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PubMed1990

Cloning of estrogen-responsive messenger RNAs in the T-47D human breast cancer cell line.

A complementary DNA library has been constructed from the polyadenylated mRNA of steroid-deprived T-47D cells which had been restimulated with estrogen for 24 h. Screening of 15,000 recombinants by sequential rounds of colony, Southern and Northern blot differential hybridization has identified eight different clones which vary in abundance and are stimulated between 2.5- and 8-fold by estrogen. Whereas five recombinants hybridize to single mRNA sequences, two clones, pSyd 2 and pSyd 8, appear to hybridize weakly to an addition mRNA sequence and one clone, pSyd 3, hybridizes to a multiple mRNA species (1.9, 1.7, 0.9, and 0.5 kilobases). Furthermore, at least one clone, pSyd 2, appears to be expressed only in ER-positive cells. While its level of expression is stimulated by estrogen approximately 4-fold in all the estrogen and progesterone receptor-positive cell lines tested (T-47D, ZR-75-1, and MCF-7), pSyd 2 levels in the estrogen and progesterone receptor-negative HBL-100 cell line were lower than the corresponding levels in estrogen-stimulated T-47D cells and were unresponsive to estradiol. These results show that we have isolated several estrogen-responsive sequences which will be useful in studying hormone regulation of gene expression and may provide additional markers of hormone responsiveness in breast cancer.

Blotting, Northern↗
PubMed1981

Efficiency of processing of viral RNA during the early and late phases of productive infection by polyoma virus.

The efficiency of processing of polyoma viral RNA and of its export from nucleus to cytoplasm was measured in primary mouse kidney cells by comparing the initial rates of incorporation of [3H]uridine into cytoplasmic and nuclear viral RNA. Appropriate methods of cell fractionation were chosen to maximize yields of cytoplasmic RNA and to minimize leakage of nuclear RNA. Incorporation of [3H]uridine into cellular 4S RNA in the cytoplasm was followed to monitor pool equilibration and maintenance of an excess of radioactive precursor throughout the experimental period. During the early phase of infection (9 to 11 h, in the presence of 5-fluorodeoxyuridine), viral RNA was rapidly and efficiently exported from nucleus to cytoplasm. Viral RNA appeared in the cytoplasm within 6 min of its synthesis, greater than half of the viral RNA synthesized in the nucleus was exported to the cytoplasm. In contrast, during the late phase of infection (28 to 30 h), viral RNA was exported more slowly, appearing in the cytoplasm 12 to 20 min after its synthesis, and much less efficiently-only 5% of late nuclear transcripts was exported. The poor efficiency of processing of late viral RNA may be, in part, a result of (i) the presence in nuclear transcripts of non-mRNA sequences which are removed during processing; (ii) the presence in nuclear transcripts of multiple copies of mRNA sequences, only one of which is incorporated into mature mRNA; and (iii) inefficient polyadenylation of viral nuclear RNA.

Animals↗
PubMed1981

Membrane-bound ribosomes of myeloma cells. V. Subcellular distribution of immunoglobulin mRNA molecules.

The subcellular distribution of the most abundant mRNA sequences, particularly those of the immunoglobulin heavy (Ig H) and light (IG L) chain mRNA sequences, of MOPC 21 (P3K) mouse myeloma cells has been examined by translating the mRNA of various subcellular fractions in a messenger-dependent reticulocyte lysate (MDL) and by identifying Ig products with the use of a specific antiserum. Analyses of the distribution of the mRNA template activity and the translation products by SDS polyacrylamide gel electrophoresis reveal that approximately 85% of the mRNA present in the free ribosomal fraction is incorporated into polysomes and that the remainder is present as mRNP particles. On the endoplasmic reticulum (ER) the mRNA is found entirely in polysomes. In general, the size class of free (F) and membrane-bound (MB) polysomes corresponds to the size of their translation products. Thus, mRNAs coding Ig H (5.0 x 10(5) daltons in size) and Ig L (2.5 x 10(5) daltons in size) are incorporated into polysomes formed of 12 and 6 ribosomes, respectively. About 10% of the Ig mRNAs are not bound to membranes. A third of these are associated with mRNPs and the remainder incorporated into F polysomes of the same size as the Ig-synthesizing MB polysomes.

