Complement Constructions Research Articles

Use of aminoglycoside 3' adenyltransferase as a selection marker for Chlamydia trachomatis intron-mutagenesis and in vivo intron stability.

BackgroundChlamydia spp. are obligate, intracellular bacteria that infect humans and animals. Research on these important pathogens has been hindered due to a paucity of genetic tools. We recently adapted a group II intron (GII) mutagenesis platform for creation of ampicillin-selectable gene insertions in C. trachomatis L2. The aims of this study were: (1) to assess the stability of the intron-insertion in an in vivo infection model to gauge the efficacy of this genetic tool for long term animal studies and (2) to expand upon the utility of the method by validating a second selection marker (aadA, conferring spectinomycin resistance) for mutant construction.ResultsIntron stability was assessed using a mouse vaginal tract infection model with a C. trachomatis L2 434/Bu incA::GII(bla) mutant. Infections were performed in the absence of selection and isolates shed into the vaginal tract were isolated and expanded in cell culture (also without selection). PCR and inclusion phenotype analysis indicated that the intron was stable for at least 27 days post-infection (at which point bacteria were no longer recovered from the mouse). The aminoglycoside 3′ adenyltransferase (aadA) gene was used to create a spectinomycin-selectable GII intron, facilitating the construction of an incA::GII[aadA] C. trachomatis L2 insertion mutant. Both the GII(aadA) intron and our previously reported GII(bla) intron were then used to create an incA::GII(aadA), rsbV1::GII(bla) double mutant. Mutants were confirmed via PCR, sequencing, inclusion morphology (incA only), and western blot.ConclusionsThe stability of the intron-insertion during in vivo growth indicates that the GII-insertion mutants can be used to study pathogenesis using the well-established mouse infection model. In addition, the validation of an additional marker for mutagenesis in Chlamydia allows for gene complementation approaches and construction of targeted, double mutants in Chlamydia. The aadA marker also could be useful for other genetic methods. Collectively, our results expand upon the rapidly growing chlamydial genetic toolkit and will aid in the implementation of studies dissecting the contribution of individual genes to infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1542-9) contains supplementary material, which is available to authorized users.

Ramsey-Based Inclusion Checking for Visibly Pushdown Automata

Checking whether one formal language is included in another is important in many verification tasks. In this article, we provide solutions for checking the inclusion of languages given by visibly pushdown automata over both finite and infinite words. Visibly pushdown automata are a richer automaton model than the classical finite-state automata, which allows one, for example, to reason about the nesting of procedure calls in the executions of recursive imperative programs. The presented solutions do not rely on explicit automaton constructions for determinization and complementation. Instead, they are more direct and generalize the so-called Ramsey-based inclusion-checking algorithms, which apply to classical finite-state automata and proved to be effective there to visibly pushdown automata. We also experimentally evaluate these algorithms, demonstrating the virtues of avoiding explicit determinization and complementation constructions.

Profile trees for Büchi word automata, with application to determinization

The determinization of Büchi automata is a celebrated problem, with applications in synthesis, probabilistic verification, and multi-agent systems. Since the 1960s, there has been a steady progress of constructions: by McNaughton, Safra, Piterman, Schewe, and others. Despite the proliferation of solutions, they are all essentially ad-hoc constructions, with little theory behind them other than proofs of correctness. Since Safra, all optimal constructions employ trees as states of the deterministic automaton, and transitions between states are defined operationally over these trees. The operational nature of these constructions complicates understanding, implementing, and reasoning about them, and should be contrasted with complementation, where a solid theory in terms of automata run dags underlies modern constructions.In 2010, we described a profile-based approach to Büchi complementation, where a profile is simply the history of visits to accepting states. We developed a structural theory of profiles and used it to describe a complementation construction that is deterministic in the limit. Here we extend the theory of profiles to prove that every run dag contains a profile tree with at most a finite number of infinite branches. We then show that this property provides a theoretical grounding for a new determinization construction where macrostates are doubly preordered sets of states. In contrast to extant determinization constructions, transitions in the new construction are described declaratively rather than operationally.

