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 1,027 records · Page 57Linked to original sources
PubMed1987

Variable region sequences of murine IgM anti-IgG monoclonal autoantibodies (rheumatoid factors). II. Comparison of hybridomas derived by lipopolysaccharide stimulation and secondary protein immunization.

We have obtained the complete variable region mRNA sequences of 11 LPS-derived and 14 secondary immunization-derived monoclonal IgM anti-IgG antibodies (rheumatoid factors, RFs). A comparative analysis of these sequences showed that monoclonal RFs derived after polyclonal activation are structurally very similar to RFs derived after secondary protein immunization. This study was undertaken to evaluate the potential relationship between two previously described phenomena: (a) during a secondary response to a protein antigen, RF is produced in quantities that equal or exceed the immunogen-specific antibody; and (b) the frequency of B cells that make RF after polyclonal activation is quite high; 3-10%. It has been unclear whether LPS-stimulated cells that produce IgM anti-IgG that is detected by an in vitro assay are related to the cells that produce RF after in vivo stimulation. The similarity of the antigen receptors found in the two types of RF, however, suggests that most or all of the RF-producing B cells detected after LPS stimulation would also be stimulated during the secondary immune response. Thus, the presence of relatively large number of B cells that can make RF after nonspecific stimulation provides an explanation for the magnitude of RF production accompanying the secondary immune response.

Amino Acid Sequence↗
PubMed1977

Direct evidence that ribosome bound RNA-dependent RNA polymerase does not play a role in globin messenger RNA replication.

The radioactively labelled product of RNA-dependent RNA polymerase+ from ribosomes of immature chicken erythrocytes was tested for the presence of newly replicated globin mRNA using unlabelled globin complementary DNA. No radioactively labelled globin mRNA sequences were found in the product, providing direct confirmation that this RNA-dependent RNA polymerase is not involved in globin mRNA amplification.

Animals↗
PubMed1994

Measurement of androgen receptor expression in adult liver, fetal liver, and Hep-G2 cells by the polymerase chain reaction.

Hepatocellular carcinoma is the most commonly fatal malignant tumour worldwide. The role of androgen receptors, which have been found in hepatocellular carcinoma, is controversial. Sequence specific polymerase chain reaction (PCR) was used to quantify, for the first time, the expression of androgen receptor in four adult liver biopsy specimens (HL-A to HL-D), fetal liver, and Hep-G2 cells. The measurement of androgen receptor is expressed as a ratio (androgen receptor: beta-actin) of the value of androgen receptor to the value of a control gene, beta-actin. The value of the androgen receptor: beta-actin ratios for HL-A, HL-B, HL-C, HL-D, fetal liver, and Hep-G2 were 0.37, 0.86, 0.37, 0.44, 0.87, and 0.66 respectively. To verify sequence specific amplification of the androgen receptor, the PCR androgen receptor fragment was sequenced. The resultant sequence data for both strands of the double stranded PCR androgen receptor fragment had 100% similarity with the published androgen receptor mRNA sequence (complete codons).

Actins↗
PubMed2006

[The effect of telomerase reverse transcriptase on 5-HT induced pulmonary artery smooth muscle cells proliferation].

OBJECTIVE: To investigate the effect of telomerase reverse transcriptase (TERT) to the proliferation of 5-HT induced pulmonary artery smooth muscle cells (PASMCs). METHODS: The PASMCs proliferation experiment was performed to detect the effort on PASMCs of 5-HT or ASODN TERT (antisense oligoribonucleotides TERT designed according to the rat TERT mRNA sequence of gene bank). The immunohistochemistry staining experiment and the in situ hybridization experiment were to detect the TERT protein and mRNA expression with 5-HT or ASODN TERT. FITC marked ASODN TERT experiment was done to research the distribution of ASODN TERT in PASMCs. RESULTS: 5-HT promoted PASMCs proliferation in a dose-dependent manner (10(-9) - 10(-5) mol/L). 5-HT also significantly increased TERT expression at protein and mRNA levels as shown by immunohistochemistry staining and the in situ hybridization studies. This effect could be blocked by ASODN TERT in a time and dose-dependent manner. CONCLUSIONS: Our experiments show TERT is one of the key factors in the procession of 5-HT induced PASMCs proliferation. ASODN TERT might be a potential therapy agent for pulmonary hypertension.

Animals↗
PubMed1980

Transferrin gene expression. Effects of nutritional iron deficiency.

