S. Rüberg1, B. Linke1, E. Krol1, S. Weidner1, U.D. Schiller2, S. Kurtz2, R. Giegerich2,
Lehrstuhl fur Genetik, Universität Bielefeld, Germany
2AG Praktische Informatik, Technische Fakultät, Bielefeld, Germany
Sequencing of the genome of the symbiotic soil bacterium S. meliloti 1021 was recently finished by an international consortium (Galibert et al. 2001). The genome consists of three replicons: the 3.5 Mb chromosome, the 1.35 Mb megaplasmid pSymA and the 1.7 Mb megaplasmid pSymB. Annotation of the genome sequence predicted 6204 protein-encoding genes (http://sequence.toulouse.inra.fr/rhime/Consortium/home.html). To construct a microarray on glass slides as well as a macroarray on nylon membranes comprising DNA fragments representing the 6204 protein-encoding ORFs, primer pairs for amplification of 6163 internal fragments of these ORFs by PCR and 41 70mer oligonucleotides were designed using the Primer-Lib software (B. Linke, Bielefeld, unpublished) that creates a primer database and allows for the design as well as the verification of primer sequences on the whole genome scale. Most of the primer pairs amplify 300 to 350 bp fragments, whereas the remaining primer pairs amplify 80 to 299 bp fragments that represent short genes. The Tm of all primers varies less than 1°C. All primers carry 5' extensions that allow the reamplification of the amplified fragments by PCR using standard primers. Currently, the amplification of fragments for 6163 different S. meliloti 1021 ORFs is under way.
Hindlll and BsrGl restriction sites in the 5' extensions that are not present in the amplified sequences of 6051 ORFs can be used for directed cloning into the mobilizable suicide vector pK19mob2HMB. For cloning 4992 fragments longer than 220 bp were selected to allow for integration of the resulting plasmids in the S. meliloti genome by homologous recombination. Since internal fragments of the predicted protein-coding ORFs were chosen, in most cases plasmid integration will result in a loss of gene function. Currently, cloning of these fragments and conjugation is carried out.
A 183 bp linker cassette and 1498 30-bp tags were designed for the construction of a set of miniTnJ transposons each containing two of these tags. The linker cassette and the tag sequences were designed using the DNA sequence compiler software (Feldkamp et al. http://lsll-www.cs.uni-dortmund.de/molcomp). All sequences were designed to be as different as possible from the S. meliloti 1021 genome sequence and from each other. The Tm of all tags that contain 24 bp variable sequence and are flanked by sequence overhangs compatible to Hin&lll and Kpnl restriction sites varies less than one 1°C. These tags are suitable to be inserted into the Kpnl and HindUl sites of transposon mTn5-GNm-L derived from transposon mTn5-GNm (Reeve et al. 1999) by insertion of the linker cassette into the Sfil site. Tag sequences inserted into the linker cassette can be amplified by quantitative PCR using the two standard primer pairs individually or together. Tags were linked to a coated glass surface after ligation of an amino modified linker and denaturation. A hybridization was carried out using Cy3- and Cy5-labeled tags that were amplified from a mixture containing 96 different tag-containing fragments in different ratios. Cross-hybridization experiments showed that the tags can be differentiated by hybridization. Mutants carrying individually tagged transposons are suitable for the analysis of mixtures of these mutants in competition experiments.
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