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At least 19 recordsLinked to original sources
PubMed1978

Visualization of the intracellular development of bacteriophage lambda, with special reference to DNA packaging.

To reveal intermediates in lambda DNA packaging, infected cells were osmotically ruptured and the cell lysates were deposited on electron microscope grids by sedimentation through a sucrose/formalin cushion. A fixation procedure that crosslinks head-related structures to DNA allowed us to study successive stages in the process of head filling. Three types of head-related structures can be distinguished: (i) empty heads (petit lambda), less angular in outline than complete lambda heads; (ii) heads partially filled with DNA (partially filled heads), having a roundish outline; and (iii) particles tightly packed with DNA (full heads), having a hexagonal outline. DNA-head complexes were bound either at the terminal end of a DNA thread or at a point intermediate along the thread. The terminal complexes were more abundant. No head-related structures could be found in an induced lambda mutant lysogen blocked in the synthesis of petit lambda (amber in lambda gene E). One type of mutant blocked in DNA packaging (amber in gene A) produces empty heads and free tails, whereas another (amber in gene D) produces partially filled heads in addition. Our data suggest that a DNA-petit lambda complex may be an early intermediate in packaging and that the lambda DNA substrate can be a cohesive-ended concatemer or a concatemer with double-stranded cohesive site sequences.

Coliphages↗
PubMed1978

Model for DNA packaging into bacteriophage T4 heads.

The mechanism of DNA packaging into bacteriophage T4 heads in vivo was investigated by glucosylation of hydroxymethylcytosine residues in a conditionally glucose-deficient host. Cytoplasmic DNA associated with partially packaged ts49 heads can be fully glucosylated, whereas DNA already packaged into these heads is shown to be resistant to glucosylation. After temperature shift and completion of arrested packaging into the reversible temperature-sensitive ts49 head, the structure of the DNA in the mature ts49 phage was investigated by restriction enzyme digestion, autoradiography, and other techniques. Such mature DNA appears to be fully glucosylated along part of its length and nonglucosylated on the remainder. Its structure suggests that the DNA is run into the head linearly and unidirectionally from one mature end and that there is little sequence specificity in that portion of the T4 DNA which first enters the capsid. This technique should be useful in investigation of the three-dimensional structure of first- and last-packaged DNA within the head; preliminary studies including autoradiography of osmotically shocked phage suggest that the DNA which first enters the head is deposited toward the center of the capsid and that the end of the DNA which first enters the head exits first upon injection. In conjunction with studies of the structure of condensed DNA, the positions and functions of T4 capsid proteins in DNA packaging, and the order of T4 packaging functions [Earnshaw and Harrison, Nature (London) 268:598-602, 1977; Hsiao and Black, Proc. Natl. Acad. Sci. U.S.A. 74:3652-3656, 1977; Müller-Salamin et al., J. Virol. 24:121-134, 1977; Richards et al., J. Mol. Biol. 78:255-259, 1973], the features described above suggest the following model: the first DNA end is fixed to the proximal apex of the head at p20 and the DNA is then pumped into the head enzymatically by proteins (p20 + p17) which induce torsion in the DNA molecule.

Coliphages↗
PubMed1977

DNA packaging and the pathway of bacteriophage T4 head assembly.

A cold-sensitive mutation in the structural gene for a minor phage T4 capsid protein (p20) leads to formation of heads containing p20 and cleaved head proteins and empty of DNA. Such heads can be filled with DNA and converted to active phages in vivo uponshift to high temperature. It appears that p20 has two distinct roles in head assembly: first, in construction of the prehead shell (blocked by ts and am mutation) and, second,in DNA packaging (blocked by cs mutation). The latter function is closely associated with gene 17 product, previously known to be required for DNA packagaing. Temperature shift studies of cs-ts double mutants and other observations allow determination of phage function required for DNA packaging. Contrary to previous proposals, we find that T4 DNA packaging is not directly coupled to and can follow DNA synthesis, protein cleavage, prehead core removal, and gene 21-mediated cleavage-induced increase in head volume. Our evidence suggests that an altered head assembly pathway exists and that DNA packaging is probably initiated by DNA-capsid (p20) interaction.

