Retrotransposons isolation

Studies of overall genome composition based on reassociation kinetics (Simpson et ai, 1982 Cox et ai, 1990 Marx et a/., 2000) and analysis of fully sequenced bacterial artificial chromosome (BAC) clones from the 5. mansoni genome project show that platyhelminth genomes contain abundant highly and moderately repetitive sequence (Fig. 2.1). Much of the repetitive DNA comprises two classes of integrated mobile elements class I elements, which include long terminal repeat (LTR) retrotransposons and retroviruses, non-LTR retro-transposons and short interspersed nuclear elements (SINES) and transpose via an RNA intermediate, and class II elements (trans-posons), which transpose as DNA (Brindley et ai, 2003). Additionally, small dispersed or tandemly repeated sequences are common. A wide variety of these sequences have been isolated and characterized from a variety of taxa (Table 2.4). 

Although a number of mammalian retrotransposons have been discovered by chance, a variety of approaches have been developed specifically to survey the genome for the presence of retrotransposons as well as to facilitate their isolation and characterization. Of the three methods outlined here in detail, two (conserved restriction sites and phylogenetic screening) could lead to isolation of all three types of retrotransposons, whereas the third method [polymerase chain reaction (PCR) amplification of reverse transcriptase] will exclude the SINEs. An unrelated method that has been particularly useful for isolation of SINEs7 will not be detailed. 

Characterize retrotransposons. Once a positive clone has been identified, it can be used as a probe to isolate full-length copies of the element from a genomic library. These clones can also be characterized by phylogenetic screening. 

The presence of retrotransposon-derived repetitive DNA in such abundance in mammalian genomes is a mystery in terms of its origin as well as its role in the evolution of the genomes it inhabits. These mysteries will best be solved by detailed comparative studies of a number of elements in an evolutionary context. Already, phylogenetic analysis has led to an improved understanding of the structure and dynamics of retrotransposi-tion of LINE and SINE elements of vertebrate genomes.4 11 12 22-24 We have detailed three useful methods for the isolation of interspersed repetitive... 

Many retrotransposons encode proteins that are homologous with the gag and pol genes of retroviruses.23 Because the function of the reverse transcriptase enzyme may be necessary for the transposition of these elements, it is important to search for sequence similarities in the repetitive elements that are newly isolated. 

---