Functional Genome Analysis  (B070)
Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580
D-69120 Heidelberg, Germany.



Archive      Transcriptional Profiling Analyses

microRNA Studies
mRNA Studies

microRNA Functions
microRNA Diagnostics

Cancer studies

Microbial Studies

    - Melanoma tumour-niche formation
        - Human pathologies
     - Saccharomyces cerevisiae
      - Trypanosoma brucei
    - Pancreatic cancer progression
        - Primary CNS lymphoma
     - Mouse 
      - Neurospora crassa

    - Variations upon chemotherapy
        - Trypanosoma infection
     - Drosophila melanogaster
      - Bacillus subtilis

     - Arabidopsis thaliana
      - Pseudomonas putida


figure of melanoma in situ  
Melanoma microRNA trafficking controls tumour primary niche formation

logo MOST                   logo DKFZ
Melanoma originates in the epidermis and becomes metastatic after invasion into the dermis. Prior interactions between melanoma cells and dermis are poorly studied. Here, we show that melanoma cells directly affect the formation of the dermal tumour niche by microRNA trafficking before invasion. Melanocytes, cells of melanoma origin, are specialized in releasing pigment vesicles, termed melanosomes. In melanoma in situ, we found melanosome markers in distal fibroblasts before melanoma invasion. The melanosomes carry microRNAs into primary fibroblasts triggering changes, including increased proliferation, migration and pro-inflammatory gene expression, all known features of cancer-associated fibroblasts (CAFs). Specifically, melanosomal microRNA-211 directly targets IGF2R and leads to MAPK signalling activation, which reciprocally encourages melanoma growth. Melanosome release inhibitor prevented CAF formation. Since the first interaction of melanoma cells with blood vessels occurs in the dermis, our data suggest an opportunity to block melanoma invasion by preventing the formation of the dermal tumour niche.
Figure legend. Melanoma tumour in situ. The melanoma cells have not yet left the epidermis; green: fibroplasts; red: melanosomes; blue: DNA in cell nuclei.
Dror, Sanders et al. (2016) Nature Cell Biol., in press.  (doi: 10.1038/ncb3399). 


Early epigenetic down-regulation of microRNA-192 expression promotes pancreatic cancer progression logo NGFN
Pancreatic ductal adenocarcinoma (PDAC) is characterized by very early metastasis, suggesting the hypothesis that metastasis-associated changes may occur prior to actual tumor formation. We identified miR-192 as an epigenetically regulated suppressor gene with predictive value in this disease. miR-192 was downregulated by promoter methylation in both PDAC and chronic pancreatitis (CP), the latter of which is a major risk factor for development of PDAC. Functional studies in vitro and in vivo in mouse models of PDAC showed that overexpression of miR-192 was sufficient to reduce cell proliferation and invasion. Mechanistic analyses correlated changes in miR-192 promoter methylation and expression with epithelial-mesenchymal transition (EMT). Cell proliferation and invasion were linked to altered expression of the miR-192 target gene SERPINE1 that is encoding the protein plasminogen activator inhibitor-1 (PAI-1), an established regulator of these properties in PDAC cells. Notably, our data suggested that invasive capacity was altered even before neoplastic transformation occurred, as triggered by miR-192 downregulation. Overall, our results highlighted a role for miR-192 in explaining the early metastatic behavior of PDAC and suggested its relevance as a target to develop for early diagnostics and therapy.
Figure legend. Two cell lines, which express miR-192 at high level (CFPAC-1) or low level (MIAPaCa-2) were transfected with constructs that suppressed or increased miR-192 expression, respectively. Xenografted into mice, strong effects on tumour growth were observed.
Botla et al. (2016) Cancer Res. 76, 4149-4159. 
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Blood-borne microRNA signatures in human pathologies logo NGFN
Beyond studies that describe microRNAs frequently as markers for specific traits, we asked whether a general pattern for microRNAs across many diseases exists that could act as an intial, non-invasive means of diagnostics. In a multicenter study, we evaluated circulating profiles of 1,049 patients suffering from 19 different cancer and non-cancer diseases as well as unaffected controls. The results were validated on 319 individuals from three different centers using qRT-PCR. We detected consistently deregulated profiles for particular diseases; pathway analysis confirmed disease association of the respective microRNAs. Overall, we discovered 34 miRNAs with strong disease association. In addition, a set of microRNAs was discovered  that act as rather stable markers, offering reasonable control microRNAs for future studies. Our study further underscores the high potential of specific blood-borne miRNA patterns as molecular biomarkers.
Figure legend. The balloon plot shows in how many diseases microRNAs are up- or down-regulated. The bubble size represents the number of microRNAs showing the respective distribution of up- and down-regulation. Orange bubbles represent microRNAs that are predominantly down-regulated, while blue bubbles stand for predominantly up-regulated microRNAs. The two green bubbles represent 9 microRNAs that were equally often up- and down-regulated in disease.
Keller et al. (2014) BMC Med. 12, 224. 
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Keller et al. (2011) Nature Meth. 8, 841-843.  pdf icon

