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PubMed2001

Genomic structure of mouse copper chaperone, COX17.

Coxl7p was first cloned as a cytoplasmic copper chaperone from yeast mutant and recent works suggested the existence of mammalian homologues. Previous report has shown that a gel filtration fraction of heart extract containing porcine Coxl7p peptide promoted the survival of NIH3T3 fibroblast cells. In the present study, we first cloned DNA fragments of the mouse COX17 gene. The mouse COX17 spans approximately 6kb and consists of three exons. It was mapped to the center of chromosome 16, using a radiation hybrid-mapping panel. The major transcription start site is 80 bp upstream of the ATG initiation codon as determined by rapid amplification of cDNA ends (5'-RACE) analysis. Two potential polyadenylation sites are 3233 and 3293 bp downstream of the termination codon, respectively. Transient transfection of reporter plasmids containing portions of the mouse COX17 5'-flanking region into AtT-20 and NIH3T3 cells allowed the localization of the essential promoter to a 0.8 kb region upstream of the transcription starting site. Furthermore, the transfected luciferase activity was much higher in AtT-20 than NIH3T3. According to sequence analysis of the approximately 0.8kb 5'-flanking region, GC rich segments including consensus sequences for binding of the transcription factor Sp1, but no TATA/CAAT boxes, exist in the region of the transcription start site. Besides the GC box, binding sites for NRF-1 and 2 known as specific transcription factors for COX subunits are also localized around the transcription starting site.

Animals↗

PubMed1998

A frameshift mutation at Gly975 in the transmembrane domain of GPIIb prevents GPIIb-IIIa expression--analysis of two novel mutations in a kindred with type I glanzmann thrombasthenia.

Two Hispanic siblings presenting with lifelong mucocutaneous bleeding were diagnosed clinically with Glanzmann thrombasthenia on the basis of a normal platelet count, prolonged bleeding time and absent platelet aggregation in response to multiple agonists. Quantitative analysis of the probands' platelets by flow cytometry showed a complete absence of GPIIb-IIIa, consistent with Type I thrombasthenia. Genetic analysis showed the probands to be compound heterozygotes for two novel mutations of GPIIb: a C1414>G mutation in exon 14, resulting in a premature termination codon replacing residue Tyr440, and the insertion of a G at position 3016 in exon 29, leading to a frameshift affecting the C-terminal half of the transmembrane domain and the cytoplasmic tail. The frameshifted sequence alters residues from Gly975 onwards and is predicted to significantly alter the hydropathy and charge profiles of the GPIIb transmembrane domain. The Type I phenotype associated with this mutation suggests that GPIIb residues 975-1008 contain critical structural motifs for heterodimer assembly, membrane retention, export from the ER and surface expression.

Amino Acid Sequence↗

PubMed1989

Molecular cloning and nucleotide sequence of the colonization factor antigen I gene of Escherichia coli.

The colonization factor antigen I (CFA/I) gene has been isolated and sequenced. The amino acid sequence of CFA/I deduced from the nucleotide sequence is composed of 170 amino acids. The first 23 amino acids are considered to be the signal peptide of the CFA/I protein since they are not present in the protein sequence. Among the remaining amino acids, only two are different from the protein sequence: amino acid position 76 is an aspartic acid instead of an asparagine, and position 97 is a serine instead of an alanine. The CFA/I gene has a typical Shine-Dalgarno sequence located 10 base pairs (bp) upstream from the initiation codon. The sequence TACAAT located 48 bp upstream from the initiation codon was tentatively designated the -10 sequence of the CFA/I gene promoter. No sequences homologous to the consensus -35 promoter sequence was found. A pair of inverted repeat sequences followed by a stretch of eight A's are located 45 bp downstream from the termination codon of the CFA/I gene; this region may be a rho-independent transcriptional terminator.

Amino Acid Sequence↗

PubMed1978

DNA sequences of promoter regions for the str and spc ribosomal protein operons in E. coli.

