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.

We measured the nonradiative fluorescence resonance energy transfer between 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled lipids (amine labeled phosphatidylethanolamine or acyl chain labeled phosphatidylcholine) and rhodamine labeled lipids in large unilamellar dioleoylphosphatidylcholine vesicles. Two new rhodamine labeled lipid analogues, one a derivative of monolauroylphosphatidylethanolamine and the other of sphingosylphosphorylcholine, were found to exchange through the aqueous phase between vesicle populations but not to be capable of rapid transbilayer movement between leaflets. Energy transfer from NBD to rhodamine was measured using liposomes with symmetric or asymmetric distributions of these new rhodamine labeled lipid analogues to determine the relative contributions of energy transfer between donor and acceptor fluorophores in the same (cis) and opposite (trans) leaflets. Since the characteristic R0 values for energy transfer ranged from 47 to 73 A in all cases, significant contributions from both cis and trans energy transfer were observed. Therefore, neither of these probes acts strictly as a half-bilayer quencher of NBD lipid fluorescence. The dependence of transfer efficiency on acceptor density was fitted to a theoretical treatment of energy transfer to determine the distances of closest approach for cis and trans transfer. These parameters set limits on the positions of the fluorescent groups relative to the bilayer center, 20-31 A for NBD and 31-55 A for rhodamine, and provide a basis for future use of these analogues in measurements of transbilayer distribution and transport.

Abstract. We have used the technique of fluorescence recovery after photobleaching to measure the diffusibility of the fluorescent lipid analogue, 1,1 '-dihexadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate on the morphologically distinct regions of the plasma membranes of mouse spermatozoa, and the changes in lipid diffusibility that result from in vitro hyperactivation and capacitation with bovine serum albumin.We found that, as previously observed on ram spermatozoa, lipid analogue diffusibility is regionalized on mouse spermatozoa, being fastest on the flagellum. The bovine serum albumin induced changes in diffusibility that occur with hyperactivation are also regionalized. Specifically, if we compare serum incubated in control medium, which maintains normal motility, with those hyperactivated in capacitating medium, we observe with hyperactivation an increase in lipid analogue diffusion rate in the anterior region of the head, the midpiece, and tail, and a decrease in diffusing fraction in the anterior region of the head. IN general, ejaculated mammalian spermatozoa cannot fertilize ova. They gain this capacity from contact with the fluids of the female reproductive system, in particular with the ampullary fluid in the fallopian tube (l, 9; for more recent reviews see references 2, 6, and 40). Capacitation (9) results in two dramatic changes in sperm physiology: hyperactivation and acquisition of the ability to undergo the acrosome reaction and fertilize an oocyte. The midpieces of ejaculated spermatozoa are relatively inflexible. Their flagella exhibit low amplitude symmetric beating, which results in linear swimming patterns. In contrast, the midpieces of hyperactivated spermatozoa are highly flexible. Their flagella exhibit large amplitude, often asymmetric, beating, which results in circular swimming patterns. Often hyperactivation also results in altered beat frequency. The acrosome reaction refers to a fusion and vesiculation between the sperm plasma membrane in the anterior region of the head and the underlying acrosome. This results in a release of the acrosomal vesicle contents both lytic components and adhesion proteins (for review see reference 61) involved in fertilization, and exposes the inner acrosomal membrane. In most mammals, subsequent fusion of egg and sperm plasma membranes occurs at the equatorial region of the sperm surface (7). The precise molecular mechanism of this process of capacitation remains unknown. However, capacitation results in dramatic changes in plasma membrane lipid and protein composition as well as in the lateral distribution or regionalization of these components (5,16,21,25,29,35,39,46,51). Indeed, it has been suggested that lipid redistributions that result from capacitation provide the fusogenic domains required for vesiculation during the acrosome reaction (5).In vitro capacitation can in some species be effected by incubation of spermatozoa in serum albumin (30). Once again, the precise molecular mechanism of this in vitro capacitation remains unknown. ...

It has been proposed that the high affinity nerve growth factor (NGF) receptor required for NGF response is a complex of two receptor proteins, gp75 and the tyrosine kinase TrkA, but direct biochemical or biophysical evidence has been lacking. We have previously shown using fluorescence recovery after photobleaching that gp75 is highly mobile on NGF-nonresponsive cells, but relatively immobile on NGF-responsive cells. In this report, we show that a physical interaction with TrkA causes gp75 immobilization. We found that gp75 is relatively mobile on TrkA negative nnr5 cells, a PC12 variant which is nonresponsive to NGF. In contrast, on T14 nnr5 cells (which bear a TrkA expression vector) gp75 is relatively immobile. Similarly, using baculoviruses to express gp75 and TrkA on Sf9 insect cells, we found that TrkA immobilizes gp75 molecules. The related receptor, TrkB, caused a more modest immobilization of gp75. Immobilization was found to require intact TrkA kinase and gp75 cytoplasmic domains, paralleling the requirements of high affinity binding of NGF. Analysis of gp75 diffusion coefficients indicates that mutated gp75 and TrkA molecules may form a complex, even in the absence of the ability to bind NGF with high affinity.