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Biomedical subjects

Caius M Rommens

Publications and source records attributed to Caius M Rommens.

7 recordsLinked to original sources
PubMed2006

Improving potato storage and processing characteristics through all-native DNA transformation.

The dominant potato (Solanum tuberosum) variety for French fry production in the United States is the 131-year-old Russet Burbank. Market penetration of the higher yielding and more uniform Ranger Russet variety is limited to about one-fifth of that of the Russet Burbank because of two storage deficits: black spot bruise sensitivity and high levels of cold-induced sweetening. Here, these trait weaknesses are turned into strengths by simultaneously lowering the expression of Ranger Russet's tuber-expressed polyphenol oxidase (Ppo), starch-associated R1, and phosphorylase-L (PhL) genes. This genetic modification was accomplished without inserting any foreign DNA into the plant genome. French fries from the intragenic potatoes also contained reduced amounts of the antinutritional compound acrylamide while, unexpectedly, displaying enhanced sensory characteristics.

DNA, Plant↗
PubMed2006

Transposition-based plant transformation.

Agrobacterium T-DNAs were used to deliver transposable Dissociation (Ds) elements into the nuclei of potato (Solanum tuberosum) cells. A double-selection system was applied to enrich for plants that only contained a transposed Ds element. This system consisted of a positive selection for the neomycin phosphotransferase (nptII) gene positioned within Ds followed by a negative selection against stable integration of the cytosine deaminase (codA) gene-containing T-DNA. Sixteen of 29 transgenic plants were found to contain a transposed element while lacking any superfluous T-DNA sequences. The occurrence of this genotype indicates that Ds elements can transpose from relatively short extrachromosomal DNA molecules into the plant genome. The frequency of single-copy Ds transformation was determined at 0.3%, which is only about 2.5-fold lower than the potato transformation frequency for backbone-free and single-copy T-DNAs. Because of the generally high expression levels of genes positioned within transposed elements, the new transformation method may find broad applicability to crops that are accessible to Agrobacterium T-DNA transfer.

DNA Transposable Elements↗
PubMed2006

Kanamycin resistance in plants: an unexpected trait controlled by a potentially multifaceted gene.

Ayalew Mentewab and C. Neal Stewart Jr recently showed that an Arabidopsis kanamycin resistance gene encodes an ATP binding cassette (ABC) transporter. This Atwbc19 protein is hypothesized to prevent ribosome inactivation by translocating kanamycin into the vacuole. Because ABC transporters often recognize multiple exogenous substrates, overexpression of Atwbc19 can result in the accumulation of unexpected compounds in transgenic plants. Another potential safety issue associated with this gene is horizontal gene transfer. Thus, commercial applications are likely to be limited to methods that allow removal of the selectable marker from the transgenic plant genome.

ATP-Binding Cassette Transporters↗
PubMed2006

New construct approaches for efficient gene silencing in plants.

An important component of conventional sense, antisense, and double-strand RNA-based gene silencing constructs is the transcriptional terminator. Here, we show that this regulatory element becomes obsolete when gene fragments are positioned between two oppositely oriented and functionally active promoters. The resulting convergent transcription triggers gene silencing that is at least as effective as unidirectional promoter-to-terminator transcription. In addition to short, variably sized, and nonpolyadenylated RNAs, terminator-free cassette produced rare, longer transcripts that reach into the flanking promoter. These read-through products did not influence the efficacy and expression levels of the neighboring hygromycin phosphotransferase gene. Replacement of gene fragments by promoter-derived sequences further increased the extent of gene silencing. This finding indicates that genomic DNA may be a more efficient target for gene silencing than gene transcripts.

Gene Silencing↗
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↗