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PubMed1991

DNA strand-specific mutations induced by (+/-)-3 alpha,4 beta-dihydroxy- 1 alpha,2 alpha-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene in the dihydrofolate reductase gene.

We previously showed that the preferred mutation induced by (+/-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy- 1,2,3,4-tetrahydrobenzo[c]phenanthrene (BcPHDE) in the dihydrofolate reductase gene in Chinese hamster ovary cells was a purine to thymine transversion on the nontranscribed strand at the sequence 5'-RRR-3' (R is a purine and the mutated base is underlined). To determine whether the observed mutational strand specificity was due to bias in the phenotypic selection, we designed a nonsense-codon reversion assay in which a triple purine target was present on both strands and all R----T transversion mutations yielded amino acid substitutions that were compatible with dihydrofolate reductase enzyme activity. From the size of the targets, a 2:1 ratio of mutations at the purines on the nontranscribed strand was expected if the DNA strands were mutationally equivalent. We isolated a total of 66 BcPHDE-induced revertants of two mutants that carry point mutations at either the 5' or the 3' end of the gene. All reversions at the 5' end arose by substitution on the nontranscribed strand; those at the 3' end showed a strand bias that favored this strand by 7:1. For both mutants, R----T transversions accounted for 88% of all the induced base changes. Thus, in this system, mutational strand bias is independent of the selection for phenotype. The results are consistent with the model of preferential repair of the transcribed strand as proposed by others. The involvement of RNA polymerase in the selective repair recruitment is discussed.

Animals↗

PubMed1991

Identification of the most common mutation within the porphobilinogen deaminase gene in Swedish patients with acute intermittent porphyria.

Acute intermittent porphyria (AIP) is a metabolic disorder characterized by a partial deficiency of the porphobilinogen deaminase (PBGD, EC 4.3.1.8) activity. Previous haplotype analysis combined with genealogical data suggested a common origin of the PBGD gene mutation in the AIP families originating from northern Sweden (Lappland), where the highest prevalence of the disease (1 in 1500) is observed. An AIP family from Lappland consisting of two patients and two unaffected subjects was investigated. The genomic DNA fragments of the PBGD gene were amplified by polymerase chain reaction (PCR) and directly sequenced, and the sequence of the coding region was compared with the normal sequence to identify the mutation. A base substitution, G to A, in exon 10 of the PBGD gene was identified. The mutation changes the codon for Trp198 to a stop codon (nonsense mutation) and creates a recognition site for the restriction enzyme Nhe I. Screening of 33 Swedish AIP families showed that 15 had this mutation. Genealogical data revealed that 12 of the 15 families were related to the northern family. This finding supports the hypothesis of a "founder effect" of the mutation in the families originating from Lappland. In addition, a method is described for detection of specific sequences in the genome by one-sided PCR using Taq polymerase. This method is simple, fast, and economical and can be substituted for hybridization analysis using allele-specific oligonucleotides.

Base Sequence↗

PubMed1997

Translational coupling by modulation of feedback repression in the IF3 operon of Escherichia coli.

A pseudoknot formed by a long-range interaction in the mRNA of the initiation factor 3 (IF3) operon is involved in the translational repression of the gene encoding ribosomal protein L35 by another ribosomal protein, L20. The nucleotides forming the 5' strand of the key stem of the pseudoknot are located within the gene for IF3, whereas those forming the 3' strand are located 280 nt downstream, immediately upstream of the Shine-Dalgarno sequence of the gene for L35. Here we show that premature termination of IF3 translation at a nonsense codon introduced upstream of the pseudoknot results in a substantial enhancement of L20-mediated repression of L35 expression. Conversely, an increase of IF3 translation decreases repression. These results, in addition to an analysis of the effect of mutations in sequences forming the pseudoknot, indicate that IF3 translation decreases L20-mediated repression of L35 expression. We propose that ribosomes translating IF3 disrupt the pseudoknot and thereby attenuate repression. The result is a novel type of translational coupling, where unfolding of the pseudoknot by ribosomes translating IF3 does not increase expression of L35 directly, but alleviates its repression by L20.

Bacterial Proteins↗

PubMed1997

Site-specific, photochemical proteolysis applied to ion channels in vivo.

