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PubMed1991

Raman spectroscopy of filamentous bacteriophage Ff (fd, M13, f1) incorporating specifically-deuterated alanine and tryptophan side chains. Assignments and structural interpretation.

Structural interpretation of the Raman spectra of filamentous bacteriophages is dependent upon reliable assignments for the numerous Raman vibrational bands contributed from coat protein and packaged DNA of the virion. To establish unambiguous assignments and facilitate structural conclusions derived from them, we have initiated a systematic study of filamentous bacteriophage Ff (fd, f1, M13) incorporating protein subunits with specifically deuterated amino-acid side chains. Here, we report and interpret the Raman spectra of fd virions which incorporate: (a) a single deuterio-tryptophan residue per coat protomer [fd(Wd5)], (b) ten deuterio-alanines per protomer [fd(10Ad3)], and (c) both deuterio-tryptophan and deuterio-alanine [fd(Wd5 + 10Ad3)]. The unambiguous assignment of coat protein Raman bands in normal and deuterated isotopomers of fd establishes the validity of earlier empirical assignments of many key Raman markers, including those of packaged ssDNA (Thomas et al., 1988). Present results confirm that deoxyguanosine residues of the packaged ssDNA molecule depart from the usual C2'-endo/anti conformation characteristic of protein-free DNA in aqueous solution, although C2'-endo/anti conformers of thymidine are not excluded by the data. The combined results obtained here on normal fd, and on fd incorporating deuterio-tryptophan [fd(Wd5) and fd(Wd5 + 10Ad3)], show also that the microenvironment of the single tryptophan residue per coat protomer (W26) can be clearly deduced as follows: (a) The indole 1-NH donor group of each protomer in fd forms a moderately strong hydrogen bond, most likely to a hydroxyl oxygen acceptor. (b) The planar indole ring exists in a hydrophilic environment. (c) The torsion angle describing the orientation of the indole ring (C3-C2 linkage) with respect to the side-chain (C alpha-C beta bond) is unusually large, i.e., magnitude of X2,1 approximately 120 degrees. With respect to alanine isotopomers, the present results show that alanine residues, and possibly other methyl-containing side chains, are significant contributors to the fd Raman spectrum. The present study provides new information on protomer side chains of fd and demonstrates a Raman methodology which should be generally useful for investigating single-site interactions and macromolecular conformations in other nucleoprotein assemblies.

Alanine↗
PubMed1977

Viral DNA sequences from incomplete particles of human adenovirus type 7.

Large pools of empty viral capsids accumulate in cells infected by subgroup B human adenoviruses. Such infected cells also yield DNA-containing incomplete particles in larger quantities than cells infected with serotypes representing other adenovirus subgroups. DNA isolated from carefully purified classes of Ad7 incomplete particles was analyzed by restriction endonuclease cleavage, gel electrophoresis and electron microscopy. At least 90% of the DNA molecules in each sample consisted of sequences that extended from the left end of the viral genome map by variable lengths toward the right end. The average length of DNA is linearly related to the average buoyant density of the incomplete particles from which the DNA is isolated. The results indicate that each capsid contains one DNA molecule. There is also a specific association of the left end of the viral genome with assembled or assembling capsids. The characteristic distributions of Ad7 incomplete particles may result from intracellular pools of assembly intermediates in which the incompletely packaged DNA has been fragmented in vivo or by shear during preparative procedures.

Adenoviruses, Human↗
PubMed2006

A microbial genetic journey.

Fortunately, I began research in 1950 when the basic concepts of microbial genetics could be explored experimentally. I began with bacteriophage lambda and tried to establish the colinearity of its linkage map with its DNA molecule. My students and I worked out the regulation of lambda repressor synthesis for the establishment and maintenance of lysogeny. We also investigated the proteins responsible for assembly of the phage head. Using cell extracts, we discovered how to package DNA inside the head in vitro. Around 1972, I began to use molecular genetics to understand the developmental biology of Myxococcus xanthus. In particular, I wanted to learn how myxococcus builds its multicellular fruiting body within which it differentiates spores. We identified two cell-to-cell signals used to coordinate development. We have elucidated, in part, the signal transduction pathway for C-signal that directs the morphogenesis of a fruiting body.

