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Analysis of transposable elements inserted in the genomes of bacteriophages Mu and P1.

We have examined the genomes of the temperate bacteriophages Mu and P1 and some of their insertion mutants for hybridization with the prokaryotic transposable elements IS1 and IS2. We used the DNA blotting-hybridization technique in which denatured DNA fragments are transferred to nitrocellulose paper directly from agarose gels and hybridized to 32P-labeled probe DNA. The 800 base pair insertion in an X mutant of Mu was found to hybridize with IS1. The chloramphenicol resistance transposon, Tn9, in Mu X cam mutants was found to be located at or close to the sites of IS1 insertion in X mutants; Tn9 also hybridized with IS1. The restriction endonuclease BalI cleaved IS1 once; it cleaved Tn9 in all Mu X cam mutants twice to release a fragment of about 1700 base pairs. These results support the conclusion that Tn9 contains one copy of IS1 at each end. In the P1cam isolate, from which Tn9 was transposed to Mu, BalI made a third cut in Tn9 giving rise to fragments of about 850 base pairs. The data further suggested that Tn9 is present in tandem copies in the P1cam isolate we examined. P1 itself was found to harbor IS1. The two P1 strains tested had a common fragment containing IS1; one strain had an additional copy of IS1. The IS1 element common to the P1 strains was shown to be the site of the Tn9 insertion in the P1cam isolate examined. No hybridization between IS2 and any of the Mu and P1 strains could be detected.

Chloramphenicol↗

A functional analysis of the inverted repeat of the gamma delta transposable element.

We have constructed a library of point mutants of the 35 base-pair terminal inverted repeat (IR) of the bacterial transposon gamma delta, a member of the Tn3 family of transposable elements. The effect of the mutant ends, both on the immunity conferred on an IR-containing target plasmid and on the transposition of model transposons, was determined. The region important for immunity was shown to be a 30 base-pair stretch of DNA, running from G8 and A9 to G38; mutations in the outermost seven or eight base-pairs did not significantly affect immunity. Positions at which mutations disrupted immunity chiefly coincided with positions previously determined to constitute three segments of the IR with which gamma delta tranposase protein interacts via major groove contacts. We conclude that sequence-specific binding contacts between gamma delta transposase and its cognate IR are limited to a specific subset of positions (those sensitive to mutation in the immunity assay) within this 30 base-pair region. We found that the innermost of the three major groove contact regions was the most susceptible to mutation, while the outermost was the least. Indications of minor groove contacts were also found. Very few point mutations within the 30 base-pair sequence-specific binding region had much effect on transposition when the mutant ends were in the "wild-type" context with the adjacent integration host factor (IHF) binding site. However, deletion of the IHF site, in some cases, revealed a transposition defect, suggesting that for transposition (but not immunity), IHF-transposase cooperation can largely overcome the effects of reduced transposase binding. Although the outer seven base-pairs were not important for immunity, mutations in the outer three or four eliminated or reduced transposition activity, suggesting that these positions are involved in a step in transposition that follows transposase binding.

Base Sequence↗

Molecular evolutionary analyses of mariners and other transposable elements in fire ants (Hymenoptera: Formicidae).

Screens of a library of genomic DNA made during a recent study of the fire ant Solenopsis invicta revealed the presence of three distinct types of transposable elements (TEs). Two of the recovered sequences showed a high similarity to long-terminal repeat (LTR) retrotransposons, while the third showed a high homology to mariner elements. To investigate the distribution and relationships of mariners in related ants, we PCR-amplified these elements from additional Solenopsis species. Phylogenetic analyses showed that they form a single group within the mauritiana subfamily that is part of a larger clade derived from hymenopteran species. We also present partial sequence data for the two LTR-retrotransposons and describe their phylogenetic affinities.

Amino Acid Sequence↗

Molecular cloning of the white locus region of Drosophila melanogaster using a large transposable element.

We report the molecular cloning of a chromosome segment including the white locus of Drosophila melanogaster. This region was isolated using a deficiency extending from the previously cloned heat-shock puff sequences at 87A7 to a large transposable element containing the loci white and roughest.FB-NOF, a 7.5 kb element with partial homology to a family of inverted repeat sequences (Potter et al., 1980), is found very near the deficiency breakpoint, and is followed by DNA originating from the white locus region. Sequences totalling 60 kb surrounding this initial entry point were obtained by the cloning of successively overlapping fragments from a wild-type strain. Several rearrangement breakpoints have been mapped relative to the cloned DNA; these define the limits of the white locus and further differentiate the "white proximal region", thought to function in gene regulation, from the remainder of the locus. Insertion of the dispersed repetitive element copia into the white locus is observed in strains carrying the white-apricot allele. Analysis of several white-apricot revertants suggests that copia insertion is responsible for the apricot eye color phenotype.

Journal Article↗

Transposon tagging of the maize Glossy2 locus with the transposable element En/Spm.

