Very high frequency of reversion to guanidine resistance in clonal pools of guanidine-dependent type 1 poliovirus.

We have carefully examined the frequency of guanidine-resistant revertants in six different clonal pools of guanidine-dependent mutants of type 1 poliovirus. The mutation frequency was (6.5 +/- 6.3) x 10(-4) (with all amino acid substitutions occurring at position 227). The minimal corrected base substitution frequency per single nucleotide site in the codon for amino acid 227 was (2.1 +/- 1.9) x 10(-4).     

Amino Acid Changes in Disease-Associated Variants Differ Radically from Variants Observed in the 1000 Genomes Project Dataset

The 1000 Genomes Project data provides a natural background dataset for amino acid germline mutations in humans. Since the direction of mutation is known, the amino acid exchange matrix generated from the observed nucleotide variants is asymmetric and the mutabilities of the different amino acids are very different. These differences predominantly reflect preferences for nucleotide mutations in the DNA (especially the high mutation rate of the CpG dinucleotide, which makes arginine mutability very much higher than other amino acids) rather than selection imposed by protein structure constraints, although there is evidence for the latter as well. The variants occur predominantly on the surface of proteins (82%), with a slight preference for sites which are more exposed and less well conserved than random. Mutations to functional residues occur about half as often as expected by chance. The disease-associated amino acid variant distributions in OMIM are radically different from those expected on the basis of the 1000 Genomes dataset. The disease-associated variants preferentially occur in more conserved sites, compared to 1000 Genomes mutations. Many of the amino acid exchange profiles appear to exhibit an anti-correlation, with common exchanges in one dataset being rare in the other. Disease-associated variants exhibit more extreme differences in amino acid size and hydrophobicity. More modelling of the mutational processes at the nucleotide level is needed, but these observations should contribute to an improved prediction of the effects of specific variants in humans.     

Structural analysis of human apolipoprotein A-I variants. Amino acid substitutions are nonrandomly distributed throughout the apolipoprotein A-I primary structure

In the course of an electrophoretic mutation screening program of 32,000 dried blood samples from newborns, 17 genetic variants of apolipoprotein A-I (apoA-I) were found and structurally analyzed. The following defects were identified by the combined use of high performance liquid chromatography, time-of-flight secondary ion mass spectrometry, and sequence analysis: Pro3----Arg (1 x), Pro4----Arg (1 x), Asp89----Glu (1 x), Lys107----0 (4 x), Lys107----Met (2 x), Glu139----Gly (2 x), Glu147----Val (1 x), Pro165----Arg (4 x), and Glu198----Lys (1 x). The distribution of point mutations in the apoA-I gene leading to these 9 and 11 other variants of apoA-I reported previously was statistically analyzed. Substitutions are overrepresented in the 10 amino-terminal amino acids (p less than 0.001, chi 2-test) and in residues 103-177 (p less than 0.025, chi 2-test) or residues 103-198 (p less than 0.05, chi 2-test), respectively. We further noted the following. (i) Prolines were substituted by arginine or histidine residues at a frequency much higher than expected on the basis of random nucleotide substitutions (5 out of 18 "electrically non-neutral" amino acid substitutions, p less than 0.001, chi 2-test). These substitutions are the result of transversions of cytosines contained within stretches of at least 5 consecutive cytosines in the apoA-I gene. The observed hypervariability of the apoA-I amino terminus, therefore, might be caused by a hot spot for mutation formed by the 7 subsequent cytosines in codons 3, 4, and 5. (ii) CpG dinucleotides were overrepresentatively affected by C----T transitions (5 out of 18 electrically nonneutral amino acid substitution, p less than 0.001, chi 2-test). The hypervariability of the apoA-I alpha-helical domain might therefore be caused by CpG dinucleotides predominantly occurring in codons 120-208 of apoA-I (82 out of 125). (iii) Comparison of mutation sites in the human apoA-I gene with sites of nonsynonymous substitutions revealed that amino acid substitutions found in human apoA-I were predominantly localized in areas that were little conserved during mammalian evolution. These regions may therefore represent areas of less structural constraint for the function of apoA-I.     

Reversion of the Frameshift hisD3018 of Salmonella typhimurium

Previous studies on histidinol dehydrogenase from His(+) revertants have shown that the frameshift mutation hisD3018 is a +1 type, resulting from inclusion of an extra cytidylate residue in messenger ribonucleic acid. Histidinol dehydrogenase from newly isolated spontaneous and N-methyl-N'-nitro-N-nitrosoguanidine (NG)-induced intragenic revertants has been examined for amino acid replacements. The results provide additional evidence that NG can delete guanine plus cytosine base pairs from deoxyribonucleic acid. One spontaneous revertant was found to result from a +2 addition of approximately 16 nucleotide residues before the +1 parent frameshift, and another by a -4 deletion about six residues before the same. Circumstantial evidence suggests the in vivo codon assignment GAG for glutamic acid. A region of histidinol dehydrogenase highly permissive of amino acid changes encoded in the minus (-) phase is now apparent.     

