Induction of Mendelian and non-Mendelian streptomycin resistant mutants during the synchronous cell cycle of Chlamydomonas reinhardtii

Summary In synchronized cultures of Chlamydomonas reinhardtii N-methyl-N-nitro-N-nitrosoguanidine (MNNG) was found to selectively mutate replicating forms of nuclear DNA. This conclusion was based on the 15- to 30-fold increase in the recovery of MNNG induced Mendelian streptomycin resistant mutants (sr-1) which was correlated with mutagenesis during the period of nuclear DNA replication. No concomitant increase in the recovery of non-Mendelian streptomycin resistant mutants (sr-2) occurred during this same period.Mutagenesis at the time of chloroplast DNA replication, however, resulted in a 1.5- to 1.6-fold increase in the recovery of both sr-1 and sr-2 induced mutants.     

Mechanism of conjugation and recombination in bacteria XVI

Summary Protein synthesis by ribosomes from several cryptopleurine-resistant yeast mutants is also resistant to emetine and tubulosine. These mutants can be classified into two different types: Class I mutants which display high levels of resistance to emetine and tubulosine and Class II mutants that are only weakly resistant to tubulosine and are slightly more sensitive to emetine than those of Class I. Apparently all mutants have similar levels of resistance to cryptopleurine. The distinct phenotypes of Class I and Class II strains are expressed through their 40S ribosomal subunit. Genetic analysis has shown that the mutations to cryptopleurine resistance are allelic and that in a particular case (strain CRY6) the pleiotropic phenotype is a result of the expression of the cryl locus. It is suggested that Class I and Class II mutants arise from two independent mutational events within the cryl allele. in heterozygous (+/cryl) diploids both the sensitive and the resistant genes are expressed as shown by studies of the action of cryptopleurine on polyphenylalanine-synthesizing system derived from each parental sensitive and resistant haploid strain and heterozygous diploid strains. The apparent dominance of sensitivity over resistance which may be observed in vivo in heterozygous (+/cryl) diploids has been explained in terms of the mode of action of the inhibitors.     

Isolation and characterization of streptomycin-resistant mutants in Nicotiana plumbaginifolia

Summary Streptomycin-resistant colonies were isolated from protoplast cultures of haploid Nicotiana plumbaginifolia based on their ability to green in medium containing 1 mg/ml streptomycin sulfate. The frequency of resistant colonies was 0.9×10^–5 in nonmutagenized culture, and increased ten-fold following treatment of culture with 10 g/ml N-methyl-N-nitro-N-nitrosoguanidine. Of a total of 52 resistant clones isolated, 2 gave rise to haploid, 15 to diploid, and 3 to tetraploid plants upon transfer of calli to differentiation medium. Leaf-segment and protoplast assays showed that all diploid regenerates were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, neomycin, and spectinomycin. Plants in most diploid clones were fertile and able to set seeds when self-fertilized and crossed reciprocally to wild-type plants. Inheritance of streptomycin resistance was studied in the diploid clones and, without exception, the resistance was transmitted maternally. Comparative studies of the ultrastructure of organelles and protein synthesis in isolated chloroplasts between wild-type and resistant clones in the presence of streptomycin suggest that streptomycin resistance is controlled by chloroplasts.     

Cytoplasmic inheritance of antimycin A resistance in Saccharomyces cerevisiae

Summary Three antimycin resistant mutants of Saccharomyces cerevisiae are characterized genetically. The mutations have been shown to be cytoplasmically inherited by four criteria. The phenotype persists in diploids formed by a cross with a ⁰ strain of yeast of the opposite mating type. Diploids heterozygous for the antimycin marker, however, show segregation of the resistance and sensitivity during mitosis. Tetrad analysis indicated a non-Mendelian segregation (4:0 and 0:4) of the mutations. The antimycin marker can be eliminated by ethidium bromide treatment under conditions that should have deleted all of the mitochondrial DNA.     