number of miRNA expression variations in diseases


MicroRNAs in the blood of patients with primary CNS lymphoma (PCNSL) act as prognostic biomarkers
Despite improved therapeutic regimens, primary CNS lymphoma (PCNSL) remains a therapeutic challenge. We characterized by next generation sequencing the microRNA fingerprints in the blood of PCNSL patients enrolled in a large randomized phase III study, comparing short-term to long-term survival. Twelve microRNAs were significantly deregulated between the two groups. The microRNAs miR-493-3p and miR-432-5p exhibited the most prominent differences. Furthermore, we identified short RNA molecules with putative microRNA function that had not been described before.
Roth et al. (2015) Eur. J. Cancer 51, 382-390.

Boxplot presentation of the pancreas classification results

Diagnosis of pancreatic ductal adenocarcinoma and chronic pancreatitis by measurement of microRNA abundance in blood and tissue logo NGFN
MicroRNAs can regulate hundreds of genes post-transcriptionally and appear to regulate virtually all cellular processes. Owing to these properties, microRNAs have a critical role not only in physiological but also in pathological processes. There is meanwhile a lot of evidence that miRNA profiles from body fluids, such as blood, and informative about the disease status of a blood donor.
A detailed analysis of microRNA profiles was performed for pancreatic ductal adenocarcinoma. A solid diagnostic process could have substantial impact on the successful treatment of the disease, for which currently mortality is nearly identical to incidence. Variations in the abundance of all microRNA molecules from peripheral blood cells and pancreas tissues were analysed. In total, 245 samples from two clinical centers were studied that were obtained from patients with pancreatic ductal adenocarcinoma or chronic pancreatitis and from healthy donors.
Thee blood test demonstrated very high sensitivity and specificity of a distinction between healthy people and patients with either cancer or chronic pancreatitis. Confirmative and partly even more discriminative diagnosis could be performed on tissue samples. In addition, discrimination between cancer and chronic pancreatitis was achieved. Several miRNAs were identified that exhibited abundance variations in both tissue and blood samples.
The results could have an immediate diagnostic value for the evaluation of tumour reoccurrence in patients, who have undergone curative surgical resection, and for people with a familial risk of pancreatic cancer.

Bauer et al. (2012) PLoS ONE 7, e34151.  pdf icon

MicroRNA signatures of peripheral blood cells in humans infected with Trypanosoma brucei gambiense logo DAAD
Human African Trypanosomiasis - sleeping sickness - still affects thousands of people a year. Control relies on diagnosis and treatment, and in the absence of this surveillance, the number of cases rapidly rebounds. Diagnosis relies on an antibody test and microscopy. The type of treatment depends on the disease stage: whether parasites have entered the central nervous system; and this can be determined only by lumber puncture and analysis of cerebrospinal fluid. The development of sensitive, simple and reliable tools for diagnosis and staging of human African Trypanosomiases would significantly ease field surveillance and enhance patient care. We investigated whether the patterns of miRNAs from peripheral blood could be used to decide whether patients are infected, or to determine the disease stage. We found that the miRNA patterns did differ between parasite-positive patients and uninfected controls with no immune reaction to trypanosomes. Also, people with immune reactions to trypanosomes, but no detectable parasites, sometimes showed patient-like profiles. The patterns were not reliable enough, however, to be used for diagnosis.
Lueong et al. (2013) PLOS ONE 8, e74555.  pdf icon