Animals↗
PubMed2003

The role of 3' UTRs in the mechanisms for segregating mitochondrial proteins.

The mitochondrial proteome, meant as the collection of proteins localized in the mitochondrion, whose fundamental function is the energy production for the life of the cell, is estimated to be constituted by at least 1000 proteins. Since the mitochondrial genome codes only for 13 polypeptides, in practice all of them are coded by nuclear genes and are imported in the mitochondrion by means of a sophisticated mechanism of sorting that is not completely known. Most classified mitochondrial proteins are synthesized as precursors with an amino-terminal extension (leader peptide), provided with peculiar physical and structural characteristics that are responsible for the recognition and import system. However, there is experimental evidence that complementary or alternative import systems exist. In particular, the 3' UnTranslated Regions (UTRs) of the mRNAs may be involved, through their secondary structure, in the mechanisms of recognition and localization. In this paper, the information content of different human mRNA sequences has been analyzed, and the results show that a significative short-term correlation exists in the 3' UTR sequences of the mRNA's coding for the proteins provided with leader peptides, probably representing the structural constraints that are necessary to the sorting machinery.

3' Untranslated Regions↗
PubMed1991

Xenopus liver ferritin H subunit: cDNA sequence and mRNA production in the liver following estrogen treatment.

In vitro translation of liver mRNA from estrogen-treated Xenopus frogs yields two abundant polypeptides in the range of 20 kDa. DNA clones for one of these translation products were isolated and shown to be complementary to mRNA for the heavy subunit of ferritin. The predicted Xenopus amino acid sequence shares about 86% identity with the ferritin heavy chain from bullfrogs and about 70% identity with the comparable mammalian and avian proteins. Clone identity was confirmed by hybridization selection followed by in vitro translation into translation products of 19.5-20 kDa. The nearly full-length cDNA clone, termed XlferH1, comprises 868 nucleotides plus 22 adenosines of the poly(A) tail, including 134 nucleotides of the 5'-untranslated region, a 528-base coding region for 176 amino acids, and a 206-nucleotide 3'-untranslated region. The clone lacks 22 nucleotides from the 5' end of the mRNA. The level of ferritin mRNA in the liver of estrogen-treated frogs was determined over time. The amount of this mRNA relative to total RNA decreased about 3-fold 14 days after estradiol-17 beta was administered. However, the hormone also elevated total RNA in the liver about 24-fold. Hence, the total ferritin mRNA content of the liver increased to about 8 times its initial amount. This pattern of gene expression was very similar to that for serum retinol binding protein. The estrogen induction of these two mRNAs appeared to parallel the overall stimulation of hepatic RNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

Isolation of a mRNA instability sequence that is cis-dominant to the ompA stability determinant in Escherichia coli.

Transcriptional fusions with ompA and bla have been used to identify a novel mRNA instability element. A 287-nucleotide (nt) sequence containing a repetitive extragenic palindrome (REP) from the chloramphenicol acetyltransferase (cat) gene was inserted into the 3' untranslated region (UTR) of the ompA gene. In one orientation, the insert had no effect on the half-life of the ompA-cat chimeric transcript. In the other orientation, however, the sequence functioned as a destabilizing element and was dominant to the 5'-UTR ompA and REP stability elements. The orientation-dependent effect of the instability sequence suggests that sequence rather than structure alone is important to the function of the instability determinant. In addition, the instability sequence also destabilized an ompA-bla fusion construct when fused to its 3'-UTR region. A sensitive RNA ligation/PCR amplification technique was developed and used to analyze RNA decay intermediates. The results indicated that degradation of the chimeric transcript initiated within the 287-nt inserted sequence.

