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At least 109 records · Page 6Linked to original sources
PubMed2025

Pentatricopeptide repeat protein targeting CUG repeat RNA ameliorates RNA toxicity in a myotonic dystrophy type 1 mouse model.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disorder caused by the expansion of a CTG-triplet repeat in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. It results in the transcription of toxic RNAs that contain expanded CUG repeats (CUGexp). Splicing factors, such as muscleblind-like 1 (MBNL1), are sequestered by CUGexp, thereby disrupting the normal splicing program that is essential for various cellular functions. Pentatricopeptide repeat (PPR) proteins, originally found in plants, regulate RNA in organelles by binding in a sequence-specific manner. Here, we designed PPR proteins that specifically bind to the hexamer of CUG repeat RNAs (CUG-PPRs) and showed that CUG-PPR1 could ameliorate RNA toxicity induced by CUGexp in cell models of DM1. A single systemic recombinant adeno-associated virus (AAV9) vector-mediated gene delivery of CUG-PPR1 demonstrated long-term therapeutic effects on myotonia and restored splicing activity in a mouse model of DM1. These results highlight the potential of PPR molecules to target pathogenic RNA sequences in DM1 and potentially other RNA-mediated disorders.

Animals↗
PubMed1992

NF-IL6, a member of the C/EBP family, regulates E1A-responsive promoters in the absence of E1A.

A cDNA encoding NF-IL6, an interleukin-6 (IL-6)-regulated human nuclear factor of the C/EBP family, is demonstrated to complement the transactivation function of E1A. The endogenous NF-IL6 level varies according to cell type and correlates positively with an IL-6-regulated cellular E1A-substituting activity that was described recently (J.M. Spergel and S. Chen-Kiang, Proc. Natl. Acad. Sci. USA 88:6472-6476, 1991). When expressed by transfection in cells which contain low levels of NF-IL6 and are incapable of complementing the function of E1A proteins, NF-IL6 also transactivates the E1A-responsive E2ae and E1B promoters, to the same magnitude as E1A. Activation by NF-IL6 is concentration dependent and sequence specific: mutational studies of the E2ae promoter suggest that the promoter-proximal NF-IL6 recognition site functions as a dominant negative regulatory site whereas the promoter-distal NF-IL6 recognition site is positively regulated at low NF-IL6 concentrations and negatively regulated when the NF-IL6 level is high. Consistent with these functions, NF-IL6 alone is sufficient to complement an E1A deletion mutant dl312 in viral infection, when expressed at appropriate concentrations. These results identify NF-IL6 as a sequence-specific cellular nuclear factor which regulates E1A-responsive genes in the absence of E1A.

Adenovirus Early Proteins↗
PubMed1992

Interaction of Epstein-Barr virus nuclear antigen 1 with the viral latent origin of replication.

The Epstein-Barr virus latent origin of replication (oriP) requires only one viral protein, the Epstein-Barr virus nuclear antigen 1 (EBNA-1), for activity. oriP consists of two spatially separated, essential sequence elements, regions I and II, both of which contain multiple EBNA-1-binding sites. Region II contains, or is close to, the site at which DNA synthesis initiates. The role of region I, a transcriptional enhancer in cells that express EBNA-1, in replication is not understood. To determine how the binding of EBNA-1 to sites in region II leads to the initiation of DNA synthesis and to investigate the role of region I, EBNA-1 has been overproduced in insect cells by using a baculovirus vector and purified to homogeneity, and the interaction of EBNA-1 with oriP has been examined. Footprinting experiments demonstrated that EBNA-1 binds to oriP in a sequence-specific manner and bends or untwists the DNA at two symmetry-related sites in region II. Distortion of region I by EBNA-1 was not detected, suggesting that differences in the spacing of binding sites in regions I and II and resulting protein-protein interactions underlie differences in their biological properties. KMnO4 footprinting experiments did not reveal significant single-stranded structures in region II, suggesting that cellular proteins may recognize the EBNA--region II complex and unwind the DNA duplex. Region I did not quantitatively or qualitatively alter the interaction of EBNA-1 with region II. The contribution of an A + T-rich sequence in region II to replication was investigated by a mutational analysis. The results indicated that the overall A + T-rich nature of this sequence is not essential for replication of oriP-bearing plasmids. Nuclease protection experiments performed with these mutagenized plasmids provided additional evidence for protein-protein interactions in region II.

