Tey

Results in galactose-containing medium

. Ty mRNA synthesis increased . Transposition of Ty elements increased

Results in galactose-containing medium

. Ty mRNAs lack intron .Transposed Ty elements lack intron

The resulting retroviral RNA genome, which lacks a complete LTR, is packaged into a virion that buds from the host cell.

After a retrovirus infects a cell, reverse transcription of its RNA genome by the retrovirus-encoded reverse transcriptase yields a double-stranded DNA containing complete LTRs (Figure 10-13). Integrase, another enzyme encoded by retroviruses that is closely related to the trans-posase of some DNA transposons, uses a similar mechanism to insert the double-stranded retroviral DNA into the host-cell genome. In this process, short direct repeats of the targetsite sequence are generated at either end of the inserted viral DNA sequence.

As noted above, LTR retrotransposons encode reverse transcriptase and integrase. By analogy with retroviruses, these mobile elements are thought to move by a "copy-and-paste" mechanism whereby reverse transcriptase converts an RNA copy of a donor-site element into DNA, which is inserted into a target site by integrase. The experiments depicted in Figure 10-14 provided strong evidence for the role of an RNA intermediate in transposition of Ty elements.

Sequencing of the human genome has revealed that the most common LTR retrotransposon-related sequences in humans are derived from endogenous retroviruses (ERVs). Most of the 443,000 ERV-related DNA sequences in the human genome consist only of isolated LTRs. These are derived from full-length proviral DNA by homologous recombination between the two LTRs, resulting in deletion of the internal retroviral sequences.

M EXPERIMENTAL FIGURE 10-14 Recombinant plasmids demonstrate that the yeast Ty element transposes 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 diagrammed at the top, researchers engineered two different plasmid vectors containing recombinant Ty elements adjacent to a galactose-sensitive promoter. These plasmids were transformed into yeast cells, which were grown in a galactose-containing and a nongalactose medium. In experiment 1, growth of cells in galactose-containing medium resulted in many more transpositions than in nongalactose medium, indicating that transcription into an mRNA intermediate is required for Ty transposition. In experiment 2, an intron from an unrelated yeast gene was inserted into the putative protein-coding region of the recombinant galactose-responsive Ty element. The observed absence of the intron in transposed Ty elements is strong evidence that transposition involves an mRNA intermediate from which the intron was removed by RNA splicing, as depicted in the box on the right. In contrast, eukaryotic DNA transposons, like the Ac element of maize, contain introns within the transposase gene, indicating that they do not transpose via an RNA intermediate. [See J. Boeke et al., 1985, Cell 40:491.]

Retrotransposons That Lack LTRs Move by a Distinct Mechanism

The most abundant mobile elements in mammals are retro-transposons that lack LTRs, sometimes called nonviral retrotransposons. These moderately repeated DNA sequences form two classes in mammalian genomes: long interspersed elements (LINEs) and short interspersed elements (SINEs). In humans, full-length LINEs are «6 kb long, and SINEs are «300 bp long. Repeated sequences with the characteristics of LINEs have been observed in protozoans, insects, and plants, but for unknown reasons they are particularly abundant in the genomes of mammals. SINEs also are found primarily in mammalian DNA. Large numbers of LINEs and SINEs in higher eukaryotes have accumulated over evolutionary time by repeated copying of sequences 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.

LINEs Human DNA contains three major families of LINE sequences that are similar in their mechanism of transposition, but differ in their sequences: L1, L2, and L3. Only members of the L1 family transpose in the contemporary human genome. LINE sequences are present at «900,000 sites in the human genome, accounting for a staggering 21 percent of total human DNA. The general structure of a complete LINE is diagrammed in Figure 10-15. LINEs usually are flanked by short direct repeats, the hallmark of mobile elements, and contain two long open reading frames (ORFs). ORF1, «1 kb long, encodes an RNA-binding protein. ORF2, «4 kb long, encodes a protein that has a long region of homology with the reverse transcriptases of retro-viruses and viral retrotransposons, but also exhibits DNA endonuclease activity.

Long interspersed element (LINE)

Long interspersed element (LINE)

A/T-rich region

Protein-coding region

Target-site direct repeat

A/T-rich region

Protein-coding region

Target-site direct repeat

▲ FIGURE 10-15 General structure of a LINE, one of the two classes of non-LTR retrotransposons in mammalian DNA. The length of the target-site direct repeats varies among copies of the element at different sites in the genome. Although the full-length L1 sequence is «6 kb long, variable amounts of the left end are absent at over 90 percent of the sites where this mobile element is found. The shorter open reading frame (ORF1), «1 kb in length, encodes an RNA-binding protein. The longer ORF2, «4 kb in length, encodes a bifunctional protein with reverse transcriptase and DNA endonuclease activity.

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 within this gene in either parent. About 1 in 600 mutations that cause significant disease in humans are due to L1 transpositions or SINE transpositions that are catalyzed by L1-encoded proteins. Later experiments similar to those just described with yeast Ty elements (see Figure 10-14) 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. I

Since LINEs do not contain LTRs, their mechanism of transposition through an RNA intermediate differs from that of LTR retrotransposons. ORF1 and ORF2 proteins are translated from a LINE RNA. In vitro studies indicate that transcription by RNA polymerase II is directed by promoter sequences at the left end of integrated LINE DNA. LINE RNA is polyadenylated by the same post-transcriptional mechanism that polyadenylates other mRNAs. The LINE RNA then is transported into the cytoplasm, where it is translated into ORF1 and ORF2 proteins. Multiple copies of ORF1 protein then bind to the LINE RNA, and ORF2 protein binds to the poly(A) tail.

The LINE RNA is then transported back into the nucleus as a complex with ORF1 and ORF2. ORF2 then makes staggered nicks in chromosomal DNA on either side of any A/T-rich sequence in the genome (Figure 10-16, step 1). Reverse transcription of LINE RNA by ORF2 is primed by the single-stranded T-rich sequence generated by the nick in the bottom strand, which hybridizes to the LINE poly(A) tail (step 2). ORF2 then reverse-transcribes the LINE RNA (step 3) and then continues this new DNA strand, switching to the single-stranded region of the upper chromosomal strand as a template (steps 4 and [5). Cellular enzymes then hydrolyze the RNA and extend the 3' end of the chromosomal DNA top strand, replacing the LINE RNA strand with DNA (step 6 ).

► FIGURE 10-16 Proposed mechanism of LINE reverse transcription and integration. Only ORF2 protein is represented. Newly synthesized LINE DNA is shown in black. See the text for explanation. [Adapted from D. D. Luan et al., 1993, Cell 72:595.]

ORF2 protein

Chromosomal DNA

ORF2 protein

Chromosomal DNA

AAATACT ♦TTTATGA

Priming of reverse transcription by chromosomal DNA

AAATACT ♦TTTATGA

Priming of reverse transcription by chromosomal DNA

Reverse transcription of LINE RNA by ORF2

AAATACT► .tttatga

AAATACT► .tttatga

Reverse transcription of LINE RNA by ORF2

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