Research - Laboratories of Core Facilities - Laboratory of Functional Genomics

László PUSKÁS
scientific adviser

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Gábor SZEBENI
research associate

Ágnes ZVARA senior research associate
Klára KITAJKA senior research associate
Nóra FARAGÓ research associate
Rozália CSAPÓNÉ TÖRÖK laboratory assistant

LABORATORY OF FUNCTIONAL GENOMICS

Using approaches of functional genomics, we are focusing on gene activity profiling, gene copy number alterations, methylation pattern analysis and protein expression profiling in different organisms, including human, rat, mouse, wheat, rice, carp, dog, wild dear. Global gene expression changes can be followed in diverse physiologic and pathologic states. This makes possible to reveal changes at the genome level to better understand diseases, stress resistance in plants and to follow the effects of drug treatments.


Microarrays are chemically activated glass slides with a large number of oligonucleotides, cDNAs, proteins or drug-like compounds spotted on their surfaces in high density. They are novel and extraordinary tools for functional molecular biology providing a rapid and comprehensive approach to simultaneously monitor the different mutations in the genome, the expression levels of thousands of known and uncharacterized genes, and protein expression differences between diverse biological samples in a comparative way at different system levels (genome, transcriptome, proteome). Since 2000, the Laboratory of Functional Genomics in BRC has successfully applied the microarray technique for genomic, transcriptomic and proteomic research. The laboratory is equipped with all the high-tech instruments, hardware and software background necessary for microarray printing, reading and data analysis and validation: arrayer robot (MicroGrid II TAS) that enables spotting high density microarrays (up to 25.000 spots/slide), a fully automated hybridization station (Ventana) with precisely regulated hybridization conditions and a confocal laser scanner (Agilent Technologies) which can read the arrays with high sensitivity and resolution, for data validation Quantitative Real Time PCR machine (Corbett Research). We are currently building a local database containing all the information about the experiments using Sun workstation (Sun Microsystem) as hardware and GenePix (spot analysis) and OmniViz (cluster analysis) as software background. This laboratory works partly as a custom-service and has several scientific cooperations with other laboratories and institutes all over Hungary and abroad.

Genome level: Changes within the chromosome: deletion or amplification is quite frequent in various diseases. Specific rearrangements, in many cases, are characteristic of the individual diseases and states. Comparative genomic hybridization (CGH) is a rapid, high throughput, DNA microarray based method that provides a lot of information about the genomic balance of cells, mono- or trisomies, amplifications and deletions in a simple experimental procedure. CGH offers a new solution for amplification/deletion analysis applied so far, because it is extremely well applicable and high throughput way for the overall analysis of the whole genome. Using this method we analyzed a clinical case. Merkel cell carcinoma (MCC) was diagnosed in a woman’s upper lip. After a long tumour-free period, an anaplastic carcinoma with neuroendocrine features developed in her palatine tonsil, raising the possibility of a late haematogenous metastasis, a second field tumour, or a second primary tumour. The regional lymph nodes were devoid of metastasis. Our aim was to reveal whether the tumours have a common origin. Using array-based CGH and an improved DOP-PCR technique we could demonstrate that our protocol preserves the original copy number of different chromosomal regions in amplified genomic DNA more accurately than standard DOP-PCR techniques. In the case of MCC the partly similar and partly different molecular patterns indicated a genetic relationship between the tumours, and excluded the possibility that the tonsillar tumor was a metastasis. The common origin was further confirmed, namely out of 3 early markers (9p, 3p and 17p), two (9p and 17p) were common in both cancer samples. These findings suggest that a genetically altered field was the reason for the development of the tonsillar cancer; thus, it can be regarded pathogenetically as a second field tumour.

