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 1,477 records · Page 82Linked to original sources
PubMedDate not supplied

129/Ola mice carrying a null mutation in PrP that abolishes mRNA production are developmentally normal.

The neural membrane glycoprotein PrP is implicated in the pathogenesis of the transmissible spongiform encephalopathies; however, the normal function of PrP and its precise role in disease are not understood. Recently, gene targeting has been used to produce mice with neo/PrP fusion transcripts, but no detectable PrP protein in the brain (1). Here we report the use of a different targeting strategy, to produce inbred mice with a complete absence of both PrP protein and mRNA sequences. At 7 mo of age, these mice show no overt phenotypic abnormalities despite the normal high levels of expression of PrP during mouse development. The mice are being used in experiments designed to address the role of PrP in the pathogenesis of scrapie and the replication of infectivity.

Aging↗
PubMed2006

Cellular regulation and molecular interactions of the ferritins.

Controlling iron/oxygen chemistry in biology depends on multiple genes, regulatory messenger RNA (mRNA) structures, signaling pathways and protein catalysts. Ferritin, a protein nanocage around an iron/oxy mineral, centralizes the control. Complementary DNA (antioxidant responsive element/Maf recognition element) and mRNA (iron responsive element) responses regulate ferritin synthesis rates. Multiple iron-protein interactions control iron and oxygen substrate movement through the protein cage, from dynamic gated pores to catalytic sites related to di-iron oxygenase cofactor sites. Maxi-ferritins concentrate iron for the bio-synthesis of iron/heme proteins, trapping oxygen; bacterial mini-ferritins, DNA protection during starvation proteins, reverse the substrate roles, destroying oxidants, trapping iron and protecting DNA. Ferritin is nature's unique and conserved approach to controlled, safe use of iron and oxygen, with protein synthesis in animals adjusted by dual, genetic DNA and mRNA sequences that selectively respond to iron or oxidant signals and link ferritin to proteins of iron, oxygen and antioxidant metabolism.

Animals↗
PubMed1994

[In situ hybridization. Theoretical principles and practical applications].

In situ hybridisation is a sensitive and reliable technique for the demonstration of DNA- or mRNA-sequences on histological tissue preparations. This review deals with the theoretical background as well as the benefits and limitations of the method; additionally the value of in situ hybridisation as a tool in routine diagnostic pathology is briefly described.

Base Sequence↗
PubMed2003

Molecular and functional evidence for a Na(+)-HCO3(-)-cotransporter in sheep ruminal epithelium.

The present study aimed to identify the HCO3(-)-dependent mechanisms contributing to the homeostasis of the intracellular pH (pHi) in ruminal epithelial cells of sheep. Therefore, pHi was measured spectrofluorometrically in primary cultured ruminal epithelial cells loaded with the pH-sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5(6')-carboxyfluorescein acetoxymethyl ester. Switching from a HEPES-buffered to a CO2/HCO3(-)-buffered solution caused a rapid intracellular acidification followed by a counter-regulation towards alkaline levels. The counter-regulation was totally dependent upon extracellular Na+, but independent of intracellular Cl-. Adding 30 microM EIPA to the solutions did not affect the pHi counter-regulation following the acidification. Presence of 500 M H2DIDS inhibited the counter-regulation of pHi by 67%. These results pointed to a Na(+)-HCO3(-)-cotransporter (NBC) as the main pHi regulatory mechanism in the presence of HCO3-. Existence of an NBC in both cultured ruminal epithelial cells and intact ruminal epithelium was verified by reverse transcription polymerase chain reaction (RT-PCR) studies. RT-PCR yielded a band of the expected molecular size of 333 bp in both cultured cells and intact epithelium. The mRNA sequences were identical and shared a homology of 62% with human kidney NBC (Genebank accession number AF007216), of 66% with rat kidney NBC (AF004017) and of 65% with mouse duodenal NBC (AF141934).

Animals↗
PubMed1996

Cellular localization of growth hormone receptors/binding proteins in immune tissues.

