SEARCH · Search PubMed

Results for “upstream 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.

SourceResearch connectionFrom yearThrough year

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

At least 415 records · Page 23Linked to original sources

PubMed1987

Structure of yeast regulatory gene LEU3 and evidence that LEU3 itself is under general amino acid control.

Determination of the nucleotide sequence of a DNA region from Saccharomyces cerevisiae previously shown to contain the LEU3 gene revealed one long open reading frame (ORF) whose 887 codons predict the existence of a protein with a molecular mass of 100,162 daltons. The codon bias index of 0.02 suggests that LEU3 encodes a low-abundance protein. The predicted amino acid sequence contains a stretch of 31 residues near the N-terminus that is rich in cysteines and basic amino acids and shows strong homology to similar regions in five other regulatory proteins of lower eukaryotes. Additional regions with a predominance of basic amino acids are present adjacent to the cysteine-rich region. A stretch of 20 residues, 19 of which are glu or asp, is found in the carboxy terminal quarter of the protein. The 5' flanking region of LEU3 contains a TATA box 111 bp upstream from the beginning of the long ORF and two transcription initiation elements (5'TCAA3') 58 and 48 bp upstream from the ORF. The 3' flanking region shows a tripartite potential termination-polyadenylation signal. The predicted 5' and 3' ends of the transcript are in very good agreement with the previously determined size of the LEU3 message. Analysis of a LEU3'-'lacZ translational fusion suggests that the LEU3 gene, whose product is involved in the specific regulation of the leucine and possibly the isoleucine-valine pathways, is itself under general amino acid control. Consistent with this observation is the finding that the 5' flanking region of LEU3 contains two perfect copies of the general control target sequence 5'TGACTC3'.

Amino Acid Sequence↗

PubMed1996

Mosaic type of the nontoxic-nonhemaggulutinin component gene in Clostridium botulinum type A strain isolated from infant botulism in Japan.

The gene encoding the nontoxic-nonhemaggulutinin (NTNH) component was amplified by the PCR technique using two primer sets and the DNA template from Clostridium botulinum type A strain 7I03-H isolated from infant botulism in Japan. The nucleotide sequence revealed that the NTNH gene was composed of 1,193 amino acids with a molecular weight of 130868.08. Furthermore, the N-terminal half side and C-terminal half side of the NTNH component were similar to the NTNH component of type C and type A, respectively. These results indicate that the NTNH component gene codes the mosaic NTNH component composed of type A and type C. The hemaggulutinin gene, aha, and ORF-22 gene, orf-22a, were undetectable in the region upstream of the NTNH component gene, ant. Therefore, orf-22a is not thought to play a key role in the expression of botulinum type A progenitor toxin gene.

Amino Acid Sequence↗

PubMed1998

Organization and transcriptional characterization of the cat1 gene cluster in Acinetobacter lwoffi K24.

Previously, we have reported that two clustered cat genes from Acenitobacter lwoffi K24 had different arrangements, catB1C1A1 and catB2A2C2 (Kim, S.I., S.-H. Leem, J.-S. Choi, Y.H. Chung, S. Kim, Y.-M. Park, Y.K. Park, Y.N. Lee, and K.-S. Ha. 1997, J. Bacteriol. 179, 5226-5231). By further analysis of the organization of the cat1 gene cluster, we obtained a complete sequence of the catB1 gene, which encoded 40.8-kDa polypeptide containing 379 amino acids, and found a open reading frame (ORF) coding a putative regulatory protein in upstream region of catB1 on plasmid pCD1-1. This ORF encoded 34.2-kDa polypeptide containing 379 amino acids and had more than 40% identity with catR, LysR family regulatory protein of Pseudomonas putida. RT-PCR, Northern blot analysis and primer extension assay for transcriptional analysis of the cat1 gene cluster revealed that the catB1C1 genes were cotranscribed and the catA1 gene was independently transcribed.

Acinetobacter↗

PubMed1989

Organization and nucleotide sequence of the genes encoding the large subunits A, B and C of the DNA-dependent RNA polymerase of the archaebacterium Sulfolobus acidocaldarius.

