The Ty1-copia families SALIRE and Cotzilla populating the Beta vulgaris genome show remarkable differences in abundance, chromosomal distribution, and age

Long terminal repeat (LTR) retrotransposons are major components of plant genomes influencing genome size and evolution. Using two separate approaches, we identified the Ty1-copia retrotransposon families Cotzilla and SALIRE in the Beta vulgaris (sugar beet) genome. While SALIRE elements are similar to typical Ty1-copia retrotransposons, Cotzilla elements belong to a lineage called Sireviruses. Hallmarks of Cotzilla retrotransposons are the existence of an additional putative env-like open reading frame upstream of the 3′LTR, an extended gag region, and a frameshift separating the gag and pol genes. Detected in a c ₀ t-1 DNA library, Cotzilla elements belong to the most abundant retrotransposon families in B. vulgaris and are relatively homogenous and evolutionarily young. In contrast, the SALIRE family has relatively few copies, is diverged, and most likely ancient. As revealed by fluorescent in situ hybridization, SALIRE elements target predominantly gene-rich euchromatic regions, while Cotzilla retrotransposons are abundant in the intercalary and pericentromeric heterochromatin. The analysis of two retrotransposons from the same subclass contrasting in abundance, age, sequence diversity, and localization gives insight in the heterogeneity of LTR retrotransposons populating a plant genome.     

Divergence of satellite DNA and interspersion of dispersed repeats in the genome of the wild beet Beta procumbens

Several repetitive sequences of the genome of Beta procumbens Chr. Sm., a wild beet species of the section Procumbentes of the genus Beta have been isolated. According to their genomic organization, the repeats were assigned to satellite DNA and families of dispersed DNA sequences.The tandem repeats are 229–246 bp long and belong to an AluI restriction satellite designated pAp11. Monomers of this satellite DNA form subfamilies which can be distinguished by the divergence or methylation of an internal restriction site. The satellite is amplified in the section Procumbentes, but is also found in species of the section Beta including cultivated beet (Beta vulgaris). The existence of the pAp11 satellite in distantly related species suggests that the AluI sequence family is an ancient component of Beta genomes and the ancestor of the diverged satellite subfamily pEV4 in B. vulgaris. Comparative fluorescent in-situ hybridization revealed remarkable differences in the chromosomal position between B. procumbens and B. vulgaris, indicating that the pAp11 and pEV4 satellites were most likely involved in the expansion or rearrangement of the intercalary B. vulgaris heterochromatin.Furthermore, we describe the molecular structure, and genomic and chromosomal organization of two repetitive DNA families which were designated pAp4 and pAp22 and are 1354 and 582 bp long, respectively. The families consist of sequence elements which are widely dispersed along B. procumbens chromosomes with local clustering and exclusion from distal euchromatic regions. FISH on meiotic chromosomes showed that both dispersed repeats are colocalized in some chromosomal regions. The interspersion of repeats of the pAp4 and pAp22 family was studied by PCR and enabled the determination of repeat flanking sequences. Sequence analysis revealed that pAp22 is either derived from or part of a long terminal repeat (LTR) of an Athila-like retrotransposon. Southern analysis and FISH with pAp4 and pAp22 showed that both dispersed repeats are species-specific and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. This was tested in the sugar beet hybrid PRO1 which contains a small B. procumbens chromosome fragment.     

A PCR-based assay to detect En/Spm-like transposon sequences in plants

Degenerate primers deduced from the TPase region of plant En/Spm-like transposons allowed the amplification of similar sequences from various plant species including sugar beet, wheat and pea. These primers are efficient tools for the detection of this family of transposons in many plant genomes irrespective of sequence knowledge or phenotypic pecularities. An efficient PCR assay was therefore developed for these class II transposons, similar to assays already available for Ty1-copia-, Ty3-gypsy- or LINEs. This approach allowed us not only to show the widespread almost-ubiquitous presence of En/Spm-elements in plant genomes, but also to characterize their genomic organization and chromosomal distribution in the genome of chickpea (Cicer arietinum L.) and its abundance in related Cicer species. This approach can be used for the detection and characterization of endogenous DNA transposable elements in plant species, their complete isolation and evaluation of their use for genome analysis.     

