The demonstration of morphological nuclear changes in pyknotic irradiated thymocytes.
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Cell Nucleus↗
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Results for “DNA packaging”
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[Bozóky's condenser in comparative measurement of the dispersion dose in roentgenotherapy of breast cancer].
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Breast↗
[The histone deacetylase activity of SIR2 and chromatin silencing].
Chromatin silencing is the inactivation of large domains of DNA by packaging them into a specialized inaccessible chromatin structure. This type of inactivation is involved in the regulation of gene expression and is also associated with the chromosome structures required for chromosome maintenance and inheritance. Silent information protein 2(SIR2) is one of the important proteins involved in chromatin silencing. It is clear that SIR2 has two coupled enzymatic activities, histone deacetylation and NAD breakdown activities, and produces a novel compound, O-acetyl-ADP-ribose in the enzymatic reactions. The histone deacetylation activity of SIR2 provides the direct link between SIR2 and the hypoacetylation of silent chromatin. Moreover, the relationship between the NAD cleavage and the deacetylase activity of SIR2 shows that the histone deacetylase activity is not its only crucial function. The breakdown of NAD C-N bond and the synthesis of O-acetyl-ADP-ribose may also be involved in chromatin silencing.
English Abstract↗
[Design and rapid evaluation of a TaqMan assay for the detection of influenza A viruses].
OBJECTIVE: To design and rapidly evaluate a TaqMan assay for detecting influenza A viruses. METHODS: The probe and the primers of the assay were designed with the software packages of DNA Star and Primer Premier 5.0. Their specificity and conservation were verified through Blast in GenBank and electronic hybridization. The assay's sensitivity was compared with the standard RT-PCR. RESULTS: The designed primers and probe were confirmed to be very specific and conserved. The assay was 3-27 folds more sensitive than the standard RT-PCR. The RT and PCR steps could be simplified into one step. CONCLUSION: The TaqMan Real-time PCR assay is specific, sensitive and easy to perform.
Animals↗
Determination of S-phase cells by in situ hybridization for histone H3 mRNA in hepatocellular carcinoma: correlation with histologic grade and other cell proliferative markers.
Cell proliferation has a crucial importance in biologic potentialities of tumor cells. Several methods to measure S-phase fraction in tumor samples have been developed, with considerable limitations in practical applications. Histones are nucleosomal proteins that are responsible for packaging chromosomal DNA into nucleosomes. The expression of histone genes is a fundamental step constituting the process of cell proliferation. Because histone H3 mRNA accumulates in the cytoplasm during S-phase, and then decreases as cells approach G2-phase, demonstration of histone H3 mRNA expression in a tumor cell population may represent responsible S-phase fraction. Thus, we have assessed S-phase fraction by nonisotopic in situ hybridization for histone H3 mRNA in paraffin sections of hepatocellular carcinomas (HCCs); then, we compared this with the histologic grades and other cell proliferative markers. In contrast to radioisotopic detection, signals were distinctly visualized, and quantitation of the stained-cells was easily carried out. Histone H3 labeling index (LI) significantly correlated with other cell proliferative markers, including Ki-67 (MIB-1) and PCNA immunostainings, and mitotic index. In addition, a statistically significant correlation was seen between histone H3 LI and histologic grades of differentiation, i.e., histone H3 LI being higher in less-differentiated HCCs. Furthermore, histone H3 LI/Ki-67 LI ratio was significantly higher in the moderately and poorly differentiated subtypes (including one case of the undifferentiated subtype) than in well-differentiated HCCs. By using this nonisotopic in situ hybridization technique, it becomes possible to assess rapidly, retrospectively, safety, and easily a malignant potentiality of HCCs in paraffin-embedded tissues.
Adult↗
[Reversal of malignant cell phenotype of human pancreatic adenocarcinoma cell line by recombinant retroviral vector expressing antisense Ki-ras].
