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PubMed1979

Down syndrome: gene dosage at the transcriptional level in skin fibroblasts.

Chromosome imbalance (aneusomy) is the leading known cause of both spontaneous abortion and mental retardation in human beings. The primary abnormality is thought to result from quantitative changes of transcription products from the unbalanced genetic material. To document this point, I compared chromosome 21-specific transcription in skin fibroblasts from subjects with monosomy 21, disomy 21 (normal), and trisomy 21 (Down syndrome). Polyadenylylated RNA [poly(A)-RNA], which is enriched in messenger and messenger-precursor RNA sequences, was isolated from the above fibroblast lines. Radioactive DNA (cDNA) complementary to these RNAs was synthesized with reverse transcriptase (RNA-dependent DNA polymerase). These cDNAs were hybridized with (i) DNA from a cell line with a mouse genome plus human chromosome 21 and (ii) mouse DNA. Subtraction of the amount of hybridization in experiment ii from that in experiment i yielded a measure of human chromosome 21-specific RNA sequences. The results were consistent with gene dosage at the transcriptional level; for monosomy 21-derived cDNA, 0.6% (of the total cDNA) hybridized specifically to human chromosome 21; for disomy 21-derived cDNA, 2% hybridized; and for trisomy 21-derived cDNA, 3% hybridized. Thus, for DNA sequences on chromosome 21 in human skin fibroblasts, transcription depends on DNA dosage. Characterization of the chromosome 21-specific RNA sequences quantitated in these experiments could help to elucidate the mechanisms by which abnormal karyotypes result in abnormal phenotypes.

Alleles↗
PubMed1996

Classification of RNA secondary structures using the techniques of cluster analysis.

An RNA molecule has a lot of suboptimal secondary structures. Some of these suboptimal structures are very similar and some are entirely different. In some cases, the free energy of these suboptimal structures does not differ much from that of the optimal structure. In order to characterize this relationship more clearly, we defined a metric among the secondary structures by the unweighted pair group method, using the arithmetic average and extended this metric to the sets of secondary structures. We report two applications of this metric. First, we developed a method for the classification of these suboptimal secondary structures of a given RNA sequence. Results of classification are presented with the secondary structures of cadang-cadang coconut viroid, potato spindle tuber viroid and polio virus as examples. Second, we applied this metric to the classification of the secondary structures derived from a set of mutant RNA sequences. We discuss that the mutation of a given RNA sequence changes not only the optimal secondary structure, but also the population of the cluster of the secondary structures.

Animals↗
PubMed1979

Initiation of translation with Pseudomonas aeruginosa phage PP7 RNA: nucleotide sequence of the coat cistron ribosome binding site.

Initiation complex formation between PP7 RNA and ribosomes of Pseudomonas aeruginosa and Escherichia coli has been investigated. The PP7 RNA fragments protected by both species of ribosome have been isolated, and their sequences have been determined. Only one binding sites is available on the intact PP7 RNA strand, and this site is recognized by ribosomes of both species. The PP7 RNA binding site is approximately 38 nucleotides long. It contains two AUG sequences and a purine-rich segment near the 5'-end that is complementary to segments near the 3'-ends of the 16S ribosomal RNA's of both P. aeruginosa and E. coli. In order to establish which of the AUG codons acts as the initiator, the H2N-terminal amino acid sequence of PP7 coat protein was determined. This sequence is compatible with the codon sequence following the second AUG codon. The extent of the reaction of PP7 RNA with E. coli ribosomes is greater than with P. aeruginosa ribosomes, but our results do not indicate a qualitative difference in the initial interaction between intact PP7 RNA and the ribosomes of either species.

Amino Acid Sequence↗
PubMed2025

Heritable virus-induced germline editing in tomato.

