Mobile DNA Elements Research Articles

Localization and functional analysis of structural and regulatory dehalogenase genes carried on DEH from Pseudomonas putida PP3.

Pseudomonas putida PP3 expressed two dehalogenases, DehI and DehII. The DehI gene (dehI) was located on a mobile DNA element (DEH) which inserted at high frequencies into target plasmids from its chromosomal location. From a recombinant TOL plasmid (pWW0) containing a 6.0-kb DEH element inserted into the plasmid's 5.6-kb EcoRI-G restriction endonuclease fragment, an 11.6-kb EcoRI fragment was cloned. Subcloning analysis and insertion mutagenesis produced a structural map of the DEH element and located the dehalogenase functions. The gene dehI was transcribed from a regulated promoter on DEH which was expressed in P. putida and Escherichia coli. The direction of transcription of dehI was determined, and it was also found to be under positive control, activated by an adjacent regulatory gene (dehRI). Expression of dehI in clones containing the intact DEH supported good growth on 2-monochloropropionate (2MCPA). Subclones lacking dehRI expressed dehI at levels which allowed only slow growth on 2MCPA, even when dehI expression was initiated from vector promoters. Expression of dehI in P. putida containing the intact DEH element required rpoN, suggesting that it was omega 54 dependent. The intact DEH element transferred to P. putida on a suicide plasmid donor pAWT34 (pBR325 replicon), and dehI was stably inherited, without vector DNA sequences, in transformants selected on 2MCPA. This indicated that the cloned DEH element contained functions associated with recombination.

Artificial mobile DNA element constructed from the EcoRI endonuclease gene.

There exist several examples of mobile group I introns. These introns appear to use a straightforward mechanism to achieve highly site-specific and efficient insertion into homologous intronless genes. Because the only intron-specific function required by the prevailing model for the mechanism of intron mobility is the introduction of a site-specific double-stranded break in the intronless recipient DNA molecule, we reasoned that it should in principle be possible to construct artificially mobile DNA sequences. We have constructed an artificial mobile element from the gene for the restriction enzyme EcoRI that is capable of site-specific insertion at rates near those of authentic mobile introns. The generality of the mobility mechanism may enable high-efficiency targeted gene replacements or disruptions in a variety of organisms.

IS5: a mobile enhancer of transcription in Escherichia coli.

The cryptic bgl operon of Escherichia coli is activated by the spontaneous insertion of mobile DNA elements. Screening of a collection of such mutations revealed insertion of the 1195-base-pair element IS5 into various positions both upstream and downstream of the bgl promoter P0. Activation of the operon was in all cases attributable to enhancement of P0 activity. Introduction of internal deletions into IS5 almost completely abolished P0 enhancement, demonstrating that enhancement is not simply the result of mutational inactivation of some inhibitory sequences. Intact copies of IS5 in trans restored the enhancing activity of the deletion derivatives. The trans-activator is encoded by IS5 gene ins5A, an essential transposition function. Activation of gene expression by means of interaction of a defective mobile element in cis with functions encoded by a nondefective element in trans has so far been described only for a maize controlling element.

Molecular cloning and analysis of forked locus in Drosophila ananassae

Drosophila ananassae, in spite of its unique genetic characters including high mutability and high frequency of male recombination has been little studied at the molecular level. Since the species is very similar to D. melanogaster, it is natural to expect that the high spontaneous mutation rate and male recombination may be caused by inserted mobile DNA elements, as in D. melanogaster. The present study was designed to determine whether or not most spontaneous mutations of the forked locus of D. ananassae are caused by insertion sequences as is found in D. melanogaster. I cloned the forked locus of D. ananassae, using forked DNA from D. melanogaster as a probe and investigated the molecular structure and transcription of the gene by Southern and Northern analyses. The results suggest that the restriction map and transcriptional patterns of the forked locus of D. ananassae are similar to those of D. melanogaster, while the spontaneous mutations available in D. ananassae are induced quite differently from those that have been described in D. melanogaster; in four (f;cd, f10-14, f49 and f86) out of eight forked alleles, neither insertions nor deletions were detected around the forked coding region. Forked transcripts are expressed in a pattern which is very similar to that of D. melanogaster and were all of normal size in these mutants. The other four mutants (f10-3, f9-10, f83i and f79b) had insertion sequences upstream of the forked coding region, while transcripts of the forked gene were of normal size. Hence, none of the eight mutations studied appear to affect the structure of the forked transcripts.

