Characterization of a transpositionally active Ty3 element and identification of the Ty3 integrase protein.

Ty3 is a Saccharomyces cerevisiae retrotransposon associated with tRNA genes. Two Ty3 elements have been cloned and characterized. The complete nucleotide sequence for one element, Ty3-2, was reported previously (L. J. Hansen, D. L. Chalker, and S. B. Sandmeyer, Mol. Cell. Biol. 9:5245-5256, 1988). However, this element is incapable of autonomous transposition. The complete DNA sequence of a transpositionally competent Ty3 element, Ty3-1, is presented here. Its sequence translates into two overlapping open reading frames, TYA3-1 and TYB3-1, which encode proteins with homology to the proteins specified by the retroviral gag and pol genes, respectively. Comparison of the Ty3-1 nucleotide sequence to Ty3-2 suggests that the TYB3-2 open reading frame of Ty3-2 is truncated by the deletion of a single nucleotide, which causes a frameshift mutation. Restoration of the reading frame with insertion of a single adenine by site-directed mutagenesis converted Ty3-2 into a transpositionally active element, Ty3-2(+ A). Western blot analysis with antibodies made against synthetic peptides identified integrase (IN) proteins in viruslike particle preparations from cells expressing Ty3 elements. Cells expressing Ty3-1 and Ty3-2 (+A) produce antibody-reactive proteins with approximate molecular masses of 61 and 58 kilodaltons (kDa), while cells expressing Ty3-2 produce reactive proteins of approximately 52 and 49 kDa. Together, these data show that the 61- or 58-kDa protein, or both, provides the integrase function of Ty3.     

Ty3 nucleocapsid controls localization of particle assembly

Expression of the budding yeast retrotransposon Ty3 results in production of viruslike particles (VLPs) and retrotransposition. The Ty3 major structural protein, Gag3, similar to retrovirus Gag, is processed into capsid, spacer, and nucleocapsid (NC) during VLP maturation. The 57-amino-acid Ty3 NC protein has 17 basic amino acids and contains one copy of the CX₂CX₄HX₄C zinc-binding motif found in retrovirus NC proteins. Ty3 RNA, protein, and VLPs accumulate in clusters associated with RNA processing bodies (P bodies). This study investigated the role of the NC domain in Ty3-P body clustering and VLP assembly. Fifteen Ty3 NC Ala substitution and deletion mutants were examined using transposition, immunoblot, RNA protection, cDNA synthesis, and multimerization assays. Localization of Ty3 proteins and VLPs was characterized microscopically. Substitutions of each of the conserved residues of the zinc-binding motif resulted in the loss of Ty3 RNA packaging. Substitution of the first two of four conserved residues in this motif caused the loss of Ty3 RNA and protein clustering with P bodies and disrupted particle formation. NC was shown to be a mediator of formation of Ty3 RNA foci and association of Ty3 RNA and protein with P bodies. Mutations that disrupted these NC functions resulted in various degrees of Gag3 nuclear localization and a spectrum of different particle states. Our findings are consistent with the model that Ty3 assembly is associated with P-body components. We hypothesize that the NC domain acts as a molecular switch to control Gag3 conformational states that affect both assembly and localization.     

Characterization of Ty3-gypsy-like elements in Hibiscus syriacus

Southern blot analysis revealed a ubiquitous distribu-tion and high copy number of Ty3-gypsy-like elements in the genome of Hibiscus syriacus. Comparative phylogenetic analysis of the large subunit of Rubisco and the integrase region of Ty3-gypsy elements in various plant species indicated that the retrotransposon-like sequences have different evolutionary histories and their own unique polymorphism in the H. syriacus population. Sequence-tagged site-restriction fragment length polymorphisms (STS-RFLP) analysis also indicated great variability in the numbers and sequences of Ty3-gypsy-like elements within H. syriacus varieties. Ty3-gypsy-like elements may still be active within H. syriacus, since Northern analysis of wounded leaves of H. syriacus variety Saehan with a probe for the integrase domain gave strong hybridization signals. The sequence heterogeneity and ubiquity of the Ty3-gypsy-like elements in H. syriacus genomes could provide reliable DNA markers for line identification as well for the analysis of genetic diversity in H. syriacus.     