The genes for the three large subunits A, B and C, of the DNA-dependent RNA polymerase of the archaebacterium Sulfolobus acidocaldarius DSM 639, were identified and characterized. The three genes follow each other immediately in the order B-A-C, which corresponds to that found in the rpoBC operon of the Escherichia coli genome. The transcription products formed in vivo were studied by Northern analysis and the start-points were determined by S1-nuclease mapping and primer directed extension analysis. The three RNA polymerase subunit genes were co-transcribed together with an open reading frame (ORF) of 88 amino acid residues length situated immediately upstream of the B gene and two ORFs of 104 and 130 amino acid residues following the C gene (together 8500 nucleotides). The following ORF, encoding a protein of 118 amino acids homologous to the ribosomal protein S12 of E. coli, was weakly transcribed with the large co-transcript and strongly from an own promoter. The derived amino acid sequence of the B-subunit was found to be homologous to the B- (second largest) subunits of the eukaryotic nuclear polymerases I, II and III and to the eubacterial beta-subunit. The combined A + C-subunits correspond to the A- (largest) subunits of the eukaryotic RNA polymerases I, II and III and to the eubacterial beta'-subunit. The amino acid sequence similarity of the Sulfolobus subunits to the eukaryotic components is clearly higher than to the E. coli subunit.

Amino Acid Sequence↗

PubMed1999

A short open reading frame terminating in front of a stable hairpin is the conserved feature in pregenomic RNA leaders of plant pararetroviruses.

In plant pararetroviruses, pregenomic RNA (pgRNA) directs synthesis of circular double-stranded viral DNA and serves as a polycistronic mRNA. By computer-aided analysis, the 14 plant pararetroviruses sequenced so far were compared with respect to structural organization of their pgRNA 5'-leader. The results revealed that the pgRNA of all these viruses carries a long leader sequence containing several short ORFs and having the potential to form a large stem-loop structure; both features are known to be inhibitory for downstream translation. Formation of the structure brings the first long ORF into the close spatial vicinity of a 5'-proximal short ORF that terminates 5 to 10 nt upstream of the stable structural element. The first long ORF on the pgRNA is translated by a ribosome shunt mechanism discovered in cauliflower mosaic (CaMV) and rice tungro bacilliform viruses, representing the two major groups of plant pararetroviruses. Both the short ORF and the structure have been implicated in the shunt process for CaMV pgRNA translation. The conservation of these elements among all plant pararetroviruses suggests conservation of the ribosome shunt mechanism. For some of the less well-studied viruses, the localization of the conserved elements also allowed predictions of the pgRNA promoter region and the translation start site of the first long ORF.

Badnavirus↗

PubMed1993

Characterization of the hemA-prs region of the Escherichia coli and Salmonella typhimurium chromosomes: identification of two open reading frames and implications for prs expression.

The prs gene, encoding phosphoribosylpyrophosphate synthetase, is preceded by a leader, which is 302 bp long in Escherichia coli and 417 bp in Salmonella typhimurium. A potential open reading frame (ORF) extends across the prs promoter and into the leader. The region between the prs coding region and an upstream gene (hemA) in E. coli and S. typhimurium was cloned, sequenced and shown to encode two ORFs of unknown function. ORF 1 encodes a 23 kDa protein and ORF 2 a 31 kDa protein, as observed by denaturing PAGE of extracts of cells bearing plasmids encoding the ORFs. Both ORFs are transcribed in the same direction as the prs gene with ORF 2 extending into the prs leader. Northern blot analysis showed that the prs message in E. coli was on 1.3 and 2.7 kb transcripts. The shorter transcript encoded the prs gene only, while the longer transcript also encoded the two ORFs. Thus, the prs gene is transcribed from two promoters, the first promoter (P1) originating upstream of ORF 1, and expressing the prs gene in a tricistronic operon and a second promoter (P2), located within the ORF 2 coding frame, which transcribes the prs gene only. The transcripts encoding prs only were 20 times as abundant as the tricistronic transcripts under all conditions examined. This was the case whether cells containing plasmid-encoded or only chromosomally encoded copies of the hemA-prs region were probed for these transcripts. Derepression of the prs gene upon pyrimidine starvation was shown to be due to an increase in the amount of message originating from the promoter P2.

