Carsten Stig Poulsen

HMG I-like proteins from leaf and nodule nuclei interact with different AT motifs in soybean nodulin promoters.

Three different nuclear factors recognizing short AT-rich DNA sequences were identified in different organs of soybean. One factor (NAT2) was found to be present in mature nodules, another factor (NAT1) was detected in roots and nodules, and a third one (LAT1) was only observed in leaves. All three factors recognized several DNA sequences in the promoter region of the soybean nodulin N23 gene. Footprinting, deletion, and point mutation analyses revealed different binding properties for all three factors and further showed that even single base pair substitutions had a dramatic effect on binding affinity. The LAT1 and NAT1 factors were released from chromatin by extraction with a low-salt buffer and were soluble in 2% trichloroacetic acid, implying a relationship to high-mobility group (HMG) proteins. DNA binding studies further indicated a functional relationship of these factors to the human HMG I protein. Purification of the LAT1 factor from leaf nuclei revealed the presence of two polypeptides with molecular masses of 21 kilodaltons and 23 kilodaltons, respectively, binding the same DNA sequence with equal affinity.

Discrete and Continuous Representations of Unobserved Heterogeneity in Choice Modeling

We attempt to provide insights into how heterogeneity has been and can be addressed in choice modeling. In doing so, we deal with three topics: Models of heterogeneity, Methods of estimation and Substantive issues. In describing models we focus on discrete versus continuous representations of heterogeneity. With respect to estimation we contrast Markov Chain Monte Carlo methods and (simulated) likelihood methods. The substantive issues discussed deal with empirical tests of heterogeneity assumptions, the formation of empirical generalisations, the confounding of heterogeneity with state dependence and consideration sets, and normative segmentation.

Gene targeting approaches using positive-negative selection and large flanking regions

We report here on strategies aimed at improving the frequency of detectable recombination in plants by increasing the efficiency of selecting double-recombinants in transgenic calli. Gene targeting was approached on the Gln1 and the Pzf loci of Lotus japonicus, using Agrobacterium tumefaciens T-DNA replacement vectors. Large flanking regions, up to 22.9 kb, surrounding a positive selection marker were presented as substrates for homologous recombination. For easier detection of putative recombinants the negative selectable marker cytosine deaminase was inserted at the outside borders of the flanking regions offered for cross-over. A combination of positive and negative selection allowing double-recombinants to grow, while counter-selecting random insertions, was used to select putative targeting events. The more than 1000-fold enrichment observed with replacement vectors designed to minimize gene silencing demonstrated the efficiency of the negative selection. Using five different replacement vectors an estimated total of 18974 transformation events were taken through the positive-negative selection procedure and 185 resistant calli obtained. Targeting events could not be verified in the survivors by PCR screening and Southern blot analysis. With this approach the frequency of detectable gene targeting in L. japonicus was below 5.3×10−5, despite the large flanking sequences offered for recombination.

Mixed Markov and latent Markov modelling applied to brand choice behaviour

Abstract Two models, the mixed Markov and the latent Markov model, are presented. Both can be seen as generalizations of Lazarsfelds latent class model. The mixed Markov model is defined as a finite mixture of (manifest) Markov chains, allowing for individual differences in transition probabilities. It generalizes the latent class model by relaxing the assumption of local independence. The latent Markov model describes a Markov chain operating at the unobservable or latent level, due to, for example, errors in the determination of the relevant states. From a marketing perspective, a partial segmentation approach is taken. This is implemented in well-defined statistical models that can be estimated and tested efficiently. The structural insights and predictions provided by the models are illustrated using a data set from Aaker (1970) on brand switching.

Quality guidance and quality formation

Abstract This paper presents an extension of the quality guidance model of Steenkamp and Van Trijp that includes consumer quality formation processes. Quality expectations and quality experiences are seen as antecedents of perceived overall product quality. The conceptual model is applied using LIS-REL to a data set on Danish butter cookies. Five plausible models of the relation between expectation, experience and perceived product quality are estimated. Finally one model is selected on the basis of three criteria: chi-square, RMSEA and AIC. The results show a model where expectations are indirectly related to perceived quality through experience.

Plant cell growth and differentiation may involve GAP regulation of Rac activity.

