PubMed Health⌕ Search

Biomedical subjects

Jan-Peter Nap

Publications and source records attributed to Jan-Peter Nap.

7 recordsLinked to original sources
PubMed2003

A self-excising Cre recombinase allows efficient recombination of multiple ectopic heterospecific lox sites in transgenic tobacco.

To study the impact of different DNA configurations on the stability of transgene expression, a variant of the cre gene was developed. This variant allows for the highly efficient in planta removal of its own loxP-flanked coding sequence as well as other DNAs flanked by ectopic heterospecific lox sites, either lox511 or lox2272 or both, in trans. The plant intron-containing cre gene, creINT, was configured in such a way that self-excision generated an intact hygromycin resistance selectable marker gene. In this combination, all selected transformants showed highly efficient excision. Plants obtained showed no indication of any chimerism, indicating a cell autonomous nature of the hygromycin selection during transformation and regeneration. The highly efficient concomitant removal of wildtype and heterospecific lox site-flanked DNA demonstrated that upon retransformation with the self-excising creINT, sufficient amounts of Cre enzyme were produced prior to its removal. Plants obtained with creINT showed much less frequently the Cre-associated phenomenon of reduced fertility than plants obtained with a continuous presence of Cre recombinase. The creINT system has therefore advantages over systems with a continuously present Cre. The creINT system was successfully used for removal of two chromatin boundary elements from transgene cassettes in tobacco. Analysis of plants with and without boundary elements on the same chromosomal location will contribute to a better evaluation of the role of such elements in the regulation of transgene expression in plants.

Anti-Bacterial Agents↗
PubMed2003

The release of genetically modified crops into the environment. Part II. Overview of ecological risk assessment.

Despite numerous future promises, there is a multitude of concerns about the impact of GM crops on the environment. Key issues in the environmental assessment of GM crops are putative invasiveness, vertical or horizontal gene flow, other ecological impacts, effects on biodiversity and the impact of presence of GM material in other products. These are all highly interdisciplinary and complex issues. A crucial component for a proper assessment is defining the appropriate baseline for comparison and decision. For GM crops, the best and most appropriately defined reference point is the impact of plants developed by traditional breeding. The latter is an integral and accepted part of agriculture. In many instances, the putative impacts identified for GM crops are very similar to the impacts of new cultivars derived from traditional breeding. When assessing GM crops relative to existing cultivars, the increased knowledge base underpinning the development of GM crops will provide greater confidence in the assurances plant science can give on the risks of releasing such crops.

Crops, Agricultural↗
PubMed2003

The release of genetically modified crops into the environment. Part I. Overview of current status and regulations.

In the past 6 years, the global area of commercially grown, genetically modified (GM) crops has increased more than 30-fold to over 52 million hectares. The number of countries involved has more than doubled. Especially in developing countries, the GM crop area is anticipated to increase rapidly in the coming years. Despite this high adoption rate and future promises, there is a multitude of concerns about the impact of GM crops on the environment. Regulatory approaches in Europe and North America are essentially different. In the EU, it is based on the process of making GM crops; in the US, on the characteristics of the GM product. Many other countries are in the process of establishing regulation based on either system or a mixture. Despite these differences, the information required for risk assessment tends to be similar. Each risk assessment considers the possibility, probability and consequence of harm on a case-by-case basis. For GM crops, the impact of non-use should be added to this evaluation. It is important that the regulation of risk should not turn into the risk of regulation. The best and most appropriate baseline for comparison when performing risk assessment on GM crops is the impact of plants developed by traditional breeding. The latter is an integral and accepted part of agriculture.

Biotechnology↗
PubMed2003

The presence of a chromatin boundary appears to shield a transgene in tobacco from RNA silencing.

We present isogenic transgenic tobacco lines that carry at a given chromosomal position a beta-glucuronidase (GUS) reporter gene either with or without the presence of the matrix-associated region known as the chicken lysozyme A element. Plants were generated with the Cre-lox site-specific recombination system using heterospecific lox sites. Analysis of GUS gene expression in plant populations demonstrates that the presence of the A element can shield against RNA silencing of the GUS gene. Protection was observed in two of three independent tobacco transformants. Plants carrying an A element 5' of the GUS gene always had stable GUS activity, but upon removal of this A element, the GUS gene became silenced over time in two lines, notably when homozygous.

Chromatin↗
PubMed2002

The promiscuity of heterospecific lox sites increases dramatically in the presence of palindromic DNA.

Heterospecific lox sites are mutated lox sites that in the presence of Cre recombinase recombine with themselves but not or much less with wildtype loxP. We here show that in Escherichia coli both lox511 and lox2272 sites become highly promiscuous with respect to loxP when in the presence of Cre one of the recombination partners is present in a larger stretch of an inverted repeat of non-lox DNA. In such a palindromic DNA configuration, also the occurrence of other DNA repeat-mediated recombination events is somewhat increased in the presence of Cre. The results indicate that in recombinase mediated cassette exchange or other double lox applications based on the exclusivity of heterospecific lox sites, or in research combining Cre-lox approaches with hairpin RNA for gene silencing, the presence of duplicated DNA around lox sites has to be taken into account. It is proposed that the presence of palindromic non-lox DNA interferes with the homology search of the Cre enzyme prior to the actual recombination event.

Binding Sites↗
PubMed2002

Assembly of two transgenes in an artificial chromatin domain gives highly coordinated expression in tobacco.

The chromatin loop model predicts that genes within the same chromatin domain exhibit coordinated regulation. We here present the first direct experimental support for this model in plants. Two reporter genes, the E. coli beta-glucuronidase gene and the firefly luciferase gene, driven by different promoters, were placed between copies of the chicken lysozyme A element, a member of the matrix-associated region (MAR) group of chromatin boundary elements, and introduced in tobacco (Nicotiana tabacum). In plants carrying A elements, quantitative enzyme activities and mRNA levels of both genes show high correlations compared to control plants. The A element thus creates an artificial chromatin domain that yields coordinated expression. Surprisingly, enzyme activities correlated poorly with their respective mRNA levels. We hypothesize that this indicates the occurrence of "error pipelines" in data generation: systematic errors of a given analytical method will point in the same direction and cancel out in correlation analysis, resulting in better correlations. In combining different methods of analysis, however, such errors do not cancel out and as a result relevant correlations can be masked. Such error pipelines will have to be taken into account when different types of (e.g., whole-genome) data sets are combined in quantitative analyses.

Chromatin↗