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At least 19 recordsLinked to original sources
PubMed1992

RNA-primed complementary-sense DNA synthesis of the geminivirus African cassava mosaic virus.

The plant DNA virus African cassava mosaic virus (ACMV) is believed to replicate by a rolling circle mechanism. To investigate complementary-sense DNA (lagging strand) synthesis, we have analysed the heterogenous form of complementary-sense DNA (H3 DNA) from infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and blot hybridisation. The presence of an RNA moeity is demonstrated by comparison of results for nucleic acids resolved on neutral/alkaline and neutral/formamide gels, suggesting that complementary-sense DNA synthesis on the virus-sense single-stranded DNA template is preceded by the synthesis of an RNA primer. Hybridisation with probes to specific parts of ACMV DNA A genome indicates that synthesis of the putative RNA primer initiates between nucleotides 2581-221, a region that includes intergenic sequences that have been implicated in geminivirus DNA replication and the control of gene expression.

DNA, Viral↗
PubMed1992

Inhibition of human immunodeficiency virus-1 proliferation by liposome-encapsulated sense DNA to the 5' tat splice acceptor site.

A liposome formulation containing a distearoylphosphatidylethanolamine analog was developed that was endocytosed by both lymphocytes and monocytes. This formulation was used to encapsulate sense and antisense 20-mer oligodeoxynucleotides to the 5' tat splice acceptor site of human immunodeficiency virus type 1. At a DNA concentration of 140 nM, the liposome-encapsulated sense DNA inhibited p24 production by as much as 84% in human peripheral blood leukocytes infected with "wild-type" virus. This treatment also reduced the number of peripheral blood leukocytes producing intracellular viral antigen by 71%. Of interest, no reduction in either parameter was observed for the antisense-containing liposomes. The results demonstrate the promise of a new liposomal delivery vehicle to inhibit human immunodeficiency virus replication by an entrapped oligodeoxynucleotide.

Antiviral Agents↗
PubMed2026

Defective RNA Polymerase III sensing of mitochondrial DNA in pulmonary epithelial cells impairs type I IFN immunity to SARS-CoV-2.

The clinical spectrum of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection ranges from asymptomatic cases to critical COVID-19 pneumonia. To investigate the role of host genetics in susceptibility to critical COVID-19 and identify pathophysiological mechanisms and pathways, we analyzed whole-exome and whole-genome sequencing data from the COVID Human Genetic Effort. We identified 10 rare, monoallelic predicted loss-of-function variants in 18 patients in POLR3A and POLR3C encoding two subunits of RNA polymerase III (POL III), a nuclear multisubunit enzyme, which has been implicated in cytosolic DNA sensing. These variants were deleterious for expression of full-length POLR3A and POLR3C proteins. We demonstrate that human pulmonary A549-hACE2 cells with reduced POLR3A or POLR3C expression exhibit impaired type I IFN responses to transfected mitochondrial DNA (mtDNA) or SARS-CoV-2 infection, together with increased viral replication. Mechanistically, we show that SARS-CoV-2 induces cellular mtDNA release via oligomerization of the mitochondrial voltage-dependent anion channel under virus-induced oxidative stress, enabling POL III-mtDNA interaction. These findings establish POL III as a sensor of endogenous mtDNA released during viral infection and indicate that autosomal dominant POL III haploinsufficiency may predispose individuals to critical COVID-19.

Humans↗
PubMed1991

DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication.

We have analysed DNA from African cassava mosaic virus (ACMV)-infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot-hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND-cellulose and their ability to hybridise to strand-specific and double-stranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virus-sense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2).

Chromatography↗
PubMed2025

Quorum sensing and DNA methylation play active roles in clinical Burkholderia phase variation.

