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PubMed1989

Insertion elements and deletion formation in a halophilic archaebacterium.

Deletion events that occur spontaneously in 36-kilobase-pair (kbp) plasmid pHH4 from the archaebacterium Halobacterium halobium were investigated. Four different deletion derivatives with sizes ranging from 5.7 to 17 kbp were isolated. Three of these deletion variants derived from pHH4 (pHH6 [17 kbp], pHH7 [16 kbp], and pHH8 [6.3 kbp]), whereas the 5.7-kbp plasmid pHH9 derived from pHH6. Strains containing pHH6, pHH7, or pHH9 each lacked the parental plasmid pHH4, while pHH8 occurred at a 1:1 ratio together with pHH4. Common to all of these plasmids was the 5.7-kbp region of pHH9 DNA. The regions containing the fusion site in the deletion derivatives were investigated and compared with the corresponding area of the parental plasmid. Each deletion occurred exactly at the terminus of an insertion element. In pHH6 and pHH7, a halobacterial insertion element (ISH2) was located at the deletion site. The DNA fused to ISH2 displayed a 7-base-pair (bp) (pHH7) or 10-bp (pHH6) sequence homology to the inverted repeat of ISH2. In the two smaller plasmids, pHH8 and pHH9, an ISH27 element was located at the deletion site. Most likely, all of these smaller plasmids resulted from an intramolecular transposition event. The ISH27 insertion sequence contains a 16-bp terminal inverted repeat and duplicates 5 bp of target DNA during the transposition with the specificity 5'ANNNT3'. Four ISH27 copies were analyzed, and two ISH27 element types were identified that have approximately 85% sequence similarity. The ISH27 insertion elements constitute a family which is related to the ISH51 family characterized for H. volcanii, another halophilic archaebacterium.

Base Composition↗

PubMed1983

Specific in vitro transcription of the insertion sequence IS2.

The insertion sequence IS2 is a small transposable element of Escherichia coli that lacks any known genetic markers. Insertion of this element in one orientation (I) within bacterial operons blocks expression of downstream genes. In the other orientation (II), IS2 has been associated with the constitutive expression of genes distal to its insertion, suggesting that IS2 might contain promoters directing transcription of IS2(II) into other genes. To test the transcription potential of IS2, we have transcribed in vitro DNA templates from gal3, a Gal- allele in which an IS2(I) is inserted between the gal promoter and the gal genes. We have detected two IS2-specific RNAs which initiate from promoters within IS2 and are transcribed in orientation II (away from the galETK genes). Though the presence and orientation of these promoters suggests that they could be responsible for the constitutive expression of genes adjacent to an IS2(II) element, an alternative role could be for transcription of IS2-encoded genes. Although IS2(I) insertions normally block expression of adjacent genes, certain altered (e.g. mutant) IS2(I) sequences lead to the constitutive expression of downstream genes. We have transcribed DNA templates from galwc5 and galc331, which are Galc alleles that contain altered IS2(I) insertions within the gal operon. For each allele, we have detected two gal-directed transcripts initiating within the IS2 sequence. These RNAs are not detected upon transcription of the unaltered IS2(I) DNA and the promoters arise as a direct consequence of the IS2(I) alterations. This result suggests that these promoters detected in vitro are responsible for the Galc phenotype of these alleles.

Autoradiography↗

PubMed1984

Transcriptional control of IS1 transposition in Escherichia coli.

The movement of the bacterial insertion sequence IS1 often generates cointegrate structures in which donor and target replicons are connected by direct repeats of IS1. The experiments reported here were designed to understand how IS1 transposition is controlled. Our physical characterization of the structures of cointegrates between an F factor ( pOX38 ) and a set of pBR322::Tn9-related plasmids indicate that the relative mobilities of the two IS1 elements of Tn9 are inversely correlated with the strength of promoters upstream in the vector DNA. This implies that transcription across the ends of an IS1 element inhibits its transposition. Transcriptional inhibition may be due to interference with either the synthesis or the action of transposase.

DNA Replication↗

PubMed1991

Retrotransposable elements R1 and R2 interrupt the rRNA genes of most insects.

