Nucleotide sequence of IS26, a new prokaryotic mobile genetic element.

The DNA sequence of a new IS element, the IS26, is 820 bp long and carries 14 bp perfect terminal inverted repeats. Upon integration, IS26 generates an 8 bp duplication of its target sequence. A large open reading frame within IS26 could code for a protein of 234 amino acids. On its reverse strand, IS26 also carries one large open reading frame, 591 bp long, which contains no stop codon within IS26.     

Nucleotide sequence of insertion sequence IS3411, which flanks the citrate utilization determinant of transposon Tn3411.

The nucleotide sequences of insertion sequences IS3411L (left) and IS3411R (right), present as direct terminal repeats in the citrate utilization of citrate utilization transposon Tn3411, and of IS3411 (generated by intramolecular recombination between IS3411L and IS3411R) were determined. The three IS3411 elements (IS3411R, IS3411L, and IS3411) were 1,309 base pairs long and identical in DNA sequence. IS3411 had 27-base-pair terminal inverted repeats with three bases mismatched and one long open reading frame (240 amino acids) that was proposed to be a transposase. Three polypeptides of 29,000, 27,000, and about 10,000 molecular weight, determined by IS3411, were identified in minicells. Since Tn3411 generates a 3-base-pair repeat upon integration, the nucleotide sequences of IS3411 were compared with those of IS3.     

IS801, an insertion sequence element isolated from Pseudomonas syringae pathovar phaseolicola

A transposable element, designated IS801, was isolated from strain LR781 of Pseudomonas syringae pathovar phaseolicola in two independent events using the entrapment plasmid, pUCD800. IS801 is 1517 base pairs in length and contains open reading frames that potentially encode proteins of 311 and 172 amino acids, as well as smaller proteins. Unlike most other prokaryotic transposable elements, IS801 lacks terminal repeats. Sequence analysis revealed two target pentamers for IS801 insertion that differ by one base pair. One copy of IS801 generated a perfect duplication of its target, TGAAC. The second copy of IS801 was flanked by the target, TGGAC, at one end, and TGAAC at the other end. A third copy of IS801 was cloned from pMMC7105, an indigenous plasmid of strain LR781, and it was flanked by copies of the pentamer TGAAC.     

IS186: an Escherichia coli insertion element isolated from a cDNA library.

Escherichia coli IS186 was isolated from cDNA libraries made from rainbow trout RNA and maintained in E. coli RR1. The element was 1,347 base pairs in length, had a perfect inverted repeat of 25 base pairs, and had an open reading frame of 375 amino acids. The hypothetical protein sequence of IS186 had limited homology to the E. coli IS4 hypothetical protein I sequence. There were three copies of IS186 in E. coli RR1.     

Identification and characterization of IS1381, a new insertion sequence in Streptococcus pneumoniae.

A new insertion sequence (IS1381) was identified in the genome of Streptococcus pneumoniae R6 as an 846-bp segment containing 20-bp terminal inverted repeats and flanked by 7-bp direct repeats. The three sequenced copies of this element have two overlapping open reading frame (ORF) genes named orfA and orfB. However, significant variations between individual copies were found, suggesting that inactivating mutations have occurred in an original single ORF. Accordingly, the consensus IS1381 element derived from the comparison of the three available copies should contain a single ORF sufficient to encode a basic protein of 267 amino acids which exhibited high similarity to the putative transposases of ISL2 from Lactobacillus helveticus and of IS702 from the cyanobacterium Calothrix sp. strain PCC 7601. A minimum of five to seven copies were detected by hybridization experiments in the R6 genome. In remarkable contrast with the two previously reported pneumococcal insertion sequences, several copies of IS1381 have been detected in all of the clinical isolates tested so far. Interestingly, Streptococcus oralis NCTC 11427 (type strain), a close relative of pneumococcus, does not contain this element, but its occurrence in the type strain of Streptococcus mitis (NCTC 12261) suggests that this species has exchanged DNA with S. pneumoniae directly or through an intermediate species.     

Nucleotide sequence of the cryptic plasmid pTT8 from Thermus thermophilus HB8 and isolation and characterization of its high-copy-number mutant

The complete nucleotide sequence of pTT8, a cryptic plasmid from Thermus thermophilus HB8, was determined. pTT8 was 9328bp long and its G+C content was 69%. pTT8 contained eight putative open reading frames, three of which showed extensive similarities to the plasmid addiction proteins PasA and PasB of pTC-F14 and pAM10.6, and the RepA protein of the ColE2-related plasmids, respectively. During the analysis of pTT8-based plasmid pPP442, which had been obtained during a promoter-screening experiment, we occasionally isolated a plasmid with a relatively high-copy-number. This plasmid, pPP442m, contained a 1025 bp fragment derived from the genome of the HB27 host strain immediately upstream of the putative repA gene. Using the ori region of pPP442m, we constructed an expression vector, pTEV131m, with an estimated high-copy-number of 30-40. This plasmid was stably maintained in T. thermophilus HB27 under nonselective conditions for at least 100 generations. Cloning of the alpha-amylase gene of Bacillus stearothermophilus DY-5 into pTEV131m gave more than twofold production of the enzyme compared with pTEV131, the parental plasmid.     