A recombinant retroviral vector expressing antisense Ki-ras was constructed. The recombinant virus DNA was packaged with packaging cell line PA317 cells by the calcium phosphate coprecipitation method. The virus supernatant was used to infect human pancreatic carcinoma cell line PC-2. After selection with Puromycin, resistant colonies were obtained. Stable expressions of retrovirus in both PA317 and PC-2 cells were exhibited by Northern blot hybridization. A down regulation of endogenous Ki-ras was found in PC-2 cells infected with antisense Ki-ras construct. It was demonstrated that the antisense Ki-ras did inhibit the cell growth rate and 3H-TdR incorporation rate of PC-2 cells. The ability of colony formation in soft agar and tumorogenicity in nude mice of PC-2 cells were significantly suppressed by the antisense Ki-ras. The results implicate that recombinant retroviral vector containing antisense Ki-ras could inhibit target gene expression and partly reverse malignant phenotype of pancreatic adenocarcinoma cells.
Adenocarcinoma↗
[Reversal of malignant phenotype of cells of human pancreatic adenocarcinoma cell line by recombinant retroviral vector expressing antisense c-myc].
A recombinant retroviral vector expressing antisense c-myc was constructed. The recombinant virus DNA was packaged with packaging cell line PA317 cells by the calcium phosphate coprecipitation method. The virus supernatant was used to infect human pancreatic carcinoma cell line PC-2. After selection with G418, resistant colonies were obtained. Stable expression of retrovirus in both PA317 and PC-2 cells were confirmed by Northern blot hybridization. A down-regulation of endogenous c-myc was found in PC-2 cells infected with antisense c-myc construct. It was demonstrated that the antisense c-myc did inhibit the cell growth rate and 3H-TdR incorporation rate of PC-2 cells. The ability of colony formation in soft agar and tumorogenicity in nude mice of PC-2 cells were significantly suppressed by the antisense c-myc. The results implicate that recombinant retroviral vector containing antisense c-myc could inhibit target gene expression and partly reverse malignant phenotype of pancreatic adenocarcinoma cells.
Adenocarcinoma↗
Nucleosomes and regulation of gene expression. Structure of the HIV-1 5'LTR.
Packaging of DNA into chromatin adds complexity to the problem of regulation of gene expression. Nucleosomes affect the accessibility of transcription factors to occupy their binding sites in chromatin of eukaryotic cells. The disruption of nucleosome structure within the enhancer/promoter region of the integrated HIV-1 proviral genome is an instructive example of a chromatin remodeling process during transcriptional activation. To investigate the mechanism responsible for generating nuclease hypersensitive sites that exist in vivo in the promoter/enhancer region of the 5'LTR (long terminal repeat) of integrated HIV-1 we have utilized an in vitro chromatin assembly system with Xenopus oocyte extracts. Chromatin assembly in the presence of Sp1 and NFkappaB transcription factors induces DNase I hypersensitive sites on either side of their binding sites and positions the adjacent nucleosomes. This structure can also be formed in a factor-induced, ATP-dependent chromatin remodeling process and closely resembles the in vivo chromatin structure. The DNase I hypersensitive sites that form within the HIV LTR are probably histone-free and remain after removal of transcription factors.
Acetylation↗
The null mutant of the U(L)31 gene of herpes simplex virus 1: construction and phenotype in infected cells.
Earlier studies have shown that the U(L)31 protein is homogeneously distributed throughout the nucleus and cofractionates with nuclear matrix. We report the construction from an appropriate cosmid library a deletion mutant which replicates in rabbit skin cells carrying the U(L)31 gene under a late (gamma1) viral promoter. The mutant virus exhibits cytopathic effects and yields 0.01 to 0.1% of the yield of wild-type parent virus in noncomplementing cells but amounts of virus 10- to 1,000-fold higher than those recovered from the same cells 3 h after infection. Electron microscopic studies indicate the presence of small numbers of full capsids but a lack of enveloped virions. Viral DNA extracted from the cytoplasm of infected cells exhibits free termini indicating cleavage/packaging of viral DNA from concatemers for packaging into virions, but analyses of viral DNAs by pulsed-field electrophoresis indicate that at 16 h after infection, both the yields of viral DNA and cleavage of viral DNA for packaging are decreased. The repaired virus cannot be differentiated from the wild-type parent. These results suggest the possibility that U(L)31 protein forms a network to enable the anchorage of viral products for the synthesis and/or packaging of viral DNA into virions.
