Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “downstream ORF”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 793 records · Page 44Linked to original sources
PubMed2004

Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells.

During cellular stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) elicits gene expression designed to ameliorate the underlying cellular disturbance. Central to this stress response is the transcriptional regulator activating transcription factor, ATF4. Here we describe the mechanism regulating ATF4 expression involving the differential contribution of two upstream ORFs (uORFs) in the 5' leader of the mouse ATF4 mRNA. The 5' proximal uORF1 is a positive-acting element that facilitates ribosome scanning and reinitiation at downstream coding regions in the ATF4 mRNA. When eIF2-GTP is abundant in nonstressed cells, ribosomes scanning downstream of uORF1 reinitiate at the next coding region, uORF2, an inhibitory element that blocks ATF4 expression. During stress conditions, phosphorylation of eIF2 and the accompanying reduction in the levels of eIF2-GTP increase the time required for the scanning ribosomes to become competent to reinitiate translation. This delayed reinitiation allows for ribosomes to scan through the inhibitory uORF2 and instead reinitiate at the ATF4-coding region. Increased expression of ATF4 would contribute to the expression of genes involved in remediation of cellular stress damage. These results suggest that the mechanism of translation reinitiation involving uORFs is conserved from yeast to mammals.

Activating Transcription Factor 4↗
PubMed2002

Expression of a unique helios isoform in human leukemia cells.

The purpose of the present study was to characterize the human Helios gene products expressed in leukemia cells. A 3.5 kb human Helios cDNA clone was isolated from a human T-cell cDNA library derived from the human T-acute lymphoblastic leukemia (ALL) cell line JURKAT. This cDNA clone had a unique open reading frame (ORF) encoding a novel 304 amino acid (AA) peptide, which was designated as Helios 3. The sequence of the 289 AA C-terminal portion of Helios 3 downstream of V-16 is identical to the corresponding sequence found in Helios 1 and 2 and contains two zinc fingers. By contrast, the 15 AA N-terminal portion of Helios 3 is unique and does not contain the N-terminal zinc finger motifs that are conserved in Helios 1 and 2 as well as other previously identified members of the Ikaros family. Southern blot analysis of genomic DNA fragments of the human Helios gene locus showed that Helios 3 is encoded by the same genomic locus as Helios 1 and 2. The expression of Helios 3 mRNA was not restricted to T-lineage ALL cells or another immunophenotypically distinct subset of leukemias. Helios 3 mRNA was expressed in freshly obtained primary leukemic cells from six of 15 children with newly diagnosed ALL. Helios 3 exhibited a unique protein interaction profile via its N-terminal portion, which may have biological significance in pathogenesis of human leukemias.

Adolescent↗
PubMed1993

Identification of a novel gene, dep, associated with depolymerization of the capsular polymer in Bacillus anthracis.

Bacillus anthracis produces a gamma-linked poly-D-glutamic acid capsule that is essential for virulence. A 6.2 kb fragment of B. anthracis DNA (cap), when present in Escherichia coli, produces a capsular polymer that is immunologically identical to that produced by B. anthracis. By immunodiffusion analysis of E. coli strains carrying varying portions of the cap region, we identified a novel gene (dep) responsible for degradation of the capsular polymer of B. anthracis. The simultaneous presence of the cap region and the dep gene caused production of low-molecular-weight, degraded capsular polymer both in E. coli and in B. anthracis, whereas the cap region alone caused production of a high-molecular-weight capsule. The dep gene mapped immediately downstream of the cap region within a 1.8 kb fragment and was transcribed in the same direction. This fragment was sequenced and a 1401 bp open reading frame (ORF) was found that is predicted to encode a peptide with molecular weight of 51,460. By in vitro transcription-translation analysis, this ORF was shown to be the dep gene product. The deduced amino acid sequence of the dep product has sequence similarity to E. coli and mammalian gamma-glutamyltranspeptidase (GGT). However, the Dep protein did not have GGT activity. The Dep protein appears to be an enzyme that catalyses the hydrolysis of the poly-D-glutamic acid capsule. Although the biological functions of the dep gene are unknown, it is possible that low-molecular-weight, diffusible polyglutamates produced through the action of the dep gene may act to inhibit host defence mechanisms.

