RNA splice site selection: evidence for a 5' leads to 3' scanning model.

Human G gamma-globin genes containing tandem duplications of the donor (5') or acceptor (3') RNA splice sites of the second intervening sequence were constructed in order to ascertain the directionality of RNA splice site selection. These genes were introduced into cultured monkey cells, and their transcripts were analyzed. Transcripts of these duplication variants were spliced only at the proximal copy of the duplicated splice sites. These data are consistent with a 5' leads to 3' model of splice site selection.

Sequence requirements in different steps of the pre-mRNA splicing reaction: analysis by the RNA modification-exclusion technique.

The stepwise assembly of splicing complexes and the subsequent splicing reaction were analyzed by the RNA modification-exclusion technique, which generates the equivalent of a complete set of point mutations in a single reaction. We found that although the sequences surrounding the 5' splice site, the branch point, and the 3' splice site, including the 3' AG, were required for presplicing complex formation, modified nucleotides at these positions were not completely excluded. The same sequences were required for splicing complex formation; however, modified nucleotides in these sequences were excluded to a much greater extent.

In vitro splicing pathways of pre-mRNAs containing multiple intervening sequences?

We analyzed the in vitro splicing pathways of three multi-intervening-sequence (IVS) pre-mRNAs: human beta-globin, which contains two IVSs (K. M. Lang, V. L. van Santen, and R. A. Spritz, EMBO J. 4:1991-1996, 1985); rat alpha-lactalbumin, which contains three IVSs; and murine interleukin-3, which contains four IVSs. We found that there are highly preferred pathways of IVS removal from these multi-IVS pre-mRNAs in vitro. The three IVSs of rat alpha-lactalbumin pre-mRNA were excised sequentially from 5' to 3'; in most molecules, IVS1 was removed first, followed by IVS2 and finally by IVS3. The splicing pathway of interleukin-3 pre-mRNA in vitro was more complex. The four IVSs were excised in a highly preferred temporal order, but the order was not strictly sequential or directional. In most molecules, IVS1 and IVS4 were removed first, either simultaneously or in rapid succession. Subsequently, IVS2 was excised, followed by IVS3. The observed splicing pathways apparently resulted from differences in lag times and maximum excision rates of the different IVSs. We detected no exon skipping during splicing of these transcripts in vitro. These observations have implication for proposed models of splice site selection.

Cloning specific complete polyadenylylated 3'-terminal cDNA segments.

A simple method for extending partial cDNA clones of specific mRNA molecules is described. The method combines plasmid-primed first-strand cDNA synthesis [Okayama and Berg, Mol. Cell. Biol. 2 (1982) 161-170] with second-strand synthesis by primer extension. This approach selects for the synthesis of specific double-stranded cDNAs with complete 3' termini and poly(A) tails. We have used this method to extend preexisting cDNA clones to isolate 3' segments of human beta- and G gamma-globin cDNAs. These recombinants are particularly useful for the construction of templates for in vitro transcription of synthetic polyadenylylated mRNAs and pre-mRNAs. In addition, nucleotide sequence analysis of the cloned gamma-globin cDNA segment defined a previously unreported sequence polymorphism in the 3' untranslated region.

A general model of the effects of sleep medications on the risk and cost of motor vehicle accidents and its application to France.

