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PubMed2003

Measurement of the number of molecules of a single mRNA species in a complex mRNA preparation.

The normalization of data obtained from hybridization experiments with DNA chips to determine mRNA expression and concentration (gene expression profiling) is an unsolved problem. Furthermore, slight changes in mRNA expression or small numbers of mRNA molecules which may be relevant to disease cannot be detected so far. We have designed a method to calculate the number of molecules of a single mRNA species in a complex mRNA preparation. The basic concept is the transformation of a quantitative problem into a qualitative problem. Individual molecules pertaining to the same molecular species (IMPSMS) are transformed to a mixture of new different molecular species (DMS) and amplified. We propose two implementations of the method. The first procedure is based on a method for cloning tagged nucleic acid molecules onto the surface of micro-beads. It should be possible to transform and determine up to 10(6) IMPSMS into new DMS. The second strategy uses multimeric linkers, a method frequently used in DNA computing to assemble random DNA. The second strategy should be easier to implement but is limited to a few hundred IMPSMS.

Cloning, Molecular↗
PubMed1999

High levels of IL-4 and IL-10 mRNA and low levels of IL-2, IL-9 and IFN-gamma mRNA in MuLV-induced lymphomas.

The expression of cytokines may influence the development of lymphoma in retrovirally infected animals in at least two ways: (1) cytokines in the tumor environment may stimulate the proliferation of tumor cells and/or (2) cytokines in the tumor environment may diminish the cell-mediated antitumor immune response. To evaluate these possibilities, a semiquantitative RT-PCR approach was utilized to permit a broad screening of cytokine mRNAs in a large number of tissue samples. Examination of MuLV-induced end-stage lymphomas revealed the absence of mRNA for cytokines known to stimulate the proliferation of T cells (i.e., IL-2, IL-9), the absence of mRNA for cytokines known to enhance cell-mediated antitumor immune responses (i.e., IL-2, IFNgamma), and the presence of mRNA for cytokines known to diminish such responses (i.e., IL-4, IL-10). Similar patterns of cytokine mRNA expression were detected in tumor-derived cell lines. Spleen and thymus from animals collected longitudinally during infection and from age-matched uninfected mice also demonstrated a similar pattern, except that IFNgamma mRNA was readily detectable. These findings do not support the hypothesis that the developing tumor depends on cytokines to provide proliferative signals. The findings suggest that cytokines in the immediate environment of the lymphoma support tumor development by acting to diminish an effective antitumor immune response.

Animals↗
PubMed1990

Stored mRNA in cotyledons of Vigna unguiculata seeds: nucleotide sequence of cloned cDNA for a stored mRNA and induction of its synthesis by precocious germination.

By differential hybridization screening, we previously selected a class of cDNA clones from a lambda gt10 cDNA library that was constructed from the total poly(A)+ RNA of mature cowpea cotyledons (Plant Cell Physiol 31: 39-44, 1990). pSAS10, a clone of this class, hybridized with a cDNA probe complementary to poly(A)+ RNA from cotyledons collected 1 day after the onset of imbibition (DAI), but not with the cDNA probe from cotyledons at developmental stage II (13 to 15 days after flowering, DAF). pSAS10 mRNA was detectable only in cotyledons at developmental stage III (17 to 19 DAF) or later, and its level began to decline when seeds germinated. We have suggested that pSAS10 mRNA is likely to belong to the class of 'stored' mRNA or the mRNA that is formed at the late stage of seed maturation, is conserved in quiescent seeds and becomes functional at the early stage of germination. We determined the nucleotide sequence of pSAS10 cDNA consisting of 459 bp and an approximately 36 bp poly(A) tract, and deduced the amino acid sequence of its product, a 10-kDa cysteine-rich polypeptide. Synthesis of pSAS10 mRNA was induced just before germination began, not only in mature seeds but also in immature seeds even at stages I (9 to 11 DAF) and II (13 to 15 DAF) if they were placed under conditions suitable for germination.

Amino Acid Sequence↗
PubMed1989

Ataxia-telangiectasia fibroblasts have less fibronectin mRNA than control cells but have the same levels of integrin and beta-actin mRNA.

