Doublet preference and gene evolution

Summary Doublet preference analysis was carried out on coding and noncoding regions ofEscherichia coli, Saccharomyces cerevisiae, and human mitochondrial and nuclear DNA. The preference pattern in 1–2 and 2–3 doublets inE. coli andS. cerevisiae correlated with that in noncoding regions. The 3-1 doublet preference inE. coli genes with low optimal codon frequency and inS. cerevisiae genes also showed a correlation with each of their noncoding doublet preference. A mechanism to explain these double preference correlations in doublet preference is presented: mutational biases, the origin of the noncoding region doublet preference, evolved so as to maintain the 1–2 and 2–3 doublet preference, which is determined by codon usage. These biases then acted on the 3-1 doublet, which was almost free of coding constraints, resulting in a similar preference in this doublet.     

Molecular cloning and expression ofBacillus subtilis bglS gene inSaccharomyces cerevisiae

A 2.7-kb EcoRI DNA fragment carrying aBacillus subtilis endo--1,3-1,4-glucanase gene (bglS) from theE. coli plasmid pFG1 was cloned into anEscherichia coli/yeast shuttle vector to construct a hybrid plasmid YCSH. The hybrid plasmid was used to transformSaccharomyces cerevisiae, and thebglS gene was expressed. Variation between levels ofbglS gene expression inS. cerevisiae was about 2.3-fold, depending on the orientation of the 2.7-kb DNA fragment. Assay of substrate specificity and optimal pH of the enzyme demonstrated that the enzyme encoded by YCSH (bglS) was identical with that found inB. subtilis, but the expression level ofbglS gene inS. cerevisiae (YCSH) was much lower than that inE. coli (YCSH).     

Adaptive mutation inEscherichia coli strain FC40

Mutations can arise in static populations of cells that are subjected to nonlethal selective pressure, a phenomenon that has been called ‘adaptive mutation’. This phenomenon has been extensively studied in FC40, a strain ofEscherichia coli that cannot metabolize lactose (Lac⁻) but that reverts to lactose utilization (Lac⁺) when lactose is its sole energy and carbon source. The adaptive Lac⁺ mutations arise by two mutational processes: a recombination-dependent process that is highly active on the episome carrying the Lac⁻ allele, and an unknown process that affects the whole genome. Most of the Lac⁺ mutations are due to the first process, which also produces nonselected mutations on the F′ episome. However, about 10% of the Lac⁺ mutations arise in a subpopulation of cells that experience a period of transient hypermutation. Although minor contributors to any one type of mutation, the hypermutators account for nearly all cases of multiple mutations. The evolutionary implications of these results are: (i) DNA synthesis associated with recombination may be an important source of spontaneous mutation, particularly in cells that are not actively growing; (ii) the efficient mutational mechanism that occurs on the episome could result in the horizontal transfer of new alleles among species that carry and exchange conjugal plasmids; and (iii) a subpopulation of transient hypermutators could be a source of multiple mutations that would allow for rapid adaptive evolution under adverse conditions.     

Spontaneous and 9-aminoacridine-induced frameshift mutagenesis: second-site frameshift mutation within the N-terminal region of the lacI gene of Escherichia coli

Summary A novel forward mutational system, based on the acquisition of an I^q-d dominant phenotype from an initial I^q− recessive state, was used to identify second-site frameshift mutation [±1(±3 _n ) events] within the N-terminal region of thelacI gene ofEscherichia coli. The DNA sequences are described of forty-six spontaneous and twenty 9-aminoacridine(9-AA)-induced second site mutations. Although −1 frameshift events dominate both spectra, the nature and site specificity of these events clearly distinguish two mutational distributions. The spontaneous distribution contains two −(A: T) frameshift hotspots; one within a monotonic A₅ run (9 occurrences), the other at a 5′-CACAACAAC-3′ sequence (12 occurrences). In contrast 17 of the 20 mutations recovered after 9-AA treatment involve the loss of a G: C pair, 14 of which occur at a single site (5′-CGGGC-3′). The striking specificity of the observed mutational hotspots is of interest since this open genetic target contains similar sequences which were infrequently recovered.     