The DNA sequences have been determined for promoter regions of two ribosomal protein operons in E. coli, the str operon and the spc operon. The site of in vitro transcription initiation within each of these promoter regions has been determined. The start site of the str operon occurs 69 bases upstream from the initiation codon of the S12 gene. The start site of the spc operon occurs 72 bases upstream from the L14 gene, and only 91 bases downstream from the termination codon of the S17 gene (which is in the preceding S10 operon). Both promoters are similar to other sequenced promoters in that they each have an identifiable "Pribnow box" sequence 5 bases upstream from the transcription start site. The spc promoter has a long sequence of 2 fold symmetry centered within the Pribnow box; the str promoter has a shorter but similar symmetry. At positions -69 through -40 in the spc operon, another long region of symmetry is present which may be the termination signal of the preceding S10 operon. Extensive sequence similarity between the str and spc promoter regions is found downstream from the Pribnow box-that is, in a transcribed region preceding the translation start sites.

Bacterial Proteins↗

PubMed1989

Structure of human osteonectin based upon analysis of cDNA and genomic sequences.

Overlapping human bone osteonectin cDNAs were obtained by screening two independent human SaOS-2 lambda gt11 libraries using antibovine osteonectin monoclonal antibodies. One clone contains a 0.54-kb insert and the other a 1.9-kb insert. Insertion fragments from lambda clones were liberated by restriction digestion and subcloned into pUC19 for sequencing. Digestion of the 1.9-kb insert with EcoRI released 0.4- and 1.5-kb fragments. Sequencing analysis revealed that the 0.54- and 0.4-kb fragments are identical except for 150 nucleotides missing at the 5' region of the 0.4-kb fragment. The composite nucleotide sequence of human osteonectin has a total length of 2091 nucleotides and is comprised of 50 nucleotides of 5'-noncoding sequence, a coding segment for 303 amino acids, a termination codon, and 1114 nucleotides of 3'-noncoding sequence. The primary transcript codes for 286 amino acids of mature protein and a 17-residue amino-terminal hydrophobic signal peptide. Outstanding properties inferred from the primary structure are putative Ca2+ binding domains located in the glutamic acid rich NH2 terminus (residues 1-52) and two "EF"-hand domains in the C-terminal half of the protein (residues 165-176 and 257-286). The mature protein also contains a cysteine-rich, highly hydrophilic region homologous to the ovomucoid serine protease inhibitors (residues 76-132). Overlapping human genomic clones in lambda EMBL3 for osteonectin have been isolated and characterized. Intron/exon junction sequencing of the human osteonectin gene shows the presence of 10 exons and 9 introns. The mature protein is encoded by nine exons separated by eight introns.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1988

Introduction of an intervening sequence into a human serine suppressor tRNA gene: effects on gene expression in vitro and in vivo.

The 13 nucleotide Xenopus laevis tyrosine tRNA gene intervening sequence was into a human serine suppressor tRNA gene which lacked an intron, by site-directed mutagenesis. Analysis of the products of in vitro transcription in a HeLa cell extract indicates that the intervening sequence is accurately removed to generate a mature sized RNA identical to that obtained from an intron-less gene. Analysis of the transcripts obtained in vitro and in vivo shows that the U in the CUA anticodon sequence is partially modified to psi. Total TRNA isolated from cells infected with recombinant SV40 viruses carrying the mutant tRNA genes is active in suppression of UAG codons in a reticulocyte cell-free system. Cotransfection of COS cells with the mutant tRNA genes and a mutant chloramphenicol acetyltransferase gene containing the termination codon UAG demonstrated that the tRNA functions as a UAG suppressor in vivo. Analysis of 32P-labeled RNA obtained from infected cells showed, however, that cells infected with the intron-containing gene accumulate less mature tRNA than cells infected with the intron-less tRNA genes.

Animals↗

PubMed1989

Remarkable intron and exon sequence conservation in human and mouse homeobox Hox 1.3 genes.

A high degree of conservation exists between the Hox 1.3 homeobox genes of mice and humans. The two genes occupy the same relative positions in their respective Hox 1 gene clusters, they show extensive sequence similarities in their coding and noncoding portions, and both are transcribed into multiple transcripts of similar sizes. The predicted human Hox 1.3 protein differs from its murine counterpart in only 7 of 270 amino acids. The sequence similarity in the 250 base pairs upstream of the initiation codon is 98%, the similarity between the two introns, both 960 base pairs long, is 72%, and the similarity in the 3' noncoding region from termination codon to polyadenylation signal is 90%. Both mouse and human Hox 1.3 introns contain a sequence with homology to a mating-type-controlled cis element of the yeast Ty1 transposon. DNA-binding studies with a recombinant mouse Hox 1.3 protein identified two binding sites in the intron, both of which were within the region of shared homology with this Ty1 cis element.

