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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 9.3Mobile DNA

Much of the discussion throughout this book deals with the functions of the gene products expressed from proteincoding genes and RNA genes. However, noncoding repetitious DNA, consisting of simple-sequence DNA and moderately repeated DNA, constitutes a significant fraction of the genomic DNA in higher eukaryotes. In this section, we focus on moderately repeated DNA sequences, or mobile DNA elements, which are interspersed throughout the genomes of higher plants and animals. Although mobile DNA elements, ranging from hundreds to a few thousand base pairs in length, originally were discovered in eukaryotes, they also are found in prokaryotes. The process by which these sequences are copied and inserted into a new site in the genome is called transposition. Mobile DNA elements (or simply mobile elements) are essentially molecular parasites, which appear to have no specific function in the biology of their host organisms, but exist only to maintain themselves. For this reason, Francis Crick referred to these sequences as “selfish DNA.”

The transposition of mobile DNA elements is believed to have resulted in their slow accumulation in eukaryotic genomes over evolutionary time. These elements also are lost at a very slow rate by deletion of segments of DNA containing them and by accumulation of mutations until they can no longer be recognized to be related to the original mobile DNA element. Since mobile elements are eliminated from eukaryotic genomes so slowly, they have accumulated to the point where they now constitute a significant portion of the genomes of many eukaryotes.

Movement of Mobile Elements Involves a DNA or RNA Intermediate

Barbara McClintock discovered the first mobile elements while doing classical genetic experiments in maize (corn) during the 1940s. She characterized genetic entities that could move into and back out of genes, changing the phenotype of corn kernels. Her theories were very controversial until similar mobile elements were discovered in bacteria, where they were characterized as specific DNA sequences, and the molecular basis of their transposition, was deciphered. When bacterial mobile elements were first discovered, researchers did not initially link them to moderately repeated DNA, which had been previously identified in reassociation experiments with eukaryotic DNA. However, as the wide variations in the amounts and chromosomal positions of eukaryotic intermediate repeats were documented, their similarity with bacterial mobile elements was recognized. Thus the study of moderately repeated DNA in eukaryotes converged with research on mobile DNA elements in bacteria, although at first there was no apparent connection between the two classes of DNA.

As research on mobile elements progressed, they were found to fall into two categories: (1) those that transpose directly as DNA and (2) those that transpose via an RNA intermediate transcribed from the mobile element by an RNA polymerase and then converted back into double-stranded DNA by a reverse transcriptase (Figure 9-10). Mobile elements that transpose through a DNA intermediate are generally referred to as transposons. (As discussed below, this term has a more specific meaning in reference to bacterial mobile elements.) Mobile elements that transpose to new sites in the genome via an RNA intermediate are called retrotransposons because their movement is analogous to the infectious process of retroviruses (see Figure 6-22). Indeed, retroviruses can be thought of as retrotransposons that evolved genes encoding viral coats, thus allowing them to transpose between cells. Both transposons and retrotransposons can be further classified based on their specific mechanism of transposition, as summarized in Table 9-3. We describe the structure and movement of the various types of mobile elements and then consider their likely role in evolution.

Figure 9-10. Classification of mobile elements into two major classes.

Figure 9-10

Classification of mobile elements into two major classes. (a) Insertion sequences and transposons (orange) move via a DNA intermediate. (b) Retrotransposons (green) are first transcribed into an RNA molecule, which then is reverse-transcribed into double-stranded (more...)

Table 9-3. Major Types of Mobile DNA Elements.

Table 9-3

Major Types of Mobile DNA Elements.

Mobile Elements That Move as DNA Are Present in Prokaryotes and Eukaryotes

Most mobile elements in bacteria transpose directly as DNA. In contrast, most mobile elements in eukaryotes are retrotransposons, but some eukaryotic transposons have been identified. Indeed, the original mobile elements discovered by Barbara McClintock are transposons.