Toxicity of and mutagenesis by chlorate are independent of nitrate reductase activity in Chlamydomonas reinhardtii

Summary Spontaneous chlorate-resistant (CR) mutants have been isolated from Chlamydomonas reinhardtii wildtype strains. Most of them, 244, were able to grow on nitrate minimal medium, but 23 were not. Genetic and in vivo complementation analyses of this latter group of mutants indicated that they were defective either at the regulatory locus nit-2, or at the nitrate reductase (NR) locus nit-1, or at very closely linked loci. Some of these nit-1 or nit-2 mutants were also defective in pathways not directly related to nitrate assimilation, such as those of amino acids and purines. Chlorate treatment of wild-type cells resulted in both a decrease in cell survival and an increase in mutant cells resistant to a number of different chemicals (chlorate, methylammonium, sulphanilamide, arsenate, and streptomycin). The toxic and mutagenic effects of chlorate in minimal medium were not found when cells were grown either in darkness or in the presence of ammonium, conditions under which nitrate uptake is drastically inhibited. Chlorate was also able to induce reversion of nit ^– mutants of C. reinhardtii, but failed to produce His ⁺ revertants or Ara^r mutants in the BA-13 strain of Salmonella typhimurium. In contrast, chlorate treatment induced mutagenesis in strain E1F1 of the phototrophic bacterium Rhodobacter capsulatus. Genetic analyses of nitrate reductase-deficient CR mutants of C. reinhardtii revealed two types of CR, to low (1.5 mM) and high (15 mM) chlorate concentrations. These two traits were recessive in heterozygous diploids and segregated in genetic crosses independently of each other and of the nit-1 and nit-2 loci. Three her loci and four lcr loci mediating resistance to high (HC) and low (LC) concentrations of chlorate were identified. Mutations at the nit-2 locus, and deletions of a putative locus for nitrate transport were always epistatic to mutations responsible for resistance to either LC or HC. In both nit ⁺ and nit ^– chlorate-sensitive (CS) strains, nitrate and nitrite gave protection from the toxic effect of chlorate. Our data indicate that in C. reinhardtii chlorate toxicity is primarily dependent on the nitrate transport system and independent of the existence of an active NR enzyme. At least seven loci unrelated to the nitrate assimilation pathway and mediating CR are thought to control indirectly the efficiency of the nitrate transporter for chlorate transport. In addition, chlorate appears to be a mutagen capable of inducing a wide range of mutations unrelated to the nitrate assimilation pathway.     

Streptomycin resistance is inherited as a recessive Mendelian trait in a Nicotiana sylvestris line

Summary The SR180 cell line has been isolated in a callus culture derived from a haploid Nicotiana sylvestris (n = X = 12) plant by its ability to proliferate on a selective medium containing 2,000 g/ml streptomycin sulphate. From the cell line diploid plants have been regenerated. The SR180 selfs are resistant to streptomycin. Streptomycin sensitivity in F1, and a 31 (sensitive to resistant) segregation in F2 indicate that resistance in the SR180 mutant is the result of a recessive Mendelian mutation.     

Direct selection of cybrids by streptomycin and valine resistance in tobacco

Summary Direct selection of cybrids by simultaneous selection for donor chloroplasts and for the recipient nuclei is described. Mesophyll protoplasts of two tobacco (Nicotiana tabacum) mutants, SR1 (streptomycin resistant) and Val^r-2 (valine resistant), were fused by polyethylene glycol treatment. Streptomycin resistance in the SR1 mutant is a maternally inherited chloroplast trait while valine resistance is a Mendelian (nuclear) digenic recessive character. The fused protoplast population was cultured and colonies were selected for resistance to valine (1 mM) and streptomycin (343 M). The efficiency of selection has been confirmed in three clones by demonstrating seed transmission of both streptomycin and valine resistances. In one subclone both streptomycin resistant and sensitive plants were obtained indicating that the streptomycin sensitive chloroplasts had not been totally eliminated by growth on the selective medium.     

