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At least 973 records · Page 54Linked to original sources
PubMed1996

Strength of translation initiation signal sequence of mRNA as studied by quantification method: effect of nucleotide substitutions upon translation efficiency in rat preproinsulin mRNA.

Concerning the translation initiation signals in vertebrate mRNAs, both the ATG initiation codon and the sequences flanking the initiation codon are required to direct the position of initiation. A consensus sequence for the signal, (GCC)GCC(A or G)CCATGG, has been proposed, but actual initiation sequences differ from it to a greater or lesser degree. In the present report, the translation initiation signal sequences of rat preproinsulin and its mutant mRNAs were analyzed using a quantification method proposed previously. In this method, each 16 nt sequence in the mRNA was characterized by its sample score, which shows strength of the signal. So far, Kozak has constructed a number of preproinsulin mutant mRNAs in which nucleotides flanking the ATG codon are systematically varied, and measured the translation initiation efficiency in terms of the proinsulin product. Her experimental results were well understood on the basis of the strength of the translation initiation signal sequence.

Animals↗
PubMed1994

Presence of human cytomegalovirus (HCMV) immediate early mRNA but not ppUL83 (lower matrix protein pp65) mRNA in polymorphonuclear and mononuclear leukocytes during active HCMV infection.

During an active human cytomegalovirus (HCMV) infection, leukocytes, harbouring the HCMV lower matrix protein pp65 (ppUL83) are present in the peripheral blood and can be detected with the HCMV antigenaemia assay. In the present study, it was investigated whether the presence of pp65 in these cells was due to transcription of the virus genome or might be the result of uptake of this viral protein. Peripheral blood leukocytes of transplant recipients and AIDS patients with an active HCMV infection were investigated for the presence of HCMV immediate early (IE) antigen and pp65 using well characterized monoclonal antibodies, and for the presence of the corresponding mRNAs using non-radioactive in situ hybridization. Both mononuclear and polymorphonuclear cells were found to contain IE antigen and pp65. However, only mRNAs encoding IE antigen were found in these cells, whereas mRNAs encoding pp65 were not detected. In contrast, both IE antigen and pp65, as well as their corresponding mRNAs, were detected in the circulating late-stage HCMV-infected endothelial cells that were also present in the leukocyte fractions. These findings demonstrate that a restricted viral gene expression (transcription of IE genes) does occur in mononuclear and polymorphonuclear leukocytes. However, the abundant presence of the early antigen pp65 without detectable presence of the corresponding mRNA in these cells strongly indicates uptake of this protein by the phagocytic leukocytes, rather than de novo synthesis.

Antigens, Viral↗
PubMed2004

Elevated levels of transferrin receptor 2 mRNA, not transferrin receptor 1 mRNA, are associated with increased survival in acute myeloid leukaemia.

Transferrin receptor 1 (TfR1) is a type II membrane protein that mediates cellular iron uptake. Transferrin receptor 2(TfR2), another receptor for transferrin (Tf), has recently been cloned. We examined expression levels of TfR1, TfR2-alpha (membrane form) and TfR2-beta (non-membrane form) transcripts in cells from 67 patients with de novo acute myeloid leukaemia (AML) using reverse transcription-polymerase chain reaction (RT-PCR), and correlated the results with a variety of clinical features and disease outcomes of these patients. Significant correlations were noted between the levels of both TfR1 and TfR2-alpha (r = 0.771, P < 0.001) and TfR1 and TfR2-beta (r = 0.534, P < 0.001). Unexpectedly, initial white blood cell (WBC) counts were inversely correlated with levels of expression of either TfR1(r = -0.357, P = 0.003), TfR2-alpha (r = -0.486, P < 0.0001), or TfR2-beta (r = -0.435, P = 0.0003). Only TfR2 expression was significantly associated with either serum iron (r = -0.270, P = 0.045) or serum ferritin (r = -0.364, P = 0.008). Multivariate analyses using Cox's proportional hazard model showed that elevated TfR2-alpha, but not TfR1 or TfR2-beta mRNA levels significantly contributed to a better prognosis for AML patients. Furthermore, a group with high expression levels of both TfR2-alpha and TfR2-beta survived significantly longer than a group without high expression of both of them (P < 0.01 by log-rank). The present study suggests that (i) TfRs-independent iron uptake might have an important role in in vivo proliferation of AML cells; (ii) expression of TfR2 (especially the alpha form) is a novel prognostic factor for patients with AML.

