A candidate gene for human neurodegenerative disorders: a rat PKC gamma mutation causes a Parkinsonian syndrome.
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Animals↗
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Results for “Codon, Nonsense”
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Comparison of the immunoglobulin gene transcripts between immature B lineage acute lymphoblastic leukemia and the normal phenotypic counterparts in the bone marrow.
Immature B lineage acute lymphoblastic leukemia (ALL) is divided into two subtypes, 'pre-B' and 'early pre-B' ALL, by the presence or absence of cytoplasmic immunoglobulin (cIg). To study their clonal origin, we compared mu-chain transcripts in six cIg+ and eight cIg- ALL samples (CD10+/- CD19+ surface Ig-) with those in the normal phenotypic counterparts (CD10+ CD19+ surface Ig-) sorted from the bone marrow (BM). Northern blot analysis showed that the cIg+ ALL samples expressed greater amounts of mu-chain transcripts than the cIg- ALL samples. In the ALL samples and their counterparts, sequence analysis of mu-chain transcripts revealed infrequent somatic mutations of the V(H) genes and the similar usage of D and J(H) gene segments, but the length of complementarity determining region (CDR)-3 in the ALL samples was longer than that in the counterparts (50.0 +/- 15.5 vs 40.8 +/- 12.7 bp, P = 0.01). The mu-chain transcripts in the six cIg+ ALL samples and the counterparts (119/120 clones) had productive sequences, whereas those in the eight cIg- ALL samples had nonsense codons and/or frame shifts in their CDR-3. Our data suggest that a phenotype of ALL, 'pre-B' or 'early pre-B', is associated with V(H)-D-J(H) gene recombinatorial events, and that the CD10+ CD19+ surface Ig- population in the BM is not simply the cellular origin of ALL.
Antigens, CD19↗
Negative and positive regulation of the non-osmoregulated ompS1 porin gene in Salmonella typhi: a novel regulatory mechanism that involves OmpR.
The Salmonella typhi ompS1 gene codes for an outer membrane protein of the OmpC/OmpF porin family. It is expressed at very low levels, relative to the major porins. However, deletion analysis of the 5' regulatory region showed that the gradual removal of nucleotides -310 to -88, upstream from the P1 major transcriptional start-point, resulted in a stepwise increase in expression, reaching levels 10-fold above those for the ompC major porin gene. Hence, this 222 bp segment contains cis-acting regulatory elements involved in negative control. Primer extension analysis revealed the presence of three promoters: P1 activity was OmpR dependent; P2 was expressed at a lower level in the absence of OmpR; and P3 had a minor constitutive activity. OmpR bound preferentially to box II, an 18 bp F1/C1 canonical site, the removal (-88 to -66) of which resulted in a decrease in expression thus supporting its role in positive control. Expression of ompS1 was not induced by a set of stress conditions, including a shift in osmolarity, nor was the IHF regulator involved in negative control. An ompS1 homologue was found in E. coli K-12, which contains a nonsense codon and a shift in the reading frame, whereas Salmonella typhimurium contains an open reading frame in this region. Thus, S. typhi ompS1 provides novel features in OmpR regulation.
Bacterial Outer Membrane Proteins↗
Construction and expression of nonsense suppressor tRNAs which function in plant cells.
An Arabidopsis thaliana L. DNA containing the tRNA(TrpUGG) gene was isolated and altered to encode the amber suppressor tRNA(TrpUAG) or the ochre suppressor tRNA(TrpUAA). These DNAs were electroporated into carrot protoplasts and tRNA expression was demonstrated by the translational suppression of amber and ochre nonsense mutations in the chloramphenicol acetyltransferase (CAT) reporter gene. DNAs encoding tRNA(TrpUAG) and tRNA(TrpUAA) nonsense suppressor tRNAs caused suppression of their cognate nonsense codons in CAT mRNAs, with the tRNA(TrpUAG) gene exhibiting the greater suppression under optimal conditions for expression of CAT. The development of these translational suppressors which function in plant cells facilitates the study of plant tRNA gene expression and will make possible the manipulation of plant protein structure and function.