Relative contributions of germline gene variation and somatic mutation to immunoglobulin diversity in the mouse

The relative contributions of germline gene variation and somatic mutation to immunoglobulin diversity were studied by comparing germline gene sequences with their rearranged counterparts for the mouse VH, V kappa, and V lambda genes. The mutation rate at the amino acid level was estimated to be 7.0% in the first and second complementarity- determining regions (CDRs) and 2.0% in the framework regions (FRs). The difference in the mutation rate at the nucleotide level between the CDRs and FRs was of the same order of magnitude as that for the amino acid level. Analysis of amino acid diversity or nucleotide diversity indicated that the contribution of somatic mutation to immunoglobulin diversity is approximately 5%. However, the contribution of somatic mutation to the number of different amino acid sequences of immunoglobulins is much larger than that estimated by the analysis of amino acid diversity, and more than 90% of the different immunoglobulins seem to be generated by somatic mutation. Examination of the pattern of nucleotide substitution has suggested that clonal selection after somatic mutation may not be as strong as generally believed.      

The histidin-loop is essential for transport activity of human MDR3. A novel mutation of MDR3 in a patient with progressive familial intrahepatic cholestasis type 3

Experimental evidence has been provided that a histidine-loop within the nucleotide binding domain of ABC transporter is essential for efficient function of this class of transporter proteins. Here we report the first patient with a mutation of the putative histidine-loop of a human ABC transporter, the multi drug resistance protein 3 (MDR3). The patient presented at the age of 4years with a history of severe pruritus, elevated serum gamma-glutamyltransferase and bile acid levels since several years suggesting the diagnosis of progressive familial intrahepatic cholestasis type 3 (PFIC-3) due to defects in MDR3. Liver biopsy demonstrated an apparently normal MDR3 expression, however, genetic analysis revealed a novel homozygous mutation in the ABCB4 gene (c.3691C>T) in the patient. This mutation was associated with a change of histidine to tyrosine at amino acid position 1231 of MDR3 (p.H1231Y). As shown by sequence alignment, this amino acid corresponds to the highly conserved histidine of the "H-loop", which is critical for ATP-hydrolysis, suggesting an essential role of histidine 1231 of human MDR3.     

输入病毒导致的十起本土疫情首例病例新冠病毒基因特征分析

2020年4月中国阻断湖北省武汉市新冠肺炎疫情传播后,中国国内报道了多起由境外输入导致的本土聚集性新冠肺炎疫情。为分析引起聚集性疫情的输入性新型冠状病毒(SARS-CoV-2)的基因组特征,本研究对2020年4-11月份十起输入相关本土疫情首例病例的SARS-CoV-2全基因组基因特征进行分析,系统阐述了相关SARS-CoV-2的全基因组和氨基酸变异特征。结果显示,与武汉参考株相比,十起本土聚集性疫情首例病例的SARS-CoV-2核苷酸突变中位数为10个(8个-26个),氨基酸突变的中位数为6个(4个-16个),且刺突(spike,S)蛋白只有D614G一个氨基酸发生突变。除分支位点外,10条SARS-CoV-2全基因组序列的65个核苷酸突变位点以及35个氨基酸突变仅出现1-2次,呈现随机性。全基因组分析表明,这十起本土疫情的首例病例基因组按照中国分型法可划分为4个型,按照Pangolin分型法可划分为7个型,与我国2020年1-3月份武汉流行的毒株属于不同基因型,不是本土SARS-CoV-2的持续传播。与2020年9-12月英国和南非变异株属于不同基因型,无相关性。本文系统分析了2020年由输入病毒导致的十起本土疫情首例病例的SARS-CoV-2核苷酸与氨基酸变异特征,为我国新冠防控策略的制定以及后续新冠疫情的溯源提供了参考依据。     

Map locations of mouse hepatitis virus temperature-sensitive mutants: confirmation of variable rates of recombination.