A method for site-specific, nitrobenzyl-induced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K+ channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the "signature" Cys128-Cys142 disulfide loop of the nAChR alpha subunit.

Animals↗

PubMed1995

A hPMS2 mutant cell line is defective in strand-specific mismatch repair.

Human cells contain several homologs of the bacterial mutL gene required for mismatch repair, including a gene on chromosome 7 designated hPMS2. We have identified an endometrial carcinoma cell line, HEC-1-A, that has a C-->T mutation in hPMS2 that generates a nonsense codon and yields a protein truncated at the C terminus. No wild-type gene or gene product was detected. The missing amino acids in hPMS2 are highly conserved among PMS homologs, suggesting that they may be critical for function. In support of this, extracts of HEC-1-A cells are defective in repairing a variety of mismatched substrates. Moreover, di-, tri-, and tetranucleotide repeated sequences are highly unstable in single cell clones of HEC-1-A cells, and HEC-1-A cells are resistant to killing by N-methyl-N'-nitro-N-nitrosoguanidine. The results provide strong experimental support for the involvement of the hPMS2 gene product in mismatch repair in human cells and support the concept that a defective hPMS2 gene may lead to predisposition to certain forms of cancer.

Adenosine Triphosphatases↗

PubMed2001

A role for the Ppz Ser/Thr protein phosphatases in the regulation of translation elongation factor 1Balpha.

In vivo 32P-labeled yeast proteins from wild type and ppz1 ppz2 phosphatase mutants were resolved by bidimensional electrophoresis. A prominent phosphoprotein, which in ppz mutants showed a marked shift to acidic regions, was identified by mixed peptide sequencing as the translation elongation factor 1Balpha (formerly eEF1beta). An equivalent shift was detected in cells overexpressing HAL3, a inhibitory regulatory subunit of Ppz1. Subsequent analysis identified the conserved Ser-86 as the in vivo phosphorylatable residue and showed that its phosphorylation was increased in ppz cells. Pull-down experiments using a glutathione S-transferase (GST)-EF1Balpha fusion version allowed to identify Ppz1 as an in vivo interacting protein. Cells lacking Ppz display a higher tolerance to known translation inhibitors, such as hygromycin and paromomycin, and enhanced readthrough at all three nonsense codons, suggesting that translational fidelity might be affected. Overexpression of a GST-EF1Balpha fusion counteracted the growth defect associated to high levels of Ppz1 and this effect was essentially lost when the phosphorylatable Ser-86 is replaced by Ala. Therefore, the Ppz phosphatases appear to regulate the phosphorylation state of EF1Balpha in yeast, and this may result in modification of the translational accuracy.

Base Sequence↗

PubMed2003

129-derived strains of mice are deficient in DNA polymerase iota and have normal immunoglobulin hypermutation.

Recent studies suggest that DNA polymerase eta (poleta) and DNA polymerase iota (poliota) are involved in somatic hypermutation of immunoglobulin variable genes. To test the role of poliota in generating mutations in an animal model, we first characterized the biochemical properties of murine poliota. Like its human counterpart, murine poliota is extremely error-prone when catalyzing synthesis on a variety of DNA templates in vitro. Interestingly, when filling in a 1 base-pair gap, DNA synthesis and subsequent strand displacement was greatest in the presence of both pols iota and eta. Genomic sequence analysis of Poli led to the serendipitous discovery that 129-derived strains of mice have a nonsense codon mutation in exon 2 that abrogates production of poliota. Analysis of hypermutation in variable genes from 129/SvJ (Poli-/-) and C57BL/6J (Poli+/+) mice revealed that the overall frequency and spectrum of mutation were normal in poliota-deficient mice. Thus, either poliota does not participate in hypermutation, or its role is nonessential and can be readily assumed by another low-fidelity polymerase.

Animals↗

PubMed1993

Molecular and genetic analyses of the Caenorhabditis elegans dpy-2 and dpy-10 collagen genes: a variety of molecular alterations affect organismal morphology.

