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 217 records · Page 12Linked to original sources
PubMed1986

Localization of specific mRNA sequences in Xenopus laevis embryos by in situ hybridization.

In situ hybridization of cloned cDNA probes to frozen sections of Xenopus laevis stage-42 tadpoles has been used to determine the tissue localization of several mRNAs. Nine out of sixteen probes tested hybridized to most or all tadpole tissues; seven probes exhibited tissue-specific hybridization. The non-tissue-specific sequences hybridized to RNA species that are also present in maternal RNA while the tissue-specific sequences hybridized to embryonic RNA species induced after gastrulation and undetectable in maternal RNA. Tissue-specific hybridization was observed with muscle (five clones), epidermis (one clone), and the nervous system (one clone). All muscle-specific sequences hybridized to somites and lateral plate muscles, but they differed in their hybridization to heart muscle.

Animals↗
PubMedDate not supplied

[Possible role of tRNA1Lys in recognition of pre-mRNA sequences involved in the control of splicing].

Previously we presented some evidence for involvement of the imported tRNA1Lys in splicing the mitochondrial mosaic genes transcripts in yeast. The new data provided here together with our previous results show that tRNA1Lys/transcripts complementarity permits accurate prediction of intron-exon donor boundaries for at least 7 (probably all 9) Dujonian 1st class introns. A computer-assisted search for homologies between mosaic genes transcripts and sequences complementary to tRNA1Lys reveals most of the control sequences previously determined as the sites of 1) cis-acting splicing blocks, 2) conserved RNA sequences and 3) conserved features of transcripts secondary structure. The sequences revealed in our study are as follows: sequences GGGGG and GGCTC in 2nd class introns and less convincing cases are R + E', E + P and IG in 1st class introns. The results obtained here together with the results of other authors suggest a series of (often weak) alternative RNA-RNA interactions needed to regulate and(or) provide a sufficiently error-free mechanism of splicing for the mosaic genes transcripts. A comparison was made of: 1) sequences near the sites of 2 different autocatalytic Tetrahymena pre-rRNA cleavage-ligation events (splicing and cyclization of the intron RNA) and 2) sequences of tRNA1Lys probably contacting donor boundaries of 1st class mitochondrial introns. The comparison reveals an obvious conservation of some basic structure regardless of the different chemical directions of the strands involved. The result suggests a possible catalytic function for the tRNA1Lys probably involved also in recognition and control processes of mitochondrial transcripts splicing.

Base Sequence↗
PubMed1981

5'-Terminal sequences of eucaryotic mRNA can be cloned with high efficiency.

A method for cloning mRNAs has been used which results in a high yield of recombinants containing complete 5'-terminal mRNA sequences. It is not dependent on self-priming to generate double-stranded DNA and therefore the S1 nuclease digestion step is not required. Instead, the cDNA is dCMP-tailed at its 3'-end with terminal deoxynucleotidyl transferase (TdT). The synthesis of the second strand is primed by oligo(dG) hybridized to the 3'-tail. Double-stranded cDNA is subsequently tailed with dCTP and annealed to dGMP-tailed vector DNA. This approach overcomes the loss of the 5'-terminal mRNA sequences and the problem of artifacts which may be introduced into cloned cDNA sequences. Chicken lysozyme cDNA was cloned into pBR322 by this procedure with a transformation efficiency of 5 x 10(3) recombinant clones per ng of ds-cDNA. Sequence analysis revealed that at least nine out of nineteen randomly isolated plasmids contained the entire 5'-untranslated mRNA sequence. The data strongly support the conclusion that the 5'-untranslated region of the lysozyme mRNA is heterogeneous in length.

Avian Myeloblastosis Virus↗
PubMed1978

Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries.

Selected regions of cloned EcoRI fragments of the chicken ovalbumin gene have been sequenced. The positions where the sequences coding for ovalbumin mRNA (ov-mRNA) are interrupted in the genome have been determined, and a previously unreported interruption in the DNA sequences coding for the 5' nontranslated region of the messenger has been discovered. Because directly repeated sequences are found at exon-intron boundaries, the nucleotide sequence alone cannot define unique excision-ligation points for the processing of a possible ov-mRNA precursor. However, the sequences in these boundary regions share common features; this leads to the proposal that there are, in fact, unique excision-ligation points common to all boundaries.

