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At least 1,513 records · Page 84Linked to original sources
PubMed1992

Identification of two critical base pairings in 5' untranslated regions affecting translation efficiency of synthetic uncapped globin mRNAs.

We observed a marked difference between the in vitro translation efficiency of two uncapped synthetic mRNAs, displaying the entire human alpha or beta globin mRNA sequences and some additional non-globin sequences in 5'. The comparison of the translation efficiencies of chimeric mRNAs indicated that the alpha 5' untranslated region (5' UTR) is responsible for a low translation efficiency that cannot be explained neither by primary sequence nor by the overall stability of 5' UTR secondary structures only. By point mutations in this alpha 5' UTR, we identified two base pairings at position -1 and -2 preceding the initiation codon which are associated with a negative effect on translation efficiency.

Base Composition↗
PubMed1991

Regulation of gene expression in parasitic flatworms.

Transcriptional regulation and the role of transcription factors are widely regarded to be the major contributors controlling gene expression in eucaryotes. Translational control is less well understood due to the complexity of the components involved in regulation of protein synthesis at this level. Nevertheless, considerable advances have been made recently in elucidating the major controlling factors within the messenger RNA (mRNA) sequence and the translation machinery. In this article, Ron Podesta and Afzal Siddiqui suggest that protein synthesis in flatworm parasites is controlled post-transcriptionally and that these intracellular regulatory mechanisms are activated/suppressed by effectors of the host's immune response.

Journal Article↗
PubMed1991

Detection of tyrosine hydroxylase mRNA and minimal neuroblastoma cells by the reverse transcription-polymerase chain reaction.

To facilitate the diagnosis of bone marrow metastasis in neuroblastoma, we have developed a method of amplifying and detecting the tyrosine hydroxylase (TH) mRNA sequence in bone marrow cells using a combination of reverse transcription and the polymerase chain reaction (RT/PCR). By this method, the sequence of TH was detected clearly in the neuroblastoma tissues of all 6 patients and not detected in the bone marrow cells of any of the 9 negative control children. In a reconstitution experiment, 1 neuroblastoma cell per 100,000 normal bone marrow cells could be detected, thus indicating the great sensitivity of this method. Based on these results, this technique may be of value in the diagnosis and treatment follow-up of bone marrow metastasis of neuroblastoma.

Adult↗
PubMed1989

Alternative splicing may be responsible for heterogeneity of thyroglobulin structure.

During the cloning of the bovine thyroglobulin cDNA, the restriction map of one of the recombinant plasmids was in disagreement with that of the full-length double-stranded thyroglobulin cDNA. When compared to the bovine Tg mRNA sequence, this cDNA clone exhibits a 333-nucleotide deletion which corresponds precisely to 2 exons of the Tg gene. It is thus likely that alternative processing of the premessenger RNA is at the origin of the deletion. The presence of giant introns in the vicinity of the dispensable exons may also reflect some error level in the splicing mechanism. Together with previous results the alternative splicing described in this study indicates that alternative processing of the Tg transcripts may be at the origin of thyroglobulin isoforms.

Base Sequence↗
PubMed1981

Effects of testosterone on protein synthesis in rat seminal vesicles analysed by two-dimensional gel electrophoresis.

Proteins synthesised by seminal vesicles of normal rats were labelled with [35S]methionine and were then separated using two-dimensional gel electrophoresis. Isoelectric focusing or nonequilibrium pH gradient electrophoresis were used in the first dimension followed by polyacrylamide gel electrophoresis under denaturing conditions. Using antiserum to total seminal vesicle secretion and running the immuno-precipitated proteins on two-dimensional gels, secretory proteins were identified and shown to be much more complex than previously thought. Proteins synthesised by seminal vesicles from rats castrated 1-2 weeks before were also labelled with [35S]methionine and separated on two-dimensional gels. Comparison of the two-dimensional protein maps from normal and castrated animals showed that a substantial number of proteins were differentially induced or repressed by testosterone. Of the secretory proteins, some were clearly regulated in a highly differential manner but others appeared to be unaffected by castration. The results are discussed in relation to previous measurements of mRNA sequence complexity and show that previous conclusions derived from using less sophisticated methods are oversimplifications of the response.

