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The nucleotide sequence of the vaccinia virus open reading frame B1 predicts a polypeptide with significant sequence similarity to the catalytic domain of known protein kinases. To determine whether the B1R polypeptide is a protein kinase, we have expressed it in bacteria as a fusion with glutathione S-transferase. Affinity-purified preparations of the fusion protein were found to undergo autophosphorylation and also phosphorylated the exogenous substrates casein and histone H1. Mutation of lysine 41 to glutamine within the conserved kinase catalytic domain II abrogated protein kinase activity on all three protein substrates, supporting the notion that the protein kinase activity is inherent to the B1R polypeptide. Casein and histone H1 were phosphorylated on serine and threonine residues. The B1R fusion protein was phosphorylated on a threonine residue(s) by an apparently intramolecular mechanism. The autophosphorylation reaction resulted in phosphorylation of the glutathione S-transferase portion of the fusion and not the protein kinase domain. The protein kinase activity of B1R was specific for ATP as the phosphate donor; GTP was not utilized to a detectable extent. Immunoblotting experiments with anti-B1R antiserum showed that the protein kinase is located in the virion particle. Chromatography of virion extracts resulted in separation of the B1R protein kinase from the bulk of the total protein kinase activity, indicating that multiple protein kinases are present in the virion particle and that B1R is distinct from the previously described vaccinia virus-associated protein kinase.

Summary We have developed a system of transposon mutagenesis for bacteriophage T4. The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24, permitting a direct selection for transposition events into a gene 24-deteted phage. The transposition occurred at a frequency of only 10-7 per progeny phage, even though a dam- host was used to increase transposition frequency. Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon-specific probe. Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions. The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome. In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred. Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.

The Neurospora CYT-18 protein, a tyrosyl-tRNA synthetase, which functions in splicing group I introns in mitochondria, promotes splicing of mutants of the distantly related bacteriophage T4 td intron. In an in vivo assay, wild-type CYT-18 protein expressed in E. coli suppressed mutations in the td intron's catalytic core. CYT-18-suppressible mutations were also suppressed by high Mg2+ or spermidine in vitro, suggesting they affect intron structure. Both the N- and C-terminal domains of CYT-18 are required for efficient splicing, but CYT-18 with a large C-terminal truncation retains some activity. Our results indicate that CYT-18 interacts with conserved structural features of group I introns, and they provide direct evidence that a protein promotes splicing by stabilizing the catalytically active structure of the intron RNA.

Three lysozyme-encoding genes, one of eukaryotic and two of prokaryotic origin, were expressed in Lactococcus lactis subsp. lactis. Hen egg white lysozyme (HEL) could be detected in L. lactis lysates by Western blotting. No lysozyme activity was observed, however, presumably because of the absence of correctly formed disulphide bonds in the L. lactis product. The functionally related lysozymes of the E. coli bacteriophages T4 and lambda were produced as biologically active proteins in L. lactis. In both cases, the highest expression levels were obtained using configurations in which the bacteriophage lysozyme genes had been translationally coupled to a short open reading frame of lactococcal origin. Both enzymes, like HEL, may prevent the growth of food-spoilage bacteria.

Two-dimensional 1H-15N NMR techniques combined with pulsed hydrogen-deuterium exchange have been used to characterize the folding pathway of T4 lysozyme. In the unfolded state, there is little differential protection of the various amides from hydrogen exchange. In the native folded structure, 84 amides of the 164 residues are sufficiently spectrally resolved and protected from solvent exchange to serve as probes of the folding pathway. These probes are located in both the N-terminal and C-terminal domains of the native folded structure of the protein. The studies described here show that at least one intermediate is formed early during refolding at low denaturant concentrations. This intermediate (or intermediates) forms very rapidly (within the 10-ms temporal resolution of our mixing device) under the conditions used and is completed at least 10 times faster than the overall folding event. The intermediate(s) protect(s) from exchange a subset of amides in the N-terminal and C-terminal regions of the protein. In the final folded states these protected regions correspond to two alpha-helices and a beta-sheet region. These amides are protected from exchange by factors between 20 and 200 as compared to the fully unfolded protein. Protection of this magnitude is consistent with the formation of somewhat exposed secondary structure in these regions and could represent a "molten globule"-like or a "framework"-like structure for the intermediate(s) in which specific parts of the sequence form isolated secondary structures that are not stabilized by extensive tertiary interactions.

Two new plasmid expression vectors, pTacT7 and pTacT7L, have been constructed, which incorporate between the tac promoter and a downstream NcoI-HindIII polylinker sequence a synthetic sequence derived from the region upstream from gene 10 of bacteriophage T7 (g10-L). This sequence was recently shown to act as a translational enhancer (Olins et al., 1988) and was termed "Epsilon" (Enhancer of Protein Synthesis Initiation) element (Olins and Rangwala, 1989). In this communication we describe in detail the construction of ptacT7 and ptacT7L. Furthermore, we present evidence that the "Epsilon" element is able to enhance 3 to 20-fold the expression levels of two poorly expressed test genes encoding the human placental proteins PP9 and PP15. On the other hand, the expression levels of two highly expressed test genes encoding the human placental proteins PP4 and FXIIIa could not be further enhanced by the presence of the "Epsilon" element. These experiments show that the T7 gene 10 leader sequence can be utilized to improve the expression yields of otherwise poorly expressed heterologous genes in Escherichia coli.