Nutritional iron deficiency was produced experimentally by raising newly hatched chicks on an iron-deficient diet for several weeks. During this time, hematocrit and hemoglobin values declined, iron stores were depleted, and the circulating level of transferrin increased 2- to 4-fold. The increase in serum transferrin was related to a similar increase in the rate of transferrin synthesis in liver. In addition, the level of transferrin mRNA sequences, as determined by hybridization to a specific cDNA, increased 2- to 3-fold, and more than 80% of the transferrin mRNA was associated with polyribosomes in both control and iron-deficient liver. These results demonstrate that the induction of transferrin synthesis in iron-deficient chicks is regulated directly by an increase in transferrin mRNA. The iron-mediated effects on transferrin also appear to be gene-specific since the rate of synthesis of serum albumin, the major secretory product of liver, was unaffected by any of the experimental conditions. Furthermore, when iron stores were rapidly replenished by the administration of iron-saturated ferritin, both the rate of transferrin synthesis and the level of transferrin mRNA returned to control values with 2 to 3 days.

Animals↗
PubMed1995

[Angiotensin receptors--organ and subtype specific regulation in cardiovascular diseases by modulation of the renin-angiotensin system. Studies of the rat model and in human myocardium].

So far, two angiotensin receptor subtypes, called AT1 and AT2, have been described in an animal model and in human. AT1 mediates almost all known effects of angiotensin II and its gene sequence and regulation is well studied. In contrast, only few data on function and regulation of AT2 are available. The complete mRNA sequence of AT2 has only recently been cloned and sequenced. The angiotensin receptors' receptor density and subtype distribution is organ specific. In the rat, lowest densities are found in the myocardium, followed by kidney, liver, adrenal medulla and cortex. The percentage of AT1 in the different organs amounts to 80, 85, 90, 57 and 10%. Angiotensin receptor subtypes have also been quantitated in human myocardium. There, the relatively unknown subtype AT2 dominates (67%). Myocardial receptor density is low, amounting to about 11 fmol/mg protein corresponding to 1/20-1/50 of the density of beta-adrenergic receptors. Angiotensin receptors in the human heart are present on cardiac fibroblasts and induce proliferation of these cells. Blockade of the renin angiotensin system by ACEI and AT1 antagonists in the rat downregulates angiotensin receptors in liver, kidney and adrenals to about 50% in an organ- and subtype specific manner, whereas cyclosporin A upregulates receptors twice. In end-stage human heart failure, but not in early stages, angiotensin receptors are downregulated to 1/3 of control values. Regulator mechanisms at transcriptional level have been elucidated by reporter gene assays; PMA, an activator of proteinkinase C, stimulates the transcription of the AT1 gene. The organ- and subtypespecific regulation of angiotensin receptors by pharmacological agents and/or cardiovascular diseases can contribute to the understanding of these drugs and of the pathophysiology of the corresponding diseases.

Animals↗
PubMed1981

Structural and functional similarities of delta-crystallin messenger ribonucleic acids from duck and chicken lenses.

delta-Crystallin of the embryonic duck lens was compared with that of the embryonic chicken lens with respect to polypeptide composition, synthesis, and messenger ribonucleic acid (mRNA) sequences. Labeling experiments with [35S]methionine revealed that the duck delta-crystallin is composed of minor amounts of polypeptides with molecular weights near 50000 (50K) and 49000 (49K) and much greater amounts of polypeptides with molecular weights near 48000 (48K) and 47000 (47K), as judged by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. All four sizes of polypeptides were synthesized in similar relative proportions as found in vivo in a rabbit reticulocytes lysate supplemented with delta-crystallin mRNA isolated from the embryonic duck lens. Synthesis of the 48K and 47K delta-crystallin polypeptides was differentially reduced in duck lenses cultured in the presence of ouabain. This is similar to the differential reduction of synthesis of the lower molecular weight delta-crystallin peptides in embryonic chicken lenses demonstrated previously. R loops formed between duck or chicken delta-crystallin mRNA and a cloned chicken delta-crystallin cDNA and heteroduplexes formed between duck or chicken delta-crystallin mRNA and cloned chicken genomic DNAs containing delta-crystallin sequences showed that, except for the putative 5' leader sequence, the duck and chicken delta-crystallin mRNAs have extremely similar nucleotide sequences. These data indicate considerable conservation of delta-crystallin throughout the approximately 100 million years of divergence between ducks and chickens. The findings also suggest a possible relationship between the structure of delta-crystallin mRNA and the differential reduction in synthesis of the lower molecular weight delta-crystallin polypeptides in ouabain-treated lenses of ducks and chickens.

