The phage 434 Cro/OR1 complex at 2.5 A resolution.

The crystal structure of phage 434 Cro protein in complex with a 20 base-pair DNA fragment has been determined to 2.5 A resolution. The DNA fragment contains the sequence of the OR1 operator site. The structure shows a bent conformation for the DNA, straighter at the center and more bent at the ends. The central base-pairs adopt conformations with significant deviations from coplanarity. The two molecules interact extensively along their common interface, both through hydrogen bonds and van der Waals interactions. The significance of these interactions for operator binding and recognition is discussed.

A rapid and simple method for screening large numbers of recombinant DNA clones.

A simple and rapid method has been described for the isolation of plasmid, phagemid and phage DNAs. Hundreds of recombinant clones can be screened in one day employing this method. It takes half an hour to prepare plasmid DNA from ten clones, and the DNA prepared from a single colony using this method is of sufficient quality and in sufficient amount to perform at least five restriction digestions. This method eliminates the need for RNase treatment and phenol chloroform extraction if the plasmids are needed only for the restriction digestion. If needed, RNAs can be removed after restriction digestion by adding RNase and incubating for two minutes at room temperature. After RNase treatment and phenol/chloroform extraction, the plasmid DNA serves as a good template for sequencing. The DNA can be stored at -20 degrees C for over eight weeks.

Temperate bacteriophages of Streptococcus pneumoniae that contain protein covalently linked to the 5' ends of their DNA.

We have characterized three temperate bacteriophages of pneumococcus (HB-3, HB-623, and HB-746). Although all the phages belong to the same family, the polypeptide composition of the virions and the DNA restriction endonuclease analysis of their DNAs revealed differences among the three phages. The genomes of these bacteriophages have been isolated as DNA-protein complexes. The protein is specifically associated with the two 5' termini of the DNA as shown by experiments carried out with exonucleases. The protein bound to the DNA in the three phages studied, iodinated in vitro with 125I, has a molecular weight of 23,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of the complexes with chaotropic agents suggested that the protein is covalently bound to the 5' termini of the DNA. Comparative pulsed-field gel electrophoresis analysis and Southern hybridization of the SmaI restriction fragments of DNAs from one lysogenic bacteria and its parental strain revealed that the prophage genome was integrated in the host chromosome.

Characterization of filamentous bacteriophage phi Lf from Xanthomonas campestris pv. campestris.

A filamentous phage, phi Lf, which specifically infects Xanthomonas campestris pv. campestris was isolated. The phage particle measured 1,000 (+/- 200) x 8 nm. It formed turbid plaques of about 1 mm in diameter. During multiplication, the progeny virions extruded into the medium without retarding host cell growth. Stocks were stable for 6 months at 4 degrees C and survived treatment at 80 degrees C for 10 min. Treatment with chloroform, ethanol or acetone completely destroyed infectivity; ethyl ether and methanol inactivated 98 to 99% of the phage. SDS-polyacrylamide gel electrophoresis showed a major coat protein band of approximate Mr 4000 whereas an immunoprecipitation test detected the existence of two coat protein species. The phage genome was shown to be a single-stranded DNA molecule. A physical map was constructed and the DNA size was calculated to be 5.9 kb. Cells treated with Tris-HCl containing CaCl2 and polyethylene glycol 6000 were transfected by replicative form DNA at a frequency of 3.4 x 10(3) p.f.u./micrograms.

Structural proteins of the Actinobacillus actinomycetemcomitans bacteriophage phi Aa.

øAa is an A1 morphotype bacteriophage which infects certain strains of Actinobacillus actinomycetemcomitans. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of dissociated, purified phi Aa particles revealed 7 major structural proteins (P1-P7) ranging in size from 17.5 to 52.7 kilodaltons (Kd). Treatment of the intact phage particles with 67% dimethyl sulfoxide (DMSO) resulted in the separation of the virion head and tail subunits. Purification of the head subunits was accomplished by sucrose density gradient centrifugation of the DMSO-treated phage particles. The purified head subunits were composed of a single protein having an electrophoretic mobility which corresponded to a 39.5 Kd protein (P3) of the intact virus. Raising the pH of a purified phi Aa suspension to 12.7 disrupted the head subunits, as well as the tail tube and tail fibers, releasing intact contractile tail sheaths. The tail sheaths were collected by centrifugation. The purified tail sheaths were analyzed by SDS-PAGE and were found to be composed of two proteins (P1 and P2) having molecular weights of 52.7 and 41.2 Kd respectively. The location of each of the 4 remaining major structural proteins in the phi Aa virion remains to be determined.

Sequencing reactions in microtiter plates.

