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At least 1,513 records · Page 84Linked to original sources
PubMed1989

Direct analysis of single nucleotide variation in human DNA and RNA using in situ dot hybridization.

Using oligonucleotide hybridization, single and multiple nucleotide differences between alleles were detected directly in genomic DNA without electrophoretic separation. The DNA was immobilized in depressions in an agarose gel (in situ dots) and hybridized with radiolabeled, allele-specific oligonucleotide probes. An oligonucleotide complementary to a unique sequence region of the human major histocompatibility complex gene HLA-B27 only hybridized with genomic DNA from an HLA-B27-positive individual. Two other oligonucleotides complementary to the normal human beta-globin gene (beta A) and to the sickle cell globin gene (beta S) were synthesized. Using competition hybridization conditions which included the presence of a 10-fold molar excess of unlabeled oligonucleotide complementary to the other beta-globin allele, DNA from individuals homozygous for the normal beta-globin gene (beta A beta A) hybridized to the beta A probe exclusively, whereas DNA from individuals homozygous for the sickle cell globin gene (beta S beta S) hybridized only with the probe for the sickle cell gene. As expected, DNA from heterozygous individuals bound to both probes. Similar results were obtained with total human RNA immobilized in in situ dots. Possible applications of this methodology include genetic disease diagnosis, population carrier screening, HLA "DNA" typing, and DNA and RNA sequence polymorphism analysis.

DNA↗
PubMed2006

Cyclohexanyl peptide nucleic acids (chPNAs) for preferential RNA binding: effective tuning of dihedral angle beta in PNAs for DNA/RNA discrimination.

[structures: see text] A serious drawback of peptide nucleic acids (PNAs) from an application perspective that has not been adequately dealt with is nondiscrimination of identical DNA and RNA sequences. An analysis of the available X-ray and NMR solution structures of PNA complexes with DNA and RNA suggested that it might be possible to rationally impart DNA/RNA duplex binding selectivity by tuning the dihedral angle beta of the flexible ethylenediamine part of the PNA backbone (II) via suitable chemical modifications. Cyclohexanyl PNAs (chPNAs) with beta approximately = 65 degrees were designed on the basis of this rationale. The chPNAs introduced remarkable differences in duplex stabilities among their DNA and RNA complexes, with melting temperatures (deltaTm(RNA-DNA) = +16-50 degrees C) depending on the number of modifications and the stereochemistry. This is a highly significant, exceptional binding selectivity of a mix sequence of PNA to RNA over the same DNA sequence as that seen to date. In contrast, cyclopentanyl PNAs (cpPNAs) with beta approximately = 25 degrees hybridize to DNA/RNA strongly without discrimination because of the ring puckering of the cyclopentane ring. The high affinity of chPNAs to bind to RNA without losing base specificity will have immediate implications in designing improved PNAs for therapeutic and diagnostic applications.

Chromatography, High Pressure Liquid↗
PubMed2000

Large-scale analysis of gene expression changes during acute and chronic exposure to [Delta]9-THC in rats.

Large-scale cDNA microarrays were employed to assess transient changes in gene expression levels following acute and chronic exposure to cannabinoids in rats. A total of 24,456 cDNA clones were randomly selected from a rat brain cDNA library, amplified by PCR, and arrayed at high density to investigate differential gene expression profiles following acute (24 h), intermediate (7 days), and chronic (21 days) exposure to Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the psychoactive ingredient of marijuana. Hippocampal mRNA probes labeled with (33)P obtained from both vehicle and Delta(9)-THC-treated animals were hybridized with identical cDNA microarrays. Results revealed a total of 49 different genes altered by Delta(9)-THC exposure; of these, 28 were identified, 10 had homologies to expressed sequence tags (ESTs), and 11 had no homology to known sequences in the GenBank database. Chronic or acute cannabinoid receptor activation altered expression of several genes (i.e., prostaglandin D synthase, calmodulin) involved in biochemical cascades of cannabinoid synthesis or cannabinoid effector systems. Other genes [i.e., neural cell adhesion molecule (NCAM), myelin basic protein], whose relation to cannabinoid system function was not immediately obvious, were also significantly altered. Verification of the changes obtained with the large-scale screen was determined by RNA dot blots in different groups of animals treated the same as those in the large-scale screen. Results are discussed in terms of the different types of genes affected at different times during chronic Delta(9)-THC exposure.

