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PubMed1990

Identification of chicken calbindin D28K pre-messenger RNA sequences by polymerase chain reaction.

A transcribed RNA sequence encompassing the junction between the first intron and the second exon of the chicken calbindin D28K gene was copied in a cDNA fragment and subsequently amplified by polymerase chain reaction. When intestinal RNA is used as template, the appearance of the 161 bp amplified fragment is strictly dependent on the vitamin D status of the animal. In fact no amplified fragment is obtained when the RNA is extracted from the intestine of vitamin D-deficient chickens, while it is easily detected when the RNA is extracted only 30 min after injection with 1,25-dihydroxycholecalciferol. Conversely, the amplified fragment is obtained, irrespectively of the vitamin D status of the animal, when the RNA template is extracted from the brain. The appearance of unspliced RNA sequences upon vitamin D induction is followed, after a 30 min lag, by the appearance of the corresponding mature mRNA sequences.

Animals↗
PubMed1980

Transcription of adenoviral genetic information in isolated nuclei. Characterization of viral RNA sequences synthesized in vitro.

The virus-specific RNA sequences synthesized in nuclei isolated from adenovirus type 2-infected HeLa cells comprise a fraction of the total RNA similar to that observed with RNA made in vivo. By 16 h after infection, for example, some 25% of the RNA made in isolated nuclei is transcribed from adenoviral DNA. Only 10 to 15% of the adenoviral RNA sequences synthesized in nuclei isolated during the late phase of infection are transcribed by form III RNA polymerase. This RNA, whose synthesis is resistant to 0.5 microgram/ml, but sensitive to 200 microgram/ml of alpha-amanitin, sediments at about 5 S in denaturing sucrose gradients and must, therefore, represent the virus-associated RNAs. The remainder of the sequences are transcribed by form II RNA polymerase and sediment as several RNA species in denaturing sucrose gradients. The largest of these exhibits the size, 55 to 60 S, expected of a complete transcript of the major, adenoviral transcriptional unit expressed during the late phase. Hybridization of RNA 32P-labeled in nuclei isolated during the late phase of infection to restriction endonuclease fragments of adenoviral DNA immobilized on nitrocellulose filters suggests that sequences of this transcriptional unit are indeed transcribed in vitro. To make a detailed assessment of the fidelity of transcription in isolated nuclei, transcription reactions were performed in the presence of 5-mercuricytidine 5'-triphosphate and the RNA mercurated in vitro separated from endogenous RNA by chromatography on sulfhydryl-agarose columns by a stringent procedure. After demercuration, RNA made in nuclei isolated 20 h following adenovirus type 2 infection was hybridized to the separated strands of restriction endonuclease fragments of 32P-labeled adenovirus type 2 DNA. Such RNA is complementary to the r strand of adenoviral DNA from 16.6 units to a point to the right of 98.3 units. These sequences comprise the major transcriptional unit expressed during the late phase (Fig. 1). It is therefore clear that the fidelity of transcription of adenoviral DNA by form II RNA polymerase is preserved in isolated nuclei. Two 1-strand transcriptional units, those of the IVa2 and ts36 genes (see Fig. 1) are also active at 20 h after infection. The results of similar analysis of RNA made in nuclei isolated 4 and 12 h after infection are also presented and discussed in terms of the mapping of individual transcriptional units within the type 2 adenoviral genome and the temporal regulation of adenoviral gene expression during productive infection.

Adenoviruses, Human↗
PubMed1992

Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis delta virus RNA sequence.

A self-cleaving RNA sequence from hepatitis delta virus was modified to produce a ribozyme capable of catalyzing the cleavage of RNA in an intermolecular (trans) reaction. The delta-derived ribozyme cleaved substrate RNA at a specific site, and the sequence specificity could be altered with mutations in the region of the ribozyme proposed to base pair with the substrate. A substrate target size of approximately 8 nucleotides in length was identified. Octanucleotides containing a single ribonucleotide immediately 5' to the cleavage site were substrates for cleavage, and cleavage activity was significantly reduced only with a guanine base at that position. A deoxyribose 5' to the cleavage site blocked the reaction. These data are consistent with a proposed secondary structure for the self-cleaving form of the hepatitis delta virus ribozyme in which a duplex forms with sequences 3' to the cleavage site, and they support a proposed mechanism in which cleavage involves attack on the phosphorus at the cleavage site by the adjacent 2'-hydroxyl group.

