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PubMed1990

Isolation and characterization of cloned cDNAs encoding human liver chlordecone reductase.

Chlordecone (Kepone), a toxic organochlorine pesticide, undergoes bioreduction to chlordecone alcohol in human liver. This reaction is controlled by a cytosolic enzyme, chlordecone reductase (CDR), which may be of the aldo-keto reductase family of xenobiotic metabolizing enzymes [Molowa et al. (1986) J. Biol. Chem. 261, 12624-12627]. To further investigate the primary structure and expression of CDR, we screened a library of human liver cDNAs cloned in the expression vector lambda gt11 and isolated an 800 bp cDNA that directed synthesis of a fusion protein recognized by polyclonal anti-CDR antibodies. Using this cDNA as a probe, we screened two human liver cDNA libraries and found several 1.2-kb cDNAs which would code for a polypeptide with 308 residues (35.8 kDa). However, a similar full-length cDNA, possibly the transcript of a pseudogene, contained an in-frame nonsense codon. The deduced protein sequence of CDR showed 65% similarity to the primary structure of human liver aldehyde reductase and 66% similarity to the inferred protein sequence of rat lens aldose reductase. A search of GenBank revealed significant nucleotide similarity to a cDNA coding for bovine lung prostaglandin f synthase and to a partial cDNA coding for frog lens rho-crystallin. Southern blot analysis of human genomic DNA displayed between 45 and 65 kilobases of DNA hybridizable to CDR cDNA and demonstrated several restriction fragment length polymorphisms among 26 individuals. Northern blot analysis of RNA from human, gerbil, rabbit, hamster, mouse, and rat livers disclosed hybridization with CDR cDNA only for the first three species.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Oxidoreductases↗
PubMed2003

A new strategy for the site-specific modification of proteins in vivo.

We recently developed a method for genetically incorporating unnatural amino acids site-specifically into proteins expressed in Escherichia coli in response to the amber nonsense codon. Here we describe the selection of an orthogonal tRNA-TyrRS pair that selectively and efficiently incorporates m-acetyl-l-phenylalanine into proteins in E. coli. We demonstrate that proteins containing m-acetyl-l-phenylalanine or p-acetyl-l-phenylalanine can be selectively labeled with hydrazide derivatives not only in vitro but also in living cells. The labeling reactions are selective and in general proceed with yields of >75%. In specific examples, m-acetyl-l-phenylalanine was substituted for Lys7 of the cytoplasmic protein Z domain, and for Arg200 of the outer membrane protein LamB, and the mutant proteins were selectively labeled with a series of fluorescent dyes. The genetic incorporation of a nonproteinogenic "ketone handle" into proteins provides a powerful tool for the introduction of biophysical probes for the structural and functional analysis of proteins in vitro or in vivo.

Amino Acyl-tRNA Synthetases↗
PubMed2006

Translational fidelity mutations in 18S rRNA affect the catalytic activity of ribosomes and the oxidative balance of yeast cells.

The function of mutations rdn1A, rdn1T, and rdn2 in 18S rRNA of Saccharomyces cerevisiae is investigated. The mutations correspond to substitutions C1054A, C1054U in helix 34, and G517A in helix 18 of 16S rRNA in Escherichia coli, respectively, in which the first and third mutations caused nonsense suppression, while C1054U caused no suppression. In yeast, rdn1A caused phenotypic suppression at nonsense codons, whereas rdn1T and rdn2 caused antisuppression. We provide in vitro evidence that, in addition, rdn1A decreases translational accuracy at sense codons as well, by a factor of 8, accompanied by extreme sensitivity to paromomycin, compatible with its error-prone character. Mutations rdn1T andrdn2 exhibit hyperaccuracy and paromomycin resistance. Thus, mutations in conserved rRNA regions may affect the same functions in the various species but in opposite directions. Mutation rdn1A, but not rdn1T or rdn2, affected also the catalytic activity of the ribosome, a 60S subunit activity. The rate of peptide bond formation was reduced to half its normal value, indicating a communication between the two subunits. Moreover, error-prone mutation rdn1A was less susceptible to oxidative modifications than wild type, indicated by decreased lipid peroxidation and nonprotein/protein disulfides, as well as by increased protein thiols. In contrast, hyperaccurate mutations rdn1T and rdn2 displayed increased oxidative stress. Our results suggest that the cells may consume more energy to achieve hyperaccuracy leading to increased oxidative modifications.

