Genome-wide, large-scale production of mutant mice by ENU mutagenesis.

In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html).

Flexibility and interchangeability of polyadenylation signals in Saccharomyces cerevisiae.

Various signal motifs have been reported to be essential for proper mRNA 3'-end formation in the yeast Saccharomyces cerevisiae. However, none of these motifs has been shown to be sufficient to direct 3'-end processing and/or transcription termination. Therefore, several structural motifs have to act in concert for efficient 3'-end formation. In the region upstream of the three polyadenylation sites of the yeast gene for alcohol dehydrogenase I (ADH1), we have identified a hitherto unknown signal sequence contained within the octamer AAAAAAAA. This motif, located 11 nucleotides upstream of the first ADH1 polyadenylation site, is responsible for the utilization of this site in vitro and in vivo, since mutational alteration drastically reduced 3'-end formation at this position. Insertion of 38 ADH1-derived nucleotides encompassing the (A)8 motif into the 3'-end formation-deficient cyc1-512 deletion mutant restored full processing capacity in vitro. Insertion of the octamer alone did not restore 3'-end formation, although mutation of the (A)8 motif in the functional construct had abolished 3'-end processing activity almost completely. This demonstrates that the sequence AAAAAAAA is a necessary, although not sufficient, signal for efficient mRNA 3'-end formation in S. cerevisiae.

Splicing of a circular yeast pre-mRNA in vitro.

We have tested the fate of a circularized synthetic pre-mRNA transcript in a whole cell splicing extract of Saccharomyces cerevisiae. Our results demonstrate that this circular precursor RNA is able to induce spliceosome formation in vitro and that the products of the following splicing reaction are the lariat-shaped intron, and a mature circular mRNA. Thus, it would appear that free 5' and/or 3' ends are not obligatory for a splicing reaction to occur, although we find its efficiency to be strongly influenced by the presence or lack of free ends. To our knowledge, this is the first demonstration that a circular pre-mRNA molecule is recognized as a suitable substrate by an eukaryotic mRNA splicing apparatus.

In vitro generation of a circular exon from a linear pre-mRNA transcript.

Recent findings have firmly established the existence of circular exons in vivo. We were interested in the possible splicing mechanism by which these unusual mRNA molecules could be created in vitro, though no biological relevance has been attached to their existence as yet. In this report we demonstrate that a modified synthetic linear yeast ACT1 transcript whose sequence begins with the 3'-part of its original intron, is continued by 247 nt of exon sequence and terminates with the 5'-part of its intron will generate a circular exon when introduced to standard in vitro splicing reactions in whole cell splice extracts from Saccharomyces cerevisiae. The formation of a circular exon was found to be independent of specific circular or secondary structures of the pre-mRNA transcript. We hypothesize that circular exons which are found in vivo may be generated from pre-mRNAs which derive from rare events of transcription initiation within an intron.

A mutant SV40 large T antigen interferes with nuclear localization of a heterologous protein.

A mutant SV40 genome carrying a frameshift at the carboxyl terminus of the large T antigen failed to replicate SV40 DNA and to transform rat2 cells, although the altered region is known to be dispensable for these functions. The mutant T antigen also failed to localize normally in the nucleus and interfered with nuclear localization of at least one other nuclear protein, adenovirus fiber. A double mutant carrying an additional lesion in the nuclear localization signal was also localized in the cytoplasm, but regained the ability to transform rat2 cells and no longer affected the nuclear localization of fiber protein. We suggest that the frameshift T antigen may disrupt a mechanism required for nuclear localization of proteins.

Comet assay as a tool to screen for mouse models with inherited radiation sensitivity.

Recent in vivo and in vitro data of patients analyzed for genetic susceptibility to radiation during cancer therapy have shown structural changes in the chromosomes to be prevalent both in the patients being treated and in their immediate family members. As structural changes in chromosomes frequently lead to activation of proto-oncogenes and elimination of tumor-suppressor genes, they represent important mechanisms for the initiation of DNA repair processes and tumorigenesis. With the exception of rare genetic syndromes such as AT (Ataxia telangiectasia) or NBS (Nijmegen Breakage Syndrome), the background for the inheritance of genetic susceptibility to radiation is unknown. Recently, a large-scale genetic screen of mouse mutants has been established within the German Human Genome Project (Hrabè de Angelis and Balling 1998). The goal of this ENU (ENU: ethylnitrosourea) mutagenesis screen is the generation of mutant mice that will serve as animal models for human diseases and genetic susceptibility. In order to fully utilize the potential of a genetic screen of this magnitude, in which exploration for genes responsible for genomic instability and radiation sensitivity is to occur, it is necessary to establish a simple assay system that is amenable to automation. Hence, we are using the single-cell gel electrophoresis (comet assay) to detect mouse mutants that display a genetic susceptibility to ionizing radiation. We have established the analysis parameters in the comet assay which are currently used to detect radiation-sensitive mouse mutants and to control the variance within the mouse population in the ENU screen. The assay can be used to isolate genes that are responsible for DNA repair and radiation sensitivity in mouse and human.