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PubMed1989

Nucleotide sequence of the FNR-regulated fumarase gene (fumB) of Escherichia coli K-12.

The nucleotide sequence of a 3,162-base-pair (bp) segment of DNA containing the FNR-regulated fumB gene, which encodes the anaerobic class I fumarase (FUMB) of Escherichia coli, was determined. The structural gene was found to comprise 1,641 bp, 547 codons (excluding the initiation and termination codons), and the gene product had a predicted Mr of 59,956. The amino acid sequence of FUMB contained the same number of residues as did that of the aerobic class I fumarase (FUMA), and there were identical amino acids at all but 56 positions (89.8% identity). There was no significant similarity between the class I fumarases and the class II enzyme (FUMC) except in one region containing the following consensus: Gly-Ser-Xxx-Ile-Met-Xxx-Xxx-Lys-Xxx-Asn. Some of the 56 amino acid substitutions must be responsible for the functional preferences of the enzymes for malate dehydration (FUMB) and fumarate hydration (FUMA). Significant similarities between the cysteine-containing sequence of the class I fumarases (FUMA and FUMB) and the mammalian aconitases were detected, and this finding further supports the view that these enzymes are all members of a family of iron-containing hydrolyases. The nucleotide sequence of a 1,142-bp distal sequence of an unidentified gene (genF) located upstream of fumB was also defined and found to encode a product that is homologous to the product of another unidentified gene (genA), located downstream of the neighboring aspartase gene (aspA).

Aconitate Hydratase↗

PubMed1998

Selenium deficiency reduces the abundance of mRNA for Se-dependent glutathione peroxidase 1 by a UGA-dependent mechanism likely to be nonsense codon-mediated decay of cytoplasmic mRNA.

The mammalian mRNA for selenium-dependent glutathione peroxidase 1 (Se-GPx1) contains a UGA codon that is recognized as a codon for the nonstandard amino acid selenocysteine (Sec). Inadequate concentrations of selenium (Se) result in a decrease in Se-GPx1 mRNA abundance by an uncharacterized mechanism that may be dependent on translation, independent of translation, or both. In this study, we have begun to elucidate this mechanism. We demonstrate using hepatocytes from rats fed either a Se-supplemented or Se-deficient diet for 9 to 13 weeks that Se deprivation results in an approximately 50-fold reduction in Se-GPx1 activity and an approximately 20-fold reduction in Se-GPx1 mRNA abundance. Reverse transcription-PCR analyses of nuclear and cytoplasmic fractions revealed that Se deprivation has no effect on the levels of either nuclear pre-mRNA or nuclear mRNA but reduces the level of cytoplasmic mRNA. The regulation of Se-GPx1 gene expression by Se was recapitulated in transient transfections of NIH 3T3 cells, and experiments were extended to examine the consequences of converting the Sec codon (TGA) to either a termination codon (TAA) or a cysteine codon (TGC). Regardless of the type of codon, an alteration in the Se concentration was of no consequence to the ratio of nuclear Se-GPx1 mRNA to nuclear Se-GPx1 pre-mRNA. The ratio of cytoplasmic Se-GPx1 mRNA to nuclear Se-GPx1 mRNA from the wild-type (TGA-containing) allele was reduced twofold when cells were deprived of Se for 48 h after transfection, which has been shown to be the extent of the reduction for the endogenous Se-GPx1 mRNA of cultured cells incubated as long as 20 days in Se-deficient medium. In contrast to the TGA allele, Se had no effect on expression of either the TAA allele or the TGC allele. Under Se-deficient conditions, the TAA and TGC alleles generated, respectively, 1.7-fold-less and 3-fold-more cytoplasmic Se-GPx1 mRNA relative to the amount of nuclear Se-GPx1 mRNA than the TGA allele. These results indicate that (i) under conditions of Se deprivation, the Sec codon reduces the abundance of cytoplasmic Se-GPx1 mRNA by a translation-dependent mechanism and (ii) there is no additional mechanism by which Se regulates Se-GPx1 mRNA production. These data suggest that the inefficient incorporation of Sec at the UGA codon during mRNA translation augments the nonsense-codon-mediated decay of cytoplasmic Se-GPx1 mRNA.

3T3 Cells↗

PubMed1991

A nonsense mutation in the tyrosinase gene of Afghan patients with tyrosinase negative (type IA) oculocutaneous albinism.

We detected a nonsense mutation in the tyrosinase gene of two Afghan sibs with classical tyrosinase negative (type IA) oculocutaneous albinism. The mutation, a single base substitution at codon 178, creates an amber termination codon that truncates the 529 amino acid tyrosinase polypeptide at this position. The patients' parents are first cousins, and the patients are therefore homoallelic for this mutation.

