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A D Henk

Publications and source records attributed to A D Henk.

3 recordsLinked to original sources

A new Ac-like transposon of Arabidopsis is associated with a deletion of the RPS5 disease resistance gene.

The RPS5 and RFL1 disease resistance genes of Arabidopsis ecotype Col-0 are oriented in tandem and are separated by 1.4 kb. The Ler-0 ecotype contains RFL1, but lacks RPS5. Sequence analysis of the RPS5 deletion region in Ler-0 revealed the presence of an Ac-like transposable element, which we have designated Tag2. Southern hybridization analysis of six Arabidopsis ecotypes revealed 4-11 Tag2-homologous sequences in each, indicating that this element is ubiquitous in Arabidopsis and has been active in recent evolutionary time. The Tag2 insertion adjacent to RFL1 was unique to the Ler-0 ecotype, however, and was not present in two other ecotypes that lack RPS5. DNA sequence from the latter ecotypes lacked a transposon footprint, suggesting that insertion of Tag2 occurred after the initial deletion of RPS5. The deletion breakpoint contained a 192-bp insertion that displayed hallmarks of a nonhomologous DNA end-joining event. We conclude that loss of RPS5 was caused by a double-strand break and subsequent repair, and cannot be attributed to unequal crossing over between resistance gene homologs.

Amino Acid Sequence↗

RSF1010-based shuttle vectors for cloning and expression in Pasteurella multocida.

The broad host-range cloning vectors, pJRD215 and pMMB67EH, were evaluated for stability and cloning efficiency in Pasteurella multocida. Transformation of P. multocida by electroporation was unreliable and poorly efficient regardless of whether the transforming DNA was isolated from E. coli or P. multocida. Both vectors contain a mob site that enabled transfer by conjugation from E. coli to P. multocida with high efficiency. Kanamycin, streptomycin, and ampicillin resistance encoded by the vectors were expressed in P. multocida. LacZ was cloned in pMMB67EH, an expression vector, and was transferred to P. multocida by conjugation. The transconjugants expressed a functional beta-galactosidase as determined by o-nitrophenyl-beta-D-galactopyranoside (ONPG) test. We propose the use of these cosmid and expression vectors as a shuttle vectors for cloning in P. multocida.

Ampicillin Resistance↗

Tn10 insertional mutagenesis in Pasteurella multocida.

The objective of this study was to identify a transposon that will randomly and stably integrate in the genome of Pasteurella multocida. Using the suicide conjugative delivery system, pLOF, containing a kanamycin resistance marked Tn10 insertion element, we determined that Tn10 is effective in producing insertional mutations in this species. Combined conjugation/transposition events occurred once per 10,000 donor cells. Only 1.4% of the isolates resulted from vector integration and the genomic insertions were random and stable as determined by DNA/DNA hybridization after 10 serial subcultures lacking antibiotic selection. Twenty-two percent of the isolates were auxotrophic, 4% were tryptophan auxotrophs. Mutants derived by this method will be useful for studying putative virulence determinants of Pasteurella.

DNA Transposable Elements↗