Direct repeats

Finally, 5' and 3' ends of DNA strands are ligated, completing the insertion (step 7 ). These last steps (6 and 7 ) probably are catalyzed by the same cellular enzymes that remove RNA primers and ligate okazaki fragments during DNA replication (see Figure 4-33). The complete process results in insertion of a copy of the original LINE retrotransposon into a new site in chromosomal DNA. A short direct repeat is generated at the insertion site because of the initial staggered cleavage of the two chromosomal DNA strands (step 1 ).

The vast majority of LINEs in the human genome are truncated at their 5' end, suggesting that reverse transcription terminated before completion and the resulting fragments extending variable distances from the poly(A) tail were inserted. Because of this shortening, the average size of LINE elements is only about 900 base pairs, whereas the full-length sequence is =6 kb long. In addition, nearly all the full-length elements contain stop codons and frameshift mutations in ORF1 and ORF2; these mutations probably have accumulated in most LINE sequences over evolutionary time. As a result of truncation and mutation, only =0.01 percent of the LINE sequences in the human genome are full-length with intact open reading frames for ORF1 and ORF2, =60-100 in total.

SINEs The second most abundant class of mobile elements in the human genome, SINEs constitute =13 percent of total human DNA. Varying in length from about 100 to 400 base pairs, these retrotransposons do not encode protein, but most contain a 3' A/T-rich sequence similar to that in LINEs. SINEs are transcribed by RNA polymerase III, the same nuclear RNA polymerase that transcribes genes encoding tRNAs, 5S rRNAs, and other small stable RNAs (Chapter 11). Most likely, the ORF1 and ORF2 proteins expressed from full-length LINEs mediate transposition of SINEs by the retrotransposition mechanism depicted in Figure 10-16.

SINEs occur at about 1.6 million sites in the human genome. Of these, =1.1 million are Alu elements, so named because most of them contain a single recognition site for the restriction enzyme AluI. Alu elements exhibit considerable sequence homology with and may have evolved from 7SL RNA, a component of the signal-recognition particle. This abundant cytosolic ribonucleoprotein particle aids in targeting certain polypeptides, as they are being synthesized, to the membranes of the endoplasmic reticulum (Chapter 16). Alu elements are scattered throughout the human genome at sites where their insertion has not disrupted gene expression: between genes, within introns, and in the 3' untranslated regions of some mRNAs. For instance, nine Alu elements are located within the human p-globin gene cluster (see Figure 10-3b). The overall frequency of L1 and SINE retrotranspo-sitions in humans is estimated to be about one new retro-transposition in very eight individuals, with =40 percent being L1 and 60 percent SINEs, of which =90 percent are Alu elements.

Similar to other mobile elements, most SINEs have accumulated mutations from the time of their insertion in the germ line of an ancient ancestor of modern humans. Like LINEs, many SINEs also are truncated at their 5' end. Table 10-1 summarizes the major types of interspersed repeats derived from mobile elements in the human genome.

In addition to the mobile elements listed in Table 10-1, DNA copies of a wide variety of mRNAs appear to have integrated into chromosomal DNA. Since these sequences lack introns and do not have flanking sequences similar to those of the functional gene copies, they clearly are not simply duplicated genes that have drifted into nonfunctionality and become pseudogenes, as discussed earlier (Figure 10-3a). Instead, these DNA segments appear to be retrotransposed copies of spliced and polyadenylated (processed) mRNA. Compared with normal genes encoding mRNAs, these inserted segments generally contain multiple mutations, which are thought to have accumulated since their mRNAs were first reverse-transcribed and randomly integrated into the genome of a germ cell in an ancient ancestor. These nonfunctional genomic copies of mRNAs are referred to as processed pseudogenes. Most processed pseudogenes are flanked by short direct repeats, supporting the hypothesis that they were generated by rare retrotransposition events involving cellular mRNAs.

Other moderately repetitive sequences representing partial or mutant copies of genes encoding small nuclear RNAs (snRNAs) and tRNAs are found in mammalian genomes. Like processed pseudogenes derived from mRNAs, these nonfunctional copies of small RNA genes are flanked by short direct repeats and most likely result from rare retro-transposition events that have accumulated through the course of evolution. Enzymes expressed from a LINE are thought to have carried out all these retrotransposition events involving mRNAs, snRNAs, and tRNAs.

Mobile DNA Elements Probably Had a Significant Influence on Evolution

Although mobile DNA elements appear to have no direct function other than to maintain their own existence, their presence probably had a profound impact on the evolution of modern-day organisms. As mentioned earlier, about half the spontaneous mutations in Drosophila result from insertion of a mobile DNA element into or near a transcription unit. In mammals, however, mobile elements cause a much smaller proportion of spontaneous mutations: «10 percent in mice and only 0.1-0.2 percent in humans. Still, mobile elements have been found in mutant alleles associated with several human genetic diseases.

In lineages leading to higher eukaryotes, homologous recombination between mobile DNA elements dispersed throughout ancestral genomes may have generated gene duplications and other DNA rearrangements during evolution (see Figure 10-4). For instance, cloning and sequencing of the ^-globin gene cluster from various primate species has provided strong evidence that the human C7 and A7 genes arose from an unequal homologous crossover between two L1 sequences flanking an ancestral globin gene. Subsequent divergence of such duplicated genes could lead to acquisition of distinct, beneficial functions associated with each member of a gene family. Unequal crossing over between mobile elements located within introns of a particular gene could lead to the duplication of exons within that gene. This process most likely influenced the evolution of genes that contain multiple copies of similar exons encoding similar protein domains, such as the fibronectin gene (see Figure 4-15).

Some evidence suggests that during the evolution of higher eukaryotes, recombination between interspersed repeats in introns of two separate genes also occurred, generating new genes made from novel combinations of preexisting exons (Figure 10-17). This evolutionary process, termed exon shuffling, may have occurred during evolution of the genes encoding tissue plasminogen activator, the Neu receptor, and epidermal growth factor, which all contain an EGF domain (see Figure 3-8). In this case, exon shuffling presumably resulted in insertion of an EGF domain-encoding exon into an intron of the ancestral form of each of these genes.

Both DNA transposons and LINE retrotransposons have been shown to occasionally carry unrelated flanking sequences when they insert into new sites by the mechanisms diagrammed in Figure 10-18. These mechanisms likely also contributed to exon shuffling during the evolution of contemporary genes.

► FIGURE 10-17 Exon shuffling via recombination between homologous interspersed repeats. Recombination between interspersed repeats in the introns of separate genes produces transcription units with a new combination of exons. In the example shown here, a double crossover between two sets of Alu repeats results in an exchange of exons between the two genes.

Gene 1 Gene 2

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