Bacterial Proteins↗
PubMed2006

[Comparison of new giant bacteriophages OBP and Lu11 of soil pseudomonads with bacteriophages of phiKZ-supergroup of Pseudomonas aeruginosa].

Study of two recently isolated giant bacteriophages Lu11 and OBP that are active on Pseudomonas putida var. Manila and Pseudomonas fluorescens, respectively, demonstrated their similarity in morphology, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of phiKZ-like phages of the family Myoviridae designated in this manner according to the best studied phage phiKZ that belongs to the species of this group widely distributed in nature. Comparison of major polypeptide sizes of mature particles suggests the similarity of certain proteins in the phages examined. In OBP particles visualized with an electron microscope, an "inner body" was detected, which points to the specific DNA package intrinsic to phages of phiKZ group. In the meantime, phages Lul11 and OBP do not exhibit resemblance among themselves or with any of earlier described phiKZ-like phages in respect to other traits; particularly, they have no detectable DNA homology. Note that phage Lu11 of P. putida var. Manila exhibits very slight homology with phage Lin68 of the family of P. aeruginosa phiKZ-like phages detected only in blot hybridization. This suggests the possible involvement of these phages in interspecies recombination ("gene shuffling") between phages of various bacterial species. Results of partial sequencing of phage genomes confirmed the phylogenetic relatedness of phage OBP to phages of the phiKZ-supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup phiKZ composition. The results of the study are discussed in terms of the evolution of these phages.

Genome, Viral↗
PubMed1995

Large deletions in antigenic variants of the chlorella virus PBCV-1.

Four spontaneously derived, antigenic variants of chlorella virus PBCV-1 contained 27- to 37-kb deletions in the left end of the 330-kb genome. Two of the mutants, which were serologically identical, had deletions that began from map position 4.9 or 16 and ended at position 42.2 kb. In total, the two deleted regions encoded 28 putative functional open reading frames (ORFs); these deletions probably arose from homologous recombination. The other two mutants, which were serologically identical but distinct from the first two mutants, lacked the entire left terminal 37 kb of the PBCV-1 genome, including an identical 2.2-kb inverted terminal repeat region present at both ends of the wild-type genome. The deleted left end region was replaced by the transposition of an inverted 7.7- or 18.5-kb copy of the right end of the PBCV-1 genome. The region deleted in these two viruses encoded 26 single-copy ORFs, of which 23 were common to those deleted in the first two mutant viruses. The junctions of the deletions/transpositions probably arose from nonhomologous recombination. Taken together, the results indicate that 40.1 kb of single-copy DNA encoding 31 ORFs at the left end of the genome are unnecessary for PBCV-1 replication in Chlorella strain NC64A in the laboratory. The results also indicate that the size of the inverted terminal repeat region in this virus can be highly variable and that the PBCV-1 DNA packaging process tolerates large changes in genome size.

Antigenic Variation↗
PubMed2005

Solution structure, stability, and flexibility of Sso10a: a hyperthermophile coiled-coil DNA-binding protein.