Animals↗
PubMed1992

Characterization of a herpes simplex virus sequence which binds a cellular protein as either a single-stranded or double-stranded DNA or RNA.

Earlier we reported that herpes simplex virus 1 DNA contains a sequence which binds a host protein in a sequence-specific manner as either a single-stranded or a double-stranded DNA or RNA and that this sequence is located in a transcriptional unit whose RNA traverses the origin of viral DNA replication (OriSRNA) (R.J. Roller, L. McCormick, and B. Roizman, Proc. Natl. Acad. Sci. USA 86:6518-6522, 1989). The protein reacts with both DNA and RNA in band-shift assays and protects the single-stranded RNA sequence from digestion by RNase. We report that the minimal cognate sequence required for these interactions consisted of [N(GTGGGTGGG)2(N less than or equal to 10)]. The ninemer repeat sequence was located at nucleotides -1 to -18 relative to the transcription initiation of the major species of OriSRNA. The activity of the cognate sequence required at least three guanines between thymines and tolerates the insertion of additional thymines, but it was inactivated by the insertion of adenines or by the substitution of some of the guanines with cytosines in one repeat. Replacement of the 10 3' nucleotides has no effect on binding activity, whereas deletion of these sequences abolished it. Among the related sequences with no demonstrable binding activity were some telomeric sequences which interact with known cognate proteins. The electrophoretic mobility of the herpes simplex virus cognate sequences in nondenaturing gels suggests that they may be able to form higher-order structures, but the conditions under which they were formed were different from the optimal conditions for binding the protein. UV light cross-linking studies of labeled RNA-protein complexes following digestion with RNases indicated that the electrophoretic mobility of the protective activity corresponded to that of a protein with an apparent molecular weight of 100,000.

Base Sequence↗
PubMedDate not supplied

Antisense oligonucleotide-mediated inhibition of metallothionein protein synthesis in neuroblastoma IMR 32 and Chang liver cells in culture.

A synthetic antisense oligodeoxyribonucleotide with sequence complementary to the messenger RNA (mRNA) coding for human metallothionein (MT) II was prepared and tested for its ability to inhibit both constitutive- and cadmium-induced MT protein synthesis in neuroblastoma-IMR and Chang liver cells in culture. The sense oligonucleotide was also prepared and tested as a control for its sequence-specific effects. Oligonucleotide entry into cells was enhanced through the use of a polybrene carrier so that nearly 30% of a 10 microM dose of oligonucleotide was shown to be associated with cells. The antisense oligonucleotide inhibition of MT protein synthesis rendered both cell types more sensitive to cadmium toxicity. However, the sense oligonucleotide had no effects on either MT protein synthesis or sensitivity to cadmium toxicity.

Base Sequence↗
PubMed1975

X inactivation, differentiation, and DNA methylation.

A model based on DNA methylation is proposed to explain the initiation and maintenance of mammalian X inactivation and certain aspects of other permanent events in eukaryotic cell differentiation. A key feature of the model is the proposal of sequence-specific DNA methylases that methylate unmethylated sites with great difficulty but easily methylate half-methylated sites. Although such enzymes have not yet been detected in eukaryotes, they are known in bacteria. An argument is presented, based on recent data on DNA-binding proteins, that DNA methylation should affect the binding of regulatory proteins. In support of the model, short reviews are included covering both mammalian X inactivation and bacterial restriction and modification enzymes.

Animals↗
PubMed1979

Cytomegalovirus strain differentiation by DNA restriction analysis.

The heterogeneity of CMV DNA obtained from standard strains and new isolates, including a vaccination strain (Towne 125), was investigated. The cleavage patterns produced by the restriction endonucleases Eco RI and Bam 1 revealed stable strain specificities of CMV. On the other hand, a remarkable homology of sequence-specific CMV DNA fragmentation was demonstrated. A CMV subtyping relevant to clinical questions seems to be improbable.

Antigens, Viral↗
PubMed1978

Characterization and application of a radioimmunoassay for reduced, carboxymethylated human luteinizing hormone alpha-subunit.