Complete nucleotide sequence of an unusual mobile element from trypanosoma brucei

The complete nucleotide sequence of a mobile element from Trypanosoma brucei is presented along with the sequence of its target site, which shows that the insertion has generated a 7 base pair direct repeat. The cloned copy of the element is a dimeric structure, one end of each monomer consisting of a stretch of 14 A residues preceded by a putative trypanosome polyadenylation signal. Six base pairs of DNA of unknown origin are found in the dimer between the two copies of the element. Evidence suggests that the element is present in the genome mainly as a monomer whose sequence is conserved across several species of trypanosome. The element contains an open reading frame encoding the same 160 amino acid protein in both sequenced copies and is extensively transcribed from both strands.     

The transposable element Uhu from Hawaiian Drosophila--member of the widely dispersed class of Tc1-like transposons

We report the complete nucleotide sequence of the transposable element Uhu from the vicinity of the alcohol dehydrogenase (Adh) gene of Drosophila heteroneura (an endemic Hawaiian Drosophila). The complete element is about 1650 base-pairs (bp) long, has 46-50 base-pair inverse imperfect repeats at it's ends, and contains a large open reading frame potentially encoding a 192 amino acid protein. We demonstrate that Uhu belongs to a class of transposable elements which includes Tc1 from Caenorhabditis elegans, Barney from Caenorhabditis briggsae, and HB1 from Drosophila melanogaster. All of these elements share significant sequence similarity, are approximately 1600 base pairs long, have short inverse terminal repeats (ITRs), contain open reading frames (ORFs) with significant sequence identity, and appear to insert specifically at TA sequences generating target site duplications.     

Identification and characterization of IS1112 and IS1113 insertion element sequences in Xanthomonas oryzae pv. oryzae.

Two new insertion sequences (IS1112 and IS1113) were identified in the genome of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice. Three copies of IS1112 were trapped, one containing 1052-bp and the other two with 1055-bp. They all have 25-bp imperfect inverted repeats with a 3-bp duplication at the site of insertion. They contain an open reading frame (ORF) of 317 and 318 amino acid residues, respectively. IS1113 is 1306-bp, contains 25-bp imperfect terminal inverted repeats, and is flanked by a 9-bp direct repeat at the site of insertion. It contains an ORF of 395 amino acid residues.     

IS431, a staphylococcal insertion sequence-like element related to IS26 from Proteus vulgaris

We present the nucleotide sequence of IS431, a new staphylococcal insertion sequence-like element flanking the mercury-resistance determinant of pI524 and associated with the methicillin-resistance determinant. IS431 left is 800 bp long and has a perfect terminal inverted repeat (IR) of 22 bp; IS431 right is 786 bp long and has a terminal IR homologous to the IR of IS431 left except that the terminal 8 bp are absent. Both IRs share a 10-bp homology with the IR of IS26 from Proteus vulgaris. No directly repeated sequences were detected immediately adjacent to the IRs. An open reading frame (ORF) of 675 bp spans most of the IS431 sequence. Its deduced amino acid (aa) sequence shows 40% homology to the 234-aa-long putative transposase coded by ORFI of IS26.     

The DNA sequence of the gene (rnc) encoding ribonuclease III of Escherichia coli.

The DNA sequence of a 1,076 base pair BglI-BamHI fragment containing the entire rnc gene for ribonuclease III (RNase III) was determined. An open reading frame of 681 base pairs was found in this region which encodes a protein of 227 amino acid residues (calculated molecular weight = 25,218). When this open reading frame was cloned into a high expression vector, pIN-III, a protein of apparent molecular weight of 26,000 was produced upon induction of the cloned gene. This product accounted for up to 5% of the total cellular protein, and comigrated with purified RNase III. RNase III enzyme activity was induced in parallel with the production of the 26,000 molecular weight protein. A putative promoter was found 170 base pairs upstream from the initiation codon. In the long leader region a very stable stem-bulge-stem structure was found which closely resembles typical RNase III cleavage sites. This structure may be cleaved by RNase III to auto-regulate the expression of the rnc gene.     

Identification and nucleotide sequence of Rhizobium meliloti insertion sequence ISRm3: similarity between the putative transposase encoded by ISRm3 and those encoded by Staphylococcus aureus IS256 and Thiobacillus ferrooxidans IST2.

