The Arabidopsis transposable element Tag1 is widely distributed among Arabidopsis ecotypes

Tag1 is an autonomous transposable element (3.3 kb in length) first identified as an insertion in the CHL1 (NRT1) gene of Arabidopsis thaliana. Tag1 has been found in the Landsberg erecta ecotype of A. thaliana but not in Columbia or WS. In this paper, 41 additional ecotypes were examined for the presence of Tag1. Using an internal Tag1 fragment as probe, we found that DNA from 19 of the 41 ecotypes strongly hybridized to Tag1. Almost all of the Tag1-containing ecotypes had only one or two copies of Tag1 per haploid genome, as determined by Southern blot analysis. The only exception, Bf-1 from Bretagny-sur-Orge, France, had four copies. Two ecotypes, Di-G and S96, gave identical Southern blot patterns to that of Landsberg erecta and were subsequently shown to contain Tag1 at the same two positions found in Landsberg erecta (loci designated as Tag1-2 and Tag1-3). Two other ecotypes, Ag-0 and Lo-1, had a Tag1 element located at Tag1-2 but not at Tag1-3. The distance between these two loci was determined to be 0.37 cM. Analysis of DNA from two related species, A. griffithiana and A. pumila, showed that both species contain sequences that hybridize to Tag1 and that could be amplified with an oligonucleotide specific to the terminal inverted repeats of Tag1. These results show that Tag1 and related elements are present, and may be useful for insertional mutagenesis, in many A. thaliana ecotypes and several Arabidopsis species. Received: 18 August 1997 / Accepted: 9 October 1997     

Tag1 is an autonomous transposable element that shows somatic excision in both Arabidopsis and tobacco.

Tag1 is a transposable element first identified as an insertion in the CHL1 gene of Arabidopsis. The chl1::Tag1 mutant originated from a plant (ecotype Landsberg erecta) that had been transformed with the maize transposon Activator (Ac), which is distantly related to Tag1. Genomic analysis of untransformed Landsberg erecta plants demonstrated that two identical Tag1 elements are present in the Landsberg erecta genome. To determine what provides transposase function for Tag1 transposition, we examined Tag1 excision in different genetic backgrounds. First, the chl1::Tag1 mutant was backcrossed to untransformed wild-type Arabidopsis plants to remove the Ac element(s) from the genome. F2 progeny that had no Ac elements but still retained Tag1 in the CHL1 gene were identified. Tag1 still excised in these Ac-minus progeny producing CHL1 revertants; therefore, Ac is not required for Tag1 excision. Next, Tag1 was inserted between a cauliflower mosaic virus 35S promoter and a beta-glucuronidase (GUS) marker gene and transformed into tobacco. Transformants showed blue-staining sectors indicative of Tag1 excision. Transgenic tobacco containing a defective Tag1 element, which was constructed in vitro by deleting an internal 1.4-kb EcoRI fragment, did not show blue-staining sectors. We conclude that Tag1 is an autonomous element capable of independent excision. The 35S-GUS::Tag1 construct was then introduced into Arabidopsis. Blue-staining sectors were found in cotyledons, leaves, and roots, showing that Tag1 undergoes somatic excision during vegetative development in its native host.     

Engineering variegated floral patterns in tobacco plants using the Arabidopsis transposable element Tag1

Variegated flower phenotypes were generated using the Arabidopsis transposon Tag1 and the maize R regulatory gene. Tag1 was inserted between the CaMV 35S promoter and the maize R gene and transformed into tobacco plants. In half of the transgenic plants, variegated flower patterns were observed. Each line had a different pattern, with varying intensities with three lines showing only tiny sectors indicative of late excision and one showing large sectors indicative of earlier excision.     

Characterization of the germinal and somatic activity of the Arabidopsis transposable element Tag1.

Tag1 is an autonomous transposon of Arabidopsis thaliana. The excision behavior of Tag1 during reproductive and vegetative development was examined using CaMV 35STag1-GUS constructs. Germinal reversion frequencies varied from 0 to 27% and correlated with Tag1 copy number. Southern blot and somatic sector analyses indicated that each revertant was derived from an independent excision event, and approximately 75% of the revertants had new Tag1 insertions. Revertants were obtained with similar frequencies from the male and female parents. In flowers, small somatic sectors were observed in siliques, carpels, petals and sepals while stemlike organs (filaments and pedicels) had larger sectors. No sectors encompassing entire flowers or inflorescences were observed, however, indicating that excision occurs late in flower development and rarely in inflorescence meristems. Late excision was also observed during vegetative development with 99.8% of leaves showing small sectors encompassing no more than 20 cells. Roots and cotyledons, however, showed larger sectors that included entire lateral roots and cotyledons. These results indicate that Tag1 can excise in the embryo and all the organs of the plant with the timing of excision being restricted to late stages of vegetative and reproductive development in the shoot.     