The Structure of Double Complement Construction and Syntactic Reflection of Logical Order between Internal Arguments

The Structure of Double Complement Construction and Syntactic Reflection of Logical Order between Internal Arguments

Dialogic syntax and complement constructions in toddlers' peer interactions

AbstractChildren start producing grammatically complex sentences during toddlerhood (Bloom et al. 1984; Diessel 2004). This study examines how toddlers use complement constructions as a communicative resource in peer interactions. The data come from an archival database, which consists of 500 hours of videorecordings of children's naturalistic interactions in a daycare center. All complement constructions produced by seven target children were identified. The data illustrate children using “format tying” (Goodwin 1990, 2006) and “dialogic syntax”. They construct utterances “based on the immediately co-present utterance[s]” (Du Bois, this issue) of their own and others (caregivers) in the discourse, as means of demonstrating positive or negative alignment with their interlocutors, thereby negotiating the participation framework (Goffman 1981; Goodwin and Goodwin 2004). As children tie to prior utterances, and transform and embed them into matrix clauses with stance-indexing verbs like I said, I want, and Let me, complex complement constructions are built up dialogically over sequences of interaction.

Hygromycin B and apramycin antibiotic resistance cassettes for use in Campylobacter jejuni.

Campylobacter jejuni genetic manipulation is restricted by the limited number of antibiotic resistance cassettes available for use in this diarrheal pathogen. In this study, two antibiotic resistance cassettes were developed, encoding for hygromycin B and apramycin resistance, for use in mutagenesis or for selection of gene expression and complementation constructs in C. jejuni. First, the marker genes were successfully modified to allow for insertional mutagenesis or deletion of a gene-of-interest, and were bracketed with restriction sites for the facilitation of site-specific cloning. These hygromycin B and apramycin markers are encoded by plasmids pAC1H and pAC1A, respectively. We also modified an insertional gene-delivery vector to create pRRH and pRRA, containing the hygromycin B and apramycin resistance genes, and 3 unique restriction sites for the directional introduction of genes into the conserved multi-copy rRNA gene clusters of the C. jejuni chromosome. We determined the effective antibiotic concentrations required for selection, and established that no harmful effects or fitness costs were associated with carrying hygromycin B or apramycin resistance under standard C. jejuni laboratory conditions. Using these markers, the arylsulfatase reporter gene astA was deleted, and the ability to genetically complement the astA deletion using pRRH and pRRA for astA gene insertion was demonstrated. Furthermore, the relative levels of expression from the endogenous astA promoter were compared to that of polycistronic mRNA expression from the constitutive promoter upstream of the resistance gene. The development of additional antibiotic resistance cassettes for use in Campylobacter will enable multiple gene deletion and expression combinations as well as more in-depth study of multi-gene systems important for the survival and pathogenesis of this important bacterium.

That-variation in German and Spanish L2 English

abstractIn certain English finite complement clauses, inclusion of the complementizerthatis optional. Previous research has identified various factors that influence when native speakers tend to produce or omit the complementizer, including syntactic weight, clause juncture constraints, and predicate frequency. The present study addresses the question to what extent German and Spanish learners of English as a second language (L2) produce and omit the complementizer under similar conditions. 3,622 instances of English adjectival, object, and subject complement constructions were retrieved from theInternational Corpus of Englishand the German and Spanish components of theInternational Corpus of Learner English. A logistic regression model suggests that L2 learners’ and natives’ production is largely governed by the same factors. However, in comparison with native speakers, L2 learners display a lower rate of complementizer omission. They are more impacted by processing-related factors such as complexity and clause juncture, and less sensitive to verb-construction cue validity.

Private Searching on Streaming Data Based on Keyword Frequency

Private searching on streaming data is a process to dispatch to a public server a program, which searches streaming sources of data without revealing searching criteria and then sends back a buffer containing the findings. From an Abelian group homomorphic encryption, the searching criteria can be constructed by only simple combinations of keywords, for example, disjunction of keywords. The recent breakthrough in fully homomorphic encryption has allowed us to construct arbitrary searching criteria theoretically. In this paper, we consider a new private query, which searches for documents from streaming data on the basis of keyword frequency, such that the frequency of a keyword is required to be higher or lower than a given threshold. This form of query can help us in finding more relevant documents. Based on the state of the art fully homomorphic encryption techniques, we give disjunctive, conjunctive, and complement constructions for private threshold queries based on keyword frequency. Combining the basic constructions, we further present a generic construction for arbitrary private threshold queries based on keyword frequency. Our protocols are semantically secure as long as the underlying fully homomorphic encryption scheme is semantically secure.