Base Sequence↗
PubMed1978

Detection of yeast ribosomal RNA sequences in E. coli infected with hybrid bacteriophage.

Yeast ribosomal DNA was inserted into Escherichia coli on a bacteriophage vector and the host cell RNA was then extracted and analyzed for the presence of yeast ribosomal RNA sequences. RNA complementary to yeast rDNA was detected by hybridization. The transcription of yeast rDNA was found to be independent of phage RNA synthesis and to occur on the same DNA strand as rRNA transcription in yeast. However, hybridization to restriction fragments of yeast rDNA suggested that the RNA species detected in E. coli differ somewhat from authentic yeast rRNA.

Base Sequence↗
PubMed1986

Localization of preangiotensinogen messenger RNA sequences in the rat brain.

Angiotensinogen (renin substrate) and its messenger RNA are known to accumulate in the rat brain. We have cloned rat preangiotensinogen cDNAs and used them as probes to measure the accumulation of preangiotensinogen messenger RNA sequences in eight regions of rat brain, as well as in liver and kidney. The brain regions examined were the cerebral cortex, hippocampus, striatum, cerebellum, diencephalon (including basal forebrain structures), midbrain, brainstem, and pituitary. On a tissue weight basis, the accumulation of preangiotensinogen RNA sequences was greatest in the liver, midbrain, and brainstem. The relative concentrations of messenger RNA were ranked as follows: liver, brainstem, midbrain greater than cerebellum, diencephalon greater than hippocampus greater than cortex, striatum, kidney greater than pituitary. Relative RNA concentrations from liver to kidney varied over a 16-fold range. Liver and brain preangiotensinogen RNA sequences were indistinguishable in size as measured by gel electrophoresis; however, the kidney sequences appeared some 100 nucleotides larger. Our data agree with previous measurements of angiotensinogen in the rat brain as assayed by renin-catalyzed angiotensin I release.

Angiotensinogen↗
PubMed2025

RNA sequencing offers new diagnostic opportunities in neurodevelopmental disorders: A systematic review.

PURPOSE: Transcriptomics by way of RNA sequencing (RNAseq) has emerged as a means to increase the diagnostic yield in genetic conditions. In this systematic review, we focus on the contribution of transcriptomics to improve the diagnostic yield in neurodevelopmental disorders. METHODS: We performed a systematic literature search in PubMed until January 2024, including articles describing diagnostic RNAseq on at least 1 individual with a primary neurodevelopmental phenotype. We extracted data on cohort size, phenotype, sample tissue, previously used diagnostic methods, added diagnostic yield of RNAseq, the use of control samples, and technical aspects of the RNA sequencing methodology. RESULTS: A total of 17 articles were eligible for inclusion in the systematic review. We found an average added diagnostic yield of 15.5% through RNA sequencing for individuals with neurodevelopmental disorders. There is heterogeneity in the tissue type, reported quality measures, and the computational pipeline. CONCLUSION: The significantly increased diagnostic yield demonstrates the value of this novel tool in the diagnostic setting of neurodevelopmental disorders. Our results offer an overview of common methodologies for RNAseq and allow us to formulate recommendations for genetic labs and clinicians when implementing RNAseq as a diagnostic tool. Lastly, we provide recommendations for future publications to increase transparency and reproducibility.

Humans↗
PubMed1994

RNAlign program: alignment of RNA sequences using both primary and secondary structures.

We have developed an algorithm and a computer program for aligning new RNA sequences with a bank of aligned homologous RNA sequences. Given a common folding structure for the bank, the program performs an alignment between the bank and a new sequence, optimal both in terms of primary and secondary structure. This method is useful to align sequences that present a common folding structure despite extensive divergence of their primary structures. It allows these preserved regions to be precisely distinguished from domains with more variable secondary structure. An optimal alignment of a sequence of length N with a bank of homologous sequences of length M is produced in O (M2N3) time and O(M2N2) space. For sequences that are too long for an algorithm of this complexity, a proposed strategy is to use a classical alignment (using only primary structure data) then improve it with the new algorithm in the regions where the bank stems are not aligned with possible stems in the new sequence. The algorithm has been implemented in Turbo Pascal on a PC, and has been used to align RNA sequences of eubacterial large ribosomal subunit.