The expression of fibronectin, integrin and beta-actin genes in skin fibroblasts from patients with the genetic disorder ataxia-telangiectasia (A-T) was studied. These three genes were selected because their protein products contribute to the shape and function of the fibroblast. Expression of mRNA by these genes was compared with that in fibroblasts from normal individuals and from patients with the genetic disorder, hereditary hemorrhagic telangiectasia (HHT). A-T fibroblasts were found to produce less fibronectin mRNA than normal and HHT fibroblasts. A-T fibroblasts senesce around passage level 15 while normal and HHT fibroblasts can be propagated for many more passages in vitro. However, the expression of the integrin gene in A-T fibroblasts was similar to that in normal fibroblasts, while the beta-actin gene was expressed at a higher level. The increased beta-actin mRNA levels were similar in fibroblasts of patients with the two genetic disorders, A-T and HHT, but higher than in normal fibroblasts. HHT fibroblasts differed markedly from A-T fibroblasts in having a high level of fibronectin and integrin mRNA expression. The results indicate that regulation of the fibronectin gene in A-T fibroblasts differs from that of the integrin and beta-actin genes, and that the decline in fibronectin mRNA may be linked to the shortened in vitro life-span of these cells.

Actins↗
PubMed1994

The loss of E-cadherin mRNA transcripts in rat prostatic tumors is accompanied by increased expression of mRNA transcripts encoding fibronectin and its receptor.

We compared the levels of mRNA transcripts encoding E-cadherin, N-cadherin, beta 1 integrin subunit, alpha 5 integrin subunit and fibronectin in the normal rat prostate gland, as well as in tumors derived from three invasive sublines (G, MatLyLu, AT-2) of the Dunning R-3227 rat prostatic adenocarcinoma. E-cadherin mRNA transcripts were only detectable in total RNA extracts prepared from normal rat prostates, whereas N-cadherin mRNA transcripts were only found in normal rat brains. In contrast, the mRNA transcripts encoding the beta 1 integrin subunit, alpha 5 integrin subunit and fibronectin were all elevated in the tumors, as compared to the levels of these transcripts in normal tissues. Our results suggest that there is an inverse correlation between cadherin and integrin mRNA levels in rat prostatic tumors.

Adenocarcinoma↗
PubMed1996

Differential mRNA decay within the transfer operon of plasmid R1: identification and analysis of an intracistronic mRNA stabilizer.

Processing of the transfer operon mRNA of the conjugative resistance plasmid R1-19 results in the accumulation of stable traA mRNAs. The stable traA transcripts found in vivo have identical 3' ends within downstream traL sequences, but vary at their 5' ends. The 3' ends determined coincide with the 3' base of a predicted large clover-leaf-like RNA secondary structure. Here we demonstrate that this putative RNA structure, although part of a coding sequences, stabilizes the upstream traA mRNA very efficiently. We also show that the 3' ends of the stable mRNAs are formed posttranscriptionally and not by transcription termination. Half-life determinations reveal the same half-lives of 13 +/- 2 min for the traA mRNAs transcribed from hybrid lac-traAL-cat test plasmids, the R1-19 plasmid, and the F plasmid. Protein expression experiments demonstrate that the processed stable traA mRNA is translationally active. Partial deletions of sequences corresponding to the predicted secondary structure within the traL coding region drastically reduce the chemical and functional half-life of the traA mRNA. The results presented here unambiguously demonstrate that the proposed secondary structure acts as an efficient intracistronic mRNA stabilizer.

Bacterial Outer Membrane Proteins↗
PubMed1992

Changes in dopa decarboxylase mRNA but not tyrosine hydroxylase mRNA levels in rat brain following antipsychotic treatment.

The effects of antipsychotic administration (1-32 days, twice per day) on the levels of mRNA coding for dopa decarboxylase (DDC) and tyrosine hydroxylase (TH) in rat brain has been assessed by a procedure utilising solution hybridisation with oligonucleotides. Saline and sulpiride (20 mg/kg/day) had no apparent effect on DDC mRNA levels. Haloperidol (3 mg/kg/day) elicited increases in DDC mRNA levels of 240% after 32 days and loxapine (4 mg/kg/day) elicited increases of 180% in DDC mRNA levels. None of the drugs affected TH mRNA levels. These results indicate that DDC may be more important than TH in the long term regulation of dopamine production.

