Morphology and current-voltage characteristics of nanostructured pentacene thin films probed by atomic force microscopy.

Atomic force microscopy was used to study the growth modes (on SiO2, MoS2, and Au substrates) and the current-voltage (I-V) characteristics of organic semiconductor pentacene. Pentacene films grow on SiO2 substrate in a layer-by-layer manner with full coverage at an average thickness of 20 A and have the highest degree of molecular ordering with large dendritic grains among the pentacene films deposited on the three different substrates. Films grown on MoS2 substrate reveal two different growth modes, snowflake-like growth and granular growth, both of which seem to compete with each other. On the other hand, films deposited on Au substrate show granular structure for thinner coverages (no crystal structure) and dendritic growth for higher coverages (crystal structure). I-V measurements were performed with a platinum tip on a pentacene film deposited on a Au substrate. The I-V curves on pentacene film reveal symmetric tunneling type character. The field dependence of the current indicates that the main transport mechanism at high field intensities is hopping (Poole-Frenkel effect). From these measurements, we have estimated a field lowering coefficient of 9.77 x 10(-6) V-1/2 m1/2 and an ideality factor of 18 for pentacene.

A common core RNP structure shared between the small nucleoar box C/D RNPs and the spliceosomal U4 snRNP.

The box C/D snoRNAs function in directing 2'-O-methylation and/or as chaperones in the processing of ribosomal RNA. We show here that Snu13p (15.5 kD in human), a component of the U4/U6.U5 tri-snRNP, is also associated with the box C/D snoRNAs. Indeed, genetic depletion of Snu13p in yeast leads to a major defect in RNA metabolism. The box C/D motif can be folded into a stem-internal loop-stem structure, almost identical to the 15.5 kD binding site in the U4 snRNA. Consistent with this, the box C/D motif binds Snu13p/ 15.5 kD in vitro. The similarities in structure and function observed between the U4 snRNP (chaperone for U6) and the box C/D snoRNPs raises the interesting possibility that these particles may have evolved from a common ancestral RNP.

Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure.

The eukaryotic nucleolus contains a large number of small nucleolar RNAs (snoRNAs) that are involved in preribosomal RNA (pre-rRNA) processing. The H box/ACA-motif (H/ACA) class of snoRNAs has recently been demonstrated to function as guide RNAs targeting specific uridines in the pre-rRNA for pseudouridine (psi) synthesis. To characterize the protein components of this class of snoRNPs, we have purified the snR42 and snR30 snoRNP complexes by anti-m3G-immunoaffinity and Mono-Q chromatography of Saccharomyces cerevisiae extracts. Sequence analysis of the individual polypeptides demonstrated that the three proteins Gar1p, Nhp2p, and Cbf5p are common to both the snR30 and snR42 complexes. Nhp2p is a highly basic protein that belongs to a family of putative RNA-binding proteins. Cbf5p has recently been demonstrated to be involved in ribosome biogenesis and also shows striking homology with known prokaryotic psi synthases. The presence of Cbf5p, a putative psi synthase in each H/ACA snoRNP suggests that this class of RNPs functions as individual modification enzymes. Immunoprecipitation studies using either anti-Cbf5p antibodies or a hemagglutinin-tagged Nhp2p demonstrated that both proteins are associated with all H/ACA-motif snoRNPs. In vivo depletion of Nhp2p results in a reduction in the steady-state levels of all H/ACA snoRNAs. Electron microscopy of purified snR42 and snR30 particles revealed that these two snoRNPs possess a similar bipartite structure that we propose to be a major structural determining principle for all H/ACA snoRNPs.

Processing of vertebrate box C/D small nucleolar RNAs in plant cells.

