A motif in the C-terminal domain of ϕC31 integrase controls the directionality of recombination

Bacteriophage ϕC31 encodes an integrase, which acts on the phage and host attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. In the absence of accessory factors, ϕC31 integrase cannot catalyse attL x attR recombination to excise the prophage. To understand the mechanism of directionality, mutant integrases were characterized that were active in excision. A hyperactive integrase, Int E449K, gained the ability to catalyse attL x attR, attL x attL and attR x attR recombination whilst retaining the ability to recombine attP x attB. A catalytically defective derivative of this mutant, Int S12A, E449K, could form stable complexes with attP/attB, attL/attR, attL/attL and attR/attR under conditions where Int S12A only complexed with attP/attB. Further analysis of the Int E449K-attL/attR synaptic events revealed a preference for one of the two predicted synapse structures with different orientations of the attL/attR sites. Several amino acid substitutions conferring hyperactivity, including E449K, were localized to one face of a predicted coiled-coil motif in the C-terminal domain. This work shows that a motif in the C-terminal domain of ϕC31 integrase controls the formation of the synaptic interface in both integration and excision, possibly through a direct role in protein–protein interactions

The chloroplast transformation toolbox: Selectable markers and marker removal

Plastid transformation is widely used in basic research and for biotechnological applications. Initially developed in Chlamydomonas and tobacco, it is now feasible in a broad range of species. Selection of transgenic lines where all copies of the polyploid plastid genome are transformed requires efficient markers. A number of traits have been used for selection such as photoautotrophy, resistance to antibiotics and tolerance to herbicides or to other metabolic inhibitors. Restoration of photosynthesis is an effective primary selection method in Chlamydomonas but can only serve as a screening tool in flowering plants. The most successful and widely used markers are derived from bacterial genes that inactivate antibiotics, such as aadA that confers resistance to spectinomycin and streptomycin. For many applications, the presence of a selectable marker that confers antibiotic resistance is not desirable. Efficient marker removal methods are a major attraction of the plastid engineering tool kit. They exploit the homologous recombination and segregation pathways acting on chloroplast genomes and are based on direct repeats, transient co-integration or co-transformation and segregation of trait and marker genes. Foreign site-specific recombinases and their target sites provide an alternative and effective method for removing marker genes from plastids

Recombinase technology: applications and possibilities

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes

Arabidopsis mutants revealing novel aspects of leaf morphogenesis

The establishment of dorsoventrality along the adaxial-abaxial axis is considered an early and crucial step of leaf development and proposed as a requirement for subsequent leaf blade expansion. Because photosynthesis mainly takes place in leaves, several key determinants are required to ensure the proper formation of mature leaves. Several efforts have been dedicated to elucidate the regulatory networks controlling leaf morphogenesis. In this thesis, characterization of novel regulators for both leaf polarity establishment and leaf blade expansion is described. Several Arabidopsis leaf polarity mutants have been identified. However, none of the single, recessive mutants display polarity defects similar to phantastica (phan), the first leaf polarity mutant in Antirrhinum majus. Characterization of filliforme (flr) indicates that flr may be the missing counterpart of phan. flr exhibited the abaxial features in the radialized organs as well as disturbed shoot apical meristem organization. Gene expression analysis revealed the alteration of expression levels of several key regulatory genes. Remarkably, FLR encodes a plastid membrane protein, underlining the roles of plastids in Arabidopsis growth and development. sensitive to red light reduced (srr) 1-2 mutants displayed a narrow-leaf phenotype in addition to defects in light perception and circadian clock regulation. This suggests a connection between leaf blade expansion and the circadian clock. Due to the lack of recognizable functional domains, molecular functions of SRR1 remain unknown. However, genetic interactions between srr1-2 and translational machinery mutants indicate the involvement of SRR1 in the translational regulation of leaf morphogenesis. Previous studies have demonstrated the importance of a translational regulation during leaf morphogenesis in Arabidopsis. Mutations in several ribosomal protein genes similarly result in a pointed-leaf phenotype. Characterization of pointed-first-leaves (pfl) 2-2 mutants, in which the ribosomal protein gene RPS13 is disrupted, revealed perturbation in both adaxial and abaxial mesophyll layers. This mutation in pfl2-2 also significantly enhanced polarity defects in several leaf polarity mutants. These results suggest that RPS13 contributes along with other regulatory genes in establishing or maintaining leaf polarity. Together, genetic approaches present in this dissertation expose unexpected contribution of plastids, the circadian clock and translational machineries on leaf morphogenesis.Ph.D.Includes bibliographical referencesIncludes vitaby Chokchai Kittiwongwattan