The Glossy2 (Gl2) locus of maize is required for the formation of the epicuticular wax layer of young plants. gl2 mutant seedlings can be visually identified because of their glossy leaf surface which is different from the dull surface of wild-type seedlings. The Gl2 locus was isolated by transposon tagging. Seven unstable mutations, gl2-m2 to gl2-m8, were induced in a parental strain carrying an active transposable Activator (Ac) element in the unstable wx-m7 allele. Genetic tests on the gl2-m2 allele indicated that it was not caused by the Ac element but by the insertion of the transposable element Enhancer/Suppressor-Mutator (En/Spm). A Sa/l restriction fragment segregating with the mutant phenotype was identified, by Southern analysis, using sequences from the En/Spm element as a probe. Part of the fragment was cloned and was shown to carry part of the unstable gl2-m2 allele. These gl2 sequences were used to identify a genomic fragment carrying the wild-type allele and to isolate its corresponding cDNA sequence. The predicted Glossy2 protein consists of 426 amino acids. No similar amino acid sequence was found in protein data banks and the biochemical function of the Gl2 gene product is still unknown. The wild-type Gl2 transcript is found predominantly in juvenile leaves. The transcript level in the leaves of seedlings homozygous for a stable recessive gl2-ref allele is hardly detectable.

Alleles↗

Behaviour of the transposable elements copia and mdg1 in hybrids between the sibling species Drosophila melanogaster and D. simulans.

The behaviour of the retrotransposons copia and mdg1 was analysed in hybrids between Drosophila melanogaster and D. simulans. Females of a highly inbred line of D. melanogaster were crossed with D. simulans males from three natural populations. The insertion site profiles for the two elements were determined in F1 hybrid larvae by in situ hybridization to polytene chromosomes, and were compared with that of their parents. No somatic transposition events were detected after this genomic stress of interspecific hybridization for the two transposable elements concerned.

Animals↗

Escherichia coli K-12 auxotrophs induced by insertion of the transposable element Tn5.

The sites of insertion of the transposable kanamycin-neomycin resistance-determining element, Tn5, in the E. coli K-12 chromosome were assessed in a collection of over 300 auxotrophs. Although mutations in at least 45 different cistrons were obtained, the distribution of insertion sites was not completely random: proA or proB; cysG; and cysH, cysD or cysC mutants were found in excess.

Chromosome Mapping↗

Progress in plant CACTA elements.

Transposable elements are DNA fragments that can insert new chromosomal locations. On the basis of the mechanism of transposition, transposable elements were divided into two classes. Class 1 elements were retroelements that used reverse transposase to transpose by an RNA intermediate. Class 2 elements or DNA transposons transposed directly from DNA to DNA. Of the Class 2 elements, CACTA superfamily, so far identified exclusively in plants and previously regarded as low-copy-transposon for the conserved mechanism of propagation, recently received considerable interest because of their increasing evidence reiterating their high copies in some plant genomes. This article aimed at outlining CACTA elements with regard to their structure, transposition, and utilization.

DNA Transposable Elements↗

The Spm (En) transposable element controls the excision of a 2-kb DNA insert at the wx allele of Zea mays.

The waxy (Wx) locus of Zea mays was cloned from strains carrying the wild-type and wx mutant alleles. The receptor component of the Suppressor-Mutator (Spm) controlling element system in the wx allele was shown to be a 2 kb long insertion within the transcribed region of the Wx gene. The insertion, termed Spm-I8, is excised during somatic reversion events induced by the autonomous controlling element Enhancer (En), which is an equivalent to Spm. Integration of Spm-I8 into the Wx gene generates a 3-bp target site duplication. Spm-I8 has a 13 bp long inverted repeat at its termini. The ends of the element can be further folded to build a large double-stranded structure consisting of five perfectly matching double-stranded regions of 9-13 bp in length, interrupted by single-stranded loops. A comparison of the wild-type and wx alleles revealed two additional insertions 6 (insert-1) and 0.25 (insert-2) kb in length. No En-induced excision of insert-1 and insert-2 could be detected so far. There is remarkable structure and sequence homology between Spm-I8 and the transposable elements Tam1 and Tam2 of Antirrhinum majus at their termini, reflecting a possible evolutionary and/or functional relationship between transposons in different plant species.

Journal Article↗

Identification of two sequence elements associated with the gene encoding the 24-kDa crystalline component in Bacillus thuringiensis ssp. fukuokaensis: an example of transposable element archaeology.

A 6.5-kb fragment of plasmid DNA from Bacillus thuringiensis (Bt) ssp. fukuokaensis that encodes a 24-kDa crystalline component was analyzed to identify additional open reading frames (orfs). A novel Bt IS240-like element was found upstream of this gene and is considered to be a vestige of a once active insertion sequence due to a stop codon that interrupts the long orf encoding the putative transposase. This element was bounded by 17-bp terminal inverted repeats that defined the length of the insertion sequence as 802 bp. Further upstream of this element two tandem overlapping and out of phase open reading frames (orfX and orfY) were identified which represent the first example of an IS150-like element in Bt containing both orfs. orfX and orfY are not bounded by terminal inverted repeats but are associated with a gene encoding a putative site-specific recombinase of a type found in Staphylococcus aureus Class II transposons but not previously in Bt.