Anticodon↗
Hemophilia A. Detection of molecular defects and of carriers by DNA analysis.
To understand the molecular basis of hemophilia A and to provide heterozygote detection and prenatal diagnosis by DNA analysis, we used cloned factor VIII:C DNA fragments to study 10 affected families. In four of these families, inhibitors of factor VIII:C had developed in affected persons. In one such family a deletion of approximately 80 kb within the factor VIII:C gene was identified. Carriers of the deletion were identified through detection of an abnormal DNA fragment located at the deletion end points. In another family a single nucleotide change in the coding region of the factor VIII:C gene produced a nonsense codon leading to premature termination of factor VIII:C synthesis. Carrier detection was performed in eight female members of this four-generation family. In a third family a small change in the size of a restriction-endonuclease fragment correlated with the presence of the mutant gene, and in the other seven families the molecular defect has not yet been identified. In addition, we used two common polymorphic sites in the factor VIII:C gene to differentiate the normal from the defective gene in four of six obligate female carriers from families with patients in whom inhibitors did not develop. Carrier detection was possible in other members of these families. These data suggest that DNA analysis of the factor VIII:C gene provides an accurate method of carrier detection and, potentially, of prenatal diagnosis in at least 50 per cent of the pedigrees affected by hemophilia A.
Chromosome Deletion↗
Addition of the keto functional group to the genetic code of Escherichia coli.
Although the keto group is the most versatile of the functional groups in organic chemistry, it is absent in the genetically encoded amino acids. To overcome this natural limitation on protein biosynthesis, we have evolved an orthogonal tRNA-synthetase pair that makes possible the efficient incorporation of a keto amino acid, p-acetyl-l-phenylalanine, into proteins in E. coli with high translational fidelity in response to the amber nonsense codon. To demonstrate the utility of this keto amino acid, we have used it to modify a protein selectively with a small molecule fluorophore and biotin derivative. This additional genetically encoded amino acid should greatly expand our ability to manipulate protein structure and function both in vitro and in living cells.
Amino Acid Substitution↗
Mutant LYS2 mRNAs retained and degraded in the nucleus of Saccharomyces cerevisiae.
We previously demonstrated that mRNAs retained in the nucleus of Saccharomyces cerevisiae are subjected to a degradation system-designated DRN (degradation of mRNA in the nucleus), that is diminished in cbc1-Delta or cbc2-Delta mutants lacking components of the cap-binding complex and in rrp6-Delta mutants lacking Rrp6p, a 3' to 5' nuclear exonuclease. Two mutants, lys2-187 and lys2-121, were uncovered by screening numerous lys2 mutants for suppression by cbc1-Delta and rrp6-Delta. Both mutants were identical and contained the two base changes, one of which formed a TGA nonsense codon. LYS2 mRNAs from the lys2-187 and related mutants were rapidly degraded, and the degradation was suppressed by cbc1-Delta and rrp6-Delta. The U1A-GFP imaging procedure was used to show that the lys2-187 mRNA was partially retained in the nucleus, explaining the susceptibility to DRN. The creation of several derivatives of lys2-187 by site-directed mutagenesis revealed that the in-frame TGA by itself was not responsible for the increased susceptibility to DRN. Thus, mRNAs susceptible to DRN can be formed by a 2-bp change. Furthermore, this "retention signal" causing susceptibility to DRN is lost by altering one of the base pairs, establishing that mRNAs susceptible and unsusceptible to DRN can be attributed to a single nucleotide in the proper context.
Base Sequence↗
Stop codons affect 5' splice site selection by surveillance of splicing.
Pre-mRNA splicing involves recognition of a consensus sequence at the 5' splice site (SS). However, only some of the many potential sites that conform to the consensus are true ones, whereas the majority remain silent and are not normally used for splicing. We noticed that in most cases the utilization of such a latent intronic 5' SS for splicing would introduce an in-frame stop codon into the resultant mRNA. This finding suggested a link between SS selection and maintenance of an ORF within the mRNA. Here we tested this idea by analyzing the splicing of pre-mRNAs in which in-frame stop codons upstream of a latent 5' SS were mutated. We found that splicing with the latent site is indeed activated by such mutations. Our findings predict the existence of a checking mechanism, as a component of the nuclear pre-mRNA splicing machine, to ensure the maintenance of an ORF. This notion is highly important for accurate gene expression, as perturbations that would lead to splicing at these latent sites are expected to introduce in-frame stop codons into the majority of mRNAs.