Aldehyde Oxidoreductases↗

PubMed1993

The predominant mRNA class in HPV16-infected genital neoplasias does not encode the E6 or the E7 protein.

Human papillomavirus (HPV) type 16 is strongly implicated in the development of progressive neoplasias of the uterine cervix. Its oncogenic potential is decisively determined by the activity of the early gene products E6 and E7. To look for changes in the expression of these genes during tumour progression we cloned subgenomic fragments of HPV16 into RNA expression vectors, which allowed the generation of 35S-labelled riboprobes specific for distinct mRNA classes. Four constructs were made to differentiate between transcripts starting upstream of the E6 ORF or the E1 ORF, and one probe was specific for unspliced E6/E7 region transcripts. Five other constructs were used to identify transcripts covering the E1, E2, E4, L1 and L2 regions. With the help of these constructs, we analyzed by in situ hybridization 2 low-grade intraepithelial neoplasias of the vulva, 1 high-grade neoplasia of the cervix as well as 4 vulvar and 3 cervical carcinomas. Transcripts from the E1, E2, E4, L1 and L2 region that were consistently detected in the differentiated layers of benign lesions were variably expressed in precancers and carcinomas. None of the investigated cases revealed detectable amounts of unspliced E6/E7 transcripts with a coding potential for a full-length E6 protein. In benign lesions, the E7 transcripts were confined to isolated nuclei of differentiated cells, whereas high-grade lesions and invasive cancers showed elevated levels of equally distributed E7-specific signals in the cytoplasm of all tumour cells. The most abundant transcripts observed in intraepithelial neoplasias and in invasive cancers appear to initiate within ORF E7 and therefore have no coding potential for full-length E6 and E7 proteins. Our data show that the actual level of E7-specific transcripts in cancers is lower than anticipated from earlier studies using an ORF E6/E7-specific probe that hybridizes with the 5'-ends of the abundant mRNA class.

Base Sequence↗

PubMed1995

The Thermoplasma acidophilum rpl15 gene encodes a homologue of eukaryotic ribosomal proteins L15/YL10.

A gene has been cloned from the archaebacterium, Thermoplasma acidophilum, which, on the basis of the deduced amino acid sequence, encodes a homologue of the eukaryotic large subunit ribosomal proteins, L15/YL10. This gene, rpl15, was identified as an open reading frame (ORF) located 2.3 kb upstream of the gene encoding the alpha-subunit of the T. acidophilum proteasome. The putative translation product of rpl15 (RPL15) contains 197 amino acid residues, with a M(r) of 22,928 and a basic pI of 11.59. The RPL15 amino acid sequence shows significant similarity (> 35% identity) to the L15/YL10 proteins of various eukaryotes.

Amino Acid Sequence↗

PubMed1995

Highly bioluminescent Streptococcus thermophilus strain for the detection of diary-relevant antibiotics in milk.

Inefficient translational initiation is often the cause of poor foreign gene expression in gram-positive organisms. The expression of bacterial luciferase (lux) genes in Streptococcus thermophilus (bioluminescence) was improved by addressing this problem in two ways; by ribosome-binding site (RBS) replacement, and by enhancing lux RBS access by polymerase chain reaction modification either alone or combined with translational coupling to a truncated upstream open- reading frame (orf') having its own RBS. Lactococcal expression signals were employed for plasmid-based lux expression. The same constructs were used to monitor bioluminescence in Lactococcus lactis, as well as two non-lactic bacterial strains, for comparison. High lux expression was achieved in all four organisms with a heterodimeric thermostable enzyme. Surprisingly, where ready access to the lux RBS was predicted, translational coupling to the lactococcal orf remained a prerequisite for detectable lux expression in L. lactis. In contrast, high bioluminescence in S. thermophilus was independent of coupling. Consistent with these observations, inspection of published gene sequences suggests that RBS "strength" may be a more important factor in translation in S. thermophilus than in L. lactis. Using reduced light production in highly bioluminescent S. thermophilus as an indicator of biocide presence in milk, test times could be significantly shortened compared with a commercial test utilizing the related non-bioluminescent strain. lux genes appear to be sensitive, exponential-phase reporters of gene activity in S. thermophilus, an organism with molecular biology and genetics that remain largely unstudied.

