A phenotype-driven ENU mutagenesis screen for the identification of dominant mutations involved in alcohol consumption

The aim of this study was the application of a phenotype-driven N-ethyl-N-nitrosourea (ENU) mutagenesis screen in mice for the identification of dominant mutations involved in the regulation and modulation of alcohol-drinking behavior. The chemical mutagen ENU was utilized in the generation of 131 male ENU-mutant C57BL/6J mice (G0). These ENU-treated mice were paired with wild-type C57BL/6J mice to generate G1 and subsequent generations. In total, 3327 mice were generated. Starting with G1, mice were screened for voluntary oral self-administration of 10% (v/v) alcohol vs. water in a two-bottle paradigm. From these mice, after a total period of 5 weeks of drinking, 43 mutants fulfilled the criteria of an “alcohol phenotype,” that is, high or low ethanol intake. They were then selected for breeding and tested in a “confirmation cross” (G2–G4) for inheritance. Although we did not establish stable high or low drinking lines, several results were obtained in the context of alcohol consumption. First, female mice drank more alcohol than their male counterparts. Second, the former demonstrated greater infertility. Third, all animals displayed relatively stable alcohol intake, although significantly different in two different laboratories. Finally, seasonal and monthly variability was observed, with the highest alcohol consumption occurring in spring and the lowest in autumn. In conclusion, it seems difficult to identify dominant mutations involved in the modulation or regulation of voluntary alcohol consumption via a phenotype-driven ENU mutagenesis screen. In accordance with the findings from knockout studies, we suggest that mainly recessive mutations contribute to an alcohol-drinking or alcohol-avoiding phenotype.     

Novel loci and a role for nitric oxide for seed dormancy and preharvest sprouting in barley

Barley is used for food and feed, and brewing. Nondormant seeds are required for malting, but the lack of dormancy can lead to preharvest sprouting (PHS), which is also undesired. Here, we report several new loci that modulate barley seed dormancy and PHS. Using genome‐wide association mapping of 184 spring barley genotypes, we identified four new, highly significant associations on chromosomes 1H, 3H, and 5H previously not associated with barley seed dormancy or PHS. A total of 71 responsible genes were found mostly related to flowering time and hormone signalling. A homolog of the well‐known Arabidopsis Delay of Germination 1 (DOG1) gene was annotated on the barley chromosome 3H. Unexpectedly, DOG1 appears to play only a minor role in barley seed dormancy. However, the gibberellin oxidase gene HvGA20ox1 contributed to dormancy alleviation, and another seven important loci changed significantly during after‐ripening. Furthermore, nitric oxide release correlated negatively with dormancy and shared 27 associations. Origin and growth environment affected seed dormancy and PHS more than did agronomic traits. Days to anthesis and maturity were shorter when seeds were produced under drier conditions, seeds were less dormant, and PHS increased, with a heritability of 0.57–0.80. The results are expected to be useful for crop improvement.     

Cloning of the genes for a 4-sulphocatechol-oxidizing protocatechuate 3,4-dioxygenase from Hydrogenophaga intermedia S1 and identification of the amino acid residues responsible for the ability to convert 4-sulphocatechol

The genes for a protocatechuate 3,4-dioxygenase (P34O-II) with the ability to oxidize 4-sulphocatechol were cloned from the 4-aminobenzenesulphonate(sulphanilate)-degrading bacterium Hydrogenophaga intermedia strain S1 (DSMZ 5680). Sequence comparisons of the deduced amino acid sequences of both subunits of the P34O-II from H. intermedia S1 (PcaH-II and PcaG-II) with those of another P34O-II, previously obtained from Agrobacterium radiobacter S2, and the corresponding sequences from the protocatechuate 3,4-dioxygenases from other bacterial genera demonstrated that seven amino acid residues, which were conserved in all previously known P34Os (P34O-Is), were different in both P34O-IIs. According to previously published structural data for the P34O of Pseudomonas putida only two of these amino acid residues were located near the catalytical centre. The respective amino acid residues were mutated in the P34O-I from A. radiobacter S2 by site-specific mutagenesis, and it was found that a single amino acid exchange enabled the protocatechuate converting P34O also to oxidize 4-sulphocatechol.