Transfer RNA genes from Dictyostelium discoideum are frequently associated with repetitive elements and contain consensus boxes in their 5' and 3'-flanking regions.

A total of 68 different tRNA genes from the cellular slime mold Dictyostelium discoideum have been isolated and characterized. Although these tRNA genes show features common to typical nuclear tRNA genes from other organisms, several unique characteristics are apparent: (1) the 5'-proximal flanking region is very similar for most of the tRNA genes; (2) more than 80% of the tRNA genes contain an "ex-B motif" within their 3'-flanking region, which strongly resembles characteristics of the consensus sequence of a T-stem/T-loop region (B-box) of a tRNA gene; (3) probably more than 50% of the tRNA genes in certain D. discoideum strains are associated with a retrotransposon, termed DRE (Dictyostelium repetitive element), or with a transposon, termed Tdd-3 (Transposon Dictyostelium discoideum). DRE always occurs 50 (+/- 3) nucleotides upstream and Tdd-3 always occurs 100 (+/- 20) nucleotides downstream from the tRNA gene. D. discoideum tRNA genes are organized in multicopy gene families consisting of 5 to 20 individual genes. Members of a particular gene family are identical within the mature tRNA coding region while flanking sequences are idiosyncratic.     

Isolation of three kinds of human endogenous retrovirus-like sequences using tRNA(Pro) as a probe.

Three kinds of human endogenous retrovirus-like sequences (HuERS-P1, 2 and 3) were isolated from a HeLa cell genomic library using the 3'-half fragment of proline tRNA as a hybridization probe. These elements contained putative primer binding sites complementary to the 3'-terminus of proline tRNA and long terminal repeats (LTRs) characteristic of retrovirus provirus. The LTR sequence of HuERS-P1 consisted of about 690 nucleotides and contained a CAT box, a TATA box and a polyadenylation signal. A complete unit of an Alu family sequence was inserted into the 5'-LTR of one of the clones. HuERS-P2 also contained a TATA box and a polyadenylation signal in its LTR (about 840 nucleotides long), but the LTR sequence of this element was quite different from that of HuERS-P1. Although clone HuERS-P3 contained only the 5'-LTR region, this LTR sequence contained a CAT box, a TATA box and a poly-adenylation signal and was quite similar to the LTR sequence of the recently isolated human retrovirus-related sequence HuRRS-P (Kr?ger, B. and Horak, I. (1987) J. Virol., 61, 2071-2075). Human and simian DNAs contain 10 to 40 copies of these elements, but mouse DNA does not contain these elements.     

Tdd-4, a DNA transposon of Dictyostelium that encodes proteins similar to LTR retroelement integrases.

Tdd-4 is the first DNA transposon to be isolated from Dictyostelium discoideum. This element was isolated by insertion into a target plasmid. Two classes of elements were identified which include a 3.8 kb version and a 3.4 kb deleted version. Sequence analysis reveals that the 145 bp inverted terminal repeats contain the 5'-TGellipsisCA-3' conserved terminal dinucleotides found in prokaryotic transposons and integrated LTR retroelement DNA sequences. Tdd-4 open reading frames are assembled by removal of six introns. Introns 1-5 conform to the GT-AG rule, whereas intron 6 appears to be an AT-AA intron. Also, intron 6 undergoes an alternative 5' splicing reaction. The alternatively spliced region encodes 15 tandem SPXX repeats that are proposed to function as a DNA binding motif. By analogy to other transposons that encode two proteins from the same gene, the full-length Tdd-4 protein is the putative transposase and the truncated Tdd-4 protein is the putative transposition inhibitor. Protein database searches demonstrate Tdd-4 encoded proteins are unique for a DNA element by containing similarities to retroviral/retrotransposon integrases. The putative Tdd-4 transposase contains the same structural relationship as integrases by possessing an N-terminal HHCC motif, a central DDE motif and a C-terminal DNA-binding domain composed of the SPXX motif.     

Non-LTR retrotransposons with unique integration preferences downstream of Dictyostelium discoideum tRNA genes

Retrotransposable elements are genetic entities which move and replicate within host cell genomes. We have previously reported on the structures and genomic distributions of two non-long terminal repeat (non-LTR) retrotransposons, DRE and Tdd-3, in the eukaryotic microorganism Dictyostelium discoideum. DRE elements are found inserted upstream, and Tdd-3 elements downstream, of transfer RNA (tRNA) genes with remarkable position and orientation specificities. The data set currently available from the Dictyostelium Genome Project led to the characterisation of two repetitive DNA elements which are related to the D. discoideum non-LTR retrotransposon Tdd-3 in both their structural properties and genomic distributions. It appears from our data that in the D. discoideum genome tRNA genes are major targets for the insertion of mobilised non-LTR retrotransposons. This may be interpreted as the consequence of a process of coevolution, allowing a viable population of retroelements to transpose without being deleterious to the small microbial host genome which carries only short intergenic DNA sequences. A new nomenclature is introduced to designate all tRNA gene-targeted non-LTR retrotransposons (TREs) in the D. discoideum genome. TREs inserted 5′ and 3′ of tRNA genes are named TRE5 and TRE3, respectively. According to this nomenclature DRE and Tdd-3 are renamed TRE5-A and TRE3-A, respectively. The new retroelements described in this study are named TRE3-B (formerly RED) and TRE3-C. Received: 27 May 1999 / Accepted: 23 July 1999     

Multiple enhancer domains in the 3'' terminus of the Prague strain of Rous sarcoma virus.

