Subnuclear localization of the active variant surface glycoprotein gene expression site in Trypanosoma brucei

In Trypanosoma brucei, transcription by RNA polymerase II and 5′ capping of messenger RNA are uncoupled: a capped spliced leader is trans spliced to every RNA. This decoupling makes it possible to have protein-coding gene transcription driven by RNA polymerase I. Indeed, indirect evidence suggests that the genes for the major surface glycoproteins, variant surface glycoproteins (VSGs) in bloodstream-form trypanosomes, are transcribed by RNA polymerase I. In a single trypanosome, only one VSG expression site is maximally transcribed at any one time, and it has been speculated that transcription takes place at a unique site within the nucleus, perhaps in the nucleolus. We tested this by using fluorescence in situ hybridization. With probes that cover about 50 kb of the active 221 expression site, we detected nuclear transcripts of this site in a single fluorescent spot, which did not colocalize with the nucleolus. Analysis of marker gene-tagged active expression site DNA by fluorescent DNA in situ hybridization confirmed the absence of association with the nucleolus. Even an active expression site in which the promoter had been replaced by an rDNA promoter did not colocalize with the nulceolus. As expected, marker genes inserted in the rDNA array predominantly colocalize with the nucleolus, whereas the tubulin gene arrays do not. We conclude that transcription of the active VSG expression site does not take place in the nucleolus.

Think globally, translate locally: What mitotic spindles and neuronal synapses have in common

Early metazoan development is programmed by maternal mRNAs inherited by the egg at the time of fertilization. These mRNAs are not translated en masse at any one time or at any one place, but instead their expression is regulated both temporally and spatially. Recent evidence has shown that one maternal mRNA, cyclin B1, is concentrated on mitotic spindles in the early Xenopus embryo, where its translation is controlled by CPEB (cytoplasmic polyadenylation element binding protein), a sequence-specific RNA binding protein. Disruption of the spindle-associated translation of this mRNA results in a morphologically abnormal mitotic apparatus and inhibited cell division. Mammalian neurons, particularly in the synapto-dendritic compartment, also contain localized mRNAs such as that encoding α-CaMKII. Here, synaptic activation drives local translation, an event that is involved in synaptic plasticity and possibly long-term memory storage. Synaptic translation of α-CaMKII mRNA also appears to be controlled by CPEB, which is enriched in the postsynaptic density. Therefore, CPEB-controlled local translation may influence such seemingly disparate processes as the cell cycle and synaptic plasticity.

Functional cell surface expression of the anion transport domain of human red cell band 3 (AE1) in the yeast Saccharomyces cerevisiae.

We expressed the 52-kDa integral membrane domain (B3mem) of the human erythrocyte anion transporter (band 3; AE1) in a protease-deficient strain of the yeast Saccharomyces cerevisiae under the control of the inducible GAL10-CYC1 promoter. Immunoblots of total protein from transformed yeast cells confirmed that the B3mem polypeptide was overexpressed shortly after induction with galactose. Cell surface expression of the functional anion transporter was detected by using a simple transport assay to measure stilbene disulfonate-inhibitable chloride influx into intact yeast cells. The B3mem polypeptide was recycled and degraded by the cells with a half-life of approximately 1-3 hr, which led to a steady-state level of expression in exponentially growing cultures. Our data suggest that 5-10% of total B3mem is functionally active at the cell surface at any one time and that overexpression of this anion transport protein does not interfere with cell growth or survival. This is one of only a few reports of the functional expression of a plasma membrane transport protein in the plasma membrane of yeast cells and to our knowledge is the first report of red cell band 3-mediated anion transport at the plasma membrane of cDNA-transformed cells. The cell surface expression system we describe will provide a simple means for future study of the functional properties of band 3 by using site-directed mutagenesis.

The 5' coding region of Paramecium surface antigen genes controls mutually exclusive transcription.

Paramecium tetraurelia stock 51 can express at least 11 different types of surface antigens, yet only a single type is expressed on the surface of an individual cell at any one time. The differential expression of stock 51 type A and B surface antigen genes (51A and 51B) is regulated at the level of transcription. Previously, we reported that nucleotide sequences upstream of position -26 (relative to the start of translation) in the 51A and 51B surface antigen genes are necessary for transcriptional activity but are not sufficient to direct differential transcriptional control. In this report we demonstrate that at least some of the critical elements necessary for differential transcription of the 51A and 51B genes lie within the 5' coding region. A hybrid gene that contains 51B upstream sequences (-475 to +1) attached to the ATG start codon of 51A is not cotranscribed with the 51B gene. In contrast, further substitution with 51B sequences (-1647 to +885) allows the chimeric gene to be coexpressed with 51B. A different hybrid gene containing a substitution of 51B sequence from -26 to +885 in the 51A gene is also coexpressed with 51B, revealing that the critical elements within the coding region of 51B do not require 51B upstream sequences for their effect. Coinjection of the 51A gene with the chimeric gene that contains 51B up to +885 showed that the same sequences that allow coexpression with 51B prevent cotranscription with 51A. Together, these results demonstrate that a region downstream of the transcriptional start site between nucleotide positions +1 and +885 (relative to translational start) is necessary to control differential transcriptional activity.

