Length determination of the terminal redundant regions in the DNA of phage T7

Abstract

Summary. The length of the terminal redundant regions in T7 DNA has been determined by two methods. One involved the specific labeling and isolation of the redundant DNA fragment and determination of the molecular weight by polyacrylamide gel electrophoresis. A value of 150_+10 nucleotide pairs was obtained. The other determination based on a correlation of the melting temperature of the redundant region to that of whole T7 DNA confirmed the result obtained by the first method.The genome of bacteriophage T7 is a linear DNA duplex of 26 x 106 dalton and has unique terminal redundant regions (TRs; see Studier, 1972). It has been speculated that the TRs play an essential role during the replication of T7 DNA (Watson, 1972). This replication apparently requires formation of linear concatemers (for ref. see Hausmann, 1976) in which unit length molecules are joined together in a head-to-tail fashion by means of the TRs (Watson, 1972;Langman et al., 1978). The length of the TRs has been determined by various techniques, including electronmicroscopy, restriction fragment analysis and biological tests, and the estimates range from 50 to 260 nucleotide pairs (Ritchie et al., 1967;Ludwig and Summers, 1975;Ehrlich et al., 1976;Langman et al., 1978;Dreiseikelmann and Wackernagel, 1978). Here we describe the length determination of the TRs of phage T7 DNA by a method which involves the direct isolation of TRs and which may be generally applied for similar determinations. Figure 1 outlines the method for the specific labeling and isolation of the TRs. 3H-labeled T7 DNA was treated with alkaline phosphatase to remove the terminal 5'phosphate groups. The DNA was then diOffprint requests to : Dr. W. Wackernagel gested about 2% with the 3'~5' exonuclease III of E. coli resulting in single-stranded terminal regions with 5'OH ends. The molecules could now be joined together by thermal annealing of the exposed complementary nucleotide sequences of the TRs. After the annealing the 5'OH ends were labeled with 32p in a kinase reaction. After removing the 32p label not linked to the DNA by gel chromatography and dialysis, the single-stranded regions flanking the duplex TRs were digested with the single strand-specific S1 endonuclease. In this step the 32p-labeled TRs are released from the concatemers. The total DNA was subjected to polyacrylamide gel electrophoresis followed by autoradiography. We used the fragments of uniformly 32p-labeled pBR322 generated by restriction endonuclease Sau3AI (prepared according to Sussenbach et al., 1976) as precise molecular weight markers. Sau3AI cleaves the same nucleotide sequence as MboI, but does this irrespective of adenosylmethylation within this sequence (Sussenbach et al., 1976;Dreiseikelmann et al., 1979). The exact size of these fragments is known from the complete nucleotide sequence of pBR322 (Sutcliffe, 1978). Figure 2 shows the autoradiogram of such a gel and reveals a single DNA band for the TRs corresponding to a size of 150 nucleotide pairs. Control experiments in which Sau3AI fragmen...

Bacteriophage T3 and bacteriophage T7 virus-host cell interactions.

“…DNA T3 and T7 DNAs possess terminal redundancies (391) which are essential for phage multiplication (22,104). The length of the terminal repetition of T7 DNA has now been estimated by independent methods to be 150 to 160 base pairs (103,405). Due to unidirectional DNA polymerase movement, unit-size DNA has no means of replicating the 3'-terminal gaps which arise by excision of primer RNA.…”