The effect of genomic position on reversion of a lac frameshift mutation (lacIZ33) during non-lethal selection (adaptive mutation)

In a system described by Cairns and Foster, starvation of a particular leaky lac mutant (lacIZ33) in the presence of lactose appears to direct mutation in non-growing cells to sites that allow growth (adaptive mutation). This behaviour requires that the lac operon be located on an F' plasmid. This position effect was investigated by placing the mutant lac operon at many sites in the genome of Salmonella enterica (Typhimurium; LT2) and testing reversion behaviour. Genomic position did not affect reversion during non-selective growth. When lac was at any of 550 chromosomal sites, starvation caused little or no enhancement of reversion. In the 28 strains with the lac on Salmonella's conjugative plasmid (pSLT), selection enhanced reversion strongly, just as seen for strains with lac on an F' plasmid. In 46 strains, the lac operon was inserted within a small chromosomal duplication, and selection stimulated RecA-dependent partial reversion by simple amplification (about 8x) of the mutant lac region. The position of lac on a conjugative plasmid is important to reversion because it allows more frequent gene duplication and amplification. These events are central to growth and reversion under selection because they increase the number of replicating lac alleles within each developing revertant clone.     

Evidence that accumulation of mutants in a biofilm reflects natural selection rather than stress-induced adaptive mutation

The accumulation of mutant genotypes within a biofilm evokes the controversy over whether the biofilm environment induces adaptive mutation or whether the accumulation can be explained by natural selection. A comparison of the virulence of two strains of the dental pathogen Streptococcus mutans showed that rats infected with one of the strains accumulated a high proportion (average, 22%) of organisms that had undergone a deletion between two contiguous and highly homologous genes. To determine if the accumulation of deletion mutants was due to selection or to an increased mutation rate, accumulations of deletion mutants within in vitro planktonic and biofilm cultures and within rats inoculated with various proportions of deletion organisms were quantified. We report here that natural selection was the primary force behind the accumulation of the deletion mutants.     

Evidence from mutation spectra that the UV hypermutability of xeroderma pigmentosum variant cells reflects abnormal, error-prone replication on a template containing photoproducts.

Xeroderma pigmentosum (XP) variant patients are genetically predisposed to sunlight-induced skin cancer. Fibroblasts derived from these patients are extremely sensitive to the mutagenic effect of UV radiation and are abnormally slow in replicating DNA containing UV-induced photoproducts. However, unlike cells from the majority of XP patients, XP variant cells have a normal or nearly normal rate of nucleotide excision repair of such damage. To determine whether their UV hypermutability reflected a slower rate of excision of photoproducts specifically during early S phase when the target gene for mutations, i.e., the hypoxanthine (guanine) phosphoribosyltransferase gene (HPRT), is replicated, we synchronized diploid populations of normal and XP variant fibroblasts, irradiated them in early S phase, and compared the rate of loss of cyclobutane pyrimidine dimers and 6-4 pyrimidine-pyrimidones from DNA during S phase. There was no difference. Both removed 94% of the 6-4 pyrimidine-pyrimidones within 8 h and 40% of the dimers within 11 h. There was also no difference between the two cell lines in the rate of repair during G1 phase. To determine whether the hypermutability resulted from abnormal error-prone replication of DNA containing photoproducts, we determined the spectra of mutations induced in the coding region of the HPRT gene of XP variant cells irradiated in early S and G1 phases and compared with those found in normal cells. The majority of the mutations in both types of cells were base substitutions, but the two types of cells differed significantly from each other in the kinds of substitutions, but the two types differed significantly from each other in the kinds of substitutions observed either in mutants from S phase (P < 0.01) or from G1 phase (P = 0.03). In the variant cells, the substitutions were mainly transversions (58% in S, 73% in G1). In the normal cells irradiated in S, the majority of the substitutions were G.C --> A.T, and most involved CC photoproducts in the transcribed strand. In the variant cells irradiated in S, substitutions involving cytosine in the transcribed strand were G.C --> T.A transversions exclusively. G.C --> A.T transitions made up a much smaller fraction of the substitutions than in normal cells (P < 0.02), and all of them involved photoproducts located in the nontranscribed strand. The data strongly suggest that XP variant cells are much less likely than normal cells to incorporate either dAMP or dGMP opposite the pyrimidines involved in photoproducts. This would account for their significantly higher frequency of mutants and might explain their abnormal delay in replicating a UV-damaged template.     