Animals↗
PubMed2002

Characterization of nontypeable rotavirus strains from the United States: identification of a new rotavirus reassortant (P2A[6],G12) and rare P3[9] strains related to bovine rotaviruses.

Among 1316 rotavirus specimens collected during strain surveillance in the United States from 1996 to 1999, most strains (95%) belonged to the common types (G1 to G4 and G9), while 5% were mixed infections of common serotypes, rare strains, or not completely typeable. In this report, 2 rare (P[9],G3) and 2 partially typeable (P[6],G?; P[9],G?) strains from that study were further characterized. The P[6] strain was virtually indistinguishable by hybridization analysis in 10 of its 11 gene segments with recently isolated P2A[6],G9 strains (e.g., U.S.1205) from the United States, but had a distinct VP7 gene homologous (94.7% a.a. and 90.2% nt) to the cognate gene from P1B[4],G12 reference strain L26. Thus, this serotype P2A[6],G12 strain represents a previously unrecognized reassortant. Three P3[9] strains were homologous (97.8-98.2% aa) in the VP8 region of VP4 to the P3[9],G3 feline-like reference strain AU-1, but had a high level of genome homology to Italian bovine-like, P3[9],G3 and P3[9],G6 rotavirus strains. Two of the U.S. P3[9] strains were confirmed to be type G3 (97.2-98.2% VP7 aa homology with reference G3 strain AU-1), while the other was most similar to Italian bovine-like strain PA151 (P3[9],G6), sharing 99.0% a.a. homology in VP7. Cross-neutralization studies confirmed all serotype assignments and represented the first detection of these rotavirus serotypes in the United States. The NSP4 genes of all U.S. P3[9] strains and rotavirus PA151 were most closely related to the bovine and equine branch within the DS-1 lineage, consistent with an animal origin. These results demonstrate that rare strains with P and G serotypes distinct from those of experimental rotavirus vaccines circulate in the United States, making it important to understand whether current vaccine candidates protect against these strains.

Animals↗
PubMed1987

Genome RNA terminus conservation and diversity among vesiculoviruses.

Sequence analysis of the RNA genome termini of various vesiculovirus standard and defective interfering (DI) particles demonstrated that some virus regulatory sequences and domains of virus N protein are highly conserved while others show considerable divergence. Clearly, distinct RNA signal sequences and protein-coding regions of these virus genomes have quite different evolutionary pressures or constraints. Terminal regions of DI-particle RNA genomes of these viruses were found to possess self-complementary stems at the RNA termini, demonstrating the conservation of this DI-particle structural feature throughout the vesiculovirus group. A high degree of conservation of the 3'-terminal sequences of recent and historic isolates of vesicular stomatitis virus New Jersey was also demonstrated.

Amino Acid Sequence↗
PubMed2006

Evaluation of sequence alignments and oligonucleotide probes with respect to three-dimensional structure of ribosomal RNA using ARB software package.

BACKGROUND: Availability of high-resolution RNA crystal structures for the 30S and 50S ribosomal subunits and the subsequent validation of comparative secondary structure models have prompted the biologists to use three-dimensional structure of ribosomal RNA (rRNA) for evaluating sequence alignments of rRNA genes. Furthermore, the secondary and tertiary structural features of rRNA are highly useful and successfully employed in designing rRNA targeted oligonucleotide probes intended for in situ hybridization experiments. RNA3D, a program to combine sequence alignment information with three-dimensional structure of rRNA was developed. Integration into ARB software package, which is used extensively by the scientific community for phylogenetic analysis and molecular probe designing, has substantially extended the functionality of ARB software suite with 3D environment. RESULTS: Three-dimensional structure of rRNA is visualized in OpenGL 3D environment with the abilities to change the display and overlay information onto the molecule, dynamically. Phylogenetic information derived from the multiple sequence alignments can be overlaid onto the molecule structure in a real time. Superimposition of both statistical and non-statistical sequence associated information onto the rRNA 3D structure can be done using customizable color scheme, which is also applied to a textual sequence alignment for reference. Oligonucleotide probes designed by ARB probe design tools can be mapped onto the 3D structure along with the probe accessibility models for evaluation with respect to secondary and tertiary structural conformations of rRNA. CONCLUSION: Visualization of three-dimensional structure of rRNA in an intuitive display provides the biologists with the greater possibilities to carry out structure based phylogenetic analysis. Coupled with secondary structure models of rRNA, RNA3D program aids in validating the sequence alignments of rRNA genes and evaluating probe target sites. Superimposition of the information derived from the multiple sequence alignment onto the molecule dynamically allows the researchers to observe any sequence inherited characteristics (phylogenetic information) in real-time environment. The extended ARB software package is made freely available for the scientific community via http://www.arb-home.de.

