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PubMed1995

Changes in the chromosomal insertion pattern of the copia element during the process of making chromosomes homozygous in Drosophila melanogaster.

In situ hybridization on polytene chromosomes of Drosophila melanogaster was used to compare the insertion patterns of copia and mdg1 transposable elements on chromosome 2 in male gametes sampled by two different methods: (i) by crossing the males tested with females from a highly inbred line with known copia and mdg1 insertion profiles; (ii) by crossing the same males with females from a marked strain, and analysing the resulting homozygous chromosomes. Crossing of the males with the inbred line led to homogeneous insertion profiles for both the copia and mdg1 elements in larvae, thus giving an accurate estimation of the patterns in the two gamete classes of each male. Crossing with the marked strain led, however, to heterogeneity in insertion patterns of the copia transposable element, while no significant polymorphism was observed for mdg1. The use of balancer chromosomes is thus not an adequate way of inferring transposable element insertion patterns of Drosophila males, at least for the copia element. This technique could, however, be powerful for investigating the control of movements of this element.

Animals↗
PubMed1992

Dosage compensation of the copia retrotransposon in Drosophila melanogaster.

Dosage compensation in Drosophila has been studied at the steady state RNA level for several single-copy genes; however, an important point is addressed by analyzing a repetitive, transposable element for dosage compensation. The two issues of gene-specific cis control and genomic position can be studied by determining the extent of dosage compensation of a transposable element at different chromosomal locations. To determine whether the multicopy copia transposable element can dosage compensate, we used the X-linked white-apricot (wa) mutation in which a copia element is present. The extent of dosage compensation was determined for the white and copia promoters in larvae and adults in two different genomic locations of the wa allele. We conclude that copia is able to dosage compensate, and that the white promoter and the copia promoter are not coordinate in their dosage compensation abilities when assayed under these various conditions. Thus, two transcriptional units, one within the other, both of which are able to dosage compensate, do so differently in response to developmental stage and genomic position.

Animals↗
PubMed1987

Heritable somatic excision of a Drosophila transposon.

A mutation in the white gene of Drosophila mauritiana that results from insertion of the transposable element mariner is genetically unstable in both germ cells and somatic cells. Somatic instability is indicated by the occurrence of animals having mosaic eyes with patches of pigmented cells on a peach-colored background. Normally uncommon, the frequency of mosaicism is so greatly enhanced in a particular mutant strain that virtually every animal in the strain is an eye-color mosaic. The molecular basis of the mosaicism is the excision of the mariner element from its location in the DNA of the white gene in somatic cells. The phenomenon results from a single dominant genetic factor located in chromosome 3. Genetic control over the excision of transposable elements may play a role in determining the persistence of transposable elements in the genome.

Alleles↗
PubMed1992

Petunia plants escape from negative selection against a transgene by silencing the foreign DNA via methylation.

Transgenic Petunia hybrida clones harbouring the T-DNA gene 2 of Agrobacterium tumefaciens were used to test a strategy for the trapping of plant transposable elements. In the Petunia line used, floral variegation is due to the presence of the non-autonomous transposable element dTph1 at the An1 locus. The gene 2 product converts the auxin precursor indole-3-acetamide and its analogue 1-naphthalene acetamide into the active auxins indole-3-acetic acid and 1-naphthalene acetic acid. Plant cells that express gene 2 can use a low concentration of the precursors as auxins and become sensitive to the toxicity of high concentrations of these compounds. By selecting protoplast-derived microcalli or seedlings able to grow on medium with high precursor concentrations, variant plants were obtained in which gene 2 was no longer expressed. Southern analysis, using gene 2-specific probes, revealed that in one variant the T-DNA was deleted. For 30 other variants no alteration in gene 2 structure was observed, indicating that transposable element insertion was not responsible for the inactivation of gene 2. Analysis with restriction enzymes allowing discrimination between methylated or non-methylated DNA sequences showed that the inactivated gene 2 sequences were methylated. Addition of the in vivo methylation inhibitor 5-azacytidine to the medium led to reactivation of gene 2 expression in some of the variants. These observations demonstrated that reversible DNA methylation was the main cause of silencing of gene 2 in this system.

