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A cryptic plasmid from a soil strain of Bacillus subtilis was found to contain a sequence having features of IS element. Homologous sequences were also found in the chromosome of this strain and in the chromosomes of some other B. subtilis strains.
LINE-1 (L1) is a mammalian family of highly repeated DNA sequences that are members of a class of transposable elements whose movement involves an RNA intermediate. Both structural and evolutionary data indicate that the L1 family consists of a small number of active transposable elements interspersed with a large number of L1 pseudogenes. In the mouse, the longest, characterized L1 sequences span about 7000 base-pairs and contain two long open reading frames. Two subfamilies of mouse L1 elements, A and F, have been defined on the basis of the type of putative transcriptional regulatory sequence found at the 5' end. In order to identify a transcribed subset of L1 elements in mouse F9 teratocarcinoma cells, we have examined the strand-specificity of L1 transcription by Northern analysis and compared the open reading frame-1 sequences of ten A-type cDNAs with fifteen genomic A-type L1 elements. Transcripts containing A-type sequence are far more abundant than those containing F-type sequence. Although the majority of L1 RNA in F9 cells appears to be transcribed non-specifically from both strands, our results provide evidence for a subpopulation of variable length, strand-specific transcripts arising from A-type transcriptional regulatory sequences. F9 cell cDNA sequences, which share greater than 99.5% sequence identity with one another, represent a homogeneous subset of the genomic L1 population. Examination of genomic mouse L1 sequences reveals three types of length polymorphism in a defined segment of the first open reading frame. Phylogenetic analysis shows a correlation between the type of length polymorphism in the first open reading frame and the relative age of an individual A-type genomic L1 element. Comparison of the cDNA and genomic sequences indicates that the youngest subgroup of A-type L1 elements is preferentially transcribed in F9 cells. This subgroup may be currently dominating the L1 dispersal process in mice.
The piggyBac transposable element was tested for transposition activity in plasmid-based excision and inter-plasmid transposition assays to determine if this element would function in Anopheles gambiae cells and embryos. In the Mos55 cell line, precise excision of the piggyBac element was observed only in the presence of a helper plasmid. Excision occurred at a rate of 1 event per 1000 donor plasmids screened. Precise excision of the piggyBac element was also observed in injected An. gambiae embryos, but at a lower rate of 1 excision per 5000 donor plasmids. Transposition of the marked piggyBac element into a target plasmid occurred in An. gambiae cells at a rate of 1 transposition event per 24,000 donor plasmids. The piggyBac element transposed in a precise manner, with the TTAA target site being duplicated upon insertion, in 56% of transpositions observed, and only in the presence of the piggyBac helper. The remaining transpositions resulted in a deletion of target sequence, a novel observation for the phenomenon of piggyBac element insertion. 'Hot spots' for insertion into the target plasmid were observed, with 25 of 34 events involving one particular site. These results are the first demonstration of the precise mobility of piggyBac in this malaria vector and suggest that the lepidopteran piggyBac transposon is a candidate element for germline transformation of anopheline mosquitoes.
ROAM mutations cause overproduction in S. cerevisiae. Overproduction of ROAM mutant gene products is less in MATa/MAT alpha diploid strains which cannot conjugate than in haplolid strains which can. Overproduction occurs in diploid strains capable of mating whether or not they are capable of sporulating. Overproduction decreases when haploid ROAM mutants also contain the ste7 mutation which prevents conjugation; other ste mutations do not affect the expression of ROAM mutations. Cloning of the ROAM mutant gene CYC7-H2 shows that a 5.5 kb sequence homologous to a transposable and reiterated Ty1 element is inserted in the 5' noncoding region of the CYC7 structural locus. The similar genetic properties of other ROAM mutations suggest that they each contain an inserted Ty element. These results also suggest that ROAM mutations respond to signals normally directed toward genes controlling conjugation functions, and that sequences present in Ty elements may be adjacent to structural loci and are the normal receptors for these signals.