MicroRNA expression profiles in peripheral blood cells of rats infected with Trypanosoma congolese and Trypanosoma brucei subspecies
To identify differentially regulated miRNAs during trypanosome infections, we analyzed the miRNA expression profiles of uninfected rats and animals infected with Trypanosoma congolense and different Trypanosoma brucei species. The potential target genes of the regulated miRNAs as well as their biological pathways and biological functions were investigated.

Simo et al. (2015) Microb. Infect. 17, 596-608. 
logo Humboldt Foundation

MicroRNA variations in cells and their exosomes upon chemotherapy
It has been convincingly proposed that exchange of molecules via exosomes is a means of eukaryotic intercellular communication, especially within the tumour microenvironment. However, there are no data on the alterations of exosomal molecular cargo upon pharmacological anticancer treatments. To approach this issue, we analysed the abundance of miRNAs and cancer-related proteins in exosomes secreted by Caco-2 (Cetuximab-responsive) and HCT-116 (Cetuximab-resistant) colorectal cancer cells before and after Cetuximab treatment. We also characterized both profiles in whole source cells.
Cetuximab significantly altered the molecular cargo of exosomes from Caco-2: we detected increased abundance of miRNAs and proteins that activate cell proliferation and proinflammatory processes; simultaneously, we observed a decrease of miRNAs and proteins related to immune suppression. These changes did not overlap entirely with those in source cells, suggesting a Cetuximab-linked distribution bias.
Molecular changes of a minor extent were also detected in exosomes from HCT-116. Transfection of exosomes from Cetuximab-treated Caco-2 into HCT-116 significantly increased HCT-116 viability; conversely, Caco-2 transfected with exosomes from treated HCT-116 did not show viability alterations. This suggests that the molecular phenotype of source cells is important for determining both the exosomal cargo as the biological effects of transferred exosomes.
Gene Ontology analysis of networks that comprise targets of differentially expressed exosomal miRNAs and proteins demonstrated a significant involvement of biological processes related to proliferation control, inflammation, immune response, and apoptosis. Our data shed light on molecular mechanisms of intercellular communication in eukaryotes.

Ragusa et al. (2014) Oncoscience 1, 132-157pdf icon

Quantitative asymmetric distribution of miRNAs in colorectal cancer cells and exosomes. Relative quantities (RQ) of the miRNAs amounts as isolated from exosomes were compared to those from the source cells Caco-2. Values are shown as log10 of RQ.

Identification of malignancy factors by analysing cystic tumours of the pancreas

logo NGFN            

The diversity in the aggressiveness of cystic tumours of the pancreas – ranging from the usually benign serous cystadenoma to lesions of variable degrees of malignancy – was utilised for the identification of molecular factors that are involved in the occurrence of malignancy. We analysed the transcript profiles of different cystic tumour types. Variations could be identified that could be critical for the regulation of malignancy and thus relevant to the treatment of also the majority of pancreatic tumours. The results were confirmed at the protein level by immunohistochemistry. Also, functional studies with siRNA silencing were performed. Expression variations at the RNA and protein level were identified that are closely correlated with the degree of malignancy. Besides, all tumours could be classified effectively by this means. Many of the identified factors had not previously been known to be associated with malignant cystic lesions. SiRNA silencing of the gene with the most prominent variation – the anti-apoptotic factor FASTK (Fas-activated Serine/Threonine Kinase) – revealed a regulative effect on several genes known to be relevant to the development of tumours.

