Altered recognition mutants of the response regulator PhoB: A new genetic strategy for studying protein–protein interactions

Two-component regulatory systems require highly specific interactions between histidine kinase (transmitter) and response regulator (receiver) proteins. We have developed a novel genetic strategy that is based on tightly regulated synthesis of a given protein to identify domains and residues of an interacting protein that are critical for interactions between them. Using a reporter strain synthesizing the nonpartner kinase VanS under tight arabinose control and carrying a promoter-lacZ fusion activated by phospho-PhoB, we isolated altered recognition (AR) mutants of PhoB showing enhanced activation (phosphorylation) by VanS as arabinose-dependent Lac+ mutants. Changes in the PhoBAR mutants cluster in a “patch” near the proposed helix 4 of PhoB based on the CheY crystal structure (a homolog of the PhoB receiver domain) providing further evidence that helix 4 lies in the kinase-regulator interface. Based on the CheY structure, one mutant has an additional change in a region that may propagate a conformational change to helix 4. The overall genetic strategy described here may also be useful for studying interactions of other components of the vancomycin resistance and Pi signal transduction pathways, other two-component regulatory systems, and other interacting proteins. Conditionally replicative oriRR6Kγ attP “genome targeting” suicide plasmids carrying mutagenized phoB coding regions were integrated into the chromosome of a reporter strain to create mutant libraries; plasmids encoding mutant PhoB proteins were subsequently retrieved by P1-Int-Xis cloning. Finally, the use of similar genome targeting plasmids and P1-Int-Xis cloning should be generally useful for constructing genomic libraries from a wide array of organisms.

Similar DNA recognition properties of alternatively spliced Drosophila POU factors

The POU-IV or “Brn-3” class of POU-domain transcription factors is represented in Drosophila by I-POU and twin-of-I-POU, alternative splice products of the I-POU gene. I-POU has been previously reported to inhibit DNA binding by the POU-III class factor drifter/Cf1a via the formation of heterodimeric complexes. Here we report that expression of the I-POU/tI-POU message is maximal late in the embryonic phase of Drosophila development, and I-POU is the preferred splice variant. Although I-POU lacks two basic amino acid residues in the POU-homeodomain found in tI-POU and Brn-3.0, these three POU-IV class proteins exhibit very similar DNA-binding specificity. In contrast to previously published reports, the results presented here show no effect of I-POU on DNA binding by drifter, and no evidence for I-POU/drifter dimerization. These results suggest that the I-POU/tI-POU gene products function by transcriptional mechanisms similar to those of the homologous POU-IV class factors expressed in other species, not by a unique inhibitory mechanism.

Using electroactive substrates to pattern the attachment of two different cell populations

This report describes the development of an electroactive mask that permits the patterning of two different cell populations to a single substrate. This mask is based on a self-assembled monolayer of alkanethiolates on gold that could be switched from a state that prevents the attachment of cells to a state that promotes the integrin-mediated attachment of cells. Monolayers were patterned into regions having this electroactive monolayer and a second set of regions that were adhesive. After Swiss 3T3 fibroblasts had attached to the adhesive regions of this substrate, the second set of regions was activated electrically to permit the attachment of a second population of fibroblast cells. This method provides a general strategy for patterning the attachment of multiple cell types and will be important for studying heterotypic cell-cell interactions.

Glucocorticoid and progestin receptors are differently involved in the cooperation with a structural element of the mouse mammary tumor virus promoter.

We have previously characterized a regulatory element located between -294 and -200 within the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). This element termed AA element cooperates with the glucocorticoid response elements (GREs) for glucocorticoid activation. Here we show that in a MMTV LTR wild type context, the deletion of this element significantly reduces both glucocorticoid and progestin activation of the promoter. Deletion of the two most distal GREs forces the glucocorticoid receptor (GR) and the progestin receptor (PR) to bind the same response elements and results in a dramatic decrease in the inducibility of the MMTV promoter by the two hormones. The simultaneous deletion of the two distal GREs and of the AA element abolishes completely the glucocorticoid-induced activation of the promoter. In contrast it restores a significant level of progestin-induced activation. This different effect of the double deletion on glucocorticoid- and progestin-induced MMTV promoter activation is not cell specific because it is also observed, and is even stronger, when either GR or PR is expressed in the same cell line (NIH 3T3). This is the first description of a mutated MMTV promoter that, although retaining GREs, is activated by progestins and not by glucocorticoids. This suggests a different functional cooperation between protein(s) interacting with the AA element and GR or PR. Cotransfections with constructs containing wild-type or mutated MMTV LTR with either PR lacking its C-terminal domain or GR/PR chimeras in which the N-terminal domains have been exchanged demonstrate that the N-terminal domains of the receptors specify the different behavior of GR and PR regarding the AA element.