mRNA-targeted fluorescent in situ hybridization (FISH) of Gram-negative bacteria without template amplification or tyramide signal amplification

Technologies are needed to study gene expression at the level of individual cells within a population or microbial community. Fluorescent in situ hybridization (FISH) supplies high-resolution spatial information and has been widely applied to study microbial communities at the rRNA level. While mRNA-targeted FISH has been popular for studying gene expression in eukaryotic cells, very little success has been achieved with prokaryotes. At present, detection of specific mRNAs in individual prokaryotic cells requires the use of in situ RT-PCR or tyramide signal amplification (TSA). In this study we used DNA oligonucleotide probes labeled with a single near-infrared dye in FISH assays to detect multi-copy plasmid-based and endogenous mRNA molecules in Escherichia coli and Shewanella oneidensis MR-1. We took advantage of the fact that there is much less background signal produced by biological materials and support matrices in the near-infrared spectrum and thus long camera exposure times could be used. In addition, we demonstrate that a combination of probes targeting both rRNA and mRNA could be successfully employed within the same FISH assay. These results, as well as ongoing R&D improvements in NIR and infrared dyes, indicate that the FISH approach we demonstrated could be applied in certain environmental settings to monitor gene expression in mixed populations.     

The processing of a Benzo(a)pyrene adduct into a frameshift or a base substitution mutation requires a different set of genes in Escherichia coli

Replication through a single DNA lesion may give rise to a panel of translesion synthesis (TLS) events, which comprise error-free TLS, base substitutions and frameshift mutations. In order to determine the genetic control of the various TLS events induced by a single lesion, we have chosen the major N2-dG adduct of (+)-anti-Benzo(a)pyrene diol epoxide [(+)-anti-BPDE] adduct located within a short run of guanines as a model lesion. Within this sequence context, in addition to the major event, i.e. error-free TLS, the adduct also induces base substitutions (mostly G --> T transversions) and -1 frameshift mutations. The pathway leading to G --> T base substitution mutagenesis appears to be SOS independent, suggesting that TLS is most probably performed by the replicative Pol III holoenzyme itself. In contrast, both error-free and frameshift TLS pathways are dependent upon SOS-encoded functions that belong to the pool of inducible DNA polymerases specialized in TLS (translesional DNA polymerases), namely umuDC (Pol V) and dinB (Pol IV). It is likely that, given the diversity of conformations that can be adopted by lesion-containing replication intermediates, cells use one or several translesional DNA polymerases to achieve TLS.     

Melt analysis of mismatch amplification mutation assays (Melt-MAMA): a functional study of a cost-effective SNP genotyping assay in bacterial models

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA), is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg). Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs) and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of Melt-MAMA, which should prove useful to the wider scientific community.     

Evidence that eukaryotic initiation factor (eIF) 2 is a cap-binding protein that stimulates cap recognition by eIF-4B and eIF-4F