Algorithms↗
PubMed2003

The chemorepellent semaphorin is expressed in the horseshoe crab, Limulus polyphemus.

Semaphorins are a family of soluble and membrane-bound proteins that play a critical role in axonal guidance and other processes of neuronal development. Currently, more than twenty semaphorins have been identified, all of which share a conserved 500 amino acid domain near the amino terminus. Semaphorins are divided into eight classes according to species of origin and structural similarities. Classes 1 and 2 are found in invertebrates, classes 3 through 7 are present in vertebrates and viruses encode class V semaphorin. Microarray analysis of Limulus CNS RNA revealed the presence of a semaphorin-like gene in Limulus polyphemus. Based on these data, we aligned 31 different sequences and designed degenerate primers for the consensus domains (WTT/SFLKA) and (DPY/VCA/GW). RT-PCR products were generated using 6 forward primers and 4 reverse primers. The expected size PCR products (750 bp) was obtained and then ligated with pCR II TOPO vector and transferred into E. coli Top 10. Five partial semaphorin cDNAs were found in Limulus: semaphorins 1a, 1b, 2a, 2b and F (now known as 5) were partially cloned. Subsequent Northern blot analyses using these Limulus specific-probes revealed hybridization with total RNAs purified from six different tissues.

Animals↗
PubMed2026

DirectASRM: uncovering allele-specific post-transcriptional RNA modifications through direct RNA sequencing.

SUMMARY: We developed DirectASRM, a comprehensive database for the systematic identification, integration, and annotation of allele-specific RNA modifications (ASRMs) from direct RNA sequencing data. DirectASRM enables single-base, transcript-level detection of ASRMs across multiple RNA modification types, diverse organisms and condition-specific contexts. The database further evaluates the confidence of each ASRM-SNP pair association within isoform context by jointly considering statistical evidence of allelic modification imbalance and independent support from external next-generation sequencing (NGS) - based RNA modification resources. DirectASRM also provides extensive functional annotations for ASRMs and their associated variants, including intra-sample transcript-level allele-specific expression (ASE) and allele-specific splicing, as well as additional post-transcriptional regulatory features such as miRNA binding, circRNA, RNA-protein interactions, and disease relevance. Overall, DirectASRM serves as a comprehensive resource that supports systematic investigation of the potential functional impact of genetic variants in epitranscriptomic regulation. AVAILABILITY AND IMPLEMENTATION: DirectASRM database is freely accessible at http://modinfor.com/DirectASRM/. DirectASRM pipeline is available at GitHub (https://github.com/jiayin1101/DirectASRM_pipeline) and Zenodo (DOI: https://doi.org/10.5281/zenodo.19876077).

Alleles↗
PubMed1995

Characterization of the pre-mRNA binding site for yeast ribosomal protein L32: the importance of a purine-rich internal loop.

The structure of the RNA binding target for Saccharomyces cerevisiae ribosomal protein L32 was examined using chemical and enzymatic probes as well as thermodynamic methods. In vivo, the production of yeast RPL32 is regulated by a feedback mechanism whereby RPL32 binds to the 5' end of its transcript and inhibits splicing. The binding site of ribosomal protein L32 on the L32 RNA transcript can be reduced to fewer than 30 nucleotides which compromise a stem-internal loop-stem structural motif. The internal loop is closed by a potential G-U pair, is asymmetric and contains mostly purines. The existence of the two helical regions was confirmed by chemical and enzymatic probing. The reactivity of the loop region suggests a structure intermediate between that of single and double-stranded RNA. Base stacking continues into the loop, but two loop bases are extremely reactive to chemical agents. The interaction between the model RNA and the protein is specific and has a dissociation constant of approximately 10 nM. Several of the loop bases are critical for protein binding, as demonstrated by mutational data and chemical protection and modification interference studies. The internal loop destabilizes the RNA, and allows the RNA to melt in an all-or-none fashion.