Animals↗
PubMed1997

Predominance of the alpha1D subunit in L-type voltage-gated Ca2+ channels of hair cells in the chicken's cochlea.

The voltage-gated Ca2+ channels that effect tonic release of neurotransmitter from hair cells have unusual pharmacological properties: unlike most presynaptic Ca2+ channels, they are sensitive to dihydropyridines and therefore are L-type. To characterize these Ca2+ channels, we investigated the expression of L-type alpha1 subunits in hair cells of the chicken's cochlea. In PCRs with five different pairs of degenerate primers, we always obtained alpha1D products, but only once an alpha1C product and never an alpha1S product. A full-length alpha1D mRNA sequence was assembled from overlapping PCR products; the predicted amino acid sequence of the alpha1D subunit was about 90% identical to those of the mammalian alpha1D subunits. In situ hybridization confirmed that the alpha1D mRNA is present in hair cells. By using a quantitative PCR assay, we determined that the alpha1D mRNA is 100-500 times more abundant than the alpha1C mRNA. We conclude that most, if not all, voltage-gated Ca2+ channels in hair cells contain an alpha1D subunit. Furthermore, we propose that the alpha1D subunit plays a hitherto undocumented role at tonic synapses.

Amino Acid Sequence↗
PubMed1986

Detection of alpha-foetoprotein messenger RNA in human hepatocellular carcinoma and hepatoblastoma tissue.

Alpha-foetoprotein (AFP) synthesis, although repressed in normal adults, is increased in the majority of patients with hepatocellular carcinoma (HCC). We have investigated whether active transcription of the AFP gene may explain raised serum AFP concentrations in patients with HCC and hepatoblastoma by assaying human tumour and non-neoplastic tissue by molecular hybridization for the presence of mRNA encoding AFP. Ten operative HCC and six autopsy HCC specimens, two HCC cell lines, and one hepatoblastoma specimen were examined. Total cellular RNA and poly-(A)+ RNA were extracted and AFP mRNA sequences sought by dot-blot and Northern blot hybridisation to a human cDNA AFP probe. Cellular AFP was localised by avidin-biotin staining. AFP mRNA was detected in 8/10 operative specimens, as well as PLC/PRF/5 nude mouse tumours. Weaker hybridization was detected in 4/6 autopsy specimens. Signals of comparable intensity to that in operative tumours were detected in non-neoplastic tissue of 6 patients. AFP mRNA from nude mouse tumours migrated as a 20S discrete band on agarose gel electrophoresis, whereas a more complex hybridization pattern was evident in human tumours. Positive cytoplasmic immuno-staining for AFP was observed in 4 tumours and 2 corresponding non-neoplastic specimens and in a HCC cell line. In non-neoplastic liver, AFP was localised in cells that appeared dysplastic. Thus steady-state levels of AFP mRNA are detectable in human HCC tissue and surrounding non-neoplastic liver. These findings may prove pertinent to an understanding of the genetic expression of AFP in malignant hepatocytes, and the sequence of events leading to uncontrolled cellular proliferation.

Carcinoma, Hepatocellular↗
PubMed2003

[Cloning and sequence analysis of active fragments of human thrombospondin-1].

OBJECTIVE: To lay a foundation for researches on the expression of gene engineering anti-tumor drugs. METHODS: On the basis of mRNA sequence of human thrombospondin-1(TSP-1), two pairs of primers were designed and synthesized. Two active fragments TSP-1, called TSP-1-I and TSP-1-III, were obtained by using reverse transcription-polymerase chain reaction(RT-PCR). The sizes of those two fragments were found to be 728 bp and 257 bp respectively. They were purified and then cloned into pMDT vector after transformation into Escherichia coli with recombined DNAs. RESULTS: Plasmid DNAs were isolated from transformants and digested with restriction enzyme. The results from agarose gel electrophorasis showed that these two fragments had been inserted into vector and their sizes were in accord with the expected values. Sequence analysis demonstrated that TSP-1-I is 728 bp in size and encodes a peptide containing 212 amino acid residues with relative molecular mass of 23.6 x 10(3) and the TSP-I III is 257 bp in size and encodes a peptide containing 86 amino acid residues with relative molecular mass of 9.5 x 10(3). CONCLUSION: The results from homology analysis indicate that the sequence of these two fragments is identified with that of TSP-1 of Genbank.