We studied the mRNA-binding properties of eukaryotic initiation factor (eIF) 2. This Met-tRNA-binding factor interacts with the cap structure of reoviral mRNA in an ATP-independent manner. Both the beta- and gamma-subunit of eIF-2 are involved in the UV-induced cross-linking of eIF-2 to the cap. The interaction of eIF-2 with a messenger is sensitive to the cap analogue 7-methyl-guanosine 5'-triphosphate as measured by cross-linking and by mRNA retention on nitrocellulose filters. The cap-binding property of eIF-2 does not conflict with the current mRNA-binding model of initiation factors eIF-4A, -4B, and -4F: cross-linking of eIF-4E and of eIF-4B is stimulated by eIF-2. The eIF-2-mediated increase of eIF-4E interaction results in a decrease of the cross-linking of the beta- and gamma-subunits of eIF-2. The presence of GTP in the cross-linking assay interferes with the interaction of eIF-2 with the cap structure but does not inhibit the eIF-2 stimulated eIF-4E and -4B cross-linking. These observations indicate a role for eIF-2 in the mRNA recognition.     

Evidence against the reversion of mutation in the Haemophilus influenzae phage HP1c1 by preinfection treatment of host cells or phage with MNNG.

N-Methyl-N'-nitrosoguanidine (MNNG) causes reversion of a temperature-sensitive mutation in a bacteriophage of Haemophilus influenzae if exposure to the mutagen takes place after infection but before lysis. However, neither pre-infection treatment of the phage DNA, host cells, or both will cause reversion. The reasons for this are discussed in relation to the somewhat different results in the Escherichia coli lambda phage system and in relation to error-prone repair and replication processes.     

Evidence that endogenous 6-(ARG or LYS)-opioid peptides can interact with kappa-receptors as agonists

Evidence is provided for the abilities of endogenous 6-(Arg or Lys)-opioid peptides to interact with kappa-receptors as agonists. Dynorphin-(1-17) and -(1-8), alpha- and beta-neo-endorphin, [Met5]-enkephalin-Arg6-Phe7 and des acetyl salmon endorphin I significantly inhibited the electrically-evoked contractions of rabbit vas deferens which had been shown to contain kappa-receptors exclusively, indicating that endogenous 6-(Arg or Lys)-opioid peptides could act on kappa-receptors as agonists. Additionally, the inhibition of contractions of rabbit vas deferens by 6-(Arg or Lys)-opioid peptides was antagonized more effectively by Mr 2266 which had a high affinity to both mu- and kappa-receptors, than naloxone which had a high affinity only to mu-receptors. This also suggested that 6-(Arg or Lys)-opioid peptides acted as kappa-receptor agonists. The rank order of the inhibitory potency of 6-(Arg or Lys)-opioid peptides against contractions of rabbit vas deferens was as follows: dynorphin-(1-17) greater than alpha-neo-endorphin greater than beta-neo-endorphin .=. dynorphin-(1-8) greater than des acetyl salmon endorphin I greater than [Met5]-enkephalin-Arg6-Phe7. Since other endogenous opioid peptides such as [Met5]- and [Leu5]-enkephalin and beta-endorphin have been shown not to act on kappa-receptors as agonist, data in the present study suggest that endogenous opioid peptides can be classified into two groups in terms of an ability to interact with kappa-receptors as an agonist.     

L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs)

A flexible, non-gel-based single nucleotide polymorphism (SNP) detection method is described. The method adopts thermostable ligation for allele discrimination and rolling circle amplification (RCA) for signal enhancement. Clear allelic discrimination was achieved after staining of the final reaction mixtures with Cybr-Gold and visualisation by UV illumination. The use of a compatible buffer system for all enzymes allows the reaction to be initiated and detected in the same tube or microplate well, so that the experiment can be scaled up easily for high-throughput detection. Only a small amount of DNA (i.e. 50 ng) is required per assay, and use of carefully designed short padlock probes coupled with generic primers and probes make the SNP detection cost effective. Biallelic assay by hybridisation of the RCA products with fluorescence dye-labelled probes is demonstrated, indicating that ligation-RCA (L-RCA) has potential for multiplexed assays.     

