A preferred origin for the replication of lambda DNA.

Abstract

It has been suggested that replication of DNA in general should start at genetically specific molecular sites.' Unique origins of replication exist in Bacillus subtilis2 and Escherichia coli.3In the present communication, I report preliminary results concerning the origin of replication in the DNA of phage lambda.Lambda DNA extracted from phage particles has regions of dissimilar nucleotide composition.4 When sheared to small fragments and banded in a density gradient of Cs2SO4-HgCl2, the DNA is resolved into two major peaks.' The denser fragments, rich in adenine and thymine (AT), come from a continuous right-hand section of the molecule representing 56 per cent of its length. The less dense fragments, rich in guanine and cytosine (GC), originate from the remaining left-hand section. A given sample of lambda DNA subjected to analysis of the sort indicated may be characterized by the ratio R _ DNA content of denser band DNA content of less-dense band which measures 1.27 for DNA extracted from phage particles. If DNA synthesis starts in the AT-rich section, replicating DNA should give a ratio R that exceeds 1.27 by an amount related to, among other things, the distance between the point of origin and the boundary between sections. However, if replication starts at random sites, R should be the same for replicating and nonreplicating DNA. The experimental approach suggested by these considerations is similar in principle to the genetic method used by Sueoka and Yoshikawa2 to locate the origin of replication in B. subtilis.The following experiments showed that lambda DNA synthesized during the first few minutes after infection (when it is reasonable to assume that most of the DNA is replicating) contains an excess of AT-rich sections. Materials and Methods.-Bacterial and phage strains: A clear-plaque (Cl) mutant of lambda was used to infect E. coli HF4704 T-, Hcr-(provided by Dr. R. L. Sinsheimer). DNA synthesis is arrested in this host by treatment with mitomycin C and restored by infection with phage 6X1746 or lambda.7 Culture medium: Experimental cultures were grown in a medium containing, per liter, 0.05 mole of tris(hydroxymethyl)aminomethane (Trizma-Base, Sigma Chemical Co.); 1.0 ml of 0.66 M KH2PO4; 1.0 ml of 1.0 M MgSO4; 1.0 ml of 1.0% CaCl2; 0.1 ml of 0.5% FeSO 6H20; 0.8 gm NH4C1; 0.2 gm Na2SO4; 1.0 gm KCl; 10 ml of 40% glycerol;and 20 ml of 10% "vitamin-free" casein hydrolysate (Nutritional Biochemicals Corp.).The pH of the medium was adjusted with HCl to 7.4. Thymidine was added as required. Experimental cultures: Bacterial cultures were grown with aeration at 370C in the specified medium supplemented with 10 Ag/ml of thymidine. When they reached 2 X 108 cells/ml, they were treated with 50 lg/ml of mitomycin C for 10 min in the dark without aeration. Then the cells were collected on a membrane filter (B4, Schleicher & Schuell Co.), washed with medium devoid of thymidine, and resuspended in culture medium 1345

Evidence for a New Endonuclease Synthesized by λ Bacteriophage

“…It is therefore possible that the initiation of DNA synthesis requires a breaking or nicking of the double helix at the point where DNA synthesis begins. Recently, it has been re-ported that replication of the phage DNA begins in the adenine-thymine-rich half of the right side of the phage genome (11). This site would differ from the site of breakage for integration, so that two or more separate enzymes may be responsible for the breaking of species I observed in these experiments.…”

Origin and Direction of Haemophilus Bacteriophage HP1 DNA Replication

“…The first round(s) of bacteriophage X DNA replication after phage infection begins at a fixed origin of about one-fifth unit from the right end (6,7,8,18) and proceeds to the left. Electron microscopy observations showed the origin to be 0.18 units from the right end (11).…”