BACKGROUND: Although various prescription drugs may be equally effective in promoting sleep, some may lead to substantial impairment in psychomotor functioning and an increased risk of motor vehicle accidents. OBJECTIVE: To develop a general model to evaluate the potential effects of sleep medications on motor vehicle accidents and costs, and apply the model to the French setting. METHODS: Impairment in driving performance, as evaluated by randomised controlled open-road studies using the standard deviation of a vehicle's lateral position (SDLP), a measure of weaving, was expressed in terms of equivalent blood alcohol (ethanol) concentration (BAC). Epidemiological data were then used to relate BAC to the excess risk of motor vehicle accidents. Although a non-impairing medication would not increase risk, a medication that produces mild impairment in driving performance (a change of 2.5 cm in SDLP, equivalent to 0.05% BAC) would increase motor vehicle accident risk by 25%. One that leads to moderate impairment (an SDLP change of 4.5 cm, equivalent to 0.08% BAC) would roughly double this risk, and a severely impairing medication (an SDLP change of 7 cm, equivalent to 0.12% BAC) would result in up to a 5-fold increase in motor vehicle accident risk. For application to the French setting, a hypothetical cohort of 100,000 adult drivers with insomnia was assumed to be treated for 14 days either with zaleplon 10 mg, a new sleep medication that has been shown not to significantly impair driving performance, or zopiclone 7.5 mg, which has been shown to cause moderate impairment. RESULTS: Compared with zaleplon, use of zopiclone over 14 days in France would be expected to result in 503 excess accidents per 100,000 drivers at an additional cost of 158 French francs (31 US dollars) per person (1996 values). CONCLUSIONS: Our model illustrates the extent to which non-impairing sleep medications could reduce the burden posed by motor vehicle accidents. Our model is designed to be general, and thus can serve as the basis for similar investigations.

Coincidence of checkerboard charge order and antinodal state decoherence in strongly underdoped superconducting Bi2Sr2CaCu2O8 + delta).

The doping dependence of nanoscale electronic structure in superconducting Bi(2)Sr(2)CaCu(2)O(8 + delta) is studied by scanning tunneling microscopy. At all dopings, the low energy density-of-states modulations are analyzed according to a simple model of quasiparticle interference and found to be consistent with Fermi-arc superconductivity. The superconducting coherence peaks, ubiquitous in near-optimal tunneling spectra, are destroyed with strong underdoping and a new spectral type appears. Exclusively in regions exhibiting this new spectrum, we find local "checkerboard" charge ordering of high energy states, with a wave vector of Q = (+/- 2pi/4.5a(0),0); (0, +/- 2pi/4.5a(0)) +/- 15%. Surprisingly, this spatial ordering of high energy states coexists harmoniously with the low energy Bogoliubov quasiparticle states.

Cleavage and polyadenylation factor CPF specifically interacts with the pre-mRNA 3' processing signal AAUAAA.

Cleavage and polyadenylation factor (CPF) is required for the cleavage as well as for the subsequent polyadenylation reaction during 3' processing of messenger RNA precursors. Here, we have investigated the interaction of CPF and poly(A) polymerase with short RNA substrates. CPF activates poly(A) polymerase to elongate RNA primers carrying the canonical hexamer recognition signal AAUAAA. CPF specifically binds to such RNA as shown by gel mobility shift assays and competition experiments. Upon binding of CPF, two polypeptides of 35 kDa and 160 kDa can be covalently crosslinked to the RNA by irradiation with UV light. These polypeptides may correspond to the smallest and the largest subunit contained in purified CPF fractions. In addition, chemical modification-exclusion experiments demonstrate that CPF interacts directly with the AAUAAA recognition signal in the RNA. The entire hexamer signal is involved in binding of CPF since modification of any of its bases interferes with complex formation.

The two intervening sequences of human beta- and gamma-globin pre-mRNAs are excised in a preferred temporal order in vitro.

Splicing of complete synthetic human beta- and gamma-globin mRNA precursors (pre-mRNAs) follows a preferred pathway in vitro. In almost all molecules, excision of the first intervening sequence (IVS) is completed before excision of the second IVS initiates. However, in a very small fraction of molecules the order of IVS excision is reversed.

Regulation of ureteric bud outgrowth by Pax2-dependent activation of the glial derived neurotrophic factor gene.