Afghanistan↗

PubMed1992

Molecular and genetic analysis of a compound heterozygote for dysprothrombinemia of prothrombin Tokushima and hypoprothrombinemia.

The molecular and genetic basis of a compound heterozygote for dys- and hypoprothrombinemia was analyzed. Abnormal nucleotide sequences of the human prothrombin gene were screened by PCR-single-strand conformation polymorphism (PCR-SSCP) with endonuclease digestion and mutated primer-mediated PCR-RFLP. A single nucleotide substitution responsible for dysprothrombinemia of prothrombin Tokushima was detected, as were three polymorphisms. The mutation for hypoprothrombinemia was detected by PCR-single-strand conformation polymorphism (PCR-SSCP) with endonuclease digestion in exon 6, near MboII-RFLP and NcoI-RFLP. Sequencing of PCR-amplified genomic DNA revealed a single base insertion of thymine (T) at position 4177. The resulting frameshift mutation caused both an altered amino acid sequence from codon 114 and a premature termination codon (i.e., TGA) at codon 174 in exon 7. Because exon 7 encodes the kringle 2 domain preceding the thrombin sequence, this frameshift leads to the null prothrombin phenotype. The inheritance of the hypoprothrombinemia gene from the father to the proband was proved by PCR-SSCP with endonuclease digestion and mutated primer-mediated PCR-RFLP.

Amino Acid Sequence↗

PubMed1990

Identification of point mutations in the alpha-galactosidase A gene in classical and atypical hemizygotes with Fabry disease.

Efforts were directed to identify the specific mutations in the alpha-galactosidase A (alpha-Gal A) gene which cause Fabry disease in families of Japanese origin. By polymerase-chain-reaction-amplification of DNA from reverse-transcribed mRNA and genomic DNA, different point mutations were found in two unrelated Fabry hemizygotes. A hemizygote with classic disease manifestations and no detectable alpha-Gal A activity had a G-to-A transition in exon 1 (codon 44) which substituted a termination codon (TAG) for a tryptophan codon (TGG) and created an NheI restriction site. This point mutation would predict a truncated alpha-Gal A polypeptide, consistent with the observed absence of enzymatic activity and a classic Fabry phenotype. In an unrelated Japanese hemizygote who had an atypical clinical course characterized by late-onset cardiac involvement and significant residual alpha-Gal activity, a G-to-A transition in exon 6 (codon 301) resulted in the replacement of a glutamine for an arginine residue. This amino acid substitution apparently altered the properties of the enzyme such that sufficient enzymatic activity was retained to markedly alter the disease course. Identification of these mutations permitted accurate molecular heterozygote diagnosis in these families.

Adult↗

PubMed1996

The genetic code and cyclic codes.

We proposed previously a cyclic code made of 22 triplets, which we now call the AB code. It is made up of the following chain: AUGGUGCCAUUCAAGACUAUGA. The letters A, U, C, G represent the classical symbols of the (purine and pyrimidine) bases of the genetic code. This chain presents the following features: (1) when it is in cyclic form, it begins with the initiation codon AUG, ends with the termination codon UGA, and it can be read triplet after triplet by choosing 1 and only 1 representative of each synonymy class in the classical degenerate genetic code made of 64 triplets. The chain, therefore, possesses 1 and only 1 codon for each amino-acid; (2) except for the doublet CG, triplets of the chain begin with the 15 other possible doublets of bases (satisfying the "wobble" hypothesis presented by Crick); (3) it corresponds (except for 1 base) to the "loop" part of the CEnothera mitochondrial Gly-tRNA; (4) it can be modified, without loss of the properties (1) and (2), in such a way as to have 15 bases in common with the loop part of other mitochondrial tRNA's considered as primitive, like Ala-, Pro- and Arg-tRNA; (5) it contains the most frequent triplets, but not the most rare ones, appearing in the genome of numerous species; (6) it exhibits a coherent internal structure with respect to the molecular weight of its triplets. This structure, also found in the loop part of mitochondrial tRNA's, contains an excess of AU bases with respect to GC bases. This fact has no explanation in the classical probabilistic model of the tRNA's. Therefore, we propose the cyclic AB code as a primitive genetic structure with the essential coding properties of the present genetic code.

Codon↗

PubMed1998

Identification of a novel mutation of the CPO gene in a Japanese hereditary coproporphyria family.

