mrna

SUMMARY
In vitro recombination techniques were used to construct a new cloning vehicle, pBR322. This plasmid, derived from pBR313, is a relaxed replicating plasmid, does not produce and is sensitive to colicin e1, and carries resistance genes to the antibiotics ampicillin 8Ap) and tetracycline (Tc). The antibiotic-resistant genes on pBR322 are no transposable. The vector pBR322 was contructedin order to have to have a plasmid with a single PstI site, located in the ampicillin-resistant gene (Apr), in addition to four unique restriction sites, EcoRI, HindIII, BamHI and SalI. Survival of Escherichia coli strain X1776 containing pBR313 and pBR322 as a function of thymine and diaminopimelic acid (DAP) starvation and sensitivity to bile salts was found to be equivalent to the non-plasmidcontainig strain. Conjugal tranfer of these plasmids in bi- and triparental matings were significantly reduce or undetectable relative to plasmid ColE1.

Introduction
Bacterial plasmids and bactetophage have a key role in recombinant DNA technology. Segments of DNA fron diverse origins can be excised with th appropiate restriction endonuclease and added to plasmids or bacteriophage (Hershfieldet al., 1974; Morrow et al. 1974; cameron et al., 1975) If these new molecules contain an intact replicon, the can be propagated in a suitable host to yiel large quantities of recombinant DNA and in some instances, specific gene products (Hershfield et al., 1974). Several bacterial plasmids have been used as cloning vector: pSC101 (Cohen et al., 1973), ColE1(Hershfield et al., 1974) and pCR1(Covey et al., 1976). Howerver, these plasmids and their derivatives (Hamer et al., 1975; Hersfield et al., 1976; So et al., 1976) have limited versatility in terms of genetic markers for selection of transformants and screening for recombinant plasmids.
We have described the construction of series of plasmids containing Ap- and Tc-resistant genes derived from pRSF2124 (so et al., 1976) and pSC101respectively in combination whit replication elements of a ColE1 like plasmid (Betlach et al., 1976; rodriguez et al., 1976). One of these plasmids pBR313, provides single cleavage sites for the HindIII, BamHI, EcoRI, hpaI, SalI and SmaI restiction endonucleases (Bolivar et al., 1877). In the case of the hindIII, BamHI and SalI endonuclease cloning sites, the insertion of DNA fragments inactivatesthe Tcr gene. In this paper, we report the construction of anotherplasmid (pBR322) which is less tan half the size of PBR313 and provide additional cloning advantages. The plasmid pBR322 contains a unique PstI clevage site located in the Apr gene as well as two HincII sites located in the Apr and Tcr genes. The PstI site can be used for molecular cloning of DNA fragments via homodeoxy polymericextensión (Lobban and Kaiser, 2972) and the HincII site for blunt-end ligation techiniques (sgaramella et al., 1970; Sugino et al., 1977). The properties of pBR313 and pBR322 in the E. coli strain X1776 are also presented.
Material and Methods
(a) Bacterial strains
E. coli K12 strain RR1 F_ pro leu thi lacY Strr rk mk was used as the recipent cells in the transformation experiments. E. coli Bstrain HB50 pro leu try his arg met thr gal lac Y Strr was used to prepare unmethylated plasmid DNA for EcoRII digestions (Yoshimori et al., 1972). E.coli K12 strain X1776 F_ tau A53 dap D8 merA1 supE42 Δ40(gal-uurB) λ_ minB2 malA25 thy A57 met C65 Δ29(bioH-asd) cysB2 cyc a1 Hsd R2 was kindly prided by R.curtiss III.


(b ) Media and buffers
For transformation RR1 was grown in either LB orM9-gluucose minimal media, before CaCl2 treatment. X1776 was also grown in LB supplemented with DAP 200 µg/ml and thymine (thy) 50 µg/ml. The BSG buffer solution used for washing X1776 in the DAP-less death experiments was 0.85% NaCI, 0.03% KH2FO4, 0.06% Na2HPO4 100 µg/ml gelatin.

(c) Preparation of plasmid DNA
Plasmid DNA was prepared by first amplifying M9-glucose-grown cultures by the...