Coliphages↗
PubMed1978

Symmetry mismatch and DNA packaging in large bacteriophages.

A model is presented for the mechanism of packaging double-stranded DNA into phage heads. The model is based on, and rationalizes, the mismatch in symmetry between the heads and tails of large bacteriophages. DNA movement is postulated to be mediated by a rotating protein structure at the tail-proximal vertex of the head.

Bacteriophages↗
PubMed1977

The problems of eukaryotic and prokaryotic DNA packaging and in vivo conformation posed by superhelix density heterogeneity.

Systems for gel electrophoresis in the presence of one of the intercalative unwinding ligands, ethidium or chloroquine, have been developed which permit the resolution of highly supercoiled closed circular DNA molecules differing by unit values of the topological winding number, alpha. All native closed circular DNAs examined, including the viral and intracellular forms of SV40 and polyoma DNA, bacterial plasmid DNAs, and the double stranded closed circular DNA genome of the marine bacteriophage, PM2, are more heterogeneous with respect to the number of superhelical turns present than are the thermal distributions observed in the limit products of the action of nicking-closing (N-C) enzyme on the respective DNAs. In the cases of SV40 and polyoma, where it has been shown that the supercoiling is a combined consequence of the binding of the four nucleosomal histones, H2a, H2b, H3 and H4, and the action of N-C enzyme, the breadth of the distributions within the form I DNAs poses specific problems since the work of other laboratories indicates that the number of nucleosomes on the respective minichromosomes falls within a narrow distribution of 21. If it is assumed that all nucleosomes have identical structures, and that the DNA within a nucleosome is not free to rotate, the native DNA would be anticipated to be less heterogeneous than the thermal equilibrium mixtures present in N-C enzyme relaxed SV40 and polyoma DNAs. The absolute number of superhelical turns (at 37 degrees C in 0.2 M NaCl) in virion polyoma DNA has been determined to be 26 +/- 1, which is the same value obtained for virion SV40 DNA. This is consistent with the observations that polyoma DNA has a higher molecular weight, a lower superhelix density, but the same number of nucleosomes as SV40 DNA. In addition, the distributions within the virion and intracellular form I DNAs of both SV40 and polyoma were found to be indistinguishable.Images

Bacteriophages↗
PubMed1979

Accumulation of bacteriophage T7 head-related particles in an Escherichia coli mutant.

Upon infection with bacteriophage T7, a newly isolated mutant strain of Escherichia coli, Y49, produces T7-specific macromolecules including DNA almost normally. However, concatemeric T7 progeny DNA molecules, synthesized in Y49 cells, are later cleaved abnormally, resulting in an accumulation of DNA molecules shorter in size than the T7 genome and a poor production of progeny phage (Y. Yamada, J. Silnutzer, and D. Nakada, J. Mol. Biol. 121:95-111, 1978). The abnormal cutting of concatemeric T7 DNA in Y49 cells is accompanied by a simultaneous accumulation of large amounts of two types of phage head-related particles, proheads and newly found "X particles." Lysates from normal T7 infection of parental cells also contain X particles, although to a lesser amount. Electron microscopic examination of phage head-related particles (i.e., proheads, X particles, and empty heads), gel electrophoretic analysis of proteins in these particles, and kinetic studies on the appearance and fate of these particles suggest that X particles are likely to be intermediary structures between proheads and phage heads probably derived from proheads during the process of T7 DNA packaging. Our data also suggest that empty heads are not precursors to phage heads but are derived from proheads as by-products probably due to an abortive attempt to package T7 DNA. The host mutation in Y49 strain appears to block a step of T7 DNA processing and packaging pathway after generation of X particles from proheads.

DNA, Viral↗
PubMed1978

New late gene, dar, involved in the replication of bacteriophage T4 DNA. II. Overproduction of DNA binding protein (gene 32 protein) and further characterization.