Bacterial Insertion Sequences

The first molecular understanding of mobile elements came from the study of certain E. coli mutations resulting from the spontaneous insertion of a DNA sequence, ≈1 – 2 kb long, into the middle of a gene. These inserted stretches of DNA — called insertion sequences, or IS elements — were first visualized by analyzing hybrids (heteroduplexes) of wild-type and mutant DNAs in the electron microscope. Because the IS element integrated into the mutant strand has no complement in the wild-type strand, it cannot hybridize and forms a visible single-stranded loop extending from the rest of the double-stranded heteroduplex. So far, more than 20 different IS elements have been found in E. coli and other bacteria.

IS elements appear to be molecular parasites of bacterial cells. Transposition of an IS element is a very rare event, occurring in only one in 105 – 107 cells per generation, depending on the IS element. Higher rates of transposition would probably result in too great a mutation rate for the host cell. At a very low rate of transposition, most host cells survive and therefore propagate the parasitic IS element. Even though many transpositions inactivate essential genes, killing the host cell and the IS elements it carries, other host cells survive. Since IS elements transpose into approximately random sites, some transposed sequences enter nonessential regions of the genome (e.g., regions between genes), thereby expanding the number of IS elements in a cell. IS elements also can insert into plasmids or lysogenic viruses, which can be transferred to other cells. When this happens, IS elements can transpose into the chromosomes of virgin cells.

The general structure of IS elements is diagrammed in Figure 9-11. An inverted repeat, usually containing ≈50 base pairs, invariably is present at each end of an insertion sequence. Between the inverted repeats is a protein-coding region, which encodes one or two enzymes required for transposition of an IS element to a new site. In either case, IS-encoded proteins are expressed at a very low rate, accounting for the very low frequency of transposition. An important hallmark of IS elements is the presence of short direct repeats, containing 5 – 11 base pairs, immediately adjacent to both ends of the inserted element. The length of the direct repeat is characteristic of each type of IS element, but its sequence depends on the target site where a particular copy of the IS element is inserted. When the sequence of a mutated gene containing an IS element is compared to the sequence of the wild-type gene before insertion, only one copy of the short direct- repeat sequence is found in the wild-type gene. Duplication of this target-site sequence to create the second direct repeat adjacent to an IS element occurs during the insertion process.

Figure 9-11. General structure of bacterial IS elements.

Figure 9-11

General structure of bacterial IS elements. The central region, which encodes one or two enzymes required for transposition, is flanked by inverted repeats whose sequence is characteristic of a particular IS element. The 5′ and 3′ short (more...)

The enzyme that catalyzes transposition of an IS element is called a transposase. In the simplest mechanism of transposition, an IS element is excised from one location and inserted at a new position in the bacterial chromosome by a nonreplicative process (Figure 9-12). In this mechanism, transposase molecules bind to the inverted-repeat sequences present at each end of the IS element in the donor DNA and cleave the DNA, precisely excising the element. Transposase molecules also bind to and make staggered cuts in a short sequence in the target DNA, generating single-stranded tails. This remarkable enzyme then ligates the 3′ termini of the IS element to the 5′ ends of the cut donor DNA. A DNA polymerase encoded by the host cell then extends the 3′ ends of the target site, filling in the single-stranded gaps and generating a short repeat of the target-site sequence at either end of the newly inserted IS element. This is the origin of the short direct repeats that flank IS elements. Some IS elements transpose by a more complicated replicative mechanism; in this case, a copy of the original IS element is generated in the target DNA and the original copy is retained in the donor DNA.

Figure 9-12. Model for nonreplicative transposition of bacterial insertion sequences.

Figure 9-12

Model for nonreplicative transposition of bacterial insertion sequences. Step 1: A transposase, which is encoded by the IS element (IS10 in this example), cleaves both strands of the donor DNA between the terminal direct repeats (light blue) and the inverted (more...)

Bacterial Transposons

In addition to IS elements, bacteria contain composite mobile genetic elements that are larger than IS elements and contain one or more protein-coding genes in addition to those required for transposition. Referred to as bacterial transposons, these elements are composed of an antibiotic-resistance gene flanked by two copies of the same type of IS element (Figure 9-13). Insertion of a transposon into plasmid or chromosomal DNA is readily detectable because of the acquired resistance to an antibiotic. Transposition produces a short direct repeat of the target site on either side of the newly integrated transposon, just as for IS elements.

Figure 9-13. General structure of bacterial transposons, such as Tn9 of E. coli.

