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Background: Murine leukemia virus (MLV) vector particles can be pseudotyped with a truncated variant of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) and selectively target gene transfer to human cells expressing both CD4 and an appropriate co-receptor. Vector transduction mimics the HIV-1 entry process and is therefore a safe tool to study HIV-1 entry. Results: Using FLY cells, which express the MLV gag and pol genes, we generated stable producer cell lines that express the HIV-1 envelope gene and a retroviral vector genome encoding the green fluorescent protein (GFP). The BH10 or 89.6 P HIV-1 Env was expressed from a bicistronic vector which allowed the rapid selection of stable cell lines. A codon-usage-optimized synthetic env gene permitted high, Rev-independent Env expression. Vectors generated by these producer cells displayed different sensitivity to entry inhibitors. Conclusion: These data illustrate that MLV/HIV-1 vectors are a valuable screening system for entry inhibitors or neutralizing antisera generated by vaccines.
Background: Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV) has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. Results: We inserted the coding sequences for green fluorescent protein (GFP) into the proline-rich region (PRR) of the ecotropic envelope protein (Env) and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP) and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. Conclusions: Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.
The 5'-terminal cloverleaf (CL)-like RNA structures are essential for the initiation of positive- and negative-strand RNA synthesis of entero- and rhinoviruses. SLD is the cognate RNA ligand of the viral proteinase 3C (3Cpro), which is an indispensable component of the viral replication initiation complex. The structure of an 18mer RNA representing the apical stem and the cGUUAg D-loop of SLD from the first 5'-CL of BEV1 was determined in solution to a root-mean-square deviation (r.m.s.d.) (all heavy atoms) of 0.59 A (PDB 1Z30). The first (antiG) and last (synA) nucleotide of the D-loop forms a novel ‘pseudo base pair’ without direct hydrogen bonds. The backbone conformation and the base-stacking pattern of the cGUUAg-loop, however, are highly similar to that of the coxsackieviral uCACGg D-loop (PDB 1RFR) and of the stable cUUCGg tetraloop (PDB 1F7Y) but surprisingly dissimilar to the structure of a cGUAAg stable tetraloop (PDB 1MSY), even though the cGUUAg BEV D-loop and the cGUAAg tetraloop differ by 1 nt only. Together with the presented binding data, these findings provide independent experimental evidence for our model [O. Ohlenschläger, J. Wöhnert, E. Bucci, S. Seitz, S. Häfner, R. Ramachandran, R. Zell and M. Görlach (2004) Structure, 12, 237–248] that the proteinase 3Cpro recognizes structure rather than sequence.
We have isolated the human protein SNEV as downregulated in replicatively senescent cells. Sequence homology to the yeast splicing factor Prp19 suggested that SNEV might be the orthologue of Prp19 and therefore might also be involved in pre-mRNA splicing. We have used various approaches including gene complementation studies in yeast using a temperature sensitive mutant with a pleiotropic phenotype and SNEV immunodepletion from human HeLa nuclear extracts to determine its function. A human–yeast chimera was indeed capable of restoring the wild-type phenotype of the yeast mutant strain. In addition, immunodepletion of SNEV from human nuclear extracts resulted in a decrease of in vitro pre-mRNA splicing efficiency. Furthermore, as part of our analysis of protein–protein interactions within the CDC5L complex, we found that SNEV interacts with itself. The self-interaction domain was mapped to amino acids 56–74 in the protein's sequence and synthetic peptides derived from this region inhibit in vitro splicing by surprisingly interfering with spliceosome formation and stability. These results indicate that SNEV is the human orthologue of yeast PRP19, functions in splicing and that homo-oligomerization of SNEV in HeLa nuclear extract is essential for spliceosome assembly and that it might also be important for spliceosome stability.
In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase a (pol alpha) and Klenow fragment (exo-) of DNA polymerase I (Escherichia coli ). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electronwithdrawing (trifluoromethyl and dinitro). Both pol a and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol a and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol a and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol {alpha} and Klenow fragment.
Background: Costly structures need to represent an adaptive advantage in order to be maintained over evolutionary times. Contrary to many other conspicuous shell ornamentations of gastropods, the haired shells of several Stylommatophoran land snails still lack a convincing adaptive explanation. In the present study, we analysed the correlation between the presence/absence of hairs and habitat conditions in the genus Trochulus in a Bayesian framework of character evolution. Results: Haired shells appeared to be the ancestral character state, a feature most probably lost three times independently. These losses were correlated with a shift from humid to dry habitats, indicating an adaptive function of hairs in moist environments. It had been previously hypothesised that these costly protein structures of the outer shell layer facilitate the locomotion in moist habitats. Our experiments, on the contrary, showed an increased adherence of haired shells to wet surfaces. Conclusion: We propose the hypothesis that the possession of hairs facilitates the adherence of the snails to their herbaceous food plants during foraging when humidity levels are high. The absence of hairs in some Trochulus species could thus be explained as a loss of the potential adaptive function linked to habitat shifts.