Animals↗

PubMed2006

Cloning and molecular characterization of three novel LMW-i glutenin subunit genes from cultivated einkorn (Triticum monococcum L.).

Three novel low molecular weight (LMW) glutenin subunits from cultivated einkorn (Triticum monococcum L., A(m)A(m), 2n = 2x = 14) were characterized by SDS-PAGE and molecular weights determined by MALDI-TOF-MS. Their coding genes were amplified and cloned with designed AS-PCR primers, revealing three complete gene sequences. All comprised upstream, open reading frame (ORF), downstream and no introns were present. The deduced amino acid sequences showed that all three genes, named as LMW-M1, LMW-M3 and LMW-M5, respectively, belonged to the LMW-i type subunits with the predicted molecular weight between 38.5206 and 38.7028 kDa. They showed high similarity with other LMW-i type genes from hexaploid bread wheats, but also displayed unique features. Particularly, LMW-M5 subunit contained an extra cysteine residue in the C-terminus except for eight conserved cysteines, which resulted from a single-nucleotide polymorphism (SNP) of the T-C transition, namely arginine --> cysteine substitution at position 242 from the N-terminal end. This is the first report that the LMW-i subunit contained nine cysteines residues that could result in a more highly cross-linked and more elastic glutenin suggesting that LMW-M5 gene may associates with good quality properties. In addition, a total of 25 SNPs and one insertions/deletions (InDels) were detected among three LMW-i genes, which could result in significant functional changes in polymer formation of gluten. It is anticipated that these SNPs could be used as reliable genetic markers during wheat quality improvement. The phylogenetic analysis indicated that LMW-i type genes apparently differed from LMW-m and LMW-s type genes and diverged early from the primitive LMW-GS gene family, at about 12.92 million years ago (MYA) while the differentiation of A(m) and A genomes was estimated at 3.98 MYA.

Amino Acid Sequence↗

PubMed1991

Transcriptional factor C/EBP binds to and transactivates the enhancer element II of the hepatitis B virus.

The human hepatitis B Virus genome (HBV) contains a liver-specific enhancer upstream of the X ORF which has been studied in detail by several investigators. A second liver-specific enhancer element, designated here as enhancer II, has been relatively recently described in the HBV genome, which is located within the core/pregenomic promoter. We have studied the interactions of transcriptional factors with this element and show here that the nuclear factor CCAAT/enhancer binding protein (C/EBP) binds at a unique site within these sequences. Further, using the transient cotransfection scheme of expression with C/EBP encoding vectors and an enhancer II-reporter gene construct, we demonstrate that the enhancer element II responds to increasing amounts of C/EBP by displaying transactivation. Evidence for the functional role of the enhancer element II in transcriptional regulation of the HBV gene expression is presented. A major influence of the enhancer II appears to be on the surface antigen expression.

Base Sequence↗

PubMed1991

The cloning and sequencing of the virion protein genes from a British isolate of porcine respiratory coronavirus: comparison with transmissible gastroenteritis virus genes.

Previous analysis of porcine respiratory coronavirus (PRCV) mRNA species showed that mRNAs 2 and 3 were smaller than the corresponding transmissible gastroenteritis virus (TGEV) mRNA species (Page et al. (1991) J. Gen. Virol. 72, 579-587). Sequence analysis showed that mRNA 3 was smaller due to the presence of a new putative RNA-leader binding site upstream of the PRCV ORF-3 gene. However, this observation did not explain the deletion observed in PRCV mRNA 2. Polymerase chain reaction (PCR) was used to generate cDNA from the 3' coding region of the putative polymerase gene to the poly (A) tail of PRCV for comparison to the equivalent region from TGEV. The PRCV S protein was found to consist of 1225 amino acids, which had 98% similarity to the TGEV S protein. However, the PRCV S gene contained a 672 nucleotide deletion, corresponding to 224 amino acids (residues 21 to 245 in TGEV S protein), 59 nucleotides downstream of the S gene initiation codon. The PRCV genome from the ORF-3 gene to the poly (A) tail was sequenced for comparison to TGEV in order to identify other potential differences between the two viruses. Four ORFs were identified that showed 98% similarity to the TGEV ORF-4, M, N and ORF-7 genes. No other deletions or any PRCV specific sequences were identified.

