TABLE 3.

Dependence on α helicase and primase activities

Plasmida	Presence of P4 sitesb:			No. of transformantsc
				pGEX4T-1 (−)		pGEX-α (α)		pGM686 (αE214Q)		pGM687 (αK507T)
	ori1	ori2	crr	30°C	42°C	30°C	42°C	30°C	42°C	30°C	42°C
pGB2ts	−	−	−	800	0	137	0	274	0	142	0
pGM633	−	−	+	2,310	0	13	0	1	0	2,320	0
pGM671	+	+	+	2,560	0	1,984	2,300	226	0	268	0
pGM669	+	−	+	1,632	0	1,680	200	421	0	864	0
pGM651	−	+	+	3,168	0	34	340	0	0	2,562	0

^apGB2ts is thermosensitive for replication (12). The other plasmids are pGB2ts derivatives in which the indicated P4 sites are cloned. 

^bCoordinates of the P4 sites are as follows: ori1, 8835 to 9465; ori2, 6186 to 6421; and crr, 4260 to 4595. The relative orientations of the fragments are the same as in the P4 genome. −, not present; +, present. 

^cThe transformed strains were C-2428 carrying the indicated plasmids. The P4 proteins, expressed as GST fusions from the plasmids, are indicated as follows: −, no protein; α, α wild type; αE214Q, α primase null; αK507T, α helicase null. Transformation was performed with 50 ng of DNA, and the transformants were selected at the indicated temperature in the presence of spectinomycin (100 μg/ml) and ampicillin (50 μg/ml).