Base Sequence↗
PubMed2002

Adding L-3-(2-Naphthyl)alanine to the genetic code of E. coli.

An unnatural amino acid, L-3-(2-naphthyl)alanine, has been site-specifically incorporated into proteins in Escherichia coli. An orthogonal aminoacyl-tRNA synthetase was evolved that uniquely aminoacylates the unnatural amino acid onto an orthogonal amber suppressor tRNA, which delivers the acylated amino acid in response to an amber nonsense codon with translational fidelity greater than 99%. This result, together with the in vivo site-specific incorporation of O-methyl-L-tyrosine reported previously, demonstrate that this methodology may be applicable to a host of amino acids. The expansion of the genetic code to include amino acids beyond the common 20 would provide an opportunity to better understand and possibly enhance protein (and perhaps organismal) function.

Alanine↗
PubMed2003

Generation of a bacterium with a 21 amino acid genetic code.

We have generated a completely autonomous bacterium with a 21 amino acid genetic code. This bacterium can biosynthesize a nonstandard amino acid from basic carbon sources and incorporate this amino acid into proteins in response to the amber nonsense codon. The biosynthetic pathway for the amino acid p-aminophenylalanine (pAF) as well as a unique pAF synthetase and cognate tRNA were added to Escherichia coli. Denaturing gel electrophoresis and mass spectrometric analysis show that pAF is incorporated into myoglobin with fidelity and efficiency rivaling those of the common 20 amino acids. This and other such organisms may provide an opportunity to examine the evolutionary consequences of adding new amino acids to the genetic repertoire, as well as generate proteins with new or enhanced biological functions.

Amino Acyl-tRNA Synthetases↗
PubMed2003

A method for the generation of glycoprotein mimetics.

A general method for preparing glycoprotein mimetics with defined glycan structure using the Z domain protein as an example is reported. An unnatural amino acid containing the keto group was site-specifically incorporated into a target protein, Z domain, in response to the amber nonsense codon with high translational fidelity and efficiency. An aminooxy saccharide derivative was then selectively coupled to this genetically encoded keto group. The resulting saccharide core was elaborated to a glycan complex with glycosyltransferases. Alternatively, aminooxy analogues of more complicated glycans were prepared and directly attached to the keto group. Homogeneous glycoprotein mimetics thus prepared should prove useful for the study of carbohydrate effects on glycoprotein structure and function. This method may also lead to the production of glycoprotein therapeutics from Escherichia coli.

Acetylglucosamine↗
PubMed2004

A genetically encoded photocaged amino acid.

We have developed a second orthogonal tRNA/synthetase pair for use in yeast based on the Escherichia coli tRNALeu/leucyl tRNA-synthetase pair. Using a novel genetic selection, we have identified a series of synthetase mutants that selectively charge the amber suppresor tRNA with the C8 amino acid, alpha-aminocaprylic acid, and the photocaged amino acid, o-nitrobenzyl cysteine, allowing them to be inserted into proteins in yeast in response to the amber nonsense codon, TAG.

Base Sequence↗
PubMed2005

Caged phosphoproteins.

We present the chemical and biological synthesis of caged phosphoproteins using the in vitro nonsense codon suppression methodology. Specifically, phosphoamino acid analogues of serine, threonine, and tyrosine with a single photocleavable o-nitrophenylethyl caging group were synthesized as the amino acyl tRNA adducts for insertion into full-length proteins. For this purpose, a novel phosphitylating agent was developed. The successful incorporation of these bulky and charged amino acids into the alpha-subunit of the nicotinic acetyl choline receptor (nAChR) and the vasodilator-stimulated phosphoprotein (VASP) using an in vitro translation system is reported.

Cyclic AMP-Dependent Protein Kinases↗
PubMed2006

The incorporation of a photoisomerizable amino acid into proteins in E. coli.