We describe a simple and inexpensive method of performing sequencing reactions for 24 single-strand M13 DNA clones in microtiter plates. To simplify elevated temperature incubations during sequencing reactions, two heating blocks were designed to accommodate microtiter plates and fit within common laboratory heating modules. With only slight modification of standard fluorescent and radioisotopic sequencing methods, the sequencing reactions for 24 clones can be done in as little as 40 minutes.

Model-building studies of Inovirus: genetic variations on a geometric theme.

Inovirus (filamentous bacteriophage) is a simple system for studying the rules by which protein primary structure (amino acid sequence) controls secondary and higher order structure, and thereby function. The virus occurs naturally as a number of different strains with similar secondary and higher order structure, but the protein subunit that assembles to form the virion coat has quite different primary structures in different virus strains. Despite these differences in primary structure, the subunits of all strains have much the same size, about 50 residues, which are distributed by type in much the same way into three domains of primary structure: a collection of acidic residues in the N-terminal region, a hydrophobic domain of about 19 residues near the middle, and a collection of basic residues near the C-terminus. Each subunit can be closely approximated by an alpha-helix with its long axis roughly parallel to the fibre axis, sloping from large to small radius in the virion and interleaving between subunits in the next turn or level. The acidic residues near the N-terminus of the subunit face outwards on the virion surface, and explain the low isoelectric point of the virion; the basic residues near the C-terminus face inwards, where they neutralize the charge on the DNA at the core of the virion; and the hydrophobic central domain is involved in interactions which bind neighbouring subunits. Detailed X-ray fibre diffraction analysis of one strain gives the subunit structure. Comparative model-building studies of different strains illustrate the common structural principles.

A CD determination of the alpha-helix contents of the coat proteins of four filamentous bacteriophages: fd, IKe, Pf1, and Pf3.

The CD spectra of four filamentous bacteriophages--fd, IKe, Pf1, and Pf3--were analyzed to determine the alpha-helix contents of their major coat proteins. Measured spectra included the 192-nm band so that analyses could be carried out over the full wavelength range of the reference spectra for protein secondary structures available (a) from globular proteins [J.T. Yang, C.S.C. Wu, and H.M. Martinez (1986) Methods in Enzymology 130, 208-269] and (b) from poly(L-lysine) [N. Greenfield and G.D. Fasman (1960) Biochemistry 8, 4108-4116]. Extended analyses were also performed with the addition of the spectrum of a model beta-turn to the Greenfield and Fasman reference set, with the spectrum of a short alpha-helix in the Yang et al. reference set, and with an estimate of the spectrum of Trp added to both reference sets. The reference set based on the simple poly(L-lysine) polypeptide, plus a spectrum of a model beta-turn or of Trp, gave reasonably good fits to the measured spectra for all four phages and yielded the largest percentages of alpha-helix. The class I phages--fd and IKe--had large percentages of alpha-helix of 98 +/- 2 and 97 +/- 5%, respectively, while the two class II phages--Pf1 and Pf3--had similar but smaller alpha-helix contents of 83 +/- 6 and 84 +/- 2, respectively. While these alpha-helix contents were within the ranges previously reported from CD spectra of these phages in solution, they were more precise, and they indicated that the coat proteins of the intact phages have CD spectra that are probably modeled better by the reference spectra of polypeptides than by those of globular proteins.

Quantitative fluorescence of DNA-intercalated ethidium bromide on agarose gels.

Techniques for analyzing DNA distributions on agarose gels are examined by both two-dimensional and one-dimensional methods. It is demonstrated that very large errors in DNA concentration occur in such analyses unless (i) the electrophoresis is performed in a careful, reproducible manner, (ii) the films are calibrated with an internal standard, (iii) high resolution densitometry is used for analyzing the films, and (iv) appropriate background controls are used to determine the baselines for integration. Two-dimensional scanning produces more accurate results than one-dimensional scanning, but in cases where the bands are relatively uniform, the one-dimensional analysis gives good results. A technique for determining accurate distributions is described.

Rapid isolation of DNA from complex biological samples using a novel capture reagent--methidium-spermine-sepharose.

We have synthesized and analyzed the functional properties of a novel DNA capture reagent containing a methidium moiety attached to a sepharose bead by a spermine linker. DNA present in a biological fluid or other complex sample binds to the reagent. The DNA-capture reagent complex is then separated from the sample by centrifugation and the DNA is released from the reagent by brief incubation in 0.1 to 0.5 N NaOH or KOH. Capture of DNA from complex samples is independent of the salt concentration of the sample, and occurs in the presence of high concentrations of EDTA, proteinase K and detergents. Many samples can be processed simultaneously. The following specific applications, in which denatured DNA is quantitated or characterized, are demonstrated: 1). Isolation of hepatitis B virus DNA from serum and quantitation by dot-blot hybridization, 2). Isolation and quantitation of DNA from urine, 3). Isolation of human genomic DNA from one microliter of blood or 100 HeLa cells followed by amplification of a specific gene sequence using the Polymerase Chain Reaction, 4). Isolation of single stranded phage M13 sequencing templates from bacterial cultures. These investigations suggest that a capture reagent containing an intercalating moiety bound to a solid support may be useful for many applications in molecular biology and molecular diagnostics.

