Tn10 insertion specificity is strongly dependent upon sequences immediately adjacent to the target-site consensus sequence.

ABSTRACTInsertion sequences (IS) are fundamental mediators of genome plasticity with the potential to generate phenotypic variation with significant evolutionary outcomes. Here, a recently active miniature inverted-repeat transposon element (MITE) was identified in a derivative ofAcinetobacter baumanniiATCC 17978 after being subjected to stress conditions. Transposition of the novel element led to the disruption of thehnsgene, resulting in a characteristic hypermotile phenotype. DNA identity shared between the terminal inverted repeats of this MITE and coresident ISAba12elements, together with the generation of 9-bp target site duplications, provides strong evidence that ISAba12elements were responsible for mobilization of the MITE (designated MITEAba12) within this strain. A wider genome-level survey identified MITEAba12in 30 additionalAcinetobactergenomes at various frequencies and oneMoraxella osloensisgenome. Ninety MITEAba12copies could be identified, of which 40% had target site duplications, indicating recent transposition events. Elements ranged between 111 and 114 bp; 90% were 113 bp in length. Using the MITEAba12consensus sequence, putative outward-facingEscherichia coliσ70 promoter sequences in both orientations were identified. The identification of transcripts originating from the promoter in one direction supports the proposal that the element can influence neighboring host gene transcription. The location of MITEAba12varied significantly between and within genomes, preferentially integrating into AT-rich regions. Additionally, a copy of MITEAba12was identified in a novel 8.5-kb composite transposon, Tn6645, in theM. osloensisCCUG 350 chromosome. Overall, this study shows that MITEAba12is the most abundant nonautonomous element currently found inAcinetobacter.IMPORTANCEOne of the most important weapons in the armory ofAcinetobacteris its impressive genetic plasticity, facilitating rapid genetic mutations and rearrangements as well as integration of foreign determinants carried by mobile genetic elements. Of these, IS are considered one of the key forces shaping bacterial genomes and ultimately evolution. We report the identification of a novel nonautonomous IS-derived element present in multiple bacterial species from theMoraxellaceaefamily and its recent translocation into thehnslocus in theA. baumanniiATCC 17978 genome. The latter finding adds new knowledge to only a limited number of documented examples of MITEs in the literature and underscores the plastic nature of thehnslocus inA. baumannii. MITEAba12, and its predicted parent(s), may be a source of substantial adaptive evolution within environmental and clinically relevant bacterial pathogens and, thus, have broad implications for niche-specific adaptation.

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IS231A insertion specificity: consensus sequence and DNA bending at the target site

Molecular Microbiology ◽

10.1111/j.1365-2958.1994.tb01273.x ◽

1994 ◽

Vol 14 (1) ◽

pp. 131-139 ◽

Cited By ~ 49

Author(s):

Bernard Hallet ◽

René Rezsöhazy ◽

Jacques Mahilton ◽

Jean Delcour

Keyword(s):

Dna Bending ◽

Consensus Sequence ◽

Target Site

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Transposition of Tn125encoding the NDM-1 carbapenemase inAcinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01755-16 ◽

2016 ◽

pp. AAC.01755-16 ◽

Cited By ~ 13

Author(s):

Séverine Bontron ◽

Patrice Nordmann ◽

Laurent Poirel

Keyword(s):

Acinetobacter Baumannii ◽

Direct Evidence ◽

Opportunistic Pathogen ◽

Target Site ◽

Gram Negative Bacteria ◽

Transposition Frequency ◽

Gram Negative ◽

Temperature Changes ◽

Site Consensus ◽

Almost All

TheblaNDM-1gene encodes a carbapenemase that confers resistance to almost all β-lactams, including latest resort carbapenems. It is increasingly reported worldwide in nosocomial and community-acquired Gram-negative bacteria.Acinetobacter baumanniiis an important opportunistic pathogen considered as an intermediate reservoir for theblaNDM-1gene. In this species, theblaNDM-1gene is located within the Tn125composite transposon. The mechanism driving the mobility of Tn125has not yet been elucidated. Here we experimentally demonstrated transposition of Tn125inA. baumannii. Systematic 3-bp duplication of the target site, being signature of transposition, was evidenced. The target site consensus for Tn125transposition was found to be GC-enriched at the duplicated 3 bp and AT-rich in the vicinity. Transposition frequency was not influenced by temperature changes or by exposure to sub-inhibitory concentrations of various antibiotics. This work is the first direct evidence of the functionality of a composite transposon inA. baumannii. It provides a mechanistic clue for the dissemination of theblaNDM-1gene inAcinetobacterspp. and subsequently among Enterobacteriaceae.

