Biomedical subjects

Jaime M Humara

Publications and source records attributed to Jaime M Humara.

3 recordsLinked to original sources

PubMed2005

Plant-derived transfer DNAs.

The transfer of DNA from Agrobacterium to plant cell nuclei is initiated by a cleavage reaction within the 25-bp right border of Ti plasmids. In an effort to develop all-native DNA transformation vectors, 50 putative right border alternatives were identified in both plant expressed sequence tags and genomic DNA. Efficacy tests in a tobacco (Nicotiana tabacum) model system demonstrated that 14 of these elements displayed at least 50% of the activity of conventional Agrobacterium transfer DNA borders. Four of the most effective plant-derived right border alternatives were found to be associated with intron-exon junctions. Additional elements were embedded within introns, exons, untranslated trailers, and intergenic DNA. Based on the identification of a single right border alternative in Arabidopsis and three in rice (Oryza sativa), the occurrence of this motif was estimated at a frequency of at least 0.8x10(-8). Modification of plasmid DNA sequences flanking the alternative borders demonstrated that both upstream and downstream sequences play an important role in initiating DNA transfer. Optimal DNA transfer required the elements to be preceded by pyrimidine residues interspaced by AC-rich trinucleotides. Alteration of this organization lowered transformation frequencies by 46% to 93%. Despite their weaker resemblance with left borders, right border alternatives also functioned effectively in terminating DNA transfer, if both associated with an upstream A[C/T]T[C/G]A[A/T]T[G/T][C/T][G/T][C/G]A[C/T][C/T][A/T] domain and tightly linked cytosine clusters at their junctions with downstream DNA. New insights in border region requirements were used to construct an all-native alfalfa (Medicago sativa) transfer DNA vector that can be used for the production of intragenic plants.

Amino Acid Sequence↗

PubMed2004

Crop improvement through modification of the plant's own genome.

Plant genetic engineering has, until now, relied on the incorporation of foreign DNA into plant genomes. Public concern about the extent to which transgenic crops differ from their traditionally bred counterparts has resulted in molecular strategies and gene choices that limit, but not eliminate, the introduction of foreign DNA. Here, we demonstrate that a plant-derived (P-) DNA fragment can be used to replace the universally employed Agrobacterium transfer (T-) DNA. Marker-free P-DNAs are transferred to plant cell nuclei together with conventional T-DNAs carrying a selectable marker gene. By subsequently linking a positive selection for temporary marker gene expression to a negative selection against marker gene integration, 29% of derived regeneration events contain P-DNA insertions but lack any copies of the T-DNA. Further refinements are accomplished by employing Omega-mutated virD2 and isopentenyl transferase cytokinin genes to impair T-DNA integration and select against backbone integration, respectively. The presented methods are used to produce hundreds of marker-free and backbone-free potato (Solanum tuberosum) plants displaying reduced expression of a tuber-specific polyphenol oxidase gene in potato. The modified plants represent the first example of genetically engineered plants that only contain native DNA.

Base Sequence↗

PubMed2003

Identification of Arabidopsis rat mutants.

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various "reverse genetic" approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.

Agrobacterium tumefaciens↗