An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: Insertion elements dominate the spontaneous spectra

A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI⁻ (am26), and carry the lacZΔM15 marker for α-complementation in β-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-α, the lactose operon is repressed (off). Furthermore, supF suppression of lacl^um26 results in a lactose repressor that has an uninducible, lacl^S genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF⁻ mutations in pUB3 prevent suppresion of lacl^am26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is 0.7 and 1.0 × 10⁻⁶ without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Element: IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for - SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively), The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.     

The molecular analysis ofbrown eye color mutations isolated from geographically discrete populations ofDrosophila melanogaster

A large proportion of spontaneous mutations inDrosophila melanogaster strains of laboratory origin are associated with insertions of mobile DNA elements. As a first step toward determining whether spontaneous laboratory mutations are predictive for mutational events occurring in the wild, recessivebrown (bw) eye color mutants were isolated. By inbreeding the progeny of wild-caughtDrosophila melanogaster females,bw mutations were isolated from seven separate geographic sites distributed among Japan, California, Siberia and Hungary. Among a total of 14 mutations studied, no case of transposon mutagenesis was found. At least 4 mutations are associated with small deletions in thebw gene. The remainder are inseparable from wild-typebw by Southern analysis and are presumed to be basepair changes or very small indels. Although only two spontaneousbw mutants of laboratory origin have been analyzed molecularly, one is a mobile element insertion.     

Systems Metabolic Engineering of Escherichia coli Improves Coconversion of Lignocellulose‐Derived Sugars

Currently, microbial conversion of lignocellulose‐derived glucose and xylose to biofuels is hindered by the fact that most microbes (including Escherichia coli [E. coli], Saccharomyces cerevisiae, and Zymomonas mobilis) preferentially consume glucose first and consume xylose slowly after glucose is depleted in lignocellulosic hydrolysates. In this study, E. coli strains are developed that simultaneously utilize glucose and xylose in lignocellulosic biomass hydrolysate using genome‐scale models and adaptive laboratory evolution. E. coli strains are designed and constructed that coutilize glucose and xylose and adaptively evolve them to improve glucose and xylose utilization. Whole‐genome resequencing of the evolved strains find relevant mutations in metabolic and regulatory genes and the mutations’ involvement in sugar coutilization is investigated. The developed strains show significantly improved coconversion of sugars in lignocellulosic biomass hydrolysates and provide a promising platform for producing next‐generation biofuels.     

Spectrum of spontaneous mutations in a cDNA of the human hprt gene integrated in chromosomal DNA

Summary Altered sequences were determined of 52 independent spontaneous mutations occuring in a cDNA of the human hypoxanthine phosphoribosyltransferase (hprt) gene, which was integrated into chromosomal DNA of the mouse cell as a part of the retroviral shuttle vector. Spontaneous mutations comprised a variety of events: base substitutions, frameshifts, deletions, duplications, and complex mutational events, and were distributed randomly over the coding region of the gene. Frameshifts were the most frequent mutational event (38%), and base substitutions were the next most frequent (25%), followed by deletions (19%). Frameshift and deletion mutations commonly occurred preferentially at sites flanked by short direct repeats. Short inverted repeats were frequently found to be associated with duplication and complex mutational events. Analysis of the sequence alterations in the mutant genes suggests that misalignment mutagenesis represents an important molecular mechanism for the generation of spontaneous mutations in eukaryotic cells.     

Cryptic genes: Evolutionary puzzles

Many microorganisms carry genes that have the potential to code for specific functions but remain inactive during the normal lifetime of the organism. Such genes have been termed cryptic genes and their activation usually requires a mutational event. They are different from pseudogenes which arise as a result of duplication of a functional gene but remain inactivated because of the accumulation of multiple mutations. This review is an attempt to examine some of the well-characterized cryptic genetic systems inEscherichia coli in an effort to understand their functional and evolutionary significance.     

Studies on possible genetic effects of microwaves in procaryotic and eucaryotic cells

Summary The biological effects of microwaves in the hyperfrequency range, 9.4 GHz, 17 GHz, and 70–75 GHz were investigated in bacteria and yeast. At power densities below 60 mW/cm² and SAR values not exceeding 28 mW/g no significant effects on survival of repair competent and deficient strains were observed inEscherichia coli andSaccharomyces cerevisiae. In addition, microwaves of 17 GHz did not induce mutations inE. coli B/r WP2trp ^– uvr ^– above the spontaneous level, and the induction of nuclear reversions, cytoplasmic petite mutations and mitotic recombination as well as the efficiency of sporulation was not affected in yeast.     

