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J P Nap

Publications and source records attributed to J P Nap.

13 recordsLinked to original sources
PubMed2001

Genetical genomics: the added value from segregation.

The recent successes of genome-wide expression profiling in biology tend to overlook the power of genetics. We here propose a merger of genomics and genetics into 'genetical genomics'. This involves expression profiling and marker-based fingerprinting of each individual of a segregating population, and exploits all the statistical tools used in the analysis of quantitative trait loci. Genetical genomics will combine the power of two different worlds in a way that is likely to become instrumental in the further unravelling of metabolic, regulatory and developmental pathways.

Animals↗
PubMed2001

The effect of MAR elements on variation in spatial and temporal regulation of transgene expression.

The level of transgene expression often differs among independent transformants. This is generally ascribed to different integration sites of the transgene into the plant genome in each independently obtained transformant (position effect). It has been shown that in tobacco transformants expressing, for example, a cauliflower mosaic virus (CaMV) 35S promoter-driven beta-glucuronidase (GUS) reporter gene, these position-induced quantitative differences among individual transformants were reduced by the introduction of matrix-associated regions (MAR elements) on the T-DNA. We have previously shown by imaging of in planta firefly luciferase (luc) reporter gene activity that quantitative differences in transgene activity can be the result of either a variation in (1) level, (2) spatial distribution and/or (3) temporal regulation of transgene expression between independent transformants. It is not known which of these three different aspects of transgene expression is affected when the transgene is flanked by MAR elements. Here we have used the firefly luciferase reporter system to analyse the influence of MAR elements on the activity of a CaMV 35S-luc transgene in a population of independently transformed tobacco plants. Imaging of in planta LUC activity in these tobacco plant populations showed that the presence of MAR elements does not result in less variation in the average level of transgene expression between individual transformants. This result is different from that obtained previously with a 35S-GUS reporter gene flanked by MAR elements and reflects the differences in the stability of the LUC and GUS reporter proteins. Also the variation in spatial patterns of in vivo LUC activity is not reduced between independent transformants when the transgene is flanked by MAR elements. However, MAR elements do seem to affect the variation in temporal regulation of transgene expression between individual transformants. The potential effects of MAR elements on the variability of transgene expression and the relation to the stability of the (trans)gene product are discussed.

Gene Expression Regulation, Developmental↗
PubMed2000

Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobacco.

Posttranscriptional gene-silencing phenomena such as cosuppression and RNA interference are associated with the occurrence of small, about 21-23 nt short RNA species homologous to the silenced gene. We here show that the small RNA present in silenced transgenic plants can easily be detected in total RNA isolated according to standard procedures. This will allow for the development of routine and early screenings for the presence of small RNA species and, therefore, gene silencing in transgenic plants. We further demonstrate that the small RNA fraction can be visualized with the SYBR Green II RNA stain, isolated from a gel, labeled and used as a specific probe. Using these approaches, we have fine-mapped the sequences of the GUS gene that are represented in the small RNA fraction of a GUS-silenced tobacco line containing an inverted repeat of the GUS gene. In this tobacco line, the silencing-associated small RNA is a mixture of fragments that cover the 3' two-thirds of the GUS coding region. The 5' coding and the 3' noncoding ends of the GUS mRNA are not represented in the small RNA fraction. The RNA fragments are not likely to be a primary synthesis product of an RNA-dependent RNA polymerase, but rather degradation products from nuclease activity. Surprisingly, RNA isolated from wild-type, untransformed plants showed the presence of a similar-sized small RNA pool. This might indicate the existence of small RNA species from putative endogenous genes that are down regulated by a similar posttranscriptional gene-silencing mechanism. The possibility of isolating and labeling the small RNA pool from wild-type plants will provide a way to identify and study such putative genes.

Fluorescent Dyes↗
PubMed1998

Flank matrix attachment regions (MARs) from chicken, bean, yeast or tobacco do not prevent homology-dependent trans-silencing in transgenic tobacco plants.

The effect of flanking matrix attachment regions (MARs) on homology-dependent trans-silencing was tested using two strong trans-silencing loci. The transgenic tobacco line 271 carries at a single locus a p35S-RiN-tNos transgene which is able to silence, in trans and at the transcriptional level, the expression of any p35S-driven transgene irrespective of its position. The transgenic tobacco line 6b8 carries at a single locus a p35S-uidA-tRbcS transgene which is able to silence in trans, at the post-transcriptional level, the expression of any uidA-expressing transgene irrespective of its position. Various transgenic tobacco lines carrying a target p35S-uidA-tNos transgene, flanked on each side by MARs from chicken, bean, yeast or tobacco, were crossed with lines carrying the 271 and 6b8 loci. Expression of the target transgene was silenced in all hybrids, irrespective of the presence or absence of MAR sequences. These results therefore demonstrate that MARs are not able to protect transgene expression from strong silencing loci that act in trans.