Acute Disease↗
PubMed1979

Wheat embryo ribonucleates. XIV. Mass isolation of mRNA from wheat germ and comparison of its translational capacity with that of mRNA from imbibing wheat embryos.

Commercially milled wheat germ is shown to be a convenient source material for facile recovery of mass (milligram) quantities of highly purified poly(A)-rich RNA. This poly(A)-rich RNA is efficiently translated in a nuclease-treated extract of rabbit reticulocytes. By sucrose density gradient fractionation of bulk poly(A)-rich RNA from wheat germ, it has been possible to show that there is a direct relationship between the molecular weights of the polypeptide products of cell-free synthesis and the molecular weights of the wheat mRNA molecules which program their synthesis. As assessed by SDS -- polyacrylamide gel electrophoresis, the same array of polypeptides is synthesized when nuclease-treated reticulocyte extract is programmed by poly(A)-rich RNA from either commercially supplied or laboratory-prepared wheat embryos. Significantly, there are gross quantitative if not qualitative differences between the translational capacities of poly(A)-rich RNA from dry and imbibing wheat embryos, and the possible importance of these differences for interpreting a changing pattern of polypeptide synthesis in imbibing wheat embryos is the subject of a brief discussion.

Animals↗
PubMed2001

Development of a method for mRNA differential display in filamentous fungi: comparison of mRNA differential display reverse transcription polymerase chain reaction and cDNA amplified fragment length polymorphism in Leptosphaeria maculans.

We modified a technique, cDNA-AFLP, for identifying differentially expressed genes in plants to work in the filamentous fungus Leptosphaeria maculans (Desmaz.) Ces. & De Not. The cDNA fragments generated by our method ranged in size from approximately 100 to 400 bps. On average, twice as many cDNA fragments were amplified per primer set with cDNA amplified fragment length polymorphism in comparison with mRNA differential display reverse transcription polymerase chain reaction. The DNA fragments of interest were excised from gels and analyzed by single-stranded conformation polymorphism to eliminate nondifferentially expressed cDNA contamination. The method was used to examine gene expression differences between cultures grown in the presence or absence of an analog of the Brassica phytoalexin brassinin. Eleven of the fourteen fragments examined were determined by reverse Northern blot to be differentially expressed. In examining gene expression differences between young cultures not producing sirodesmins and older cultures that were producing these phytotoxins, we found 17 of 25 fragments were differentially expressed. Northern blots with these fragments confirmed the results.

Ascomycota↗
PubMed2005

Chunghyuldan activates NOS mRNA expression and suppresses VCAM-1 mRNA expression in human endothelial cells.

Chunghyuldan (CHD), a combinatorial drug that has antihyperlipidemic and anti-inflammatory activities, has been shown to improve arterial stiffness and inhibit stroke recurrence in clinical study. To understand the molecular basis of CHD's clinical effects, we explored its effect on cell proliferation and expression of nitric oxide synthase (NOS) and vascular cell adhesion molecule (VCAM-1) in human umbilical vein endothelial cells (HUVECs). Cell number counting and [3H]thymidine incorporation assay demonstrated that nontoxic doses of CHD have an inhibitory effect on DNA synthesis and suppress cell cycle progression of HUVECs. CHD treatment led to a marked induction of NO production through up-regulation of NOS mRNA expression in a dose- and time-dependent manner, whereas it suppressed VCAM-1 expression. CHD inhibition of VCAM-1 expression was totally blocked by pretreatment with the NO synthesis inhibitor L-NMMA, whereas pretreatment with the NO donor DETA-NO further decreased VCAM-1 level in CHD-treated HUVECs, indicating that VCAM-1 regulation by CHD is mediated through increased NO synthesis by CHD. In addition, TNF-alpha-mediated VCAM-1 activation was substantially impeded by CHD treatment. Collectively, our data suggest that anti-inflammatory or anti-hyperlipidemic effects of CHD might be associated with its ability to activate NO production and suppress VCAM-1 expression in human endothelial cells.

