Strategies of Loop Recombination in Ciliates

Gene assembly in ciliates is an extremely involved DNA transformation process, which transforms a nucleus, the micronucleus, to another functionally different nucleus, the macronucleus. In this paper we characterize which loop recombination operations (one of the three types of molecular operations that accomplish gene assembly) can possibly be applied in the transformation of a given gene from its micronuclear form to its macronuclear form. We also characterize in which order these loop recombination operations are applicable. This is done in the abstract and more general setting of so-called legal strings.Comment: 22 pages, 14 figure

Maximal Pivots on Graphs with an Application to Gene Assembly

We consider principal pivot transform (pivot) on graphs. We define a natural variant of this operation, called dual pivot, and show that both the kernel and the set of maximally applicable pivots of a graph are invariant under this operation. The result is motivated by and applicable to the theory of gene assembly in ciliates.Comment: modest revision (including different latex style) w.r.t. v2, 16 pages, 5 figure

"Going back to our roots": second generation biocomputing

Researchers in the field of biocomputing have, for many years, successfully "harvested and exploited" the natural world for inspiration in developing systems that are robust, adaptable and capable of generating novel and even "creative" solutions to human-defined problems. However, in this position paper we argue that the time has now come for a reassessment of how we exploit biology to generate new computational systems. Previous solutions (the "first generation" of biocomputing techniques), whilst reasonably effective, are crude analogues of actual biological systems. We believe that a new, inherently inter-disciplinary approach is needed for the development of the emerging "second generation" of bio-inspired methods. This new modus operandi will require much closer interaction between the engineering and life sciences communities, as well as a bidirectional flow of concepts, applications and expertise. We support our argument by examining, in this new light, three existing areas of biocomputing (genetic programming, artificial immune systems and evolvable hardware), as well as an emerging area (natural genetic engineering) which may provide useful pointers as to the way forward.Comment: Submitted to the International Journal of Unconventional Computin

Telomere structure and telomerase in health and disease

Telomerase is the enzyme responsible for maintenance of the length of telomeres by addition of guanine-rich repetitive sequences. Telomerase activity is exhibited in gametes and stem and tumor cells. In human somatic cells, proliferation potential is strictly limited and senescence follows approximately 50-70 cell divisions. In most tumor cells, on the contrary, replication potential is unlimited. The key role in this process of the system of the telomere length maintenance with involvement of telomerase is still poorly studied. Undoubtedly, DNA polymerase is not capable of completely copying DNA at the very ends of chromosomes; therefore, approximately 50 nucleotides are lost during each cell cycle, which results in gradual telomere length shortening. Critically short telomeres cause senescence, following crisis and cell death. However, in tumor cells the system of telomere length maintenance is activated. Much work has been done regarding the complex telomere/telomerase as a unique target, highly specific in cancer cells. Telomeres have additional proteins that regulate the binding of telomerase. Telomerase, also associates with a number of proteins forming the sheltering complex having a central role in telomerase activity. This review focuses on the structure and function of the telomere/telomerase complex and its altered behavior leading to disease, mainly cancer. Although telomerase therapeutics are not approved yet for clinical use, we can assume that based on the promising in vitro and in vivo results and successful clinical trials, it can be predicted that telomerase therapeutics will be utilized soon in the combat against malignancies and degenerative diseases. The active search for modulators is justified, because the telomere/telomerase system is an extremely promising target offering possibilities to decrease or increase the viability of the cell for therapeutic purposes.Fil: Gomez, Daniel Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Armando, Romina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Farina, Hernán Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Lorenzano Menna, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Cerrudo, Carolina Susana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular; ArgentinaFil: Ghiringhelli, Pablo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular; ArgentinaFil: Alonso, Daniel Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentin

Regulation of Antigenic Variation by Trypanosoma brucei Telomere Proteins Depends on Their Unique DNA Binding Activities

Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, Variant Surface Glycoprotein (VSG), to evade the host immune response. Such antigenic variation is a key pathogenesis mechanism that enables T. brucei to establish long-term infections. VSG is expressed exclusively from subtelomere loci in a strictly monoallelic manner, and DNA recombination is an important VSG switching pathway. The integrity of telomere and subtelomere structure, maintained by multiple telomere proteins, is essential for T. brucei viability and for regulating the monoallelic VSG expression and VSG switching. Here we will focus on T. brucei TRF and RAP1, two telomere proteins with unique nucleic acid binding activities, and summarize their functions in telomere integrity and stability, VSG switching, and monoallelic VSG expression. Targeting the unique features of TbTRF and TbRAP10 s nucleic acid binding activities to perturb the integrity of telomere structure and disrupt VSG monoallelic expression may serve as potential therapeutic strategy against T. brucei

Models of natural computation : gene assembly and membrane systems

This thesis is concerned with two research areas in natural computing: the computational nature of gene assembly and membrane computing. Gene assembly is a process occurring in unicellular organisms called ciliates. During this process genes are transformed through cut-and-paste operations. We study this process from a theoretical point of view. More specifically, we relate the theory of gene assembly to sorting by reversal, which is another well-known theory of DNA transformation. In this way we obtain a novel graph-theoretical representation that provides new insights into the nature of gene assembly. Membrane computing is a computational model inspired by the functioning of membranes in cells. Membrane systems compute in a parallel fashion by moving objects, through membranes, between compartments. We study the computational power of various classes of membrane systems, and also relate them to other well-known models of computation.Netherlands Organisation for Scientific Research (NWO), Institute for Programming research and Algorithmics (IPA)UBL - phd migration 201

Telomere-associated proteins in Arabidopsis thaliana

Telomeres comprise the physical ends of chromosomes. Essential functions of telomeres include protecting the terminus from being recognized as a DNA doublestrand break and facilitating the complete replication of the physical end of the DNA. Telomere functions are mediated by a large array of telomere-associated proteins. Mutations in telomere-related genes cause immediate telomere dysfunction, activation of DNA damage response, and accumulation of end-to-end chromosome fusions. In addition, changes in telomere complex composition may affect the ability of the telomerase enzyme to maintain telomeres in vivo. Here, we describe the characterization of telomere-associated proteins in the flowering plant, Arabidopsis thaliana. Using a bioinformatics approach, we identified twelve proteins with sequence similarity to vertebrate duplex telomere DNA binding proteins TRF1 and TRF2. We showed that, like their vertebrate counterparts, some of the Arabidopsis TRFL (TRF-LIKE) proteins can homodimerize and bind telomeric DNA in vitro, indicating that Arabidopsis encodes a large family of double-strand telomeric DNA binding proteins. We have also characterized three Arabidopsis POT1 proteins whose homologs in yeast and vertebrates associate with the single-stranded portion of telomeric DNA. Unexpectedly, we found that unlike POT1 protein in other organisms, Arabidopsis AtPOT1a protein associates with telomeres only in the S phase of the cell cycle and is a physical component of the active telomerase RNP complex, providing positive telomere length regulation. Our data implicated AtPOT1b, another Arabidopsis POT1 protein, in chromosome end protection. Finally, we showed that Arabidopsis thaliana has evolved a third POT1 protein, AtPOT1c, which contributes to both telomere length regulation and telomerase activity, and maintenance of the structure of the chromosome terminus. Thus, Arabidopsis has evolved a set of POT1 proteins that make distinct and novel contributions to telomere biology. Finally, we describe the identification and characterization of a novel Arabidopsis protein CIT1 (Critical for Integrity of Telomeres 1), and show that CIT1 deficiency leads to an immediate and profound telomere dysfunction and chromosome end deprotection. Altogether, these data provide new insight into plant telomereassociated factors and significantly improve our understanding of the overall architecture and evolution of telomeric complex in Arabidopsis

Read, write, adapt:Challenges and opportunities during kinetoplastid genome replication

The genomes of all organisms are read throughout their growth and development, generating new copies during cell division and encoding the cellular activities dictated by the genome’s content. However, genomes are not invariant information stores but are purposefully altered in minor and major ways, adapting cellular behaviour and driving evolution. Kinetoplastids are eukaryotic microbes that display a wide range of such read–write genome activities, in many cases affecting critical aspects of their biology, such as host adaptation. Here we discuss the range of read–write genome changes found in two well-studied kinetoplastid parasites, Trypanosoma brucei and Leishmania, focusing on recent work that suggests such adaptive genome variation is linked to novel strategies the parasites use to replicate their unconventional genomes