The recent isolation of a number of plant box C/D small nucleolar (sno)RNAs demonstrates the conservation in plants of sequence and structural elements of processed box C/D snoRNAs. Boxes C and D, and terminal inverted repeats are known to be essential for accumulation and processing in vertebrates and yeast. Processing of vertebrate box C/D snoRNAs was examined by expression of various mouse hsc70 intron 5-U14 constructs in tobacco protoplasts. Full-length U14 and internally deleted U14 accumulated in the plant cells. Human U3 and U8 fragments, consistent with processing to internal box C/C' sequences, also accumulated in the plant cells. The similarity of processing behaviour of the vertebrate box C/D constructs in tobacco protoplasts and Xenopus oocytes suggests the mechanism of processing, involving recognition and association of proteins, is conserved in plants.

In vitro assembly of the mouse U14 snoRNP core complex and identification of a 65-kDa box C/D-binding protein.

The eukaryotic nucleolus contains a diverse population of small nucleolar RNAs (snoRNAs) that have been categorized into two major families based on evolutionarily conserved sequence elements. U14 snoRNA is a member of the larger, box C/D snoRNA family and possesses nucleotide box C and D consensus sequences. In previous studies, we have defined a U14 box C/D core motif that is essential for intronic U14 snoRNA processing. These studies also revealed that nuclear proteins that recognize boxes C/D are required. We have now established an in vitro U14 snoRNP assembly system to characterize protein binding. Electrophoretic mobility-shift analysis demonstrated that all the sequences and structures of the box C/D core motif required for U14 processing are also necessary for protein binding and snoRNP assembly. These required elements include a base paired 5',3' terminal stem and the phylogenetically conserved nucleotides of boxes C and D. The ability of other box C/D snoRNAs to compete for protein binding demonstrated that the box C/D core motif-binding proteins are common to this family of snoRNAs. UV crosslinking of nuclear proteins bound to the U14 core motif identified a 65-kDa mouse snoRNP protein that requires boxes C and D for binding. Two additional core motif proteins of 55 and 50 kDa were also identified by biochemical fractionation of the in vitro-assembled U14 snoRNP complex. Thus, the U14 snoRNP core complex is a multiprotein particle whose assembly requires nucleotide boxes C and D.

Identification of specific nucleotide sequences and structural elements required for intronic U14 snoRNA processing.

Vertebrate U14 snoRNAs are encoded within hsc70 pre-mRNA introns and U14 biosynthesis occurs via an intron-processing pathway. We have shown previously that essential processing signals are located in the termini of the mature U14 molecule and replacement of included boxes C or D with oligo C disrupts snoRNA synthesis. The experiments detailed here now define the specific nucleotide sequences and structures of the U14 termini that are essential for intronic snoRNA processing. Mutagenesis studies demonstrated that a 5', 3'-terminal stem of at least three contiguous base pairs is required. A specific helix sequence is not necessary and this stem may be extended to as many as 15 base pairs without affecting U14 processing. The spatial positioning of boxes C and D with respect to the terminal stem is also important. Detailed analysis of boxes C and D revealed that both consensus sequences possess essential nucleotides. Some, but not all, of these critical nucleotides correspond to those required for the stable accumulation of nonintronic yeast U14 snoRNA. The presence of box C and D consensus sequences flanking a terminal stem in many snoRNA species indicates the importance of this "terminal core motif" for snoRNA processing.

Coating of human decay accelerating factor (hDAF) onto medical devices to improve biocompatibility.

