Presence Of rRNA Research Articles

Indole-based cyanine as a nuclear RNA-selective two-photon fluorescent probe for live cell imaging.

We have demonstrated that the subcellular targeting properties of the indole-based cyanines can be tuned by the functional substituent attached onto the indole moiety in which the first example of a highly RNA-selective and two-photon active fluorescent light-up probe for high contrast and brightness TPEF images of rRNA in the nucleolus of live cells has been developed. It is important to find that this cyanine binds much stronger toward RNA than DNA in a buffer solution as well as selectively stains and targets to rRNA in the nucleolus. Remarkably, the TPEF brightness (Φσmax) is dramatically increased with 11-fold enhancement in the presence of rRNA, leading to the record high Φσmax of 228 GM for RNA. This probe not only shows good biocompatibility and superior photostability but also offers general applicability to various live cell lines including HeLa, HepG2, MCF-7, and KB cells and excellent counterstaining compatibility with commercially available DNA or protein trackers.

Temporal study of Helicobacter pylori presence in coastal freshwater, estuary and marine waters

Helicobacter pylori, a gastric pathogen, is believed to be transmitted via the fecal-oral route as well as the oral-oral route. Its presence and viability in environmental waters is not well characterized. The goals of this study were to test H. pylori presence via molecular methods in freshwater, estuarine and beach sites in Delaware over both short and long time scales and to establish whether fecal indicator bacteria, including total Enterococcus and human-specific Bacteroidetes species, are predictive of the pathogen in these waters. The presence of Helicobacter pylori was initially tested by PCR with newly designed 23S rRNA gene primers against Helicobacter spp. and confirmed by sequencing. Two coastal beach sites were repeatedly positive in 2007. Clone library analysis indicated the persistence of one operational taxonomic unit (OTU) over time at one site. Detection of H. pylori was also determined by PCR assays from DNA and RNA for the 16S rRNA gene, as well as DNA for the ureA and cagA genes. Approximately 21% of the samples were positive for H. pylori 16S rRNA gene and 80% of those were also positive for H. pylori 16S rRNA, indicating that this potential pathogen is not only present in natural waters, but also probably viable. There was no correlation between the occurrence of H. pylori and fecal indicator bacteria, suggesting that standard water quality tests are ineffective in predicting the presence of this pathogen in natural waters. These results demonstrate the widespread presence of potentially viable H. pylori in coastal marine and estuarine waters. Additionally, the repeatedly positive samples indicate either a continual contamination source or persistence of H. pylori in marine waters.

Detection of Campylobacter jejuni and Campylobacter coli in chicken meat samples by real-time nucleic acid sequence-based amplification with molecular beacons

A nucleic acid sequence-based amplification (NASBA) assay based on molecular beacons was used for real-time detection of Campylobacter jejuni and Campylobacter coli in samples of chicken meat. A set of specific primers and beacon probe were designed to target the 16S rRNA of both species. The real-time NASBA protocol including the RNA isolation was valid for both of the cell suspensions in buffered saline and the artificially contaminated chicken meat samples. The presence of rRNA could be correlated with cellular viability, following inactivation of the bacteria by heating, in inoculated chicken meat samples but not in RNase-free cell suspensions.

RRNA stability in heat-killed and UV-irradiated enterotoxigenic Staphylococcus aureus and Escherichia coli O157:H7.

Differentiation of viable cells from nonviable cells is of considerable importance in the development of methods to detect foodborne pathogens. To study the suitability of 16S rRNA as an indicator of cell viability in nucleic acid-based detection assays, we examined rRNA stability in two representative foodborne pathogens, Escherichia coli O157:H7 and enterotoxigenic Staphylococcus aureus, which were inactivated by extreme heat, moderate heat, and UV irradiation. Cell death under all conditions was confirmed by a failure to grow in brain heart infusion broth after incubation for 48 h at 37 degrees C. rRNA stability was monitored by a Northern blot analysis, and detection was evaluated by using reverse transcription (RT)-PCR performed with two primer sets (which produced 325- and 1, 400-bp amplicons). rRNA of neither pathogen was detected by Northern blot analysis and RT-PCR after cells were killed by autoclaving at 121 degrees C for 15 min. In contrast, intact rRNA of both pathogens were detected by Northern blotting and could be amplified by RT-PCR up to 48 h after cells were killed by heat treatment at 80 degrees C and UV irradiation at 254 nm. rRNA was a suitable target molecule for monitoring bacterial viability under extreme heat conditions, but the presence of rRNA was not correlated with viability following moderate heat inactivation or UV irradiation of cells.