We have established a double antibody RIA using a rabbit antiserum prepared against reduced, carboxymethylated (RCXM) human LH alpha-subunit, with RCXM-alpha as tracer and standard. This antiserum did not cross-react with any native gonadotropins or subunit, and reacted only weakly with RCXM-alpha. A tryptic digest of RCXM alpha-subunit was completely reactive, while chymotryptic digestion abolished all immunoreactivity. By testing with separate tryptic fragments, the recognition site could be localized to a segment close to the amino-terminus of the peptide chain. When applied to measurement of serum and urine, an immunoreactive species, parallel to RCXM alpha-subunit by serial dilution, was found in concentrations of 1-2 ng/ml in serum and 3-4 ng/ml in urine. Similar levels of the immunoreactive component were found in conditions of elevated gonadotropins (e.g. pregnancy) as well as gonadotropin deficiency(panhypopituitarism and Kallmann's syndrome). After stimulation with LHRH, no rise was noted at times up to 6 h despite the fact that both LH and LH-alpha were elevated. The data indicate that the sequence-specific antiserum may be detecting an immunoreactive form of alpha-subunit of LH whose kinetics of appearance and disappearance differs from those of the native subunit.

Amino Acid Sequence↗
PubMed1978

Carboxyl-terminal fragments of human parathyroid hormone in parathyroid tumors: unique new source of immunogens for the production of antisera potentially useful in the radioimmunoassay of parathyroid hormone in human serum.

We have found large quantities of immunoreactive carboxyl-terminal fragments of human parathyroid hormone )hPTH) in a previously discarded fraction [the 7.5% trichloroacetic acid (TCA)supernate] generated during extraction of intact hPTH from hyperfunctioning parathyroid tissue by the urea-TCA procedure. It is well established that serum RIAs directed toward the carboxyl-terminal region of hPTH are superior to those directed toward the amino-terminal region in the differential diagnosis of patients with suspected chronic parathyroid dysfunction. However, antisera that react with the carboxyl-terminal region of hPTH are not yet available for general use for these assays because of a lack of suitable hPTH immunogens. We immunized seven guinea pigs and two goats with the desalted 7.5% TCA supernate (containing about 2% carboxyl-terminal hPTH fragments); three of the guinea pigs and one goat produced high affinity antisera with predominant specificity for the carboxyl-terminal region of PTH. One of the guinea pig antisera had affinity for hPTH equal to that of our laboratory's best antiserum (GP1M) used in diagnostic RIAs for serum PTH. The use of this byproduct fraction as an immunogen should permit a large scale immunization program in large animals to provide standardized, species-and sequence-specific antisera potentially useful in RIAs for diagnosis of parathyroid disease.

Adenoma↗
PubMed2025

An updated compendium and reevaluation of the evidence for nuclear transcription factor occupancy over the mitochondrial genome.

In most eukaryotes, mitochondrial organelles contain their own genome, usually circular, which is the remnant of the genome of the ancestral bacterial endosymbiont that gave rise to modern mitochondria. Mitochondrial genomes are dramatically reduced in their gene content due to the process of endosymbiotic gene transfer to the nucleus; as a result most mitochondrial proteins are encoded in the nucleus and imported into mitochondria. This includes the components of the dedicated mitochondrial transcription and replication systems and regulatory factors, which are entirely distinct from the information processing systems in the nucleus. However, since the 1990s several nuclear transcription factors have been reported to act in mitochondria, and previously we identified 8 human and 3 mouse transcription factors (TFs) with strong localized enrichment over the mitochondrial genome using ChIP-seq (Chromatin Immunoprecipitation) datasets from the second phase of the ENCODE (Encyclopedia of DNA Elements) Project Consortium. Here, we analyze the greatly expanded in the intervening decade ENCODE compendium of TF ChIP-seq datasets (a total of 6,153 ChIP experiments for 942 proteins, of which 763 are sequence-specific TFs) combined with interpretative deep learning models of TF occupancy to create a comprehensive compendium of nuclear TFs that show evidence of association with the mitochondrial genome. We find some evidence for chrM occupancy for 50 nuclear TFs and two other proteins, with bZIP TFs emerging as most likely to be playing a role in mitochondria. However, we also observe that in cases where the same TF has been assayed with multiple antibodies and ChIP protocols, evidence for its chrM occupancy is not always reproducible. In the light of these findings, we discuss the evidential criteria for establishing chrM occupancy and reevaluate the overall compendium of putative mitochondrial-acting nuclear TFs.

Genome, Mitochondrial↗
PubMed2026

OligoSeq: Rapid nanopore-sequencing of single-stranded oligonucleotides.

