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Abstract: Spontaneous mutations are stochastic events. The mutation rate, defined as mutations per genome per replication, is generally very low, and it is widely accepted that spontaneous mutations occur at defined, but different, rates in bacteriophage and in bacterial, insect, and mammalian cells. The calculation of mutation rates has proved to be a significant problem. Mutation rates can be calculated by following mutant accumulation during growth or from the distribution of mutants obtained in parallel cultures. As Luria and Delbrück described in 1943, the number of mutants in parallel populations of bacterial cells varies widely depending on when a spontaneous mutation occurs during growth of the culture. Since 1943, many mathematical refinements to estimating rates, called estimators, have been described to facilitate determination of the mutation rate from the distribution or frequency of mutants detected following growth of parallel cultures. We present a rigorous mathematical solution to the mutation rate problem using an unbiased and consistent estimator. Using this estimator we demonstrate experimentally that mutation rates can be easily calculated by determining mutant accumulation, that is, from the number of mutants measured in two successive generations. Moreover, to verify the consistency of our estimator we conduct a series of simulation trials that show a surprisingly rapid convergence to the targeted mutation rate (reached between 25th and 30th generations). [Copyright &y& Elsevier]

Abstract: Nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) plays a central role in virus replication. In this study, serial N- and C-terminal truncations of N protein were performed in the context of type 2 PRRSV infectious cDNA clone, and our results revealed that a stretch of inter-genotypic variable N terminal residues aa 5–13 (5NGKQQKKK13K) and the last four inter-genotypic variable aa residues (120SPS123A) at the C terminus of N protein were dispensable for type 2 PRRSV infectivity. All the recovered deletion mutant viruses had spontaneous mutations in the N coding region, including substitution, deletion and insertion. We re-engineered the additional internal deletion with or without the original C-terminal deletion back into wild-type APRRS and found that the internal domain spanning the inter-genotypic variable residues 39–42 (39KGP42G) and conserved residues 48–52 (48KNPE52K), respectively, were dispensable for type 2 PRRSV viability. These results demonstrated that N protein contains non-essential regions for virus viability in cell culture. Such dispensable regions could be utilized as insertion site for foreign tag expression and the rescued viruses could be the candidates for marker vaccine. [Copyright &y& Elsevier]

Azoxymethane (AOM) administration to F344 male rats is a widely used model of human colon carcinogenesis. The current study investigates quantitatively the accumulation of K- Ras codon 12 mutations following AOM exposure. Male, 6-week-old F344 rats were treated subcutaneously with 30 mg/kg body weight of AOM, and colon tissue was collected at 1, 8, 24, and 32 weeks after treatment. The K- Ras codon 12 GGT to GAT and GGT to GTT mutant fractions (MFs) were measured using allele-specific competitive blocker polymerase chain reaction (ACB-PCR). Between 1 and 32 weeks after AOM treatment, the K- Ras codon 12 GGT to GAT geometric mean MF in the rat colon increased significantly from 12.9 × 10 to 145 × 10, and the GGT to GTT geometric mean MF increased significantly from 5.26 × 10 to 180 × 10. K- Ras codon 12 GGT to GAT MF also increased significantly in AOM-treated rat colon tissue at 1 week compared to controls (4.44 × 10). The accumulation of the GGT to GAT MF long after the DNA adduct repair phase suggests that a portion of cells containing this mutation have a proliferative advantage, allowing them to accumulate as nascent tumors progress. Also, the GGT to GAT background MF increased in untreated rats, indicating that there is accumulation with age. The ACB-PCR assay generates quantitative data of cancer-related mutations and thus provides insight into pathological processes following carcinogen exposure. Environ. Mol. Mutagen., 2011. © Published 2011 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Spectinomycin resistant mutant carrot ( Daucus carota L.) callus lines detected in the experiments on biolistic transformation of plastome were analyzed. It has been found that this antibiotic resistance is determined by point nucleotide substitutions at two distinct sites of the chloroplast gene rrn16, coding for 16S rRNA, namely, G1012T, G1012C, and A1138G. The detected mutations are localized to the 16S rRNA region forming helix h34, which contains spectinomycin binding site, and lead to its destabilization by several kilocalories per mole. Comparative analysis of rrn16 gene sequences has demonstrated conservation of the positions of the nucleotide substitutions determining this antibiotic resistance in carrot ( D. carota L.), tobacco ( Nicotiana tabacum L.), and bladder pod ( Lesquerella fendleri L.), as well as in Escherichia coli. [ABSTRACT FROM AUTHOR]

