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At least 145 records · Page 8Linked to original sources
PubMed1992

Triple-helix formation by oligonucleotides containing the three bases thymine, cytosine, and guanine.

A homopurine-homopyrimidine sequence of human immunodeficiency virus (HIV) proviral DNA was chosen as a target for triple-helix-forming oligonucleotides. An oligonucleotide containing three bases (thymine, cytosine, and guanine) was shown to bind to its target sequence under physiological conditions. This oligonucleotide is bound in a parallel orientation with respect to the homopurine sequence. Thymines recognize A.T base pairs to form T.A.T base triplets and guanines recognize a run of G.C base pairs to form G.G.C base triplets. A single 5-methylcytosine was shown to stabilize the triple helix when incorporated in a stretch of thymines; it recognizes a single G.C base pair in a run of A.T base pairs. These results provide some of the rules required for choosing the more appropriate oligonucleotide sequence to form a triple helix at a homopurine-homopyrimidine sequence of duplex DNA. A psoralen derivative attached to the oligonucleotide containing thymine, 5-methylcytosine, and guanine was shown to photoinduce cross-linking of the two DNA strands at the target sequence in a plasmid containing part of the HIV proviral DNA sequence. Triplex formation and cross-linking were monitored by inhibition of Dra I restriction enzyme cleavage. The present results provide a rational basis for the development of triplex-forming oligonucleotides targeted to specific sequences of the HIV provirus integrated in its host genome.

Base Sequence↗
PubMed1991

The target DNA sequence for resolution of poxvirus replicative intermediates is an active late promoter.

The linear double-stranded genomes of poxviruses such as Shope fibroma virus (SFV) replicate autonomously within the cytoplasm of infected cells, and it is believed that all of the replication functions are virally encoded. During DNA replication the incompletely base-paired terminal hairpin loops of the viral genome transiently exist in the form of inverted repeat replicative intermediates. These inverted repeat structures form the target for telomere resolution events that include sequence-specific cleavage and directed strand exchange to form the hairpin termini of progeny virus genomes. The terminal sequence domain which forms the telomere resolution target (TRT) shares considerable sequence similarity with viral late promoters. In this study we demonstrate that the TRT of SFV is capable of functioning as a strong viral promoter late in infection. A spectrum of TRT mutations affects telomere resolution and late transcription in a strictly concordant fashion, suggesting that the two activities may be inextricably linked. Further support for this concept comes from the demonstration that a late SFV promoter sequence designated cryptic TRT, which differs substantially from the native TRT in terms of sequence, can support telomere resolution when placed in the correct spatial context. The proposed model for telomere resolution invokes directed unwinding of the TRT double helix by a transcription initiation complex and processing of the resulting secondary structure by viral late-gene products.

Animals↗
PubMed1991

Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA.

Two crystal structures of the glucocorticoid receptor DNA-binding domain complexed with DNA are reported. The domain has a globular fold which contains two Zn-nucleated substructures of distinct conformation and function. When it binds DNA, the domain dimerizes, placing the subunits in adjacent major grooves. In one complex, the DNA has the symmetrical consensus target sequence; in the second, the central spacing between the target's half-sites is larger by one base pair. This results in one subunit interacting specifically with the consensus target half-site and the other nonspecifically with a noncognate element. The DNA-induced dimer fixes the separation of the subunits' recognition surfaces so that the spacing between the half-sites becomes a critical feature of the target sequence's identity.

Amino Acid Sequence↗
PubMed1975

Acquisition of viral DNA sequences in target organs of chickens infected with avian myeloblastosis virus.

The distribution of oncornavirus DNA sequences in various tissues of normal chickens and of chickens with leukemia or kidney tumors induced by avian myeloblastosis virus (AMV) was analyzed by DNA-RNA hybridization using 35S AMV RNA as a probe. All the tissues from normal chickens which were tested contained the same average cellular concentration of endogenous oncornavirus DNA. In contrast, different tissues from lekemic chickens and from chickens bearing kidney tumors contained different concentrations of AMV homologous DNA: in some tissues there was no increase whereas other tissues acquired additional AMV-specific DNA sequences. The increase was the greatest in tissues which can become neoplastic after infection, such as myeloblasts, erythrocytes, and kidney cells. It was directly demonstrated that DNA from AMV-induced kidney tumor contains AMV sequences which are absent in DNA from normal cells. A similar finding had been previously obtained with leukemic cells (15). 3H-labeled 35S RNA from purified AMV was exhaustively hybridized with an excess of normal chicken DNA to remove all the viral RNA sequences which are complementary to DNA from uninfected cells. The 3H-labeled RNA which failed to hybridize was isolated by hydroxylapatite column chromatography which separates DNA-RNA hybrids from single-stranded RNA. The residual RNA hybridized to chicken kidney tumor DNA but did not rehybridize with normal chicken DNA.