Amino Acid Sequence↗
PubMed1999

Cloning, sequence analyses, expression, and distribution of ampC-ampR from Morganella morganii clinical isolates.

Shotgun cloning experiments with restriction enzyme-digested genomic DNA from Morganella morganii 1, which expresses high levels of cephalosporinase, into the pBKCMV cloning vector gave a recombinant plasmid, pPON-1, which encoded four entire genes: ampC, ampR, an hybF family gene, and orf-1 of unknown function. The deduced AmpC beta-lactamase of pI 7.6 shared structural and functional homologies with AmpC from Citrobacter freundii, Escherichia coli, Yersinia enterocolitica, Enterobacter cloacae, and Serratia marcescens. The overlapping promoter organization of ampC and ampR, although much shorter in M. morganii than in the other enterobacterial species, suggested similar AmpR regulatory properties. The MICs of beta-lactams for E. coli MC4100 (ampC mutant) harboring recombinant plasmid pACYC184 containing either ampC and ampR (pAC-1) or ampC (pAC-2) and induction experiments showed that the ampC gene of M. morganii 1 was repressed in the presence of ampR and was activated when a beta-lactam inducer was added. Moreover, transformation of M. morganii 1 or of E. coli JRG582 (delta ampDE) harboring ampC and ampR with a recombinant plasmid containing ampD from E. cloacae resulted in a decrease in the beta-lactam MICs and an inducible phenotype for M. morganii 1, thus underlining the role of an AmpD-like protein in the regulation of the M. morganii cephalosporinase. Fifteen other M. morganii clinical isolates with phenotypes of either low-level inducible cephalosporinase expression or high-level constitutive cephalosporinase expression harbored the same ampC-ampR organization, with the hybF and orf-1 genes surrounding them; the organization of these genes thus differed from those of ampC-ampR genes in C. freundii and E. cloacae, which are located downstream from the fumarate operon. Finally, an identical AmpC beta-lactamase (DHA-1) was recently identified as being plasmid encoded in Salmonella enteritidis, and this is confirmatory evidence of a chromosomal origin of the plasmid-mediated cephalosporinases.

Amino Acid Sequence↗
PubMed2026

A novel peptide encoded by circTLL1 drives osimertinib resistance in lung cancer by modulating the NT5C2/Ras/PI3K axis.

BACKGROUND: Acquired resistance to osimertinib, a third-generation EGFR tyrosine kinase inhibitor, remains a major clinical challenge in the treatment of non-small cell lung cancer (NSCLC). Although circular RNAs (circRNAs) have been increasingly implicated in drug resistance, most studies have focused on their canonical role as microRNA sponges, while their capacity to encode functional micropeptides remains largely unexplored. This study aimed to identify novel circRNAs involved in osimertinib resistance and to characterize their regulatory functions at the protein level. METHODS: Osimertinib-resistant (OR) NSCLC cell lines were established and validated. High-throughput RNA sequencing was performed to compare the circRNA expression profiles between parental and OR cells. The function of the candidate circRNA was assessed through a series of in vitro and in vivo experiments, including cell viability assays, apoptosis analysis, and xenograft mouse models. Mechanistic investigations involved mass spectrometry, co-immunoprecipitation and western blotting to explore its protein-coding potential and downstream signaling pathways. RESULTS: We identified a novel circRNA, termed circTLL1, that was stably and significantly upregulated in OR-NSCLC cells. Functionally, overexpression of circTLL1 promoted osimertinib resistance, whereas its knockdown restored drug sensitivity both in vitro and in vivo. Mechanistically, we discovered that circTLL1 harbors an open reading frame (ORF) that is translated into a novel 90-amino-acid protein, which we designated circTLL1-90aa. Further investigation revealed that circTLL1-90aa directly interacts with and promotes the degradation of 5'-nucleotidase, cytosolic II (NT5C2), thereby uncoupling nucleotide metabolism from its normal regulatory constraints. The consequent downregulation of NT5C2 leads to elevated GTP levels and leading to the sustained activation of the downstream Ras/PI3K/AKT signaling pathway. CONCLUSION: Our findings unveil a previously unrecognized circRNA/micropeptide/metabolism cascade underlying osimertinib resistance. The identification of the circTLL1-90aa/NT5C2/Ras/PI3K axis not only expands the functional repertoire of the non-coding genome but also provides new insights into the complexity of drug resistance. Given its selective upregulation in resistant cells, circTLL1-90aa holds promise both as a predictive biomarker for treatment stratification and as an actionable therapeutic target, offering a novel strategy to overcome osimertinib resistance in NSCLC patients.