Bauer et al. (2009) Pancreatology 9, 34-44.  pdf icon

Figure legend: correspondence cluster analysis of transcript profiles. In the resulting biplot, each hybridisation of an individual sample is depicted as a coloured square. Genes that exhibited significantly differential transcription levels are shown as black dots. The closer the co-localisation of two spots (both genes and tumours) the higher is the degree of association between them. Also, guidelines are displayed in the diagram. They are calculated from the data and point to the positions of virtual genes, which exhibit a variation in one tumour entity only. The closer a depicted gene lies to one of these guidelines and the further its distance to the centroid the better its expression is described by the respective ideal profile. All genes that are not significantly differentially transcribed are located close to the centroid of the lines but are not shown for clarity. In (a), a cluster analysis is shown of normal pancreatic tissue, all cystic tumours combined and ductal adenocarcinoma. Panel (b) presents the results obtained for the cystic tumours alone. As a consequence of the normalisation process, only the median of the controls is shown in the diagram as a single red circle instead of the individual hybridisation events. In (d), a close-up of the data is shown, which were generated with the IPMC, MCA and MCAC samples. Panel (c), finally, presents a combination of the data with a colour-code added that indicated the tendency of malignancy of the respective tumour types: blue, non-malignant; red, highly malignant.



Frank Sauer &
Renato Paro

Functional analysis of gene and protein networks in Drosophila with microarray based expression studies

The complete genomic sequence of several metazoan organisms such as Drosophila melanogaster and Caenorhabditis elegans was available. The next task arising from this sequence data was (and still is) the deciphering of the role and function of the identified genes and their corresponding protein products in the context of an entire organism. As often the expression pattern of a gene provides clues to its function, we produced a DNA-microarray that enabled us to monitor gene expression in the context of the entire Drosophila genome. The system enabled us to identify genes whose activities are required for the execution of complex developmental gene networks and signal transduction pathways. As such networks and pathways are evolutionary highly conserved among metazoans, the analyses of gene and protein function in Drosophila also provided valuable clues for a better knowledge of corresponding pathways in vertebrates.

While the genome sequences for a variety of organisms are now available, the precise number of genes encoded is sometimes still a matter of debate. We based our whole-transcriptome microarray, the Heidelberg FlyArray, on the combination of the BDGP annotation and a novel ab initio gene prediction of lower stringency using the Fgenesh software. A microarray was established with altogether some 24,000 different features, each actually present in duplicate. The primer set used to produce the PCR-products was from Eurogentec.

Apart from being used for the production of the microarray, the very primer set was also applied to the generation of a genome-wide dsRNA library, the actual work being performed in the laboratory of Norbert Perrimon at Harvard Medical School in Boston (USA). This molecule set allowed the identification of gene functions by cell-based RNAi-screens.

To assess the overall quality of our array design as well as to validate the novel predictions, we performed developmental profiling of the Drosophila lifecycle using 9 different stages.
We were able to provide evidence for the transcription of ~2,600 additional genes predicted by Fgenesh. Validation of the developmental profiling data by RT-PCR and in situ hybridization indicated a lower limit of 2,000 novel annotations, thus substantially raising the number of genes that make a fly. The successful design and application of this Drosophila microarray confirmed our expectation that mere in silico approaches will always tend to be incomplete. The identification of at least 2,000 novel genes highlights the importance of gathering experimental evidence to discover all genes within a genome. Moreover, as such an approach is independent of homology criteria, it will allow the discovery of novel genes unrelated to known protein families or which have not been strictly conserved between species.

Figure to the right. Correspondence cluster analysis of the developmental profiling. Samples from 9 different stages of the Drosophila lifecycle were hybridised. Each hybridisation of an individual developmental stage is depicted as coloured square. They all form distinct clusters ­– but for the larval stage – indicating the degree of reproducibility and specificity between them. As a consequence of the normalisation process, only the median of all control hybridisations (0-4 h) is shown in the diagram as a single red square. Genes are shown as grey dots, if they exhibited significant differential transcription levels. The distance between dots is low when their expression profiles show similar shape, independent of their absolute values.

Diehl et al. (2002) Nucleic Acids Res. 30, e79. link to pdf file

Hild et al. (2003) Genome Biol. 5, R3.

Boutros et al. (2004) Science 303, 382-385.

