Conditionally Expressed Missense Mutations: The Basis for the Unusual Phenotype of an Apparent trpD Nonsense Mutant of SALMONELLA TYPHIMURIUM

Tryptophan auxotroph trp-28 is anomalous since preliminary mapping and suppression studies indicate the presence of a single amber nonsense mutation either late in trpE or early in trpD, but enzymological tests indicate the complete inactivation of both genes in this strain. Since the trpE and trpD genes are contiguous and encode the two subunits of a multifunctional enzyme complex, it was of interest to learn the mechanism of action of this apparent pleiotropic nonsense mutation. Our study has revealed that the phenotype of this strain derives not from a single mutation, but from the presence and interaction of multiple mutations. Besides the recognized amber mutation (designated trpD28), this strain carries two additional, conditionally expressed missense mutations (designated trpE1651 and trpD1652). The trpD28 amber codon maps in the promoter-proximal region 1 of trpD and eliminates the glutamine amidotransferase activity of the bifunctional trpD polypeptide. The trpD1652 mutation maps in the promoter-distal region 2 of trpD and severely reduces (but does not eliminate) the phosphoribosyl transferase activity of the trpD polypeptide. The trpE1651 mutation maps in the anterior part of trpE and causes a rapid loss of activity of the trpE polypeptide, but only when it exists as an uncomplexed subunit. The existence of the two missense mutations escaped prior notice in standard recombinational tests since the nature of each mutation is such that neither is detectable by the nutritional screens normally used in such tests unless an unsuppressed chainterminating mutation, such as trpD28, is also present.     

The reversion of trp frameshift mutations in mut,polA, lig and dnaE mutant strains of Escherichia coli

Mutator mutations mutL25, mutR34, and mutU4 had similar effects on the reversion of 4 trp frameshift mutations of known sequence. The mutation trpE9777, which resulted from the addition of an A–T base-pair to a run of 5 A–T base-pairs, was most strongly reverted by the 4 mutators. Reversion of trpE9777 was also increased by mutation polA1 (DNA polymerase I) and dnaE486 and dnaE511 (DNA polymerase III). No effect was found with the ligase mutations, lig-4 or lig-ts7. Mutations polAex1 and polA107, both deficient in the 5′ → 3′ exonuclease activity of DNA polymerase I, had different mutator effects; the factor increase in reversion of trpE9777 was 28-fold for polAex1, 6-fold for polA107, and 21-fold for polA1. The trpE9777 mutation is a useful indicator of frameshift mutator activity.     

Amino Acid Replacements Resulting from Suppression and Missense Reversion of a Chain-Terminator Mutation in Neurospora

The Neurospora crassa super-suppressor mutation, ssu-1, suppresses the auxotrophic phenotype of the mutant am(17) by inserting tyrosine at residue 313 of NADP-specific glutamate dehydrogenase, a position occupied in the wild type by glutamate. Two classes of am(17) revertants due to further mutation within the am gene have, respectively, tyrosine and leucine at residue 313. These replacements are consistent with a chain-terminating codon in am(17) of either the amber (UAG) or the ochre type (UAA), but are inconsistent with UGA. The Leu³¹³ and Tyr³¹³ variants of the enzyme have effective activity but are grossly different from the wild type in Michaelis constants (especially for ammonium) and heat stabilities at two different pH values. They show smaller but significant differences in these respects from each other.     

New Mutational Variants of Neurospora Nadp-Specific Glutamate Dehydrogenase

The am locus of Neurospora codes for NADP-dependent glutamate dehydrogenase (GDH). Four new am mutants that produced mutationally altered GDH have been characterized. Mutant am₁₁₉ is a CRM-negative, complementing mutant that maps between am₂ and am₁. The other three mutants are CRM formers that produce varieties of GDH that can be activated by glutamate or succinate. The GDH of am₁₃₀ and am₁₃₁ is similar in terms of activation properties to that of am₃. The GDH of am₁₂₂ requires very high concentrations of dicarboxylate for activity. The mutation in am₁₃₀ maps between am₁₄ and am₂ and resulted in a replacement at residue 75 of the GDH (pro → ser). The mutation in am₁₂₂ maps near am₁₁ and apparently resulted in the replacement of the tryptophan residue at position 389 with an unknown amino acid. The mutation in am₁₃₁ maps between am₂ and am₁.     