The pattern of amplification and differentiation of Ty1-copia and Ty3-gypsy retrotransposons in Brassicaceae species

One of the causes of genome size expansion is considered to be amplification of retrotransposons. We determined nucleotide sequences of 24 PCR products for each of six retrotransposons in Brassica rapa and Brassica oleracea. Phylogenetic trees of these sequences showed species-specific clades. We also sequenced STF7a homologs and Tto1 homologs, 24 PCR products each, in nine diploids and three allopolyploids, and constructed phylogenetic trees. In these phylogenetic trees, species-specific clades of diploid species were also formed, but retrotransposons of allopolyploids were clustered into the clades of their original genomes, indicating that these two retrotransposons amplified after speciation of the nine diploids. Genetic variation in these retrotransposons may have arisen before emergence of allopolyploid species. There was a positive correlation between the genome size and the average number of substitutions of STF7a and Tto1 homologs in at least seven diploids. The implications of these results in the genome evolution of Brassicaceae are herein discussed.     

Distribution of Ty3-gypsy- and Ty1-copia-like DNA sequences in the genus Helianthus and other Asteraceae.

Two repeated DNA sequences, pHaS13 and pHaS211, which revealed similarity to the int gene of Ty3-gypsy retrotransposons and the RNAse-H gene of Ty1-copia retroelements, respectively, were surveyed in Asteraceae species and within the genus Helianthus. Southern analysis of the genome of selected Asteraceae that belong to different tribes showed that pHaS13- and pHaS211-related subfamilies of gypsy- and copia-like retroelements are highly redundant only in Helianthus and, to a lesser extent, in Tithonia, a Helianthus strict relative. However, under low stringency posthybridization washes, bands were observed in almost all the other Asteraceae tested when pHaS13 was used as a probe, and in several species when pHaS211 was hybridized. FISH analysis of pHaS13 or pHaS211 probes was performed in species in which labelling was observed in Southern hybridizations carried out under high stringency conditions (Helianthus annuus, Tithonia rotundifolia, Ageratum spp., Leontopodium spp., Senecio vulgaris for pHaS13, and H. annuus, Tithonia rotundifolia, and S. vulgaris for pHaS211). Scattered labelling was observed over all metaphase chromosomes, indicating a large dispersal of both Ty3-gypsy- and Ty1-copia-like retroelements. However, preferential localization of Ty3-gypsy-like sequences at centromeric chromosome regions was observed in all of the species studies but one, even in species in which pHaS13-related elements are poorly represented. Ty1-copia-like sequences showed preferential localization at the chromosome ends only in H. annuus. To study the evolution of gypsy- and copia-like retrotransposons in Helianthus, cladograms were built based on the Southern blot hybridization patterns of pHaS13 or pHaS211 sequences to DNA digests of several species of this genus. Both cladograms agree in splitting the genomes studied into annuals and perennials. Differences that occurred within the clades of perennial and annual species between gypsy- and copia-like retroelements indicated that these retrotransposons were differentially active during Helianthus speciation, suggesting that the evolution of the 2 retroelement families was, within limits, independent.     

The cotton centromere contains a Ty3-gypsy-like LTR retroelement

The centromere is a repeat-rich structure essential for chromosome segregation; with the long-term aim of understanding centromere structure and function, we set out to identify cotton centromere sequences. To isolate centromere-associated sequences from cotton, (Gossypium hirsutum) we surveyed tandem and dispersed repetitive DNA in the genus. Centromere-associated elements in other plants include tandem repeats and, in some cases, centromere-specific retroelements. Examination of cotton genomic survey sequences for tandem repeats yielded sequences that did not localize to the centromere. However, among the repetitive sequences we also identified a gypsy-like LTR retrotransposon (Centromere Retroelement Gossypium, CRG) that localizes to the centromere region of all chromosomes in domestic upland cotton, Gossypium hirsutum, the major commercially grown cotton. The location of the functional centromere was confirmed by immunostaining with antiserum to the centromere-specific histone CENH3, which co-localizes with CRG hybridization on metaphase mitotic chromosomes. G. hirsutum is an allotetraploid composed of A and D genomes and CRG is also present in the centromere regions of other AD cotton species. Furthermore, FISH and genomic dot blot hybridization revealed that CRG is found in D-genome diploid cotton species, but not in A-genome diploid species, indicating that this retroelement may have invaded the A-genome centromeres during allopolyploid formation and amplified during evolutionary history. CRG is also found in other diploid Gossypium species, including B and E2 genome species, but not in the C, E1, F, and G genome species tested. Isolation of this centromere-specific retrotransposon from Gossypium provides a probe for further understanding of centromere structure, and a tool for future engineering of centromere mini-chromosomes in this important crop species.     

Proteolytic processing of Ty3 proteins is required for transposition.