Sso10a is one of a number of DNA-binding proteins from the hyperthermophile Sulfolobus solfataricus that has been associated with DNA packaging and chromatin regulation. Sequence analysis indicates that it is a member of a conserved group of archaeal transcription regulators (COG3432). We have determined the solution structure of Sso10a and show that it is a homodimer of winged-helix DNA-binding domains. The dimer interface consists of an extended antiparallel coiled coil, with the globular DNA-binding domains positioned at opposite ends of a solvent-exposed coiled-coil rod. NMR structure refinement of the elongated structure benefited not only from the inclusion of residual dipolar couplings from partially aligned samples but also the influence of anisotropic rotational diffusion on heteronuclear relaxation. An analysis of backbone mobility using (15)N relaxation rates indicated that the overall tertiary and quaternary structure is largely inflexible on the nanosecond to picosecond time scale. Amide hydrogen exchange data demonstrated that the most stable region of the protein extends from the core of the winged helices into the coiled coil. The positions of the globular heads relative to the coiled coil in solution deviate only slightly from that observed in a crystal structure. The most significant difference between the solution and crystal structures occurs in the putative DNA-binding helix-turn-helix (HTH) motif. This is the region of lowest stability in solution and a point of protein-protein contact in the crystal. Alternative conformations of the HTH motif may permit adjustment of the structure for optimal DNA binding.

Amino Acid Sequence↗
PubMed2004

Detection of equine herpesvirus 3 in equine skin lesions by polymerase chain reaction.

During a recent breeding season, ulcerative, pustular skin lesions were observed on the external genitalia of 2 mares and 1 stallion within a small herd. Based on the location and description of the skin lesions plus the clinical history, equine coital exanthema, caused by equine herpesvirus 3 (EHV3), was the primary differential diagnosis. Scrapings of skin lesions from the perineum of 2 mares were submitted for diagnostic evaluation. Virus isolation was attempted by inoculation of several cell lines of equine origin, but no cytopathic agent was detected. The skin scrapings were processed for DNA extraction, and polymerase chain reaction (PCR) amplification was performed for herpesvirus DNA polymerase and DNA-packaging protein (terminase) genes using nested, degenerate primers targeted to conserved regions of the herpesvirus genome. Products of the expected sizes were generated for both assays, and subsequent nucleotide sequencing of the amplification products established that EHV3 had been detected in DNA extracted from the skin lesions. Detection of EHV3 was confirmed using an EHV3-specific PCR assay targeted to the gC gene. Using the novel EHV3 nucleotide sequence identified in this report, a sensitive and specific PCR assay targeted to the highly conserved DNA polymerase gene was developed.

Animals↗
PubMed2004

Compositional regulation of poly(lysine-g-(lactide-b-ethylene glycol))-DNA complexation and stability.

The search for an effective nonviral gene therapy vector has revealed several significant hurdles, such as transient expression and cytotoxicity, that impede the success of these systems. A terpolymer of poly(lysine-g-(lactide-b-ethylene glycol)) [pK-g-p(LL-b-EG)] has been developed, which is capable of stably packaging DNA with significantly less polycation than unmodified polylysine (pK) systems. A comparison of unmodified pK (DP=14, M(w)=2930) to pK grafted with p(LL-b-EG) (M(w tot)=7500) showed that the minimum amine/phosphate ratio (N:P(min)) needed for complete DNA condensation was reduced by 50%. However, when the molecular weight of pK was reduced (DP=4, M(w)=838), there was evidence of terpolymer interference with DNA condensation. Increasing the number of grafted p(LL-b-EG) chains produced a similar result of incomplete DNA condensation. All terpolymer formulations produced complexes with DNA that had greater resistance to salt-induced dissociation and short-term exchange with excess DNA. Terpolymer-DNA complexes exhibited approximately zero-order plasmid release profiles over a period of 6 weeks. The rate of release was dependent on the complex N:P ratio as well as the molecular weights of pLL and pK. The ability to use terpolymer composition to control complex stability and controlled release can provide a means for system optimization for sustained expression profiles of exogenous DNA.

DNA↗
PubMed2004

The prophages of Lactobacillus johnsonii NCC 533: comparative genomics and transcription analysis.

