Rapid selection of accessible and cleavable sites in RNA by Escherichia coli RNase P and random external guide sequences.

A method of inhibiting the expression of particular genes by using external guide sequences (EGSs) has been improved in its rapidity and specificity. Random EGSs that have 14-nt random sequences are used in the selection procedure for an EGS that attacks the mRNA for a gene in a particular location. A mixture of the random EGSs, the particular target RNA, and RNase P is used in the diagnostic procedure, which, after completion, is analyzed in a gel with suitable control lanes. Within a few hours, the procedure is complete. The action of EGSs designed by an older method is compared with EGSs designed by the random EGS method on mRNAs from two bacterial pathogens.

The AmpC phenotype in Norwegian clinical isolates of Escherichia coli is associated with an acquired ISEcp1-like ampC element or hyperproduction of the endogenous AmpC.

OBJECTIVES: The aim of the study was to examine resistance mechanisms associated with an AmpC phenotype in Norwegian clinical isolates of Escherichia coli. METHODS: Clinical E. coli isolates (n = 106) with reduced susceptibility to third-generation cephalosporins without clavulanic acid synergy were collected from 12 Norwegian laboratories from 2003 to 2005. Twenty-two isolates with an AmpC phenotype were selected for further characterization by PFGE, isoelectric focusing, different PCR-based techniques, DNA sequencing, AmpC qRT-PCR, transfer studies and plasmid analyses. RESULTS: The 22 isolates were not clonally related by the PFGE analysis. All isolates expressed a beta-lactamase with a pI of 9.0-9.2. Ten isolates contained a bla(CMY) gene, which was linked to an ISEcp1-like element in all cases. Twelve isolates had mutations or insertions in the promoter or the attenuator regions, leading to increased expression of the chromosomal ampC gene. One of these isolates had an ISEc10 element inserted upstream of the chromosomal ampC gene. CONCLUSIONS: This is the first molecular study of Norwegian clinical E. coli isolates with an AmpC phenotype. Resistance was mediated either by expression of bla(CMY) from acquired ISEcp1-like-bla(CMY) elements, or by mutations or insertions in the chromosomal ampC gene control region leading to hyperproduction of the endogenous AmpC enzyme. There was no correlation between the level of ampC mRNA and the MICs of cephalosporins.

RNA reprogramming of alpha-mannosidase mRNA sequences in vitro by myxomycete group IC1 and IE ribozymes.

Trans-splicing group I ribozymes have been introduced in order to mediate RNA reprogramming (including RNA repair) of therapeutically relevant RNA transcripts. Efficient RNA reprogramming depends on the appropriate efficiency of the reaction, and several attempts, including optimization of target recognition and ribozyme catalysis, have been performed. In most studies, the Tetrahymena group IC1 ribozyme has been applied. Here we investigate the potential of group IC1 and group IE intron ribozymes, derived from the myxomycetes Didymium and Fuligo, in addition to the Tetrahymena ribozyme, for RNA reprogramming of a mutated alpha-mannosidase mRNA sequence. Randomized internal guide sequences were introduced for all four ribozymes and used to select accessible sites within isolated mutant alpha-mannosidase mRNA from mammalian COS-7 cells. Two accessible sites common to all the group I ribozymes were identified and further investigated in RNA reprogramming by trans-splicing analyses. All the myxomycete ribozymes performed the trans-splicing reaction with high fidelity, resulting in the conversion of mutated alpha-mannosidase RNA into wild-type sequence. RNA protection analysis revealed that the myxomycete ribozymes perform trans-splicing at approximately similar efficiencies as the Tetrahymena ribozyme. Interestingly, the relative efficiency among the ribozymes tested correlates with structural features of the P4-P6-folding domain, consistent with the fact that efficient folding is essential for group I intron trans-splicing.

Optimization and application of the group I ribozyme trans-splicing reaction.