Transfer of the maize transposable element MU1 into Arabidopsis thaliana

The maize transposable element Mu1 was transferred to Arabidopsis thaliana by Ti plasmid-mediated transformation and a fertile line containing Mu1 was regenerated. Southern analysis of transformed tissue indicated that the Mu1 DNA remained entirely within a segment of T-DNA during three sexual generations. The results of a search for spontaneous mutations in a large number of Mu1-containing seedlings suggests that the presence of Mu1 did not cause a major increase in the spontaneous mutation frequency.     

The transposable element Uhu from Hawaiian Drosophila--member of the widely dispersed class of Tc1-like transposons

We report the complete nucleotide sequence of the transposable element Uhu from the vicinity of the alcohol dehydrogenase (Adh) gene of Drosophila heteroneura (an endemic Hawaiian Drosophila). The complete element is about 1650 base-pairs (bp) long, has 46-50 base-pair inverse imperfect repeats at it's ends, and contains a large open reading frame potentially encoding a 192 amino acid protein. We demonstrate that Uhu belongs to a class of transposable elements which includes Tc1 from Caenorhabditis elegans, Barney from Caenorhabditis briggsae, and HB1 from Drosophila melanogaster. All of these elements share significant sequence similarity, are approximately 1600 base pairs long, have short inverse terminal repeats (ITRs), contain open reading frames (ORFs) with significant sequence identity, and appear to insert specifically at TA sequences generating target site duplications.     

Functional dissection of the cis-acting sequences of the Arabidopsis transposable element Tag1 reveals dissimilar subterminal sequence and minimal spacing requirements for transposition

The Arabidopsis transposon Tag1 has an unusual subterminal structure containing four sets of dissimilar repeats: one set near the 5' end and three near the 3' end. To determine sequence requirements for efficient and regulated transposition, deletion derivatives of Tag1 were tested in Arabidopsis plants. These tests showed that a 98-bp 5' fragment containing the 22-bp inverted repeat and four copies of the AAACCX (X = C, A, G) 5' subterminal repeat is sufficient for transposition while a 52-bp 5' fragment containing only one copy of the subterminal repeat is not. At the 3' end, a 109-bp fragment containing four copies of the most 3' repeat TGACCC, but not a 55-bp fragment, which has no copies of the subterminal repeats, is sufficient for transposition. The 5' and 3' end fragments are not functionally interchangeable and require an internal spacer DNA of minimal length between 238 and 325 bp to be active. Elements with these minimal requirements show transposition rates and developmental control of excision that are comparable to the autonomous Tag1 element. Last, a DNA-binding activity that interacts with the 3' 109-bp fragment but not the 5' 98-bp fragment of Tag1 was found in nuclear extracts of Arabidopsis plants devoid of Tag1.     

The transposition frequency of Tag1 elements is increased in transgenic Arabidopsis lines.

Tag1 was identified as a highly active endogenous transposable element in transgenic Arabidopsis thaliana Landsberg erecta plants carrying the maize transposable element Activator (Ac). Here, we describe experiments designed to determine the basis for the high activity of Tag1. The frequency of transposition of Tag1 elements was compared in lines containing or lacking Ac transposase to assess the effect of Ac transposase on Tag1 activity. Three populations of nontransgenic plants, including nontransformed regenerants, were also analyzed. The high level of activity of Tag1 did not correlate with the presence or absence of Ac transposase but was significantly higher in transgenic lines. This result was maintained through at least six generations after transformation. These data suggest that Tag1 transposition is stimulated by processes that occur during the Agrobacterium transformation and that thereafter remain active. Two Tag1 elements are tightly linked in the Landsberg erecta genome and map to the lower arm of chromosome 1. Tag1 elements were found in only a few A. thaliana ecotypes but were present in four other Arabidopsis species.     

Behaviour of the maize transposable element Ac in Arabidopsis thaliana

The somatic and germinal activity of the maize transposable element, Ac, has been analysed in progeny of 43 transformants of A. thaliana using a streptomycin resistance assay to monitor Ac excision. The ability to assay somatic activity enabled, for the first time, a detailed analysis of Ac activity in individual A. thaliana seedlings to be made. The effects of T-DNA copy number, generation, dosage at each locus, flanking sequences and orientation of the element were compared. The most striking observation was the variability in Ac activity in genotypically identical individuals and the poor penetrance of the variegated phenotype. In general, increasing Ac dosage increased both somatic and germinal excision frequencies. The majority of families from individuals selected as inheriting an excision event carried transposed Ac elements re-integrated in different positions in the genome.     