Animals↗
A bipartite bacteriophage T4 SOC and HOC randomized peptide display library: detection and analysis of phage T4 terminase (gp17) and late sigma factor (gp55) interaction.
HOC and SOC are dispensable T4 capsid proteins that can be used for phage display of multiple copies of peptides and proteins. A bipartite phage T4 peptide library was created by displaying on tetra-alanine linker peptides five randomized amino acids from the carboxyl-terminus of SOC and five randomized amino acids from the amino terminus of HOC. The bipartite library was biopanned against the phage T4 terminase large subunit gp17 to identify T4 gene products that may interact with the terminase. The sequences of selected phages displayed matches to those T4 gene products previously known by genetic and biochemical criteria to interact with gp17: gp20 (portal protein), gp32 (single-stranded DNA binding protein), gp16 (terminase small subunit), and gp17 (self). In addition, matches were found to gp55 (T4 late sigma factor), gp45 (sliding clamp), gp44 (clamp loader), gp2 (DNA end protein), and gp23 (major capsid protein). Abundant amino acid sequence matches were found to aa region 118-134 of gp55. Immunoprecipitation and affinity column chromatography demonstrated direct binding of gp17 and gp55; moreover, gp17 bound specifically to a column-coupled peptide corresponding to gp55 residues 111-136. Measurements of gene 17 and other mRNA levels in mutant-infected bacteria did not support a role of gp17-gp55 interaction in regulation of terminase or other late gene transcription. However, whereas DNA concatemers that accumulate in prohead and terminase defective phage T4 infections could be packaged in vitro to approximately 10% wild-type efficiency, 55am33am defective concatemeric DNA was packaged at least 100-fold less efficiently. Moreover, gp55 residues 111-136 peptide specifically blocked DNA packaging in vitro. These results suggest that the T4 terminase interaction with T4 late sigma factor gp55 plays a role in DNA packaging in vivo. The gp55 interaction may function to load the terminase onto DNA for packaging.
Amino Acid Sequence↗
Insights into specific DNA recognition during the assembly of a viral genome packaging machine.
Terminase enzymes mediate genome "packaging" during the reproduction of DNA viruses. In lambda, the gpNu1 subunit guides site-specific assembly of terminase onto DNA. The structure of the dimeric DNA binding domain of gpNu1 was solved using nuclear magnetic resonance spectroscopy. Its fold contains a unique winged helix-turn-helix (wHTH) motif within a novel scaffold. Surprisingly, a predicted P loop ATP binding motif is in fact the wing of the DNA binding motif. Structural and genetic analysis has identified determinants of DNA recognition specificity within the wHTH motif and the DNA recognition sequence. The structure reveals an unexpected DNA binding mode and provides a mechanistic basis for the concerted action of gpNu1 and Escherichia coli integration host factor during assembly of the packaging machinery.
Adenosine Triphosphate↗
Protein IX, a minor component of the human adenovirus capsid, is essential for the packaging of full length genomes.