The volume changes of lithium and sodium under pressure are discussed with respect to the packing density of the atoms and their valence. In densely packed Li I (bcc), Li II (fcc), and Li III (alpha-Hg ype), valence increases from 1 at ~ 5 GPa to ~ 2.5 at 40 GPa. The maximum valence 3 is attained in Li IV (body-centered cubic, 16 atoms per cell, packing density q = 0.965) at 47 GPa. In densely packed Na I (bcc) a linear increase of valence from 1 at ~ 10 GPa to 2.9 at 65 GPa is found which continues in Na II (fcc) up to 4.1 at 103 GPa.
A new approach to optimize multilevel logic circuits is introduced. Given a multilevel circuit, the synthesis method optimizes its area while simultaneously enhancing its random pattern testability. The method is based on structural transformations at the gate level. New transformations involving EX-OR gates as well as Reed–Muller expansions have been introduced in the synthesis of multilevel circuits. This method is augmented with transformations that specifically enhance random-pattern testability while reducing the area. Testability enhancement is an integral part of our synthesis methodology. Experimental results show that the proposed methodology not only can achieve lower area than other similar tools, but that it achieves better testability compared to available testability enhancement tools such as tstfx. Specifically for ISCAS-85 benchmark circuits, it was observed that EX-OR gate-based transformations successfully contributed toward generating smaller circuits compared to other state-of-the-art logic optimization tools.
Channel routing is an NP-complete problem. Therefore, it is likely that there is no efficient algorithm solving this problem exactly.In this paper, we show that channel routing is a fixed-parameter tractable problem and that we can find a solution in linear time for a fixed channel width.We implemented our approach for the restricted layer model. The algorithm finds an optimal route for channels with up to 13 tracks within minutes or up to 11 tracks within seconds.Such narrow channels occur for example as a leaf problem of hierarchical routers or within standard cell generators.
We present a theoretical analysis of structural FSM traversal, which is the basis for the sequential equivalence checking algorithm Record & Play presented earlier. We compare the convergence behaviour of exact and approximative structural FSM traversal with that of standard BDD-based FSM traversal. We show that for most circuits encountered in practice exact structural FSM traversal reaches the fixed point as fast as symbolic FSM traversal, while approximation can significantly reduce in the number of iterations needed. Our experiments confirm these results.
We present the FPGA implementation of an algorithm [4] that computes implications between signal values in a boolean network. The research was performed as a masterrsquos thesis [5] at the University of Frankfurt. The recursive algorithm is rather complex for a hardware realization and therefore the FPGA implementation is an interesting example for the potential of reconfigurable computing beyond systolic algorithms. A circuit generator was written that transforms a boolean network into a network of small processing elements and a global control logic which together implement the algorithm. The resulting circuit performs the computation two orders of magnitudes faster than a software implementation run by a conventional workstation.
One of the most severe short-comings of currently available equivalence checkers is their inability to verify integer multipliers. In this paper, we present a bit level reverse-engineering technique that can be integrated into standard equivalence checking flows. We propose a Boolean mapping algorithm that extracts a network of half adders from the gate netlist of an addition circuit. Once the arithmetic bit level representation of the circuit is obtained, equivalence checking can be performed using simple arithmetic operations. Experimental results show the promise of our approach.
This paper argues that short (clause-internal) scrambling to a pre-subject position has A properties in Japanese but A'-properties in German, while long scrambling (scrambling across sentence boundaries) from finite clauses, which is possible in Japanese but not in German, has A'-properties throughout. It is shown that these differences between German and Japanese can be traced back to parametric variation of phrase structure and the parameterized properties of functional heads. Due to the properties of Agreement, sentences in Japanese may contain multiple (Agro- and Agrs-) specifiers whereas German does not allow for this. In Japanese, a scrambled element may be located in a Spec AgrP, i.e. an A- or L-related position, whereas scrambled NPs in German can only appear in an AgrP-adjoined (broadly-L-related) position, which only has A'-properties. Given our assumption that successive cyclic adjunction is generally impossible, elements in German may not be long scrambled because a scrambled element that is moved to an adjunction site inside an embedded clause may not move further. In Japanese, long distance scrambling out of finite CPs is possible since scrambling may proceed in a successive cyclic manner via embedded Spec- (AgrP) positions. Our analysis of the differences between German and Japanese scrambling provides us with an account of further contrasts between the two languages such as the existence of surprising asymmetries between German and Japanese remnant-movement phenomena, and the fact that unlike German, Japanese freely allows wh-scrambling. Investigation of the properties of Japanese wh-movement also leads us to the formulation of the "Wh-cluster Hypothesis", which implies that Japanese is an LF multiple wh-fronting language.
