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PubMed1995

Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector.

Adenoviral vectors are widely used as highly efficient gene transfer vehicles in a variety of biological research strategies including human gene therapy. One of the limitations of the currently available adenoviral vector system is the presence of the majority of the viral genome in the vector, resulting in leaky expression of viral genes particularly at high multiplicity of infection and limited cloning capacity of exogenous sequences. As a first step to overcome this problem, we attempted to rescue a defective human adenovirus serotype 5 DNA, which had an essential region of the viral genome (L1, L2, VAI + II, pTP) deleted and replaced with an indicator gene. In the presence of wild-type adenovirus as a helper, this DNA was packaged and propagated as transducing viral particles. After several rounds of amplification, the titer of the recombinant virus reached at least 4 x 10(6) transducing particles per ml. The recombinant virus could be partially purified from the helper virus by CsCl equilibrium density-gradient centrifugation. The structure of the recombinant virus around the marker gene remained intact after serial propagation, while the pBR sequence inserted in the E1 region was deleted from the recombinant virus. Our results suggest that it should be possible to develop a helper-dependent adenoviral vector, which does not encode any viral proteins, as an alternative to the currently available adenoviral vector systems.

Adenoviruses, Human↗
PubMed2003

Protecting the terminus: t-loops and telomere end-binding proteins.

Telomeric DNA is composed of a region of duplex telomeric tract followed by a single-strand overhang on the 3' G-rich strand. The DNA is packaged by proteins that associate directly with the single- and double-strand regions of the telomeric tract and by their associated proteins. This review discusses the evidence that G-strand overhangs are present on both ends of eukaryotic chromosomes and the steps needed to generate these overhangs. The overhangs are protected by specialized G-overhang-binding protein and/or invasion by the overhang of the duplex region of the telomeric tract to form a structure called a 't-loop'. The G-overhang-binding proteins identified from different species are described, and their properties compared. The data supporting the existence of t-loops at native telomeres is discussed, and the conditions required to promote their in vitro formation are presented.

Animals↗
PubMed2004

The SNF2 domain protein family in higher vertebrates displays dynamic expression patterns in Xenopus laevis embryos.

All eukaryotes share a common nuclear infrastructure, in which DNA is packaged into nucleosomal chromatin. Its functional states, in particular the accessibility of the chromatin fiber to trans-acting factors, are determined by two classes of evolutionarily conserved enzymes, i.e. histone modifying enzymes and ATP-driven nucleosome remodeling machines. Browsing the annotated human genome database, we establish here a family of SNF2-like nuclear ATPases, which are the core enzymatic subunits of chromatin remodeling protein complexes. Homologues of those human genes are also to a large extent found in the Xenopus laevis genome, indicating a high degree of sequence conservation of this family among vertebrates. Expression analyses of the ATPase family of proteins reveal stage- and tissue-specific domains of peak RNA expression during early frog embryogenesis. These dynamic expression profiles suggest specific functional requirements for individual members of this family throughout early stages of vertebrate development.

Adenosine Triphosphatases↗
PubMed1998

A new system for stringent, high-titer vesicular stomatitis virus G protein-pseudotyped retrovirus vector induction by introduction of Cre recombinase into stable prepackaging cell lines.

We report here on stable prepackaging cell lines which can be converted into packaging cell lines for high-titer vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovirus vectors by the introduction of Cre recombinase-expressing adenovirus. The generated prepackaging cell lines constitutively express the gag-pol genes and contain an inducible transcriptional unit for the VSV-G gene. From this unit, the introduced Cre recombinase excised both a neomycin resistance (Neo(r)) gene and a poly(A) signal flanked by a tandem pair of loxP sequences and induced transcription of the VSV-G gene from the same promoter as had been used for Neo(r) expression. By inserting an mRNA-destabilizing signal into the 3' untranslated region of the Neo(r) gene to reduce the amount of Neo(r) transcript, we were able efficiently to select the clones capable of inducing VSV-G at high levels. Without the introduction of Cre recombinase, these cell lines produce neither VSV-G nor any detectable infectious virus at all, even after the transduction of a murine leukemia virus-based retrovirus vector encoding beta-galactosidase. They reproducibly produced high-titer virus stocks of VSV-G-pseudotyped retrovirus (1.0 x 10(6) infectious units/ml) from 3 days after the introduction of Cre recombinase. We also present evidence that VSV-G-producing cells are still fully susceptible to transduction by VSV-G pseudotypes. However, in this vector-producing system, which regulates VSV-G pseudotype production in an all-or-none manner, the integration of vector DNA into packaging cell lines would be minimized. We further show that heparin significantly inhibits retransduction of VSV-G pseudotypes in the culture fluids of packaging cell lines, leading to a two- to fourfold increase in the yield of the pseudotypes after induction. This vector-producing system was very stable and should be advantageous in human gene therapy.