We have previously shown that the arrested DNA synthesis of mutant defective in T4 phage gene 59 can be reversed by a mutation in dar. In this paper, we have examined the effect of the dar mutation on the kinetics of gene 32 protein (DNA binding protein) synthesis, DNA packaging, progeny formation, and several other porcesses. Several lines of evidence are presented showing that the regulation of synthesis of gene 32 protein is abnormal in dar 1-infected cells. In these cells, gene 32 protein, an early protein, is also expressed late in the infectious cycle. Our data also indicate that the packaging og DNA into T4 phage heads is delayed in dar mutant-infected cells, and this in turn results in a 6- to 8-min delay in intracellular progeny formation, although the synthesis of late proteins appears to be normal, as shown by gel electrophoresis. We have also studied the phenotypes of the double mutant dar-amC5 (gene 59). The increased sensitivity to hydroxyurea caused by a mutation in the dar gene can be alleviated by a second mutation in gene 59, but an increased sensitivity to UV irradiation caused by a mutation in gene 59 cannot be alleviated by a second mutation in the dar gene. Therefore, the double mutant still exhibits abnormalities in the repair of UV lesions.

Coliphages↗
PubMed1977

Packaging recombinant DNA molecules into bacteriophage particles in vitro.

Recombinant phage genomes made in reactions with purified enzymes may be recovered directly by packaging into phage heads in vitro. The process is efficient and nonselective and offers containment in initial stages of handling recombinant DNA. Ligase [poly(deoxyribonucleotide):poly-(deoxyribonucleotide) ligase (AMP-forming), EC 6.5.1.1] reaction products can recombine with endogenous phage DNA during packaging, but UV-irradiation eliminates the biological activity of the endogenous DNA.

Coliphages↗
PubMed1978

Packaging of ColE1 DNA having a lambda phage cohesive end site.

The mechanism of lambda phage-mediated transduction of hybrid colicin E1 DNAs of various lengths was studied, and factors influencing the formation of these transducing particles were investigated. The results were as follows: 1. The presence of a cohesive end site of lambda phage (coslambda) on colicin E1 DNA was essential for packaging of the DNA. 2. Packaging of colicin E1 DNAs, which carry coslambda with molecular sizes corresponding to 68% of that of lambda phage DNA, was observed in the absence of all known recombination functions of E. coli K-12 and of lambda phage. 3. Hybrid colicin E1 DNAs having coslambda with molecular sizes corresponding to 28% of that of lambda phage DNA were packaged within lambda phage particles as trimers; hybrid DNAs with coslambda of 40 and 47% of the length of lambda phage DNA were packaged as dimers; and those with molecular sizes of 68% of that of lambda phage DNA were packaged mostly as monomers. These results demonstrated that two factors are essential for the packaging of DNAs within lambda phage particles; the presence of coslambda on the DNA molecule and an appropriate size of DNA.

Bacteriocin Plasmids↗
PubMed1978

Bacteriophage P22-mediated specialized transduction in Salmonella typhimurium: high frequency of aberrant prophage excision.

The temperate bacteriophage P22 mediates both generalized and specialized transduction in Salmonella typhimurium. Specialized transduction by phage P22 is different from, and less restricted than, the well characterized specialized transduction by phage lambda, due to differences in the phage DNA packaging mechanisms. Based on the properties of the DNA packaging mechanism of phage P22 a model for the generation of various types of specialized transducing particles is presented that suggests generation of substantial numbers of specialized transducing genomes which are heterogeneous but only some of which have terminally redundant ends. The primary attachment site, ataA, for phage P22 in S. typhimurium is located between the genes proA,B and supQ newD. (The newD gene is a substitute gene for the leuD gene, restoring leucine prototrophy of leuD mutant strains.) The proA,B and supQ newD genes are very closely linked and thus cotransducible by generalized transducing particles. Specialized transducing particles can carry either proA,B or supQ newD but not both simultaneously, and thus cannot give rise to cotransduction of the proA,B and supQ newD genes. This difference is used to calculate the frequency of generalized and specialized transducing particles from the observed cotransduction frequency in phage lysates. By this method, very high frequencies of supQ newD (10(-2)/PFU)- and proA,B (10(-3)/PFU)-specialized transducing particles were detected in lysates produced by induction of lysogenic strains. These transducing particles most of which would have been produced by independent aberrant excision events (which include in situ packaging), were of various types.