In this article, I discuss some important properties of wh-questions and wh-scrambling in Japanese. The questions I will address are (i) which instances of (wh-) scrambling involve reconstruction and (ii) how the undoing effects of scrambling can be derived. First I will discuss the claim that (wh-) scrambling is semantically vacuous and is therefore undone at LF (Saito 1989, 1992). Then I consider the data that led Takahashi (1993) to the conclusion that at least some instances of wh-scrambling have to be analyzed as instances of "full wh-movement" i.e., overt movement of the wh-phrase in its scopal position. It will be argued that these examples are not instances of full wh-movement in Japanese, but that they also represent semantically vacuous scrambling. Those instances of scrambling that apprently cannot be undone are best explained with recourse to parsing effects. I conclude that wh-scrambling in Japanese is always triggered by a ([-wh]-) scrambling feature. In addition, long distance scrambling (scrambling out of finite CPs) is analyzed as adjunction movement, whereas short distance scrambling is movement to a specifier position of IP. Turning to the mechanisms of undoing, I will argue that only long distance scrambling is undone. This is shown to follow from Chomsky's (1995) bare phrase structure analysis, according to which multi-segmental categories derived by adjunction movement are not licensed at LF. The article is organized as follows. In section 2, the wh-scrambling phenomenon is described. In section 3, I discuss the reconstruction properties of scrambling. In addition, this section provides some basic assumptions about my analysis of Japanese scrambling in general. In section 4, I turn to the analysis of wh-scrambling as an instance of full wh-movement in Japanese. Section 5 provides discussion of multiple wh-questions in Japanese, and section 6 gives the conclusion.
Die Doppelobjekt-Konstruktion bildet einen Untersuchungsgegenstand, der in der Vergangenheit die Theoriebildung in der Syntaxforschung wesentlich beeinflusst hat. Untersuchungen zu Doppelobjekt-Konstruktionen sind u.a. folgenreich gewesen für die Kasustheorie sowie für Analysen der Verbbewegung, Satz-, VP- und Argument-Struktur. In diesem Aufsatz stelle ich eine Analyse einiger wichtiger Aspekte der Doppelobjekt-Konstruktion im Deutschen vor. Untersucht wird, in welcher Position die Objekte des Verbs basisgeneriert werden und in welchen abgeleiteten Positionen sie erscheinen. Die Beantwortung dieser Fragen liefert eine Erklärung für das asymmetrische Verhalten der beteiligten Objekte in Bezug auf ihr Bindungs- und Extraktionsverhalten.
In diesem Aufsatz diskutiere ich die Distribution von kohärenten Kontroll-Infinitiven im Deutschen. Es werden die Verbklassen bestimmt, die kohärente Infinitive lizenzieren. Dabei zeigt sich, dass ausschließlich Infinitive 'kohärent' konstruiert werden können, die die Position der Akkusativ NP (bzw. die Position des direkten Objekts) einnehmen. Kontroll-Infinitive in anderen strukturellen Positionen sind zwangsläufig 'inkohärent'. Transparente Infinitive in Sprachen wie dem Polnischen und Spanischen sind in derselben Weise in ihrer Distribution beschränkt. Ich schlage eine einheitliche Analyse der relevanten Daten vor, die weitere distributionelle Generalisierungen bezüglich des Auftretens kohärenter Infinitive korrekt prognostiziert. Für die idiolektale Variation, die unter Sprechern in Bezug auf bestimmte Verben existiert, die kohärente Infinitive lizenzieren, wird eine Erklärung formuliert, die auf der Idee basiert, dass die Bildung dieser Infinitive an die Präsenz eines Inkorporations-Merkmals gebunden ist, das beim Spracherwerb auf der Grundlage positiver Evidenz erworben wird. In this article, I discuss the distribution of so-called 'coherent (control) infinitives' in German. In section 2, I will argue that both coherent as well as incoherent control-infinitives have a sentential status. In section 3, I argue that only infinitives occupying the position of the direct object show the well-known properties associated with coherent infinitives. Control infinitives in other structural positions represent incoherent infinitives. This situation is not limited to German infinitives. Transparent infinitives in Polish and Spanish show the same structural asymmetry. In section 4, I propose a unified analysis for the data that in addition, correctly predicts further restrictions for the distribution of coherent infinitives. In section 5, I propose an account for idiolectal variation in the class of verbs that license coherent infinitives. This account is based on the idea that coherent infinitives require an incorporation-feature in their lexical entry that is acquired on the basis of positive evidence.