Two non-inducible, but apparently complete prophages were identified in the genome of the sequenced Lactobacillus johnsonii strain NCC 533. The 38- and 40-kb-long prophages Lj928 and Lj965 represent distinct lineages of Sfi11-like pac-site Siphoviridae unrelated at the DNA sequence level. The deduced structural proteins from Lj928 demonstrated aa sequence identity with Lactococcus lactis phage TP901-1, while Lj965 shared sequence links with Streptococcus thermophilus phage O1205. With the exception of tRNA genes, inserted between DNA replication and DNA packaging genes, the transcription of the prophage was restricted to the genome segments near both attachment sites. Transcribed genes unrelated to phage functions were inserted between the phage repressor and integrase genes; one group of genes shared sequence relatedness with a mobile DNA element in Staphylococcus aureus. A short, but highly transcribed region was located between the phage lysin and right attachment site; it lacked a protein-encoding function in one prophage.

Attachment Sites, Microbiological↗
PubMed1976

Prokaryotic DNA in nucleoid structure.

Recent studies of the structure of the bacterial nucleoid are reviewed. In the past 4 to 5 years results of electron microscopic and physical-chemical investigations of the isolated bacterial nucleoids have greatly advanced our understanding of this comparatively simple chromosome. Evidence for both long-range and short-range conformational organization of the packaged DNA has emerged, and preliminary characterization of the molecular interactions organizing this structure has been accomplished.

Amino Acids↗
PubMed1994

Molecular characterization of lactococcal bacteriophage Tuc2009 and identification and analysis of genes encoding lysin, a putative holin, and two structural proteins.

Bacteriophage Tuc2009 is a temperate bacteriophage with a small isometric head and is isolated from Lactococcus lactis subsp. cremoris UC509. The phage genome is packaged by a headful mechanism, giving rise to circularly permuted molecules with terminal redundancy. The unit genome size is approximately 39 kb. A map of the phage genome on which several determinants could be localized was constructed: pac, the site of initiation of DNA packaging; lys (1,287 bp), specifying the phage lysin; S (267 bp), specifying a putative holin; and mp1 (522 bp) and mp2 (498 bp), each specifying one of the phage's structural proteins. lys, S, mp1, and mp2 were further characterized. lys and S are partially overlapping and appear to be part of one operon. The lysin shows homology to the lysins of the Streptococcus pneumoniae phages Cp-9, Cp-1, and Cp-7. The putative holin, which is thought to be involved in the release of lysin from the cytoplasm, contains two strongly hydrophobic presumptive transmembrane domains and a highly charged C-terminal domain.

Amino Acid Sequence↗
PubMed1988

The bacteriophage lambda cohesive end site: isolation of spacing/substitution mutations that result in dependence on Escherichia coli integration host factor.

Substitution, insertion and deletion mutations have been constructed at the XmnI restriction site in cos lambda. The XmnI site is located between cosB, the site where terminase binds lambda DNA; and cosN, the site where terminase introduces staggered nicks to generate cohesive ends. Substitution mutations and deletion of a base pair (a -1 change) do not obviously affect lambda growth and DNA packaging. Changes of -2, +2 and -3 render lambda unable to grow on host cells lacking integration host factor (IHF). The -3 mutant has a reduced burst size in IHF+ cells, due to a defect in the initiation of packaging. A -7 deletion mutation is lethal. Models for the basis of these mutational effects are discussed.

Bacterial Proteins↗
PubMed1998

Regulation of gene expression at the beginning of mammalian development and the TEAD family of transcription factors.

In mouse development, transcription is first detected in late 1-cell embryos, but translation of newly synthesized transcripts does not begin until the 2-cell stage. Thus, the onset of zygotic gene expression (ZGE) is regulated at the level of both transcription and translation. Chromatin-mediated repression is established after formation of a 2-cell embryo, concurrent with the developmental acquisition of enhancer function. The most effective enhancer in cleavage stage mouse embryos depends on DNA binding sites for TEF-1, the prototype for a family of transcription factors that share the same TEA DNA binding domain. Mice contain at least four, and perhaps five, genes with the same TEA DNA binding domain (mTEAD genes). Since mTEAD-2 is the only one expressed during the first 7 days of mouse development, it is most likely responsible for the TEAD transcription factor activity that first appears at the beginning of ZGE. All four mTEAD genes are expressed at later embryonic stages and in adult tissues; virtually every tissue expresses at least one family member, consistent with a critical role for TEAD proteins in either cell proliferation or differentiation. The 72-amino acid TEA DNA binding domains in mTEAD-2, 3, and 4 are approximately 99% homologous to the same domain in mTEAD-1, and all four proteins bind specifically to the same DNA sequences in vitro with a Kd value of 16-38 nM DNA. Since TEAD proteins appear to be involved in both activation and repression of different genes and do not appear to be functionally redundant, differential activity of TEAD proteins must result either from association with other proteins or from differential sensitivity to chromatin-packaged DNA binding sites.