Bacterial Outer Membrane Proteins↗
PubMed1996

Subtype 2 and atypical angiotensin receptors in the human heart.

Angiotensin receptors have been described in the human heart and are suspected to play a central role in remodeling after myocardial infarction and in cardiac hypertrophy. Two subtypes, AT1 and AT2, have so far been described in humans, with AT2 being the dominant subtype in human atria. We have now determined subtype numbers and distribution by binding in ventricular myocardium from patients with end-stage heart failure. We found about 50-80% of subtype AT2 in the right and left ventricles from patients with end-stage heart failure due to coronary artery disease and cardiomyopathy, indicating that AT2 is the dominant angiotensin receptor subtype in the whole human heart. To determine the cellular localization of angiotensin receptors in human myocardium in addition to the known localization on myocytes, smooth muscle cells and endothelial cells, we investigated cardiac fibroblasts. They express an angiotensin receptor with yet incompletely understood binding characteristics which is coupled to proliferation and DNA synthesis. As AT2 is the dominant angiotensin receptor subtype in human heart, we cloned the complete mRNA sequence by a rapid amplification of cDNA ends (RACE) procedure and thereafter the promoter sequence from a human genomic library. Once the sequence of the mRNA and thus exon 1 was obtained by the RACE-PCR, a probe was constructed for the most 5' region of exon 1 and used for screening of a human genomic DNA bank. After cutting of the positive clones with EcoR1 and Not1, a 4000 bp fragment hybridized with the probe and was further sequenced. A functional AT2 promoter, with > 90% homology with the mouse promoter and 35% homology with the human AT1 promoter containing numerous cis-acting sequences for basal (TFIID) and inducible (AP-1, PEA-3, CBF) transcription factors in the first 1000 bp was identified.

DNA↗
PubMed2003

An in-silico method for prediction of polyadenylation signals in human sequences.

This paper presents a machine learning method to predict polyadenylation signals (PASes) in human DNA and mRNA sequences by analysing features around them. This method consists of three sequential steps of feature manipulation: generation, selection and integration of features. In the first step, new features are generated using k-gram nucleotide acid or amino acid patterns. In the second step, a number of important features are selected by an entropy-based algorithm. In the third step, support vector machines are employed to recognize true PASes from a large number of candidates. Our study shows that true PASes in DNA and mRNA sequences can be characterized by different features, and also shows that both upstream and downstream sequence elements are important for recognizing PASes from DNA sequences. We tested our method on several public data sets as well as our own extracted data sets. In most cases, we achieved better validation results than those reported previously on the same data sets. The important motifs observed are highly consistent with those reported in literature.

Base Sequence↗
PubMed1991

Detection of polyadenylated RNA in hepatitis B virus-infected peripheral blood mononuclear cells by polymerase chain reaction.

Polymerase chain reaction (PCR) with a reverse transcriptase step characterized a specific transcription activity in hepatitis B virus (HBV)-infected peripheral blood mononuclear cells (PBMC) in two patients (1 and 2) with chronic hepatitis positive for antibody to hepatitis B core antigen (HBc). Patient 1 was also coinfected with human hepatitis delta virus. A patient who cleared HBV replication after antiviral treatment with vidarabine served as negative control. HBV-specific RNA poly A sequences were detected by PCR in PBMC of patients 1 and 2 even without detectable HBV DNA (patient 2) as shown by dot blot and PCR assays. RNA sequences were found in both the nucleus and cytoplasm. The demonstration of HBV mRNA sequences within PBMC suggests the transcription of viral DNA, in agreement with the findings of HBV surface antigen in PBMC. The results in patient 1 demonstrated HBV mRNA sequences in leukocytes even without PCR-detectable HBV DNA sequences, likely due to ongoing hepatitis delta virus replication.

Base Sequence↗
PubMed1980

Characterisation of polyoma late mRNA leader sequences by molecular cloning and DNA sequence analysis.