Nanopore-based DNA sequencing technology has achieved remarkable success in sequencing increasingly long DNA strands (e.g., over a million nucleotides long) for genomics research and biotechnology applications. However, the same level of progress has not been achieved for DNA oligonucleotides (usually ≤ 300 nucleotides long). Oligonucleotides play a crucial role in genome engineering efforts through oligo library generation and in DNA data storage, where they are used to encode computer information, such as binary (digital) data in DNA libraries. To enable these applications, accurate sequencing of oligonucleotides in a way that allows to assess for sequence variability, quality and length is essential. But sequencing solutions for oligonucleotides - particularly DNA primers for PCR, oligo DNA libraries used for mutagenesis or cDNA libraries used in gene expression analysis - remain inadequate. To address this gap, OligoSeq is presented as an innovative approach that integrates two complementary techniques: AmpliSeq (based on PCR) and RevSeq (based on reverse complementation with sequence-specific or random primers) to facilitate sequencing of single-stranded oligonucleotides using reference sequence anchor matches of more than ≥ 90% identity spanning from about 70% to 10% with AmpliSeq or RevSeq with random nonamers, respectively, and resolving the final reference sequence based on the most likely candidate from basecall frequencies, regardless of length and double-stranding method. OligoSeq can be integrated with nanopore sequencing technology pipelines and can be used as a reference for other sequencing platforms requiring double-stranded adapters, offering a practical and scalable alternative for standard quality control in single-stranded oligonucleotide synthesis. The use of nanopore technology, compatible with the double-stranding methods showcased, is shown to be the most cost-effective method for resolving original DNA sequences of different length and quality, and to assess its sequence variability, compared to other methods such as Illumina, PacBio or HPLC/MS.

Sequence Analysis, DNA↗
PubMed2026

Virus-induced gene silencing as a tool for functional genomics in weeds: Challenges and future directions.

Virus-induced gene silencing (VIGS) has evolved from a conceptual demonstration of antiviral defense into a pivotal reverse-genetics platform for plant functional genomics. By exploiting engineered DNA- or RNA-based viral vectors, VIGS enables rapid, sequence-specific transcript knockdown through RNA-mediated degradation of target transcripts. Recent refinements in vector design, inoculation strategies, and viral species selection, such as TRV, BSMV, and FoMV, have expanded its application to previously recalcitrant plants, including major crops and emerging weed models. In weeds, functional genomics remains particularly challenging due to high genetic variability, limited genomic resources, and incompatibility with conventional viral vectors and transformation systems. In this context, VIGS provides a tractable approach to investigate genes associated with herbicide resistance, metabolic adaptation, and stress tolerance. Beyond weed biology, its application to studies of immune signaling, hormonal crosstalk, and secondary metabolism highlights VIGS as a versatile biotechnology for elucidating gene function and supporting next-generation strategies in plant improvement and integrated pest management.

Journal Article↗
PubMed1992

Regulation of Myc: Max complex formation and its potential role in cell proliferation.

The myc family of proto-oncogenes encodes short-lived nuclear phosphoproteins (Myc) involved in the control of cell proliferation and differentiation. Here we discuss the evidence for Myc's involvement in normal and abnormal cell proliferation and review recent information on Max, a novel protein that forms a sequence-specific DNA-binding complex with Myc. The properties of the Myc: Max heterodimeric complex suggest a model for how Myc may function in the cell.

Animals↗
PubMed2016

Toxin-Antitoxin Systems of Staphylococcus aureus.

Toxin-antitoxin (TA) systems are small genetic elements found in the majority of prokaryotes. They encode toxin proteins that interfere with vital cellular functions and are counteracted by antitoxins. Dependent on the chemical nature of the antitoxins (protein or RNA) and how they control the activity of the toxin, TA systems are currently divided into six different types. Genes comprising the TA types I, II and III have been identified in Staphylococcus aureus. MazF, the toxin of the mazEF locus is a sequence-specific RNase that cleaves a number of transcripts, including those encoding pathogenicity factors. Two yefM-yoeB paralogs represent two independent, but auto-regulated TA systems that give rise to ribosome-dependent RNases. In addition, omega/epsilon/zeta constitutes a tripartite TA system that supposedly plays a role in the stabilization of resistance factors. The SprA1/SprA1AS and SprF1/SprG1 systems are post-transcriptionally regulated by RNA antitoxins and encode small membrane damaging proteins. TA systems controlled by interaction between toxin protein and antitoxin RNA have been identified in S. aureus in silico, but not yet experimentally proven. A closer inspection of possible links between TA systems and S. aureus pathophysiology will reveal, if these genetic loci may represent druggable targets. The modification of a staphylococcal TA toxin to a cyclopeptide antibiotic highlights the potential of TA systems as rather untapped sources of drug discovery.

