Elimination of active tad elements during the sexual phase of the Neurospora crassa life cycle.

Tad is an active LINE-like retrotransposon isolated from the Adiopodoumé strain of Neurospora crassa. Extensive analysis of other Neurospora strains has revealed no other strain with active Tad, but all strains tested have multiple copies of defective Tad elements. We have examined the ability of Tad to survive during the sexual cycle of Neurospora and find that active Tad is rapidly eliminated. The characteristics of this elimination suggest that the repeat-induced point mutation (RIP) mechanism was responsible. By the use of transformation to switch the mating type of the Adiopodoumé strain we concluded that this strain is not defective in the RIP process. Analysis of defective Tad elements isolated from a variety of strains indicates that the major difference between these elements and active Tad is due to the presence of a large number of G-C to A-T transition mutations. This would be expected if the changes were due primarily to the RIP process. Mapping of a selection of defective Tad elements reveals that they are present on all of the chromosomes; however, many of the elements are not widely shared among strains. This suggests that repeated introduction and elimination of Tad elements has occurred. Mechanisms that might be responsible for this repeated introduction are discussed.     

Tad, a Line-like Transposable Element of Neurospora, Can Transpose between Nuclei in Heterokaryons

The Tad transposon of Neurospora crassa appears to be a LINE-like element with very restricted distribution within the genus Neurospora. When forced heterokaryons were constructed between strains which did and did not contain Tad, the nuclei of the naive nuclear type rapidly acquired Tad elements. The elements acquired by naive nuclei are active, since they can pass Tad to other naive nuclei in subsequent heterokaryons. When heterokaryons are passaged by serial transfer, the load of acquired Tad elements appears to increase, indicating that transposition is continuing in these heterokaryons, even after all of the naive nuclei have acquired Tad. In normal heterokaryons of Neurospora, nuclei do not fuse. An experiment to test for the possibility that Tad promotes nuclear fusion gave negative results. Thus Tad appears to have a cytoplasmic intermediate in its transposition. When heterokaryon incompatible strains were cocultured, there was no indication that Tad elements could be transferred to the naive strain, suggesting that Tad is not a virus. These data are consistent with the transposition of Tad via RNA and cDNA intermediates, as has been postulated to occur with LINE-like elements.     

The Neurospora Transposon Tad Is Sensitive to Repeat-Induced Point Mutation (Rip)

RIP (repeat-induced point mutation) efficiently mutates repeated sequences in the sexual phase of the Neurospora crassa life cycle. Nevertheless, an active LINE-like retrotransposon, Tad, was found in a N. crassa strain from Adiopodoume. The possibility was tested that Tad might be resistant to RIP, or that the Adiopodoume strain might be incompetent for RIP. Tad elements derived from the Adiopodoume strain were found to be susceptible to RIP. In addition, strains lacking active Tad elements, including common laboratory strains and strains representing seven species of Neurospora, were found to have sequences closely related to Tad but with numerous mutations of the type resulting from RIP (G:C to A:T). Even the Adiopodoume strain showed Tad-like elements with mutations characteristic of RIP. Results of crossing of an Adiopodoume transformant with progeny of Adiopodoume suggest that the Adiopodoume strain is proficient at RIP. We conclude that Tad is an old transposable element that has been inactivated by RIP in most strains. Finding relics of RIP in both heterothallic and homothallic species of Neurospora implicates RIP across the genus.     

Requirements for polyadenylation at the 3' end of LINE-1 elements

LINE-1 (L1) is the only active, autonomous, non-LTR, human retroelement. There are about 5 × 10⁵ L1 copies in the human genome, the majority of which are truncated at their 5′ ends. Both truncated and full-length L1 insertions contain a polyadenylation (polyA) signal at their 3′ ends. A typical polyA site consists of the three main cis-acting elements: a conserved hexamer, cleavage site, and a GU-rich downstream region. A newly inserted L1 copy contains the conserved AATAAA hexamer at the end of its sequence. However, the GU-rich downstream region has to be provided by the neighboring genomic sequences and therefore it would vary for every L1 copy. Using northern blot analysis of transiently transfected L1 expression vectors we demonstrate that L1 element contain sequence that allow efficient polyadenylation at the L1 3′ end upon retrotransposition into a new genomic location independent of the base composition downstream of the insertion site. The strategy of polyadenylation at the 3′ end of L1 parallels the approach the element employs at its 5′UTR by having an unusual internal polymerase II promoter, making new insertions less dependent on the properties of the flanking sequences at the new locus.