Animals↗
PubMed1993

Mutations in liver DNA of lacI transgenic mice (Big Blue) following subchronic exposure to 2-acetylaminofluorene.

2-Acetylaminofluorene (2-AAF) was administered at levels of 0, 300 and 600 ppm in the diet for 28 days to female transgenic mice bearing the lacI gene in a lambda vector (Big Blue mice). The lambda vector was excised from liver DNA and packaged in vitro into bacteriophage particles which were allowed to infect E. coli bacteria, forming plaques on agar plates. Approximately 10(5) plaques were screened per animal for the appearance of a blue colour, indicative of mutations in the lacI gene which had resulted in an inactive gene product. Background mutation rate was 2.7 x 10(-5) (pooled results of two animals, 8 mutant plaques/289,530 plaques). At 300 ppm in the diet, the rate of 3.5 x 10(-5) (8/236,300) was not significantly increased over background. At 600 ppm in the diet, the rate increased approximately 3 fold to 7.7 x 10(-5) (17/221,240). In comparison to the usual single or 5-day carcinogen exposure regimes, the 4-week exposure protocol allowed the use of much lower dose levels (10-1000 fold lower). Overt toxicity could thus be avoided. The daily doses used were somewhat higher than those required in 2-year carcinogenicity studies with 2-AAF.

2-Acetylaminofluorene↗
PubMed2001

Structure of bacteriophage P22 portal protein in relation to assembly: investigation by Raman spectroscopy.

Salmonella phage P22, which serves as an assembly paradigm for icosahedral double-stranded DNA viruses, packages its viral genome through a capsid channel (portal) comprising 12 copies of a 725-residue subunit. Secondary and tertiary structures of the portal subunit in monomeric and dodecameric states have been investigated by Raman spectroscopy using a His6-tagged recombinant protein that self-assembles in vitro [Moore, S. D., and Prevelige, P. E., Jr. (2001) J. Biol. Chem. 276, 6779-6788]. The portal protein exhibits Raman secondary structure markers typical of a highly alpha-helical subunit fold that is little perturbed by assembly. On the other hand, Raman markers of subunit side chains change dramatically with assembly, an indication of extensive changes in side chain environments. The cysteinyl Raman signature of the portal consists of a complex pattern of sulfhydryl stretching bands, revealing diverse hydrogen-bonding states for the four S-H groups per subunit (Cys 153, Cys 173, Cys 283, and Cys 516). Upon assembly, the population of strongly hydrogen-bonded S-H groups decreases, while the population of weakly hydrogen-bonded S-H groups increases, implying that specific intrasubunit S-H.X hydrogen bonds must be weakened to effect dodecamer assembly and that the molecular mechanism involves reorganization of subunit domains without appreciable changes in domain conformations. Comparison with other viral protein assemblies suggests an assembly process not requiring metastable intermediates. The recently published X-ray structure of the phi29 portal [Simpson, A. A., et al. (2000) Nature 408, 745-750] shows that residues 125-225 lining the channel surface form alpha-helical modules spaced by short beta-strands and turns; a surprisingly close secondary structure homology is predicted for residues 240-350 of the P22 portal, despite no apparent sequence homology. This motif is proposed as an evolutionarily conserved domain involved in DNA translocation.