In eukaryotes, together with the Mre11/Rad50/Xrs2 (or Nbs1) complex, a family of related protein kinases (the ATM family) is involved in checkpoint activation in response to DNA double-strand breaks. In Saccharomyces cerevisiae, two members of this family, MEC1 and TEL1, have functionally redundant roles in DNA damage repair. Strains with mutations in their mec1 as well as mre11 genes are very sensitive to DNA damaging agents, show defective induction of damage-induced cell-cycle checkpoints, and defective damage-induced homologous recombination. However, the fact that both the mec1Δ and mre11Δ strains exhibit the spontaneous hyper-recombination phenotype is paradoxical in light of the homologous recombination defects in these strains. In this study, we constructed yeast mec1, tel1, and mre11 null mutations and characterized their genome stability properties. Spontaneous and methylmethane sulfonate (MMS)-induced point mutations, base-substitutions, and frameshifts occurred to an almost equal extent in the wild-type, mec1Δ, tel1Δ, and mre11Δ strains. Thus, Mec1, Tel1, and Mre11 do not play roles in the point mutation response. We then found that the mec1Δ, mre11Δ, and mec1Δ tel1Δ strains demonstrated increased rates of spontaneous loss of heterozygosity (LOH), which includes crossover, gene conversion, and chromosome loss, compared with the wild-type strain. In the tel1Δ strain, the rate of spontaneous LOH was as low as that in the wild-type strain. Finally, no induction of LOH by MMS was observed in the mec1Δ, mre11Δ, or mec1Δ tel1Δ strain; however, it was detected in the wild-type and tel1Δ strains upon exposure to MMS. The elevated level of spontaneous LOH but not MMS-induced LOH in the mec1Δ, mre11Δ, and mec1Δ tel1Δ strains suggests the presence of high levels of spontaneous recombinogenic DNA damage, which differs from the damage induced by MMS treatment, in these strains. [ABSTRACT FROM AUTHOR]

Abstract: Very few human genes can be used to identify spontaneous inactivating somatic mutations. We hypothesized that because the XK gene is X-linked, it would be easy to identify spontaneously arising red cells with a phenotype resembling the McLeod syndrome, which results from inherited XK mutations. Here, by flow cytometry, we detect such phenotypic variants at a median frequency of 9×10−6 in neonatal cord blood samples and 39×10−6 in healthy adults (p =0.004). It may be possible to further investigate the relationship between aging, mutations, and cancer using this approach. [Copyright &y& Elsevier]

Abstract: In an attempt to evaluate the role of the Xpc gene in maintaining genomic stability in vivo under normal conditions, the age-dependent accumulation of spontaneous mutations in different tissues was analyzed in Xpc-deficient lacZ-transgenic mice. Brain, testis, and small intestine revealed no effects from the Xpc-deficiency, whereas liver, spleen, heart, and lung showed an enhanced age-related accumulation of mutations in Xpc-deficient mice. In the spleen, the effect was not obvious at 2 and 12 months of age, but became apparent at 23 months. The magnitude of the observed effect at an advanced age was similar in the liver, spleen and heart, but was comparatively smaller in the lung. Haploinsufficiency was observed in liver and spleen but not in heart and lung. Analysis of DNA sequences in the mutants revealed that the frequency of G:C to T:A changes were elevated in the liver and heart of Xpc-deficient aged mice, supporting the possible involvement of XPC in base excision repair of oxidized guanine. The occurrence of two or more mutations within a single lacZ gene was termed a multiple mutation and was also elevated in old Xpc-deficient mice. Among the clones examined, two mutant clones showed as many as four mutations within a short stretch of DNA. This is the first demonstration to support suggestions for the existence of a role for XPC in the suppression of multiple mutations. These multiple mutations could conceivably be generated by error-prone trans-lesional DNA synthesis. Overall, these results indicate that there may be diverse roles or mechanisms through which XPC participates in genome maintenance in different tissues. [Copyright &y& Elsevier]

Abstract: CYP21A2 mutations resulting from microconversions of the CYP21A1P sequence in congenital adrenal hyperplasia (CAH) commonly appear in all populations. However, it has not often been described as being due to the gene founder effect. Herein, we investigated two spontaneous mutations of IVS2+1G>A and R316X in ethnic Chinese (Taiwanese) CAH patients to determine whether they share the same haplotype of ancient origin by the analysis of sequence-specific oligonucleotide (SSO) for HLA class I B and sequence-based typing (SBT) for HLA class II DRB1 gene-typing methods. From over 200 CAH families, eight unrelated CAH patients were found and examined: five had the IVS2+1G>A mutation and three had the R316X mutation. Based on HLA typing data, five alleles in five patients with the IVS2+1G>A mutation were consistent with a shared haplotype of the B∗3909–DRB1∗160201 allele, and the three alleles in the three patients with the R316X mutation were all the B∗460101–DRB1∗080302 allele. The evidence indicates that the haplotype of single-base substitutions of either the IVS2+1G>A or R316X mutation came from the same allele rather than a mutational hot spot, suggesting that the gene founder effect has occurred in the Taiwanese population. This is the first report of the gene founder effect of the CYP21A2 mutation occurring in ethnic Chinese (Taiwanese) CAH patients with 21-hydroxylase deficiency. [Copyright &y& Elsevier]

The article presents a study evaluating the novel lacl mutation (C354T), a transgenic mouse mutation detection system that aims to allow the assessment of in vivo mutation frequency and pattern in individual tissues. In an assay of spontaneous mutations, there was a reappearance of C354T in six of seven mutants with a second mutation. It is observed that the frequency of spontaneous doublets was advanced by 2.7-fold and there was greater average spacing between mutations in the doublets. Furthermore, the study reveals the coherence of C354T as a hitch-hiker mutation in one of 38 mutation targets in the Big Blue mouse genome with germline mutation. Results suggest the essence of determining all mutations and analyzing new mutations in murine mutations.