A large number of insect species have been screened for the presence of the retrotransposable elements R1 and R2. These elements integrate independently at specific sites in the 28S rRNA genes. Genomic blots indicated that 43 of 47 insect species from nine orders contained insertions, ranging in frequency from a few percent to greater than 50% of the 28S genes. Sequence analysis of these insertions from 8 species revealed 22 elements, 21 of which corresponded to R1 or R2 elements. Surprisingly, many species appeared to contain highly divergent copies of R1 and R2 elements. For example, a parasitic wasp contained at least four families of R1 elements; the Japanese beetle contained at least five families of R2 elements. The presence of these retrotransposable elements throughout Insecta and the observation that single species can harbor divergent families within its rRNA-encoding DNA loci present interesting questions concerning the age of these elements and the possibility of cross-species transfer.

Amino Acid Sequence↗

PubMed2006

Properties of the various Botmar1 transcripts in imagoes of the bumble bee, Bombus terrestris (Hymenoptera: Apidae).

Botmar1 elements are mariner-like elements (MLEs), class II transposable elements that occur in the genome of the bumble bee, Bombus terrestris. Each haploid B. terrestris genome contains about 230 Botmar1, consisting entirely of 1.3-kb and 0.85-kb elements. During their evolution in the B. terrestris genome, two Botmar1 lineages have been differentiated in terms of their nucleic acid sequences and the differences found in their 5' untranslated regions suggest that they could be transcribed differently in B. terrestris. Here, we show that small amounts of Botmar1 mRNA occur in RNA extracts purified from B. terrestris imagoes. This indicates that the Botmar1 transcription is either weak in imagoes, or is restricted to very few cells. The cloning of several mRNAs reveals that only lineage-2 Botmar1 elements are transcribed. This transcription is specific, and uses cardinal initiators and terminators of eukaryotic elements in the Botmar1 elements. The intrastrand stem-loop folds in the mRNA theoretically synthesized by elements of the first lineage suggest that mRNA maintenance in cells might be self-regulated by RNA interference.

3' Untranslated Regions↗

PubMed1992

Sequence of ISRm4 from Rhizobium meliloti strain GR4.

ISRm4, an IS-like sequence structurally similar to Pseudomonas cepacia insertion element IS402, was identified by sequence analysis. This 933-bp element carries 17-bp putative terminal inverted repeats with five mismatches and a putative direct target duplication of 3 bp.

Base Sequence↗

PubMed1994

Characterization and distribution of insertion sequence IS1239 in Streptococcus pyogenes.

The human pathogenic bacterium Streptococcus pyogenes causes pharyngitis, acute rheumatic fever, glomerulonephritis and toxic-shock-like syndrome. The bacterium synthesizes several extracellular products, including the recently described streptococcal superantigen SSA, a molecule that shares considerable homology with several Staphylococcus aureus enterotoxins. While studying allelic variation at the ssa locus, six isolates expressing serotypes M4, M23, M33, M41, M43, and provisional type PT4854, were identified that had PCR products about 40-bp larger than expected, and one isolate (M15) had an amplified fragment that was more than 1-kb larger than expected. All six isolates have a 34-bp insert located 103 bp 5' of the ssa start codon. The larger product is a result of a 1110-bp insertion at the analogous location. The complementary strand of this insert has a 981-bp open reading frame that potentially encodes a 326-amino-acid polypeptide with substantial homology to the Escherichia coli IS30 transposase. Results of Southern blot analysis showed that at least twelve copies of the sequence are present in the serotype M15 S. pyogenes isolate. This element, designated IS1239, is the first simple insertion sequence described in group-A streptococci. Results of PCR screening showed that 26 of 78 (33%) S. pyogenes isolates expressing distinct M protein serotypes contained sequences with homology to IS1239, which means that the element is widely distributed in the species.

Amino Acid Sequence↗

PubMed2005

Occurrence of insertion sequences within the genomes and Tn1546-like elements of glycopeptide-resistant enterococci isolated in Brazil, and identification of a novel element, ISEfa5.