Amino Acid Sequence↗

PubMed1985

The traM gene of the resistance plasmid R1: comparison with the corresponding sequence of the Escherichia coli F factor.

The 7.7-kb EcoRI fragment of the resistance plasmid R1 contains the gene for the TraM protein. The sequence was identified by the presence of an open reading frame (ORF) which is preceded upstream by two promoter sequences. Both these promoters were found to be active, although the more distant one predominates, as was judged by the relative abundance of mRNA of the expected length. The TraM protein could be synthesized in an in vitro DNA-dependent protein synthesis system if the DNA of the corresponding region was supplied as template. Comparison of the traM genes of R1 and the F factor showed a high degree of similarity, although a number of mutations, especially near the 5' terminus, introduce specific amino acid (aa) changes. Two-thirds of the 3' sequences differ mainly in silent mutations; hence the aa sequence of the corresponding carboxy-terminal portion of the protein is highly conserved. The 5' and 3' untranslated regions of the mRNAs show little homology. One of the promoter regions, the ribosome-binding sequences, and the transcription termination sites are located at comparable positions but differ in details.

Amino Acid Sequence↗

PubMed1994

Sequence analysis of the Rickettsia prowazekii gyrA gene.

The Rickettsia prowazekii (Rp) gyrA gene, which codes for a subunit of DNA gyrase in this obligate intracellular bacterium, has been isolated and characterized. Nucleotide sequence analysis revealed an open reading frame (ORF), initiating with a GTG start codon, of 2718 bp that could encode a protein of 905 amino acids (aa) with a calculated M(r) of 101,048. The Rp gyrase subunit A (GyrA), when compared to GyrA analogs of other bacterial species, exhibited 43 to 50% identity. Alignment of the Rp GyrA aa sequence with the other analogs revealed the presence of a span of additional aa within the putative DNA-binding domain. The lack of an ORF within 865 bp upstream from the Rp gyrA demonstrates a Rp gene organization different from that of characterized gyrA from other species. Despite the similarity to Escherichia coli GyrA, Rp GyrA did not complement an E. coli gyrA temperature-sensitive mutant. However, Rp gyrA was dominant to an E. coli gyrA96 nalidixic-acid-resistant (NalR) mutant, conferring Nal sensitivity when introduced into the NalR E. coli strain.

Amino Acid Sequence↗

PubMed2004

Genetic and metabolic analysis of folate salvage in the human malaria parasite Plasmodium falciparum.

Antifolate drugs that target the biosynthesis and processing of essential folate cofactors are widely used for treatment of chloroquine-resistant falciparum malaria. Salvage of pre-formed folate can strongly compromise the efficacy of these drugs in vitro and the availability of folate from the human host in natural infections also influences therapeutic outcomes. To investigate how different parasite lines respond to the presence of exogenous folate, we measured the effect of the latter on the susceptibility of parasites to sulfa-drug blockage of folate biosynthesis, utilising the parents and 22 progeny of the HB3-Dd2 genetic cross of Plasmodium falciparum, together with selected unrelated lines. Complete linkage of the folate utilisation phenotype was observed to a DNA sequence of 48.6 kb lying between nucleotide positions 738,489 and 787,058 of chromosome 4 and encompassing the dihydrofolate reductase-thymidylate synthase (dhfr-ts) gene locus. Examination of the putative ORFs on this fragment upstream (3) and downstream (4) of dhfr-ts revealed no plausible candidate genes for folate processing. Similarly, a marked heterogeneity in the 5'-UTR regions of Dd2 and HB3, manifest as a directly repeated 256 bp sequence in the former, also did not correlate with the folate utilisation phenotype nor apparently influence levels of dhfr-ts transcripts or protein products. By contrast, the nature of the coding sequence of the dhfr domain appeared to play a direct role, with the single mutant (S108N) HB3-type utilising folic acid much less efficiently than other allelic variants. We also compared the processing of exogenous folic acid, folinic acid and p-aminobenzoic acid (pABA) in metabolic labelling studies of HB3 and Dd2. These support the view that DHFR is likely to have a low-level folate reductase activity as well as its normal function of reducing dihydrofolate to tetrahydrofolate, and that a significant hurdle in the utilisation of exogenous folic acid is the initial reduction of fully oxidised folic acid to dihydrofolate, an activity that the single mutant enzyme found in HB3 is postulated to perform particularly poorly. This would mirror earlier studies indicating that the DHFR activity of HB3 is also compromised relative to other variants.

