Cross-species hybridisation of human and bovine orthologous genes on high density cDNA microarrays

BACKGROUND: Cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The usefulness of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a specific developmental, metabolic, or disease-related gene pathway to be evaluated in experimental models of human diseases. The objective of this study was to investigate the feasibility and reproducibility of cross-species analysis employing a human cDNA microarray as probe. RESULTS: As a proof of principle, total RNA derived from human and bovine fetal brains was used as a source of labelled targets for hybridisation onto a human cDNA microarray composed of 349 characterised genes. Each gene was spotted 20 times representing 6,980 data points thus enabling highly reproducible spot quantification. Employing high stringency hybridisation and washing conditions, followed by data analysis, revealed slight differences in the expression levels and reproducibility of the signals between the two species. We also assigned each of the genes into three expression level categories- i.e. high, medium and low. The correlation co-efficient of cross hybridisation between the orthologous genes was 0.94. Verification of the array data by semi-quantitative RT-PCR using common primer sequences enabled co-amplification of both human and bovine transcripts. Finally, we were able to assign gene names to previously uncharacterised bovine ESTs. CONCLUSIONS: Results of our study demonstrate the harnessing and utilisation power of comparative genomics and prove the feasibility of using human microarrays to facilitate the identification of co-expressed orthologous genes in common tissues derived from different species

Clinical, histopathological and molecular features of dedifferentiated melanomas:An EORTC Melanoma Group Retrospective Analysis

PURPOSE: Dedifferentiated melanoma (DedM) poses significant diagnostic challenges. We aimed to investigate the clinical, histopathological and molecular features of DedM. Methylation signature (MS) and copy number profiling (CNP) were carried out in a subgroup of cases.PATIENTS AND METHODS: A retrospective series of 78 DedM tissue samples from 61 patients retrieved from EORTC (European Organisation for Research and Treatment of Cancer) Melanoma Group centres were centrally reviewed. Clinical and histopathological features were retrieved. In a subgroup of patients, genotyping through Infinium Methylation microarray and CNP analysis was carried out.RESULTS: Most patients (60/61) had a metastatic DedM showing most frequently an unclassified pleomorphic, spindle cell, or small round cell morphology akin to undifferentiated soft tissue sarcoma, rarely associated with heterologous elements. Overall, among 20 successfully analysed tissue samples from 16 patients, we found retained melanoma-like MS in only 7 tissue samples while a non-melanoma-like MS was observed in 13 tissue samples. In two patients from whom multiple specimens were analysed, some of the samples had a preserved cutaneous melanoma MS while other specimens exhibited an epigenetic shift towards a mesenchymal/sarcoma-like profile, matching the histological features. In these two patients, CNP was largely identical across all analysed specimens, in line with their common clonal origin, despite significant modification of their epigenome.CONCLUSIONS: Our study further highlights that DedM represents a real diagnostic challenge. While MS and genomic CNP may help pathologists to diagnose DedM, we provide proof-of-concept that dedifferentiation in melanoma is frequently associated with epigenetic modifications.</p

Two Dimensional Screening : Towards Establishing a Novel Technique to Study Biomolecular Interactions

In all combinatorial approaches used so far one (macro-) molecule of a given structure is used to select the best binding partner from a library of potential ligands or acceptors. Therefore, only a certain, limited, part of the structure activity space is evaluated. By combining a library of molecules derived from one particular acceptor structure with a library of potential ligands, a significantly larger part of the structure activity space of this particular pair of molecules is accessed. The insight gained in the interaction of interest in this way is expected to be more detailed. This thesis aimed at establishing practical ways to combine a library of peptide ligands and a library of protein acceptors in the context of one particular model system. A library of cellulose bound peptides and a library of bacteriophage displayed variants of one protein were combined. Calmodulin (CaM) was chosen as a model protein. In binding tests aiming at the design of a peptide ligand library remarkable short CaM binding peptides were found. Furthermore, it was possible to present biological active CaM on the surface of M13-bacteriophage. A highly mutated CaM-library was created using the error prone PCR and displayed on the surface of bacteriophage. Using this CaM-library in affinity enrichment experiments with peptides and peptide libraries new variants of CaM were enriched. The results presented in this thesis show that two libraries can be combined. Furthermore it was demonstrated that Two Dimensional Screening is with the chosen approach not only feasible but resulted in novel results, which would be difficult to reach with a different approach.In allen bisher etablierten kombinatorischen Ansätzen wird ein (Makro-) Molekül verwendet, um den besten Bindungspartner aus einer Bibliothek von potentiellen Bindungspartnern auszuwählen. Mit diesen Ansätzen wird nur ein begrenzter Teil des Struktur-Aktivitätsraumes erkundet. Durch die Kombination einer Bibliothek von Akzeptoren mit einer Bibliothek von Liganden wird ein wesentlich größerer Teil des Struktur-Aktivitätsraumes beschrieben. So kann ein besserer Einblick in die untersuchte Wechselwirkung gewonnen werden. Diese Dissertation zielt ab auf die methodische Etablierung der Kombination einer Peptidbibliothek mit einer Proteinbibliothek. Es wurde eine Bibliothek synthetischer, an Cellulose gekoppelter Peptide mit einer auf Bakteriophagen präsentierten Proteinbibliothek kombiniert. Calmodulin (CaM) wurde als Modell-Protein ausgewählt. Bindungstests, die zur Charakterisierung der Liganden-Bibliothek durchgeführt wurden, ergaben bemerkenswert kurze CaM bindende Peptide. Des weiteren konnte biologisch aktives Calmodulin auf der Oberfläche von Phagen präsentiert werden. Mittels der 'Error-Prone-PCR” wurde eine hoch mutierte CaM-Bibliothek erzeugt. Bei Affinitätsanreicherungsversuchen mit dieser Bibliothek präsentiert auf Bakteriophagen an Peptid-Bibliotheken wurden neue Varianten von CaM angereichert. Die Ergebnisse dieser Dissertation zeigen, daß zwei Bibliotheken miteinander kombiniert werden können und daß ein 'Zweidimensionales Screening' mit dem gewählten Ansatz nicht nur möglich ist, sondern auch zu neuen Ergebnissen führt, die mit anderen Ansätzen nur schwerlich zu erreichen sind

