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PubMed1989

The molecular defect in a COOH-terminal-modified and shortened mutant of human serum albumin.

Albumin Venezia is a fast migrating genetic variant of human serum albumin which, in heterozygous subjects, represents about 30% of the circulating protein. The molecular defect in this variant was studied in a subject possessing an atypical level of the mutant (80% of the total protein) and in other members of his family. Albumins, isolated from the sera of the propositus and his heterozygous relatives, were treated with CNBr and the resulting fragments analyzed by isoelectric focusing. The peptides were then isolated in a homogeneous form by reverse-phase high performance liquid chromatography and submitted to sequence analysis. The results show that albumin Venezia possesses a shortened polypeptide chain, 578 residues instead of 585, completely variant from residue 572 to the COOH-terminal end: sequence: (see text). This extensive modification may be accounted for by the deletion of exon 14 and translation to the first terminator codon of exon 15, which normally does not code for protein. The absence of a basic COOH-terminal dipeptide in the mature molecule can be explained by the probable action of serum carboxypeptidase N. Additional support for such action comes from examination of the remaining 20% of the total albumin of the propositus, which is found to contain an extra Arg at its COOH terminus, probably due to partial digestion by carboxypeptidase N. The low serum level of the variant in heterozygous subjects suggests that the COOH-terminal end of the molecule is critical for albumin stability.

Amino Acid Sequence↗

PubMed1986

Complete amino acid sequence of human thyroxine-binding globulin deduced from cloned DNA: close homology to the serine antiproteases.

Antibodies directed against thyroxine-binding globulin (TBG) have been used to screen a human liver lambda gt11 expression library. A 1.46-kilobase clone was identified which encodes nearly the complete amino acid sequence, beginning at amino acid 17 of the mature protein. To complete the protein sequence, the cDNA clone was used to identify a genomic clone coding for TBG in a human X chromosome library. The overlapping recombinant clones contained an open reading frame coding for 415 amino acids followed by a polyadenylylation signal (AATAAA) located 275 nucleotides from a TAG termination codon. Beginning at residue 21, the deduced amino acid sequence agrees closely with the known NH2-terminal sequence of the mature peptide. The preceding 20 amino acid residues are hydrophobic in character and presumably represent a leader sequence. Four glycosylation sites were identified, corresponding to the number determined for the purified protein. DNA blot hybridization revealed a single-copy gene, which by chromosomal analysis was found to be located on the long arm of the X chromosome. Unexpectedly, the nucleotide sequence of TBG is closely homologous to those encoding the plasma serine antiproteases alpha 1-antichymotrypsin and alpha 1-antitrypsin. However, there is little overall homology between TBG and transthyretin (prealbumin), the other major thyroxine-binding protein of human plasma.

Amino Acid Sequence↗

PubMed1996

Characterization of Perina nuda nucleopolyhedrovirus (PenuNPV) polyhedrin gene.

The Perina nuda nucleopolyhedrovirus (PenuNPV) polyhedrin gene was located in EcoRI-G (6.3 kilobase pairs; kbp) and PstI-G (4.3 kbp) fragments of its genomic DNA. A portion of 1333 nucleotides (nt) containing this gene was sequenced. An open reading frame of 735 nt encoded a 245-amino-acid-long polyhedrin. A conserved TAAG motif which is associated with transcriptional start sites was identified 51 nt upstream of the translation initiation codon of PenuNPV polyhedrin gene. A putative polyadenylation signal, AATAAA, was found 116 nt downstream of the termination codon (TAA). Comparison of the amino acid sequences of PenuNPV polyhedrin with those of other NPVs showed that PenuNPV polyhedrin was most closely related to Orgyia pseudotsugata multiple NPV (OpMNPV) polyhedrin.

Amino Acid Sequence↗

PubMed1995

Readthrough protein associated with virions of barley yellow dwarf luteovirus and its potential role in regulating the efficiency of aphid transmission.