Bacteriophage P22↗
PubMed1999

Transcriptional repression of the yeast CHA1 gene requires the chromatin-remodeling complex RSC.

In eukaryotes, DNA is packaged into chromatin, a compact structure that must be disrupted when genes are transcribed by RNA polymerase II. For transcription to take place, chromatin is remodeled via nucleosome disruption or displacement, a fundamental transcriptional regulatory mechanism in eukaryotic organisms. Here we show that the yeast chromatin-remodeling complex, RSC (remodels the structure of chromatin), isolated on the basis of homology to the SWI/SNF complex, is required for proper transcriptional regulation and nucleosome positioning in the highly inducible CHA1 promoter. In the absence of Sth1p/Nps1p (a homolog of Swi2p/Snf2p) or of Swh3p (a homolog of Swi3p), expression of CHA1 in non-induced cells is increased to a level comparable with that of fully induced cells. Furthermore, in non-induced cells depleted for Sth1p/Nps1p or Swh3p, a nucleosome positioned over the TATA box of the CHA1 promoter is disrupted, an architectural change normally only observed during transcriptional induction. In addition, deletion of the gene-specific activator Cha4p did not affect derepression of CHA1 in cells depleted for Swh3p. Thus, CHA1 constitutes a target for the RSC complex, and we propose that RSC is essential for maintaining a repressive chromatin structure at the CHA1 promoter.

Chromatin↗
PubMed1996

Efficient transcriptional silencing in Saccharomyces cerevisiae requires a heterochromatin histone acetylation pattern.

Heterochromatin in metazoans induces transcriptional silencing, as exemplified by position effect variegation in Drosophila melanogaster and X-chromosome inactivation in mammals. Heterochromatic DNA is packaged in nucleosomes that are distinct in their acetylation pattern from those present in euchromatin, although the role these differences play in the structure of heterochromatin or in the effects of heterochromatin on transcriptional activity is unclear. Here we report that, as observed in the facultative heterochromatin of the inactive X chromosome in female mammalian cells, histones H3 and H4 in chromatin spanning the transcriptionally silenced mating-type cassettes of the yeast Saccharomyces cerevisiae are hypoacetylated relative to histones H3 and H4 of transcriptionally active regions of the genome. By immunoprecipitation of chromatin fragments with antibodies specific for H4 acetylated at particular lysine residues, we found that only three of the four lysine residues in the amino-terminal domain of histone H4 spanning the silent cassettes are hypoacetylated. Lysine 12 shows significant acetylation levels. This is identical to the pattern of histone H4 acetylation observed in centric heterochromatin of D. melanogaster. These two observations provide additional evidence that the silent cassettes are encompassed in the yeast equivalent of metazoan heterochromatin. Further, mutational analysis of the amino-terminal domain of histone H4 in S. cerevisiae demonstrated that this observed pattern of histone H4 acetylation is required for transcriptional silencing. This result, in conjunction with prior mutational analyses of yeast histones H3 and H4, indicates that the particular pattern of nucleosome acetylation found in heterochromatin is required for its effects on transcription and is not simply a side effect of heterochromatin formation.

Acetylation↗
PubMed1991

[Construction of Echinococcus granulosus genomic library].

Using EMBL3 phage DNA as a vector, a perfect genomic library of Echinococcus granulosus from sheep from Xinjiang has been constructed, which contains 1.2 x 10(6) independent recombinants. The main procedure of construction comprised: 1. extraction of Echinococcus granulosus genomic DNA, 2. partial digestion of the extracted E. granulosus genomic DNA with restriction enzyme Sau3Al, 3. harvest of 15-23 kb DNA fragments by electric elution equipment, 4. preparation of EMBL3 phage vector DNA, 5. cleavage of EMBL3 vector DNA with two restriction enzymes Bam HI and Eco RI to remove the central fragment, 6. ligation of the harvested 15-23 kb E. granulosus genomic DNA and the cleavaged vector DNA with T4 DNA ligase, 7. the package reaction of ligated recombinant DNA in vitro with the package protein and with Q359 strain (Spi-) to screen recombinants, 8. identification of the genomic library, 9. hybridization in situ by pSM889 probe, obtaining 6 positive recombinant clones. It was estimated that the E. granulosus genome was about 1.5 x 10(8) bp. A perfect E.g. genomic library is expected to contain 4.6 x 10(4) independent recombinants at minimum. The constructed genomic library has enough independent recombinants (1.2 x 10(6)). This is the first time to establish an E. granulosus genomic library in China. Previous work showed that there existed differences in many aspects, including pathogenicity, among various isolates of E. granulosus from different hosts and areas. We plan to employ this library to screen the E. granulosus intraspecies DNA probe by hybridization in situ. This kind of probe is envisaged to be of advantage for epidemiological investigation of the hydatid disease in China. It also provides a base for researching E. granulosus at the molecular level.