We have identified and cloned the Caenorhabditis elegans dpy-2 and dpy-10 genes and determined that they encode collagens. Genetic data suggested that these genes are important in morphogenesis and possibly other developmental events. These data include the morphologic phenotypes exhibited by mutants, unusual genetic interactions with the sqt-1 collagen gene, and suppression of mutations in the glp-1 and mup-1 genes. The proximity of the dpy-2 and dpy-10 genes (3.5 kilobase) and the structural similarity of their encoded proteins (41% amino acid identity) indicate that dpy-2 and dpy-10 are the result of a gene duplication event. The genes do not, however, appear to be functionally redundant, because a dpy-10 null mutant is not rescued by the dpy-2 gene. In addition, full complementation between dpy-2 and dpy-10 can be demonstrated with all recessive alleles tested in trans. Sequence analysis of several mutant alleles of each gene was performed to determine the nature of the molecular defects that can cause the morphologic phenotypes. Glycine substitutions within the Gly-X-Y portion of the collagens can result in dumpy (Dpy), dumpy, left roller (DLRol), or temperature-sensitive DLRol phenotypes. dpy-10(cn64), a dominant temperature-sensitive DLRol allele, creates an Arg-to-Cys substitution in the amino non-Gly-X-Y portion of the protein. Three dpy-10 alleles contain Tc1 insertions in the coding region of the gene. dpy-10(cg36) (DRLol) creates a nonsense codon near the end of the Gly-X-Y region. The nature of this mutation, combined with genetic data, indicates that DLRol is the null phenotype of dpy-10. The Dpy phenotype results from reduced function of the dpy-10 collagen gene. Our results indicate that a variety of molecular defects in these collagens can result in severe morphologic changes in C. elegans.

Alleles↗

PubMed1986

Frameshift suppression by thyA mutants of Escherichia coli K-12.

We have extended our previous study on the suppression of frameshift mutants by Escherichia coli thyA mutants by assaying suppression of 15 rIIB frameshift mutants of bacteriophage T4 on one of our suppressing thyA mutant strains. The majority of insertion mutants were suppressible, whereas none of the deletion mutants tested was suppressible. Frameshift suppression could be inhibited by adding thymidine to the assay medium, but was not affected by the presence of a restrictive rpsL mutation in the host strain. We suggest that the frameshift suppression event occurs at a nonsense codon generated by the frameshift mutation.

Amino Acids↗

PubMed1990

Isolation of omnipotent suppressors in an [eta+] yeast strain.

Omnipotent suppressors decrease translational fidelity and cause misreading of nonsense codons. In the presence of the non-Mendelian factor [eta+], some alleles of previously isolated omnipotent suppressors are lethal. Thus the current search was conducted in an [eta+] strain in an effort to identify new suppressor loci. A new omnipotent suppressor, SUP39, and alleles of sup35, sup45, SUP44 and SUP46 were identified. Efficiencies of the dominant suppressors were dramatically reduced in strains that were cured of non-Mendelian factors by growth on guanidine hydrochloride. Wild-type alleles of SUP44 and SUP46 were cloned and these clones were used to facilitate the genetic analyses. SUP44 was shown to be on chromosome VII linked to cyh2, and SUP46 was clearly identified as distinct from the linked sup45.

Alleles↗

PubMed1991

Intragenic suppressors of folding defects in the P22 tailspike protein.

Within the amino acid sequences of polypeptide chains little is known of the distribution of sites and sequences critical for directing chain folding and assembly. Temperature-sensitive folding (tsf) mutations identifying such sites have been previously isolated and characterized in gene 9 of phage P22 encoding the tailspike endorhamnosidase. We report here the isolation of a set of second-site conformational suppressors which alleviate the defect in such folding mutants. The suppressors were selected for their ability to correct the defects of missense tailspike polypeptide chains, generated by growth of gene 9 amber mutants on Salmonella host strains inserting either tyrosine, serine, glutamine or leucine at the nonsense codons. Second-site suppressors were recovered for 13 of 22 starting sites. The suppressors of defects at six sites mapped within gene 9. (Suppressors for seven other sites were extragenic and distant from gene 9.) The missense polypeptide chains generated from all six suppressible sites displayed ts phenotypes. Temperature-sensitive alleles were isolated at these amber sites by pseudoreversion. The intragenic suppressors restored growth at the restrictive temperature of these presumptive tsf alleles. Characterization of protein maturation in cells infected with mutant phages carrying the intragenic suppressors indicates that the suppression is acting at the level of polypeptide chain folding and assembly.

Alleles↗

PubMed1998

The spectrum of acridine resistant mutants of bacteriophage T4 reveals cryptic effects of the tsL141 DNA polymerase allele on spontaneous mutagenesis.