The outgrowth of the ureteric bud from the posterior nephric duct epithelium and the subsequent invasion of the bud into the metanephric mesenchyme initiate the process of metanephric, or adult kidney, development. The receptor tyrosine kinase RET and glial cell-derived neurotrophic factor (GDNF) form a signaling complex that is essential for ureteric bud growth and branching morphogenesis of the ureteric bud epithelium. We demonstrate that Pax2 expression in the metanephric mesenchyme is independent of induction by the ureteric bud. Pax2 mutants are deficient in ureteric bud outgrowth and do not express GDNF in the uninduced metanephric mesenchyme. Furthermore, Pax2 mutant mesenchyme is unresponsive to induction by wild-type heterologous inducers. In normal embryos, GDNF is sufficient to induce ectopic ureter buds in the posterior nephric duct, a process inhibited by bone morphogenetic protein 4. However, GDNF replacement in organ culture is not sufficient to stimulate ureteric bud outgrowth from Pax2 mutant nephric ducts, indicating additional defects in the nephric duct epithelium of Pax2 mutants. Pax2 can activate expression of GDNF in cell lines derived from embryonic metanephroi. Furthermore, Pax2 protein can bind to upstream regulatory elements within the GDNF promoter region and can transactivate expression of reporter genes. Thus, activation of GDNF by Pax2 coordinates the position and outgrowth of the ureteric bud such that kidney development can begin.

A four unit cell periodic pattern of quasi-particle states surrounding vortex cores in Bi2Sr2CaCu2O8+delta.

Scanning tunneling microscopy is used to image the additional quasi-particle states generated by quantized vortices in the high critical temperature superconductor Bi2Sr2CaCu2O8+delta. They exhibit a copper-oxygen bond-oriented "checkerboard" pattern, with four unit cell (4a0) periodicity and a approximately 30 angstrom decay length. These electronic modulations may be related to the magnetic field-induced, 8a0 periodic, spin density modulations with decay length of approximately 70 angstroms recently discovered in La1.84Sr0.16CuO4. The proposed explanation is a spin density wave localized surrounding each vortex core. General theoretical principles predict that, in the cuprates, a localized spin modulation of wavelength lambda should be associated with a corresponding electronic modulation of wavelength lambda/2, in good agreement with our observations.

Imaging quasiparticle interference in Bi2Sr2CaCu2O8+delta.

Scanning tunneling spectroscopy of the high-Tc superconductor Bi2Sr2CaCu2O8+delta reveals weak, incommensurate, spatial modulations in the tunneling conductance. Images of these energy-dependent modulations are Fourier analyzed to yield the dispersion of their wavevectors. Comparison of the dispersions with photoemission spectroscopy data indicates that quasiparticle interference, due to elastic scattering between characteristic regions of momentum-space, provides a consistent explanation for the conductance modulations, without appeal to another order parameter. These results refocus attention on quasiparticle scattering processes as potential explanations for other incommensurate phenomena in the cuprates. The momentum-resolved tunneling spectroscopy demonstrated here also provides a new technique with which to study quasiparticles in correlated materials.

Imaging the granular structure of high-Tc superconductivity in underdoped Bi2Sr2CaCu2O8+delta.

Granular superconductivity occurs when microscopic superconducting grains are separated by non-superconducting regions; Josephson tunnelling between the grains establishes the macroscopic superconducting state. Although crystals of the copper oxide high-transition-temperature (high-Tc) superconductors are not granular in a structural sense, theory suggests that at low levels of hole doping the holes can become concentrated at certain locations resulting in hole-rich superconducting domains. Granular superconductivity arising from tunnelling between such domains would represent a new view of the underdoped copper oxide superconductors. Here we report scanning tunnelling microscope studies of underdoped Bi2Sr2CaCu2O8+delta that reveal an apparent segregation of the electronic structure into superconducting domains that are approximately 3 nm in size (and local energy gap <50 meV), located in an electronically distinct background. We used scattering resonances at Ni impurity atoms as 'markers' for local superconductivity; no Ni resonances were detected in any region where the local energy gap Delta > 50 +/- 2.5 meV. These observations suggest that underdoped Bi2Sr2CaCu2O8+delta is a mixture of two different short-range electronic orders with the long-range characteristics of a granular superconductor.