Animals↗
PubMed1999

BAC-VAC, a novel generation of (DNA) vaccines: A bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1.

This study aimed to exploit bacterial artificial chromosomes (BAC) as large antigen-capacity DNA vaccines (BAC-VAC) against complex pathogens, such as herpes simplex virus 1 (HSV-1). The 152-kbp HSV-1 genome recently has been cloned as an F-plasmid-based BAC in Escherichia coli (fHSV), which can efficiently produce infectious virus progeny upon transfection into mammalian cells. A safe modification of fHSV, fHSVDeltapac, does not give rise to progeny virus because the signals necessary to package DNA into virions have been excluded. However, in mammalian cells fHSVDeltapac DNA can still replicate, express the HSV-1 genes, cause cytotoxic effects, and produce virus-like particles. Because these functions mimic the lytic cycle of the HSV-1 infection, fHSVDeltapac was expected to stimulate the immune system as efficiently as a modified live virus vaccine. To test this hypothesis, mice were immunized with fHSVDeltapac DNA applied intradermally by gold-particle bombardment, and the immune responses were compared with those induced by infection with disabled infectious single cycle HSV-1. Immunization with either fHSVDeltapac or disabled infectious single cycle HSV-1 induced the priming of HSV-1-specific cytotoxic T cells and the production of virus-specific antibodies and conferred protection against intracerebral injection of wild-type HSV-1 at a dose of 200 LD(50). Protection probably was cell-mediated, as transfer of serum from immunized mice did not protect naive animals. We conclude that BAC-VACs per se, or in combination with genetic elements that support replicative amplification of the DNA in the cell nucleus, represent a useful new generation of DNA-based vaccination strategies for many viral and nonviral antigens.

Animals↗
PubMed1996

A new transgenic mouse mutagenesis test system using Spi- and 6-thioguanine selections.

A new transgenic mouse mutagenesis test system has been developed for the efficient detection of point mutations and deletion mutations in vivo. The mice carry lambda EG10 DNA as a transgene. When the rescued phages are infected into Escherichia coli YG6020-expressing Cre recombinase, the phage DNA is converted into plasmid pYG142 carrying the chloramphenicol-resistance gene and the gpt gene of E. coli. The gpt mutants can be positively detected as colonies arising on plates containing chloramphenicol and 6-thioguanine. The EG10 DNA carries a chi site along with the red and gam genes so that the wild-type phages display Spi- (sensitive to P2 interference) phenotype. Mutant phages lacking both red and gam genes can be positively detected as plaques that grow in P2 lysogens of E. coli. These mutant phages are called lambda Spi-. The spontaneous gpt mutation frequencies of five independent transgenic lines were 1.7 to 3.3 x 10(-5) in bone marrow. When the mice were treated with ethylnitrosourea (single i.p. treatments with 150 mg/kg body weight; killed 7 days after the treatments), mutation frequencies were increased four- to sevenfold over the background in bone marrow. The average rescue efficiencies were more than 200,000 chloramphenicol-resistant colonies per 7.5 micrograms bone marrow DNA per packaging reaction. In contrast to gpt mutation frequencies, spontaneous Spi- mutation frequencies were 1.4 x 10(-6) and 1.1 x 10(-6) in bone marrow and sperm, respectively. No spontaneous Spi- mutants have been detected so far in spleen, although 930,000 phages rescued from untreated mice were screened. In gamma-ray-treated animals, however, induction of Spi- mutations was clearly observed in spleen, at frequencies of 1.4 x 10(-5) (5 Gy), 1.2 x 10(-5) (10 Gy), and 2.0 x 10(-5) (5O Gy). These results suggest that the new transgenic mouse "gpt delta" could be useful for the efficient detection of point mutations and deletion mutations in vivo.