Characterization of the Arabidopsis Telomerase Negative Regulator: A TE-Containing lncRNA TER2- Defining Its Role in and beyond Telomere Biology

In eukaryotes the ends of chromosomes are constituted by nucleoprotein complexes termed telomeres. Telomeres represses a DNA damage response and, more importantly, facilitate the maintenance of the terminal DNA sequence by telomerase. Telomerase activity can be reconstituted by its two core subunits, the catalytic reverse transcriptase TERT and the telomerase RNA TER. The Shippen lab developed Arabidopsis as a model for telomere studies. Previous work in Shippen lab showed that an alternative copy of telomerase RNA, TER2, serves as a negative regulator of the telomerase in response to DNA damage. In this study I characterized the evolution and function of TER2 and explored its biological significance. TER2 possesses an intron and analysis of sequences from the 1001 genome project showed that the TER2 intron is derived from a transposable element (TE), specifically long terminal repeat (LTR) of a Copia-like retrotransposon. I verified that in most A. thaliana accessions the TER2 TE is intact, while in about 10% of accessions it is missing. The TE within TER2 destabilizes this RNA, enabling the plant to down regulate telomerase activity by modulating TER2 abundance. This RNA stability control mechanism contributes to the accumulation of TER2 after DNA damage, and thus links telomerase regulation directly to the DNA damage response. My results also showed that TER2 is developmentally regulated, but only in accessions that contain the TER2 TE, suggesting the exaptation of the TE endows TER2 with a function in reproductive development. Indeed, plants lacking TER2 have reduced seed production efficiency. In addition, ter2 mutants have lower pollen viability than wild type, though not as low as in tert mutants. These results defined a novel function of TER2 in plant reproduction. My work unexpectedly revealed that TER2 processing and/or stability is influenced by the small RNA processing pathway. In plants lacking Dicer-like2 (DCL2), TER2 abundance increases and the expression profile change during flower development. I further discovered that DCL2 affects TER2 in a post-transcriptional manner. Together, these data uncovered unexpected complexity of TER2 RNA processing and its regulation. Finally, I found that TER1, the canonical TER in A. thaliana, has lessons to teach. Single nucleotide polymorphisms (SNPs) in TER1 telomere templating domain were found. I showed that the SNPs do not change the newly synthesized telomere repeats. This observation provided new insight into the mechanisms of template utilization and how this is evolving. In summary, my research revealed evidence for evolution in two telomerase RNA genes in A. thaliana, and provided several novel insights into lncRNA structure, evolution and metabolism that impact telomerase regulation and benefit plant growth and reproduction

First molecular evidence of hybridization in endosymbiotic ciliates (Protista, Ciliophora)

Hybridization is an important evolutionary process that can fuel diversification via formation of hybrid species or can lead to fusion of previously separated lineages by forming highly diverse species complexes. We provide here the first molecular evidence of hybridization in wild populations of ciliates, a highly diverse group of free-living and symbiotic eukaryotic microbes. The impact of hybridization was studied on the model of Plagiotoma, an obligate endosymbiont of the digestive tube of earthworms, using split decomposition analyses and species networks, 2D modeling of the nuclear rRNA molecules and compensatory base change analyses as well as multidimensional morphometrics. Gene flow slowed down and eventually hampered the diversification of Lumbricus-dwelling plagiotomids, which collapsed into a single highly variable biological entity, the P. lumbrici complex. Disruption of the species boundaries was suggested also by the continuum of morphological variability in the phenotypic space. On the other hand, hybridization conspicuously increased diversity in the nuclear rDNA cistron and somewhat weakened the host structural specificity of the P. lumbrici complex, whose members colonize a variety of phylogenetically closely related anecic and epigeic earthworms. By contrast, another recorded species, P. aporrectodeae sp. n., showed no signs of introgression, no variability in the rDNA cistron, and very high host specificity. These contrasting eco-evolutionary patterns indicate that hybridization might decrease the alpha-diversity by dissolving species boundaries, weaken the structural host specificity by broadening ecological amplitudes, and increase the nuclear rDNA variability by overcoming concerted evolution within the P. lumbrici species complex