Antisense RNA-mediated transcriptional attenuation is a regulatory mechanism operating in the replication control of two groups of plasmids in gram-positive bacteria, the pT181 group and the inc18 family, represented by pIP501. In contrast, this control mechanism has so far not been identified in gram-negative bacteria or their plasmids. In this work we asked whether such a mechanism can be sup...

In order to examine gene function in Streptococcus mutans, we have recently initiated an antisense RNA strategy. Toward this end, we have now constructed and evaluated three Escherichia coli-S. mutans shuttle expression vectors with the fruA and scrB promoters from S. mutans, as well as the tetR-controlled tetO promoter from Staphylococcus aureus. Among these, the tetO/tetR system proved to be ...

The use of antisense oligodeoxyribonucleotides (ODN) or ribozymes to specifically suppress gene expression is simple in concept and relies on efficient binding of the antisense strand to the target RNA. Although the identification of target sites accessible to base pairing is gradually being overcome by different techniques, it remains a major problem in the antisense and ribozyme approaches. I...

Antisense RNA, transcribed intracellularly from constitutive expression cassettes, inhibits the replication of the human immunodeficiency virus type 1 (HIV-1) as demonstrated by a quantitative microinjection assay in human SW480 cells. Infectious proviral HIV-1 DNA was co-microinjected together with a fivefold molar excess of plasmids expressing antisense RNA complementary to a set of ten diffe...

The putative primase gene and other genes associated with the Sfi21-prototype genome replication module are highly conserved in Streptococcus thermophilus bacteriophages. Expression of antisense RNAs complementary to the putative primase gene (pri3.1) from S. thermophilus phage kappa 3 provided significant protection from kappa 3 and two other Sfi21-type phages. Expression of pri3.10-AS, an ant...

The effects of inducible expression of poly(ADP-ribose) polymerase (PADPRP) antisense RNA in HeLa cells were determined in order to gain further insight into the biological roles of the poly(ADP-ribosyl)ation modification of nuclear proteins. A recombinant expression plasmid was prepared with the mouse mammary tumor virus (MMTV) promoter upstream of the antisense-oriented PADPRP cDNA. Expressio...

The non-coding transcriptome of the hyperthermophilic archaeon Pyrococcus abyssi is investigated using the RNA-seq technology. A dedicated computational pipeline analyzes RNA-seq reads and prior genome annotation to identify small RNAs, untranslated regions of mRNAs, and cis-encoded antisense transcripts. Unlike other archaea, such as Sulfolobus and Halobacteriales, P. abyssi produces few leade...

RNA-centric biochemical purification is a general approach for studying the functions and mechanisms of noncoding RNAs. Here, we describe the experimental procedures for RNA antisense purification (RAP), a method for selective purification of endogenous RNA complexes from cell extracts that enables mapping of RNA interactions with chromatin. In RAP, the user cross-links cells to fix endogenous ...

Single-stranded regions in RNA secondary structure are important for RNA-RNA and RNA-protein interactions. We present a probability profile approach for the prediction of these regions based on a statistical algorithm for sampling RNA secondary structures. For the prediction of phylogenetically-determined single-stranded regions in secondary structures of representative RNA sequences, the proba...