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A symmetrical six-base-pair target site sequence determines Tn10 insertion specificity

Cell ◽

10.1016/0092-8674(82)90385-3 ◽

1982 ◽

Vol 28 (1) ◽

pp. 155-163 ◽

Cited By ~ 95

Author(s):

Shirley M. Halling ◽

Nancy Kleckner

Keyword(s):

Base Pair ◽

Target Site ◽

Tn10 Insertion

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A 3′ splice site consensus sequence mutation in the intron 3 of the α-galactosidase a gene in a patient with Fabry disease

The Japanese Journal of Human Genetics ◽

10.1007/bf01910542 ◽

1991 ◽

Vol 36 (3) ◽

pp. 245-250 ◽

Cited By ~ 15

Author(s):

Tohru Yokoi ◽

Kazuko Shinoda ◽

Ichiro Ohno ◽

Kimitaka Kato ◽

Toshio Miyawaki ◽

...

Keyword(s):

Fabry Disease ◽

Splice Site ◽

Consensus Sequence ◽

Site Consensus

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Molecular Characterization of PK-LR Gene in Pyruvate Kinase–Deficient Italian Patients

Blood ◽

10.1182/blood.v89.10.3847.3847_3847_3852 ◽

1997 ◽

Vol 89 (10) ◽

pp. 3847-3852 ◽

Cited By ~ 2

Author(s):

Alberto Zanella ◽

Paola Bianchi ◽

Luciano Baronciani ◽

Manuela Zappa ◽

Elena Bredi ◽

...

Keyword(s):

Pyruvate Kinase ◽

Splice Site ◽

Stop Codon ◽

Consensus Sequence ◽

Missense Mutations ◽

Frequent Mutation ◽

Unstable Gene ◽

Site Consensus ◽

Conserved Amino Acids

We studied the PK-LR gene in 15 unrelated Italian patients with congenital hemolytic anemia associated with erythrocyte pyruvate kinase (PK) deficiency. Fourteen different mutations were detected among 26 mutated alleles identified: a five-nucleotide (nt) deletion (227 to 231), two splice-site (1269C and IVS3(−2)c), 10 missense (514C, 787T, 823A, 993A, 994A, 1168A, 1456T, 1529A, 1552A, and 1594T) and one nonsense mutation(s) (721T). Eight of these (deletion 227-231, 1269C, IVS3(−2)c, 514C, 787T, 823A, 1168A, and 1552A) were novel. Moreover, a new polymorphic site was detected in the 3′ untranslated region of the mRNA (C/T, nucleotide 1738). The deletion 227-231 causes a stop codon after amino acid 77, probably resulting in an unstable gene product. Mutations 1269C and IVS3(−2)c lead to an alteration of the 5′ and 3′ splice-site consensus sequence, respectively; cDNA analysis failed to reveal any abnormal transcript, suggesting that these mutations generate an unstable mRNA that is rapidly degraded. Of the five new missense mutations, 823A (Gly275-Arg) and 1168A (Asp390-Asn) involve highly conserved amino acids, 514C (Glu172-Gln) and 1552A (Arg518-Ser), although found in less conserved regions, affect the balance of the electric charges of the protein. Mutation 787T (Gly263-Trp) is likely to determine strong modifications in the local structure of the molecule. The most frequent mutation in Italy appears to be 1456T (seven of 30 alleles), followed by 1529A (three of 30) and 994A (three of 30). A correlation was found between mutations, biochemical characteristics of the enzyme, and clinical course of the disease.