Altenhein et al.
Develop. Biol. 296, 545-560.
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figure of data analysis

Creation of a minimal tiling path of genomic clones for Drosophila melanogaster;
provision of a common resource

logo-Berkeley-lab         logo EMBL            logo-Oregon-university

On the basis of shotgun subclone libraries used in the sequencing of the Drosophila melanogaster genome, a minimal tiling path of subclones across much of the genome was determined. About 320,000 shotgun clones for chromosomes X(12-20), 2R, 2L, 3R and 4 were available from the Berkeley Drosophila Genome Project. The clone inserts have an average length of 3.4 kb and are amenable to standard PCR-amplification. The resulting tiling path covers 86.2% of chromosome X(12-20), 86.2% of chromosomal arm 2R, 79.0% of 2L, 89.6% of 3R and 80.5% of chromosome 4. In total, the 25,135 clones represent 76.7 Mb – equivalent to about 67% of the genome – and would be suitable for producing a microarray on a single slide.

This work was performed in collaboration with Susan Celniker (Berkeley), Eric Johnson (University of Oregon) and Eileen Furlong (EMBL).

Hollich et al. (2004) Biotechniques 37, 282-284.  
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figure of shotgun clone genome coverage

Schematic representation of the tiling path’s genome coverage. Horizontal lines indicate the chromosomes of the 115 Mb Drosophila genome. The genomic regions that are covered by the minimal tiling path of 25,135 shotgun clones are represented as blue and green coloured areas. Interruption of the colouring depicts large gaps. Any change in colour from blue to green or visa versa indicates the existence of a gap that is too small to be visible. Below, a table presents the relevant numbers.


Transcriptional profiling of Saccharomyces cerevisiae

 - EUROFAN       .       ..

Based on our involvement in the EU yeast genome sequencing, expression profiling on all yeast genes was started as part of the German and European (EUROFAN) yeast functional analysis networks. The relatively small number of some 6200 genes makes the unravelling of the basic processes of expression control in a eukaryotic cell much easier or even at all possible. Since there exists a surprising degree of structural and partially even functional homology between some human (disease and cancer) genes and their yeast equivalents, an analysis of the expression patterns of this complete gene set is not only very informative for the analysis of yeast gene expression and regulation itself but also very much of relevance to the grasp of such mechanisms in higher eukaryotes.

  - Eurocellwall          .

The main objective of this project was the exploitation of the molecular knowledge of the Saccharomyces cerevisiae cell wall for high throughput screening of anti-microbial agents. To this end, a consortium of 10 laboratories was converting the molecular data on essential gene targets involved in cell wall cross-linking, remodelling and chitin pathways into assays amenable to drug-discovery programmes. Also, genomics, proteomics and bio-informatics were applied to identify new targets through the characterisation of the cell wall compensatory mechanism, which is induced when cell wall is weakened by drug treatment, stress or mutations.

Hauser et al. (1998) Yeast 14, 1209-1221.
Hauser et al. (2002) Screening 3/02, 28-31.

Hauser et al. (1998) Meth. Microbiol.28, 193-204.

Yin et al. (2003) Mol. Microbiol. 48, 713-724.

Beissbarth et al. (2000) Bioinformatics 16, 1014-1022.
Lagorce et al. (2003) J. Biol. Chem. 278, 20345-20357.

Hoheisel & Vingron (2000) Res. Microbiol. 151, 113 -119.
Fellenberg et al. (2003) Perspect. Gene Expression, Eaton Publ., 307-343.

Fellenberg et al. (2001) Proc. Natl. Acad. Sci. USA 98, 10781-10786.
Becerra et al. (2003) Comp. Funct. Genomics 4, 366-375. pdf

Hauser et al. (2001) Comp. Funct. Genomics 2, 69-79.
Hagen et al. (2004) Mol. Microbiol. 52, 1413 -1425. pdf

Fellenberg et al. (2002) Bioinformatics 18, 423-433.
Busold et al. (2005) Bioinformatics 21, 2424-2429. pdf icon

Becerra et al. (2002) Mol. Microbiol. 43, 545-555.
Fellenberg et al. (2006) BMC Genomics 7, 319. pdf icon

Lombardia et al (2002) Cell Calcium 32, 83-91.
Hauser et al. (2007) FEMS Yeast Res. 7, 84-92.

"MouseExpress": In silico analysis of expression profiles in mouse-mutants

     .       ..    

