Nonsense Mutations in Eukaryotes

Biochemistry (Moscow) - Nonsense mutations are a type of mutations which results in a premature termination codon occurrence. In general, these mutations have been considered to be among the most...     

Nonsense Mutations in Essential Genes of Saccharomyces Cerevisiae

A new method for isolating nonsense mutations in essential yeast genes has been used to develop a collection of 115 ochre mutations that define 94 complementation groups. The mutants are isolated in a genetic background that includes an ochre suppressor on a metastable plasmid and a suppressible colony-color marker on a chromosome. When the parental strain is plated on a rich medium, the colonies display a pattern of red, plasmid-free sectors on a white background. Mutants containing an ochre mutation in any essential yeast gene give rise to nonsectoring, white colonies, since cell growth is dependent on the presence of the plasmid-borne suppressor. Analysis of the data suggests that mutations are being recovered from a pool of approximately 250 genes.     

Nonsense Mutations in the ADE3 Locus of SACCHAROMYCES CEREVISIAE

Fifty seven mutations at the ade3 locus have been crossed to ochre, amber and ochre-amber suppressors. 70% (39/56) of the mutations at this locus are nonsense mutations; 61% (34/56) are ochre mutations and 9% (5/56) are amber mutations. The frequency of nonsense mutations among ade3 alleles recovered is very high and raises the interesting possibility that only polar mutations at this locus are recovered. An hypothesis to explain these genetical findings as well as physiological properties of these mutations is proposed.     

Nonsense Mutations in AAGAB Cause Punctate Palmoplantar Keratoderma Type Buschke-Fischer-Brauer

Punctate palmoplantar keratodermas (PPKPs) are rare autosomal-dominant inherited skin diseases that are characterized by multiple hyperkeratotic plaques distributed on the palms and soles. To date, two different loci in chromosomal regions 15q22-15q24 and 8q24.13-8q24.21 have been reported. Pathogenic mutations, however, have yet to be identified. In order to elucidate the genetic cause of PPKP type Buschke-Fischer-Brauer (PPKP1), we performed exome sequencing in five affected individuals from three families, and we identified in chromosomal region 15q22.33-q23 two heterozygous nonsense mutations—c.370C>T (p.Arg124^∗) and c.481C>T (p.Arg161^∗)—in AAGAB in all affected individuals. Using immunoblot analysis, we showed that both mutations result in premature termination of translation and truncated protein products. Analyses of mRNA of affected individuals revealed that the disease allele is either not detectable or only detectable at low levels. To assess the consequences of the mutations in skin, we performed immunofluorescence analyses. Notably, the amount of granular staining in the keratinocytes of affected individuals was lower in the cytoplasm but higher around the nucleus than it was in the keratinocytes of control individuals. AAGAB encodes the alpha-and gamma-adaptin-binding protein p34 and might play a role in membrane traffic as a chaperone. The identification of mutations, along with the results from additional studies, defines the genetic basis of PPKP1 and provides evidence that AAGAB plays an important role in skin integrity.     

Suppression of Nonsense Mutations in the Dystrophin Gene by a Suppressor tRNA Gene

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10–15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing -galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.     

Efficacy of a Fluorescent-Antibody Procedure for Identifying Bacillus cereus in Foods

One hundred and seventeen strains of Bacillus were examined by the fluorescent-antibody technique by using the globulin fraction of serum prepared against spores of B. cereus T. All but one strain of the 59 B. cereus tested fluoresced at the exosporium surface. Fluorescent staining of B. anthracis, B. thuringiensis, and B. mycoides was also observed. Absorption of the globulin fraction with B. anthracis and B. mycoides resulted in the elimination of staining of these organisms. Absorption with B. thuringiensis ATCC 10792 removed antibodies reacting with 6 of the strains of B. thuringiensis tested. Absorption with B. thuringiensis var. galleriae removed antibodies against B. cereus to such a degree that the globulin fraction was unusable.     

Nonsense Mutations in the Maltose A Region of the Genetic Map of Escherichia coli

The isolation of one amber mutation in malQ, one ochre mutation in malP, and seven amber mutations in malT is reported. A study of their phenotypic expressions in the presence of the amber suppressor su(III) and the ochre suppressor su(c) suggests that (i) malQ is the structural gene for amylomaltase; (ii) malQ and the structural gene for maltodextrin phosphorylase, malP, belong to the same operon; (iii) the malT product, which promotes the expression of the malP-malQ operon, is a protein synthesized in limiting amounts by the wild-type strain.     