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Early commitment of yeast pre-mRNA to the spliceosome pathway

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3755-3760.1988 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3755-3760 ◽

Cited By ~ 1

Author(s):

P Legrain ◽

B Seraphin ◽

M Rosbash

Keyword(s):

Consensus Sequence ◽

Rnase H ◽

Small Nuclear Rna ◽

Spliceosome Assembly ◽

U2 Snrna ◽

Consensus Sequences ◽

U2 Snrnp ◽

Site Consensus ◽

Sequence Requirements

Pre-mRNA splicing in vitro is preceded by complex formation (spliceosome assembly). U2 small nuclear RNA (snRNA) is found in the earliest form of the spliceosome detected by native gel electrophoresis, both in Saccharomyces cerevisiae and in metazoan extracts. To examine the requirements for the formation of this early complex (band III) in yeast extracts, we cleaved the U2 snRNA by oligonucleotide-directed RNase H digestion. U2 snRNA depletion by this means inhibits both splicing and band III formation. Using this depleted extract, we were able to design a chase experiment which shows that a pre-mRNA substrate is committed to the spliceosome assembly pathway in the absence of functional U2 snRNP. Interactions occurring during the commitment step are highly resistant to the addition of an excess of unlabeled substrate and require little or no ATP. Sequence requirements for this commitment step have been analyzed by competition experiments with deletion mutants: both the 5' splice site consensus sequence and the branch point TACTAAC box sequence are necessary. These experiments strongly suggest that the initial assembly process requires a trans-acting factor(s) (RNA and/or proteins) that recognizes and stably binds to the two consensus sequences of the pre-mRNA prior to U2 snRNP binding.

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Molecular Characterization of PK-LR Gene in Pyruvate Kinase–Deficient Italian Patients

Blood ◽

10.1182/blood.v89.10.3847 ◽

1997 ◽

Vol 89 (10) ◽

pp. 3847-3852 ◽

Cited By ~ 48

Author(s):

Alberto Zanella ◽

Paola Bianchi ◽

Luciano Baronciani ◽

Manuela Zappa ◽

Elena Bredi ◽

...

Keyword(s):

Pyruvate Kinase ◽

Splice Site ◽

Stop Codon ◽

Consensus Sequence ◽

Missense Mutations ◽

Frequent Mutation ◽

Unstable Gene ◽

Site Consensus ◽

Conserved Amino Acids

Abstract We studied the PK-LR gene in 15 unrelated Italian patients with congenital hemolytic anemia associated with erythrocyte pyruvate kinase (PK) deficiency. Fourteen different mutations were detected among 26 mutated alleles identified: a five-nucleotide (nt) deletion (227 to 231), two splice-site (1269C and IVS3(−2)c), 10 missense (514C, 787T, 823A, 993A, 994A, 1168A, 1456T, 1529A, 1552A, and 1594T) and one nonsense mutation(s) (721T). Eight of these (deletion 227-231, 1269C, IVS3(−2)c, 514C, 787T, 823A, 1168A, and 1552A) were novel. Moreover, a new polymorphic site was detected in the 3′ untranslated region of the mRNA (C/T, nucleotide 1738). The deletion 227-231 causes a stop codon after amino acid 77, probably resulting in an unstable gene product. Mutations 1269C and IVS3(−2)c lead to an alteration of the 5′ and 3′ splice-site consensus sequence, respectively; cDNA analysis failed to reveal any abnormal transcript, suggesting that these mutations generate an unstable mRNA that is rapidly degraded. Of the five new missense mutations, 823A (Gly275-Arg) and 1168A (Asp390-Asn) involve highly conserved amino acids, 514C (Glu172-Gln) and 1552A (Arg518-Ser), although found in less conserved regions, affect the balance of the electric charges of the protein. Mutation 787T (Gly263-Trp) is likely to determine strong modifications in the local structure of the molecule. The most frequent mutation in Italy appears to be 1456T (seven of 30 alleles), followed by 1529A (three of 30) and 994A (three of 30). A correlation was found between mutations, biochemical characteristics of the enzyme, and clinical course of the disease.

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