Animals↗
PubMed1997

Osteogenic protein-1 stimulates mRNA levels of BMP-6 and decreases mRNA levels of BMP-2 and -4 in human osteosarcoma cells.

Bone morphogenetic proteins (BMPs) are novel growth and differentiation factors that act on mesenchymal stem cells to initiate new bone formation in vivo and promote the growth and differentiation of cells in the osteoblastic lineage. In the present study, we examined the effects of recombinant human osteogenic protein-1 (also known as BMP-7) on the expression of related members of the BMP family using SaOS-2 and U2-OS, two human osteosarcoma cell strains. Evaluation of BMP-2, -4, and -6 mRNA expression indicates that OP-1 stimulated the mRNA levels of BMP-6 in both SaOS-2 cells (threefold) and U2-OS cells (fivefold) after 24 hours of treatment, while decreasing the mRNA levels of BMP-4 in SaOS-2 cells (80%) and BMP-2 and BMP-4 in U2-OS cells by 50% and 72%, respectively. BMP-2 mRNA expression, as examined by Northern blot analysis, was below detectable limits in SaOS-2 cultures. These results demonstrate that OP-1 modulates the mRNA expression of related members of the BMP family, suggesting a possible mode of action of OP-1 on the growth and differentiation of cells in the osteoblastic lineage in vitro.

Blotting, Northern↗
PubMed1996

The gene fl(2)d is needed for the sex-specific splicing of transformer pre-mRNA but not for double-sex pre-mRNA in Drosophila melanogaster.

In Drosophila melanogaster, regulation of the sex determination genes throughout development occurs by sex-specific splicing of their products. The first gene is Sex-lethal(Sxl). The downstream target of Sxl is the gene transformer (tra): the Sxl protein controls the female-specific splicing of the Tra pre-mRNA. The downstream target of the gene tra is the gene double-sex (dsx): the Tra protein of females, controls the female-specific splicing of the Dsx pre-mRNA. We have identified a gene, female-lethal-2-d fl(2) d, whose function is required for the female-specific splicing of Sxl pre-mRNA. In this report we analyze whether the gene fl(2)d is also required for the sex-specific splicing of both Tra and Dsx pre-mRNAs. We found that the Sxl protein is not sufficient for the female-specific splicing of Tra pre-mRNA, the fl(2)d function also being necessary. This gene, however, is not required for the female-specific splicing of Dsx pre-mRNA.

Animals↗
PubMed1985

Elicitor-induced phytoalexin synthesis in soybean cells: changes in the activity of chalcone synthase mRNA and the total population of translatable mRNA.

Rapid changes in the mRNA activity encoding chalcone synthase, a central enzyme involved in isoflavonoid phytoalexin synthesis, were induced in cultured cells of soybean (Glycine max) after treatment with a glucan elicitor from the cell walls of the fungus, Phytophthora megasperma f. sp. glycinea, a soybean pathogen. Two-dimensional gel electrophoresis of the in vitro- and in vivo-synthesized chalcone synthase showed that it consisted of a group of proteins of similar molecular weights of about 41,000, but with differing isoelectric points between pH 6.1 and pH 7.1. Total activity of chalcone synthase mRNA increased as early as 40 to 60 min after the onset of elicitor induction, and reached a peak at about 4 h. Treatment with the fungal elicitor caused major changes in the population of total translatable RNA as indicated by two-dimensional electrophoresis of the translation products. The mRNA activities for at least 16 proteins were increased and for at least 4 proteins were decreased. The elicitor-induced changes in the population of translatable mRNA occurred at a rate similar to that observed for chalcone synthase mRNA activity. Our results suggest that soybean cells respond to the glucan elicitor by major metabolic changes at the RNA level including the enhanced capacity for phytoalexin synthesis.

Acyltransferases↗
PubMed1978

The relationship between hnRNA+-poly(A) and mRNA+-poly (A) in non-dividing human lymphocytes. Evidence for distinct synthetic pathways for mRNA precursor- and nonprecursor-hnRNA.