Sentential complements and false belief understanding in Chinese Mandarin-speaking preschoolers: A training study

We conducted a training study to better understand how Chinese Mandarin-speaking preschoolers’ facility with sentential complement grammatical constructions affects performance on false belief tasks. Eighty-four Mandarin-speaking Chinese 3–4-year-olds who were initially unsuccessful on false belief tasks were randomly assigned to four training conditions. Two involved training on sentential complement structures, one involved training on understanding of false representations, and one was a control condition that involved no specific training. Participants who received training on sentential complements with communication verbs performed significantly better on false belief posttests than those in the control group. Children in the false representation training group did not show improvement in the sentential complement tests. The findings suggest facility with sentential complement grammatical structures can promote false belief reasoning. However, explicit false belief understanding can emerge even when children have little competence with sentential complement constructions.

Independent complement constructions in Swedish and Danish: Insubordination or dependency shift?

The aim of this paper is to analyze the grammatical status of independent complement constructions in Swedish and Danish, i.e. constructions introduced by the complementizer att or at but without an accompanying main clause. These constructions can be used with two main functions: to express the speaker's evaluation of a presupposed state of affairs, or to elaborate on an aspect of the preceding discourse. In recent literature on these and similar constructions in other languages, both types have been analyzed as instances of the category of insubordination (Evans, 2007), i.e. constructions that combine subordinate marking with main clause use. We will argue that this analysis works well for the ‘expressive’ type, but that it cannot account for some of the typical properties of the ‘elaborative’ constructions, like the fact that they are pragmatically dependent and inconsistent in their use of subordinate marking. As an alternative, we will show that elaborative constructions can be dealt with much more naturally in terms of an existing model of dependency shift in clause combining, like the subordinate–coordinate shifts observed for many other types of subordinators.

On the Grammaticalization of the Potential Complement Constructions ‘V de(得)/bu(不)C’ in Chinese

On the Grammaticalization of the Potential Complement Constructions ‘V de(得)/bu(不)C’ in Chinese

Causal Role of Single Nucleotide Polymorphisms within the mprF Gene of Staphylococcus aureus in Daptomycin Resistance

Single nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) have been commonly observed in daptomycin-resistant (DAP(r)) Staphylococcus aureus strains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if such mprF SNPs are causal in DAP(r) strains or are merely a biomarker for this phenotype. In this study, we used an isogenic set of S. aureus strains: (i) Newman, (ii) its isogenic ΔmprF mutant, and (iii) several in trans plasmid complementation constructs, expressing either a wild-type or point-mutated form of the mprF ORF cloned from two isogenic DAP-susceptible (DAP(s))-DAP(r) strain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprF strain with singly point-mutated mprF genes (mprFS295L or mprFT345A) revealed that (i) individual and distinct point mutations within the mprF ORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding to S. aureus by a charge repulsion mechanism. Thus, for these two DAP(r) strains, the defined mprF SNPs appear to be causally related to this phenotype.

Profile Trees for Büchi Word Automata, with Application to Determinization

The determinization of Buchi automata is a celebrated problem, with applications in synthesis, probabilistic verification, and multi-agent systems. Since the 1960s, there has been a steady progress of constructions: by McNaughton, Safra, Piterman, Schewe, and others. Despite the proliferation of solutions, they are all essentially ad-hoc constructions, with little theory behind them other than proofs of correctness. Since Safra, all optimal constructions employ trees as states of the deterministic automaton, and transitions between states are defined operationally over these trees. The operational nature of these constructions complicates understanding, implementing, and reasoning about them, and should be contrasted with complementation, where a solid theory in terms of automata run DAGs underlies modern constructions. In 2010, we described a profile-based approach to Buchi complementation, where a profile is simply the history of visits to accepting states. We developed a structural theory of profiles and used it to describe a complementation construction that is deterministic in the limit. Here we extend the theory of profiles to prove that every run DAG contains a profile tree with at most a finite number of infinite branches. We then show that this property provides a theoretical grounding for a new determinization construction where macrostates are doubly preordered sets of states. In contrast to extant determinization constructions, transitions in the new construction are described declaratively rather than operationally.

