The medium-term objective is to determine, before the end of 1997, the entire genome sequence of Bacillus subtilis, which is both of industrial importance and a model organism for Gram-positive bacteria. An international agreement has been reached allowing the allocation of regions of the chromosome among the groups of the European and Japanese Bacillus subtilis genome sequencing networks. This international project currently includes 18 European and 7 Japanese laboratories, together with 2 biotechnology companies (Genencor International and Novo Nordisk) and 1 Korean laboratory.
The European sequencing groups have obtained support from the European Commission (this contract and contract BIO2 CT-942011). The initial objective of contract BIO2 CT-930272 was to determine the DNA sequence of about 23% (0.97 Mbp) of the Bacillus subtilis genome.
A cloning strategy for the construction of gene banks representative of the entire Bacillus subtilis chromosome is not available. This is, at least in part, due to the generally high level of expression of Bacillus subtilis genes observed in Escherichia coli, leading to toxic effects in this commonly used cloning host. However, participating groups have employed a variety of strategies to overcome this limitation. They include: (1) cloning into a range of plasmid, lambda and YAC vectors; (2) the use of an Escherichia coli strain that lowers the copy number of ColE1-based plasmids and which correspondingly reduces the potential toxic effects of cloned genes; (3) the use of integrative plasmids and marker rescue in genome walking experiments; (4) in vitro amplification using standard, long-range (LR PCR) and inverse PCR techniques.
MAJOR SCIENTIFIC BREAKTHROUGHS AND INDUSTRIAL APPLICATIONS:
During this contract 1.16 Mbp of Bacillus subtilis DNA has been sequenced. The European sequencing network has presently deposited a total of 1.9 Mbp of new Bacillus subtilis DNA sequence at the database maintained by the Institut Pasteur. In the current EU framework, a relational database containing 3.6 Mbp of non-redundant Bacillus subtilis DNA sequences, representing 86% of the total genome, has been constructed at the Institut Pasteur. The data are available at a new World Wide Web site: http://www.pasteur.fr/Bio/SubtiList.html.
An industrial platform has been set up to facilitate contacts between project participants and European biotechnology companies. The following companies have agreed to this mode of cooperation: DuPont de Nemours (France, USA), Genencor (Finland, USA), F. Hoffmann-La Roche AG (Switzerland), Novo Nordisk (Denmark), Frimond (Belgium), Roussel Uclaf (France), SmithKline Beecham (United Kingdom). Moreover, a confidential document containing a list of similarities of putatively identified B. subtilis proteins with known gene products is forwarded by the coordinator, at regular intervals, to the companies mentioned above.
Funding SchemeCSC - Cost-sharing contracts
20097 Milano - San Donato Milanese
9751 NN Haren
OX1 3RE Oxford
NE2 4HH Newcastle - Upon Tyne