Search

The expression pattern and regulatory functions of microRNAs (miRNAs) are intensively investigated in various tissues, cell types and disorders. Differential miRNA expression signatures have been revealed in healthy and unhealthy tissues using high-throughput profiling methods. For further analyses of miRNA signatures in biological samples, we describe here a simple and efficient method to detect multiple miRNAs simultaneously in total RNA. The size-coded ligation-mediated polymerase chain reaction (SL-PCR) method is based on size-coded DNA probe hybridization in solution, followed-by ligation, PCR amplification and gel fractionation. The new method shows quantitative and specific detection of miRNAs. We profiled miRNAs of the let-7 family in a number of organisms, tissues and cell types and the results correspond with their incidence in the genome and reported expression levels. Finally, SL-PCR detected let-7 expression changes in human embryonic stem cells as they differentiate to neuron and also in young and aged mice brain and bone marrow. We conclude that the method can efficiently reveal miRNA signatures in a range of biological samples.

A recently developed and powerful strategy for site directed mutagenesis is to use two successive polymerase chain reactions (1, 2, 3, 4). The first reaction introduces a specific mutation into the target sequence, normally, by using a mutagenic primer and a primer that anneals beyond a bordering restriction site. The second reaction extends the first product beyond a restriction site adjacent to the opposite border. Restriction endonuclease cleavage and DNA splicing is then used to subclone the mutated cassette for further study. In practice we have found that the second PCR is often weak or even fails entirely, particularly when the product of the first reaction is long or forms a very stable secondary structure. Presumably, this is due to poor extension of the first PCR product, possibly because secondary structure in the large primer disrupts initiation. We have isolated this step of the thermal cycle by labelling the first PCR product of an unsuccessful mutagenesis experiment, and have found that it is indeed weak under typical PCR conditions (results not shown). Successful second reactions have been obtained previously by using room temperature annealing, but the use of reduced temperatures increases the incidence of non-specific amplification (5). Without lowering the annealing temperature, we have greatly improved the second PCR by instead lengthening the annealing period at 55°C to 15 minutes. This modification has been used for various different mutagenesis experiments on ribosomal RNA genes, including simple base pair substitutions in the yeast 5S RNA gene (6), larger sequence substitutions in the Schizosaccharomyces pombe internal transcribed spacers (data submitted but not shown) and an insertion (413 bp) of Tetrahymena thermophila ribosomal DNA into the Schizosaccharomycespombe 25S RNA gene (Figure IA). The results of this last experiment (Figure iB) clearly demonstrate an improved reaction efficiency and the nearly complete extension of the first PCR product that can be achieved when the annealing period is lengthened to 15 minutes. In all cases, the mutated DNA fragments were subcloned and confirmed by sequence analysis. The PCR conditions were as follows: The reaction volume was 50 Al containing 50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25°C), 1.5 mM MgCl, 0.1% Triton X-100, 200 itM of each dNTP, 100 /Ag/ml bovine serum albumin, 70 ng of each primer, 50 ng template DNA and 2 units Taq Polymerase, all covered with 50 t1l mineral oil. An initial denaturation step at 94°C for 3 minutes was followed by 30 cycles of denaturation at 940C for 1 minute, annealing at 550C for 1 minute and extension at 72°C for 2 minutes. For the second PCR, the first PCR products were purified using agarose gel electrophoresis and a unidirectional electroeluter (International Biotechnologies, Inc., New Haven, CT) and the annealing period of the thermal cycle was lengthened to 15 minutes.

Background A majority of bacterial genes belong to tight clusters and operons, which complicates gene functional studies using conventional knock-out methods. Antisense agents can down-regulate the expression of genes without disrupting the genome because they bind mRNA and block its expression. However, it is unclear how antisense inhibition affects expression from genes that are cotranscribed with the target.

Essential bacterial genes located within operons are particularly challenging to study independently because of coordinated gene expression and the nonviability of knockout mutants. Essentiality scores for many operon genes remain uncertain. Antisense RNA (asRNA) silencing or in-frame gene disruption of genes may help establish essentiality but can lead to polar effects on genes downstream or upstream of the target gene. Here, the Escherichia coli ribF-ileS-lspA-fkpB-ispH operon was used to evaluate the possibility of independently studying an essential gene using expressed asRNA and target gene overexpression to deregulate coupled expression. The gene requirement for growth in conditional silencing strains was determined by the relationship of target mRNA reduction with growth inhibition as the minimum transcript level required for 50% growth (MTL 50 ). Mupirocin and globomycin, the protein inhibitors of IleS and LspA, respectively, were used in sensitization assays of strains containing both asRNA-expressing and open reading frame-expressing plasmids to examine deregulation of the overlapping ileS-lspA genes. We found upstream and downstream polar silencing effects when either ileS or lspA was silenced, indicating coupled expression. Weighted MTL 50 values (means and standard deviations) of ribF , ileS , and lspA were 0.65 ± 0.18, 0.64 ± 0.06, and 0.76 ± 0.10, respectively. However, they were not significantly different ( P = 0.71 by weighted one-way analysis of variance). The gene requirement for ispH could not be determined due to insufficient growth reduction. Mupirocin and globomycin sensitization experiments indicated that ileS-lspA expression could not be decoupled. The results highlight the inherent challenges associated with genetic analyses of operons; however, coupling of essential genes may provide opportunities to improve RNA-silencing antimicrobials.

Background Antibiotic resistance genes are widely used for selection of recombinant bacteria, but their use risks contributing to the spread of antibiotic resistance. In particular, the practice is inappropriate for some intrinsically resistant bacteria and in vaccine production, and costly for industrial scale production. Non-antibiotic systems are available, but require mutant host strains, defined media or expensive reagents. An unexplored concept is over-expression of a host essential gene to enable selection in the presence of a chemical inhibitor of the gene product. To test this idea in E. coli, we used the growth essential target gene fabI as the plasmid-borne marker and the biocide triclosan as the selective agent. Results The new cloning vector, pFab, enabled selection by triclosan at 1 μM. Interestingly, pFab out-performed the parent pUC19-ampicillin system in cell growth, plasmid stability and plasmid yield. Also, pFab was toxic to host cells in a way that was reversed by triclosan. Therefore, pFab and triclosan are toxic when used alone but in combination they enhance growth and plasmid production through a gene-inhibitor interaction. Conclusion The fabI-triclosan model system provides an alternative plasmid selection method based on essential gene over-expression, without the use of antibiotic-resistance genes and conventional antibiotics.