ENU-induced allele of brachyury (Tkt1) exhibits a developmental lethal phenotype similar to the original brachyury (T) mutation

New alleles of brachyury (Tkt1, Tkt4) were induced in the mouse complete tw5 haplotype by ethylnitrosourea (ENU). Like the original brachyury (T) mutation, the new alleles cause a short-tailed phenotype in heterozygotes, and interact with the t complex tail interaction factor (tct) in trans to cause phenotypically tailless mice. Because ENU is mainly a point mutagen, it is important to determine that the new alleles are homozygous embryonic lethal mutations like the original T allele, and to characterize their embryonic lethal phenotype. Moreover, the Tkt1 mutation maps to an inverted position relative to quaking (qk) in t haplotypes as compared with its position on normal chromosome 17. The Tkt1 allele was separated from the resident tw5 lethal gene, tclw5, by recombination, allowing embryology studies to be performed. Embryological analyses show that the Tkt1 allele is nearly identical to the classic T allele. At 9 and 10 days of development, homozygous Tkt1/Tkt1 embryos are grossly abnormal with properties including 1) irregular, disorganized somite pairs, 2) a shortened posterior end of the embryo, 3) an irregular neural tube, and 4) an abnormal notochord. In addition, 10 day-old abnormal embryos have anterior limb buds that point dorsally rather than ventrally, and are smaller than normal littermates. We conclude that the Tkt1 mutation is a valuable allele for both mapping and molecular characterization of the brachyury locus.     

The phosphorylase kinase deficiency (Phk) locus in the mouse: Evidence that the mutant allele codes for an enzyme with an abnormal structure

Female (I/St X C57BL/St) F1 mice heterozygous at the sex-linked phosphorylase kinase deficiency locus (Phk) have phosphorylase kinase activities averaging 86% that of mice homozygous for the wild-type allele (C57BL/St), i.e., 72% greater than the sum of one-half the activities of the parental strains. Approximately one-half the phosphorylase kinase activity in the (I X C57BL) F1 muscle extracts had a stability at 42.5 C similar to that of the activity in C57BL extracts (t1/2 = 13.2 min); the other half of the activity in the F1 extracts was more labile (t1/2 = 3.9 min). Two species of phosphorylase kinase activity in F1 muscle extracts were also differentiated with an antiserum prepared in guinea pigs against purified rabbit skeletal muscle phosphorylase kinase. This anti-serum cross-reacted with phosphorylase kinase in C57BL muscle extracts but did not cross-react with skeletal muscle extracts of mice hemi- or homozygous for the mutant allele (I/LnJ). The guinea pig antiserum precipitated 52% as much protein from (I X C57BL)F1 muscle extracts compared to those of C57BL. However, an antiserum prepared against purified rabbit skeletal muscle phosphorylase kinase in the goat cross-reacted with the mutant phosphorylase kinase. The ratio C57BL:(I X C57BL)F1:I of immunoprecipitated protein from skeletal muscle extracts with this antiserum was 1:0.97:1.08. Polyacrylamide gel electrophoresis of the immunoprecipitates in the presence of 0.1% sodium dodecylsulfate showed three subunits for mouse phosphorylase kinase with molecular weights of 139,000, 118,000, and 41,000; these values are similar to the ones obtained with purified rabbit skeletal muscle phosphorylase kinase. These three subunits were also observed in immunoprecipitates from I/LnJ muscle extracts. These results offer substantial evidence (1) that in skeletal muscle extracts of mice heterozygous at the Phk locus the mutant phosphorylase kinase is active, (2) that the gene product of the mutant allele is an enzyme with an abnormal structure, and (3) that the phosphorylase kinase deficiency in I/LnJ skeletal muscle extracts is not the result of the absence of phosphorylase kinase or one of its subunits.     

Evidence for a diurnal variation in the carotenoid content of acanthodiaptomus denticornis (Crustacea,copepoda) in Lac Pavin (Auvergne,France)

In Lac Pavin Acanthodiaptomus denticornis was found to be intensely reddish-orange coloured by keto-carotenoids of the astaxanthin type. Such pigments are not normally found in phytoplankton algae, and apparently these carotenoids result from the metabolism of pigments of dietary origin. The carotenoid content of the zooplankton showed a distinct 2.5-fold diurnal variation, with a minimum at night time and a maximum in the early morning. The possible impact of a diurnal difference in zooplankton feeding activity is discussed.     