A simple method to assay the major properties of DNA polymerases such as template binding, polymerase and exonuclease activities in one step is exemplified with the DNA polymerases of E. coli, bacteriophage T4 and herpes simplex virus. Combining the advantages of the band-shift assay with the resolving power of polyacrylamide gradient gel electrophoresis, the procedure is particularly useful for a rapid functional analysis of mutant polymerases as well as inhibitors of DNA replication.

Bacteriophage T4 baseplate gene 26 codes for two in-frame overlapping peptides with identical C-terminal regions. By site-directed mutagenesis we have now determined that an internal AUU, codon 114 of gene 26, is used as the initiation codon for the synthesis of a smaller peptide (gp26*). Thus gene 26* gives rise to a peptide of 95 amino acid residues with an Mr of 10,873, while the complete gene 26 encodes a peptide of 208 amino acid residues of M(r) 23,880. Expression of gene 26* is shown to depend on the RNA secondary structure in the translational initiation region of this gene.

This report provides a purification method for the two proteins, 51 kDa and 33 kDa, both encoded by the same mcrB gene of the McrBC restriction system in Escherichia coli K-12. The two proteins were produced in large quantity using a T7 expression system and copurified to near homogeneity by DEAE-Sepharose and Affi-Gel blue column chromatography. The N-terminal amino acid sequences of these purified McrB proteins were the same as those predicted from the mcrB DNA sequence by Ross et al. [J. Bacteriol. 171 (1989b) 1974–1981]. The 33-kDa protein totally overlaps the C-terminal part of the 51-kDa protein.

A new 3D 1H-15N-13C triple resonance experiment is presented that provides in-phase absorptive cross peaks between amide protons and alpha-protons of the same and the sequentially preceding residue. The experiment yields similar connectivities as those described previously by Montelione and Wagner (1990a) (J. Magn. Reson., 87, 183-188) and Kay et al. (1991) (J. Magn. Reson., 91, 84-92). However, the pulse sequence was designed to minimize the time that transverse coherence of the 13Calpha nucleus is present, since this nucleus has the shortest transverse relaxation time of all the nuclei involved in these experiments. This is achieved by using a coherence transfer pathway from 1HN to 15N, 13Calpha, 1Halpha and back to the 1HN. In the sequence described, transverse 13Calpha coherence is present only for a length of ca. 1/1J(Calpha-Halpha). This reduces loss of signal due to transverse relaxation. We tested the technique on uniformly 15N- and 13C-enriched T4 lysozyme.

Characterization of a mutant T7 RNA polymerase (RNAP) that is active on non-promoter templates but has lost the ability to selectively utilize the T7 promoter led to the finding that wild-type T7 RNAP initiates transcription at a high rate on non-promoter templates but that most (approximately 90%) of these initiation events lead to synthesis of dinucleotides only. The anomalously high activity of T7 RNAP on poly(dC) templates (relative to other non-promoter templates) is due to a reduction in the rate of transcription abortion after dinucleotide synthesis rather than an increase in initiation. Evidence is presented that the transition from abortive to processive transcription is associated with a conformational change in T7 RNAP. The stability of the nascent chain in a ternary complex is shown to increase with increasing chain length in the 2 to 14 base range even when the size of the complementary RNA-DNA hybrid remains constant and small (2 to 3 base-pairs). Two mutant polymerases that show increased release of transcripts during abortive transcription and a proteolytically nicked polymerase that exhibits reduced RNA binding are shown to have reduced ability to read-through a T7 RNAP hairpin U-stretch transcription terminator. Single-stranded nucleic acids are shown to bind more tightly than double-stranded nucleic acids to T7 RNAP. These observations and a large set of published studies on T7 RNAP structure and mechanism are accommodated in a relatively simple model of T7 RNAP transcription initiation and termination in which a T7 RNAP that has initiated transcription is proposed to be capable of assuming two functionally distinct conformations: an abortive conformer characterized by a loose association with the nascent RNA and an inability to translocate along the template; and a processive conformer characterized by the stable retention of the nascent RNA and the ability to process stably along the template. The equilibrium between these two conformations is shifted towards the processive form when the nascent chain binds at a site located at least partly on the T7 RNAP N-terminal domain. The interaction requires that the RNA be more than approximately nine bases and this RNAP-RNA interaction plays a primary role in retaining the RNA within the ternary complex.(ABSTRACT TRUNCATED AT 400 WORDS)

We have isolated and characterized a number of bacteriophage T7 RNAP (RNA polymerase) null mutants. Most of the mutants found to be completely inactive in vitro map to one of the well-conserved blocks of residues in the family of RNAPs homologous to T7 RNAP. The in vitro phenotypes of a smaller number of partially active T7 RNAP mutants, mapping outside these well-conserved regions, support the following assignment of functions in T7 RNAP: (1) the N-terminal region of T7 RNAP contains a nascent RNA binding site that functions to retain the nascent chain within the ternary complex; (2) the region surrounding residue 240 is involved in binding the initiating NTP; (3) residues at the very C terminus of T7 RNAP are involved in binding the elongating NTP.

The gene (cytA) coding for the 27 kDa polypeptide of the Bacillus thuringiensis var. israelensis mosquito larvicidal delta-endotoxin, was cloned into a plasmid containing the T7 bacteriophage promoter. The plasmid was used to transform an Escherichia coli strain containing the T7 RNA polymerase gene 1, under the control of lacP. Loss of colony-forming ability without substantial lysis, associated with immediate inhibition of DNA synthesis, was observed after induction of transformed cells. The cytA gene product may kill E. coli cells by disrupting their chromosome replicating apparatus.

