Ciliate Gene Unscrambling with Fewer Templates

One of the theoretical models proposed for the mechanism of gene unscrambling in some species of ciliates is the template-guided recombination (TGR) system by Prescott, Ehrenfeucht and Rozenberg which has been generalized by Daley and McQuillan from a formal language theory perspective. In this paper, we propose a refinement of this model that generates regular languages using the iterated TGR system with a finite initial language and a finite set of templates, using fewer templates and a smaller alphabet compared to that of the Daley-McQuillan model. To achieve Turing completeness using only finite components, i.e., a finite initial language and a finite set of templates, we also propose an extension of the contextual template-guided recombination system (CTGR system) by Daley and McQuillan, by adding an extra control called permitting contexts on the usage of templates.Comment: In Proceedings DCFS 2010, arXiv:1008.127

Two Refinements of the Template-Guided DNA Recombination Model of Ciliate Computing

To solve the mystery of the intricate gene unscrambling mechanism in ciliates, various theoretical models for this process have been proposed from the point of view of computation. Two main models are the reversible guided recombination system by Kari and Landweber and the template-guided recombination (TGR) system by Prescott, Ehrenfeucht and Rozenberg, based on two categories of DNA recombination: the pointer guided and the template directed recombination respectively. The latter model has been generalized by Daley and McQuillan. In this thesis, we propose a new approach to generate regular languages using the iterated TGR system with a finite initial language and a finite set of templates, that reduces the size of the template language and the alphabet compared to that of the Daley-McQuillan model. To achieve computational completeness using only finite components we also propose an extension of the contextual template-guided recombination system (CTGR system) by Daley and McQuillan, by adding an extra control called permitting contexts on the usage of templates. Then we prove that our proposed system, the CTGR system using permitting contexts, has the capability to characterize the family of recursively enumerable languages using a finite initial language and a finite set of templates. Lastly, we present a comparison and analysis of the computational power of the reversible guided recombination system and the TGR system. Keywords: ciliates, gene unscrambling, in vivo computing, DNA computing, cellular computing, reversible guided recombination, template-guided recombination

Inter- and Intramolecular recombinations in the Cucumber Mosaic Virus genome related to adaptation to Alstroemeria

In four distinct alstroemeria-infecting cucumber mosaic virus (CMV) isolates, additional sequences of various lengths were present in the 3' nontranslated regions of their RNAs 2 and 3, apparently the result of intra- and intermolecular recombination events. Competition experiments revealed that these recombined RNA 2 and 3 segments increased the biological fitness of CMV in alstroemeri

A conserved filamentous assembly underlies the structure of the meiotic chromosome axis.

The meiotic chromosome axis plays key roles in meiotic chromosome organization and recombination, yet the underlying protein components of this structure are highly diverged. Here, we show that 'axis core proteins' from budding yeast (Red1), mammals (SYCP2/SYCP3), and plants (ASY3/ASY4) are evolutionarily related and play equivalent roles in chromosome axis assembly. We first identify 'closure motifs' in each complex that recruit meiotic HORMADs, the master regulators of meiotic recombination. We next find that axis core proteins form homotetrameric (Red1) or heterotetrameric (SYCP2:SYCP3 and ASY3:ASY4) coiled-coil assemblies that further oligomerize into micron-length filaments. Thus, the meiotic chromosome axis core in fungi, mammals, and plants shares a common molecular architecture, and likely also plays conserved roles in meiotic chromosome axis assembly and recombination control

CRISPR/Cas9-induced (CTG⋅CAG)n repeat instability in the myotonic dystrophy type 1 locus: implications for therapeutic genome editing

Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5′ or 3′ unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1

Generating and repairing genetically programmed DNA breaks during immunoglobulin class switch recombination