Hereditary coproporphyria (HCP) is an autosomal dominant disease characterized by a deficiency of coproporphyrinogen oxidase (CPO) caused by a mutation in the CPO gene. Only 11 mutations of the gene have been reported in HCP patients. We report another mutation in a Japanese family. Polymerase chain reaction-single strand conformational polymorphism and direct sequence analyses demonstrated a C to T substitution in exon 1 of the CPO gene at nucleotide position 85, which lies in the putative presequence for targeting to mitochondria. This mutation changes the codon for glutamine to a termination codon at amino acid position 29. MaeI restriction analysis showed two other carriers in the family. The C-T mutation is located within a recently proposed putative alternative translation initiation codon (TIC-1), supporting that TIC-1 is the real TIC rather than TIC-2.

Adult↗

PubMed1981

Nonrandom patterns of codon usage and of nucleotide substitutions in human alpha- and beta-globin genes: an evolutionary strategy reducing the rate of mutations with drastic effects?

Nucleotide substitutions within a structural gene can cause two principal "drastic" phenotypic effects at the protein level: translatable leads to untranslatable and nonpolar hydrophobic in equilibrium hydrophilic amino acid substitutions. The sequence of nucleotides in the structural human alpha- and beta-globin genes and their variants were examined to determine whether codon usage, patterns of nucleotide substitutions, or both, reduced the relative and absolute rates of these unfavorable mutations. Based on translation of abnormal hemoglobins, it is likely that all 61 nontermination codons are potentially translatable, though only 47 are normally used. Moreover, codons that can mutate to a termination codon are never used whenever the corresponding amino acid is specified also by triplets that cannot mutate to termination by a single-step mutation. Thus, the number of opportunities to mutate to an untranslatable codon is reduced to the minimum compatible with the amino acid composition of these chains. The relative rates of U in equilibrium non-U substitutions were much lower than those of other substitutions. Because U residues must be involved in most termination mutations and in all nonpolar hydrophobic in equilibrium hydrophilic amino acid substitutions, there is a considerable reduction of mutational events, causing drastic phenotypic effects. These findings are likely to be the end result of evolutionary selection by yet unknown mechanisms.

Base Sequence↗

PubMed2006

Unconventional translation of mammalian LINE-1 retrotransposons.

Long Interspersed Element-1 (LINE-1 or L1) retrotransposons encode proteins required for their mobility (ORF1p and ORF2p), yet little is known about how L1 mRNA is translated. Here, we show that ORF2 translation generally initiates from the first in-frame methionine codon of ORF2, and that both ORF1 and the inter-ORF spacer are dispensable for ORF2 translation. Remarkably, changing the ORF2 AUG codon to any other coding triplet is compatible with retrotransposition. However, introducing a premature termination codon in ORF1 or a thermostable hairpin in the inter-ORF spacer reduces ORF2p translation or L1 retrotransposition to approximately 5% of wild-type levels. Similar data obtained from "natural" and codon optimized "synthetic" mouse L1s lead us to propose that ORF2 is translated by an unconventional termination/reinitiation mechanism.

Animals↗

PubMed2004

Nonsense-mediated mRNA decay: terminating erroneous gene expression.

Nonsense-mediated mRNA decay is a surveillance pathway that reduces errors in gene expression by eliminating aberrant mRNAs that encode incomplete polypeptides. Recent experiments suggest a working model whereby premature and normal translation termination events are distinct as a consequence of the spatial relationship between the termination codon and mRNA binding proteins, a relationship partially established by nuclear pre-mRNA processing. Aberrant termination then leads to both translational repression and an increased susceptibility of the mRNA to multiple ribonucleases.

3' Untranslated Regions↗

PubMed2006

Human SNPs resulting in premature stop codons and protein truncation.

Single nucleotide polymorphisms (SNPs) constitute the most common type of genetic variation in humans. SNPs introducing premature termination codons (PTCs), herein called X-SNPs, can alter the stability and function of transcripts and proteins and thus are considered to be biologically important. Initial studies suggested a strong selection against such variations/mutations. In this study, we undertook a genome-wide systematic screening to identify human X-SNPs using the dbSNP database. Our results demonstrated the presence of 28 X-SNPs from 28 genes with known minor allele frequencies. Eight X-SNPs (28.6 per cent) were predicted to cause transcript degradation by nonsense-mediated mRNA decay. Seventeen X-SNPs (60.7 per cent) resulted in moderate to severe truncation at the C-terminus of the proteins (deletion of >50 per cent of the amino acids). The majority of the X-SNPs (78.6 per cent) represent commonly occurring SNPs, by contrast with the rarely occurring disease-causing PTC mutations. Interestingly, X-SNPs displayed a non-uniform distribution across human populations: eight X-SNPs were reported to be prevalent across three different human populations, whereas six X-SNPs were found exclusively in one or two population(s). In conclusion, we have systematically investigated human SNPs introducing PTCs with respect to their possible biological consequences, distributions across different human populations and evolutionary aspects. We believe that the SNPs reported here are likely to affect gene/protein function, although their biological and evolutionary roles need to be further investigated.