Amino Acid Sequence↗

Plasmids and transposable elements in Salmonella wien.

The plasmids from six clinical strains of Salmonella wien have been characterized. All the S. wien strains were found to carry three types of plasmids: an IncFI R-Tc Cm Km Ap (resistance to tetracycline, chloramphenicol, kanamycin, and ampicillin) plasmid, either conjugative or nonconjugative, of large size (90 to 100 megadaltons); an R-Ap Su Sm (resistance to ampicillin, sulfonamide, and streptomycin) plasmid of 9 megadaltons; and a very small (1.4 megadaltons) cryptic plasmid. The characteristics of conjugative R plasmids, recombinant between F'lac pro and the FI nonconjugative plasmid, indicated that regions coding for the donor phenotype were present on this plasmid. The molecular and genetic features of the R plasmids were very close to those described for the R plasmids isolated from S. wien strains of different origin. This fact supported the hypothesis of a clonal distribution of this serotype in Algeria and Europe. The analysis used to identify transposable elements showed the presence of only TnA elements, which were located on both the R-Tc Cm Km Ap and R-Ap Su Sm plasmids. They contained the structural gene for a TEM-type beta-lactamase and had translocation properties analogous to those reported for other TnA's.

Ampicillin↗

The relationship between the rate of transposition and transposable element copy number for copia and Doc retrotransposons of Drosophila melanogaster.

We present data on the relationship between the rate of transposition and copy number in the genome for the copia and Doc retrotransposons of Drosophila melanogaster. copia and Doc transposition rates were directly measured in sublines of the isogenic 2b line using individual males or females, respectively, with a range of copia copy numbers from 49 to 103 and Doc copy numbers from 112 to 235 per genome. Transposition rates varied from 3 x 10(-4) to 2 x 10(-2) for copia and from 2 x 10(-4) to 2 x 10(-3) for Doc. A positive relationship between transposition rate and copy number was found both for copia and for Doc when the data were analysed across all the 2b individuals; no significant correlation was found when the data were analysed across the subline means for both retrotransposons tested. Overall, correlation between copia and Doc transposition rate and their copy number in the genome, if any, was not negative, which would be expected if transposable elements (TEs) self-regulate their copy number. Thus, for copia and Doc no evidence for self-regulation was provided, and at least for these two TEs this hypothesis is not favoured for explaining the maintenance of the stable copy number that is characteristic for natural populations. The transposition rate of copia was measured twice, and a strong positive correlation between copy number and transposition rate both across individuals and subline means was found in 1994, while in 1995 no correlation was found. This fact is in agreement with the hypothesis that a positive correlation between the rate of transposition and TE copy number may be a default starting point for future host-TE coevolution.

Animals↗

Interactions between the transposable element IS21 on R68.45 and TN7 in Pseudomonas aeruginosa PAO.

Tn7 transposes from the chromosome of Pseudomonas aeruginosa into the plasmid R68.45 with tandem IS21, at up to 400 times the frequency that it transposes into R68, which has only one copy of IS21. While R68::TN7 derivatives are stable, R68.45::Tn7 isolates undergo frequent deletions. Instability of R68.45 occurs whether Tn7 is inserted into the plasmid (cis configuration) or into the bacterial chromosome (trans configuration). The deletions of R68.45 start at the junction between the tandem IS21 copies and proceed clockwise, ending in the region of oriT. It appears that Tn7 and IS21 can mutually stimulate transposition of each other.

Chromosome Deletion↗

The genetics and evolution of the mariner transposable element in Drosophila simulans: worldwide distribution and experimental population dynamics.

We have studied both the frequency and biogeographical distribution of the transposable DNA element mariner in natural populations of Drosophila simulans and the short-term evolutionary characteristics of mariner in experimental populations. The mariner element has been identified in natural populations of D. simulans from Africa, Europe, the Middle East, Japan, Australia, several Pacific islands, North America, and South America. Only four lines out of 296 were devoid of active mariner elements, as measured by the presence of functional mariner transposase. A slight correlation was found between the latitudinal coordinate of the collection sites and the level of mariner activity in the populations; this correlation became highly significant in Australia where a cline in mariner activity was observed along the eastern coast of the continent. We also observed that wild-type laboratory strains kept for several years as small populations might lose mariner activity over time. Using experimental populations, we modeled what might happen when naturally occurring populations exhibiting high and low levels of mariner activity encounter one another. We found that active mariner elements either will tend to lose their activity over time and gradually become inactive or possibly will be lost from the population; in either case, this will lead to the pattern seen in this experiment of a significant loss of mariner activity over time.

Animals↗