Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “mRNA sequencing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,009 records · Page 56Linked to original sources
PubMed1983

Cloning and sequence analysis of cDNA for rat angiotensinogen.

A mixture of tetradecamer oligodeoxyribonucleotides complementary to the codons specifying the carboxyl-terminal sequence, Ile-His-Pro-Phe-His, of angiotensin was chemically synthesized as two pools and used for the isolation of a cDNA clone specific for angiotensinogen from a cDNA bank of rat liver mRNA sequences. The two pools (oligo 1 and oligo 2), each containing 24 oligodeoxyribonucleotides, were first used as primers to initiate reverse transcription of rat liver mRNA. One of the pools (oligo 1) was found to prime a specific 32P-labeled cDNA of approximately 160 nucleotides that contained the anticoding sequence corresponding exactly to the amino acid sequence of rat angiotensin. This cDNA, in turn, was used to rescreen cDNA clones that were isolated by initially selecting the rat liver cDNA bank by hybridization with the oligo 1 mixture. One clone thus obtained, designated pRag16, was subjected to nucleotide sequence analysis and verified to contain a nearly full-length cDNA sequence coding for rat angiotensinogen precursor. The deduced amino acid sequence indicates that the precursor molecular consists of angiotensinogen of 453 amino acid residues and a putative signal peptide of 24 amino acid residues. The predicted molecular weight and amino acid composition of angiotensinogen agree well with those determined by using the purified protein. An angiotensin moiety is located at the amino-terminal part of angiotensinogen, preceded directly by the signal peptide and followed by a large carboxyl-terminal sequence that contains two internally homologous sequences and three potential glycosylation sites.

Amino Acid Sequence↗
PubMed1977

Spliced segments at the 5' terminus of adenovirus 2 late mRNA.

An mRNA fraction coding for hexon polypeptide, the major virion structural protein, was purified by gel electrophoresis from extracts of adenovirus 2-infected cells late in the lytic cycle. The mRNA sequences in this fraction were mapped between 51.7 and 61.3 units on the genome by visualizing RNA-DNA hybrids in the electron microscope. When hybrids of hexon mRNA and single-stranded restriction endonuclease cleavage fragments of viral DNA were visualized in the electron microscope,branched forms were observed in which 160 nucleotides of RNA from the 5' terminus were not hydrogen bonded to the single-stranded DNA. DNA sequences complementary to the RNA sequences in each 5' tail were found by electron microscopy to be located at 17, 20, and 27 units on the same strand as that coding for the body of the hexon mRNA. Thus, four segments of viral RNA may be joined together during the synthesis of mature hexon mRNA. A model is presented for adenovirus late mRNA synthesis that involves multiple splicing during maturation of a larger precursor nuclear RNA.

Adenoviridae↗
PubMed1990

Identification of cis-acting sequences required for translational autoregulation of the ermC methylase.

ermC methylase gene expression has been shown to be limited by translational autorepression, presumably due to methylase binding to ermC mRNA. It was found that this repression occurs in trans, yielding a 50% reduction in translation of an ermC-lacZ fusion mRNA. We investigated the ermC mRNA sequences required for translational repression in vivo. A series of deletions identified sequences in the 5' regulatory region that were required for translational repression. These included sequences of the 5' stem-loop structure that were not required for induction, as well as some that were required. The implications of these results for regulation are discussed.

Bacillus subtilis↗
PubMed1986

[Effect of monovalent cations on the ratio of RNA transcripts from DNA with different repetitive sequences in leukemia P-388 cells].

Using leukemic P-388 cells, it was demonstrated that alterations of the intracellular Na+/K+ ratio results in qualitative changes of newly synthesized mRNA, which manifests itself as changes in the kinetics of hybridization of heterogeneous nuclear RNA (hnRNA) with DNA. The decrease of the Na+/K+ value from 3.8:1 to 1:1 leads to inhibition of mRNA synthesis in mRNA cells hybridized at 10(3) greater than Dot greater than 10(4), but weakly affects the transcription of mRNA sequences hybridized with DNA within the Dot interval of 10(3.4)-10(3.8). A similar phenomenon is observed during hybridization of hnRNA with homogeneous fractions of unique and averagely repeating sequences of DNA. The hybridization rate constants of hnRNA synthesized by cells at different values of Na+/K+ and the limit values of hybridization (H infinity) were calculated. It was shown that the rate constants for RNA hybridization with DNA decrease by more than two orders of magnitude during the transition of the averagely repeating DNA fraction to the unique one; however, in both cases these constants give equal values for the RNA synthesized by cells at different cationic balance. The H infinity values for the RNA synthesized by cells at higher Na+ ratio appeared to be 1.5-2.0 times as high as compared with those for the RNA in the cells, in which the Na+/K+ ratio was 1:1. Thus, the monovalent cation ratio seems to exert a strong influence on the expression of sequences of different repeatedness in the genome and to play a role in the regulation of proliferative activity of the cell.