In passing blood through an artificial circulatory system, the blood is exposed to surfaces that result in activation of the complement system. The consequences of the activation of complement can be extremely serious for the patient ranging from mild discomfort to respiratory distress and even anaphylaxis. An entirely novel approach was to express recombinant GPI anchored human decay accelerating factor (hDAF) using the baculovirus system and then coat the recombinant protein onto the surfaces of these materials to reduce complement activation. Expression of hDAF in Sf9 cells was shown by ELISA, FACS analysis, and Western blot. Functional activity was tested by CH50 assay. For the coating experiments a small scale model of a cardiovascular bypass circuit constructed from COBE tubing was used. hDAF was either coated onto the circuit using adsorption or covalently linked via the photoreactive crosslinker, p-azidobenzoyl hydrazide. After coating, heparinised human blood was pumped around the circuit and samples were collected into EDTA collection tubes at different time points. Complement activation was measured using a Quidel C3a-des-arg EIA. The photolinked circuits gave a reduction in C3a production of 20-50%, compared to 10-20% seen with an absorbed hDAF circuit. Furthermore, the inhibition of complement was seen over the whole time scale of the photolinked circuit, 60-90 min, whilst in the adsorbed circuit inhibition was not seen to a significant degree after 60 min. The time scale of a standard cardiac bypass is 45-90 min, therefore, the photolinked circuit results are encouraging, as significant inhibition of complement activation is seen within this time frame.

Elements essential for processing intronic U14 snoRNA are located at the termini of the mature snoRNA sequence and include conserved nucleotide boxes C and D.

Essential elements for intronic U14 processing have been analyzed by microinjecting various mutant hsc70/Ul4 pre-mRNA precursors into Xenopus oocyte nuclei. Initial truncation experiments revealed that elements sufficient for U14 processing are located within the mature snoRNA sequence itself. Subsequent deletions within the U14 coding region demonstrated that only the terminal regions of the folded U14 molecule containing con- served nucleotide boxes C and D are required for processing. Mutagenesis of either box C or box D completely blocked U14 processing. The importance of boxes C and D was confirmed with the excision of appropriately sized U3 and U8 fragments containing boxes C and D from an hsc7O pre-mRNA intron. Competition studies indicate that a trans-acting factor (protein?) is binding this terminal motif and is essential for U14 processing. Competition studies also revealed that this factor is common to both intronic and non-intronic snoRNAs possessing nucleotide boxes C and D. Immunoprecipitation of full-length and internally deleted U14 snoRNA molecules demonstrated that the terminal region containing boxes C and D does not bind fibrillarin. Collectively, our results indicate that a trans-acting factor (different from fibrillarin) binds to the box C- and D-containing terminal motif of U14 snoRNA, thereby stabilizing the intronic snoRNA sequence in an RNP complex during processing.

Functional redundancy of promoter elements ensures efficient transcription of the human 7SK gene in vivo.

Deletion and mutation studies of the human 7SK gene transfected into HeLa cells have identified three functional regions of the promoter corresponding to the TATA box at -25, the proximal sequence element (PSE) between -49 and -65 and the distal sequence element (DSE) between -243 and -210. These elements show sequence homology to equivalent regions in other snRNA genes and are functionally analogous. Unlike the DSEs of many snRNA genes however, the 7SK DSE does not contain a consensus binding site for the transcription factor Oct-1 but rather, contains two non-consensus Oct-1 binding sites that can function independently of one another to enhance transcription. Unusually, the 7SK PSE can retain function even after extensive mutation and removal of the conserved TGACC of the PSE has little effect in the context of the whole promoter. However, the same mutation abolishes transcription in the absence of the DSE suggesting that protein/protein interactions between DSE and PSE binding factors can compensate for a mutant PSE. Mutation of the 7SK TATA box allows snRNA type transcription by RNA polymerase II to occur and this is enhanced by the DSE, indicating that both the DSE and the PSE can also function with pol II. In addition, mutation of the TATA box does not abolish pol III dependent transcription, suggesting that other sequence elements may also play a role in the determination of polymerase specificity. Although the human 7SK gene is transcribed efficiently in Xenopus oocytes, analysis of the 7SK wild-type gene and mutants in Xenopus oocytes gives significantly different results from the analysis in HeLa cells indicating that the recognition of functional elements is not the same in the two systems.

Calcium and the generation of plant form.