Johannes Beckers , Martin Hrabe de Angelis , GSF, Munich; Werner Mewes , GSF, Munich; Martin Vingron , MPIMG, Berlin.

The sequencing of the mouse and human genomes have basically been accomplished. The next step for the integration of this genomic information into biological and biomedical research will be the systematic analysis of gene function. The similarities between man and mouse in their genomes, molecular pathways, physiology and developmental mechanisms make the mouse the most important model organism for the study of inherited diseases in man.
To discover new mutants that serve as models for human diseases or that have developmental defects, mice that have been subjected to ENU mutagenesis are routinely examined for clinical parameters, behaviour and dysmorphologies (Mouse ENU Mutagenesis Screen, Institute of Experimental Genetics, GSF, Neuherberg-Munich, Germany). In the 'MouseExpress' project, we extended phenotypic description to a molecular level. Combining DNA microarrays and appropriate phenotypical data, we compared RNA expression profiles of thousands of genes from tissues or embryos of mutant and wildtype mice.
We used the RNA expression profiles to identify molecular pathways that are affected in disease and analysed interdependencies between pathways within a molecular network. Expression profiles of mutant and wildtype mouse strains are filed in a database and will be linked to the phenotype and mutant databases of the Mouse ENU Mutagenesis Screen of the GSF.

overall picture of mouse array

Beckers et al. (2002) Curr. Genet. 3, 121-129.  .

Transcriptional profiling of Arabidopsis thaliana

                 logo BMBF        .

Global transcriptional profiling in Arabidopsis thaliana was started in the EU-funded PPMdb-network and extended as part of the German ZIGIA consortium. Analyses were initially performed on a set of some 13,000 non-redundant EST-clones combined from the EST-clone collection of the Institut National de la Recherche Agronomique (Versailles, France) and the MSU EST-clone collection obtained from the Arabidopsis Biological Resource Center at the Ohio State University (Columbus, USA). Subsequently, analyses were performed on a set of 50mer oligonucleotides, representing the Arabidopsis gene set.

Various conditions were studied. One initial area of emphasis was the analysis of pathogen responses, done in collaboration with Nikolaus Schlaich and Alan Slusarenko of the RWTH Aachen.

Further studies were performed in a local collaboration with Florian Haas and Rüdiger Hell of the Heidelberg Institute for Plant Science (HIP) at Heidelberg University. They aimed at the elucidation of the effects of mitochondrial serine acetyltransferase functions on cysteine synthesis in plant cells. At the cellular level, cysteine synthesis in plants is entirely different from that in non-photosynthetic eukaryotes.

Scheideler et al. (2002) J. Biol. Chem. 277, 10555-10561.   .
Wambutt et al. (2000) J. Biotechnol. 78, 281-292.   .
Haas et al. (2008) Plant Physiol. 148, 1055-1067.   .
other publications and patents


Analyses in Trypanosoma brucei 
logo DFG              logo DAAD

The life cycle of Trypanosoma brucei involves adaptation to a variety of conditions in the host and Tsetse fly. The successive changes in morphology, biochemistry and plasma membrane proteins, some of which also involve cell cycle arrest, are still poorly understood. Many of these changes are likely to be directed by changes in mRNA abundance and translation, and over two decades of effort have now been expended in the identification of stage-specific mRNAs. Because of technical limitations, however, most of the transcripts identified have been rather abundant, and the regulation studied has mainly been restricted to the rapidly-dividing long slender bloodstream and procyclic forms.
Given the relatively small size of the T. brucei genome, there is a good prospect of a complete exploration of its genome by microarray analyses. This will allow identification of lower-abundance regulated transcripts, and (using amplification methods) the study of the transcriptome of the less accessible forms found in the Tsetse fly. One format for the analysis is to perform genome-wide expression studies on genomic instead of gene-specific fragments. Such arrays have some intrinsic advantages. Overall genome representation is usually good in shotgun libraries with comparatively little variation across the genome. Thus, even if randomly selected clone inserts are used as probes, there should be good coverage and relatively little redundancy. Also, not only coding but also intergenic regions can be studied, for example in chromatin immunoprecipitation experiments. Insert amplification can be performed with a single primer pair and functional analyses can actually precede sequencing.