Spontaneous deleterious mutation has been measured in a handful of organisms, always under laboratory conditions and usually employing inbred species or genotypes. We report the results of a mutation accumulation experiment with an outbred annual plant, Raphanus raphanistrum, with lifetime fitness measured in both the field and the greenhouse. This is the first study to report the effects of spontaneous mutation measured under field conditions. Two large replicate populations ( Ne≈ 600) were maintained with random mating in the greenhouse under relaxed selection for nine generations before the field assay was performed and ten generations before the greenhouse assay. Each generation, every individual was mated twice, once as a pollen donor and once as a pollen recipient, and a single seed from each plant was chosen randomly to create the next generation. The ancestral population was maintained as seeds at 4°C. Declines in lifetime fitness were observed in both the field (1.7% per generation; P= 0.27) and the greenhouse (0.6% per generation; P= 0.07). Significant increases in additive genetic variance for fitness were found for stems per day, flowers per stem, fruits per flower and seeds per fruit in the field as well as for fruits per flower in the greenhouse. Lack of significance of the fitness decline may be due to the short period of mutation accumulation, the use of outbred populations, or both. The percent declines in fitness are at the high end of the range observed in other mutation accumulation experiments and give some support to the idea that mutational effects may be magnified under harsher field conditions. Thus, measurement of mutational parameters under laboratory conditions may underestimate the effects of mutations in natural populations. [ABSTRACT FROM AUTHOR]

Frequencies of spontaneous mutation from inducible resistance to constitutive resistance were determined for the four clinical isolates of erythromycin-resistant enterococci, including one isolate with ermB gene and three clinical isolates with ermA gene. The rate of ermB mutation was higher than that of ermA mutation by more than 10 fold. Sequence analysis of the regulatory regions of erm genes revealed that mutation type of ermB was just point mutation, by contraries the mutation type of ermA was either deletion or tandem duplication. These results showed distinct characteristics in mutation patterns of ermB and ermA. [ABSTRACT FROM AUTHOR]

It has recently become clear that the classical notion of the random nature of mutation does not hold for the distribution of mutations among genes: most collections of mutants contain more isolates with two or more mutations than predicted by the mutant frequency on the assumption of a random distribution of mutations. Excesses of multiples are seen in a wide range of organisms, including riboviruses, DNA viruses, prokaryotes, yeasts, and higher eukaryotic cell lines and tissues. In addition, such excesses are produced by DNA polymerases in vitro. These "multiples" appear to be generated by transient, localized hypermutation rather than by heritable mutator mutations. The components of multiples are sometimes scattered at random and sometimes display an excess of smaller distances between mutations. As yet, almost nothing is known about the mechanisms that generate multiples, but such mutations have the capacity to accelerate those evolutionary pathways that require multiple mutations where the individual mutations are neutral or deleterious. Examples that impinge on human health may include carcinogenesis and the adaptation of microbial pathogens as they move between individual hosts. [ABSTRACT FROM AUTHOR]

Adaptive mutation is a generic term for processes that allow individual cells of nonproliferating cell populations to acquire advantageous mutations and thereby to overcome the strong selective pressure of proliferation-limiting environmental conditions. Prerequisites for an occurrence of adaptive mutation are that the selective conditions are nonlethal and that a restart of proliferation may be accomplished by some genetic change in principle. The importance of adaptive mutation is derived from the assumption that it may, on the one hand, result in an accelerated evolution of microorganisms and, on the other, in multicellular organisms may contribute to a breakout of somatic cells from negative growth regulation, i.e., to cancerogenesis. Most information on adaptive mutation in eukaryotes has been gained with the budding yeast Saccharomyces cerevisiae. This review focuses comprehensively on adaptive mutation in this organism and summarizes our current understanding of this issue. [ABSTRACT FROM AUTHOR]

Abstract: In infected cells, replication errors during viral proliferation generate mutations in adenoviruses (Ads), and the mutant Ads proliferate and evolve in the intracellular environment. Genetically fiber-modified recombinant Ads (rAd variants) were generated, by modification of the fiber gene, for therapeutic applications in host cells that lack or express reduced levels of the Coxsackievirus and adenovirus receptor. To assess the genetic modifications of rAd variants that might induce the instability of Ad virions, we examined the frequencies of mutants that accumulated in propagated stocks. Seven of 41 lines of Ad variants generated mutants in the stocks and all mutants were infectious. Moreover, all the mutations occurred in the modified region that had been added at the 3′ end of the fiber gene. Our results show that some genetic modifications at the carboxyl terminus of Ad fiber protein lead to the instability of Ad virions. [Copyright &y& Elsevier]

The classical model of epidermal proliferative units (EPUs) postulates that each EPU is composed of a single column of corneocytes plus epidermal cells directly below the column and is maintained by a single stem cell within the unit. Using the stop-enhanced green fluorescent protein (stop-EGFP) transgenic mouse system, we previously showed epidermal stem cell clonal lineages could produce multiple adjacent corneocytes (i.e. epidermal cells belonging to multiple adjacent EPUs), contradicting the classical EPU model. One possible problem with our earlier study was that N-ethyl- N-nitrosourea (ENU) was used to generate mutations for clonal analysis. This could alter the normal environment of the epidermal tissue and might lead to an artificial expansion of stem cell clonal lineages. In this study, we replicate our earlier findings using untreated stop-EGFP mice and relying on spontaneous mutations to generate clonal cell lineages. We propose an alternative to the classical EPU model to explain the dynamic nature of epidermal proliferation. Our niche-sharing model of EPUs allows epidermal cells to horizontally migrate among EPUs, so that multiple stem cells cooperatively maintain a larger proliferative compartment. [ABSTRACT FROM AUTHOR]