Change through recombination: blending and analogy

Blending is generally seen as a marginal source of linguistic innovations in language change. However, the impact of blending is likely underestimated, because blends may occur under the guise of analogical extension. In such cases, blending is covert and cannot be detected synchronically in the innovative structure. In this paper, the relation between analogy and blending is analyzed. Next, the role of covert blending is demonstrated in two diachronic case studies. The first case study addresses the use of the English verbs want and need in the Passival Participle Construction (e.g. you need your eyes testing). The pattern could have analogically extended from perception verbs, as claimed by Visser (1963–1973), but distributional, chronological and semantic evidence indicate that the extension happened through blending between two other constructions, the Passival Gerund Construction (your eyes need testing) and the Object Complement Construction (you need your eyes tested). The second case study deals with the development of the Dutch downtoner allesbehalve (‘not at all’). It is shown that once allesbehalve had adopted the syntactic status of a downtoner it spread to new syntactic contexts. Since this brings allesbehalve in line with other downtoners, the process can be seen as an instance of analogical extension. Quantitative evidence, however, shows that the developing syntactic behaviour of the downtoner continues to be influenced by the syntax of its composing elements, alles (‘everything’) and behalve (‘except’). Change is thus partly driven by blending between the downtoner and its own historical source. In both cases, apparent analogical extensions hide an underlying blend. These findings show that blending may be more pervasive than generally recognized, supplementing rule-based strategies for coining new utterances.

Unifying B\"uchi Complementation Constructions

Complementation of B\"uchi automata, required for checking automata containment, is of major theoretical and practical interest in formal verification. We consider two recent approaches to complementation. The first is the rank-based approach of Kupferman and Vardi, which operates over a DAG that embodies all runs of the automaton. This approach is based on the observation that the vertices of this DAG can be ranked in a certain way, termed an odd ranking, iff all runs are rejecting. The second is the slice-based approach of K\"ahler and Wilke. This approach tracks levels of "split trees" - run trees in which only essential information about the history of each run is maintained. While the slice-based construction is conceptually simple, the complementing automata it generates are exponentially larger than those of the recent rank-based construction of Schewe, and it suffers from the difficulty of symbolically encoding levels of split trees. In this work we reformulate the slice-based approach in terms of run DAGs and preorders over states. In doing so, we begin to draw parallels between the rank-based and slice-based approaches. Through deeper analysis of the slice-based approach, we strongly restrict the nondeterminism it generates. We are then able to employ the slice-based approach to provide a new odd ranking, called a retrospective ranking, that is different from the one provided by Kupferman and Vardi. This new ranking allows us to construct a deterministic-in-the-limit rank-based automaton with a highly restricted transition function. Further, by phrasing the slice-based approach in terms of ranks, our approach affords a simple symbolic encoding and achieves the tight bound of Schewe's construction

On the cross-linguistic distribution of same-subject and different-subject 'want' complements: Economic vs. iconic motivation

This paper presents the results of a world-wide typological study of same-subject and different-subject complements of the verb ‘want’ (sample size: 80 languages). I am interested in whether the subject is overtly expressed only in different-subject complements (e.g. English, Mandarin Chinese, Coptic) or whether it is expressed also in same-subject complements (e.g. Modern Greek, Mixtec, Arabic, where the literal translation of ‘I want to go home’ is ‘I want that I go home’). Not surprisingly, the more economical English pattern is more widespread in the sample languages. I argue that this pattern should be explained in terms of economic motivation, not in terms of iconic motivation or conceptual closeness (as has also been suggested in the literature). The economic explanation turns out to be more general and to make more accurate predictions. 1. Subject omission and formal simplicity in ‘want’ complements In this paper, I propose a usage-based explanation of a typological trend in two types of complement constructions of the verb ‘want’: There is often an asymmetry in the way that same-subject (SS) and different-subject (DS) ‘want’ complement clauses are encoded across languages. I argue that the asymmetry can be explained by a frequency bias (economic motivation in Haiman’s 1983 sense). ‘Want’ complements overwhelmingly show 1 Earlier versions of this paper were presented at the International Cognitive Linguistics Conference (Stockholm 1999) and at the DGfS Annual Meeting in Marburg (2000). Many people have made helpful suggestions to me at different stages, and they are all gratefully acknowledged here. MARTIN HASPELMATH 42 referential identity between the wanter argument and the subject (the A or S argument) of the complement clause. Sentences like (1a) are much more frequent than sentences like (1b), apparently in all languages. (Here the symbol O is meant to show that the notional subject of the complement clause is not expressed overtly.) (1) a. Kim1 wants [O1 to go home]. b. Kim1 wants [him2 to go home]. I claim that two aspects of same-subject complement clauses are economically motivated: (i) The fact that the notional complement subject is unexpressed in many languages (as illustrated in (1a), where the notional subject of ‘to go home’ cannot be expressed overtly in its clause), and (ii) the fact that different-subject complement clauses often show a formally more complex pattern, as illustrated by German:

Sentential specifiers in the Korean clause structure

The Korean double nominative construction exhibits various properties distinguished not only from ordinary subject-object clauses but also from nominative complement constructions. Particularly, the second NP, not the initial NP, triggers the honorific agreement with the verb. I argue that the first NP of the construction is identified as a sentential specifier which exists in addition to the subject (cf. Major subject in Yoon 2004). The sentential specifier can be justified as the characteristic of the topic-prominent language in the sense of Li and Thompson (1976). Specifically I claim that any elements that satisfy the aboutness condition can be the sentential specifier. Finally, I show that HPSG's valence value and an optional lexical rule provides an elegant treatment of the construction; SPR list in a sentence level can be utilized for the sentential specifier (cf. Kim et al. 2007).