Animals↗
PubMed1994

Modulation of hepatocyte growth factor gene expression by estrogen in mouse ovary.

Hepatocyte growth factor (HGF) is expressed in a variety of tissues and cell types under normal conditions and in response to various stimuli such as tissue injury. In the present study, we demonstrate that the transcription of the HGF gene is stimulated by estrogen in mouse ovary. A single injection of 17 beta-estradiol results in a dramatic and transient elevation of the levels of mouse HGF mRNA. Sequence analysis has found that two putative estrogen responsive elements (ERE) reside at -872 in the 5'-flanking region and at +511 in the first intron, respectively, of the mouse HGF gene. To test whether these ERE elements are responsible for estrogen induction of HGF gene expression, chimeric plasmids containing variable regions of the 5'-flanking sequence of HGF gene and the coding region for chloramphenicol acetyltransferase (CAT) gene were transiently transfected into both human endometrial carcinoma RL 95-2 cells and mouse fibroblast NIH 3T3 cells to assess hormone responsiveness. Transfection results indicate that the ERE elements of the mouse HGF gene can confer estrogen action to either homologous or heterologous promoters. Nuclear protein extracts either from RL95-2 cells transfected with the estrogen receptor expression vector or from mouse liver bound in vitro to ERE elements specifically, as shown by band shift assay. Therefore, our results demonstrate that the HGF gene is transcriptionally regulated by estrogen in mouse ovary; and such regulation is mediated via a direct interaction of the estrogen receptor complex with cis-acting ERE elements identified in the mouse HGF gene.

3T3 Cells↗
PubMed1994

Functional coupling of the 5-HT2C serotonin receptor to G proteins in Xenopus oocytes.

The serotonin 2c (5-HT2C) receptor mediates its cellular effects by interacting with heterotrimeric guanine nucleotide binding proteins (G proteins). To characterize which G proteins are involved in functional coupling to the receptor, a mouse 5-HT2C receptor was expressed in Xenopus oocytes, and antisense oligoncleotides complementary to the mRNA sequence of the endogenous Xenopus G protein alpha subunits were used to inhibit G protein synthesis. Antisense oligonucleotide against the Xenopus G(o) alpha subunit inhibited the 5-HT2C receptor function, and coexpression of a rat G(o) alpha subunit reversed the inhibition by the anti-Xenopus G(o) oligonucleotide. Furthermore, antisense oligonucleotides against both the G(o) and Gi1 alpha subunits inhibited the electrophysiologic response induced by stimulation of the 5-HT2C receptor. These data suggest that both G(o) and Gi1 are involved in functional coupling of the 5-HT2C receptor to phospholipase C in Xenopus oocytes.

Animals↗
PubMed1993

Attenuation of microtubule-associated protein 1B expression by antisense oligodeoxynucleotides inhibits initiation of neurite outgrowth.

Microtubule-associated protein 1B, formerly also known as microtubule-associated protein 5, is the first structural microtubule accessory protein to appear in outgrowing axons. In PC12 pheochromocytoma cells microtubule-associated protein 1B levels increase several-fold after the addition of nerve growth factor and this increase is correlated with the initiation of process formation. To determine whether microtubule-associated protein 1B is essential for neurite outgrowth, we used antisense oligodeoxynucleotides to inhibit its expression in nerve growth factor-treated PC12 cells in the rat. The application of several different antisense oligodeoxynucleotides to the microtubule-associated protein 1B mRNA sequence inhibited both microtubule-associated protein 1B expression and neurite extension. Specificity was shown by the lack of effect of control sense oligonucleotides and by the lack of effect of the microtubule-associated protein 1B antisense oligodeoxynucleotides on the expression of either tubulin or microtubule-associated protein 3, another microtubule-associated protein whose synthesis is stimulated by nerve growth factor treatment of PC12 cells. After removal of the antisense oligodeoxynucleotides, microtubule-associated protein 1B expression recovered to normal levels and the cells grew normal neurites with the timing and morphological characteristics of normal nerve growth factor-induced outgrowth, indicating that the blockade was not because of non-specific toxic effects. These results indicate that microtubule-associated protein 1B is an essential component of the molecular mechanism underlying the formation of neuronal processes.

