Cut-and-Paste DNA Insertion with Engineered Type V-K CRISPR-associated Transposases

CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. Type V-K CASTs offer distinct technological advantages over type I CASTs given their smaller coding size, fewer components, and unidirectional insertions. However, the utility of type V-K CASTs is hindered by a replicative transposition mechanism that results in a mixture of desired simple cargo insertions and undesired plasmid co-integrate products. Here, we overcome this limitation by engineering new CASTs with dramatically improved product purity. To do so, we compensate for the absence of the TnsA subunit in multiple type V-K CASTs by engineering a Homing Endonuclease-assisted Large-sequence Integrating CAST compleX, or HELIX system. HELIX utilizes a nicking homing endonuclease (nHE) fused to TnsB to restore the 5-prime nicking capability needed for dual-nicking of the DNA donor. By leveraging distinct features of both type V-K and type I systems, HELIX enables cut-and-paste DNA insertion with up to 99.3% simple insertion product purity, while retaining robust integration efficiencies on genomic targets. Furthermore, we demonstrate the versatility of this approach by generating HELIX systems for other CAST orthologs. We also establish the feasibility of creating a minimal, 3-component HELIX, simplifying the number of proteins that must be expressed. Together, HELIX streamlines and improves the application of CRISPR-based transposition technologies, eliminating barriers for efficient and specific RNA-guided DNA insertions.

Metonymy in scientific linguistic discourse

The purpose of the article was to show the features of the functioning of different types of metonymy in scientific linguistic discourse, which is understood as a verbalized epistemic situation common to the scientific sphere of communication, taken in the entire totality of linguistic and extralinguistic factors and enshrined in the form of texts (oral and written ones). The article deals with metonymy from the point of view of langue / parole: lexicalized metonymy in langue is a semantic transposition mechanism on contiguity and carries out a terminological nomination; discursive metonymy in parole becomes the result of syntagmatic contiguity of syntactic constructions. Linguistic metonymic terms are grouped by types of knowledge: declarative and procedural ones. The shifts of meaning between the logical terms “object”, “subject”, “general” and “specific”, “abstract” and “concrete”, “form”, “content”, etc., directed towards each other, are observed in metonymic terms of declarative type. Metonymy can reflect the processes due to the causality between adjacent objects. Transitional phenomena between lexicalized (linguistic) and discursive (speech) metonymy reflect those models that contain onyms; they are related to the designation of the subject of knowledge (linguist) and his scientific discovery.

The IS6 family, a clinically important group of insertion sequences including IS26

The IS6 family of bacterial and archaeal insertion sequences, first identified in the early 1980s, has proved to be instrumental in the rearrangement and spread of multiple antibiotic resistance. Two IS, IS26 (found in many enterobacterial clinical isolates as components of both chromosome and plasmids) and IS257 (identified in the plasmids and chromosomes of gram-positive bacteria), have received particular attention for their clinical impact. Although few biochemical data are available concerning the transposition mechanism of these elements, genetic studies have provided some interesting observations suggesting that members of the family might transpose using an unexpected mechanism. In this review, we present an overview of the family, the distribution and phylogenetic relationships of its members, their impact on their host genomes and analyse available data concerning the particular transposition pathways they may use. We also provide a mechanistic model that explains the recent observations on one of the IS6 family transposition pathways: targeted cointegrate formation between replicons.

Indexation of the zone of infinitive-modal structures in Russian language

The relevance of this topic is substantiated by the need to objectivize the results of analysis of transposition mechanism of modalation of verbs in finite and attributive forms using quantitative methods of material processing. The goal of this article is to examine the levels of correspondence of differential signs of infinite forms of verbs in the zone of hybrid structures to the signs of nuclear representatives of the initial and final elements of inter-categorical introductory-modal transposition. The subject of this research became the infinitive-modal structures in transitive sentences that combine the attributes of conjunctionless complex sentences and simple expanded sentence (with unattached introductory-modal component). On the example of word form, the author demonstrates the combinatorial structure and proportion of attributes of verbs and introductory-modal units interacting with each other within the hybrid structure. The conducted research illustrates that the level of modalation of hybrids is determined by the proportion of differential properties of nuclear infinitive and nuclear introductory-modal words. It is established that in transitive structure, which balance on the border of conjunctionless complex and simple expanded sentence, hybrids of the type “to know”  show 68% of correspondence to nuclear infinitives (“know”) and 45% of correspondence to introductory-modal words (“must be”). In syntactic constructs with such hybrid the external syntactic position of modus framework of conjunctionless complex is combined with internal syntactic position of introductory-modal component of the expression.

Unprecedented Alkene Transposition in Phthalate–Amino Acid Adducts

A detailed account on the outcome of the thermal reaction between benzylidene phthalides and various amino acid derivatives is reported. It was discovered that the tricyclic pyrroles as previously described are not the products formed in these reactions. Instead under high-temperature conditions decarboxylated phthalamide adducts are formed within 5-10 minutes. Additionally, an unprecedented alkene transposition mechanism has been identified leading to the final products of these reactions.

The Birth and Demise of the ISApl1-mcr-1-ISApl1 Composite Transposon: the Vehicle for Transferable Colistin Resistance

ABSTRACT The origin and mobilization of the ~2,609-bp DNA segment containing the mobile colistin resistance gene mcr-1 continue to be sources of uncertainty, but recent evidence suggests that the gene originated in Moraxella species. Moreover mcr-1 can be mobilized as an ISApl1-flanked composite transposon (Tn6330), but many sequences have been identified without ISApl1 or with just a single copy (single ended). To further clarify the origins and mobilization of mcr-1, we employed the Geneious R8 software suite to comprehensively analyze the genetic environment of every complete mcr-1 structure deposited in GenBank as of this writing (September 2017) both with and without associated ISApl1 (n = 273). This revealed that the 2,609-bp mcr-1 structure was likely mobilized from a close relative of a novel species of Moraxella containing a chromosomal region sharing >96% nucleotide identity with the canonical sequence. This chromosomal region is bounded by AT and CG dinucleotides, which have been described on the inside ends (IE) of all intact Tn6330 described to date and represent the ancestral 2-bp target site duplications (TSDs) generated by ISApl1 transposition. We further demonstrate that all mcr-1 structures with just one ISApl1 copy or with no ISApl1 copies were formed by deletion of ISApl1 from the ancestral Tn6330, likely by a process related to the “copy-out–paste-in” transposition mechanism. Finally, we show that only the rare examples of single-ended structures that have retained a portion of the excised downstream ISApl1 including the entire inverted right repeat might be capable of mobilization. IMPORTANCE A comprehensive analysis of all intact mcr-1 sequences in GenBank was used to identify a region on the chromosome of a novel Moraxella species with remarkable homology to the canonical mcr-1 structure and that likely represents the origin of this important gene. These data also demonstrate that all mcr-1 structures lacking one or both flanking ISApl1 were formed from ancestral composite transposons that subsequently lost the insertion sequences by a process of abortive transposition. This observation conclusively shows that mobilization of mcr-1 occurs as part of a composite transposon and that structures lacking the downstream ISApl1 are not capable of mobilization.