The involvement of mechanical signals (tension and compression) in the determination of the form of living organisms has been speculated upon for many years. These mechanical signals (both environmental and those generated within the plant itself) have significant effects on plant development and thus morphology. Plants respond to externally applied mechanical signals (touch and wind) by an immediate elevation of cytosolic calcium concentration ([Ca2+]eyt) in stimulated cells. This response requires the movement of plant tissues to cause tension and compression. Some of the more longer-term responses to mechanical signals, e.g. TCH gene expression and reduction in hypocotyl growth, show a calcium-dependency. It seems likely, therefore, that the effects of mechanical signals on plant development are mediated by the second messenger, calcium. This raises the exciting possibility that this simple ion plays a central role in the determination of plant form itself.

Free calcium transients in chemotactic and non-chemotactic strains of Escherichia coli determined by using recombinant aequorin.

Intracellular Ca2+ has been previously implicated in the chemotactic response of Escherichia coli. However, no correlative measurements of intracellular free Ca2+ have been made during bacterial chemotaxis, essential if this is to be established. In order to monitor internal free Ca2+ in E. coli during challenge with chemotactic agents, the Ca(2+)-activated photoprotein aequorin was expressed in a chemotactic strain (AB1157) and a non-chemotactic strain [BL21(DE3)] of E. coli. Repellents were found to cause an increase (50-150 nM) in intracellular free Ca2+, whereas attractants caused a small but consistent decrease in intracellular free Ca2+. These data are in agreement with the proposed model that an increase in intracellular free Ca2+ causes tumbling. The effect of increasing external Ca2+ on the regulation of intracellular free Ca2+ in both strains was monitored by using aequorin. The resting level of free Ca2+ in E. coli (AB1157) was found to be 100 nM, which agrees with previous data [Gangola and Rosen (1987) J. Biol. Chem. 262, 12570-12574]. As these results also show differences in the regulation of intracellular free Ca2+ between the two strains in the presence of high external Ca2+ concentrations, this may have implications for the effect of high-Ca2+ environments on E. coli.

Requirement of the C-terminal proline residue for stability of the Ca(2+)-activated photoprotein aequorin.

cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2 h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca(2+)-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine.

The Xenopus U7 snRNA-encoding gene has an unusually compact structure.

A subclone containing a single Xenopus borealis U7 snRNA-encoding gene has been microinjected into X. laevis oocyte nuclei to examine its expression using [32P]GTP as an in vivo label. Only two U7 snRNA bands were detected after incubation, and subsequent fractionation of the oocyte showed that only the larger transcript is present in the nucleus. The sequence of this functional U7 gene shows that, in addition to the coding region, it contains, in the appropriate locations, the 3'-box and proximal sequence element (PSE) which are typical of Pol II-transcribed snRNA genes. Surprisingly, the Xenopus U7 gene contains two adjacent octamer-binding motifs located only 12 and 24 bp upstream from the PSE, instead of the usual location around 150-200 bp upstream. No other cis-acting elements appear to be present. A 5' deletion analysis shows that the transcription level of this U7 gene remains constant if sequences upstream of the two octamer motifs are removed, yet is undetectable when an additional 34 bp containing both octamers and the PSE are removed. This confirms that the Xenopus U7 gene is the most compact snRNA-encoding gene isolated to date. A comparison of U7 sequences shows there is a much greater conservation in the 5' half of the molecule, which contains sequences that base-pair with target pre-mRNA, than in the 3' half which can form a single stem-loop structure that varies in size.

Kremens v. Bartley: the case for the state.

The authors, co-counsel for the state of Pennsylvania in the child-commitment case to be heard this fall by the Supreme Court, outline the background of the case and present the state's arguments for preserving the rights of parents to commit their children to mental institutions. They warn of the potential psychological harm to both family and child in pitting one against the other in a formal commitment hearing. They also content that the lower-court ruling that granted due-process rights to children causes more problems than it solves. The decision, for example, effectively abolishes the state's program of respite care for mentally retarded youths.