PRINCIPAL INVESHGATORPROGRAM DIRECTOR. 
Sandra Handwerger, M.D. 
Cloning of the resistance trait: The general approach to be 
used is that of shotgun cloning of the chromosome of strain VR1. 
However, shotgun cloning of chromosomal genes into S. sanguis is 
frequently accompanied by deletions in the introduced genetic 
material. This observation has led to the suggestion that in 
S. sanguis , as in S. pneumoniae , plasmid replicons are assembled 
from two monomers when single stranded fragments recombine via 
homologous sequences (64). Based on this model, several 
strategies have been developed for efficient cloning into S. 
sanguis . 
The initial strategy for cloning the vancomycin resistance 
trait into S. sanguis will use a "helper plasmid", in which the 
host contains a homologous plasmid allowing recombinational 
rescue of incoming chimera monomers (64) . S. sanguis strain 
Challis V288 will be transformed with pVA736, which confers 
erythromycin resistance (Em r ) , and a resulting transformant will 
be used as the host strain (41) . Hindlll digested chromosomal 
DNA prepared from VR1 and Hindlll digested pVA736 are mixed, 
ligated with T4 DNA ligase, and used to transform the S. sanguis 
host. Alkaline phosphatase treatment of the vector is omitted, 
as this may inhibit recovery of recombinants (53) . Transformants 
will be screened for both constituitive resistance (direct 
plating on vancomycin agar 20 ug/ml) and inducible resistance 
(overnight growth on vancomycin agar 0.5 ug/ml, followed by 
overlayer of 20 ug/ml) . Hindlll digests of purified plasmids 
from vancomycin resistant transformants will be analyzed by 
Southern blot hybridization using a biotinylated probe to 
confirm their origin from the plasmid vector. In the event no 
transformants are obtained, digestions will be performed with 
other restriction enzymes for which pVA736 harbors a unique site 
(Kpnl , EcoRl) . 
The advantage of cloning via helper plasmids is its 
relative ease when isolating directly selectable markers. 
However, deletions may still occur. For this reason, in the 
event no transformants are obtained by helper plasmid cloning, 
and alternative method will be employed: use of an 
E. coli-S. sanguis shuttle vector, and construction of a 
chromosomal library. The E. coli plasmid preparations contain 
oligomers and high copy numbers of monomers, and thus transform 
S. sanguis with fewer deletion events (40) . The shuttle vector 
pVA838 (Em r and Cm r ) will be used for these experiments (40) . 
EcoRl digested bulk DNA from VR1 and EcoRl digested pVA838 are 
mixed, ligated with T4DNA ligase, and the mixture used to 
transform E. coli V850. Transformants are selected on 
erythromcyin agar (10 ug/ml) . The library of colonies is stored 
on replica plates and nitrocellulose filters at -20°C. 
Since no probe is available for screening desired clones in 
the library, a "brute force" approach to screening of 
recombinants is required. According to the equation of Clark 
and Carbon, screening of approximately 3000 clones will yield a 
99% probability of encountering the desired trait. Therefore 
portions of the library will be screened by transformation of 
the mixed population into S. sanguis and screening for 
vancomycin resistance. Populations containing positive 
recombinants will be subdivided and screened in smaller 
[ 434 ] 
Recombinant DNA Research, Volume 13 
