ness of studying null mutants is worth mentioning in the context of developmental toxicology. Their phenotypes match the toxicologist’s ideal of what the “perfect” toxicant would generate for observation if it completely inhibited just one target component of the organism.

  1. If possible, establish the order of function of the identified genes by constructing double mutants to determine which of two distinguishable phenotypes takes precedence (epistasis test).

  2. Identify additional modifier genes by using suppressor and enhancer screens in a sensitized genetic background for secondary mutations that make the defective phenotype of an existing mutant less or more severe.

  3. Using fine-structure genetic mapping and positional cloning, obtain genomic clones of each gene for molecular analysis and verify their identities by demonstrating that each gene in the corresponding mutant animal carries a DNA sequence alteration.

  4. From suitable complementary (c) DNA libraries of cloned cDNA copies of the animal’s messenger (m) RNA population, isolate cDNAs corresponding to each gene, sequence them to determine the predicted amino acid sequence of each encoded protein, and carry out a similarity search, comparing those sequences with the sequences available in databases, which often can be used to discern motifs and reveal the functional class to which a protein belongs. (Function was initially deduced for the class from other kinds of studies—biochemical, cellular, developmental, and physiological.)

  5. Determine when and where the mRNA and the protein encoded by each gene are found during development by using, respectively, nucleic acid probes and antibodies made to fusion proteins. A faster but sometimes less reliable alternative is to make reporter constructs, which carry the promoter region of the cloned gene fused to a gene encoding a reporter protein that can be detected by its activity (e.g., the E. coli β-galactosidase gene lacZ) or fluorescence (e.g., green fluorescent protein (GFP)). Embryos into which such a construct has been introduced (by DNA transformation) can be observed at various stages to determine when and in what cells and tissues the promoter is active. Generally (but not always), an active provider will reflect the expression pattern of the normal gene.

  6. Supplement that information with genetic mosaic analysis, by producing animals in which only certain cells or tissues are mutant, to discover where a gene must normally function and whether its functions are cell autonomous (i.e., intracellular) or cell nonautonomous (i.e., intercellular).

  7. Isolate and biochemically analyze proteins encoded by the mutationally identified genes to study further the function of the proteins.

All these steps are not always carried out. The most important and difficult step, once mutants have been obtained, has been positional cloning of the gene. However, shortcuts are becoming available with the accumulation of genomic mapping and sequence information and the development of new technologies



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