Cost-meetings Eden Roc, esp may 2007

II.TransCost-Meeting ARRAY

Involved COST Actions:

853 Agricultural biomarkers for array technology

858 Viticulture: Biotic and abiotic stress – grapevine defence mechanism and grape development

861 European network for pig genomics

863 Euroberry research: From genomics to sustainable production, quality and health

926 Impact of the new technologies on the health benefits and safety of bioactive plant compounds

In 2005 it was suggested to have a follow-up meeting of the successul TransCost-Meeting held 2000 in Lisboa, Portugal. The idea took until 2007 to become eventually realized at the location Eden Roc in Sant Feliu De Guixols, Spain, linked with the closing session of the COST Action 853. Here participants of five different Cost Actions, interested in and using microarray technology, met for a two day symposium to discuss their results obtained with this new, fast growing technology.

The session was started by J. Frey who welcomed everybody and handed over the floor to J. Williams, who explained that despite it was planned, no other official from Brussels could attend the meeting, however, he wanted to submit their warmest greetings and wishes for a successful conference. He expressed sincere thanks to the organisers of the event and announced prices for the best three posters instead of the cancelled welcoming speaches.

A. Session 1

Session 1: Technological developments- Past and Future
Chair: Jürg E. Frey (COST853)

Rapporteur: Günter Adam (COST853),

Keynote Speaker: Christian Oste (Nimblegen Systems Inc.)

High-resolution analysis of prokaryotic genomes using oligo arrays
C. C. Oste

International Scientific Support, NimbleGen Systems, Inc.

[e-mail]
The fine details of the structure of prokaryotic genomes can be elucidated by using high-density oligos probes microarrays. A variety of approaches will be described in this presentation, based on several techniques all implying the design and use of unique probes. Comparative Genome Sequencing (CGS) is one powerful such example: through a combination of direct sequence comparison, similar to the well-known CGH technique, and a subsequent array-based sequencing step, minute differences between related genomes can be detected and characterized. Gene expression analysis is also a very useful method to gather information about the adaptability of prokaryotes to adjust to changing environmental conditions. Tiling designs have proven to perform well for this application, in particular when associated with the search for strand-specific transcripts. ChIP-on-chip is yet another useful method, since it is designed to provide information about the function of transcription factors. In summary: the combination of a variety of methods, all based on the use of high-density oligos arrays, enables the in-depth analysis of fine structural details of prokaryotic genomes.
Unmasking species specific genes of Agrobacterium with pangenomic microarrays
X. Nesme

Microbial Ecology, Université Lyon 1, Lyon, France

[e-mail]
Bacterial species are genomically coherent clusters of strains adapted to the same or very similar ecological niches. Our project aims at finding "species specific" genes involved in the ecological specialisations of a model species (Agrobacterium tumefaciens) by comparative genome studies. The chosen tool for such a genomic study are 29- and 50-mer microarrays covering the entire genome of the reference strain C58 against which was hybridized DNA from strains belonging to the same species. Results showed a frequent organization in genomic islands of the targeted genes (especially on the linear chromosome) in which transport systems are often found.


Detection of bacterial plant pathogens based on a genome-wide microarray approach
M. Aittamaa, L. Mattinen, P. Somervuo, M. Pirhonen, P. Auvinen¹ and J.P.T. Valkonen

Department of Applied Biology and ¹Institute of Biotechnology, University of Helsinki, Finland

[e-mail]
A microarray with ca. 11,000 probes was designed for detection of the potato pathogens Pectobacterium atrosepticum, Clavibacter michiganensis subsp. sepedonicus, Streptomyces scabies and S. turgidiscabies. Probes were designed to target all known genes of P. atrosepticum, C. michiganensis and S. scabies. Probe design was carried out using the OligoArray 2.0 software. The whole-genome information was utilized to minimize the possibility that a probe would hybridize to more than one gene (locus) in the bacterial species. The majority of the probes contained 40 target-specific nucleotides (GC content 45-55 %). A poly(T) tail was added to the 3'-end to reach the final probe length of 60 nt. By these means, annealing temperatures of all probes on the array were equalized to the extent possible (T_m 82-90°C). Attention was also paid to prevent formation of stable secondary structures by the probes. Subsequently, the custom arrays were ordered from Agilent Technologies. Results from hybridization of these arrays with the genomic DNA of the aforementioned bacterial species and species related to them will be presented. The microarrays were also used for analysis of gene expression in P. atrosepticum to detect genes induced by potato extracts. These microarray profiling results and their verification with real-time PCR analysis will be presented.