An orthogonal aminoacyl tRNA synthetase/tRNA pair has been evolved that allows the incorporation of the photoisomerizable amino acid phenylalanine-4'-azobenzene (AzoPhe) into proteins in E. coli in response to the amber nonsense codon. Further, we show that AzoPhe can be used to photoregulate the binding affinity of catabolite activator protein to its promoter. The ability to selectively incorporate AzoPhe into proteins at defined sites should make it possible to regulate a variety of biological processes with light, including enzyme, receptor, and ion channel activity.

Amino Acyl-tRNA Synthetases↗
PubMed2006

The genetic incorporation of a distance probe into proteins in Escherichia coli.

The unnatural amino acid p-nitrophenylalanine (pNO2-Phe) was genetically introduced into proteins in Escherichia coli in response to the amber nonsense codon with high fidelity and efficiency by means of an evolved tRNA/aminoacyl-tRNA synthetase pair from Methanocuccus jannaschii. It was shown that pNO2-Phe efficiently quenches the intrinsic fluorescence of Trp in a distance-dependent manner in a model GCN4 basic region leucine zipper (bZIP) protein. Thus, the pNO2-Phe/Trp pair should be a useful biophysical probe of protein structure and function.

Amino Acid Sequence↗
PubMed2006

A genetically encoded infrared probe.

An orthogonal tRNA/aminoacyl-tRNA synthetase pair has been evolved that makes it possible to selectively and efficiently incorporate para-cyanophenylalanine (pCNPhe) into proteins in E. coli at sites specified by the amber nonsense codon, TAG. Substitution of pCNPhe for histidine-64 in myoglobin (Mb) affords a sensitive vibrational probe of ligand binding. This methodology provides a useful infrared reporter of protein structure, biomolecular interactions, and conformational changes.

Models, Molecular↗
PubMed1998

Misacylated Transfer RNAs Having a Chemically Removable Protecting Group.

The 4-pentenoyl group and a number of derivatives have been studied as protecting groups for N(alpha) of the aminoacyl moiety in misacylated tRNAs. The unsubstituted 4-pentenoyl group itself was found to function as efficiently as any of the derivatives studied. Four different N-(4-pentenoyl)aminoacyl-tRNA(CUA)s were prepared and shown to undergo deprotection readily upon admixture of aqueous iodine; the derived misacylated tRNAs all functioned well as suppressors of a nonsense codon in an in vitro protein biosynthesizing system. Also prepared were four N(alpha)-(4-pentenoyl)aspartyl-tRNA(CUA)s that were protected on the side chain carboxylate as the nitroveratryl ester. Following treatment with aqueous iodine, the misacylated suppressor tRNAs incorporated the aspartate derivatives into position 27 of dihydrofolate reductase by suppression of a UAG codon in the mRNA. The suppression yields were significantly better than those obtained when side chain protection was absent. The resulting "caged proteins" were inactive, but full catalytic potential was restored by irradiation under conditions sufficient to effect deprotection of the side chain carboxylate moiety.

Journal Article↗
PubMed1997

Regulated expression of plant tRNA genes by the prokaryotic tet and lac repressors.

The prokaryotic tet operator (tetO) sequence was inserted at positions upstream and downstream of sequences encoding the Arabidopsis thaliana tRNA(Lys)AUC or tRNA(Trp)AUC suppressor tRNAs, and tRNA expression in carrot protoplasts was measured by translational suppression of a nonsense codon in a luciferase reporter gene. Regulation of tRNA expression by the tetracycline repressor (tetR) occurred from genes with the tetO inserted at position -1 (for the tRNA(Trp)AUC gene), or at positions -2, -6 and -10 (for the tRNA(Lys)AUC gene), and repression reached 90%. The inducer tetracycline (Tc) restored tRNA expression. Similarly, carrot protoplasts transfected with human tRNA(Ser)AUC genes containing the lac operator (lacO) in their 5'-flanking sequence with or without the lac repressor (lacI) gene, conditionally expressed tRNAs which suppressed the luciferase reporter. Up to 30-fold repression occurred by the lactose repressor when lacO was located at position -1 of the tRNA(Ser)AUC coding sequence. In the presence of the inducer isopropyl-beta-thiogalactoside (IPTG), repression was relieved. These results demonstrate that sequences flanking tRNA genes can strongly influence tRNA expression in plants, and in a conditional fashion when bound by inducible proteins.

Arabidopsis↗
PubMed1979

Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli.

