Retrotransposons in Higher Cells

Retrotransposons in Higher Cells

The genomes of higher cells contain substantial numbers of repeated sequences. For example, the Alu sequence of 300 base pairs constitutes about 5% of the human genome. Among these repeated sequences are two main classes of transposable elements. One is similar to Tn10 in its DNA transposition mode. This includes the Ac element in maize, Tc1 in nematodes, and the P element in Drosophila. Members in the other class transpose by means of an RNA intermediate, and they include the Ty1 factor in yeast, the copia-like elements in Drosphila and the long inter-spersed, LI elements in mammals. These elements are retrotransposons and are closely related to retroviruses, if not identical to retroviruses in some cases.

Hybridization and sequencing of Ty1 and the regions into which it inserts have revealed its structure. It duplicates five bases upon insertion

and consists of two flanking regions, called delta, of 330 base pairs oriented as direct repeats around a 5,600-base-pair central region that contains considerable homology to retroviruses. Not all delta elements found in yeast are identical, nor are the central regions identical, for some Ty elements are able to block expression of nearby yeast genes while others stimulate expression of genes near the point of integration.

A recombination event between the two delta elements deletes the central region and one delta element. Hence it is not surprising that the yeast genome contains about 100 of these solo delta elements. Recombination between different Ty elements can create various chromosomal rearrangements. Although the consequences of recombination can be determined easily in yeast, similar chromosome rearrangements catalyzed by recombination between repeated sequences must also occur in other organisms. Consequently transposons may be of positive value to an organism because they may speed chromosome rearrangements that may directly generate new proteins and new schemes of gene regulation.

Retroviruses, whose study began long before their discovery as a part of the yeast Ty1 factors, are particles that contain single-stranded RNA. This is both translated upon infection as well as converted via an RNA-DNA hybrid to a DNA-DNA duplex that is often integrated into the genome of the infected cell and is called a provirus. This form of the virus and defective variants can be considered the retrotransposon. The generation of the RNA-DNA duplex is catalyzed by reverse transcriptase. This enzyme is packaged within the virus particle. Upon their insertion into the chromosome, retroviruses duplicate a small number of bases as a result of generating staggered nicks in the target sequence. At each end of the retrovirus sequence is an inverted repeat of about 10 base pairs that is part of a few-hundred-base-pair direct repeat. Between the direct repeats, which are named long terminal repeats or LTRs, are sequences of about 5,000 base pairs that code for viral coat protein and other proteins. Transcription begins near the end of one LTR, proceeds through the central region, and ends within the other LTR.

Some of the DNA that is found in repeated sequences in Drosophila has been analyzed, and its properties suggest that it too is related to proviruses. One such family is copia. Small virus-like particles contain-ing RNA homologous to copia DNA sequences can even be isolated from the nucleus of Drosophila cells. This RNA is translatable into one of the proteins that coats the RNA.

Retroviruses have been much harder to demonstrate in humans than in other animals. Nonetheless, they have been found. One was found to have inserted a copy of itself into the gene encoding factor VIII which is necessary for blood coagulation.

A more easily observed element in the human genome is the Alu family of sequences. Humans contain 100,000 to 500,000 copies of this sequence. The name derives from the fact that the restriction enzyme Alu cleaves more than once within the sequence. Consequently, diges-tion of human DNA with Alu yields 100,000 identical fragments, which upon electrophoresis generate a unique band in addition to the faint smear generated by the heterogeneity of the remainder of the DNA. The Alu sequences look like direct DNA copies of mRNA molecules becausethey contain a stretch of poly deoxyadenosine at their 3’ ends. Like transposons, the Alu sequences also are flanked by direct repeats of chromosomal sequences of 7 to 20 base pairs. It is not possible to tell from their structure whether retroviruses and Alu sequences evolved from transposable elements or the reverse.