Figure 9-13

General structure of bacterial transposons, such as Tn9 of E. coli. This transposon consists of a chloramphenicol-resistance gene (dark blue) flanked by two copies of IS1 (orange), one of the smallest IS elements. Other copies of IS1, without the drug-resistance (more...)

Transposons are very valuable tools for the bacterial geneticist. They can be introduced into cells on plasmids or viral genomes. Once transferred into a cell, transposons can act as mutagens that affect only a single cellular gene. Although transposition is a rare event, mutagenized cells are readily isolated because of their newly acquired antibiotic resistance gene. The site of the transposon-generated mutation can be determined readily by restriction-enzyme mapping, which reveals the insertion of the large transposon DNA. The precise sequence of bacterial DNA at the site of insertion can then be determined by dideoxy DNA sequencing using a primer complementary to the known sequence of the inverted repeats at the ends of the transposon (see Figure 7-29).

Eukaryotic Transposons

McClintock’s original discovery of mobile elements came from observation of certain spontaneous mutations that affect production of any of the several enzymes required to make anthocyanin, a purple pigment. Mutant kernels are white, and wild-type kernels are purple. One class of these mutations is revertible at high frequency, whereas a second class of mutations does not revert unless they occur in the presence of the first class of mutations. McClintock called the agent responsible for the first class of mutations the activator (Ac) element and those responsible for the second class dissociation (Ds) elements because they also tended to be associated with chromosome breaks.

Many years after McClintock’s pioneering discoveries, cloning and sequencing revealed that Ds elements are deleted forms of the Ac element in which a portion of the sequence encoding transposase is missing. Because it does not encode a functional transposase, a Ds element cannot move by itself. However, in plants that carry the Ac element and thus express a functional transposase, Ds elements can move. The structure of these eukaryotic elements are similar to bacterial IS elements, and they appear to move by the nonreplicative mechanism shown in Figure 9-12.

Since McClintock’s early work on mobile elements in corn, transposons have been identified in other eukaryotes. For instance, approximately half of all the spontaneous mutations observed in Drosophila are due to the insertion of mobile elements. Although most of the mobile elements in Drosophila function as retrotransposons, at least one —  the P element — functions as a transposon, moving by a nonreplicative mechanism similar to that used by bacterial insertion sequences. Current methods for constructing transgenic Drosophila depend on engineered, high-level expression of the P-element transposase and use of the terminal repeats as targets for transposition (see Figure 8-37).

Viral Retrotransposons Contain LTRs and Behave Like Retroviruses in the Genome

All eukaryotes studied from yeast to humans contain retrotransposons, mobile DNA elements that transpose through an RNA intermediate utilizing a reverse transcriptase (see Figure 9-10b). These mobile elements are divided into two major categories, viral and nonviral retrotransposons. Viral retrotransposons, which we discuss in this section, are abundant in yeast (e.g., Ty elements) and in Drosophila (e.g., copia elements). In mammals, nonviral retrotransposons are the most common type of mobile element; these are described in the next section. Still, viral retrotransposons are estimated to account for ≈4 percent of human DNA.

The general structure of viral retrotransposons found in eukaryotes is depicted in Figure 9-14. In addition to short 5′ and 3′ direct repeats typical of all mobile elements, viral retrotransposons are marked by the presence of ≈250- to 600-bp long terminal repeats (LTRs) flanking the central protein-coding region. LTRs are characteristic of integrated retroviral DNA and are critical to the life cycle of retroviruses. Moreover, Ty elements and copia encode three of the four proteins encoded by retroviral DNA. These similarities suggest that transposition of mobile elements like Ty and copia involves mechanisms similar to those whereby retroviral DNA is integrated into a host-cell genome and the retroviral RNA genome is generated (see Figure 6-22).

Figure 9-14. General structure of eukaryotic viral retrotransposons.

Figure 9-14

General structure of eukaryotic viral retrotransposons. The central protein-coding region is flanked by two long terminal repeats (LTRs), which are element-specific direct repeats. LTRs, the hallmark of these mobile elements, also are present in retroviral DNA. (more...)