Agrobacterium tumefaciens↗
PubMed1982

Genomic clones of a wild-type allele and a transposable element-induced mutant allele of the sucrose synthase gene of Zea mays L.

In an attempt to isolate the transposable genetic element Ds from Zea mays L., we cloned DNA fragments hybridizing to a cDNA clone derived from the sucrose synthase gene in a lambda vector (lambda::Zm Sh). The fragments cloned from wild-type and from the Ds-induced mutant sh-m5933 (lambda::Zm sh-m5933) share a segment 6 kb long while a contiguous segment of 15 kb of lambda::Zm sh-m5933 (mutant-derived DNA) does not hybridize to the DNA segment cloned from the wild-type. Restriction maps are given, and the junction point between the two DNA segments in the mutant clone was determined. Hybridization of DNA fragments, present in the wild-type DNA of lambda::Zm Sh, but not in the mutant clone, lambda::Zm sh-m5933, to genomic DNA of sh-m5933 showed that no part of this DNA is deleted. It cannot be said whether the DNA found in the mutant, but not in the wild-type clone, has been brought there by Ds insertion or by another Ds-dependent DNA rearrangement. The mutant-derived DNA was hybridized to genomic DNA of various maize lines digested by several restriction endonucleases. Approximately 40 bands were detected. The mutant-derived DNA contains two pairs of inverted repeats several hundred nucleotide pairs long, one of which is located at the junction to wild-type-derived DNA.

Journal Article↗
PubMed1982

Mutator mutations in Escherichia coli induced by the insertion of phage mu and the transposable resistance elements Tn5 and Tn10.

Mutator mutations in the mutS gene induced by the insertion of phage Mu or the transposable resistance elements Tn5 or Tn10 and those in the mutL gene induced by Tn5 or Tn10 gave mutagenic activities similar to that of the previously described mutS3 and mutL25 mutations. Various combinations of mutS::Tn5, muL::Tn5, uvrE156, and the deletion mutation delta mutH2 did not produce an additive effect. This supports the idea that the products of these genes function in the same pathway of error correction during DNA synthesis.

Coliphages↗
PubMed1984

Cloning of the bronze locus in maize by a simple and generalizable procedure using the transposable controlling element Activator (Ac).

The bronze (bz) locus of maize has been cloned by an indirect procedure utilizing the cloned transposable controlling element Activator (Ac). Restriction endonuclease fragments of maize DNA were cloned in bacteriophage lambda and recombinant phage with homology to the center of the Ac element were isolated. The cloned fragments were analyzed to determine which contained sequences that were structurally identical to a previously isolated Ac element. Two such fragments were identified. Sequences flanking the Ac element were subcloned and used to probe genomic DNA from plants with well-defined mutations at the bz locus. By this means, it was established that one of the genomic clones contained a bz locus sequence. The subcloned probe fragment was then used to clone a nonmutant Bz allele of the locus. The method described here should prove useful in cloning other loci with Ac insertion mutations.

Journal Article↗
PubMed1999

Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system.

In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analysed in order to evaluate the gene-tagging efficiency. The 3' end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3' end of the Ds in rice. Nearly 80% of Ds elements were excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds which underwent secondary transposition in the later cultures. Eight per cent of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybridization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds-mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a large scale mutagenesis using a heterologous Ac/Ds family in rice.

Base Sequence↗
PubMed1980

Eucaryotic transposable genetic elements with inverted terminal repeats.

DNA carrying inverted repeats was tested for transposition within the Drosophila genome. Five Bam HI segments containing related inverted repeats were isolated from D. melanogaster and analyzed by electron microscopy and restriction mapping. Southern blot experiments using single-copy flanking sequences as probes allowed the study of DNA arrangements at specific sites in the genomes of five closely related strains. We found that in some genomes the sequences with inverted repeats were present at a particular site, whereas in other genomes they were absent from this site. These results indicated that three of the sequences are transposable genetic elements. In one case we have purified the two corresponding DNA segments, with and without the sequence containing inverted repeats, thereby confirming the mobility of this sequence. These DNA elements were found to be distinct in two ways from copia and others previously described: first, they contain inverted terminal repeats, and second, they have a more heterogeneous construction.

