Synthetic mRNAs for manipulating cellular phenotypes: an overview

Availability of high quality synthetic mRNAs (syn-mRNAs) has enabled progress in their applications. Important structural features and quality requirements are discussed. Developments in the application of mRNA-mediated manipulation of cells are presented (i) mRNA-directed expression of antigens in dendritic cells for vaccination projects in oncogenesis, infectious disease and allergy prevention; (ii) reprogramming of human fibroblasts to induced pluripotent stem cells with their subsequent differentiation to the desired cell type; (iii) applications in gene therapy

Telomerase activity in `immortal' fish1We dedicate this publication to E.S. Quabius. This study would have been impossible without her initiating ideas.1

AbstractEukaryotic chromosome termini consist of telomeres, short sequence repeats. According to the telomere hypothesis, DNA replication leads to telomere shortening, resulting in a cellular mitotic clock. Telomerase resets it by telomere synthesis. In mammals with a limited growth phase, telomerase activity in somatic tissues is restricted to stem cell derivatives with high proliferation potential. But other animals, like some fish, grow throughout their life with little senescence. All somatic cells require a high proliferation capacity and telomerase should be active in all cells, irrespective of fish age. Indeed, we detected high telomerase activities in all analyzed organs of rainbow trout (Oncorhynchus mykiss)

Longevity of lobsters is linked to ubiquitous telomerase expression

AbstractMammals have high growth rates in embryonic and juvenile phases and no growth in adult and senescent phases. We analyzed telomerase activity in a fundamentally different animal which grows indeterminately. Lobsters (Homarus americanus) grow throughout their life and the occurrence of senescence is slow. A modified TRAP assay was developed and the lobster telomeric repeat sequence TTAGG was determined. We detected telomerase activities which were dependent on RNA and protein components, required dGTP, dATP and dTTP, but not dCTP. Telomerase products with a five nucleotide periodicity were generated. High telomerase activities were detected in all lobster organs. We conclude that telomerase activation is a conserved mechanism for maintaining long-term cell proliferation capacity and preventing senescence, not only in cellular models or embryonic life stages but also in adult multicellular organisms

Enzymatic RNA synthesis with deoxynucleoside 5'-O-(1-thiotriphosphates)

AbstractWe have investigated the incorporation of 2'-deoxynucleoside-5'-O-(1-thiotriphosphates) into RNA transcripts using T7 RNA polymerase. With the exception of [α-S]dGTP, we obtained full-length transcripts of pre-tRNAphe and pre-tRNAtyr using an appropriate mixture of 2'-deoxynucleoside 5'-O-(1-thiotriphosphate) and the corresponding normal nucleoside triphosphate. The yields of the transcripts were comparable to those obtained with unmodified NTPs. Both substrates, [α-S]dTTP and [α-S]dATP, were inserted specifically. However, [α-S]dCTP was excluded at specific sites. We could not obtain transcripts using the deoxyguanosine derivative

Direct sequencing of double-stranded polymerase chain reaction-amplified 16S rDNA

A number of different procedures have been developed for direct sequence analysis of PCR products. These methods rely on the cumbersome isolation of specific PCR products from agarose gels or the production of single-stranded template DNAs. In the approach presented here, we describe primers for the amplification of 16-S rDNA and a simple preparation of PCR product for sequencing

Development of Diagnostic Oligonucleotide Probes for Four Lactobacillus Species Occurring in the Intestinal Tract

Partial sequences of the 16S rRNA from Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus plantarum and Lactobacillus ruminis were used to design diagnostic species-specific DNA oligonucleotide probes. The specificity of the probes was tested against crude rRNA of 30 strains from 12 Lactobacillus species and 18 Gram-positive representatives from various genera. The detection limit of rRNA from homologous strains in mixed cultures (10 cells of a heterologous species) and in 0.6 g minced beef meat corresponded to 3 × 10 cells, and 1 × 10 cells respectively. A rapid lysis technique of cells was developed that facilitates the isolation of nucleic acids directly in 1.5 ml screw-top vials

Altering gene expression by aminocoumarins: the role of DNA supercoiling in Staphylococcus aureus

BACKGROUND: It has been shown previously that aminocoumarin antibiotics such as novobiocin lead to immediate downregulation of recA expression and thereby inhibit the SOS response, mutation frequency and recombination capacity in Staphylococcus aureus. Aminocoumarins function by inhibiting the ATPase activity of DNA gyrase subunit B with a severe impact on DNA supercoiling. RESULTS: Here, we have analysed the global impact of the DNA relaxing agent novobiocin on gene expression in S. aureus. Using a novobiocin-resistant mutant, it became evident that the change in recA expression is due to gyrase inhibition. Microarray analysis and northern blot hybridisation revealed that the expression levels of a distinct set of genes were increased (e.g., recF-gyrB-gyrA, the rib operon and the ure operon) or decreased (e.g., arlRS, recA, lukA, hlgC and fnbA) by novobiocin. The two-component ArlRS system was previously found to decrease the level of supercoiling in S. aureus. Thus, downregulation of arlRS might partially compensate for the relaxing effect of novobiocin. Global analysis and gene mapping of supercoiling-sensitive genes did not provide any indication that they are clustered in the genome. Promoter fusion assays confirmed that the responsiveness of a given gene is intrinsic to the promoter region but independent of the chromosomal location. CONCLUSIONS: The results indicate that the molecular properties of a given promoter, rather than the chromosomal topology, dictate the responsiveness to changes in supercoiling in the pathogen Staphylococcus aureus