Amino Acid Sequence↗
PubMed1986

In vitro encapsidation of plasmid DNA into human adenovirus empty capsids.

Plasmid DNA, added to extracts of human adenovirus type 3-infected HeLa cells, binds to empty viral capsids and can be purified using cesium density gradient centrifugation. The fraction of DNA bound depends on the amount of DNA added to the extract, and the capsid partially protects the bound DNA from digestion by DNase I. This capsid binding of plasmid DNA does not require the presence of the adenovirus DNA packaging sequence. However, the presence of the adenovirus packaging sequence in the plasmid results in better protection of the bound plasmid molecule from cellular nucleases.

Adenoviruses, Human↗
PubMed2002

Biophysical and transfection studies of the diC(14)-amidine/DNA complex.

Liposomes of the synthetic cationic lipid, N-t-butyl-N'-tetradecylamino-propionamidine (diC(14)-amidine), efficiently ports DNA into mammalian cells in the absence of other (neutral) lipids. The compositional simplicity of this transfection mix makes it attractive from a formulation perspective. We have used low- and wide-angle x-ray diffraction and polarized light microscopy to characterize the thermotropic phase behavior and microstructure of diC(14)-amidine and of the lipid/DNA (circular plasmid, 5.4 kb) complex with a view to understanding the structure of the complex and its role in transfection. Upon heating, the lipid in buffer undergoes a lamellar crystalline (L(c), d(001) = 41.7 A)-to-lamellar liquid crystal (L(c)(alpha), d(001) depends on hydration and T) transition at approximately 40 degrees C. Sonicated lipid vesicles with a reported transition temperature of approximately 23 degrees C complex with DNA. Complex formation is complete at a DNA/lipid mole ratio (rho) of 0.8. Adding DNA to the lipid causes d(001) of the multilayered complex to drop from 52 to 49 A as rho rises from 0.03 to 1.64. The minimal DNA-DNA duplex separation observed is 26 A, consistent with the close packing of B-DNA. Lipid bilayers in the complex undergo a lamellar gel (L(c)(beta))-to-L(c)(alpha) (superscript c refers to complex) transition at approximately 23 degrees C. Transfection efficiency was maximized at rho = 0.4. The structure and transfection data combined suggest that densely packaged DNA in a net positively charged complex is essential for transfection.

3T3 Cells↗
PubMed2001

Structure determination of the head-tail connector of bacteriophage phi29.

The head-tail connector of bacteriophage phi29 is composed of 12 36 kDa subunits with 12-fold symmetry. It is the central component of a rotary motor that packages the genomic dsDNA into preformed proheads. This motor consists of the head-tail connector, surrounded by a phi29-encoded, 174-base, RNA and a viral ATPase protein, both of which have fivefold symmetry in three-dimensional cryo-electron microscopy reconstructions. DNA is translocated into the prohead through a 36 A diameter pore in the center of the connector, where the DNA takes the role of a motor spindle. The helical nature of the DNA allows the rotational action of the connector to be transformed into a linear translation of the DNA. The crystal structure determination of connector crystals in space group C2 was initiated by molecular replacement, using an approximately 20 A resolution model derived from cryo-electron microscopy. The model phases were extended to 3.5 A resolution using 12-fold non-crystallographic symmetry averaging and solvent flattening. Although this electron density was not interpretable, the phases were adequate to locate the position of 24 mercury sites of a thimerosal heavy-atom derivative. The resultant 3.2 A single isomorphous replacement phases were improved using density modification, producing an interpretable electron-density map. The crystallographically refined structure was used as a molecular-replacement model to solve the structures of two other crystal forms of the connector molecule. One of these was in the same space group and almost isomorphous, whereas the other was in space group P2(1)2(1)2. The structural differences between the oligomeric connector molecules in the three crystal forms and between different monomers within each crystal show that the structure is relatively flexible, particularly in the protruding domain at the wide end of the connector. This domain probably acts as a bearing, allowing the connector to rotate within the pentagonal portal of the prohead during DNA packaging.

