Valine-Resistance, a Potential Marker in Plant Cell Genetics. I. Distinction between Two Types of Valine-Resistant Tobacco Mutants Isolated from Protoplast-Derived Cells

In previous experiments, seven lines of valine-resistant plants were regenerated from protoplast-derived haploid tobacco mesophyll cells which had been UV mutagenized and submitted to selection by toxic concentrations of valine. In this study we described the transmission of valine-resistance to progeny and a preliminary phenotypical and biochemical characterization of the resistant plants.—Two types were thus distinguished among the seven mutant lines. Valine-resistance of the mutants of the first type (three lines) was transmitted as a single Mendelian dominant character (Vr1), whereas valine-resistance of the second type (four lines) was transmitted as a digenic recessive character (vr2 and vr3). Allelism tests revealed that the four recessive mutant lines yielded resistant progeny when intercrossed and, therefore, bear recessive mutant alleles at the same two unlinked loci.—When cultured at a density of 100 cell/ml, protoplast-derived cells of mutants of the first type had a low level of resistance to valine, whereas protoplast-derived cells of mutants of the second type displayed a high level of resistance to valine and to other amino acids.—According to the results of ¹⁴C-labelled amino acid uptake experiments, the amino acid resistance of mutants of the second type, but not valine-resistance of the first type, could be accounted for by reduced uptake of several amino acids. Possible uses of valine-resistance as a marker in plant cell genetics are discussed.     

A cycloheximide-resistant mutant of Tetrahymena pyriformis

A mutant of Tetrahymena pyriformis, syngen 1, resistant to cycloheximide was obtained after mutagenesis (with N-methyl-N′-nitro-N-nitrosoguanidine) followed by a cross (to obtain macro-nuclear expression of the mutant phenotype). A genetic analysis has shown that cycloheximide resistance in the mutant strain is due to a dominant nuclear allele, designated chx-1. Heterozygotes (chx-1/chx⁺) are initially resistant but segregate stable, sensitive cell lines during vegetative growth, demonstrating that allelic exclusion occurs with this determinant, as with many others in syngen 1. This feature, coupled with the selective advantage conferred by the chx-1 allele in the presence of cycloheximide, makes this mutation a useful genetic tool. A strain homozygous for the chx-1 allele exhibits an exponential growth rate identical to that of the wild type in proteose peptone-yeast extract medium in the absence of cycloheximide. In 10 μg/ml of the drug, the resistant cells grow at a somewhat lower rate, after an initial lag and adaptation to the presence of the drug. This concentration causes complete inhibition of growth and eventual lysis of wild-type cells. The cellular basis for cycloheximide resistance and adaptation in the mutant is presently under investigation.     

Isolation and Genetic Characterization of a Mutation Affecting Ribosomal Resistance to Cycloheximide in Tetrahymena

A dominant mutation at a new locus affecting resistance to cycloheximide has been isolated by exploiting a synergistic relationship with a previously known mutation for cycloheximide resistance in Tetrahymena. The new mutation (ChxB) was induced in a line homozygous for ChxA and was recovered from that background by a new technique termed interrupted genomic exclusion. Segregation data from the interrupted genomic exclusion suggest that ChxA and ChxB are separate, linked loci showing 30% recombination. Minimal lethal doses of cycloheximide for the four possible combinations of the wild-type and mutant alleles of these two genes are: wild type 6 µg/ml, ChxA 125 µg/ml, ChxB 10 µg/ml, ChxA-ChxB 175 µg/ml.     

Genetics and biochemistry of cycloheximide resistance in Physarum polycephalum

Summary Cycloheximide-resistant mutants of Physarum polycephalum were induced in the haploid myxamoebae by the combined action of UV¹ and caffeine (Haugli and Dove, 1972) or by treatment with NMG². Eight independent mutants segregated in a Mendelian fashion (Table 1). Crosses between 6 of the mutants revealed 2 loci, actA and actB, for cycloheximide resistance (Table 2).All mutants are expressed in the plasmodium and are recessive in heterozygotes (Fig. 1 and 2). One mutation, conferring resistance to high levels of cycloheximide, was studied in heterokaryons and found to be incompletely recessive.An in vitro peptide synthesizing system was constructed from ribosomes from Physarum and supernatant factors from Saccharomyces cerevisiae. Cycloheximide strongly inhibited the activity of ribosomes derived from either wild type or mutants at the actB locus. In contrast, ribosomes from mutants at the actA locus were resistant to cycloheximide. Thus, the actA locus operates through the ribosomes.     