DNA, Viral↗
PubMed2024

Gene transfer agents: The ambiguous role of selfless viruses in genetic exchange and bacterial evolution.

Gene transfer agents (GTAs) are genetic elements derived from ancestral bacteriophages that have become domesticated by the host. GTAs are present in diverse prokaryotic organisms, where they can facilitate horizontal gene transfer under certain conditions. Unlike typical bacteriophages, GTAs do not exhibit any preference for the replication or transfer of the genes encoding them; instead, they exhibit a remarkable capacity to package chromosomal, and sometimes extrachromosomal, DNA into virus-like capsids and disseminate it to neighboring cells. Because GTAs resemble defective prophages, identification of novel GTAs is not trivial. The detection of candidates relies on the genetic similarity to known GTAs, which has been fruitful in α-proteobacterial lineages but challenging in more distant bacteria. Here we consider several fundamental questions: What is the true prevalence of GTAs in prokaryote genomes? Given there are high costs for GTA production, what advantage do GTAs provide to the bacterial host to justify their maintenance? How is the bacterial chromosome recognized and processed for inclusion in GTA particles? This article highlights the challenges in comprehensively understanding GTAs' prevalence, function and DNA packaging method. Going forward, broad study of atypical GTAs and use of ecologically relevant conditions are required to uncover their true impact on bacterial chromosome evolution.

Gene Transfer, Horizontal↗
PubMed1976

Studies on the maturation of the head of bacteriophage T4.

The presentation focuses on the structural rearrangements of the subunits and the processing of the various protein constituents which accompany the maturation events of the head of bacteriophage T4. The major features of the maturation steps of the head are the following: (a) the viral DNA is pulled into an empty head in a series of events; (b) cleavage of two core proteins, P22 (mol. mass = 31000), to small fragments and the internal protein IPIII (mol. mass = 23000) to IPIII (mol. mass = 21000) appears to be intimately linked to the DNA packaging event, whereas the cleavage of the major head protein of the viral coat, P23 (mol. mass = 55000), to P23 (mol. mass = 45000) precedes the DNA packaging event. Recently, we have obtained information about the mechanism by which the viral DNA is pulled into a preformed empty head. Our evidence suggests that the DNA becomes attached to the inside of the empty head and is subsequently collapsed in the interior by the so-called internal peptides. These are highly acidic and derived from a large precursor protein by cleavage.

Coliphages↗
PubMed2010

Gene therapy of mdx mice with large truncated dystrophins generated by recombination using rAAV6.

Recombinant adeno-associated viral (rAAV) vector-mediated gene transfer represents a promising approach for many diseases. However, the applicability of rAAV vectors has long been hindered by the small (~4.8 kb) DNA packaging capacity. This limitation can hamper the packaging and delivery of critical regulatory elements and/or larger coding sequences, such as the ~14-kb dystrophin complementary DNA (cDNA) that is of interest for gene therapy of Duchenne muscular dystrophy (DMD). Here, we have demonstrated reconstitution of an expression cassette (7.3 kb) encoding a highly functional "minidystrophin" protein (ΔH2-R19, 222 kd) in vivo following intravascular co-delivery of two independent rAAV6 vectors sharing a central homologous recombinogenic region of 372 nucleotides. Similar to previously reported trans-splicing approaches, one rAAV vector provides the promoter with the ~1/2 initial portion of minidystrophin, while the second vector provides the remaining minidystrophin cDNA followed by the polyadenylation signal. Significantly, administering a modest dose [2 × 10(12) vector genomes (vg)] of the two minidystrophin-encoding rAAV vectors to dystrophic mice elicited an improvement of physiological performance indicative of prevention or amelioration of the disease state. These studies provide evidence that functional dystrophin transgenes larger than that typically carried by a single rAAV genome can be reconstituted in vivo by homologous recombination (HR) following intravascular co-delivery with rAAV6.