Animals↗
PubMed1993

Pulsed field agarose gel electrophoresis in the study of morphogenesis: packaging of double-stranded DNA in the capsids of bacteriophages.

To understand how comparatively simple macromolecular components become biological systems, studies are made of the morphogenesis of bacteriophages. Pulsed field agarose gel electrophoresis (PFGE) has contributed to these studies by: (i) improving the length resolution of both mature, linear, double-stranded bacteriophage DNAs and the concatemers formed both in vivo and in vitro by the end-to-end joining of these mature bacteriophage DNAs, (ii) improving the resolution of circular conformers of bacteriophage DNAs, (iii) improving the resolution of linear single-stranded bacteriophage DNAs, (iv) providing a comparatively simple technique for analyzing protein-DNA complexes, and (v) providing a solid-phase quantitative assay for all forms of bacteriophage DNA; solid-phase assays are both less complex and more efficient than liquid-phase assays such as rate zonal centrifugation. Conversely, studies of bacteriophages have contributed to PFGE the DNA standards used for determining the length of nonbacteriophage DNAs. Among the solid-phase assays based on PFGE is an assay for excluded volume effects.

Bacteriophages↗
PubMed2005

Analysis of biological motors via multidimensional fractionation: a strategy.

Past strategies for the analysis of ATP-fueled motors include single-motor analysis. Single-motor analysis bypasses limitations caused by motor asynchrony during the traditional ensemble averaging analysis. The present communication describes revised ensemble averaging analysis that also can bypass asynchrony-derived limitations. This revised analysis makes measurements of one motor variable dependent on the others. One example is nondenaturing gel electrophoresis with more than one dimension. Each dimension measures one of the motor variables. This multidimensional procedure is used to obtain the values of "conformational" motor variables as a function of a "clock" motor variable. In theory, the cycle of the motor can be analyzed from a single multidimensional analysis of a collection of asynchronous motors sampled at only one time. That is to say, motor asynchrony becomes an asset, rather than a liability.

DNA Packaging↗
PubMed2005

Developmental control via GATA factor interplay at chromatin domains.

Despite the extraordinary task of packaging mammalian DNA within the constraints of a cell nucleus, individual genes assemble into cell type-specific chromatin structures with high fidelity. This chromatin architecture is a crucial determinant of gene expression signatures that distinguish specific cell types. Whereas extensive progress has been made on defining biochemical and molecular mechanisms of chromatin modification and remodeling, many questions remain unanswered about how cell type-specific chromatin domains assemble and are regulated. This mini-review will discuss emerging studies on how interplay among members of the GATA family of transcription factors establishes and regulates chromatin domains. Dissecting mechanisms underlying the function of hematopoietic GATA factors has revealed fundamental insights into the control of blood cell development from hematopoietic stem cells and the etiology of pathological states in which hematopoiesis is perturbed.

Animals↗
PubMedDate not supplied

Non-viral, integrin-mediated gene transfer into fibroblasts from patients with lysosomal storage diseases.