Overexpression of glycoprotein-encoding genes in Escherichia coli sometimes results in toxicity to the host and low protein yields. One possible explanation for this phenomenon is the presence of hydrophobic amino acid (aa) domains approx. 15–20 aa in length in the overproduced protein. As an initial test of this hypothesis, regions of hydrophobicity located within the envelope glycoproteins of HIV-1 and HTLV-1 were identified by computer analysis, and subsequently deleted by site-directed mutagenesis. The parent and modified envelope genes were expressed in bacteria using both λ p L and T7 inducible expression systems. Removal of the hydrophobic domains reduced the apparent toxicity and significantly increased the accumulation of recombinant protein from undetectable levels to approx. 10–15% of total cellular protein.

To assess the involvement of the RNA cleavage site-proximal 2′ hydroxyl group in the RNase III catalytic mechanism, a specific processing substrate was chemically synthesized to contain a 2′-deoxyribose residue at the scissile phosphodiester bond. The RNA substrate, corresponding to the phage T7 R1.1 primary processing signal, can be accurately cleaved in vitro by RNase III. A fully deoxyribose-substituted R1.1 processing signal is not cleaved by RNase III, nor does it excess inhibit cleavage of unmodified substrate. These results show that the 2′ hydroxyl group proximal to the scissile bond is not an essential participant in the RNase III processing reaction; however, other 2′ hydroxyl groups are important for substrate reactivity, and may be involved in establishing proper double helical conformation, and/or specific substrate contacts with RNase III.

An experimental method complete with theoretical considerations is presented for the measurement of different biological UV doses. The method is based on the high sensitivity of phage T7 activity to UV light. A precisely determined T7 inactivation action spectrum is presented over a wide optical range (240-514 nm). Using the T7 spectral sensitivity in relation to the minimal erythema dose (MED) and the effective spectral irradiance from solar radiation for the MED, an example is given to determine the MED value based on the measurement of T7 inactivation for a given case. The advantages and applicability of the method are discussed.

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.

Coliphage WPK was originally isolated from sewage in Kiel, Germany, because its plaque diameter continued to expand for days. Electron microscopy revealed an isometric capsid with dimensions of 54 nm between opposite apices, and a short, noncontractile tail 16 nm long, placing phage WPK into morphogroup C1. The nucleic acid of phage WPK was linear double stranded DNA. The host ranges of phages WPK and T3 were identical. Of ten E. coli strains tested for host range, two were resistant and of eighteen other Enterobacteriaceae only four were susceptible. Seven gram-negative species which are not members of the Enterobacteriaceae were refractory. However, there were differences in plaque morphology and plaque expansion between the two phages. Phage T3 plaques expanded for at least seven days on E. coli B only, while phage WPK plaques expanded for at least seven days on four strains of E. coli. The buoyant density of WPK, determined by isopycnic density gradient centrifugation in CsCl, was 1,508 g/ml which was significantly different than that of T3 at 1.493 g/ml (P less than 0.05). Phage-encoded proteins were examined for each phage using [35S]methionine incorporation, SDS-PAGE, and autoradiography. Of thirty proteins identified in phage WPK and twenty-eight in phage T3, only fourteen were of the same size in both. We concluded that phage WPK was distinct, but related to T3.

Phage T5 injects its DNA into Escherichia coli cells in two steps; 8% of the chromosome is first injected, and then there is a pause during which proteins encoded by this DNA fragment are synthesized allowing the remaining DNA to be injected. Using a potassium-selective electrode, we show that the injection of the two DNA fragments is associated with an efflux in two steps, of cytoplasmic potassium. The rate of efflux is linearly related to the number of added phages suggesting that each phage induces the formation of at least one channel in the inner membrane. The first efflux occurs even in depolarized cells suggesting that the insertion and the opening of the channel can take place in the absence of the electrochemical gradient of protons (delta mu H+). The channel is in a closed configuration during the time required for the synthesis of the phage-encoded proteins; this closing and the second efflux are prevented by the depolarization of the cell. The insertion of the channel in the inner membrane requires a fluid membrane. The results obtained suggest that the function of this channel is to translocate phage T5 DNA.

The DNA replication system of bacteriophage T4 serves as a relatively simple model for the types of reactions and protein-protein interactions needed to carry out and coordinate the synthesis of the leading and lagging strands of a DNA replication fork. At least 10 phage-encoded proteins are required for this synthesis: T4 DNA polymerase, the genes 44/62 and 45 polymerase accessory proteins, gene 32 single-stranded DNA binding protein, the genes 61, 41, and 59 primase-helicase, RNase H, and DNA ligase. Assembly of the polymerase and the accessory proteins on the primed template is a stepwise process that requires ATP hydrolysis and is strongly stimulated by 32 protein. The 41 protein helicase is essential to unwind the duplex ahead of polymerase on the leading strand, and to interact with the 61 protein to synthesize the RNA primers that initiate each discontinuous fragment on the lagging strand. An interaction between the 44/62 and 45 polymerase accessory proteins and the primase-helicase is required for primer synthesis on 32 protein-covered DNA. Thus it is possible that the signal for the initiation of a new fragment by the primase-helicase is the release of the polymerase accessory proteins from the completed adjacent fragment.