Animals↗
PubMed1984

Tandem arrangement of the albumin and alpha-fetoprotein genes in the human genome.

A genomic clone containing human albumin mRNA sequences was isolated from a lambda phage gene library derived from a human hepatoma cell line that produces alpha-fetoprotein (AFP) and albumin. This clone was also found to hybridize with the 5'-flanking region of the human AFP gene. Restriction mapping and nucleotide sequencing revealed that the albumin and AFP genes are present in tandem, in the same transcriptional orientation, with the albumin gene 14.5 kb upstream of the AFP gene. We have also isolated a genomic clone carrying both the albumin and AFP gene sequences from human fibroblasts, which produce neither AFP nor albumin. This DNA showed a restriction map that was indistinguishable from that of the clone obtained from the hepatoma described above, demonstrating that no gross rearrangements of the intergenic DNA sequence are involved in control of expression of the AFP and albumin genes.

Bacteriophage lambda↗
PubMed1990

The FUR1 gene of Saccharomyces cerevisiae: cloning, structure and expression of wild-type and mutant alleles.

The FUR1 gene of Saccharomyces cerevisiae encodes uracil phosphoribosyltransferase (UPRTase) which catalyses the conversion of uracil into uridine 5'-monophosphate (UMP) in the pyrimidine salvage pathway. The FUR1 gene is included in a 2.1 kb genomic segment of DNA and is transcribed into a 1 kb poly(A)+mRNA. Sequencing has determined a 753 bp open reading frame capable of encoding a protein of 251 amino acids. The FUR1 genes for three recessive fur1 alleles, having different sensibilities to 5-fluorouridine (5-FUR) but identical levels of resistance to 5-fluorouracil (5-FU), were cloned and sequenced. Single bp changes located in different regions of the gene were found in each mutant. Two in vitro-constructed deletions of the FUR1 gene have been integrated at the chromosomal locus, giving strains with 5-FURR and 5-FURR mutant phenotype. Assays of UPRTase, uridine kinase, uridine ribohydrolase and uridine 5'-monophosphate nucleotidase enzymatic activities, in extracts of strains where the FUR1 gene is overexpressed or deleted, indicate that the FUR1 encoded protein possesses only UPRTase activity.

Alleles↗
PubMed1990

Coupled reverse transcription-polymerase chain reaction (RT-PCR) as a sensitive and rapid method for isozyme genotyping.

We have developed a highly sensitive and rapid coupled reverse transcription-polymerase chain reaction (RT-PCR) technique for detection of alpha-amylase-encoding gene transcripts and for distinguishing between the human salivary (AMY1) and pancreatic (AMY2) gene transcripts. The two genes are 93-94% homologous. However, the AMY1 gene has an additional exon known as exon S, and an extra 32 bp in exon 1. Genotyping of the different AMYs by RT-PCR was based on this unique feature of the AMY1 mRNA sequence. Detection of AMY gene (AMY1 and AMY2) transcripts in cellular RNA was achieved with a set of primers common to both human AMY1 and AMY2 genes and derived from the exon 3-4 regions. In contrast, AMY1 gene transcripts were distinguished from the pancreatic AMY2 gene transcripts by use of primers specific to the exon S-1 regions of the AMY1 gene. To distinguish AMY1 transcripts from a mixture of AMY1 and AMY2, use was made of the differences in the ethidium bromide-stained agarose gel patterns obtained after digestion of the amplified exon 3-4 fragments with TaqI. AMY gene transcripts were detectable by autoradiography in RT-PCR amplified DNA obtained from as little as 5 pg of human pancreatic or parotid total RNA. A comparison of sensitivity of Northern blotting vs. RT-PCR suggested that the RT-PCR method is about 3-6 x 10(3)-fold more sensitive than Northern blotting in detecting AMY gene transcripts in human pancreatic total RNA.

