RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes, enabling loss-of-function studies in living cells. Hairpin (hp) RNA expression cassettes are typically constructed on binary plasmids and delivered into plant cells by Agrobacterium-mediated genetic transformation. Realizing the importan...

Biological risk assessment of food containing recombinant DNA has exposed knowledge gaps related to the general fate of DNA in the gastrointestinal tract (GIT). Here, a series of experiments is presented that were designed to determine if genetic transformation of the naturally competent bacterium Acinetobacter baylyi BD413 occurs in the GIT of mice and rats, with feed-introduced bacterial DNA ...

Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial produ...

In crop research programs that implement transgene-based strategies for trait improvement it is necessary to distinguish between transgene homozygous and hemizygous individuals in segregating populations. Direct methods for determining transgene zygosity are technically challenging, expensive, and require specialized equipment. In this report, we describe a standard PCR-based protocol coupled w...

We constructed small high-yielding binary Ti vectors with a bacterial tetracycline resistance gene to facilitate efficient cloning afforded by the Gateway Technology (Invitrogen) for Agrobacterium tumefaciens-mediated transformation of higher plants. The Gateway Technology vectors are kanamycin-based, thus tetracycline-based destination and expression vectors are easily selected for the antibio...

A small microcosm, based on optimized in vitro transformation conditions, was used to study the ecological factors affecting the transformation of Acinetobacter calcoaceticus BD413 in soil. The transforming DNA used was A. calcoaceticus homologous chromosomal DNA with an inserted gene cassette containing a kanamycin resistance gene, nptII. The effects of soil type (silt loam or loamy sand), bac...

This study assessed the impact of Pinus radiata (D. Don) genetically modified (GM) by biolistic insertion of the LEAFY and nptII genes on rhizosphere microbial communities of field grown trees. Rhizosphere soil was sampled quarterly for two consecutive years. A culture-independent approach was used to characterise the microbial communities based on PCR and denaturing gradient gel electrophoresi...

A tripartite resistance-nodulation-cell division (RND) transporter system, called the PseABC efflux system, was identified at the left border of the syr-syp genomic island of Pseudomonas syringae pv. syringae strain B301D. The PseABC efflux system was located within a 5.7-kb operon that encodes an outer membrane protein (PseA), a periplasmic membrane fusion protein (PseB), and an RND-type cytop...

BACKGROUND In the Arabidopsis 26S proteasome mutant rpn12a- 1, an exon-trap T-DNA is inserted 531 base pairs downstream of the RPN12a STOP codon. We have previously shown that this insertion activates a STOP codon-associated latent 5' splice site that competes with the polyadenylation signal during processing of the pre-mRNA. As a result of this dual input from splicing and polyadenylation in t...

In the 26S proteasome mutant , an Background: Arabidopsis rpn12a-1 exon-trap T-DNA is inserted 531 base pairs downstream of the STOP RPN12a codon. We have previously shown that this insertion activates a STOP codon-associated latent 5' splice site that competes with the polyadenylation signal during processing of the pre-mRNA. As a result of this dual input from splicing and polyadenylation in ...