Animals↗
PubMed1989

Assay formats involving acridinium-ester-labeled DNA probes.

We describe the development of several hybridization assay formats involving acridinium-ester-labeled DNA probes. The simplest of these is a homogeneous assay procedure that requires only three steps to complete, including a 5-s detection step. Using this format, we have detected target sequences in the 10(-16) to 10(-17) mol range; when rRNA is the target, this translates to 3000 to 300 bacterial organisms. The entire assay can be carried out in less than 30 min. This is the first homogeneous DNA probe assay to be of practical use in the clinical laboratory, and it represents a major simplification of hybridization formats. We also demonstrate the use of this homogeneous assay format to discriminate single-base differences between two closely related target sequences and to detect DNA as well as RNA target molecules. By combining homogeneous hybrid discrimination with solid-phase separation, we have been able to decrease background readings from unhybridized probe to only a few parts per million. This enhances assay sensitivity about 10-fold, to a range of 10(-17) to 10(-18) mol of target. We are in the process of further improving the performance of these assays.

Acridines↗
PubMed1985

IS50-mediated inverse transposition: specificity and precision.

The IS50 elements, which are present as inverted repeats in the kanamycin-resistance transposon, Tn5, can move in unison carrying with them any interstitial DNA segment. In consequence, DNA molecules such as a lambda::Tn5 phage genome are composed of two overlapping transposons - the kan segment bracketed by IS50 elements (Tn5), and lambda bracketed by IS50 elements. During direct transposition, mediated by IS50 "O" (outside) ends, the kan gene is moved and the lambda vector is left behind. During inverse transposition, mediated by the "I" (inside) ends of the IS50 elements, the lambda vector segment is moved and the kan gene is left behind. Direct transposition is several orders of magnitude more frequent than inverse transposition (Isberg and Syvanen, 1981; Sasakawa and Berg, 1982). We assessed the specificity and precision of the rare events mediated by pairs of I ends by mapping and sequencing independent inverse transpositions from a lambda::Tn5 phage into the amp and tet genes of plasmid pBR322. Using restriction analyses, 32 and 40 distinct sites of insertion were found among 46 and 72 independent inverse transpositions into the amp and tet genes, respectively. Eleven sites were used in two or more insertion events, and the two sites in tet used most frequently corresponded to major hotspots for the insertion of the Tn5 (by direct transposition). The sequences of 22 sites of inverse transposition (including each of the sites used more than once) were determined, in eleven cases by analyzing both pBR322-IS50 junctions, and in eleven others by sequencing one junction. The sequence of the "I" end of IS50 was preserved and 9-bp target sequence duplications were present in every case analyzed. GC pairs were found at each end of the target sequence duplication in ten of the eleven sites used more than once, and also in seven of the other eleven sites. Our data indicate that transposition mediated by pairs of "I" ends is similar in its specificity and precision to the more frequent transposition mediated by IS50 "O" ends.

Base Sequence↗
PubMed1986

Towards a comparative anatomy of N-terminal topogenic protein sequences.

A comparative study of three kinds of eukaryotic N-terminal topogenic sequences, viz signal peptides, N-terminal transmembrane anchors, and mitochondrial targeting sequences, suggests: that the sign of the N-terminal charge might influence the orientation of an N-terminal hydrophobic segment relative to the membrane and give rise to N-terminally anchored proteins with their main mass exposed either on the cytosolic or extra-cytosolic side of the membrane; and that N-terminal transmembrane segments in mitochondrial targeting sequences have a relatively low overall hydrophobicity, probably in order to avoid being recognized by the endoplasmic reticulum export machinery.

Amino Acid Sequence↗
PubMed1991

The amino terminus of the adenovirus fiber protein encodes the nuclear localization signal.