Alternative Splicing↗
Biological and molecular evidence for the transgenosis of genes from bacteria to plant cells.
Specialized transducing phages (lambda and varphi80) have been used as vectors in the transfer of genes (wild type and mutant) from the bacterium Escherichia coli to haploid cell lines of the plants Lycopersicon esculentum and Arabidopsis thaliana. The overall phenomenon of transfer, gene maintenance, transcription, translation, and function has been termed transgenosis. Transgenosis of galactose and lactose operon genes was detected by survival and growth of the plant cells on defined medium with galactose and lactose as sole sources of bulk carbon. Phages carrying a defective operon, unrelated bacterial genes, or no bacterial genes, do not affect the normal result of death on these media, nor do they prevent growth on optimal growth media. Transgenosis of the E. coli gene z (lac operon) was confirmed by a biochemical-immunological test specific for E. coli beta-galactosidase. Plant cells were unable to effectively suppress an E. coli nonsense mutation. The E. coli mutant suppressor gene, supF(+), specifies insertion of tyrosine at amber (UAG) nonsense codons. Introduction of supF(+) results in a lethal transgenosis on medium normally optimal for plant cell growth. It is concluded that amber codons are vital to the life of plant cells. Differentiating cells of A. thaliana were not affected by supF(+).
Journal Article↗
Interference of nonsense mutations with eukaryotic messenger RNA stability.
The fine structure map of the yeast URA 3 gene was established by meiotic recombination, and amber nonsense mutations were located at different points on the map. The effect of the length of the labeling time on the specific radioactivity of ura 3 messenger RNA and on its repartition between poly(A)-RNA and RNA not containing poly(A) has been followed in nonsense mutants. Nonsense mutations reduce the messenger level without lowering its instantaneous rate of synthesis. The strength of the reduction depends on the position of the nonsense codon within the locus and concerns essentially the accumulation of polyadenylylated ura 3 mRNA.
Drug Stability↗
Model for regulation of the histidine operon of Salmonella.
A model is proposed that accounts for regulation of the histidine operon by a mechanism involving alternative configuration of mRNA secondary structure (the alternative stem model). New evidence for the model includes sequence data on three regulatory mutations. The first (hisO1242) is a mutation that deletes sequences needed to form the attenuator mRNA stem and causes constitutive operon expression. The second mutation (hisO9654) is a His- ochre (UAA) mutation in the leader peptide gene; the existence of this mutation constitutes evidence that the leader peptide gene is translated. The third mutation (hisO9663) is remarkable. It neither generates a nonsense codon nor affects a translated sequence; yet, it is suppressible by amber suppressors. We believe this mutation causes a His- phenotype by interfering with mRNA secondary structure. The suppressibility of the mutation is probably due to disruption of the attenuator stem by ribosomes that read through the terminator codon of the leader peptide gene. This explanation is supported by the observation of derepression of a wild-type control region in the presence of an amber suppressor. Evidence is presented that hisT mutants (which lack pseudouridine in the anticodon arm of histidine tRNA) may cause derepression of the his operon by slowing protein synthesis in the leader peptide gene.
Bacterial Proteins↗
Nucleotide sequence of the thrA gene of Escherichia coli.
The thrA gene of Escherichia coli codes for a single polypeptide chain having two enzymatic activities required for the biosynthesis of threonine, aspartokinase I and homoserine dehydrogenase I. This gene was cloned in a bacterial plasmid and its complete nucleotide sequence was established. It contains 2460 base pairs that encode for a polypeptide chain of 820 amino acids. The previously determined partial amino acid sequence of this protein is in good agreement with that predicted from the nucleotide sequence. The gene contains an internal sequence that resembles the structure of bacterial ribosome-binding sites, with an AUG preceded by four triplets, each of which can be converted to a nonsense codon by a single mutation. This suggests that the single polypeptide chain was formed by the fusion of two genes and that initiation of translation may occur inside the gene to give a protein fragment having only the homoserine dehydrogenase activity.