Using standard genetic recombination techniques, studies in our laboratory suggest that recombination rates are very high and vary in different portions of the mouse hepatitis virus (MHV) genome. To determine the actual recombination frequencies in the MHV genome and localize the nucleotide boundaries of individual viral genes, we have sequenced temperature-sensitive and revertant viruses to identify the location of specific mutant alleles. Complementation group F RNA+ ts mutants (LA7, NC6, and NC16) each contained a unique mutation which was tightly linked to the ts phenotype and resulted in a conservative or nonconservative amino acid change in the MHV S glycoprotein gene. In agreement with previous recombination mapping studies, the mutation in LA7 and NC6 mapped within the S1 domain while NC16 mapped within the S2 domain. To determine the map coordinates of the MHV polymerase genes, several RNA- mutants and their revertants belonging to complementation groups C (NC3 and LA9) and E (LA18 and NC4) were also sequenced. Mutations were identified in each virus that were tightly linked to the ts phenotype and resulted in either a conservative or nonconservative amino acid change. The group C allele spanned the ORF 1a/ORF 1b junction, while the group E mutants mapped at the C terminus of ORF 1b about 20 to 22 kb from the 5' end of the genome. Mutation rates, calculated from the reversion frequencies of plaque-purified ts viruses requiring a single nucleotide alteration for reversion, approached 1.32 (+/- 0.89) x 10(-4) substitutions per nucleotide site per round of template copying. Detailed recombination mapping studies across known distances between these different ts alleles has confirmed that homologous recombination rates approached 25% and varied within different portions of the MHV genome.     

结核分枝杆菌耐吡嗪酰胺分子机制研究

吡嗪酰胺(PZA)是结核病短程化疗中的一线抗结核药物,由吡嗪酰胺酶转换成为活性形式吡嗪酸而生效。吡嗪酰胺酶由pncA基因编码,pncA基因突变会导致该酶活性丧失,与PZA耐药性产生有关。为了进一步明确PZA耐药性产生的基因学基础和PZA耐药株的pncA基因突变率,对中国100株结核分枝杆菌临床分离株进行了DNA序列测定,其中85株为PZA耐药株,15株为PZA敏感株。PZA耐药株有27%(23/85)发生了pncA基因突变,从而导致吡嗪酰胺酶基本氨基酸序列的改变,突变分布在pncA基因开读框架17-546位的核苷酸。其中有一株突变位于pncA基因的调节区域-11位处。同时发现20%(3/15)pncA敏感株也发生了pncA基因突变。敏感株发生突变可能是由于PZA敏感性实验不准确或存在其它耐药机制。实验表明,pncA基因突变是PZA耐药的主要机制之一,中国PZA耐药临床分离株尚存在其它耐药分子机制。     

Nonrandom amino acid substitution and estimation of the number of nucleotide substitutions in evolution

Summary A method of estimating the number of nucleotide substitutions from amino acid sequence data is developed by using Dayhoff's mutation probability matrix. This method takes into account the effect of nonrandom amino acid substitutions and gives an estimate which is similar to the value obtained by Fitch's counting method, but larger than the estimate obtained under the assumption of random substitutions (Jukes and Cantor's formula). Computer simulations based on Dayhoff's mutation probability matrix have suggested that Jukes and Holmquist's method of estimating the number of nucleotide substitutions gives an overestimate when amino acid substitution is not random and the variance of the estimate is generally very large. It is also shown that when the number of nucleotide substitutions is small, this method tends to give an overestimate even when amino acid substitution is purely at random.     

Site-directed mutagenesis of the GTP-binding domain of beta-tubulin.

Tubulin binds guanine nucleotides with high affinity and specificity. GTP, an allosteric effector of microtubule assembly, requires Mg2+ for its interaction with beta-tubulin and binds as the MgGTP complex. In contrast, GDP binding does not require Mg2+. The structural basis for this difference is not understood but may be of fundamental importance for microtubule assembly. We investigated the interaction of beta-tubulin with guanine nucleotides using site-directed mutagenesis. Acidic amino acid residues have been shown to interact with nucleotide in numerous nucleotide-binding proteins. In this study, we mutated seven highly conserved aspartic acid residues and one highly conserved glutamic acid residue in the putative GTP-binding domain of beta-tubulin (N-terminal 300 amino acids) to asparagine and glutamine, respectively. The mutants were synthesized in vitro using rabbit reticulocyte lysates, and their affinities for nucleotide determined by an h.p.l.c.-based assay. Our results indicate that the mutations can be placed in six separate categories on the basis of their effects on nucleotide binding. These categories range from having no effect on nucleotide binding to a mutation that apparently abolishes nucleotide binding. One mutation at Asp224 reduced the affinity of beta-tubulin for GTP in the presence but not in the absence of Mg2+. The specific effect of this mutation on nucleotide binding is consistent with an interaction of this amino acid with the Mg2+ moiety of MgGTP. This residue is in a region sharing sequence homology with the putative Mg2+ site in myosin and other ATP-binding proteins. As a result, tubulin belongs to a distinct class of GTP-binding proteins which may be evolutionarily related to the ATP-binding proteins.     