Arabidopsis MBP1 gene encodes a conserved ubiquitin recognition component of the 26S proteasome.

Multiubiquitin chain attachment is a key step leading to the selective degradation of abnormal polypeptides and many important regulatory proteins by the eukaryotic 26S proteasome. However, the mechanism by which the 26S complex recognizes this posttranslational modification is unknown. Using synthetic multiubiquitin chains to probe an expression library for interacting proteins, we have isolated an Arabidopsis cDNA, designated MBP1, that encodes a 41-kDa acidic protein exhibiting high affinity for chains, especially those containing four or more ubiquitins. Based on similar physical and immunological properties, multiubiquitin binding affinities, and peptide sequence, MBP1 is homologous to subunit 5a of the human 26S proteasome. Structurally related proteins also exist in yeast, Caenorhabditis, and other plant species. Given their binding properties, association with the 26S proteasome, and widespread distribution, MBP1, S5a, and related proteins likely function as essential ubiquitin recognition components of the 26S proteasome.

The Role of Passage Topic Knowledge in Typical and Poor Comprehenders' Recall

This dissertation examines the role of topic knowledge (TK) in comprehension among typical readers and those with Specifically Poor Comprehension (SPC), i.e., those who demonstrate deficits in understanding what they read despite adequate decoding. Previous studies of poor comprehension have focused on weaknesses in specific skills, such as word decoding and inferencing ability, but this dissertation examined a different factor: whether deficits in availability and use of TK underlie poor comprehension. It is well known that TK tends to facilitate comprehension among typical readers, but its interaction with working memory and word decoding is unclear, particularly among participants with deficits in these skills. Across several passages, we found that SPCs do in fact have less TK to assist their interpretation of a text. However, we found no evidence that deficits in working memory or word decoding ability make it difficult for children to benefit from their TK when they have it. Instead, children across the skill spectrum are able to draw upon TK to assist their interpretation of a passage. Because TK is difficult to assess and studies vary in methodology, another goal of this dissertation was to compare two methods for measuring it. Both approaches score responses to a concept question to assess TK, but in the first, a human rater assigns a score whereas in the second, a computer algorithm, Latent Semantic Analysis (LSA; Landauer & Dumais, 1997) assigns a score. We found similar results across both methods of assessing TK, suggesting that a continuous measure is not appreciably more sensitive to variations in knowledge than discrete human ratings. This study contributes to our understanding of how best to measure TK, the factors that moderate its relationship with recall, and its role in poor comprehension. The findings suggest that teaching practices that focus on expanding TK are likely to improve comprehension across readers with a variety of abilities.

What are the effects of the EU budget: Driving force or drop in the ocean? CEPS Special Report No. 86/April 2014

The study presents an overview of the impact of the main investment tools of the EU budget. The focus is on the increasing role of the financial instruments, which are fundamentally changing the budget’s nature and reach. Through these instruments, the EU can invest more efficiently in more areas and mobilise a multiple of funds. The EU budget has the potential to influence the European economy much more than its modest size in terms of GDP may suggest.

What are the effects of the EU budget: Driving force or drop in the ocean? CEPS Special Report No. 86, April 2014

The study presents an overview of the impact of the main investment tools of the EU budget. The focus is on the increasing role of the financial instruments, which are fundamentally changing the budget’s nature and reach. Through these instruments, the EU can invest more efficiently in more areas and mobilise a multiple of funds. The EU budget has the potential to influence the European economy much more than its modest size in terms of GDP may suggest.

In a state of necessity. How has Orban changed Hungary. OSW Point of View Number 41, April 2014

Viktor Orban’s sweeping victory in the 2010 election ensured his party, Fidesz, a constitutional two-thirds majority in parliament. The party took over the rule of the country from the discredited political left when Hungary was plunged in political and economic crisis. Claiming that the circumstances were unusual and that it had a strong electoral mandate, Fidesz introduced radical changes in the country and thus challenged the previous economic and political order. These changes have led to an unprecedented concentration of power and provoked a discussion on the limits of democracy and the rule of law in the European Union. The state’s economic role has strengthened. The Orban government has been unable to overcome economic stagnation but it has managed to stabilise Hungary’s budget situation, which needs to deal with the high debt. Hungary’s relations with most partners in the EU and NATO have cooled due to controversial moves made by its government. As regards foreign policy and economic co-operation, Orban has granted high priority to the ‘Eastern opening’, where Russia has assumed the leading role.