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PubMed1994

Comparative analysis of DNA mutations in lacI transgenic mice with age.

Transgenic mice have been developed recently that contain copies of the well-defined mutagenesis reporter gene, lacI, in an integrated bacteriophage-based shuttle vector. The lacI gene, which is present in all cells of the mouse, can be excised specifically from isolated genomic DNA and efficiently packaged into bacteriophage particles after the addition of packaging extracts. Mutations of the lacI gene are easily detected by the derepression of beta-galactosidase resulting in blue plaques in the presence of X-gal. Originally developed as a short-term in vivo mutagenesis assay to screen genotoxic agents, we have used this system to measure naturally occurring DNA mutations as a function of chronological age. There was a linear increase in the presence of phenotypic lacI mutants from birth to 24 months (r = 0.731, P < 0.001), such that 24-month-old mice have accumulated approximately fourfold more mutants than newborn pups. Molecular analysis of these spontaneously arising DNA mutations showed them to result predominantly from base substitutions (80.6-98.5%) that were equally distributed between transitions and transversions. However, lacI mutations in animals > 3 months of age demonstrated a higher percentage of mutations (12-19.4% vs. 1.2%) resulting from discriminable size changes (> 20 bp) than was observed for mice 1-2 months old. Sequence analysis of mutations resulting from a > 20 bp size change revealed them to be due to a duplication of adjacent lacI sequence. These results indicate that there is a gradual accumulation of DNA mutations with age and that the types of mutations also are influenced by the age of the animal.

Aging↗

PubMed2004

Transduction of multiple cell types using improved conditions for gene delivery and expression of SV40 pseudovirions packaged in vitro.

This comprehensive study demonstrates highly efficient transduction of a wide variety of human, murine, and monkey cell lines, using a procedure for in vitro packaging of plasmid DNA in recombinant simian virus 40 (SV40) capsid proteins to form pseudovirions. The pseudovirions are encapsidated by the VP1 major capsid protein, with no SV40 sequence requirement, and are able to carry up to 17.7 kb of supercoiled plasmid DNA. We developed a procedure to scale-up production of SV40 pseudovirions, as well as an efficient protocol to concentrate the virions with no loss of activity. We also developed a method that allows transduction of 10 times more cells than the original protocol. This protocol was tested using supercoiled in vitro-packaged plasmid carrying the human multidrug-resistance gene (MDR1 encoding P-glycoprotein; P-gp), or the enhanced green fluorescent protein reporter gene (EGFP) in .45 human lymphoblastoid cells and in K562 human erythroleukemia cells. Multiple transductions at 24-h intervals were shown to increase expression using the EGFP reporter gene. The protocols developed in this study establish in vitro-packaged SV40 pseudovirions as one of the most efficient gene delivery systems.

Animals↗

PubMed2005

Encapsidation of minute virus of mice DNA: aspects of the translocation mechanism revealed by the structure of partially packaged genomes.

Minute virus of mice (MVM) packages a single, negative-sense copy of its linear single-stranded DNA genome, but a chimeric virus, MML, in which >95% MVM sequence was fused to the right-hand terminus of LuIII, packages >40% positive-sense DNA. While encapsidation of both MML strands begins efficiently, genome translocation frequently stalls at specific sites in positive-sense DNA. Internalized sequences, derived from the 3' end of the strand, ranged from 1 to 5 kb in length, with species of around 2 kb predominating. When nuclease activity during isolation was minimized, these truncated species were found to be part of pre-excised 5 kb single-strands. Similarly, some partially encapsidated negative-sense DNAs were observed, forming a continuum of protected 3' sequences between 1 and 3 kb in length, but these were less abundant and more uniformly distributed than their positive-sense counterparts, indicating that the negative strand has evolved for efficient internalization. The paucity of protected DNAs shorter than 1-2 kb suggests that translocation is biphasic, proceeding efficiently through the first (3') third of the genome, but prone to stall thereafter. Sequences with conspicuous secondary structure, including stem-loop and guanidine rich regions, were found to interrupt packaging, especially when positioned near the 5' end of the strand. Since VP2 amino-terminal peptides were exposed at the particle surface in all packaging intermediates, extrusion of this peptide precedes translocation of the full-length strand.

Cell Line↗

PubMed1997

Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme.

