J.P.H. Nap

Sequence Polymorphisms Cause Many False cis eQTLs

Many investigations have reported the successful mapping of quantitative trait loci (QTLs) for gene expression phenotypes (eQTLs). Local eQTLs, where expression phenotypes map to the genes themselves, are of especially great interest, because they are direct candidates for previously mapped physiological QTLs. Here we show that many mapped local eQTLs in genetical genomics experiments do not reflect actual expression differences caused by sequence polymorphisms in cis-acting factors changing mRNA levels. Instead they indicate hybridization differences caused by sequence polymorphisms in the mRNA region that is targeted by the microarray probes. Many such polymorphisms can be detected by a sensitive and novel statistical approach that takes the individual probe signals into account. Applying this approach to recent mouse and human eQTL data, we demonstrate that indeed many local eQTLs are falsely reported as “cis-acting” or “cis” and can be successfully detected and eliminated with this approach.

Tentacles of in vitro-grown round-leaf sundew (Drosera rotundifolia L.) show induction of chitinase activity upon mimicking the presence of prey

Induction of plant-derived chitinases in the leaves of a carnivorous plant was demonstrated using aseptically grown round-leaf sundew (Drosera rotundifolia L.). The presence of insect prey was mimicked by placing the chemical inducers gelatine, salicylic acid and crustacean chitin on leaves. In addition, mechanical stirring of tentacles was performed. Chitinase activity was markedly increased in leaf exudates upon application of notably chitin. Application of gelatine increased the proteolytic activity of leaf exudates, indicating that the reaction of sundew leaves depends on the molecular nature of the inducer applied. In situ hybridization of sundew leaves with a Drosera chitinase probe showed chitinase gene expression in different cell types of non-treated leaves, but not in the secretory cells of the glandular heads. Upon induction, chitinase mRNA was also present in the secretory cells of the sundew leaf. The combined results indicate that chitinase is likely to be involved in the decomposition of insect prey by carnivorous plants. This adds a novel role to the already broad function of chitinases in the plant kingdom and may contribute to our understanding of the molecular mechanisms behind the ecological success of carnivorous plants in nutritionally poor environments.

A verification protocol for the probe sequences of Affymetrix genome arrays reveals high probe accuracy for studies in mouse, human and rat

BackgroundThe Affymetrix GeneChip technology uses multiple probes per gene to measure its expression level. Individual probe signals can vary widely, which hampers proper interpretation. This variation can be caused by probes that do not properly match their target gene or that match multiple genes. To determine the accuracy of Affymetrix arrays, we developed an extensive verification protocol, for mouse arrays incorporating the NCBI RefSeq, NCBI UniGene Unique, NIA Mouse Gene Index, and UCSC mouse genome databases.ResultsApplying this protocol to Affymetrix Mouse Genome arrays (the earlier U74Av2 and the newer 430 2.0 array), the number of sequence-verified probes with perfect matches was no less than 85% and 95%, respectively; and for 74% and 85% of the probe sets all probes were sequence verified. The latter percentages increased to 80% and 94% after discarding one or two unverifiable probes per probe set, and even further to 84% and 97% when, in addition, allowing for one or two mismatches between probe and target gene. Similar results were obtained for other mouse arrays, as well as for human and rat arrays. Based on these data, refined chip definition files for all arrays are provided online. Researchers can choose the version appropriate for their study to (re)analyze expression data.ConclusionThe accuracy of Affymetrix probe sequences is higher than previously reported, particularly on newer arrays. Yet, refined probe set definitions have clear effects on the detection of differentially expressed genes. We demonstrate that the interpretation of the results of Affymetrix arrays is improved when the new chip definition files are used.

Rhizobium nod genes are involved in the induction of two early nodulin genes in Vicia sativa root nodules.

Nodulin gene expresison was studied in Vicia sativa (common vetch) root nodules induced by several Rhizobium and Agrobacterium strains. An Agrobacterium transconjugant containing a R. leguminosarum symplasmid instead of its Ti-plasmid, that was previously shown to form “empty” nodules on pea, induced nodules on Vicia roots in which nodule cells were infected with bacteria. In the Vicia nodules induced by this transconjugant, two so-called early nodulin genes were found to be expressed, whereas in the nodules formed on pea the expression of only one early nodulin gene was detected. In both cases the majority of the nodulin genes was not expressed.Apparently, an intracellular location of the bacteria is not sufficient for the induction of the majority of the nodulin genes. All nodulin genes were expressed in nodules induced by cured Rhizobium strains containing cosmid clones that have a 10 kb nod region of the sym-plasmid in common. Since in tumours no nodulin gene expression was found at all, the Agrobacterium chromosome does not contribute to the induction of nodulin genes. Therefore it is concluded that the signal for the induction of the expression of the two Vicia early nodulin genes is encoded by the nod-region, and the signal involved in the induction of all other nodulin genes has to be located outside the sym-plasmid, on the Rhizobium chromosome. The apparent difference in early nodulin gene expression between pea and Vicia is discussed in the light of the usefulness of Agrobacterium transconjugants in the study of nodulin gene expression.

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.

Nodulin Gene Expression in Pisum Sativum

The symbiotic association of rhizobia and leguminous plants leads to the formation of specialized root nodules in which both the plant and bacterial cells are highly differentiated. Compared with other plant differentiation processes root nodule formation does not appear a very complex process. Goldberg et al. (Kamalay, Goldberg, 1980, 1984; Goldberg et al., 1981) using hybridization techniques for analyzing messenger RNA populations arrived at the conclusion that 15-25 thousand genes are involved in the formation of a leaf, a stem or a root. 15-40% of which are organ-specific. Verma et al. (Auger, Verma, 1981; Verma, 1982) at the other hand showed by similar hybridization experiments that less than 100 different genes are specifically involved in root nodule formation and maintenance.

Nodulins Involved in Early Stages of Pea Root Nodule Development

In nodules formed on the roots of pea plants at least 25 plant genes are specifically expressed, the nodulin genes. The majority of these genes are first expressed 13 days after sowing and inoculation with Rhizobium leguminosarum (Govers et al 1985). Histological examination of longitudinal sections of infected roots of 8, 10 and 13 day old plants showed that a complete nodule structure is already present at day 10 (Figure 1A). From the apical meristem infected as well as uninfected cells, the two cell types that are present in the symbiotic zone of the mature nodule, have been formed (Figure 1A, B). Vascular bundles are present at the periphery of the nodule, an uninfected cortex surrounds the central part of the nodule and in the zone with the differentiating cells a great number of infection threads is present. At day 8 this differentiation process has started, but the difference between infected and uninfected cells is not yet clear (Figure 1B), and at 13 days the infected cells are fully packed with rhizobia (Figure 1B). Since the complete nodule structure is already present before the majority of the nodulin genes is expressed these nodulin genes cannot be involved in the morphogenesis of this new organ.

Expression of Host Specific Sequences during Development of Root Nodules in Pea

In root nodules obtained after infection of leguminous plants with Rhizobium a number of host-encoded nodule-specific proteins, nodulins, are found. Previous studies of the symbiosis between pea (Pisum sativum) and Rhizobium leguminosarum have shown that during nodule development about 30 nodulins can be detected by means of a nodule specific antiserum (Bisseling et al., 1983). Some of these nodulins appear before nitrogen fixation starts, whereas the majority is synthesized during stages of active nitrogen fixation.