It is well established that the activity and proliferation of lymphoid cells and lymphoid organs are stimulated by growth hormone. These actions on lymphoid cells may be direct or mediated by actions on the epithelial and non-immune tissue cells that regulate immune function. The occurrence and cellular localization of growth hormone receptors in immune tissues has therefore been investigated to determine the target-sites of growth hormone action. Growth hormone receptor mRNA was first detected by Northern blotting in the spleen, bursa of Fabricius, and thymus of domestic fowl. In addition to the 4.4-kb transcript thought to encode the full-length growth hormone receptor, smaller transcripts of 2.8 kb and 1.0 kb, which may encode growth hormone-binding proteins, were also occasionally observed. Further analysis using the polymerase chain reaction revealed that mRNA sequences encoding the extracellular and intracellular domains of the growth hormone receptor were present in all tissues and highly homologous with hepatic transcripts. Translation of these transcripts also occurs in immune tissues, since immunoreactive growth hormone-binding proteins or growth hormone receptors of approximately 56 kDa were detected in hepatic, splenic, thymic, and bursal extracts. Immunocytochemistry of these tissues subsequently revealed that macrophages probably contain the bulk of this immunoreactivity, although some thymic medullary epithelial cells (including Hassall's corpuscles) and splenic ellipsoids and interdigitating cells were also immunoreactive. This immunoreactivity is present in immune tissues of newly hatched and adult chickens. Importantly, B-lymphocytes were rarely, if ever, immunoreactive, and T-lymphocytes containing growth hormone receptors or binding proteins were not observed. These results suggest that a number of primary (thymus and bursa) and secondary (spleen) lymphoid tissues in the chicken contain growth hormone receptors and are thus target-sites for growth hormone action. The distribution of growth hormone receptor/growth hormone-binding protein immunoreactivity in these tissues would further suggest that growth hormone plays a major role in macrophage proliferation and/or activity and may indirectly affect lymphocyte maturation and storage via effects on thymic and splenic stromal cells.

Animals↗
PubMed2007

Leucine-rich repeat kinase 1: a paralog of LRRK2 and a candidate gene for Parkinson's disease.

Leucine-rich repeat kinase 1 gene (LRRK1) on chromosome 15q26.3 is a paralog of LRRK2 in which multiple substitutions were recently linked to Parkinson's disease. We have examined the exon-intron structure of the gene and the expressed mRNA sequence in brain. LRRK1 sequencing analysis in 95 probands from families with autosomal dominant Parkinson's disease identified 23 variants, 14 of which are novel, with four resulting in non-synonymous amino acid substitutions. These four substitutions are rare and do not clearly segregate with disease within our families or associate with sporadic Parkinson's disease in a US case-control series. Subsequent sequencing of exon 26 encoding the kinase activation segment in an additional 360 probands identified one further synonymous variant, suggesting that LRRK1 variants are not a frequent cause of Parkinson's disease. The relative absence of substitutions within LRRK1 highlights a greater conservation of sequence than observed for LRRK2. Comparison of evolutionary interspecies sequences of LRRK1 and LRRK2 suggests they diverged from a common founder gene.

Alternative Splicing↗
PubMed2006

TGF-beta2 inhibition augments the effect of tumor vaccine and improves the survival of animals with pre-established brain tumors.

TGF-beta2 secretion by high grade gliomas has been implicated as one of the major factors contributing to tumor growth, alterations in the host immune response to tumor, and failure of gliomas to respond to current immunotherapy strategies. We hypothesized that targeted delivery and inhibition of TGF-beta2 by TGF-beta2 antisense oligonucleotides (AS-ODNs) would overcome tumor-induced immunosuppression and enhance the capacity of tumor vaccines to eradicate established brain tumors. Utilizing the mRNA sequences of TGF-beta2, specific AS-ODNs were constructed and tested for their ability to inhibit TGF-beta2 production in 9L glioma cells. The effect of combining local intracranial administration of antisense ODNs with systemic tumor vaccine was examined. Fisher 344 rats were vaccinated subcutaneously with irradiated 9L tumor cells 3 days after intracranial tumor implantation. Four days after vaccination, ODNs were administered into the tumor mass and survival was followed. ODNs delivered locally distributed widely within the brain tumor mass and inhibited TGF-beta2 expression. Survival of tumor-bearing rats treated with the combination of local antisense and systemic tumor vaccine was significantly enhanced (mean survival time (MST): 48.0 days). In contrast, MST for animals treated with nonsense plus vaccine, vaccine alone, antisense alone or PBS showed no survival advantage and no statistical differences between groups (33.5 days, 29.0 days, 37.5 days, and 31.5 days, respectively). Our data supports the hypothesis that local administration of antisense TGF-beta2 ODNs combined with systemic vaccination can increase efficacy of immunotherapy and is a novel, potentially clinically applicable, strategy for high-grade glioma treatment.