Antitoxins↗
PubMed2026

miR-197 Targets NLRP3 3' UTR and Correlates with NLRP3/Caspase-1/IL-18 Signaling in Hyperoxia-Stimulated Neonatal BPD Mouse Model.

Reduced circulating miR-197 was previously observed in preterm infants who later developed bronchopulmonary dysplasia (BPD), but its relationship with NLRP3 inflammasome signaling remains unclear. This study examined miR-197 expression, NLRP3 inflammasome-related markers, and the interaction between miR-197 and the NLRP3 3' UTR in a neonatal hyperoxia model. Neonatal C57BL/6J mice were exposed to 60% oxygen or room air from birth, and lung tissues were collected on postnatal days 1, 7, 14, and 21. Lung injury and alveolar development were assessed by histology, radial alveolar count, mean linear intercept, and lung wet-to-dry ratio. miR-197 and NLRP3 expression and NF-κB-, caspase-1-, and IL-18-related proteins were evaluated by RT-qPCR and Western blotting. A dual-luciferase reporter assay in MLE12 cells tested the interaction between miR-197 and the NLRP3 3' UTR. Hyperoxia increased lung wet-to-dry ratios and mean linear intercept, reduced radial alveolar count, and caused progressive alveolar simplification. miR-197 expression decreased, whereas NLRP3 mRNA increased, at all examined time points; NLRP3 protein and inflammasome-related protein changes were most evident from postnatal day 7 onward. The miR-197 mimic reduced luciferase activity in the wild-type but not mutant NLRP3 3' UTR reporter. These findings show that neonatal hyperoxia is associated with reduced miR-197 and increased NLRP3/inflammasome-related signaling and support a sequence-specific interaction between miR-197 and the NLRP3 3' UTR, although causal regulation in vivo requires further investigation.

Animals↗
PubMed1992

[Gene amplification with PCR and sequence specific HLA oligonucleotide typing].

Genetic polymorphism in the HLA class II region has been identified by the analysis of the polymerase chain reaction (PCR) products using sequence-specific oligonucleotide (SSO). The PCR-SSO method permits precise and direct analysis of allelic variations with as little as 1 microgram of genomic DNA. The standardized, uniform hybridization and critical wash protocol of the Eurotransplant typing kit enables HLA typing independent of gene expression and quality of lymphocytes. One of the advantages of this technique is that the definition of splits is much better than typing by serology.

Base Sequence↗
PubMed1992

Tachyplesin I as a model peptide for antiparallel beta-sheet DNA binding motif.

In this study, we present a model compound for antiparallel beta-sheet-DNA interaction. Tachyplesin I, cationic antimicrobial peptide, interacts through contacts with the minor groove. Secondary structure of tachyplesin I, antiparallel beta-sheet constrained by two disulfide bridges and connected by beta-turn, contributes significantly to its DNA binding. The present results give valuable information for design of sequence-specific DNA binding peptide based on antiparallel beta-sheet.

Amino Acid Sequence↗
PubMed1992

Interaction of a protooncogene product, Myb with DNAs.

The DNA-binding domain of Myb consists of three imperfect tandem repeats and the third one which is essential for sequence-specific binding was established to have a helix-turn-helix-related motif. DNA sequences recognized by Myb have been reported to contain TAACPy sequence. Here we have examined the details of Myb-binding sequence. Using DNAs with a single mutation on the various sites of two specific DNAs and some fragments of the DNA-binding domain of Myb, we have found that (i) in a specific DNA which contains only one AAC sequence, each AAC nucleotide is found to be essential for the specific binding of Myb, while any other mutations cause no serious binding loss, (ii) in a specific DNA which contains two AAC sequences separately, one AAC is not so important in the binding, and (iii) for the specific binding with DNA, at least both repeats 2 and 3 of Myb are required. These findings suggest that repeat 3 containing a helix-turn-helix-related structure recognizes the core AAC sequence and repeat 2 supports this recognition by interactions with phosphate groups of DNA.

Base Sequence↗