Bacteriophage T7 lysozyme is known to inhibit transcription by T7 RNA polymerase. Lysozyme present before initiation inhibited the synthesis of long RNA chains but did not inhibit elongation when added shortly after chains were initiated. A combination of gel-shift and transcription assays showed that lysozyme and polymerase form a 1:1 complex that binds promoter DNA and makes abortive transcripts, indicating that lysozyme has little effect on the early steps of transcription. Extension of stalled transcription complexes suggested that a transcribing polymerase becomes resistant to lysozyme inhibition after synthesis of an RNA chain as short as 15 nucleotides. It seems likely that bound lysozyme prevents an initiating polymerase from converting to an elongation complex. This conversion is thought to involve both a conformational change in the polymerase and the binding of nascent RNA. Gel-shift experiments indicated that lysozyme does not interfere with the binding of RNA, so it probably prevents a necessary conformational change in the polymerase. Lysozyme also increased pausing or termination at two sites in lambda DNA and at a site near the right end of the concatemer junction of T7 DNA. If pausing at these sites involves a reversal from the elongation to the initiation conformation, lysozyme may increase pausing or termination by "locking in" the initiation conformation. The arrest of transcription complexes near promoters and near the right end of the concatemer junction almost certainly must relate to lysozyme's ability to stimulate replication, maturation and packaging of T7 DNA during T7 infection.

Bacteriophage T7↗

PubMed2006

Compaction dynamics of single DNA molecules under tension.

Using transverse magnetic tweezers, we studied the dynamics of DNA compaction induced by hexaammine cobalt chloride under constant forces. Discontinuous DNA compaction events were found for forces ranging from 0.5 to 1.7 pN, with approximately 270 nm DNA adsorbed in each compaction event. Forces larger than 6 pN were found able to unravel the toroid in a similar intermittent stepwise manner. The observations indicate that the folding/unfolding events are transitions between two metastable structural states which are separated by a tension-dependent energy barrier. Analysis of the waiting time revealed that the degree of the package ordering of DNA in a toroid depends on the compaction kinetics.

Bacteriophage lambda↗

PubMed1995

Consideration of the liver of embryonic lacZ transgenic mice as an analogue of the mouse coat colour spot test: preliminary data and technical problems.

Pregnant lacZ+ transgenic mice (Muta Mouse) were treated with ENU (25 mg/kg) by oral gavage on day 10.5 of pregnancy. This dose of ENU is the optimal dose observed by other investigators for activity in the mouse coat colour spot test. Day 10.5 of pregnancy represents the stage when the embryonic liver first becomes visually discernable. By day 15.5, when the maternal and embryonic livers were analysed for lacZ- mutation frequency, the embryonic liver is the largest tissue in the embryo (approximately 25 mg). These experiments therefore represent treatment of a small pool of progenitor hepatocytes just as they are entering into an intense wave of cell division--optimum conditions for the fixation of mutations. Exposure to ENU led to an average fourfold, and a maximum tenfold increase in mutation frequency in the embryonic livers. This relatively weak response is consistent with lacZ- mutants not having a growth advantage; unlike in the mouse spot test, clonal amplification of lacZ- mutants cannot be separately scored. The level of mutation in the control embryonic livers was lower than that of the maternal control livers, but the group sizes were too small to conclude this definitively. Embryonic livers from each individual mother showed a range of mutation frequencies that were not obviously related to that of the maternal liver. On a treatment group basis, ENU was non-mutagenic to maternal livers. Half of the embryonic livers yielded DNA that failed to package, despite repeated attempts and re-isolation of the DNA from the liver. The cause of this unexpected finding is not clear. What is clear is that it was not due to the ENU treatment because eight of 22 control embryonic livers behaved similarly. These preliminary results suggest that further research is required in order to establish a practical transgenic analogue of the mouse coat colour spot test.

Animals↗

PubMed1978

Heteroduplex electron microscopy of phage Mu mutants containing IS1 insertions and chloramphenicol resistance transposons.