Pyrimidines↗
PubMed1994

Genome characterization and identification of viral-associated dsDNA component of banana bunchy top virus.

The single-stranded (ss) and double-stranded (ds) viral-associated DNAs (VADs) of banana bunchy top virus (BBTV) were characterized. The ssVADs were shown to be the viral genomic DNA and the dsVADs are the likely replication forms. Both dsVADs and ssVADs contain DNA species that cross-hybridize to each other. Under the electron microscope, both circular and linear DNAs were observed from these VAD species. Libraries were constructed from VADs to further characterize them. Two overlapping VAD clones were isolated and their combined sequence indicated that their corresponding VAD, named as BBTV component 2, is circular with a size of 1095 nt. The largest ORF spans from nucleotides 82 to 390, which is sandwiched by a putative TATA box, 54 nt upstream from the first ATG codon, and a AATAAA motif, 7 nt downstream from the UAA codon. Although the sequence of this VAD is different from the previously reported BBTV component 1 (Harding et al., J. Gen. Virol. 74, 323-328, 1993) and the genome of coconut foliar decay virus (Rohde et al., Virology 176, 648-651, 1990), stretches of highly homologous sequences were found between BBTV component 2 and the other two viral genomes by computer analysis. The sequence of BBTV component 1 is highly homologous to one of the clones, pBTs26, in the VAD library. Nevertheless, our libraries still contain many clones which hybridized with ss viral genomic DNAs but not with pBTs26 and BBTV component 2, indicating that the viral genome consists of more than two components.

Base Sequence↗
PubMed1993

A beta-ketoacyl-acyl carrier protein synthase III gene (fabH) is encoded on the chloroplast genome of the red alga Porphyra umbilicalis.

DNA sequencing of a region of the chloroplast genome of the red alga Porphyra umbilicalis revealed an open reading frame of 326 amino acids. Databank searches indicated that this ORF is 34% identical to an E. coli gene (fabH) encoding beta-ketoacyl-carrier protein synthase III. In addition, a leucine tRNA gene (trnL(GAG)) was detected just downstream. Neither of these genes are encoded on the chloroplast genomes of land plants.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed1990

Expression, nucleotide sequence and mutational analysis of two open reading frames in the nif gene region of Anabaena sp. strain PCC7120.

A 1.8 kb transcript corresponding to a region of the Anabaena 7120 chromosome 4 kb downstream of the nifHDK operon appears 12-18 h after heterocyst induction. The DNA corresponding to this transcript was sequenced and found to contain two open reading frames, designated ORF 1 and ORF 2. Two polypeptides, of 30 kDa and 13 kDa, encoded by these ORFs were expressed in Escherichia coli. An apparent start site for the transcript, detected by S1 nuclease protection, was located 42 bp upstream of the ATG start codon of ORF 1. ORF 2 shows strong sequence similarity to ORF 6 in the nif gene region of Azotobacter vinelandii. ORF 1 was interrupted using a 1.4 kb neomycin resistance cassette and the resulting mutant grew very slowly on medium lacking combined nitrogen. The mutant had 45% of wild-type acetylene reduction activity, which could be complemented by a 2.8 kb EcoRI fragment of wild-type Anabaena DNA containing only ORF 1 and ORF 2. Thus, one or both of these ORFs is required for efficient nitrogen fixation in Anabaena.

Amino Acid Sequence↗
PubMed1994

Sequence and characterization of three genes within the hydrogenase gene cluster of Bradyrhizobium japonicum.