The leader sequences of two of the three polyoma virus late mRNAs were characterised by molecular cloning and DNA sequence analysis. A short single-stranded DNA fragment complementary to the 5' end of the body of mVP1 was used to prime cDNA synthesis, and double-stranded cDNA was inserted into a derivative of pAT 153. Analysis of fourteen mVP1 cDNAs and two mVP3 cDNAs allowed the precise determination of the leader-body joints, and demonstrated that the majority of polyoma late leader sequences consist of exact tandem repeats of a 57 nucleotide sequence present only once in the genomic DNA at 66-67 m.u. The sequences in the genomic DNA borderline this unit are typical of those found at RNA splice points. Leader sequences contained on average three to four repeat units. Nuclease S1 mapping of total late mRNA demonstrated that most mRNA 5' ends map heterogeneously in the 50 nucleotides 5' to the repeated sequence unit. The structures of the leader sequences strongly suggest that they are generated by appropriate splicing events from a tandemly repeated transcript of the entire circular viral genome.

Base Sequence↗
PubMed1983

Complete mRNA coding sequence of the acetylcholine binding alpha-subunit of Torpedo marmorata acetylcholine receptor: a model for the transmembrane organization of the polypeptide chain.

A 1,350-base-pair-long cDNA clone, named p alpha-2, was isolated by hybridization to the previously characterized clone p alpha-1 and found to be specific for the alpha-subunit of the Torpedo marmorata acetylcholine receptor. The nucleotide sequences of both cDNA inserts were analyzed and the sequence of the complete coding region and part of the 5' and 3' untranslated regions of the alpha-chain mRNA was determined. The complete amino acid sequence of the alpha-chain precursor is presented and used to develop a model for the transmembrane organization of the polypeptide.

Acetylcholine↗
PubMed1987

Hen vitellogenin genes: a study of the distribution of phosvitin phosphoprotein coding sequences using mRNA hybridization with synthetic complementary oligonucleotide probes.

Pentadecamer DNA probes were synthesized, having complementary codons for selected unique pentapeptide sequences of low codon degeneracy present in hen phosvitin minor phosphoprotein, hen phosvitin major phosphoprotein, both phosvitin phosphoproteins. These probes were 5'-32P-labelled. Vitellogenin mRNA was isolated from estrogenized chick liver, fractionated by electrophoresis using formaldehyde/agarose gels and blot transferred to nitrocellulose paper. Relative yields of the two vitellogenin mRNAs differed with the extraction method used. The minor phosphoprotein DNA probe formed a hybrid with a 1.6 megadalton component. The remaining two probes hybridized to a 2.3 megadalton component, this being the expected size of a full-length message. The smallest polyadenylated fragment to which the major phosphoprotein DNA probe hybridized was 1.0 megadalton. The remaining two probes hybridized to fragments of 0.7 megadalton and possibly smaller. Phosvitin major phosphoprotein is concluded to be coded for by part of the larger vitellogenin mRNA, while the minor phosphoprotein is coded for by part of the smaller vitellogenin mRNA. Estimates of the distances of the hybridization sites from polyadenylated tails are also given.

Animals↗
PubMed2006

Naturally occurring adenines within mRNA coding sequences affect ribosome binding and expression in Escherichia coli.

Translation initiation requires the precise positioning of a ribosome at the start codon. The major signals of bacterial mRNA that direct the ribosome to a translational start site are the Shine-Dalgarno (SD) sequence within the untranslated leader and the start codon. Evidence for the presence of many non-SD-led genes in prokaryotes provides a motive for studying additional interactions between ribosomes and mRNA that contribute to translation initiation. A high incidence of adenines has been reported downstream of the start codon for many Escherichia coli genes, and addition of downstream adenine-rich sequences increases expression from several genes in E. coli. Here we describe site-directed mutagenesis of the E. coli aroL, pncB, and cysJ coding sequences that was used to assess the contribution of naturally occurring adenines to in vivo expression and in vitro ribosome binding from mRNAs with different SD-containing untranslated leaders. Base substitutions that decreased the downstream adenines by one or two nucleotides decreased expression significantly from aroL-, pncB-, and cysJ-lacZ fusions; mutations that increased downstream adenines by one or two nucleotides increased expression significantly from aroL- and cysJ-lacZ fusions. Using primer extension inhibition (toeprint) and filter binding assays to measure ribosome binding, the changes in in vivo expression correlated closely with changes in in vitro ribosome binding strength. Our data are consistent with a model in which downstream adenines influence expression through their effects on the mRNA-ribosome association rate and the amount of ternary complex formed. This work provides evidence that adenine-rich sequence motifs might serve as a general enhancer of E. coli translation.