Identification and nucleotide sequence of the Acinetobacter calcoaceticus encoded trpE gene

Summary The trpE gene from Acinetobacter calcoaceticus encoding the anthranilate synthase component I was cloned, identified by deletion analysis and sequenced. It encodes a predicted polypeptide of 497 amino acids with a calculated molecular weight of 55323. Its primary structure shows 49% identical amino acids with the enzyme from Clostridium thermocellum, 45% with that of Thermus thermophilus and only 35% with that of Escherichia coli. The codon usage of the trpE genes encoding the most homologous enzymes differs greatly indicating selection for amino acid maintainance. The homologies are clustered in the C-terminal 200 amino acids of the sequences indicating that this part is important for enzymic activity.     

Photoreactivation reverses ultraviolet radiation induced premutagenic lesions leading to frameshift mutations in Escherichia coli

Summary The effect of photoreactivation of the ultraviolet radiation induced reversion of a trpE9777 frameshift mutation was studied in a uvrA6 derivative of Escherichia coli K12. Two different photoreactivation treatments were used, one providing a single flash of photoreactivating light and another providing 10 min of light from fluorescent lamps. The reversion frequency of the trpE9777 frameshift mutation was strongly reduced when subsequently exposed to visible light. The dose modification factor (the ratio of equally effective doses), for cells challenged with single-flash photoreactivation, for survival and induction of reversion to Trp⁺ was 3.6 and 3.4, respectively. UV induction of RecA protein synthesis was not reversed by a single flash of photoreactivation. The dose modification factor for 10 min of fluorescent lamp photoreactivation for survival and for induction of reversion to Trp⁺ was 6.5 and 6.3, respectively. The dose modification factor for 10 min of photoreactivation for induction of RecA protein was 1.7–2.5. Photoreactivation decreased the reversion of trpE9777 and increased survival to the same extent. We concluded that cyclobutyl pyrimidine dimers are the premutagenic lesions of UV mutagenesis of the trpE9777 allele in a uvrA6 background.     

Frameshift mutations affecting the N-terminal sequence of Neurospora NADP-specific glutamate dehydrogenase

A series of ultraviolet light-induced revertants from the mutant am6, mapping at the left-hand (“N-terminal”) end of the structural gene for NADP-specific glutamate dehydrogenase, have been shown to have amino acid substitutions in the N-terminal tryptic peptide. Only a few were found to have the wild-type sequence; the great majority had the replacement Ser5 → Pro and most had a further altered sequence extending one, two, three or four residues to the left. The most extensively altered revertant had a sequence with the extra residue Met at the N-terminus: Met-Leu-Thr-Phe-Pro-Pro- instead of the normal sequence N-acetyl-Ser-Asn-Leu-Pro-Ser-. The results are interpreted as meaning that am6 is a frameshift mutant, with the insertion of a base in the Ser5 codon, and that the revertants are all deletions at various positions to the left. Most of the revertants can be explained as single-base deletions, but some appear to have arisen by a more complex type of event. One revertant is a four-base deletion. The longest double-frameshifted sequence, on the basis of the simplest hypothesis as to its origin, defines the first 17 bases of the messenger RNA coding sequence. The altered sequences do not appear to affect the enzyme activity, except that they do, to different extents depending on the sequence, affect its sensitivity to heat.     

Functional analysis of three amino acid residues of purR repressor, Trpl47, Gln-218 and Gln-292 inSalmonella typhimurium

The amber mutation sites of 6 purR(am) mutants were determined by cloning and DNA sequencing. The results showed that the mutations were distributed at three different sites in PurR coding region, G⁷²¹(→A), C⁹³³(→T) and C¹¹⁵⁵(→T), which respectively turn Trp-147,Gln-218 and Gln-292 of PurR into TAG terminal codon. To determine the effect of the three amino acid residues on regulatory function of PurR protein 5 different kinds of tRNA suppressor genes, Su3, Su4, Su6, Su7 and Su9 were used for creating the PurR protein variants with single amino acid substitution. The results indicated that Cys, Glu, Gly, His and Arg which substituted Trp-147 respectively all could not recover the regulation function of PurR. It confirmed that Trp-147 is a critical amino acid for the PurR function. Gln-292 substituted respectively by the same amino acids also could not recover the PurR function, demonstrating that Gln-292 is also an important amino acid residue in PurR.     