Multiple LTR-retrotransposon families in the asexual yeast Candida albicans

We have begun a characterization of the long terminal repeat (LTR) retrotransposons in the asexual yeast Candida albicans. A database of assembled C. albicans genomic sequence at Stanford University, which represents 14.9 Mb of the 16-Mb haploid genome, was screened and >350 distinct retrotransposon insertions were identified. The majority of these insertions represent previously unrecognized retrotransposons. The various elements were classified into 34 distinct families, each family being similar, in terms of the range of sequences that it represents, to a typical Ty element family of the related yeast Saccharomyces cerevisiae. These C. albicans retrotransposon families are generally of low copy number and vary widely in coding capacity. For only three families, was a full-length and apparently intact retrotransposon identified. For many families, only solo LTRs and LTR fragments remain. Several families of highly degenerate elements appear to be still capable of transposition, presumably via trans-activation. The overall structure of the retrotransposon population in C. albicans differs considerably from that of S. cerevisiae. In that species, retrotransposon insertions can be assigned to just five families. Most of these families still retain functional examples, and they generally appear at higher copy numbers than the C. albicans families. The possibility that these differences between the two species are attributable to the nonstandard genetic code of C. albicans or the asexual nature of its genome is discussed. A region rich in retrotransposon fragments, that lies adjacent to many of the CARE-2/Rel-2 sub-telomeric repeats, and which appears to have arisen through multiple rounds of duplication and recombination, is also described.     

Genomic evolution of the long terminal repeat retrotransposons in hemiascomycetous yeasts

We identified putative long terminal repeat- (LTR) retrotransposon sequences among the 50,000 random sequence tags (RSTs) obtained by the Génolevures project from genomic libraries of 13 Hemiascomycetes species. In most cases additional sequencing enabled us to assemble the whole sequences of these retrotransposons. These approaches identified 17 distinct families, 10 of which are defined by full-length elements. We also identified five families of solo LTRs that were not associated with retrotransposons. Ty1-like retrotransposons were found in four of five species that are phylogenetically related to Saccharomyces cerevisiae (S. uvarum, S. exiguus, S. servazzii, and S. kluyveri but not Zygosaccharomyces rouxii), and in two of three Kluyveromyces species (K. lactis and K. marxianus but not K. thermotolerans). Only multiply crippled elements could be identified in the K. lactis and S. servazzii strains analyzed, and only solo LTRs could be identified in S. uvarum. Ty4-like elements were only detected in S. uvarum, indicating that these elements appeared recently before speciation of the Saccharomyces sensu stricto species. Ty5-like elements were detected in S. exiguus, Pichia angusta, and Debaryomyces hansenii. A retrotransposon homologous with Tca2 from Candida albicans, an element absent from S. cerevisiae, was detected in the closely related species D. hansenii. A complete Ty3/gypsy element was present in S. exiguus, whereas only partial, often degenerate, sequences resembling this element were found in S. servazzii, Z. rouxii, S. kluyveri, C. tropicalis, and Yarrowica lipolytica. P. farinosa (syn. P. sorbitophila) is currently the only yeast species in which no LTR retrotransposons or remnants have been found. Thorough analysis of protein sequences, structural characteristics of the elements, and phylogenetic relationships deduced from these data allowed us to propose a classification for the Ty1/copia elements of hemiascomycetous yeasts and a model of LTR-retrotransposon evolution in yeasts.     

Local definition of Ty1 target preference by long terminal repeats and clustered tRNA genes

LTR-containing retrotransposons reverse transcribe their RNA genomes, and the resulting cDNAs are integrated into the genome by the element-encoded integrase protein. The yeast LTR retrotransposon Ty1 preferentially integrates into a target window upstream of tDNAs (tRNA genes) in the yeast genome. We investigated the nature of these insertions and the target window on a genomic scale by analyzing several hundred de novo insertions upstream of tDNAs in two different multicopy gene families. The pattern of insertion upstream of tDNAs was nonrandom and periodic, with peaks separated by ~80 bp. Insertions were not distributed equally throughout the genome, as certain tDNAs within a given family received higher frequencies of upstream Ty1 insertions than others. We showed that the presence and relative position of additional tDNAs and LTRs surrounding the target tDNA dramatically influenced the frequency of insertion events upstream of that target.     