Animals↗
PubMed1982

The complete nucleotide sequence of the chicken ovotransferrin mRNA.

The nucleotide sequence of an almost double-stranded cDNA copy [Cochet, M., Perrin, F., Gannon, F., Krust, A., Chambon, P., McKnight, G. S., Lee, D. C., Mayo, K. E., and Palmiter, R. D. (1979) Nucleic Acids Res. 6, 2435-2452] of chicken ovotransferrin (conalbumin) mRNA has been determined. Taking into account the previously reported 5'-end sequence [Cochet, M., Gannon, F., Hen, R., Maroteaux, L., Perrin, F., and Chambon, P. (1979) Nature (Lond.) 282, 567-574] we present the complete nucleotide sequence of the ovotransferrin mRNA from which the amino acid sequence of the protein is inferred. A computer and statistical analysis of the nucleotide sequence reveals a pattern of internal homology which confirms that the present-day chicken ovotransferrin gene (and by extrapolation the transferrin genes of other species) has evolved by duplication and gives some support to the quadruplication hypothesis of transferrin evolution.

Animals↗
PubMed1984

A multigene family of intron lacking and containing genes, encoding for mouse ribosomal protein L7.

Mouse ribosomal protein L7 is encoded by a multigene family. Screening of two mouse genomic libraries with cloned L7 cDNA, has resulted in the isolation of nine independent lambda Charon 4A recombinant phages which include seven different L7 genes. Restriction enzyme mapping of six of these genes (L7-1, L7-16, L7-18, L7-28, L7-35 and L7- 16b ) reveals dissimilarity in sites within the L7 sequences as well as in the flanking regions. Electron microscopic analysis of heteroduplex and S1 nuclease mapping demonstrate that the first five genes contain the entire L7 mRNA sequence but lack introns. Based on these features we propose that these are processed genes. Of the L7 genes described here only one (L7- 16b ) exhibits a high degree of homology with L7 mRNA and contains introns. We discuss the possibility that this low representation of intron containing L7 genes may reflect the proportion of functional L7 genes in this multigene family.

Animals↗
PubMed1997

The structural organization of the human Na+/myo-inositol cotransporter (SLC5A3) gene and characterization of the promoter.

The genomic structure, transcription start site, polyadenylation signals, and promoter of the human Na+/ myo-inositol cotransporter (SLC5A3) gene have been elucidated through cloning, sequencing, mRNA analyses, and reporter gene assays. The gene consists of one promoter and two exons spanning approximately 26 kb. Exon 1 contains 175 bp of 5' untranslated sequence and is 15 kb upstream of exon 2. The 9.5-kb exon 2 contains the entire 2157-bp open reading frame and a large 3' untranslated sequence with seven putative polyadenylation signals. Multiple messages with different-sized 3' untranslated regions can be detected on Northern blots. Hypertonic stress caused mRNA levels, and primarily that of the full-length 9.5-kb transcript, to increase in cultured melanoma cells; ribonuclease protection analysis demonstrated that the transcription start site was the same in stressed as in control cells. The SLC5A3 gene functions in cellular osmoregulation and is expressed in many human tissues including the brain, kidney, and placenta. It is localized to chromosome 21q22.1. An overexpression of the SLC5A3 gene deserves consideration as a factor in the pathophysiology of Down syndrome.

Animals↗
PubMed1977

Human placental lactogen mRNA and its structural genes during pregnancy: quantitation with a complementary DNA.

A complementary DNA (cDNA) strand was transcribed from human placental lactogen (hPL) mRNA. Based on alkaline sucrose gradient centrifugation, the size of the cDNA was about 8 S, which would represent at least 80% of the hPL mRNA. Previously we showed that four to five times more hPL was synthesized in cell-free extracts derived from term as compared to first trimester placentas. Hybridization of the cDNA with RNA derived from placental tissue revealed that there was about four times more hPL mRNA sequences in total RNA from term placenta than in a comparable quantity of total first trimester RNA. Only background hybridization was observed when the cDNA was incubated with RNA prepared from human kidney. To test if this differential accumulation of hPL mRNA was the result of an amplification of hPL genes, we hybridized the labeled cDNA with cellular DNA from first trimester and term placentas and with DNA isolated from human brain. In all cases, the amount of hPL sequences was approximately two copies per haploid genome. Thus, the enhanced synthesis of hPL mRNA appears to result from a transcriptional activation rather than an amplification of the hPL gene. The increase likely reflects placental differentiation in which the proportion of syncytial trophoblast increases at term.