Ultraviolet mutagenesis and genetic analysis of resistance to methylglyoxal-bis (guanylhydrazone) in tobacco

Summary We have isolated cell lines of Nicotiana tabacum resistant to methylglyoxal-bis(guanylhydrazone) (MGBG), a potent inhibitor of S-adenosylmethionine decarboxylase. We obtained 31 resistant lines from ultraviolet light mutagenized cultures, representing at least 13 independent events. No resistant lines were obtained from non-mutagenized control cultures. The increase in mutation rate due to the ultraviolet light treatment was 22 to 62 fold increased over an estimate of the maximum possible spontaneous rate. In reconstruction experiments we reselected resistant cell lines from varying dilutions into a background of a constant concentration of wild type cells; at the minimum ratio of resistant cells to wild type cells, 1:125,000, we recovered resistant colonies at an estimated plating efficiency of 12.5%. A number of resistant lines have been regenerated into plants. All of the ones that flowered are male sterile, sometimes associated with morphological transformations. Some are female sterile as well. Meiotic genetic analysis of one resistant line, Mgr12, suggests that the MGBG resistance is segregating as a nuclear dominant trait. The male sterility and abnormal floral development of Mgr12 cosegregate with the MGBG resistance, suggesting the two phenotypes are coincident.     

Genetic and enzymatic characterization of conditional lethal mutants of the yeast Schizosaccharomyces pombe with a temperature-sensitive DNA ligase.

The DNA ligase activities of wild type and temperature-sensitive lethal cdc 17 mutants of Schizosaccharomyces pombe have been studied by measuring effects on the conversion of relaxed DNA circles containing a single nick to a closed circular form. Such assays have revealed that all cdc 17 mutants have a thermosensitive DNA ligase deficiency, that this deficiency cosegregates 2:2 with their temperature-sensitive cdc-lethality in three tetrads derived from a cross against wild type, and that genetic reversion of the temperature-sensitive cdc^? phenotype is accompanied by a restoration of DNA ligase activity; all of which implies that the temperature-sensitive cdc^? phenotype of cdc 17 mutants is due to a single nuclear mutation causing a DNA ligase deficiency. Both wild type and mutant enzymes have been partially purified by chromatography in heparin/agarose columns. The wild-type enzyme is completely stable in vitro at both permissive (25 °C) and restrictive (35 °C) temperatures, whereas that of two different mutants, though completely stable at 25 °C, is rapidly inactivated at 35 °C, implying that their mutations are located in the structural gene for DNA ligase.     

Studies on the expression of a mutant with abnormal cell division in Chlamydomonas reinhardi

A mutant of Chlamydomonas reinhardi, in which cell and nuclear division are no longer synchronised, has been compared with wild type with the aim of clarifying the nature of the difference between the two strains. On entry into stationary phase, wild type cultures show a marked increase in protein, RNA and chlorophyll per cell, whereas mutant cultures do not show a comparable increase. The effect of chemicals which may interfere with particular aspects of the cell division process on the expression of the mutant have been studied. Vitamin B12 and the related compounds, benzimidazole, 5,6,dimethylbenzimidazole and cobaltous chloride increase the asynchrony between cell and nuclear division and consequently lead to the accumulation of large multinucleate cells. The mutant is less resistant than wild type to the inhibitory effects of caffeine.     

Dictyostelium discoideum cobB mutants show reduced heavy metal accumulation associated with gene amplification

Wild-type Dictyostelium discoideum cells grow- ing on non-toxic levels of nickel chloride or cobaltous chloride accumulate 2–3.5 times as much nickel and at least 1.5 times as much cobalt as cobB mutants. The cobB trait is dominant, confers unstable cobalt and nickel resistance and is correlated with the presence of up to 50 copies of a linear extrachromosomal DNA, approximately 100?kb in length, derived from linkage group III. Independent cobB mutants can be obtained by selection on medium containing either cobalt or nickel. The amplified DNA can be transferred to wild-type strains by electroporation. Strains with mutations at a second cobalt resistance locus, cobA, accumulate the same amount of cobalt, but more nickel than wild-type strains. Our results are consistent with the cobA mutant phenotype being due to internal sequestration of cobalt, and the cobB mutant phenotype being due to reduced net uptake of cobalt and nickel. Energy-dependent nickel export was detectable in wild-type and cobB mutant strains but its role in heavy metal resistance has not yet been proved.     