Diversification of the rice Waxy gene by insertion of mobile DNA elements into introns.

The waxy (wx) gene of Oryza glaberrima was cloned, and its nucleotide sequence was determined. A waxy mutant of O. glaberrima showing a glutinous phenotype was found to contain a substitution mutation generating a termination codon in the coding region of the wx gene. The Wx sequence of O. glaberrima was different from that of Oryza sativa by substitutions and insertions/deletions, among which only a few substitutions occurred in several exons not to severely alter the amino acid sequence of the Wx protein. The most striking difference observed in introns was a 139-bp deletion (or insertion) in intron 10 of O. glaberrima (or O. sativa). In O. sativa, 125 bp of the 139-bp sequence was flanked by direct repeats of a 14-bp sequence. A sequence homologous to the 125-bp sequence was found in the region preceding exon 2; this sequence was also flanked by direct repeats of another 14-bp sequence. This result and the observation that the 125-bp sequence was interspersed in rice genomes indicate that they are SINEs (short interspersed elements) in the plant system. We also identified a DNA sequence with long terminal inverted repeats in intron 13 of both O. glaberrima and O. sativa. This sequence was present in multiple copies in rice genomes, suggesting that it is a transposable element. These results obtained suggest that mobile DNA elements have diversified the rice Waxy gene by inserting into introns, each of which may originally have a length of about 100 bp.

Structural and functional characterization of tnpI, a recombinase locus in Tn21 and related beta-lactamase transposons

A novel discrete mobile DNA element from Tn21 from the plasmid R100.1 is described, and its mobilization function was confirmed experimentally. In addition, the element behaves as a recombinase-active locus (tnpI) which facilitates insertions of antibiotic resistance genes as modules or cassettes at defined hot spots or integration sites. A similar tnpI sequence was detected by DNA hybridization in a series of beta-lactamase transposons and plasmids and localized on their physical maps. The genetic function of the locus cloned from Tn21 into pACYC184 was tested for conduction and integration into the plasmids R388 and pOX38Km, and the results suggested recombinase-integrase activity and recA independence. DNA sequence analysis of the tnpI locus revealed no inverted or direct terminal repeats or transposition features of class I and class II transposons. The coding capacity revealed three putative open reading frames encoding 131, 134, and 337 amino acids. Orf3 encoded a putative polypeptide product of 337 amino acids that shared highly significant identity with the carboxyl region of integrase proteins. A comparison and an alignment of the tnpI locus from Tn21 and its flanking sequences identified similar sequences in plasmids and in transposons. The alignment revealed discrete nucleotide changes in these tnpI-like loci and a conserved 3' and 5' GTTA/G hot spot as a duplicated target site. Our data confirm the remarkable ubiquity of tnpI associated with antibiotic resistance genes. We present a model of transposon modular evolution into more complex multiresistant units via tnpI and site-specific insertions, deletions, and DNA rearrangements at this locus.

UV Light and Ionizing Radiations Cause Programmed Death of Rat Chloroleukaemia Cells by Inducing Retropositions of a Mobile DNA Element (L1Rn)

The long interspersed repetitive DNA, L1 or LINE, is a class of mobile genetic elements which can amplify in the cell genome by retroposition, i.e. by a mechanism similar to that of retroviruses. We have shown before that in rat chloroleukaemia cells, maintained in suspension culture in vitro, this element is spontaneously transcriptionally activated at about half of the maximal population density. About 24 h later an explosive amplification of the L1 element is seen in DNA: about 300,000 copies are inserted into apparently random locations in the cell genome, thus creating an outburst of lethal mutations. Dead cells display morphological features typical to programmed death. The present results show that UV light and ionizing radiation induce rapid, premature activation of the L1Rn element during the fast exponential growth of chloroleukaemia cells, and that also this exogenously induced activation is followed by programmed cell death. Transcriptional activation of the L1Rn element can be very strong after the UV exposure: at least 70-fold. Severe hyperthermia, lethal to the cells, does not lead to L1Rn activation (actually a marked suppression is seen) and the mode of phenomic death is necrosis. Some biological implications of the results are discussed.