Transcriptome level: The most important and most informative application of DNA microarrays is the parallel study of gene expression from different biological samples that focuses on the functionally active parts of the genome. The method has enabled large numbers of genes, from specific cell populations, to be studied in a single experiment. A primary goal of expression profiling studies is to characterize genes that are expressed abnormally. Global gene expression changes can be followed in diverse physiological and pathological states. DNA microarrays with sets of synthetic oligonucleotides on their surface can be used to obtain a molecular fingerprint of the gene expression of cells. We have successfully applied the microarray technique to analyze transcriptome changes due to many different environmental effects. To study the dietary effects of essential fatty acids on gene expression, we have monitored expression profiles of tissues obtained from transgenic animals, osteoporosis and inflammation animal models, bacteria and plants under different stress conditions, human cell lines treated with different drugs, and human tissues of different pathological states. We selected many different stress-response genes to create a special microarray for analyzing different toxicological stresses in different organisms. Using this very specialized microarray a toxicological profile of many small molecules, drug-like compounds or any other biological or non-biological sample can be analyzed.

Proteome level: Although transcript profiling offers a good opportunity to identify genes that play a role in diseases, even the complete mRNA fingerprints have their limitations, since proteins carry out cellular functions. Numerous protein modifications, such as RNA splicing and posttranslational modification (e.g. phosphorylations, glycosylations) are known that protein functions are dependent on. The genomic or the transcript sequence does not give full information about the different protein-protein interactions, how and where these interaction occur inside the cells under various conditions. To obtain detailed information about a complex biological sample, information about many proteins and protein-protein interactions is required. Protein chips are also used in our laboratory (commercially available – Sigma, or in-house made) for screening protein expression and protein modifications in a high throughput manner. Focused protein microarrays are planned to be developed in the future.

Figure 1. DNA microarrays A: hybridized with Cy5 labeled cDNA probe obtained from human lymphocyte total RNA B: hybridized with Cy5 labeled cDNA (obtained from human healthy tissue total RNA) and Cy3 labeled cDNA (obtained from human tumor tissue total RNA).

Selected publications

Kitajka, K., Puskás, L.G., Zvara, A., Hackler, L. Jr,, Barceló-Coblijn, G., Yeo, Y.K. and Farkas, T. (2002). The role of n-3 polyunsaturated fatty acids in brain: modulation of rat brain gene expression by dietary n-3 fatty acids. Proc. Natl. Acad. Sci. U.S.A. 99(5): 2619-2624.

Gu, R., Fonseca, S., Puskás, L.G., Hackler, L. Jr., Zvara, A., Dudits, D. and Pais, M.S. (2004). Transcript identification and profiling during salt stress and recovery of Populus euphratica. Tree Physiol. 24(3): 265-276.

Kitajka, K., Sinclair, A.J., Weisinger, R.S., Weisinger, H.S., Mathai, M., Jayasooriya, A.P., Halver, J.E. and Puskás, L.G. (2004). Effects of dietary omega-3 polyunsaturated fatty acids on brain gene expression. Proc. Natl. Acad. Sci. U.S.A. 101(30): 10931-10936.

Jayasooriya, A.P., Ackland, M.L., Mathai, M.L., Sinclair, A.J., Weisinger, H.S., Weisinger, R.S., Halver, J.E., Kitajka, K. and Puskás L.G. (2005). Perinatal omega-3 polyunsaturated fatty acid supply modifies brain zinc homeostasis during adulthood. Proc. Natl. Acad. Sci. U.S.A. 102(20): 7133-7138.

Kelemen, J.Z., Kertész-Farkas, A., Kocsor, A. and Puskás, L.G. (2006). Kalman filtering for disease-state estimation from microarray data. Bioinformatics 22(24): 3047-3053.

Tímár, J., Mészáros, L., Ladányi, A., Puskás, L.G. and Rásó, E. (2006). Melanoma genomics reveals signatures of sensitivity to bio- and targeted therapies. Cell. Immunol.244(2): 154-157.

Faragó, N., Kocsis, G.F., Fehér, L.Z., Csont, T., Hackler, L. Jr., Varga-Orvos, Z., Csonka, C., Kelemen, J.Z., Ferdinandy, P. and Puskás L.G. (2008). Gene and protein expression changes in response to normoxic perfusion in mouse hearts. J. Pharmacol. Toxicol. Methods. 57(2): 145-154.