A 2.0-kb DNA fragment downstream from the hydrogenase-encoding structural genes within the hydrogenase gene cluster of Bradyrhizobium japonicum was sequenced. Analysis of the nucleotide (nt) sequence revealed three open reading frames (ORFs), designated hupC, hupD and hupF, which encode polypeptides of 28, 21 and 10.7 kDa, respectively. Based on analysis of the nt sequence and physiological studies, hupSL (hydrogenase structural genes) and hupCDF are organized as a single transcriptional unit. Plasmid pRY12 carrying hupSL genes did not complement (restore) hydrogenase activity of the hupSL deletion mutant strain (JHCS2), whereas the activity of the mutant was considerably restored by pLD22 harboring the entire hydrogenase operon (hupSLCDF genes). Western blots revealed a very low level of hydrogenase protein in JHCS2 containing pRY12. The results suggest that the products of the hupCDF genes may be involved in either stabilizing the hydrogenase peptides (i.e., from degradation) or in post-translational regulation of hydrogenase production. The products of hupC and hupD were successfully expressed in Escherichia coli by a phage T7 promoter system, although the apparent sizes of the gene products were slightly larger than those calculated from the deduced amino-acid sequences.

Amino Acid Sequence↗
PubMed1995

Cloning and sequencing of the Bordetella pertussis cpn10/cpn60 (groESL) homolog.

The nucleotide sequence downstream from the Bordetella resistance to killing (brk) locus in Bordetella pertussis was determined. Analysis of the sequence revealed an operon consisting of two highly predicted open reading frames (ORFs). The deduced amino-acid sequence of each ORF has strong homologies to the heat-shock proteins/chaperonins Cpn60 and Cpn10. The promoter contains consensus sequences for both sigma 32 and sigma 70 binding, and it possesses the CIRCE regulatory inverted repeat. A potential Rho-independent terminator was identified and appears to be shared with the brkA gene.

Amino Acid Sequence↗
PubMed1996

Accumulation of the angucycline antibiotic rabelomycin after disruption of an oxygenase gene in the jadomycin B biosynthetic gene cluster of Streptomyces venezuelae.

DNA from a region downstream of and overlapping the polyketide synthase (PKS) gene cluster for jadomycin B biosynthesis in Streptomyces venezuelae was cloned and sequenced. Analysis of the nucleotide sequence located one complete ORF (ORF6), an incomplete one representing the 3' region of ORF4 in the PKS cluster, and a second incomplete one (ORF7). The deduced amino acid sequences for ORFs 6 and 7 resemble those of oxygenases. Since a plausible biosynthetic pathway for jadomycin B includes an angular polyketide intermediate that undergoes oxidative ring fission before condensation with an amino acid, we subcloned one of the presumptive oxygenase genes (ORF6) in a segregationally unstable shuttle vector (pHJL400) and disrupted it by inserting the gene for apramycin resistance. Transformation of S. venezuelae with the disruption vector and selection for apramycin resistance gave mutants blocked in jadomycin biosynthesis. Southern hybridization confirmed that gene replacement had occurred. Cultures of the mutants accumulated a metabolite identified by comparison with an authentic sample as rabelomycin, a non-nitrogenous polyketide-derived antibiotic originally isolated from Streptomyces olivaceus.

Amino Acid Sequence↗
PubMed1992

Analysis of bph operon from the polychlorinated biphenyl-degrading strain of Pseudomonas pseudoalcaligenes KF707.

The entire nucleotide sequences (6.8 kilobase pairs) of the bphABC genes and their products involved in the initial dioxygenation and ring-meta-cleavage of biphenyls and polychlorinated biphenyls were determined. The first bphA gene starts about a 100 base pairs downstream from the transcriptional initiation site. The bphA region, which encodes a cluster of enzymes including biphenyl dioxygenase catalyzing the initial catabolic step, consists of five open reading frames (ORFs). Five proteins corresponding to these ORFs in the molecular masses were detected by in vitro protein synthesis, of which four ORFs are very similar to the recently reported todC1C2BA genes coding for the corresponding enzymes catalyzing the initial dioxygenation reactions of toluene (Zylstra, G.J., and Gibson, D. T. (1989) J. Biol. Chem. 264, 14940-14946). The third open reading frame (ORF3) of the bphA region, missing its counterpart in the toluene dioxygenase gene cluster, was site-specifically deleted, and the resulting enzymatically active mutant reveals that this ORF3 is not mandatory for the catabolism of biphenyls. Thus the biphenyl degradation pathway and the responsible enzymes/genes are very similar to those of toluene degradation despite their discrete substrate specificity.