Ty3 is a retroviruslike element found in Saccharomyces cerevisiae. It encodes GAG3 and GAG3-POL3 polyproteins which are processed into mature proteins found in the Ty3 viruslike particle. In this study, the region encoding a protease that is homologous to retroviral aspartyl proteases was identified and shown to be required for production of mature Ty3 proteins and transposition. The Ty3 protease has the Asp-Ser-Gly consensus sequence found in copia, Ty1, and Rous sarcoma virus proteases, rather than the Asp-Thr-Gly found in most retroviral proteases. The Asp-Ser-Gly consensus is flanked by residues similar to those which flank the active sites of cellular aspartyl proteases. Mutations were made in the Ty3 active-site sequence to examine the role of the protease in Ty3 particle maturation and to test the functional significance of the Ser active-site variant in the consensus sequence. Mutation of the active-site Asp blocked processing of Gag3 and Gag3-Pol3 and allowed identification of a GAG3-POL3 polyprotein. This protein was turned over rapidly in cells expressing the mutant Ty3. Changing the active-site Ser to Thr caused only a modest reduction in the levels of certain Ty3 proteins. Five putative cleavage sites of this protease in Ty3 GAG3 and GAG3-POL3 polyproteins were defined by amino-terminal sequence analysis. The existence of an additional protein(s) of unknown function, encoded downstream of the protease-coding region, was deduced from the positions of these amino termini and the sizes of known Ty3 proteins. Although Ty3 protease cleavage sites do not correspond exactly to known retroviral protease cleavage sites, there are similarities. Residues P3 through P2' in the regions encompassing each of the five sites are uncharged, and no P1 position is occupied by an amino acid with a branched beta carbon.     

Ogre elements--a distinct group of plant Ty3/gypsy-like retrotransposons

Ogre elements are a group of LTR retrotransposons recently discovered in legume plants, where they constitute almost 40% of the genome in some species. They are exceptional in their size (reaching 25 kb) and possess several specific features, including an intron within a polyprotein-coding region, and an extra open reading frame (ORF1) encoding a protein of unknown function located upstream of the gag gene. Although these features make Ogres interesting for further research, identification of additional elements from a broader range of plant taxa has been complicated by the divergence of their sequences, preventing their detection using similarity-based searches. Here we report the results of structure-based computational searches for Ogre elements in available plant genomic sequences, which proved to be more efficient and revealed occurrences of Ogres in three families of dicot plants (Leguminosae, Solanaceae and Salicaceae). In addition, a representative set of 85 elements was retrieved from a model legume species Medicago truncatula. All identified full-length elements were used for comparative analysis, which showed that in spite of only little conservation of their nucleotide sequences, their protein domains were highly conserved, including several regions within ORF1. Further, the elements shared the same functional regions, including a primer binding site complementary to tRNA_arg, a conserved motif within a polypurine tract, and a putative intron between the pro and rt/rh coding domains. These findings, together with analysis of their phylogenetic relationship to other retrotransposons based on similarities of rt domains suggest that Ogre elements from different plant taxa have a common origin and thus constitute a distinct group of Ty3/gypsy retrotransposons.     

Site-specific integration of heterologous genes in yeast via Ty3 retrotransposition

The Ty3 retrotransposon of Saccharomyces cerevisiae was employed for the site-specific integration of heterologous genes into the yeast genome. A GAL-regulated promoter allowed induction of the retrotransposition process, and a bacterial neo(r) gene inserted in the Ty3 element was used as a selectable model heterologous gene. The frequency of transposition of this neo(r)-marked element was found to be comparable to that of an unmarked element. Three amplification systems were constructed; the systems varied with respect to the location and number of the GAL-regulated helper and neo(r)-marked Ty3 elements. For all three systems, neo(r) integrations were readily selected with a maximum of two insertions obtained per round of amplification. A sequential amplification strategy was effective for further increasing the number of integrated cloned genes, and families of strains varying by only one neo(r) insertion were easily obtained. Resistance to the antibiotic G418 correlated well with the number of integrated neo(r) genes, and Northern blots verified the relationship between cloned gene number (up to four) and neo(r) expression. Structural stability of the integrated genes was also demonstrated. By controlling the number of rounds of amplification and the level of G418 selection, precise numbers of integrated heterologous genes could be obtained. Because the amplification process can be repeated using different cloned genes inserted in the Ty3 element, these results demonstrate the potential of retrotransposition for the regulated integration of a series of different genes at nondeleterious chromosomal locations.