We have examined by electron microscopy the DNA heteroduplexes of six bacteriophage Mu mutants, Mu X cam, generated by the insertion of the Tn9 transposon for chloramphenicol resistance. Tn9 was found to be 2.8 +/- 0.2 kilobases (kb) in length and to consist of a cam determinant flanked by two IS1 sequences arranged in a direct order. In two of the six Mu X cam mutants, the Tn9 insertion was at a fixed location, 3.9 kb from the left, or c, end. In the other four mutants, the position of the insertion varied, even though the lysogenic cultures induced were grown from single colonies. The insertion was located at either 3.3 kb, 3.9 kb, or, less frequently, at 4.4 kb from the left end of the DNA. Furthermore, at low frequencies, the insertions were found to be in an orientation opposite to what predominated in the preparation. Thus, Tn9 in the Mu X cam mutants examined could appear to undergo rapid rearrangements during Mu growth or over a few generations of cell growth. One of the Tn9 insertion sites was apparently the same as that for a 0.8 kb insertion found in a Mu X mutant. This latter insertion was identified as an IS1 sequence. The DNA molecules from all the Mu X cam mutant phage particles were found to be missing the bacterial DNA at the S (right) end, along with a variable amount of the adjoining Mu DNA in the beta region. This observation supports the headful packaging model for Mu DNA.

Chloramphenicol↗

PubMed1992

Cloning and integration of DNA fragments in human cells via the inverted terminal repeats of the adeno-associated virus 2 genome.

In current systems for molecular cloning of eukaryotic genes, bacterial cells are routinely utilized as intermediate hosts. We investigated the possibility of using a viral system for cloning DNA fragments independent of bacterial cell usage. In this report, we provide an alternative approach for molecular cloning of DNA fragments in eukaryotic cells by utilizing the inverted terminal repeats (ITRs) of the genome of a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). We constructed a series of chimeric linear duplex DNA molecules, ranging in length from 1.8 to 7.2 kb, containing the cruciform structures of AAV-ITRs at both ends. These 'no-end' (NE) DNA structures, when transfected into adenovirus-infected human cells in the presence of AAV replication proteins (Rep), underwent DNA replication. Furthermore, in the presence of AAV capsid proteins (Cap), all replicated DNA molecules of less than 5.0 kb were packaged into mature, biologically active AAV progeny virions. When a chimeric NE DNA (NE-neo) containing a gene (neo) encoding resistance to neomycin was transfected into human cells, neoR clones could be readily isolated in the presence of G418 (Geneticin). Southern-blot analysis of genomic DNA of several independently isolated neoR clones suggested stable integration of the NE-neo DNA into the host chromosomal DNA. AAV-ITRs, therefore, offer an alternative system for molecular cloning, as well as packaging of DNA fragments in mammalian cells independent of bacterial cell usage.

Blotting, Southern↗

PubMed1986

Methidiumpropyl-EDTA-iron(II) cleavage of ribosomal DNA chromatin from Dictyostelium discoideum.

We have used methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)] in parallel with micrococcal nuclease to investigate the chromatin structure of the extrachromosomal palindrome ribosomal RNA genes of Dictyostelium. Confirming our earlier results with micrococcal nuclease (1,2), MPE.Fe(II) digested the coding region of rapidly transcribing rRNA genes as a smear, indicating the absence or severe disruption of nucleosomes, whereas in slowly transcribing rRNA genes, a nucleosomal ladder was produced. In the central non-transcribed spacer region of the palindrome, MPE.Fe(II) digestion resulted in a normal nucleosomal repeat, whereas micrococcal nuclease gave a complex banding pattern. The difference is attributed to the lower sequence specificity of MPE.Fe(II) compared to micrococcal nuclease. In the terminal region of the palindrome, however, both substances gave a complex chromatin digestion pattern. In this region the DNA appears to be packaged in structures strongly positioned with respect to the underlying DNA sequence.

Cell Nucleolus↗

PubMed2006

Bacterial ghosts as a novel advanced targeting system for drug and DNA delivery.

Although there are powerful drugs against infectious diseases and cancer on the market, delivery systems are needed to decrease serious toxic and noncurative side effects. In order to enhance compliance, several delivery systems such as polymeric micro- and nanoparticles, liposomal systems and erythrocyte ghosts have been developed. Bacterial ghosts representing novel advanced delivery and targeting vehicles suitable for the delivery of hydrophobic or water-soluble drugs, are the main focus of this review. They are useful nonliving carriers, as they can carry different active substances in more than one cellular location separately and simultaneously. Bacterial ghosts combine excellent natural or engineered adhesion properties with versatile carrier functions for drugs, proteins and DNA plasmids or DNA minicircles. The simplicity of both bacterial ghost production and packaging of drugs and/or DNA makes them particularly suitable for the use as a delivery system. Further advantages of bacterial ghost delivery vehicles include high bioavailability and a long shelf life without the need of cold-chain storage due to the possibility to freeze-dry the material.

Animals↗

PubMed2005

Effect of mutating the two cysteines required for HBe antigenicity on hepatitis B virus DNA replication and virion secretion.

