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We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells.

The nucleoprotein filament formed by the RecA protein of Escherichia coli on single-stranded DNA catalyzes the hybridization of RNA transcripts with single-stranded DNA sequences at 37 degrees C, in vitro. RecA protein rapidly promotes hybridization, even when noncomplementary RNA is in a millionfold nucleotide excess over hybridizing RNA, and in a thousandfold nucleotide excess over hybridizing single-stranded DNA. Heterologous double-stranded DNA and RecA-coated noncomplementary single-stranded DNA are also poor competitors of RNA transcripts produced in vitro. Since large excesses of noncomplementary RNA fail to inhibit sharply the hybridization reaction by RecA protein under mild, non-degradative conditions, the reaction may be useful in the identification and isolation of transcripts produced in vivo.

The increased level of IL-1 mRNA was discovered in neutrophils of patients suffering from pollinosis for a long time and responding poorly to specific immunotherapy. Moreover, neutrophils from patients with exacerbation of pollinosis responded to stimuli less than cells from healthy donors. In remission, the activated neutrophils from pollinosis patients and healthy donors had the same levels of IL-1b mRNA. Tumor necrosis factor alpha expression was unchanged in neutrophils of pollinosis patients. Changes in the status of cells from patients were detected with a simple procedure of dot hybridization. Peripheral blood cells were used as available material. This allows one to hope that the approach can be used for analyzing the patients' immune status in choosing the treatment course as well as for controlling the treatment effect.

The distribution of mRNA expression for three types of voltage gated neuronal sodium-channels was studied in the rat brain at different developmental stages (embryonal day E18, postnatal day P5 and adult). With the in-situ hybridization technique, using synthetic DNA-oligomer probes, pronounced regional and temporal variations in the expression levels of the different channel subtypes could be detected. In comparison with types I and III, sodium channel II mRNA was the most abundant subtype at all developmental stages. Maximal expression of sodium channel II mRNA was seen at P5 in virtually all parts of the grey matter, except for the cerebellum. In adult rat brain in contrast, sodium channel II mRNA levels were maximal in the granular layer of the cerebellum, whereas in all other regions expression had decreased to roughly 50% of postnatal levels. Na channel I expression was virtually absent at E18 and showed highest levels at P5, with maxima in the caudate nucleus and hippocampus. In the adult brain, expression of Na-channel I was nearly absent in the neocortex, but well detectable in the cerebellum and, at lower levels in the striatum and thalamus. Sodium channel III was mainly expressed at the embryonal stage and showed a decrease to very low levels with little regional preferences in the adult.

We studied distribution of mRNA for nuclear protooncogene c-myc and nuclear protein P-53 in mature oocytes and embryos of Xenopus laevis from the stage of fertilization up to the stage of hatching by in situ hybridization with histological sections. mRNA for c-myc was present in all cells of the embryo at all studied developmental stages. Between the stage of fertilization and up to the late blastula, mRNA concentration for c-myc decreased progressively in all embryonic cells. During gastrulation a local increase in the concentration of this messenger was found in dorsal mesoderm and ectoderm. At the stage of neurula increased concentration of mRNA for c-myc was observed in all cells of the embryo but the hybridization signal increased particularly distinctly in cells of the neural tube. In studies of P-53 mRNA distribution hybridization signal was detected only in brain cells after stage 20 of development (after closure of the neural folds) and up to the stage of hatching.

In situ hybridization histochemistry technology was developed for future application to neuro-otologic research. This method allowed the detection of cellular mRNA in tissue sections from the temporal bone or brainstem after cRNA/mRNA hybridization. To produce specific cRNA, single-stranded 35S-labeled cRNA (complimentary to target mRNA) is transcribed from commercially available plasmid vectors. These vectors contain promotor sequences for specific synthesis of RNA, and polylinker regions that will accept cloned DNA inserts for virtually any target nucleic acid sequence of interest. The protocol used in this research was optimized for studies that included concomitant immunohistochemical evaluation. The combination of in situ hybridization and immunohistochemistry provides the only method to correlate molecular information (gene expression) with biochemical or molecular markers, such as peptides or proteins (mRNA translation products) on individual cells in the temporal bone or brainstem. Using these techniques, we examined the distribution of the neuropeptide calcitonin gene-related peptide in rat temporal bone and brainstem sections using calcitonin gene-related peptide (CGRP) antisera and CGRP cRNA probes. We used in situ hybridization histochemistry with a cRNA probe complementary to the 3'-end noncoding sequence of the alpha CGRP mRNA and immunohistochemistry with a polyclonal antibody to the (TYR)CGRP23-37 to study the distribution of CGRP mRNA and CGRP-like immunoreactivity in the central and peripheral facial nerve. Numerous motoneuron cell bodies in the facial nucleus and accessory seventh nucleus and cell bodies in the gustatory geniculate ganglion were found to contain CGRP mRNA and the CGRP peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of 1,25(OH)2D3 and dexamethasone on cellular proliferation and gene expression of the HTLV-I-infected T-cell line, KH-2, established from a patient with adult T-cell leukemia, endemic in the south-west Japanese islands and the Caribbean, were examined. KH-2 cells are integrated by HTLV-I proviral DNA and expressed mRNA for c-myc, IL-2 receptor alpha-chain (IL-2R alpha), and T-cell receptor beta-chain (TCR beta) while it did not express IL-2 mRNA. 1,25(OH)2D3 and dexamethasone did not suppress the mRNA levels of HTLV-I, IL-2R alpha or TCR beta but reduced the c-myc mRNA level. The reduction of c-myc mRNA level was marked in 1,25(OH)2D3-treated cells but relatively weak in dexamethasone-treated cells. This inhibitory effect of the steroid hormones correlated with the inhibition of KH-2 cell proliferation.