Adenine↗
PubMed1986

Insulin-like growth factor I regulates growth hormone secretion and messenger ribonucleic acid levels in human pituitary tumor cells.

GH secretion and mRNA levels were measured in cultured human GH adenoma cells incubated in serum-free medium for up to 48 h. A human recombinant insulin-like growth factor I (IGF-I) analog, Thr-59-IGF-I (6.5 nM), inhibited basal GH secretion by up to 60% in tumor cell cultures. The 30-50% stimulation of GH secretion by GH-releasing hormone (GHRH) was prevented by simultaneous exposure of the cells to IGF-I (6.5 nM). Gel electrophoresis of total RNA derived from GH cell adenoma tissue, followed by transfer and hybridization with 32P-labeled human GH cDNA, revealed a distinct mRNA species of about 1.0 kilobases. Using cytoplasmic dot blot hybridization, IGF-I inhibited the levels of human GH mRNA sequences in these cells and also prevented the GHRH-induced stimulation of GH mRNA. A monoclonal antibody to the type I IGF-I receptor (alpha IR3) prevented the inhibitory effects of IGF-I on basal and GHRH-stimulated GH secretion. This antibody also prevented the IGF-I-induced suppression of GH mRNA sequences. PRL secretion in these cells was not altered by IGF-I. Furthermore, relative levels of beta-actin mRNA were unaltered by IGF-I. Thus, IGF-I suppresses basal and GHRH-stimulated GH secretion and GH mRNA levels in pituitary adenoma cells, indicating that IGF-I acts selectively on the somatotroph to directly regulate GH gene expression.

Adenoma↗
PubMed2002

Yersinia enterocolitica type III secretion: mutational analysis of the yopQ secretion signal.

Pathogenic Yersinia spp. secrete Yop proteins via the type III pathway. yopQ codons 1 to 15 were identified as a signal necessary and sufficient for the secretion of a fused reporter protein. Frameshift mutations that alter codons 2 to 15 with little alteration of yopQ mRNA sequence do not abolish type III transport, suggesting a model in which yopQ mRNA may provide a signal for secretion (D. M. Anderson and O. Schneewind, Mol. Microbiol. 31:1139-1148, 2001). In a recent study, the yopE signal was truncated to codons 1 to 12. All frameshift mutations introduced within the first 12 codons of yopE abolished secretion. Also, multiple synonymous mutations that changed the mRNA sequence of yopE codons 1 to 12 without altering the amino acid sequence did not affect secretion. These results favor a model whereby an N-terminal signal peptide initiates YopE into the type III pathway (S. A. Lloyd et al., Mol. Microbiol. 39:520-531, 2001). It is reported here that codons 1 to 10 of yopQ act as a minimal secretion signal. Further truncation of yopQ, either at codon 10 or at codon 2, abolished secretion. Replacement of yopQ AUG with either of two other start codons, UUG or GUG, did not affect secretion. However, replacement of AUG with CUG or AAA and initiating translation at the fusion site with npt did not permit Npt secretion, suggesting that the translation of yopQ codons 1 to 15 is a prerequisite for secretion. Frameshift mutations of yopQ codons 1 to 10, 1 to 11, and 1 to 12 abolished secretion signaling, whereas frameshift mutations of yopQ codons 1 to 13, 1 to 14, and 1 to 15 did not. Codon changes at yopQ positions 2 and 10 affected secretion signaling when placed within the first 10 codons but had no effect when positioned in the larger fusion of yopQ codons 1 to 15. An mRNA mutant of yopQ codons 1 to 10, generated by a combination of nine synonymous mutations, was defective in secretion signaling, suggesting that the YopQ secretion signal is not proteinaceous. A model is discussed whereby the initiation of YopQ polypeptide into the type III pathway is controlled by properties of yopQ mRNA.

