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PubMed2003

Insertion sequence-like elements associated with putative polyhydroxybutyrate regulatory genes in Azotobacter sp. FA8.

The genes phaR, phaP, and phaF, encoding putative regulatory proteins, were found in the poly (3-hydroxybutyrate) (PHB) gene cluster of the free nitrogen-fixing bacteria Azotobacter sp. FA8. These genes were flanked by the insertion sequence ISAzsp1, belonging to the IS3 family, and a region highly homologous to insertion sequences of the IS630 family. These are the first site-specific recombination elements to be described in association with genes involved in the metabolism of polyhydroxyalkanoates (PHAs). A possible role for ISs in the assembly of pha genes is presented.

3-Hydroxybutyric Acid↗

PubMed1999

Insertion of sequences into the 3' untranslated region of a replication-competent spleen necrosis virus vector disrupts env gene expression.

The genomes of all replication-competent retroviruses contain cis-acting elements that regulate gene expression. However, the identities of many of these elements remain to be characterized. Inserting sequences into the 3' untranslated region of a replication-competent spleen necrosis virus (SNV) vector disrupted its ability to replicate. Inserts varying in sequences and sizes (0.4-kb to 1.6-kb) all resulted in this defect. Genetic compensation experiments and immunostaining revealed that env gene expression was deficient. Northern analysis indicated the presence of spliced viral mRNA of the correct size although at a reduced level compared to a wildtype vector. It is likely that the block in env expression occurs at a post-transcriptional step. These results suggest that the function of a cis-acting element distinct from the constitutive transport element is disrupted by the inserted sequences into the 3' untranslated region of SNV.

3' Untranslated Regions↗

PubMed2001

Oligonucleotide-mediated, PCR-independent cloning by homologous recombination.

We have developed an oligonucleotide-mediated cloning technique based on homologous recombination in Saccharomyces cerevisiae that allows precise DNA sequences to be transferred independent of restriction enzymes and PCR. In this procedure, linear DNA sequences are targeted to a chosen site in a yeast vector by DNA linkers, which consist of two partially overlapping oligonucleotides. The linkers contain relatively short regions of both yeast vector sequences and insert sequences, which stimulate homologous recombination between the vector and the insert. The linkers can also contain sequences not found in either the vector or the insert (e.g., sequences that encode ribosome binding sites, epitope tags, preferred codons, etc.), thus allowing modification of the transferred DNA. Linkers can be designed such that DNA sequences can be transferred with just two reusable universal oligonucleotides and two gene-specific oligonucleotides. This cloning method, which is performed by co-transforming yeast with linear vector, substrate DNA, and unannealed oligonucleotides, has been termed the yeast-based, oligonucleotide-mediated gap repair technique (YOGRT).

Cloning, Molecular↗

PubMed1982

The making of strand-specific M13 probes.

A novel approach has been developed for the preparation of highly radioactive, strand-specific M13 probes. A universal primer, complementary to the region 5' to the multiple cloning sites of M13mp7, was used to initiate the DNA synthesis of the complementary strand of the M13 sequence downstream from the inserted sequence. The synthesis of the (-) strand, which was labeled with a radioactively labeled precursor, did not proceed to completion so that the inserted sequence was kept single-stranded. Thus, a partially double-stranded probe that had the specificity of this inserted sequence was obtained. As an example for the application of single-stranded specific hybridization probes, an M13mp7 subclone of a zein cDNA clone of maize (A30) was labeled and used in a dot hybridization test to select from the hundreds of M13mp7 subclones of the zein genomic clone, Z4, the sequences complementary to the probe. The specificity of the probe was confirmed by dideoxy chain terminator sequencing experiments.

Base Sequence↗

PubMed1992

Sequence analysis of an insertion element, IS1131, isolated from the nopaline-type Ti plasmid of Agrobacterium tumefaciens.

