Combining signals from spotted cDNA microarrays obtained at different scanning intensities.

MOTIVATION: The analysis of spotted cDNA microarrays involves scanning of color signals from fluorescent dyes. A common problem is that a given scanning intensity is not usually optimal for all spotted cDNAs. Specifically, some spots may be at the saturation limit, resulting in poor separation of signals from different tissues or conditions. The problem may be addressed by multiple scans with varying scanning intensities. Multiple scanning intensities raise the question of how to combine different signals from the same spot, particularly when measurement error is not negligible. RESULTS: This paper suggests a non-linear latent regression model for this purpose. It corrects for biases caused by the saturation limit and efficiently combines data from multiple scans. Combining multiple scans also allows reduction of technical error particularly for cDNA spots with low signal. The procedure is exemplified using cDNA expression data from maize. AVAILABILITY: All methods were implemented using standard procedures available in the SAS/STAT module of the SAS System. Programming statements are available from the first author upon request. CONTACT: piepho@uni-hohenheim.de SUPPLEMENTARY INFORMATION: The supplementary data are available at Bioinformatics online.

Designing a microarray experiment to estimate dominance in maize (Zea mays L.).

Experiments using cDNA microarrays for the identification of genes with certain expression patterns require a thoughtfully planned design. This study was conducted to determine an optimal design for a microarray experiment to estimate differential gene expression between hybrids and their parental inbred lines in maize (i.e. dominance). It has two features: the contrasts of interest contain more than two genotypes and the procedure may be customised to other microarray experiments where different effects may influence hybridisation signals. A mixed model was used to include all important effects. Impacts during growth of the plant material were taken into consideration as well as those occurring during hybridisation. The results of a preliminary experiment were used to determine which effects were to be included in the model, and data from another microarray experiment were used to estimate variance components. In order to select good designs, an optimality criterion adapted to the problem of differential gene expression between hybrids and their parental inbred lines was defined. Two approaches were used to determine an optimal design: the first one simplifies the problem by dividing it into several subproblems, whereas the second is more sophisticated and uses a simulated annealing (SA) algorithm. We found that the first approach constitutes a useful means for designing microarray experiments to study this problem. Using the more sophisticated SA approach the design can be further improved.

Protein kinase C isoforms involved in the transcriptional activation of cyclin D1 by transforming Ha-Ras.

Transcriptional activation of the cyclin D1 by oncogenic Ras appears to be mediated by several pathways leading to the activation of multiple transcription factors which interact with distinct elements of the cyclin D1 promoter. The present investigations revealed that cyclin D1 induction by transforming Ha-Ras is MEK- and Rac-dependent and requires the PKC isotypes epsilon, lambda, and zeta, but not cPKC-alpha. This conclusion is based on observations indicating that cyclin D1 induction by transforming Ha-Ras was depressed in a dose-dependent manner by PD98059, a selective inhibitor of the mitogen-activated kinase kinase MEK-1, demonstrating that Ha-Ras employs extracellular signal-regulated kinases (ERKs) for signal transmission to the cyclin D1 promoter. Evidence is presented that PKC isotypes epsilon and zeta, but not lambda are required for the Ras-mediated activation of ERKs. Expression of kinase-defective, dominant negative (DN) mutants of nPKC-epsilon or aPKC-zeta inhibit ERK activation by constitutively active Raf-1. Phosphorylation within the TEY motif and subsequent activation of ERKs by constitutively active MEK-1 was significantly inhibited by DN aPKC-zeta, indicating that aPKC-zeta functions downstream of MEK-1 in the pathway leading to cyclin D1 induction. In contrast, TEY phosphorylation induced by constitutively active MEK-1 was not effected by nPKC-epsilon, suggesting another position for this kinase within the cascade investigated. Transformation by oncogenic Ras requires activation of several Ras effector pathways which may be PKC-dependent and converge on the cyclin D1 promoter. Therefore, we investigated a role for PKC isotypes in the Ras-Rac-mediated transcriptional regulation of cyclin D1. We have been able to reveal that cyclin D1 induction by oncogenic Ha-Ras is Rac-dependent and requires the PKC isotypes epsilon, lambda, and zeta, but not cPKC-alpha. Evidence is presented that aPKC-lambda acts upstream of Rac, between Ras and Rac, whereas the PKC isotypes epsilon and zeta act downstream of Rac and are required for the activation of ERKs.

Complex formation and cooperation of protein kinase C theta and Akt1/protein kinase B alpha in the NF-kappa B transactivation cascade in Jurkat T cells.

Protein kinase C theta (PKC theta) is known to induce NF-kappa B, an essential transcriptional element in T cell receptor/CD28-mediated interleukin-2 production but also T cell survival. Here we provide evidence that PKC theta is physically and functionally coupled to Akt1 in this signaling pathway. First, T cell receptor/CD3 ligation was sufficient to induce activation as well as plasma membrane recruitment of PKC theta. Second, PKC theta selectively cooperated with Akt1, known to act downstream of CD28 co-receptor signaling, in activating a NF-kappa B reporter in T cells. Third, Akt1 function was shown to be required for PKC theta-mediated NF-kappa B transactivation. Fourth, PKC theta co-immunoprecipitated with Akt1; however, neither Akt1 nor PKC theta served as a prominent substrate for each other in vitro as well as in intact T cells. Finally, plasma membrane targeting of PKC theta and Akt1 exerted synergistic transactivation of the I-kappa B kinase beta/inhibitor of NF-kappa B/NF-kappa B signaling cascade independent of T cell activation. Taken together, these findings suggest a direct cross-talk between PKC theta and Akt1 in Jurkat T cells.

