Cooperative control of translational fidelity by ribosomal proteins in Escherichia coli

Summary Two spontaneous mutants of Escherichia coli strain KMBL-146 selected for resistance to the aminoglycoside antibiotic neamine show severe restriction of amber suppressors in vivo. Purified ribosomes from the mutant strains exhibit low neamine-induced misreading in vitro and a decreased affinity for the related antibiotic streptomycin.Biochemical analysis shows that the mutants each have two modified 30S ribosomal proteins, S12 and S5. In agreement with these results, genetic analysis shows that two mutations are present, neither of which confers resistance to neamine by itself; the mutation located in gene rpxL (the structural gene for protein S12) confers streptomycin dependence but this dependence is suppressed in the presence of the second mutation, located in gene rpxE (the structural gene for protein S5).     

Localization of the structural gene for ribosomal protein L19 (rplS) in Escherichia coli

Summary A temperature-sensitive mutant derived from an E. coli K12 strain, PA3092, was found to have an alteration in the ribosomal protein L19 (Isono et al., 1977). This mutant is a double mutant with a temperature-sensitivity mutation and a mutation leading to the structural alteration of L19 protein. Crosses with various Hfr strains and transductions with P1kc have revealed that the latter mutation maps at 56.4 min, between pheA and alaS. From the fact that two other mutations causing different types of alterations in L19 protein also map at this locus, the gene affected by these mutations was concluded to be the structural gene for the ribosomal protein L19 (rplS).     

Nucleotide sequence of dihydrofolate reductase genes from trimethoprim-resistant mutants of Escherichia coli. Evidence that dihydrofolate reductase interacts with another essential gene product

Summary We report the construction of recombinant plasmids containing the dihydrofolate reductase structural gene (fol) from several trimethoprim-resistant mutants of Escherichia coli. Strains carrying some of these plasmids produced approximately 6% of their soluble cell protein as dihydrofolate reductase and are therefore excellent sources of the purified enzyme for inhibitor binding or mechanistic studies. The nucleotide sequence of the fol region from each of the plasmids was determined. A plasmid derived from a K_i mutant which produced a dihydrofolate reductase with lowered affinity for trimethoprim contained a mutation in the structural gene that altered the sequence of the polypeptide in a conserved region which is adjacent to the dihydrofolate binding site. Two other independently-isolated mutants which overproduced dihydrofolate reductase had a mutation in the-35 region of the fol promoter. One of them, strain RS35, was also temperature-sensitve for growth in minimal medium. This phenotype was shown to be the result of an additional mutation in a locus unlinked to fol by P1 transduction. The fol regions from two temperature-independent revertants of strain RS35 were sequenced. One of these had a mutation within the dihydrofolate reductase structural gene which altered some properties of the enzyme. This confirmed some previous enzymological data which suggested that some revertants of strain RS35 had mutations in fol (Sheldon 1977). These results suggest that dihydrofolate reductase interacts physically with some other essential gene product in E. coli.     

A nonsense mutation in the structural gene for glutamine synthetase leading to loss of nitrogen regulation in Klebsiella aerogenes

Summary An amber mutation (glnA3711), the first nonsense mutation isolated in Klebsiella aerogenes, is described. When amber suppressors were present, the mutant made active glutamine synthetase which was more thermolabile than wild type, showing that glnA3711 lies in the structural gene for glutamine synthetase. Strains carrying the glnA3711 allele were unable to express nitrogen regulation of genes coding for histidase, asparaginase, and glutamate dehydrogenase unless amber suppressors were also present. These results support a model that expression of gene(s) from the glnA promoter is required for nitrogen regulation in K. aerogenes.     