The 3' terminal region of the Prague strain of Rous sarcoma virus (PrRSV) contains at least three distinct domains that comprise two functional enhancer elements. Two of these domains (designated B and C) are found in the U3 region of the 3' long terminal repeat (LTR) while the third (designated A) is located in the sequences immediately preceding the LTR termed XSR sequences. Combinations of adjacent domains [e.g., (A + B or B + C)] are capable of activating the expression of the SV40 early promoter (21 bp repeats and TATA box) coupled to coding sequences from the prokaryotic gene chloramphenicol acetyltransferase (CAT) while a single domain is inactive. Furthermore, duplication or triplication of the central domain B restores activity. The related, Schmidt-Ruppin, strain of RSV, contains an almost identical 3' LTR element, but differs in the enhancer sequences immediately preceding the 3' LTR. A model is presented in which the sequence differences may contribute to the difference in disease spectrum of transformation defective (td) variants of these viruses.     

Genomic organization of the transposable element Tdd-3 from Dictyostelium discoideum.

The transposable element Tdd-3 from D. discoideum has been described originally in 1984 (Poole and Firtel, 1984). Additional copies of this element were discovered in the course of a recent study on tRNA gene organization in D. discoideum. Five out of 24 independently isolated tRNA genes proved to be associated with Tdd-3 elements. The surprising observation that all the elements always occurred within the 3'-flanking sequences of the Dictyostelium tRNA genes suggested the possibility of a general position specific integration of Tdd-3 elements upon transposition. Therefore we isolated additional Tdd-3 elements from various genomic D. discoideum libraries in order to test this hypothesis. Several new Tdd-3 elements were found associated with various tRNA genes. Additionally we identified Tdd-3 elements organized in tandem array or in association with RED (Repetitive Element of Dictyostelium), another repetitive element recently identified by our laboratory. In all cases a B-box equivalent of the eukaryotic gene-internal RNA polymerase III promoter was identified upstream of all Tdd-3 elements.     

''Solo'' large terminal repeats (LTR) of an endogenous retrovirus-like gene family (VL30) in the mouse genome.

VL30 genetic elements constitute a murine multicopy gene family that is retrovirus-like, despite the lack of sequence homology with any known retrovirus. Over one hundred copies of VL30 units are dispersed throughout the mouse genome. We report here that the mouse genome also contains 'solo' VL30 long terminal repeats (LTRs). These are structures which contain the LTR detached from the rest of the VL30 sequences. The isolation of solo LTRs from a mouse embryonic gene library with the aid of sub-genomic VL30 probes is described. Direct DNA sequencing established that the solo LTR unit is grossly similar to a standard VL30 LTR and that the LTR is flanked by a 4-base pair duplication. The analogy to the occurrence of solitary LTR units of transposable elements is discussed.     

Nonsense suppression in Dictyostelium discoideum

We describe the generation of Dictyostelium discoideum cell lines that carry different suppressor tRNA genes. These genes were constructed by primer-directed mutagenesis changing a tRNA(Trp)(CCA) gene from D. discoideum to a tRNA(Trp)(amber) gene and changing a tRNA(Glu)(UUC) gene from D. discoideum to a tRNA(Glu)(ochre) as well as a tRNA(Glu)(amber) gene. These genes were stably integrated into the D. discoideum genome together with a reporter gene. An actin 6::lacZ gene fusion carrying corresponding translational stop signals served as a reported. Active beta-galactosidase is expressed only in D. discoideum strains that contain, in addition to the reporter, a functional suppressor tRNA. Both amber suppressors are active in D. discoideum without interfering significantly with cell growth and development. We failed, however, to establish cell lines containing a functional tRNA(Glu)(ochre) suppressor. This may be due to the fact that nearly every message from D. discoideum known so far terminates with UAA. Therefore a tRNA capable of reading this termination codon may not be compatible with cell growth.     

Mobile dispersed genetic element MDG1 of Drosophila melanogaster: nucleotide sequence of long terminal repeats.

Long terminal repeats (LTRs) of two members of mdg1 family were sequenced. In the both cases, they are represented by perfect direct repeats 442 and 444 bp in length. Sixteen nucleotides in the LTRs of two different mdg1 elements are different. Each LTR contains slightly mismatched 16-nucleotide inverted repeats located at the ends of the LTR. Six base pairs closest to the termini of LTR form perfect inverted repeats. On the gene-distal sides of LTRs, short 4-nucleotide direct repeats are located, probably representing the duplication of a target DNA sequence arising from insertion of mdg. They are different in the two cases analyzed. Just as the other analyzed eukaryotic transposable elements, mdg1 starts with TGT and ends with ACA. Within the both strands of LTR, the sequences similar to Hogness box (a putative signal for RNA initiation, or a selector) and AATAAA blocks (putative polyadenylation signals) are present. The LTR of mdg1 contains many short direct and inverted repetitive sequences. These include a 10-nucleotide sequence forming a perfect direct repeat with the first ten nucleotides of the LTR. A region of LTR about 70 bp long is represented by simple repetitive sequences (TAT).