The structure of a partial duplication in the integron of plasmid pDGO100

A family of novel potentially mobile DNA elements called integrons, has recently been described (H. W. Stokes and R. M. Hall, 1989, Mol. Microbiol. 3, 1669-1683). The integrons present in the plasmids pDGO100 and pSa are unusual in that they include a duplication of the sulI gene which is located in one of the two conserved segments that make up these elements. In order to define the nature of the duplication in pDGO100, we have sequenced the sulI gene region located between the aadB and the dhfr genes of pDGO100. This region includes the first 1355 bases of the 2026-base 3'-conserved segment present in the integrons of Tn21, R46 and R388, and the sequence identity in pDGO100 ceases 24 bases beyond the end of the sulI gene. This position corresponds to the center of a 59-base element, a remnant of which is located at the end of sulI. This finding suggests that the 59-base element may have been involved in the event which gave rise to the partial duplication.

A novel family of potentially mobile DNA elements encoding site‐specific gene‐integration functions: integrons

A family of novel mobile DNA elements is described, examples of which are found at several independent locations and encode a variety of antibiotic resistance genes. The complete elements consist of two conserved segments separated by a segment of variable length and sequence which includes inserted antibiotic resistance genes. The conserved segment located 3' to the inserted resistance genes was sequenced from Tn21 and R46, and the sequences are identical over a region of 2026 bases, which includes the sulphonamide resistance gene sull, and two further open reading frames of unknown function. The complete sequences of both the 3' and 5' conserved regions of the DNA element have been determined. A 59-base sequence element, found at the junctions of inserted DNA sequences and the conserved 3' segment, is also present at this location in the R46 sequence. A copy of one half of this 59-base element is found at the end of the sull gene, suggesting that sull, though part of the conserved region, was also originally inserted into an ancestral element by site-specific integration. Inverted or direct terminal repeats or short target site duplications, both of which are characteristics of class I and class II transposons, are not found at the outer boundaries of the elements described here. Furthermore, the conserved regions do not encode any proteins related to known transposition proteins, except the DNA integrase encoded by the 5' conserved region which is implicated in the gene insertion process. Mobilization of this element has not been observed experimentally; mobility is implied from the identification of the element in at least four independent locations, in Tn21, R46 (IncN), R388 (IncW) and Tn1696. The definitive features of these novel elements are (i) that they include site-specific integration functions (the integrase and the insertion site); (ii) that they are able to acquire various gene units and act as an expression cassette by supplying the promoter for the inserted genes. As a consequence of acquiring different inserted genes, the element exists in a variety of forms which differ in the number and nature of the inserted genes. This family of elements appears formally distinct from other known mobile DNA elements and we propose the name DNA integration elements, or integrons.

The nature of spontaneous mutations

Tbe induction of mutations is often expressed in relation to spontaneous mutations and designed for instance by the doubling-dose concept. A problem in that context is the fact that the mutational spectrum for spontaneous and induced mutations is not the same and it can furthermore vary considerably between loci. This is illustrated by molecular characterization of spontaneous and ionizing-radiotion-induced mutations in mammalian cells. Futhermore changes of the genetic machinery ar not limited to those endpoints usually measured in mutational assays, that is, base substitutions, frameshifts, classical chromosomal aberrations and numerical alterations of chrosomes. Additional alterations of which far less is known, include insertion mutations, recombinogenic events and disproportionate replication of DNA giving rise to gene amplifications, and changes of gene expression through methylation of cytosine. There are reasons to believe that these endpoints are of imporatance in the development of tumors. Amplificition of oncogenes is a well-known phenomenon in tumorigenicity and lately mutations by the insertion of mobile DNA elements have demonstrated in cancer cells. Often these alterations are induced by strees and they constitute a manifestation of the dynamics and instability of DNA and the genetic system revelead in recent years. It is of interest in that context that the flow of genetic information does not only occur in one direction that is, DNA-RNA-protien, but also from RNA to DNA through reverse transcription and possibly even from the protein end of the sequence.