Purified particles of barley yellow dwarf luteovirus (BYDV) contain a major 22-kDa protein and a minor protein of approximately 58 kDa. The 22-kDa capsid protein is encoded by open reading frame (ORF) 3. ORF 5 is immediately downstream and in frame with ORF 3 and a 72-kDa protein can be translated via a readthrough suppression of the ORF 3 termination codon. Antibodies were produced against two Escherichia coli expressed polypeptides that represent the amino- and carboxyl-terminal halves of a putative 50-kDa protein encoded by ORF 5. Immunological analyses indicated that the 58-kDa protein associated with purified virions contained sequences encoded by ORF 3 and ORF 5. The carboxyl terminal portion of the full-length (72 kDa) readthrough protein was absent from the 58-kDa protein. The full-length readthrough protein was detected in infected oat protoplasts and plant tissue, but was not associated with virus particles purified from plants. The carboxyl-terminal portion of the 72-kDa readthrough protein was not required for aphid transmission; however, virus was transmitted more efficiently from protoplast extracts containing virions and soluble 72-kDa readthrough protein than from mock-inoculated protoplast extracts to which plant purified virus was added. The full-length readthrough protein, although not required for transmission, may increase the transmission efficiency of BYDV by aphids.

Animals↗

PubMed2006

A chemiluminescence-based reporter system to monitor nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that mediates rapid degradation of transcripts bearing premature translation termination codons (PTCs) and thereby limits the expression of unproductively processed mRNAs and the synthesis of C-terminally truncated peptides. Both its importance as a means to control gene expression and in the context of genetic and acquired human diseases call for an exploration of the mammalian NMD pathway using chemical biology approaches. Here, we describe a novel cell-based chemiluminescence reporter system that recapitulates the hallmark features of mammalian NMD. The assay is characterized by its high sensitivity, robustness, and its potential for automated handling. Limiting NMD efficiency by RNAi-mediated depletion of the essential NMD factor UPF1 markedly and specifically increased the NMD reporter mRNA level and resulted in a proportional increase in protein expression reflected by Renilla luminescence. The PI 3-kinase inhibitor wortmannin has previously been found to up-modulate PTC-containing transcripts by inhibiting the UPF1 kinase SMG1. Wortmannin treatment enhanced NMD reporter expression in our system in a dose-dependent way, illustrating its utility for small molecule screening.

Androstadienes↗

PubMed2003

Site of functional interaction of release factor 1 with the ribosome.

Ribosomal protein L11 consists of a C-terminal and an N-terminal domain. To determine the importance of each domain for interaction with release factor 1, which works specifically at the UAG termination codon, we constructed Escherichia coli strains lacking either the entire L11 protein or just the N-terminal portion. Strains lacking L11 exhibited UAG suppression, defective growth, and high-temperature lethality, phenotypes that were reversed by expression of L11 protein from a plasmid. Strains lacking only the N-terminal portion of L11 grew well at physiological temperatures and survived at high temperature, but they were defective in UAG-dependent termination. Our results show for the first time that it is precisely the N-terminal part of ribosomal protein L11 that is required for the functional interaction of release factor 1 with the ribosome in the cell.

Binding Sites↗

PubMed1999

mRNA surveillance in eukaryotes: kinetic proofreading of proper translation termination as assessed by mRNP domain organization?

In the last few years it has become clear that a conserved mRNA degradation system, referred to as mRNA surveillance, exists in eukaryotic cells to degrade aberrant mRNAs. This process plays an important role in checking that mRNAs have been properly synthesized and functions, at least in part, to increase the fidelity of gene expression by degrading aberrant mRNAs that, if translated, would produce truncated proteins. A critical issue is how normal and aberrant mRNAs are distinguished and how that distinction leads to differences in mRNA stability. Recent results suggest a model with three main points. First, mRNPs have a domain organization that is, in part, a reflection of the completion of nuclear pre-mRNA processing events. Second, the critical aspect of distinguishing a normal from an aberrant mRNA is the environment of the translation termination codon as determined by the organization of the mRNP domains. Third, the cell distinguishes proper from improper termination through an internal clock that is the rate of ATP hydrolysis by Upf1p. If termination is completed before ATP hydrolysis, the mRNA is protected from mRNA degradation. Conversely, if termination is slow, then ATP hydrolysis and a structural rearrangement occurs before termination is completed, which affects the fate of the terminating ribosome in a manner that fails to stabilize the mRNA. This proposed system of distinguishing normal from aberrant transcripts is similar to, but distinct from other systems of kinetic proofreading that affect the accuracy of other biogenic processes such as translation accuracy and spliceosome assembly.