Algorithms↗
PubMed1990

Spliced leader RNA sequences can substitute for the essential 5' end of U1 RNA during splicing in a mammalian in vitro system.

L. collosoma or C. elegans SL RNA sequences joined to an adenovirus intron and 3' exon are spliced highly efficiently and accurately in HeLa nuclear extract. After inactivation of U1 snRNPs using RNAase H and a deoxyoligonucleotide complementary to the first 12 nucleotides of U1, splicing of SL RNA-containing constructs continues undiminished, whereas control substrates no longer splice. Since neither binding of U1 snRNPs nor inhibition of splicing is detected using anti-(U1)RNP antibodies, splicing of SL RNA-containing constructs may be entirely U1 snRNP independent. Analyses of altered L. collosoma constructs revealed that the sequence surrounding the 5' splice site is not sufficient to confer U1-independent splicing; the smallest U1-independent region identified so far retains only the first stem-loop of the SL RNA. That sequences responsible for recognition of the 5' splice site can be relocated within the splicing substrate itself reinforces the similarity between group II self-splicing and spliceosome-mediated pre-mRNA splicing.

Animals↗
PubMed1996

Sequence heterogeneities among 16S ribosomal RNA sequences, and their effect on phylogenetic analyses at the species level.

We have analyzed what phylogenetic signal can be derived by small subunit rRNA comparison for bacteria of different but closely related genera (enterobacteria) and for different species or strains within a single genus (Escherichia or Salmonella), and finally how similar are the ribosomal operons within a single organism (Escherichia coli). These sequences have been analyzed by neighbor-joining, maximum likelihood, and parsimony. The robustness of each topology was assessed by bootstrap. Sequences were obtained for the seven rrn operons of E. coli strain PK3. These data demonstrated differences located in three highly variable domains. Their nature and localization suggest that since the divergence of E. coli and Salmonella typhimurium, most point mutations that occurred within each gene have been propagated among the gene family by conversions involving short domains, and that homogenization by conversions may not have affected the entire sequence of each gene. We show that the differences that exist between the different operons are ignored when sequences are obtained either after cloning of a single operon or directly from polymerase chain reaction (PCR) products. Direct sequencing of PCR products produces a mean sequence in which mutations present in the most variable domains become hidden. Cloning a single operon results in a sequence that differs from that of the other operons and of the mean sequence by several point mutations. For identification of unknown bacteria at the species level or below, a mean sequence or the sequence of a single nonidentified operon should therefore be avoided. Taking into account the seven operons and therefore mutations that accumulate in the most variable domains would perhaps increase tree resolution. However, if gene conversions that homogenize the rRNA multigene family are rare events, some nodes in phylogenetic trees will reflect these recombination events and these trees may therefore be gene trees rather than organismal trees.

Bacteria↗
PubMed1987

Two transfer RNA sequences abut the large ribosomal RNA gene in Tetrahymena mitochondrial DNA: tRNA(leu) (anticodon UAA) and tRNA(met) (anticodon CAU).

The sequence of a 1,427 base pair restriction fragment, HaeIII fragment 6, of the ciliate protozoan Tetrahymena mitochondrial DNA, is presented. The first 780 nucleotide sequence aligns well with the terminal segment of the large rDNA sequence of Paramecium mitochondria. Immediately abutting this rDNA termination sequence, a tRNA sequence was found with anticodon UAA for leucine. The derived tRNA sequence is 81 bases long without the 3' CCA end, has a high G + C content of 48.1%, and can be folded into a normal cloverleaf structure with mostly conserved bases and normal stems and loops. The tRNA sequence found at an analogous position of the Paramecium mitochondrial DNA is tRNA(tyr). Following a highly A + T rich sequence of 300 base pairs, another tRNA-like sequence is present; this putative tRNA has only 67 bases with anticodon CAT (Met) and forms standard aminoacyl, anticodon and T psi C stems with a conventional T psi C loop. However, the DHU loop and stem are unusually short and irregular; the base at position 8 is G instead of T; and the base following the anticodon, which is normally a purine, is T. The significance of these tRNA structures is discussed.