Amylases↗
PubMed1990

Amylase synthesis as a simple model system for translation and hybrid arrest in Xenopus oocytes.

A human alpha-amylase-encoding cDNA has been cloned in a transcription vector. When messenger RNA (mRNA) made in vitro from this construct was injected into Xenopus oocytes, amylase (AMY) activity was detected both in oocyte homogenates and in the incubation medium, indicating that the oocyte machinery correctly translated and processed the protein. Because AMY activity is easy to detect with a blue-starch assay, this expression system was used to determine the parameters of antisense oligodeoxyribonucleotide (oligo) inhibition of translation in the oocytes. Unique oligos complementary to the AMY mRNA sequence were effective in arresting translation, at approximately stoichiometric levels. Mixed oligos also inhibited translation, at levels that suggest that some mismatches may be tolerated in the formation of DNA-RNA hybrids. The AMY system provides a convenient probe of oocyte protein synthesis and processing machinery and can serve as a control substrate in investigations of other mRNAs.

Amylases↗
PubMed1996

Identification of a maize endosperm-specific cDNA encoding farnesyl pyrophosphate synthetase.

Farnesyl pyrophosphate synthetase (FPS; EC 2.5.1.10) produces the 15-carbon farnesyl pyrophosphate which is utilized in the synthesis of sterols, carotenoids, dolichols, coenzyme Q, heme a and farnesylated proteins. We have cloned this mRNA sequence from a maize endosperm cDNA library and determined the 1378-nucleotide (nt) sequence of the DNA fragment. This sequence specifies an open reading frame of 1050 nt encoding FPS. The deduced amino acid sequence shows a high degree of similarity to FPS from a wide range of organisms. Southern blot analysis indicated that there are at least two FPS gene copies in the maize genome. The cloned FPS is expressed preferentially in maize endosperm and is up-regulated in the endosperm mutants, o2 and fl2.

Alkyl and Aryl Transferases↗
PubMed1992

Aberrant oestrogen receptor species in human meningioma tissue.

Meningiomas are very rich in progestin receptors (PR) whereas oestrogen receptors (ER) are seldomly found and only at low concentrations. These tumours might possess an ER which is defective in oestrogen binding but still functional in stimulating oestrogen-responsive genes such as PR. In human meningiomas a polymerase chain reaction fragment including the DNA binding domain, the hinge region and the ligand binding domain of ER was amplified. The size of the fragment obtained was as expected from wild type mRNA sequences. Moreover, a variant, which was overexpressed in meningiomas, with a major deletion in exons 2-6 was detected.

Base Sequence↗
PubMed1996

Endothelial-monocyte-activating polypeptide II.

Endothelial-monocyte-activating polypeptide II (EMAP II) was initially identified as a product of murine methylcholanthrene A-induced fibrosarcoma cells. The deduced mRNA sequence indicates that EMAP II is synthesised as a 34 kDa precursor molecule (proEMAP) and enzymatically cleaved to produce a biologically active 22 kDa mature polypeptide, which has been isolated and characterised. It modulates a range of properties of endothelial cells, monocytes, and neutrophils in vitro, and induces an acute inflammatory reaction and tumour regression in vivo. Recent evidence suggests that EMAP II can induce apoptosis in endothelial cells, and as such may act in an anti-angiogenic role. The question arises whether EMAP II is primarily a pro-inflammatory cytokine or a novel mediator of programmed cell death.

