APPENDIX A 
4 
for insertion into plasmids or utilized as a scaffold to integrate other 
heterologous DNA into the chromosome of B_. subtil is . Alternatively, it 
is possible to purify fragments of the chromosome by gel electrophoresis 
(21, 22), for insertion into bacteriophage <f>3T or SP02. At present, un- 
fortunately, only the former carries a selective marker, i.e., the gene 
for thymidylate synthetase, thyP . 
C. Development of vehicles 
B_. subtil is is a Gram-positive sporulating rod that usually inhabits 
soil. Although it can exist on cutaneous surfaces of man (23) and experi- 
mental animals, it rarely produces disease. To develop a suitable vehicle 
it is imperative to have a host that is asporogenic. The most appropriate 
deletion mutation is deletion 29 (cit D). In addition to a deficiency in 
sporulation this mutant rapidly lyses when it has reached the end of its 
growth cycle. Presumably this is due to the failure to inactivate one of 
the autolytic enzymes (24). Through the introduction of a D-alanine 
requirement (34 ug/ml ) it is possible to block transport of compounds that 
are transported by active transport (25,26). The further introduction of 
thymine auxotrophy (defects in the thy A thyB loci) will enable the strain 
to survive only with a plasmid vector carrying the purified thyP gene from 
bacteriophage <}>3T or a defective bacteriophage <J>3T carrying the thyP gene 
but attached to the chromosome at an alternative site (due to the presence 
of deletion 29 in the host). We have recently isolated temperature-sensitive 
thyP mutants. If we can isolate a temperature-dependent lysogen that will 
grow only at 48 C it should be possible to make an unusual vehicle. 
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