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At least 703 records · Page 39Linked to original sources
PubMed1993

Cloning of the 5' mRNA for the 230-kD bullous pemphigoid antigen by rapid amplification of cDNA ends.

The 230-kD bullous pemphigoid antigen (BPAG1), defined by autoantibodies in patient sera, is a hemidesmosomal plaque protein in the same gene family as the intracellular proteins desmoplakin I/II and plectin. We had previously isolated, from a lambda gt11 library, overlapping cDNA clones with 6921 bp of mRNA sequence for BPAG1. The coding sequence encoded by these clones included the 3' stop codon but not the 5' coding and non-coding region of the mRNA. To obtain these sequences we used the polymerase chain reaction (PCR) method called rapid amplification of cDNA ends (RACE). The PCR products were cloned into plasmids and sequenced. With five PCR primers we were able to obtain overlapping clones containing the 5' region of the mRNA. An upstream stop codon in frame with the rest of the coding sequence demonstrates that the full 5' coding sequence is obtained. Four different PCR products from two separate reactions had the same 5' end, suggesting that this 5' end is near, or at, the transcription start site. No alternatively spliced clones were found and no transmembrane site was predicted, confirming that BPAG1 is an intracellular hemidesmosomal plaque protein.

Amino Acid Sequence↗
PubMed1998

Translational inhibition of messenger RNA of the human pi class glutathione S-transferase by antisense oligodeoxyribonucleotides.

In this study, a T7 plasmid expression vector containing the cDNA of a variant human GST-pi gene, hGSTP1*C, was used to examine the translational inhibition of the GST-pi mRNA with antisense deoxyribonucleotides (AS-ONs), and to investigate the dependency of the inhibition on ribonuclease (RNAse) H, AS-ON and target mRNA sequence specificity and AS-ON back bone modification. Translational inhibition of hGSTP1*C mRNA showed significant AS-ON concentration-dependency and was both target mRNA and AS-ON sequence specific. Fully modified phosphoromonthioate AS-ONs were less inhibitory than their partial phosphoromonthioate analogs; unmodified AS-ONs were inactive. RNAse H enhanced the translational inhibition by AS-ON specific to the translation initiation region mRNA, and was associated with cleavage of the target mRNA at the site of AS-ON:mRNA hybridization. AS-ONs directed to the A-->G and C-->T transitions, unique to hGSTP1*C, were more RNAse H-dependent than AS-ONs directed against the translation initiation site, indicating a greater involvement of RNAse H-dependent mRNA cleavage in the mechanism of translational inhibition by AS-ON at the polymorphic site. These data suggest that AS-ONs provide a potentially effective means of specific down-regulation of the human GST-pi gene, and demonstrate that the sites of GST-pi gene allelo-polymorphism can be targeted to translationally down-regulate the different GST-pi gene variants, specifically and differentially targeted.

Base Sequence↗
PubMed2025

Identification of potential cell surface targets in patient-derived cultures toward photoimmunotherapy of high-grade serous ovarian cancer.

Tumor-targeted, activatable photoimmunotherapy (taPIT) has shown promise in preclinical models to selectively eliminate drug-resistant micrometastases that evade standard treatments. Moreover, taPIT has the potential to resensitize chemo-resistant tumor cells to chemotherapy, making it a complementary modality for treating recurrent high-grade serous ovarian cancer (HGSOC). However, the established implementation of taPIT relies on the overexpression of EGFR in tumor cells, which is not universally observed in HGSOCs. Motivated by the need to expand taPIT applications beyond EGFR, we conducted mRNA-sequencing and proteomics to identify alternative cell surface targets for taPIT in patient-derived HGSOC cell cultures with weak EGFR expression and lacking expression of other cell surface proteins commonly reported in the literature as overexpressed in ovarian cancers, such as FOLR1 and EpCAM. Our findings highlight TFRC and LRP1 as promising alternative targets. Notably, TFRC was overexpressed in 100% (N = 5) of the patient-derived HGSOC models tested, whereas only 60% of models had high EpCAM expression, suggesting that future larger cohort studies should include TFRC. While this study focuses on target identification, future work will expand the approaches developed here to larger HGSOC biopsy repositories and will also develop and evaluate antibody-photosensitizer conjugates targeting these proteins for taPIT applications.

Humans↗
PubMed1990

Complementary DNA cloning and regulation of expression of the messenger RNA encoding a pregnancy-associated porcine uterine protein related to human antileukoproteinase.

