Formation of chimeric DNA primer extension products by template switching onto an annealed downstream oligonucleotide.

Oligodeoxynucleotide sequences are described that anneal to a template downstream of a priming site. During polymerase-catalyzed extension of the primer, the extending primer shifts from the original template to a segment of the annealed oligonucleotide that acts as an alternative template. The resulting chimeric extended primer has one segment that is complementary to the template and a second segment that is complementary to the oligonucleotide. The influence of the sequence elements of the oligonucleotide and the reaction conditions on template switching have been explored. The sequence requirements for template switching are compared to those for transposon excision.

Expression of the human acute myeloid leukemia gene AML1 is regulated by two promoter regions.

The human chromosome 21 AML1 gene is expressed predominantly in the hematopoietic system. In several leukemia-associated translocations AML1 is fused to other genes and transcription of the fused regions is mediated by upstream sequences that normally regulate the expression of AML1. The 5' genomic region of AML1 was cloned and sequenced. The two 5' untranslated regions (UTRs) previously identified in AML1 cDNAs were located in this region and the distance between them was established. The distal 5' UTR maps over 7 kb upstream of the proximal one. Using primer extension with mRNA, transcription start sites were identified at two distinct sites above these 5' uTRs. Sequence analysis revealed the absence of a TATA motif and the presence of Sp1, PU.1, Oct, CRE, Myb, Ets, and Ets-like binding sites in both upstream regions. Several initiator elements (Inr) that overlap the transcription start sites were also identified. These proximal and distal upstream regions and their deletion mutants were cloned in front of a luciferase reporter gene and used in transfection assays. We demonstrate that both upstream regions function as promoters in hematopoietic (Jurkat) and nonhematopoietic (HEK) cell lines. The activity of both promoters was orientation dependent and was enhanced, in a cell-type specific manner, by a heterologous enhancer sequence. These results indicate that additional control elements, either negative or positive, regulate the tissue-specific expression of AML1.

Molecular cloning of the transcription factor TFIIB homolog from Sulfolobus shibatae.

The Archaea (archaebacteria) constitute a group of prokaryotes that are phylogenetically distinct from Eucarya (eukaryotes) and Bacteria (eubacteria). Although Archaea possess only one RNA polymerase, evidence suggests that their transcriptional apparatus is similar to that of Eucarya. For example, Archaea contain a homolog of the TATA-binding protein which interacts with the TATA-box like A-box sequence upstream of many archaeal genes. Here, we report the cloning of a Sulfolobus shibatae gene that encodes a protein (transcription factor TFB) with striking homology to the eukaryotic basal transcription factor TFIIB. We show by primer extension analysis that transcription of the S. shibatae TFB gene initiates 27 bp downstream from a consensus A-box element. Significantly, S. shibatae TFB contains an N-terminal putative metal-binding region and two imperfect direct repeats--structural features that are well conserved in eukaryotic TFIIBs. This suggests that TFB may perform analogous functions in Archaea and Eucarya. Consistent with this, we demonstrate that S. shibatae TFB promotes the binding of S. shibatae TBP to the A-box element of the Sulfolobus 16S/23S rRNA gene. Finally, we show that S. shibatae TFB is significantly more related to TFB of the archaeon Pyrococcus woesei than it is to eukaryotic TFIIBs. These data suggest that TFB arose in the common archaeal/eukaryotic ancestor and that the lineages leading to P. woesei and S. shibatae separated after the divergence of the archaeal and eukaryotic lines of descent.

DNA polymerase beta bypasses in vitro a single d(GpG)-cisplatin adduct placed on codon 13 of the HRAS gene.