Animals↗
PubMed2003

The RING-H2 protein RNF11 is differentially expressed in breast tumours and interacts with HECT-type E3 ligases.

A breast cancer-associated mRNA originally cloned as a 475-bp partial cDNA from a library enriched for tumour cDNAs [Oncogene 16 (1998) 327] is expressed at high levels in breast and prostate cancer cells. Immunohistochemical analysis indicates that the protein is expressed in primary breast tumours. We used RT-PCR to generate a full-length 2852 nt mRNA sequence that includes the hypothetical open reading frame (ORF) for human RNF11. Our analysis shows that RNF11 encodes modular domains and motifs likely to interact with other proteins involved in oncogenesis. Chief among these are the RING-H2 finger domain that could facilitate the degradation of specific substrate(s) involved in oncogenesis and the PY motif which binds to WW-domain proteins, several of which are known to be E3 ubiquitin ligases. Our GST-pulldown and immunoprecipitation results indicate that RNF11 interacts with the E3 ligase AIP4 when coexpressed with RNF11 in mammalian cells.

Amino Acid Sequence↗
PubMed2003

Induction of anti-melanoma CTL response using DC transfected with mutated mRNA encoding full-length Melan-A/MART-1 antigen with an A27L amino acid substitution.

Modification of the parental immunodominant Melan-A/MART-1 peptide (MART-1(26-35)) by replacing the alanine with leucine (A27L) enhances its immunogenicity. Because of the reported advantages of RNA over peptides in DC vaccines, we sought to mutate the MART-1 gene to encode a full-length MART-1 antigen with an A27L amino acid substitution. Human DC were transfected with A27L-mutated MART-1 RNA (A27L RNA) or native MART-1 RNA, and then used to stimulate autologous T cells from a series of 8 HLA-A2+ volunteers. After three stimulations, all CTL induced with DC/A27L RNA exhibited more tetramer+ cells, and demonstrated stronger antigen-specific IFNgamma-secreting activity compared to CTL induced with DC/native RNA. A potent MART-1-specific, and predominantly class-I-restricted lysis was detected in most CTL induced with DC/A27L RNA, while native RNA-induced CTL showed minimal and non-specific lysis. HLA-A2+ DC and MART-1 negative/A2+ melanoma cells transfected with the A27L RNA were recognized and killed by MART-1-specific CTL, suggesting that these APC efficiently processed the A27L RNA and presented correct MART-1-specific epitope(s). In summary, introducing an A27L mutation into the MART-1 full-length mRNA sequence enhanced the immunogenicity of the encoded MART-1 Ag. The ease with which such a mutation can be made in RNA presents another potential advantage of using RNA for immunotherapy. Our results support considering this strategy for enhancing the immunogenicity of DC-based RNA vaccines.

Amino Acid Substitution↗
PubMed2004

Molecular therapy to inhibit NFkappaB activation by transcription factor decoy oligonucleotides.

Molecular therapy is emerging as a potential strategy for the treatment of various diseases for which few known effective therapies exist. One strategy for combating disease processes has been to target the transcriptional process. Two approaches have been used to accomplish this: the use of antisense complimentary to the mRNA of interest and the use of ribozymes, a unique class of RNA molecules that not only store information but also process catalytic activity. Ribozymes are known to catalytically cleave specific target RNA, leading to its degradation, whereas antisense molecules inhibit translation by binding to mRNA sequences on a stoichiometric basis. More recently, small interfering RNA has been shown to inhibit target gene expression. The application of oligonuclotide technology, such as antisense, to regulate the transcription of disease-related genes in vivo has important therapeutic potential. Transfection of cis-element double-stranded oligodeoxynucleotides has been reported as a powerful tool in a new class of anti-gene strategies for molecular therapy.

DNA↗