Cooperative action of SLR1 and SLR2 is required for lateral root-specific cell elongation in maize.

Lateral roots play an important role in water and nutrient uptake largely by increasing the root surface area. In an effort to characterize lateral root development in maize (Zea mays), we have isolated from Mutator (Mu) transposon stocks and characterized two nonallelic monogenic recessive mutants: slr1 and slr2 (short lateral roots1 and 2), which display short lateral roots as a result of impaired root cell elongation. The defects in both mutants act specifically during early postembryonic root development, affecting only the lateral roots emerging from the embryonic primary and seminal roots but not from the postembryonic nodal roots. These mutations have no major influence on the aboveground performance of the affected plants. The double mutant slr1; slr2 displays a strikingly different phenotype than the single mutants. The defect in slr1; slr2 does not only influence lateral root specific cell elongation, but also leads to disarranged cellular patterns in the primary and seminal roots. However, the phase-specific nature of the single mutants is retained in the double mutant, indicating that the two loci cooperate in the wild type to maintain the lateral root specificity during a short time of early root development.

Novel membrane-targeted ERK1 and ERK2 chimeras which act as dominant negative, isotype-specific mitogen-activated protein kinase inhibitors of Ras-Raf-mediated transcriptional activation of c-fos in NIH 3T3 cells.

Expression of constructs encoding fusion proteins of ERK1 and ERK2 containing a C-terminal farnesylation motif (CAAX) is predominantly localized at the cell membrane and was activated by coexpression of constitutively active Ha-RasL61 and epidermal growth factor. Both fusion proteins significantly inhibit the transcriptional activation of a c-fos-chloramphenicol acetyltransferase reporter induced by RasL61, constitutively active MEK1, or constitutively active RafBXB. The corresponding SAAX chimeras or overexpression of the wild-type ERKs did not interfere with the transcriptional activation of c-fos. The inhibition of the Ras-mediated c-fos induction by ERK2-CAAX can in part be rescued by coexpression of a wild-type ERK2 but not by wild-type ERK1. We find that ERK1-CAAX acts in the same fashion, indicating that mitogen-activated protein kinase (MAPK)-CAAX chimeras interact in an isotype-specific manner. It is demonstrated that both ERK1-CAAX and ERK2-CAAX associate with the corresponding endogenous ERKs, which explains the isotype-specific inhibitory effects of the ERK-CAAX chimeras. Evidence is presented that expression of ERK-CAAX fusion proteins inhibits the nuclear translocation of the corresponding endogenous ERKs. Disruption of MAPK translocation by membrane targeting provides additional, independent proof that nuclear translocation of ERKs is essential for the transcriptional activation of c-fos.

Early post-embryonic root formation is specifically affected in the maize mutant lrt1.

The isolation and detailed characterisation of the maize mutant lrt1 , which is completely deficient in the initiation of lateral roots at the primary and seminal lateral roots and of the crown roots at the coleoptilar node is described. The monogenic and recessive mutant was isolated from a segregating EMS mutagenised population, maps to the short arm of chromosome 2, and acts independently of the nodal root deficient rtcs locus. Histological analysis revealed that the mutation acts at a very early stage of root initiation, as indicated by the absence of primordia formation in the affected roots. At later stages of plant development lateral and crown root initiations recover leading to fertile plants. If grown in the dark, the mutant does not form an elongated mesocotyl, although the photomorphogenic response appears to be normal in the mutant. Furthermore, the wild-type cannot be rescued from mutants by the application of auxin to germinating kernels. The gene impaired in lrt1 seems to be of great importance for the general mechanism of early post-embryonic root initiation, both from root and nodal tissues, since lateral and crown root initiation are both affected to the same extent and in the same transient time pattern.

Differential Ca2+ signaling induced by activation of the epidermal growth factor and nerve growth factor receptors.

Stimulation by epidermal growth factor (EGF) of NIH3T3 cells overexpressing the EGF receptor (EGFR) results in a release of Ca2+ from internal stores. Ca2+ release is followed by an influx of extracellular calcium which can be recorded by the influx of the calcium surrogate Mn2+. Both Ca2+ release and Mn2+/Ca2+ influx are inhibited by expression of the dominant negative Asn17-Ras mutant and abrogated by microinjected neutralizing anti-Ras antibody Y13-259, whereas microinjection of the anti-Ras antibody Y13-238 which does not interact with the effector binding domain of Ras is without any effect on the EGF-induced Ca2+ transient. Neither Asn17-Ha-Ras nor the Y13-259 antibody interferes with the thapsigargin-induced Mn2+/Ca2+ influx. The nerve growth factor receptor (Trk)-mediated Ca2+ transient was found to be unaffected by the dominant negative Ras mutant or microinjected neutralizing anti-Ras antibodies. Substitution of the phospholipase Cgamma1 (PLCgamma1) binding site of the EGFR by the PLCgamma binding domain of Trk renders the EGFR-induced Ca2+ influx insensitive to the expression of Asn17-Ha-Ras, whereas the Ca2+ signal induced by Trk carrying the PLC binding site of EGFR is Ras-dependent and abrogated by the dominant negative Ras mutant. It is concluded that the Ca2+ transient induced by the activated EGFR, not, however, the Ca2+ transient elicited by the activated NGFR/Trk, is a Ras-mediated phenomenon and that the role of Ras in regulating EGFR-induced Ca2+ influx depends on the structure of the PLCgamma binding domain.