Three approaches were used to study hybridization of complementary oligodeoxynucleotides by nonradiative fluorescence resonance energy transfer. (i) Fluorescein (donor) and rhodamine (acceptor) were covalently attached to the 5' ends of complementary oligodeoxynucleotides of various lengths. Upon hybridization of the complementary oligodeoxynucleotides, energy transfer was detected by both a decrease in fluorescein emission intensity and an enhancement in rhodamine emission intensity. In all cases, fluorescein emission intensity was quenched by about 26% in the presence of unlabeled complement. Transfer efficiency at 50C decreased from 0.50 to 0.22 to 0.04 as the distance between donor and acceptor fluorophores in the hybrid increased from 8 to 12 to 16 nucleotides. Modeling of these hybrids as double helices showed that transfer efficiency decreased as the reciprocal of the sixth power of the donor-acceptor separation R, as predicted by theory with a corresponding Ro of 49 A. (u) Fluorescence resonance energy transfer was used to study hybridization of two fluorophore-labeled oligonucleotides to a longer, unlabeled oligodeoxynucleotide. Two 12-mers were prepared that were complementary to two adjacent sequences separated by four bases on a 29-mer. The adjacent 5' and 3' ends of the two 12-mers labeled with fluorescein and rhodamine exhibited a transfer efficiency of -0.60 at 50C when they both hybridized to the unlabeled 29-mer. (Wi) An intercalating dye, acridine orange, was used as the donor fluorophore to a single rhodamine covalently attached to the 5' end of one oligodeoxynucleotide in a 12-base-pair hybrid. Under these conditions, the transfer efficiency was =0.47 at 50C. These results establish that fluorescence modulation and nonradiative fluorescence resonance energy transfer can detect nucleic acid hybridization in solution. These techniques, with further development, may also prove useful for detecting and quantifying nucleic acid hybridization in living cells.In this paper we describe how fluorescently labeled oligodeoxynucleotides (ODNTs) and the process of nonradiative fluorescence resonance energy transfer (FRET) can be used to study nucleic acid hybridization. When two fluorophores whose excitation and emission spectra overlap are in sufficiently close proximity, the excited-state energy of the donor molecule is transferred by a resonance dipole-induced dipole interaction to the neighboring acceptor fluorophore. The results are a decrease in donor lifetime, a quenching of donor fluorescence, an enhancement of acceptor fluorescence intensity, and a depolarization of fluorescence intensity. The efficiency of energy transfer, Et, falls off rapidly with the distance between donor and acceptor molecule, R, and is expressed as Et = 1/[1 + (R/R0)6], [1] where Ro is a value that depends upon the overlap integral of the donor emission spectrum and the acceptor excitation spectrum, the index of refraction, the quantum yield of the donor, and the orientation of the donor emission and the acce...

Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by f luorescence correlation spectroscopy (FCS) and f luorescence recovery after photobleaching (FRAP). FCS revealed that a large fraction of both intranuclear oligo(dT) (43%) and oligo(dA) (77%) moves rapidly with a diffusion coefficient of 4 ؋ 10 ؊7 cm 2 ͞s. Interestingly, this rate of intranuclear oligo movement is similar to their diffusion rates measured in aqueous solution. In addition, we detected a large fraction (45%) of the intranuclear oligo(dT), but not oligo(dA), diffusing at slower rates (<1 ؋ 10 ؊7 cm 2 ͞s). The amount of this slower-moving oligo(dT) was greatly reduced if the oligo(dT) was prehybridized in solution with (unlabeled) oligo(dA) prior to introduction to cells, presumably because the oligo(dT) was then unavailable for subsequent hybridization to endogenous poly(A) RNA. The FCS-measured diffusion rate for much of the slower oligo(dT) population approximated the diffusion rate in aqueous solution of oligo(dT) hybridized to a large polyadenylated RNA (1.0 ؋ 10 ؊7 cm 2 ͞s). Moreover, this intranuclear movement rate falls within the range of calculated diffusion rates for an average-sized heterogeneous nuclear ribonucleoprotein particle in aqueous solution. A subfraction of oligo(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients obtained from FRAP experiments were in agreement with the FCS data. These results demonstrate that oligos can move about within the nucleus at rates comparable to those in aqueous solution and further suggest that this is true for large ribonucleoprotein complexes as well.An understanding of the physical environment inside the cell nucleus is central to a coherent view of gene expression. It is important to know how the viscosity and molecular diffusion rates in the nucleus of a living cell compare with experimental conditions in vitro, where interactions between nucleic acids and proteins are studied at high dilution in aqueous solution. For example, it is not clear whether ribonucleoprotein (RNP) complexes can diffuse freely about the nucleus, impeded only by locally high concentrations of macromolecules or, alternatively, are bound or compartmentalized in such a way as to constrain their motion. Some observations suggest that premRNA transcripts are tethered to elements of the transcriptional, splicing, and͞or polyadenylation machinery (1-3), and it has been proposed that processed mRNAs make their way out of the nucleus by molecular diffusion (4, 5). However, mediated processes have not been ruled out, and the functional relationships between RNA export and nuclear structure remain unclear (6).Recently, Politz et al. (7) characterized nucleic acid uptake and hybridization in living cells by in situ reverse transcription and found that fluorescently labeled oligo(dT) can be taken up by living cells and form hybrid...