Animals↗
PubMed2006

Efficacy of siRNA nanocapsules targeted against the EWS-Fli1 oncogene in Ewing sarcoma.

The EWS-Fli1 fusion gene encodes for a chimeric oncogenic transcription factor considered to be the cause of the Ewing sarcoma. The efficiency of small interfering RNAs (siRNAs) targeted toward the EWS-Fli1 transcript (at the junction point type 1) was studied, free or encapsulated into recently developed polyisobutylcyanoacrylate aqueous core nanocapsules. Because this mRNA sequence is only present in cancer cells, it therefore constituted a relevant target. Studies of the intracellular penetration by confocal microscopy in NIH/3T3 EWS-Fli1 cells showed that nanocapsules improved the intracellular penetration of siRNA with mainly a cytoplasmic localization. These biodegradable siRNA-loaded nanocapsules were then tested in vivo on a mice xenografted EWS-Fli1-expressing tumor; they were found to trigger a dose-dependant inhibition of tumor growth after intratumoral injection. A specific inhibition of EWS-Fli1 was observed, too. These findings now open new prospects for the treatment of experimental cancers with junction oncogenes.

Animals↗
PubMed1990

Comparison of cathepsin L synthesized by normal and transformed cells at the gene, message, protein, and oligosaccharide levels.

The major excreted protein of transformed mouse fibroblasts (MEP) has recently been identified as the lysosomal cysteine protease, cathepsin L. The synthesis and intracellular trafficking of this protein in mouse fibroblasts are regulated by growth factors and malignant transformation. To further define the basis for this regulation, a cDNA encoding MEP/cathepsin L was isolated from a mouse liver cDNA library and used to compare cathepsin L of normal and Kirsten sarcoma virus-transformed NIH 3T3 fibroblasts. Although cathepsin L message levels were elevated 20-fold in the transformed fibroblasts, normal and transformed cells displayed similar cathepsin L genomic DNA digest patterns and gene copy numbers, and cathepsin L mRNA sequences appeared identical by RNase protection analysis. These findings indicate that (i) cathepsin L is synthesized from the same gene in normal and transformed cells and (ii) cathepsin L polypeptides made by these cells are translated with the same primary sequence. Cathepsin L polypeptides synthesized by quiescent, growing, and transformed cells displayed similar isoelectric focusing patterns, suggesting similar post-translational modification. Site-directed mutagenesis of the mouse liver cDNA and expression in COS monkey cells was used to examine the glycosylation of mouse cathepsin L. The results indicated that only one of the two potential N-linked glycosylation sites (the one at Asn221) is glycosylated. Analysis by ion exchange chromatography on QAE-Sephadex, and affinity chromatography on mannose 6-phosphate receptor-Affi-Gel 10, indicated that the cathepsin L oligosaccharide was phosphorylated similarly in normal and transformed cells. Although several phosphorylated oligosaccharide species were observed, the major species contained two phosphomonoester moieties and bound efficiently to the receptor. These findings suggest that cathepsin L made by normal and transformed mouse fibroblasts are identical and substantiate the hypothesis that trafficking of cathepsin L in these cells is regulated by growth-induced changes in the lysosomal protein transport system.

Amino Acid Sequence↗
PubMed1981

Acute effects of ethanol intake on albumin and total protein synthesis in free and membrane-bound polyribosomes of rat liver.