Isolation, characterization and physiological properties of an autolytic-deficient mutant of Streptococcus pneumoniae

Summary A spontaneous mutation in the gene lyt encoding the pneumococcal autolysin has been characterized. This mutation, named lyt-32, which behaves as a high-efficiency marker in pneumococcal transformation, is a single base pair GC deletion causing the appearance of two consecutive termination codons in the amino terminal part of the sequence of the autolysin gene. The mutant lyt gene did not code for a polypeptide of relative molecular mass corresponding to the pneumococcal E form amidase in Escherichia coli maxicells. Pneumococcal cells containing the lyt-32 mutation (M32) were fully transformable, multiplied at a normal growth rate forming small chains and showed a tolerant response when treated with beta-lactam antibiotics. Strain M32 represents the first example of a mutant of Streptococcus pneumoniae completely lacking amidase as a consequence of an alteration in the structural gene coding for the pneumococcal autolysin.     

Origin and evolution of the waxy phenotype in Amaranthus hypochondriacus: evidence from the genetic diversity in the Waxy locus

The existence of polymorphism in the Waxy locus in a large gene pool of 53 strains with various waxy phenotypes from samples of Amaranthus hypochondriacus collected from different regions was investigated in an origin-and-evolution study. First, we screened all strains for a mutation point (G–A polymorphism in exon 6) by using PCR–RFLP and/or direct sequence analysis. The results showed that the nonsense mutation in the coding region (exon 6) of the Waxy gene was responsible for the change in perisperm starch, leading to a waxy phenotype in all strains. Second, phylogenetic analysis, which was based on the Waxy variation, indicated diverse waxy types occurring separately and independently in certain domesticated regions in Mexico. Finally, we designated nine molecular types by comparing obvious structural variations in the coding region of the Waxy gene. Among the molecular types, A. hypochondriacus contained Type III in three subtypes with the waxy phenotype, with evolutionary routes that could originate from Type II in accordance with G–A polymorphism. In addition, these types had the same mutation points by which the Waxy gene was converted into the waxy phenotype. Therefore, the present results showed that the nonsense mutation is a unique event in the evolution of waxy phenotypes in this crop. This study will provide useful information for understanding the evolutionary process of the waxy phenotype.     

Identification of the structural gene for the S9 ribosomal protein in the fungus Podospora anserina: a new protein involved in the control of translational accuracy

Summary AS9-1 was isolated as a mutation restoring growth in a strain carrying the ribosomal mutation su12-1. The AS9-1 mutation confers a weak antisuppressor effect and a low level of resistance to paromomycin. Two-dimensional polyacrylamide gel electrophoresis patterns of the ribosomal proteins from AS9-1 strains show an altered S9 protein which is more basic than the wild-type form. The presence of the two forms of the protein (wild-type and mutant) in heterocaryotic strains strongly suggests that AS9 is the structural gene for the ribosomal protein S9.     

Mutational alteration of the breakage/resealing subunit of bacteriophage T4 DNA topoisomerase confers resistance to antitumor agent m-AMSA

Summary Bacteriophage T4 provides a simple model system in which to examine the mechanism of action of antitumor agents that have been proposed to attack type II DNA topoisomerases. Prior results demonstrated that T4 type II DNA topoisomerase is the target of antitumor agent 4-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in phage-infected Escherichia coli: a point mutation in topoisomerase structural gene 39 was shown to confer both m-AMSA-resistant phage growth and m-AMSA-insensitive topoisomerase activity. We report here that a point mutation in T4 topoisomerase structural gene 52 can also independently render both phage growth and topoisomerase activity resistant to m-AMSA. The DNA relaxation and DNA cleavage activities of this newly isolated mutant topoisomerase were significantly insensitive to m-AMSA. The drug-resistance mutation in gene 52, as well as that in gene 39, alters the DNA cleavage site specificity of wild-type T4 topoisomerase. This fording is consistent with a mechanism of drug action in which both topoisomerase and DNA participate in formation of the drug-binding site.     

PET1402, a nuclear gene required for proteolytic processing of cytochrome oxidase subunit 2 in yeast

The nuclear mutation pet ts1402 prevents proteolytic processing of the precursor of cytochrome oxidase subunit 2 (cox2) in Saccharomyces cerevisiae. The structural gene PET1402 was isolated by genetic complementation of the temperature-sensitive mutation. DNA sequence analysis identified a 1206-bp open reading frame, which is located 215 by upstream of the PET122 gene. The DNA sequence of PET1402 predicts a hydrophobic, integral membrane protein with four transmembrane segments and a typical mitochondrial targeting sequence. Weak sequence similarity was found to two bacterial proteins of unknown function. Haploid cells containing a null allelle of PET1402 are respiratory deficient.     