Insertion sequences (IS) occur widely within the Tn1546-like elements responsible for VanA glycopeptide resistance in enterococci from several countries. As such insertions can be used as epidemiological markers and for studying horizontal transfer of gene clusters, we investigated the distribution of IS6770, IS1542, IS1216V, IS1476, and IS1251 elements in 26 VanA Enterococcus faecium and 21 VanA Enterococcus faecalis from Brazil. PCR, using genomic DNA as a template, indicated that most of the isolates contained IS6770 (97%), IS1216V (87%) and IS1476 (72%) elements. IS1251 was also detected, but at a higher frequency in E. faecium (80%) than in E. faecalis (14%). None of the isolates harboured IS1542. Only two of 47 isolates had IS elements within their Tn1546-like elements; one possessed IS1251 between vanS and vanH, as reported in the United States; another possessed a novel IS element, designated ISEfa5, located between vanX and van Y. This novel element was found in the genomic DNA of 25 (96%) E. faecium and II (52%) E. faecalis. In stability studies, no IS-mediated changes were detected in the Tn1546-like elements of 25 vancomycin-resistant enterococci (VRE) monitored over 11 months. These results suggest that the occurrence of IS in Brazilian isolates is similar to that reported in American isolates, but that these elements occur rarely within the vanA gene clusters. As patterns of IS carriage did not correlate with the PFGE type of the VRE, the prevalence of IS elements in genomic DNA of VRE is not a useful epidemiological marker. However, the presence of IS-modified Tn1546-like elements, which appear to be rare in Brazil, could be a useful molecular marker in local epidemiological studies to monitor the evolution and horizontal transmission of VanA elements.

Anti-Bacterial Agents↗

PubMed2005

A novel insertion sequence element, IS Yen2, as an epidemiological marker for weakly pathogenic bioserotypes of Yersinia enterocolitica.

The structure and distribution of IS Yen2, a new Yersinia enterocolitica insertion sequence element, were investigated. ISYen2 is related to IS elements of the IS21 family and is present in two isoforms in Y. enterocolitica serotype O:3. Analysis of all copies of both isoforms, IS Yen2A and IS Yen2B, by PCR and sequencing indicated that they are not flanked by direct repeats which are typical of the IS21 family of repetitive elements. IS Yen2 is present in multiple copies in Y. enterocolitica O:3 and O:9 and in a single copy in Y. enterocolitica O:1 and O:2 serotypes. The probe for IS Yen2 efficiently detected all weakly pathogenic Y. enterocolitica bioserotypes investigated, whereas it did not hybridize with other strains. This indicates that IS Yen2 can serve as an additional tool for Y. enterocolitica differentiation and epidemiological studies. Distribution of the different groups of IS elements in two Y. enterocolitica pathotypes is in favor of the parallel evolution of American and European Y. enterocolitica strains.

Amino Acid Sequence↗

PubMed2000

Genomic exploration of the hemiascomycetous yeasts: 17. Yarrowia lipolytica.

A total of 4940 random sequence tags of the dimorphic yeast Yarrowia lipolytica, totalling 4.9 Mb, were analyzed. BLASTX comparisons revealed at least 1229 novel Y. lipolytica genes 1083 genes having homology with Saccharomyces cerevisiae genes and 146 with genes from various other genomes. This confirms the rapid sequence evolution assumed for Y. lipolytica. Functional analysis of newly discovered genes revealed that several enzymatic activities were increased compared to S. cerevisiae, in particular, transport activities, ion homeostasis, and various metabolism pathways. Most of the mitochondrial genes were identified in contigs spanning more than 47 kb. Matches to retrotransposons were observed, including a S. cerevisiae Ty3 and a LINE element. The sequences have been deposited with EMBL under the accession numbers AL409956-AL414895.

DNA Transposable Elements↗

PubMed1992

Turnover of R1 (type I) and R2 (type II) retrotransposable elements in the ribosomal DNA of Drosophila melanogaster.