Antileukoproteinase (ALP) is a low mol wt mucosal secretory protein which, in human tissues, inhibits the activities of the neutral serine lysosomal proteinases elastase and cathepsin-G. In this study a number of recombinant cDNA clones corresponding to porcine ALP (pALP) were isolated from a cDNA library prepared from porcine endometrial poly(A)+ RNAs. The combined nucleotide sequences of the cDNA clones, representing the entire pALP mRNA sequence, are approximately 600 nucleotides long and encode a protein of 114 amino acids. The deduced amino acid sequence of pALP is 68% similar in primary structure to that of human ALP, is cysteine and proline rich, and exhibits a two-domain structure which, in the human protein, is involved in binding trypsin/cathepsin-G and elastase, respectively. However, pALP appears to lack the internal signal sequence of the corresponding human protein. Northern blot analysis of uterine RNAs using pALP cDNAs as probe demonstrated a single mRNA species approximately 0.8 kilobase in length. Uterine expression of pALP mRNA was highest in mid- and late pregnancy and very low or undetectable in early pregnancy. Estrogen and progesterone increased the levels of uterine pALP mRNA in prepubertal gilts, but not to the levels obtained at mid- and late gestation. pALP mRNA was also abundant in adult pig lung, where its expression was constitutive. Lower levels of pALP were found in fetal and neonatal lung and small intestine and in maternal cervix, spleen, and small intestine. Our study on the molecular cloning and analysis of pALP mRNA represents the first report on the porcine proteinase inhibitor and extends the identification of pregnancy-associated uterine proteins, which may play important functions in embryo or fetal development. The control of expression of pALP mRNA, which is distinct from those of other porcine uterine proteins studied to date, should provide additional insights into the mechanisms of regulation of uterine secretory activity.

Amino Acid Sequence↗
PubMed1986

A major internal initiation site for the in vitro translation of the adenovirus DNA polymerase.

An open reading frame which encodes at least 90% of the adenovirus type 2 DNA polymerase gene was cloned behind the SP6 promoter and transcribed in vitro using the SP6 RNA polymerase. The resultant RNA was translated in a rabbit reticulocyte cell free system. In addition to the translation of a 120-kDa protein corresponding to the size of the complete open reading frame, the synthesis of a 62-kDa polypeptide was demonstrated. Data is presented to show that the synthesis of the 62-kDa polypeptide resulted from internal initiation of translation in frame in the middle of the message at the 11th or 12th AUG. Capping of the mRNA resulted in an increase in synthesis of the 120-kDa protein and a concordant decrease of the internally initiated polypeptide. We propose that there may be competition between the binding of the translational preinitiation complex at or near the 5' end of the mRNA and at the internal initiation site. Because of inhibition of synthesis of the 120-kDa but not the 62-kDa polypeptide by hybrid arrested translation using DNA complementary to approximately one third of the 5' Ad Pol mRNA sequences, scanning of the ribosome from the 5' end of the mRNA to the internal initiation site seemed unlikely. The sequence proximal to the 12th AUG is ACCCACCCCAUG which is similar to a noncontinuous sequence 5'AUCCACC(X)nAUG complementary to the 3' end of the 18 S rRNA. This sequence is a favored ribosome binding site based on the observation that it is the most commonly observed one at or near the 5' end of 162 mRNA's analyzed (D. R. Sargan, S. P. Gregory, and P. H. W. Butterworth, 1982, FEBS Lett. 147, 133-136).

Adenoviruses, Human↗
PubMed1977

Changes in the sequence diversity of polyadenylated cytoplasmic RNA during testis differentiation in rainbow trout (Salmo gairdnerii).

We have compared the sequence diversity of polyadenylated cytoplasmic RNA derived from naturally maturing trout testis at three different stages of spermatogenesis, by performing hybridization experiments between cDNA synthesized on a template of polyadenylated RNA and a vast excess of polyadenylated RNA. Polyadenylated RNA from early testis has a base sequence complexity of 7.2x10(9) daltons. As testis maturation proceeds, there is a decrease in the complexity of polyadenylated mRNA sequences in testis cells and the relative abundance of individual mRNAs varies over a more narrow range. Heterologous hybridization reactions demonstrate that a substantial fraction of polyadenylated RNA sequences present in early testis cytoplasm is absent from late testis cytoplasm. Nevertheless, all the sequences of late testis mRNA are represented in the population of early testis mRNA molecules.

Aging↗
PubMed1981

Nucleotide sequences of the mRNA's encoding the vesicular stomatitis virus N and NS proteins.