Bacillus Phages↗
PubMed1997

Cloning and characterization of bacteriophage-like DNA from Haemophilus somnus homologous to phages P2 and HP1.

In an attempt to identify and characterize components of a heme uptake system of Haemophilus somnus, an Escherichia coli cosmid library of H. somnus genomic DNA was screened for the ability to bind hemin (Hmb+). The Hmb+ phenotype was associated with a 7,814-bp HindIII fragment of H. somnus DNA that was subcloned and sequenced. Thirteen open reading frames (orfs) were identified, all transcribed in one direction, and transposon mutagenesis identified orf7 as the gene associated with the Hmb+ phenotype. Orf7 (178 amino acids) has extensive homology with the lysozymes of bacteriophages P-A2, P21, P22, PZA, phi-29, phi-vML3, T4, or HP1. The orf7 gene complemented the lytic function of the K gene of phage P2 and the R gene of phage lambda. A lysozyme assay using supernatants from whole-cell lysates of E. coli cultures harboring plasmid pRAP501 or pGCH2 (both of which express the orf7 gene product) exhibited significant levels of lysozyme activity. The orf6 gene upstream of orf7 has the dual start motif common to the holins encoded by lambdoid S genes, and the orf6 gene product has significant homology to the holins of phages HP1 and P21. When expressed from a tac promoter, the orf6 gene product caused immediate cell death without lysis, while cultures expressing the orf7 gene product grew at normal rates but lysed immediately after the addition of chloroform. Based on this data, we concluded that the Hmb+ phenotype was an artifact resulting from the expression of cloned lysis genes which were detrimental to the E. coli host. The DNA flanking the cloned lysis genes contains orfs that are similar to structural and DNA packaging genes of phage P2. Polyclonal antiserum against Orf2, which is homologous to the major capsid precursor protein (gpN) of phage P2, detected a 40,000-M(r) protein expressed from pRAP401 but did not detect Orf2 in H. somnus, lysates. The phage-like DNA was detected in the serum-susceptible preputial strains HS-124P and HS-127P but was absent from the serum-resistant preputial strains HS-20P and HS-22P. Elucidation of a potential role for this cryptic prophage in the H. somnus life cycle requires more study.

Amino Acid Sequence↗
PubMed1976

Analysis of gene function of bacteriophage phi 29 of Bacillus subtilis: identification of cistrons essential for viral assembly.

Restrictive infection of Bacillus subtilis by suppressor-sensitive (sus) mutants of phi 29 has been used to search for cistrons that function in viral assembly. The products of cistrons 7, 9, 10, and 16 are necessary for head morphogenesis. The neck upper collar protein P10 and the tail protein P9 must be present for DNA packaging to occur. The protein P7 must be present for phage-related particles to form. A prohead-like particle has been isolated during 16-restrictive infection. The particle is composed of the proteins Hd, P10, F, and P7. P16 must function for DNA-filled particles to accumulate. A DNA-containing particle produced in the absence of the cistron 11 product may be an intermediate in the phi 29 assembly pathway. The protein P13 interacts with P9 and P11 to form a stable DNA-filled particle. The products of cistrons 2 and 3 are essential for viral DNA synthesis, and in their absence virus-related particles are not detected.

Bacillus subtilis↗