Hepatitis B virus (HBV) variants with impaired expression of e antigen (HBeAg) frequently arise at the chronic stage of infection, as exemplified by precore and core promoter mutants. Since an intramolecular disulfide bond maintains the secondary structure of HBeAg, we explored effect of missense mutations of either cysteine codon. Consistent with earlier reports, substitution of each cysteine rendered HBeAg nearly undetectable. With underlying nucleotide changes at the loop of pregenome encapsidation signal, the C-7 mutants were severely impaired in pregenomic RNA packaging and hence DNA replication. Although none of the missense mutations at C61 reduced DNA replication, replacement with arginine, but not alanine, aspartic acid, phenylalanine, or serine, blocked virion secretion. Consistent with the detection of C61R genome from a patient serum, secretion block of the C61R mutant could be overcome by co-expression of wild-type core protein. In conclusion, point mutations of the C61 codon may generate viable HBeAg-negative variants.

Codon↗

PubMed1981

Replication of herpes simplex virus DNA: localization of replication recognition signals within defective virus genomes.

Serially passaged herpes simplex virus type 1 (HSV-1) strain Justin was previously shown to contain defective virus genomes consisting of head-to-tail reiterations of sequences derived from the end of the S component of the standard virus DNA. Cotransfection of purified monomeric defective genome repeat units with foster helper virus DNAs onto rabbit skin cells resulted in regeneration and replication of concatemeric defective DNA molecules which were successfully encapsidated. Thus, defective HSV-1 (Justin) genomes contain, within their limited DNA sequences, a sufficient set of recognition sites required for HSV DNA replication and packaging. The arrangement of repeat units within the regenerated defective virus genomes was consistent with their replication by a rolling circle mechanism in which a single repeat unit served as the circularized template. This replication occurred most actively late after infection and could be shown to be inhibited by low concentrations of phosphonoacetate known to inhibit the HSV-specified viral DNA polymerase selectively. The resultant concatemers were shown to be cleaved to Mr 100 X 10(6) DNA molecules which were terminated at one end with the proper ac end sequence of the parental standard virus DNA.

Animals↗

PubMed1990

Constraints in simian virus 40 (SV40) encapsidation, as determined by SV40-based shuttle viruses.

Simian virus 40 (SV40)-based shuttle vectors, containing the SV40 late genes, can be packaged as infectious pseudovirions. In terms of their function as bacterial plasmids, modifications in the overall size of these plasmids can be tolerated within a very wide range, which has allowed us to determine the requirements for SV40 encapsidation, free of the more stringent limitations of SV40 virus. Monkey COS7 cells were transfected with over- and undersized SV40-based shuttle virus plasmids and their progeny have been analysed to follow the stability and evolution of these genomes. Two of the three plasmids analysed undergo recombination, generating molecules with sizes of between 4.0 and to 4.8 kb which were selected after multiple lytic cycles. This size range may correspond to the DNA lengths preferentially packaged in SV40 capsids. The structure of the rearranged plasmids indicates that there is a strong selective pressure for genomes that retain the functions necessary for replication and virus production. Depending on the parent DNA, two main classes of rearrangements were generated: duplications in tandem with the SV40 origin of replication and deletions. Both classes are probably a result of selective size and replicative advantages, which are then biologically amplified during plasmid transmission as virus particles.

Animals↗

PubMed1988

Immunogenic (tum-) variants of mouse tumor P815: cloning of the gene of tum- antigen P91A and identification of the tum- mutation.

Mutagen treatment of mouse P815 tumor cells produces tum- variants that are rejected by syngeneic mice because these variants express new surface antigens. These "tum- antigens" are recognized by cytolytic T lymphocytes but induce no detectable antibody response. Transfection of P815 cell line P1.HTR with DNA of tum- variant P91 yielded transfectants expressing tum- antigen P91A. They were detected by their ability to stimulate proliferation of cytolytic T lymphocytes [Wölfel, T., Van Pel, A., De Plaen, E., Lurquin, C., Maryanski, J. L. & Boon, T. (1987) Immunogenetics 26, 178-187]. A cosmid library of a cell line expressing antigen P91A was transfected into P1.HTR. Transfectants expressing the antigen were obtained. By packaging directly the DNA of a transfectant with lambda phage extracts, we obtained a small cosmid population containing as major component a cosmid that transferred the expression of P91A. The assay of various restriction fragments of this cosmid led to the isolation of an 800-base-pair fragment containing the P91A sequence required for transfection. Comparison with a homologous cDNA showed that this fragment contained only one of the several exons of the P91A gene. The normal and the tum- forms of the gene differ by one nucleotide located in this 137-base-pair exon. The essential role of this mutation, which produces an amino acid change, was confirmed by site-directed mutagenesis. No significant sequence similarity was found between the 800-base-pair fragment and any recorded gene.