In diesem Aufsatz gehe ich der Frage nach, in wie viel unterschiedlichen Positionen Verben im deutschen Satz vorkommen können. Anhand syntaktischer Tests wird gezeigt, daß das Verb im Deutschen in insgesamt drei unterschiedlichen Positionen auftritt und nicht, wie in der traditionellen Grammatik angenommen wird, in nur zwei Positionen (in der rechten und in der linken Satzklammer). Es wird dafür argumentiert, daß die Anwendung des abstrakten Satzschemas, wie es heute gängigerweise in der generativen Grammatikforschung als universelles Satzmodell angenommen wird, die Erklärung einer Vielzahl syntaktischer Phänomene im Deutschen ermöglicht, die mit der traditionellen Verbstellungsanalyse, die von nur zwei Verbpositionen ausgeht, nicht erklärt werden können. Gemäß des universellen Satzschemas repräsentiert die Infl(ection)-Position eine Verbposition im Satz. Diese Position ist identisch mit der rechten Satzklammer. Eine weitere Verbposition ist die V-Position innerhalb des Mittelfelds und eine dritte potenzielle Position für das Verb entspricht der C(omplementizer-) Position (bzw. der linken Satzklammer). Der Aufsatz ist folgendermaßen gegliedert. In der Einleitung schildere ich kurz die unterschiedlichen Auffassungen, die in der Vergangenheit zur Verbstellungsproblematik im Deutschen vertreten wurden. In Abschnitt 2 nenne ich die wichtigsten Argumente, die gegen die Annahme vorgebracht wurden, daß im deutschen Satz für Verben insgesamt drei Positionen zur Verfügung stehen. Im Anschluss daran werden in den Abschnitten 3.1 bis 3.2 Argumente diskutiert, die für drei Verbpositionen sprechen. Abschnitt 3.3 behandelt die Frage, wie vor diesem Hintergrund die Daten aus Abschnitt 2, die sich als problematisch für diese Analyse erwiesen haben, erklärt werden können. In Abschnitt 4 wende ich mich weiterer unabhängiger Evidenz aus dem Bereich der historischen Syntax zu, die für drei Verbpositionen im Deutschen spricht. In Abschnitt 5 gebe ich eine kurze Zusammenfassung der wichtigsten Ergebnisse.
Ausgangspunkt der folgenden Untersuchung ist die Überlegung, daß verschiedene Versionen der Prinzipien- und Parametertheorie unterschiedliche Prognosen bezüglich strukturell ambiger Wortfolgen in Passiv-Konstruktionen des Deutschen machen. Im Rahmen einer Theorie, in der Move-alpha frei appliziert, wie etwa in der Rektions- und Bindungstheorie (Chomsky 1981, 1986a, 1986b), können multiple Derivationen für derartige Abfolgen nicht ausgeschlossen werden, wohingegen eine andere Situation vorliegt, wenn man die entsprechenden Konstruktionen im Rahmen des Minimalistischen Programms analysiert. Hier kann die Anzahl möglicher (und mit einer Wortfolge verträglicher) Derivationen mit Hilfe von Ökonomieprinzipien beschränkt werden. Auf Grundlage verschiedener syntaktischer Tests wird im weiteren gezeigt, daß bestimmte Wortfolgen nur mit einer Derivation verträglich sind, was im Einklang mit einer minimalistischen Analyse der Daten steht. Der Aufsatz ist folgendermaßen gegliedert. In Abschnitt 2 erläutere ich das Grundproblem der multiplen Derivationen, das sich im Deutschen z. B. bei Passiv-Konstruktionen ergibt, wenn man annimmt, daß NP-Bewegung und Scrambling optional erfolgen und ferner keine Beschränkungen für potentielle Derivationen gelten. In Abschnitt 3 diskutiere ich die Voraussetzungen für die Überprüfung der Prognosen der verschiedenen Varianten des Prinzipien- und Parametermodells und versuche anschließend auf Grundlage syntaktischer Tests zu belegen, daß die diskutierten Beispiele tatsächlich nicht strukturell ambig, sondern strukturell eindeutig sind, wie es die Analyse der entsprechenden Konstruktionen im Rahmen des Minimalistischen Programms vorhersagt. Abschnitt 4 beschreibt die Konsequenzen der Analyse für weitere Sprachen wie Niederländisch und Japanisch und zusätzliche Bewegungstypen. Abschnitt 5 enthält die Konklusion.
Homing in with GPS
(2000)