DNA, Complementary↗
PubMed2006

Increased maternal cortisol in late-gestation ewes decreases fetal cardiac expression of 11beta-HSD2 mRNA and the ratio of AT1 to AT2 receptor mRNA.

Moderately elevated maternal cortisol levels late in gestation cause enlargement of the fetal sheep heart. We have used quantitative real-time PCR to examine expression of candidate genes in fetal hearts from mothers in whom cortisol levels were increased (by infusion of 1 mg cortisol.kg(-1).day(-1)) or decreased (by adrenalectomy and replacement to 0.5 mg cortisol.kg(-1).day(-1)) from 115 to 130 days gestation. Control ewes were not treated with steroid. Expression of mineralocorticoid receptor (MR), glucocorticoid receptor (GR), 11beta-hydroxysteroid dehydrogenases 1 and 2 (11beta-HSD1 and -2), IGF I and II, IGF receptors 1 and 2 (IGF-1R and IGF-2R), endothelial nitric oxide synthase, VEGF, myotrophin, angiotensinogen, the angiotensin receptors 1 and 2 (AT1R and AT2R), and the angiotensin converting enzymes 1 and 2 were measured. MR mRNA abundance in fetal hearts was found to be similar to that in adult kidney and hippocampus. Although there were no significant changes in most genes, 11beta-HSD2 and IGF-1R expression were significantly decreased in the high cortisol group and 11beta-HSD2 expression negatively correlated to left ventricular wall thickness. There was also a significant change in the ratio of AT receptor expression, with increased AT2R and decreased AT1R in the high cortisol group. MR, GR, and 11beta-HSD1 immunoreactivity was found in cardiomyocytes and cardiac blood vessels in 126-128 day fetal sheep; in contrast 11beta-HSD2 staining was predominantly in blood vessels. These results indicate that cortisol could indeed act in the fetal heart to induce enlargement and suggest that the renin-angiotensin system may play a role.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed2006

Autoregulation of the mRNA export factor Yra1p requires inefficient splicing of its pre-mRNA.

Yra1p is an essential RNA-binding protein that couples transcription to export. The YRA1 gene is one of only approximately 5% of genes that undergo splicing in budding yeast, and its intron is unusual in several respects, including its large size and anomalous branchpoint sequence. We showed previously that the intron is required for autogenous regulation of Yra1p levels, which cause a dominant negative growth phenotype when elevated. The mechanism of this regulation, however, remains unknown. Here we demonstrate that growth is inversely correlated with splicing efficiency. Substitution of a canonical branchpoint moderately improves splicing but compromises autoregulation. Shortening the intron from 766 to approximately 350 nt significantly improves splicing but abolishes autoregulation. Notably, proper regulation can be restored by insertion of unrelated sequences into the shortened intron. In that the current paradigm for regulated splicing involves the binding of protein factors to specific elements in the pre-mRNA, the regulation of YRA1 expression appears to occur by a novel mechanism. We propose that appropriate levels of Yra1p are maintained by inefficient cotranscriptional splicing.

Base Pairing↗
PubMed1996

Anterior pituitary vasoactive intestinal peptide mRNA is colocalised with prolactin mRNA in hyperoestrogenised rats.

It is well established that oestrogens can stimulate prolactin (PRL) secretion as well as the expression of the vasoactive intestinal peptide (VIP) gene whose product is also a potent PRL secretagogue. Previous evidence has supported both an autocrine and a paracrine role for pituitary VIP in PRL release in vitro; however, the cellular origin of VIP in pituitary tissue still remains poorly defined. In these studies, we have demonstrated by in situ hybridisation that VIP RNA is detected in the anterior pituitaries of chronically hyperoestrogenised rats, but not in those of untreated animals. Using a double-probe labelling procedure, VIP RNA has been shown to be present in a subpopulation of PRL-producing cells, while colocalisation of VIP and GH RNA was not observed. VIP gene expression in the rat anterior pituitary gland was characterised by the presence of two alternatively polyadenylated transcripts, 1.7 kb and 1.0 kb in size. We have generated a probe specific for the 1.7 kb transcript and double-labelling studies also showed definitive colocalisation with PRL mRNA. Our results demonstrating the presence of VIP RNA in PRL-producing cells thus suggest that VIP may play an autocrine role in PRL hypersecretion under conditions of oestrogen-induced hyperplasia.

