Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “DNA packaging”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 451 records · Page 25Linked to original sources
PubMed1992

Molecular analysis of the Bacillus subtilis bacteriophage SPP1 region encompassing genes 1 to 6. The products of gene 1 and gene 2 are required for pac cleavage.

Packaging of Bacillus subtilis phage SPP1 DNA into viral capsids is initiated at a specific DNA site termed pac. Using an in vivo assay for pac cleavage, we show that initiation of DNA synthesis and DNA packaging are uncoupled. When the DNA products of pac cleavage were analyzed, we could detect the pac end that was destined to be packaged, but we failed to detect the other end of the cleavage reaction. SPP1 conditional lethal mutants, which map adjacent to pac, were analyzed with our assay. This revealed that the products of gene 1 and gene 2 are essential for pac cleavage. SPP1 mutants that are affected in the genes necessary for viral capsid formation (gene 41) or involved in headful cleavage (gene 6) remain proficient in pac site cleavage. Analysis of the nucleotide sequence (2.769 x 10(3) base-pairs) of the region of the genes required for pac cleavage revealed five presumptive genes. We have assigned gene 1 and gene 2 to two of these open reading frames (orf), giving the gene order gene 1-gene 2-orf 3-orf 4-orf 5. The direction of transcription of the gene 1 to orf 5 operon and the length of the mRNAs was determined. We have identified, upstream from gene 1, the major transcriptional start point (P1). Transcription originating from P1 requires a phage-encoded factor for activity. The organization of gene 1 and gene 2 of SPP1 resembles the organization of genes in the pac/cos region of different Escherichia coli double-stranded DNA phages. We propose that the conserved gene organization is representative of the packaging machinery of a primordial packaging system.

Amino Acid Sequence↗
PubMed1998

Inter-RNA interaction of phage phi29 pRNA to form a hexameric complex for viral DNA transportation.

Ds-DNA viruses package their DNA into a preformed protein shell (procapsid) during maturation. Bacteriophage phi29 requires an RNA (pRNA) to package its genomic DNA into the procapsid. We report here that the pRNA upper and lower loops are involved in RNA/RNA interactions. Mutation in only one loop results in inactive pRNAs. However, mixing of two, three and six inactive mutant pRNAs restores DNA packaging activity as long as an interlocking hexameric ring can be predicted to form by base pairing of the mutated loops in separate RNA molecules. The stoichiometry of pRNA for the packaging of one viral DNA genome is six. Homogeneous pRNA purified from a single band in denaturing gels showed six bands when rerun in native gels. These results suggest that six pRNAs form a hexameric ring by the intermolecular interaction of two RNA loops to serve as part of the DNA transportation machinery.

Bacillus Phages↗
PubMed2005

Translocation of nicked but not gapped DNA by the packaging motor of bacteriophage phi29.

The biomolecular mechanism that the double-stranded DNA viruses employ to insert and package their genomic DNA into a preformed procapsid is still elusive. To better characterize this process, we investigated packaging of bacteriophage phi29 DNA with structural alterations. phi29 DNA was modified in vitro by nicking at random sites with DNase I, or at specific sites with nicking enzyme N.BbvC IA. Single-strand gaps were created by expanding site-specific nicks with T4 DNA polymerase. Packaging of modified phi29 DNA was studied in a completely defined in vitro system. Nicked DNA was packaged at full genome length and with the same efficiency as untreated DNA. Nicks were not repaired during packaging. Gapped DNA was packaged only as a fragment corresponding to the DNA between the genome terminus and gap. Thus the phi29 DNA packaging machinery tolerated nicks, but stopped at gaps. The packaging motor did not require a nick-free DNA backbone, but the presence of both DNA strands, for uninterrupted packaging.

Bacillus Phages↗
PubMed1999

Single-event analysis of the packaging of bacteriophage T7 DNA concatemers in vitro.

Bacteriophage T7 packages its double-stranded DNA genome in a preformed protein capsid (procapsid). The DNA substrate for packaging is a head-to-tail multimer (concatemer) of the mature 40-kilobase pair genome. Mature genomes are cleaved from the concatemer during packaging. In the present study, fluorescence microscopy is used to observe T7 concatemeric DNA packaging at the level of a single (microscopic) event. Metabolism-dependent cleavage to form several fragments is observed when T7 concatemers are incubated in an extract of T7-infected Escherichia coli (in vitro). The following observations indicate that the fragment-producing metabolic event is DNA packaging: 1) most fragments have the hydrodynamic radius (R(H)) of bacteriophage particles (+/-3%) when R(H) is determined by analysis of Brownian motion; 2) the fragments also have the fluorescence intensity (I) of bacteriophage particles (+/-6%); 3) as a fragment forms, a progressive decrease occurs in both R(H) and I. The decrease in I follows a pattern expected for intracapsid steric restriction of 4',6-diamidino-2-phenylindole (DAPI) binding to packaged DNA. The observed in vitro packaging of a concatemer's genomes always occurs in a synchronized cluster. Therefore, the following hypothesis is proposed: the observed packaging of concatemer-associated T7 genomes is cooperative.