Codon, Terminator↗

PubMed1982

Primary structure of the ompF gene that codes for a major outer membrane protein of Escherichia coli K-12.

The nucleotide sequence of the ompF gene coding for a major outer membrane protein of Escherichia coli K-12 has been determined and the amino acid sequence of the OmpF protein was deduced from it. The OmpF protein contains 340 amino acid residues, and is produced from a precursor having 22 extra amino acid residues, the signal peptide, at the amino terminus. The expected secondary structure of the OmpF protein had a high beta-sheet content with a low alpha-helix content. The promoter region and the transcription termination region of the ompF gene had a significantly high AT content, while the AT content of the coding region was about the same as the average AT content of the E. coli chromosome. Following the termination codon, a typical rho-independent transcription termination signal was observed. The codon usage in the ompF gene was highly nonrandom; the codons preferably utilized are those recognized by the most abundant species of isoaccepting tRNAs or those, among synonymous codons recognized by the same tRNA, that can interact more properly with the anticodon.

Amino Acid Sequence↗

PubMed1980

Polyoma virus DNA: complete nucleotide sequence of the gene which codes for polyoma virus capsid protein VP1 and overlaps the VP2/VP3 genes.

The nucleotide sequence of part of the late region of the polyoma virus genome was determined. It contains coding information for the major capsid protein VP1 and the C-terminal region of the minor proteins VP2 and VP3. In the sequence with the same polarity as late mRNA's, all coding frames are blocked by termination codons in a region around 48 units on the physical map. This is the region where the N-terminus of VP1 and the C-termini of VP2 and VP3 have been located (T. Hunter and W. Gibson, J. Virol. 28:240-253, 1978; S. G. Siddell and A. E. Smith, J. Virol. 27:427-431, 1978; Smith et al., Cell 9:481-487, 1976). There are two long uninterrupted coding frames in the late region of polyoma virus DNA. One lies at the 5' end of the sequence and contains potential coding sequences for VP2 and VP3. The other contains 383 consecutive sense codons starting with the ATG at nucleotide position 1,218, extends from 47.5 to 25.8 units counterclockwise on the physical map, and is located where the VP1 gene has been mapped. The VP1 gene overlaps the genes for proteins VP2/VP3 by 32 nucleotides and uses a different coding frame. From the DNA sequence, the amino acid sequence of VP1 was predicted. The proposed VP1 sequence is in good agreement with other data, namely, with the partial N-terminal amino acid sequence and the total amino acid composition. The VP1 coding frame terminates with a TAA codon at 25.8 map units. This is followed by an AATAAA sequence, which may act as a processing signal for the viral late mRNA's. When both nucleotide and amino acid sequences are compared with their counterparts in the related simian virus 40, extensive homologies are found over the entire region of the two viral genomes. Maximum homology appears to occur in those regions which code for the C-termini of the VP1 proteins. The overlap region of VP1 with VP2/VP3 of polyoma virus is shorter by 90 nucleotides than is that of simian virus 40 and shows very limited homology with the simian virus 40 sequence. This leads to the suggestion that the overlap segments of both viruses have been freed from stringency imposed on drifting during evolution and that proteins VP2 and VP3 of polyoma virus may have been truncated by the appearance of a termination codon within the sequence.

Amino Acid Sequence↗

PubMed1998

Elimination of an HuIFN alpha 2b readthrough species, produced in Escherichia coli, by replacing its natural translational stop signal.

When human interferon-alpha 2b (HuIFN alpha 2b) was expressed intracellularly in Escherichia coli as insoluble aggregates, a HuIFN alpha 2b molecular species of high molecular weight was detected, even after immunoaffinity chromatography and characterized by mass spectrometry and automatic sequencing. This HuIFN alpha 2b species was synthesized by an inefficient reading of the UGA natural stop codon, stopping the translation at another UGA in frame placed 10 codons downstream of the HuIFN alpha 2b stop signal. To avoid this translational readthrough process the UGA termination codon was replaced by UAA, which is frequently used in highly expressed E. coli genes. Simultaneously, almost all the HuIFN alpha 2b gene 3' noncoding region was removed. Analysis by SDS-PAGE and enzyme-linked immunosorbent assay revealed the elimination of the undesired HuIFN alpha 2b molecular species and an almost twofold increase in the expression level. These results indicate that both factors, the stop codon used and the length of the transcription unit should be taken into account when the expression in E. coli of heterologous proteins is desired.