Animals↗
PubMed1984

Changes in the sequence content of albumin mRNA and in its translational activity in the rat liver with age.

To investigate the regulation of age-related changes in albumin synthesis in the rat liver, total postnuclear RNA and polyribosomes, both membrane-bound and free, were prepared from livers of rats of different ages. By the use of a specific complementary DNA probe, the albumin mRNA sequence content was quantitated in these RNA fractions. These studies showed a specific increase in albumin mRNA sequence content in total postnuclear RNA and membrane-bound polyribosomes at between 12 and 24 months of age. Between 24 and 36 months of age, the increase in the amount of albumin mRNA in these two fractions was due only to an increase in liver weight. The increase in albumin mRNA sequence content was not found in the poly(A)+ fraction but in the RNA extracted from the void of oligo(dT)-cellulose column chromatography. The isolated polyribosomes were translated in a cell-free system to assess age-related changes in total protein and albumin synthesis due to translational control. No changes with age were found in the translational capacity of membrane-bound and free polyribosomes per RNA unit. Immunoprecipitation of the synthesized albumin in the translation products revealed that albumin synthesis in the cell-free system is not increased proportionally with the elevated albumin mRNA level between 12 and 24 months of age. This indicates that albumin mRNAs present in the livers of old rats are biologically less active than those found in younger animals.

Aging↗
PubMed1981

Bacteriorhodopsin: partial sequence of mRNA provides amino acid sequence in the precursor region.

mRNA for bacteriorhodopsin from Halobacterium halobium has been partially purified. By using this mRNA as template in the presence of reverse transcriptase RNA-dependent DNA nucleotidyltransferase and a 5'-[32P] synthetic oligodeoxyribonucleotide corresponding to amino acids 9-12 of bacteriorhodopsin as primer, we have isolated the major 5'-[32P]cDNA product, approximately 80 nucleotides long, and determined its sequence. Based on the cDNA sequence, the 5'-proximal sequence of bacteriorhodopsin mRNA is G-C-A-U-G-U-U-G-G-A-G-U-U-A-U-U-G-C-C-A-A-C-A-G-C-A-G-U-G-G-A-G-G-G-G-G-U-A-U-C -G-C-A-G-G-C-C-C-A-G-A-U-C-A-C-C-G-G-A-C-G-U-C-C-G. This includes the expected sequence for amino acids 1-8 and shows that bacteriorhodopsin is synthesized as a precursor that is at least 13 amino acids longer (Met-Leu-Glu-Leu-Leu-Pro-Thr-Ala-Val-Glu-Gly-Val-Ser) at the NH2 terminus. Agarose/urea gel electrophoresis of the partially purified mRNA showed several bands; of these, a major one hybridized with 5'-[32P]cDNA. These results suggest that the bacteriorhodopsin mRNA in the partially purified preparation is homogeneous in size and that it constitutes a substantial portion of the RNA preparation subjected to electrophoresis.

Amino Acid Sequence↗
PubMed2001

Thermodynamics and kinetics of Hsp70 association with A + U-rich mRNA-destabilizing sequences.

Rapid mRNA degradation directed by A + U-rich elements (AREs) is mediated by the interaction of specific RNA-binding proteins to these sequences. The protein chaperone Hsp70 has been identified in a cellular complex containing the ARE-binding protein AUF1 and has also been detected in direct contact with A + U-rich RNA substrates, indicating that Hsp70 may be involved in the regulation of ARE-directed mRNA turnover. By using gel mobility shift and fluorescence anisotropy assays, we have determined that Hsp70 directly and specifically associates with U-rich RNA substrates in solution. With the ARE from tumor necrosis factor alpha (TNFalpha) mRNA, Hsp70 forms a dynamic complex consistent with a 1:1 association of protein:RNA but demonstrates cooperative binding behavior on polyuridylate substrates. Unlike AUF1, the RNA binding activity of Hsp70 is not regulated by ion-dependent folding of the TNFalpha ARE, suggesting that AUF1 and Hsp70 recognize distinct binding determinants on this RNA substrate. Binding of Hsp70 to the TNFalpha ARE is driven entirely by enthalpy at physiological temperatures, indicating that burial of hydrophobic surfaces is likely the principal mechanism stabilizing the Hsp70.RNA complex. Potential roles for the interaction of Hsp70 with ARE-containing mRNAs in the regulation of mRNA turnover and/or translational efficiency are discussed.