Mutations in Chlamydomonas reinhardtii Conferring Resistance to the Herbicide Sulfometuron Methyl

Chlamydomonas reinhardtii mutants resistant to the herbicide sulfometuron methyl (SM) were isolated and characterized. Growth of C. reinhardtii is sensitive to inhibition by SM at a concentration of 1 micromolar. Four mutants resistant to 10- to 100-fold higher concentrations were isolated. All possess a form of acetolactate synthase (ALS) whose specific activity in cell extracts is 100- to 1000-fold more resistant to SM than is the specific activity of wild-type enzyme. Only one mutant had abnormally low ALS specific activity in the absence of SM. All mutations were inherited as single lesions in the nuclear genome and were expressed in heterozygous diploids. The mutations in two strains mapped to linkage group IX about 30 centimorgans from streptomycin resistance and on the same side of the centromere, and in genetic crosses between mutants no segregation was observed. Accordingly, all mutations are tentatively assigned to gene smr-1. Herbicide resistance appears to be suitable as a selectable marker for molecular transformation in this organism.     

Complementation analysis at the ery-M1 locus in Chlamydomonas reinhardi

Summary Previous studies with haploid erythromycin-resistant mutants mapping to the Mendelian locus ery-M1 in Chlamydomonas reinhardi have revealed the presence of an altered chloroplast ribosomal protein (LC6) (Mets and Bogorad, 1971, 1972; Davidson et al., 1974). Vegetative diploids of C. reinhardi heterozygous at the ery-M1 locus have now been constructed. Chloroplast ribosomes from such diploids contain 60–70% wild-type form of protein LC6 and 30–40% altered form of LC6. Growth assays show that these diploids are partially resistant to erythromycin. Whether the diploids are grown in the presence or absence of erythromycin, the same ratio of wild-type: altered form of LC6 in chloroplast ribosomes is observed. Therefore, resistant chloroplast ribosomes must be able to carry out protein synthesis even when many of the sensitive chloroplast ribosomes are blocked by erythromycin.The presence of both the altered and wild-type forms of LC6 in diploids heterozygous at the ery-M1 locus is further evidence that a nuclear gene codes directly for a chloroplast ribosomal protein.An abstract of this work appeared in Genetics 80, S40 (1975)     

Genetic analysis of Aspergillus niger: Isolation of chlorate resistance mutants, their use in mitotic mapping and evidence for an eighth linkage group

Summary This paper describes the use of chlorate resistant mutants in genetic analysis of Aspergillus niger. The isolated mutants could be divided into three phenotypic classes on the basis of nitrogen utilization. These were designated nia, nir and cnx as for Aspergillus nidulans. All mutations were recessive to their wild-type allele in heterokaryons as well as in heterozygous diploids. The mutations belong to nine different complementation groups. In addition a complex overlapping complementation group was found. Evidence for the existence of eight linkage groups was obtained. Two linked chlorate resistance mutations and two tryptophan auxotrophic markers, which were unlinked to any of the known markers (Goosen et al. 1989), form linkage group VIII. We used the chlorate resistance mutations as genetic markers for the improvement of the mitotic linkage map of A. niger. We determined the linear order of three markers in linkage group VI as well as the position of the centromere by means of direct selection of homozygous cnxA1 recombinants. In heterozygous diploid cultures diploid chlorate resistant segregants appeared among conidiospores with a frequency of 3.9×10^–2 (cnxG13 in linkage group I) to 2.1 × 10^–2 (cnxD6 in linkage group 111). The mean frequency of haploid chlorate resistant segregants was 1.3 × 10^–3. The niaD1 and niaD2 mutations were also complemented by transformation with the A. niger niaD ⁺ gene cloned by Unkles et al. (1989). Mitotic stability of ten Nia⁺ transformants was determined. Two distinct stability classes were found, showing revertant frequencies of 5.0 × 10^–3 and 2.0 × 10^–5 respectively.     