The complete nucleotide sequence of hepatitis B virus genome (subtype ayw) cloned in Escherichia coli has been determined using the Maxam and Gilbert method and the dideoxynucleotide method. This sequence is 3,182 nucleotides long. Location of the nonsense codons shows that the coding capacity of the L chain is larger than the coding capacity of the S chain. Eight open regions, able to code for polypeptide chains larger than 100 amino acids, have been located. Region 6, which is the largest, covers more than 80% of the genome. The gene S which codes for polypeptide I of the Hbs Ag and was previously located between coordinates 95.1 and 73.6 is contained in region 7.

Base Sequence↗
PubMedDate not supplied

Recurrent mutations in haemophilia A give evidence for CpG mutation hotspots.

Haemophilia A is a common disorder of blood coagulation caused by a deficiency of factor VIII. It is inherited as an X-linked recessive trait, and one-third of all cases are thought to result from de novo mutations. The clinical severity of haemophilia A varies markedly among different families and a subset of the patients with severe disease develop antibodies against factor VIII, called inhibitors. Because of this heterogeneity, it is likely that many different molecular lesions result in haemophilia A. Indeed, of the nine mutations described to date, all appear to be unique changes. However in this study of 83 patients with haemophilia A we have identified two different point mutations, one in exon 18 and one in exon 22, that have recurred independently in unrelated families. Each mutation produces a nonsense codon by a change of CG to TG, and each occurred de novo on the X-chromosome donated by the maternal grandfather. These observations strongly support the view that CpG dinucleotides are mutation hotspots.

Base Composition↗
PubMed1988

Gene for a novel tRNA species that accepts L-serine and cotranslationally inserts selenocysteine.

The biological requirement of the trace element selenium was recognized 40 years ago. Selenium is incorporated into several enzymes and transfer RNA species of both prokaryotic and eukaryotic origin. In enzymes which contain a selenopolypeptide, selenium is present as covalently bound selenocysteine which participates in the catalytic reaction. Sequence analysis of the genes coding for two selenoproteins, formate dehydrogenase H from Escherichia coli and glutathione peroxidase from mouse and man, demonstrated that an in-frame UGA opal nonsense codon directs the incorporation of selenocysteine. In the case of formate dehydrogenase incorporation occurs cotranslationally. Recently, we identified four genes whose products are required for selenocysteine incorporation in E. coli. We report here that one of these genes codes for a tRNA species with unique properties. It possesses an anticodon complementary to UGA and deviates in several positions from sequences, until now, considered invariant in all tRNA species. This tRNA is aminoacylated with L-serine by the seryl-tRNA ligase which also charges cognate tRNASer. Selenocysteine, therefore, is synthesized from a serine residue bound to a natural suppressor tRNA which recognizes UGA.

Base Sequence↗
PubMed2001

Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice.

Pigmentary glaucoma is a significant cause of human blindness. Abnormally liberated iris pigment and cell debris enter the ocular drainage structures, leading to increased intraocular pressure (IOP) and glaucoma. DBA/2J (D2) mice develop a form of pigmentary glaucoma involving iris pigment dispersion (IPD) and iris stromal atrophy (ISA). Using high-resolution mapping techniques, sequencing and functional genetic tests, we show that IPD and ISA result from mutations in related genes encoding melanosomal proteins. IPD is caused by a premature stop codon mutation in the Gpnmb (GpnmbR150X) gene, as proved by the occurrence of IPD only in D2 mice that are homozygous with respect to GpnmbR150X; otherwise, similar D2 mice that are not homozygous for GpnmbR150X do not develop IPD. ISA is caused by the recessive Tyrp1b mutant allele and rescued by the transgenic introduction of wildtype Tyrp1. We hypothesize that IPD and ISA alter melanosomes, allowing toxic intermediates of pigment production to leak from melanosomes, causing iris disease and subsequent pigmentary glaucoma. This is supported by the rescue of IPD and ISA in D2 eyes with substantially decreased pigment production. These data indicate that pigment production and mutant melanosomal protein genes may contribute to human pigmentary glaucoma. The fact that hypopigmentation profoundly alleviates the D2 disease indicates that therapeutic strategies designed to decrease pigment production may be beneficial in human pigmentary glaucoma.

Animals↗