Alan H. Rosenberg

Encapsidated Conformation of Bacteriophage T7 DNA

The structural organization of encapsidated T7 DNA was investigated by cryo-electron microscopy and image processing. A tail-deletion mutant was found to present two preferred views of phage heads: views along the axis through the capsid vertex where the connector protein resides and via which DNA is packaged; and side views perpendicular to this axis. The resulting images reveal striking patterns of concentric rings in axial views, and punctate arrays in side views. As corroborated by computer modeling, these data establish that the T7 chromosome is spooled around this axis in approximately six coaxial shells in a quasi-crystalline packing, possibly guided by the core complex on the inner surface of the connector.

Genetic and physical mapping in the early region of bacteriophage T7 DNA

Abstract A detailed physical map of the early region of bacteriophage T7 DNA has been constructed. This map contains: locations for all the cuts made by the restriction endonucleases Hin dII, Hpa II, Hae III and Hae II, and many of the cuts by Hha I; the approximate end points for each of 61 different deletions; initiation sites and the termination site for RNAs made by Escherichia coli RNA polymerase; an initiation site for RNA made by T7 RNA polymerase; the five primary RNase III cleavage sites of the early region; and the coding sequences for perhaps nine different early proteins. Virtually all of the non-overlapping coding capacity of the five early messenger RNAs is used, except for untranslated stretches of perhaps 30 or so nucleotides at the ends. It seems likely that each of the nine early proteins is made from its own ribosome-binding and initiation site. The mapped restriction cuts provide fixed reference points, and allow DNA fragments containing specific genetic signals to be identified and isolated. The nucleotide sequences around the ends of three different T7 deletions have been determined. Each deletion eliminated a segment of DNA between repeated sequences of seven, eight or ten base-pairs, located 578 to 2100 base-pairs apart in the wild-type sequence. In each case, one copy of the repeated sequence was retained in the deletion mutant. This is consistent with the deletions having arisen by a genetic crossover between the repeated sequences. The approximate frequency of genetic recombination per base-pair has been estimated within two early genes; in both cases, the value was close to 0.01% recombination per base-pair, consistent with the value expected from the total length of the T7 genetic map. Genetic recombination between non-overlapping deletions appears to be severely depressed when the distance between the deletions is closer than about 40 to 50 base-pairs, but recombination between a point mutation and a deletion does not appear to be similarly depressed. This suggests that efficient genetic recombination in T7 may require a base-paired “synapse” of some minimum size between the recombining DNA molecules.

Utilization of bacteriophage T7 late promoters in recombinant plasmids during infection

Abstract When bacteriophage T7 infects a cell that carries a recombinant plasmid having a promoter for T7 RNA polymerase, the cloned promoter is utilized in the plasmid. A simple hybridization test has been used to screen recombinant plasmids for T7 promoter activity in vivo and to analyze the kinetics of utilization of the promoters in the plasmids. At least 13 different T7 promoters have been found to be active in plasmids, and all can be correlated with promoters identified by other means. During infection, promoters in T7 DNA appear to be activated sequentially from left to right over a period of several minutes; however, regardless of their original position in the T7 DNA molecule, T7 promoters in plasmids are all activated at the same time as the first promoters in T7 DNA. A likely explanation for this difference is that T7 DNA enters the cell in stages, and that not all promoters are accessible at the time T7 RNA polymerase is first made; promoters in plasmids, on the other hand, would be immediately accessible. Phased entry of the T7 DNA molecule could also explain why class II promoters are utilized before the stronger class III promoters during T7 infection. Utilization of a promoter in a plasmid seems to shut off with the same kinetics as the equivalent promoter in T7 DNA, and a mutation in T7 lysozyme that causes prolonged utilization of class II promoters in T7 DNA has a similar effect on class II promoters in plasmids. Transcription of promoter-containing plasmid DNAs by purified T7 RNA polymerase terminates at specific sites in pBR322 DNA, but the efficiency of termination is not high, and RNAs that result from transcription several times around the plasmid DNA are made in both directions. The ability to utilize T7 promoters in plasmids suggests that this system could be developed for high efficiency of expression of cloned genes in bacteria.