Controversial results have been reported in the last few years concerning the effects of ethanol on hepatic protein synthesis. In most of the studies no distinction has been made between the synthetic capabilities of the polyribosomes and the secretory product of labelled protein by the hepatocytes. In order to assess the influence of a single feeding of ethanol on the synthesis of albumin and total protein by the polyribosomes of rat liver, free and membrane-bound polyribosomes were isolated quantitatively from rats given 4--8 g ethanol per kg body weight 3--5 h before killing. The following results were obtained: (1) No difference was found in yield and size of free and membrane-bound polyribosomes isolated from control and ethanol-treated rats. The abilities to synthesize albumin and total protein were also equal for polyribosomes from both groups. (2) Addition of 1% ethanol to the incubation mixture of protein synthesis lowered albumin and total protein synthesis by 20%. No effect was observed with 0.5% ethanol. (3) Cell sap prepared from ethanol-treated rats contains a factor or factors which stimulate protein synthesis (10--15%). (4) The albumin mRNA sequence content was not changed in free and membrane-bound polyribosomal RNA fractions of ethanol-treated rats as compared to the control animals.

Animals↗
PubMed1981

Specificity of chromatin transcription in vitro. Asymmetric transcription of the globin gene by Escherichia coli RNA polymerase.

The transcription of globin genes in mouse foetal liver chromatin and nuclei by exogenous Escherichia coli RNA polymerase is prone to artifacts due to RNA-dependent transcription of endogenous mRNA sequences. This is particularly evident when Mn2+ is used as divalent cation in the RNA transcription reaction. However, substitution or supplementation with Mg2+ eliminates this artifact and gives essentially asymmetric DNA-dependent transcription by the polymerase. In this paper we discuss a number of general criteria which can be applied to test the validity of specific gene transcription in vitro.

Animals↗
PubMed1985

Conservation from rat to human of cytosolic phosphoenolpyruvate carboxykinase and the control of its gene expression.

Structural conservation of cytosolic phosphoenolpyruvate carboxykinase protein and mRNA sequence was found in all species examined from rodents to human. The mitochondrial isoenzyme, in all species tested, represents a distinct protein. Moreover, irrespective of the ratio of cytosolic to mitochondrial isoenzyme, cytosolic phosphoenolpyruvate carboxykinase activity in the human as in the rat is controlled at the level of gene expression and through the same multiple hormonal stimulation. This evolutionary conservation of the cytosolic phosphoenolpyruvate carboxykinase structure and mode of regulation supports the enzymes' physiological importance in mammals.

Animals↗
PubMed1993

PCR-based assignment of two myosin heavy chain cDNA clones to biochemically and histochemically defined single type IIB and IID fibers of rabbit muscle.

The present study assigns two as yet unidentified fast myosin cDNA clones to specific myosin heavy chain (MHC) isoforms and their mRNAs in different fiber types of rabbit skeletal muscle. Specific oligonucleotide primers were used for reverse transcription and polymerase chain reaction (PCR) to yield products of defined lengths. The method was sensitive enough to detect specific mRNA sequences in total RNA extracts from microdissected, freeze-dried, single-fiber fragments down to 16 ng dry weight. The fibers were typed histochemically and biochemically by their electrophoretically assessed MHC complement. The following results were obtained: clone pMHC20-40 was assigned to type IIB fibers and clone pMHC24-79 to type IID fibers.

Animals↗
PubMed1996

Fetal myosin heavy chain increases in human masseter muscle during aging.

Biochemical, immunohistochemical and molecular biological methods were used to detect fetal myosin heavy chain (MyHC) in the human masseter of elderly and young subjects. Samples from the elderly subjects contained larger amounts of fetal MyHC than those of young adults. Only a very small amount of embryonic MyHC could be detected in both age groups. Embryonic and fetal MyHCs were never detected in the control adult orofacial, limb and trunk muscles. Polymerase chain reaction (PCR) analysis revealed the presence of fetal mRNA sequences in elderly and young masseter muscles. We conclude that fetal MyHC is present in the human masseter throughout the life span and that there is an increase in the relative amount of this protein with age.

Adult↗
PubMed1988

Accumulation of abundant messenger ribonucleic acids during postnatal development of mouse small intestine.