Abstract: Under stressful conditions mechanisms that increase genetic variation can bestow a selective advantage. Bacteria have several stress responses that provide ways in which mutation rates can be increased. These include the SOS response, the general stress response, the heat-shock response, and the stringent response, all of which impact the regulation of error-prone polymerases. Adaptive mutation appears to be process by which cells can respond to selective pressure specifically by producing mutations. In Escherichia coli strain FC40 adaptive mutation involves the following inducible components: (i) a recombination pathway that generates mutations; (ii) a DNA polymerase that synthesizes error-containing DNA; and (iii) stress responses that regulate cellular processes. In addition, a subpopulation of cells enters into a state of hypermutation, giving rise to about 10% of the single mutants and virtually all of the mutants with multiple mutations. These bacterial responses have implications for the development of cancer and other genetic disorders in higher organisms. [Copyright &y& Elsevier]

Natural variations of the urea content of milk have a detrimental effect on the regularity of acidification by Streptococcus thermophilus strains used in dairy processes. The aim of the present study was to select urease-deficient mutants of S. thermophilus and to investigate their properties. Using an improved screening medium on agar plates, mutants were selected from 4 different parent strains after mutagen treatment and by spontaneous mutation. Most mutants were stable and had a phage sensitivity profile similar to that of their parent strain. Some of them contained detrimental secondary mutations, as their acidifying activity was lower than that of the parent strain cultivated in the presence of the urease inhibitor flurofamide. The proportion of this type of mutant was much lower among spontaneous mutants than among mutants selected after mutagen treatment. Utilization of urease-deficient mutants in dairy processes may have several advantages, such as an increase in acidification, an improved regularity of acidification, and a lower production of ammonia in whey. [ABSTRACT FROM AUTHOR]

Reversions of an auxotrophy-causing frameshift allele during prolonged starvation of yeast cells were used as a means to elucidate the mechanisms concerned with the generation of spontaneous adaptive mutations in cell cycle-arrested cells. Whereas about 50% of these reversions were previously shown to depend on the non-homologous end joining pathway of DNA double-strand break repair, the origin of the residual 50% remains unknown. In search for a mechanism for generation of the latter fraction of reversions we examined the role of the translesion synthesis (TLS) polymerases ζ, η and Rev1p in cells with wild-type or impaired nucleotide excision repair (NER) capacity. The basal level of adaptive mutations in the repair-proficient wild type was not influenced by disruptions of the genes coding for these three TLS polymerases. Intriguingly, a deficiency in NER by disruption of RAD14, RAD16 or RAD26 resulted in a significantly higher frequency of adaptive mutation, yet this increase was strictly dependent on an intact REV3 gene, coding for the catalytic subunit of polymerase ζ. Furthermore, we observed that intact REV3 was also required for the occurrence of increased frequencies of adaptive mutants in the NER-proficient wild type following UV irradiation. While in proliferating cells the translesion synthesis function of polymerase ζ is connected to DNA replication, our data suggest that in cell cycle-arrested cells this enzyme is able to carry out either TLS or error-prone polymerization along an undamaged template in the course of repair processes. Such a hitherto unappreciated activity of polymerase ζ in non-replicating cells may contribute to the incidence of mutations in evolution, aging and cancer. [Copyright &y& Elsevier]

Genetic selection assays were developed to measure rates of deletion of one or more (CAG)·(CTG) repeats, or an entire repeat tract, in Escherichia coli. In-frame insertions of ≥25 repeats in the chloramphenicol acetyltransferase (CAT) gene of pBR325 resulted in a chloramphenicol-sensitive (Cms) phenotype. When (CAG)25 comprised the leading template strand, deletion of one or more repeats resulted in a chloramphenicol resistant (Cmr) phenotype at a rate of 4×10−2 revertants per cell per generation. The mutation rates for plasmids containing (CAG)43 or (CAG)79 decreased significantly. When (CTG)n comprised the leading template strand the Cmr mutation rates were 100–1000 lower than for the opposite orientation. As an initial application of this assay, the effects of mutations influencing mismatch repair and recombination were examined. The methyl directed mismatch repair system increased repeat stability only when (CTG)n comprised the leading template strand. Replication errors made with the opposite repeat orientation were apparently not recognized. For the (CAG)n leading strand orientation, mutation rates were reduced as much as 3000-fold in a recA− strain. In a second assay, out-of-frame mutation inserts underwent complete deletion at rates ranging from about 5×10−9 to 1×10−7 per cell per generation. These assays allow careful quantitation of triplet repeat instability in E. coli and provide a way to examine the effects of mutations in replication, repair, and recombination on repeat instability. [Copyright &y& Elsevier]

Escherichia coli has provided an important model system for understanding the molecular basis for genetic instabilities associated with repeated DNA. Changes in triplet repeat length during growth following transformation in E. coli have been used as a measure of repeat instability. However, very little is known about the molecular and biological changes that may occur on transformation. Since only a small proportion of viable cells become competent, uncertainty exists regarding the nature of these transformed cells. To establish whether the process of transformation can be inherently mutagenic for certain DNA sequences, we used a genetic assay in E. coli to compare the frequency of genetic instabilities associated with transformation with those occurring in plasmid maintained in E. coli. Our results indicate that, for certain DNA sequences, bacterial transformation can be highly mutagenic. The deletion frequency of a 106 bp perfect inverted repeat is increased by as much as a factor of 2×105 following transformation. The high frequency of instability was not observed when cells stably harboring plasmid were rendered competent. Thus, the process of transformation was required to observe the instability. Instabilities of (CAG)·(CTG) repeats are also dramatically elevated upon transformation. The magnitude of the instability is dependent on the nature and length of the repeat. Differences in the methylation status of plasmid used for transformation and the methylation and restriction/modification systems present in the bacterial strain used must also be considered in repeat instability measurements. Moreover, different E. coli genetic backgrounds show different levels of instability during transformation. [Copyright &y& Elsevier]