Adenosine↗
PubMed1991

Simian virus 40 late mRNA leader sequences involved in augmenting mRNA accumulation via multiple mechanisms, including increased polyadenylation efficiency.

We have examined the contribution of 5' leader sequences to expression directed by the simian virus 40 (SV40) late promoter. These studies showed that addition of sequences which contain the late leader 3' splice site to the late promoter led to an increase in the accumulation of mRNA expressed by the promoter. No other sequences within the leader region, between SV40 positions 334 and 560, exhibited a substantial influence on mRNA accumulation. The increase was due, at least in part, to the creation of a spliceable mRNA transcript, since mutation of either the 5' or 3' splice site could attenuate the effect. However, sequences at or near the 3' splice site appeared to play a more important role than did the 5' splice site in bringing about this increase. In many instances, mutation of the 3' splice site also led to the accumulation of extended transcripts, whereas mutation of the 5' splice site did not produce this result in any instance. Analysis of these extended transcripts showed that they retained sequences normally lost upon cleavage and polyadenylation. This finding suggested that mutation of the 3' splice site sequence led to decreases in the efficiency of polyadenylation. We propose that the SV40 late leader sequences positively contribute to expression of the viral late genes by increasing mRNA accumulation via multiple mechanisms, including the enhancement of pre-mRNA polyadenylation efficiency.

Animals↗
PubMed2000

Mutants of Arabidopsis defective in a sequence-specific mRNA degradation pathway.

One of the ways a cell can rapidly and tightly regulate gene expression is to target specific mRNAs for rapid decay. A number of mRNA instability sequences that mediate rapid mRNA decay have been identified, particularly from multicellular eukaryotes, but pinpointing the cellular components that play critical roles in sequence-specific decay in vivo has been more difficult. In contrast, general pathways of mRNA degradation in yeast have been well established through the analysis of mutants affecting the general mRNA decay machinery. Strategies to isolate mutants in sequence-specific mRNA decay pathways, although extremely limited so far, have the potential to be just as powerful. In the study reported here, a selection in transgenic plants allowed the isolation of rare mutants of Arabidopsis thaliana that elevate the abundance of mRNAs that contain the plant mRNA instability sequence called DST (downstream element). This instability sequence is highly conserved in unstable small auxin up RNA (SAUR) transcripts. Genetic analysis of two dst mutants isolated via this selection showed that they are incompletely dominant and represent two independent loci. In addition to affecting DST-containing transgene mRNAs, mutations at both loci increased the abundance of the endogenous DST-containing SAUR-AC1 mRNA, but not controls lacking DST sequences. That these phenotypes are caused by deficiencies in DST-mediated mRNA decay was supported by mRNA stability measurements in transgenic plants. Isolation of the dst mutants provides a means to study sequence-specific mRNA degradation in vivo and establishes a method to isolate similar mutants from other organisms.

Arabidopsis↗
PubMed1975

Cell-free transcription of mammalian chromatin. Quantitative measurement of newly synthesized globin messenger RNA sequences.

Transcription of globin mRNA sequences from rabbit marrow chromatin was detected by hybridization with globin complementary DNA (cDNA). The presence of newly synthesized RNA in cDNA-globin mRNA hybrids isolated by Cs2SO4 density equilibrium centrifugation required the addition of both rabbit marrow chromatin and DNA-dependent RNA polymerase (Escherichia coli) to the transcription reaction. No globin mRNA sequences were detected in RNA transcribed from rabbit liver chromatin or from rabbit marrow DNA. Selective transcription of globin mRNA sequences was therefore tissue-specific and dependent on the presence of chromosomal proteins. Globin mRNA sequences synthesized by E. coli RNA polymerase were distinguished from those synthesized by chromatin-bound (endogenous) RNA polymerases by the use of alpha-amanitin. A typical reaction with rabbit marrow chromatin yielded 100 mug of purified RNA which contained approximately 5 ng (0.005%) of globin mRNA sequences synthesized by E. coli RNA polymerase, 1 ng (0.001%) of globin mRNA seqeences synthesized by endogenous RNA polymerases, and 4 ng (0.004%) of globin mRNA sequences derived from chromatin-associated (endogenous) RNA. Forty per cent of the globin mRNA sequences derived from endogenous RNA could be removed by poly(U)-Sepharose chromatography. The accurate measurement of globin mRNA sequences required improved conditions for the purification and hybridization of RNA transcribed from chromatin.

Animals↗
PubMed1979

Parvovirus RNA transcripts containing sequences not present in mature mRNA: a method for isolation of putative mRNA precursor sequences.

We report here a method of RNA preparation that may enrich for precursor RNA sequences and the results of an investigation of adeno-associated virus (AAV) RNA transcription that used this method. Whole cells were lysed with detergent and high salt and separated into supernatant and pellet (crude chromatin) fractions. These fractions were then separately deproteinized by proteolytic digestion and phenol extractions. DNA was removed from the preparation by two cycles of pancreatic DNase digestion and phenol extraction. Hybridization analyses of the RNA obtained from AAV/adenovirus-infected KB (human) cells revealed some AAV-specific RNA sequences that were not present in the mature 20S mRNA. These additional sequences were contained in AAV RNA molecules present in the pellet fraction, whereas the 20S AAV mRNA accumulated in the supernatant. A species of AAV-specific RNA (about 22S), which was associated only with the pellet fraction and was labeled only after a short pulse, appeared to have a kinetic relationship with the more stable cytoplasmic 20S mRNA. These putative AAV mRNA "precursors" and precursor sequences were not observed previously when conventional methods were used to obtain RNA from either whole cells or isolated nuclei.

Base Sequence↗
PubMed1983

5'-Terminal sequence of the mRNA of mouse whey acidic protein contains three possible sites of interaction with 18S rRNA.

The mRNA sequence of whey acidic protein, a major mouse milk protein, was cloned in full length. The sequence analysis of the cDNA clones revealed that the mRNA contains a 28 nucleotide-long 5'-noncoding region. Three different portions of this region were identified as possible sites of interaction with the 3'-end of 18S rRNA that may facilitate efficient rate of translation of the mRNA.

Animals↗
PubMed1986

Human parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein: nucleotide sequence of mRNA and limited amino acid sequence of the purified protein.

The nucleotide sequence of mRNA for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 3 virus obtained from the corresponding cDNA clone had a single long open reading frame encoding a putative protein of 64,254 daltons consisting of 572 amino acids. The deduced protein sequence was confirmed by limited N-terminal amino acid microsequencing of CNBr cleavage fragments of native HN that was purified by immunoprecipitation. The HN protein is moderately hydrophobic and has four potential sites (Asn-X-Ser/Thr) of N-glycosylation in the C-terminal half of the molecule. It is devoid of both the N-terminal signal sequence and the C-terminal membrane anchorage domain characteristic of the hemagglutinin of influenza virus and the fusion (F0) protein of the paramyxoviruses. Instead, it has a single prominent hydrophobic region capable of membrane insertion beginning at 32 residues from the N terminus. This N-terminal membrane insertion is similar to that of influenza virus neuraminidase and the recently reported structures of HN proteins of Sendai virus and simian virus 5.

Amino Acid Sequence↗
PubMed1993

Leader-mRNA junction sequences are unique for each subgenomic mRNA species in the bovine coronavirus and remain so throughout persistent infection.