Here, we report the successful implementation of heritable virus-induced genome editing (VIGE) in tomato (Solanum lycopersicum). We generated three transgenic tomato lines expressing Streptococcus pyogenes Cas9 (SpCas9) under the control of Cauliflower mosaic virus 35S (35S), S. lycopersicum ribosomal protein S5A (SlRPS5A), or S. lycopersicum YAO promoters (SlYAO). These three lines were tested for somatic and heritable editing using the tobacco rattle virus (TRV)-based system carrying guide RNAs (gRNAs) fused with mobile RNA sequences. TRV with gRNA targeted to Phytoene desaturase (SlPDS) and Downy mildew resistance 6 (SlDMR6) genes fused to mobile RNA sequences showed significant somatic editing efficiency in all three tomato lines expressing SpCas9. However, the progenies from the SlYAO promoter-driven SpCas9 tomato infected with TRV with gRNA targeted to SlDMR6 fused to the mobile RNA sequence resulted in monoallelic mutations with a frequency of 3%. Optimization of environmental conditions, such as reduced light intensity, significantly increased heritable editing frequencies, from 0% to 86% at the SlPDS and from 3% to 100% at the SlDMR6, including biallelic mutations. These findings underscore the use of appropriate promoters to express Cas nucleases and optimized environmental conditions to enhance heritable genome editing efficiency in tomato using VIGE. Furthermore, our method enables the generation of mutants without additional tissue culture or transformation once a SpCas9-expressing tomato line is established.

Solanum lycopersicum↗
PubMed1981

Further characterization of virus-like 30S (VL30) RNA of mice: initiation of reverse transcription and intracellular synthesis.

We have studied the virus-like 30S (VL30) RNA sequences of mice. Previous work has shown that these sequences are coded in the mouse genome, expressed in some normal cells and released as pseudotypic particles from cells producing murine C-type retroviruses. VL30 sequences have some similarities to standard retrovirus RNA, but differences also exist. To further assess the similarities and differences, several aspects of VL30-specific metabolism were investigated. We studied the initiation of VL30-specific DNA synthesis during an endogenous reverse transcriptase reaction. Short initial VL30-specific cDNA transcripts were covalently attached to RNA as measured by equilibrium banding in caesium sulphate density gradients. Therefore, reverse transcription of VL30-specific cDNA is initiated by an RNA primer. The intracellular synthesis of VL30 RNA was investigated by pulse labelling uninfected JLS-V9 cells with 3H-uridine. Hybridization of the pulse-labelled nuclear RNA indicated that the major VL30-specific RNA evident after a 15 min label was the same size as the mature VL30 RNA. Thus, VL30 RNA is apparently not synthesized via a higher mol. wt. precursor. Both of these results demonstrate similarity of VL30 RNA sequences to standard retroviruses. One unique feature of VL30 RNA was detected. JLS-V9 cells contained both the monomeric VL30 RNA and a hydrogen-bonded 38S form which yielded the monomer when denatured. This contrasts with standard murine leukaemia virus which is only found as a monomer within cells.

Animals↗
PubMed1989

Two sequences participating in the autolytic processing of satellite tobacco ringspot virus complementary RNA.

Circular and multimeric forms of the satellite RNA of tobacco ringspot virus and their autolytic processing reactions are well known. They suggest replication models in which key elements are rolling circle transcription and the processing of the resulting multimeric RNA to generate the unit, 'monomeric' satellite RNA sequence. We prepared plasmids bearing two distinct sequences of the satellite RNA. Each was arranged to allow transcription of an oligoribonucleotide (r-oligo) of the polarity that is complementary to encapsidated satellite RNA. One sequence has the autolytic processing phosphodiester bond, ApG, and the other is located at a distance of about 150 nucleotide (nt) residues. The second r-oligo accomplished cleavage of the first, in a catalytic fashion. Analysis of truncated forms showed that 10 nt of the ApG junction-containing r-oligo and 46 of the endoribonucleolytic r-oligo were sufficient for recognition in the cleavage reaction. These results map the sequences involved in autolytic processing of the complementary polarity satellite RNA to two regions.

Base Sequence↗
PubMed2004

Small-subunit ribosomal RNA gene sequences of Phaeodarea challenge the monophyly of Haeckel's Radiolaria.