Aniline Compounds↗
PubMed1995

An aromatase pseudogene is transcribed in the bovine placenta.

The present investigation is focused on a new, truncated bovine aromatase cytochrome P-450-encoding transcript (bCyp19) which was repeatedly isolated from a placental lambda gt10 cDNA library. Different cDNA probes derived from bCyp19 were used during screenings. The deviant transcript contains areas of considerable sequence homology (89-98%) corresponding to exons II, III, V, VIII and IX of bCyp19. Exons VI and VII are missing. Exon IV is replaced by a bovine repeat sequence motif. Numerous translation stop codons occur within all reading frames, thus suggesting that the transcript does not encode a functional protein. The gene was detected by PCR in the genomes of all animals investigated (n = 38). The experiments demonstrate that the bovine genome contains a non-functional copy (pseudogene, Cyp19 phi) of bCyp19 that is transcribed together with the bCyp19 messenger in the bovine post-parturition placenta.

Animals↗
PubMed2005

Regulating gene expression through RNA nuclear retention.

Multiple mechanisms have evolved to regulate the eukaryotic genome. We have identified CTN-RNA, a mouse tissue-specific approximately 8 kb nuclear-retained poly(A)+ RNA that regulates the level of its protein-coding partner. CTN-RNA is transcribed from the protein-coding mouse cationic amino acid transporter 2 (mCAT2) gene through alternative promoter and poly(A) site usage. CTN-RNA is diffusely distributed in nuclei and is also localized to paraspeckles. The 3'UTR of CTN-RNA contains elements for adenosine-to-inosine editing, involved in its nuclear retention. Interestingly, knockdown of CTN-RNA also downregulates mCAT2 mRNA. Under stress, CTN-RNA is posttranscriptionally cleaved to produce protein-coding mCAT2 mRNA. Our findings reveal a role of the cell nucleus in harboring RNA molecules that are not immediately needed to produce proteins but whose cytoplasmic presence is rapidly required upon physiologic stress. This mechanism of action highlights an important paradigm for the role of a nuclear-retained stable RNA transcript in regulating gene expression.

3' Untranslated Regions↗
PubMed2025

ChemPerturb-seq screen identifies a small molecule cocktail enhancing human beta cell survival after subcutaneous transplantation.

Traditional chemical screens have focused on a single assay per screen, making them labor intensive and costly. Here, we combined a chemical screen with single-cell RNA sequencing (scRNA-seq) to perform Chemical Perturb-seq (ChemPerturb-seq), enabling a systematic analysis of the molecular changes of human beta cells upon individual small molecule treatments. Using this platform, we performed an in vivo barcoded screen and discovered a small molecule cocktail, including beta-lipotropin 61-91, insulin growth factor-1, and prostaglandin E2, with which preconditioning human beta cells and primary islets significantly enhanced function and survival when transplanted subcutaneously to female, but not to male, mice. We identified two additional molecules, serotonin and histamine, that promote islet function when transplanted subcutaneously to male mice using ChemPerturb-seq. Such small molecule cocktails could be applied to improve the current FDA-approved islet transplantation procedure. Finally, we developed an artificial intelligence (AI)-powered website, ChemPerturbDB, which provides user-friendly open access analysis of the extensive ChemPerturb-seq dataset.

Humans↗
PubMed2004

Cloning of cDNA encoding a regeneration-associated muscle protease whose expression is attenuated in cell lines derived from Duchenne muscular dystrophy patients.

In the dystrophin-mutant mdx mouse, an animal model for Duchenne muscular dystrophy (DMD), damaged skeletal muscles are efficiently regenerated and thus the animals thrive. The phenotypic differences between DMD patients and the mdx mice suggest the existence of factors that modulate the muscle wasting in the mdx mice. To identify these factors, we searched for mRNAs affected by the mdx mutation by using cDNA microarrays with newly established skeletal muscle cell lines from mdx and normal mice. We found that in the mdx muscle cell line, 12 genes, including L-arginine:glycine amidinotransferase and thymosin beta4, are up-regulated, whereas 7 genes, including selenoprotein P and a novel regeneration-associated muscle protease (RAMP), are down-regulated. Northern blot analysis and in situ hybridization revealed that RAMP mRNA is predominantly expressed in normal skeletal muscle and brain, and its production is enhanced in the regenerating area of injured skeletal muscle in mice. RAMP expression was much lower in individual muscle cell lines derived from biopsies of six DMD patients compared to a normal muscle cell line. These results suggest that RAMP may play a role in the regeneration of skeletal muscle and that its down-regulation could be involved in the progression of DMD in humans.