A number of unnatural amino acids and amino acid analogs with modified backbone structures were substituted for alanine-82 in T4 lysozyme. Replacements included alpha,alpha-disubstituted amino acids, N-alkyl amino acids, and lactic acid, an isoelectronic analog of alanine. The effects of these electronic and structural perturbations on the stability of T4 lysozyme were determined. The relatively broad substrate specificity of the Escherichia coli protein biosynthetic machinery suggests that a wide range of backbone and side-chain substitutions can be introduced, allowing a more precise definition of the factors affecting protein stability.

A 3382 bp fragment containing a gene for a DNA ligase from the extremely thermophilic, acidophilic, and facultatively anaerobic archaeon (archaebacterium) Desulfurolobus ambivalens was cloned and sequenced. The deduced amino acid sequence (600 amino acids, 67619 molecular weight) showed 30-34% sequence identity with the ATP-dependent eucaryal (eukaryotic) DNA ligases of Schizosaccharomyces pombe, Saccharomyces cerevisiae, the human DNA ligase I, and with the Vaccinia DNA ligase. Distant similarity to the DNA ligases from the bacteriophages T3, T4, T6, T7 and the African swine fever virus was found, whereas no similarities were detectable to the NAD-dependent DNA ligases from the bacteria (eubacteria) Escherichia coli and Thermus thermophilus, to the ATP-dependent RNA-ligase of bacteriophage T4, and to the tRNA-Ligase from S.cerevisiae. A detailed comparison of the phylogenetic relationship of the amino acid sequences of all known DNA and RNA ligases is presented including a complete alignment of the ATP-dependent DNA ligases. The in vivo-transcription initiation and termination sites of the D.ambivalens gene were mapped. The calculated transcript length was 1904-1911 nt.

A partial duplex DNA substrate containing the Lac repressor binding site, within the duplex region, was constructed to examine the effect of bound Lac repressor on the unwinding reaction catalyzed by several DNA helicases. The substrate contained 90 base pairs of double-stranded DNA and, in the absence of Lac repressor, was effectively unwound by each of the seven helicases tested. The unwinding reactions catalyzed by Escherichia coli Rep protein, bacteriophage T4 Dda protein and E. coli DNA helicase I were not inhibited by the presence of bound Lac repressor. Both SV40 T antigen and E. coli helicase II were partially inhibited by bound repressor at the highest repressor concentrations tested. The helicase reactions catalyzed by E. coli DnaB protein and helicase IV were substantially inhibited by the presence of bound protein. When the length of the duplex region was increased to 323 base pairs the inhibition spectrum caused by bound Lac repressor on the unwinding reactions catalyzed by DnaB protein, helicase I and helicase II was essentially the same as that observed using the shorter partial duplex molecule. Inhibition of the unwinding reaction was due to the presence of bound Lac repressor as evidenced by the substantially weaker inhibition of helicase IV by Lac repressor in the presence of IPTG. In addition, we have shown that Rep protein displaces the bound repressor protein during the course of an unwinding reaction.

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, the product of the Escherichia coli aroA gene, has been overproduced in E. coli BL21(lambda DE3) under the control of the T7 gene 10 promoter and ribosome binding site, to a level of approximately 50% of total cell protein. EPSP synthase is the primary target of the post-emergence herbicide, glyphosate, commonly known as Roundup. A simple two step purification is described, which results in 99% pure homogeneous protein (as determined by PAGE). The integrity of the protein has been compared with previously characterized protein from E. coli AB2829(pKD501) by determination of its kinetic parameters, N-terminal protein and DNA sequences, amino acid analysis and 13C-NMR spectroscopy. This new overproducing strain readily provides the gram quantities of highly pure protein required for NMR studies of the active site and the development of novel time-resolved solid-state NMR techniques currently underway in this laboratory.

The derived amino-acid sequences of the proteins encoded by E. coli genes sbcC and sbcD have been compared with other protein sequences using computer assisted methods. This work has shown that SbcC and D, which inhibit the propagation of replicons containing long palindromic DNA sequences, are distantly related to two putative bacteriophage nucleases. These nucleases both comprise two polypeptide chains which are the products of genes 46 and 47 of bacteriophage T4 (gp 46 and gp 47) and genes D13 and D12 of bacteriophage T5 (gp D13 and gp D12). The comparisons reveal that SbcC, gp 46 and gp D13 are more closely related to each other than are SbcD, gp 47 and gp D12. SbcC appears to have undergone a partial duplication of an ancestral sequence. These proteins all contain motifs common to the superfamily of nucleotide-binding proteins that includes UvrA and the cystic fibrosis transmembrane regulator CFTR.

A double-strand break in a bacteriophage T7 genome significantly reduced the ability of that DNA to produce viable phage when the DNA was incubated in an in vitro DNA replication and packaging system. When a homologous piece of T7 DNA (either a restriction fragment or T7 DNA cloned into a plasmid) that was by itself unable to form a complete phage was included in the reaction, the break was repaired to the extent that many more viable phage were produced. Moreover, repair could be completed even when a gap of about 900 nucleotides was put in the genome by two nearby restriction cuts. The repair was accompanied by acquisition of a genetic marker that was present only on the restriction fragment or on the T7 DNA cloned into a plasmid. These data are interpreted in light of the double-strand gap repair mode of recombination.