In his grand monograph of Radiolaria, Ernst Haeckel originally included Phaeodarea together with Acantharea and Polycystinea, all three taxa characterized by the presence of a central capsule and the possession of axopodia. Cytological and ultrastructural studies, however, questioned the monophyly of Radiolaria, suggesting an independent evolutionary origin of the three taxa, and the first molecular data on Acantharea and Polycystinea brought controversial results. To test further the monophyly of Radiolaria, we sequenced the complete small subunit ribosomal RNA gene of three phaeodarians and three polycystines. Our analyses reveal that phaeodarians clearly branch among the recently described phylum Cercozoa, separately from Acantharea and Polycystinea. This result enhances the morphological variability within the phylum Cercozoa, which already contains very heterogeneous groups of protists. Our study also confirms the common origin of Acantharea and Polycystinea, which form a sister-group to the Cercozoa, and allows a phylogenetic reinterpretation of the morphological features of the three radiolarian groups.

Animals↗
PubMed1990

Homologies between the genomes of a carlavirus (lily symptomless virus) and a potexvirus (lily virus X) from lily plants.

cDNA clones complementary to the 3'-terminal regions of the genomic RNAs of the carlavirus lily symptomless virus and the potexvirus lily virus X (LVX) have been sequenced. The carlavirus RNA sequence contains five open reading frames (ORFs) coding for proteins of Mr 25,374, 11,631, 6960, 32,041 (coat protein) and 16,121, which are all very similar in size, amino acid sequence and relative position in the genome to proteins encoded by two different potato carlaviruses. The first four of these proteins also show considerable amino acid sequence similarity to proteins encoded by RNA of potexviruses, and the relative position of the ORFs on the carlavirus genome strongly resembles that in the potexvirus genomes. The LVX cDNA clone contains three ORFs encoding proteins of Mr 23,574, 11,767 and 21,569 (coat protein). A small ORF immediately 5' of the coat protein ORF, which has been found in other potexvirus genomes, is not present in the LVX genome. Thus, the data confirm the close taxonomic relationship between carlaviruses and potexviruses and reveal some differences in genome organization among the potexviruses.

Amino Acid Sequence↗
PubMed2005

Model for codon position bias in RNA editing.

RNA editing can be crucial for the expression of genetic information via inserting, deleting, or substituting a few nucleotides at specific positions in an RNA sequence. Within coding regions in an RNA sequence, editing usually occurs with a certain bias in choosing the positions of the editing sites. In the mitochondrial genes of Physarum polycephalum, many more editing events have been observed at the third codon position than at the first and second, while in some plant mitochondria the second codon position dominates. Here we propose an evolutionary model that explains this bias as the basis of selection at the protein level. The model predicts a distribution of the three positions rather close to the experimental observation in Physarum. This suggests that the codon position bias in Physarum is mainly a consequence of selection at the protein level.

Animals↗
PubMed2025

Molecular hallmarks of excitatory and inhibitory neuronal resilience to Alzheimer's disease.

BACKGROUND: A significant proportion of individuals maintain cognition despite extensive Alzheimer's disease (AD) pathology, known as cognitive resilience. Understanding the molecular mechanisms that protect these individuals could reveal therapeutic targets for AD. METHODS: This study defines molecular and cellular signatures of cognitive resilience by integrating bulk RNA and single-cell transcriptomic data with genetics across multiple brain regions. We analyzed data from the Religious Order Study and the Rush Memory and Aging Project (ROSMAP), including bulk RNA sequencing (n = 631 individuals) and multiregional single-nucleus RNA sequencing (n = 48 individuals). Subjects were categorized into AD, resilient, and control based on β-amyloid and tau pathology, and cognitive status. We identified and prioritized protected cell populations using whole-genome sequencing-derived genetic variants, transcriptomic profiling, and cellular composition. RESULTS: Transcriptomics and polygenic risk analysis position resilience as an intermediate AD state. Only GFAP and KLF4 expression distinguished resilience from controls at tissue level, whereas differential expression of genes involved in nucleic acid metabolism and signaling differentiated AD and resilient brains. At the cellular level, resilience was characterized by broad downregulation of LINGO1 expression and reorganization of chaperone pathways, specifically downregulation of Hsp90 and upregulation of Hsp40, Hsp70, and Hsp110 families in excitatory neurons. MEF2C, ATP8B1, and RELN emerged as key markers of resilient neurons. Excitatory neuronal subtypes in the entorhinal cortex (ATP8B+ and MEF2Chigh) exhibited unique resilience signaling through activation of neurotrophin (BDNF-NTRK2, modulated by LINGO1) and angiopoietin (ANGPT2-TEK) pathways. MEF2C+ inhibitory neurons were over-represented in resilient brains, and the expression of genes associated with rare genetic variants revealed vulnerable somatostatin (SST) cortical interneurons that survive in AD resilience. The maintenance of excitatory-inhibitory balance emerges as a key characteristic of resilience. CONCLUSIONS: We have defined molecular and cellular hallmarks of cognitive resilience, an intermediate state in the AD continuum. Resilience mechanisms include preserved neuronal function, balanced network activity, and activation of neurotrophic survival signaling. Specific excitatory neuronal populations appear to play a central role in mediating cognitive resilience, while a subset of vulnerable interneurons likely provides compensation against AD-associated hyperexcitability. This study offers a framework to leverage natural protective mechanisms to mitigate neurodegeneration and preserve cognition in AD.