Animals↗
PubMedDate not supplied

[Chromatin structure, heterochromatin, and transposable genetic elements--are they from one team?].

Gene content proved to be less than expected in completely sequenced eukaryotic genomes. Moreover, gene number differs only three times between such distant organisms as human and Drosophila. Hence it is likely that the essential functional and structural differences between the two species mostly depend on the regulation of gene activity than on the set and quality of genes themselves. New data demonstrate that changes in chromatin structure play a greater role in the fine gene activity regulation than considered before. R.B. Khesin had foresaw many chromatin functions that only recently came to be recognized. Khesin was interested in genome inconstancy over his last years. A higher content of several important chromosomal proteins was recently revealed in chromatin of transposable genetic elements (TGE). The possible role of TGE in chromatin organization in the nucleus is considered.

Animals↗
PubMed1999

A P element has induced intron formation in Drosophila.

We report evidence supporting the hypothesis that some introns could be originated from transposable elements. In the Drosophila montium species subgroup, we recently described a novel example of domestication by the host genome of a P transposable element. The element is a unique truncated P sequence transcribed into a polyadenylated RNA encoding a putative 66-kDa transposition repressor-like protein. Here, we analyze the genomic modifications associated with this transition of a transposable element into a stationary gene that is useful for the host. Study of the transcription modalities of this neogene reveals that the new transcriptional unit harbors a de novo synthesis of a new exon and a new intron upstream of the original P sequence initiation site. The new exon was constructed from the genomic flanking sequence of the P sequence, whereas the first half of the new intron is composed of genomic flanking sequence and the second half is composed of P sequence. This domestication event has involved the capture of a new promoter. An investigation of a large number of species belonging to the melanogaster species group revealed that this P element domestication is restricted to the species of the montium subgroup and that the new exon-intron structure is present in at least three other species. From sequence data, we hypothesize that cryptic acceptor and donor splicing sites present on the P element and flanking sequences have been under selective constraints which have led to the emergence of a new intron.

Animals↗
PubMed2001

A new basal subfamily of mariner elements in Ceratitis rosa and other tephritid flies.

Several copies of highly related transposable elements, Crmar2, Almar1, and Asmar1, are described from the genomes of Ceratitis rosa, Anastrepha ludens, and A. suspensa, respectively. One copy from C. rosa, Crmar2.5, contains a full-length, uninterrupted ORF. All the other copies, from the three species contain a long deletion within the putative ORF. The consensus Crmar2 element has features typical of the mariner/Tc1 superfamily of transposable elements. In particular, the Crmar2 consensus encodes a D,D41D motif, a variant of the D,D34D catalytic domain of mariner elements. Phylogenetic analysis of the relationships of these three elements and other members of the mariner/Tc1 superfamily, based on their encoded amino acid sequences, suggests that they form a new basal subfamily of mariner elements, the rosa subfamily. BLAST analyses identified sequences from other diptera, including Drosophila melanogaster, which appear to be members of the rosa subfamily of mariner elements. Analyses of their molecular evolution suggests that Crmar2 entered the genome of C. rosa in the recent past, a consequence of horizontal transfer.

Amino Acid Sequence↗
PubMed1991

Targeted gene replacement in Drosophila via P element-induced gap repair.

Transposable elements of the P family in Drosophila are thought to transpose by a cut-and-paste process that leaves a double-strand gap. The repair of such gaps resulted in the transfer of up to several kilobase pairs of information from a homologous template sequence to the site of P element excision by a process similar to gene conversion. The template was an in vitro-modified sequence that was tested at various genomic positions. Characterization of 123 conversion tracts provided a detailed description of their length and distribution. Most events were continuous conversion tracts that overlapped the P insertion site without concomitant conversion of the template. The average conversion tract was 1379 base pairs, and the distribution of tract lengths fit a simple model of gap enlargement. The conversion events occurred at sufficiently high frequencies to form the basis of an efficient means of directed gene replacement.