A Mutation Causing Imidazolinone Resistance Maps to the Csr1 Locus of Arabidopsis thaliana

A mutant of Arabidopsis thaliana, two hundred times more resistant to the imidazolinone herbicide imazapyr than wild-type plants, was isolated by direct selection of seedlings from a mutagenized population. Genetic analysis showed that resistance is due to a single dominant nuclear mutation that could not be separated by recombination from a mutation in the CSR1 gene encoding acetohydroxy acid synthase. Acetohydroxy acid synthase activity in extracts isolated from the mutant was 1000-fold more resistant to inhibition by imazapyr than that of the wild type. The resistant enzyme activity cosegregated with whole plant resistance. These data strongly suggest that the mutation is an allele of CSR1 encoding an imazapyr-resistant AHAS.     

Induced Resistance to 6-Methylpurine and Cycloheximide in Tetrahymena. I. Germ Line Mutants of T. THERMOPHILA

Two new mutant lines of Tetrahymena thermophila ( T. pyriformis, syngen 1), each conferring resistance to a different agent, are described. Resistance to cycloheximide and 6-methylpurine are each determined by dominant genes, ChxA2 and Mpr; the traits show phenotypic assortment. The method used to select these mutations, the critical importance of backcrossing to wild type following mutagenesis, and the utility of these marker genes in further mutagenic selection schemes and studies of the sexual cycle of Tetrahymena are noted.     

Modification and Inheritance of Pleiotropic Cross Resistance and Collateral Sensitivity in SACCHAROMYCES CEREVISIAE

A meiotic segregant (oli^PR1) was isolated with a phenotype of multiple cross resistance and collateral sensitivity. Strain oli^PR1 has increased sensitivity to ethidium bromide, dequalinium chloride, acriflavin, paromomycin and neomycin, and increased resistance to oligomycin, rutamycin, venturicidin, triethyltin bromide, antimycin, carbonylcynamide-m-chlorophenylhydrazone, tetra-N-butylammonium bromide, dibenzyldimethylammonium chloride, triphenylmethylphosphonium bromide, chloramphenicol, carbomycin, tetracycline, triton-X-165 and cycloheximide. Single gene inheritance of the cross resistance and collateral sensitivity was shown by 2:2 parental ditype segregation and reversion of the complete phenotype by a spontaneous revertant. The locus conferring the oli^PR1 phenotype was mapped 11.7 units from an unspecified centromere. Antibiotic resistance showed incomplete dominance, with the level of hybrid resistance dependent upon the inhibitor tested. Resistant diploids that produced four resistant ascospores were the result of mitotic recombination prior to meiosis. A partial revertant phenotype (sensitive to all inhibitors except oligomycin, antimycin and carbonylcyanide-m-chlorophenylhydrazone) was shown to be due to a single nuclear gene causing partial suppression of oli^PR1. Anaerobic pretreatment, 37° and 0.5 M KCl were observed to reduce the growth of oli^PR1 when challenged with seven diverse inhibitors (antimycin, carbonylcyanide-m-chlorophenylhydrazone,-chloramphenicol, cycloheximide, oligomycin, triethyltin bromide, and triphenylmethylphosphonium bromide). Resistance to cycloheximide was not altered by the [rho—] state. A revertant of oli^PR1 (sensitive to the above inhibitors but resistant to ethidium bromide, paromycin and neomycin) showed anaerobic and temperature sensitization to ethidium bromide, paromomycin and neomycin. Continuous monitoring of oxygen uptake by the revertant after anaerobic pretreatment revealed that anaerobiosis sensitized respiratory adaptation of the revertant to neomycin. It is proposed that oli^PR1 is a mutation resulting in the alteration of plasma membrane premeability to many diverse inhibitors.     