Properties and partial genetic characterization of Nepean phage and other lytic phages of Brucella species.

Nepean (Np), a new brucellaphage, was associated with atypical Brucella abortus strains from Ontario cattle. Carriage of Np was associated with loss of smooth lipopolysaccharide, changes in some protein bands in acrylamide gel electrophoresis profiles, increased susceptibility to colistin, and increased resistance to ultraviolet killing. Nepean (Np) was compared with brucellaphages Tb, Fi, Wb, Iz and R/C. All were morphologically identical, with icosahedral capsids (50-65 nm diameter) and short tails (15-25 nm long), but Np had a more restricted host range, replicating only in smooth strains of B. abortus. All six brucellaphages were generally similar in resistance to chemical and physical agents. Brucellaphage DNA was double stranded and unmethylated; its molecular size was 38 kilobase pairs. The DNAs of Tb, Fi, Wb, Iz and R/C could not be differentiated by restriction endonuclease digest profiles produced by BgII, EcoRI, HindIII or PvuII. Nepean (Np) DNA was very similar to that of the other brucellaphages, but with every enzyme used its profile differed in the number and/or position of at least one fragment. However, there was complete cross-hybridization of Tb and Np DNAs. Hybridization techniques failed to detect Brucella DNA in Dp or Tb phages, or phage DNA in Brucella cells. Extrachromosomal plasmid DNA was not detected.

[Variability of Bacillus thuringiensis bacteriophages with C2-morphology]. / Variabel'nost' fagov Bacillus thuringiensis C2-morfologii.

Biological as well as physicochemical properties of Bacillus thuringiensis bacteriophages "17" and "7/13" having C2-morphology and isolated from factory phagolysates were studied. The bacteriophages are identical in the lytic spectrum++, morphology, size, GC-content, have the same buoyant density. The physical map for restriction endonucleases EcoRI, HindIII, SalGI and MvaI has been constructed of the bacteriophages DNA. Heteroduplex analysis has revealed the nonhomologous region of the deletion-insertion type as a 0.8 Md loop. The bacteriophages "17" and "7/13" are concluded to be closely related but not identical.

Purification in an active form of the phage phi 29 protein p4 that controls the viral late transcription.

The phage phi 29 protein p4, that controls viral late transcription, was highly purified from Escherichia coli cells harbouring a gene 4-containing plasmid. This protein, representing about 6% of the total cellular protein, was obtained in a highly purified form. The protein was characterized as p4 by amino acid analysis and NH2-terminal sequence determination. The purified protein was active in an in vitro transcription assay, allowing specific initiation of transcription at the phi 29 A3 late promoter in the presence of Bacillus subtilis sigma 43-RNA polymerase holoenzyme.

[Restriction analysis of the DNA of the toxigenic corynephages BF, phi 984, phi 9 and C]. / Restriktsionnyi analiz DNK toksigennykh korinefagov BF, fi 984, fi 9 i C.

The results of restriction analysis of toxigenic corynephages BF, phi 984, phi 9, and C are presented. Phage BF was shown to be a deletion derivative of phage beta vir, phage phi 984 to be omega-like. Toxigenic corynephages phi 9 and C belong to a new group of corynephages designated phi. DNA of phages phi 9 and C as well as chromosomal DNA of the indicator strains was not hydrolysed by specific Hind III endonuclease, that is likely to be associated with the presence of a modification system in host strains.

Solution hybridization of crosslinkable DNA oligonucleotides to bacteriophage M13 DNA. Effect of secondary structure on hybridization kinetics and equilibria.

Several DNA oligonucleotides have been photochemically modified with the furocoumarin 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) such that each contained a single HMT furan side monoadduct to thymidine at a unique 5' TpA 3' sequence. When these oligonucleotides were hybridized to their respective complements, the HMT adduct could be driven to form an interstrand crosslink by irradiation of the hybrid with 360 nm light. The ability to crosslink probe-target complexes has allowed us to determine the kinetics and the extent of hybridization in solution between these oligonucleotides and their complementary sequences in single-stranded bacteriophage M13 DNA. Our data indicate that these parameters are strongly influenced by the existence of local as well as global secondary structure in the viral DNA. During hybridization, rearrangement of this secondary structure so as to expose the target sequence can be rate-limiting. Upon attainment of equilibrium, only a portion of the target sequence may be hybridized to the probe with the remainder involved in intrastrand base-pairing. Using crosslinkable oligonucleotide probes hybridized and irradiated near the melting temperature of the respective probe-target complex one can partially overcome these secondary structure effects.