Alport syndrome caused by inversion of a 21 Mb fragment of the long arm of the X-chromosome comprising exon 9 through 51 of the COL4A5 gene

The X-linked form of Alport syndrome (AS) is caused by mutation in the COL4A5 gene located at Xq22.3 and encoding the α5-chain of type IV-collagen. More than 400 different mutations have so far been detected in the COL4A5 gene. Not all mutations, however, will be detected using an exon-by-exon mutation detection strategy such as SSCP analysis or direct sequencing. We have previously reported the results of SSCP analysis of 81 patients suspected of X-linked AS. Genomic DNA from these 81 patients was also analyzed for larger genomic rearrangements, using Southern blotting analysis. Abnormal band patterns were found in three patients, two of which were caused by single base substitutions in the coding region, also detected by the SSCP analysis. Here we report the results of the analysis of a larger structural COL4A5 rearrangement that escaped the SSCP analysis. The rearrangement was found to be an inversion of a 21 Mb fragment of the COL4A5 gene comprising exon 9 through 51 with proximal breakpoint within intron 8 at Xq22.3 and a distal breakpoint 56 kb upstream to the initiation codon in the RAB33A gene at Xq25. The inversion of exon 9 through 51 is expected to result in a truncated or absent α5(IV)-chain and has not previously been associated with AS. These findings emphasize the need for a supplement to mutation detection strategies such as SSCP analysis and direct sequencing, in order to detect more complicated structural COL4A5 rearrangements. Larger structural rearrangements constitute 2.3% (1/43) of the mutations in the present material.     

Isolation and characterization of further cis- and trans-acting regulatory elements involved in the synthesis of glucose-repressible alcohol dehydrogenase (ADHII) in Saccharomyces cerevisiae.

Summary Starting with yeast cells lacking the constitutive alcohol dehydrogenase activity (ADHI), mutants with partially glucose-insensitive formation of ADHII were isolated. Genetic analysis showed that four mutants (designated ADR3 ^c) were linked to the ADHII-structural gene, ADR2, and were cis-dominant. On derepression, two of them produced elevated ADHII-levels, indicating a promotor function of the altered controlling site. The other ADR3 ^c-mutant alleles affected the ADHII-subunit association in diploids carrying two electrophoretically distinct alleles of the structural gene ADR2. Twelve semidominant constitutive mutants could be attributed to gene ADR1 (ADR1 ^c-alleles) previously identified by recessive mutants with blocked derepression. This suggested a positive regulatory role of the ADR1 gene product on the expression of the ADHII-structural gene. A pleiotropic mutation ccr1 (Ciriacy, 1977) was epistatic over glucose-resistant ADHII-formation caused by ADR1 ^c-alleles. From this it was concluded that CCR1 specifies for a product co-activating the structural gene or modifying the ADR1-gene product. A further regulatory element (gene designation ADR4) not linked to the structural gene could be identified upon isolation of recessive constitutive mutants adr4 from a ccr1 ADR1 ^c-double mutant.     

Characterization of tryptophan synthase alpha subunit mutants of Arabidopsis thaliana

 Three mutations in the Arabidopsis thaliana gene encoding the alpha subunit of tryptophan synthase were isolated by selection for resistance to 5-methylanthranilate or 5-fluoroindole, toxic analogs of tryptophan pathway intermediates. Plants homozygous for trp3-1 and trp3-2 are light-conditional tryptophan auxotrophs, while trp3-100 is a more leaky mutant. Genetic complementation crosses demonstrated that the three mutations are allelic to each other, and define a new complementation group. All three mutants have decreased steady-state levels of tryptophan synthase alpha protein, and the trp3-100 polypeptide exhibits altered electrophoretic mobility. All three mutations were shown to be in the TSA1 (tryptophan synthase alpha subunit) structural gene by several criteria. Firstly, the trp3-1 mutation is linked to TSA1 on the bottom of chromosome 3. Secondly, the trp3-1 mutation was complemented when transformed with the wild-type TSA1 gene. Finally, DNA sequence analysis of the TSA1 gene revealed a single transition mutation in each trp3 mutant. Received: 28 May 1996 / Accepted: 19 June 1996     

The gene for ribosomal protein L25 (rplY) maps at 47.3 min near nalA in Escherichia coli K-12.