Transferred DNA (T-DNA) from several nopaline-type Ti plasmids of Agrobacterium tumefaciens was previously shown to be variable in size due to the insertion of extra DNA segments. We found an insertion sequence (named IS1131) in the T-DNA of the strain PO22 and determined the nucleotide (nt) sequence. IS1131 is 2773 bp long, contains four open reading frames, and is flanked by 12-bp perfect inverted repeats (IR). An 8-bp direct repeat was found immediately outside the IR, and represents a target site of integration. Although the IS1131 nt sequence showed a limited degree of similarity to those of the previously reported IS elements of A. tumefaciens and to the central region of T-DNA, the terminal IR of IS1131 were highly homologous (up to 83%) to those of IS66 and IS866, suggesting that these three IS elements are related to each other. A number of IS1131-related copies were found in several pathogenic, as well as nonpathogenic, nopaline-type strains from different plant sources, and were distributed on both chromosomes and plasmids.

Agrobacterium tumefaciens↗

PubMed1981

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.

Animals↗

PubMed1996

Characterization of IS199 from Streptococcus mutans V403.

The complete nucleotide sequence of the insertion sequence IS199 has been determined. This novel element was found in Streptococcus mutans V403 where DNA hybridization studies suggested that it was present in multiple copies on the genome. IS199 is 1220 bp and appears unique to the mutans streptococci. A limited epidemiological survey revealed this element to occur infrequently in this group (4/50 strains examined). IS199 belongs to the IS3 family of procaryotic insertion sequence elements. It contained tandem open reading frames (ORFs) reminiscent of the structure of the IS3 class. Additionally, the larger of the two ORF products (ORFB) exhibited amino acid sequence similarities to comparable proteins from IS3 and other elements of this family. Transposition of IS199 gave rise to a 3-bp target site duplication. We were able to insert an antibiotic resistance gene into IS199 enabling us to follow the transposition of this element in S. mutans V403.

Amino Acid Sequence↗

PubMed1987

The obtention of simian virus 40 recombinants carrying d(CG.GC)n, d(CA.GT)n and d(CT.GA)n sequences. Stability of the inserted simple repeating sequences.

A general strategy for the introduction of simple repeating DNA sequences into the simian virus 40 (SV40) has been developed. SV40 recombinants carrying d(CG.GC)5, d(CA.GT)30 or d(CT.GA)22 insertions at either the TaqI site (position 4739) or the HpaII site (position 346) were obtained and the stability of the inserted DNA sequences studied. The palindromic potentially Z-DNA-forming d(CG.GC)n sequence was found to be highly unstable when compared to either d(CA.GT)n or d(CT.GA)n.

Base Sequence↗

PubMed1997

Identification and characterization of IS-like elements in Mycobacterium gordonae.

A new insertion sequence, IS1512, from Mycobacterium gordonae was cloned and sequenced. This element is present in up to 10 copies which provides a high diversity for restriction fragment length polymorphism analysis. We have also identified truncated IS1512-like elements, including a truncated IS1512 and another truncated insertion sequence which displays homology with IS1512 and was designated IS1511. Sequences homologous to the previously described transposon Tn554 are inserted into these truncated insertion sequences. Insertion loci of IS1511/IS1512 are shown to be highly related to those described for IS256-like elements in other species. Alignment of the putative transposases from Rhodococcus and Mycobacterium suggests these insertion sequences may form a distinct closely homologous subclass within the IS256 family. Analysis comparing phylogenetic divergence of these elements with that of 16S rRNA and superoxide dismutase genes suggests horizontal transfer of a IS1511/IS1512 precursor into M. gordonae, and the occurrence of horizontal transfer between Mycobacteriaceae and Rhodococcaceae.

Actinomyces↗

PubMed1996

Identification of Tnr3, a suppressor-mutator/enhancer-like transposable element from rice.

We isolated members of the retroposon family p-SINE1 in rice and found that one member contained an insertion.Aa 3-bp sequence at the insertion site within p-SINE1 appeared duplicated. The insertion sequence, 1539 bp in length, carried imperfect inverted repeats of about 13 bp at its termini which begin with 5'-CACTA---3'; these repeats are similar to those found in members of the En/Spm transposable element family. These results indicate that the insertion sequence is a transposable element belonging to the En/Spm family and is thus named Tnr3 (transposable element in rice no. 3). In fact, Tnr carried long subterminal regions containing direct and inverted repeats of short DNA sequences of 15 bp, another characteristic of the En/Spm family. The subterminal repeat sequences in Tnr3 are, however, of two kinds, although they share homology with each other. Tnr3 and its relatives were present in multiple copies in rice. considering the length of Tnr3, it cannot represent an autonomous type element, but is a non-autonomous element probably derived by deletion from an autonomous transposon.

