Genes can be isolated by the conservation of exons

KEY TERMS:

• A zoo blot describes the use of Southern blotting to test the ability of a DNA probe from one species to hybridize with the DNA from the genomes of a variety of other species.
• Exon trapping inserts a genomic fragment into a vector whose function depends on the provision of splicing junctions by the fragment.

KEY CONCEPTS:

• Conservation of exons can be used as the basis for identifying coding regions by identifying fragments whose sequences are present in multiple organisms. 

Some major approaches to identifying genes are based on the contrast between the conservation of exons and the variation of introns. In a region containing a gene whose function has been conserved among a range of species, the sequence representing the protein should have two distinctive properties:

• it must have an open reading frame;
• and it is likely to have a related sequence in other species.

These features can be used to isolate genes.

Suppose we know by genetic data that a particular genetic trait is located in a given chromosomal region. If we lack knowledge about the nature of the gene product, how are we to identify the gene in a region that may be (for example) >1 Mb?

A heroic approach that has proved successful with some genes of medical importance is to screen relatively short fragments from the region for the two properties expected of a conserved gene. First we seek to identify fragments that cross-hybridize with the genomes of other species. Then we examine these fragments for open reading frames.

The first criterion is applied by performing a zoo blot. We use short fragments from the region as (radioactive) probes to test for related DNA from a variety of species by Southern blotting. If we find hybridizing fragments in several species related to that of the probe—the probe is usually human—the probe becomes a candidate for an exon of the gene.

The candidates are sequenced, and if they contain open reading frames, are used to isolate surrounding genomic regions. If these appear to be part of an exon, we may then use them to identify the entire gene, to isolate the corresponding cDNA or mRNA, and ultimately to identify the protein.

This approach is especially important when the target gene is spread out because it has many large introns. This proved to be the case with Duchenne muscular dystrophy (DMD), a degenerative disorder of muscle, which is X-linked and affects 1 in 3500 of human male births. The steps in identifying the gene are summarized in Figure 2.10.

Linkage analysis localized the DMD locus to chromosomal band Xp21. Patients with the disease often have chromosomal rearrangements involving this band. By comparing the ability of X-linked DNA probes to hybridize with DNA from patients and with normal DNA, cloned fragments were obtained that correspond to the region that was rearranged or deleted in patients' DNA.

Once some DNA in the general vicinity of the target gene has been obtained, it is possible to "walk" along the chromosome until the gene is reached (see 32.12 Genome mapping). A chromosomal walk was used to construct a restriction map of the region on either side of the probe, covering a region of >100 kb. Analysis of the DNA from a series of patients identified large deletions in this region, extending in either direction. The most telling deletion is one contained entirely within the region, since this delineates a segment that must be important in gene function and indicates that the gene, or at least part of it, lies in this region (Kunkel et al., 1985; Monaco et al., 1985).

Having now come into the region of the gene, we need to identify its exons and introns. A zoo blot identified fragments that cross-hybridize with the mouse X chromosome and with other mammalian DNAs. As summarized in Figure 2.11, these were scrutinized for open reading frames and the sequences typical of exon-intron junctions. Fragments that met these criteria were used as probes to identify homologous sequences in a cDNA library prepared from muscle mRNA.

The cDNA corresponding to the gene identifies an unusually large mRNA, ~14 kb. Hybridization back to the genome shows that the mRNA is represented in >60 exons, which are spread over ~2000 kb of DNA. This makes DMD the longest gene identified; in fact, it is 10× longer than any other known gene (van Ommen et al., 1986; Koenig et al., 1987).

The gene codes for a protein of ~500 kD, called dystrophin, which is a component of muscle, present in rather low amounts. All patients with the disease have deletions at this locus, and lack (or have defective) dystrophin.

Muscle also has the distinction of having the largest known protein, titin, with almost 27,000 amino acids. Its gene has the largest number of exons (178) and the longest single exon in the human genome (17,000 bp).

Another technique that allows genomic fragments to be scanned rapidly for the presence of exons is called exon trapping (Buckler et al., 1991). Figure 2.12 shows that it starts with a vector that contains a strong promoter, and has a single intron between two exons. When this vector is transfected into cells, its transcription generates large amounts of an RNA containing the sequences of the two exons. A restriction cloning site lies within the intron, and is used to insert genomic fragments from a region of interest. If a fragment does not contain an exon, there is no change in the splicing pattern, and the RNA contains only the same sequences as the parental vector. But if the genomic fragment contains an exon flanked by two partial intron sequences, the splicing sites on either side of this exon are recognized, and the sequence of the exon is inserted into the RNA between the two exons of the vector. This can be detected readily by reverse transcribing the cytoplasmic RNA into cDNA, and using PCR to amplify the sequences between the two exons of the vector. So the appearance in the amplified population of sequences from the genomic fragment indicates that an exon has been trapped. Because introns are usually large and exons are small in animal cells, there is a high probability that a random piece of genomic DNA will contain the required structure of an exon surrounded by partial introns. In fact, exon trapping may mimic the events that have occurred naturally during evolution of genes (see 2.9 How did interrupted genes evolve?).