Estimate of the mutation rate per nucleotide in humans

Many previous estimates of the mutation rate in humans have relied on screens of visible mutants. We investigated the rate and pattern of mutations at the nucleotide level by comparing pseudogenes in humans and chimpanzees to (i) provide an estimate of the average mutation rate per nucleotide, (ii) assess heterogeneity of mutation rate at different sites and for different types of mutations, (iii) test the hypothesis that the X chromosome has a lower mutation rate than autosomes, and (iv) estimate the deleterious mutation rate. Eighteen processed pseudogenes were sequenced, including 12 on autosomes and 6 on the X chromosome. The average mutation rate was estimated to be approximately 2.5 x 10(-8) mutations per nucleotide site or 175 mutations per diploid genome per generation. Rates of mutation for both transitions and transversions at CpG dinucleotides are one order of magnitude higher than mutation rates at other sites. Single nucleotide substitutions are 10 times more frequent than length mutations. Comparison of rates of evolution for X-linked and autosomal pseudogenes suggests that the male mutation rate is 4 times the female mutation rate, but provides no evidence for a reduction in mutation rate that is specific to the X chromosome. Using conservative calculations of the proportion of the genome subject to purifying selection, we estimate that the genomic deleterious mutation rate (U) is at least 3. This high rate is difficult to reconcile with multiplicative fitness effects of individual mutations and suggests that synergistic epistasis among harmful mutations may be common.     

Advantages of a mechanistic codon substitution model for evolutionary analysis of protein-coding sequences

BACKGROUND: A mechanistic codon substitution model, in which each codon substitution rate is proportional to the product of a codon mutation rate and the average fixation probability depending on the type of amino acid replacement, has advantages over nucleotide, amino acid, and empirical codon substitution models in evolutionary analysis of protein-coding sequences. It can approximate a wide range of codon substitution processes. If no selection pressure on amino acids is taken into account, it will become equivalent to a nucleotide substitution model. If mutation rates are assumed not to depend on the codon type, then it will become essentially equivalent to an amino acid substitution model. Mutation at the nucleotide level and selection at the amino acid level can be separately evaluated. RESULTS: The present scheme for single nucleotide mutations is equivalent to the general time-reversible model, but multiple nucleotide changes in infinitesimal time are allowed. Selective constraints on the respective types of amino acid replacements are tailored to each gene in a linear function of a given estimate of selective constraints. Their good estimates are those calculated by maximizing the respective likelihoods of empirical amino acid or codon substitution frequency matrices. Akaike and Bayesian information criteria indicate that the present model performs far better than the other substitution models for all five phylogenetic trees of highly-divergent to highly-homologous sequences of chloroplast, mitochondrial, and nuclear genes. It is also shown that multiple nucleotide changes in infinitesimal time are significant in long branches, although they may be caused by compensatory substitutions or other mechanisms. The variation of selective constraint over sites fits the datasets significantly better than variable mutation rates, except for 10 slow-evolving nuclear genes of 10 mammals. An critical finding for phylogenetic analysis is that assuming variable mutation rates over sites lead to the overestimation of branch lengths.     

Frequency of spontaneous mutations in an avian hepadnavirus infection

In this study, we measured the frequency of revertants of a cytopathic strain of the duck hepatitis B virus that bears a single nucleotide substitution in the pre-S envelope protein open reading frame, resulting in the amino acid substitution G133E. Cytopathic virus mixed with known amounts of a genetically marked wild-type virus was injected into ducklings. Virus outgrowth was accompanied by a coselection of wild-type and spontaneous revertants during recovery of the ducklings from the acute liver injury caused by death of the G133E-infected cells. The frequency of individual revertants in the selected noncytopathic virus population was estimated by determining the ratio of each revertant to the wild-type virus. Spontaneous revertants were found to be present at frequencies of 1 x 10(-5) to 6 x 10(-5) per G133E genome inoculated. A mathematical model was used to estimate that the mutation rate was 0.8 x 10(-5) to 4.5 x 10(-5) per nucleotide per generation.     