Aspartokinase Homoserine Dehydrogenase↗
The Escherichia coli supX locus is topA, the structural gene for DNA topoisomerase I.
Mutations in the supX locus, which result in the absence of DNA topoisomerase I enzyme activity in both Salmonella typhimurium and Escherichia coli, are all selected as suppressors of the leu-500 promoter mutation in S. typhimurium. To determine whether the supX locus is the structural gene topA for the DNA topoisomerase I enzyme or is a positive-acting regulator/activator gene for a nearby topA structural gene, nonsense mutations were selected in the E. coli supX gene carried on an F' episome in S. typhimurium cells. The cysB-topA region of the episomes with nonsense-mutant supX alleles were then cloned onto plasmid pBR322 and transformed into E. coli cells lacking a chromosomal supX gene. Three such E. coli strains, each carrying cloned DNA from episomes with different nonsense-mutant supX alleles, all lacked DNA topoisomerase I activity but expressed antigenic determinants specific to the enzyme; control cells lacked both enzyme activity and antigenic determinants. Maxicell studies of plasmid-coded proteins demonstrated the absence of the DNA topoisomerase I protein (100 kDa) in the three strains but the appearance of a new smaller peptide in each (36, 47, and 64 kDa). These new peptides must represent fragments of the enzyme resulting from translation termination at the supX nonsense codons and confirm the interpretation that the supX gene is topA, the structural gene for DNA topoisomerase I.
Cloning, Molecular↗
Involvement of ribosomal protein L7/L12 in control of translational accuracy.
The effects of two mutations, which map at the rplL locus and both give a changed 50S ribosomal protein L7/L12, were studied. Both mutations are associated with an increased misreading of all three nonsense codons in vivo and ribosomes from the mutants give an increased misreading of the phenylalanine codon UUU by tRNALeu in vitro. The rplL-associated misreading in vitro is not limited to a particular type of mRNA or tRNA. Results from a translational proofreading assay, using mutant ribosomes, suggest that protein L7/L12 is involved in the control of translational accuracy by contributing to the efficiency of a translational proofreading step(s).
Bacterial Proteins↗
Translation of a synthetic two-cistron mRNA in Escherichia coli.
A synthetic two-cistron expression system was constructed for the high-level expression of eukaryotic genes in Escherichia coli. This system was designed to overcome translational inhibition of mRNAs containing eukaryotic sequences. The first cistron in this system is a 31-base A + T-rich synthetic sequence that provides for efficient translation initiation. The second cistron contains the protein coding sequence for the eukaryotic gene. Insertion of the first cistron between the 5' untranslated region of the mRNA and the protein coding region separates the two and thereby potentially minimizes the formation of local secondary structures that might prevent ribosomes from binding and initiating translation. The 31-base cistron contains three nonsense codons (TAA), one in each of the three translational reading frames, and an 8-base Shine-Dalgarno sequence that is complementary to the 3' end of the 16S rRNA. The effects of translation of the first cistron in all three reading frames on the expression of the second cistron was examined. The most efficient expression of the second cistron seemed to occur when the stop codon that terminates translation of the first cistron is located 3' to the Shine-Dalgarno sequence and close to the AUG start codon for the second cistron. When the Shine-Dalgarno sequence was deleted from the first cistron, no detectable expression of the second cistron was observed. This two-cistron system has been used to express the gene encoding methionylalanyl bovine growth hormone with its native codons and the gene encoding methionyl human growth hormone at a level greater than 20% of total cell protein. In the case of human growth hormone, we show that the amount of gene product is not significantly diminished by placing a "functional" first cistron in front of a gene that can be expressed without a cistron.
Animals↗
Exonucleolytic proofreading by calf thymus DNA polymerase delta.