We first consider the distinct functions of the two LTRs of integrated retroviral DNA in generating retroviral genomic RNA, which corresponds to the RNA intermediate in transposition of Ty elements and copia. As depicted in Figure 9-15, the leftward LTR functions as a promoter that directs host-cell RNA polymerase II to initiate transcription at the 5′ nucleotide of the R sequence. After the entire retroviral DNA has been transcribed, the RNA sequence corresponding to the rightward LTR directs host-cell RNAprocessing enzymes to cleave the primary transcript and add a poly(A) tail at the 3′ end of the R sequence. The resulting retroviral RNA genome lacks complete LTRs. However, after a virus infects a cell, reverse transcription of the RNA genome by virus-encoded reverse transcriptase yields a double-stranded DNA containing LTRs (Figure 9-16). Integrase, another enzyme encoded by retroviruses, then inserts the double-stranded retroviral DNA into the host-cell genome; in this process, short direct repeats of the target-site sequence are generated at either end of the inserted viral DNA sequence. Like retroviral DNA, Ty elements and copia encode reverse transcriptase and integrase; these enzymes are thought to function in transposition by converting the RNA intermediate into DNA and inserting the DNA into the target site in a manner similar to retroviruses.

Figure 9-15. Generation of retroviral genomic RNA from integrated retroviral DNA.

Figure 9-15

Generation of retroviral genomic RNA from integrated retroviral DNA. The short direct repeat sequences (light blue) of target-site DNA are generated during integration of the retroviral DNA into the host-cell genome. The left LTR directs cellular RNA (more...)

Figure 9-16. Generation of LTRs during reverse transcription of retroviral genomic RNA.

Figure 9-16

Generation of LTRs during reverse transcription of retroviral genomic RNA. A complicated series of nine events generates a double-stranded DNA copy of the single-stranded RNA genome of a retrovirus (top). The genomic RNA is packaged in the virion with (more...)

Although these considerations imply that Ty elements transpose through an RNA intermediate, the experiments depicted in Figure 9-17 provided strong functional evidence for this conclusion. Ty elements normally transpose at a very low rate, probably because random insertion of Ty elements into the yeast genome, which contains relatively little spacer and intron DNA, would often inactivate genes. However, when yeast cells were transformed with plasmids containing a Ty element cloned next to a galactose-sensitive promoter, the production of Ty mRNA and Ty-element transposition was much higher in the presence of galactose than in its absence (experiment 1). This increased Ty transposition resulted from an increase in the amount of Ty mRNA, which could function as a template for reverse transcription, and in the amount of reverse transcriptase and integrase expressed from the Ty element. An even more revealing result was observed when an unrelated intron was inserted into the Ty DNA sequence (experiment 2). Addition of galactose to the medium stimulated Ty transposition, as in experiment 1, but the resulting newly integrated Ty elements all lacked the inserted intron. Presumably, the intron was spliced out of the Ty mRNA before it was reverse-transcribed into a double-stranded DNA copy, which subsequently inserted into the host-cell genome. The observed removal of the intron from transposed Ty elements strongly implies that transposition occurs by reverse transcription of mRNA produced by transcription of Ty DNA.

Figure 9-17. Experimental demonstration that yeast Ty element moves through an RNA intermediate.

Figure 9-17

Experimental demonstration that yeast Ty element moves through an RNA intermediate. When yeast cells are transformed with a Ty-containing plasmid, the Ty element can transpose to new sites, although normally this occurs at a low rate. Using the elements (more...)

In contrast to Ty elements, the coding region of maize Ac elements contains introns. The presence of introns in Ac elements supports the conclusion that they transpose via direct movement of DNA sequences, not by reverse transcription of an RNA intermediate.