The complete nucleotide sequences of the vesicular stomatitis virus (VSV) mRNA's encoding the N and NS proteins have been determined from the sequences of cDNA clones. The mRNA encoding the N protein is 1,326 nucleotides long, excluding polyadenylic acid. It contains an open reading frame for translation which extends from the 5'-proximal AUG codon to encode a protein of 422 amino acids. The N and mRNA is known to contain a major ribosome binding site at the 5'-proximal AUG codon and two other minor ribosome binding sites. These secondary sites have been located unambiguously at the second and third AUG codons in the N mRNA sequence. Translational initiation at these sites, if it in fact occurs, would result in synthesis of two small proteins in a second reading frame. The VSV and mrna encoding the NS protein is 815 nucleotides long, excluding polyadenylic acid, and encodes a protein of 222 amino acids. The predicted molecular weight of the NS protein (25,110) is approximately one-half of that predicted from the mobility of NS protein on sodium dodecyl sulfate-polyacrylamide gels. Deficiency of sodium dodecyl sulfate binding to a large negatively charged domain in the NS protein could explain this anomalous electrophoretic mobility.

Amino Acid Sequence↗
PubMed2002

Identification of the alternative splice products encoded by the human protein phosphatase inhibitor-1 gene.

We have isolated three major cDNA fragments of protein phosphatase inhibitor-1 from human brain and liver by RT-PCR. The 536 bp fragment encoded the wild-type of inhibitor-1 while two other fragments were alternative splice products of the inhibitor-1 gene, which was confirmed by partial genomic DNA sequencing. The 380 bp fragment encoded an in-frame 51-residue-deleted inhibitor-1, named inhibitor-1alpha, and the deletion occurred from residue 84 to 134 of inhibitor-1. The 316 bp fragment termed inhibitor-1beta was derived from an internal deletion of 536 bp fragment. This deletion resulted in an out of frame shift, allowing the 316 bp fragment that encoded the partial sequence of inhibitor-1. Based on the reported mRNA sequence of inhibitor-1 and evidence from our RT-PCR, we suggested that inhibitor-1beta consisted of 132 amino acids of which the N-terminal 61 amino acid sequences were identical to inhibitor-1 while the sequence after residue-61 was markedly different.

Alternative Splicing↗
PubMed1987

Regulation of cardiac myosin synthesis: studies of RNA content in cultured heart cells.

Contraction regulates the myosin content and the rate of myosin synthesis in cultured neonatal rat heart cells. To further explore the mechanism for this regulation we examined various parameters of RNA content and RNA synthesis in contacting versus noncontracting myocytes. While contraction stimulated myosin heavy chain (MHC) synthesis by 72% compared to that of KCl-arrested cells, simultaneous analyses of polysome profiles were no different under the two culture conditions. Incorporation of [3H]uridine monophosphate into cellular RNA revealed no change in the rate of total RNA or ribosomal subunits synthesis. In vitro translation of cellular RNA yielded similar incorporation of [35S]methionine into trichloroacetic acid precipitable protein. Specific transcription of the MHC gene was examined by dot-blot analysis and was unaltered by contraction. Northern blot analysis of the MHC sequences detected by a cDNA probe revealed an mRNA sequence corresponding to a molecular weight of approximately 30 S. These data suggest that RNA synthesis and RNA content are unaltered by contraction in cultured heart cells and therefore the changes in myosin synthesis may be mediated at a post-transcriptional control level.

Animals↗
PubMed1974

Chemical synthesis of a primer and its use in the sequence analysis of the lysozyme gene of bacteriophage T4.

We have developed a general approach for determining the nucleotide sequence of a gene, with the aid of a deoxyribonucleotide primer of defined sequence. The selection of the primer sequence was based on a short segment of mRNA sequence of T4 phage lysozyme. A tetradecadeoxyribonucleotide primer was chemically synthesized and its sequence verified by sequence analysis. This primer was found to bind to the single-stranded region of the exonuclease III-treated T4 DNA, and specific nucleotides were incorporated to its 3' end. The result indicated that this primer was bound to the expected location on the T4 DNA. Therefore, long sequences of the T4 lysozyme gene can now be determined from this specific starting point.

Base Sequence↗
PubMed1986

Proenkephalin mRNA in rat heart.

The distribution of preproenkephalin mRNA in rat tissues was investigated using a homologous cDNA probe for detection. The heart was found to contain larger amounts of the mRNA than any other tissue including brain, which heretofore had been considered the richest source. The identity of the message in heart was verified by hybridizing RNA blots with a synthetic oligodeoxynucleotide that recognizes a different region of the preproenkephalin mRNA sequence than does the cDNA probe. The preproenkephalin mRNA extracted from both heart and brain contained approximately equal to 1500 bases. Dissection of heart revealed that essentially all of the message is contained within the ventricles. In contrast to the large amounts of preproenkephalin mRNA in rat heart, the opioid peptide contents is only 3% of the amount in brain. The rat heart may be a useful model for the investigation of translation control of protein synthesis.