Daidara: A gigantic Gypsy LTR retrotransposon lineage in the springtail Allacma fusca genome

Long terminal repeat (LTR) retrotransposons are the major contributor to genome size expansion, as in the cases of the maize genome or the axolotl genome. Despite their impact on the genome size, the length of each retrotransposon is limited, compared to DNA transposons, which sometimes exceed over 100 kb. The longest LTR retrotransposon known to date is Burro-1 from the planarian Schmidtea medierranea, which is around 35.7 kb long. Here through bioinformatics analysis, a new lineage of gigantic LTR retrotransposons, designated Daidara, is reported from the springtail Allacma fusca genome. Their entire length (25–33 kb) rivals Burro families, while their LTRs are shorter than 1.5 kb, in contrast to other gigantic LTR retrotransposon lineages Burro and Ogre, whose LTRs are around 5 kb long. Daidara encodes three core proteins corresponding to gag, pol, and an additional protein of unknown function. The phylogenetic analysis supports the independent gigantification of Daidara from Burro or Ogre.     

Sequence organization and evolutionary dynamics of Brachypodium-specific centromere retrotransposons

Brachypodium distachyon is a wild annual grass belonging to the Pooideae, more closely related to wheat, barley, and forage grasses than rice and maize. As an experimental model, the completed genome sequence of B. distachyon provides a unique opportunity to study centromere evolution during the speciation of grasses. Centromeric satellite sequences have been identified in B. distachyon, but little is known about centromeric retrotransposons in this species. In the present study, bacterial artificial chromosome (BAC)-fluorescence in situ hybridization was conducted in maize, rice, barley, wheat, and rye using B. distachyon (Bd) centromere-specific BAC clones. Eight Bd centromeric BAC clones gave no detectable fluorescence in situ hybridization (FISH) signals on the chromosomes of rice and maize, and three of them also did not yield any FISH signals in barley, wheat, and rye. In addition, four of five Triticeae centromeric BAC clones did not hybridize to the B. distachyon centromeres, implying certain unique features of Brachypodium centromeres. Analysis of Brachypodium centromeric BAC sequences identified a long terminal repeat (LTR)-centromere retrotransposon of B. distachyon (CRBd1). This element was found in high copy number accounting for 1.6 % of the B. distachyon genome, and is enriched in Brachypodium centromeric regions. CRBd1 accumulated in active centromeres, but was lost from inactive ones. The LTR of CRBd1 appears to be specific to B. distachyon centromeres. These results reveal different evolutionary events of this retrotransposon family across grass species.     

Distinct chromosomal distributions of highly repetitive sequences in maize

The majority of genomic DNA in most plant species is made up of repetitive elements including satellites and retrotransposons. The maize genome is intermediate in size and abundance of repetitive elements between small genomes such as Arabidopsis and rice and larger genomes such as wheat. Although repetitive elements are present throughout the maize genome, individual families are non-randomly distributed along chromosomes. In this work we use fluorescence in-situ hybridization (FISH) to examine the distribution of abundant LTR retroelement families and satellites contained in heterochromatic blocks called knobs. Different retroelement families have distinct patterns of hybridization. Prem1 and Tekay, two very closely related elements, both hybridize along the length of all chromosomes but do so with greater intensity near the centromeres, although subtle differences are detectable between the hybridization patterns. Opie, Prem2/Ji, and Huck are enriched away from the centromeres and Grande is distributed uniformly along the chromosomes. Double labeling with proximally and distally enriched elements on pachytene chromosomes produces alternating blocks of element enrichment. The maize elements hybridized in the same general patterns to chromosomes of maize relatives including Zea diploperennis and Tripsacum dactyloides. Additionally, abundant Tripsacum LTR retroelements are enriched in similar chromosomal regions among the different species. The 180 bp knob satellite is present in large blocks at interstitial locations on chromosome arms. With long exposures, smaller sites of hybridization are detected at the ends of chromosomes, adjacent to the telomere tract. This distal position for knob satellites is conserved among Zea and Tripsacum species. Electronic supplementary material Supplementary material to this paper is available in electronic form at and is accessible for authorized users.     

