A 3.2-kb fragment encoding five genes, parCBA/DE, in two divergently transcribed operons promotes stable maintenance of the replicon of the broad-host-range plasmid RK2 in a vector-independent manner in Escherichia coli. The parDE operon has been shown to contribute to stabilization through the postsegregational killing of plasmid-free daughter cells, while the parCBA operon encodes a resolvase, ParA, that mediates the resolution of plasmid multimers through site-specific recombination. To date, evidence indicates that multimer resolution alone does not play a significant role in RK2 stable maintenance by the parCBA operon in E. coli. It has been proposed, instead, that the parCBA region encodes an additional stability mechanism, a partition system, that ensures that each daughter cell receives a plasmid copy at cell division. However, studies carried out to date have not directly determined the plasmid stabilization activity of the parCBA operon alone. An assessment was made of the relative contributions of postsegregational killing (parDE) and the putative partitioning system (parCBA) to the stabilization of mini-RK2 replicons in E. coli. Mini-RK2 replicons carrying either the entire 3.2-kb (parCBA/DE) fragment or the 2.3-kb parCBA region alone were found to be stably maintained in two E. coli strains tested. The stabilization found is not due to resolution of multimers. The stabilizing effectiveness of parCBA was substantially reduced when the plasmid copy number was lowered, as in the case of E. coli cells carrying a temperature-sensitive mini-RK2 replicon grown at a nonpermissive temperature. The presence of the entire 3.2-kb region effectively stabilized the replicon, however, under both low-and high-copy-number conditions. In those instances of decreased plasmid copy number, the postsegregational killing activity, encoded by parDE, either as part of the 3.2-kb fragment or alone played the major role in the stabilization of mini-RK2 replicons within the growing bacterial population. Our findings indicate that the parCBA operon functions to stabilize by a mechanism other than cell killing and resolution of plasmid multimers, while the parDE operon functions solely to stabilize plasmids by cell killing. The relative contribution of each system to stabilization depends on plasmid copy number and the particular E. coli host.

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and Summary.-Lambda-specific RNA extracted from lysogenic bacteria hybridizes specifically with fragments of X DNA containing 43 per cent GC (guanine plus cytosine). Therefore genes known to function in the prophage state (c, and rex) lie 0.38 i 0.08 fractional molecular length from the right end of the X DNA molecule, according to the compositional map of Skalka, Burgi, and Hershey.Only two functions are known to be expressed by prophage X: immulilty and exclusion of rII mutants of phage T4. It jsg/ml of thymidine and 20 Mg/ml of uridine. The high concentration of uridine served to repress endogenous uridine synthesis. The culture (700 ml at 8 X 107/ml) was chilled and the cells were harvested, washed, and resuspended in 7 ml of the same medium without uridine. The suspension was aerated for 2 min at 370C, H3-uracil was added (500 ,Pc, 9.65 c/mmole), and, after another 2 min, the culture was poured over an equal volume of frozen 0.9% saline. The cells were washed twice with 0.01 M Tris buffer (pH 7.4) containing 5 X 10-3 M MgC12, and then frozen in 2 ml of the same solution. After the addition of 100 jug of yeast RNA, cellular RNA was extracted as described by Skalka.6 After phenol extraction, the aqueous phase was washed several times with ether. Uptake of H3-uracil from the medium, measured as counts precipitable by trichloroacetic acid, was 50-80% of the input. DNA preparations: Lambda DNA was prepared from a clear-plaque mutant7 of that phage propagated on E. coli W3110. DNAof Ximm434 (see ref. 1) was extracted from phage produced after induction of the appropriate lysogenic culture with 2 Mg/ml of mitomycin C.The phages were grown in a peptone medium6 which contained P32 (specific activity 1 c/gm P) and yielded DNA with about 105 cpm/ug. E. coli DNA was made by the Marmur8 procedure, and phage DNA's were prepared as described by Burgi.9 Methods for shearing DNA and fractionating DNA-mercury complexes have been described.10Membrane filters were coated with DNA or DNA fractions by the method of Gillespie and Spiegelman11 (see legend, Fig. 2), and except for modifications employed in the preparatory steps detailed below, hybridization tests were performed as described by them.

The Raman spectrum of long chain biological polymers displays a broad band which is distinct from conventional molecular Raman lines.