Is70

9-bp target-site direct repeats

▲ FIGURE 10-10 Model for transposition of bacterial insertion sequences. Step 1: Transposase, which is encoded by the IS element (IS 10 in this example), cleaves both strands of the donor DNA next to the inverted repeats (dark red), excising the IS 10 element. At a largely random target site, transposase makes staggered cuts in the target DNA. In the case of IS 10, the two cuts are 9 bp apart. Step 2|: Ligation of the 3' ends of the excised IS element to the staggered sites in the target DNA also is catalyzed by transposase. Step 3: The 9-bp gaps of single-stranded DNA left in the resulting intermediate are filled in by a cellular DNA polymerase; finally cellular DNA ligase forms the 3'^5' phosphodiester bonds between the 3' ends of the extended target DNA strands and the 5' ends of the IS 10 strands. This process results in duplication of the target-site sequence on each side of the inserted IS element. Note that the length of the target site and IS 10 are not to scale. [See H. W. Benjamin and N. Kleckner, 1989, Cell 59:373, and 1992, Proc. Nat'l. Acad. Sci. USA 89:4648.]

ment to a new site. The transposase is expressed at a very low rate, accounting for the very low frequency of transposition. An important hallmark of IS elements is the presence of a short direct-repeat sequence, 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 with 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.

As depicted in Figure 10-10, transposition of an IS element is similar to a "cut-and-paste" operation in a word-processing program. Transposase performs three functions in this process: it (1) precisely excises the IS element in the donor DNA, (2) makes staggered cuts in a short sequence in the target DNA, and (3) ligates the 3' termini of the IS element to the 5' ends of the cut donor DNA. Finally, a host-cell DNA polymerase fills in the single-stranded gaps, generating the short direct repeats that flank IS elements, and DNA ligase joins the free ends.

Eukaryotic DNA Transposons McClintock's original discovery of mobile elements came from observation of certain spontaneous mutations in maize that affect production of any of the several enzymes required to make anthocyanin, a purple pigment in maize kernels. 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 Ac elements are equivalent to bacterial IS elements. Like IS elements, they contain inverted terminal repeat sequences that flank the coding region for a transposase, which recognizes the terminal repeats and catalyzes transposition to a new site in DNA. 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.

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 DNA transposon, moving by a cut-and-paste mechanism similar to that used by bacterial insertion sequences. Current methods for constructing trans-genic Drosophila depend on engineered, high-level expression of the P-element transposase and use of the P-element inverted terminal repeats as targets for transposition.

DNA transposition by the cut-and-paste mechanism can result in an increase in the copy number of a transposon when it occurs during S phase, the period of the cell cycle when DNA synthesis occurs. This happens when the donor DNA is from one of the two daughter DNA molecules in a region of a chromosome that has replicated and the target DNA is in the region that has not yet replicated. When DNA replication is complete at the end of the S phase, the target DNA in its new location is also replicated. This results in a net increase by one in the total number of these transposons in the cell. When this occurs during the S phase preceding meiosis, two of the four germ cells produced have the extra copy. Repetition of this process over evolutionary time has resulted in the accumulation of large numbers of DNA transposons in the genomes of some organisms. Human DNA contains about 300,000 copies of full-length and deleted DNA tranposons, amounting to «3 percent of human DNA.

Some Retrotransposons Contain LTRs and Behave Like Intracellular Retroviruses

The genomes of all eukaryotes studied from yeast to humans contain retrotransposons, mobile DNA elements that transpose through an RNA intermediate utilizing a reverse transcriptase (see Figure 10-8b). These mobile elements are divided into two major categories, those containing and those lacking long terminal repeats (LTRs). LTR retrotrans-posons, which we discuss in this section, are common in yeast (e.g., Ty elements) and in Drosophila (e.g., copia elements). Although less abundant in mammals than non-LTR retrotransposons, LTR retrotransposons nonetheless constitute «8 percent of human genomic DNA. Because they exhibit some similarities with retroviruses, these mobile elements sometimes are called viral retrotransposons. In mammals, retrotransposons lacking LTRs are the most common type of mobile element; these are described in the next section.

LTR retrotransposon (=6-11 kb)

LTR retrotransposon (=6-11 kb)

Protein-coding region

Target-site direct repeat (5-10-bp)

Protein-coding region

Target-site direct repeat (5-10-bp)

▲ FIGURE 10-11 General structure of eukaryotic LTR retrotransposons. The central protein-coding region is flanked by two long terminal repeats (LTRs), which are element-specific direct repeats. Like other mobile elements, integrated retrotransposons have short target-site direct repeats at each end. Note that the different regions are not drawn to scale. The protein-coding region constitutes 80 percent or more of a retrotransposon and encodes reverse transcriptase, integrase, and other retroviral proteins.

The general structure of LTR retrotransposons found in eukaryotes is depicted in Figure 10-11. In addition to short 5' and 3' direct repeats typical of all mobile elements, these retrotransposons are marked by the presence of LTRs flanking the central protein-coding region. These long direct terminal repeats, containing «250-600 base pairs, are characteristic of integrated retroviral DNA and are critical to the life cycle of retroviruses. In addition to sharing LTRs with retroviruses, LTR retrotransposons encode all the proteins of the most common type of retroviruses, except for the envelope proteins. Lacking these envelope proteins, LTR retro-transposons cannot bud from their host cell and infect other cells; however, they can transpose to new sites in the DNA of their host cell.

A key step in the retroviral life cycle is formation of retrovi-ral genomic RNA from integrated retroviral DNA (see Figure 4-43). This process serves as a model for generation of the RNA intermediate during transposition of LTR retrotransposons. As depicted in Figure 10-12, the leftward retroviral 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 downstream retroviral DNA has been transcribed, the RNA sequence corresponding to the rightward LTR directs host-cell RNA-processing enzymes to cleave the primary transcript and add a poly(A) tail at the 3' end of the R sequence.

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