Optimization of ENU mutagenesis of Caenorhabditis elegans.

Chemical mutagenesis of Caenorhabditis elegans has relied primarily on EMS to produce missense mutations. The drawback of EMS mutagenesis is that the molecular lesions are primarily G/C --> A/T transitions. ENU has been shown to produce a different spectrum of mutations, but its greater toxicity to C. elegans makes it a difficult mutagen to use. We describe here methods for minimizing ENU toxicity in C. elegans. Methods include preparing ENU stocks in absolute ethanol and storing stock solutions for not more than 2 weeks at -20 degrees C. To maintain reasonable brood sizes of mutagenized animals, mutagenic solutions should not exceed 1.0mM ENU. We provide data which suggest ENU is degraded or altered to more toxic products in aqueous solution, but less so in solvents such as absolute ethanol.

Characterization of revertants of unc-93(e1500) in Caenorhabditis elegans induced by N-ethyl-N-nitrosourea.

Phenotypic reversion of the rubber-band, muscle-defective phenotype conferred by unc-93(e1500) was used to determine the utility of N-ethyl-N-nitrosourea (ENU) as a mutagen for genetic research in Caenorhabditis elegans. In this system, ENU produces revertants at a frequency of 3 x 10(-4), equivalent to that of the commonly used mutagen, EMS. The gene identity of 154 ENU-induced revertants shows that the distribution of alleles between three possible suppressor genes differs from induced by EMS. A higher percentage of revertants are alleles of unc-93 and many fewer are alleles of sup-9 and sup-10. Three revertants complement the three known suppressor genes; they may therefore identify a new gene product(s) involved in this system of excitation-contraction coupling in C. elegans. Molecular characterization of putative unc-93 null alleles reveals that the base changes induced by ENU are quite different from those induced by EMS; specifically we see an increased frequency of A/T-->G/C transitions. The frequency of ENU-induced intragenic deletions is found to be 13%. We suggest that ENU, at concentrations below 5 mM, will be a superior mutagen for studies of protein function in C. elegans.

Interaction between 16S ribosomal RNA and ribosomal protein S12: differential effects of paromomycin and streptomycin.

Strains containing a series of restrictive and non-restrictive mutations in ribosomal protein S12 have been transformed with plasmids carrying the rrnB operon with mutations at positions 1409 and 1491 in 16S rRNA. The effects of the double-mutant constructs have been measured by growth rate, paromomycin and streptomycin sensitivity, resistance and dependence. The results demonstrate a functional interaction between the 1409-1491 region of rRNA and ribosomal protein S12.

Effects of mutagenesis of C912 in the streptomycin binding region of Escherichia coli 16S ribosomal RNA.

Four different mutations were produced at position 912 of Escherichia coli 16S rRNA in the multicopy plasmid pKK3535. Cells transformed with the mutant plasmids were assayed for growth in steptomycin. The U912 mutant conferred low level streptomycin resistance as reported originally by Montandon and co-workers (EMBO J 1986; 5:3705-3708). The G912 mutant also gave low level resistance but, unlike U912, caused significant retardation in growth rate and tended to select for fast-growing revertants. The A912 mutant was without effect on growth rate or streptomycin sensitivity, while deletion of C912 was lethal. Cells with U912 were selected for increased streptomycin resistance (MIC up to 160 micrograms/ml) and then cured of the plasmid. The cured cells retained a higher level of streptomycin resistance (MIC: 80 micrograms/ml) than the original wild type strain (MIC: 10 micrograms/ml), but sequencing by reverse transcriptase showed no evidence of U912 in the cellular 16S rRNA. Thus, recombination of the plasmid-coded U912 mutation into host rrn operons was not the mechanism by which increased streptomycin resistance occurred. The plasmid with U912 was transformed into three different streptomycin-dependent strains to determine whether the rRNA mutation, which presumably alters streptomycin binding, was compatible with S12 mutations which require bound streptomycin in order to function properly. In one strain, no transformants could be isolated, indicating that the plasmid was lethal. The two other streptomycin-dependent strains were transformed, but ribosomes containing the mutant rRNA were non-functional.

Effects of mutagenesis of a conserved base-paired site near the decoding region of Escherichia coli 16 S ribosomal RNA.

Eleven of 15 possible single and double mutations were constructed in a cloned copy of Escherichia coli 16 S rDNA at a base-paired site, 1409-1491. Expression of any of these mutations was detrimental to the growth of E. coli. Mutations that substituted unpaired purine bases were lethal in the system described. Otherwise, the degree of detrimental effect on growth-rate was not directly correlated with specific rRNA primary or secondary structures. Using reverse transcription of rRNA isolated from subunits or 70 S ribosomes, we were able to determine the amount of mutant rRNA used in translation. From these experiments, we found that the lethal mutations appeared to be selectively excluded from the pool of 70 S ribosomes following expression from a repressible plasmid. In contrast, a non-lethal mutation was present in subunits, ribosomes and polysomes in approximately equal amounts. Mutations that disrupted base-pairing were found to confer varying levels of resistance to nine aminoglycosides, including four neomycins, two kanamycins, gentamicin, apramycin and hygromycin. A high frequency of reversion from resistant and slow-growth phenotypes due to a host mutation was observed.

Mutations in 16S ribosomal RNA disrupt antibiotic--RNA interactions.

Two of six mutations at a base-paired site in Escherichia coli 16S rRNA confer resistance to nine different aminoglycoside antibiotics in vivo. Chemical probing of mutant and wild-type ribosomes in the presence of paromomycin indicates that interactions between the antibiotic and 16S rRNA in mutant ribosomes are disrupted. The altered interactions measured in vitro correlate precisely with resistance seen in vivo and may be attributable to specific structural changes observed in the mutant rRNA.