A full lengthTy3/gypsy-type retrotransposon in the fernAdiantum

Ty3/gypsy-type LTR-retrotransposons have been found only in lily and maize but not in cryptogam. In fernAdiantum, we recently found a full-lengthTy3/gypsy-type LTR-retrotransposon (ARET-1; 8284 bp). This retrotransposon has both 5′ and 3′ LTRs (1.2 kb), a primer binding site, a polypurine tract, and an RNA binding motif and its domain arrangement in thepol region is the same as that ofTy3/gypsy-type retrotransposon. These results suggest thatTy3/gypsy-type retrotransposons are widespread among vascular plants. The nucleotide sequence data reported will appear in the EMBL, DDBJ and GenBank Nucleotide Sequence Databases under the accession number AB003364.     

skippy,a retrotransposon from the fungal plant pathogen Fusarium oxysporum

A retrotransposon from the fungal plant pathogen Fusarium oxysporum f. sp. lycopersici has been isolated and characterized. The element, designated skippy (skp) is 7846 by in length, flanked by identical long terminal repeats (LTR) of 429 by showing structural features characteristic of retroviral and retrotransposon LTRs. Target-site duplications of 5 bp were found. Two long overlapping open reading frames (ORF) were identified. The first ORF, 2562 by in length, shows homology to retroviral gag genes. The second ORF, 3888 bp in length, has homology to the protease, reverse transciptase. RNase H and integrase domains of retroelement pol genes in that order. Sequence comparisons and the order of the predicted proteins from skippy indicate that the element is closely related to the gypsy family of LTR-retrotransposons. The element is present in similar copy numbers in the two races investigated, although RFLP analysis showed differences in banding patterns. The number of LTR sequences present in the genome is higher than the number of copies of complete elements, indicating excision by homologous recombination between LTR sequences.     

Presence ofenv-like sequences inQuercus suber retrotransposons

The main difference between LTR retrotransposons and retroviruses is the presence of theenvelope (env) gene in the latter, downstream of thepol gene. Theenv gene is involved in their infectious capacity. Here we report the presence ofenv-like sequences in the genome ofQuercus suber (cork oak), one of the most economically important Portuguese species. These gene sequences were isolated through DNA amplification betweenRNaseH conserved motifs and 3’ LTR, based on the structure ofcopia retrotransposons. Phylogenetic analysis revealed that almost all the clones isolated are clustered withCyclops-2, aTy3-gypsy element identified inPisum sativum, except one clustered withgypsy andcopia retroelements found in different species. This suggests the existence of a potential ancestral sequence of theenv gene, prior to the separation ofTy3-gypsy andTy1-copia retrotransposons. Additionally, the isolatedenv-like sequences showed 26–39% of homology withenv-like sequences characterized in viruses. The origin ofenv-like sequences in retrotransposons from host plant taxa is discussed.     

Identification and genomic distribution of<Emphasis Type="Italic"> gypsy</Emphasis> like retrotransposons in<Emphasis Type="Italic"> Citrus</Emphasis> and<Emphasis Type="Italic"> Poncirus</Emphasis>

Transposable elements might be importantly involved in citrus genetic instability and genome evolution. The presence of gypsy like retrotransposons, their heterogeneity and genomic distribution in Citrus and Poncirus, have been investigated. Eight clones containing part of the POL coding region of gypsy like retrotransposons have been isolated from a commercial variety of Citrus clementina, one of the few sexual species in Citrus. Four of the eight clones might correspond to active elements given that they present all the conserved motifs described in the literature as essential for activity, no in-frame stop codon and no frame-shift mutation. High homology has been found between some of these citrus elements and retroelements within a resistance-gene cluster from potato, another from Poncirus trifoliata and two putative resistance polyproteins from rice. Nested copies of gypsy like elements are scattered along the Citrus and Poncirus genomes. The results on genomic distribution show that these elements were introduced before the divergence of both genera and evolved separately thereafter. IRAPs based on gypsy and copia types of retrotransposons seem to distribute differently, therefore gypsy based IRAPs prove a new, complementary set of molecular markers in Citrus to study and map genetic variability, especially for disease resistance. Similarly to copia-derived IRAPs, the number of copies and heterozygosity values found for gypsy derived IRAPs are lower in Poncirus than in Citrus aurantium, which is less apomictic and the most usual rootstock for clementines until 1970.Communicated by C. Möllers     