Interplay of magnetism and high-Tc superconductivity at individual Ni impurity atoms in Bi2Sr2CaCu2O8+delta.

Magnetic interactions and magnetic impurities are destructive to superconductivity in conventional superconductors. By contrast, in some unconventional macroscopic quantum systems (such as superfluid 3He and superconducting UGe2), the superconductivity (or superfluidity) is actually mediated by magnetic interactions. A magnetic mechanism has also been proposed for high-temperature superconductivity. Within this context, the fact that magnetic Ni impurity atoms have a weaker effect on superconductivity than non-magnetic Zn atoms in the high-Tc superconductors has been put forward as evidence supporting a magnetic mechanism. Here we use scanning tunnelling microscopy to determine directly the influence of individual Ni atoms on the local electronic structure of Bi2Sr2CaCu2O8+delta. At each Ni site we observe two d-wave impurity states of apparently opposite spin polarization, whose existence indicates that Ni retains a magnetic moment in the superconducting state. However, analysis of the impurity-state energies shows that quasiparticle scattering at Ni is predominantly non-magnetic. Furthermore, we show that the superconducting energy gap and correlations are unimpaired at Ni. This is in strong contrast to the effects of non-magnetic Zn impurities, which locally destroy superconductivity. These results are consistent with predictions for impurity atom phenomena derived from a magnetic mechanism.

Decoherence in josephson phase qubits from junction resonators.

Although Josephson junction qubits show great promise for quantum computing, the origin of dominant decoherence mechanisms remains unknown. Improving the operation of a Josephson junction based phase qubit has revealed microscopic two-level systems or resonators within the tunnel barrier that cause decoherence. We report spectroscopic data that show a level splitting characteristic of coupling between a two-state qubit and a two-level system. Furthermore, we show Rabi oscillations whose "coherence amplitude" is significantly degraded by the presence of these spurious microwave resonators. The discovery of these resonators impacts the future of Josephson qubits as well as existing Josephson technologies.

Nanoscale one-dimensional scattering resonances in the CuO chains of YBa(2)Cu(3)O(6+x).

We present scanning tunneling spectroscopy measurements of the CuO chain plane in YBa(2)Cu(3)O(6+x), showing an approximately 25 meV gap in the local density of states (LDOS) filled by numerous intragap resonances: intense peaks in LDOS spectra associated with one-dimensional, Friedel-like oscillations. We discuss how these phenomena shed light on recent results from other probes, as well as their implications for phenomena in the superconducting CuO(2) plane.

Microscopic electronic inhomogeneity in the high-Tc superconductor Bi2Sr2CaCu2O8+x.

The parent compounds of the copper oxide high-transition-temperature (high-Tc) superconductors are unusual insulators (so-called Mott insulators). Superconductivity arises when they are 'doped' away from stoichiometry. For the compound Bi2Sr2CaCu2O8+x, doping is achieved by adding extra oxygen atoms, which introduce positive charge carriers ('holes') into the CuO2 planes where the superconductivity is believed to originate. Aside from providing the charge carriers, the role of the oxygen dopants is not well understood, nor is it clear how the charge carriers are distributed on the planes. Many models of high-Tc superconductivity accordingly assume that the introduced carriers are distributed uniformly, leading to an electronically homogeneous system as in ordinary metals. Here we report the presence of an electronic inhomogeneity in Bi2Sr2CaCu2O8+x, on the basis of observations using scanning tunnelling microscopy and spectroscopy. The inhomogeneity is manifested as spatial variations in both the local density of states spectrum and the superconducting energy gap. These variations are correlated spatially and vary on the surprisingly short length scale of approximately 14 A. Our analysis suggests that this inhomogeneity is a consequence of proximity to a Mott insulator resulting in poor screening of the charge potentials associated with the oxygen ions left in the BiO plane after doping, and is indicative of the local nature of the superconducting state.