Base Sequence↗

PubMed2001

Specificity of insertion sequence-based PCR assays for mycobacterium tuberculosis complex.

OBJECTIVE: To determine the specificity of different insertion sequence-targeted polymerase chain reaction (PCR) tests for Mycobacterium tuberculosis complex. DESIGN: One M. bovis BCG strain, two M. tuberculosis strains and ten species of mycobacteria other than tuberculosis (MOTT) were tested by three PCR assays based on the repetitive elements IS6110, IS1081 and IS990 under variable amplification conditions (different temperatures of primer annealing and numbers of reaction cycles). RESULTS: DNA amplifications based on the three insertion sequences yielded fragments of expected sizes only in DNA from M. tuberculosis complex strains when the tests were conducted at high stringency (65 degrees C). At the annealing temperature of 60 degrees C the PCR assay with IS6110-specific primers yielded a 245 bp fragment also in nine MOTT strains tested. This could result from previously reported homology between non-tuberculous mycobacteria and a central region of IS6110. Amplification assays based on IS1081 and IS990 gave false-positive results in some MOTT isolates only under very low stringency (55 degrees C), which could be due to non-specific priming of the target DNA at that temperature. CONCLUSION: Repetitive elements IS1081 and IS990 may represent a more reliable alternative to the more widely used IS6110 PCR target for tuberculosis diagnosis.

DNA Transposable Elements↗

PubMed2001

Isolation and diagnostic potential of ISMav2, a novel insertion sequence-like element from Mycobacterium avium subspecies paratuberculosis.

A novel Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) specific insertion sequence has been identified by representational difference analysis and designated as ISMav2. ISMav2 has no similarity to known mycobacterial IS elements but shows more than 50% identity to a non-composite transposon of Streptomyces coelicolor at the DNA and protein level. ISMav2 is present in at least three copies on the genome as assessed by Southern blot analysis and its potential value as a diagnostic tool was confirmed by PCR analyses on 79 M. paratuberculosis field isolates, nine M. avium ssp. avium isolates, and the reference strains of nine other mycobacterial species.

Base Sequence↗

PubMed1997

Identification of an unusual intein in chloroplast ClpP protease of Chlamydomonas eugametos.

The proteasome-like ClpP protease is widely distributed and structurally conserved among bacteria and eukaryotic cell organelles. In Chlamydomonas eugametos, however, the chloroplast clpP gene predicted a much larger ClpP protein containing large insertion sequences (ISs). One insertion sequence, IS2, is 456 amino acid residues long and not similar to known proteins. Here we show that IS2 is an unusual intein, and its protein splicing activity in Escherichia coli cells can be activated by a single amino acid substitution. Analysis of IS2 sequence revealed short sequence motifs that are similar to known intein motifs, including putative LAGLI-DADG endonuclease motifs. But a histidine residue conserved at the C terminus of known inteins is replaced in the IS2 sequence by a glycine residue (Gly455), rendering the IS2 sequence incapable of detectable protein splicing when tested in E. coli cells. Changing Gly455 to histidine activated the ability of IS2 to undergo protein splicing in E. coli cells. The IS2 sequence (intein) was precisely excised from a precursor protein, with the flanking sequences (exteins) joined together by a normal peptide bond.

Adenosine Triphosphatases↗

PubMed2005

Periodic extinctions of transposable elements in bacterial lineages: evidence from intragenomic variation in multiple genomes.

Most previous work on the evolution of mobile DNA was limited by incomplete sequence information. Whole genome sequences allow us to overcome this limitation. I study the nucleotide diversity of prominent members of five insertion sequence families whose transposition activity is encoded by a single transposase gene. Eighteen among 376 completely sequenced bacterial genomes and plasmids carry between 3 and 20 copies of a given insertion sequence. I show that these copies generally show very low DNA divergence. Specifically, more than 68% of the transposase genes are identical within a genome. The average number of amino acid replacement substitutions at amino acid replacement sites is Ka = 0.013, that at silent sites is Ks = 0.1. This low intragenomic diversity stands in stark contrast to a much higher divergence of the same insertion sequences among distantly related genomes. Gene conversion among protein-coding genes is unlikely to account for this lack of diversity. The relation between transposition frequencies and silent substitution rates suggests that most insertion sequences in a typical genome are evolutionarily young and have been recently acquired. They may undergo periodic extinction in bacterial lineages. By implication, they are detrimental to their host in the long run. This is also suggested by the highly skewed and patchy distribution of insertion sequences among genomes. In sum, one can think of insertion sequences as slow-acting infectious diseases of cell lineages.