Functional analysis of three amino acid residues of purR repressor, Trpl47, Gln-218 and Gln-292 in Salmonella typhimurium

The amber mutation sites of 6 purR(am) mutants were determined by cloning and DNA sequencing. The results showed that the mutations were distributed at three different sites in PurR coding region, G⁷²¹(→A), C⁹³³(→T) and C¹¹⁵⁵(→T), which respectively turn Trp-147, Gln-218 and Gln-292 of PurR into TAG terminal codon. To determine the effect of the three amino acid residues on regulatory function of PurR protein 5 different kinds of tRNA suppressor genes, Su3, Su4, Su6, Su7 and Su9 were used for creating the PurR protein variants with single amino acid substitution. The results indicated that Cys, Glu, Gly, His and Arg which substituted Trp-147 respectively all could not recover the regulation function of PurR. It confirmed that Trp-147 is a critical amino acid for the PurR function. Gln-292 substituted respectively by the same amino acids also could not recover the PurR function, demonstrating that Gln-292 is also an important amino acid residue in PurR.     

A frameshift mutation that elongates the penicillinase protein of Bacillus licheniformis

A penicillinase mutant penP102, isolated after ICR (acridine mustard) mutagenesis of Bacillus licheniformis strain 749/C, retains about 50% of the wild-type penicillinase specific activity. The penicillinase produced by this mutant differs from the wild-type protein in its sensitivity to pH and its electrophoretic behaviour. The penP102 mutation appears to have several other phenotypic effects, including an increase in the efficiency of release of the extracellular form of the enzyme.The penP102 penicillinase has been purified and its amino acid sequence compared to that of the wild-type enzyme. The mutation has resulted in the replacement of the last three amino acids of the wild-type enzyme and the addition of 17 residues at the carboxy-terminus. Comparison of the wild-type and mutant amino acid sequences shows that the mutational event is a single nucleotide deletion from the codon for asparagine²⁶⁵. Consideration of the possible nucleotide sequence for the region beyond the carboxy-terminus of the wild-type protein shows that there are no possible termination codons until four and six triplets beyond the codon for the carboxy-terminal lysine, indicating that the carboxy-terminus of the wild-type extracellular penicillinase is generated by proteolytic cleavage of a larger precursor protein.     

Analysis of a Mouse α-Globin Gene Mutation Induced by Ethylnitrosourea

A DBA/2 mouse treated with ethylnitrosourea sired an offspring whose hemoglobin showed an extra band following starch gel electrophoresis. The variant hemoglobin migrated to a more cathodal position in starch gel. Isoelectric focusing indicated that chain 5 of the mutant hemoglobin migrated to a more cathodal position than the normal chain 5 from DBA/2 mice and that the other α-globin, chain 1, was not affected. On focusing gels the phenotype of the mutant allele, Hba^y9, was expressed without dominance to normal chain 5, and Hba^y9/Hba^y9 homozygotes were fully viable in the laboratory. The molecular basis for the germinal mutation was investigated by analyzing the amino acid sequence of chain 5^y9, the mutant form of α-chain 5. A single amino acid substitution (His → Leu) at position 89 was found in chain 5^y9. We propose that ethylnitrosourea induced an A → T transversion in the histidine codon at position 89 (CAC → CTC). This mutation has apparently not been observed previously in humans, mice or other mammals, and its novel occurrence may be indicative of other unusual mutational events that do not ordinarily occur in the absence of specific mutagen exposure.     

Mutational variants of the Neurospora crassa NADP-specific glutamate dehydrogenase altered in a conformational equilibrium

Seven defective variants of the NADP-specific glutamate dehydrogenase of Neurospora crassa, resulting from missense mutations in the am gene, are quantitatively different from the wild type enzyme in the allosteric equilibrium between enzymically active A and inactive I conformations, and in the kinetics of conformational transitions between these states. These abnormalities have been defined using measurements of enzymic activity and of the intrinsic tryptophan fluorescence emission of the proteins.The protein from am¹(Ser336 → Phe) is hyperstable in the A conformation but this state is enzymically inactive because it fails to bind coenzyme. The other six variants are potentially active but are, to different extents, hyperstable in the I conformation. They form a series of analogues, those of am¹³¹ (substitution not determined), am¹³⁰(Pro75 → Ser), am³(Glu393 → Gly), am²(His142 → Gln), am¹⁹(Lys141 → Met) in order of increasing abnormality of the equilibrium position. am¹²²(Trp389 changed to an undetermined residue) resembles am¹⁹. The hyperstability is sufficient to explain the auxotrophy of am The proteins of am¹³¹ and am¹³⁰ are, in addition, abnormally prone to denaturation. These hyperstabilities of the I state are small in free energy terms, consistent with the fact that the defects of some variants may be corrected or partially corrected by second site substitutions or by complementation in hybrid hexamers with am¹ protein.Five out of seven amino acid substitutions known to affect this equilibrium (including Gln391 → Arg of revertant am¹⁹24) involve charged residues clustered around positions 141 and 391. Interactions between these two parts of the polypeptide are implicated in stabilizing the A state of the enzyme, possibly by providing protonatable groups or part of the dicarboxylate binding site, and in affecting the environment of a tryptophan residue responsible for the fluorescence difference of the two conformations.     