R1 and R2 are distantly related non-long terminal repeat retrotransposable elements each of which inserts into a specific site in the 28S rRNA genes of most insects. We have analyzed aspects of R1 and R2 abundance and sequence variation in 27 geographical isolates of Drosophila melanogaster. The fraction of 28S rRNA genes containing these elements varied greatly between strains, 17-67% for R1 elements and 2-28% for R2 elements. The total percentage of the rDNA repeats inserted ranged from 32 to 77%. The fraction of the rDNA repeats that contained both of these elements suggested that R1 and R2 exhibit neither an inhibition of nor preference for insertion into a 28S gene already containing the other type of element. Based on the conservation of restriction sites in the elements of all strains, and sequence analysis of individual elements from three strains, nucleotide divergence is very low for R1 and R2 elements within or between strains (less than 0.6%). This sequence uniformity is the expected result of the forces of concerted evolution (unequal crossovers and gene conversion) which act on the rRNA genes themselves. Evidence for the role of retrotransposition in the turnover of R1 and R2 was obtained by using naturally occurring 5' length polymorphisms of the elements as markers for independent transposition events. The pattern of these different length 5' truncations of R1 and R2 was found to be diverse and unique to most strains analyzed. Because recombination can only, with time, amplify or eliminate those length variants already present, the diversity found in each strain suggests that retrotransposition has played a critical role in maintaining these elements in the rDNA repeats of D. melanogaster.

Animals↗

PubMed1993

A ubiquitin-conjugating enzyme, RAD6, affects the distribution of Ty1 retrotransposon integration positions.

A galactose-inducible Ty1 element was used to generate 59 independent Ty1 inserts that inactivate the CAN1 gene. As found in previous studies, the distribution of these elements shows a gradient of insertion frequency from highest to lowest between the 5' and 3' ends of the gene. However, 53 independent Ty1 and Ty2 insertions isolated by an identical procedure in an isogenic rad6 deletion strain do not show this bias. In this strain, the Ty elements insert randomly throughout CAN1. These results show that the ubiquitin-conjugating enzyme, RAD6, alters the integration site preferences of Ty1 retrotransposons.

Alleles↗

PubMed1985

Complete sequence of IS3.

We have determined the nucleotide sequence of IS3. Our IS3 isolate has 39 bp inverted repeats (IR's) with 6 mismatches, and is 1258 bp long. IS3 contains a large open reading frame (ORF) of 288 codons with a smaller, partially overlapping ORF of 91 codons on the opposite strand in codon-codon register. The large ORF is preceded by and has a 4 bp overlap with a 99 codon ORF that has potential transcriptional and translational start signals. Thus, IS3 could encode a bicistronic mRNA. The Shine-Dalgarno sequence for this 99 codon ORF could be sequestered in a stem-loop structure, but only if the transcript began outside IS3, as was first seen with IS10. This could be a means for preventing fortuitous activation of IS3 by outside promoters. No DNA sequence homology was found between IS3 and other prokaryotic IS elements, but there is slight amino acid sequence homology and significant conservation of hydropathicity patterns between the putative transposases of IS3 and IS2.

Base Sequence↗

PubMed1988

Mys retrotransposons in Peromyscus leucopus and transgenic Mus musculus.

The mys family of retrotransposons exhibits an interesting phylogenetic distribution with 500-1000 copies per haploid genome in the white-footed mouse Peromyscus leucopus and no copies detectable in the house mouse Mus musculus, even though most other repeated sequences are shared by these two species. Comparison of the DNA sequences from the 3' ends of five mys elements show that insertion occurs just upstream of a well-conserved 11 bp target sequence. Transcription patterns of the elements in brain, liver, heart, kidneys and gonads in Peromyscus leucopus are analyzed. Transcripts are found in all tissues examined, but they are remarkably heterogeneous in size. When four cloned elements are introduced into transgenic Mus musculus, however, discrete patterns of expression are revealed. Furthermore, a study of the structure of the concatemers of mys elements in the transgenic mice demonstrates that recircularization of injected linear molecules is an important event in concatemer generation.

Animals↗

PubMed1990

Precise excision and instability of the transposon Tn5 in Pseudomonas aeruginosa.