NIH shift in flavin-dependent monooxygenation: Mechanistic studies with 2-aminobenzoyl-CoA monooxygenase/reductase

The flavoprotein 2-aminobenzoyl-CoA monooxygenase/reductase from the eubacterium Azoarcus evansii catalyzes the dearomatization of 2-aminobenzoyl-CoA. The reaction consists in an O(2)-dependent monooxygenation at the benzene position 5, which is followed immediately by an NADH-dependent hydrogenation of the intermediate at the same catalytic locus. The reaction was studied by (1)H, (2)H, and (13)C NMR spectroscopy of the products. The main product was characterized as 5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA by two-dimensional NMR spectroscopy. Thus, [5-(2)H]2-aminobenzoyl-CoA was converted into [6-(2)H]5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA, indicating a 5 → 6 shift of the [5-(2)H] label. Label from NAD(2)H was transferred to the 3 position of the cyclic eneamine, whereas label from solvent D(2)O was incorporated into the 4 and the 6 positions of 5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA. The labeling pattern is compatible with the monooxygenation proceeding via what is formally an NIH shift, yielding 5-oxo-2-aminocyclohex-1,3-diene-1-carboxyl-CoA as a protein-bound intermediate. It is suggested that this shift in flavin-dependent monooxygenation may have general validity

Substrate-Dependent Regulation of Anaerobic Degradation Pathways for Toluene and Ethylbenzene in a Denitrifying Bacterium, Strain EbN1

Anaerobic biodegradation of toluene and ethylbenzene is of environmental concern and biochemical interest due to toxicity and novel reactions, respectively. The denitrifying strain EbN1 is unique in anaerobically degrading both alkylbenzenes via different pathways which converge at benzoyl coenzyme A. The organization of genes involved in both pathways was only recently determined for strain EbN1. In the present study, global expression analysis (DNA microarray and proteomics) indicated involvement of several thus-far-unknown proteins in the degradation of both alkylbenzenes. For example, orf68 and orf57, framing the ebd operon, are implicated in ethylbenzene degradation, and the ebA1932 and ebA1936 genes, located 7.2 kb upstream of the bbs operon, are implicated in toluene degradation. In addition, expression studies were now possible on the level of the complete pathways. Growth experiments demonstrated that degradative capacities for toluene and ethylbenzene could be simultaneously induced, regardless of the substrate used for adaptation. Regulation was studied at the RNA (real-time reverse transcription-PCR and DNA microarray) and protein (two-dimensional-difference gel electrophoresis) level by using cells adapted to anaerobic growth with benzoate, toluene, ethylbenzene, or a mixture of toluene and ethylbenzene. Expression of the two toluene-related operons (bss and bbs) was specifically induced in toluene-adapted cells. In contrast, genes involved in anaerobic ethylbenzene degradation were induced in ethylbenzene- and toluene-adapted cells, suggesting that toluene may act as a gratuitous inducer. In agreement with the predicted sequential regulation of the ethylbenzene pathway, Ebd proteins (encoding subunits of ethylbenzene dehydrogenase) were formed in ethylbenzene- but not in acetophenone-adapted cells, while Apc proteins (subunits of predicted acetophenone carboxylase) were formed under both conditions

High throughput identification of potential Arabidopsis mitogen-activated protein kinases substrates

Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogenactivated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtaine