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Results for “mRNA sequencing”

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At least 181 records · Page 10Linked to original sources
PubMed1993

PCR amplification of partial mRNA sequences encoding the alpha- and beta-globin chains of the bivalve mollusc Anadara trapezia: correction of the C-terminal amino acid sequence of the alpha-chain.

1. PCR was carried out on cDNA which had been synthesized from poly(A)+ RNA extracted from A. trapezia red blood cells. 2. A 348 bp product encoding the C-terminal region of the alpha-globin chain and another product encoding a portion of the beta-globin chain were isolated. 3. The C-terminal alpha-chain sequence, encoded by the cDNA, differs from the previously published sequence and these changes improve the alignment of this chain with the other globin chains of this organism.

Amino Acid Sequence↗
PubMed1992

A rat beta-interferon-induced mRNA: sequence characterization.

Polymerase chain reaction amplification of a cDNA derived from rat aortic smooth muscle cells, using sequences from conserved regions of the intramembrane domains of adrenergic receptors as primers, yielded the clone, rat8. This clone possesses a high degree of sequence similarity to a series of human interferon (IFN)-inducible genes. The rat8 sequence is 70% similar to that derived from the human alpha-IFN-induced gene, 9-27; there is 66% similarity between the deduced amino acid sequences encoded by the rat and the human genes. The rat homologue hybridizes with many bands in Southern analysis of rat DNA, suggesting that it is a member of a large multigene family.

Amino Acid Sequence↗
PubMed2004

mRNA sequences for Haemonchus contortus intestinal cathepsin B-like cysteine proteases display an extreme in abundance and diversity compared with other adult mammalian parasitic nematodes.

Cathepsin B-like cysteine protease (cbl) genes produce the most abundant mRNAs ( approximately 16%) detected in the adult female intestine of the parasitic nematode Haemonchus contortus. CBL enzymes appear to digest host proteins and are vaccine candidates for immune control of H. contortus and potentially other parasitic nematodes. Hence, it is important to quantify the extent of diversity of H. contortuscbl genes. Here, expressed sequence tags (ESTs) were used to assess both the size and diversity of the H. contortuscbl gene family. Contig analysis of 686 cbl ESTs from a USA isolate resolved 123 clusters. ESTs were grouped into discrete sets and analyzed using an additive model. Discovery of new cbl clusters increased with each set and reached a terminal rate of about 1 per 10 ESTs. The extreme diversity was unique to cbls relative to other genes investigated and was ascribed to specific cbl clades. Sixty percent of cbl clusters from a UK isolate were shared with those identified in the USA isolate, suggesting conservation of cbl gene repertoires across regions, although minor to moderate geographic variation cannot be excluded. Sequence comparisons also suggested high potential for antigenic diversity among CBL proteins, which is relevant to vaccine strategies. Compared to other parasitic nematodes of mammals, the extreme abundance and diversity of intestinal cbl transcripts appear to be relative specializations for H. contortus. Therefore, adaptations related to nutrient acquisition may vary markedly among these parasitic nematodes.

Amino Acid Sequence↗
PubMed2004

Biological significance of the U residue at the -3 position of the mRNA sequences of influenza A viral segments PB1 and NA.

The levels of viral proteins in infected cells are thought to be regulated by a variety of mechanisms. The initiation codons for the PB1 and NA proteins of A/WSN/33 (H1N1) influenza virus are in a suboptimal Kozak sequence for translation. To determine the significance of these suboptimal Kozak sequences, model vRNAs, whose coding regions were replaced with the reporter SEAP gene (for secreted alkaline phosphatase) and recombinant viruses with optimal Kozak sequences for PB1 and NA were constructed. Conversion of the upstream sequence of the PB1 and NA initiation codon to an optimal Kozak sequence was reflected in the level of reporter protein expression, but not the level of PB1 and NA protein expression. The recombinant viruses that had optimal Kozak sequences for PB1, NA, or both genes had similar replicative properties, both in cell culture and in mice, to those of the wild-type virus. These results suggest that expression of the PB1 and NA proteins is regulated by a mechanism other than that controlling the initiation of translation of these proteins.

Adenine↗
PubMed1996

Translational recoding induced by G-rich mRNA sequences that form unusual structures.

We investigated a herpesvirus mutant that contains a single base insertion in its thymidine kinase (tk) gene yet expresses low levels of TK via a net +1 translational recoding event. Within this mutant gene, we defined a G-rich signal that is sufficient to induce recoding. Unlike other translational recoding events, downstream RNA structures or termination codons did not stimulate recoding, and paused ribosomes were not detected. Mutational analysis indicated that specific tRNAs or codon-anticodon slippage were unlikely to account for recoding. Rather, recoding efficiency correlated with the G-richness of the signal and its ability to form unusual structures. These findings identify a mechanism of translational recoding with unique features and potential implications for clinical drug resistance and other biological systems.

Amino Acid Sequence↗
PubMed2001

mRNA sequence of the Xenopus laevis paxillin gene and its expression.

Paxillin is a cytoskeletal protein found in structures of focal adhesions where cells adhere to the extracellular matrix. We isolated paxillin cDNA from the Xenopus laevis ovary. The cDNA sequence encodes a protein of 539 amino acids with four LIM and five LD motifs. 80% of the amino acids of frog paxillin are shared by human and chicken paxillins. Northern analysis showed that the frog gene is expressed in the spleen, kidney, testis and ovary. Immunocytochemistry showed that paxillin protein is accumulated in the nucleus as well as in the periphery of the cytoplasm of the A6 cell. This intriguing result shows that paxillin, which has been characterized as a cytoskeletal protein, is capable of translocating to the nucleus.

Amino Acid Sequence↗
PubMed2003

Alternative ways to think about mRNA sequences and proteins that appear to promote internal initiation of translation.

Translation of some mRNAs is postulated to occur via an internal initiation mechanism which is said to be augmented by a variety of RNA-binding proteins. A pervasive problem is that the RNA sequences to which the proteins bind were not rigorously proven to function as internal ribosome entry sites (IRESs). Critical examination of the evidence reveals flaws that leave room for alternative interpretations, such as the possibility that IRES elements might function as cryptic promoters, splice sites, or sequences that modulate cleavage by RNases. The growing emphasis on IRES-binding proteins diverts attention from these fundamental unresolved issues. Many of the putative IRES-binding proteins are heterogeneous nuclear ribonucleoproteins that have recognized roles in RNA processing or stability and no recognized role in translation. Thus the mechanism whereby they promote internal initiation, if indeed they do, is not obvious. Some recent experiments were said to support the idea that IRES-binding proteins cause functionally important changes in folding of the RNA, but the evidence is not convincing when examined closely. The proteins that bind to some (not all) viral IRES elements include a subset of authentic initiation factors. This has not been demonstrated with any candidate IRES of cellular origin, however; and even with viral RNAs, the required chase experiment has not been done to prove that a pre-bound initiation factor actually mediates subsequent entry of ribosomes. In short, the focus on IRES-binding proteins has gotten us no closer to understanding the mechanism of internal initiation. Given the aforementioned uncertainty about whether other mechanisms (splicing, cryptic promoters) might underlie what-appears-to-be internal initiation, a temporary solution might be to redefine IRES to mean "internal regulatory expression sequence." This compromise would allow the sequences to be used for gene expression studies, for which they sometimes work, without asserting more than has been proven about the mechanism.

Animals↗
PubMed1986

Cloning of mRNA sequences from the human colon: preliminary characterisation of defined mRNAs in normal and neoplastic tissues.

RNA has been extracted from the normal human colon, converted into cDNA and cloned in the bacterial plasmid pBR322. About 4,000 sequences from this library were screened with probes derived from normal mucosa, familial polyposis mucosa, colonic adenocarcinomas and the colon tumour cell line HT29. Some mucosal sequences showed greatly reduced levels of transcription in neoplastic conditions, while a few showed elevated transcription. These have been further characterised by Northern and RNA dot-blot analysis.

Base Sequence↗
PubMed1996

Comparison of the mRNA sequences for Pi class glutathione transferases in different hamster species and the corresponding enzyme activities with anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide.

Glutathione S-transferase (GST) of class Pi (GST Pi) is known to detoxify the mutagenic and carcinogenic (+)-anti-benzo[a]pyrene-7, 8-dihydrodiol 9,10-epoxide [(+)-anti-BPDE] by conjugation with glutathione. Previously, we have shown that Chinese hamster V79 cells contain GST Pi, but seem to lack the capacity to conjugate (+)-anti-BPDE, although these cells do conjugate other substrates with GSH [Romert, Dock, Jenssen and Jernström (1989) Carcinogenesis 10, 1701-1707; Swedmark, Romert, Morgenstern and Jenssen (1992) Carcinogenesis 13, 1719-1723; Swedmark and Jenssen (1994) Gene 139, 251-256]. In the present study we have compared four cell lines derived from different hamster species with respect to GST cDNA sequences and capacity to conjugate (+)-or(-)-anti-BPDE. The cell lines were V79 and Chinese hamster ovary cells (CHO), Armenian hamster lung (AHL) cells and baby hamster kidney (BHK) cells. The sequencing revealed a complete homology between the V79 and CHO cDNA for GST Pi, whereas the corresponding amino acid sequences predicted from the corresponding AHL and BHK cDNAs differed by six and nine amino acids, respectively, from the predicted V79 sequence. None of these changes alone was found to influence the xenobiotic substrate-binding site. The cytosolic fractions from BHK and AHL cells were found to catalyse conjugation of (+)-anti-BPDE with GSH, whereas the corresponding activity in CHO cells was non-detectable. As shown previously, V79 cells were devoid of activity towards (+)-anti-BPDE. All the cell lines studied demonstrated appreciable GST activity towards 1-chloro-2,4-dinitrobenzene, but no activity with (-)-anti-BPDE. The latter result suggests that GST Pi is the sole or predominant GST in these cell lines. This was confirmed by HPLC analysis of purified enzymes obtained by affinity chromatography. However, when the catalytic activities of the pure enzymes were determined, all four different GST Pi enzymes were found to be highly capable of conjugating (+)-anti-BPDE with GSH. This observation indicates the existence of an intracellular factor that selectively inhibits conjugation of (+)-anti-BPDE, but not of 1-chloro-2,4-dinitrobenzene in the V79 and CHO cell lines. This new phenomenon seems to be specific for Chinese hamster, since both these cell lines originate from this species.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2003

Glutamate transporters in the guinea-pig cochlea: partial mRNA sequences, cellular expression and functional implications.

In the cochlea, glutamate plays a major role in synaptic transmission between the inner hair cell and the primary auditory neurons. Extracellular glutamate concentration must be regulated to prevent excitotoxicity. This regulation is mediated by excitatory amino acid transporters, membrane proteins that remove glutamate from the synaptic cleft. In this study, we investigated the distribution and activity of three excitatory amino acid transporters subtypes in the guinea-pig cochlea: glutamate aspartate transporter, glutamate transporter and excitatory amino acid carrier. A partial messenger ribonucleic acid sequence was determined for each of these transporters, by polymerase chain reaction with degenerate primers, using guinea-pig brain complementary deoxyribonucleic acid as the template. Primers specific for each transporter were then designed and used to screen a dissected organ of Corti complementary deoxyribonucleic acid library. The cellular distribution of each transporter was examined by immunocytochemistry. We investigated the functional consequences of inhibiting glutamate uptake by recording cochlear potentials during intracochlear perfusion with either l-trans-pyrrolidine-2,4-dicarboxylic acid or dihydrokainate. At the end of the electrophysiological session, cochleas were processed for electron microscopy. Only the glutamate aspartate transporter messenger ribonucleic acid was detected in the organ of Corti. Consistently, glutamate aspartate transporter protein was detected in the inner hair cell-supporting cells and in the ganglion of Corti satellite cells. Glutamate transporter and excitatory amino acid carrier were found in the afferent auditory neurons. Only intracochlear perfusions with l-trans-pyrrolidine-2,4-dicarboxylic acid resulted in a dose-dependent decrease in the amplitude of the cochlear compound action potential, leaving cochlear microphonic potential unaffected. After l-trans-pyrrolidine-2,4-dicarboxylic acid perfusion, cochleas displayed a swelling of the afferent endings typical of excitotoxicity. [(-)1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-4,5-dihydro-3-methylcarbamyl-2,3-benzodiazepine], a selective alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist protects the cochlea against l-trans-pyrrolidine-2,4-dicarboxylic acid effect.

Afferent Pathways↗