Humans↗
PubMed1994

Revision of the nucleotide sequence and RNA splicing pathway of the Neurospora mitochondrial gene encoding ATPase subunit 6.

Previous sequence analysis of the Neurospora oli2 (ATP6) mitochondrial gene suggested that, in addition to a typical Group-I intron, it contained an unusual, mostly-palindromic, 93-nucleotide intron. We report here revisions of the nucleotide sequence and analysis of the size and sequence of reverse-transcriptase PCR products that show: (1) the Group-I intron splice sites are located as predicted by previous DNA sequence analysis; (2) the putative 93-nt intron is not excised from the mature mRNA, and most of this sequence is actually in the 5' untranslated region. We conclude that the Neurospora ATP6 gene contains only one intron. Analysis of the cDNA sequence also confirms the non-universal nature of the Neurospora mitochondrial genetic code: a TGA codon inferred from the DNA sequence is present as UGA in the mRNA. This provides direct evidence that this codon is not altered, for example by RNA editing, to conform to the universal code.

Adenosine Triphosphatases↗
PubMed1979

Identification of transcribed regions of Epstein-Barr virus DNA in Burkitt lymphoma-derived cells.

RNA was extracted from the Burkitt lymphoma-derived cell line Raji and from Burkitt lymphoma tumor biopsies, isotope labeled in vitro by iodination with 125I, and hybridized to electrophoretically separated restriction endonuclease fragments of Epstein-Barr virus DNA on nitrocellulose membranes. The results indicated that only certain parts of the Epstein-Barr virus genome are represented as polyribosomal RNA in Raji cells, with a pronounced dominance of RNA sequences complementary to a 2.0 x 10(6)-dalton segment of Epstein-Barr virus DNA located close to the left end of the viral genome. A map of virus-specific polyribosomal RNA sequences was constructed, which indicated that a minimum of three regions of the Epstein-Barr virus genome are expressed in Raji cells. Total-cell RNA preparations from five Burkitt lymphoma biopsies contained RNA sequences homologous to the same regions of Epstein-Barr virus DNA as polyribosomal RNA from Raji cells, albeit at different relative proportions.

Base Sequence↗
PubMed1994

Dimerization of human immunodeficiency virus type 1 RNA involves sequences located upstream of the splice donor site.

The retroviral genome consists of two homologous RNA molecules associated close to their 5' ends. We studied the spontaneous dimerization of four HIV-1 RNA fragments (RNAs 1-707, 1-615, 311-612, and 311-415) containing the previously defined dimerization domain, and a RNA fragment (RNA 1-311) corresponding to the upstream sequences. Significant dimerization of all RNAs is observed on agarose gels when magnesium is included in the electrophoresis buffer. In contrast to dimerization of RNAs 311-612 and 311-415, dimerization of RNAs 1-707, 1-615 and 1-311 strongly depends on the size of the monovalent cation present in the incubation buffer. Also, dimerization of RNAs 1-707, 1-615, and 1-311 is 10 times faster than that of RNAs 311-612 and 311-415. The dimers formed by the latter RNAs are substantially more stable than that of RNA 1-615, while RNA 1-311 dimer is 5-7 degrees C less stable than RNA 1-615 dimer. These results indicate that dimerization of HIV-1 genomic RNA involves elements located upstream of the splice donor site (position 305), i.e. outside of the previously defined dimerization domain.