Nonviral Retrotransposons Lack LTRs and Move by an Unusual Mechanism

The most abundant mobile elements in mammals are nonviral retrotransposons, which lack LTRs. Many of these belong to the two classes of moderately repeated DNA sequences found in mammalian genomes: long interspersed elements (LINES) and short interspersed elements (SINES). In humans, full-length LINES are ≈6 – 7 kb long, and SINES are ≈300 bp long. One major class of SINES and perhaps about ten classes of LINES have been identified in mammals; each class may be present in thousands of copies, which may not be exact repeats. Repeated sequences with characteristics of LINES have been observed in protozoa, insects, and plants, but for unknown reasons they are particularly abundant in the genomes of mammals. SINES also are found primarily in mammalian DNA. The large numbers of LINES and SINES in higher eukaryotes have accumulated over evolutionary time by repeated copying of a sequence at a few positions in the genome and insertion of the copies into new positions. Although these mobile elements do not contain LTRs, the available evidence indicates that they transpose through an RNA intermediate.

L1 LINE Elements

The most common LINE elements constitute the L1 LINE family. Some 600,000 copies of L1 elements occur in the human genome, accounting for ≈15 percent of total human DNA. The general structure of L1 elements, based on a “consensus” (average) sequence, is diagrammed in Figure 9-18. L1 elements usually are flanked by short direct repeats, the hallmark of mobile elements. The consensus sequence contains two long open reading frames (ORFs), one ≈1 kb long and the other ≈4 kb long. ORF1 encodes an RNA-binding protein. The protein encoded by ORF2 is similar in sequence to the reverse transcriptases of retroviruses and viral retrotransposons.

Figure 9-18. General structure of an L1 LINE element, a common type of eukaryotic nonviral retrotransposon.

Figure 9-18

General structure of an L1 LINE element, a common type of eukaryotic nonviral retrotransposon. The length of the flanking direct repeats varies among copies of the element at different sites in the genome. The sequence of the direct repeats appears to (more...)

Evidence for the mobility of L1 elements first came from analysis of DNA cloned from humans with certain genetic diseases. DNA from these patients was found to carry mutations resulting from insertion of an L1 element into a gene, whereas no such element occurred in the DNA of either parent. Later experiments similar to those just described with yeast Ty elements (see Figure 9-17) confirmed that L1 elements transpose through an RNA intermediate. In these experiments, an intron was introduced into a cloned mouse L1 element, and the recombinant L1 element was stably transformed into cultured hamster cells. After several cell doublings, a PCR-amplified fragment corresponding to the L1 element but lacking the inserted intron was detected in the cells. This finding strongly suggests that over time the recombinant L1 element containing the inserted intron had transposed to new sites in the hamster genome through an RNA intermediate that underwent RNA splicing to remove the intron.

Since L1 elements do not contain LTRs, their mechanism of transposition through an RNA intermediate must differ from viral retrotransposition in which the LTRs play a crucial role. In vitro studies indicate that transcription of L1 is directed by promoter sequences located within the left end of the element. L1 elements also contain an A/T-rich region near their right end, which generates a stretch of A residues in their RNA transcripts. Based on these properties of L1 elements, the model of L1 retrotransposition shown in Figure 9-19 has been proposed.

Figure 9-19. Proposed mechanism of nonviral retrotransposition of L1 elements.

Figure 9-19

Proposed mechanism of nonviral retrotransposition of L1 elements. According to this model, transcription of the bottom L1 strand containing a 3′ T-rich region terminates after the first T/A-rich sequence in the flanking DNA. Folding and hybridization (more...)

In the L1 element diagrammed in Figure 9-18, the length of the open reading frames has been maximized. The vast majority of L1 sequences, however, contain stop codons and frameshift mutations in ORF1 and ORF2; these mutations probably have accumulated in most L1 sequences over evolutionary time. Maintenance of L1 transposition requires that only one L1 sequence in the genome maintain intact open reading frames encoding the reverse transcriptase and other proteins required for L1 retrotransposition. Mutations could accumulate in other L1 elements without interfering with their retrotransposition, which can be directed by the enzymes expressed from only one or a few intact L1 elements in the cell.

SINES and Alu Sequences

Several hundred examples of SINES, the second major class of moderately repeated DNA in mammals, have been cloned and sequenced. Although no two copies of these intermediate repeats are identical, their general sequence similarity shows that an ancestral relationship exists between these elements both within and among mammalian species. The sequence conservation is about 80 percent within a species, but falls to only about 50 – 60 percent among different species. Because many of these repetitive sequences in human DNA were found to contain a recognition site for the restriction enzyme AluI, they were collectively called the Alu family. However, since these short interspersed elements are not precisely identical, many lack the Alu site; nonetheless, the name is widely used to refer to the most abundant type of human SINE.