Mutation is often said to be random. Although it must be true that mutation is ignorant about the adaptive needs of the organism and thus is random relative to them as a rule, mutation is not truly random in other respects. Nucleotide substitutions, deletions, insertions, inversions, duplications and other types of mutation occur at different rates and are effected by different mechanisms. Moreover the rates of different mutations vary from organism to organism. Differences in mutational biases, along with natural selection, could impact gene and genome evolution in important ways. For instance, several recent studies have suggested that differences in insertion/deletion biases lead to profound differences in the rate of DNA loss in animals and that this difference per se can lead to significant changes in genome size. In particular, Drosophila melanogaster appears to have a very high rate of deletions and the correspondingly high rate of DNA loss and a very compact genome. To assess the validity of these studies we must first assess the validity of the measurements of indel biases themselves. Here I demonstrate the robustness of indel bias measurements in Drosophila, by comparing indel patterns in different types of nonfunctional sequences. The indel pattern and the high rate of DNA loss appears to be shared by all known nonfunctional sequences, both euchromatic and heterochromatic, transposable and non-transposable, repetitive and unique. Unfortunately all available nonfunctional sequences are untranscribed and thus effects of transcription on indel bias cannot be assessed. I also discuss in detail why it is unlikely that natural selection for or against DNA loss significantly affects current estimates of indel biases. [ABSTRACT FROM AUTHOR]

In Pseudomonas fluorescens strain CHA0, the response regulator gene gacA controls expression of extracellular enzymes and antifungal secondary metabolites, which are important for this strain's biocontrol activity in the plant rhizosphere. Two Tn 5 insertion mutants of strain CHA0 that had the same pleiotropic phenotype as gacA mutants were complemented by the gacS sensor kinase gene of P. syringae pv. syringae as well as that of P. fluorescens strain Pf-5, indicating that both transposon insertions had occurred in the gacS gene of strain CHA0. This conclusion was supported by Southern hybridisation using a gacS probe from strain Pf-5. Overexpression of the wild-type gacA gene partially compensated for the gacS mutation, however, the overexpressed gacA gene was not stably maintained, suggesting that this is deleterious to the bacterium. Strain CHA0 grown to stationary phase in nutrient-rich liquid media for several days accumulated spontaneous pleiotropic mutants to levels representing 1.25% of the population; all mutants lacked key antifungal metabolites and extracellular protease. Half of 44 spontaneous mutants tested were complemented by gacS, the other half were restored by gacA. Independent point and deletion mutations arose at different sites in the gacA gene. In competition experiments with mixtures of the wild type and a gacA mutant incubated in nutrient-rich broth, the mutant population temporarily increased as the wild type decreased. In conclusion, loss of gacA function can confer a selective advantage on strain CHA0 under laboratory conditions. [ABSTRACT FROM AUTHOR]

It has been hypothesized that a large fraction of 24% noncoding DNA in R. prowazekii consists of degraded genes. This hypothesis has been based on the relatively high G+C content of noncoding DNA. However, a comparison with other genomes also having a low overall G+C content shows that this argument would also apply to other bacteria. To test this hypothesis, we study the coding potential in sets of genes, pseudogenes, and intergenic regions. We find that the correlation function and the χ2-measure are clearly indicative of the coding function of genes and pseudogenes. However, both coding potentials make almost no indication of a preexisting reading frame in the remaining 23% of noncoding DNA. We simulate the degradation of genes due to single-nucleotide substitutions and insertions/deletions and quantify the number of mutations required to remove indications of the reading frame. We discuss a reduced selection pressure as another possible origin of this comparatively large fraction of noncoding sequences. [ABSTRACT FROM AUTHOR]

We report the unusual features of a female patient who had MELAS‐specific A3243G mutation in mitochondrial DNA (mtDNA) and diabetes mellitus (DM). The patient showed mitochondrial myopathy, encephalopathy, lactic acidosis, and deafness but lacked the stroke‐like episode. Acute hyperglycemia was noted after one attack of status epilepticus. Molecular genetic analysis demonstrated a heteroplasmic A3243G point mutation in the mtDNAs of muscle, blood cells and hair follicles. Glucagon stimulation test exhibited marked depression of pancreatic β‐cell function. However, in a further study neither this mutation, nor MELAS syndrome or DM, was found in all of her maternal relatives. A series of follow‐up studies for β‐cell function also showed gradual improvement. The pedigree study led us to believe that this A3243G mutation arose from the germ line cells or occurred later in somatic tissues of the patient. We also suggest that the A3243G mutation of mtDNA may elicit the pathogenesis of a subtype of DM. Nevertheless, environmental stress may be another important factor for provocation of the disease. [ABSTRACT FROM AUTHOR]

