Sequence of bacteriophage T3 DNA from gene 2.5 through gene 9

The nucleotide sequence of bacteriophage T3 DNA, from gene 2.5 through gene 9 has been determined. In addition to regulatory sites, the sequence predicts 19 close-packed genes plus two genes that overlap, in a different reading frame, another gene. The majority of these genes are highly homologous to those in the corresponding region of bacteriophage T7. However, there are some genes that are present in one, but not the other, phage. These apparent deletions are almost exactly gene size and thus the close-packed organization of genes remains the same in T3 as in T7. The varying levels of homology between T3 and T7 DNAs, first noted by Davis and Hyman in their study of DNA heteroduplexes, are also demonstrated here by a comparison of T3 and T7 nucleotide sequences. Many regions of extremely high homology immediately abut sequences that have no apparent homology. These data suggest that bacteriophages T3 and T7 have recombined, both with each other and with other members of a pool of T7-like phages, during their co-evolution.     

T7 and E. coli share homology for replication-related gene products

Recently, the complete nucleotide sequence of the bacteriophage T7 genome was determined and 50 genes were identified on the genome. We compared amino acid sequences of all the gene products of T7 and replication-related gene products of E. coli. As a result, we found that T7 and E. coli share homology for each pair of exonuclease, DNA primase and helix-destabilizing protein. For E. coli, these gene products are known to be involved in the process of discontinuous DNA replication. These observations suggest that T7 and E. coli have a common origin for a part of their replication systems.     

Mutants of bacteriophage T7 that escape F restriction

Mutants of bacteriophage T7 that escape F restriction have been isolated. Two mutations in gene 10, which codes for the capsid protein, and one mutation in gene 1.2 are required for these phages to grow on F-containing strains. The products of these two genes are the two targets of the exclusion system; the presence of either wild-type product results in an abortive infection. Phages that grow normally in male hosts still lead to membrane dysfunction and nucleotide efflux from the infected cell. This type of membrane damage and the abortive infection are therefore separable phenomena.     

Nucleotide sequence of the three major early promoters of bacteriophage T7.

I have determined the nucleotide sequences of the three major early promoters of bacteriophage T7 (A1, A2, A3). The sequences confirm the two main homologies found between other known promoters for E. coli RNA polymerase (nucleoside triphosphate:RNA nucleotidyl transferase, E.C. 2. 7. 7. 6). In particular, all three T7 promoters show a very good match with the -35 region homology; the A2 and A3 promoters share a 17 basepair sequence in this region. On the other hand, the match with the Pribnow Box homology is much less pronounced and different for each T7 promoter.     

The genome of bacteriophage K1F, a T7-like phage that has acquired the ability to replicate on K1 strains of Escherichia coli

Bacteriophage K1F specifically infects Escherichia coli strains that produce the K1 polysaccharide capsule. Like several other K1 capsule-specific phages, K1F encodes an endo-neuraminidase (endosialidase) that is part of the tail structure which allows the phage to recognize and degrade the polysaccharide capsule. The complete nucleotide sequence of the K1F genome reveals that it is closely related to bacteriophage T7 in both genome organization and sequence similarity. The most striking difference between the two phages is that K1F encodes the endosialidase in the analogous position to the T7 tail fiber gene. This is in contrast with bacteriophage K1-5, another K1-specific phage, which encodes a very similar endosialidase which is part of a tail gene "module" at the end of the phage genome. It appears that diverse phages have acquired endosialidase genes by horizontal gene transfer and that these genes or gene products have adapted to different genome and virion architectures.     

Effect of thermoinduced changes in T4 bacteriophage structure on the process of molecular recognition of 'host' cells.

By means of high-precision acoustic measurements and by methods of fluorescent and electron microscopy, investigations have been performed of thermoinduced conformational changes in T4 bacteriophage and its thermolabile mutants altered in baseplate proteins (gene products 7, 8, 10). A relationship was found between the conformational changes in T4 bacteriophage structure in the temperature range of 33-45 degrees C and the efficiency of bacteriophage adsorption and the changes in the orientation of long tail fibers. The possibility of heat regulation of 'recognition' of 'host' cells by bacterial viruses is suggested.     

Five genes at the 3' end of the Klebsiella pneumoniae pulC operon are required for pullulanase secretion

The nucleotide sequence of a 5082bp fragment of chromosomal DNA from Klebsiella pneumoniae strain UNF5023 is reported. The sequence includes the last four genes of an operon of genes specifically required for the secretion of the enzyme pullulanase. All four genes (puIL, puIM, puIN and puIO) are shown to be required for pullulanase secretion, as is a fifth gene (puIK) which extends beyond the 5′ end of the sequenced DNA. The products of the puIL, puIM, puIN and puIO genes (44kD, 18kD, 27kD and 24kD, respectively) are all predicted to have one or more hydrophobic domains typical of signal sequences and/or membrane anchors, and were all found mainly associated with the inner membranes of subfractionated cells in which the corresponding genes had been expressed from the bacteriophage T7 gene 10 promoter. The results of this study increase the number of genes which have been identified as required for pullulanase secretion to eight, in addition to genes coding for components of the general export pathway.     