In the presence of the non-hydrolyzable analog of ATP, ATP gamma S, RecA protein can polymerize on an oligodeoxy-ribonucleotide to form a stable oligonucleoprotein filament that can find its homologous sequence in double-stranded DNA. The homologous joint formed by the oligonucleotide and duplex DNA is stable only if RecA protein is not removed. Such a nucleoprotein joint, covering a part or all of the promoter region of T3 or T7 phage RNA polymerase, blocked transcription directed by those polymerases. The same kind of joint, located downstream of the RNA polymerase promoter, also inhibited elongation of transcription and caused accumulation of truncated transcripts. These observations suggest that RecA protein can be used to shut off transcription from any promoter of known sequence.

Host-induced modification of phage T2 to T*2 was discovered in 1952. This phenomenon, a reversible alteration in viral host range resulting from a single growth cycle in certain bacterial hosts, is an ‘epigenetic’ change. In 1963 the chemical basis for the T* modification was shown to be the loss of DNA glucosylation, which resulted from T-even phage growth in cells lacking the glucosyl donor UDPG. Thus, DNA glucosylation of T-even phages was the first recognized epigenetic signal.

The interest in the TonA protein, now called FhuA, has endured since the beginnings of phage genetics and molecular biology. The functions of this energy-coupled transporter and receptor in the outer membrane of Escherichia coli cells are fascinating. The historical perspective of this protein

To determine the energetic and structural consequences of placing a charged group within the core of a protein, two "buried charge" mutants, Met 102----Lys (M102K) and Leu 133----Asp (L133D) were constructed in phage T4 lysozyme. Both proteins fold at neutral pH, although they are substantially less stable than wild type. The activity of M102K is about 35% that of wild type, while that of L133D is about 4%. M102K could be crystallized, and its structure was determined at high resolution. The crystal structure (at pH 6.8) of the mutant is very similar to that of wild type except for the alpha-helix that includes residues 108-113. In wild-type lysozyme, one side of this helix is exposed to solvent and the other contacts Met 102. In the M102K structure this alpha-helix becomes much more mobile, possibly allowing partial access of Lys 102 to solvent. The stability of M102K, determined by monitoring the unfolding of the protein with CD, is pH-dependent, consistent with the charged form of the substituted amino acid being more destabilizing than the uncharged form. The pKa of Lys 102 was estimated to be 6.5 both by differential titration and also by NMR analysis of isotopically labeled protein with 13C incorporated at the C epsilon position of all lysines. As the pH is lowered below pH 6.5, the overall three-dimensional structure of M102K at room temperature appears to be maintained to pH 3 or so, although there is evidence for some structural adjustment possibly allowing solvent accessibility to the protonated form of Lys 102.

This paper presents a method to observe the motions and configurations of large DNA molecules undergoing capillary electrophoresis (CE). A simple device to perform CE horizontally under microscopic observation is designed and images of single DNA molecules inside the capillary are obtained using an epi-fluorescence microscope. DNA molecules moved towards the negative electrode when an electric field was applied. The mobilities of three types of DNA (T4 and lambda bacteriophage DNA and PBR322 plasmid DNA) were measured at different electric field strength. The mobility vs. electric field strength curves of these three large DNAs showed that the mobility remained constant at high electric field strength (200-600 Volt/cm) and increased significantly at low electric field strength (less than or equal to 50 Volt/cm.). The apparent mobilities of the large DNA molecules were independent of molecular weight. At electric field strengths greater than or equal to 400 Volt/cm., big aggregates (snowballs) of DNA molecules formed and moved upstream towards the positive electrode. When the field was turned off, the aggregates dissociated into a cloud of single DNA molecules, and diffused into the solution.

Three different genes (trpR+, tyrR+ and phi (trpR-lacZ)) were inserted into pET3a, a multicopy transcription-translation vector designed by Rosenberg et al. (1) for the T7 RNA polymerase-driven overexpression of proteins in Escherichia coli. Gene orientation was in the anticlockwise ("silent") direction. Gene expression in the absence of T7 RNA polymerase was evaluated either directly using lacZ reporter systems or indirectly by observing the susceptibility of plasmid-bearing tester strains to inhibition by an aromatic amino acid analog. The production of repressor proteins and of a Trp repressor-LacZ chimera was readily detected, at levels comparable to those of haploid trpR+ or tyrR+ E. coli strains. Such T7 vector constructs thus have two especially useful properties: first, they provide a means for the high-level production of various proteins in E. coli; second, they offer a technically advantageous point of departure for structure-function studies of genes whose overexpression from multicopy plasmids would normally be cytotoxic.

The authors have used a self-cleaving RNA molecule (a hammerhead') to study the length-dependent folding of RNA produced during transcription by T7 RNA polymerase. Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues, 3{prime}-deoxynucleoside triphosphates. When the nascent transcription attains the minimum length required for the hammerhead' domain of the transcript to fully emerge from the ternary complex, the hammerhead' structure forms and self-cleaves, producing a truncated product. The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible: the sequencing ladder is compared to that generated by using a noncleaving control template. They have shown that 13 nucleotides past the cleavage point must be synthesized before the transcript can self-cleave in the ternary complex whereas RNA freed from the complex by heating can cleave with only 3 or more nucleotides present beyond the cleavage site. The results indicate that RNA in T7 RNA polymerase is not free of steric interactions in the ternary complex and not available for structure formation until it is at least 10 bases away from the site of polymerization. The results suggest that the maximum possible length of the RNA-DNA hybrid in the ternary complexes is more » 10. The relevance of the results in comparisons with other RNA polymerases, especially Escherichia coli RNA polymerase, is discussed. « less