The processing of hnRNA+-poly(A) to mRNA+-poly(A) has been studied in resting lymphocytes from human peripheral blood. In pulse-chase experiments, two types of hnRNA+-poly(A) have been distinguished: the first is labeled predominantly with exogenous radioactive precursors supplied during the pulse, and the second incorporates primarily scavenged labeled precursors made available during a chase incubation. When the disappearance of both types of hnRNA+-poly(A) was quantitatively compared with the appearance of stable and labile mRNA+-poly(A), only 10% of the anticipated cytoplasmic material was actually obtained. Statistically, 90% of the poly(A)-bearing hnRNA molecules processed were degraded. The two types of hnRNA+-poly(A) were found to be functionally different. Pulse-labeled material was processed to poly(A)-bearing mRNA; "chase-labeled" molecules did not leave the nucleus and never served as precursors for cytoplasmic mRNA. The data fit a model in which there are distinct pathways for mRNA precursor- and nonprecursor-hnRNA+-poly(A).

Cell Nucleus↗
PubMed1995

The effect of a unilateral inflammation at the rat's ankle joint on the expression of preprotachykinin-A mRNA and preprosomatostatin mRNA in dorsal root ganglion cells--a study using non-radioactive in situ hybridization.

In rats with an acute (2 days) and chronic (20 days) unilateral ankle joint inflammation (induced by Freund's complete adjuvant), the proportion of dorsal root ganglion cells containing preprotachykinin-A mRNA or preprosomatostatin mRNA was determined using non-radioactive in situ hybridization. At the acute stage of inflammation, the proportion of neurons containing preprotachykinin-A mRNA was similar to that in control rats. At the chronic stage, the proportion of neurons expressing preprotachykinin-A mRNA was significantly higher on the inflamed side than on the contralateral side. The proportion of dorsal root ganglion cells containing preprosomatostatin mRNA did not change. These data suggest that inflammation influences the synthesis of substance P but not of somatostatin in afferent neurons.

Acute Disease↗
PubMed1994

Cholinergic differentiation of cultured sympathetic neurons induced by retinoic acid. Induction of choline acetyltransferase-mRNA and suppression of tyrosine hydroxylase-mRNA levels.

Here we show that retinoic acid (RA) has the ability to alter the transmitter phenotype of cultured sympathetic neurons from newborn rats superior cervical ganglia (SCG). In the presence of RA, the level of choline acetyltransferase (ChAT) mRNA was increased, while the level of tyrosine hydroxylase (TH) mRNA was reduced in the cultured SCG neurons. Selective PCR amplification of different upstream regions of the ChAT-mRNA indicates that RA promotes the transcription of ChAT gene from R and M exons. The RA-induced upregulation of ChAT-mRNA level was significantly diminished by the chronic treatment with phorbol ester, suggesting that PKC has an important role in the induction of ChAT-mRNA in this system.

Animals↗
PubMed1985

Biosynthesis of reovirus-specified polypeptides. The s1 mRNA synthesized in vivo is structurally and functionally indistinguishable from in vitro-synthesized s1 mRNA and encodes two polypeptides, sigma 1a and sigma 1bNS.

The structural and functional properties of the reovirus serotype 1 (Lang strain) s1 mRNA were examined. Reovirus s-class mRNAs, synthesized either in vivo within infected mouse L cells or in vitro by chymotrypsin-derived cores of purified virions, were purified by filter-hybridization using cDNA clones of the S-class genome segments. S1 cDNA-selected mRNA encoded the synthesis of the Mr approximately 12,000 nonstructural polypeptide designated sigma 1bNS in addition to the well-established structural polypeptide sigma 1, now designated sigma 1a. The coding properties of in vivo- and in vitro-synthesized s1 mRNA were equivalent: both encoded sigma 1a and sigma 1bNS. Primer extension analysis of s1 mRNA revealed a single major 5' terminus for both in vivo- and in vitro-synthesized s1 mRNA. These results suggest that there is a single transcript of the reovirus S1 genome segment which is functionally dicistronic, and likely encodes both sigma 1a and sigma 1bNS.

Animals↗
PubMed1979

Production of mRNA in Chinese hamster cells: relationship of the rate of synthesis to the cytoplasmic concentration of nine specific mRNA sequences.

