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2 gene banks of B. subtilis DNA were constructed using lambda FixII. A partial Sau3A digestion and a partial fill-in reaction were performed on Bacillus subtilis chromosomal DNA producing fragments with 5'-GA ends which were then separated using agarose gel electrophoresis. Fragments in the size range of 9-20 kbp were electroeluted and purified. This DNA was ligated in vitro to lambda FixII DNA and packaged, creating a bank of 10000 to 50000 independent recombinant phages per ug of vector DNA. Given the number of phages obtained, both banks should be representative of the total B. subtilis genome.

A collection of large segments of the B. subtilis genome in yeast artificial chromosomes (YAC) was constructed. The following considerations led to this choice : 1) large DNA segments (>50 kbp) can be cloned in YAC, so that a representative collection requires a relatively low nomber of clones; 2) Certain B. subtilis genes which are toxic to E. coli may not be toxic for yeast, since the likelihood of their expression in this latter host is low. To clone large DNA segments it is necessary to minimize DNA breakage. All the biochemical steps necessary for cloning (DNA preparation, partial cleavage with EcoRI in the presence of the cognate methylase and ligation ot the vector) were therefore performed in agarose blocks. A highly transformable yeast strain SX4-6A was used as the cloning host. 800 yeast clones were obtained carrying B. subtilis DNA segments, as ascertained by hybridizing B. Subtilis total DNA probe with yeast chromosomes separated by pulse field electrophoresis. 363 YAC had inserts of more than 90 kb which is 9 genome equivalents and should be representative with a probability of more than 99%. Comparison of the restriction maps of several randomly chosen YAC carrying a total of about 1200 kb of B. subtilis DNA (25% of the genome) with that of the B. subtilis chromosome suggests that there are no rearrangements of the bacterial DNA in yeast. Efforts have been directed towards ordering the collection, by using probes provided by several colleagues. An efficient strategy for cloning in B. subtilis, using vectors based on bacteriophage theta 105 was previously developed. The most recent improvements involved the development of a system in which defective phage vectors capable of incorporating inserts of at least 11 kbp were generated. The system is based on the prophage transformation method with several improvements. The flanking DNA segments used to direct insertion into the prophage are provided by specific cloned DNA segments in E. coli. The inserts in plasmids pSG521 and pSG523 correspond to flanking regions of a 6.9 kbp dispensable DNA fragment in the theta 105 genome. Each cloned DNA segment ends in a polylinker, allowing the choice of various different restriction endonucleases for cloning. Between these restriction sites and one of the phage DNA segments is a selectable chloramphenicol resistance determinant. The ligation reaction generates some molecules in which a fragmentof target DNA is flanked by fragments of phage DNA in the correct orientation (partial fill-in reactions are used to minimize the proportion of non-recombinant molecules). Selection for chloramphenicol resistant transformants of a strain containing the theta 105J124 prophage generates recombinants that have replaced the region of dispensable phage DNA with a segment of theta 105 DNA. The library of recombinants that the collection of transformants constitutes can be stored as frozen cells, or amplified by prophage induction and screened for specific recombinants using standard methods. The problem of the poor transformability of theta 105 lysogens has been overcome by incorporating an ind mutation. The poor inducibility of ind mutants has been avoided by the use of an ind cts double mutant, which can be thermo-induced. The system has been further improved in 2 ways. First, by incorporating both upstream and downstream fragments of homologous DN A used for insertion into the prophage into a single plasmid, pSG421-cat. Secondly, by constructing a recipient prophage with 2 markers aphA3 (kanamycin resistance) and lacZ that are replaced by the incoming DNA, thus providing a screening method for the presence of insert DNA. It was observed that using competent cells there is an upper limit of about 9 kbp on the size of DNA that can be inserted successfully, which may reflect the size of DNA fragments taken up by the cell during competent cell transformation.
European participation to the complete sequencing of the genome of Bacillus subtilis. The present application corresponds to the first steps of the project. It includes the establishment of a complete physical map, its correlation with the genetic map and the beginning of the cloning of the entire genome. In between, the cloning and sequencing of part of the genome starting from known regions will be achieved, with a correlative evaluation of the most appropriate techniques for each step of the project, cloning, sub-cloning, sequencing, computer follow-up of the processes, and computer treatment of the data.


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