The Importance of TM3-4 Loop Subdomains for Functional Reconstitution of Glycine Receptors by Independent Domains

Truncated glycine receptors that have been found in human patients suffering from the neuromotor disorder hyperekplexia or in spontaneous mouse models resulted in non-functional ion channels. Rescue of function experiments with the lacking protein portion expressed as a separate independent domain demonstrated restoration of glycine receptor functionality in vitro. This construct harbored most of the TM3-4 loop, TM4, and the C terminus and was required for concomitant transport of the truncated α1 and the complementation domain from the endoplasmic reticulum toward the cell surface, thereby enabling complex formation of functional glycine receptors. Here, the complementation domain was stepwise truncated from its N terminus in the TM3-4 loop. Truncation of more than 49 amino acids led again to loss of functionality in the receptor complex expressed from two independent domain constructs. We identified residues 357-418 in the intracellular TM3-4 loop as being required for reconstitution of functional glycine-gated channels. All complementation constructs showed cell surface protein expression and correct orientation according to glycine receptor topology. Moreover, we demonstrated that the truncations did not result in a decreased protein-protein interaction between both glycine receptor domains. Rather, deletions of more than 49 amino acids abolished conformational changes necessary for ion channel opening. When the TM3-4 loop subdomain harboring residues 357-418 was expressed as a third independent construct together with the truncated N-terminal and C-terminal glycine receptor domains, functionality of the glycine receptor was again restored. Thus, residues 357-418 represent an important determinant in the process of conformational rearrangements following ligand binding resulting in channel opening.

B\"uchi Complementation and Size-Change Termination

We compare tools for complementing nondeterministic B\"uchi automata with a recent termination-analysis algorithm. Complementation of B\"uchi automata is a key step in program verification. Early constructions using a Ramsey-based argument have been supplanted by rank-based constructions with exponentially better bounds. In 2001 Lee et al. presented the size-change termination (SCT) problem, along with both a reduction to B\"uchi automata and a Ramsey-based algorithm. The Ramsey-based algorithm was presented as a more practical alternative to the automata-theoretic approach, but strongly resembles the initial complementation constructions for B\"uchi automata. We prove that the SCT algorithm is a specialized realization of the Ramsey-based complementation construction. To do so, we extend the Ramsey-based complementation construction to provide a containment-testing algorithm. Surprisingly, empirical analysis suggests that despite the massive gap in worst-case complexity, Ramsey-based approaches are superior over the domain of SCT problems. Upon further analysis we discover an interesting property of the problem space that both explains this result and provides a chance to improve rank-based tools. With these improvements, we show that theoretical gains in efficiency of the rank-based approach are mirrored in empirical performance.

New complementation constructs for inducible and constitutive gene expression in Neisseria gonorrhoeae and Neisseria meningitidis.

We have created new complementation constructs for use in Neisseria gonorrhoeae and Neisseria meningitidis. The constructs contain regions of homology with the chromosome and direct the insertion of a gene of interest into the intergenic region between the genes iga and trpB. In order to increase the available options for gene expression in Neisseria, we designed the constructs to contain one of three different promoters. One of the constructs contains the isopropyl-β-d-thiogalactopyranoside-inducible lac promoter, which has been widely used in Neisseria. We also designed a construct that contains the strong, constitutive promoter from the gonococcal opaB gene. The third construct contains a tetracycline-inducible promoter, a novel use of this promoter in Neisseria. We demonstrate that anhydrotetracycline can be used to induce gene expression in the pathogenic Neisseria at very low concentrations and without negatively affecting the growth of the organisms. We use these constructs to complement an arginine auxotrophy in N. gonorrhoeae as well as to express a translational fusion of alkaline phosphatase with TraW. TraW is a component of the gonococcal type IV secretion system, and we demonstrate that TraW localizes to the periplasm.