Characterization of Vibrio eltor typing phages: properties of the Eltor phage e4.

Biophysical characteristics of Vibrio eltor phage e4, a key phage in the Vibrio cholerae typing scheme were studied. This icosahedral phage was found to contain 12 structural polypeptides with mol. wt. ranging from 25,000 to 120,000. One of these polypeptides of mol. wt. 50,000 accounted for most of the structural proteins present and was probably the major phage capsid protein. The phage genome comprised a single linear, double-stranded DNA molecule, 69.2 kbp in length (45.6 X 10(6) mol. wt.) as determined by electron microscopy and restriction fragment analyses. The G + C content was 34.6%. Electron microscopy data indicated that unlike the DNAs of other cholera phages, phage e4 DNA is not circularly permuted. Adsorption under normal conditions was biphasic with rate constants of 1.02 X 10(-9)/ml/min up to 60% adsorption and 3 X 10(-10)/ml/min thereafter. Intracellular phage multiplication was characterized by a latent period of 27 min. The burst size was approximately 100 phage particles per infected cell.

A preliminary structure for the DNA binding protein from bacteriophage IKe.

A modelling procedure has been utilized to obtain a preliminary three-dimensional structural model for the bacteriophage IKe DNA binding protein (IKe-DBP) based on the known high resolution X-ray diffraction structure of a functionally related protein (G5BP) from bacteriophage fd. The degree of structural homology observed is much higher than the 44% primary sequence identity between these proteins would indicate. These studies suggest IKe-DBP, like G5BP, is composed of a central three-stranded beta sheet from which protrude three extended beta loops. Furthermore, the IKe-DBP structural model can easily form, without conformational rearrangements, the compact dimer unit that is the functionally active species of G5BP. Structural comparisons show residues conserved in the primary sequence of both proteins tend to cluster in two regions. The first being essential for the maintenance of dimer association. The second about the two DNA binding channels which cross the face of each dimer. Based upon an earlier characterized G5BP-DNA complex, a model for DNA complexation to IKe-DBP is also presented.

Genome homology and divergence in the SP beta-related bacteriophages of Bacillus subtilis.

Chromosomal organization in related temperate Bacillus subtilis bacteriophages SP beta, phi 3T, rho 11, Z, and E was compared. DNA-DNA hybridization studies done in conjunction with available restriction fragment maps of SP beta, phi 3T, and rho 11 demonstrated that DNA homology between these three phages extended over most of their respective genomes, although each contained unique chromosomal segments, phi 3T, rho 11, Z, and E, but not SP beta, possessed apparently homologous structural genes (thyP) for thymidylate synthetase. DNA from all thyP-containing phages transformed thymine auxotrophs of B. subtilis SP beta lysogens to prototrophy. This transformation commonly involved incorporation of the thyP gene into SP beta prophage within a region corresponding to the middle of the viral chromosome. Chimeric plasmids containing the thyP gene from phi 3T or cloned fragments of SP beta DNA were used in DNA-DNA hybridization studies to locate the thymidylate synthetase gene near the center of the phi 3T chromosome, and to demonstrate that the organization of this region resembled the analogous portion of the SP beta genome. Profiles of virion structural proteins from the five phages were also very similar, further suggesting functional homology between these viruses. However, despite these evidences of relatedness, populations of fragments generated by digesting SP beta, phi 3T, rho 11, Z, and E DNA with restriction enzymes were quite dissimilar.

The morphology and nucleotide composition of DNA of Citrobacter phages.

Citrobacter phages 38/37, 31/37, 40/1 and 8/5, isolated from lysogenic cultures, were concentrated and purified by 2 cycles of differential centrifugation. Electron microscopy of the phages has shown that their particles have similar morphology and that they relate to the morphological group A1. The heads of the phages are hexagonal, 50 +/- 2 nm in diameter. The tail of the phage is straight, 112-152 nm in length, with a contracting sheath 11.5-12.5 nm wide. The tails of the phages 38/37 and 40/1 were found to be slightly longer in comparison with the phages 31/37 and 8/5. Chromatographic investigation of DNA preparations of the phages revealed the presence of 4 nitrous bases. Identification of the latter permitted us to relate them to common nitrous bases. DNA of the phages is double-stranded and belongs to a weakly expressed guanine-cytosine type. The content of guanine and cytosine in DNA of the phage 38/37 amounts to 56.68%, that of the phage 31/37 to 56.75, of the phage 40/1 to 57.36% and of the phage 8/5 to 55.58%. No substantial variations were observed in the DNA composition of the phages.