The fidelity of DNA synthesis by calf thymus DNA polymerase delta (pol delta) in vitro has been determined using an M13lacZ alpha nonsense codon reversion assay. Pol delta is highly accurate, producing on average less than 1 single-base substitution error for each 10(6) nucleotides polymerized. This accuracy is 10- and 500-fold greater than that of DNA polymerases alpha and beta, respectively, in the same assay. Three observations suggest that this higher fidelity results in part from proofreading of misinserted bases by the 3' to 5' exonuclease associated with pol delta. First, the exonuclease efficiently excises terminally mismatched bases. Second, both terminal mismatch excision and the fidelity of DNA synthesis by pol delta are reduced with increasing concentration of deoxynucleoside triphosphates in the synthesis reaction. These effects result from increasing the rate of polymerization relative to the rate of exonucleolytic excision and are hallmarks of exonuclease proofreading. Third, both terminal mismatch excision and fidelity decrease upon addition to the reaction mixture of adenosine monophosphate, a compound known to selectively inhibit the exonuclease but not the polymerase activity of pol delta. These results suggest that 3' to 5' exonuclease-dependent proofreading enhances the fidelity of DNA synthesis by a mammalian DNA polymerase in vitro.
Animals↗
Specificity of mismatch repair following transformation of Saccharomyces cerevisiae with heteroduplex plasmid DNA.
A method is described for genetic detection of mismatch repair products following transformation of Saccharomyces cerevisiae. The method is based on the detection of beta-galactosidase activity in clonal derivatives of cells transformed with heteroduplex plasmid DNA. Heteroduplex plasmid substrates were constructed by insertion of an oligonucleotide heteroduplex into the coding sequence of the Escherichia coli lacZ gene. The plasmid and oligonucleotides were designed so that one strand of the construct would code for a functional beta-galactosidase and the other strand would contain an in-frame nonsense codon. The frequencies of transformed clones containing only Lac+ cells, only Lac- cells, or a mixture of the two Lac phenotypes provided information on the efficiency of the repair reaction. With this method, plasmids carrying single-base substitution mismatches, a single-base frameshift mismatch (T/delta), or a 3-base-pair substitution mismatch (TGA/GAA) were tested. A/C, G/T, G/A, G/G, and T/delta mismatches were repaired with significantly greater efficiencies than C/C, A/A, T/T, and TGA/GAA. T/C was repaired with an intermediate efficiency. The frequencies of products obtained with G/G, G/A, and T/delta mismatches suggested modest inequality of repair in the two possible directions. Strains carrying the repair-deficient pms1-1 mutation were also tested. The efficiencies of repair of A/C, G/T, G/G, and A/A mismatches were reduced in pms1-1 cells compared with wild-type cells. In addition, a change in repair inequality was detected when transformation of the two strains with an A/C mismatch was compared.
DNA Repair↗
Molecular definition of bovine argininosuccinate synthetase deficiency.
Citrullinemia is an inborn error of metabolism due to deficiency of the urea cycle enzyme, argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5]. The disease was first described in humans but was recently reported in dairy cattle in Australia. Here we report the nucleotide sequence of the normal bovine cDNA for argininosuccinate synthetase and the mutation present in animals with citrullinemia. Analysis of DNA from affected animals by Southern blotting did not readily identify the mutation in the bovine gene. RNA (Northern) blotting revealed a major reduction in the steady-state amount of mRNA in the liver of affected animals to less than 5% of controls. The bovine cDNA was cloned and sequenced and revealed 96% identity with the deduced human sequence at the amino acid level. Starting with mutant bovine liver, the mRNA was reverse-transcribed; the cDNA product was amplified with the polymerase chain reaction, cloned, and sequenced. The sequence revealed a C----T transition converting arginine-86 (CGA) to a nonsense codon (TGA). A second C----T transition represented a polymorphism in proline-175 (CCC----CCT). The mutation and the polymorphism were confirmed by amplification of genomic DNA and demonstration with restriction endonuclease enzymes of both the loss of an Ava II site in DNA from mutant animals at codon 86 and the presence or absence of a Dde I site at codon 175. The loss of the Ava II site can be used for rapid, economical, nonradioactive detection of heterozygotes for bovine citrullinemia.
Amino Acid Metabolism, Inborn Errors↗