DNA↗
PubMed2005

Pokemon expression in malignant glioma: an application of bioinformatics methods.

OBJECT: In this report the authors review the role of bioinformatics in the design of a research project in which the molecular genetics of malignant glioma were studied. A project to characterize Pokemon expression in malignant glioma was developed, refined, and implemented using bioinformatics methods. METHODS: Using the resources available from the National Center for Biotechnology Information, the messenger RNA (mRNA) sequence for Pokemon was determined. With this information and online primer design tools, novel primers were designed that would specifically amplify Pokemon mRNA by using reverse transcription-polymerase chain reaction assays. CONCLUSIONS: The promise of bioinformatics is in the rapid and widespread dissemination and analysis of genomic information. This information is then used in research investigating the genetic basis of disease. In this paper the authors review the bioinformatics methods used in their study of Pokemon expression in malignant glioma.

Base Sequence↗
PubMed1986

The nucleotide sequence of the mRNA encoding the fusion protein of measles virus (Edmonston strain): a comparison of fusion proteins from several different paramyxoviruses.

Membrane fusion is the primary cytopathic effect observed in cells infected with measles virus. The viral protein responsible for this process has previously been defined as the fusion (F) protein. Fusion is activated by the proteolytic cleavage of a precursor molecule (F0) to yield two disulfide-linked polypeptides (F1 and F2). In this paper the mRNA for the membrane fusion protein has been cloned and the resulting cDNAs were sequenced. A mRNA composed of 2377 nucleotides was found to contain one open reading frame which could potentially code for a protein of 550 amino acids. This corresponding gene product was identified as the fusion protein through use of antibodies directed against a synthetic peptide which was constructed from the deduced amino acid sequence. A long and rather G-C rich 5' terminus was found on the mRNA and this noncoding region may play some role in regulation of protein synthesis at the translational level. Protein sequence data derived from the cDNA clones revealed a highly conserved F1 amino terminus which is characteristic of most paramyxoviruses. Very little amino acid homology (except for the conservation of the F1 terminus and 9 cysteines) was evident when the sequence was compared to other paramyxovirus fusion proteins. However an overall hydrophobic nature was characteristic of all the F proteins and hydrophobicity plots for the fusion proteins of 4 different paramyxoviruses were very similar. Computer analysis was also employed to analyze the secondary structure of the measles virus F protein. Large stretches of alpha helix were characteristic of the regions which purportedly interact with membranes. The functional domains of the F protein and their similarity to those of the influenza hemagglutinin protein are discussed in this communication. We concluded that the distribution of hydrophobic regions capable of spanning biological membranes determines the fusogenic nature of the F protein.

Amino Acid Sequence↗
PubMed1994

Identification of an activity that interacts with the 3'-untranslated region of c-myc mRNA and the role of its target sequence in mediating rapid mRNA degradation.

Single or multiple copies of the pentameric motif, AUUUA, are found in the 3'-untranslated domain of most immediate early mRNAs. A broad body of data links this 5-base element to the selective degradation of these mRNAs. However, AUUUA motifs are neither always necessary nor always sufficient to tag an mRNA for rapid degradation. Here, we describe a cytoplasmic, protease-sensitive factor in Balb/c3T3 cells that interacts with the 3'-untranslated region of c-myc mRNA. The factor recognizes a 39-base uridine-rich domain adjacent to, but distinct from, the c-myc AUUUA motif. In chimeric mRNA constructs, the 39-base binding element cooperates with adjacent sequence material to destabilize beta-globin mRNA.

3T3 Cells↗
PubMed2026

Clinical and functional characterization of a novel homozygous non-canonical splice mutation (c.1910-15_1910-11delinsTTACA) in CEP290 causing Joubert syndrome.