Mutations in the ac gene of bacteriophage T4 confer resistance to acridine-inhibition of phage development. Previous studies had localized the ac gene region; we show that inactivation of T4 Open Reading Frame 52.2 confers the Acr phenotype. Thus, 52.2 is ac. The resistance mechanism is unknown. The ac gene provides a convenient forward mutagenesis assay. Its compact size (156 bp) simplifies mutant sequencing and diverse mutant types are found: base substitutions leading to missense or nonsense codons, in-frame deletions or duplications within the coding sequence, deletion or duplication frameshifts, insertions, complex mutations, and large deletions extending into neighboring sequences. Comparisons of spontaneous mutagenesis between phages bearing the wild-type or tsL141 alleles of DNA polymerase demonstrate that the impact of the mutant polymerase is cryptic when total spontaneous mutant frequencies are compared, but the DNA sequences of the ac mutants reveal a substantial alteration of fidelity by the mutant polymerase. The patterns of base substitution mutagenesis suggest that some site-specific mutation rate effects may reflect hotspots for mutagenesis arising by different mechanisms. A new class of spontaneous duplication mutations, having sequences inconsistent with misaligned pairing models, but consistent with nick-processing errors, has been identified at a hotspot in ac.

Acridines↗

PubMed1999

Analysis of mutations in the yeast mRNA decapping enzyme.

A major mechanism of mRNA decay in yeast is initiated by deadenylation, followed by mRNA decapping, which exposes the transcript to 5' to 3' exonucleolytic degradation. The decapping enzyme that removes the 5' cap structure is encoded by the DCP1 gene. To understand the function of the decapping enzyme, we used alanine scanning mutagenesis to create 31 mutant versions of the enzyme, and we examined the effects of the mutations both in vivo and in vitro. Two types of mutations that affected mRNA decapping in vivo were identified, including a temperature-sensitive allele. First, two mutants produced decapping enzymes that were defective for decapping in vitro, suggesting that these mutated residues are required for enzymatic activity. In contrast, several mutants that moderately affected mRNA decapping in vivo yielded decapping enzymes that had at least the same specific activity as the wild-type enzyme in vitro. Combination of alleles within this group yielded decapping enzymes that showed a strong loss of function in vivo, but that still produced fully active enzymes in vitro. This suggested that interactions of the decapping enzyme with other factors may be required for efficient decapping in vivo, and that these particular mutations may be disrupting such interactions. Interestingly, partial loss of decapping activity in vivo led to a defect in normal deadenylation-dependent decapping, but it did not affect the rapid deadenylation-independent decapping triggered by early nonsense codons. This observation suggested that these two types of mRNA decapping differ in their requirements for the decapping enzyme.

Alleles↗

PubMed1976

Marker effects on reversion of T4rII mutants.

The frequencies of 2-aminopurine- and 5-bromouracil-induced A:T leads to G:C transitions were compared at nonsense sites throughout the rII region of bacteriophage T4. These frequencies are influenced both by adjacent base pairs within the nonsense codons and by extracodonic factors. Following 2AP treatment, they are high in amber (UAG) and lower in opal (UGA) codons than in allelic ochre (UAA) codons. In general, 5BU-induced transitions are more frequent in both amber and opal codons than in the allelic ochre codons. 2AP- and 5BU-induced transition frequencies in the first and third positions of opal codons are correlated with those in the corresponding positions of the allelic ochre codons. Similarly, the frequencies of 2AP-induced transition in the first and second positions of amber codons and their ochre alleles are correlated. However, there is little correlation between the frequencies of 5BU-induced transitions in the first and second positions of allelic amber and ochre codons.

2-Aminopurine↗

PubMed2000

Mutations in SCO2 are associated with a distinct form of hypertrophic cardiomyopathy and cytochrome c oxidase deficiency.

