cDNA of the Yeast Retrotransposon Ty5 Preferentially Recombines with Substrates in Silent Chromatin

The yeast retrotransposon Ty5 preferentially integrates into regions of silent chromatin. Ty5 cDNA also recombines with homologous sequences, generating tandem elements or elements that have exchanged markers between cDNA and substrate. In this study, we demonstrate that Ty5 integration depends upon the conserved DD(35)E domain of integrase and cis-acting sequences at the end of the long terminal repeat (LTR) implicated in integrase binding. cDNA recombination requires Rad52p, which is responsible for homologous recombination. Interestingly, Ty5 cDNA recombines at least three times more frequently with substrates in silent chromatin than with a control substrate at an internal chromosomal locus. This preference depends upon the Ty5 targeting domain that is responsible for integration specificity, suggesting that localization of cDNA to silent chromatin results in the enhanced recombination. Recombination with a telomeric substrate occasionally generates highly reiterated Ty5 arrays, and mechanisms for tandem element formation were explored by using a plasmid-based recombination assay. Point mutations were introduced into plasmid targets, and recombination products were characterized to determine recombination initiation sites. Despite our previous observation of the importance of the LTR in forming tandem elements, recombination cannot simply be explained by crossover events between the LTRs of substrate and cDNA. We propose an alternative model based on single-strand annealing, where single-stranded cDNA initiates tandem element formation and the LTR is required for strand displacement to form a looped intermediate. Retrotransposons are increasingly found associated with chromosome ends, and amplification of Ty5 by both integration and recombination exemplifies how retroelements can contribute to telomere dynamics.     

Localization of sequences required in cis for yeast Ty1 element transposition near the long terminal repeats: analysis of mini-Ty1 elements.

In order to identify and characterize sequences within Ty1 elements which are required in cis for transposition, a series of mini-Ty1 plasmids were constructed and tested for transposition. Mini-Ty1s are deletion mutants of the Ty1-H3 element; Ty1 gene products required for transposition are supplied in trans from a helper Ty1 which has intact open reading frames but lacks a 3' long terminal repeat (LTR) and therefore cannot transpose itself. Up to 5 kilobase pairs of internal sequences of the 6-kilobase-pair-long Ty1 element can be deleted without a significant effect on transposition. The smallest mini-Ty1 element capable of transposition contains the 3' LTR and the transcribed portion of the 5' LTR, 285 base pairs (bp) of internal sequence 3' to the 5' LTR, and 23 bp of internal sequence 5' to the 3' LTR. We conclude that Ty1-encoded proteins can act in trans and that cis-acting sequences in Ty1-H3 are all within or near the LTRs. Further deletion of the 285-bp internal sequence adjacent to the 5' LTR significantly reduced transposition frequency, and the mini-Ty1 RNA produced failed to be packaged into the viruslike particles efficiently. Surprisingly, several nonhomologous cellular mRNAs were also associated with viruslike particles.     

An element with long terminal repeats and its variant arrangements in the genome of Lilium henryi

Summary A 9.35 kbp element with long terminal direct repeats (LTRs) of 2.4 kbp has been characterized from the large genome of Lilium henryi. The organization of the element, named del, was examined in 20 fragments from a genomic DNA library constructed using phage EMBL3. Five fragments apparently contained full 9.35 kbp elements while in 11 del was recovered in part. Two clones carried tandem arrangements of del, with single LTRs and internal sequences alternating, and one insert contained a solo LTR. Evidence suggests that these arrangements, which can arise by unequal crossing over, have a genomic rather than a cloning origin. Finally, one cloned fragment had a complex del arrangement yet to be fully defined. Limited sequencing of five LTRs (from a full del, a tandem arrangement and a solo LTR) indicates that the consensus termini are 5TGT ... ACA 3, with del sequences flanked by a 5 bp tandem repeat. Thus del shares properties with retrotransposons such as the copia family of Drosophila and Ty of yeast. However with more than 13000 copies per genome it seems del has been amplified more than is usual for such elements.     