A Simple and Rapid PCR-based Procedure for Identifying Homozygous Transgenic Rice Plants

We present a simple and rapid method for screening second-generation transgenic rice plants (T₁) to identify homozygous plants. The plasmid (pfd11) used for rice transformation contains a partially deleted cytochrome c gene (cyc) for comparing with the endogenous cyc for copy number. After polymerase chain reaction (PCR) amplification of a segment of the cyc in transgenic rice DNA followed by agarose gel electrophoresis, two specific bands are obtained. The upper band represents the endogenous cyc, and the lower band represents the partially deleted cyc in the transgene. The first-generation plants (T₀) that harbor a single copy of the transgene are selected based on the fact that the density of the lower band is half as dense as the upper band. Next, only plants harboring a single copy of the transgene are advanced to the second generation (T₁). The same PCR procedure is used again, and homozygous T₁ plants are easily identified from samples in which the intensity of the two bands is the same.     

Identifying Mutations in Duplicated Functions in Saccharomyces cerevisiae: Recessive Mutations in Hmg-Coa Reductase Genes

The two yeast genes for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, HMG1 and HMG2, each encode a functional isozyme. Although cells bearing null mutations in both genes are inviable, cells bearing a null mutation in either gene are viable. This paper describes a method of screening for recessive mutations in the HMG1 gene, the gene encoding the majority of HMG-CoA reductase activity in the cell. This method should be applicable to the isolation of mutations in other recovered in HMG1. These mutations exhibited intragenic complementation: one allele is in one complementation group and three alleles are in a second complementation group. Assays of HMG-CoA reductase activity indicated that the point mutations destroy most if not all of the activity encoded by HMG1. Intragenic complementation occurred with partial restoration of enzymatic activity. HMG1 was mapped to the left arm of chromosome XIII near SUP79, and HMG2 was mapped to the right arm of chromosome XII near SST2. A slight deleterious effect of a null mutation in either HMG-CoA reductase gene was detected by a co-cultivation experiment involving the wild-type strain and the two single mutants.     

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.LacI transgenic mice carry a recoverable λ phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of β-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin^-IL7R^-Sca-1⁺cKit⁺⁺(LSK) cells and other subpopulations of the hematopoietic system.     

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.     

OptForce: An Optimization Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions

Computational procedures for predicting metabolic interventions leading to the overproduction of biochemicals in microbial strains are widely in use. However, these methods rely on surrogate biological objectives (e.g., maximize growth rate or minimize metabolic adjustments) and do not make use of flux measurements often available for the wild-type strain. In this work, we introduce the OptForce procedure that identifies all possible engineering interventions by classifying reactions in the metabolic model depending upon whether their flux values must increase, decrease or become equal to zero to meet a pre-specified overproduction target. We hierarchically apply this classification rule for pairs, triples, quadruples, etc. of reactions. This leads to the identification of a sufficient and non-redundant set of fluxes that must change (i.e., MUST set) to meet a pre-specified overproduction target. Starting with this set we subsequently extract a minimal set of fluxes that must actively be forced through genetic manipulations (i.e., FORCE set) to ensure that all fluxes in the network are consistent with the overproduction objective. We demonstrate our OptForce framework for succinate production in Escherichia coli using the most recent in silico E. coli model, iAF1260. The method not only recapitulates existing engineering strategies but also reveals non-intuitive ones that boost succinate production by performing coordinated changes on pathways distant from the last steps of succinate synthesis.     

Suppression of Nonsense and Frameshift Mutations Obtained by Different Methods for Inactivating the Translation Termination Factor eRF3 in Yeast Saccharomyces cerevisiae

Mutations in genes of omnipotent nonsense suppressors SUP35 and SUP45 in yeast Saccharomyces cerevisiae encoding translation termination factors eRF3 and eRF1, respectively, and prionization of the eRF3 protein may lead to the suppression of some frameshift mutations (CPC mutations). Partial inactivation of the translation termination factor eRF3 was studied in strains with unstable genetically modified prions and also in transgenic yeast S. cerevisiae strains with the substitution of the indigenous SUP35 gene for its homolog from Pichia methanolica or for a recombinant S. cerevisiae SUP35gene. It was shown that this partial inactivation leads not only to nonsense suppression, but also to suppression of the frameshift lys2-90 mutation. Possible reasons for the correlation between nonsense suppression and suppression of the CPC lys2-90 mutation and mechanisms responsible for the suppression of CPC mutations during inactivation of translation termination factors are discussed.