The sunY gene of bacteriophage T4 contains a self-splicing group I intron. The ligated exons encode an open reading frame of 605 amino acids, whose inferred molecular mass is 68 kDa. However, none of the proteins made following T4 infection have been assigned to the sunY gene, and no mutations have been mapped to this locus. We show here that the primary product of the sunY gene is a protein with an apparent molecular mass of 64 kDa, which is processed to a protein approximately 4 kDa smaller. Unlike most other processed T4 proteins, cleavage occurs independently of both the T4 processing protease, the product of gene 21, and late phage protein synthesis. Insertional mutagenesis demonstrated that the sunY protein is not necessary for normal T4 growth under the conditions tested.

Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber mutants were tested for plaque formation on amber suppressor strains of Salmonella typhimurium. Experiments with other hybrid phages engineered to produce different amounts of wild-type T4 lysozyme have shown that, to score as deleterious, a mutation must reduce lysozyme activity to less than 3% of that produced by wild-type P22 e416. Plating the collection of amber mutants covering 163 of the 164 codons of T4 lysozyme, on 13 suppressor strains that each insert a different amino acid substitutions at every position in the protein (except the first). Of the resulting 2015 single amino acid substitutions in T4 lysozyme, 328 were found to be sufficiently deleterious to inhibit plaque formation. More than half (55%) of the positions in the protein tolerated all substitutions examined. Among (N-terminal) amber fragments, only those of 161 or more residues are active. The effects of many of the deleterious substitutions are interpretable in light of the known structure of T4 lysozyme. Residues in the molecule that are refractory to replacements generally have solvent-inaccessible side-chains; the catalytic Glu11 and Asp20 residues are notable exceptions. Especially sensitive sites include residues involved in buried salt bridges near the catalytic site (Asp10, Arg145 and Arg148) and a few others that may have critical structural roles (Gly30, Trp138 and Tyr161).

Infections of F plasmid-containing strains of Escherichia coli by bacteriophage T7 result in membrane damage that allows nucleotides to exude from the infected cell into the culture medium. Only pifA of the F pif operon is necessary for "leakiness" of the T7-infected cell. Expression of either T7 gene 1.2 or gene 10 is sufficient to cause leakiness, since infections by phage containing null mutations in both of these genes do not result in permeability changes of the F-containing cell. Even in the absence of phage infection, expression from plasmids of either gene 1.2 or 10 can cause permeability changes, particularly of F plasmid-containing cells. In contrast, gene 1.2 of the related bacteriophage T3 prevents leakiness of the infected cell. In the absence of T3 gene 1.2 function, expression of gene 10 causes membrane damage that allows nucleotides to leak from the cell. Genes 1.2 and 10 of both T3 and T7 are the two genes involved in determining resistance or sensitivity to F exclusion; F exclusion and leakiness of the phage-infected cell are therefore closely related phenomena. However, since leakiness of the infected cell does not necessarily result in phage exclusion, it cannot be used as a predictor of an abortive infection.

Bacteriophage T4 lysozyme is a basic molecule with an isoelectric point above 9·0, and an excess of nine positive charges at neutral pH. It might be expected that it would be energetically costly to bring these out-of-balance charges from the extended, unfolded, form of the protein into the compact folded state. To determine the contribution of such long-range electrostatic interactions to the stability of the protein, five positively charged surface residues, Lys16, Arg119, Lys135, Lys147 and Arg154, were individually replaced with glutamic acid. Eight selected double, triple and quadruple mutants were also constructed so as to sequentially reduce the out-of-balance formal charge on the molecule from +9 to +1 units. Each of the five single variant proteins was crystallized and high-resolution X-ray analysis confirmed that each mutant structure was, in general, very similar to the wild-type. In the case of R154E, however, the Arg154 to Glu replacement caused a rearrangement in which Asp127 replaced Glu128 as the capping residue of a nearby α-helix. The thermal stabilities of all 13 variant proteins were found to be fairly similar, ranging from 0·5 kcal/mol more stable than wild-type to 1·7 kcal/mol less stable than wild-type. In the case of the five single charge-change variants, for which the structures were determined, the changes in stability can be rationalized in terms of changes in local interactions at the site of the replacement. There is no evidence that the reduction in the out-of-balance charge on the molecule increases the stability of the folded relative to the unfolded form, either at pH 2·8 or at pH 5·3. This indicates that long-range electrostatic interactions between the substituted amino acid residues and other charged groups on the surface of the molecule are weak or non-existent. Furthermore, the relative stabilities of the multiple charge replacement mutant proteins were found to be almost exactly equal to the sums of the relative stabilities of the constituentn single mutant proteins. This also clearly indicates that the electrostatic interactions between the replaced charges are negligibly small. The activities of the charge-change mutant lysozymes, as measured by the rate of hydrolysis of cell wall suspensions, are essentially equal to that of the wild-type lysozyme, but on a lysoplate assay the mutant enzymes appear to have higher activity. This observation can be rationalized by assuming that the reduction of positive charge on T4 lysozyme does not affect the rate of catalysis, but does weaken the interaction between the negatively charged substrate and the positively charged C-terminal domain of the protein.