Human adenovirus type 5 (Ad5) contains a 36-kb double-stranded DNA molecule in an icosahedral capsid. Attempts to construct Ad5 insertion mutants containing DNA of more than about 105% of the genome size resulted in viral progeny in which deletions had occurred suggesting the existence of severe constraints on the size of packageable DNA molecules. To partially circumvent these constraints we used an adenovirus vector, Ad5dlE1,3, with deletions in early regions 1 (E1) and 3 for a total net reduction in genome size of 5349 bp and an expected capacity for inserts of greater than 7 kb. To use this vector efficiently we generated a circular form of dlE1,3 DNA which could be propagated as an infectious bacterial plasmid. When this plasmid was used as a recipient for inserts of various sizes it was found that its capacity was much less than expected and that dlE1,3 virion capsids could not even package DNA as large as the wt genome. Because the E1 deletion of dlE1,3 extends into the coding sequences for protein IX, a minor capsid component known to affect the heat stability of adenovirions, the possibility that absence of this polypeptide might also affect the DNA capacity of the virion was investigated. It was found that when the coding sequences for protein IX were restored the packaging capacity of the vector was also restored to that of wt virions. Thus protein IX is an essential constituent of virion capsids dispensable only for virions containing DNA of less than genomic size.
Adenovirus Early Proteins↗
CIRCE: a scalable Python package to predict cis-regulatory DNA interactions from single-cell chromatin accessibility data.
MOTIVATION: Chromatin 3D folding creates numerous DNA interactions, participating in gene expression regulation. Single-cell chromatin-accessibility assays now profile hundreds of thousands of cells, challenging existing methods for mapping cis-regulatory interactions. RESULTS: We present CIRCE, a fast and scalable Python package to predict cis-regulatory DNA interactions from single-cell chromatin accessibility data. CIRCE re-implements the Cicero workflow to analyse single-cell atlases, cutting runtime and memory use by several orders of magnitude. We also provide new options to compute metacells, grouping similar cells to reduce data sparsity. We benchmarked CIRCE against Cicero on two datasets of different sizes and demonstrated the improvement from CIRCE's metacells' strategy with promoter capture Hi-C data. We also evaluated how DNA interaction predictions are impacted by different pre-processing. We observed a negative impact of Cicero's count normalization, and the best performance was obtained with the single-cell count matrix directly. Finally, we demonstrated the scalability of CIRCE by processing a dataset of more than 700 000 cells and 1 million DNA regions in less than an hour. CIRCE should greatly facilitate the prediction of DNA region interactions for scverse and Python users, while providing new and up-to-date pre-processing insights. AVAILABILITY AND IMPLEMENTATION: CIRCE is released as an open-source software under the AGPL-3.0 licence. The package source code is available on GitHub at https://github.com/cantinilab/CIRCE, and its documentation is accessible at https://circe.readthedocs.io. The code to reproduce the presented results is available as a Snakemake pipeline at https://github.com/cantinilab/circe_reproducibility.s.
Software↗
Cre/loxP-mediated adenovirus type 5 packaging signal excision demonstrates that core element VI is sufficient for virus packaging.
Previous analyses have demonstrated that packaging of the adenovirus type 5 (Ad5) genome is dependent on at least seven cis-acting elements, called AI to AVII, which are located in the left-end region of the genome. These elements have different packaging efficiencies, and without AI through AV, viral DNA cannot be packaged. Here we report the identification of the cis-acting Ad5 packaging domain in vivo by using the Cre/loxP system. We found that an adenoviral DNA fragment (nt 192 to nt 358), which includes elements AI to AV, is excised by Cre recombinase and packaged into capsids. Furthermore, this mutant adenovirus replicated so efficiently by repetitive propagation that its purification by CsCI equilibrium gradient was possible. This study clarified that the region from nt 358 to nt 454 on the viral genome is sufficient for packaging. Recently, the helper-dependent adenoviral vector (HDAd) construction system has been developed for the purpose of gene therapy. This system uses a helper virus with two parallel loxP sites flanking the packaging signal. This region is eliminated by Cre-mediated excision, which prevents helper virus packaging. Our data provide useful information regarding factors affecting efficient elimination.
Adenoviruses, Human↗
Identification of cis-acting sequences required for selective packaging of bovine adenovirus type 3 DNA.