Mutations in SCO2, a cytochrome c oxidase (COX) assembly gene located on chromosome 22, have recently been reported in patients with fatal infantile cardio-encephalomyopathy and severe COX deficiency in heart and skeletal muscle. The Sco2 protein is thought to function as a copper chaperone. To investigate the extent to which mutations in SCO2 are responsible for this phenotype, a complete sequence analysis of the gene was performed on ten patients in nine families. Mutations in SCO2 were found in three patients in two unrelated families. We detected two missense mutations, one of which (G1541A) results in an E140K substitution adjacent to the highly conserved CxxxC metal-binding site. The other (C1634T) results in an R171W substitution more distant from the copper-binding site. A nonsense codon was found on one allele in two siblings presenting with a rapidly progressive fatal cardio-encephalomyopathy. Interestingly, all patients so far reported are compound heterozygotes for the G1541A mutation, suggesting that this is either an ancient allele or a mutational hotspot. The COX deficiency in patient fibroblasts (approximately 50%) did not result in a measurable decrease in the steady-state levels of COX complex polypeptide subunits and could be rescued by transferring chromosome 22, but not other chromosomes. These data indicate that mutations in SCO2 cause a fatal infantile mitochondrial disorder characterized by hypertrophic cardiomyopathy and encephalopathy, and point to the presence of one or more other genes, perhaps in the copper delivery pathway, in this clinical phenotype.

Amino Acid Sequence↗

PubMed2003

Single nucleotide polymorphisms in the protamine-1 and -2 genes of fertile and infertile human male populations.

Although various genetic factors have been implicated in human male infertility, the causative genes for the different types of idiopathic male infertility have not been elucidated. Protamines, which are the major DNA-binding proteins in the sperm nucleus, package the DNA into the sperm head. Analysis of the human protamine-1 (PRM1) and -2 (PRM2) gene sequences in 226 sterile male patients and in 270 proven-fertile male volunteers revealed four single nucleotide polymorphisms (SNPs) in the PRM1 coding region, which did not cause any amino acid substitutions, and one SNP in the PRM2 gene, which produced translation termination. We also observed one SNP in the 3' non-coding region of the PRM1 gene, and two SNPs within the intron of the PRM2 gene. The prevalence of these SNPs was similar in both infertile patients and in proven-fertile volunteers, except that the c248t alteration in the PRM2 gene induced a nonsense codon under conditions of heterozygosity in one infertile patient. Although the PRM1 and PRM2 genes are highly conserved, the single SNP in the PRM2 gene that induces translation termination may result in male infertility due to haploinsufficiency of PRM2.

Amino Acid Sequence↗

PubMed1987

Characterization of rat c-myc and adjacent regions.

Rat genomic regions covering c-myc were cloned from the DNA of both normal liver and two lines of Morris hepatomas, one of which had c-myc amplification. The three restriction maps showed perfect agreement within the overlapping regions. The 7 kb regions, which included the entire normal rat c-myc and the region 2.2 kb upstream, and one from the hepatomas, were sequenced and found to be identical. The coding regions of exons 2 and 3 were highly conserved between rat, mouse and man, but some differences in amino acids were noted. Exon 1 and the non-coding region of exon 3 showed limited homology between the three species. Rat exon 1 contained several nonsense codons in each frame and no ATG codon, indicating there to be no coding capacity in this exon. The 2.2 kb upstream regions and the introns compared showed unusual conservation between the rat and human genes. Some motifs, previously proposed as having a functional role in human c-myc, were also found in equivalent positions of the rat sequence. Nucleas S1 protection mapping revealed the second promoter to be preferentially used in most tissues or in hepatoma cells, and the second poly A addition signal to be the only one functional in all the RNA sources examined.

Amino Acid Sequence↗

PubMed1989

The selenocysteine-inserting opal suppressor serine tRNA from E. coli is highly unusual in structure and modification.

Selenocysteine is cotranslationally incorporated into selenoproteins in a unique pathway involving tRNA mediated suppression of a UGA nonsense codon (1-3). The DNA sequence of the gene for this suppressor tRNA from Escherichia coli predicts unusual features of the gene product (4). We determined the sequence of this serine tRNA (tRNA(UCASer]. It is the longest tRNA (95 nt) known to date with an acceptor stem of 8 base pairs and lacks some of the 'invariant' nucleotides found in other tRNAs. It is the first E. coli tRNA that contains the hypermodified nucleotide i6A, adjacent to the UGA-recognizing anticodon UCA. The implications of the unusual structure and modification of this tRNA on recognition by seryl-tRNA synthetase, by tRNA modifying enzymes, and on codon recognition are discussed.

Amino Acyl-tRNA Synthetases↗