The goal of this study is to investigate the probable intermediate hosts and the allergenicity of the notorious virus SARS-CoV-2 to understand how this virus emerged. The phylogenetic analysis of the virus spike proteins indicates that SARS-CoV-2 falls into various small subclades that include a bat coronavirus RaTG13, suggesting bats as a likely natural origin. Refined alignment of the spike protein in NCBI found several fragments that are specific to SARS-CoV-2 and/or SARS-CoV are specific to Rattus norvegicus and/or Mus musculus, suggesting that rodents are the intermediate reservoir of SARS-CoV-2 and SARS-CoV. To evaluate the allergenicity values, the binding affinities of human leukocyte antigen (HLA) class I or II molecules with the spike proteins were calculated, and the results showed that both SARS-CoV-2 and SARS-CoV are predicted to bind to fourteen HLA class I and II molecules with super-high HLA allele-peptide affinities. The infection rate of individuals who have HLA alleles with very high binding affinities who might become infected and develop into refractory patients if there were no medical or non-medical interventions is about 7.36% and 4.78% of Chinese and Americans, respectively. Extremely high temperature and exceptionally low precipitation, the common climate factors between the outbreak sites of COVID-19 in Wuhan in 2019 and SARS in Guangdong in 2002, might have promoted coronavirus evolution into more virulent forms. Our hypothesis suggests that early immunization with an allergenically-engineered virus, in combination with continued surveillance of meteorological factors and viral mutations, may be one of the most powerful prophylactic modalities to fight this virus. [ABSTRACT FROM AUTHOR]

Among adult BALB mice fewer than 20% usually have a small or absent corpus callosum (CC) and inheritance is polygenic. In the fetus at the time when the CC normally forms, however, almost all BALB mice show a distinct bulge in the interhemispheric fissure and grossly retarded commissure formation, and inheritance appears to result from two autosomal loci, provided the overall maturity of fetuses is equated. Most fetuses recover from the early defect when the CC axons manage to cross over the hippocampal commissure, and thus there is developmental compensation for a genetic defect rather than arrested midline development. The pattern of interhemispheric connections when the adult CC is very small is topographically normal in most respects, despite the unusual paths of the axons. The proportion of mice which fail to recover completely can be doubled by certain features of the maternal environment, and the severity of defects in adults can also be exacerbated by new genetic mutations which create new BALB substrains. The behavioral consequences of absent CC in mice are not known, nor have electrophysiological patterns been examined. The mouse provides an important model for prenatal ontogeny and cortical organization in human CC agenesis, because these data are not readily available for the human condition. [ABSTRACT FROM AUTHOR]

mms4-1 is one of several Saccharomyces cerevisiae mutants that exhibit an increased sensitivity to methyl methanesulfonate (MMS), but not to UV or X-rays. We have isolated the MMS4 gene by functional complementation of the MMS-sensitive phenotype in the mms4-1 strain. The MMS4 gene encodes a 691-amino acid, 78.7-kDa protein. The deduced Mms4 protein does not show significant homology to any of the known proteins in the database. However, several putative functional domains suggest that it may be a nuclear protein capable of interacting with other proteins. Examination of the mms4Δ mutant phenotype indicates that the mutation not only sensitizes DNA to methylating and ethylating agents, but also to other DNA damage that blocks DNA replication. However, the mms4Δ mutant appears to be more sensitive to chronic treatment than to acute treatment by DNA-damaging agents. Furthermore, the spontaneous mutation rate increases significantly in the mms4Δ mutant. Mms4 alone, when fused to a Gal4 DNA-binding domain, is able to activate P GAL1- lacZ and P GAL1 -HIS3 reporter genes in a two-hybrid system; the Mms4 transactivation domain maps to the highly acidic N-terminal region. These results collectively suggest that Mms4 may function as a transcriptional (co)activator and play an important role in DNA repair and/or synthesis. [ABSTRACT FROM AUTHOR]

A retrospective review of the familial adenomatous polyposis registry at the Cleveland Clinic Foundation revealed an incidence of spontaneous mutation in familial adenomatous polyposis (FAP) of 22 percent of family kindreds. These patients were reviewed retrospectively and compared with the total FAP population followed at The Cleveland Clinic Foundation with respect to the onset of disease, the incidence of carcinoma in the resected colon, and incidence of extracolonic manifestations. Review of the characteristics and presentations of these patients suggested that these individuals may harbor a more severe form of FAP. This may be due, in part, to the delay in diagnosis and, therefore, a higher rate of development of colorectal carcinoma and possibly duodenal adenomas. There is also a demonstrable higher rate of extracolonic manifestations of FAP present in this subset of patients. When selecting the initial type of prophylactic colonic resection the surgeon should bear in mind the increased incidence of extracolonic manifestations of the disease in this group of patients and their potential for complications. [ABSTRACT FROM AUTHOR]