Group I ribozymes are naturally occurring catalytic RNAs that are able to excise themselves as introns (group I introns) from a precursor RNA, and to ligate the flanking exons. Group I ribozymes can be engineered to act in trans by recognizing a separate RNA molecule in a sequence specific manner, and to covalently link an RNA sequence to this separate RNA molecule. This ribozyme transesterification reaction has potential in molecular biology and in medicine as a new approach to gene therapy. Here we describe detailed optimized protocols where trans-splicing group I ribozymes are applied in mapping accessible sites in target messenger RNA, and in messenger RNA-repair by correcting mutations.

Inhibition of expression in Escherichia coli of a virulence regulator MglB of Francisella tularensis using external guide sequence technology.

External guide sequences (EGSs) have successfully been used to inhibit expression of target genes at the post-transcriptional level in both prokaryotes and eukaryotes. We previously reported that EGS accessible and cleavable sites in the target RNAs can rapidly be identified by screening random EGS (rEGS) libraries. Here the method of screening rEGS libraries and a partial RNase T1 digestion assay were used to identify sites accessible to EGSs in the mRNA of a global virulence regulator MglB from Francisella tularensis, a Gram-negative pathogenic bacterium. Specific EGSs were subsequently designed and their activities in terms of the cleavage of mglB mRNA by RNase P were tested in vitro and in vivo. EGS73, EGS148, and EGS155 in both stem and M1 EGS constructs induced mglB mRNA cleavage in vitro. Expression of stem EGS73 and EGS155 in Escherichia coli resulted in significant reduction of the mglB mRNA level coded for the F. tularensis mglB gene inserted in those cells.

The molecular evolution and structural organization of group I introns at position 1389 in nuclear small subunit rDNA of myxomycetes.

The number of nuclear group I introns from myxomycetes is rapidly increasing in GenBank as more rDNA sequences from these organisms are being sequenced. They represent an interesting and complex group of intervening sequences because several introns are mobile (or inferred to be mobile) and many contain large and unusual insertions in peripheral loops. Here we describe related group I introns at position 1389 in the small subunit rDNA of representatives from the myxomycete family Didymiaceae. Phylogenetic analyses support a common origin and mainly vertical inheritance of the intron. All S1389 introns from the Didymiaceae belong to the IC1 subclass of nuclear group I introns. The central catalytic core region of about 100 nt appears divergent in sequence composition even though the introns reside in closely related species. Furthermore, unlike the majority of group I introns from myxomycetes the S1389 introns do not self-splice as naked RNA in vitro under standard conditions, consistent with a dependence on host factors for folding or activity. Finally, the myxomycete S1389 introns are exclusively found within the family Didymiaceae, which suggests that this group I intron was acquired after the split between the families Didymiaceae and Physaraceae.

Trans-splicing of a mutated glycosylasparaginase mRNA sequence by a group I ribozyme deficient in hydrolysis.

RNA reprogramming represents a new concept in correcting genetic defects at the RNA level. However, for the technique to be useful for therapy, the level of reprogramming must be appropriate. To improve the efficiency of group I ribozyme-mediated RNA reprogramming, when using the Tetrahymena ribozyme, regions complementary to the target RNA have previously been extended in length and accessible sites in the target RNAs have been identified. As an alternative to the Tetrahymena model ribozyme, the DiGIR2 group I ribozyme, derived from a mobile group I intron in rDNA of the myxomycete Didymium iridis, represents a new and attractive tool in RNA reprogramming. We reported recently that the deletion of a structural element within the P9 domain of DiGIR2 turns off hydrolysis at the 3' splice site (side reaction) without affecting self-splicing [Haugen, P., Andreassen, M., Birgisdottir, A.B. & Johansen, S.D. (2004) Eur. J. Biochem. 271, 1015-1024]. Here we analyze the potential of the modified ribozyme, deficient in hydrolysis at the 3' splice site, for application in group I ribozyme-mediated trans-splicing of RNA. The improved ribozyme catalyses both cis-splicing and trans-splicing in vitro of a human glycosylasparaginase mRNA sequence with the same efficiency as the original DiGIR2 ribozyme, but without detectable levels of the unwanted hydrolysis.