Protein chips in analysis of potato, plant and insect biomarkers.
F. Cimaglia, T. Erban, I. Krizkova-Kudlikova, A. Santino, A.B. Shevelev, P. Poltronieri

ISPA-CNR, Lecce, Italy

[e-mail]
Kunitz-type proteinase inhibitors belong to an array of both constitutive and inducible plant defence genes. Many of the proteins induced by pathogen infection in potato are Kunitz-type protease inhibitors (KPI). Based on primary structure analysis and on biochemical characterization, KPIs have been divided into three groups named A, B and C. The KPI-A group includes aspartic protease inhibitors that can also inhibit trypsin, or chymotrypsin. The KPI-B group includes mainly serine protease (trypsin, chymotrypsin, elastase) inhibitors. The KPI-C group includes cysteine protease (cathepsin B, bromelain, ficin) inhibitors and also invertase inhibitors. His-tagged group A group B and group C belonging to cultivated and wild potato species (S. tuberosum, S. palustre, and S. stoloniferum) were expressed in E. coli and purified using Ni²⁺-sepharose. The protein chip, containing ten group A-, B- and C- KPI inhibitors, were used to screen differential binding of proteases to each KPI to individuate their specificity. Proteins were diluted to concentrations of 0.1, 0.2, and 0.4 mg/ml in NaHCO₃ buffer (0.1M, pH 9) with glycerol (40% w/v) and arrayed on epoxy slides (Nexterion, Schott, Jena, DE) using a SpotArray 24 (Perkin Elmer) for miniaturization of the reactions. The KPI spots were recognised and visualised by cyanine-3 labelled anti-KPI antibodies. We investigated the extent to which closely related protein interaction domains vary when printed as microarrays, i.e. to individuate diversity of proteins when used on glass arrays, a problem when using these assays for quantitative analysis. To this intent, we performed hybridization with decreasing amounts of trypsin, to find which KPi remained functional bound to the lowest protease concentration. Highest sensibility was achieved with KPI-A group proteins that interacted with trypsin at 10 nm concentration. The KPI chip could be a valuable tool for capturing various types of proteases from plants, insects and pathogens, as agricultural biomarkers. Trials are ongoing to verify the effectiveness of this assay and its application in the allergen field. In a similar approach, we used a protease chip in the screening of KPIs expressed in potato tubers, in order to apply the protease-chip in the analysis of pest-induced potato protease inhibitors. We printed a set of proteases (trypsin, chymotrypsin, elastase, cathepsin D, cathepsin B, ficin, bromelain) with the function of capture probes for constitutive KPIs and for the KPIs induced by pest infection in potato tubers. Group-A and group-C KPIs are highly induced during fungal infection. To visualize the KPIs bound onto the array, we used an anti-KPI antibody labelled with cyanine-3. Difference in spot-bound inhibitors and preference for specific proteases was observed, confirming the induction of specific groups of KPIs during fungal infection. We propose that the protease chip could be used for measuring the expression of protease inhibitors in the screening of potatoes attacked by pests.

Electrochemical methods for detection of proteins and DNA
A. Ramanavicius

Vilnius University, Vilnius, Lithuania

[e-mail]
Conducting polymers can be exploited as an excellent tool for the preparation of nanocomposites with nano-scaled biomolecules. Polypyrrole (Ppy) is one of the most extensively used conducting polymers in design of bioanalytical sensors. During this work significant attention was paid to immobilization of biologically active molecules (proteins and single-stranded DNA) within Ppy during electrochemical deposition of this polymer. Such unique properties of this polymer as prevention of some undesirable electrochemical interactions and facilitation of electron transfer from some redox enzymes are important for improvement of biosensors. Recent advances in application of polypyrrole in immunosensors and DNA sensors will be discussed in presented poster. Some new electrochemical target DNA and target protein detection methods based on changes of semiconducting properties of electrochemically generated Ppy doped by affinity agents is introduced. Pulsed amperometric detection (PAD) of target proteins and DNA with platinum electrodes modified by affinity exhibiting proteins and single-stranded DNA (ssDNA) entrapped within polypyrrole is reported. Single-stranded DNA 20-mers complementary to the target DNA were used to construct the DNA biosensors. Electrochemical impedance spectroscopic (EIS) investigation of modified electrodes before and after incubation in target protein or DNA-containing sample revealed significant changes in terms of an imaginary (Z″) vs. a real (Z′) component. The PAD results were in good agreement with EIS investigations. The PAD method was selected, because it does not require such sophisticated equipment as it is used to perform EIS and the results obtained can be more easily estimated. Optimum conditions for performing PAD and evaluating an analytical signal were elaborated. No label-binding step was necessary for detection of target proteins or DNA. The detection duration was reduced down to 30-35 min. Recent progress and problems in development of molecularly imprinted polypyrrole will be considered in this presentation as alternative method for possible detection of some target analytes.

Transfer technology from COST 853 to the new COST 873
B. Duffy

Agroscope Changins-Waedenswil Research Station ACW, Laboratory for Molecular Diagnostics, Waedenswil, Switzerland

[e-mail]
COST Action 873 'Bacterial diseases of stone fruits and nuts' has recently been launched (www.cost873.ch). This Action will is aimed at improving plant protection and disease control efforts in Europe against all the bacterial diseases of all stone fruits and nuts. This includes pathogen diversity/diagnostics and host resistance as cornerstones. We are interested in applying the advances made in COST 853 for achieving our aims. Specific applications already identified include developing microarrays to rapidly characterise pathogens in orchards and to characterise plant host genotypes for suitability in integrated pest management approaches. We look forward to future utilisation of COST 853 discoveries.