Phenotypic diversity in bacteria often results from adaptation to changing environmental conditions, exemplified by variable colony morphotypes. In Burkholderia pseudomallei, discrete genomic alterations and modulation of gene expression facilitate adaptation. Adapted variants of species within the Burkholderia cepacia complex (Bcc) often lose the pC3 virulence megaplasmid, impacting their colony morphology and their production of virulence factors. In this study, we characterize variants arising in Burkholderia ambifaria clinical isolates using proteomics and phenotypic tests and show that some of them have retained the pC3, indicating a distinct phase variation mechanism at play in this Bcc species. Interestingly, variants of B. ambifaria strains CEP0996 (pC3-null) and HSJ1 (pC3-positive) still share similarities in phenotypes controlled by the Cep quorum-sensing (QS) system. We further investigated the role of QS in B. ambifaria HSJ1 phase variation and confirmed that the Cep QS system is important for the emergence of variants. Given that DNA methylation is a key epigenetic factor regulating virulence factors in Burkholderia cenocepacia, we hypothesized that adenosine DNA methylation also governs phase variation in B. ambifaria HSJ1. By deleting the genes encoding putative adenosine DNA methyltransferases, we discovered that an orphan type II DNA methyltransferase inhibits the emergence of phase variants. This study is the first to demonstrate that quorum sensing and adenosine DNA methylation are two antagonistic systems independently controlling phase variation in B. ambifaria.IMPORTANCESome Burkholderia species are pathogenic to plants, animals, or humans. In immunocompromised individuals, and people suffering from cystic fibrosis, infection from the Burkholderia cepacia complex (Bcc) can lead to "cepacia syndrome." In northern Australia and southeast Asia, melioidosis caused by Burkholderia pseudomallei is prevalent among native population, particularly among people with diabetes, chronic lung or kidney disease or alcoholism. Burkholderia's phenotypic plasticity, including colony morphotype variation (CMV), enables rapid adaptation to diverse environments, enhancing its survival and pathogenicity. This study reveals phase variation as a new CMV mechanism within the Bcc group and is the first to report that quorum sensing and DNA methylation are involved in phase variation. Understanding the underlying mechanisms of CMV could lead to the development of targeted therapies against these highly antibiotic-tolerant bacteria.

Quorum Sensing↗
PubMed1979

Split genes and RNA splicing.

A number of genes in higher organisms and in their viruses appear to be split. That is, they have "nonsense" stretches of DNA interspersed within the sense DNA. The cell produces a full RNA transcript of this DNA, nonsense and all, and then appears to splice out the nonsense sequences before sending the RNA to the cytoplasm. In this article what is known about these intervening sequences and about the processing of the RNA is outlined. Also discussed is their possible use and how they might have arisen in evolution.

Animals↗
PubMed1992

Geminivirus replication proteins are related to prokaryotic plasmid rolling circle DNA replication initiator proteins.

It is demonstrated, by means of computer-assisted analysis, that C1 protein involved in the replication of geminivirus DNA is related to the rolling circle replication initiator proteins of eubacterial plasmids, particularly the plasmids of the pMV158 family. Three sequence motifs conserved in the geminivirus and plasmid replication proteins were delineated, one of them encompassing the Tyr residue that presumably forms a covalent linkage to DNA. These findings are compatible with the results of recent analyses of geminivirus replicative intermediates suggesting a rolling circle mechanism for geminivirus DNA replication. It is hypothesized that C1 protein initiates the rolling circle replication of geminivirus DNA by nicking a specific site in the virus-sense DNA and covalently linking to the 5' side of the nick. The putative rolling circle replication initiator domain comprises the N-terminal portion of C1, whereas its C-terminal part is a putative helicase domain. By analogy with prokaryotic systems, it is speculated that the replication initiator domain and the helicase domain function coordinately. The possibility of the origin of geminiviruses from prokaryotic circular ssDNA replicons is discussed.

Amino Acid Sequence↗
PubMed1992

E-cadherin expression: a counterbalance for cancer cell invasion.

Invasion, eventually leading to metastasis, is presented as the result of a balance between the activation of 2 sets of genes, coined i+ (invasion promotor) and i- (invasion suppressor) genes. Experiments in vitro have indicated that the homotypic homophilic epithelial cell--cell adhesion molecule E-cadherin (L-CAM; uvomorulin; cell CAM 120/80; Arc-1; rrl antigen) is an i- gene product. In several cell families, manipulation of E-cadherin at the level of the protein by antibody-mediated inactivation, at the level of the mRNA by antisense DNA transfection, and at the level of the genome by sense DNA transfection respectively resulted in induction and suppression of invasiveness. Nude mouse tumors from non-invasive homogeneously E-cadherin-positive cell populations were found to be invasive and metastatic. These tumors expressed E-cadherin in a heterogeneous manner, the undifferentiated cells being negative; but tumor-derived cells in culture were again E-cadherin-positive, indicating downregulation of this protein by host factors. Several types of human cancers showed a similar heterogeneity suggesting a relationship between downregulation of E-cadherin and invasion. Our current research focus is on the factors responsible for E-cadherin downregulation in experimental and human cancers.

Animals↗
PubMed1992

Cyclin A is required in S phase in normal epithelial cells.

We have investigated cyclin A expression in a primary culture of normal rat hepatocytes and during rat liver regeneration after partial hepatectomy. In both cases, cyclin A mRNA and protein accumulate as the cells enter S phase. To investigate the potential implication of cyclin A accumulation at S phase, we microinjected anti-sense DNA constructs for cyclin A, resulting in effective inhibition of S phase entry. These effects were specific for cyclin A since anti-sense cyclin B construct had no similar effects. These results therefore, obtained in normal epithelial cells, indicate that cyclin A is involved in S phase and thus should not be only considered as a mitotic cyclin.

