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Changes in gene regulation underlie much of phenotypic evolution.sup.1. However, our understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations.sup.2. Using an automated robotics pipeline, we surveyed an unbiased mutation library for a developmental enhancer in Drosophila melanogaster. We found that almost all mutations altered gene expression and that parameters of gene expression--levels, location, and state--were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution. A robotic pipeline is used to survey a library of mutations in a Drosphila gene enhancer, showing that most mutations altered gene expression and had widespread pleiotropic effects that are likely to constrain regulatory evolution., Author(s): Timothy Fuqua [sup.1] [sup.2] , Jeff Jordan [sup.3] , Maria Elize van Breugel [sup.1] , Aliaksandr Halavatyi [sup.1] , Christian Tischer [sup.1] , Peter Polidoro [sup.3] , Namiko Abe [...]

Forward genetic analysis is one of the principal advantages of the zebrafish model system. However, managing zebrafish mutant lines derived from mutagenesis screens and mapping the corresponding mutations and integrating them into the larger collection of mutations remain arduous tasks. To simplify and focus these endeavors, we developed an approach that facilitates the rapid mapping of new zebrafish mutations as they are generated through mutagenesis screens. We selected a minimal panel of 149 simple sequence length polymorphism markers for a first-pass genome scan in crosses involving C32 and SJD inbred lines. We also conducted a small chemical mutagenesis screen that identified several new mutations affecting zebrafish embryonic melanocyte development. Using our first-pass marker panel in bulked-segregant analysis, we were able to identify the genetic map positions of these mutations as they were isolated in our screen. Rapid mapping of the mutations facilitated stock management, helped direct allelism tests, and should accelerate identification of the affected genes. These results demonstrate the efficacy of coupling mutagenesis screens with genetic mapping.

Using selectable genes as proof of principle, a new high-through-put genotype-based mutation screen in mouse embryonic stem (ES) cells was developed [Chen et al. (2002) Nat. Genet. 24, 314-317]. If expanded to nonselectable genes, this approach would allow one to proceed quickly from sequence to whole-animal phenotypes. Here data are presented showing that a screen of a cryopreserved library of clonal, germ line competent, N-ethyl-N-nitrosurea (ENU) mutagenized ES cells can identify a large series of allelic mutations in Smad2 and Smad4, two nonselectable genes of the transforming growth factor [beta] superfamily of signaling molecules. Whole animal phenotypic analyses of some of these alleles provided evidence for novel developmental processes mediated by these components of transforming growth factor [beta] signaling, demonstrating the utility of non-null alleles created by chemical mutagens. The accurately assessed mutation load of the ES cell library indicates that it is a valuable resource for developing mouse lines for genetic and functional studies. This methodology can conceptually be applied for the generation of an allelic series of subtle mutations at any locus of interest in the mouse.

The detection of a point mutation using dual-signaling probes on CMS-DNA arrays coupled to an electrochemical reader is described. The results show that rapid and accurate detection of a single-base mismatch is achieved by using these dual-signaling probes on CMS-DNA chips.

Thirteen flouride-resistant mutants of nematode Caenorhabditis elegans were mapped to five genes, wherein the mutants of class 1 genes flr-1X, flr-3 IV and flr-4X are immune to 400 micrograms per milliliter of NaF. Phenotypes of double mutants revealed that class 2 mutations are epistatic to Class I mutations with regard to brood size and growth rate, and hypostatic with regard to fluoride resistance. Analyses of these mutants will facilitate the discovery of signal transductions that regulate the development of C. elegans.

Forty five Caenorhabditis elegans mutants were identified using lectins from wheatgerm and soybeans. The recessive mutations spans six genes, namely srf-2, srf-3, srf-4, srf-5, srf-8 and srf-9. srf-2, -3 and -5 mutants are essentially normal except for their different lectin-binding reaction. On the other hand, the other three mutants possess various structural abnormalities in addition to aberant lectin reaction. Among these defects are aberant egg-laying, movement incoordination and abnormal morphogenesis of the copulatory bursa.