Amino Acid Sequence↗
PubMed1989

Two group I introns with long internal open reading frames in the chloroplast psbA gene of Chlamydomonas moewusii.

We report the nucleotide sequence of the chloroplast psbA gene encoding the 32 kilodalton protein of photosystem II from Chlamydomonas moewusii. Like its land plant homologues, this green algal protein consists of 353 amino acids. The C. moewusii psbA gene is composed of three exons containing 252, 11 and 90 codons and of two group I introns containing 2363 and 1807 nucleotides. Each of the introns features an internal open reading frame (ORF) that potentially encodes a basic protein of more than 300 residues. The primary sequences of the putative intron-encoded proteins are unrelated and none of them shares conserved elements with any of the proteins predicted from the group I intron sequences published so far. The first C. moewusii intron is inserted at the same position as the fourth intron of the psbA gene from Chlamydomonas reinhardtii; the second intron lies at a novel site downstream of this position. On the basis of their RNA secondary structures, the C. moewusii introns 1 and 2 can be assigned to subgroups IA and IB, respectively. However, intron 1 is not typical of subgroup IA introns, its most unusual feature being the location of the ORF in the "loop L5" region. To our knowledge, this is the first time that an ORF is located in this region of the group I intron structure.

Amino Acid Sequence↗
PubMed2002

A mammalian homolog of unc-53 is regulated by all-trans retinoic acid in neuroblastoma cells and embryos.

The vitamin A metabolite, all-trans retinoic acid (atRA), plays an important role in neuronal development, including neurite outgrowth. However, the genes that lie downstream of atRA and its receptors in neuronal cells are largely unknown. By using the human neuroblastoma cell line, SH-SY5Y, we have identified an atRA-responsive gene (RAINB1: retinoic acid inducible in neuroblastoma cells) that is induced within 4 h after exposure of SH-SY5Y cells to atRA. RAINB1 mRNA is highly expressed in the nervous system (10.5- to 11-kb transcript) in both developing embryos and adults. Its expression is perturbed in developing rat embryos exposed to excess or insufficient atRA. RAINB1 is present on chromosome 11 and is spread over 38 exons, resulting in a putative ORF of 2,429 amino acids. The RAINB1 protein shows high similarity to a gene in Caenorhabditis elegans, unc-53, that is required for axonal elongation of mechanosensory neurons, suggesting that these proteins are orthologs. Thus, RAINB1 may represent a critical downstream gene in atRA-mediated neurite outgrowth.

Aging↗
PubMed1993

Analysis of the role of 5' promoter elements and 3' flanking sequences on the expression of a baculovirus polyhedron envelope protein gene.

The polyhedron envelope protein gene of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) is the third in a series of five open reading frames (ORFs 1-5) oriented in the same direction. Individual mRNAs initiate at conserved late gene promoter/mRNA start site (A/GTAAG) sequences located upstream of each ORF and the mRNAs co-terminate after the fifth ORF. To examine the influence of transcription from upstream promoter elements and the presence of an extensive 3' flanking sequence on the expression of the polyhedron envelope protein gene, the region was cloned into a phagemid vector and a BamHi site was inserted downstream of the ATG by site-directed mutagenesis and used for the insertion of a chloroamphenicol acetyl transferase (CAT) reporter gene. A set of clones were constructed in which individual or combinations of the late promoter elements from OFs 1, 2, and 3 were destroyed by site-directed mutagenesis. These plasmid constructs were transfected into Lymantria dispar cells infected with OpMNPV and cell extracts were assayed for CAT activity. Inactivation of the late promoter element immediately 5' of the polyhedron envelope protein gene led to a 96% decrease in CAT expression. Destruction of the ORF 2 late promoter element, or both the ORF 1 and ORF 2 late promoter elements, or deletion of the entire region containing ORFs 1 and 2 resulted in a 17 to 35% increase in CAT expression. In contrast, inactivation of the ORF 1 promoter alone resulted in no increase in CAT expression. Deletions of 3' flanking sequences of the polyhedron envelope protein gene caused major reduction in both CAT activity and steady-state levels of CAT mRNA.