Two Rac GTPase cDNAs, LjRac1 and LjRac2, were identified in the legume Lotus japonicus. Two‐hybrid screening with dominant‐constitutive mutations in the two Rac GTPases target three plant cDNAs, LjRacGAP1, LjRacGAP2 and LjRacGAP3, that encode putative GTPase activating proteins of Rho‐GTPase subfamily members. Employing Rac antiserum, purified recombinant LjRac GTPases and recombinant LjRacGAP1, for ligand overlay assays, in vitro GAP affinity assays and GTPase activation, we confirmed that eukaryote Rac/RacGAP interplay is conserved in plants. In this investigation we have developed some tools that can be used to characterize the role of enhanced LjRac2 expression in developing root nodules.

A protein binding AT-rich sequence in the soybean leghemoglobin c3 promoter is a general cis element that requires proximal DNA elements to stimulate transcription.

A nodule nuclear factor, NAT2, interacts with two AT-rich binding sites (NAT2 BS1 and NAT2 BS2) in the soybean leghemoglobin (lb) c3 promoter. In transgenic Lotus corniculatus nodules, an oligonucleotide containing NAT2 BS1 activated an inactive -159 lbc3 promoter when placed immediately upstream of the promoter. The activation was independent of the orientation of NAT2 BS1 but was dependent on its position in the promoter. The abilities of different mutated binding sites to activate expression in vivo were correlated to their respective in vitro affinities for binding NAT2. This suggested that the interaction between NAT2 and NAT2 BS1 is responsible for the observed reactivation. Further activation experiments with the lbc3 and the leaf-specific Nicotiana plumbaginifolia ribulose bisphosphate carboxylase/oxygenase small subunit (rbcS-8B) promoter suggested that another specific cis element(s) is required for the function of NAT2 BS1. Thus, the -102 lbc3 promoter lacking the organ-specific element (-139 to -102) was not reactivated by the presence of the binding site, and the rbcS-8B promoter required sequences between -312 and -257 to be activated by NAT2 BS1. This implies that NAT2 has to work in combination with other trans-acting factor(s) to increase expression. The finding of NAT2-like binding activities in different plant organs and the specific expression of the hybrid NAT2 BS1/-312 rbcS-8B promoter in leaves suggest that NAT2 is a general activator of transcription.

Consumers’ Quality Perception

Much has been said about food quality, and the discussion is endless because the notion of quality changes along with the changes in our life and society. This underlines the complexity of the issue of food quality. Today food production in Europe is highly concentrated, and the global market is rapidly becoming a reality. On the other hand, still many consumers are in favour of local production, resulting in fierce competition between the multinational suppliers and the local producers of food.

Expressed sequence tags from roots and nodule primordia of Lotus japonicus infected with Mesorhizobium loti.

Messenger RNA from young Lotus japonicus roots carrying root nodule primordia appearing after inoculation with Mesorhizobium loti bacteria were used to construct a cDNA expression library. Single-pass sequencing employing colony-polymerase chain reaction (PCR) and analysis of PCR products established a total of 2,397 new expressed sequence tags (ESTs). We have putatively identified 1,236 known and 484 hypothetical proteins coded by the corresponding mRNAs. The remaining cDNAs are unknown (316) or redundant overlapping cDNAs (361). We hope that this batch of ESTs will assist in the recognition of plant genes involved during development of nitrogen-fixing root nodules.

Molecular analysis of two Ypt/Rab-related sequences isolated from soybean (Glycine max) DNA libraries

From nodule and seedling cDNA libraries we isolated cDNA copies of two mRNAs, derived from the genes gmrl and gmr2, encoding members of the Ypt/Rab family of small GTP-binding proteins. Two deduced protein products, GMR1 and GMR2, were found to be nearly identical differing by only four amino acids in the analysed parts. The two putative proteins are 79% identical to the previously described ARA small GTPase from Arabidopsis thaliana. The GMR proteins may thus be the counterpart of the ARA protein and may perform a related biological function in Glycine max. The gmr2 genomic sequence was isolated and structurally analysed. Expression analyses by northern and cDNA-based PCR showed that the gmr1 and gmr2 genes are constitutively expressed in different plant organs, although at a slightly higher level in callus culture. The classification of the gmr sequences as relatives of the Ypt/Rab family suggests that the deduced GMR proteins are involved in control of processes related to vesicle trafficking in plant cells.