3' Untranslated Regions↗

PubMed1999

The product of the cph oncogene is a truncated, nucleotide-binding protein that enhances cellular survival to stress.

Cph was isolated from neoplastic Syrian hamster embryo fibroblasts initiated by 3-methylcholanthrene (MCA), and was shown to be a single copy gene in the hamster genome, conserved from yeast to human cells, expressed in fetal cells and most adult tissues, and acting synergistically with H-ras in the transformation of murine NIH3T3 fibroblasts. We have now isolated Syrian hamster full-length cDNAs for the cph oncogene and proto-oncogene. Nucleotide sequence analysis revealed that cph was activated in MCA-treated cells by a point-mutational deletion at codon 214, which caused a shift in the normal open reading frame (ORF) and brought a translation termination codon 33 amino acids downstream. While proto-cph encodes a protein (pcph) of 469 amino acids, cph encodes a truncated protein (cph) of 246 amino acids with a new, hydrophobic C-terminus. Similar mechanisms activated cph in other MCA-treated Syrian hamster cells. The cph and proto-cph proteins have partial sequence homology with two protein families: GDP/GTP exchange factors and nucleotide phosphohydrolases. In vitro translated, gel-purified cph proteins did not catalyze nucleotide exchange for H-ras, but were able to bind nucleotide phosphates, in particular ribonucleotide diphosphates such as UDP and GDP. Steady-state levels of cph mRNA increased 6.7-fold in hamster neoplastic cells, relative to a 2.2-fold increase in normal cells, when they were subjected to a nutritional stress such as serum deprivation. Moreover, cph-transformed NIH3T3 cells showed increased survival to various forms of stress (serum starvation, hyperthermia, ionizing radiation), strongly suggesting that cph participates in cellular mechanisms of response to stress.

3T3 Cells↗

PubMed1995

A novel amber mutation in a beta zero-thalassaemia gene (beta 37TGG-->TAG), with direct detection by mapping the restriction fragments in amplified genomic DNA.

A novel amber mutation, a G to A substitution at the second position of codon 37 in the beta-globin gene that changes the tryptophan coding triplet (TGG) to a termination codon (TAG), was found in a Chinese beta-thalassaemia carrier. The mutant gene creates an additional Dde I recognition site and eliminates the Ava II site, so this point mutation can be directly identified by restriction enzyme analysis.

Adult↗

PubMed1993

Organization and transcription of the principal sigma gene (rpoDA) of Pseudomonas aeruginosa PAO1: involvement of a sigma 32-like RNA polymerase in rpoDA gene expression.

S1 nuclease mapping and Northern (RNA) hybridization revealed that the rpoDA gene encoding the principal sigma subunit of Pseudomonas aeruginosa PAO1 is transcribed as a monocistronic mRNA of 2 kb and that the transcription from the rpoDA promoter (PC) starts 32 bases upstream from the first nucleotide of the initiation codon during the steady-state growth condition at a low temperature (30 degrees C). The transcript terminates 31 bases downstream from the last nucleotide of the termination codon. When the growth temperature was shifted to 42 degrees C, the synthesis of rpoDA mRNA from a heat shock promoter was transiently induced, although transcription was still occurring from PC during the heat shock period. The transcription initiation site of the heat shock promoter (PHS) is located about 220 bases upstream of the initiation codon of rpoDA. In addition, both promoters were utilized in vitro by RNA polymerase partially purified from heat-shocked cells of P. aeruginosa PAO1. When the rpoDA was introduced into Escherichia coli, the transcription patterns of rpoDA at 30 and 42 degrees C were similar to those observed for P. aeruginosa. These results suggested that the transcription of rpoDA in P. aeruginosa is regulated by the principal RNA polymerase and the heat shock RNA polymerase in response to the environmental temperature.