Nucleotide sequence from the genetic left end of bacteriophage T7 DNA to the beginning of gene 4

The nucleotide sequence running from the genetic left end of bacteriophage T7 DNA to within the coding sequence of gene 4 is given, except for the internal coding sequence for the gene 1 protein, which has been determined elsewhere. The sequence presented contains nucleotides 1 to 3342 and 5654 to 12,100 of the approximately 40,000 base-pairs of T7 DNA. This sequence includes: the three strong early promoters and the termination site for Escherichia coli RNA polymerase: eight promoter sites for T7 RNA polymerase; six RNAase III cleavage sites; the primary origin of replication of T7 DNA; the complete coding sequences for 13 previously known T7 proteins, including the anti-restriction protein, protein kinase, DNA ligase, the gene 2 inhibitor of E. coli RNA polymerase, single-strand DNA binding protein, the gene 3 endonuclease, and lysozyme (which is actually an N-acetylmuramyl-l-alanine amidase); the complete coding sequences for eight potential new T7-coded proteins; and two apparently independent initiation sites that produce overlapping polypeptide chains of gene 4 primase. More than 86% of the first 12,100 base-pairs of T7 DNA appear to be devoted to specifying amino acid sequences for T7 proteins, and the arrangement of coding sequences and other genetic elements is very efficient. There is little overlap between coding sequences for different proteins, but junctions between adjacent coding sequences are typically close, the termination codon for one protein often overlapping the initiation codon for the next. For almost half of the potential T7 proteins, the sequence in the messenger RNA that can interact with 16 S ribosomal RNA in initiation of protein synthesis is part of the coding sequence for the preceding protein. The longest non-coding region, about 900 base-pairs, is at the left end of the DNA. The right half of this region contains the strong early promoters for E. coli RNA polymerase and the first RNAase III cleavage site. The left end contains the terminal repetition (nucleotides 1 to 160), followed by a striking array of repeated sequences (nucleotides 175 to 340) that might have some role in packaging the DNA into phage particles, and an A · T-rich region (nucleotides 356 to 492) that contains a promoter for T7 RNA polymerase, and which might function as a replication origin.     

A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase

A simple and rapid method for determining nucleotide sequences in single-stranded DNA by primed synthesis with DNA polymerase is described. It depends on the use of Escherichia coli DNA polymerase I and DNA polymerase from bacteriophage T4 under conditions of different limiting nucleoside triphosphates and concurrent fractionation of the products according to size by ionophoresis on acrylamide gels. The method was used to determine two sequences in bacteriophage φX174 DNA using the synthetic decanucleotide A-G-A-A-A-T-A-A-A-A and a restriction enzyme digestion product as primers.     

Amino acid changes coded by bacteriophage T4 DNA polymerase mutator mutants. Relating structure to function

Previous studies on the selection of bacteriophage T4 mutator mutants have been extended and a method to regulate the mutator activity of DNA polymerase mutator strains has been developed. The nucleotide changes of 17 bacteriophage T4 DNA polymerase mutations that confer a mutator phenotype and the nucleotide substitutions of several other T4 DNA polymerase mutations have been determined. The most striking observation is that the distribution of DNA polymerase mutator mutations is not random; almost all mutator mutations are located in the N-terminal half of the DNA polymerase. It has been shown that the T4 DNA polymerase shares several regions of homology at the protein sequence level with DNA polymerases of herpes, adeno and pox viruses. From studies of bacteriophage T4 and herpes DNA polymerase mutants, and from analyses of similar protein sequences from several organisms, we conclude that DNA polymerase synthetic activities are located in the C-terminal half of the DNA polymerase and that exonucleolytic activity is located nearer the N terminus.     

Expression of gene 1.2 and gene 10 of bacteriophage T7 is lethal to F plasmid-containing Escherichia coli.

Plasmids expressing bacteriophage T7 gene 1.2 or gene 10 DNA transform F plasmid-containing strains of Escherichia coli only at low efficiency, though they transform plasmid-free strains normally. The gene products T7 gp1.2 and T7 gp10 appear to be the toxic agents, and their effects are directed towards the product of the F pifA gene, PifA. T7 gp1.2 and gp10 are also the two targets of the pif exclusion system of F, and their synthesis normally triggers the abortive infection of T7 in pifA+ hosts. The properties of plasmids containing T7 gene 1.2 or 10 suggest that they can be used to study the molecular mechanisms of phage exclusion in model systems that avoid the pleiotropic dysfunctions associated with an abortive infection.     

Expression of bacteriophage T4 minor baseplate proteins in the bacteriophage T7 promoter/RNA polymerase expression system.

The central part of bacteriophage T4 baseplate is built of several proteins which are present in only a few copies per phage particle. Only some of these minor baseplate components have been identified previously as distinct protein species by biochemical analysis. We have used the bacteriophage T7 RNA polymerase expression system to identify and overexpress the minor baseplate proteins. The products of genes 25, 26 and 51 were identified on the autoradiographs after selective labelling with [35]S methionine. The overexpression of gene 25 and 51 products was high enough to make possible undertaking their purification and studies of their properties.     

Nucleotide sequence of the cloned gene for bacteriophage T7 RNA polymerase

The coding sequence of the gene for bacteriophage T7 RNA polymerase has been cloned into the PstI site of plasmid pBR322. This cloned DNA extends from T7 nucleotide (Dunn & Studier, 1981) 3127 to 5821 or T7 coordinate 7.82 to 14.55. The nucleotide sequence is given, as well as the predicted amino acid sequence of T7 RNA polymerase. This peptide sequence is comprised of 883 amino acid residues with a total molecular weight of 98,092.     

λZLG6: a phage lambda vector for high-efficiency cloning and surface expression of cDNA libraries on filamentous phage

We describe a vector, λZLG6, combining the high efficiency of cDNA library cloning in bacteriophage λ, with filamentous phage display of cDNA-encoded products. The cDNAs are expressed as fusions to the 3′ end of M13 gene VI. The λZLG6 library is converted to a pZLG6-cDNA phagemid library by in vivo mass excision. Helper phage infection generates a library of phagemid particles displaying the cDNA-encoded products and containing the corresponding nucleotide sequences within.