The COP9/signalosome (CSN) is known to remove the stimulatory NEDD8 modification from cullins. The activity of the fission yeast cullins Pcu1p and Pcu3p is dramatically stimulated when retrieved from csn mutants but inhibited by purified CSN. This inhibition is independent of cullin deneddylation but mediated by the CSN-associated deubiquitylating enzyme Ubp12p, which forms a complex with Pcu3p in a CSN-dependent manner. In ubp12 mutants, as in csn mutants, Pcu3p activity is stimulated. CSN is required for efficient targeting of Ubp12p to the nucleus, where both cullins reside. Finally, the CSN/Ubp12p pathway maintains the stability of the Pcu1p-associated substrate-specific adaptor protein Pop1p. We propose that CSN/Ubp12p-mediated deubiquitylation creates an environment for the safe de novo assembly of cullin complexes by counteracting the autocatalytic destruction of adaptor proteins.

We have examined the phase partition preferences of the even chain length (n = 10-22) diacyl-3'3'-indocarbo-cyanine iodides (Cn diI) incorporated in disaturated lecithin (PC) vesicles. Two parameters were used to determine this phase preference: (i) the direction of shift of the phase transition temperature (Tm) induced by the dyes and (ii) the self-quenching of fluorescence due to aggregation in the gel phase of those dyes which preferentially partition into the fluid. Dyes that lower Tm preferentially partition into the fluid phase; those that raise Tm preferentially partition into the gel. By these criteria in dimyristoyl-PC, C10 diI and C12 diI preferentially partition into the fluid phase, C14 diI and C16 diI show no preferential partition, C18 diI preferentially partitions into the gel, and C20 diI and C22 diI preferentially partition into the fluid. In dipalmitoyl-PC, the pattern of preference is identical with that observed in dimyristoyl-PC, only shifted to longer chain length diI's by two carbons. Diffusion measurements by fluorescence photobleaching recovery of these dyes in gel-phase multilayers showed tham all to be immobile, D less than 10(-10) cm2/s, while in fluid-phase multilayers thay all had diffusion coefficients of D approximately 10(-8) cm2/s independent of chain length. In mixed-phase multilayers, however, each Cn diI showed mobile fraction which reflected its phase-partition preference.

MRI was used to target and evaluate the tissue effects of focused ultrasound ablation on tumors implanted in the skeletal muscle of rabbits in vivo. First, MRI was used to localize the tumors and plan the ultrasound therapy. Second, temperature-sensitive phase-difference images were acquired to monitor the location of the ultrasound focus and to estimate the effects of temperature rise. After the treatment, the spatial and temporal temperature profiles for defining boundaries of tissue coagulation were calculated. Finally, these boundaries were compared to T2-weighted and contrast-enhanced T1-weighted images obtained immediately after therapy. The results indicate that using MRI for planning and evaluating focused ultrasound surgery is feasible. We showed a linear relationship between applied power and shifts in the proton resonant frequency. Fluctuations in the location of the focus about the target location were on the order of the resolution of the MR images. The temperature rise and lesion size varied significantly. Regions of tissue coagulation calculated from MR data correlated well with post-therapy imaging.