Using a recombinant vaccinia virus vector, the fiber protein from adenovirus serotype 2 has been expressed in human cells; the protein expressed was correctly assembled into trimers, glycosylated, and transported to the nucleus. Deletion of amino acids 2-5 (KRAR) resulted in accumulation of fiber in the cytoplasm; fusion of the sequence TKRVRL, found at the beginning of Ad7 fiber, to the N-terminus of this mutant restored correct targeting. Changing the charge of amino acids 91 and 92 within another potential targeting sequence (LKKTK to LEETK) had little effect on nuclear targeting. When fused to the N-terminus of beta-galactosidase and expressed in recombinant vaccinia virus, neither MKRARP nor MTKRVRL (from Ad2 and Ad7 fibers, respectively), were sufficient for efficient transport of the hybrid protein to the nucleus; on the other hand, fusions of either MKRARPSEDTF (from Ad2 fiber) or of MKRPRP (a known targeting sequence from the C-terminus of Ad2 E1A proteins) to beta-galactosidase were localized to the nucleus. These results suggest that sequences at the N-terminus of Ad2 and Ad7 fiber are required for correct nuclear targeting.

Adenoviridae↗
PubMed1992

Quantification of gene expression over a wide range by the polymerase chain reaction.

We investigated the usefulness of the polymerase chain reaction (PCR) method for the relative quantification of gene expression using a simultaneously amplified sequence of beta-actin mRNA as an internal control for the target sequence of tax/rex mRNA of human T-cell leukemia virus type I. The PCR product of the internal control was reduced by delaying the addition of the primers for its sequence. The photostimulated luminescence of the bands was measured with a laser image analyzer, and the values were plotted against the cycle number. The cycle differences between the logarithmic phase of the curves for the target sequence and for beta-actin (delta cycle) showed a linear correlation with the initial concentration of the sample. This method is highly sensitive for evaluating gene expression over a wide range.

Actins↗
PubMed2026

A noncontiguous code for RNA-guided DNA recognition at the origin of CRISPR-Cas.

CRISPR-Cas provides RNA-mediated adaptive immunity, but how its first RNA-guided effector arose is unclear. In this study, we report the discovery of Viral Interference Programmable Repeat (VIPR) systems consisting of a Vipr protein ancestral to the earliest CRISPR-Cas effectors and VIPR RNAs (vrRNAs) comprising alternating GGY/NN motifs. Unlike canonical guide RNAs that pair with target nucleic acids through contiguous complementarity, vrRNAs recognize double-stranded DNA through a noncontiguous code in which the variable NN dinucleotides collectively specify a gapped target sequence. Natural vrRNA targets suggest that VIPR systems act against competing phages, and we demonstrate programmable phage defense by redirecting the complex for transcriptional repression. These results suggest that adaptive immunity originated from ancient warfare between viruses, revealing a previously unidentified logic for encoding information in sequence.

CRISPR-Cas Systems↗
PubMed1992

[Site-specific photomodification of nucleic acids with arylazide and perfluoroarylazide oligonucleotide derivatives. II. Specificity in relation to nucleosides].

Oligonucleotide reagents bearing aromatic azido groups of different structures were shown to be suitable for nucleoside specific photomodification of nucleic acids. Modification of the pentadecanucleotide targets d(TAAGTGGAGTTTGGC), d(TAAGTGGAAAAAAAA), d(TAAGTGGACCCCCCC) and d(TAAGTGGATTTTTTT) was investigated with reagents d(UCH2OCH2CH2NHCORCCACTT) carrying a photoactive group R(R1-n-azidotetrafluorophenyl-reagent (I), R2-2-nitro-5-azidophenyl-reagent (II) and R3-n-azidophenyl-reagent (III)) at C-5-modified deoxyuridine. Photomodification did not exceed 5% for the targets in case of reagent (III); the modification extent was 25-50% depending on the target sequence for reagent (II); reagent (I) with perfluoro azido group was the most effective, that provided 60-70% of modification. Reagents (I) and (II) were found to be sensitive to the nucleoside sequence of the target: the most vulnerable sites for reagent (I) and (II) were guanine and cytosine residues, respectively. These bases were modified predominantly when being adjacent to the addressed site of the target.

