Retrotransposon Tto1: functional analysis and engineering for insertional mutagenesis

Retrotransposons are genomic parasites activated by stress conditions that can be seriously detrimental for their host. In this work I demonstrate that Tto1, a typical plant LTR retrotransposon with insertion preference into genes can be turned into a synthetic molecular tool for gene tagging in plants and can be used to predict models for its replication steps. Although retrotransposons have been already used in plant mutagenesis, such application always required establishing protocols for tissue cultures and regeneration in vitro. Here, I show that sequence engineering of Tto1 provides the possibility to obtain transposition in vivo, with a simple screening method based on PCR and with the advantage to skip all in vitro manipulations. An artificial -estradiol inducible promoter has been used to obtain transposition “on demand” in Arabidopsis plants, which generates stable unlinked insertions that follow mendelian segregation in the progeny. Comparing serial deletions of 3’ LTR of the engineered inducible Tto1 (iTto1), I have mapped its two natural terminators and identified the “minimal” R (redundant) region required to achieve the complete reverse transcription of the genomic mRNA into a new cDNA copy. Interestingly, the transcripts ending at the major “early” terminator cannot support reverse transcription, suggesting a mechanism of natural control on the expression. Transcripts with a more extended termination point contain 100 essential nucleotides that define the active nucleus of the R region. This sequence promotes the formation of a stable hairpin structure that “kisses” a complementary identical hairpin on the cDNA and determines the formation of the characteristic cDNA/mRNA heteroduplex. Since the LTR is a repeated sequence the definition of a minimal redundant region has also the important implication to reduce the only possible target for sequence-based gene silencing, which should lead to an increase of the mutagenic efficiency of iTto1. Additional investigations have been carried out in attempt to identify points of improvement of iTto1 performances. By sequence alignment I identified different versions of the integrase that might have influence on insertion efficiency. Furthermore I tested the pOp6/LhGR-N system that will provide higher expression levels in different host plants. The final goal of my work is to extend the application of iTto1 to crop mutagenesis, therefore a big part of my work has been spent to develop Tto1 constructs with activity in barley. Transgenic plants have been obtained, however the constructs still need further experimentation

Classification of maximal transitive prolongations of super-Poincar\'e algebras

Let $V$ be a complex vector space with a non-degenerate symmetric bilinear form and $\mathbb S$ an irreducible module over the Clifford algebra $Cl(V)$ determined by this form. A supertranslation algebra is a $\mathbb Z$-graded Lie superalgebra $\mathfrak m=\mathfrak{m}_{-2}\oplus\mathfrak{m}_{-1}$, where $\mathfrak{m}_{-2}=V$ and $\mathfrak{m}_{-1}=\mathbb S\oplus\cdots\oplus\mathbb{S}$ is the direct sum of an arbitrary number $N\geq 1$ of copies of $\mathbb S$, whose bracket $[\cdot,\cdot]|_{\mathfrak{m}_{-1}\otimes \mathfrak{m}_{-1}}:\mathfrak{m}_{-1}\otimes\mathfrak{m}_{-1}\rightarrow\mathfrak{m}_{-2}$ is symmetric, $\mathfrak{so}(V)$-equivariant and non-degenerate (that is the condition "$s\in\mathfrak{m}_{-1}, [s,\mathfrak{m}_{-1}]=0$" implies $s=0$). We consider the maximal transitive prolongations in the sense of Tanaka of supertranslation algebras. We prove that they are finite-dimensional for $\dim V\geq3$ and classify them in terms of super-Poincar\'e algebras and appropriate $\mathbb Z$-gradings of simple Lie superalgebras.Comment: 32 pages, v2: general presentation improved, corrected several typos. Proofs and results unchanged. Final version to appear in Adv. Mat

A Reconstruction Procedure for Microwave Nondestructive Evaluation based on a Numerically Computed Green's Function

This paper describes a new microwave diagnostic tool for nondestructive evaluation. The approach, developed in the spatial domain, is based on the numerical computation of the inhomogeneous Green’s function in order to fully exploit all the available a-priori information of the domain under test. The heavy reduction of the computational complexity of the proposed procedure (with respect to standard procedures based on the free-space Green’s function) is also achieved by means of a customized hybrid-coded genetic algorithm. In order to assess the effectiveness of the method, the results of several simulations are presented and discussed

Percolation and lack of self-averaging in a frustrated evolutionary model

We present a stochastic evolutionary model obtained through a perturbation of Kauffman's maximally rugged model, which is recovered as a special case. Our main results are: (i) existence of a percolation-like phase transition in the finite phase space case; (ii) existence of non self-averaging effects in the thermodynamic limit. Lack of self-averaging emerges from a fragmentation of the space of all possible evolutions, analogous to that of a geometrically broken object. Thus the model turns out to be exactly solvable in the thermodynamic limit.Comment: 22 pages, 1 figur

Operator Product Expansion on the Lattice: a Numerical Test in the Two-Dimensional Non-Linear Sigma-Model

We consider the short-distance behaviour of the product of the Noether O(N) currents in the lattice nonlinear sigma-model. We compare the numerical results with the predictions of the operator product expansion, using one-loop perturbative renormalization-group improved Wilson coefficients. We find that, even on quite small lattices (m a \approx 1/6), the perturbative operator product expansion describes that data with an error of 5-10% in a large window 2a \ltapprox x \ltapprox m^{-1}. We present a detailed discussion of the possible systematic errors.Comment: 53 pages, 11 figures (26 eps files