Division of Functional Genome Analysis


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

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Genome Mapping and Sequencing

For de novo large-scale sequencing projects, the availability of physical maps is desirable. The existence of a reliable clone map is extremely helpful even in shotgun sequencing projects. All of them have taken into account some sort of mapping information for contig alignment and control of colinearity. Based on earlier results on Drosophila melanogaster, Schizosaccharomyces pombe, Saccharomyces cerevisiae, Arabidopsis thaliana, Trypanosoma cruzi and human, mapping projects were pursued to provide a scaffold for subsequent or parallel sequence analysis or directly for the preparation of probe molecules for functional studies.

In the projects on Pseudomonas putida, for example, the shotgun clones were immediately used for transcriptional profiling analysis. To this end, a minimal tiling path was identified and placed on microarrays in form of PCR-products. Since this microarray does exhibit all coding regions of the genome, transcript analyses are performed by definition on a complete gene representation of the organism, irrespective of the status of the sequence annotation. Similar work was done in other projects, such as studies on Trypanosoma brucei and Drosophila melanogaster.

FINISHED PROJECT:
Directed gap closure in large-scale sequencing projects

A problem in many sequencing projects is the final closure of gaps left in the clone libraries, which serve as templates for sequencing, because of uncloned or unclonable genomic areas. Using the Xylella fastidiosa genome as a test system, a technique was established to generate in a directed manner sequence information from those gaps. We used the complete clone library as a competitor against the genomic DNA of interest in a subtractive hybridisation procedure similar to representational difference analysis (RDA). The resulting sequence information serves directly for gap closure or can be used to screen selectively other clone resources.

Figure.

(A) Variance in genome coverage of clone library.

(B) Gel separation of the difference products from a comparison of genomic DNA versus library DNA. The enzymes Sau3AI and BamHI had been used for the initial restriction digests; M: 100 bp marker ladder.

(C) Individual fragments were picked at random and end-sequenced. 74% of the sequences were not contained in the clone library but in the final X. fastidiosa genome sequence. The relatively high portion of false positives is probably the result of the single step of subtraction enrichment, done in an attempt to reach a compromise between specificity and representation.

Frohme et al. (2001) Genome Res. 11, 901-903. .

FINISHED PROJECT:
Physical mapping and sequencing of Neurospora crassa

In a collaboration coordinated by the University of Düsseldorf, chromosomes 2 and 5 of Neurospora crassa were sequenced in addition to a limited shotgun sequencing of the entire genome. Within this network, we provided a physical map of both chromosomes to act as a scaffold for sequencing.

In addition, we were producing and using cDNA-microarrays for transcriptional studies.

Aign et al. (2001) Genetics 157, 1015-1020.
Mannhaupt et al. (2003) Nucleic Acids Res. 31, 1944-1954.

Hybridisation of complex DNA-sample
on genomic clone library for mapping purposes.

FINISHED PROJECT:
Physical mapping and transcriptional profiling analyses of the 6.1 Mb genome of Pseudomonas putida.

In a network made up by the Medizinische Hochschule Hannover, the GBF in Braunschweig, QIAGEN in Hilden and us - and in collaboration with TIGR (

U.S.A.

) - the entire genome of P. putida was sequenced. Within this network, we provided a physical clone map and analysed transcriptional changes and genomic differences between strains using microarrays produced from a minimal tiling path of shotgun sequencing clones.

Nelson et al. (2002) Environ. Microbiol. 4, 799-808.

Stjepandic et al. (2002) Environ. Microbiol. 4, 819-823.

Reva et al. (2006) J. Bacteriol. 188, 4079-4092.

FINISHED PROJECT:
Mapping and sequencing of the Xyllela fastidiosa genome.

. .

The complete genome sequence of X. fastidiosaclone 9a5c, which causes citrus variegated chlorosis – a serious disease of orange trees, was mapped and sequenced. Our contribution was the provision of a clone map used for both directed sequencing and as a scaffold. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. Putative functions to 47% of the 2,904 predicted coding regions could be assigned. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium–bacterium and bacterium–host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host.

Simpson et al. (2000) . Nature 406, 151-157.

Frohme et al. (2000) . Nucleic Acids Res. 28, 3100-3104.

Heber et al. (2000) . Genomics 69, 235-241.

Heber et al. (2000) .J. Comput. Biol. 7, 395-408.

Frohme et al. (2001) .Genome Res. 11, 901-903.

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