Transposable elements may eventually be used as transformation vectors of anti-Plasmodium genes in natural populations of Anopheles mosquitoes. The mariner transposable element is widespread in most animal phyla and we wished to test for its presence in the important Latin American malaria vector, Anopheles albimanus. Degenerate primers were designed from the consensus of insect mariner elements and used in a polymerase chain reaction to amplify a fragment of the predicted size from An. albimanus. The fragment was cloned, sequenced, and determined to be a mariner-like element through multiple alignment with known insect mariner elements. Dot blot and Southern blot analyses showed only one or a few elements per haploid genome. The cloned fragment was used as a probe to isolate similar sequences from an An. albimanus genomic DNA library. A 659-base-pair clone was 57% similar at the DNA level with mariner elements from Anopheles gambiae, Chrysoperla ptorapunda, and Haematobia irritans. This low sequence similarity is comparable to that found among mariner-like elements in all insects. However, conserved amino acid motifs were not discovered nor was a single open reading frame found. These aspects suggest that the An. albimanus mariner-like element may represent an ancient transposition event but that the element is no longer active, the typical open reading frame having been disrupted through nucleotide insertions and deletions.
Cell surface-located sialic acids of the capsule and the lipooligosaccharide (LOS) are both pivotal virulence factors in Neisseria meningitidis, promoting survival and dissemination of this pathogen which can cause both sepsis and meningitis. With the aid of a unique set of isogenic meningococcal mutants defective in the expression of cell surface-located sialic acids, we have demonstrated that encapsulation hinders the primary event in the development of the disease, but the spontaneous switching of encapsulated wild-type bacteria to a capsule-negative phenotype promotes meningococcal adherence and invasion into mucosal epithelial cells. Genetic analysis of the capsule-negative, invasive bacteria revealed a unique mechanism for modulation of capsule expression based on the reversible inactivation of an essential sialic acid biosynthesis gene, siaA, by insertion/excision of a naturally occurring insertion sequence element, IS1301. Inactivation of siaA regulates both capsule expression and endogenous LOS sialylation. This is the first example of an insertion sequence element-based genetic switch mechanism in the pathogenic bacterium and is an important step in the understanding of bacterial virulence.
A new type of chromosomal rearrangements associated with a transposable element has been described for IS4. These rearrangements are deletions, in which the transposable element IS4 and DNA adjacent to it on either side is lost. These deletions are at least ten times more frequent in the presence of IS4, than when the element is absent from that region. The formation of bidirectional deletions is more than 1,000 times more frequent than precise excision of IS4.
The maize P gene is required for the production of red phlobaphene pigments in the pericarp and cob. The P-vv allele, which specifies variegated pericarp and cob, contains an insertion of the transposable element Ac in the P gene. A new P-ovov allele (orange variegated pericarp and cob) was obtained as a single event mutation from P-vv. In contrast to the progenitor P-vv allele, P-ovov provides substantial pericarp and cob pigmentation and produces significant amounts of normal-sized P transcripts. The Ac element is not detectably altered in the P-ovov allele, but it has transposed to a new position within P that is 161 bp distant and inserted in the opposite orientation. This example provides molecular confirmation for the hypothesis that changes in expression of genes bearing insertions of transposable elements can occur via movement of the element to new sites within the gene.
Molecular domestication of a transposable element is defined as its functional recruitment by the host genome. To date, two independent events of molecular domestication of the P transposable element have been described: in the Drosophila obscura species group and in the Drosophila montium species subgroup. These P neogenes consist of stationary, nonrepeated sequences, potentially encoding 66-kDa repressor-like (RL) proteins. Here we investigate the function of the montium P neogenes. We provide evidence for the presence of RL proteins in two montium species (D. tsacasi and D. bocqueti) specifically expressed in adult and larval brain and gonads. We tested the hypothesis that the montium P neogenes' function is related to the repression of the transposition of distantly related mobile P elements which coexist in the genome. Our results strongly suggest that the montium P neogenes are not recruited to downregulate the P element transposition. Given that all the proteins encoded by mobile or stationary P homologous sequences show a strong conservation of the DNA binding domain, we tested the capacity of the RL proteins to bind DNA in vivo. Immunostaining of polytene chromosomes in D. melanogaster transgenic lines strongly suggests that montium P neogenes encode proteins that bind DNA in vivo. RL proteins show multiple binding to the chromosomes. We suggest that the property recruited in the case of the montium P neoproteins is their DNA binding property. The possible functions of these neogenes are discussed.