Genetic Diversity for Resistance to Heterodera glycines Race 5 in Soybean

Heterodera glycines is a serious pest of soybean in the United States. Plant introductions 90763 and 424595 are reported to be resistant to H. glycines race 5; however their genetic relationship for resistance is unknown. Crosses between these two lines and the susceptible cultivar Essex were studied in the F₁, F₂, and F₃ generations to determine the number of genes involved in inheritance of resistance. The plants were screened using conventional techniques based on the index of parasitism. The data were subjected to analyses using chi-square test to determine goodness of fit between observed and expected genetic ratios. The cross PI 424595 x Essex segregated 1 resistant:63 susceptible in the F₂ generation, which indicated the presence of three recessive genes controlling resistance to race 5. In the cross PI 90763 x Essex, resistance was conditioned by one dominant and two recessive genes. The cross between PI 424595 and PI 90763 segregated into 13 resistant:3 susceptible. The data fit a four-gene model with two recessive and two dominant genes with epistasis. PI 90763 has a dominant gene, whereas PI 424595 has a recessive gene; both share two additional recessive genes for resistance to race 5. This information is important to geneticists and soybean breeders for the development of cultivars resistant to H. glycines.     

Macrolide and aminoglycoside antibiotic resistance mutations in the Bacillus subtilis ribosome resulting in temperature-sensitive sporulation

Summary Mutants of Bacillus subtilis resistant to various macrolide antibiotics have been isolated and characterized with respect to their sporulation phenotype and the electrophoretic mobility of their ribosomal proteins (r-proteins). Two types of major alterations of r-protein L17, one probably due to a small deletion, are found among mutants exhibiting high-level macrolide resistance. These mutants are all temperature-sensitive for sporulation (Spo^ts). Low-level resistance to some macrolides is found to be associated with minor alterations in r-protein L17. These mutations do not cause a defective sporulation phenotype. All of the macrolide resistance mutations map at the same locus within the Str-Spc region of the B. subtilis chromosome. Hence, changes in a single ribosomal protein can result in different sporulation phenotypes.Mutants resistant to the aminoglycoside antibiotics neomycin and kanamycin have been isolated. Approximately 5% of these are Spo^ts. Representative mutations, neo 162 and kan25, cause concomitant drug resistance and sporulation temperature-sensitivity and map as single-site lesions in the Str-Spc region of the chromosome. Strains bearing neo162 or kan25 are equally cross-resistant to several aminoglycoside antibiotics but show no resistance to streptomycin or spectinomycin. These mutations define a new B. subtilis drug resistance locus at which mutation can cause defective sporulation.     

CRISPR/Cas9 mediated G4946E substitution in the ryanodine receptor of Spodoptera exigua confers high levels of resistance to diamide insecticides

Diamide insecticides selectively activate insect ryanodine receptors (RyRs), inducing uncontrolled release of calcium ions, and causing muscle contraction, paralysis and eventually death. The RyR^G4946E substitution associated with diamide resistance has been identified in three lepidopteran pests, Plutella xylostella, Tuta absoluta and Chilo suppressalis. Recently, the T. absoluta RyR^G4946V mutation was knocked into the model insect Drosophila melanogaster by CRISPR/Cas9 mediated genome editing and provided in vivo functional confirmation for its role in diamide resistance. In the present study, we successfully introduced the RyR^G4946E mutation with CRISPR/Cas9 technology into a lepidopteran pest of global importance, Spodoptera exigua. The genome-edited strain (named 4946E) homozygous for the SeRyR^G4946E mutation exhibited 223-, 336- and >1000-fold resistance to chlorantraniliprole, cyantraniliprole and flubendiamide, respectively when compared to the wild type strain (WHS) of S. exigua. Reciprocal crossing experiments revealed that the target-site resistance in strain 4946E underlies an autosomal and almost recessive mode of inheritance for anthranilic diamides, whereas it was completely recessive for flubendiamide. Our results not only provided in vivo functional validation of the RyR^G4946E mutation in conferring high levels of resistance to diamide insecticides for the first time in a controlled genetic background of a lepidopteran pest, but also revealed slight differences on the level of resistance between anthranilic diamides (chlorantraniliprole and cyantraniliprole) and flubendiamide conferred by the SeRyR^G4946E mutation.     