We constructed cloned DNA sequences complementary to unselected mRNAs [poly(A)+ cytoplasmic RNA] from Chinese hamster ovary cells and used them in RNA:DNA hybridization experiments. Each cloned DNA hybridized a single mRNA from 1.3-3.5 kb in length. The relative rates of labeling (transcription rates) of nuclear RNA complementary to each individual DNA segment varied approximately 10 fold. The relative cytoplasmic concentration of the same specific RNA sequences in the mRNA after an equilibrium labeling of the cells varied approximately 100 fold. In addition, we estimated the sizes of the nuclear RNA precursor molecules to these cytoplasmic mRNAs. Four main conclusions arise from these studies. First, the primary RNA transcripts, which range in size from 2.4-13.5 kb, are 2-6 times larger than the mRNAs; second, each cloned DNA segment is complementary to only one species of mRNA; third, for the RNA complementary to at least three of the nine cloned DNA segments, the relative cytoplasmic content is considerably different from the relative rate of nuclear RNA synthesis, suggesting the post-transcriptional events are involved in the determination of the cytoplasmic concentrations of some mammalian mRNAs; and fourth, the fraction of total nonribosomal nuclear RNA complementary to the nine cloned DNA segments is in most cases 10 fold less than the fraction of cytoplasmic mRNA complementary to the same cloned DNA segments, suggesting the synthesis of many hnRNA molecules that are qualitatively different from those which eventually contribute mRNA to the cytoplasm.

Animals↗
PubMed1988

An intercistronic stem-loop structure functions as an mRNA decay terminator necessary but insufficient for puf mRNA stability.

Segmental differences in stability within the polycistronic transcripts of the puf operon contribute to differential expression of photosynthesis genes in R. capsulatus. The comparatively stable 5' segment of these transcripts ends in a large intercistronic stem-loop structure. We show here that deletion of this RNA hairpin destabilizes the 5' puf mRNA segment but that its insertion at the 3' end of the puf operon transcripts fails to stabilize the labile 3' puf mRNA segment. Evidence is presented that decay of the 3' segment begins with endonucleolytic cleavage in which the intercistronic stem-loop structure does not participate. We conclude that this RNA hairpin is necessary but insufficient for the stability of mRNA upstream of it, and that it functions in message degradation solely as an mRNA decay terminator that protects upstream mRNA segments from degradation by 3' exoribonucleases.

Bacterial Proteins↗
PubMed1992

Response of pituitary and spleen pro-opiomelanocortin mRNA, and spleen and thymus interleukin-1 beta mRNA to adjuvant arthritis in the rat.

During development of adjuvant-induced arthritis (AA) in the rat, pituitary pro-opiomelanocortin (POMC) mRNA expression was increased. Pituitary POMC mRNA was much higher following adrenalectomy and AA. Spleen POMC mRNA also increased with a similar time kinetics, although the levels in the spleen were much lower than those in the pituitary. In control animals, spleen interleukin-1 beta (IL-1 beta mRNA) was undetectable, whereas AA led to the accumulation of IL-1 beta mRNA and the highest levels were seen in the adrenalectomised AA group. Thymic IL-1 beta expression was also increased in AA animals. These results suggest that AA leads to the activation of both the neuroendocrine and the immune systems and the interaction between these systems may play a role in this disease state.

Adrenalectomy↗
PubMed1988

Transient increases and individual variations in preprocholecystokinin gene expression in rat telencephalic regions during postnatal development: a quantitative comparison between preprocholecystokinin mRNA and preproenkephalin mRNA.

Postnatal development of the tissue levels of mRNAs encoding preprocholecystokinin and preproenkephalin in rat telencephalic regions was analyzed by quantitative dot blot hybridization using synthetic RNAs as standards. Preprocholecystokinin mRNA levels per tissue weight showed distinct peaks in the frontal cortex and hippocampus on postnatal days 10 and 20, respectively, while preproenkephalin mRNA levels in these regions showed no apparent peaks. In the striatum, preproenkephalin mRNA abundance linearly increased 8-fold from day 5-43, while a transient increase in the preprocholecystokinin mRNA abundance between days 10 and 20 was noted. Furthermore, the striatal levels of preprocholecystokinin mRNA showed marked individual variations during the early period of postnatal development. These results suggest transient and variable increases in cell density and/or in gene expression of cholecystokinin-synthesizing cells in the developing telencephalon.

Aging↗