Animals↗
PubMedDate not supplied

[Possible relation between terminal modification of pre-mRNA and mRNA and discrete organization of genes].

A suggestion of the importance of end modifications of pre-mRNA and RNA for selective defence of RNA coding sequences in the course of processing allows postulating the relationship between RNA modifications and presence of introns in genes. This conclusion was verified by comparing the presence of introns with RNA end modifications in different types of organisms, subcellular organelles and some genes; in the whole an obvious correlation of these phenomena was shown.

Animals↗
PubMed1987

Identification of glutathione S-transferase Yb1 mRNA as the androgen-repressed mRNA by cDNA cloning and sequence analysis.

Androgens, while stimulating the growth of the rat ventral prostate, can also repress the levels of a limited number of mRNAs. The cDNA for one of the androgen-repressed mRNAs has been identified by nucleotide sequence analysis as coding for the glutathione S-transferase Yb1 subunit. The prostate cDNA is 1071 nucleotides long, and only 2 or 4 bases of this sequence do not match the two published sequences of the cDNA for the Yb1 subunit of rat liver glutathione S-transferase. The amino acids in the protein encoded by the prostate cDNA matched completely with that for one of the liver cDNAs and differ with the other cDNA only in two of 218 amino acids. The identification of the androgen-repressed mRNA as a glutathione S-transferase subunit may indicate that some of the cellular actions of the enzyme may be important in the control of androgen-dependent growth of the prostate. Since Yb forms of the transferases have been colocalized with uridylic acid-rich small nuclear RNAs at interchromatinic regions of the cell nucleus, autoregulation of prostate growth by androgens may be carried out through the modulation of RNA production or processing in this target organ.

Amino Acid Sequence↗
PubMed1983

Regulation of tyrosine hydroxylase mRNA by glucocorticoid and cyclic AMP in a rat pheochromocytoma cell line. Isolation of a cDNA clone for tyrosine hydroxylase mRNA.

Treatment of a subclone of the PC12 pheochromocytoma cell line, PC8b, with either dexamethasone or 8-bromo cyclic AMP resulted in increased translational activity of tyrosine hydroxylase mRNA (mRNATH). Poly(A+)-containing RNA from cells treated with both inducers was used to construct a cDNA library. Double-stranded cDNA was inserted into the PstI site of pBR322 using GC tailing, and plasmids were used to transform Escherichia coli HB101. Colonies containing plasmids with inserted sequences were initially screened by DNA dot hybridization, and those positive colonies were then screened by hybrid selected translation. One plasmid, pTH.4, was identified as containing a 400-base pair sequence complementary to mRNATH. Nick-translated pTH.4 DNA was used to identify mRNATH as containing approximately 1800 nucleotides by Northern blot analysis. PC8b cells treated with either dexamethasone or 8-bromo cyclic AMP yielded greater mRNATH hybridization on Northern blot analysis and accumulated higher molecular weight tyrosine hydroxylase RNA species. Following treatment of cells with inducers, the temporal increase in tyrosine hydroxylase enzyme activity was associated in all cases with an increase in the translational activity and relative amount of mRNATH, and the fold increase in the latter two parameters was equal to or greater than the increase in enzyme activity.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1983

[Study of the ribosome mRNA-binding region during different stages of translation. I. Functional activity of mRNA analogs, AUGU6 and its benzylidene derivatives, in ribosome-dependent protein synthesis].

Hexaribouridylic acid, prepared by digestion of poly(U) with cobra venom endonuclease, and trinucleotide AUG synthesized chemically by triester approach were joined by RNA-ligase to yield a nonaribonucleotide AUGU6 bearing the initiation codon at its 5'-terminus. 2',3'-O-(4-[N-(2-chloro(or hydroxy) ethyl-N-Methylamino])- benzylidene residues were introduced at the 3'-terminus of oligonucleotide AUGU6 and its benzylidene derivatives AUGU6CHRCl or AUGU6CHROH were obtained. The mRNA analogs synthesized were tested for their template activity in the formation of 70S initiation complex. AUGU6, AUGU6CHRC1 and AUGU6CHROH were shown to stimulate factor-dependent binding of fMet-tRNA to ribosomes. The effect of benzylidene fragment on the template activity of AUGU6CHROH in the course of translation process was studied. It was shown that AUGU6CHROH stimulates synthesis of di- and tripeptides with the same efficiency as AUGU6.

