Think tanks must think more about issues of national interest, not self-interest

US think tanks have grown into a species primarily concerned with illustrating their influence on policy-makers. Professor Donald Abelson writes that it is time for these institutions to focus on their original intention: to bring bright minds together to promote solutions to the nation’s ills

How mothers handle their feelings toward children with congenital anomalies: a study of cases seen in psychiatry department of the children's medical center

Thesis (M.S.)--Boston Universit

The subnuclear localization of tRNA ligase in yeast

Yeast tRNA ligase is an enzyme required for tRNA splicing. A study by indirect immune fluorescence shows that this enzyme is localized in the cell nucleus. At higher resolution, studies using indirect immune electron microscopy show this nuclear location to be primarily at the inner membrane of the nuclear envelope, most likely at the nuclear pore. There is a more diffuse, secondary location of ligase in a region of the nucleoplasm within 300 nm of the nuclear envelope. When the amount of ligase in the cell is increased, nuclear staining increases but staining of the nuclear envelope remains constant. This experiment indicates that there are a limited number of ligase sites at the nuclear envelope. Since the other tRNA splicing component, the endonuclease, has the characteristics of an integral membrane protein, we hypothesize that it constitutes the site for the interaction of ligase with the nuclear envelope

PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex

The 2'-5' RNA Ligase of Escherichia coli: Purification, Cloning, and Genomic Disruption

An RNA ligase previously detected in extracts of Escherichia coli is capable of joining Saccharomyces cerevisiae tRNA splicing intermediates in the absence of ATP to form a 2-5 phosphodiester linkage (Greer, C., Javor, B., and Abelson, J. (1983) Cell 33, 899-906). This enzyme specifically ligates tRNA half-molecules containing nucleoside base modifications and shows a preference among different tRNA species. In order to investigate the function of this enzyme in RNA metabolism, the ligase was purified to homogeneity from E. coli lysate utilizing chromatographic techniques and separation of proteins by SDS-polyacrylamide gel electrophoresis. A single polypeptide of approximately 20 kilodaltons exhibited RNA ligase activity. The amino terminus of this protein was sequenced, and the open reading frame (ORF) encoding it was identified by a data base search. This ORF, which encodes a novel protein with a predicted molecular mass of 19.9 kDa, was amplified from E. coli genomic DNA and cloned. ORFs coding for highly similar proteins were detected in Methanococcus jannaschii and Bacillus stearothermophilus. The chromosomal gene encoding RNA ligase in E. coli was disrupted, abolishing ligase activity in cell lysates. Cells lacking ligase activity grew normally under laboratory conditions. However, moderate overexpression of the ligase protein led to slower growth rates and a temperature-sensitive phenotype in both wild-type and RNA ligase knockout strains. The RNA ligase reaction was studied in vitro using purified enzyme and was found to be reversible, indicating that this enzyme may perform cleavage or ligation in vivo

tRNA splicing

Introns interrupt the continuity of many eukaryal genes, and therefore their removal by splicing is a crucial step in gene expression. Interestingly, even within Eukarya there are at least four splicing mechanisms. mRNA splicing in the nucleus takes place in two phosphotransfer reactions on a complex and dynamic machine, the spliceosome. This reaction is related in mechanism to the two self-splicing mechanisms for Group 1 and Group 2 introns. In fact the Group 2 introns are spliced by an identical mechanism to mRNA splicing, although there is no general requirement for either proteins or co-factors. Thus it seems likely that the Group 2 and nuclear mRNA splicing reactions have diverged from a common ancestor. tRNA genes are also interrupted by introns, but here the splicing mechanism is quite different because it is catalyzed by three enzymes, all proteins and with an intrinsic requirement for ATP hydrolysis. tRNA splicing occurs in all three major lines of descent, the Bacteria, the Archaea, and the Eukarya. In bacteria the introns are self-splicing (1-3). Until recently it was thought that the mechanisms of tRNA splicing in Eukarya and Archaea were unrelated as well. In the past year, however, it has been found that the first enzyme in the tRNA splicing pathway, the tRNA endonuclease, has been conserved in evolution since the divergence of the Eukarya and the Archaea. Surprising insights have been obtained by comparison of the structures and mechanisms of tRNA endonuclease from these two divergent lines