Amino Acid Sequence↗

PubMed1998

Identifying the right stop: determining how the surveillance complex recognizes and degrades an aberrant mRNA.

The nonsense-mediated mRNA decay (NMD) pathway functions by checking whether translation termination has occurred prematurely and subsequently degrading the aberrant mRNAs. In Saccharomyces cerevisiae, it has been proposed that a surveillance complex scans 3' of the premature termination codon and searches for the downstream element (DSE), whose recognition by the complex identifies the transcript as aberrant and promotes its rapid decay. The results presented here suggest that translation termination is important for assembly of the surveillance complex. Neither the activity of the initiation ternary complex after premature translation termination has occurred nor the elongation phase of translation are essential for the activity of the NMD pathway. Once assembled, the surveillance complex is active for searching and recognizing a DSE for approximately 200 nt 3' of the stop codon. We have also identified a stabilizer sequence (STE) in the GCN4 leader region that inactivates the NMD pathway. Inactivation of the NMD pathway, as a consequence of either the DSE being too far from a stop codon or the presence of the STE, can be circumvented by inserting sequences containing a new translation initiation/termination cycle immediately 5' of the DSE. Further, the results indicate that the STE functions in the context of the GCN4 transcript to inactivate the NMD pathway.

Codon↗

PubMed1991

RNA editing makes mistakes in plant mitochondria: editing loses sense in transcripts of a rps19 pseudogene and in creating stop codons in coxI and rps3 mRNAs of Oenothera.

An intact gene for the ribosomal protein S19 (rps19) is absent from Oenothera mitochondria. The conserved rps19 reading frame found in the mitochondrial genome is interrupted by a termination codon. This rps19 pseudogene is cotranscribed with the downstream rps3 gene and is edited on both sides of the translational stop. Editing, however, changes the amino acid sequence at positions that were well conserved before editing. Other strange editings create translational stops in open reading frames coding for functional proteins. In coxI and rps3 mRNAs CGA codons are edited to UGA stop codons only five and three codons, respectively, downstream to the initiation codon. These aberrant editings in essential open reading frames and in the rps19 pseudogene appear to have been shifted to these positions from other editing sites. These observations suggest a requirement for a continuous evolutionary constraint on the editing specificities in plant mitochondria.

Amino Acid Sequence↗

PubMed1981

Position of the lacZX90 mutation and hybridization between complete and incomplete beta-galactosidase.

The position of the termination codon in lacZX90 was determined by isolation of a lac+ revertant. Lysine was found to replace tyrosine at position 1,012 of beta-galactosidase, indicating that X90 protein lacked the carboxyl-terminal 10 residues. A heat- and urea-sensitive hybrid enzyme was formed in vivo when supC, which supplies tyrosine to the position in the polypeptide corresponding to the nonsense codon, was used to suppress lacZX90. This result shows that suppression that adds back the original amino acid may not lead to the production of the wild-type enzyme if the latter is multimeric, because incomplete chains can be incorporated into the oligomer.

Amino Acid Sequence↗

PubMed2003

Rapid deadenylation triggered by a nonsense codon precedes decay of the RNA body in a mammalian cytoplasmic nonsense-mediated decay pathway.

Nonsense-mediated mRNA decay (NMD) is an RNA surveillance pathway that detects and destroys aberrant mRNAs containing nonsense or premature termination codons (PTCs) in a translation-dependent manner in eukaryotes. In yeast, the NMD pathway bypasses the deadenylation step and directly targets PTC-containing messages for decapping, followed by 5'-to-3' exonuclease digestion of the RNA body. In mammals, most PTC-containing mRNAs are subject to active nucleus-associated NMD. Here, using two distinct transcription-pulsing approaches to monitor mRNA deadenylation and decay kinetics, we demonstrate the existence of an active cytoplasmic NMD pathway in mammalian cells. In this pathway, a nonsense codon triggers accelerated deadenylation that precedes decay of the PTC-containing mRNA body. Transcript is stabilized when accelerated deadenylation is impeded by blocking translation initiation; by ectopically expressing two RNA-binding proteins, UNR and NSAP1; or by ectopically expressing a UPF1 dominant-negative mutant. These results are consistent with the notion that the nonsense codon can function in the cytoplasm by promoting rapid removal of the poly(A) tail as a necessary first step in the decay process.

3T3 Cells↗

PubMed1993

HTLV-1 gene expression by defective proviruses in an infected T-cell line.