Benzylidene Compounds↗
PubMed1983

Adjuvants to immunological methods for mRNA purification. Application to the isolation of mRNA for carbonic anhydrase I from rabbit reticulocytes.

In order to generate the molecular probes needed to investigate the seemingly coordinate expression of carbonic anhydrase (CA I) and beta-globin within erythrocytes during human development, CA I-containing polyribosomes have been isolated from rabbit reticulocytes by reaction with purified antibodies to CA I followed by immunoadsorption of immune complexes with formalin-fixed protein A-bearing bacteria. In the course of such isolation, a series of maneuvers were seen to have a markedly favorable influence on the level of purity attained. These maneuvers include the use of 5 mg/ml of heparin concentrations to attenuate nonspecific binding and entrapment of unwanted polyribosomes, the addition of 200 units/ml of placental ribonuclease inhibitor to augment recovery in reactions which by test already appeared RNase-free, and the preadsorption of polyribosomes with formalin-fixed bacteria prior to immunological reaction so as to remove a subset of polyribosomes seemingly predisposed to nonspecific binding. In the absence of all of the maneuvers, attained purity was no greater than a few per cent. When all were employed, CA I-mRNA derived from immunopurified polyribosomes was recovered with more than 80% purity and 20% yield, as evident from both immunoassays and electrophoresis of its cell-free products.

Animals↗
PubMedDate not supplied

[Animal and viral mRNA translation in a mRNA-dependent cell-free system and rabbit reticulocytes].

A system of cell-free synthesis from rabbit reticulocytes is described. A method of repeated bloodlettings for induction of reticulocytosis was used. The lysate of washed reticulocytes was treated with micrococcus nuclease enabling effective inhibition of synthesis in endogenous templates. The translating activity is manifested most completely in the presence of the following compounds: ATP, GTP, ATP-regenerating system, SH-compounds, polyamines, tRNA. This system must be optimized in K+ and Mg2+ concentrations for different templates. The system is tolerant to high concentrations of rRNA which allows the utilization of total RNA preparations in the experiments. This system may be satisfactorily used for translation of mRNA of different origins.

Adenoviridae↗
PubMed1993

Differential recovery of mRNA transcripts using acid organic extraction techniques: quantification of abl mRNA abundance in the blowfly, Calliphora erythrocephala.

A commonly used RNA isolation technique that employs a single-step acid phenol-chloroform extraction (Chomczynski and Sacchi, 1987) produces a different distribution pattern of C. erythrocephala c-abl (Ceabl) transcripts following Northern blot analysis than methods that rely on phenol-chloroform extraction at basic pH (Jowett, 1986). Only Ceabl transcripts of 10.5 kb and 6.5 kb are recovered, while Ceabl transcripts of 9 kb and 4.4 kb are lost, when acidic phenol-chloroform methods are used. All tested extraction methods which do not rely on phenol-chloroform extraction under acidic conditions can recover Ceabl transcripts of 10.5 kb, 9 kb, 6.5 kb and 4.4 kb. The reason for this discrepancy is apparently due to differential loss of mRNA at the phenol phase and/or interphase boundary during the acidic extractions.

Acids↗
PubMed1988

Lymphokine mRNA and T cell multireceptor mRNA of the Ig super gene family are reciprocally modulated during human T cell activation.

The expression of genes, implicated in T cell proliferation as well as those coding for the three chains of the T cell receptor (TcR) and for the CD4 and CD8 molecules, has been investigated during activation of various T cell populations maintained in long-term culture. We show that a minimal activation signal, capable of inducing interleukin 2 (IL 2) receptor expression without the expression of IL2 and interferon-gamma, was not associated with a down-regulation of the messengers coding for the T cell multireceptor molecules. In contrast, a strong activation signal via the TcR/CD3 complex or via the CD2 molecule resulted in a reciprocal transient up-regulation of the messengers implicated in T cell proliferation and a down-regulation of the messengers encoding the molecules of the TcR complex of the immunoglobulin super gene family.

Antigens, Differentiation, T-Lymphocyte↗