Bacteriophage T7↗
PubMed1985

In vitro packaging of plasmid DNA oligomers by Salmonella phage P22: independence of the pac site, and evidence for the termination cut in vitro.

In vitro packaging experiments with phage P22 using artificially ligated plasmid concatemers have shown that the pac site is not necessary for DNA packaging although in vivo this initiation signal is indispensable. This indicates that the phage-coded protein gp3 also executes other important functions during phage maturation in addition to the recognition of pac, or that its site specificity is lost in vitro. It has been shown previously that gp3 is necessary for in vitro packaging. Further, it was demonstrated that DNA which is only 74% of headful size cannot be packaged. Oversized DNA, however, is cut in vitro to unit length.

DNA Transposable Elements↗
PubMed1997

In vivo packaging of bacteriophage lambda monomeric chromosomes.

There is an apparent paradox between the reported requirements for lambda DNA packaging in vivo and in vitro. In vivo, DNA concatemers are required for packaging. On the other hand, in vitro, packaging extracts can encapsidate either linear or circular monomeric lambda DNA. Perhaps cellular nucleases restrict the in vivo ability of monomers to package by degrading a free double chain end present as an intermediate in the packaging reaction. Consistent with this hypothesis, enhanced packaging of monomers was found in an ExoV- host. No additional enhancement was noted in a host also mutant for sbcB and sbcC. We isolated a mutant phage for which in vivo packaging of monomeric lambda chromosomes is increased about 10(3)-fold. The responsible mutation (plm1 for packages lambda monomers) was mapped to cro, sequenced, and found to cause a change from Ala29 to Ser in the alpha3 helix of Cro's DNA binding domain. Density transfer experiments showed that packaging of both plm1 and wild-type lambda was aided by allowing some DNA synthesis. However, the packaged chromosomes had not themselves undergone a full round of replication and therefore were not part of a canonical concatemer made by replication. Other tests showed that packaged phage had not been part of concatemers made by recombination or by annealing at cos. Our results with wild-type lambda also favor models in which two cos sites are needed for packaging, but these sites need not be in cis. In lambda plm1, replication intermediates may serve as substrates for encapsidation.

Bacteriophage lambda↗
PubMed1990

Production of lambda-phi 29 phage chimeras.

Proheads of bacteriophage lambda which carry the connector of phage phi 29 instead of that of lambda have been produced in vitro. These hybrid proheads have a structure similar to that of normal lambda proheads. Furthermore, the chimeric proheads can package both lambda and phi 29 DNA. These data show that the connector domains involved in both head assembly and DNA packaging are functionally similar. The DNA-containing lambda-phi 29 proheads can be complemented in vitro with phi 29 tails to yield infective particles capable of DNA transfer.

Bacteriophage lambda↗
PubMed1995

Self-fitting and self-modifying properties of the B-DNA molecule.

X-ray structure analysis of oligonucleotides shows that self-fitting of B-DNA molecules by groove-backbone interaction can trigger modification of the secondary structure of the double helix in a sequence-dependent manner, leading to a "pre-melted" transition state. This work reveals that some sequences respond to the DNA-DNA intermolecular interactions by forming rearranged H-bonding schemes which stabilize the transition states. This study suggests that the close and specific approach of DNA segments occurring in genome packaging, DNA looping, synapsis formation or supercoiling can contribute directly to the secondary structure changes needed for DNA processing.

Base Composition↗
PubMed1993

Atomic force microscopy of mammalian sperm chromatin.

We have used the atomic force microscope (AFM) to image the surfaces of intact bull, mouse and rat sperm chromatin and partially decondensed mouse sperm chromatin attached to coverglass. High resolution AFM imaging was performed in air and saline using uncoated, unfixed and unstained chromatin. Images of the surfaces of intact chromatin from all three species and of an AFM-dissected bull sperm nucleus have revealed that the DNA is organized into large nodular subunits, which vary in diameter between 50 and 100 nm. Other images of partially decondensed mouse sperm chromatin show that the nodules are arranged along thick fibers that loop out away from the nucleus upon decondensation. These fibers appear to stretch or unravel, generating narrow smooth fibers with thicknesses equivalent to a single DNA-protamine complex. High resolution AFM images of the nodular subunits suggest that they are discrete, ellipsoid-shaped DNA packaging units possibly only one level of packaging above the protamine-DNA complex.