Mutation leading to gene fusion in the histidine operon of Salmonella typhimurium

A spontaneous polar mutation located in the region of an intercistronic border in the hiatidine operon of Salmonella was isolated in our laboratory. The mutant, R81, tests as a frameshift in reversion experiments but is prototrophic, capable of growth without histidine supplements despite lowered levels of certain histidine enzymes. The mutation affects the operator distal end of the D gene, causing production of an active histidinol dehydrogenase enzyme with an altered C-terminus. The mutation severely affects expression of the immediately succeeding gene in the translation sequence, hisC, suggesting either that the D–C border and possibly hisC are physically altered or that their normal function in translation is seriously impaired. We have previously described the fortuitous production from R81 of a non-polar derivative with fused D and C genes. This strain produces a bifunctional enzyme with normally separate dehydrogenase and aminotransferase activities present on dimers or multimers of a single fused polypeptide chain. We have now investigated in greater detail the R81 mutation by amino acid sequencing of the C-terminus of altered histidinol dehydrogenase. We find that the R81 mutation causes the addition of a “tail” of four amino acid residues to an otherwise normal dehydrogenase polypeptide chain. The results support our previous suggestion that the R81 mutation profoundly effects the D–C gene border and that this effect is prerequisite to gene fusion.     

Characterization of the L683P mutation of SLC26A9 in Xenopus oocytes

In the present study, we characterized a STAS-domain amino acid mutation of SLC26A9 having a significant impact on ion transport. We focused on the sole conserved L- leucine residue of the STAS domain identified among SLC26 members. We therefore characterized the L683P mutation of SLC26A9 in Xenopus oocytes by monitoring the protein functional expression (two-electrode technique for voltage-clamp analysis) and its presence at the cell membrane (surface protein biotinylation technique). This mutation was found to reduce Cl⁻ transport through SLC26A9 as well as the positive interaction exerted by SLC26A9 on CFTR ion transport activity. The origin of this effect is discussed in the light of the presence of the SLC26A9–L683P mutant at the plasma membrane.     

Gene structure in the tryptophan operon of Escherichia coli: Nucleotide sequence of trpC and the flanking intercistronic regions

We have determined the DNA sequence for the portion of the Escherichia coli tryptophan (trp) operon spanning trpC, which codes for the bifunctional enzyme N-(5′-phosphoribosyl)-anthranilic acid isomerase/indole-3-glycerol phosphate synthetase. The coding region consists of 1356 nucleotides, directing the synthesis of a polypeptide 452 amino acids in length. The predicted protein sequence is consistent with the amino acid composition of the pure enzyme, and with all known partial peptide sequences derived from this molecule. The enzyme is of particular functional interest, because it contains the catalytic activities for two sequential reactions in tryptophan biosynthesis in a single polypeptide chain.The nucleotide sequences of the junctions between trpC and its flanking genes, trpD and trpB, have also been determined. The trpD-trpC junction consists of six untranslated nucleotides and translation of trpC initiates at the second of two adjacent AUG codons. The trpC termination codon is separated from trpB by 11 nucleotides. The short non-translated regions flanking trpC distinguish it from trpA and trpD, whose initiation codons overlap the termination codons of the preceding genes (trpB and trpE), respectively. These differences in the intercistronic regions may reflect functional relationships between the products of adjacent genes in the operon.     