We report here on the identification in Arabidopsis thaliana of a new family of transposable elements named Hairpin. These elements are related to foldback transposons (FTs), a large and heterogeneous group of transposable elements first described in Drosophila and recently in Solanaceae. Hairpin elements are the first family of FTs reported in Arabidopsis thaliana and the first family of FTs of type 3 to be described in the plant kingdom. In contrast to previous FTs described, Hairpin appears to be a homogeneous family in size (238 +/- 7 bp) as well as in structure. Hairpin elements are dispersed in the Arabidopsis genome and Southern hybridization revealed that they are present in relatively low copy numbers. Finally, we discuss the potential usefulness of these elements in studying the phylogenetic relationship between Arabidopsis ecotypes.
Sigma is a recently described family of transposable elements in yeast (Saccharomyces cerevisiae). The most striking feature of the seven sigma elements that have been previously identified is that all are located 16-18 base pairs upstream from tRNA-encoding regions. Because these cases were all encountered in the process of studying specific tRNA genes, the full extent of the association between sigma elements and tRNA genes could not be assessed. In this paper, we report a more global characterization of the sigma family in a typical laboratory yeast strain: of the 30 copies of sigma that we estimate to be present in the haploid genome, we have cloned and analyzed 25 loci. Although in two cases a pair of sigma elements were found within several kilobases of each other, the majority occur as individual elements at widely dispersed sites. Moreover, in all 25 cases analyzed, the sigma elements are closely associated with tRNA genes. Thus, the sigma transposable element has been shown to have an absolute association with another gene family.
Genome size differences among crop plants are largely due to unequal accumulation of repetitive DNA sequences, mainly transposable elements (TEs). Over the past decade, many families of miniature inverted-repeat transposable elements (MITEs) have been identified and characterized in a variety of organisms including animals and plants. MITEs are characterized by short terminal inverted repeats (TIRs) (10-15 bp), small size (approx 100 to 500 bp), high-copy-number (approx 1000 to 15,000 per haploid genome), and a preference for insertion into 2-bp to 3-bp targets that are rich in A and T residues. In this chapter, we present a modified transposon display procedure based on the maize MITE family Heartbreaker (Hbr). This technique is similar to AFLP in which AFLP adaptors are ligated to compatible ends of digested genomic DNA. Subsets of Hbr-containing fragments are then amplified using one AFLP primer and another primer complementary to an internal sequence of the Hbr element. Like AFLP, the Hbr display method permits the simultaneous analysis of numerous DNA fragments. Given the plethora of available marker systems, the major advantage of Hbr markers, and perhaps most MITE-based markers, is a preference for insertion in or near transcriptionally active genomic regions. This feature may be especially valuable in the large genomes of agriculturally important plants like maize, wheat, and barley where gene-rich islands are thought to exist in a sea of retrotransposons. Having a class of markers that are enriched in genic regions, coupled with the ease of isolating MITE markers, could expedite chromosome walks and map-based cloning protocols in these organisms.
Living organisms have to fight against the invasion of many parasites. Among them are viruses and transposable elements that are able to integrate in the genome of their host. After integration, they can replicate and propagate. The defence mechanisms against these invaders are still largely unknown but are widely studied in plants as well as in fungi and animals, particularly Caenorhabditis elegans and Drosophila melanogaster. The compilation of recent data allows us to draw a general scheme for these mechanisms. In particular, it seems that the propagation of viruses, transposable elements and repeated sequences is controlled by mechanisms repressing the expression of these sequences at both transcriptional and post-transcriptional levels. Post-transcriptional regulation of repeated sequences and transposable elements is related to RNA interference (RNAi), an evolutionary conserved silencing process induced by the presence of double-stranded RNAs (dsRNAs). Many genes and molecular intermediates have now been identified, revealing the different steps of the mechanism underlying this process. Finally, different results suggest that these post-transcriptional silencing processes are involved in the development of organisms. DICER is a nuclease responsible for the processing of dsRNA into short RNA molecules (siRNAs) considered as the interfering agent. siRNAs interact with the transcript of the targeted gene and this interaction induces degradation of the transcript. DICER is also involved in the processing of small temporal RNAs (stRNA) involved in the timing of development. stRNAs have the same structure as siRNAs. They regulate their target genes by interacting with elements present in their 3'UTR and blocking translation. RNAi appears to be an universal regulatory mechanism that was still unknown a few years ago. It is now largely used in large scale inactivation of genes to determine their function, and some recent studies indicate that it might be used in human therapy.
