A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse.

As the human genome project approaches completion, the challenge for mammalian geneticists is to develop approaches for the systematic determination of mammalian gene function. Mouse mutagenesis will be a key element of studies of gene function. Phenotype-driven approaches using the chemical mutagen ethylnitrosourea (ENU) represent a potentially efficient route for the generation of large numbers of mutant mice that can be screened for novel phenotypes. The advantage of this approach is that, in assessing gene function, no a priori assumptions are made about the genes involved in any pathway. Phenotype-driven mutagenesis is thus an effective method for the identification of novel genes and pathways. We have undertaken a genome-wide, phenotype-driven screen for dominant mutations in the mouse. We generated and screened over 26,000 mice, and recovered some 500 new mouse mutants. Our work, along with the programme reported in the accompanying paper, has led to a substantial increase in the mouse mutant resource and represents a first step towards systematic studies of gene function in mammalian genetics.

A missense mutation in the non-neural G-protein alpha-subunit isoforms modulates susceptibility to obesity.

OBJECTIVE: The Gnas transcription unit located within an imprinting region encodes several proteins, including the G-protein alpha-subunit, Gsalpha, its isoform XLalphas and their variant truncated neural forms GsalphaN1 and XLN1. Gsalpha and GsalphaN1 are expressed predominantly from the maternally derived allele in some tissues, whereas XLalphas and XLN1 are expressed exclusively from the paternally derived allele. The relative contribution of full-length Gsalpha and XLalphas, and truncated forms GsalphaN1 and XLN1 to phenotype is unknown. The edematous-small point mutation (Oed-Sml) in exon 6 of Gnas lies downstream of GsalphaN1 and XLN1, but affects full-length Gsalpha and XLalphas, allowing us to address the role of full-length Gsalpha and XLalphas. The aim of this study was therefore to determine the metabolic phenotypes of Oed and Sml mice, and to correlate phenotypes with affected transcripts. METHODS: Mice were fed standard or high-fat diets and weighed regularly. Fat mass was determined by DEXA analysis. Indirect calorimetry was used to measure metabolic rate. Glucose was measured in tolerance tests and biochemical parameters in fasted plasma samples. Histological analysis of fat and liver was carried out post mortem. RESULTS: Oed mice are obese on either diet and have a reduced metabolic rate. Sml mice are lean and are resistant to a high-fat diet and have an increased metabolic rate. CONCLUSION: Adult Oed and Sml mice have opposite metabolic phenotypes. On maternal inheritance, the obese Oed phenotype can be attributed to non-functional full-length Gsalpha. In contrast, on paternal inheritance, Sml mice were small and resistant to the development of obesity on a high-fat diet, effects that can be attributed to mutant XLalphas. Thus, the neural isoforms, GsalphaN1 and XLN1, do not appear to play a role in these metabolic phenotypes.

Investigation of lung tumour induction in BALB/cJ mice following paternal X-irradiation.

Evidence of an enhanced incidence of lung tumours (benign adenomas and adenocarcinomas) was sought in the BALB/cJ mouse following paternal germ cell X-irradiation. In a series of replicate studies spanning approximately 1 year, males were exposed to single, acute X-ray doses of 0, 250 and 500 cGy. In each of the 2 consecutive weeks immediately thereafter they were placed with two females to generate progeny that were derived from irradiated post-meiotic cells (spermatozoa to late spermatids). These animals were then examined at 8 or 12 months for lung tumours. While the proportion of fertile females and mean litter size was affected by the radiation, showing a dose-dependent, dominant lethal response, and while cases of mutant offspring were detected, the paternal radiation did not affect lung tumour incidence in the offspring. The incidence did not vary significantly between germ cell stages irradiated (week of mating), sex of offspring, or radiation dose. However, significant differences between lung tumour incidence (mostly representing benign adenomas) were found between different replicates, these being high at the start of the study, declining and then rising to yet higher levels at its close. The finding that lung tumour incidence in BALB/cJ mice is not affected by paternal germ cell irradiation does not accord with Nomura's reports using other strains of mice. This, in turn, weakens biological support for a causal association between the raised incidence of childhood leukaemia and non-Hodgkin lymphoma near Sellafield and the father's recorded radiation exposure during employment by the nuclear industry.

Investigation of lung tumour induction in C3H/HeH mice, with and without tumour promotion with urethane, following paternal X-irradiation.

In series of papers Nomura has reported that parental irradiation can lead to an enhanced incidence of lung and other tumours. However, in a recent study with BALB/cJ mice, using optimum conditions as defined by Nomura, we were unable to confirm this. We have now repeated the investigation using a different inbred strain, C3H/HeH, with and without tumour promotion in the F1 by urethane, again using protocols defined by Nomura. In a series of replicate studies spanning over 2 years, males were exposed to single, acute doses of 0, 250 and 500 cGy X-rays and thereafter placed with two females each in each of two consecutive weeks. Half the offspring from each treatment group and each week of mating were given 5 mmol/kg body weight of the urethane, while the remainder remained untreated. Most of the offspring produced were killed and scored for lung tumours at 6 months of age, while the rest were examined at 12 months of age. The proportion of fertile females and litter size provided evidence of a dose-dependent mutational response to the paternal irradiation, but no trace of a radiation-enhanced lung tumour incidence was detected among the progeny, whether in the urethane or non-urethane groups at 6 or 12 months of age, and whether assessed by numbers of mice with tumours, clusters of tumours, or cluster size. As seen in the BALB/cJ study, significant differences among different replicates were found, again suggesting a cyclical or seasonal variation in tumour incidence, but the variations seen with the two strains were not the same. The need for concurrent controls for tumour work was, nevertheless, again indicated. The overall findings do not therefore accord with those of Nomura. Furthermore, they do not support the causal association between the raised incidence of childhood leukaemia and non-Hodgkins lymphoma near Sellafield and the father's recorded radiation exposure during employment in the nuclear industry, as suggested by the Gardner report.

Eggs as energy: revisiting the scaling of egg size and energetic content among echinoderms.

Marine organisms exhibit substantial life-history diversity, of which egg size is one fundamental parameter. The size of an egg is generally assumed to reflect the amount of energy it contains and the amount of per-offspring maternal investment. Egg size and energy are thought to scale isometrically. We investigated this relationship by updating published datasets for echinoderms, increasing the number of species over those in previous studies by 62%. When we plotted egg energy versus egg size in the updated dataset we found that planktotrophs have a scaling factor significantly lower than 1, demonstrating an overall trend toward lower energy density in larger planktotrophic eggs. By looking within three genera, Echinometra, Strongylocentrotus, and Arbacia, we also found that the scaling exponent differed among taxa, and that in Echinometra, energy density was significantly lower in species with larger eggs. Theoretical models generally assume a strong tradeoff between egg size and fecundity that limits energetic investment and constrains life-history evolution. These data suggest that the evolution of egg size and egg energy content can be decoupled, possibly facilitating response to selective factors such as sperm limitation which could act on volume alone.