Amino Acid Sequence↗
PubMed2003

A permutation-based genetic algorithm for the RNA folding problem: a critical look at selection strategies, crossover operators, and representation issues.

This paper presents a Genetic Algorithm (GA) to predict the secondary structure of RNA molecules, where the secondary structure is encoded as a permutation. More specifically, the proposed algorithm predicts which specific canonical base pairs will form hydrogen bonds and build helices, also known as stems. Since RNA is involved in both transcription and translation and also has catalytic and structural roles in the cell, determining the structure of RNA is of fundamental importance in helping to determine RNA function. We introduce a GA where a permutation is used to encode the secondary structure of RNA molecules. We discuss results on RNA sequences of lengths 76, 210, 681, and 785 nucleotides and present several improvements to our algorithm. We show that the Keep-Best Reproduction operator has similar benefits as in the traveling salesman problem domain. In addition, a comparison of several crossover operators is provided. We also compare the results of the permutation-based GA with a binary GA, demonstrating the benefits of the newly proposed representation.

Algorithms↗
PubMed1998

Arenavirus phylogeny: a new insight.

Arenaviridae is a worldwide distributed family, of enveloped, single stranded, RNA viruses. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as the geographic distribution. In this study the phylogenetic relationship among the members of the Arenaviridae was examined, using the reported genomic sequences. The comparison of the aligned nucleotide sequences of the S RNA and the predicted amino acid sequences of the GPC and N proteins, together with the phylogenetic analysis, strongly suggest a possible kinship of Pichindé and Oliveros viruses, with the Old World arenavirus group. This analysis points at the evolutive relationships between the arenaviruses of the Americas and can be used to evaluate the different hypotheses about their origin.

Arenavirus↗
PubMed2002

Identification of a novel stress-responsive gene Hi95 involved in regulation of cell viability.

cDNA microarray hybridization was used in an attempt to identify novel genes participating in cellular responses to prolonged hypoxia. One of the identified novel genes, designated Hi95 shared significant homology to a p53-regulated GADD family member PA26. In addition to its induction in response to prolonged hypoxia, the increased Hi95 transcription was observed following DNA damage or oxidative stress, but not following hyperthermia or serum starvation. Whereas induction of Hi95 by prolonged hypoxia or by oxidative stress is most likely p53-independent, its induction in response to DNA damaging treatments (gamma- or UV-irradiation, or doxorubicin) occurs in a p53-dependent manner. Overexpression of Hi95 full-length cDNA was found toxic for many types of cultured cells directly leading either to their apoptotic death or to sensitization to serum starvation and DNA damaging treatments. Unexpectedly, conditional overexpression of the Hi95 cDNA in MCF7-tet-off cells resulted in their protection against cell death induced by hypoxia/glucose deprivation or H(2)O(2). Thus, Hi95 gene seems to be involved in complex regulation of cell viability in response to different stress conditions.

Amino Acid Sequence↗
PubMed1999

Molecular characterization and in situ localization of a full-length cyclic nucleotide-gated channel in rat brain.

Ion channels gated directly by cyclic nucleotides are required for the transduction of sensory signals in photoreceptor cells and olfactory cells. Cyclic nucleotide-gated (CNG) channels may also be expressed in the central nervous system because partial transcripts that share homology with CNG channels have been found therein. We have now isolated and cloned a full-length CNG channel cDNA from adult rat brain. The sequence is identical to that of the alpha-subunit originally found in the olfactory epithelium (CNCalpha3). In situ hybridization, using probes specific for the CNCalpha3 mRNA, suggest that this channel is expressed widely in the rat brain, albeit mostly at relatively low levels. Certain neuronal populations, however, such as deep cerebellar nuclei, olfactory bulb mitral cells and cerebellar Purkinje neurons, appeared specially enriched. The study demonstrates for the first time that a full-length CNG channel mRNA is expressed in the brain, supporting the possibility that CNG channels are involved in central neural communication and plasticity. The sequence reported in this paper has been deposited in the GenBank data base (accession no. AF126808).

Animals↗