The transposable element Tam1 of Antirrhinum majus is 17 kb long

Summary The nivea locus of Antirrhinum majus encodes chalcone synthase, a key enzyme in anthocyanin biosynthesis. Genetic instability is known to occur at this locus. In vitro cloning and characterization of genomic DNA fragments from the unstable nivea-recurrens allele T53 revealed that the instability of nivea-recurrens is due to the presence of a 17 kb DNA insert in the nivea locus. Somatic instability leading to the variegated phenotype, i.e. highly spotted flowers, is due to frequent excision of the 17 kb Tam1 element (Transposon antirrhinum majus) during development of the plant. Excision of Tam1 is not tissue specific, but occurs with similar frequencies in leaves and blossoms.     

Genetic variation among endosymbionts of widely distributed vestimentiferan tubeworms

Vestimentiferan tubeworms thriving in sulfidic deep-sea hydrothermal vents and cold seeps are constrained by their nutritional reliance on chemoautotrophic endosymbionts. In a recent phylogenetic study using 16S ribosomal DNA, we found that endosymbionts from vent and seep habitats form two distinct clades with little variation within each clade. In the present study, we used two different approaches to assess the genetic variation among biogeographically distinct vestimentiferan symbionts. DNA sequences were obtained for the noncoding, internal transcribed spacer (ITS) regions of the rRNA operons of symbionts associated with six different genera of vestimentiferan tubeworms. ITS sequences from endosymbionts of host genera collected from different habitats and widely distributed vent sites were surprisingly conserved. Because the ITS region was not sufficient for distinguishing endosymbionts from different habitats or locations, we used a DNA fingerprinting technique, repetitive-extragenic-palindrome PCR (REP-PCR), to reveal differences in the distribution of repetitive sequences in the genomes of the bacterial endosymbionts. Most of the endosymbionts displayed unique REP-PCR patterns. A cladogram generated from these fingerprints reflected relationships that may be influenced by a variety of factors, including host genera, geographic location, and bottom type.     

The capacity of UL49.5 proteins to inhibit TAP is widely distributed among members of the genus Varicellovirus

The lifelong infection by varicelloviruses is characterized by a fine balance between the host immune response and immune evasion strategies used by these viruses. Virus-derived peptides are presented to cytotoxic T lymphocytes by major histocompatibility complex (MHC) class I molecules. The transporter associated with antigen processing (TAP) transports the peptides from the cytosol into the endoplasmic reticulum, where the loading of MHC-I molecules occurs. The varicelloviruses bovine herpesvirus 1 (BoHV-1), pseudorabies virus, and equid herpesviruses 1 and 4 have been found to encode a UL49.5 protein that inhibits TAP-mediated peptide transport. To investigate to what extent UL49.5-mediated TAP inhibition is conserved within the family of Alphaherpesvirinae, the homologs of another five varicelloviruses, one mardivirus, and one iltovirus were studied. The UL49.5 proteins of BoHV-5, bubaline herpesvirus 1, cervid herpesvirus 1, and felid herpesvirus 1 were identified as potent TAP inhibitors. The varicella-zoster virus and simian varicellovirus UL49.5 proteins fail to block TAP; this is not due to the absence of viral cofactors that might assist in this process, since cells infected with these viruses did not show reduced TAP function either. The UL49.5 homologs of the mardivirus Marek's disease virus 1 and the iltovirus infectious laryngotracheitis virus did not block TAP, suggesting that the capacity to inhibit TAP via UL49.5 has been acquired by varicelloviruses only. A phylogenetic analysis of viruses that inhibit TAP through their UL49.5 proteins reveals an interesting hereditary pattern, pointing toward the presence of this capacity in defined clades within the genus Varicellovirus.     

Genetic similarity among Arabidopsis thaliana ecotypes estimated by DNA sequence comparison

DNA polymorphisms among Arabidopsis thaliana ecotypes are widely used as genetic markers in map-based cloning strategies. New PCR-based molecular markers do not only facilitate molecular mapping, but can also be used to obtain reliable sequence information for cladistic analyses. We have used CAPS (cleaved amplified polymorphic sequences) markers and a direct sequencing strategy to estimate genetic similarity among eighteen Arabidopsis ecotypes. Sequences at four loci, two from the nuclear and two from a non-nuclaar genome, were analysed. For each ecotype more than 1000pb of sequence information was obtained, and genetic similarity was calculated from a total of 35 polymorphic sites using a character-based approach. Divergence ranged from zero up to 50 discordant characters among the 72 characters defined by the polymorphisms. Separate calculations based on the nuclear and the non-nuclear sequences were performed and revealed a number of common features, including the existence of small clusters of very closely related ecotypes separated from each other by extensive sequence divergence. Our results provide information useful especially to investigators setting up crosses for chromosome landing strategies.