The common leader sequence on bovine coronavirus subgenomic mRNAs and genome was determined. To examine leader-mRNA junction sequences on subgenomic mRNAs, specific oligodeoxynucleotide sets were used in a polymerase chain reaction to amplify junction sequences from either the positive-strand mRNA (eight of nine total identified species) or the negative-strand anti-mRNA (six of the nine species), and sequenced. The mRNA species studied were those for the N, M, S, and HE structural proteins and the 9.5-, 12.7-, 4.8-, and 4.9-kDa putative nonstructural proteins. By defining the leader-mRNA junction sequence as the sequence between (i) the point of mismatch between the leader and genome and (ii) the 3' end of the consensus heptameric intergenic sequence [(U/A)C(U/C)AAAC)], or its variant, a unique junction sequence was found for each subgenomic mRNA species studied. In one instance (mRNA for the 12.7-kDa protein) the predicted intergenic sequence UCCAAAC was not part of the junction region, and in its place was the nonconforming sequence GGTAGAC that occurs just 15 nt downstream in the genome. Leader-mRNA junction sequences found after 296 days of persistent infection were the same as those found during acute infection (< 18 hr postinfection). These data indicate that, in contrast to the closely related mouse hepatitis virus, the bovine coronavirus maintains a stable leader-mRNA junction sequence for each mRNA. Interestingly, this stability may be related to the fact that a UCUAA sequence element, postulated by others to be a regulator of the leader-mRNA fusion event, occurs only once within the 3' flanking sequence of the genomic leader donor and once at intergenic sites in the bovine coronavirus genome, whereas it occurs two to four times at these sites in the mouse hepatitis coronavirus.

Animals↗
PubMed1994

Sequence of the mRNA for a glutathione transferase Pi with a different substrate specificity in V79 Chinese hamster lung cells.

The mRNA sequence for a glutathione transferase (GST) belonging to the Pi class has been determined. This was a first step towards elucidating, at the molecular level, why V79 Chinese hamster lung cells lack the capacity to conjugate the benzo[a]pyrene (BP) derivative BPDE, but nonetheless contain the GST pi gene, express GST pi mRNA and contain a protein that binds to antibodies directed against the human GST Pi enzyme. The sequencing strategy involved synthesis of a cDNA library, circularization of the GST pi cDNA for PCR amplification and subsequent DNA sequencing. The coding sequence for the GST Pi protein of V79 cells, designated CLOGSTP1, consisted of 627 bp coding for 209 amino acids (aa), corresponding to a 23-kDa protein. The cDNA sequence obtained demonstrated extensive homology to those from other species, especially rat and mouse, where this homology was 92 and 91%, respectively. Upon comparing the aa sequence predicted from CLOGSTP1 to those of rat, mouse, pig, cow and man, the most striking differences were found in aa positions 19, 39, 40, 110, 113 and 151. Consequently, the explanation for the lower capacity for GST Pi-catalyzed conjugation in V79 cells, as compared to other species, remains a matter of speculation, since none of these aa positions coincides with positions involved in the xenobiotic substrate-binding site of GST Pi from pig and human or of GST Mu from rat. The most likely candidates for causing the observed change in substrate specificity might be Lys110 and Glu113, which are the altered residues closest to this binding site and which might, thus, exclude BPDE as a substrate for the Chinese hamster enzyme.

Amino Acid Sequence↗
PubMed1984

alpha-Amanitin-insensitive transcription of mouse beta major-globin 5'-flanking and structural gene sequences correlates with mRNA expression.

A small proportion of the RNAs of mouse reticulocytes consists of beta major-globin mRNA sequences linked to sequences transcribed from the 5'-flanking region of the beta major-globin gene. These upstream RNAs are polyadenylylated and contain 700-800 nucleotides, and their 5' regions are heterogeneous. RNAs with similar or identical 5' regions are transcribed in cell-free extracts from a circular mouse beta major-globin gene template. Synthesis of most of the upstream RNAs in vitro is not inhibited by low levels (1 microgram/ml) of alpha-amanitin, indicating that they are transcribed by an enzyme(s) different from RNA polymerase II. During culture of mouse erythroleukemia cells with dimethyl sulfoxide, globin mRNA and upstream RNAs accumulate with similar kinetics. In contrast, upstream RNAs are not detected in hemin-treated cells.

Amanitins↗