Animals↗
PubMed1992

Inhibition of class II MHC gene expression by anti-sense RNA in transgenic mice.

We have established transgenic mice carrying the anti-sense DNA to the gene encoding beta chain of the class II major histocompatibility complex (I-A) molecule. The amount of I-A molecule on splenic B lymphocytes from the mice was reduced in the presence of a large amount of the exogenous anti-sense RNA. The amount of I-A beta chain RNA was selectively reduced and inversely correlated with the amount of anti-sense RNA in the spleens. These results suggest that the I-A beta chain RNA is rapidly degraded by duplex formation with the anti-sense RNA in splenic B cells from the transgenic mice.

Animals↗
PubMed2026

cGAS-STING signaling in aging and age-related diseases: therapeutic promise and precaution.

Endogenous cytoplasmic DNA (cytoDNA) is increasingly recognized as a mediator of tissue dysfunction and disease progression during aging. As a major cytosolic DNA-sensing pathway, the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway can translate aging-associated cytoDNA accumulation into innate immune and inflammatory programs. This review summarizes the evolutionary and signaling features of the cGAS-STING cascade and critically discusses its crosstalk with aging-associated intracellular molecular threats, including nuclear genomic and chromatin stress, mitochondrial dysfunction, oxidative-metabolic stress, and defective clearance of nucleic acids or damaged organelles. We further synthesize evidence linking dysregulated cGAS-STING activation to inflammatory remodeling, senescence-associated changes, cell injury, fibrosis, and tissue dysfunction, while highlighting the context-dependent roles of this pathway across physiological aging and ARDs. Finally, we discuss the therapeutic potential and limitations of cGAS-STING modulation, emphasizing that successful translation will require context-defined therapeutic windows, tissue- and cell-specific targeting, subcellular compartmentalization, and long-term safety assessment.

Humans↗
PubMed2026

Proteomics-based analysis of the defense mechanisms of disease-resistant grass carp against Aeromonas veronii.

Sustainable aquaculture of grass carp (Ctenopharyngodon idella, GC) is consistently threatened by bacterial diseases, particularly those caused by Aeromonas veronii. A disease-resistant grass carp (DR-GC) has been developed by backcrossing female gynogenetic GC with normal male GC, exhibiting improved resistance. However, the systemic molecular mechanisms of DR-GC defending against Aeromonas veronii infection remain largely unexplored. Here, a label-free quantitative proteomics approach was employed to systematically compare proteomic profiles across five tissues (intestine, liver, muscle, skin, and kidney) in DR-GC and GC under healthy and infected conditions. The intestine was identified as the central defense tissue, exhibiting the highest number of differentially abundant proteins (DAPs). In DR-GC, A0A3N0YEK7 (small ribosomal subunit protein eS28), A0A3N0YGT8 (ATP synthase-coupling factor 6) and A0A3N0YNS7 (apolipoprotein A-I) were significantly upregulated in intestine, while D5KZW6 (GCHV-induced protein), A0A3N0Z0A1 and Q8JH84 (hemoglobin subunit alpha) were significantly dysregulated across multiple tissues, which playing the critical roles in defense mechanisms at the protein level. Furthermore, cytochrome P450-associated pathways, cytosolic DNA-sensing and RIG-I-like receptor signaling pathways were identified as crucial coordinators mediating immune and metabolic responses. This study provides the first comprehensive proteomic view of multi-tissue defense mechanisms in DR-GC, and identifies key DAPs and pathways for subsequent functional validation.

Animals↗
PubMed1990

Deregulation of DNA polymerase beta by sense and antisense RNA expression in mouse 3T3 cells alters cell growth.

DNA polymerase beta (beta-pol) and its mRNA are maintained at constitutive levels during the cell cycle and during stages of cell growth in culture. To study biological consequences of variations in the level of this DNA repair enzyme and/or its mRNA, we prepared expression vectors in which cDNA for human beta-pol is inserted under the control of a metallothionein promoter (pMT) in the sense and antisense orientation, respectively, and these vectors then were used for stable transformation of mouse 3T3 cells. Vectors also contained the mouse DHFR gene, such that culture of transformants in medium with increasing concentrations of methotrexate resulted in amplification of inserted DNA. The levels of sense and antisense transcripts are strongly increased by culture of transformants in medium with 65 microM Zn2+, although some expression is detected even without Zn2+ induction. After five days of induction, the beta-pol level was about threefold higher in sense cells and about 10-fold lower in antisense cells than in parallel cultures without induction. The antisense line has a threefold increased cell doubling time in the presence of 65 microM Zn2+ compared with the absence of Zn2+. Zn2+ (65 microM) induction for the sense line results in normal growth for the first three days and, thereafter, a complete cessation of growth. Yet, these blocked cells remain fully viable. The results indicate that sudden deregulation of beta-pol expression alters cell growth in mouse 3T3 cells.