Amino Acid Sequence↗

PubMed2000

Molecular mechanisms of replication of herpes simplex virus 1.

The present review focuses on recent development in our understanding of the molecular mechanisms of DNA replication of herpes simplex virus 1 (HSV-1). Progress made in the characterization of the early events of viral DNA synthesis and of virus-host cell interactions, especially in the context with the formation of viral DNA replication sites, is highlighted. An up-dated overview is presented on important stages of lytic infection cycle, such as virion entry, viral DNA synthesis, viral DNA cleavage and packaging into preformed capsids; and maturation (nucleocapsid envelopment) and egress of virions. Many novel interactions are discussed that extend not only our knowledge of the biology of this virus but may represent possible new targets for antiherpesviral therapy.

Capsid↗

PubMed2001

Zinc finger proteins: new insights into structural and functional diversity.

Zinc finger proteins are among the most abundant proteins in eukaryotic genomes. Their functions are extraordinarily diverse and include DNA recognition, RNA packaging, transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding. Zinc finger structures are as diverse as their functions. Structures have recently been reported for many new zinc finger domains with novel topologies, providing important insights into structure/function relationships. In addition, new structural studies of proteins containing the classical Cys(2)His(2) zinc finger motif have led to novel insights into mechanisms of DNA binding and to a better understanding of their broader functions in transcriptional regulation.

Carrier Proteins↗

PubMedDate not supplied

DNA spatial considerations in the arrangement of G/C and A/T blocks.

Intensive computations have been carried out on eukaryotic and prokaryotic DNA sequences present in the GenBank database. These calculations were aimed at studying particular sequence patterns. Previously we have observed a trend in the relative positioning of DNA oligomers with respect to each other. Specifically, we have shown that there is a preference for A/T stretches to be inserted inside G/C blocks, partitioning the latter. This arrangement is preferred over having a longer G/C block with an A/T stretch next to it. That is (G/C)n(A/T)m(G/C)2 greater than (G/C)n + 2(A/T)m. Previously we have attributed this preferred pattern to nucleosome packaging of the DNA. Since the average length of the DNA involved in a single nucleosome formation is 200 bp, oligomers were scored in the analysis only if two identical ones occurred within that distance. Since, in addition, the total length of the oligomers studied is n + m + 2 less than or equal to 7, the counts of the longer oligomers were quite low. Here we have repeated the analysis on the newer, larger database, with that restriction removed. Our new analysis confirms and strengthens the older findings. Moreover, we have carried out DNA structural analysis of these trends, focusing on the twist and roll angular parameters. We have obtained these values from detailed structural computations on the Cray super computer. Some correlations between these values, DNA flexibility and the sequence trends are observed. We suggest that the positioning of the (A/T)m sequence stretch between the two (G/C) blocks affords greater flexibility in the spatial positioning of the G/C sequence elements.

Base Sequence↗

PubMed2006

Recruitment of transcription complexes to the beta-globin locus control region and transcription of hypersensitive site 3 prior to erythroid differentiation of murine embryonic stem cells.

Eukaryotic chromosomal DNA is densely packaged in the nucleus and organized into discrete domains of active and inactive chromatin. Gene loci that are activated during the process of cell differentiation undergo changes that result in modifications of specific histone tail residues and in loosening of chromatin structure. The beta-globin genes are expressed exclusively in erythroid cells. High-level expression of these genes is mediated by a locus control region (LCR), a powerful DNA regulatory element composed of several DNase I hypersensitive (HS) sites and located far upstream of the beta-globin genes. Here we show that RNA polymerase II and specific histone modifications that mark transcriptionally active chromatin domains are associated with the LCR core elements HS2 and HS3 in murine embryonic stem cells prior to differentiation along the erythroid lineage. At this stage HS3 is abundantly transcribed. After in vitro differentiation, RNA Polymerase II can also be detected at the embryonic epsilon- and adult beta-globin genes. These results are consistent with the hypothesis that activation of the beta-globin gene locus is initiated by protein complexes recruited to the LCR.

Animals↗