Transposable elements make up a significant fraction of many eukaryotic genomes. Although both classes of transposable elements, the DNA transposons and the retrotransposons, show substantial expansion in plants and invertebrates, the DNA transposons are thought to have become inactive in mammalian genomes long ago. Here, we report the first evidence for recent activity of DNA transposons in a mammalian lineage, the bat genus Myotis. Six recently active families of nonautonomous hobo/Activator/TAM transposons were identified in the Myotis lucifugus genome using computational tools. Low sequence divergence among the individual sequences and between individual sequences and their respective consensus sequences suggest their recent expansion in the M. lucifugus genome. Furthermore, amplification and sequencing of polymorphic insertion loci in a related taxon, M. austroriparius, confirms their recent activity. Myotis is one of the largest mammalian genera with 103 species. The discovery of DNA transposon activity in this genus may therefore influence our understanding of genome evolution and diversification in bats and in mammals in general. Furthermore, the identification of a likely autonomous element may lead to new approaches for mammalian genetic manipulation.
In our studies on the regulation of adjacent-gene expression by Ty sequences, we demonstrated that a single-base-pair change (T-A----C-G) in the epsilon sequence of Ty917-derived elements is primarily responsible for enhancement of beta-galactosidase expression from lacZ fusion plasmids. Using an electrophoretic gel mobility assay, we showed that the same base pair transition is required for binding of a trans-acting factor, TyBF, from crude cell extracts in vitro. We identified the site of TyBF binding and determined the guanine nucleotide contact sites required for TyBF interaction. We propose that TyBF binding to cis-acting Ty2 sequences activates adjacent-gene transcription.
We estimated the genome size of Korean ginseng (Panax ginseng C.A. Meyer), a medicinal herb, constructed a HindIII BAC library, and analyzed BAC-end sequences to provide an initial characterization of the library. The 1C nuclear DNA content of Korean ginseng was estimated to be 3.33 pg (3.12 x 10(3) Mb). The BAC library consists of 106,368 clones with an average size of 98.61 kb, amounting to 3.34 genome equivalents. Sequencing of 2167 BAC clones generated 2492 BAC-end sequences with an average length of 400 bp. Analysis using BLAST and motif searches revealed that 10.2%, 20.9% and 3.8% of the BAC-end sequences contained protein-coding regions, transposable elements and microsatellites, respectively. A comparison of the functional categories represented by the protein-coding regions found in BAC-end sequences with those of Arabidopsis revealed that proteins pertaining to energy metabolism, subcellular localization, cofactor requirement and transport facilitation were more highly represented in the P. ginseng sample. In addition, a sequence encoding a glucosyltransferase-like protein implicated in the ginsenoside biosynthesis pathway was also found. The majority of the transposable element sequences found belonged to the gypsy type (67.6%), followed by copia (11.7%) and LINE (8.0%) retrotransposons, whereas DNA transposons accounted for only 2.1% of the total in our sequence sample. Higher levels of transposable elements than protein-coding regions suggest that mobile elements have played an important role in the evolution of the genome of Korean ginseng, and contributed significantly to its complexity. We also identified 103 microsatellites with 3-38 repeats in their motifs. The BAC library and BAC-end sequences will serve as a useful resource for physical mapping, positional cloning and genome sequencing of P. ginseng.
Three derivatives of mutant ga10P-308::IS2-I of Escherichia coli were characterized by DNA sequence analysis. Deletions and DNA sequence rearrangements were observed which apparently were initiated at short A-T rich inverted repeats within IS2. Two of the mutants carried newly synthesized DNA sequences which were inverted copies of already existing IS2 sequences. Thus long stretches with twofold symmetry were formed. It is discussed whether these inverted repeats were formed by DNA repair synthesis which was initiated at the A-T rich palindromes of IS2.
We have cloned the B breakpoint in Drosophila melanogaster using DNA from a P-M-induced revertant of B, which has a P element inserted at the B breakpoint. The analysis of the B DNA reveals that there is a transposable element, B104, right at the breakpoint. This suggests that this element may have been involved in the generation of the B breakpoint and the associated tandem duplication. One possible mechanism to generate the B duplication is a recombination event between two B104 elements, one at 16A1 and the other at 16A7. DNA sequencing data of the junctions of the B104 element support this model. Four partial revertants of B are the result of insertions of transposable elements very close to the B breakpoint. This supports the hypothesis that the breakpoint is the cause of the B mutation. The clones from B were used to isolate wild-type clones from 16A1, the location of the Bar gene. Four rearrangement breakpoints associated with various Bar mutations map within a 37-kb region, suggesting that the Bar gene is very large.