In collaboration with the group of Christine Clayton, we produced DNA-microarrays containing more than 21,000 PCR-products of 2 to 2.5 kb long genomic fragments of T. brucei strain TREU927/4, which were used in several projects. We also applied oligonucleotide microarrays provided by TIGR.
figure of T. brucei chip

Diehl et al.  (2002)
Mol. Biochem. Paras. 123, 115-123.
Brems et al.  (2005) Mol. Biochem. Paras. 139, 163-172. pdf icon
Van-Duc et al.  (2006) Mol. Biochem. Paras. 150, 340-349. pdf icon
Denninger et al.  (2007) Exp. Cell. Res. 313, 1805-1819. pdf icon
Hartmann et al.  (2007) Eurcaryotic Cell 6, 1964-1978. pdf icon
Kramer et al.  (2008) J. Cell. Sci. 121, 3002-3014. pdf icon
Clayton et al. .(2008) Biochem. Soc. Trans. 36, 520-521. pdf icon
Wurst et al. .(2009) Mol. Biochem. Paras. 163, 61-65. pdf icon
Queiroz et al. .(2009) BMC Genomics 10, 495 pdf icon
Archer et al. .(2009) PLoS Pathogen 5, e1000565. pdf icon
Kramer et al. .(2010) J. Cell. Sci. 123, 699-711. pdf icon

logo Humboldt-StiftungFINISHED PROJECT:
Analysis of gene expression in Trypanosoma brucei gambiense

Human African Trypanosomiasis (HAT) is a disease that exists in two forms. The acute form is caused by T. b. rhodesiense (observed in East Africa) while the chronic form is due to T. b. gambiense (found in West and central Africa). Around 60 million persons are exposed to this disease with some 500,000 current infections. Despite a relentless fight against HAT during the last century, it is currently resurgent in epidemic form in several countries.

The low sensitivity of the diagnostic techniques associated to the low parasitaemia that characterizes T. b. gambiense infections lead permanently to residual human reservoir of trypanosomes in HAT foci after medical surveys. Moreover, the treatment of T. b. gambiense infections requires toxic drugs that are also less effective in the late stage of the disease. This ineffective treatment is strengthens by the emergency of parasite strains resistant to the most available drug (Melarsoprol). Since treatment of HAT is harsh and sometimes ineffective, the vaccine approach is probably a good perspective for trypanosomiasis prevention. Until now, there is no prospective vaccine for HAT despite the fact that investigations on vaccine have been a goal for nearly a century.

With the resurgence of HAT, it is important to improve the control of this disease by undertaking studies that may lead to the discovery of new genes essential for the survival of trypanosomes. These genes could be targeted further for drugs, vaccine or diagnostic tools. The considerable differences between T. b. gambiense and the others T. brucei sub-species should be most likely the result of changes at the nucleotide sequences or in gene expression. We were analysing gene expression in T. b. gambiense using DNA-microarrays, which were produced on the basis of the shotgun clones made for sequencing. The identification of sub-species specific gene expression variations could greatly facilitate the generation and testing of hypotheses on the mechanism of human serum resistance in T. b. gambiense, the key factors to its ability to infect human.

Simo et al. (2010) Infect. Genet. Evol. 10, 229-237.  pdf icon


Transcription analysis in microbial organisms

Bacillus subtilis

Within a 'Leitmotiv Medizin' project, comparative studies were performed in collaboration with Michael Hecker of the University of Greifswald on the variation of all transcripts of Bacillus subtilis - carried out by microarray analysis - and the actual protein levels as identified in 2D-electrophoresis. 
Petersohn et al. (2001) J. Bacteriol. 183, 5617-5631.   .