3' Untranslated Regions↗
PubMedDate not supplied

Analyzing the evolution of the opioid/orphanin gene family.

Advances in molecular biology have made it possible to rapidly obtain the amino acid sequence of neuropeptide precursors-either by cloning and sequencing the cDNA that encodes the precursor, or by reconstructing the arrangement of exons and introns in a neuropeptide-coding gene through genomic approaches. The databases generated from these molecular approaches have been used to design probes to identify the cells that express the gene, or to ascertain the rate of expression of the gene, and even to predict the post-translational modifications that can generate functional neuropeptides from a biologically inert precursor. Although the power of these approaches is substantial, it is appreciated that a gene sequence or an mRNA sequence reflects the potential products that may be assembled in a secretory cell. To understand the functional capabilities of the secretory cell, the molecular genetics approaches must be combined with procedures that actually characterize the end-products generated by the secretory cell. Recent advances in two-dimensional gel electrophoresis and mass spectrometry now make it possible to analyze neuropeptides from a relatively small amount of tissue. These procedures can reveal novel end-products, tissue-specific endoproteolytic cleavage events, and developmental shifts in post-translational processing schemes. A gene family that illustrates all of these processes and the advantages of combining genomics with proteomics is the opioid/orphanin gene family.

Amino Acid Sequence↗
PubMed1979

Evidence for endonucleolytic cleavage at the 5'-proximal segment of the trp messenger RNA in Escherichia coli.

The 5'-proximal trp leader RNA segment (about 5S) decays at 2 to 3 times slower rates than the distal trp mRNA sequence. This has been demonstrated by employing the deletion mutants which lack a large portion of the structural genes but retain the promoter-proximal region of the trp operon. Relative stability of the leader RNA is not merely due to the presence of an untranslatable region in the segment; the internal untranslatable segment of trp mRNA downstream from the nonsense alteration site of a double mutant trpAD28.trpE9758 decays as fast as the normal trp mRNA sequence. These results suggest that the trp mRNA is endonucleolytically cleaved to yield the small 5'-proximal leader RNA segment before the distal mRNA decays and that the leader RNA sequence is not subject to usual mode of mRNA decay in the 5' to 3' direction.

DNA, Viral↗
PubMed2001

Amitriptyline and clomipramine activate Gi-protein signaling pathway in the induction of analgesia.

The post-receptorial mechanisms of the analgesic action of amitriptyline and clomipramine, two tricyclic antidepressants, were investigated in the mouse hot plate test by using an antisense strategy. Mice were injected i.c.v. with antisense oligonucleotides (aODN), complementary to the sequence of the mRNA sequence of the alpha-subunit of Gi1, Gi2 and Gi3-proteins, 18-24 h prior to the hot plate test. Treatment with aODN against Gi1alpha, Gi2alpha and Gi3alpha dose-dependently reduced the analgesia induced by both amitriptyline (15 mg/kg s.c.) and clomipramine (25 mg/kg s.c.). This antagonistic effect disappeared 7 days after the end of the i.c.v. treatment, indicating the absence of irreversible damage or toxicity. Treatment with aODN against Gi1alpha, Gi2alpha and Gi3alpha, at the active doses, did not modify the animals' pain threshold, indicating the absence of any hyperalgesic effect. Amitriptyline, clomipramine and the aODN employed, at the maximal effective doses, did not produce any alteration of motor coordination of the mice, as revealed by rotarod experiments, and spontaneous motility, as revealed by the Animex apparatus. These results indicate that amitriptyline and clomipramine induce their analgesic effect by activating all three subtypes of the Gi-proteins.

Amitriptyline↗