The Rad27 (Fen-1) nuclease inhibits Ty1 mobility in Saccharomyces cerevisiae

Although most Ty1 elements in Saccharomyces cerevisiae are competent for retrotransposition, host defense genes can inhibit different steps of the Ty1 life cycle. Here, we demonstrate that Rad27, a structure-specific nuclease that plays an important role in DNA replication and genome stability, inhibits Ty1 at a post-translational level. We have examined the effects of various rad27 mutations on Ty1 element retrotransposition and cDNA recombination, termed Ty1 mobility. The point mutations rad27-G67S, rad27-G240D, and rad27-E158D that cause defects in certain enzymatic activities in vitro result in variable increases in Ty1 mobility, ranging from 4- to 22-fold. The C-terminal frameshift mutation rad27-324 confers the maximum increase in Ty1 mobility (198-fold), unincorporated cDNA, and insertion at preferred target sites. The null mutation differs from the other rad27 alleles by increasing the frequency of multimeric Ty1 insertions and cDNA recombination with a genomic element. The rad27 mutants do not markedly alter the levels of Ty1 RNA or the TyA1-gag protein. However, there is an increase in the stability of unincorporated Ty1 cDNA in rad27-324 and the null mutant. Our results suggest that Rad27 inhibits Ty1 mobility by destabilizing unincorporated Ty1 cDNA and preventing the formation of Ty1 multimers.     

Increased Length of Long Terminal Repeats Inhibits Ty1 Transposition and Leads to the Formation of Tandem Multimers

The Ty1 retrotransposon of Saccharomyces cerevisiae is bounded by long-terminal repeats (LTRs). We have constructed a variety of Ty1 elements in which the LTR length has been increased from the normal length of 334 bp to >2 kb. Although small insertions in the LTR have minimal effects on transposition frequency, larger insertions dramatically reduce it. Nevertheless, elements with long LTRs are incorporated into the genome at a low frequency. Most of these rare insertion events represent Ty1 tandem (head to tail) multimers.     

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.     

Ty1 copy number dynamics in Saccharomyces

To understand long terminal repeat (LTR)-retrotransposon copy number dynamics, Ty1 elements were reintroduced into a "Ty-less" Saccharomyces strain where elements had been lost by LTR-LTR recombination. Repopulated strains exhibited alterations in chromosome size that were associated with Ty1 insertions, but did not become genetically isolated. The rates of element gain and loss under genetic and environmental conditions known to affect Ty1 retrotransposition were determined using genetically tagged reference elements. The results show that Ty1 retrotransposition varies with copy number, temperature, and cell type. In contrast to retrotransposition, Ty1 loss by LTR-LTR recombination was more constant and not markedly influenced by copy number. Endogenous Ty1 cDNA was poorly utilized for recombination when compared with LTR-LTR recombination or ectopic gene conversion. Ty1 elements also appear to be more susceptible to copy number fluctuation in haploid cells. Ty1 gain/loss ratios obtained under different conditions suggest that copy number oscillates over time by altering the rate of retrotransposition, resulting in the diverse copy numbers observed in Saccharomyces.     

Multimeric arrays of the yeast retrotransposon Ty.

We have identified a novel integrated form of the yeast retrotransposon Ty consisting of multiple elements joined into large arrays. These arrays were first identified among Ty-induced alpha-pheromone-resistant mutants of MATa cells of Saccharomyces cerevisiae which contain Ty insertions at HML alpha that result in the expression of that normally silent cassette. These insertions are multimeric arrays of both the induced genetically marked Ty element and unmarked Ty elements. Structural analysis of the mutations indicated that the arrays include tandem direct repeats of Ty elements separated by only a single long terminal repeat. The Ty-HML junction fragments of one mutant were cloned and shown to contain a 5-base-pair duplication of the target sequence that is characteristic of a Ty transpositional insertion. In addition, the arrays include rearranged Ty elements that do not have normal long terminal repeat junctions. We have also identified multimeric Ty insertions at other chromosomal sites and as insertions that allow expression of a promoterless his3 gene on a plasmid. The results suggest that Ty transposition includes an intermediate that can undergo recombination to produce multimers.     