Animals↗
PubMed1998

Biosafety of E. coli beta-glucuronidase (GUS) in plants.

The beta-glucuronidase (GUS) gene is to date the most frequently used reporter gene in plants. Marketing of crops containing this gene requires prior evaluation of their biosafety. To aid such evaluations of the GUS gene, irrespective of the plant into which the gene has been introduced, the ecological and toxicological aspects of the gene and gene product have been examined. GUS activity is found in many bacterial species, is common in all tissues of vertebrates and is also present in organisms of various invertebrate taxa. The transgenic GUS originates from the enterobacterial species Escherichia coli that is widespread in the vertebrate intestine, and in soil and water ecosystems. Any GUS activity added to the ecosystem through genetically modified plants will be of no or minor influence. Selective advantages to genetically modified plants that posses and express the E. coli GUS transgene are unlikely. No increase of weediness of E. coli GUS expressing crop plants, or wild relatives that might have received the transgene through outcrossing, is expected. Since E. coli GUS naturally occurs ubiquitously in the digestive tract of consumers, its presence in food and feed from genetically modified plants is unlikely to cause any harm. E. coli GUS in genetically modified plants and their products can be regarded as safe for the environment and consumers.

Ecology↗
PubMed1997

Dissection of a synthesized quantitative trait to characterize transgene interactions.

Six transgenic tobacco lines, each homozygous for the beta-glucuronidase (GUS) gene at a different locus, and wild type were selfed and intercrossed to evaluate GUS activity in all possible hemizygous, homozygous and dihybrid combinations of GUS alleles. The transgenic lines are characterized by their GUS activity (two low, three intermediate, one high), T-DNA complexity (four single-copy, two more complex single-locus) and the presence of the chicken lysozyme matrix-associated region (MAR) around the full T-DNA (two lines). Gene action and interaction was analyzed by weighted linear regression with parameters for additivity, dominance and epistasis. The analysis showed that each of the four single-copy lines acted fully additively. In contrast, the two complex single-locus lines showed classical single-locus overdominance and were epistatic dominant over all other GUS alleles. The latter is manifested in severe suppression of GUS activity in dihybrid lines, irrespective of the presence of MAR elements around the GUS gene. Such elements apparently do not protect against epistatic dominance. The quantitative data suggested that the epistatic dominance and overdominance are based on the same molecular mechanism. Our approach of a genetic analysis of quantitative variation in well-characterized transgenic lines provides a powerful tool to gain insight into complex plant traits.

Alleles↗
PubMed1995

pBINPLUS: an improved plant transformation vector based on pBIN19.

We describe the construction of a new plant transformation vector, pBINPLUS, based on the popular pBIN19 vector. Improvements over pBIN19 include location of the selectable marker gene at the left T-DNA border, a higher copy number in E. coli, and two rare restriction sites around the multiple cloning site for easier cloning and analysis of T-DNA insertions in plant genomes.

Agrobacterium tumefaciens↗
PubMed1993

Activity of the promoter of the Lhca3.St.1 gene, encoding the potato apoprotein 2 of the light-harvesting complex of Photosystem I, in transgenic potato and tobacco plants.

We have isolated cDNA and genomic clones for the potato (Solanum tuberosum) apoprotein 2 of the light harvesting complex of Photosystem I, designated Lhca3.St.1. The protein shows all characteristics of the family of chlorophyll a/b-binding proteins. Potato Lhca3.1 gene expression occurs predominantly in leaves, and is transcriptionally regulated by light. One gene copy is present per haploid genome. The sequence of the 5' upstream region was determined. Most boxes identified in the promoter sequences of genes whose expression is light-regulated recur in the Lhca3.St.1 sequence. Functional analyses of the Lhca3.St.1 promoter and two deletion derivatives in transgenic potato transformed with a promoter-GUS fusion show high promoter activity in leaves and other green parts of the plant, which depends on light. Activity is absent in roots and potato tubers. The 500 bp promoter fragment is as active as the full 2.0 kb sequence, showing that all regulatory elements are present on the smallest deletion derivative. In transgenic tobacco (Nicotiana tabacum) plants carrying the largest promoter derivative a similar distribution of activity is found. Promoter activity is not restricted to the phloem, but also prominent in the xylem of the young stem, which contrasts with promoters of other photosynthesis-associated genes.