We have examined human T-lymphotropic virus type I (HTLV-I) gene expression in the human T-cell line, C8166-45 (C81), as a model to define the gene products expressed from defective proviruses. C81 cells contain one complete and two different deleted proviral genomes. The internal deletions of the latter encompass most of the gag to env region. All three proviruses are transcriptionally active as evidenced by the presence of three unspliced nuclear mRNAs. Unspliced genomic mRNAs and singly spliced env mRNAs were present in the nucleus, but were not detected in the cytoplasm, suggesting a defect in Rex function. Three small cytoplasmic mRNAs were observed and are likely to correspond to the normal 1.8-kb Tax1/Rex1 mRNA, a 1.6-kb mRNA formed by splicing the 5'LTR to the pX region, and a 2.1-kb mRNA of unknown origin. Consistent with the subcellular mRNA distribution pattern, the viral structural proteins encoded by gag and env genes were not detected. The transcriptional transactivator protein, Tax1 (p40), was abundantly expressed in C81 cells; in addition, a 42-kDa Tax1 protein, unique to this cell line, was also detected. Although Tax1 and Rex1 (p27) are translated from overlapping open reading frames in the same mRNA, Rex1 was not detected in C81 cells. The presence of a premature termination codon in the Tax1/Rex1 mRNA encoded by the full-length provirus was inferred from the presence of small Rex1-related polypeptides lacking C-terminal sequences and confirmed by sequence analysis. Furthermore, a p21X protein lacking the N-terminus of Rex1 was expressed at high levels; our data indicate that p21X is translated from the 1.6-kb mRNA which is derived primarily from deleted proviruses.

Blotting, Northern↗

PubMed2005

Stable deletions arising in the readthrough region of Soil-borne wheat mosaic virus RNA2 define the 5' limit of the functional promoter for the p19 subgenomic RNA.

The appearance of de novo deletion mutations in the readthrough (RT) region (nucleotide positions 861-2591) downstream of the capsid protein (CP) gene of a Japanese strain of Soil-borne wheat mosaic virus RNA2 was examined using infectious transcripts. Mutant RNA2s with different deletions predominated in independent serial passage experiments but all best-adapted mutants retained the 3'-terminal portion of the RT gene in frame with the CP gene. The longest best-adapted mutation deleted the 1434 nucleotides between positions 1061 and 2494. When the RT protein was truncated by insertion of a termination codon plus an additional nucleotide to give a +1 frame-shift, after serial passages the progeny viruses regained the ability to express the C-terminal region of RT by an internal deletion. The 5' terminus of the p19 subgenomic RNA was identified at position 2598 and an essential transcription signal for this mRNA mapped between positions 2534 and 2563. A mutant in which this essential promoter element has been deleted cannot transcribe the p19 subgenomic RNA and has lost infectivity in planta. These results indicate that the 3'-terminal region of the RT gene has a major function in cis for expression of p19, which is essential for infecting plants. A reason for retaining the RT C-terminal region in stable deletion mutants is still unknown.

3' Flanking Region↗

PubMed1991

Nucleotide sequence and transcriptional mapping of the major capsid protein gene of pseudorabies virus.

The gene encoding the 142-kDa major capsid protein (MCP142) of pseudorabies virus (PrV) was isolated and sequenced. Nucleotide sequence analysis revealed that the MCP142 gene has a single open reading frame of 3993 nucleotides (nt) encoding 1330 amino acids. The 4400-nt major RNA from the MCP142 gene was detected in PrV-infected cells. The 5' end of the transcript was located 60 nt upstream of the initiation codon. The 3' end of the transcript was located 18 nt downstream of a putative poly(A) signal sequence TATAAA and 133 nt downstream of the termination codon. In comparing amino acid sequence homology between MCP142 of PrV and other available herpesviruses MCP was shown to have 58% homology with herpes simplex virus type 1 and varicella-zoster virus, 27% with Epstein-Barr virus, and 24% with human herpesvirus 6 and human cytomegalovirus. It has greater homology with those of the alpha-herpesviruses than with those of the beta-herpesviruses and the gamma-herpesviruses.

Amino Acid Sequence↗

PubMed2003

RNA editing in hornwort chloroplasts makes more than half the genes functional.