Adaptive immune responses require the generation of a diverse repertoire of immunoglobulins (Igs) that can recognize and neutralize a seemingly infinite number of antigens. V(D)J recombination creates the primary Ig repertoire, which subsequently is modified by somatic hypermutation (SHM) and class switch recombination (CSR). SHM promotes Ig affinity maturation whereas CSR alters the effector function of the Ig. Both SHM and CSR require activation-induced cytidine deaminase (AID) to produce dU:dG mismatches in the Ig locus that are transformed into untemplated mutations in variable coding segments during SHM or DNA double-strand breaks (DSBs) in switch regions during CSR. Within the Ig locus, DNA repair pathways are diverted from their canonical role in maintaining genomic integrity to permit AID-directed mutation and deletion of gene coding segments. Recently identified proteins, genes, and regulatory networks have provided new insights into the temporally and spatially coordinated molecular interactions that control the formation and repair of DSBs within the Ig locus. Unravelling the genetic program that allows B cells to selectively alter the Ig coding regions while protecting non-Ig genes from DNA damage advances our understanding of the molecular processes that maintain genomic integrity as well as humoral immunity

DNA repair in cancer: emerging targets for personalized therapy

Genomic deoxyribonucleic acid (DNA) is under constant threat from endogenous and exogenous DNA damaging agents. Mammalian cells have evolved highly conserved DNA repair machinery to process DNA damage and maintain genomic integrity. Impaired DNA repair is a major driver for carcinogenesis and could promote aggressive cancer biology. Interestingly, in established tumors, DNA repair activity is required to counteract oxidative DNA damage that is prevalent in the tumor microenvironment. Emerging clinical data provide compelling evidence that overexpression of DNA repair factors may have prognostic and predictive significance in patients. More recently, DNA repair inhibition has emerged as a promising target for anticancer therapy. Synthetic lethality exploits intergene relationships where the loss of function of either of two related genes is nonlethal, but loss of both causes cell death. Exploiting this approach by targeting DNA repair has emerged as a promising strategy for personalized cancer therapy. In the current review, we focus on recent advances with a particular focus on synthetic lethality targeting in cancer

Formal Model and Simulation of the Gene Assembly Process in Ciliates

The construction process of the functional macronucleus in certain types of ciliates is known as the ciliate gene assembly process. It consists of a massive amount of DNA excision from the micronucleus and the rearrangement of the rest of the DNA sequences (in the case of stichotrichous ciliates). While several computational models have tried to represent certain parts of the gene assembly process, the real process remains not completely understood. In this research, a new formal model called the Computational 2JLP model is introduced based on the recent biological 2JLP model. For justifying the formal model, a simulation is created and tested with real data. Several parameters are introduced in the model that are used to test ambiguities or edge cases of the biological model. Parameters are systematically tested from the simulation to try to find their optimal values. Interestingly, a negative correlation is found between a parameter (which is used to filter out scnRNAs that are similar to IES specific sequences from the macronucleus) and the outcome of the simulation. It indicates that if a scnRNA consists of both an MDS and IES, then from the perspective of maximizing the outcome of the simulation, it is desirable to filter out this scnRNA. The simulator successfully performs the gene assembly process whether the inputs are scrambled or unscrambled DNA sequences. It is desirable for this model to serve as a foundation for future computational and mathematical study, and to help inform and refine the biological model

The Pathway to Detangle a Scrambled Gene

Programmed DNA elimination and reorganization frequently occur during cellular differentiation. Development of the somatic macronucleus in some ciliates presents an extreme case, involving excision of internal eliminated sequences (IESs) that interrupt coding DNA segments (macronuclear destined sequences, MDSs), as well as removal of transposon-like elements and extensive genome fragmentation, leading to 98% genome reduction in Stylonychia lemnae. Approximately 20-30% of the genes are estimated to be scrambled in the germline micronucleus, with coding segment order permuted and present in either orientation on micronuclear chromosomes. Massive genome rearrangements are therefore critical for development.To understand the process of DNA deletion and reorganization during macronuclear development, we examined the population of DNA molecules during assembly of different scrambled genes in two related organisms in a developmental time-course by PCR. The data suggest that removal of conventional IESs usually occurs first, accompanied by a surprising level of error at this step. The complex events of inversion and translocation seem to occur after repair and excision of all conventional IESs and via multiple pathways.This study reveals a temporal order of DNA rearrangements during the processing of a scrambled gene, with simpler events usually preceding more complex ones. The surprising observation of a hidden layer of errors, absent from the mature macronucleus but present during development, also underscores the need for repair or screening of incorrectly-assembled DNA molecules