Autoradiography↗
PubMed2026

Epigenetic Profiling for Early Detection and Treatment Response Monitoring in Non-Small Cell Lung Cancer: Protocol for a Prospective Translational Biomarker Study.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide and continues to have poor survival outcomes, with most patients diagnosed at advanced stages of disease. In New Zealand, NSCLC contributes substantially to cancer inequities, with Māori communities experiencing disproportionately high incidence and mortality rates. Although low-dose computed tomography screening can improve early detection, major limitations remain, including false-positive findings, overdiagnosis, high infrastructure costs, and limited accessibility for rural and underserved populations. Liquid biopsy approaches using circulating tumor DNA (ctDNA), particularly DNA methylation profiling, have emerged as promising, minimally invasive strategies for improving cancer detection, treatment monitoring, and precision oncology. OBJECTIVE: This study aims to establish integrated genomic and epigenomic predictive and prognostic biomarkers using ctDNA, tumor tissue, and transcriptomic profiling to improve early detection, risk stratification, treatment selection and response prediction, and longitudinal monitoring, with particular emphasis on identifying molecular mechanisms associated with treatment resistance and disease progression. METHODS: This prospective observational translational biomarker study is being conducted through the University of Otago and associated respiratory and oncology services in New Zealand. The study will recruit participants with NSCLC (including squamous and nonsquamous subtypes), individuals referred to fast-track lung nodule assessment clinics, and nonmalignant respiratory controls. Serial peripheral blood sampling will be performed in selected participants at predefined clinical follow-up time points to evaluate treatment response and disease progression. The availability of formalin-fixed paraffin-embedded archival tissues will be recorded, but will not be mandatory for enrollment. Genome-scale DNA methylation profiling will be performed using cell-free reduced representation bisulfite sequencing (cfRRBS), while targeted genomic profiling and transcriptomic analyses will be conducted using targeted sequencing panels and RNA sequencing. Integrative bioinformatic analyses will be used to identify molecular biomarkers associated with early-stage disease, advanced disease, treatment response, and therapeutic resistance. RESULTS: Ethics approval for the study has been obtained from the New Zealand Health and Disability Ethics Committee (2022 EXP 12566). This study commenced in 2022, and recruitment and biospecimen collection are ongoing. The study aims to recruit approximately 450 participants, including patients with NSCLC, individuals referred through respiratory diagnostic pathways, and nonmalignant controls. As of July 31, 2026, 205 participants have been recruited, with recruitment continuing until the target sample size is reached. Molecular and data analyses are ongoing, with additional publications expected as the cohort matures. CONCLUSIONS: This study will generate one of the first integrated genomic, epigenomic, and transcriptomic liquid biopsy datasets for NSCLC in New Zealand. The findings are expected to support the development of sensitive, accessible, and equitable blood-based biomarkers for NSCLC detection and treatment monitoring while also contributing to improved precision oncology approaches and reducing NSCLC inequities among Māori populations.

Humans↗
PubMed1989

Sequence and structural requirements of a mitochondrial protein import signal defined by saturation cassette mutagenesis.

The Saccharomyces cerevisiae F1-ATPase beta subunit precursor contains redundant mitochondrial protein import information at its NH2 terminus (D. M. Bedwell, D. J. Klionsky, and S. D. Emr, Mol. Cell. Biol. 7:4038-4047, 1987). To define the critical sequence and structural features contained within this topogenic signal, one of the redundant regions (representing a minimal targeting sequence) was subjected to saturation cassette mutagenesis. Each of 97 different mutant oligonucleotide isolates containing single (32 isolates), double (45 isolates), or triple (20 isolates) point mutations was inserted in front of a beta-subunit gene lacking the coding sequence for its normal import signal (codons 1 through 34 were deleted). The phenotypic and biochemical consequences of these mutations were then evaluated in a yeast strain deleted for its normal beta-subunit gene (delta atp2). Consistent with the lack of an obvious consensus sequence for mitochondrial protein import signals, many mutations occurring throughout the minimal targeting sequence did not significantly affect its import competence. However, some mutations did result in severe import defects. In these mutants, beta-subunit precursor accumulated in the cytoplasm, and the yeast cells exhibited a respiration defective phenotype. Although point mutations have previously been identified that block mitochondrial protein import in vitro, a subset of the mutations reported here represents the first single missense mutations that have been demonstrated to significantly block mitochondrial protein import in vivo. The previous lack of such mutations in the beta-subunit precursor apparently relates to the presence of redundant import information in this import signal. Together, our mutants define a set of constraints that appear to be critical for normal activity of this (and possibly other) import signals. These include the following: (i) mutant signals that exhibit a hydrophobic moment greater than 5.5 for the predicted amphiphilic alpha-helical conformation of this sequence direct near normal levels of beta-subunit import (ii) at least two basic residues are necessary for efficient signal function, (iii) acidic amino acids actively interfere with import competence, and (iv) helix-destabilizing residues also interfere with signal function. These experimental observations provide support for mitochondrial protein import models in which both the structure and charge of the import signal play a critical role in directing mitochondrial protein targeting and import.

Amino Acid Sequence↗
PubMed1991

HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency.