RNA editing in chloroplasts alters the RNA sequence by converting C-to-U or U-to-C at a specific site. During the study of the complete nucleotide sequence of the chloroplast genome from the hornwort Anthoceros formosae, RNA editing events have been systematically investigated. A total of 509 C-to-U and 433 U-to-C conversions are identified in the transcripts of 68 genes and eight ORFs. No RNA editing is seen in any of the rRNA but one tRNA suffered a C-to-U conversion at an anticodon. All nonsense codons in 52 protein-coding genes and seven ORFs are removed in the transcripts by U-to-C conversions, and five initiation and three termination codons are created by C-to-U conversions. RNA editing in intron sequence suggests that editing can precede intercistronic processing. The sequence complementary to the edited site is proposed as a distant cis-recognition element.

Amino Acid Substitution↗

PubMed1990

Epitope mapping of recombinant HeLa SmB and B' peptides obtained by the polymerase chain reaction.

SmB and SmB' are the major antigenic proteins contained within small nuclear RNP particles that are recognized by both human SLE and MRL mouse anti-Sm sera. We amplified cDNA obtained from HeLa cells by using the polymerase chain reaction and identified two clones, U2 and L13, that encode SmB and SmB', respectively. The nucleotide sequences of these two clones were identical except for the insertion of a 145-bp sequence in U2 that contained an early in frame termination codon and a potential 3' consensus splice site. The predicted amino acid sequences of HeLa SmB and B' proteins were therefore identical except for the COOH terminal 2 (U2) and 11 (L13) amino acids. U2, L13, and four subclones of U2 (F-B, B-R, F-X, and X-B) were ligated to pATH vectors and expressed as trpE fusion proteins. Epitope mapping with 12 human SLE and 12 MRL/lpr mouse anti-SmB/B' sera revealed that antibodies directed against the X-B peptide accounted for most (65.5 +/- 15.4 and 63.2 +/- 25.3%), B-R intermediate levels (51.5 +/- 30.8 and 18 +/- 19.6%), and F-X none of the anti-SmB activity in human and mouse sera, respectively. Ten human and two mouse sera contained antibodies that cross-reacted with epitopes located within the proline-rich, COOH-terminal, 27-residue peptide encoded by B-R and the NH2-proximal F-B peptide. These observations suggest that a) the polymerase chain reaction is a powerful ancillary method to synthesize autoantigens, b) SmB and B' in HeLa cells are derived from alternative splicing of a common RNA transcript, and c) both SLE and MRL anti-SmB/B' sera recognize multiple epitopes (some shared and some unique) on these proteins.

Amino Acid Sequence↗

PubMed1991

Point mutations in the beta-subunit of cytochrome b558 leading to X-linked chronic granulomatous disease.

The NADPH:O2 oxidoreductase of phagocytic leukocytes is an important enzyme for the bactericidal activity of these cells. Cytochrome b558 is a membrane component of this enzyme. In X-linked chronic granulomatous disease (Xb- CGD) the phagocytes are defective in the beta-subunit (gp91-phox) of this cytochrome. We have studied the genetic defect in a group of six X-linked CGD patients characterized by complete or partial loss of cytochrome b558 with the use of the polymerase chain reaction. All patients had a different single point mutation in the gp91-phox gene, indicating that the genetic defect in Xb- CGD is very heterogeneous. In one patient the mutation leads to a premature termination codon. In the other five cases these mutations predict incorporation of a different amino acid. The mutations were with one exception found in the N-terminal half of the protein, suggesting that this part of cytochrome b558 is important for the binding of the heme or for formation of a stable complex with p22-phox. Two histidyl residues were found that might be ligands of the heme iron.

Alleles↗

PubMed1999

Resuming translation on tmRNA: a unique mode of determining a reading frame.

The bacterial ribosome switches from an mRNA lacking an in-frame stop codon and resumes translation on a specialized RNA known as tmRNA, SsrA or 10Sa RNA. We find that the ribosome can reach and use the extreme 3' terminal codon of the defective mRNA prior to switching. The first triplet to be translated in tmRNA (the resume codon) is determined at two levels: distant elements in tmRNA restrict resume codon choice to a narrow window and local upstream elements provide precision. Insights from a randomization-selection experiment secure the alignment of tmRNA sequences from diverse species. The triplet UA(A/G) (normally recognized as a stop codon by release factor-1) is strongly conserved two nucleotides upstream of the resume codon. The central adenosine of this triplet is essential for tmRNA activity. The reading frame of tmRNA is determined differently from all other known reading frames in that the first translated codon is not specified by a particular tRNA anticodon.

Amino Acid Sequence↗