Baculoviridae↗
PubMed1997

The sequences of hypF, hypC and hypD complete the hyp gene cluster required for hydrogenase activity in Bradyrhizobium japonicum.

A region of DNA 6 kb downstream of the hydrogenase (H2ase) structural genes and directly downstream of the hypB gene of Bradyrhizobium japonicum was shown by mutational analysis to be necessary for H2ase synthesis. Sequencing of this region revealed two complete open reading frames, and the 5' fragment of a third ORF. They encode proteins with homologies to the HypF, HypC and the N-terminus of HypD from other H2ase-containing organisms. The hypF of B. japonicum encodes a 753-aa protein with a predicted molecular mass of 80.3 kDa that contains the two zinc-finger motifs characteristic of other HypF proteins. The hypC encodes a 85-aa protein with a predicted molecular mass of 8.4 kDa. The 5' portion of hypD, which encodes the first 35 aa, upon combining with the previously reported C-terminus of HypD, designated HypD' (Van Soom et al. (1993) Mol. Gen. Genet. 239, 235-240) encodes a protein with a predicted molecular mass of 42.4 kDa. Complementation studies on a H2 uptake defective strain of B. japonicum containing a polar mutation in the hyp operon revealed that the products of the hyp F, C, D, E genes are required for H2ase production. Evidence is also presented that the hyp genes are co-transcribed from a large operon together with the downstream genes hupGHIJK, making a polycistronic message of 11 genes.

Amino Acid Sequence↗
PubMed2003

Cloning and sequence analysis of the TRP1 gene encoding the phosphoribosyl anthranilate isomerase from Pichia anomala (strain K).

Pichia anomala (strain K) is an efficient biocontrol agent against post-harvest diseases affecting apples. To study the role of strain K genes in biocontrol activity, it is useful to identify selectable markers on which to base a gene disruption strategy. The Pichia anomala TRP1 gene (PaTRP1) was isolated by complementation of the multi-auxotrophic S. cerevisiae strain FY-1679-18b. DNA sequence analysis revealed the presence of a 699 bp ORF encoding a 233 amino acid protein showing the typical conserved structure of proteins of the phosphoribosyl anthranilate isomerase (PRAI) family. Codon analysis revealed a high number of unused codons. Downstream from PaTRP1 was found the 3' extremity of a gene highly similar to the IPP1 gene (coding for the inorganic pyrophosphatase).

Aldose-Ketose Isomerases↗
PubMed1990

Nucleotide sequence of the Synechococcus sp. PCC7942 branching enzyme gene (glgB): expression in Bacillus subtilis.

The nucleotide sequence of the Synechococcus sp. PCC7942 glgB gene has been determined. The gene contains a single open reading frame (ORF) of 2322 bp encoding a polypeptide of 774 amino acids (aa) with an Mr of 89,206. Extensive sequence similarity exists between the deduced aa sequence of the Synechococcus sp. glgB gene product and that of the Escherichia coli branching enzyme in the middle portions of the proteins (62% identical aa). In contrast, the N-terminal portions shared little homology. The sequenced region which follows glgB contains an ORF encoding 79 aa of the N terminus of a polypeptide that shares extensive sequence similarity (41% identical aa) with human and rat uroporphyrinogen decarboxylase. This suggests that the region downstream from glgB contains the hemE gene and, therefore, that the organization of genes involved in glycogen biosynthesis in Synechococcus sp. is different from that described for E. coli. A fusion gene was constructed between the 5' end of the Bacillus licheniformis penP gene and the Synechococcus sp. glgB gene. The fusion gene was efficiently expressed in the Gram+ micro-organism Bacillus subtilis and specified a branching enzyme with an optimal temperature for activity similar to the wild-type enzyme.

1,4-alpha-Glucan Branching Enzyme↗