Base Sequence↗

PubMed1980

Sequence of 1060 3'-terminal nucleotides of poliovirus RNA as determined by a modification of the dideoxynucleotide method.

The dideoxynucleotide method for sequencing DNA developed by Sanger et al. [Sanger, F., Nicklen, S. & Coulson, A. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467] was modified to allow sequence analysis of poliovirus RNA without recourse to cloning. Our method involves reverse transcription of poliovirus RNA followed by cDNA-dependent DNA synthesis in the presence of unlabeled dNTPs and 2',3'-dideoxynucleoside triphosphates, with Escherichia coli DNA polymerase I (Klenow) used to catalyze the reaction. DNA synthesis is primed by 5'-32P-labeled RNase T1- or RNase A-resistant oligonucleotides generated from poliovirus RNA. The sequence of 1060 nucleotides preceding the 3'-terminal poly(A) is presented. Based on the position of termination codons we propose that viral translation terminates at nucleotide -562.

Base Sequence↗

PubMed1996

Invertebrate synapsins: a single gene codes for several isoforms in Drosophila.

Vertebrate synapsins constitute a family of synaptic proteins that participate in the regulation of neurotransmitter release. Information on the presence of synapsin homologs in invertebrates has been inconclusive. We have now cloned a Drosophila gene coding for at least two inferred proteins that both contain a region with 50% amino acid identity to the highly conserved vesicle- and actin-binding "C" domain of vertebrate synapsins. Within the C domain coding sequence, the positions of two introns have been conserved exactly from fly to human. The positions of three additional introns within this domain are similar. The Drosophila synapsin gene (Syn) is widely expressed in the nervous system of the fly. The gene products are detected in all or nearly all conventional synaptic terminals. A single amber (UAG) stop codon terminates the open reading frame (ORF1) of the most abundant transcript of the Syn gene 140 amino acid codons downstream of the homology domain. Unexpectedly, the stop codon is followed by another 443 in-frame amino acid codons (ORF2). Using different antibodies directed against ORF1 or ORF2, we demonstrate that in the adult fly small and large synapsin isoforms are generated. The small isoforms are only recognized by antibodies against ORF1; the large isoforms bind both kinds of antibodies. We suggest that the large synapsin isoform in Drosophila may be generated by UAG read-through. Implications of such an unconventional mechanism for the generation of protein diversity from a single gene are discussed.

Amino Acid Sequence↗

PubMed1987

Identification and nucleotide sequence of a gene encoding 5'-phosphoribosylglycinamide transformylase in Escherichia coli K12.

5'-Phosphoribosylglycinamide transformylase (EC 2.1.2.2), encoded by the purN gene of Escherichia coli, catalyzes the synthesis of 5'-phosphoribosylformylglycinamide from 5'-phosphoribosylglycinamide (GAR). The mature protein, as deduced from the purN structural gene sequence, contains 212 amino acid residues and has a calculated Mr of 23,241. The purN gene is located adjacent to and immediately downstream from the purM gene encoding 5'-phosphoribosyl-5-aminoimidazole (AIR) synthetase where the initiation codon for GAR transformylase overlaps the termination codon of AIR synthetase. Based on polarity studies, the expression of the purN gene originates from the purM control region and thus forms a purMN operon. The E. coli GAR transformylase shows greater homology to the GAR transformylase domain of the trifunctional Gart polypeptide of Drosophila than to the single GAR transformylase of Saccharomyces. Immediately downstream from the purN gene of the purMN operon is a region of dyad symmetry capable of forming a hairpin stem and loop structure characteristic of a rho-independent terminator.

Acyltransferases↗

PubMed2002

Intranuclear degradation of nonsense codon-containing mRNA.

Most vertebrate mRNAs with premature termination codons (PTCs) are specifically recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD) while still associated with the nucleus. However, it is still a matter of debate whether PTCs can be identified by intranuclear scanning or only by ribosomes on the cytoplasmic side of the nuclear envelope. Here we show that inhibition of mRNA export by two independent approaches does not affect the downregulation of PTC-containing T-cell receptor beta transcripts in the nuclear fraction of mammalian cells, providing strong evidence for intranuclear NMD. Our results are fully consistent with recently reported evidence for nuclear translation and suggest that an important biological role for nuclear ribosomes is the early elimination of nonsense mRNA during a pioneer round of translation.

Active Transport, Cell Nucleus↗