Base Sequence↗
PubMed1976

Determining the base sequence of RNA molecules using continuous degradation kinetics.

A kinetic approach to sequence analysis is presented. A polymer chain composed of a small number of monomer types is degraded continuously with degradation proceeding in only one direction of the chain. Ordinarily, the direct determination of the sequence from the amount of degraded monomers is hampered by the rapid loss of reaction synchrony. However, it is shown, that if the monomers can be measured reasonably accurately, one can solve a system of linear equations with the unknowns giving the positions of the monomers in the chain, and the syn chrony loss due to the stochastic nature of the degradation process can be accounted for. Model calculations are presented.

Base Sequence↗
PubMed1980

Identification of a cytidine-specific ribonuclease from chicken liver.

Rapid RNA sequencing technology was used to determine if the base specificities of an RNase recently purified from chicken liver would prove useful for RNA sequence analysis. Escherichia coli 5 S [5'-32P]rRNA or yeast 5.8 S [5'-32P]rRNA was digested with the enzyme and this digest, along with digests derived from RNases of known specificity (U2, T1, T2) were subjected to electrophoresis through denaturing polyacrylamide slab gels. Following autoradiography, the banding patterns arising from the activity of each enzyme were compared, and the base specificity of the unknown RNase was established. The chicken liver RNase was found to have a marked preference for phosphodiester bonds containing cytidylic acid residues, a property which should make the enzyme useful for distinguishing between pyrimidines in RNA sequencing.

Animals↗
PubMed1994

Presence of hepatitis C virus (HCV) genomic RNA and viral replicative intermediates in bone marrow and peripheral blood mononuclear cells from HCV-infected patients.

The cellular tropism of hepatitis C virus (HCV) was studied in vivo in samples from patients with persistent HCV infection. Plasma, liver, peripheral blood mononuclear cell (PBMC), and bone marrow cell (BMC) samples from 15 subjects positive for anti-HCV antibodies were tested for the presence of HCV RNA sequences by reverse transcription PCR. Virus-specific RNA sequences were found to be present in liver samples from all subjects (100%), in plasma samples from 13 of 15 patients (86.7%), in PBMC samples from 3 patients (20%), and in BMC samples from 9 (60%) of the 15 anti-HCV-positive patients enrolled in this study. The presence of the molecular intermediate of HCV replication (the negative-stranded HCV RNA) was evident in the two of the three PBMC and in five of the nine BMC HCV RNA-positive samples. Finally, we studied the nucleotide sequence of a large portion (-270 to -59) of the 5'untranslated region of HCV amplified from plasma samples of 12 of the 15 patients with and without HCV in BMCs; the degree of heterogeneity compared with the prototype HCV sequence was similar in both groups. The data principally indicate that HCV infection of PBMCs and BMCs is frequent in persistently infected patients, as shown by the occurrence of positive- and negative-stranded HCV RNA, thus suggesting the possibility of extrahepatic replication of HCV.

Adult↗
PubMed1999

Complete sequence of RNA 1 and the presence of tRNA-like structures in all RNAs of Potato mop-top virus, genus Pomovirus.