Identification of Five Putative Yeast RNA Helicase Genes

The RNA helicase gene family encodes a group of eight homologous proteins that share regions of sequence similarity. This group of evolutionarily conserved proteins presumably all utilize ATP (or some other nucleoside triphosphate) as an energy source for unwinding double-stranded RNA. Members of this family have been implicated in a variety of physiological functions in organisms ranging from Escherichia coli to human, such as translation initiation, mitochondrial mRNA splicing, ribosomal assembly, and germinal line cell differentiation. We have applied polymerase chain reaction technology to search for additional members of the RNA helicase family in the yeast Saccharomyces cerevisiae. Using degenerate oligonucleotide primers designed to amplify DNA fragments flanked by the highly conserved motifs V L D E A D and Y I H R I G, we have detected five putative RNA helicase genes. Northern and Southern blot analyses demonstrated that these genes are single copy and expressed in yeast. Several members of the RNA helicase family share sequence identity ranging from 49.2% to 67.2%, suggesting that they are functionally related. The discovery of such a multitude of putative RNA helicase genes in yeast suggests that RNA helicase activities are involved in a variety of fundamentally important biological processes

In Vitro Studies of the Prp9·Prp11·Prp21 Complex Indicate a Pathway for U2 Small Nuclear Ribonucleoprotein Activation

Pre-mRNA splicing takes place on a large ribonucleoprotein particle, the spliceosome which contains the five small nuclear ribonucleoproteins (snRNPs), U1, U2, U4, U5, and U6. In Saccharomyces cerevisiae the mRNA splicing factors, Prp9, Prp11, and Prp21, are necessary for addition of the U2 snRNP to the pre-mRNA in an early step of spliceosome assembly. This paper describes a study of interactions between these proteins and their role in spliceosome assembly. The proteins were expressed in Escherichia coli. Prp9 and Prp11 were purified by metal affinity chromatography. Prp21 was purified using a solubilization/renaturation protocol. We have combined these separately purified proteins and present direct evidence of a Prp9·Prp11·Prp21 protein complex that is functional in in vitro splicing assays. Characteristics of this Prp9·Prp11·Prp21 complex were further investigated using proteins synthesized in vitro. In addition, we found that Prp9, Prp11, and Prp21 influence the structure of the U2 snRNP in a manner that alters the accessibility of the branch point pairing region of the U2 snRNA to oligonucleotide-directed RNaseH cleavage. We present a model, based on the data presented here and in the accompanying paper, for a combined role of Prp9, Prp11, Prp21, and Prp5 in activating the U2 snRNP for assembly into the pre-spliceosome

Relationship between respiratory, endocrine, and cognitive-emotional factors in response to a pharmacological panicogen

Background : The cholecystokinin agonist pentagastrin has been used to study panic attacks in the laboratory and to investigate hypothalamic–pituitary–adrenal axis activity. Its mechanism of panicogenesis remains unclear. Data from other models suggest that respiratory stimulation itself may induce panic, but pentagastrin's effects on respiration are not well established. Data from another model also suggest links between respiratory and HPA axis reactivity and cognitive modulation of both. To further explore these phenomena, we added respiratory measures to a study of cognitive modulation of HPA and anxiety responses to pentagastrin. Methods : Healthy subjects received pentagastrin and placebo injections, with measurement of cortisol and subjective responses, on two different laboratory visits. They were randomly assigned to receive standard instructions or one of two versions of previously studied cognitive interventions (to either facilitate coping or increase sense of control), given before each visit. Capnograph measures of heart rate (HR), respiratory rate (RR), and end-tidal pCO 2 were obtained on 24 subjects. Results : Relative to placebo, pentagastrin induced a significant decline in pCO 2 with no change in RR. Cortisol and HR increased, as expected. Cognitive intervention reduced the hyperventilatory response to pentagastrin. Conclusions : Pentagastrin stimulates respiration, likely via increases in tidal volume. Respiratory stimulation could play a role in its panicogenic potency, though perhaps indirectly. As with HPA axis responses, higher-level brain processes may be capable of modulating pentagastrin-induced hyperventilation. This model may be useful for further study of cortical/cognitive control of interacting emotional, respiratory, and neuroendocrine sensitivities, with potential relevance to panic pathophysiology. Depression and Anxiety, 2010.  © 2010 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78296/1/20725_ftp.pd