Base Sequence↗

PubMed1983

Homologous nucleotide sequences at the 5' termini of messenger RNAs synthesized from the yeast enolase and glyceraldehyde-3-phosphate dehydrogenase gene families. The primary structure of a third yeast glyceraldehyde-3-phosphate dehydrogenase gene.

Genomic DNA containing a third yeast glyceraldehyde-3-phosphate dehydrogenase structural gene has been isolated on a bacterial plasmid designated pgap11. The complete nucleotide sequence of this structural gene was determined. The gene contains no intervening sequences, codon usage is highly biased, and the nucleotide sequence of the coding portion of this gene is 90% homologous to the other two glyceraldehyde-3-phosphate dehydrogenase genes (Holland, J. P., and Holland, M. J. (1980) J. Biol. Chem. 255, 2596-2605). Based on the extent of nucleotide sequence divergence among the three glyceraldehyde-3-phosphate dehydrogenase genes, it is likely that they arose as a consequence of two duplication events and the gene contained on the hybrid plasmid designated pgap11 is a product of the first duplication event. All three structural genes share extensive nucleotide sequence homology in the 5'-noncoding regions adjacent to the three respective translational initiation codons. The gene contained on pgap11 is not homologous to the others downstream from the respective translational termination codon, however. The 5' termini of messenger RNAs synthesized from the three glyceraldehyde-3-phosphate dehydrogenase and two yeast enolase genes have been mapped to sites ranging from 36 to 82 nucleotides upstream from the respective translational initiation codons. In each case the 5' terminus of the mRNA maps to a region of strong nucleotide sequence homology which is shared by all five structural genes. These latter data confirm that all five structural genes are expressed during vegetative cell growth and further support the hypothesis that a portion of the 5'-noncoding flanking region of the yeast glyceraldehyde-3-phosphate dehydrogenase and enolase genes evolved from a common precursor sequence.

Base Sequence↗

PubMed1998

ATP is a cofactor of the Upf1 protein that modulates its translation termination and RNA binding activities.

The nonsense-mediated mRNA decay pathway decreases the abundance of mRNAs that contain premature termination codons and prevents suppression of nonsense alleles. The UPF1 gene in the yeast Saccharomyces cerevisiae was shown to be a trans-acting factor in this decay pathway. The Upf1p demonstrates RNA-dependent ATPase, RNA helicase, and RNA binding activities. The results presented here investigate the binding affinity of the Upf1p for ATP and the consequences of ATP binding on its affinity for RNA. The results demonstrate that the Upf1p binds ATP in the absence of RNA. Consistent with this result, the TR800AA mutant form of the Upf1p still bound ATP, although it does not bind RNA. ATP binding also modulates the affinity of Upf1p for RNA. The RNA binding activity of the DE572AA mutant form of the Upf1p, which lacks ATPase activity, still bound ATP as efficiently as the wild-type Upf1p and destabilized the Upf1p-RNA complex. Similarly, ATPgammaS, a nonhydrolyzable analogue of ATP, interacted with Upf1p and promoted disassociation of the Upf1p-RNA complex. The conserved lysine residue (K436) in the helicase motif Ia in the Upf1p was shown to be critical for ATP binding. Taken together, these findings formally prove that ATP can bind Upf1p in the absence of RNA and that this interaction has consequences on the formation of the Upf1p-RNA complex. Further, the results support the genetic evidence indicating that ATP binding is important for the Upf1p to increase the translation termination efficiency at a nonsense codon. Based on these findings, a model describing how the Upf1p functions in modulating translation and turnover and the potential insights into the mechanism of the Upf1p helicase will be discussed.