Animals↗
PubMed1984

Preparation and analysis of the nucleocapsid proteins of vesicular stomatitis virus and sendai virus, and analysis of the sendai virus leader-NP gene region.

A procedure is presented for isolating the nucleocapsid proteins, N and NP from vesicular stomatitis virus and Sendai virus respectively, in soluble form. These proteins were suitable for the determination of their blocked amino-terminal peptide sequences by gas-liquid chromatography/mass spectrometry at the low nanomole level. The N protein prepared by this procedure was previously shown to retain some of its expected biological activity. The sequence of 626 nucleotides from the 3' end of the Sendai virus genome, which includes the first one-third of the NP gene, was determined. Using this information, primer extension studies on intracellular Sendai virus mRNAs allowed the determination of the structure of the leader-NP intervening sequence and the 5' end of the NP mRNA. Comparison of the amino termini of the nucleocapsid proteins with their respective mRNA sequences revealed that these proteins are similarly processed in vivo.

Amino Acid Sequence↗
PubMed1995

Inhibition by modified oligodeoxynucleotides of the expression of type-1 plasminogen activator inhibitor in human endothelial cells.

Antisense phosphodiester and phosphorothioate oligodeoxynucleotides (23-residue or 24-residue oligodeoxynucleotides) were constructed for sequences of type-1 plasminogen-activator-inhibitor mRNA to assess their capability to modulate type-1 plasminogen-activator-inhibitor-mediated fibrinolysis. Antisense oligodeoxynucleotides were targeted at the mRNA sequence coding a signal peptide, at a part of the reactive center Ile342-Pro349, and at an internally translated segment Asn265-Leu272. The effect of antisense oligonucleotides on the concentration of type-1 plasminogen activator inhibitor in conditioned media and human endothelial cells was determined by the activity test with fibrin as a substrate, and by immunoprecipitation after metabolic labelling of cells with [35S]methionine. Three phosphorothioate oligodeoxynucleotides were specifically inhibitory while phosphodiester oligodeoxynucleotides with the same sequence did not show any activity. Phosphorothioate oligodeoxynucleotides 2, 4 and 6 inhibited the synthesis of type-1 plasminogen activator inhibitor in endothelial cells in a time-dependent and concentration-dependent manner. These data suggest that antisense oligodeoxynucleotides may be useful in the design of antithrombolytic therapeutics.

Base Sequence↗
PubMed1985

Family of developmentally regulated, maternally expressed Drosophila RNA species detected by a v-myc probe.

Drosophila melanogaster genomic sequences that hybridize with v-myc have been reported (B.-Z. Shilo and R. A. Weinberg, Proc. Natl. Acad. Sci. U.S.A. 78:6789-6792, 1981). We have detected Drosophila RNA sequences that also hybridize with v-myc. In an attempt to characterize these RNA sequences, we used v-myc hybridization probes to isolate Drosophila genomic segments. None of the Drosophila genomic or cDNA clones that we have isolated hybridize with the 3' exon of v-myc. Preliminary nucleotide sequence analyses have revealed sufficient homology to account for the observed hybridization between v-myc and the Drosophila clones but have failed to detect significant amino acid sequence homology. Thus is seems unlikely that the mRNA sequences or the genomic sequences that we have isolated by hybridization with v-myc represent homologs of the vertebrate myc gene. Despite the lack of structural homology between the cloned Drosophila sequences and v-myc, we have investigated the pattern of expression of those RNA species that hybridize with v-myc. Polyadenylic acid-containing transcripts of 2.7, 2.2, and 1.7 kilobases (kb) in embryos, pupae, adults, and Kc cells and an additional 1.4-kb transcript in adults were complementary to the Drosophila genomic clones and to v-myc. The 1.7- and 2.2-kb transcripts were localized on polyribosomes in Kc cells. The 1.7- and 2.2-kb transcripts were present after 45 min, 2 h, and 4 h of embryonic development, but by 16 h of development their levels had decreased by more than sixfold. During metamorphosis, two peaks of expression of the 1.7- and 2.2-kb transcripts were observed, at 6 and 72 h postpupariation. The 1.4-kb RNA species was first detected at 72 h postpupariation. In adults, the 1.7- and 2.2-kb transcripts were detected only in ovaries in females, whereas the 1.4-kb transcript was present in female nonovarian RNA and in males. These results suggest that the transcripts in early embryos are of maternal origin.

Age Factors↗
PubMed2002

Translation initiation in cancer: a novel target for therapy.