Mining Attribute Profiles - An improved road-MAP to the discovery of co-regulated gene expression modules
U. Wagner

Functional Genomics Center Zürich, Uni/ETH Zürich, Switzerland

[e-mail]
Association pattern discovery (APD) methods have been successfully applied to gene expression data. They find groups of co-regulated genes in which the genes are either up- or down-regulated throughout the identified conditions. Traditional APD algorithms, developed for binary (or Boolean) attributes, can be applied to such data with a prerequisite of transforming non-binary (continuous or categorical) attribute domains into binary ones. As a consequence of this binarization, the discovered patterns no longer reflect the associations between attributes but the relations between their binned independent values, and thus, interactions between the original attributes may be lost. Consequently, these methods fail to identify similarly expressed genes whose expressions change between up- and down-regulation from one condition to another. In order to discover these hidden patterns, we propose the concept of mining co-regulated gene profiles, also called the concept of "Mining Attribute Profiles" (MAP). Co-regulated gene profiles contain two gene sets such that genes within the same set behave identically (up or down) while genes from different sets display contrary behaviour. To reduce and group the large number of similar resulting patterns, we propose a new similarity measure that can be applied together with hierarchical clustering methods. We tested our proposed method on two well-known yeast microarray datasets. As a results we observed that our implementation of the MAP method mined the data effectively and discovered patterns of co-regulated genes that are hidden to traditional APD methods. The high content of biologically relevant information in these patterns is demonstrated by the significant enrichment of co-regulated genes with similar functions. Our experimental results show that the MAP method is an efficient tool for the analysis of gene expression data and competitive with bi-clustering techniques.

1. Rapporteurs Remarks 1

The first session, which was devoted to the technical developments that we have witnessed during the running Action 853, started with a keynote lecture where the business philosophy of Nimblegen Systems Inc. was explained as to be highly devoted towards applied development of a technology for practical use. After showing the developmental stages on a time scale C. Oste explained the different degrees of services his company is offering and explained the synthesis technology the company applies to produce oligonucleotide arrays. He then switched to his speciality, the Comparative Genome Sequencing (CGS) method, leading to increased speed in whole genome sequencing and mapping of mutations which allows to study evolutionary changes in almost real time. He also points out in this direction that obviously ribosomal populations exist due to the presence of up to seven different operons of rDNA. This seems to support more flexibility in the expression system of proteins.

In the second talk X. Nesme used the CGS method with Nimblegen technology to explore the reasons for the great genetic heterogeneity of Agrobacterium tumefaciens isolates and strains. From comparisons of the different genomic traits of the plant pathogenic bacterium it became evident, that the linear genome was the most diverse between different isolates when compare with the circular genome part. This gave first indications about the genetic background for biovars and the genes involved in pathogenicity and host range.

The next report by J. Valkonen described the results obtained with diagnostic oligonu-cleotide chips to detect plant pathogenic bacteria threatening potatoes, i.e. Erwinia spec and Streptomyces spec. Each probe was selected from whole genome information and was 40 nucleotides long with a 3’-based poly(T)-tail. These arrays, made by Agilent, were used for detection and differentiation of the different pathogens as well as for expression profiling of genes being induced by potato extract in Pectobacterium atrosepticum. The results were confirmed by qPCR.

In a first talk about protein based chips P. Poltronieri reports the application of chips where either Kunitz-type-proteaseinhibitors (KPIs) or the respective interacting proteases were spotted to study their interaction. This could facilitate the study of proteases from various plants, insects and pathogens as biomarkers, as well as to identify the constitutively expressed or induced KPIs in plants after plant pathogen attacks. The protease-chips could also be useful to study the allergene formation via mite protases which could be prevented by the different KPIs.

The different ways to read out array results without the need to label reaction partners was the topic of the talk from A. Ramanavicius, who demonstrated via animation how catalytic sensors, affinity sensors and further on electrochemical methods using conducting polymers like Polypyrroles to construct biosensors. Especially the application of Pulsed Amperometric Detection (PAD) was described in detail as a method for detection of target proteins or DNA.

The talk by B. Duffy aimed for two goals, first describing how the achievements of the finished action 853 have been taken up by the newly started Action 873 and second asking for support and initiation of cooperation in order to support the development of more homogeneous diagnostic tools for plant pathogens in order to help plant health organisations, as well as breeders and growers via genotyping and plant host transcriptomics to reveal plant microbe interactions.

In a very convincing and clear talk U. Wagner explained how he replaced hierarchical clustering approach for gene expression by a new and better bioinformatic method. The newly developed method originated from market analyses, asking what is significantly more often bought together. This question is now addressed to gene expression data from array experiments and leads to more defined and function supported results.