RIRE2, a novel gypsy-type retrotransposon from rice

The 441-bp DNA segment in a PCR-amplified fragment from Oryza sativa cv. IR36 was found to have a sequence with features characteristic of LTRs of retroelements, which was named RIRE2 (Rice retroelement #2) and further analyzed. Cloning and sequencing analyses of the DNA segments connected to LTR-like sequence showed that RIRE2 has a long internal region almost 10 kb long that is flanked by LTR-like sequences. This internal region carries a primer binding site (PBS) and polypurine tract (PPT) which are necessary for cDNA synthesis of retroelements. The PBS sequence is complementary to the 3' end region of tRNA(Arg). The internal region has an rt gene homologous to that of gypsy-type retrotransposons, evidence that RIRE2 is indeed a retrotransposon related to gypsy from Drosophila. RIRE2 has an extra sequence more than 4 kb long in the region downstream of gag-pol. Phylogenetic analysis of the putative amino-acid sequences of the rt gene as well as the int gene showed that RIRE2 is related to a group of gypsy-type retrotransposons of a large size that include Grande1-4 of teosinte, Tat4-1 and Athila1-1 of Arabidopsis thaliana, and Cyclops-2 of pea, but distantly related to any other group of gypsy-type retrotransposons, including RIRE3 and RIRE8 of rice. RIRE2 and Grande1-4 had the highest homology in the gag-pol region, but the nucleotide sequences of the LTR regions differed. Both elements had significant homology in the middle area of the extra regions downstream of gag-pol, in which they had an open reading frame encoding a protein with no known function on the opposite strand from that coding for gag-pol.     

skippy, a retrotransposon from the fungal plant pathogen Fusarium oxysporum

A retrotransposon from the fungal plant pathogen Fusarium oxysporum f. sp. lycopersici has been isolated and characterized. The element, designated skippy (skp) is 7846 by in length, flanked by identical long terminal repeats (LTR) of 429 by showing structural features characteristic of retroviral and retrotransposon LTRs. Target-site duplications of 5 bp were found. Two long overlapping open reading frames (ORF) were identified. The first ORF, 2562 by in length, shows homology to retroviral gag genes. The second ORF, 3888 bp in length, has homology to the protease, reverse transciptase. RNase H and integrase domains of retroelement pol genes in that order. Sequence comparisons and the order of the predicted proteins from skippy indicate that the element is closely related to the gypsy family of LTR-retrotransposons. The element is present in similar copy numbers in the two races investigated, although RFLP analysis showed differences in banding patterns. The number of LTR sequences present in the genome is higher than the number of copies of complete elements, indicating excision by homologous recombination between LTR sequences.     

Repetitive sequences in the genome ofAnemone blanda: Identification of tandem arrays and of dispersed repeats

A tandemly repetitive sequence family (AbS1) and a repetitive sequence (Hd) forming part of a larger dispersed element (dorf-1) ofAnemone blanda were characterised. TheAbS1 satellite sequence family is located in all 4,6-diamidino-2-phenylindole (DAPI) positive intercalary heterochromatic bands and in the DAPI positive heterochromatic terminal region of chromosome 3, while the dispersedHd homologous sequences are preferentially associated with euchromatic chromosome regions. The major component of theAbS1 satellite isAbS1-H1 with a basic repeat unit of 1640 bp; a minor fraction (AbS1-H5) consists of 320 bp units. A subsection of theAbS1-H1 repeat unit exhibits homologies to the 25S rRNA gene of flowering plants suggesting that the 1.64 kb satellite was generated by amplification of a precursor satellite and/or single copy sequence together with an rDNA fragment. The rDNA homologous region is considered to evolve at a rate similar to pseudogenes and thus the age of this satellite DNA fraction can be roughly estimated as about 27 million years.The dispersed repeated sequenceHd (about 1300 bp) is associated with the 8 kb elementdorf-1. A. blanda dorf-1 constitutes about 0.2% of the genome (3×10⁴ copies), is bounded by identical long terminal repeats, and exhibits partial homology to theLilium gypsy-type elementdell, but has yet to be confirmed as a retrotransposon. In contrast to theAbS1 satellite sequence family,Hd homologous sequences were found not only inA. apennina, the closest relative ofA. blanda, but also inA. nemorosa andA. ranunculoides indicating that a progenitor sequence ofdorf-1 was present in a common ancestor before speciation ocurred.     

水稻逆转录转座子RIRE10拷贝数与LTR区转录活性的鉴定

在水稻第四号染色体的长臂上鉴定了一个结构完整的Ty3型逆转录转座子RIRE10。RIRE10两LTR间的中间区域在gag pol的上游还包含另一个开放阅读框。通过RT PCR与Northern印迹杂交检测到来自LTR区的转录产物 ;根据点杂交结果 ,鉴定出包含中间区域的RIRE10成员的个数以及LTR区的拷贝数。除了 6 5个完整的逆转录转座子所具备的两个LTR外 ,水稻基因组还含有近 90 0个RIRE10的solo LTR。LTR区的转录以及导致solo LTR产生的同源重组可能影响了RIRE10成员在水稻基因组中的转座活性     

ThehapC gene ofAspergillus nidulans is involved in the expression of CCAAT-containing promoters

The 5′ regulatory region of theamdS gene ofAspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence which is required for setting the basal level ofamdS expression. Mobility shift studies have identified a factor inA. nidulans nuclear extracts which binds to this CCAAT sequence. InSaccharomyces cerevisiae theHAP3 gene encodes one component of a multisubunit complex that binds CCAAT sequences. A search of the EMBL and SwissProt databases has revealed anA. nidulans sequence with significant homology to theHAP3 gene adjacent to the previously cloned regulatory geneamdR. Sequencing of the remainder of this region has confirmed the presence of a gene, designatedhapC, with extensive homology toHAP3. The predicted amino acid sequence of HapC shows extensive identity to HAP3 in the central conserved domain, but shows little conservation in the flanking sequences. A haploid carrying ahapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially slowly on acetamide as a sole carbon and nitrogen source, indicating thathapC plays a role inamdS expression. In agreement with this notion, it has been shown that thehapC deletion results in reduced levels of expression of anamdS::lacZ reporter gene and this effect is particularly evident under conditions of carbon limitation. Nuclear extracts prepared from thehapC deletion mutant show no CCAAT binding activity to theamdS orgatA promoters, indicating thathapC may encode a component of the complex binding at this sequence.     

Sequence organization of barley centromeres

By sequencing, fingerprinting and in situ hybridization of a centromere-specific large insert clone (BAC 7), the sequence organization of centromeric DNA of barley could be elucidated. Within 23 kb, three copies of the Ty3/gypsy-like retroelement cereba were present. Two elements of ~7 kb, arranged in tandem, include long terminal repeats (LTRs) (~1 kb) similar to the rice centromeric retrotransposon RIRE 7 and to the cereal centromeric sequence family, the primer binding site, the complete polygene flanked by untranslated regions, as well as a polypurine tract 5′ of the downstream LTR. The high density (~200 elements/centromere) and completeness of cereba elements and the absence of internally deleted elements and solo LTRs from the BAC 7 insert represent unique features of the barley centromeres as compared to those of other cereals. Obviously, the conserved cereba elements together with barley-specific G+C-rich satellite sequences constitute the major components of centromeric DNA in this species.     

Gypsy homologous sequences inDrosophila subobscura (gypsyDS)

Summary Characterization of sequences homologous to theDrosophila melanogaster gypsy transposable element was carried out inDrosophila subobscura (gypsyDS). They were found to be widely distributed among natural populations of this species. From Southern blot and in situ analyses, these sequences appear to be mobile in this species.GypsyDS sequences are located in both euchromatic and heterochromatic regions. A completegypsyDS sequence was isolated from aD. subobscura genomic library, and a 1.3-kb fragment which aligns with the ORF2 of theD. melanogaster gypsy element was sequenced. Comparisons of this sequence in three species (D. subobscura, D. melanogaster, and D. virilis) indicate that there is greater similarity between theD. subobscura-D. virilis sequences than betweenD. subobscura andD. melanogaster. Molecular divergence ofgypsy sequences betweenD. virilis andD. subobscura is estimated at 16 MY, whereas the most likely divergence time of these two species is more than 60 MY. These data strongly suggest thatgypsy sequences have been horizontally transferred between these species.Offprint requests to: T.M. Alberola     

AgTHR4, a new selection marker for transformation of the filamentous fungusAshbya gossypii,maps in a four-gene cluster that is conserved betweenA. gossypii andSaccharomyces cerevisiae

Single-read sequence analysis of the termini of eight randomly picked clones ofAshbya gossypii genomic DNA revealed seven sequences with homology toSaccharomyces cerevisiae genes (15% to 69% on the amino acid level). One of these sequences appeared to code for the carboxy-terminus of threonine synthase, the product of theS. cerevisiae THR4 gene (52.4% identity over 82 amino acids). We cloned and sequenced the complete putativeAgTHR4 gene ofA. gossypii. It comprises 512 codons, two less than theS. cerevisiae THR4 gene. Overall identity at the amino acid sequence level is 67.4%. A continuous stretch of 32 amino acids displaying complete identity between these two fungal threonine synthases presumably contains the pyridoxal phosphate attachment site. Disruption of theA. gossypii gene led to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying theAgTHR4 gene and variousS. cerevisiae ARS elements. Using these plasmids only very weak complementation of aS. cerevisiae thr4 mutation was observed. Investigation of sequences adjacent to theAgTHR4 gene identified three additional ORFs. Surprisingly, the order and orientation of these four ORFs is conserved inA. gossypii andS. cerevisiae.