BACKGROUND: Joubert syndrome (JS) is a rare, predominantly autosomal recessive neurodevelopmental disorder characterized by hypotonia, motor delay, intellectual disability, oculomotor apraxia, and the hallmark "molar tooth sign" on axial view of MRI. JS is genetically heterogeneous, with pathogenic variants identified in more than 40 genes involved in primary cilia function. Among these, CEP290 is one of the most frequently mutated genes. RESULTS: In this study, we investigated two children-an 11-year-old boy (the proband) and his 5-year-old sister-both presenting with a similar phenotype consistent with JS. The parents, who self-identified as Chechen, reported distant consanguinity. The family also included a healthy 13-year-old daughter. The proband had previously been evaluated by a neurologist and underwent whole-genome sequencing (WGS); however, no causative variants were identified initially. After phenotype reassessment by a clinical geneticist, we performed a reanalysis of the raw WGS data and identified a novel homozygous intronic variant of uncertain significance (VUS), c.1910-15_1910-11delinsTTACA in CEP290 (NM_025114.4). Sanger sequencing confirmed that both the proband and his affected sister were homozygous for this variant, which they inherited from their heterozygous parents. Their healthy sister did not carry the variant. mRNA-sequencing and targeted cDNA sequencing (read depth ~ 100,000x) demonstrated that this intronic variant causes completely aberrant splicing of CEP290 pre-mRNA. Predominantly this variant causes the skipping of exon 20 in the main CEP290 transcript. Alternatively, the variant results in partial inclusion of intron 19 into the mRNA, elongation of exon 20 by 58 nucleotides, and a homozygous substitution chr12:88114573 (ACTGTGTA> TTACAGTA). No canonical mRNA isoform was detected when the variant was homozygous. Both the predicted severe truncation and the likely degradation of aberrant transcripts through nonsense-mediated decay (NMD) would correspond to complete loss of CEP290 function. Following the reclassification of this VUS to likely pathogenic, the family was able to pursue in vitro fertilization (IVF) with preimplantation genetic testing for monogenic disorders (PGT-M). CONCLUSION: Our study highlights the critical importance of proper phenotyping prior to referral for WES/WGS as well as of combining NGS with functional mRNA studies to achieve a molecular diagnosis for patients with predicted splice-site mutations in JS-associated genes. It also emphasizes the need for functional reassessment of VUS when genomic data are expected to guide reproductive decision-making within affected families.

Humans↗
PubMed1988

Characterization of two divergently transcribed Dictyostelium gene pairs and identification of G-rich sequence element lying between them with the characteristics of a basal promoter element.

The cysteine proteinase 1 (CP1) and cysteine proteinase 2 (CP2) genes of Dictyostelium discoideum encode coordinately expressed mRNA sequences that are inducible by extracellular cAMP. Both genes form part of divergently transcribed gene pairs. The gene proximal to CP1 is coordinately regulated and encodes a protein containing several potential zinc binding domains of the kind found in DNA binding proteins. The gene proximal to CP2 is a constitutively transcribed gene of unknown function. There are multiple, short, G-rich sequence elements between both gene pairs, and deletion of the pair of elements 200 nucleotides upstream from the CP2 gene abolishes cAMP-inducibility. A synthetic oligonucleotide, containing two copies of the G-rich element from the CP1 gene, will reconstitute cAMP-inducibility in the deletion mutant of the CP2 gene. This shows that the elements in the two genes are functionally homologous. Efficient induction requires at least two copies of the CP1 element, but their relative orientation is unimportant. Two copies in an inverted orientation are, however, inactive when moved upstream of their normal position and are incapable of conferring cAMP-inducibility on a heterologous gene. These observations suggest that these sequences are either essential promoter elements, not themselves interacting with the inducer, or that their interaction with a separate class of control sequences is necessary for inducible expression.

Amino Acid Sequence↗
PubMed1991

Insertion of cellular sequences in the genome of bovine viral diarrhea virus.

The genomic sequences of four pestiviruses, two BVDV strains (Osloss and NADL, both of which are cytopathogenic) and two HCV strains, were analyzed. Comparative studies revealed the presence of small insertions of cellular sequences in the genomes of both BVDV strains; the insertions are located in a region coding for a nonstructural protein. Such insertions are not present in the HCV sequences. The insertion identified in BVDV Osloss encodes a complete ubiquitin-like element. The sequence inserted in the BVDV NADL genome shows no homology to a ubiquitin gene but is almost identical with another bovine mRNA sequence. Molecular characterization of a BVDV "pair", isolated from an animal with mucosal disease, led to the detection of a ubiquitin-like sequence in the genome of the cytopathogenic strain, but not of the noncytopathogenic strain. It is proposed that recombination between viral and cellular RNA leads to formation of cpBVDV genomes. This hypothesis has direct implications for the pathogenesis of mucosal disease.

Amino Acid Sequence↗
PubMed1976

Characterization of the 5' termini of hn RNA in mouse L cells: implications for processing and cap formation.

