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

Complete Nucleotide Sequence and Organization of the Atrazine Catabolic Plasmid pADP-1 from Pseudomonas sp. Strain ADP

The complete 108,845-nucleotide sequence of catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP was determined. Plasmid pADP-1 was previously shown to encode AtzA, AtzB, and AtzC, which catalyze the sequential hydrolytic removal of s-triazine ring substituents from the herbicide atrazine to yield cyanuric acid. Computational analyses indicated that pADP-1 encodes 104 putative open reading frames (ORFs), which are predicted to function in catabolism, transposition, and plasmid maintenance, transfer, and replication. Regions encoding transfer and replication functions of pADP-1 had 80 to 100% amino acid sequence identity to pR751, an IncPβ plasmid previously isolated from Enterobacter aerogenes. pADP-1 was shown to contain a functional mercury resistance operon with 99% identity to Tn5053. Complete copies of transposases with 99% amino acid sequence identity to TnpA from IS1071 and TnpA from Pseudomonas pseudoalcaligenes were identified and flank each of the atzA, atzB, and atzC genes, forming structures resembling nested catabolic transposons. Functional analyses identified three new catabolic genes, atzD, atzE, and atzF, which participate in atrazine catabolism. Crude extracts from Escherichia coli expressing AtzD hydrolyzed cyanuric acid to biuret. AtzD showed 58% amino acid sequence identity to TrzD, a cyanuric acid amidohydrolase, from Pseudomonas sp. strain NRRLB-12227. Two other genes encoding the further catabolism of cyanuric acid, atzE and atzF, reside in a contiguous cluster adjacent to a potential LysR-type transcriptional regulator. E. coli strains bearing atzE and atzF were shown to encode a biuret hydrolase and allophanate hydrolase, respectively. atzDEF are cotranscribed. AtzE and AtzF are members of a common amidase protein family. These data reveal the complete structure of a catabolic plasmid and show that the atrazine catabolic genes are dispersed on three disparate regions of the plasmid. These results begin to provide insight into how plasmids are structured, and thus evolve, to encode the catabolism of compounds recently added to the biosphere

Transposons Tn1696 and Tn21 and Their Integrons In4 and In2 Have Independent Origins

The first 13.6 kb of the mercury and multidrug resistance transposon Tn1696, which includes the class 1 integron In4, has been sequenced. In4 is 8.33 kb long and contains the 5′-conserved segment (5′-CS) and 2.24 kb of the 3′-conserved segment (3′-CS) flanking four integrated cassettes. The 3′-CS region is followed by one full copy and an adjacent partial copy of the insertion sequence IS6100 flanked, in inverse orientation, by two short segments (123 and 152 bp) from the outer right-hand end of class 1 integrons. This structure is representative of a distinct group of class 1 integrons that differs from In2, found in Tn21, and other related class 1 integrons. In4 does not include transposition genes but is bounded by characteristic 25-bp inverted repeats and flanked by a direct duplication of 5 bp of the target sequence, indicating that it was inserted by a transpositional mechanism. In4 lies between the resII and resI sites of a backbone mercury resistance transposon which is >99.5% identical to Tn5036. Although Tn21 and Tn1696 are both classified as members of the Tn21 subfamily of the Tn3 transposon family, the backbone mercury resistance transposons are only 79 to 96% identical. Tn21 also contains a region of about 0.7 kb not found in Tn1696. The integrons In2 and In4 carrying the antibiotic resistance genes have been inserted at different locations into distinct ancestral mercury resistance transposons. Thus, Tn21 and Tn1696 have independent histories and origins. Other transposons (Tn1403 and Tn1412) that include a class 1 integron also have independent origins. In all except Tn21, the integron is located within the res region of the backbone transposon