Mutations conferring lincomycin, spectinomycin, and streptomycin resistance in Solanum nigrum are located in three different chloroplast genes

A number of Solanum nigrum mutants resistant to the antibiotics spectinomycin, streptomycin and lincomycin have been isolated from regenerating leaf strips after mutagenesis with nitroso-methylurea. Selection of streptomycin- and spectinomycin-resistant mutants has been described earlier. Lincomycin-resistant mutants show resistance to higher levels of the antibiotic than used in the initial selection, and in the most resistant mutant (Ll7A1) maternal inheritance of the trait was demonstrated. The lincomycin-resistant mutant L17A1 and a streptomycin plus spectinomycin resistant double mutant (StSpl) were chosen for detailed molecular characterisation. Regions of the plastid DNA, within the genes encoding 16S and 23S rRNA and rps12 (3) were sequenced. For spectinomycin and lincomycin resistance, base changes identical to those in similar Nicotiana mutants were identified. Streptomycin resistance is associated with an A C change at codon 87 of rps 12 (converting a lysine into a glutamine), three codons upstream from a mutation earlier reported for Nicotiana. This site has not previously been implicated in streptomycin resistance mutations of higher plants, but has been found in Escherichia coli. The value of these mutants for studies on plastid genetics is discussed.     

Analysis of L-glycerol-3-phosphate dehydrogenase mutants in Drosophila melanogaster: Complementation for intracellular degradation of the mutant polypeptide

Summary Null and low activity alleles at the genetic locus coding for L-Glycerol-3-phosphate dehydrogenase (-GPDH, NAD⁺ oxidoreductase, E.C. 1.1.1.8) in Drosophila melanogaster have been analyzed by a combination of rocket immunoelectrophoresis, interallelic complementation, and two-dimensional gel electrophoresis. In addition to providing information on the molecular weight, charged state, and steady state level of CRM in each of these mutants, it is suggested that each mutation has resulted in a genetic lesion within the structural element, Gpdh ⁺. CRM levels appear to be the result of a differential sensitivity to the normal intracellular degradative process and the CRM^- mutants represent hypersensitive alleles, such that the mutant polypeptide does not accumulate in the intracellular environment.This investigation was supported in part by NIH Research Grants No. GM-23617, AG-01739, and by NIH Training Grant No. GM 296. Paper No. 6192 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27650     

Mitochondrial genome transmission in Chlamydomonas diploids obtained by sexual crosses and artificial fusions: Role of the mating type and of a 1 kb intron

Summary The linear mitochondrial DNAs of the two infertile algal species Chlamydomonas smithii and C. reinhardtii are co-linear with the exception of a 1 kb intron ( intron) located in the cytochrome b gene of C. smithii. C. smithii also possesses an additional HpaI restriction site (H marker) located in the COXI gene, about 5 kb from the intron. In reciprocal crosses, C. smithii (H ⁺⁺) × C. reinhardtii (H ^––), the intron is transmitted to all diploid progeny, whereas the H marker is frequently transmitted either biparentally or paternally depending on whether the C. smithii parent is maternal (mt ⁺) or paternal (mt ^–). In diploids resulting from artificial fusion between vegetative cells, the absolute transmission of a is accompanied by the frequent transmission of the H ⁺ marker, irrespective of the mating type of the parental strains. Finally, in reciprocal crosses between C. smithii (H ⁺⁺) and recombinant H ^–+ clones, the transmission of the H marker is predominantly paternal or biparental. These results allow us to conclude that (1) the a intron behaves as a group I intron whose unidirectional conversion influences the transmission of the H marker; and (2) the mt ^– paternal mitochondrial genome is transmitted more often than the mt ⁺. The mating type has no effect in diploids obtained by artificial fusion.     

Isolation and characterization of biochemical and morphological mutants in chlamydomonas smithii

Mutants of Chlamydomanas smithii resistant to chlorate were induced after UV irradiation. Some were unable to grow on nitrate and suspected to be defective in nitrate reductase. One of them (nit-a) was analyzed and shown to complement nit-1 nit-2 of C. reinhardtii.Mutants resistant to streptomycin, spectinomycin or erythromycin selected from nit-a pre-grown in the presence of 5-fluorodeoxyridine. Two spectinomycin resistant strains were genetically analyzed from crosses with C. reinhardtii carrying different chloroplast mutations. The transmission of resistance was shown to be non-Mendelian and similar to that obtained in intraspecific crosses involving C. reinhardtii chloroplast mutants.Mutants lacking a cell wall (CW⁻) were selected after UV treatment on the basis of the particular flat amoeboid shape of the colonies. Seven of them were tested for complementation with CW15 and CW_d mutants of C. reinhardtii: none was found to be allelic with these two mutations.     