Genetic and physical mapping of the late region of bacteriophage T7 DNA by use of cloned fragments of T7 DNA

Abstract Specific fragments of bacteriophage T7 DNA that account for about 99% of the total molecule have been cloned in the plasmid pBR322. This set of plasmids was used to map individual point mutations of T7, by measuring recombination between T7 mutants and cloned fragments of wild-type T7 DNA. Cloned fragments that complement mutants defective in one or more of genes 2, 3.5, 8, 9, 10, 11, 13, 14 and 18 were also obtained. All but one of the plasmids that provide T7 functions carry a promoter for T7 RNA polymerase, a feature that is probably needed for efficient expression from the plasmid during infection. However, the promoter need not be immediately ahead of the gene; the polymerase can apparently transcribe around the entire plasmid DNA before transcribing the T7 gene. The major protein of the T7 phage head is among those that can be provided from a plasmid, indicating that substantial amounts of plasmid-specified proteins can be made. Using a combination of nucleotide sequence and cloning information the locations of 41 known or potential genes in T7 DNA have now been identified, at least 34 of which are known to specify a protein. T7 genes appear to be closely packed but essentially non-overlapping. The only places left in T7 DNA where undiscovered T7 genes are likely to lie are between genes 6 and 8, and to one or both sides of gene 19. The physical and genetic locations of the promoters and termination site for T7 RNA polymerase have also been defined. Certain fragments of T7 DNA cannot be cloned intact, and the lethality of at least some such fragments appears to be due to weak promoters for Escherichia coli RNA polymerase (in the T7 DNA) linked to T7 genes that are lethal if expressed. Separating the promoter from the lethal gene allows the intact gene to be cloned, but only in the silent orientation, where the predominant transcription from promoters in the pBR322 DNA crosses the inserted T7 DNA in the opposite direction from transcription in wild-type T7 DNA.

Survey and mapping of restriction endonuclease cleavage sites in bacteriophage T7 DNA

Abstract A survey of restriction endonucleases having different cleavage specificities has identified 10 that do not cut wild-type bacteriophage T7 DNA, 11 that cut at six or fewer sites, four that cut at 18 to 45 sites, and 12 that cut at more than 50 sites. All the cleavage sites for the 13 enzymes that cut at 26 or fewer sites have been mapped. Cleavage sites for each of the 10 enzymes that do not cut T7 DNA would be expected to occur an average of 9 to 10 times in a random nucleotide sequence the length of T7 DNA. A possible explanation for the lack of any cleavage sites for these enzymes might be that T7 encounters enzymes having these specificities in natural hosts, and that the sites have been eliminated from T7 DNA by natural selection. Five restriction endonucleases were found to cut within the terminal repetition of T7 DNA; one of these, Kpn I, cuts at only three additional sites in the T7 DNA molecule. The length of the terminal repetition was estimated by two independent means to be approximately 155 to 160 base-pairs.

T7 RNA polymerase can direct expression of influenza virus cap-binding protein (PB2) in Escherichia coli

Influenza virus cap-binding protein (PB2; Mr 85,000) is made in Escherichia coli when the cloned cDNA is transcribed by T7 RNA polymerase. Translation begins at the probable natural start codon and also from at least five internal sites in the same reading frame. The eukaryotic initiation site is not typical of protein initiation sites of E. coli, in that the closest potential Shine-Dalgarno sequence is far (15 nucleotides) from the start codon. Nevertheless, protein synthesis initiates efficiently at this site even in competition with a strong upstream prokaryotic initiation site. PB2 is somewhat unstable in the cell, but accumulates to a level where it is easily detectable in electrophoresis patterns of total cell protein. The full-length protein and various subfragments of it are insoluble in crude extracts, but have been useful for producing antibodies.