Animals↗
PubMed1986

Mitotic recombination in germ cells generated two major histocompatibility complex mutant genes shown to be identical by RNA sequence analysis: Kbm9 and Kbm6.

RNA sequencing represents a major procedural simplification for nucleotide sequence analysis of a transcribed gene. Using newly adapted mRNA and cDNA sequencing procedures, we have sequenced 855 nucleotides of Kbm9 mRNA, corresponding to the codons for the aminoterminal 285 amino acids. The inferred DNA sequence of the Kbm9 gene differs from the parental Kb sequence by single nucleotide alterations in each of codons 116 and 121, resulting in Tyr----Phe and Cys----Arg substitutions, respectively. The Kbm9 sequence is identical to that of another independently arising MHC mutant gene, Kbm6. As both the Kbm9 and Kbm6 genes were generated by recombination between the Kb and Q4 genes, our data indicate that the identical genetic interactions have occurred at least twice. The relatively large extent of identity between Q4 and Kb may be responsible for frequent recombination between the two genes. The parents of the original bm9 mutant mice had five identical mutant offspring, which can be explained by mitotic recombination in the germ cells, producing gonadal mosaicism in the C57BL/6 mother. Thus, mitotic recombination, and not meiotic recombination, appears to be responsible for the formation of at least some of the Kb mutants. Such a mechanism probably plays a major role in the generation of diversity in the major histocompatibility complex.

Animals↗
PubMed1989

Rapid determination of bacterial ribosomal RNA sequences by direct sequencing of enzymatically amplified DNA.

Ribosomal RNA sequences are an appealing target for bacterial classification as well as for development of group- or species-specific DNA probes. Using the polymerase chain reaction and synthetic primers, the feasibility of this gene amplification technique for rapid sequence determination of the major 16S ribosomal RNA domains from small amounts of input DNA is demonstrated. Information useful for phylogenetic classification as well as for construction of specific DNA probes may be obtained by comparison with known sequences.

Bacteria↗
PubMed1999

Recombination of hepatitis D virus RNA sequences and its implications.

Recombination between RNA sequences plays a role in the fast evolution of a few viruses. There has been no report on hepatitis D virus (HDV) recombination. In this study, we analyzed genetic recombination of HDV and its possible impact on evolution and clinical course. The aligned HDV sequences allowed us to construct a phylogenetic tree which supported the notion of distinct lineages of HDV. The tree was also used in the analysis of recombination using partial likelihoods assessed through optimization. Nine segments of the HDV genome with significant levels of genetic recombination were detected. Five segments were in the hypervariable region, and four were in the delta-antigen- coding region. None could be found in the well-conserved autocleavage region that is essential for replication. Recombination occurred both between and within types. The results of this study indicated that the remarkable variation in HDV genomic sequences, particularly in the hypervariable region, among different genotypes may at least partly result from multiple episodes of genetic recombination during evolution. Genetic recombination may play a significant role in increasing genetic diversity. Importantly, a genetic recombination (nt 1082-1093) occurred in one of the immunogenic domains of hepatitis delta virus antigen recognized by human and woodchuck antibodies (amino acids 174-195). Genetic recombination also occurred at another segment between nt 1517 and 1535, which was close to one of the predicted T-cell epitopes (amino acids 26-41). In longitudinal analysis of HDV genomes at different time points during chronic infection, novel dominant HDV strains with amino acid changes at these epitopes usually emerged after severe hepatitis attacks. In the comparison of HDV clones during or shortly after flare-up of liver disease, Ka/Ks ratios of > 1 were frequently found, suggesting Darwinian positive selection. Therefore, recombination in these two segments may play an important role for HDV in the evasion of immunity.

Amino Acid Sequence↗
PubMed1990

Heterogeneity and evolution rates of delta virus RNA sequences.

