Internally located and oppositely oriented polymerase II promoters direct convergent transcription of a LINE-like retroelement, the Dictyostelium repetitive element, from Dictyostelium discoideum.

Abstract

The Dictyostelium discoideum NC4 genome harbors approximately 150 individual copies of a retrotransposable element called the Dictyostelium repetitive element (DRE). This element contains nonidentical terminal repeats (TRs) consisting of conserved building blocks A and B in the left TR and B and C in the right TR. Seven different-sized classes of RNA transcripts from these elements were resolved by Northern (RNA) blot analysis, but their combined abundance was very low. When D. discoideum cells were grown in the presence of the respiratory chain blocker antimycin A, steady-state concentrations of these RNA species increased 10-to 20-fold. The D. discoideum genome contains two DRE subtypes, the full-length 5.7-kb DREa and the internally deleted 2.4-kb DREb. Both subtypes are transcribed, as confirmed by analysis of cloned cDNA. Primary transcripts from the sense strand originate at nucleotide +1 and terminate at two dominant sites, located 21 or 28 nucleotides upstream from the 3' end of the elements. The activity of a reasonably strong polymerase II promoter in the 5'-terminal A module is slightly upregulated by the tRNA gene located 50 ± 4 nucleotides upstream and drastically reduced by the adjacent B module of the DRE. Transcripts from the opposite DNA strand (complementary-sense transcripts) were also detected, directed by an internally located polymerase II promoter residing within the C module. This latter transcription was initiated at multiple sites within the oligo(dA12) stretch which terminates DREs.Retrotransposable Dictyostelium repetitive elements (DREs) are present in approximately 150 to 200 copies in the genomes of different Dictyostelium discoideum strains (28-31).Most remarkably, this element always integrates at a distance of 50 ± 4 nucleotides upstream from tRNA genes but in the opposite transcriptional orientation. Upon integration, a 14 + 2-nucleotide target site duplication is created (29). The 5.7-kb DREs contain an internal coding portion with two open reading frames (ORFs) which is flanked by nonidentical terminal repeats (TRs) (see Fig. IA). These TRs are composed of three distinct modules, termed A, B, and C. The 5' TR consists of one or several A modules followed by a 290-bp B module which provides the AUG translation initiation codon. The 3' TR consists of a B module followed by a terminal C module.Approximately half of the DREs in NC4-derived strains show a variant organization (30). They are only 2.4 kb long and are termed DREbs, as opposed to the 5.7-kb DREas (Fig. IA). DREbs carry an extensive internal deletion encompassing nearly the entire ORF2. They also contain several small deletions within the right TR and a variant A module (termed Ab), which always differs from the Aa module of the 5.7-kb DREas by the same three distinct point mutations (30). A similar situation has been described for jockey elements of Drosophila melanogaster, which occur as full-size (5-kb) copies

The Two Drosophila Telomeric Transposable Elements Have Very Different Patterns of Transcription

“…Similar conserved repeats have been reported for other non-LTR retrotransposons, most notably DER from Dictyostelium discoideum (30) and TOC1 from Chlamydomonas reinhardtii (11). In these elements a 5Ј repeat is completely identical with a 3Ј repeat that, like the TART repeats, is some distance from the 3Ј end of the element.…”

“…In addition, the 5Ј repeat of TART elements extends some distance into ORF1, another unusual characteristic of this group (30). Our preliminary studies indicate that the placement of TART promoters differs from that of HeT-A promoters and instead may resemble that of the promoters described for the imperfect repeat element, DER (30).…”

“…These repeats resemble those found in a small subgroup of non-LTR retrotransposons that have been described as having unequal, or nonidentical, terminal repeats (30). HeT-A does not have repeats of this type.…”

“…In addition, the 5Ј repeat of TART elements extends some distance into ORF1, another unusual characteristic of this group (30). Our preliminary studies indicate that the placement of TART promoters differs from that of HeT-A promoters and instead may resemble that of the promoters described for the imperfect repeat element, DER (30). In earlier work we showed that the promoter of HeT-A is in an unusual position (8): it is at the 3Ј end of the element and serves to promote transcription of its downstream neighbor.…”

Role of RNA Polymerase III Transcription Factors in the Selection of Integration Sites by the Dictyostelium Non-Long Terminal Repeat Retrotransposon TRE5-A

“…Of these clones 2 ϫ 10 7 logarithmically growing cells were pelleted and stored at Ϫ80°C for further use. ␤-Galactosidase activity of 10 7 cells was measured using chlorophenol red-␤-D-galactoside (CPRG) as described previously (34). One katal is defined as the enzyme activity that hydrolyzes 1 mol of CPRG per second at 22°C.…”