Amino Acid Sequence↗
PubMed1992

Analysis of the region in between two closely linked patatin genes: class II promoter activity in tuber, root and leaf.

From a potato genomic library a phage lambda clone was isolated that carried nucleotide sequences of two patatin genes, thus demonstrating a close physical linkage between these two members of the patatin gene family. Sequence and restriction analysis showed the genes to be oriented in tandem. The more upstream gene was a pseudogene truncated at the 3' end, whereas the downstream gene was a class II patatin gene. In addition to a 208 bp fragment also present in patatin class I promoters, the region in between both genes contained various direct repeats also found in other patatin genes. To study the promoter activity of this intergenic region, a 2.78 kb fragment was transcriptionally fused to the beta-glucuronidase gene and reintroduced into potato cultivar Bintje. Histochemical analysis revealed expression in the outermost layer of cells of the cortex, in the tuber phellogen, in or around the root vascular system, and also in the abaxial phloem layer of the vascular bundle in leaves.

Amino Acid Sequence↗
PubMed1992

Biosafety of kanamycin-resistant transgenic plants.

Kanamycin resistance is one of the most frequently used selection markers for obtaining transgenic plants. The introduction of these transgenic plants into agricultural practice will cause the kanamycin resistance gene and the gene product to be present on a large scale. The desirability of this situation is analysed. The nature, properties and applications of the antibiotic kanamycin are briefly reviewed, as are the mechanisms of kanamycin resistance. It is argued that the gene used for resistance is an excellent choice because of the high substrate specificity of the enzyme encoded. Human or veterinary antibiotic therapies will not be compromised. Also, the physico-chemical characteristics of the antibiotic exclude the existence of selective conditions in the environment. Therefore, a transgenic plant or any other organism that might have acquired the gene will not get any selective advantage because of this gene. Evidence further suggests there is no toxicity or predictable harm of both gene or gene product for human or animal consumption. Full legislative clearance of this transgenic trait is therefore acceptable.

Acetyltransferases↗
PubMed1987

Characterization of cDNA for nodulin-75 of soybean: A gene product involved in early stages of root nodule development.

Establishment of a nitrogen-fixing root nodule is accompanied by a developmentally regulated expression of nodulin genes, only some of which, the so-called early nodulin genes, are expressed in stages preceding actual nitrogen fixation. We have isolated soybean cDNA clones representing early nodulin genes and have studied clone pENOD2 in detail. The cDNA insert of this clone hybridizes to nodule-specific RNA of 1200 nucleotides in length. The RNA that was hybrid-selected by the cloned ENOD2 DNA was in vitro translated to produce two nodulins with an apparent M(r) of 75,000, the N-75 nodulins. These two nodulins differ slightly in charge and one does not contain methionine. The amino acid sequence deduced from the DNA sequence shows that proline accounts for 45% of the 240 residues in these nodulins and the sequence contains at least 20 repeating heptapeptide units. The amino acid composition of none of the (hydroxy)proline-rich (glyco)proteins described in plants resembles the composition of the N-75 nodulins, especially with respect to the high glutamic acid and the low serine content. This suggests that the N-75 nodulins belong to a hitherto unidentified class of presumably structural proteins. The genes encoding the N-75 nodulins were found to be expressed in nodule-like structures devoid of intracellular bacteria and infection threads, indicating that these nodulins do not function in the infection process but more likely function in nodule morphogenesis.

Journal Article↗
PubMedDate not supplied

The relationship between nodulin gene expression and the Rhizobium nod genes in Vicia sativa root nodule development.

The role of the Rhizobium nod genes in the induction of nodulin gene expression was examined by analyzing nodules formed on vetch roots by bacterial strains containing only the nod region. Introduction of an 11-kb cloned nod region of the R. leguminosarum sym plasmid pRL1JI into sym plasmid-cured rhizobia conferred on the recipient strains the ability to induce nodules in which all nodulin genes were expressed. This proves that from the sym plasmid only the nod region is involved in the induction of nodulin gene expression. A transconjugant of Agrobacterium carrying the same nod region induces nodules in which only early nodulin gene expression is detected. Thus, the nod region is essential for the induction of early nodulin gene expression. In this case, nodule cytology may indicate that a defense response of the plant interferes with the induction of late nodulin gene expression. Indirect evidence is presented that indeed the Rhizobium nod genes are also in some way involved in the induction of the expression of late noduling genes. The combination between histological data and pattern of nodulin gene expression furthermore reveals a correlation between nodule structure and nodulin gene expression. This correlation may aid in speculations about the functions of nodulins.

Blotting, Western↗