Expression of the structural proteins of HIV-1 requires the direct interaction of the viral Rev trans-activator with its cis-acting RNA target sequence, the Rev response element or RRE. Here, we demonstrate that this specific RNA-binding event is, as expected, mediated by the conserved arginine-rich motif of Rev. However, we also show that amino acid residues located proximal to this basic domain that are critical for in vivo Rev function are dispensable for sequence-specific binding to the RRE. Instead, these sequences are required for the multimerization of Rev on the viral RRE target sequence. The observation that Rev function requires the sequential binding of multiple Rev molecules to the RRE provides a biochemical explanation for the observed threshold effect for Rev function in vivo and suggests a molecular model for the high incidence of latent infection by HIV-1.

Cloning, Molecular↗
PubMed1992

The Drosophila homolog of the immunoglobulin recombination signal-binding protein regulates peripheral nervous system development.

The J kappa RBP binds to the immunoglobulin recombination signal sequence flanking the kappa-type J segment. We previously isolated the highly conserved homolog of the J kappa RBP gene from D. melanogaster, which is not thought to have immunoglobulin molecules. Using many deficiency mutants and in situ hybridization, we mapped the Drosophila J kappa RBP gene in a region containing two recessive lethal mutations, i.e., br26 and br7, which shows the dominant Suppressor of Hairless (Su(H)) phenotype in heterozygotes. All six Su(H) alleles analyzed at the DNA level contained mutations in the Drosophila J kappa RBP gene. Since the Su(H) mutation affects peripheral nervous system development, the Drosophila J kappa RBP gene product is involved in gene regulation of peripheral nervous system development. The results also imply that the immunoglobulin recombination signal sequence and the target sequence of the Drosophila J kappa RBP protein might have a common evolutionary origin.

Alleles↗
PubMed1989

Cloning of a housekeeping-type gene (MER5) preferentially expressed in murine erythroleukemia cells.

DNA complementary to mRNA preferentially produced in murine erythroleukemia (MEL) cells was cloned from a cDNA library of anemic mouse spleen mRNAs. An open reading frame was noted in the cloned DNA, and was tentatively designated MER5. The MER5 mRNA is abundant in three MEL cell lines, but less in other tissues or cell lines. The levels of the MER5 mRNA changed periodically during MEL cell differentiation and decreased as globin mRNA accumulated. The MER5 promoter region contained no typical TATA-like sequence, but possible target sequences for AP1, AP2, SP1 and octamer-binding protein. More interestingly, this promoter contained the duplicated CACCC boxes, which are common in the adult beta-globin promoter from many species, but uncommon for promoters of other eukaryotic genes.

Amino Acid Sequence↗
PubMed1991

M.H2I, a multispecific 5C-DNA methyltransferase encoded by Bacillus amyloliquefaciens phage H2.

Bacillus amyloliquefaciens phage H2 codes for a multispecific cytosine-5-DNA- methyltransferase (MTase), M.H2I, which methylates GGCC, GCNGC and [sequence: see text] target sequences. The gene coding for M.H2I was cloned in Escherichia coli and its nucleotide (nt) sequence was determined. It consists of 1509 bp, corresponding to a protein of 503 amino acids (aa) with a calculated Mr of 57,166. A comparison of the aa sequence of M.H2I with those of the multispecific MTases encoded by Bacillus subtilis phages SPR, phi 3T and rho 11S, revealed that M.H2I is closely related to these enzymes. A very high degree of homology was observed between M.H2I and M.rho 11SI, with 96.2% aa identity and 97.8% nt identity of the corresponding genes.

Amino Acid Sequence↗
PubMed1988

5' flanking sequence and genomic structure of Egr-1, a murine mitogen inducible zinc finger encoding gene.

Egr-1 is a murine zinc finger encoding cDNA whose expression is modulated by a variety of ligand-receptor interactions and is often coregulated with c-fos (1). This study reports the isolation of a mouse Egr-1 genomic clone, its intron-exon structure, and 935 bp of 5' flanking sequence. The gene spans about 3.8 kb and consists of 2 exons and one 700 bp intron. S1 nuclease protection and primer extension analysis were used to define the transcription initiation site. "TATA" and "CCAAT" sequences were located at nucleotides -26 and -337 respectively. In addition, there exist five elements whose sequence is nearly identical to the inner core 10 nucleotide region (CCATATTAGG) of the c-fos serum response element, four Sp1 consensus sequences, two AP1 target sequence analogs, and two potential cAMP response elements. These results will ultimately lead to a detailed definition of the intracellular events regulating Egr-1 expression.

Animals↗