Bacteria↗

PubMed1995

Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking.

Isolation of DNA segments adjacent to known sequences is a tedious task in genome-related research. We have developed an efficient PCR strategy that overcomes the shortcomings of existing methods and can be automated. This strategy, thermal asymmetric interlaced (TAIL)-PCR, utilizes nested sequence-specific primers together with a shorter arbitrary degenerate primer so that the relative amplification efficiencies of specific and nonspecific products can be thermally controlled. One low-stringency PCR cycle is carried out to create annealing site(s) adapted for the arbitrary primer within the unknown target sequence bordering the known segment. This sequence is then preferentially and geometrically amplified over nontarget ones by interspersion of high-stringency PCR cycles with reduced-stringency PCR cycles. We have exploited the efficiency of this method to expedite amplification and sequencing of insert end segments from P1 and YAC clones for chromosome walking. In this study we present protocols that are amenable to automation of amplification and sequencing of insert end sequences directly from cells of P1 and YAC clones.

Automation↗

PubMed2000

Local hopping of IS3 elements into the A+T-rich part of the high-pathogenicity island in Yersinia enterocolitica 1B, O:8.

The high-pathogenicity island (Yen HPI) of Yersinia enterocolitica biogroup (BG) 1B strains is associated with mouse virulence. Three repeated sequences are clustered on the A+T-rich part of the Yen HPI downstream of the fyuA yersiniabactin receptor gene in Y. enterocolitica O:8 strains WA-314 and 8081. In addition to IS1328 and IS1400, the RS3 repeated sequence consists of a novel insertion sequence, IS1329, inserted into the remnants of IS1222. This partial IS retains both 44-bp inverted terminal repeats (ITRs) of IS1222 but has suffered deletions of different sizes in strains WA-314 and 8081. IS1329 is 1243-bp long, carries 25-bp imperfect ITRs and two consecutive orfs capable to encode 110-amino acid (aa) and 249-aa proteins, respectively. IS1329 is present only in BG 1B Y. enterocolitica strains. Similarly to IS1400, IS1329 and IS1222 belong to the IS3 group of mobile elements and seem to have preference for the 'local hopping' into the A+T-rich part of the Yen HPI. These insertion sequences may be responsible for the imprecise deletions of the Yen HPI in strain WA-314.

AT Rich Sequence↗

PubMed1993

Transposable elements in Lactococci: a review.

Genetic studies have identified the presence of transposable elements within the genus Lactococcus, which includes industrially important microorganisms used in the production of fermented dairy products. Three insertion sequences have been fully characterized in addition to several reports of transpositionlike events. The three insertion sequence elements, ISS1, IS904, and IS981, exhibit the physical and genetic properties characteristic of known insertion sequences. They are closely related to insertion sequences isolated from a wide variety of microorganisms. In lactococci, insertion sequence elements are associated with lactose and sucrose metabolism, proteinase activity, nisin production and immunity, conjugal transfer determinants, and bacteriophage resistance, which are attributes significant for growth in a milk environment. The characteristics, involvement in lactococcal evolution, and recent developments as tools for genetic engineering of the lactococcal elements are discussed.

Base Sequence↗

PubMed2005

Novel insertion sequence-like elements in phytoplasma strains of the aster yellows group are putative new members of the IS3 family.

Novel insertion sequence (IS)-like elements were isolated and characterized from phytoplasma strains in the aster yellows (AY) group (16SrI). The IS-like elements were cloned from phytoplasma strains AY1 and NJAY or PCR-amplified from 15 additional strains representing nine subgroups in the AY group using primers based on sequences of the putative transposases (Tpases). All IS-like elements contained sequences encoding similar Tpases of 321 amino acids (320 for strain CPh). Substantial amino acid sequence variability suggested multiple species of Tpases or IS-like elements exist in the AY phytoplasma group. These Tpases have an identical DDE motif that is most similar to the DDE consensus of Tpases in the IS3 family.

Amino Acid Sequence↗