To describe the differentiation of the small bowel at the molecular level, intestinal messenger ribonucleic acids (mRNAs) from mice at different stages of fetal and postnatal development were investigated. On the basis of cell-free translation and complementary deoxyribonucleic acid cloning experiments, abundant mRNAs coding for small polypeptides of 6-12 kilodaltons (low-molecular-weight mRNAs) were found in adult small intestine but not in the fetal gut. These developmentally regulated low-molecular-weight mRNAs are uniquely abundant in jejunum and ileum of adult mice, but they are absent or occur only at low levels in the duodenum, colon, stomach, and all other mouse organs examined. Low-molecular-weight mRNAs begin accumulating in the small bowel at approximately 3 wk of age, coinciding with weaning and with profound changes in intestinal differentiation. One complementary deoxyribonucleic acid clone of a low-molecular-weight mRNA (asb4/134) is specific to the distal small bowel, specifically accumulates at weaning, and hybridizes to RNA from mouse testis and brain at approximately 2%-5% of the intestinal level. Low-molecular-weight mRNA sequences may provide important markers of intestinal differentiation at the genetic level, leading to a better understanding of the factors that contribute to its postnatal maturation.

Animals↗
PubMed1987

Translational repression: biological activity of plasmid-encoded bacteriophage T4 RegA protein.

The RegA protein of bacteriophage T4 is a translational repressor that regulates expression of several phage early mRNAs. We have cloned wild-type and mutant alleles of the T4 regA gene under control of the heat-inducible, plasmid-borne leftward promoter (PL) of phage lambda. Expression of the cloned regA+ gene resulted in the synthesis of a protein that closely resembled phage-encoded RegA protein in biological properties. It repressed its own synthesis (autogenous translational control) as well as the synthesis of specific T4-encoded proteins that are known from other studies to be under RegA-mediated translational control. Cloned mutant alleles of regA exhibited derepressed synthesis of the mutant regA gene products and were ineffective in trans against RegA-sensitive mRNA targets. The effects of plasmid-encoded RegA proteins were also demonstrated in experiments using two compatible plasmids in uninfected Escherichia coli. The two-plasmid assays confirm the sensitivities of several cloned T4 genes to RegA-mediated translational repression and are well-suited for genetic analysis of RegA target sites. Repression specificity in this system was demonstrated by using wild-type and operator-constitutive translational initiation sites of T4 rIIB fused to lacZ. The results show that no additional T4 products are required for RegA-mediated translational repression. Additional evidence is provided for the proposal that uridine-rich mRNA sequences are preferred targets for the repressor. Surprisingly, plasmid-generated RegA protein represses the synthesis of some E. coli proteins and appears to enhance selectively the synthesis of others. The RegA protein may have multiple functions, and its binding sites are not restricted to phage mRNAs.

Base Sequence↗
PubMed1991

Influence of mRNA determinants on translation initiation in Escherichia coli.

We have studied the classic initiation elements of mRNA sequence and structure to better understand their influence on translation initiation rates in Escherichia coli. Changes introduced in the initiation codon, the Shine and Dalgarno sequence, the spacing between those two elements, and in the secondary structures within initiation domains each change the rate of 30 S ternary complex formation. We measured these differences using extension inhibition analysis, a technique we have called "toeprinting". The rate of 30 S initiation complex formation in the absence of initiation factors agrees well with in vivo translation rates in some instances, although in others a regulatory role of initiation factors in 30 S complex formation is likely. Nucleotides 5' to the Shine and Dalgarno domain facilitate ternary complex formation.

Anticodon↗
PubMed1987

Estradiol stimulates prolactin gene transcription in primary cultures of rat anterior pituitary cells.

Dispersed cells were prepared from the anterior pituitary glands of 6-week old male and retired breeder female rats and were cultured for 3 days in a serum-containing medium which lacked estrogen. Following short-term treatments (1-24 h) with estradiol at a concentration of 10(-9) M, the levels of prolactin gene transcription were assayed by quantitating the amount of radiolabeled uridine monophosphate incorporated into prolactin mRNA sequences by pituitary cell nuclei prepared from these cultures. Estradiol significantly stimulated the transcription of the prolactin gene in these primary cultures within 6 h. Similar levels of stimulation were observed in cells from both male and female rats. Estradiol treatment had no effect on the transcription of the growth hormone gene, suggesting its stimulatory effects on prolactin gene transcription are specific. Estradiol also significantly stimulated the transcription of the prolactin gene in cultures in which protein synthesis was grossly inhibited by cycloheximide. These data illustrate that estrogen regulates the expression of the prolactin gene at the transcriptional level, through a direct action on the cells of the anterior pituitary gland, and by a mechanism which does not require the synthesis of any intermediary proteins.

Animals↗