We present a comparison of spacer and coding sequences of histone gene repeats from four Stronglycocentrotus purpuratus individuals. Sequences of two previously cloned units (pCO2 and pSp2) were compared with three new histone gene clones, two of them from a single individual. Within a 1.7-kb region, 59 polymorphic sites were found in spacers, in mRNA nontranslated stretches, and at silent sites in codons of the H4 gene. The permitted silent-site changes were as frequent as in any other region studied. The most abundant polymorphisms were single-base substitutions. The ratio of transitions: tranversions: single-base-pair insertions/deletions was 3∶2∶2. A number of larger insertions/deletions were found, as well as differences in the length of (CTA) and (CT) runs. Two of the five cloned repeats contained an insertion of a 195-bp element that is also present at many other sites in the genomes of every S. purpuratus individual studied. Pairwise comparisons of the different clones indicate that the variation is not uniformly divergent, but ranges from a difference of 0.34% to 3.0% of all nucleotide sites. A parsimonious tree of ancestry constructed from the pariwise comparisons indicates that recombination between the most distantly related repeats has not occurred in the 1-2 million years necessary for accumulation of the variation. The level of sequence variation found within the S. purpuratus population, for both tandemly repeated and single-copy genes, is 25%-50% of that found between S. purpuratus and S. drobachiensis. [ABSTRACT FROM AUTHOR]

Aspergillus niger var. awamori contains multiple copies of a transposable element, Vader. This element was detected as a 437-bp insertion in four independently isolated spontaneous mutants of the niaD (nitrate reductase) gene. The Vader element is present in approximately 15 copies in both A. niger var. awamori and A. niger. A single copy of Vader was detected from only one of the two laboratory strains of A. nidulans which were also examined. Insertion of the Vader element into the niaD gene of A. niger var. awamori caused a 2-bp duplication (TA) of the target sequence. The Vader element is flanked by a 44-bp inverted repeat. The genetic stabilities of the inserted Vader elements at niaD were examined by studying reversion frequencies resulting in colonies able to grow on nitrate as a sole nitrogen source. Mutants niaD392 and niaD436 reverted at a frequency of 9x10 and 4x10, respectively. Two of the mutants, niaD587 and niaD410, reverted at a lower frequency of 6x10. [ABSTRACT FROM AUTHOR]

The antimutator phenotype, reportedly conferred by disruption of the Saccharomyces cerevisiae DDR48 gene, was suggested to affect only a specific spontaneous mutational pathway. We attempted to identify the types of mutation that are DDR48-dependent by determining the specificity of the ddr48 antimutator. However, disruption of DDR48 did not decrease the rates of spontaneous forward mutation in a plasmid-borne copy of the yeast SUP4-o gene, the reversion or suppression of the lys2-1 allele, or forward mutation at the CAN1 locus. Interestingly, the latter gene had been reported previously to be subject to the antimutator effect. DNA sequence analysis of spontaneous SUP4-o mutations arising in DDR48 and ddr48 backgrounds provided no evidence for a reduction in the rates of individual mutational classes. Thus, we were unable to verify that disruption of DDR48 causes an antimutator phenotype. [ABSTRACT FROM AUTHOR]

We have employed the analysis of spontaneous forward mutations that confer the ability to utilize L-α-aminoadipate as a nitrogen source (α-Aa) to discern the events that contribute to mitotic segregation of spontaneous recessive mutations by diploid cells. α-Aa diploid cells yield α-Aa mutants at a rate of 7.8±3.6×10. As in haploid strains, approximately 97% (30/31) of α-Aa mutants are spontaneous lys2-x recessive mutations. α-Aa mutants of diploid cells reflect mostly the fate of LYS2/lys2-x heterozygotes that arise by mutation within LYS2/LYS2 populations at a rate of 1.2±0.4×10. Mitotic recombination occurs in nonrandom association with forward mutation of LYS2 at a rate of 1.3±0.6×10. This mitotic recombination rate is tenfold higher than that of a control LYS2/lys2-1 diploid. Mitotic segregation within LYS2/lys2-x subpopulations yields primarily lys2-x/lys2-x diploids and a minority of lys2-x aneuploids. Fifteen percent of lys2-x/lys2-x diploids appear to have arisen by gene conversion of LYS2 to lys2-x; 85% of lys2-x/lys2-x diploids appear to have arisen by mitotic recombination in the CENII-LYS2 interval. lys2-1/lys2-1 mitotic segregants of a control LYS2/lys2-1 diploid consist similarly of 18% of lys2-1/lys2-1 diploids that appear to have arisen by gene conversion of LYS2 to lys2-1 and 82% of lys2-1/lys2-1 diploids that appear to have arisen by mitotic recombination in the CENII-LYS2 interval. The methods described can be used to simultaneously monitor the effects of yeast gene mutations and carcinogens on the principal parameters affecting the genomic stability of diploid mitotic cells: mutation, gene conversion, intergenic recombination, and chromosomal loss or rearrangement. [ABSTRACT FROM AUTHOR]

There are 2 categories of spontaneously occurring avian and mammalian RNA tumour virus mutants: conditional and non-conditional. 1) Conditional mutants are able to replicate in or transform cells only under certain physiological conditions or in certain cells. RNA tumour virus temperature-sensitive mutants, focus-morphology mutants, and host range mutants are spontaneously formed. Some of these conditional mutants probably arise by point mutations in the viral genome. 2) Non-conditional mutants have genetic lesions that render them inactive under all conditions. There are non-conditional spontaneous RNA tumour virus mutants that are missing either the virion envelope glycoprotein or both the envelope glycoprotein and the virion DNA polymerase. These mutants cannot replicate or transform cells. Other spontaneous non-conditional mutants can replicate but are defective in their ability to transform fibroblastoid cells. These spontaneous transformation-defective mutants can have deletions in 10-20 % of the genomic RNA. Conditional mutants with an altered host range occur at a high rate of approximately 1 mutation/50 infected cell generations during DNA-to-DNA information transfer. This type of conditional mutation requires cell replication but does not occur frequently either during the original synthesis of viral DNA (RNA-to-DNA information transfer) or during the transcription of progeny viral RNA from the DNA (DNA-to-RNA information transfer). Temperature-sensitive and focus-morphology mutants also have a high rate of spontaneous formation. Non-conditional mutants missing the viral envelope glycoprotein, DNA polymerase, or transformation gene also appear to be spontaneously formed at a high rate. Normal avian and mammalian cells contain RNA tumour virus-related genes in their DNA. It is hypothesized that these endogenous RNA tumour virus-related genes in normal cells also have a high rate of spontaneous mutation and are involved in neoplastic processes. [ABSTRACT FROM AUTHOR]

