Long-term retention of surgically implanted radio transmitters in pikeperch

Radio-tagged pikeperch Stizostedion lucioperca (55–74 cm L _T) were recaptured in a reservoir by anglers 52–55 months after tagging. A total of four fish were recaptured during the summer of 2001. These recaptures are remarkable because of the long period between tagging and recapture and because only one (of 15 potential) tagged pikeperch had been caught in the long period since tagging.     

R-prime plasmids from Bradyrhizobium japonicum and Rhizobium fredii

The formation of R-prime plasmids was selected in crosses involving soybean microsymbionts with genomic Tn5 insertions and carrying plasmid pJB3JI (with one IS2) copy as donors and Escherichia coli HB101 as recipient. Whereas the parent plasmid was 60 kb, recombinant plasmids between 76 kb and 121 kb were obtained. Restriction and Southern analyses confirmed the mobilization of Tn5 on four R-primes from Bradyrhizobium japonicum I-110 and on an R-prime plasmid from Rhizobium fredii HH303. The largest R-prime plasmid was obtained from the rescue of two symbiotically defective R. fredii mutant strains that required adenosine.Non-standard abbreviation TDP transposon donor pool Scientific article number A-4728 and contribution number 7724 of the Maryland Agricultural Experiment Station     

Transposable element-induced mutations of the viviparous-1 gene in maize

The viviparous-1 (vp1) locus in maize is a developmental gene that controls diverse aspects of the maturation phase of seed development. Mutations of vp1 alter embryo sensitivity to the hormone abscisic acid and block formation of anthocyanin pigment. Molecular cloning of a Robertson Mutator-induced mutant allele, vp1-mum-1, by transposable element tagging has allowed analysis of several transposon-induced vp1 mutants. In the vp1-Mc mutation, the gene is disrupted by 4.0 kbp insertion, which results in expression of a 3′ truncated mRNA. Phenotypically, this allele is at least partially functional in causing embryo dormancy, but is ineffective in controlling anthocyanin expression. This result suggests that disruption of the C-terminal domain of the Vp1 protein specifically affects regulation of the anthocyanin pathway. A second Mutator- derived allele, vp1-mum2, exhibits an unusual form of somatic mutability in which endosperm cells revert from wild-type vp1 expression to a mutant condition. The vp1-mum2 allele contains a 1.5 kbp Insertion that has no detectable homology to known Mu elements. This element is retained In wild-type germinal revertants derived from vp1-mum2 An apparent DNA modification affecting cleavage at an internal Sstl restriction site in the element correlates with vp1-mum2 states that exhibit wild-type Vp1 expression. A model involving mitotic assortment of modified and unmodified DNA strands during development is proposed for vp1-mum2 somatic mutation.     

The maize autonomous element Activator (Ac) shows a minimal germinal excision frequency of 0.2%–0.5% in transgenic Arabidopsis thaliana plants

Summary The autonomous mobile element Activator from Zea mays was introduced into Arabidopsis thaliana via Agrobacterium-mediated gene transfer. The use of a chimaeric construct, where the Ac element is located in the leader of the neomycin phosphotransferase (NPT II) gene, enabled the excision of Ac to be monitored by assaying for the reconstitution of NPT II gene activity. Using this approach, the transpositional activity of AC was initially studied in primary transformants. About 50% of the regenerating Ac transformants showed evidence for excision of the element. Reintegration of Ac was confirmed by Southern blot analysis. Transposition events are transmitted to the F1 generation with a minimal frequency of 0.3%. In a few exceptional cases they are detected in a high proportion of the F1 generation. Seedlings from the F2 and F3 generations were assayed for the rate of germinal excisions by scoring for kanamycin resistance. The minimal frequency of germinal excision events amounts to 0.2%–0.5% and hence allows the use of the Ac element for gene tagging purposes in A. thaliana.     

Regulation of pyrimidine biosynthesis in Pseudomonas cepacia

Pyrimidine biosynthesis was investigated in Pseudomonas cepacia ATCC 17759. The presence of the de novo pyrimidine biosynthetic pathway enzyme activities was confirmed in this strain. Following transposon mutagenesis of the wild-type cells, a mutant strain deficient for orotidine 5-monophosphate decarboxylase activity (pyrF) was isolated. Uracil, cytosine or uridine supported the growth of this mutant. Uracil addition to minimal medium cultures of the wild-type strain diminished the levels of the de novo pyrimidine biosynthetic enzyme activities, while pyrimidine limitation of the mutant cells increased those de novo enzyme activities measured. It was concluded that regulation of pyrimidine biosynthesis at the lelel of enzyme synthesis in P. cepacia was present. Aspartate transcarbamoylase activity was found to be regulated in the wild-type cells. Its activity was shown to be controlled in vitro by inorganic pyrophosphate, adenosine 5-triphosphate and uridine 5-phosphate.     

Precise excision of bacterial transposon Tn5 in yeast

Summary We have demonstrated that precise excision of bacterial transposon Tn5 can occur in the yeast, Saccharomyces cerevisiae. Tn5 insertions in the yeast gene LYS2 were generated by transposon mutagenesis made in Escherichia coli by means of a ::Tn5 vector. Nine insertions of Tn5 into the structural part of the yeast LYS2 gene situated in a shuttle epsiomal plasmid were selected. All the plasmids with a Tn5 insertion were used to transform yeast strains carrying a deletion of the entire LYS2 gene or a deletion of the part of LYS2 overlapping the point of insertion.All insertions inactivated the LYS2 gene and were able to revert with low (about 10^-8) frequencies to lysine prototrophy. Restriction analysis of revertant plasmids revealed them to be indistinguishable from the original plasmid without Tn5 insertion. DNA sequencing of the regions containing the points of insertions, made for two revertants, proved that Tn5 excision was completely precise.     

Site-specific integration of genes into hot spots for recombination flanking aadA in Tn21 transposons.

Summary Tn21-related transposons are widespread among bacteria and carry various resistance determinants at preferential sites, hs1 and hs2. In an in vivo integrative recombination assay it was demonstrated that these hot spots direct the integration of aminoglycoside resistance genes like aadB from Klebsiella pneumoniae and aacAI from Serratia marcescens, in a recA ^– background. The maximum required recognition sequence which must be present in both the donor and recipient plasmids is 5 CTAAAACAAAGTTA 3 (hs2). The double-site-specific recombination occurred with a frequency of 10^–5–10^–6. The resulting structures include not only replicon fusion products but also more complex structures carrying two copies of the donor plasmid or simply the donor gene flanked by hs elements. hs1 and hs2 are thought to act as recognition sites for a trans-acting site-specific recombinase. By the use of Tn21 deletion derivatives, it has been shown that the recombinase is not encoded by Tn21. This new integrative recombination system is involved in the acquisition of new genes by Tn21-related transposons and their spread among bacterial populations.     

Insertional mutagenesis in Arabidopsis thaliana

Recently, a number of mutant gene loci in the Arabidopsis thaliana plant genome have been identified through insertional mutagenesis. In this review, we evaluate different methods used for Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis with regard to their mutation frequencies and conclude that a major breakthrough in the isolation of genes involved in plant development has been acheived. To provide a specific example, we summarize recent progress made in the understanding of flower morphogenesis at the molecular level through the study of homeotic genes obtained via gene tagging. T-DNA gene fusion vectors are being discussed that will allow the isolation of plant regulatory sequences with particular cell or tissue specificity, or that are controlled by specific external stimuli. Finally, we report on the approaches followed to convert the maize transposons Ac/Ds into valuable gene tags for use in a heterologous host such as Arabidopsis.