4-Aminobenzoic Acid↗

PubMed2006

Translation initiation at an upstream CUG codon regulates the expression of Hibiscus chlorotic ringspot virus coat protein.

Viruses depend heavily on host cells for replication and exploit the host translation machinery for its gene expression using various unorthodox translation mechanisms. According to the conventional scanning model, only the 5'-proximal gene in the viral RNA is accessible to the ribosomes whereas other genes are silent. In this study, we use a model plant RNA virus, Hibiscus chlorotic ringspot virus (HCRSV), to investigate various translation mechanisms involved in regulation of the expression of internal genes. The 3'-end 1.2kb region of HCRSV genomic and subgenomic RNAs were shown to encode four polypeptides of 38, 27, 25 and 22.5kDa. Mutagenesis studies revealed that a CUG codon ((2570)CUG) is the initiation codon for p27, the longest of the three co-C-terminal products (p27, p25 and p22.5), and translation of p25 and p22.5 was initiated at (2603)AUG and (2666)AUG, respectively. Translation initiation of the p27 expression at the (2570)CUG codon regulates the expression of p38, the viral coat protein through a leaky scanning mechanism and mutational analysis of an upstream open reading frame (ORF) demonstrated that initiation of the p27 expression at this CUG codon (instead of an AUG) may play a role in maintaining the ratio of p27 and p38. In addition, a previously identified internal ribosome entry site was shown to control the expression of p27 and p38 in the subgenomic RNA 2.

Blotting, Western↗

PubMed2000

Genetic structure and functional implication of the fcb gene cluster for hydrolytic dechlorination of 4-chlorobenzoate from Pseudomonas sp. DJ-12.

The fcb gene cluster responsible for the hydrolytic dechlorination of 4-chlorobenzoate (4CBA) was cloned from the chromosomal DNA of Pseudomonas sp. DJ-12, and its nucleotide sequence analyzed. The gene cluster was organized in the order fcbB-fcbA-fcbT1-fcbT2-cbT3-fcbC, which is different from that reported in other bacteria. A promoter-like sequence (-35 and -10 region) is located upstream of the fcbB gene and putative ribosome-binding sequences were found upstream of the respective orfs. A stem-loop transcription terminator structure is found downstream of fcbC. This suggests that the six orfs are transcribed into a polycistronic mRNA. The FcbA, FcbB, and FcbC enzymes for dechlorination of 4CBA have a relationship in common with the enzymes involved in fatty acid metabolism on the basis of their deduced amino acid sequences. The proteins encoded by fcbT1, fcbT2, and fcbT3 show similarity to those encoded by dctP, dctQ, and dctM of Rhodobacter capsulatus respectively, which encode transporter proteins for C4-dicarboxylate. It is likely, therefore, that these proteins of DJ-12 play a role in transport of 4CBA into the cell.

Biological Transport↗

PubMed1996

The aroA gene of Campylobacter jejuni.

The gene for 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (aroA) cloned from Campylobacter jejuni (Cj) strain 81116 was identified by complementation of an Escherichia coli (Ec) auxotrophic aroA mutant. The Cj aroA gene has been sequenced. It encodes an enzyme of 428 amino acids (aa), that is homologous to other bacterial EPSP synthases, especially that of Bacillus subtilis with which it has a 39% aa identity. The transcriptional start point was mapped. It is present in an upstream open reading frame (ORF) that has a strong homology to the gene encoding phenylalanine tRNA synthetase (pheS). Downstream from aroA another ORF is present which is homologous to the lytB gene of Ec. The stop codon of the aroA gene overlaps the start codon of lytB.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