Adenocarcinoma↗
PubMed1978

Variable and constant regions are separated in the 10-kbase transcription unit coding for immunoglobulin kappa light chains.

UV transcription mapping with recombinant DNA probes containing immunoglobulin kappa light chain mRNA sequences has been used to determine the size of the transcription unit coding for kappa light chain m RNA and to establish the arrangement of variable and constant regions in this transcription unit. In relation to ribosomal RNA standards, the transcription of kappa light chain constant region sequences into nuclear RNA exhibits a UV target size of 9.6 kbases (kb). The kappa light chain variable region exhibits a UV target size of 7.6 kb indicating that it is separated by approximately 2.0 kb from the constant region in the kappa light chain transcription unit. The size of the primary transcript (i.e., the direct, unprocessed RNA product of transcription) predicted from the constant region target size concurs with our previous pulse-labeling results which showed that the largest presumptive nuclear RNA precursor to kappa light chain mRNA is approximately 10 kb. In addition, the UV target size of cytoplasmic kappa mRNA is indistinguishable from the target size of constant region sequences in nuclear RNA. These results suggest that the kappa light chain transcription unit is copied directly into a 10-kb nuclear RNA precursor in which the kappa variable and constant regions are separated by approximately 2 kb. Accordingly, it is proposed that the joining of immunoglobulin kappa light chain variable and constant regions occurs in the post-transcriptional processing of this large nuclear RNA precursor into kappa light chain mRNA.

Base Sequence↗
PubMed1985

Messenger RNA conformation and ribosome selection of translational reinitiation sites in the lac repressor mRNA.

In the early region of the Escherichia coli lac repressor mRNA, the pattern of cleavage by nucleases specific for single or double-stranded RNA confirms the presence of secondary structures previously proposed to influence the pattern of translational reinitiation. These are positioned so as to mask a potential restart site centered on an in-phase GUG triplet corresponding to repressor amino acid position Val38. Our finding that a restart polypeptide initiated at the Val38 GUG codon is observed only in situations that that preclude base-pairing of adjacent mRNA sequences establishes a functional role for these structures in vivo. This evidence for structure, considered with the overall pattern of reinitiation events, suggests that local mRNA conformation is the major determinant that dictates ribosomal selection of restart sites within the early region of the repressor cistron.

Amino Acid Sequence↗
PubMed1995

Antisense phosphorothioate oligodeoxynucleotides alter HIV type 1 replication in cultured human macrophages and peripheral blood mononuclear cells.

The use of antisense oligodeoxynucleotides as antiviral drugs to combat HIV-1 infection may offer an alternative to traditional pharmacological therapies. We compared the effects of two 28-mer antisense phosphorothioate oligodeoxynucleotides [PS-oligo(dN)] with non-sequence-specific controls on HIV-1 replication in long-term human monocyte/macrophage and PBMC cultures. The anti-rev PS-oligo(dN) was complementary to the messenger RNA (mRNA) sequences derived from the overlapping region of the HIV-1 regulatory genes tat and rev, while anti-gag targeted the translational initiation site of the gag mRNA. In vitro cytotoxicity of the PS-oligo(dN) was evaluated at concentrations ranging from 0.1 to 10.0 microM for a period of 20 days. Cell survival was 100% at 0.1 microM, but decreased to 5% at 10.0 microM in relation to the untreated control cultures. Our data demonstrate that replication of both the T cell-tropic and macrophage-tropic HIV-1 strains in primary cells can be inhibited by PS-oligo(dN) in a sequence-specific and dose-dependent manner at concentrations achievable in vivo. However, the sequence-dependent antiviral activity of the utilized PS-oligo(dN) was limited to a window of specificity at concentrations between 0.25 and 1.0 microM.

Antiviral Agents↗
PubMed1981

RNA metabolism in isolated nuclei: processing and transport of immunoglobulin light chain sequences.

Transport of prelabeled RNA from isolated myeloma nuclei is studied using conditions that permit RNA synthesis. Cytosol and spermidine are not required to maintain nuclear stability and inhibited RNA release. Omission of ATP or GTP decreased release 25 to 40%. The stimulatory effect of ATP or GTP is not due to hydrolysis of the triphosphates by the nuclear envelope NTPase, since addition of quercetin (an inhibitor of this NTPase) has no effect on the quantity of RNA released. The size distribution and percentage of poly A-containing species released from nuclei incubated with or without ATP or the other rNTPs are identical. Hybridization analysis of nuclear RNA before the transport assay revealed mature and precursor k light chain mRNA sequences. Following the transport assay, a significant fraction of k mRNA precursors is chased into mature k mRNA which is found both in nuclear-retained and released RNA.