Increased spontaneous mutation rates in mutants of E. coli with altered DNA polymerase III

Summary Two temperature-sensitive mutants in dnaE, the structural gene for DNA polymerase III of Escherichia coli, show increased spontaneous mutation rates at permissive temperatures. Studies of the reversion of well-characterized trpA mutations in dnaE strains show that the mutagenic effect of altered DNA polymerase III applies to several different base substitution events, but not to frameshifts. The results suggest that DNA polymerase III is involved in base-selection during DNA replication.MRC Molecular Genetics Unit     

New advances in coenzyme Q biosynthesis

Summary Coenzyme Q (or ubiquinone) is the product of two distinct biosynthetic pathways: the lipid tail of coenzyme Q is formed via the isoprene biosynthetic pathway, and the quinone ring derives from the metabolism of either shikimic acid or tyrosine. In general, eukaryotic organisms use the classical mevalonate pathway to form isopentenyl- and dimethylallyl-diphosphate, the five carbon building blocks of the polyisoprenoid tail, and prokaryotes use 1-deoxy-D-xylulose-5-phosphate, formed via the Rohmer pathway. The quinone ring precursor is 4-hydroxybenzoic acid, which is formed directly from chorismate inSaccharomyces cerevisiae andEscherichia coli, or from tyrosine in animal cells. Ring modification steps including prenylation, decarboxylation, and successive hydroxylation and methylation steps form the fully substituted benzoquinone ring of coenzyme Q. Many of the genes and polypeptides involved in coenzyme Q biosynthesis have been isolated and characterized by utilizing strains ofE. coli andS. cerevisiae with mutations in theubi andCOQ genes, respectively. This article reviews recent progress in characterizing the biosynthesis of coenzyme Q inE. coli, S. cerevisiae, and other eukaryotic organisms.     

Participation of translesion synthesis DNA polymerases in the maintenance of chromosome integrity in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We employed a genetic assay based on illegitimate hybridization of heterothallic Saccharomyces cerevisiae strains (the α-test) to analyze the consequences for genome stability of inactivating translesion synthesis (TLS) DNA polymerases. The α-test is the only assay that measures the frequency of different types of mutational changes (point mutations, recombination, chromosome or chromosome arm loss) and temporary changes in genetic material simultaneously. All these events are manifested as illegitimate hybridization and can be distinguished by genetic analysis of the hybrids and cytoductants. We studied the effect of Polζ, Polη, and Rev1 deficiency on the genome stability in the absence of genotoxic treatment and in UV-irradiated cells. We show that, in spite of the increased percent of accurately repaired primary lesions, chromosome fragility, rearrangements, and loss occur in the absence of Polζ and Polη Our findings contribute to further refinement of the current models of translesion synthesis and the organization of eukaryotic replication fork.     

An Escherichia coli mutant refractory to nitrosoguanidine mutagenesis

Summary A newly-isolated Escherichia coli mutant suffers only about 10% as many mutations as normal strains on exposure to nitrosoguanidine¹. The responsible mutation, inm-1, maps at approximately minute 79 in the current E. coli genetic map. The mutant is normal for overall growth, nitrosoguanidine lethality, spontaneous mutagenesis, ultraviolet light lethality and mutagenesis, ethyl methanesulfonate lethality and mutagenesis, and the adaptive repair induced by alkylating agents. The existence of this mutation proves that nitrosoguanidine mutagenesis is not merely the result of reactions between the chemical and DNA, but requires specific cellular function(s), and underscores the peculiarity of nitrosoguanidine as a mutagen.     

Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells

Summary We have compared the suppression of nonsense mutations by aminoglycoside antibiotics inEscherichia coli and in human 293 cells. Six nonsense alleles of the chloramphenicol acetyl transferase (cat) gene, in the vector pRSVcat, were suppressed by growth in G418 and paromomycin. Readthrough at UAG, UAA and UGA codons was monitored with enzyme assays for chloramphenicol acetyl transferase (CAT), in stably transformed bacteria and during transient expression from the same plasmid in human 293 tissue culture cells. We have found significant differences in the degree of suppression amongst three UAG codons and two UAA codons in different mRNA contexts. However, the pattern of these effects are not the same in the two organisms. Our data suggest that context effects of nonsense suppression may operate under different rules inE. coli and human cells.     