Adenosine Triphosphate↗

PubMed1990

The involvement of base 1054 in 16S rRNA for UGA stop codon dependent translational termination.

The deletion of the highly conserved cytidine nucleotide at position 1054 in E. coli 16S rRNA has been characterized to confer an UGA stop codon specific suppression activity which suggested a functional participation of small subunit rRNA in translational termination. Based on this structure-function correlation we constructed the three point mutations at site 1054, changing the wild-type C residue to an A, G or U base. The mutations were expressed from a complete plasmid encoded rRNA operon (rrnB) using a conditional expression system with the lambda PL-promoter. All three altered 16S rRNA molecules were expressed and incorporated into 70S ribosomal particles. Structural analysis of the protein and 16S rRNA moieties of the mutant ribosomes showed no differences when compared to wild-type particles. The phenotypic analysis revealed that only the 1054G base change led to a significantly reduced generation time of transformed cells, which could be correlated with the inability of the mutant ribosomes to specifically stop at UGA stop codons in vivo. The response towards UAA and UAG termination codons was not altered. Furthermore, in vitro RF-2 termination factor binding experiments indicated that the association behaviour of mutant ribosomes was not changed, enforcing the view that the UGA stop codon suppression is a direct consequence of the rRNA mutation. Taken together, these results argue for a direct participation of that 16S rRNA motif in UGA dependent translational termination and furthermore, suggest that termination factor binding and stop codon recognition are two separate steps of the termination event.

Base Composition↗

PubMed1981

Structure and properties of a bovine liver UGA suppressor serine tRNA with a tryptophan anticodon.

A bovine liver serine tRNA with a variety of unusual features has been sequenced and characterized. This tRNA is aminoacylated with serine, although it has a tryptophan anticodon CmCA. In ribosome binding assays, this tRNA (tRNASERCmCA) binds to the termination codon UGA and shows little or no binding in response to a variety of other codons including those for tryptophan and serine. The unusual codon recognition properties of this molecule were confirmed in an in vitro assay where this tRNA suppressed UGA termination. This is the first naturally occurring eucaryotic suppressor tRNA to be so characterized. Other unusual features, possibly related to the ability of this tRNA to read UGA, are the presence of two extra nucleotides, compared to all other tRNAs, between the universal residues U at position 8 and A at position 14 and the presence of an extra unpaired nucleotide within the double-stranded loop IV stem. This tRNA is also the largest eucaryotic tRNA sequenced to date (90 nucleotides). Despite its size, however, it contains only six modified residues, tRNASerCmCA shows extremely low homology to other mammalian serine (47-52% homology) or tryptophan (49% homology) tRNAs.

Animals↗

PubMed1996

Analysis of eukaryotic mRNA structures directing cotranslational incorporation of selenocysteine.

Translation of an mRNA encoding a selenoprotein requires that at least one UGA codon in the reading frame is recoded as a site for the insertion of selenocysteine. In eukaryotes, the termination codon recoding event is directed by a cis-acting signal element located in the 3' untranslated region of the gene. This 'selenocysteine insertion sequence' (SECIS) comprises conserved sequences in a region of extensive base-pairing. In order to study the structure-function relationships of the SECIS structure, we have applied a newly developed reporter gene system which allows analysis of stop codon suppression in animal cell lines. This system obviates the need for enzymatic or immunological estimation of selenoprotein synthesis, relying instead on the simple quantification of translational readthrough from the lacZ gene into the luciferase gene. The 3'-UTR of the phospholipid hydroperoxide glutathione peroxidase (PHGPx) gene was shown to contain a highly active SECIS element. Mutations in the base-paired sequences of other SECIS elements were used to analyse the significance of primary structure, secondary structure and pairing stability in the stem regions. The results demonstrate that the exact sequences of the paired nucleotides are comparatively unimportant, provided that a consensus combination of length and thermodynamic stability of the base-paired structures is maintained.

Animals↗

PubMed1995

BAP2, a gene encoding a permease for branched-chain amino acids in Saccharomyces cerevisiae.