Animals↗
PubMed1992

Overexpression of SPARC in stably transfected F9 cells mediates attachment and spreading in Ca(2+)-deficient medium.

The Ca(2+)-binding protein SPARC is one of a group of proteins that function in vitro to promote the rounding of cells. To assess whether the modulation of cell shape by SPARC is affected by extracellular Ca2+, we used F9 cell lines that had been stably transfected with sense or antisense SPARC DNA. Sense-transfected (S) lines that overexpress SPARC are aggregated and rounded, whereas antisense (AS) lines that express low levels of the protein are flat and spread. We tested whether the cell lines would exhibit these altered morphologies in Ca(2+)-deficient media. When cultured under these conditions, S lines attached and spread, whereas AS lines attached but remained round, with no subsequent spreading. Addition of CaCl2 or purified SPARC to the Ca(2+)-deficient medium resulted in spreading of the AS and control lines and a reappearance of the altered morphologies. Expression of the Ca(2+)-binding cadherin uvomorulin by the cell lines correlated with neither their morphology nor their level of SPARC expression. We conclude that the altered phenotypes of the transected lines reflect, in part, the concentration of extracellular Ca2+ and that the spreading exhibited by the S lines under Ca(2+)-deficient conditions is directly related to their enhanced expression of SPARC. SPARC might, therefore, mediate interactions between cells and matrix that are permissive for adhesion when levels of extracellular Ca2+ are diminished.

Animals↗
PubMed2026

Gabija restricts phage circularization and DNA replication.

Anti-bacteriophage systems such as restriction-modification and CRISPR-Cas have DNA substrate specificity mechanisms that enable the identification of invaders. How Gabija, a highly prevalent nuclease-helicase antiphage system, limits phage replication while executing self- vs. non-self-discrimination remains unknown. Here, we show that phage-encoded DNA end-binding proteins that antagonize host RecBCD sensitize phages to Gabija. When targeting a temperate lambda-like phage in Pseudomonas aeruginosa, Gabija prevents phage genome circularization and subsequent replication. DNA end-binding complexes, including a phage exonuclease and a single-stranded DNA (ssDNA)-annealing protein or GamMu dimers that prevent loading of the host repair complex RecBCD, are necessary and sufficient to license phage and plasmid sensitivity to Gabija. Mutant escape phages lacking these DNA end-binding proteins become protected from Gabija by RecBCD translocation activities. RecBCD activity on the bacterial genome, presumably whenever it is linearized, also prevents Gabija from targeting self-DNA. Therefore, we propose that Gabija antagonizes the circularization and replication of linear DNA devoid of RecBCD as a mechanism to identify and antagonize foreign invaders.

DNA Replication↗
PubMed1991

Transcript mapping of Abutilon mosaic virus, a geminivirus.

The transcripts of the DNA of Abutilon mosaic virus, a geminivirus with a bipartite genome (DNA A and DNA B), were characterized by Northern blot hybridization, S1 nuclease assay, primer extension analysis, and sequencing of the 3' termini of cDNA clones. It was shown that transcription is bidirectional and that the transcripts are polyadenylated. Two overlapping transcripts of 1.6 and 0.7 kb were mapped to the complementary strand of DNA A and two of 1.3 and 1.2 kb to the complementary strand of DNA B. One transcript of 0.9 kb was mapped to the viral sense in DNA A and one of 1.0 kb to the viral sense in DNA B. The ends of complementary and viral mRNA overlapped in both genome parts in regions rich in polyadenylation signals.

Amino Acid Sequence↗
PubMed1991

Analysis of the potential promoter sequences of African cassava mosaic virus by transient expression of the beta-glucuronidase gene.

DNA fragments from promoter regions of the geminivirus, African cassava mosaic virus, were cloned into pG1, a vector based on pUC18, producing transcriptional fusions with the beta-glucuronidase (GUS) gene and nopaline synthase termination sequence. The activity of each promoter construct was assessed by analysing the transient expression of GUS in Nicotiana clevelandii protoplasts. The results demonstrated that constructs containing the common region of DNA A showed much stronger promoter activity in the complementary sense than in the viral sense. These results were supported by the analysis of promoter activity in transgenic N. benthamiana plants. In comparison, in protoplasts a region upstream of the AC2 open reading frame was shown to have moderate promoter activity. Unlike DNA A, the complementary sense DNA B promoter constructs had weak activity; the viral sense DNA B promoter constructs appeared to be regulated by host factors. The implications of these results for the regulation of early and late genes are discussed.

Base Sequence↗