The insertion sequence ISRm3 was discovered simultaneously in different Rhizobium meliloti strains by probing Southern blots of total cellular DNA with 32P-labeled pTA2. This plasmid is indigenous to strain IZ450 and fortuitously contained four copies of ISRm3. By using an internal EcoRI fragment as a specific probe (pRWRm31), homology to ISRm3 was subsequently detected in over 90% of R. meliloti strains tested from different geographical locations around the world. The frequency of stable nonlethal ISRm3 transpositions was estimated to be 4 x 10(-5) per generation per cell in strain SU47 when grown in liquid culture. The entire nucleotide sequence of ISRm3 in R. meliloti 102F70 is 1,298 bp and has 30-bp terminal inverted repeats which are perfectly matched. Analysis of six copies of ISRm3 in two strains showed that a variable number of base pairs (usually eight or nine) were duplicated and formed direct repeats adjacent to the site of insertion. On one DNA strand, ISRm3 contains an open reading frame spanning 93% of its length. Comparison of the putative protein encoded with sequences derived from the EMBL and GenBank databases showed significant similarity between the putative transposases of ISRm3 from R. meliloti, IS256 from Staphylococcus aureus, and IST2 from Thiobacillus ferroxidans. These insertion sequences appear to be distantly related members of a distinct class.     

Evolutionarily conserved regions in Caenorhabditis transposable elements deduced by sequence comparison.

In this paper we present the sequence of an intact Caenorhabditis briggsae transposable element, Tcb2. Tcb2 is 1606 base pairs in length and contains 80 base pair imperfect terminal repeats and a single open reading frame. We have identified blocks of T-rich repeats in the regions 150-200 and 1421-1476 of this element which are conserved in the Caenorhabditis elegans element Tc1. The sequence conservation of these regions in elements from different Caenorhabditis species suggests that they are of functional importance. A single open reading frame corresponding to the major open reading frame of Tc1 is conserved among Tc1, Tcb1, and Tcb2. Comparison of the first 550 nucleotides of the sequence among the three elements has allowed the evaluation of a model proposing an extension of the major open reading frame. Our data support the suggestion that Tc1 is capable of producing a 335 amino acid protein. A comparison of the sequence coding for the amino and carboxy termini of the 273 amino acid transposase from Caenorhabditis Tc1-like elements and Drosophila HB1 showed different amounts of divergence for each of these regions, indicating that the two functional domains have undergone different amounts of selection. Our data are not compatible with the proposal that Tc1-related sequences have been acquired via horizontal transmission. The divergence of Tc1 from the two C. briggsae elements, Tcb1 and Tcb2, indicated that all three elements have been diverging from each other for approximately the same amount of time as the genomes of the two species.     

Identification of a new insertion element, similar to gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3.

In Streptococcus lactis ML3, the lactose plasmid (pSK08) forms cointegrates with a conjugal plasmid (pRS01). It has been proposed that cointegration is mediated by insertion sequences (IS) present on pSK08 (D. G. Anderson and L.L. McKay, J. Bacteriol. 158:954-962, 1984). We examined the junction regions of the cointegrate pPW2 and the corresponding regions of pSK08 (donor) and pRS01 (target) and identified a new IS element on pSK08 (ISS1S) which was involved in and duplicated during formation of pPW2. ISS1S was 808 base pairs (bp) in size, had 18-bp inverted repeats (GGTTCTGTTGCAAAGTTT) at its ends, contained a single long open reading frame encoding a putative protein of 226 amino acids, and generated 8-bp direct repeats of target DNA during cointegrate formation. An iso-IS element, ISS1T, which is duplicated in some other cointegrate plasmids, was also found on pSK08. ISS1T was also 808 bp in size and was identical to ISS1S in sequence except for 4 bp, none of which altered the inverted repeats or amino acid sequence of the open reading frame. Comparison of ISS1 with gram-negative IS26 revealed strong homologies in size (820 bp), sequence of inverted repeats (GGCACTGTTGCAAA), size of direct repeats generated after cointegration (8 bp), and number, size, and amino acid sequence (44.5% identical) of the open reading of frame.     

Characterization of IS1001, an insertion sequence element of Bordetella parapertussis.

By analysis of repetitive DNA in Bordetella parapertussis, an insertion sequence element, designated IS1001, was identified. Sequence analysis revealed that IS1001 comprised 1,306 bp and contained inverted repeats at its termini. Furthermore, several open reading frames that may code for transposition functions were identified. The largest open reading frame coded for a protein comprising 406 amino acid residues and showed homology to TnpA, which is encoded by an insertion sequence element (IS1096) found in Mycobacterium smegmatis. Examination of flanking sequences revealed that insertion of IS1001 occurs preferentially in stretches of T's or A's and results in a duplication of target sequences of 6 to 8 bases. IS1001 was found in about 20 copies in 10 B. parapertussis strains analyzed. No restriction fragment length polymorphism was observed in B. parapertussis when IS1001 was used as a probe. An insertion sequence element similar or identical to IS1001 was found in B. bronchiseptica strains isolated from pigs and a rabbit. In these strains, about five copies of the IS1001-like element were present at different positions in the bacterial chromosome. Neither B. pertussis nor B. bronchiseptica strains isolated from humans and dogs contained an IS1001-like element. Therefore, IS1001 may be used as a specific probe for the detection of B. parapertussis in human clinical samples.     

Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci.

An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS). IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase. IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion. IS981 was present on the chromosome of Lactococcus lactis subsp. lactis LM0230 from where it transposed to pGBK17 during transformation. Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L. lactis subsp. cremoris strains containing the highest number of copies. Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, IS51, IS150, IS600, IS629, IS861, IS904, and ISL1.