Alu sequences containing ≈300 base pairs are present at ≈1 million sites in the human genome, accounting for about 10 percent of the total genomic DNA; similar sequences are abundant in other vertebrates. In addition to full-length Alu sequences, many partial Alu-like sequences, clearly related to the Alu family but as short as 10 base pairs, have been found scattered between genes and within introns in human DNA.

Alu sequences are remarkably homologous to 7SL RNA, a small cellular RNA that is part of the signal-recognition particle. This cytoplasmic ribonucleoprotein particle aids in the secretion of newly formed polypeptides through the membranes of the endoplasmic reticulum (Chapter 17). The 7SL sequence is highly conserved even in species as diverse as Drosophila, mouse, and man. The discovery of a small (≈100-nucleotide) E. coli RNA whose sequence is similar to eukaryotic 7SL RNA indicates that this molecule has existed since early in evolution. However, neither Drosophila nor single-celled organisms have any Alu-type intermediate repeats (at least in large numbers). These findings suggest that 7SL RNA genes existed before Alu sequences and that Alu sequences somehow arose fairly late in evolution from the 7SL sequences.

Image med.jpgThe initial evidence for the mobility of SINES came from analysis of DNA from a patient with neurofibromatosis, a genetic disorder marked by the occurrence of multiple neuronal tumors called neurofibromas due to mutation in the NF1 gene. Like the retinal tumors that occur in hereditary retinoblastoma (see Figure 8-7), neurofibromas develop only when both NF1 alleles carry a mutation. In one individual with neurofibromatosis, one NF1 allele contains an inactivating Alu sequence; inactivating somatic mutations in the other NF1 allele in peripheral neurons lead to the development of neurofibromas. Several other inherited recessive mutations causing disease in humans also have been found to result from insertion of Alu sequences in exons, thereby disrupting protein-coding regions.

Like all other mobile elements, Alu sequences usually are flanked by direct repeats. Although Alu sequences do not encode proteins, they are transcribed by RNA polymerase III and contain an A/T-rich region at one end, similar to L1 elements. Consequently, Alu sequences are thought to be retrotransposed by a mechanism similar to that proposed for L1 elements (see Figure 9-19), possibly by the reverse transcriptase and other required proteins expressed from functional L1 elements.

Alu sequences appear to have retrotransposed widely through the human genome and are tolerated, in both possible orientations, at sites where they do not disrupt gene function: flanking solitary genes (see Figure 9-4) and between duplicated genes (see Figure 9-3b), as well as within introns and the regions transcribed into the 5′ and 3′ untranslated regions of mRNAs. Although once postulated to function in controlling gene expression, Alu sequences are now thought to have no function, like other mobile elements, despite their widespread occurrence in mammalian genomes.

Retrotransposed Copies of Cellular RNAs Occur in Eukaryotic Chromosomes

In addition to SINES and LINES, which constitute the bulk of the moderately repeated DNA in mammals, other moderately repetitive sequences have been identified. Many of these represent mutated DNA copies of a wide variety of mRNAs that have integrated into chromosomal DNA. These are not duplicates of whole genes that have drifted into nonfunctionality (i.e., the pseudogenes discussed earlier in this chapter) because they lack introns and do not have flanking sequences similar to those of the functional gene copies. 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 reversetranscribed 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, these nonfunctional copies of small RNA genes are flanked by short direct repeats and most likely result from rare retrotransposition events that have accumulated through the course of evolution. Enzymes expressed from a LINE or viral retrotransposon are thought to have carried out the retrotransposition of 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, many spontaneous mutations in Drosophila result from insertion of a mobile DNA element into or near a transcription unit, and mobile elements also have been found in mutant human genes. In addition, homologous recombination between mobile DNA elements dispersed throughout ancestral genomes may have been important in generating gene duplications and other DNA rearrangements during evolution (see Figure 9-5). Cloning and sequencing of the β-globin gene cluster from various primate species have provided strong evidence that the human Gγ and Aγ genes (see Figure 9-3) arose from an unequal homologous crossover between two L1 sequences. Such duplications and DNA rearrangements contributed greatly to the evolution of new genes. As discussed in an earlier section, gene duplication probably preceded the evolution of a new member of a gene family, which subsequently acquired distinct, beneficial functions.