Evidence that the tertiary structure of 20(S)-ginsenoside Rg(3) with tight hydrophobic packing near the chiral center is important for Na(+) channel regulation

Ginsenosides are the active ingredients of Panax ginseng. Ginsenoside Rg(3) exists as two stereoisomers of carbon-20: 20-S-protopanaxatriol-3-[O-beta-d-glucopyranosyl (1-->2)-beta-glucopyranoside] (20(S)-Rg(3)) and 20-R-protopanaxatriol-3-[O-beta-d-glucopyranosyl (1-->2)-beta-glucopyranoside] (20(R)-Rg(3)). Recently, we reported that 20(S)-Rg(3) regulates voltage-dependent Ca(2+) channel activity and several types of ligand-gated ion channels, whereas 20(R)-Rg(3) does not have this activity. In this study, we investigated the structure-activity relationship of these two stereoisomers by NMR spectroscopy and by measurement of the current in Xenopus oocytes expressing the mouse cardiac voltage-dependent Na(+) channel (Na(v)1.5). We found that 20(S)-Rg(3) but not 20(R)-Rg(3) inhibited Na(+) channel current in a dose- and voltage-dependent manner. The difference between Rg(3) epimers in voltage-dependent ion channel regulation indicates that the structure of 20(S)-Rg(3) may be geometrically better aligned than that of 20(R)-Rg(3) for interaction with receptor regions in Na(+) channels. The (1)H and (13)C NMR chemical shifts, including all hydroxyl protons of 20(S)-Rg(3) and 20(R)-Rg(3), were completely assigned, and their tertiary structures were determined. 20(S)-Rg(3) has more tight hydrophobic packing near the chiral center than 20(R)-Rg(3). Tertiary structures and activities of 20(S)-Rg(3) and 20(R)-Rg(3) indicate that 20(S)-Rg(3) may have stronger interactions with the receptor region in ion channels than 20(R)-Rg(3). This may result in different stereoselectivity of Rg(3) stereoisomers in the regulation of voltage-dependent Na(+) channel activity. This is the first structural approach to ginsenoside action on ion channel.     

The mammalian mid-pachytene checkpoint: meiotic arrest in spermatocytes with a mutation in Atm alone or in combination with a Trp53 (p53) or Cdkn1a (p21/cip1) mutation

ATM, the protein product of the gene mutated in the human autosomal recessive disorder ataxia telangiectasia, is involved in detection of double strand breaks (DSBs) and is a key component of the damage surveillance network of cell cycle proteins. In somatic cells ATM phosphorylates many other proteins including p53, an important regulator of cell cycle control. Mice deficient for Atm are male sterile with arrest and apoptosis occurring at testis epithelial stage IV, which in normal spermatocytes corresponds to mid-pachynema. Unlike the situation in somatic cells, we find no evidence that disruption of the Trp53 (p53) gene, or its down-stream target Cdkn1a (p21/Cip1) results in even a partial rescue of the Atm defect.     

Comparison of full genome sequences between two hepatitis B virus strains with or without preC mutation (A1896) from a single Korean hepatocellular carcinoma patient

This report describes the full-length sequences of 2 HBV clones from a hepatocellular carcinoma (HCC) patient, one with preC mutation (1896A) and the other without preC mutation. The high level of discrepancy in mutation frequency between these 2 strains was observed in the Core (C) region among 4 ORFs. These data support previous results that Korean HBV strains, belonging to genotype C2, are prone to mutations. It is possible that the mutations (BCP and preC mutations) associated with the HBeAg defective production might contribute to the diversity of mutations related to HBV persistence, playing an important role in hepatocarcinogenesis in this patient.