The nucleoid-associated DNA-binding protein H-NS is required for the efficient adaptation of Escherichia coli K-12 to a cold environment

The hns gene is a member of the cold-shock regulon, indicating that the nucleoid-associated, DNA-binding protein H-NS plays an important role in the adaptation of Escherichia coli to low temperatures. We show here that the ability to cope efficiently with a cold environment (12°C and 25°C) is strongly impaired in E. coli strains carrying hns mutations. Growth inhibition is much more pronounced in strains carrying the hns-206 allele (an ampicillin resistance cassette inserted after codon 37) than in those carrying the hns-205 mutation (a Tn10 insertion located in codon 93). A protein fragment (H-NS^*) is synthesized in strains carrying the hns-205::Tn10 mutation, suggesting that this truncated polypeptide is partially functional in the cold adaptation process. Analysis of the growth properties of strains harbouring four different low-copy-number plasmid-encoded hns genes that result in the production of C-terminally truncated H-NS proteins supports this proposal. H-NS^* proteins composed of 133, 117 or 94 amino-terminal amino acids partially complemented the severe cold-sensitive growth phenotype of the hns-206 mutant. In contrast, synthesis of a truncated H-NS protein with only 75 amino-terminal amino acids was insufficient to restore growth at low temperature.     

Expression in Escherichia coli and characterization of human growth-hormone-releasing factor

A chemically synthesized DNA sequence, coding for the 44 amino acid residues of human growth-hormone-releasing factor (GRF) preceded by a tryptophan codon, was cloned in frame with Escherichia coli trpE gene within a pBR322-derived plasmid. GRF was expressed in E. coli as a fused polypeptide chain (TrpE-GRF) and then the GRF amino acid sequence was released from the fused protein by specific chemical cleavage at the tryptophan residue using o-iodosobenzoic acid. The thioether group of the methionine residue of GRF was converted in the sulfonium salt derivative, in order to prevent irreversible oxidation of methionine to the sulfone derivative by the o-iodosobenzoic acid reagent. GRF was purified by HPLC and characterized in terms of amino acid composition after acid hydrolysis, protein sequencing and gel electrophoretic behaviour. These data clearly established that the biosynthetic GRF was identical to the natural one, except for the lack of amidation at the carboxyl-terminal amino acid. Far-ultraviolet circular dichroism measurements established that both biosynthetic and natural GRF are devoid of secondary structure in aqueous solution at neutral pH, whereas both peptide samples achieve a high percentage of helical structure in the presence of trifluoroethanol.     

Isolation and Characterization of the Azospirillum brasilense trpE(G) Gene,Encoding Anthranilate Synthase

The Azospirillum brasilense trpE gene has been isolated by DNA hybridization and by genetic complementation of an Escherichia coli trpE deletion mutant. DNA sequence analysis of a 3.1-kb PstI restriction fragment of A. brasilense revealed the presence of an open reading frame encoding a putative TrpE(G) fusion protein. Previously an A. brasilense clone containing trpGDC was identified (Zimmer et al. Mol Gen Genet 229:41–51, 1991). It can, therefore, be concluded that A. brasilense contains two trpG genes. A putative leader peptide is found upstream of trpE(G), containing three consecutive tryptophan residues. Putative terminator and anti-terminator loops have also been identified. The LLESX₁₀S motif, which is responsible for feedback inhibition by tryptophan in other TrpE proteins, is absent in the A. brasilense TrpE(G) fused protein. Received: 29 May 1996 / Accepted: 5 July 1996     

New Frame Shift Mutation in Puroindoline B in Indian Wheat Cultivars Hyb65 and NI5439

Puroindoline genes pinA and pinB are the main components of the 15 kD friabilin protein reported to be associated with kernel softness. However, grain hardness of Hyb65 and NI5439, the two Indian wheat varieties, could not be explained based on the earlier identified alleles in puroindolines in wheat. Hyb65 and NI5439 are hard but based on the earlier identified allelic forms of puroindolines both the varieties could have been soft. In this investigation, puroindolines (a and b) from Hyb65 and NI5439 were characterised to understand their role in determining grain hardness. The sequence of puroindoline genes from both the varieties indicated that there was no mutation in pinA. However, there was frame shift mutation in pinB generated by insertion of a guanine residue 126 bp downstream from the start codon in both the varieties. This created new hardness allele of pinB designated as pinb-D1h. Frame shift also culminated into stop codon (TGA) 231 bp downstream from the start codon terminating protein synthesis at 77^th amino acid position. Five more stop codons (4TGA & 1TAG) were also created to the downstream positions of the first stop codon because of frame shift. There was additional point mutation in NI5439 (transition from A to G) resulting into change of amino acid residue from thymine to arginine at 205^th nucleotide position. Thus single nucleotide change in pinB resulted into truncated pin B and consequently the harder texture.