Gene targeting approaches to complex genetic diseases: atherosclerosis and essential hypertension.

Gene targeting allows precise, predetermined changes to be made in a chosen gene in the mouse genome. To date, targeting has been used most often for generation of animals completely lacking the product of a gene of interest. The resulting "knockout" mice have confirmed some hypotheses, have upset others, but have rarely been uninformative. Models of several human genetic diseases have been produced by targeting--including Gaucher disease, cystic fibrosis, and the fragile X syndrome. These diseases are primarily determined by defects in single genes, and their modes of inheritance are well understood. When the disease under study has a complex etiology with multiple genetic and environmental components, the generation of animal models becomes more difficult but no less valuable. The problems associated with dissecting out the individual genetic factors also increases substantially and the distinction between causation and correlation is often difficult. To prove causation in a complex system requires rigorous adherence to the principle that the experiments must allow detection of the effects of changing only a single variable at one time. Gene targeting experiments, when properly designed, can test the effects of a precise genetic change completely free from the effects of differences in any other genes (linked or unlinked to the test gene). They therefore allow proofs of causation.

Mutational analysis of the conserved region 2 site of adenovirus E1A and its effect on binding to the retinoblastoma gene product: use of the "double-tagging" assay.

We have explored the feasibility of using a "double-tagging" assay for assessing which amino acids of a protein are responsible for its binding to another protein. We have chosen the adenovirus E1A-retinoblastoma gene product (pRB) proteins for a model system, and we focused on the high-affinity conserved region 2 of adenovirus E1A (CR2). We used site-specific mutagenesis to generate a mutant E1A gene with a lysine instead of an aspartic acid at position 121 within the CR2 site. We demonstrated that this mutant exhibited little binding to pRB by the double-tagging assay. We also have shown that this lack of binding is not due to any significant decrease in the level of expression of the beta-galactosidase-E1A fusion protein. We then created a "library" of phage expressing beta-galactosidase-E1A fusion proteins with a variety of different mutations within CR2. This library of E1A mutations was used in a double-tagging screening to identify mutant clones that bound to pRB. Three classes of phage were identified: the vast majority of clones were negative and exhibited no binding to pRB. Approximately 1 in 10,000 bound to pRB but not to E1A ("true positives"). A variable number of clones appeared to bind equally well to both pRB and E1A ("false positives"). The DNA sequence of 10 true positive clones yielded the following consensus sequence: DLTCXEX, where X = any amino acid. The recovery of positive clones with only one of several allowed amino acids at each position suggests that most, if not all, of the conserved residues play an important role in binding to pRB. On the other hand, the DNA sequence of the negative clones appeared random. These results are consistent with those obtained from other sources. These data suggest that a double-tagging assay can be employed for determining which amino acids of a protein are important for specifying its interaction with another protein if the complex forms within bacteria. This assay is rapid and up to 1 x 10(6) mutations can be screened at one time.

Aprendizagem de substantivos e adjetivos por meio de leitura compartilhada para crianças com desenvolvimento típico e com Síndrome de Down

A leitura compartilhada de livros para crianças é uma atividade que tem sido estudada como forma de ensino incidental de vocabulário, que envolve, dentre outros processos, o responder por exclusão. O objetivo do presente trabalho foi investigar a ocorrência de aprendizagem de relações entre estímulos visuais (figuras) com seus respectivos estímulos auditivos (palavras) a partir de diferentes condições de leitura compartilhada de livros para crianças com Síndrome de Down (SD) e com desenvolvimento típico (DT). Para a pesquisa foram desenvolvidos dois estudos. No Estudo 1, participaram seis crianças com SD com seis a sete anos, e seis crianças com DT com três a quatro anos (amostras pareadas em função do nível de vocabulário). Foi utilizado um livro de história produzido pela pesquisadora, no qual havia dois substantivos e dois adjetivos desconhecidos (estímulos visuais S1, S2, A1, A2), apresentados uma única vez na história. Esse livro foi lido para cada criança duas vezes em sequência por sessão e em cada sessão foi realizada uma condição de leitura diferente. Foram apresentadas três condições de leitura e cada criança passou por todas, mas em diferentes ordens (contrabalanceamento). Na Condição 1, o livro foi lido para a criança sem intervenções. Na Condição 2, o livro foi lido para a criança e ela tinha que repetir o nome dos estímulos desconhecidos. Na Condição 3, o livro foi lido e foram realizadas perguntas relacionadas aos estímulos-alvo. Ao final de cada sessão foram realizadas sondas de aprendizagem (sondas de emparelhamento ao modelo e nomeação), e após uma semana da última sessão foi aplicada uma sonda de manutenção e uma de generalização. As crianças com DT apresentaram maior número de acertos que as com SD, e os acertos foram mais relacionados ao estímulo S1. As crianças não aprenderam a relação nome-cor. A análise dos resultados sugeriu que o número de estímulos-alvo era excessivo e com apresentações insuficientes no livro. No Estudo 2 participaram seis crianças com DT de 3 a 4 anos e seis crianças com SD, de 5 a 8 anos. O procedimento utilizado no Estudo 2 foi semelhante ao primeiro com as seguintes alterações no livro: utilização de apenas duas relações-alvo (um substantivo-alvo e um adjetivo-alvo - S2 e A3), cada uma sendo apresentada três vezes ao longo da história, em figuras que possibilitavam o responder por exclusão. Também foi acrescentada uma tentativa de exclusão nas sondas de aprendizagem. Nesse estudo, todas as crianças com DT conseguiram selecionar e nomear estímulo S2 e duas mostraram indícios de aprendizagem do estímulo A3. As crianças com SD apresentaram um menor número de acertos nas sondas de emparelhamento, mas apresentaram algumas nomeações corretas, o que não foi observado no Estudo 1. Os dados sugerem que as mudanças realizadas no livro melhoram o desempenho das crianças com DT, mas não o das crianças com SD. Não foram encontradas diferenças entre as condições de leituras nos dois estudos. No entanto, são necessários estudos adicionais para avaliar essas diferentes condições e as variáveis envolvidas na aprendizagem de palavras a partir da leitura compartilhada de livro.