It was shown previously that the introduction of a negatively charged amino acid at the N-terminus of an alpha-helix could increase the thermostability of phage T4 lysozyme via an electrostatic interaction with the "helix dipole" [Nicholson, H., Becktel, W. J., & Matthews, B. W. (1988) Nature 336, 651-656]. The prior report focused on the two stabilizing substitutions Ser 38----Asp (S38D) and Asn 144----Asp (N144D). Two additional examples of stabilizing mutants, T109D and N116D, are presented here. Both show the pH-dependent increase in thermal stability expected for the interaction of an aspartic acid with an alpha-helix dipole. Control mutants were also constructed to further characterize the nature of the interaction with the alpha-helix dipole. High-resolution crystal structure analysis was used to determine the nature of the interaction of the substituted amino acids with the end of the alpha-helix in both the primary and the control mutants. Control mutant S38N has stability essentially the same as that of wild-type lysozyme but hydrogen bonding similar to that of the stabilizing mutant S38D. This confirms that it is the electrostatic interaction between Asp 38 and the helix dipole, rather than a change in hydrogen-bonding geometry, that gives enhanced stability. Structural and thermodynamic analysis of mutant T109N provide a similar control for the stabilizing replacement T109D. In the case of mutant N116D, there was concern that the enhanced stability might be due to a favorable salt-bridge interaction between the introduced aspartate and Arg 119, rather than an interaction with the alpha-helix dipole. The additivity of the stabilities of N116D and R119M seen in the double mutant N116D/R119M indicates that favorable interactions are largely independent of residue 119. As a further control, Asp 92, a presumed helix-stabilizing residue in wild-type lysozyme, was replaced with Asn. This decreased the stability of the protein in the manner expected for the loss of a favorable helix dipole interaction. In total, five mutations have been identified that increase the thermostability of T4 lysozyme and appear to do so by favorable interactions with alpha-helix dipoles. As measured by the pH dependence of stability, the strength of the electrostatic interaction between the charged groups studied here and the helix dipole ranges from 0.6 to 1.3 kcal/mol in 150 mM KCl. In the case of mutants S38D and N144H, NMR titration was used to measure the pKa's of Asp 38 and His 144 in the folded structures.(ABSTRACT TRUNCATED AT 400 WORDS)

Four-way DNA junctions are thought to be important intermediates in a number of recombination processes. Resolution of these junctions occurs by cleavage of two strands of DNA to generate two duplex molecules. The interaction between DNA junctions and resolving enzymes appears to be largely structure-specific, reflecting a molecular recognition on a significant scale. We propose a working model for this interaction that takes account of the present state of knowledge of the structure of the DNA junction, and the substrate requirements of the enzymes. We note that three different enzymes introduce cleavages at phosphodiester bonds that are presented on one side of the molecule, suggesting that the enzymes selectively interact with this face of the junction. By forcing a junction of constant sequence to adopt one or other of the two possible antiparallel isomers, we show that the junction is cleaved in such a way as to suggest a constant mode of interaction with the protein that is dependent on structure rather than sequence. We propose that the feature that is recognized is a mutual inclination of two DNA helices at approximately 120 °. We show that a number of DNA substrates that contain similar inclined helices, such as a three-way junction, bulged duplexes and a duplex that is curved because of repeated runs of oligoadenine sequences, are each cleaved by phage T4 endonuclease VII. This mode of DNA-protein interaction could be significant in either recombination or DNA repair processes.

A general mechanism by which proteins locate their target sites within large domains of DNA is a one-dimensional facilitated diffusion process in which the protein scans DNA in a nonspecifically bound state. An electrostatic contribution to this type of mechanism has been previously established. This study was designed to question whether other characteristics of a protein's structure might contribute to the scanning mechanism of target site location. In this regard, T4 endonuclease V was shown to establish an ionic strength dependent monomer-dimer equilibrium in solution. A protein dimer interaction site was postulated to exist along a putative alpha-helix containing amino acid residues 54-62. The conservative substitutions of Phe-60----Leu-60 and Phe-59, Phe-60----Leu-59, Leu-60 resulted in mutant enzymes which remained in the monomeric state independent of the ionic strength of the solution. The target site location mechanism of these mutants has also been altered. Under conditions where wild-type endonuclease V processively scans nontarget DNA, the target location mechanism of the monomeric mutant proteins was shifted toward a less processive search. This decrease in the processivity of the mutants was especially surprising because the nontarget DNA binding affinity was found to be significantly increased. Thus, an additional component of the endonuclease V DNA scanning mechanism appears to be the formation of a stable endonuclease V dimer complex.

This study evaluated bacteriophages phi X174, T7, PRD1, and phi 6 as possible surrogates for pathogenic human viruses to challenge barrier materials and demonstrated some important factors for their use. Chemical incompatibility with test material was demonstrated when lipid-enveloped phi 6 was inactivated by an aqueous eluate of vinyl gloves, but 0.5% calf serum protected phi 6 from the eluate. Low concentrations (2%) of calf serum also prevented the exaggerated binding of the bacteriophages to filters. Recovery of viruses from surfaces decreased with increasing time before recovery. Penetration through punctures displayed different types of kinetics. The combined data indicate that (i) some bacteriophages may serve as surrogate viruses, (ii) experimental conditions determine whether a particular virus is appropriate as a challenge, and (iii) phi X174 is an excellent choice as a surrogate virus to test barrier materials. The data further indicate that before barrier materials are challenged with viruses, adequate tests should be performed to ensure that the virus is compatible with the test material and test conditions, so that meaningful data will result.