Retrotransposon-adenovirus hybrid vectors: efficient delivery and stable integration of transgenes via a two-stage mechanism

It has long been recognized that the mechanisms mediating retrotransposition might be adapted for genomic integration and long-term expression of foreign genes. In particular, long interspersed nuclear elements (LINEs), an abundant class of retrotransposons that are the most active mobile genetic elements in the human genome, have been largely ignored as candidates for development as an integrating vector system because there has been no suitable method for efficiently introducing them into target cells. We have recently developed a LINE-based retrotransposon-adenovirus hybrid vector, in which a helper-dependent adenovirus (HDAd) is utilized as the platform for delivery of a human L1 element and its linked heterologous transgene cassette into the host cell nuclei. While a major drawback to the use of HDAd vectors has been their lack of specific mechanisms to achieve permanent integration into the host genome, the inserted retrotransposon sequences overcome this limitation. The L1-HDAd hybrid thus represents a single vector capable of mediating long-term gene expression by a two-stage mechanism: in the first (adenovirus) stage, the helper-dependent adenovirus serves as a carrier for efficient delivery and transient expression of its encoded L1/transgene cassette, and in the second (retrotransposon) stage, the L1 retro-element and its associated transgene then permanently integrate into the genome of the adenovirus-transduced cells. We propose that this novel retrotransposon-adenovirus hybrid vector system will be useful both as a vehicle for efficient delivery and long-term stable transduction of therapeutic genes, as well as a tool to elucidate aspects of retrotransposon biology that have previously been difficult to study.     

Mobilization and evolutionary history of miniature inverted-repeat transposable elements (MITEs) in Beta vulgaris L.

We have identified three families of miniature inverted-repeat transposable elements (VulMITEs) in the genome of sugar beet (Beta vulgaris L.), evidently derived from a member of the Vulmar family of mariner transposons. While VulMITEs I are typical stowaway-like MITEs, VulMITEs II and VulMITEs III are rearranged stowaway elements of increased size. The integration of divergent moderately and highly repetitive sequences into VulMITEs II and, in particular in VulMITEs III, respectively, shows that amplification of repetitive DNA by MITEs contribute to the increase of genome size with possible implications for plant genome evolution. Fluorescent in-situ hybridization (FISH), for the first time visualizing stowaway MITE distribution on plant chromosomes, revealed a dispersed localization of VulMITEs along all B. vulgaris chromosomes. Analysis of the flanking sequences identified a dispersed repeat as target site for the integration of the stowaway element VulMITE I. Recent transposition of VulMITE I, which most likely occurred during the domestication of cultivated beets, was concluded from insertional polymorphisms between different B. vulgaris cultivars and species. ^†Sequence data from this article have been deposited in the EMBL/GenBank Data Library under the accession nos. AM231630-AM231653 and AM259123-AM259125.     

Thermal blockage of viruslike particle formation for the yeast retrotransposon Ty3 reveals differences in the cellular stress response

Summary.  The long terminal repeat (LTR) retrotransposons of the yeast Saccharomyces cerevisiae are similar in their structures and life cycles to animal retroviruses. The yeast LTR retrotransposon Ty3 does not transpose under conditions where the cellular stress response is activated. During stress, mature Ty3 proteins, indicative of the formation of intracellular Ty3 viruslike particles (VLPs), do not accumulate. In order to examine the role of stress proteins in Ty3 transposition, a sensitive genetic assay was developed to measure VLP formation. The assay employs a Ty3 element marked with a mutant allele of the yeast HIS3 gene (his3AI). To create a stable His⁺ phenotype, Ty3 must form VLPs, reverse transcribe Ty3 RNA into cDNA, and then insert the cDNA into either chromosomal or plasmid DNA. Using this assay, thermal inhibition of Ty3 transposition was evident at temperatures as low as 30 °C. The level of production of mature Ty3 proteins parallels the transposition frequency. Although overexpression of the yeast UBP3 gene allows VLPs to form and transposition to occur in the constitutively stressed ssa1 ssa2 strain, it does not alleviate the inhibition of these processes during stress induced by heat or ethanol. This suggests that the genetic and physical modes of stress response induction are not equivalent. Accepted April 11, 2001 Received February 2, 2001