Asking For Help: Vulnerability within Consultative Assessment A Couples Therapy Example.

In many different ways psychologists are challenged to evaluate their work and be accountable when therapy is not progressing. One time honored approach to addressing the lack of progress is to seek formal psychological assessment to gain a better diagnostic understanding as well as treatment recommendations. The perspective on the purpose of the assessment seems to have changed however. Historically, the presumption was that there was some kind of pathology that the clinician was not seeing, where currently the perspective seems to be shifting more towards a focus on what might not be working in the therapeutic process. This shift in focus has led to new styles of collaborative and consultative assessment such as Therapeutic Assessment. Therapeutic Assessment was developed by Stephen Finn and has shown a great deal of promise helping clients and therapists gain valuable insight and move toward meaningful life changes (1998). Amidst the promise of consultative assessment also lie potential challenges for the referring clinician. This paper will explore the nature of consultative relationships in the field of mental health and the current thoughts about the advantages and challenges of these relationships. Using a case study format, it will discuss and illustrate issues of professional vulnerability that can arise during these relationships and postulate that a similar vulnerability could be present during a consultative assessment. Recommendations are made for maximizing the success of this type of assessment, and ideas for future research are reviewed. Through the lens of a couples Therapeutic Assessment case, this paper will examine what it is about this unique kind of professional relationship that garners such powerful potential and pitfalls.

The Significance of the Disputed Territory in Bechuanaland 1887-1893

British Imperial policy in Southern Africa in the last three decades of the nineteenth century oscillated between two extremes. It began in the early 1870's with Lord Kimberley's attempt to effect confederation as a means of devolving Imperial responsibility and expenditure. It ended in 1899 with Britain's active intervention against the Boers. For most of the remaining years of those decades a middle course was adopted while the British Government struggled to reconcile its diverse political interests. Strategy, supremacy, economy, humanitarianism, and recognition of colonial aspirations were all at one time or another, in varying degrees, motivating forces behind Imperial policy. Many historians have pointed out how incompatible many of these ends were and how the attempt to pursue them all at once almost inevitably ended in at least one of them being sacrificed on the way. This study focusses on a relatively minor problem over a period of about seven years. It attempts to show how the British Government tried to reconcile, in this case, the predominant motives of economy and supremacy. The problem of the Disputed Territory now seems like a small fish in a big ocean because non the great hopes and fears that it raised were ever realized. But the anticlimactic nature of the outcome of events should not be allowed to conceal two important points: first, that the problem loomed large at the time in the eyes of the Imperial Government; and second, that in the case of its policy towards the Disputed Territory, the Government gained a greater degree of success in trying to reconcile seemingly incompatible ends than it did in many other instances.

Minty and the cavalry. A history of cavalry campaigns in the western armies.

Includes rosters of officers of the 4th Mich., 7th Pa., 4th U.S. and 9th Pa. cavalry. A dozen or more other regiments were, at one time or another attached to the brigade.

Pastor bonus.

Merged with: Oberrheinisches Pastoralblatt; and: Ostdeutsches Pastoralblatt, to form a one-time issue of: Pastoral Blatt.

The building of the Horace H. Rackham School of Graduate Studies, University of Michigan, Ann Arbor, Mich.

Sign in front of building during construction. Smith, Hinchman & Grylls, architects. W.B. Wood Co., construction. Image was folded at one time.

The audiences of 5 magazines: size, characteristics, possessions.

"Supplement. Readers per dollar figures based on publisher's 1963 one-time black & white page rates": (20 p.) inserted.

Sleep Patterns and Parents’ Experience in Caring for Young Children Newly Diagnosed with Juvenile Idiopathic Arthritis

Thesis (Ph.D.)--University of Washington, 2016-05

PLACE AND URBAN EXPERIENCE IN THE SUBURBS: MICROSOFT’S CAMPUS LOCATION AND ITS LONG-TERM COMPETITIVENESS

Thesis (Master's)--University of Washington, 2016-06