BACKGROUND: Non-viral vectors consisting of Lipofectin/integrin-targeting peptide/DNA (LID) complexes have great potential for gene therapy, as they are safe, simple, and able to package large DNA molecules. In this study, these vectors were evaluated in vitro for the therapy of lysosomal storage disorders. METHODS: Non-viral vectors were designed to deliver therapeutic genes by integrin-mediated uptake into fibroblasts from patients with the lysosomal storage disorders fucosidosis and Fabry disease, which result from deficiencies of alpha-L-fucosidase and alpha-galactosidase A, respectively. The vectors consisted of a complex (LID) of Lipofectin and a peptide containing an integrin-targeting domain and a poly-lysine domain to which was bound plasmid DNA, containing alpha-L-fucosidase (LID-alpha-Fuc) or alpha-galactosidase A (LID-alpha-Gal). RESULTS: Patients' fibroblasts transfected with LID-alpha-Fuc and LID-alpha-Gal produced the corresponding enzyme at levels which were 10-40% of the total activity in cultures of normal fibroblasts. However, 95-98% of this activity was secreted. Transfection of endothelial cells, the main target cells in Fabry disease, with an LID-alpha-Gal produced a total alpha-galactosidase activity 65% higher than that in untransfected cultures after 6 days, 67% of the activity being secreted. Although transfection of fibroblasts with LID complexes also caused small changes in the distribution of endogenous lysosomal enzymes, it did not appear to affect the viability of the cells. CONCLUSIONS: The integrin-mediated transfer of genes encoding lysosomal enzymes into cells results in the secretion of large amounts of normal enzyme that could be taken up by other cells. This could be a useful strategy for enzyme-replacement therapy.

Cell Line↗
PubMed2001

In vitro assembly of the herpes simplex virus procapsid: formation of small procapsids at reduced scaffolding protein concentration.

The herpes simplex virus 1 capsid is formed in the infected cell nucleus by way of a spherical, less robust intermediate called the procapsid. Procapsid assembly requires the capsid shell proteins (VP5, VP19C, and VP23) plus the scaffolding protein, pre-VP22a, a major component of the procapsid that is not present in the mature virion. Pre-VP22a is lost as DNA is packaged and the procapsid is transformed into the mature, icosahedral capsid. We have employed a cell-free assembly system to examine the role of the scaffolding protein in procapsid formation. While other reaction components (VP5, VP19C, and VP23) were held constant, the pre-VP22a concentration was varied, and the resulting procapsids were analyzed by electron microscopy and SDS-polyacrylamide gel electrophoresis. The results demonstrated that while standard-sized (T = 16) procapsids with a measured diameter of approximately 100 nm were formed above a threshold pre-VP22a concentration, at lower concentrations procapsids were smaller. The measured diameter was approximately 78 nm and the predicted triangulation number was 9. No procapsids larger than the standard size or smaller than 78-nm procapsids were observed in appreciable numbers at any pre-VP22a concentration tested. SDS-polyacrylamide gel analyses indicated that small procapsids contained a reduced amount of scaffolding protein compared to the standard 100-nm form. The observations indicate that the scaffolding protein concentration affects the structure of nascent procapsids with a minimum amount required for assembly of procapsids with the standard radius of curvature and scaffolding protein content.

Capsid↗
PubMed2005

Seed development and genomic imprinting in plants.

Genomic imprinting refers to an epigenetic phenomenon where the activity of an allele depends on its parental origin. Imprinting at individual genes has only been described in mammals and seed plants. We will discuss the role imprinted genes play in seed development and compare the situation in plants with that in mammals. Interestingly, many imprinted genes appear to control cell proliferation and growth in both groups of organisms although imprinting in plants may also be involved in the cellular differentiation of the two pairs of gametes involved in double fertilization. DNA methylation plays some role in the control of parent-of-origin-specific expression in both mammals and plants. Thus, although imprinting evolved independently in mammals and plants, there are striking similarities at the phenotypic and possibly also mechanistic level.

Animals↗
PubMed2005

How to pack the genome for a safe trip.

The transformation of the somatic chromatin into a unique and highly compact structure occurring during the post-meiotic phase of spermatogenesis is one of the most dramatic known processes of chromatin remodeling. Paradoxically, no information is available on the mechanisms controlling this specific reorganization of the haploid cell genome. The only existing hints suggest a role for histone variants, as well as for stage-specific post-translational histone modifications,before and during the incorporation of testis-specific basic nuclear proteins. Moreover, the exact functions of the latter remain obscure. This chapter summarizes the major chromatin-associated events taking place during the post-meiotic differentiation of male haploid cells in mammals and discusses some of the basic issues that remain to be solved to finally understand chromatin remodeling during spermatogenesis.

Animals↗