The nucleotide sequence of any genome can be determined rapidly and simply by using highly automated techniques based on several different strategies. Comparison of the genome sequences of different organisms allows us to trace the evolutionary relationships among genes and organisms, and it has proved valuable for discovering new genes and predicting their functions.
Taken together, these techniques for analyzing and manipulating DNA have made it possible to sequence, identify, and isolate genes from any organism of interest. Related technologies allow scientists to produce the protein products of these genes in the large quantities needed for detailed analyses of their structure and function, as well as for medical purposes.
Ultimately, one wishes to determine how genes—and the proteins they encode— function in the intact organism. Although it may seem counterintuitive, one of the most direct ways to find out what a gene does is to see what happens to the organism when that gene is missing. Studying mutant organisms that have acquired changes or deletions in their nucleotide sequences is a time-honored practice in biology and forms the basis of the important field of genetics. Because mutations can disrupt cell processes, mutants often hold the key to understanding gene function. In the classical genetic approach, one begins by isolating mutants that have an interesting or unusual appearance: fruit flies with white eyes or curly wings, for example. Working backward from the phenotype—the appearance or behavior of the individual—one then determines the organism’s genotype, the form of the gene responsible for that characteristic (Panel 8–2).
Today, with numerous genome sequences available, the exploration of gene function often begins with a DNA sequence. Here, the challenge is to translate sequence into function. One approach, discussed earlier in the chapter, is to search databases for well-characterized proteins that have similar amino acid sequences to the protein encoded by a new gene. From there, the protein (or for noncoding genes, the RNA molecule) can be overexpressed and purified and the methods described in the first part of this chapter can be employed to study its three-dimensional structure and its biochemical properties. But to determine directly a gene’s function in a cell or organism, the most effective approach involves studying mutants that either lack the gene or express an altered version of it. Determining which cell processes have been disrupted or compromised in such mutants will usually shed light on a gene’s biological role.
In this section, we describe several approaches to determining a gene’s function, starting either from an individual with an interesting phenotype or from a DNA sequence. We begin with the classical genetic approach, which starts with a genetic screen for isolating mutants of interest and then proceeds toward identification of the gene or genes responsible for the observed phenotype. We then describe the set of techniques that are collectively called reverse genetics, in which one begins with a gene or gene sequence and attempts to determine its function. This approach often involves some intelligent guesswork—searching for similar sequences in other organisms or determining when and where a gene is expressed—as well as generating mutant organisms and characterizing their phenotype.
Before the advent of gene cloning technology, most genes were identified by the abnormalities produced when the gene was mutated. Indeed, the very concept of the gene was deduced from the heritability of such abnormalities. This classical genetic approach—identifying the genes responsible for mutant phenotypes—is most easily performed in organisms that reproduce rapidly and are amenable to genetic manipulation, such as bacteria, yeasts, nematode worms, and fruit flies. Although spontaneous mutants can sometimes be found by examining extremely
PANeL 8–2: Review of Classical Genetics
GENES AND PHENOTYPES Gene: a functional unit of inheritance, usually corresponding to the segment of DNA coding for a single protein. Genome: all of an organism’s DNA sequences. locus:the site of the gene in the genome
Wild-type: the normal, Mutant: differing from the alleles:alternative forms of a gene naturally occurring type wild-type because of a genetic change (a mutation)
GENOTYPE: the specifc set of alleles forming the genome of an individual
PHENOTYPE: the visible character of the individual allele A is dominant (relative to a); allele a is recessive (relative to A)