The detection of monosomic colonies produced by mitotic chromosome non-disjunction in the yeast Saccharomyces cerevisiae

A diploid yeast strain, D6, is described which monitors mitotic non-disjunction by the phenotypic expression of a set of coupled and recessive markers flanking the centromere of chromosome VII. These markers are not expressed in the heterozygous condition prevailing in D6. The left arm of chromosome VII carries a tightly centromere linked marker, leu1 (leucine requirement), distal to leu1 in this order: trp5 (trytophan requirement), cyh2 (recessive resistance to cycloheximide) and met 13 (requirement for methionine). The right arm is marked with ade3 (simultaneous requirement for adenine and histidine). D6 is homozygous for ade2 and consequently, forms red rather than the normally white colonies. It shows no requirement for the above amino acids and it is sensitive to cycloheximide.Unmasking of all the markers on chromosome VII leads to colonies that are white because ade3 sets a block preceding the ade2 block (which causes the accumulation of a precursor of the red pigment), they require leucine, tryptophan and methionine, and grow on media with cycloheximide. Cells are plated on a cycloheximide medium where red and white colonies are formed. Colonies of spontaneous origin were tested. The majority of the white colonies expressed all the recessive markers whereas only few of the red colonies expressed all the markers on the left arm of chromosome VII.Basically expression of recessive markers on both sides of the centromere can be explained as a result of two coincident events of mitotic crossing over. However, the frequency of colonies expressing centromere linked leu1 was 14 times higher among the white types than the red ones. This suggested that the white, cycloheximide resistant, leucine requiring colonies arose by mitotic non-disjunction and not only by two coincident mitotic crossing over events.Presumptive spontaneous monosomic segregants were placed on sporulation medium. Only 8 out of 30 isolates sporulated, which showed that these eight segregants were diploid at the time of sporulation. They could have arisen by two coincident crossover events or through restoration of a normal disomic condition after non-disjunction had occurred. The genetic data thus leaves us with only its statistical argument in favour of non-disjunction. Further confirmation of monosomic nature of the white cycloheximide resistant colonies was provided by the estimates of their DNA contents. Compared to the stock wild type diploids the presumptive monosomics showed a reduction in DNA content.We have utilized D6 to investigate the possible induction of mitotic non-disjunction after treatment with gamma rays, heat shock at 52°C and ultraviolet irradiation. In all cases white, cycloheximide resistant colonies were produced at levels significantly higher than that found in untreated cultures. In order to detect the production of monosomic cells, treated cultures were grown for 48 h in non-selective medium after exposure to allow for “expression” of the monosomic condition.     

Mutant ribosomes can generate dominant kirromycin resistance.

Mutations in the two genes for EF-Tu in Salmonella typhimurium and Escherichia coli, tufA and tufB, can confer resistance to the antibiotic kirromycin. Kirromycin resistance is a recessive phenotype expressed when both tuf genes are mutant. We describe a new kirromycin-resistant phenotype dominant to the effect of wild-type EF-Tu. Strains carrying a single kirromycin-resistant tuf mutation and an error-restrictive, streptomycin-resistant rpsL mutation are resistant to high levels of kirromycin, even when the other tuf gene is wild type. This phenotype is dependent on error-restrictive mutations and is not expressed with nonrestrictive streptomycin-resistant mutations. Kirromycin resistance is also expressed at a low level in the absence of any mutant EF-Tu. These novel phenotypes exist as a result of differences in the interactions of mutant and wild-type EF-Tu with the mutant ribosomes. The restrictive ribosomes have a relatively poor interaction with wild-type EF-Tu and are thus more easily saturated with mutant kirromycin-resistant EF-Tu. In addition, the mutant ribosomes are inherently kirromycin resistant and support a significantly faster EF-Tu cycle time in the presence of the antibiotic than do wild-type ribosomes. A second phenotype associated with combinations of rpsL and error-prone tuf mutations is a reduction in the level of resistance to streptomycin.     

Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology

Summary Mutant lines of Arabidopsis thaliana resistant to the artificial auxin 2,4-dichloro phenoxyacetic acid (2,4-D) were isolated by screening for growth of seedlings in the presence of toxic levels of 2,4-D. Genetic analysis of these resistant lines indicated that 2,4-D resistance is due to a recessive mutation at a locus we have designated Axr-1. Mutant seedlings were resistant to approximately 50-fold higher concentrations of 2,4-D than wild-type and were also resistant to 8-fold higher concentrations of indole-3-acetic acid (IAA) than wild-type. Labelling studies with (¹⁴C)2,4-D suggest that resistance was not due to changes in uptake or metabolism of 2,4-D. In addition to auxin resistance the mutants have a distinct morphological phenotype including alterations of the roots, leaves, and flowers. Genetic evidence indicates that both auxin resistance and the morphological changes are due to the same mutation. Because of the pleiotropic morphological effects of these mutations the Axr-1 gene may code for a function involved in auxin action in all tissues of the plant.