Biochemical studies with bi-resistant mutants (ethambutol plus streptomycin and isoniazid plus streptomycin) ofMycobacterium smegmatis ATCC 607

Biochemical characteristics of bi-resistant mutants (resistant to ethambutol plus streptomycin or isoniazid plus streptomycin) of mycobacteria isolated by replica plating fromMycobacterium smegmatis ATCC were compared with those of the drug-susceptible strains. Reduced incorporation of [¹⁴C]uracil, [³H]lysine and [¹⁴C]acetate into RNA, protein and phospholipids respectively was seen in the resistant mutants. Total phosphorlipids were enhanced in ethambutol plus streptomycin resistant mutant and decreased in isoniazid plus streptomycin resistant mutant. There were similar changes in levels of individual phospholipids. The resistant mutants revealed an accumulation of phospholipids in the cell wall, and a marked decrease of phospholipids in the cell membrane in comparison to the susceptible strain. Several qualitative alterations in the polypeptide profile (with respect to number and molecular weight) of the crude protein extract and of different subcellular compartments were seen in the resistant mutants.     

The cytoplasmic ribosomes of Chlamydomonas reinhardtii: characterization of antibiotic sensitivity and cycloheximide-resistant mutants

Summary In vitro protein synthesis was used to characterize the antibiotic sensitivity of cytoplasmic ribosomes from wild-type and antibiotic-resistant strains of Chlamydomonas reinhardtii. Cytoplasmic ribosomes from two cycloheximide-resistant mutants, act-1 and act-2, were resistant to the antibiotic in vitro. The alteration effected by the act-1 mutation, which was dominant in diploids, was localized to the large subunit of the cytoplasmic ribosomes, but no ribosomal protein alterations were detected using two-dimensional gel electrophoresis. The act-2 mutation, which was semidominant in diploids, was frequently associated with a charge alteration in the large subunit ribosomal protein (r-protein) cyL38 that segregated independently from the antibiotic-resistant phenotype in crosses.     

New tools for mitochondrial genetics of Chlamydomonas reinhardtii: manganese mutagenesis and cytoduction

Summary A novel and efficient genetic procedure is described for generating mitochondrial mutants of the green alga Chlamydomonas reinhardtii. The development of a mutagenesis procedure using manganese cations and the application of cytoduction techniques resulted in a combined approach for the generation and analysis of mitochondrial mutants. Although mitochondrial mutations are inherited in sexual crosses from the minus mating type parent, the cytoduction technique can be used to transfer mitochondrial mutations into recipient strains with different genetic backgrounds, irrespective of their mating type. Cytoduction allows the transfer of mitochondrial markers from diploid to haploid cells also, which is of great benefit since diploid cells do not germinate in C. reinhardtii. We report here the isolation and characterisation of eight mutants, which are resistant to the antibiotics myxothiazol and mucidin. The mutants all have point mutations in the mitochondrial gene for apocytochrome b. Using in vitro-amplified cytb gene fragments as probes for direct DNA sequencing, three different types of single base pair substitutions were revealed in all mutants tested. In particular, amino acid substitutions in the mutant apocytochrome b polypeptide have been identified at residues 129, 132 and 137, which have been implicated in forming part of an antibiotic-binding niche. The amino acid substitution at position 132 has not been so far described for mutant apocytochrome b in any other organism, prokaryotic or eukaryotic. The genetic approach presented here confirms C. reinhardtii as a model system that is unique among plant cells.     