To investigate the geographical divergence of delta virus RNA sequences, 868 nucleotides (nt), including the delta antigen-coding region, were determined in isolates from two Japanese patients, M and S, by polymerase chain reaction and direct sequencing and compared with three previously reported nucleotide sequences. The sequence obtained for hepatitis delta virus RNA from patient M was approximately 92% identical to sequences previously obtained for two other strains of hepatitis delta virus, whereas the sequence of hepatitis delta virus RNA obtained from patient S was approximately 81% identical to the previously sequenced strains. This suggests that delta agent in Japan has a heterogeneous origin and the delta virus RNA sequence from Japanese patient S is the most divergent delta virus isolate yet analyzed. To study the evolution rate of delta virus RNA, viral isolates obtained 3 and 4 years apart from each of two patients were also sequenced. It was estimated that the substitution rate of viral RNA was 0.57 x 10(-3) nt per site per year in patient M and 0.64 x 10(-3) nt per site per year in patient S for the delta antigen gene.

Amino Acid Sequence↗
PubMed1985

Oligomerization of intervening sequence RNA molecules in the absence of proteins.

The intervening sequence RNA excised from the ribosomal RNA precursor of Tetrahymena forms linear and circular oligomers when exposed to a heating-cooling treatment in vitro. The reactions require no protein or external energy source. Oligomerization is different from other self-catalyzed reactions of the intervening sequence RNA in that it involves intermolecular rather than intramolecular recombination, producing RNA molecules that are substantially larger than the original. The observation that RNA molecules can catalyze their own oligomerization has possible implications for the evolution of chromosomes and for the replicative cycle of plant viroids and virus-associated RNA's.

Animals↗
PubMed1977

Genome analysis of Peromyscus (Rodentia, Cricetidae) VII. Localization of satellite DNA sequences and cytoplasmic poly(A) RNA sequences of P. eremicus on metaphase chromosomes.

A satellite DNA fraction from P. eremicus, having a buoyant density of 1.705 g/ml in neutral CsCl density gradients, was isolated. In situ hybridization experiments, using 3H-RNA complementary to this DNA fraction indicated that the short (heterochromatic) arms of most of the autosomes contained this sequence. Conversely, in situ hybridization using 3H-complementary DNA (cDNA) synthesized from the cytoplasmic poly (A) RNA of P. eremicus (comprising a substantial fraction of total messenger RNA) showed that the number of silver grains in the long arms (euchromatin) was significantly higher than that in the short arms. The X chromosomes showed a distinct localization pattern of both sequences.

Animals↗
PubMed1981

Poliovirus replication proteins: RNA sequence encoding P3-1b and the sites of proteolytic processing.

A partial amino-terminal amino acid sequence of each of the major proteins encoded by the replicase region (P3) of the poliovirus genome has been determined. A comparison of this sequence information with the amino acid sequence predicted from the RNA sequence that has been determined for the 3' region of the poliovirus genome has allowed us to locate precisely the proteolytic cleavage sites at which the initial polyprotein is processed to create the poliovirus products P3-1b (NCVP1b), P3-2 (NCVP2), P3-4b (NCVP4b), and P3-7c (NCVP7c). For each of these products, as well as for the small genome-linked protein VPg, proteolytic cleavage occurs between a glutamine and a glycine residue to create the amino terminus of each protein. This result suggests that a single proteinase may be responsible for all of these cleavages. The sequence data also allow the precise positioning of the genome-linked protein VPg within the precursor P3-1b just proximal to the amino terminus of polypeptide P3-2.

Amino Acid Sequence↗
PubMed1997

Automated alignment of RNA sequences to pseudoknotted structures.

Seq7 is a new program for generating multiple structure-based alignments of RNA sequences. By using a variant of Dijkstra's algorithm to find the shortest path through a specially constructed graph, Seq7 is able to align RNA sequences to pseudoknotted structures in polynomial time. In this paper, we describe the operation of Seq7 and demonstrate the program's abilities. We also describe the use of Seq7 in an Expectation-Maximization procedure that automates the process of structural modeling and alignment of RNA sequences.

Algorithms↗