We have examined the capacity of calf thymus DNA polymerases alpha, beta, delta, and epsilon to perform in vitro translesion synthesis on a substrate containing a single d(GpG)-cisplatin adduct placed on codon 13 of the human HRAS gene. We found that DNA synthesis catalyzed by DNA polymerases alpha, delta, and epsilon was blocked at the base preceding the lesion. Addition of proliferating cell nuclear antigen to DNA polymerase delta and replication protein A to DNA polymerase alpha did not restore their capacity to elongate past the adduct. On the other hand, DNA polymerase beta efficiently bypassed the cisplatin adduct. Furthermore, we observed that DNA polymerase beta was the only polymerase capable of primer extension of a 3'-OH located opposite the base preceding the lesion. Likewise, DNA polymerase beta was able to elongate the arrested replication products of the other three DNA polymerases, thus showing its capacity to successfully compete with polymerases alpha, delta, and epsilon in the stalled replication complex. Our data suggest (i) a possible mechanism enabling DNA polymerase beta to bypass a d(GpG)-cisplatin adduct in vitro and (ii) a role for this enzyme in processing DNA damage in vivo.

Identification of two flavivirus-like genomes in the GB hepatitis agent.

A subtractive PCR methodology known as representational difference analysis was used to clone specific nucleotide sequences present in the infectious plasma from a tamarin infected with the GB hepatitis agent. Eleven unique clones were identified, seven of which were examined extensively. All seven clones appeared to be derived from sequences exogenous to the genomes of humans, tamarins, Saccharomyces cerevisiae, and Escherichia coli. In addition, sequences from these clones were not detected in plasma or liver tissue of tamarins prior to their inoculation with the GB agent. These sequences were detected by reverse transcription-PCR in acute-phase plasma of tamarins inoculated with the GB agent. Probes derived from two of the seven clones detected an RNA species of > or = 8.3 kb in the liver of a GB-agent-infected tamarin by Northern blot hybridization. Sequence analysis indicated that five of the seven clones encode polypeptides that possess limited amino acid identity with the nonstructural proteins of hepatitis C virus. Extension of the sequences found in the seven clones revealed that plasma from an infected tamarin contained two RNA molecules > 9 kb long. Limited sequence identity with various isolates of hepatitis C virus and the relative positions of putative RNA helicases and RNA-dependent RNA polymerases in the predicted protein products of these molecules suggested that the GB agent contains two unique flavivirus-like genomes.

Strength of the Iberian intraplate lithosphere: Cenozoic deformations and seismicity

Previous studies about the strength of the lithosphere in the center of Iberia fail to resolve the depth of earthquakes because of the rheological uncertainties. Therefore, new contributions are considered (the crustal structure from a density model) and several parameters (tectonic regime, mantle rheology, strain rate) are checked in this paper to properly examine the role of lithospheric strength in the intraplate seismicity and the Cenozoic evolution. The strength distribution with depth, the integrated strength, the effective elastic thickness and the seismogenic thickness have been calculated by a finite element modelling of the lithosphere across the Central System mountain range and the bordering Duero and Madrid sedimentary basins. Only a dry mantle under strike-slip/extension and a strain rate of 10-15 s-1, or under extension and 10-16 s-1, causes a strong lithosphere. The integrated strength and the elastic thickness are lower in the mountain chain than in the basins. This heterogeneity has been maintained since the Cenozoic and determine the mountain uplift and the biharmonic folding of the Iberian lithosphere during the Alpine deformations. The seismogenic thickness bounds the seismic activity in the upper–middle crust, and the decreasing crustal strength from the Duero Basin towards the Madrid Basin is related to a parallel increase in Plio–Quaternary deformations and seismicity. However, elasto–plastic modelling shows that current African–Eurasian convergence is resolved elastically or ductilely, which accounts for the low seismicity recorded in this region.

The Battle of the Bulge: The Surprising Last Stand Against State Marijuana Legalization

Although marijuana possession remains a federal crime, twenty-three states now allow use of marijuana for medical purposes and four states have adopted tax-and-regulate policies permitting use and possession by those twenty-one and over. In this article, I examine recent developments regarding marijuana regulation. I show that the Obama administration, after initially sending mixed signals, has taken several steps indicating an increasingly accepting position toward marijuana law reform in states; however the current situation regarding the dual legal status of marijuana is at best an unstable equilibrium. I also focus on what might be deemed the last stand of marijuana-legalization opponents, in the form of lawsuits filed by several states, sheriffs, and private plaintiffs challenging marijuana reform in Colorado (and by extension elsewhere). This analysis offers insights for federalism scholars regarding the speed with which marijuana law reform has occurred, the positions taken by various state and federal actors, and possible collaborative federalism solutions to the current state-federal standoff.