To select the gene coding for an isoleucine permease, an isoleucine dependent strain (ilv1 cha1) was transformed with a yeast genomic multicopy library, and colonies growing at a low isoleucine concentration were selected. Partial sequencing of the responsible plasmid insert revealed the presence of a previously sequenced 609 codon open reading frame of chromosome II with homology to known permeases. Deletion, extra dosage and C-terminal truncation of this gene were constructed in a strain lacking the general amino acid permease, and amino acid uptake was measured during growth in synthetic complete medium. The following observations prompted us to name the gene BAP2 (branched-chain amino acid permease). Deletion of BAP2 reduced uptake of leucine, isoleucine and valine by 25-50%, while the uptake of 8 other L-alpha-amino acids was unaltered or slightly increased. Introduction of BAP2 on a centromere-based vector, leading to a gene dosage of two or slightly more, caused a 50% increase in leucine uptake and a smaller increase for isoleucine and valine. However, when the 29 C-terminal codons of the plasmid-borne copy of BAP2 were substituted, the cells more than doubled the uptake of leucine, isoleucine and valine, while no or little increase in uptake was observed for the other 8 amino acids.

Amino Acid Sequence↗

PubMed2001

A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes.

Point mutations can generate defective and sometimes harmful proteins. The nonsense-mediated mRNA decay (NMD) pathway minimizes the potential damage caused by nonsense mutations. In-frame nonsense codons located at a minimum distance upstream of the last exon-exon junction are recognized as premature termination codons (PTCs), targeting the mRNA for degradation. Some nonsense mutations cause skipping of one or more exons, presumably during pre-mRNA splicing in the nucleus; this phenomenon is termed nonsense-mediated altered splicing (NAS), and its underlying mechanism is unclear. By analyzing NAS in BRCA1, we show here that inappropriate exon skipping can be reproduced in vitro, and results from disruption of a splicing enhancer in the coding sequence. Enhancers can be disrupted by single nonsense, missense and translationally silent point mutations, without recognition of an open reading frame as such. These results argue against a nuclear reading-frame scanning mechanism for NAS. Coding-region single-nucleotide polymorphisms (cSNPs) within exonic splicing enhancers or silencers may affect the patterns or efficiency of mRNA splicing, which may in turn cause phenotypic variability and variable penetrance of mutations elsewhere in a gene.

Amino Acid Motifs↗

PubMed2004

Rare autosomal recessive cardiac valvular form of Ehlers-Danlos syndrome results from mutations in the COL1A2 gene that activate the nonsense-mediated RNA decay pathway.

Splice site mutations in the COL1A2 gene of type I collagen can give rise to forms of Ehlers-Danlos syndrome (EDS) because of partial or complete skipping of exon 6, as well as to mild, moderate, or lethal forms of osteogenesis imperfecta as a consequence of skipping of other exons. We identified three unrelated individuals with a rare recessively inherited form of EDS (characterized by joint hypermobility, skin hyperextensibility, and cardiac valvular defects); in two of them, COL1A2 messenger RNA (mRNA) instability results from compound heterozygosity for splice site mutations in the COL1A2 gene, and, in the third, it results from homozygosity for a nonsense codon. The splice site mutations led to use of cryptic splice donor sites, creation of a downstream premature termination codon, and extremely unstable mRNA. In the wild-type allele, the two introns (IVS11 and IVS24) in which these mutations occurred were usually spliced slowly in relation to their respective immediate upstream introns. In the mutant alleles, the upstream intron was removed, so that exon skipping could not occur. In the context of the mutation in IVS24, computer-generated folding of a short stretch of mRNA surrounding the mutation site demonstrated realignment of the relationships between the donor and acceptor sites that could facilitate use of a cryptic donor site. These findings suggest that the order of intron removal is an important variable in prediction of mutation outcome at splice sites and that folding of the nascent mRNA could be one element that contributes to determination of order of splicing. The complete absence of pro alpha 2(I) chains has the surprising effect of producing cardiac valvular disease without bone involvement.

Adult↗