Nucleophosmin/B23 is a nucleolar phosphoprotein which forms oligomers. To determine the domain essential for oligomer formation, various deletion and point mutation clones of nucleophosmin/B23 were constructed. Nucleophosmin/B23 and the mutant proteins were produced by (a) coupled in vitro transcription and translation and (b) expression in Escherichia coli with T7 RNA polymerase expression vector (pET-8c). Nucleophosmin/B23 synthesized in vitro has the same peptide map as that synthesized in HeLa cells. Similarly, it formed oligomers which could be detected in SDS/PAGE and were cross-linked with nitrogen mustard in vivo. Substitution of Met5, Met7, and Met9 with Leu or deletion of five amino acids at the C-terminus abolished the oligomerization. Deletion of portions of amino acids in the middle of the molecule (amino acid residues 83-152, 117-186 and 185-240) had little effect on the oligomerization. Co-expression of the N- and C-terminal mutant clones in vitro did not produce oligomers. These results indicate that intra-molecular interactions with both the N- and C-terminal domains are essential for oligomer formation.

The 139 kb circular maize chloroplast genome was cloned in a linear form in a single yeast artificial chromosome (YAC), and several such clones were then analyzed to study the stability of large DNA sequences in YACs. The YAC clones were isolated from a partial maize DNA YAC library. The library was constructed by Bal-31 nuclease digestion of high-molecular-weight total maize genomic DNA followed by blunt-end ligation into a modified YAC vector, pYAC11. All chloroplast genome-containing YACs (cpYACs) were characterized by comparing their restriction enzyme digestion patterns with that of purified maize chloroplast DNA (MCD). Comparison of the restriction patterns of four cpYACs, MY503, MY504, MY518 and MY526, with that of MCD did not show any change in the size of the restriction fragments except at the chloroplast DNA-vector junction fragments. These altered size fragments were confirmed as junction fragments by hybridization of the pYAC11 border fragments. The end-specific T3 and T7 transcripts synthesized from MY503 also identified the same junction fragments as did the pYAC11 border fragments. Most cpYACs were completely stable through the analyzed one hundred generations.

RNA-based amplification systems that have been recently described are dependent upon the presence of more than one enzyme. In an attempt to minimize the number of polymerases required for efficient amplification, we have studied the template specificity of bacteriophage T3 RNA polymerase. A synthetic bacteriophage T3 promoter was covalently attached to an RNA template. The T3 promoter-RNA complex was found to be selective for its native polymerase, and dependent upon the presence of all four ribonucleoside precursors. The product of the RNA-directed transcription is complementary to the initial template.

The bacteriophage T4 DNA recombination-repair geneuvsY located at or near an origin of DNA replication and adjacent to the late base plate genes 25 and 26. Our present results reveal a complex transcription pattern in the region encompassing these genes. Most significantly, uvsY and two ORFs, downstream of it, all of which are transcribed from a middle promoter before the onset of DNA replication, are also part of a larger late transcription unit which includes the base plate genes 25 and 26. The late genes 25 and 26 are transcribed not only late, but also early from one or several early promoters further upstream. Translation, however, is inhibited by secondary structures which sequester the ribosome binding site in the early transcript. We discuss possible advantages of these transcriptional patterns for T4 DNA recombination, replication, and repair. The predicted andin vivo-expressed 23.9-kDa product of gene 26 is smaller than the reported size of gene 26 protein isolated from base plates, suggesting that nascent gp26 might be processed to a larger protein during assembly.

We have developed an efficient method for the expression of heterologous genes during infection by T4, a bacteriophage known to inhibit the proteolytic systems of Escherichia coli. This system enables us to clone genes in a plasmid expression vector and move them readily into T4. We have used lacZ as a reporter gene to show that both plasmid and phage exhibit low basal expression or high-level expression under the control of a T7 promoter. This system promises a possible solution to the problem of degradation and/or toxicity of overproduced proteins.

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.

In order to evaluate the contributions that histidine residues might play both in the catalytic activities of endonuclease V and in binding to nontarget DNA, the technique of oligonucleotide site directed mutagenesis was used to create mutations at each of the four histidine residues in the endonuclease V gene. Although none of the histidines were shown to be absolutely required for the pyrimidine dimer specific DNA glycosylase activity or the apurinic lyase activity, conservative amino acid changes at His16 produced enzymes with little or no catalytic activity. In addition, the evaluation of conservative and radical amino acid substitutions at positions 34, 56, and 107 is consistent with the interpretation that each of these histidines may be involved in nontarget DNA binding. The data supporting this conclusion are that histidine changes to lysine at positions 34 and 107 enhance the nontarget DNA binding activity of the mutant enzymes while neutralization of charge at His56 reduces nontarget DNA binding.

A system for the quantitative estimation of T4 regulatory signals in bacteriophage T4 was developed. A transmitter-reporter plasmid vector, pCV22, which is able not only to fuse transcriptional and translational signals to the coding region of lacZ gene (reporter) but also to transmit the fused gene into T4 phage genome (transmitter). The regulatory signals of T4 phage genes fused with the reportor gene was transmitted efficiently into T4 phage (T4dC-lac phage; a receiver phage) by a replacement type of recombination in the uvsY gene region. This type of chimeric phage was demonstrated to be able to quantitatively estimate the late regulatory signal of T4 phage in vivo .