Animals↗
PubMed2003

Gene packaging with lipids, peptides and viruses inhibits transfection by electroporation in vitro.

To develop improved methods of gene delivery, packaging DNA in chemical or viral vectors could increase electroporation-mediated transfection. To test this hypothesis, electroporation was applied to DU145 prostate cancer cells incubated with green fluorescent protein-encoded DNA plasmid either naked or packaged with cationic lipid (Lipofectin), polycationic peptide (salmon protamine) or retroviral vectors (Moloney murine leukemia viruses) and then assayed for gene expression and cell viability. Cationic lipid or electroporation alone each significantly increased transfection, but their combination was less effective. Addition of protamine peptide during electroporation was also less effective than electroporation alone. The combination of retroviral vectors and electroporation transfected fewer cells than retrovirus alone. We conclude that the combination of electroporation with chemical or viral vectors does not improve gene transfection in vitro.

Animals↗
PubMed1989

DNA curvature in front of the human mitochondrial L-strand replication origin with specific protein binding.

DNA bending has been suggested to play a role in the regulation of gene expression, initiation of DNA-replication, site specific recombination, and DNA packaging. In the human mitochondrial DNA we have found a DNA curvature structure within the 3'-region of ther URF2 sequence in front of the L-strand origin of replication. This structure interacts specifically with a protein factor isolated from mitochondria. Based on the localization of this DNA curvature structure and the known function of such structures the data suggest a model in which this DNA signal sequence and its specific protein binding is involved in the regulatory initiation event of L-strand replication.

Base Sequence↗
PubMed2006

Post-meiotic shifts in HSPA2/HSP70.2 chaperone activity during mouse spermatogenesis.

HSPA2 (formerly HSP70.2) is a testis-specific member of the HSP70 family known to play a critical role in the completion of meiosis during male germ cell differentiation. Although abundantly present in post-meiotic cells, its function during spermiogenesis remained obscure. Here, using a global proteomic approach to identify genome-organizing proteins in condensing spermatids, we discovered an unexpected role for HSPA2, which acquires new functions and becomes tightly associated with major spermatid DNA-packaging proteins, transition proteins 1 and 2. Hence, HSPA2 is identified here as the first transition protein chaperone, and these data shed a new light on the yet totally unknown process of genome-condensing structure assembly in spermatids.

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↗
PubMed2005

Contribution of hydrophobicity to thermodynamics of ligand-DNA binding and DNA collapse.

The importance of understanding the dynamics of DNA condensation is inherent in the biological significance of DNA packaging in cell nuclei, as well as for gene therapy applications. Specifically, the role of ligand hydrophobicity in DNA condensation has received little attention. Considering that only multivalent cations can induce true DNA condensation, previous studies exploring monovalent lipids have been unable to address this question. In this study we have elucidated the contribution of the hydrophobic effect to multivalent cation- and cationic lipid-DNA binding and DNA collapse by studying the thermodynamics of cobalt hexammine-, spermine-, and lipospermine-plasmid DNA binding at different temperatures. Comparable molar heat capacity changes (DeltaC(p)) associated with cobalt hexammine- and spermine-DNA binding (-23.39 cal/mol K and -17.98 cal/mol K, respectively) suggest that upon binding to DNA, there are insignificant changes in the hydration state of the methylene groups in spermine. In contrast, the acyl chain contribution to the DeltaC(p) of lipospermine-DNA binding (DeltaC(p ) = DeltaC(p lipospermine) - DeltaC(p spermine)) is significant (-220.94 cal/mol K). Although lipopermine induces DNA ordering into "tubular" suprastructures, such structures do not assume toroidal dimensions as observed for spermine-DNA complexes. We postulate that a steric barrier posed by the acyl chains in lipospermine precludes packaging of DNA into dimensions comparable to those found in nature.

Binding Sites↗
PubMed2000

Visualization of the maturation transition in bacteriophage P22 by electron cryomicroscopy.