A High Density of rRNA in the Generative Cells and Sperm Cells of Pollen Grains of Five Angiosperm Species.

To examine whether cytoplasmic rRNA are present at high density in the cytoplasm of generative and sperm cells (generative/sperm cells) in pollen grains of various plant species, in situ hybridization of 18S/25S rRNA was performed in thin sections of samples embedded in Technovit 7100 resin. The five plant species studied were Lilium longiflorum, Pharbitis nil, Silene latifolia, Pelargonium zonale and Plumbago auriculata. The timing and mode of pollen mitosis II (PM II) and the distribution of organelle nucleoids in generative/sperm cells differ in these species. In situ hybridization revealed that the density of rRNA was the same as or higher in generative/sperm cells than in vegetative cells, suggesting that the presence of rRNA in the generative/sperm cells at high density may be a general characteristic of mature pollen grains of various plant species.

Activation of protease-constitutive recA proteins of Escherichia coli by rRNA and tRNA

The RecA proteins of the unusually strong protease-constitutive mutants recA1202 and recA1211 can use RNA in addition to single-stranded DNA (ssDNA) as a cofactor in the cleavage of the LexA repressor in vitro. In the presence of rRNA or tRNA, the effectiveness of these proteins decreased in the order RecA1202 greater than RecA1211 much greater than RecA+, which is also the order of their in vivo constitutive protease activities. The effectiveness of rRNA was comparable to that of ssDNA in the cleavage of the LexA repressor by either mutant protease. Although all the common nucleoside triphosphates can act as positive effectors for LexA cleavage by the two mutant proteins in the presence of ssDNA (W. B. Wang, M. Sassanfar, I. Tessman, J. W. Roberts, and E. S. Tessman, J. Bacteriol. 170:4816-4822, 1988), only dATP, ATP, and ATP-gamma-S were effective in the presence of RNA. Our results explain more fully why certain recA mutants have high constitutive protease activities in vivo.

Presence of rRNA in the heavy bodies of sea urchin eggs: An in situ hybridization study with the electron microscope

Experimental conditions have been found, in which the presence of rRNA can be demonstrated by in situ hybridization at the electron microscope level in the heavy bodies of sea urchin eggs. The specificity of hybridization has been controlled by ribonuclease digestion and by competition experiments with unlabelled rRNA.

Association of nascent ribosomal ribonucleic acid with polyribosomes in Escherichia coli.

Sedimentation distributions of rapidly labeled ribonucleic acid (RNA) in extracts prepared from cells labeled for 60 s or less with [3H]uridine showed that the immature ribosome precursor particles themselves have an RNA precursor which sediments with polyribosomes and 70s ribosomes. In extracts prepared from cultures labeled for 30 s, the fraction of labeled RNA which did not sediment with polyribosomes was much less than the fraction which was ribosomal RNA (rRNA). Rapidly labeled RNA associated with polyribosomes had a nucleotide base composition consistent with the presence of rRNA, and was shown by RNA-deoxyribonucleic acid hybridization to contain 30 to 40% rRNA. Nascent rRNA is thus associated with polyribosomes during its transcription. The sedimentation distribution of rapidly labeled 16s and 23s rRNA showed that these molecules are separated during transcription and associated with different polyribosomes.

On the relationship between the streptomycin and ribosomal RNA loci

1. Ribosomal RNA (rRNA) extracted from a prototroph, streptomycin-resistant strain of Bacillus subtilis was annealed with homologous DNA. The formation of the hybrid and the saturation of most available DNA sites were controlled by the nitrocellulose filter technique. 2. The hybridization mixture was used to transform a streptomycin sensitive strain of B. subtilis requiring tryptophan and histidine. 3. The extent of DNA renaturation was followed by determining the recovery of transforming activity for the tryptophan marker, which is known to map apart from the rRNA locus. At low DNA concentrations, where renaturation was scarce, and at high DNA concentrations, where renaturation was relatively high, no difference was observed between the two markers for the recovery of their biological activity. The recovery of the transforming efficiency for streptomycin resistance and tryptophan independence was shown to be approximately the same and unaffected by the presence of rRNA in the annealing mixture. 4. These findings are only consistent with the hypothesis that the genes for rRNA and streptomycin are in different portions of the DNA. 5. Furthermore, the hybrid was fractionated from the bulk of the DNA by equilibrium density-gradient centrifugation in CsCl. The transforming activities for both streptomycin resistance and tryptophan independence were found to band in the DNA peak and in a position distinct from the hybrid one, thus confirming the non-identity of the two loci.