An analysis of the phosphorylated and capped 5' termini of the heterogenous nuclear RNA of mouse L cells has revealed four types of structure: pppXp..., ppXp..., pXp..., and m7/GpppXmp.... The 5'triphosphate termini consists exclusively of pppGp... and pppAp..., whereas a large proportion of the 5' monophosphate termini are pUp.... The 5'diphosphate termini contain all four species of nucleotide in relative proportions that are roughly similar to those found at the Xm position of cap structures. These results indicate that initiation of hnRNA transcription occurre exclusively with purine nucleotides, and consequently that the hnRNA molecules containing pyrimidines at the 5' termini very probably arise by cleavages at internal sites of larger primary transcripts. Taken together with previous results relating cap structures of hnRNA and mRNA, the data favor a model in which some mRNA sequences are located at transcriptionally initiated proportions and others in internal regions of their precursors. According to this model, both the mRNA segments derived from initial 5' end, and those derived by cleavage at internal sites could be converted to diphosphate-terminated derivatives, which then condense with GTP to form cap structures according to the mechanism previously described for vaccinia and reovirus mRNA.

Adenine↗
PubMed1996

Influenza A virus RNA-dependent RNA polymerase cleaves influenza mRNA in vitro.

We have investigated the endonuclease activity of the influenza A virus RNA polymerase in an in vitro assay with an artificial influenza-like mRNA containing a cap structure at its 5' terminus, followed by a 10 nt beta-globin mRNA sequence, and the 5' and 3' conserved termini of a truncated nucleoprotein (NP) cRNA influenza sequence. Results showed that partially purified virion ribonucleoprotein complexes (RNPs) and micrococcal nuclease treated RNPs cleaved the artificial influenza-like mRNA substrate specifically at positions near the 5' terminus to generate capped 14 and 15 nucleotide long RNA fragments which subsequently served as primers to initiate transcription. The endonuclease activity was completely blocked by addition of cap analog and competitively inhibited by added globin mRNA. Furthermore, an in vitro reconstituted influenza RNA transcription reaction containing a truncated NP vRNA as template, micrococcal nuclease treated RNPs and globin mRNA as primer, synthesized capped and uncapped full length (+) sense products. Enzyme kinetics showed that capped RNA was made earlier in the reaction; it reached a peak at 120 min and then declined. However, uncapped cRNA synthesis appeared later and remained as the dominant product later in the reaction. The nature of these products was confirmed by ribonuclease protection assays and by primer extension.

Globins↗
PubMed1977

Cloning and amplification of alpha and beta mouse globin gene sequences synthesised in vitro.

New chimeric Escherichia coli plasmids containing alpha or beta globin gene sequences of the mouse were constructed. Double-stranded DNA, synthesised in vitro in a 2-step reaction from mouse globin mRNA was inserted into E. coli plasmid pCR1, after tailing of the 2 DNAs with dG and dC respectively. Some of the mouse globin plasmids described contain at least 90% of the globin mRNA sequence and therefore contain the entire translated sequence of the globin genes. Some possible uses of these recombinant plasmids are described.

Animals↗
PubMed1986

Expression of insulin-like growth factor II in human placentas from normal and diabetic pregnancies.

The growth and development of the placenta is critical to fetal growth and development; however, little is known regarding the mechanisms controlling placental growth and development. Human placental membranes are known to possess receptors for insulin-like growth factors I and II (IGF-I and IGF-II) from early gestation, and increasing evidence supports a major role for IGF-I and/or IGF-II in fetal growth and development. Therefore, the IGFs may also play a significant role in regulating placental growth and development. We report here that an adult human liver IGF-II cDNA hybridizes to poly(A)+ RNAs of human placentas from different gestational ages. There are four placental poly(A)+ RNA species that hybridize to IGF-II cDNA, the major one of which is about 6000 bases. The sizes of the hybridized transcripts are the same for placentas of different gestational ages. Furthermore, the IGF-II sequences expressed in the human placenta were quantitated by dot blot hybridization. The second trimester placenta expresses more IGF-II mRNA sequences than placenta of first trimester and term. Interestingly, the term placentas from diabetic pregnancies also express more of these sequences than those from normal pregnancies. These results suggest that there are developmental changes in the expression of the IGF-II gene in the placenta and that IGF-II may promote placental growth by way of an autocrine and/or paracrine mechanism. Moreover, fetuses developing in diabetic pregnancies receive a large influx of glucose, which in turn may stimulate the expression of IGF-II sequences in placenta, resulting in higher utilization of glucose and overgrowth of placenta. This may explain the macrosomia and high incidence of malformations and stillbirths known to result from pregnancies in diabetics.

Female↗