Host factor for coliphage Q beta RNA replication: presence in procaryotes and association with the 30S ribosomal subunit in Escherichia coli.

Summary The Host Factor required for in vitro coliphage Q RNA replication, a heat-stable RNA binding protein present in uninfectedEscherichia coli, has been detected by both immunological and functional tests inAcinetobacter calcoaceticus, Klebsiella pneumoniae, Pseudomonas aeruginosa andPseudomonas putida. It was not detectable by these criteria inBacillus stearothermophilus, Bacillus subtilis, Caulobacter crescentus, Micrococcus lysodeikticus, Rhodopseudomonas capsulata orSaccharomyces cerevisiae. InEscherichia coli the Host Factor protein has been shown to be associated with ribosomes. It is demostrated here that this association is specific for the 30S ribosomal subunit.     

Modulation of allele leakiness and adaptive mutability inEscherichia coli

It is shown that partial phenotypic suppression of two ochre mutations (argE3 andlacZU118) and an amber mutation (inargE) by sublethal concentrations of streptomycin in anrpsL ⁺ (streptomycin-sensitive) derivative of theEscherichia coli strain AB1157 greatly enhances their adaptive mutability under selection. Streptomycin also increases adaptive mutability brought about by theppm mutation described earlier. Inactivation ofrecA affects neither phenotypic suppression by streptomycin nor replication-associated mutagenesis but abolishes adaptive mutagenesis. These results indicate a causal relationship between allele leakiness and adaptive mutability.     

Cloning of endoglucanase genes fromCellulomonas biazotea intoE. coli andS. cerevisiae using shuttle vector YEp24

We constructed aSmaI genomic library ofCellulomonas biazotea DNA inE. coli and in theS. cerevisiae shuttle vector, YEP 24. Three clone were identified that conferred the ability forE. coli orS. cerevisiae transformants to produce carboxymethylcellulase (CMCase). Cells transformed with these clones were compared with one another and with nontransformed cells for hyper-production of CMCase.In vivo andin vitro studies indicated that the CMCase genes were fully expressed and the enzyme activity was located extracellularly. The optimum pH and temperature for the CMCase thus cloned were pH 7 and 50°C, respectively, as was the case for the donor.     

A new experimental system for study on adaptive mutations

A super-repressed mutant of purR (purR^S), which encodes a repressor protein controlling expression of purine biosynthetic genes inSalmonella typhimurium, grew very slowly on NCE medium with 10 μg/mL Ade and lactose as sole carbon source (cannot form colonies). However, a phenomenon of late-arising mutations was observed when purR^S mutants were spread on NCE+lactose plates and subjected to a prolonged non-lethal selection. The reconstruction experiments of revertants showed that the late-arising “lac⁺” mutants are not slow growing mutants. Statistical analysis indicated that the distribution of late-arising mutants is Poisson distribution, showing that reversion occurred after plating. The result of co-transductional analysis preliminarily showed that late-arising mutation occurred at selected genepurR or 16 bp PUR box,cis element of structural genepurD. The above results suggest that the phenomenon of late-arising mutation observed by our system is a result of adaptive mutations which are different from random mutations. This is the first time to extend target genes at which adaptive mutations could occur from structural genes involved in carbon metabolism and amino acid biosynthesis totrans regulatory gene coding repressor protein. Our results have provided not only a new proof for generality of adaptive mutations but also a new system for study on adaptive mutations.     

A new experimental system for study on adaptive mutations

A super-repressed mutant of purR (purRs), which encodes a repressor protein controlling expression of purine biosynthetic genes in Salmonella typhimurium, grew very slowly on NCE medium with 10 μg/mL Ade and lactose as sole carbon source (cannot form colonies). However, a phenomenon of late-arising mutations was observed when purRs mutants were spread on NCE+lactose plates and subjected to a prolonged non-lethal selection. The reconstruction experiments of revertants showed that the late-arising "lac+" mutants are not slow growing mutants. Statistical analysis indicated that the distribution of late-arising mutants is Poisson distribution, showing that reversion occurred after plating. The result of co-transductional analysis preliminarily showed that late-arising mutation occurred at selected gene purR or 16 bp PUR box, cis element of structural gene purD. The above results suggest that the phenomenon of late-arising mutation observed by our system is a result of adaptive mutations which are different