Translation initiation is regulated in response to nutrient availability and mitogenic stimulation and is coupled with cell cycle progression and cell growth. Several alterations in translational control occur in cancer. Variant mRNA sequences can alter the translational efficiency of individual mRNA molecules, which in turn play a role in cancer biology. Changes in the expression or availability of components of the translational machinery and in the activation of translation through signal transduction pathways can lead to more global changes, such as an increase in the overall rate of protein synthesis and translational activation of the mRNA molecules involved in cell growth and proliferation. We review the basic principles of translational control, the alterations encountered in cancer, and selected therapies targeting translation initiation to help elucidate new therapeutic avenues.

Animals↗
PubMed1989

Structure of the canine pancreatic lipase gene.

Identification of three overlapping clones in a canine genomic lambda phage library allowed us to determine a detailed restriction enzyme map of the primary transcriptional unit of the pancreatic lipase gene (15.5 kilobase pairs) as well as 15 and 6 kilobase pairs of 5'- and 3'-flanking regions, respectively. DNA sequence analysis provided the primary structure of (a) 1,345 nucleotides (nt) of 5'-flanking sequence including CAAT and TATA boxes at positions -112 and -35, respectively, and a class 2 glucocorticoid receptor binding sequence at position -97, (b) 13,127 out of approximately 15,500 nt of the transcriptional unit which is organized into 13 exon sequences, and (c) 1,270 nt of 3'-flanking sequence. Exon 1 encodes the entire 5'-nontranslated mRNA sequence; exon 2, the ATG initiation codon and the hydrophobic portion of the signal peptide; and exon 6, Ser154 which shows homology to the active Ser152 in the porcine enzyme. Comparison of the amino acid sequences of human lipoprotein lipase, rat hepatic lipase, and Drosophila yolk proteins 1, 2, and 3 with canine pancreatic lipase shows that the central region of highest homology (encoded by exons 6-8 in the dog gene) contains four highly conserved subregions which may play a critical role in enzyme-substrate and protein-ligand binding for lipases and yolk proteins, respectively. Comparison of the sequences of 10 lipases from prokaryotes and eukaryotes identifies a 9-residue consensus sequence surrounding the active serine which includes the previously identified sequence Gly-X-Ser-X-Gly. The hydrophobic nature of this sequence in the 10 lipases contrasts with the hydrophilic nature of the corresponding sequences in serine proteases and thus defines an active site serine consensus sequence specific for lipases. An analysis of 5'- and 3'-flanking and intron 1-4 sequences in transient expression studies with AR4-2J and 266-6 cells was unable to reveal tissue-specific promoter or enhancer sequences.

Amino Acid Sequence↗
PubMed2006

Cloning of cyclooxygenase-1b (putative COX-3) in mouse.

OBJECTIVES: To clone and sequence cyclooxygenase-1b (COX-1b, also known as COX-3) mRNA and to generate an antibody against the mouse COX-1b protein and to demonstrate its existence in vivo in mouse tissues. ANIMALS: 10 C57BL/6 mice, 4 COX-1 knockout mice and 4 COX-1 wild type mice were used. METHODS: COX-1b mRNA sequence was determined by RT-PCR amplification using specific primers followed by DNA sequencing. COX-1b protein expression was determined by Western blotting. RESULTS: The mouse COX-1b mRNA is a splice variant of the COX-1 mRNA generated by the retention of intron-1. COX-1b mRNA encodes a 127 amino acid protein with no similarity with known COX sequences. We generated an anti-mouse COX-1b antibody and demonstrated the existence of COX-1b protein in vivo with the highest expression in kidney, heart, and neuronal tissues. We also detected COX-1b mRNA and protein expression in COX-1 knockout mice. CONCLUSIONS: In mouse, COX-1b encodes a protein with a completely different amino acid sequence than COX-1 or COX-2; therefore it is improbable that COX-1b in this species plays a role in prostaglandin-mediated fever and pain. In addition, the COX-1(-/-) mouse is not a COX-1b(-/-) mouse, therefore it cannot be used to elucidate the function of the COX-1b protein.

Alternative Splicing↗
PubMed1979

A nuclear precursor to human gamma-globin messenger RNA.

RNA isolated from human foetal liver at an erythroid stage of development was transferred from methyl mercury hydroxide agarose gels to diazobenzyloxymethyl paper. A 16S RNA species containing gamma-globin mRNA sequences was identified by hybridisation to 32P-labelled human gamma-globin cDNA recombinant plasmid pHgamma GI. This RNA is found only in the cell nucleus and is polyadenylated. Human globin gene transcription is thus analagous to that in mouse: interventing sequences are transcribed and processed from the pre-mRNA in the nucleus after polyadenylation.

Adult↗