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

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   Mapping and Sequencing of Pseudomonas putida
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Physical map of the Pseudomonas putida genome.

In the two-dimensional matrix, 362 hybridisation probes are arranged as columns, while 3620 clones are arranged in rows. A positive hybridisation signal is represented by a black spot at the respective cross-section. The entire data set is presented, including all false positive or negative results. The maps starts and ends at the origin of replication (ori). Gaps in the contig coverage are indicated by lines across the diagonal. 

On the right margin, the results obtained from hybridising genomic restriction fragments are shown; they are ordered left to right according to their position in the macro-restriction maps produced with SwaI and I-CeuI, respectively. These data were not immediately taken into account for the actual clone ordering process but served as an independent control of co-linearity. Hybridising a short fragment resulting from the SwaI-digest of genomic DNA, the positions of the ribosomal operon were highlighted (rDNA: A*, A-F). 

The position of four other repetitive sequences (rep1 to rep4) and the cross-hybridisation patterns produced by three chimeric clones (chimera) are indicated. The reason for a cross-hybridisation of a genomic fragment with a specific but unrelated area (rep5) is unknown; no such effect could be observed in any of the relevant cosmid probe hybridisations. 

Circular representation of the
P. putida KT2440 genome.

Outer circle, predicted coding regions on the plus strand colour coded by role categories: salmon, amino acid biosynthesis; light blue, biosynthesis of cofactors, prosthetic groups and carriers; light green, cell envelope; red, cellular processes; brown, central intermediary metabolism; yellow, DNA metabolism; green, energy metabolism; purple, fatty acid and phospholipid metabolism; pink, protein fate/synthesis; orange, purines, pyrimidines, nucleosides, nucleotides; blue, regulatory functions; grey, transcription; teal, transport and binding proteins; black, hypothetical and conserved hypothetical proteins. Second circle, predicted coding regions on the minus strand colour coded by role categories. Third circle, atypical trinucleotide composition of the genome. Fourth circle, top hits to the P. aeruginosa genome (P < 10-60). Fifth circle, transposable elements (green), phage regions (blue), pyocins (yellow). Sixth circle, tRNAs in red. Seventh circle,rRNAs in blue, and structural RNAs in black.

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


Preparation of a Pseudomonas putida microarray.

From the complete genomic sequence, a minimal set of shotgun clones was defined that represents a minimal tiling path accross the entire genome.

Microarray analysis of genomic DNA. (a) Only one half of a microarray is shown. PCR-products were spotted in duplicate onto poly-L-lysine slides. Cy5- and Cy3-labelled DNA-target was made from genomic DNA of two samples of strain KT2440 kindly provided by Soeren Molin (BioCentrum-DTU, Lyngby, Denmark) and Kenneth Timmis and Edward Moore (GBF, Braunschweig, Germany), respectively. Any genomic difference between the two samples should show up as a red or green signal, respectively. As can be judged by the yellow colour of all spots, no discernal difference in genomic representation was identified. Overall signal intensity varied across the microarray due to differences in the amount of PCR-product present at the individual positions. However, on close scrutiny, all positions with PCR-product gave rise to a signal. In (b), difference in genome content can be seen for one square of the microarray comparing samples of strain P. pudita KT2440, labelled green, and P. fluorescens, labelled red. Since the microarray represents the KT2440 genome, only the lack of such sequence in the P. fluorescens genome could be detected, indicated by a green signal.

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


 

 
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Information on the entire genome sequence:
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http://www.ncbi.nlm.nih.gov/





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