenine. The implications of these results—that yellowish lacks tryptophan oxygenase, thus failing to convert tryptophan to kynurenine, and that yellow lacks kynurenine hydroxylase (blocked in the conversion of kynurenine to 3-hydroxykynurenine)—have been confirmed. This has involved in vitro assays of tryphophan oxygenase and precursor feeding experiments. The precursor accumulation patterns are less clear for the other mutants.     

BtubA-BtubB Heterodimer Is an Essential Intermediate in Protofilament Assembly

Background

BtubA and BtubB are two tubulin-like genes found in the bacterium Prosthecobacter. Our work and a previous crystal structure suggest that BtubB corresponds to α−tubulin and BtubA to β−tubulin. A 1∶1 mixture of the two proteins assembles into tubulin-like protofilaments, which further aggregate into pairs and bundles. The proteins also form a BtubA/B heterodimer, which appears to be a repeating subunit in the protofilament.

Methodology/Principal Findings

We have designed point mutations to disrupt the longitudinal interfaces bonding subunits into protofilaments. The mutants are in two classes, within dimers and between dimers. We have characterized one mutant of each class for BtubA and BtubB. When mixed 1∶1 with a wild type partner, none of the mutants were capable of assembly. An excess of between-dimer mutants could depolymerize preformed wild type polymers, while within-dimer mutants had no activity.

Conclusions

An essential first step in assembly of BtubA + BtubB is formation of a heterodimer. An excess of between-dimer mutants depolymerize wild type BtubA/B by sequestering the partner wild type subunit into inactive dimers. Within-dimer mutants cannot form dimers and have no activity.     

Three <Emphasis Type="Italic">nth</Emphasis> homologs are all required for efficient repair of spontaneous DNA damage in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Deinococcus radiodurans is a bacterium that can survive extreme DNA damage. To understand the role of endonuclease III (Nth) in oxidative repair and mutagenesis, we constructed nth single, double and triple mutants. The nth mutants showed no significant difference with wild type in both IR resistance and H₂O₂ resistance. We characterized these strains with regard to mutation rates and mutation spectrum using the rpoB/Rif^r system. The Rif^r frequency of mutant MK1 (△dr0289) was twofold higher than that of wild type. The triple mutant of nth (ME3)generated a mutation frequency 34.4-fold, and a mutation rate 13.8-fold higher than the wild type. All strains demonstrated specific mutational hotspots. Each single mutant had higher spontaneous mutation frequency than wild type at base substitution (G:C → A:T). The mutational response was further increased in the double and triple mutants. The higher mutation rate and mutational response in ME3 suggested that the three nth homologs had non-overlapped and overlapped substrate spectrum in endogenous oxidative DNA repair.     

Selective Allele Loss in Mixed Infections with T4 Bacteriophage

Evidence is presented that when E. coli B is mixedly infected with T4D wild type and rII deletion mutants, the excess DNA of the wild type allele is lost. No loss is seen in mixed infections with rII point mutants and wild type. In similar experiments with lysozyme addition mutants, the mutant allele is lost. We believe these results demonstrate a repair system which removes "loops" in heteroduplex DNA molecules. A number of phage and host functions have been tested for involvement in the repair of the excess DNA, and T4 genes x and v have been implicated in this process.     

Evidence for a Ca-channel mutation in the K+-resistant mutants ofParamecium

Summary The K⁺-resistant mutants ofParamecium tetraurelia were isolated for their ability to survive high concentrations of K⁺ that kill wild type (Shusterman et al. (1978)Proc. Natl. Acad. Sci. USA 755645). These mutants have a normal turnover of the K-analog⁸⁶Rb and do not appear to be defective in their K-regulation. Instead, the following evidence suggests that these mutants have a defective Ca channel that carries a lower-than-normal current: (1) Two K-resistant mutants survive longer than wild type in Ba⁺⁺, which entersParamecium through the Ca channel during an action potential. (2) The most resistant mutant swims backward longer in Ba than wild type and has a much slower uptake of¹³³Ba per second backward swimming. (3) Only the influx of Ba⁺⁺ is altered in this mutant; the efflux of Ba⁺⁺ is normal. All of the phenotypic differences of these mutants, including their lack of adaptation to high K⁺, can be explained by the postulated Ca-channel defect.