Exception or extension? : reorienting the Peacock Room among James McNeill Whistler's interior designs

"Although famous for his paintings and etchings today, James McNeill Whistler (1834-1903) was also an important interior designer in the nineteenth-century British Aesthetic movement. Whistler‘s most famous and only extant interior design is Harmony in Blue and Gold: The Peacock Room (1876-77). It is also his most puzzling interior. Long considered an exception to the rule of Whistler‘s other interiors, the Peacock Room has often been overlooked in the few studies of the artist‘s interior designs"

Multilingual extension of a temporal expression normalizer using annotated corpora

This paper presents the automatic extension to other languages of TERSEO, a knowledge-based system for the recognition and normalization of temporal expressions originally developed for Spanish. TERSEO was first extended to English through the automatic translation of the temporal expressions. Then, an improved porting process was applied to Italian, where the automatic translation of the temporal expressions from English and from Spanish was combined with the extraction of new expressions from an Italian annotated corpus. Experimental results demonstrate how, while still adhering to the rule-based paradigm, the development of automatic rule translation procedures allowed us to minimize the effort required for porting to new languages. Relying on such procedures, and without any manual effort or previous knowledge of the target language, TERSEO recognizes and normalizes temporal expressions in Italian with good results (72% precision and 83% recall for recognition).

Application of Text Summarization techniques to the Geographical Information Retrieval task

Automatic Text Summarization has been shown to be useful for Natural Language Processing tasks such as Question Answering or Text Classification and other related fields of computer science such as Information Retrieval. Since Geographical Information Retrieval can be considered as an extension of the Information Retrieval field, the generation of summaries could be integrated into these systems by acting as an intermediate stage, with the purpose of reducing the document length. In this manner, the access time for information searching will be improved, while at the same time relevant documents will be also retrieved. Therefore, in this paper we propose the generation of two types of summaries (generic and geographical) applying several compression rates in order to evaluate their effectiveness in the Geographical Information Retrieval task. The evaluation has been carried out using GeoCLEF as evaluation framework and following an Information Retrieval perspective without considering the geo-reranking phase commonly used in these systems. Although single-document summarization has not performed well in general, the slight improvements obtained for some types of the proposed summaries, particularly for those based on geographical information, made us believe that the integration of Text Summarization with Geographical Information Retrieval may be beneficial, and consequently, the experimental set-up developed in this research work serves as a basis for further investigations in this field.

Algorithm for the detection of outliers based on the theory of rough sets

Outliers are objects that show abnormal behavior with respect to their context or that have unexpected values in some of their parameters. In decision-making processes, information quality is of the utmost importance. In specific applications, an outlying data element may represent an important deviation in a production process or a damaged sensor. Therefore, the ability to detect these elements could make the difference between making a correct and an incorrect decision. This task is complicated by the large sizes of typical databases. Due to their importance in search processes in large volumes of data, researchers pay special attention to the development of efficient outlier detection techniques. This article presents a computationally efficient algorithm for the detection of outliers in large volumes of information. This proposal is based on an extension of the mathematical framework upon which the basic theory of detection of outliers, founded on Rough Set Theory, has been constructed. From this starting point, current problems are analyzed; a detection method is proposed, along with a computational algorithm that allows the performance of outlier detection tasks with an almost-linear complexity. To illustrate its viability, the results of the application of the outlier-detection algorithm to the concrete example of a large database are presented.

On the Analytic Solutions of the Functional Equations w 1 f(a 1 z) + w 2 f(a 2 z) + ... + w n f(a n z) = 0

In this paper, it is showed that, given an integer number n ≥ 2, each zero of an exponential polynomial of the form w1az1+w2az2+⋯+wnazn, with non-null complex numbers w 1,w 2,…,w n and a 1,a 2,…,a n , produces analytic solutions of the functional equation w 1 f(a 1 z) + w 2 f(a 2 z) + ... + w n f(a n z) = 0 on certain domains of C, which represents an extension of some existing results in the literature on this functional equation for the case of positive coefficients a j and w j.