Summary A temperature sensitive mutant, termed JE1306, derived from Escherichia coli strain PA3092 was found to have an alteration in the ribosomal protein L25. Crosses with various Hfr strains and transductions with P1kc phage have revealed that the mutation maps at 47.3 min between nalA and fpk, in a region where no ribosomal protein gene has so far been located. The gene affected by this mutation is most probably the structural gene for protein L25 (rplY), because a strain heteromerozygous for the region shows both wild type and mutant forms of protein L25.     

Mutants that show increased sensitivity to hydrogen peroxide reveal an important role for the pentose phosphate pathway in protection of yeast against oxidative stress

We have isolated several mutants ofSaccharomyces cerevisiae that are sensitive to oxidative stress in a screen for elevated sensitivity to hydrogen peroxide. Two of the sixteen complementation groups obtained correspond to structural genes encoding enzymes of the pentose phosphate pathway. Allelism of thepos10 mutation (POS forperoxidesensitivity) to thezwf1/met1 mutants in the structural gene for glucose 6-phosphate dehydrogenase was reported previously. The second mutation,pos18, was complemented by transformation with a yeast genomic library. The open reading frame of the isolated gene encodes 238 amino acids. No detectable ribulose 5-phosphate epimerase activity was found in thepos18 mutant, suggesting that the corresponding structural gene is affected in this mutant. For that reason the gene was renamedRPE1 (forribulose 5-phosphateepimerase).RPE1 was localized to chromosome X. The predicted protein has a molecular mass of 25 966 Daltons, a codon adaptation index (CAI) of 0.32, and an isoelectric point of 5.82. Database searches revealed 32 to 37% identity with ribulose 5-phosphate epimerases ofEscherichia coli, Rhodospirillum rubrum, Alcaligenes eutrophus andSolanum tuberosum. We have characterizedRPE1 by testing enzyme activities inrpe1 deletion mutants and in strains that overexpressRPE1, and compared the hydrogen peroxide sensitivity ofrpe1 mutants to that of other mutants in the pentose phosphate pathway. Interestingly, all mutants tested (glucose 6-phosphate dehydrogenase, gluconate 6-phosphate dehydrogenase, ribulose 5-phosphate epimerase, transketolase, transaldolase) are sensitive to hydrogen peroxide.     

A missense mutation in the gene coding for ribosomal protein S17 (rpsQ) leading to ribosomal assembly defectivity in Escherichia coli.

Summary The conditionally lethal mutation, 286lmis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and was found to cotransduce at 97% with rpsE (S5). The 2861mis mutation leads to thermosensitivity and impaired assembly in vivo of 30S ribosomal particles at 42°C. The strain carrying the mutation has an altered S17 ribosomal protein; the mutational alteration involves a replacement of serine by phenylalanine in protein S17. Spontaneous reversion to temperature independence can restore the normal assembly in vivo of 30S ribosomal subunits at 42°C and the normal chromatographical sehaviour of the S17 ribosomal protein in vitro. We conclude therefore that the 2861mis mutation affects the structural gene for protein S17 (rpsQ).     