Mobile DNA most likely also influenced the evolution of genes that contain multiple copies of similar exons encoding similar protein domains (e.g., the fibronectin gene). Homol-ogous recombination between mobile elements inserted into introns probably contributed to the duplication of introns within such genes. Some evidence suggests that during the evolution of higher eukaryotes, recombination between introns of distinct genes occurred, generating new genes made from novel combinations of preexisting exons. For example, tissue plasminogen activator, the Neu receptor, and epidermal growth factor all contain an EGF domain (see Figure 3-10). Evolution of the genes encoding these proteins may have involved recombinations between mobile DNA elements that resulted in the insertion of an EGF-encoding exon into an intron of the ancestral form of each of these genes. The term exon shuffling has been coined to refer to this type of evolutionary process.

Recombination between mobile elements also may have played a role in determining which specific genes are expressed in particular cell types and the amount of the encoded protein produced. As noted earlier, eukaryotic genes have transcription-control regions, called enhancers, that can operate over distances of tens of thousands of base pairs. Moreover, as we will learn in the next chapter, the transcription of a gene can be controlled through the combined effects of several enhancers. Recombination between mobile elements inserted randomly near enhancers probably contributed to the evolution of the combinations of enhancers that control gene expression in modern organisms.

So, the early view of mobile DNA elements as completely selfish molecular parasites appears to be premature. Rather, they have probably indirectly made profound contributions to the evolution of higher organisms by serving as sites of recombination, leading to the evolution of novel genes and new controls on gene expression.

SUMMARY

  •  Most of the moderately repeated DNA sequences interspersed at multiple sites throughout the genomes of higher eukaryotes arose from mobile DNA elements.
  •  Mobile DNA elements encode enzymes that can insert their sequence into new sites in genomic DNA.
  •  Mobile DNA elements that transpose to new sites directly as DNA are called transposons; those that first are transcribed into an RNA copy of the element, which then is reverse-transcribed into DNA, are called retrotransposons (see Figure 9-10 and Table 9-3). Both types generally produce short direct repeats at the site of insertion, which flank the mobile element. The length of the direct repeats depends on the type of mobile element.
  •  The mobile DNA elements in bacteria — IS elements and bacterial transposons — move via DNA intermediates. Both encode transposase, but the longer transposons also contain at least one other protein-coding gene, generally including a drug-resistance gene.
  •  Although transposons, similar in structure to bacterial IS elements, occur in eukaryotes (e.g., Drosophila P element), retrotransposons generally are much more abundant, especially in higher eukaryotes.
  •  Viral retrotransposons are flanked by long terminal repeats (LTRs), similar to those in retroviral DNA, and, like retroviruses, encode reverse transcriptase and integrase. They move in the genome by being transcribed into RNA, which then undergoes reverse transcription and integration into the host-cell chromosome (see Figure 9-16).
  •  Nonviral retrotransposons lack LTRs and have an A/T-rich stretch at one end. These mobile elements are thought to move by an unusual nonviral retrotransposition mechanism (see Figure 9-19).
  •  The most abundant mobile elements in vertebrates are two types of nonviral retrotransposons called LINES and SINES. Both types appear to have caused mutations associated with human genetic diseases.
  •  SINES exhibit extensive homology with small cellular RNAs transcribed by RNA polymerase III. The most common SINES in humans frequently contain a site for the restriction enzyme AluI and consequently are called Alu sequences. These ≈300-bp sequences are scattered throughout the human genome, constituting ≈5 percent of the total DNA.
  •  Some moderately repeated DNA sequences are derived from cellular RNAs that were reversetranscribed and inserted into genomic DNA at some time in evolutionary history. Those derived from mRNAs, called processed pseudogenes, lack introns, a feature that distinguishes them from pseudogenes, which arose by sequence drift of duplicated genes.
  •  Although mobile DNA elements appear to serve no beneficial function to an individual organism, they most likely influenced evolution significantly.
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