The future of the Eurozone and the role of the German Constitutional Court. Research Papers in Law, 05/2012

[Introduction.] Necessary reforms towards a deepened and increased European shaped economic, financial and budgetary policy, paraphrased with the term “fiscal union”, could possibly reach constitutional limits. In its EFSF judgment1, the German Constitutional Court, following the Lisbon judgment in which certain government tasks were determined as being part of the “constitutional identity”2, connected the budget right of the parliament via the principle of democracy to the eternity clause of Art. 79 para 3 Basic Law. A transfer of essential parts of the budget right of the German Bundestag, which would be in conflict with the German constitution, is said to exist when the determination of the nature and amount of the tax affecting the citizens is largely regulated on the supranational level and thereby deprived of the Bundestag’s right to disposition. A reform of the Economic and Monetary Union that touches the core of the budget right can, according to the German Federal Court, with regard to Art. 79 (3) of the Basic Law only be realized by way of Art. 146 of the Basic Law, thus with a new constitution given by the people that replaces the Basic Law.3

The Judicial ‘Bail Out’ of the European Stability Mechanism: Comment on the Pringle Case. Research Paper in Law 09/2013

Since the beginning of the crisis, many responses have been taken to stabilise the European markets. Pringle is the awaited judicial response of the European Court of Justice on the creation of the European Stability Mechanism (ESM), a crisis-related intergovernmental international institution which provides financial assistance to Member States in distress in the Eurozone. The judgment adopts a welcome and satisfactory approach on the establishment of the ESM. This article examines the feasibility of the ESM under the Treaty rules and in light of the Pringle judgment. For the first time, the Court was called to appraise the use of the simplified revision procedure under article 48 TEU with the introduction of a new paragraph to article 136 TFEU as well as to interpret the no bail out clause under article 125 TFEU. The final result is rather positive as the Court endorses the establishment of a stability mechanism of the ESM-kind beyond a strict reading of the Treaty rules. Pringle is the first landmark ECJ decision in which the Court has endorsed the use of new and flexible measures to guarantee financial assistance between Member States. This judgment could act as a springboard for more economic, financial and, possibly, political interconnections between Member States.

The EU-Ukraine Action Plan on Visa Liberalisation: an assessment of Ukraine's readiness. OSW Commentary No. 45, 2011-01-17

The Action Plan on visas adopted during the recent EU-Ukraine summit is a success for Ukraine. It is the first time that Kyiv has succeeded in obtaining a definition of the conditions and criteria whose fulfilment will enable Ukraine to apply for the lifting of EU visas for its citizens. Ukraine's strong point has been its political will; the lifting of this visa regime has been a priority for all Ukrainian governments since 2005. Since Viktor Yanukovych became president, Ukraine has adopted or prepared key legal acts that brought it nearer to European standards in the area of border and migration management. One of Kyiv's strengths is also its relatively well reformed and efficiently managed border service. Moreover, illegal transit migration via Ukraine is decreasing, and fewer Ukrainians are trying to enter or stay in the EU illegally. Also, Kyiv has efficiently implemented the EU-Ukraine readmission agreement. The hardest task for Ukraine will be to meet the EU’s expectations concerning values, the condition of Ukrainian democracy, and the rule of law. Corruption remains the main barrier to Ukraine's development and modernisation; the courts are weak and the judicial system inefficient. The main undertaking of the new migration service that is being formed at the moment will be to create a civil system of registration, monitoring and regulating the stays of foreign nationals. This may prove difficult, as the supervisory authority (the Ministry of the Interior) remains an unreformed, police-type bureaucratic institution. Ukraine is lagging behind countries such as Russia, Belarus and Moldova when it comes to the introduction of biometric documents. Another problem is the lack of an electronic information system on foreign nationals, visas and border crossings which would be accessible to all the relevant services and institutions. For these reasons, the complete abolition of visas seems to be a longterm perspective, especially considering that many EU countries, which themselves are faced with the problem of migrants’ integration, are rather sceptical about the further liberalisation of movement of people with their eastern neighbours. In the immediate future, if Ukraine meets some of the requirements set by the EU, it will be able to seek the extension of the visa facilitations that have been in operation since 2008.