Insertional DNA and spontaneous mutation at the white locus in Drosophila simulans

A large body of data on molecular analyses of several multiallelic loci in Drosophila melanogaster has demonstrated a high incidence of mobile DNA element insertions among spontaneous mutations. In the sibling species D. simulans, the dispersed, middle repetitive, nomadic sequences are reduced to about one-seventh that of its sibling species (Dowsett and Young 1982). Does this reduced amount of middle repetitive DNA (or mobile DNA sequences) mean that in D. simulans the occurrence of insertion mutants will be rare compared with that of D. melanogaster? To test this possibility, we collected seven different spontaneous white mutants of D. simulans and studied their molecular gene structures. Five out of seven mutants had insertion sequences which varied in length from 0.4 kb to 16 kb. One bore a deletion spanning the w region and another showed no gross structural alteration. Thus the proportion of insertional mutations at the white locus in D. simulans is equivalent to that observed in D. melanogaster. Among the five insertional mutants, one, wmky, showed genetic instability; the other four were stable. wmky was found to mutate at a frequency of 2.1 x 10(-5) in meiotic cells and may also be unstable in somatic cells.

Plasmid distribution among unicellular and filamentous cyanobacteria: Occurrence of large and mega-plasmids

The plasmid content of 7 unicellular and 4 filamentous cyanobacterial strains has been analyzed. All strains were found to carry small plasmids while some strains harbor large plasmids up to approx. 400 kb. In addition, at least one megaplasmid of about 1000 kb was detected in the unicellular strain Synechococcus PCC7942. In the filamentous strain Calothrix PCC7601, 2 different distribution patterns of plasmids were observed in different subcultures, suggesting the presence of mobile DNA elements in this strain.

Mobile DNA elements in Drosophila melanogaster: a retrospective on serendipity.

Mobile DNA elements in Drosophila melanogaster: a retrospective on serendipity.

The role of mobile DNA elements in unequal and intrachromosomal crossing-over in Drosophila melanogaster.

During the past decade the existence of mobile DNA elements, first deduced from genetic experiments in maize by McClintock, has been confirmed by their molecular cloning and characterization (3). In Drosophila melanogaster a parallel, albeit somewhat later, demonstration of the occurrence of mobile elements took place. The impetus of the genetic experiments stimulated a detailed molecular study of mobile DNA elements in D. melanogaster, the main points of which can be summarized as follows. More than 10% of the D. melanogaster genome consists of moderately repetitive DNA, the bulk of which is made up of mobile DNA elements. Several distinguishable classes of elements occur, the classification stemming from distinctive biochemical structures (for a detailed discussion, see Ref. 11). Considering the middle, repetitive DNA collectively and assuming mobile DNA elements average 5 kb in length and occur on the average in 25 copies per genome, the conclusion follows that approximately 50 discrete families of mobile DNA elements occur.

The Alu family of dispersed repetitive sequences.

A family of related sequences that includes approximately 500,000 members is the most prominent short dispersed repeat family in primate and rodent DNA's. The primate sequence is approximately 300 base pairs in length and is composed of two imperfectly repeated monomer units, whereas the rodent repeat consists of only a single monomer. Properties of this repeat sequence, its flanking sequences in chromosomal DNA, and RNA's transcribed from it suggest that it may be a mobile DNA element inserted at hundreds of thousands of different chromosomal locations.

Chromosomal locations of two DNA segments that flank ribosomal insertion-like sequences in Drosophila: flanking sequences are mobile elements.

We report the chromosomal locations of two repetitive DNA sequences that flank ribosomal insertion-like sequences in Drosophila melanogaster. The chromocentric region of D. melanogaster contains many copies of sequences that are homologous to type 1 ribosomal insertions. These insertion-like elements are interspersed with other DNA segments that we call flanking sequences. Two distinct flanking sequences derived from the same cloned DNA molecule pDmI 101, the HindIII fragments 101E and 101F, were studied. Whole genome Southern blots with DNA from the D. melanogaster stocks Oregon R (P2), gt-1, and gt-X11 showed complex restriction patterns that differed substantially between the three stocks. This and other data show that flanking sequences are members of diverged repetitive sequence families. In situ hybridization to salivary gland chromosomes of gt-1 and gt-X11 showed that both sequences are homologous to the chromocenter and to about 5 to 8 (101E) or 25 to 30(101F) euchromatic sites in each stock. Most, if not all, of these sites differed in gt-1 and gt-X11. Both 101E and 101F are homologous to he chromocenter and very few euchromatic bands in D. simulans, but 101F is homologous to numerous bands in D. mauritiana. We conclude that the flanking sequences represented by 101E and 101F are mobile elements within the genome of Drosophila. These two sequences differ in several structural features from mobile DNA elements previously described in this organism.