Animals↗
PubMed1979

Gene 2 protein of bacteriophage T7: purification and requirement for packaging of T7 DNA in vitro.

The gene 2 protein of bacteriophage T7 is required for a late stage of T7 DNA replication because T7 gene 2 mutants fail to form normal concatemeric structures during the processing of newly synthesized T7 DNA. Extracts of gene 2 mutant phage-infected cells are unable to package T7 DNA into phage heads to form viable phage, as determined by an in vitro packaging assay for T7 DNA. Packaging activity can be stimulated greater than 100-fold in mutant extracts by the addition of extract prepared from cells infected with phage carrying a wild-type T7 gene 2, thus providing a complementation assay for the gene 2 protein. With this assay, the gene 2 protein has been purified to approximately 50% homogeneity. Purified preparations of the protein inhibit the activity of Escherichia coli RNA polymerase but have little effect on the activity of T7 RNA polymerase but have little effect on the activity of T7 RNA polymerase. The requirement for the gene 2 protein during T7 DNA replication may involve inactivation of E. coli RNA polymerase because the antibiotic rifampicin, a specific inhibitor of E. coli RNA polymerase, can substitute for the gene 2 protein in the in vitro packaging assay.

DNA, Viral↗
PubMed1977

In vitro packaging of UV radiation-damaged DNA from bacteriophage T7.

When DNA from bacteriophage T7 is irradiated with UV light, the efficiency with which this DNA can be packaged in vitro to form viable phage particles is reduced. A comparison between irradiated DNA packaged in vitro and irradiated intact phage particles shows almost identical survival as a function of UV dose when Escherichia coli wild type or polA or uvrA mutants are used as the host. Although uvrA mutants perform less host cell reactivation, the polA strains are identical with wild type in their ability to support the growth of irradiated T7 phage or irradiated T7 DNA packaged in vitro into complete phage. An examination of in vitro repair performed by extracts of T7-infected E.coli suggests that T7 DNA polymerase may substitute for E. coli DNA polymerase I in the resynthesis step of excision repair. Also tested was the ability of a similar in vitro repair system that used extracts from uninfected cells to restore biological activity of irradiated DNA. When T7 DNA damaged by UV irradiation was treated with an endonuclease from Micrococcus luteus that is specific for pyrimidine dimers and then was incubated with an extract of uninfected E. coli capable of removing pyrimidine dimers and restoring the DNA of its original (whole genome size) molecular weight, this DNA showed a higher packaging efficiency than untreated DNA, thus demonstrating that the in vitro repair system partially restored the biological activity of UV-damaged DNA.

Cell-Free System↗
PubMed2025

Trypanosomatid histones: the building blocks of the epigenetic code of highly divergent eukaryotes.

Histones play a fundamental role in eukaryotic organisms not only as scaffolding proteins in DNA packaging but also in regulating gene expression. They constitute the protein reel around which DNA wraps forming nucleosomes. This initial packing gives rise to the chromatin fiber which is next folded into three-dimensional arrangements. Additionally, histones have expanded their functions through the emergence of histone variants which have specialized purposes and can deeply affect chromatin organization and dynamics. Moreover, both canonical histones and histone variants comprise the building blocks of the histone code by being targets of different post-translational modifications (PTMs) that occur in a highly regulated manner both in place and time. Most of the above-mentioned about chromatin organization is conserved among eukaryotes. However, trypanosomatid histones have many peculiarities that entail a special description. In this review, we compile the current knowledge of canonical core histones, histone variants, and their PTMs in trypanosomatids. We highlight the similarities and differences between histone variants and their canonical counterparts in trypanosomatids, and we compare them with those from model organisms. Finally, we discuss the crosstalk between different histone marks and their genomic distribution underlying the uniqueness of trypanosomatids.

Histones↗