Mapping of chloroplast genes involved in chloroplast ribosome biogenesis in Chlamydomonas reinhardtii

Summary Chloroplast gene mutations which confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii have been tested for allelism and mapped by recombination analysis of progeny from biparental zygote clones. Thirty-one independently isolated streptomycin resistant mutants have chloroplast ribosomes which are resistant to this drug in an assay based on misreading of isoleucine in response to a poly U template, and comprise one nuclear and four chloroplast gene loci. Four mutants resistant to spectinomycin, and three mutants resistant to neamine and kanamycin, which have chloroplast ribosomes resistant to their respective antibiotics in poly U directed phenylalanine incorporation, appear to map in a single chloroplast gene locus. Representative alleles of this nr/spr locus, the four streptomycin resistance loci, and two chloroplast gene loci for erythromycin resistance, have been analyzed in a series of parallel crosses to establish the following map order for these seven genes in the chloroplast genome: er-u-la-er-u-37-nr-u-2-1/spr-u-1-H-4-sr-u-2-23-sr-u-2-60-sr-u-sm3-sr-u-sm2. These seven genes may constitute a ribosomal region within the chloroplast genome of Chlamydomonas comparable to the ribosomal gene clusters in bacteria.     

Genetics of CHLAMYDOMONAS REINHARDTII Diploids I. Isolation and Characterization and Meiotic Segregation Pattern of a Homozygous Diploid

A strain of Chlamydomonas reinhardtii has been investigated which, when mated with known wild-types, produces very few viable germination products and transmits its Mendelian markers to more than half of those products. Cytogenetic observations, fluorometric measurements of DNA and genetic data all suggest that the strain, d mt^-ery-M3a sr-u-1 is a stable homozygous diploid. This strain has twice as many nuclear chromatin bodies at metaphase and twice as much DNA as its haploid progenitor, and the phenotypes of its meiotic progeny are consistent with predictions based on triploid meiosis. Data from crosses involving d mt^-ery-M3a sr-u-1 and from crosses involving hybrid diploids indicate that the frequency of second division segregation increases in triploid zygotes and that mitotic segregation following triploid meiosis is a frequent event which may more often result from mitotic recombination than from chromosome loss.     

Effects of elevation of strain-ploidy on transmission and recombination of mitochondrial drug resistance genes in Saccharomyces cerevisiae

Summary In order to study the effects of strainploidy on the transmission and recombination of the mitochondrial genes C, E and O conferring the resistance to chloramphenicol, erythromycin and oligomycin, respectively, haploids were crossed to diploids and the results of genetic analysis were compared with those from haploidxhaploid crosses. All haploidx diploid crosses showed an increased transmission of diploid derived alleles, relative to haploid derived ones, but the pattern of increase differed between homosexual and heterosexual crosses. In ^– haploid x ^– diploid homosexual crosses, the increase was of roughly equal magnitude at the C, E and O loci: there was a polar co-transmission of the diploid derived alleles. In ⁺ haploid x ^– diploid heterosexual crosses, on the contrary, a differential increase was observed at the different loci, the magnitude being the smallest at the C locus and the largest at the O locus. As a result, there was a preferential transmission in favor of the haploid derived C alleles and of the diploid derived O alleles. A near equal transmission from both parents was observed for the E alleles. A decrease and an increase in the recombination frequency were noticed in the above haploidxdiploid homosexual and heterosexual crosses, respectively.The above phenomena were ascribed to different dosages of mitochondrial genomes from parents. Experimental data were well accorded with the theoretical expectations which were obtained on the assumptions that diploids contain twice as many mitochondrial genomes as haploids, and that random pairing and recombination would occur among mitochondrial genomes from parents. The elevation of strain-ploidy did not affect the recombination polarity which is under the control of the gene.It was theoretically predicted that a preferential transmission in favor of diploid derived alleles at all the C, E and O loci would be seen in ^– haploid x ⁺ diploid heterosexual crosses as well as in ^+; haploid x ^+; diploid homosexual crosses, but that the magnitude of the polar transmission would vary depending upon the loci in the former crosses, while it would be the same at all the loci in the latter ones. The recombination frequency was predicted to decrease in both of these crosses.