Neurospora crassa logo DFG

Ever since Tatum and Beadle formulated their one-gene-one-enzyme hypothesis on the basis of studies with Neurospora crassa, this filamentous fungus served as a model organism not only in genetics but also many other fields of basic research. Despite a lot of successful research, only about one tenth of the genes of Neurospora crassa had been described and localised on the seven chromosomes prior to genome initiatives. Genome analysis started by ordering cosmid and BAC clones along individual chromosomes. Based on the physical clone maps of linkage groups II and V, sequencing of the two chromosomes was done as part of the German Neurospora Genome Project. Simultaneously, a whole-genome shotgun approach was taken at the Whitehead Genome Center, Cambridge, USA, recently yielding the complete genomic sequence.
For an initial insight into transcriptional variations in Neurospora crassa, we started with the creation of a microarray prior to sequence assembly and annotation, however. Some 4,700 EST-clones were arrayed on glass slides and used to monitor nutrient-dependent functional phenomena in Neurospora crassa. Upon availability of the sequence, also arrays made by in situ synthesis of oligonucleotides were used in other analyses.

Aign & Hoheisel (2003) Fungal Genet. Biol. 40, 225-233.   .

Pseudomonas putida

As part of our participation in the sequencing ofthe Pseudomonas putida genome, we selected in collaboration with our partners a tiling path of shotgun clones across the entire genome. DNA-microarrays were produced with PCR-amplified material of these genomic fragments and used in transcriptional studies.

Stjepandic et al. (2002) Environ. Microbiol. 4, 819-823.   .
Nelson et al. (2002) Environ. Microbiol. 4, 799-808.   .
Rewa et al. (2006) J. Bacteriol. 188, 4079-4092.   .

Vingron & Hoheisel   
J. Mol. Med. 77, 3-7.
Beissbarth et al.
Bioinformatics 16, 1014-1022.
Fellenberg et al.
Proc. Natl. Acad. Sci. USA 98, 10781-10786.
Hoheisel et al.
Bioforum 12/01, 908-910.
Fellenberg et al.
Bioinformatics 18, 423-433.
Brazma et al.
Adv. Biochem. Biotechnol. 77, 113-139.
Fellenberg et al.
Perspectives in Gene Expression, Eaton Publishing, Westborough, 307-343.
Busold et al.
Bioinformatics 21, 2424-2429.
pdf icon
Fellenberg et al.
BMC Genomics 7, 319.
pdf icon
Moghaddas Gholami & Fellenberg
Bioinformatics 26, 1082-1090. pdf icon
other publications and patents

         logo BMBF        ..

Multi-Conditional Hybridisation Intensity Processing System (M-CHiPS)

Initial data analysis software tools and an appropriately structured database were developed by Kurt Fellenberg in close collaboration with Martin Vingron and went live in 1999. From this work resulted the M-CHiPS data warehouse and analysis software package, which was designed and implemented by Kurt Fellenberg. Apart from our own projects, the package is being used by various external partners and other groups elsewhere.
Eventually, the data warehouse held results of more than 13,000 experiments.
The Multi-Conditional Hybridisation Intensity Processing System (M-CHiPS) is a data warehouse, which provides a structure suitable for statistical analysis of a microarray database's entire content, including components such as the experimental and clinical annotations, for example. The storage concept is flexible and accounts for future developments. For each organism, there is a specific database. Although these databases may contain different ontologies of experimental and other annotations, they share the same structure and therefore can be accessed by the very same statistical algorithms. An ontology-independent structure enables ontology-updates during normal database operation, avoiding structure-alterations.
For overall data analyses as well as the identification of associations between transcriptional variations and annotated factors, including clinical information, GO-terms, mapping data and such alike, correspondence analysis is used extensively. It is an explorative computational method for the study of associations between variables and proved its usefulness for identifying factors, which are associated to certain phenotypes, for example. Much like principle component analysis, it displays a low-dimensional projection of the overall data matrix. One major advantage of the process is its ability to present different parameters of a multi-dimensional data matrix (e.g., genes and experimental conditions) in a single plot. Localisation of genes and individual experiments is an indicator for an association between them. Moreover, additional information, such as GO-term or clinical annotations, can be displayed also, permitting an immediate identification of regulated functional groups or pathways). In addition, algorithms have been established to identify from the annotated data the factors, which are likely to be causative for the establishment of certain sub-groups (clusters) of factors (e.g., genes or patient groups) by statistical data evaluation. 

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