Large-scale conformational transitions are involved in the life-cycle of many types of virus. The dsDNA phages, herpesviruses, and adenoviruses must undergo a maturation transition in the course of DNA packaging to convert a scaffolding-containing precursor capsid to the DNA-containing mature virion. This conformational transition converts the procapsid, which is smaller, rounder, and displays a distinctive skewing of the hexameric capsomeres, to the mature virion, which is larger and more angular, with regular hexons. We have used electron cryomicroscopy and image reconstruction to obtain 15 A structures of both bacteriophage P22 procapsids and mature phage. The maturation transition from the procapsid to the phage results in several changes in both the conformations of the individual coat protein subunits and the interactions between neighboring subunits. The most extensive conformational transformation among these is the outward movement of the trimer clusters present at all strict and local 3-fold axes on the procapsid inner surface. As the trimer tips are the sites of scaffolding binding, this helps to explain the role of scaffolding protein in regulating assembly and maturation. We also observe DNA within the capsid packed in a manner consistent with the spool model. These structures allow us to suggest how the binding interactions of scaffolding and DNA with the coat shell may act to control the packaging of the DNA into the expanding procapsids.

Bacteriophage P22↗
PubMed2003

Gene cloning, purification, and stoichiometry quantification of phi29 anti-receptor gp12 with potential use as special ligand for gene delivery.

Bacterial virus phi29 is the most efficient in vitro DNA packaging system, with which up to 90% of the added DNA can be packaged into purified recombinant procapsid in vitro. The findings that phi29 virions can be assembled with the exclusive use of cloned gene products have bred a thought that phi29 has a potential to be a gene delivery vector since it is a nonpathogenic virus. gp12 of bacterial virus phi29 has been reported to be the anti-receptor that is responsible for binding the virus particle to the host cell. We cloned the gene coding gp12, overexpressed it in Escherichia coli, and purified the gene product to study the properties and functions of gp12 in virus assembly. According to SDS PloyAcrylamide Gel Electrophoresis (SDS-PAGE) analysis and N-terminal sequencing, recombinant gp12 isolated from E. coli had a molecular mass of 80 kDa, and 24 amino acids at N-terminal were cleaved after expression. The purified recombinant gp12 was incorporated into phi29 particles and converted the gp12-lacking assembly intermediates of phi29 into infectious virions in vitro. This purified protein gp12 was able to compete with infectious phi29 virions for binding to the host cell, thus inhibiting the infection by phi29. Scanning Transmission Electron Microscopy (STEM) analysis and sedimentation studies revealed that recombinant gp12 products were assembled into biologically active dimers. Analysis of the dose-response curve showed that 12 dimeric gp12 complexes were assembled onto viral particles and that each virion contained 24 copies of gp12 molecules. The results provide a basis for future research into bacteriophage-host interaction by modifying the anti-receptor protein. The ultimate goal is to re-target the bacteriophage to new host cells for the purpose of gene delivery.

Amino Acid Sequence↗
PubMed2002

The clinical value of sperm nuclear DNA assessment.

Traditional semen analysis is essential for the diagnosis of male infertility. A number of studies over the past decade have reported that a significant contributing factor to male fertility that is not revealed as part of semen analysis is sperm DNA, specifically its composition and organization. Exogenous and endogenous factors can cause damage to sperm DNA. For example, topoisomerase II activity, which is necessary for sperm DNA packaging, can adversely influence the competence of sperm DNA if the activity of the enzyme is abnormal. Germ cell apoptosis can be induced by oxygen radicals produced from environmental (for example cigarette smoke) or testicular (for example localized ischaemia) sources. Several assays have been developed that are useful for assessing sperm DNA composition and organization. To date, each of these assays has revealed that when sperm DNA has been damaged or packaged improperly there is a concomitant and often significant decline in male fertility.

Cell Nucleus↗
PubMed1991

Regulation of filamentous bacteriophage length by modification of electrostatic interactions between coat protein and DNA.

Bacteriophage fd gene VIII, which encodes the major capsid protein, was mutated to convert the serine residue at position 47 to a lysine residue (S47K), thereby increasing the number of positively charged residues in the C-terminal region of the protein from four to five. The S47K coat protein underwent correct membrane insertion and processing but could not encapsidate the viral DNA, nor was it incorporated detectably with wild-type coat proteins into hybrid bacteriophage particles. However, hybrid virions could be constructed from the S47K coat protein and a second mutant coat protein, K48Q, the latter containing only three lysine residues in its C-terminal region. K48Q phage particles are approximately 35% longer than wild-type. Introducing the S47K protein shortened these particles, the S47K/K48Q hybrids exhibiting a range of lengths between those of K48Q and wild-type. These results indicate that filamentous bacteriophage length (and the DNA packaging underlying it) are regulated by unusually flexible electrostatic interactions between the C-terminal domain of the coat protein and the DNA. They strongly suggest that wild-type bacteriophage fd makes optimal use of the minimum number of coat protein subunits to package the DNA compactly.

Amino Acid Sequence↗