The assembly of adenovirus particles is a multistep process, in which viral genomic DNA is selected and subsequently inserted into preformed empty capsids. The selective encapsidation of the adenovirus genome is directed by cis-acting packaging motifs, termed A repeats due to their AT-rich character in DNA sequence. A repeats are usually located at the left end of the viral genome. In this report, the construction and analysis of bovine adenovirus type 3 (BAdV-3) mutants containing deletion mutations introduced into the AT-rich regions are described. The main cis-acting packaging domains of BAdV-3 were localized between nt 224 and 540 relative to the left end of the viral genome. They displayed a functional redundancy and followed a hierarchy of importance. In addition, the results demonstrated that not all of the AT-rich units functioned as cis-acting packaging motifs.
Base Sequence↗
[Insertion of the cos-site into DNA of phage M13 and its packing in proteins of phage lambda].
The cos-site of lambda phage from pHC79 cosmide is transferred to DNA from M13 mp18 phage. The recombinant DNA thus obtained (MC18) is efficiently packaged into lambda proteins in vitro. The BamHI-HindIII fragment of pGP588 (a pBR322 derivatives containing fragment of human DNA) is subcloned into MC18. Although this pGP588 fragment contains numerous Alu repeats, no essential rearrangements of the insert were revealed. The efficiency infection by recombinant DNA packaged with lambda proteins is about 1 X 10(5) pfu/microgram DNA, whereas in the similar conditions the efficiency of lambda EMBL3A was 1 X 10(6) pfu/microgram. It is assumed that the MC vectors might be suitable for cloning and sequencing large fragments either with cohesive or blunt ends. It opens also the way to construct genomic libraries in single-stranded phages.
Bacteriophage lambda↗
Morphological variation in folded duplex DNA chains.
I have monitored the packaging process of giant DNA chains with various condensing agents, such as polyamines, polyethylene glycol having pendant aminogroups and a lipospermine, under fluorescence microscopy. The detail morphological structure of the compacted DNA has been observed using electron microscopy. It has become clear that the difference in the folding process results in a large polymorphism of compacted DNA. I discuss the biological importance of controlling the compacted structure.
Bacteriophage T4↗
Herpes simplex virus type 1 cleavage and packaging proteins UL15 and UL28 are associated with B but not C capsids during packaging.
At least seven viral genes encode proteins (UL6, UL15, UL17, UL25, UL28, UL32, and UL33) that are required for DNA cleavage and packaging of herpes simplex virus type 1 (HSV-1) DNA. Sequence analysis reveals that UL15 shares homology with gp17, the large catalytic subunit of the bacteriophage T4 terminase. Thus, UL15 may play a direct role in the cleavage of viral DNA replication intermediates into monomers. In this study, we asked whether UL15 and other cleavage and packaging proteins could be detected in capsids isolated from infected cells. Consistent with previous studies showing that UL6 and UL25 are minor protein constituents of the capsids, we detected these proteins in both B and C capsids. In contrast, the previously identified full-length version (81 kDa) of UL15 was found predominantly in B capsids and in much smaller amounts in C capsids. In addition, the UL28 protein was found predominantly in B but not C capsids in a distribution similar to that of the 81-kDa version of UL15. These results suggest that UL28 and the 81-kDa form of UL15 are transiently associated with capsid intermediates during the packaging process. Surprisingly, however, a previously unidentified 87-kDa form of UL15 was found in the B and C capsids and in virions. Analysis of cells infected with mutants individually lacking UL6, UL15, UL25, UL28, or UL32 demonstrates that the lack of one cleavage and packaging protein does not affect the expression of the others. Furthermore, this analysis, together with guanidine HCl extraction analysis of purified capsids, indicates that UL6, UL25, and UL28 are able to associate with B capsids in the absence of other DNA cleavage and packaging proteins. On the other hand, the two UL15-related proteins (81 and 87 kDa) do not associate efficiently with B capsids in cells infected with UL6 and UL28 mutants. These results suggest that the ability of the UL15-related proteins to bind to B capsids may be mediated through interactions with UL6 and UL28.
Animals↗