Classification of perA sequences and their correlation with autoaggregation in typical enteropathogenic Escherichia coli isolates collected in Japan and Thailand

Enteropathogenic Escherichia coli (EPEC) strains produce a bundle‐forming pilus (BFP) that mediates localized adherence (LA) to intestinal epithelial cells. The major structural subunit of the BFP is bundlin, which is encoded by the bfpA gene located on a large EAF plasmid. The perA gene has been shown to activate genes within the bfp operon. We analyzed perA gene polymorphism among typical (eae‐ and bfpA‐ positive) EPEC strains isolated from healthy and diarrheal persons in Japan (n= 27) and Thailand (n= 26) during the period 1995 to 2007 and compared this with virulence and phenotypic characteristics. Eight genotypes of perA were identified by heteroduplex mobility assay (HMA). The strains isolated in Thailand showed strong autoaggregation and had an intact perA, while most of those isolated in Japan showed weak or no autoaggregation, and had a truncated perA due to frameshift mutation. The degree of autoaggregation was well correlated with adherence to HEp‐2 cells, contact hemolysis and BFP expression. Our results showed that functional deficiency due to frameshift mutation and subsequent nonsense mutation in perA reduced BFP expression in typical EPEC strains isolated in Japan.     

Gene cloning in Aspergillus nidulans: isolation of the isocitrate lyase gene (acuD)

Summary An Aspergillus nidulans gene library was constructed in a high-frequency transformation vector, pDJB3, based on the Neurospora crassa pyr4 gene. This gene library was used to isolate the structural gene for isocitrate lyase (acuD) by complementation of a deficiency mutation following transformation of A. nidulans. Plasmids rescued in Escherichia coli were able to transform five different A. nidulans acuD mutants. Transformation using plasmids containing the cloned fragment resulted in integration at the acuD locus in six of nine transformants.     

Characterization of the cyrl-2 UGA mutation in Saccharomyces cerevisiae

Summary cyrl-2 is a temperature-sensitive mutation of the yeast adenylate cyclase structural gene, CYR1. The cyrl-2 mutation has been suggested to be a UGA mutation since a UGA suppressor SUP201 has been isolated as a suppressor of the cyrl-2 mutation. Construction of chimeric genes restricted the region containing the cyrl-2 mutation, and the cyrl-2 UGA mutation was identified at codon 1282, which lies upstream of the region coding for the catalytic domain of adenylate cyclase. Alterations in the region upstream of the cyrl-2 mutation site result in null mutations. The complete open reading frame of the cyrl-2 gene expressed under the control of the GAL1 promoter complemented cyrl-dl in a galactose-dependent manner. These results suggest that at the permissive temperature weak readthrough occurs at the cyrl-2 mutation site to produce low levels of active adenylate cyclase. An endogenous suppressor in yeast cells is assumed to be responsible for this readthrough.     

DNA sequencing of the Escherichia coli ribonuclease III gene and its mutations

Summary A 0.7 kb DNA fragment of the Escherichia coli K12 chromosome was shown to contain the structural gene for RNAse III (rnc). The DNA sequence of the gene was determined and its alteration in an RNAse III defective mutant, AB301-105, was identified. DNA sequence analysis also showed that a secondary-site suppressor of a temperature-sensitive mutation in the E. coli ribosomal protein gene, rpsL, occurred within the rnc gene, providing genetic evidence for the interaction of ribosomal proteins with RNAse III, which in turn acts on the nascent ribosomal RNA during assembly of ribosomes in E. coli.     

Characterization of an iron sensitive Mud1 mutant in E. coli lacking the ribonucleotide reductase subunit B2

The mutant, generated by a Mud1 insertion, formed long non-viable filaments in the presence of iron and air. Under anaerobic conditions normal growth in the presence of iron was observed. The mutation was mapped by P1 transductions at 48 min on the genetic map of Escherichia coli. By Southern blotting the insertion point was determined to be in nrdB, the structural gene for the ribonucleotide reductase subunit B2. The mutation could be complemented by the cloned nrdB gene. Up to now it was assumed that E. coli possesses only one enzyme for the synthesis of deoxyribunucleotides and only conditional lethal (temperature sensitive) mutants were isolated in nrdB. The insertion of Mud1 in nrdB should lead to a complete loss of the essential B2 subunit. Since the strain was able to grow under anaerobic conditions on minimal medium lacking deoxyribonucleotides an additional pathway for the synthesis of deoxyribonucleotides is postulated.