Animals↗
PubMed1984

Organization of the HPRT gene and related sequences in the human genome.

Comparative Southern hybridization of cDNA probes to DNA from cells carrying either one or four X chromosomes has been used to distinguish sequences derived from the functional locus for hypoxanthine-guanine phosphoribosyltransferase (HPRT) on the X chromosome from four independent HPRT-like autosomal sequences in the human genome. Subfragments of cDNA were then used to orient fragments from the HPRT locus with respect to the mRNA sequence. The chromosomal origin of each of the autosomal sequences was determined by Southern analysis using DNA from a panel of human-Chinese hamster somatic cell hybrids. Two of the HPRT-like sequences were localized to chromosome 11, the third to chromosome 3, and the fourth to the region between p13 and q11 on chromosome 5. Three of these four autosomal sequences were isolated from genomic recombinant libraries and subcloned fragments from each were used as probes to study restriction fragment length polymorphisms (RFLP) at these loci. A RFLP for MspI was found at the HPRT-like locus on chromosome 5 with a 1.3-kb major allele (frequency = 0.8) and a 3.6-kb minor allele (frequency = 0.2).

Animals↗
PubMed1997

An intronic deletion in TP53 gene causes exon 6 skipping in breast cancer.

Six hundred and thirty primary breast cancer were screened for abnormalities in exons 5, 6, 7 and 8 of the TP53 tumour suppressor gene. Analysis of the structure of the TP53 gene exons was performed with the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method and with direct sequencing of amplified DNA. In a breast tumour case from a postmenopausal patient, we found a deletion of 36 bp in intron 5 and no immunohistochemical staining for p53. We amplified and sequenced the cDNA region between exons 4 and 7 and showed that the deletion causes the skipping of exon 6. The resulting mRNA sequence had a frameshift that yields an inactive protein with a truncated C terminus. These results show the first example of intronic deletion causing exon skipping at the TP53 gene level.

Base Sequence↗
PubMed1978

Immunoglobulin light chain mRNA is processed from large nuclear RNA.

Recombinant DNA probes, produced by the molecular cloning of immunoglobulin kappa light chain mRNA, have been used to analyze heterogenous nuclear RNA for presumptive precursors to cytoplasmic kappa light chain mRNA, Three discrete classes of nuclear RNA containing kappa mRNA sequences were detected after pulse-labeling of immunoglobulin-producing P3 myeloma cells. Two of these were substantially larger than kappa mRNA (approximately 10 and 4 times larger); the third was similar in size to kappa mRNA. Beginning with the largest, the sequential appearance of these three classes of nuclear RNA preceded the first appearance of newly synthesized kappa light chain mRNA in the cytoplasm. The results presented here suggest that immunoglobulin kappa light chain mRNA is generated by the stepwise cleavage and processing of a large nuclear RNA transcript.

Cell Line↗
PubMed1995

Structural and functional studies of the intracellular tyrosine kinase MATK gene and its translated product.

We recently cloned the cDNA which encodes a novel megakaryocyte-associated tyrosine kinase termed MATK. In this study, we have cloned and characterized the human MATK gene as well as the murine homolog of human MATK cDNA and performed functional studies of its translated product. Comparison of the deduced amino acid sequences of human and murine MATK cDNAs revealed 85% homology, indicating that MATK is highly conserved in mouse and human. The human gene consists of 13 exons interrupted by 12 introns. The genetic units which encode the SH3 and SH2 domains are located on separate exons. The putative ATP binding site (GXGXXG) is localized on exon 7, and the entire catalytic domain is subdivided into seven exons (7-13). Somatic cell hybrid analysis indicated that human MATK gene is located on chromosome 19 while the murine Matk gene is located on chromosome 10. The immediate 5'-flanking region was highly rich in GC sequences, and potential cis-acting elements were identified including several SP1, GATA-1, APRE, and APRE1. Antisense oligonucleotides directed against MATK mRNA sequences significantly inhibited megakaryocyte progenitor proliferation. Functional studies indicated that MATK can phosphorylate the carboxyl-terminal conserved tyrosine of the Src protein. These results support the notion that MATK acts as a regulator of p60c-src in megakaryocytic cells and participates in the pathways regulating growth of cells of this lineage.

Adenosine Triphosphate↗