Animals↗
PubMed1980

Duplicated genes producing transposable controlling elements for the mating-type differentiation in Saccharomyces cerevisiae.

Mutation of the two homothallic genes, HML alpha/HMLa and HMRa/HMR alpha, in homothallic strains of Saccharomyces cerevisiae was studied. Of 11 mutants of the HML alpha gene, eight were due to a phenotypic mutation from HML alpha to HMLa, i.e., a mutation causing a change in function of the original HML allele to that of the other HML allele (functional mutation), and three were due to a defective mutation at the HML alpha gene, i.e., a mutation causing a nonfunctional allele (nonfunctional mutation). All 14 mutants of the HMRa gene, on the other hand, were due to a phenotypic mutation from HMRa to HMR alpha i.e., a functional mutation. Phenotypic reverse mutations, i.e., HMLa to HML alpha and HMR alpha to HMRa, were also observed in the cultivation of EMS (ethyl methanesulfonate) treated spores having the HO HMR alpha HMLa genotype. Mutation from heterothallic cells to homothallism was observed in a nonfunctional mutant of the HML alpha gene, by mutagenesis with EMS, but not in the functional mutants of the HML alpha and HMRa genes or in the authentic strains having the alpha HO HMR alpha HML alpha (alpha Hp) and a HO HMRa HMLa (a Hq) genotypes. These observations suggest that the functional mutation is not caused by the direct mutation from a homothallic allele to the opposite, but by replacement of a transposable genic element produced from a homothallic locus with a region of a different homothallic locus. These observations also support the controlling-element model and the cassette model, which have been proposed to explain the mating-type differentiation by the homothallic genes.

DNA Transposable Elements↗
PubMedDate not supplied

An unusual transposon with long terminal inverted repeats in the sea urchin Strongylocentrotus purpuratus.

A 3-kilobase DNA segment characteristic of a transposable element was found within a histone H2B pseudogene in a higher eukaryote, the sea urchin Stronglyocentrotus purpuratus. The inserted segment (TU1) is flanked by 8-base pair (bp) direct repeats of the H2B sequence. TU1 has long terminal inverted repeats approximately 840 bp long with an outer domain of 15-bp tandem repeats and a non-repeating inner domain, and is a member of a heterogeneous family of transposable elements. TU1 differs from most previously characterized eukaryotic transposable elements with terminal direct repats, but resembles the foldback transposon family in Drosophila.

Animals↗
PubMed1993

Rapid spread of a P element/Adh gene construct through experimental populations of Drosophila melanogaster.

Transposable elements may be potential tools for the dispersal of engineered DNA through target insect populations. The utility of this hypothesis is predicted on the ability of transposable elements carrying a large DNA insert to rapidly disperse through a population. In addition, the inserted DNA must be replicated with a high degree of fidelity during this dispersal. We have monitored the ability of a transposable element with an inserted gene to spread through experimental populations and tested whether the passenger gene retains its ability to encode an active protein. Several Drosophila melanogaster laboratory populations were initiated with female flies that were null for alcohol dehydrogenase activity and contained no P elements. Most of the females were mated to males of the same strain; however, 1 or 10% of the females were mated to males from a strain that had previously been transformed with a helper P element and a P element/Adh gene construct. The dispersal of P elements to new genomes was monitored at each generation by randomly selecting females and performing DNA hybridization assays on dissected ovarian tissue. In addition, each female was tested for alcohol dehydrogenase activity using a simple histochemical assay. We find that, despite an approximate threefold increase in size, the P element constructs containing a functioning gene are still capable of rapid dispersal through the experimental populations. We also show that many of the inserted Adh genes still encode an active product.

Alcohol Dehydrogenase↗