Excision of the transposon Tn5 from sites of insertion in plasmid DNA was shown to occur at high frequency in Pseudomonas aeruginosa. Plasmids with Tn5 insertions conjugated poorly into P. aeruginosa and adversely affected growth compared to the respective parental plasmids. The kanamycin-resistance phenotype of Tn5 was expressed poorly in P. aeruginosa and kanamycin-sensitive strains were common during the manipulation of the P. aeruginosa transconjugants. Examination of plasmid DNA isolated from kanamycin-sensitive P. aeruginosa transconjugants revealed excision of Tn5 sequences. A plasmid containing a selectable marker (mercury resistance) inactivated by a Tn5 insertion was constructed, and Tn5 excised precisely, permitting the expression of the mercury-resistance marker at high frequency (10(-3) in P. aeruginosa and at the expected low frequency (10(-7] in Escherichia coli. The recombinational mechanism that promotes frequent Tn5 excision in P. aeruginosa operated in the absence of the P. aeruginosa recA gene product. Fragments of Tn5 were also examined for excision and instability in P. aeruginosa. A plasmid containing the terminal 485 bp of inverted repeat sequences from Tn5, but lacking the transposase or kanamycin-resistance genes, also showed precise excision of Tn5 DNA at high frequency (10(-2] in P. aeruginosa. Unlike plasmids containing a complete Tn5 insertion, this plasmid transferred to P. aeruginosa at normal frequencies and growth of the host was not severely impaired. In contrast, plasmids containing either IS50 element transferred to P. aeruginosa at greatly reduced frequencies, and transconjugants containing the IS50R element (which contains the active transposase gene) were small and especially difficult to maintain. P. aeruginosa transconjugants harbouring a plasmid containing only the DNA between the IS50 elements (which included the kanamycin-resistance gene) were of normal size and stably maintained.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗

PubMed1995

Complete nucleotide sequence of a skin element excised by DNA rearrangement during sporulation in Bacillus subtilis.

As part of the Bacillus subtilis genome sequencing project, we have determined the complete nucleotide sequence of a skin element which is located between spoIVCB and spoIIIC. The entire sequence of this element is 48,032 bp in length, and contains 57 ORFs with putative ribosome-binding sites. Two of them correspond to previously sequenced and characterized genes, cwIA and spoIVCA. Furthermore, seven ORF products identified in this element show interesting similarities with known proteins present in data banks, including the phi 105 immunity repressor, the phi 105 Cro-like protein and the SPP1 terminase. These results indicate the possibility that the skin element is a cryptic remnant of an ancestral temperate phage.

Amino Acid Sequence↗

PubMed2005

Generation of an erythromycin-sensitive derivative of Clostridium difficile strain 630 (630Deltaerm) and demonstration that the conjugative transposon Tn916DeltaE enters the genome of this strain at multiple sites.

Erythromycin resistance in Clostridium difficile strain 630 is conferred by a genetic element termed Tn5398 which contains two erm(B) genes: erm1(B) and erm2(B). An erythromycin-sensitive derivative of strain 630 (designated 630Deltaerm) was generated by spontaneous mutation after continuous subculture for 30 days. This strain had lost the erm2(B) gene from within Tn5398 but retained erm1(B). However, the strain could revert to erythromycin resistance at a frequency of 2.79 x 10(-8), although it still contained the deletion of erm2(B). The availability of C. difficile 630Deltaerm allowed the behaviour of Tn916DeltaE to be investigated in this strain. This element entered the genome at multiple sites indicating that it could be useful as an insertional mutagen.

Blotting, Southern↗

PubMed1996

Identification and sequencing of a novel insertion sequence, IS1471, in Burkholderia cepacia strain 2a.

A novel insertion sequence (IS), IS1471, has been identified which is inserted into IS element IS1071 possessed by plasmid pIJB1 in Burkholderia cepacia strain 2a. Nucleotide sequencing has revealed that IS1471 is 1112 bp in length and is flanked by 22/21-bp imperfect inverted repeats with a 3-bp duplication of the target sequence IS1471 contains a single open reading frame encoding a putative polypeptide of 345 amino acids with molecular mass of 39406 Da. Searches of DNA and protein sequence databases did not result in the detection of any homologous IS elements, suggesting that IS1471 is novel and may not belong to any known IS groups.

Amino Acid Sequence↗