The complete nucleotide sequence (6043 nt) of RNA 1 from Potato mop-top virus (PMTV-Sw), the type member of the genus Pomovirus, was determined. The first (5'-terminal) open reading frame (ORF 1) encodes a predicted protein of 148 kDa. ORF 2 extends through the opal stop codon of ORF 1 producing a predicted readthrough protein of 206 kDa which resembles the RNA-dependent RNA polymerases (RdRp) of other fungal-transmitted viruses. It includes a methyltransferase, a helicase and a GDD RdRp motif, respectively. Phylogenetic analyses of RdRps indicated that PMTV is most closely related to Beet soil-borne virus (genus Pomovirus), Broad bean necrosis virus (genus Pomovirus) and Soil-borne wheat mosaic virus (genus Furovirus), and is more distantly related to the other viruses of the former furovirus group. The 5' and 3' termini of RNA 1 in PMTV contained untranslated regions (UTR) of 114 nt and 489 nt, respectively. The 3'-UTR of RNA 1 contained a tRNA-like structure, which has previously been reported in the 3'-UTR of RNA 2 but not RNA 3. However, in this study, the tRNA-like structure was also found in the 3'-UTR of RNA 3, which confirms its presence in the 3'-UTRs of all three RNAs of PMTV.

3' Untranslated Regions↗
PubMed2025

Growth Hormone Alleviates Atherosclerosis Through Regulating the Activity of PI3K/AKT Pathway: Insights From Single-Cell Sequence and Mechanism Exploration.

PURPOSE: This research sought to investigate the impact and underlying mechanisms of growth hormone (GH) on atherosclerosis (AS) based on the analysis of single-cell RNA sequencing (scRNA-seq) data. METHODS: We analyzed the impact of GH on arterial vascular smooth muscle cells (VSMCs) by utilizing scRNA-seq data obtained from both atherosclerotic and healthy vascular tissues in mice. AS was induced in C57BL/6 and ApoE-/- mice through hypophysectomy performed via the parapharyngeal approach, followed by a high-fat diet (HFD), resulting in the C57-Hx and ApoE-/--Hx models. AS was evaluated by measuring arterial lipid deposition, plaque progression, collagen loss, vascular inflammation, and oxidative stress. Serum metabolite alterations were assessed using liquid chromatography-mass spectrometry (LC-MS). RNA sequencing was employed to examine the underlying mechanisms of GH in the context of AS treatment, with findings further confirmed through western blot analysis. In vitro experiments involved treating VSMCs with oxidized low-density lipoprotein (ox-LDL) to simulate atherosclerotic injury. The formation of foam cells was evaluated by measuring lipid accumulation, inflammatory responses, apoptosis, and the expression levels of foam cell-related markers. Finally, the PI3K/AKT inhibitor LY294002 confirmed that GH alleviates AS via the PI3K/AKT signaling pathway. RESULTS: scRNA-seq data analysis showed that growth hormone signaling was reduced in VSMCs of atherosclerotic arteries. HFD led to elevated levels of serum total cholesterol (TC), triglycerides (TGs), and low-density lipoprotein cholesterol (LDL-C), accompanied by increased lipid deposition, inflammatory responses, and oxidative stress. In contrast, high-density lipoprotein cholesterol (HDL-C) and insulin-like growth factor 1 (IGF-1) levels were lower in C57-Hx mice. GH treatment improved HFD-induced AS in ApoE-/--Hx mice. LC-MS analysis revealed that GH altered lipid metabolism in serum samples from C57-sham, C57-Hx, ApoE-/--Hx, and ApoE-/--Hx-GH(3) mice. GH maintained lipid balance by increasing 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), PE-NMe2(18:1(9z)/18:1(9z)) (DMPE), and 4-chloro-2-nitrobenzylalcohol levels and decreasing 1-heptadecanoyl-sn-glycerol-3-phosphocholine. RNA sequencing showed significant gene expression differences in the aortas of C57-sham and C57-Hx mice. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that GH inhibited the progression of AS by modulating the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway, a finding that was validated through western blotting. Further in vitro studies demonstrated that GH exerted protective effects on VSMCs against ox-LDL-induced damage through activation of the PI3K/AKT pathway, as evidenced by experiments using the specific PI3K inhibitor LY294002. CONCLUSION: GH alleviates the development of AS through the activation of the PI3K/AKT pathway. The findings of this research emphasize the therapeutic potential of GH in inhibiting AS and highlight the importance of the PI3K/AKT pathway as a promising target for clinical intervention.

PI3K/AKT signaling pathway↗