Twelve Group I introns in the same pre-rRNA transcript of the myxomycete Fuligo septica: RNA processing and evolution.

The ribosomal DNA region of the myxomycete Fuligo septica was investigated and found to contain 12 group I introns (four in the small subunit and eight in the large subunit ribosomal RNAs). We have performed molecular and phylogenetic analyses to provide insight into intron structure and function, intron-host biology, and intron origin and evolution. The introns vary in size from 398 to 943 nt, all lacking detectable open reading frames. Secondary structure models revealed considerable structural diversity, but all, except one (subclass IB), represent the common group IC1 intron subclass. In vitro splicing analysis revealed that 10 of the 12 introns were able to self-splice as naked RNA, but all 12 introns were able to splice out from the precursor rRNA in vivo as evaluated by reverse transcription PCR analysis on total F. septica RNA. Furthermore, RNA processing analyses in vitro and in vivo showed that 10 of 12 introns perform hydrolytic cleavage at the 3' splice site, as well as intron circularization. Full-length intron RNA circles were detected in vivo. The order of splicing was analyzed by a reverse transcription PCR approach on cellular RNA, but no strict order of intron excision could be detected. Phylogenetic analysis indicated that most Fuligo introns were distantly related to each other and were independently gained in ribosomal DNA during evolution.

Nonconjugative transposition of the vanB-containing Tn5382-like element in Enterococcus faecium.

The vanB2 operon encoding glycopeptide resistance is an integral part of the putative conjugative transposon Tn5382. Characterization of clinical glycopeptide resistant derivatives from an epidemic ampicillin-resistant Enterococcus faecium strain showed precise chromosomal or plasmid insertions of a vanB2-containing Tn5382-like element. Conjugative transposition of the Tn5382-like element was not demonstrated in retransfer studies.

Genetic linkage of the vanB2 gene cluster to Tn5382 in vancomycin-resistant enterococci and characterization of two novel insertion sequences.

VanB-type vancomycin resistance is encoded by the vanB gene cluster, which disseminates by horizontal gene transfer and clonal spread of vancomycin-resistant enterococci (VRE). Genetic linkage of the vanB gene cluster to transposon Tn5382 and the insertion sequences IS16 and IS256-like has previously been shown. In this study linkage of defined vanB gene cluster subtypes to these elements was examined. All the vanB2 subtype strains studied (n=14) revealed co-hybridization of vanB and Tn5382, whereas the strains of vanB1 (n=8) and vanB3 (n=1) subtypes were Tn5382 negative. Conjugative cotransfer of the vanB2 gene cluster and Tn5382 was demonstrated for two strains. DNA sequencing of the vanX(B)-ORFC region in vanB2 strains confirmed that the vanB2 gene cluster is an integral part of Tn5382. No general pattern of linkage was observed with regard to IS16 and IS256-like. Two novel insertion sequences were identified in specific vanB2 subtype strains. (i) A 1611 bp element (ISEnfa110) was detected in the left flank of Tn5382. Its insertion site, lack of terminal inverted and direct repeats, and two conserved motifs in its putative transposase all conform to the conventions of the IS110 family. (ii) A 787 bp element (ISEnfa200) was detected in the vanS(B)-vanY(B) intergenic region. Its ORF encoded a putative protein with 60-70% identity to transposases of the IS200 family. No further copies of ISEnfa110 were found by colony hybridization of 181 enterococcal isolates, whereas ISEnfa200 was found in four additional vanB2 strains from the USA. The five strains had identical ISEnfa200 element insertion sites, and Tn5382 was located downstream from a pbp5 gene conferring high-level ampicillin resistance. These isolates showed related PFGE patterns, suggesting possible clonal spread of a VRE strain harbouring a Tn5382-vanB2-ISEnfa200 element linked to a pbp5 gene conferring ampicillin resistance.