Recombination of Ty Elements in Yeast Can Be Induced by a Double-Strand Break

The Ty retrotransposons are the main family of dispersed repeated sequences in the yeast Saccharomyces cerevisiae. These elements are flanked by a pair of long terminal direct repeats (LTRs). Previous experiments have shown that Ty elements recombine at low frequencies, despite the fact that they are present in 30 copies per genome. This frequency is not highly increased by treatments that cause DNA damage, such as UV irradiation. In this study, we show that it is possible to increase the recombination level of a genetically marked Ty by creating a double-strand break in it. This break is repaired by two competing mechanisms: one of them leaves a single LTR in place of the Ty, and the other is a gene conversion event in which the marked Ty is replaced by an ectopically located one. In a strain in which the marked Ty has only one LTR, the double-strand break is repaired by conversion. We have also measured the efficiency of repair and monitored the progression of the cells through the cell-cycle. We found that in the presence of a double-strand break in the marked Ty, a proportion of the cells is unable to resume growth.     

Rapid Evolution of Recombinant Saccharomyces cerevisiae for Xylose Fermentation through Formation of Extra-chromosomal Circular DNA

Circular DNA elements are involved in genome plasticity, particularly of tandem repeats. However, amplifications of DNA segments in Saccharomyces cerevisiae reported so far involve pre-existing repetitive sequences such as ribosomal DNA, Ty elements and Long Terminal Repeats (LTRs). Here, we report the generation of an eccDNA, (extrachromosomal circular DNA element) in a region without any repetitive sequences during an adaptive evolution experiment. We performed whole genome sequence comparison between an efficient D-xylose fermenting yeast strain developed by metabolic and evolutionary engineering, and its parent industrial strain. We found that the heterologous gene XylA that had been inserted close to an ARS sequence in the parent strain has been amplified about 9 fold in both alleles of the chromosomal locus of the evolved strain compared to its parent. Analysis of the amplification process during the adaptive evolution revealed formation of a XylA-carrying eccDNA, pXI2-6, followed by chromosomal integration in tandem arrays over the course of the evolutionary adaptation. Formation of the eccDNA occurred in the absence of any repetitive DNA elements, probably using a micro-homology sequence of 8 nucleotides flanking the amplified sequence. We isolated the pXI2-6 eccDNA from an intermediate strain of the evolutionary adaptation process, sequenced it completely and showed that it confers high xylose fermentation capacity when it is transferred to a new strain. In this way, we have provided clear evidence that gene amplification can occur through generation of eccDNA without the presence of flanking repetitive sequences and can serve as a rapid means of adaptation to selection pressure.     

Homologous recombination between the LTRs of a human retrovirus-like element causes a 5-kb deletion in two siblings.

The RTVL-H family of human endogenous retrovirus-like elements consists of approximately 1,000 intact members and of a similar number of solitary long terminal repeats (LTRs). In this study, the genetic heterogeneity of these elements has been investigated using unique flanking probes isolated from cDNA clones containing RTVL-H sequences. Four such probes were used to screen a panel of human DNA samples for genetic differences. One of these probes detected a 5.0-kb deletion in two related individuals. Cloning and DNA hybridization analysis indicated that the nondeleted common allele contained an intact RTVL-H element, whereas the deleted variant allele contained only a single LTR. DNA sequence comparisons strongly suggest that the deletion is due to homologous recombination between the 5' and 3' LTRs of the RTVL-H sequence. This is the first reported case of a DNA variation in humans that is due to an LTR-LTR excision event.