Background Smallpox remains a high-priority threat due to its potential for re-emergence through events including bioterrorism and spontaneous mutation. While traditional replicating smallpox vaccines such as ACAM2000 are associated with serious side effects, the non-replicating MVA BN smallpox vaccine was developed as a safer alternative. Methods This phase 3 non-inferiority study compared indicators of efficacy between the MVA-BN smallpox vaccine and ACAM2000. The co-primary endpoints were (1) to compare vaccine-induced serum neutralizing antibodies (geometric mean titer [GMT]) at predefined Peak Visits, as measured by plaque reduction neutralization tests (PRNT) and (2) to assess the attenuation of ACAM2000-induced takes after MVA-BN administration by measuring maximum lesion area (MLA). Early neutralizing antibody GMTs at Day 14, a timepoint considered protective for traditional replicating smallpox vaccines, were also compared following single doses of either vaccine. Results A total of 440 subjects were evenly randomized to receive either 2 doses of MVA-BN followed by 1 dose of ACAM2000 at 4 week intervals (Group 1) or a single dose of ACAM2000 (Group 2). Peak neutralizing antibody GMTs were significantly higher following 2 MVA-BN doses (153.5) compared with ACAM2000 (79.3), with a ratio of 1.935 (95% CI: 1.562, 2.397). At Day 14, neutralizing antibody GMTs were equal following a single dose of either MVA BN or ACAM2000 (16.2, ratio of 0.997, 95% CI: 0.738, 1.348), with similar seroconversion rates (90.8% vs. 91.8%, respectively). The median MLA induced by ACAM2000 was significantly reduced when subjects received prior MVA-BN in Group 1 (0 mm2) compared with Group 2 (76.0 mm2), suggesting protection against orthopoxvirus. MVA BN was well tolerated, demonstrating a better safety profile than ACAM2000. Conclusion Two doses of MVA-BN induce significantly higher peak neutralizing antibody responses compared with ACAM2000. A single dose induces an early neutralizing antibody response equal to ACAM2000 at Day 14, demonstrating the suitability of MVA BN in both pre- and post-outbreak scenarios. This study was partly funded by BARDA under contract HHSO100200700034C. Disclosures Jane Maclennan, BSc MRPharmS, Bavarian Nordic (Employee), Bavarian Nordic (Employee, Shareholder), Heinz Weidenthaler, MD, Bavarian Nordic (Employee, Shareholder), Phillip Pittman, MD, Bavarian Nordic (Scientific Research Study Investigator), Darja Schmidt, PhD, Bavarian Nordic (Employee, Shareholder), Paul Chaplin, PhD, Bavarian Nordic (Board Member, Employee, Shareholder), Bavarian Nordic (Employee, Shareholder). [ABSTRACT FROM AUTHOR]

Trehalose biosynthesis pathway is a potential target for antifungal drug development, and trehalose 6-phosphate (T6P) accumulation is widely known to have toxic effects on cells. However, how organisms maintain a safe T6P level and cope with its cytotoxicity effects when accumulated have not been reported. Herein, we unveil the mechanism by which the rice blast fungus Magnaporthe oryzae avoids T6P accumulation and the genetic and physiological adjustments it undergoes to self-adjust the metabolite level when it is unavoidably accumulated. We found that T6P accumulation leads to defects in fugal development and pathogenicity. The accumulated T6P impairs cell wall assembly by disrupting actin organization. The disorganization of actin impairs the distribution of chitin synthases, thereby disrupting cell wall polymer distribution. Additionally, accumulation of T6P compromise energy metabolism. M. oryzae was able to overcome the effects of T6P accumulation by self-mutation of its MoTPS3 gene at two different mutation sites. We further show that mutation of MoTPS3 suppresses MoTps1 activity to reduce the intracellular level of T6P and partially restore Δ Motps2 defects. Overall, our results provide insights into the cytotoxicity effects of T6P accumulation and uncover a spontaneous mutation strategy to rebalance accumulated T6P in M. oryzae . IMPORTANCE M. oryzae , the causative agent of the rice blast disease, threatens rice production worldwide. Our results revealed that T6P accumulation, caused by the disruption of MoTPS2 , has toxic effects on fugal development and pathogenesis in M. oryzae uses a spontaneous mutation to restore T6P cytotoxicity. Seven spontaneous mutation sites were found, and a mutation in M. oryzae was further identified. The spontaneous mutation in MoTPS3 was further identified. The spontaneous mutation in MoTPS3 can partially rescue Δ Motps2 defects by suppressing MoTps1 activity to alleviate T6P cytotoxicity. This study provides clear evidence for better understanding of T6P cytotoxicity and how the fungus protects itself from T6P's toxic effects when it has accumulated to severely high levels.