Intragenic suppression in the uvrD gene of Escherichia coli. I. Temperature-sensitive uvrD mutations

A temperature-sensitive uvrD mutant, HD323 uvrD4, was isolated from the uvrD mutant HD4 uvrD3. The temperature sensitivity of the uvrD4 gene product was reversible. The suppressor mutation uvrD44 which rendered the uvrD3 mutant temperature-sensitive could be separated from the uvrD3 mutation by replacing the PstI fragment, which encodes the C-terminal half of the UvrD protein. The uvrD44 mutation was found to make host bacteria lethal at non-permissive temperatures only when cloned on a low copy vector pMF3. The nucleotide sequence of the uvrD3 and uvrD4 mutant genes was determined. The nucleotide change found in the uvrD3 at +1235, GAA to AAA, only alters the amino acid sequence from Glu at 387 to Lys. The uvrD44 has another nucleotide change at +1859, GAA to AAA (Glu at 595 to Lys), which is considered to be the suppressor mutation uvrD44.     

Nucleotide sequence of the gene encoding adenovirus type 2 DNA binding protein.

We have determined the nucleotide sequence of the gene encoding adenovirus type 2 (Ad2) DNA binding protein (DBP). From the nucleotide sequence the complete amino acid sequence of Ad2 DBP has been deduced. A comparison of the amino acid sequences of Ad2 and Ad5 DBP, both 529 residues long, reveals that the C-terminal 354 residues of both sequences are identical. Within the N-terminal 175 amino acid residues Ad2 and Ad5 show nine differences. The site of mutation in Ad2 ND1ts23, a mutant with a temperature-sensitive DNA replication, was mapped at the nucleotide level. A single nucleotide alteration in the DBP gene, resulting in a leucine leads to phenylalanine substitution at position 282 in the amino acid sequence is responsible for the temperature-sensitive character of this mutant. Previously, we localized the mutation of another DBP mutant with a temperature-sensitive DNA replication (H5ts125) at position 413 in the amino acid sequence of the DBP molecule (Nucleic Acids Res. 9 (1981) 4439-4457). These mapping data are discussed in relation to the structure and function of the DBP molecule.     

Site-directed mutagenesis of rabbit LAT1 at amino acids 219 and 234

The availability of amino acids in the brain is regulated by the blood-brain barrier (BBB) large neutral amino acid transporter type 1 (LAT1) isoform, which is characterized by a high affinity (low Km) for substrate large neutral amino acids. The hypothesis that brain amino acid transport activity can be altered with single nucleotide polymorphisms was tested in the present studies with site-directed mutagenesis of the BBB LAT1. The rabbit has a high Km LAT1 large neutral amino acid transporter, as compared to the low Km neutral amino acid transporter at the human or rat BBB. The rabbit LAT1 was cloned from a rabbit brain capillary cDNA library. Alignment of the amino acid sequences of rabbit, human, and rat LAT1 revealed two radical amino acid residues that differ in the rabbit relative to the rat or human LAT1. The G219D mutation had a modest effect on the Km and Vmax of tryptophan transport via cloned rabbit LAT1 in frog oocytes, but the W234L variant reduced the Km by 64% and the Vmax by 96%. Conversely, LAT1 transport of either tryptophan or phenylalanine was nearly normalized when the double mutation W234L/G219D variant was produced. These studies show that marked changes in the affinity and capacity of the LAT1 are caused by single nucleotide polymorphisms and that phenotype can be restored with a double mutation.     

Gain or loss of diabetogenicity resulting from a single point mutation in recombinant encephalomyocarditis virus.

Molecular pathogenic mechanisms for virus-induced disease have received considerable attention. Encephalomyocarditis (EMC) virus-induced diabetes in mice has been extensively studied to elucidate the cellular and molecular mechanisms involved in the development of this disease. In this study, we report for the first time that a single point mutation at nucleotide position 3155 or 3156 of the recombinant EMC viral genome, located on the major capsid protein VP1, which causes an amino acid change, results in the gain or loss of viral diabetogenicity. A G base at nucleotide position 3155 (alanine at amino acid position 776 of the EMC virus polyprotein [Ala776]; GCC) results in viral diabetogenicity, whereas the substitution of other bases at the same or next position results in a loss of viral diabetogenicity. This finding provides clear evidence that a point mutation at a critical site in a viral genome affects the ability of the virus to cause a cell-specific disease.     

A thermodynamic model of protein structure evolution explains empirical amino acid substitution matrices

Proteins evolve under a myriad of biophysical selection pressures that collectively control the patterns of amino acid substitutions. These evolutionary pressures are sufficiently consistent over time and across protein families to produce substitution patterns, summarized in global amino acid substitution matrices such as BLOSUM, JTT, WAG, and LG, which can be used to successfully detect homologs, infer phylogenies, and reconstruct ancestral sequences. Although the factors that govern the variation of amino acid substitution rates have received much attention, the influence of thermodynamic stability constraints remains unresolved. Here we develop a simple model to calculate amino acid substitution matrices from evolutionary dynamics controlled by a fitness function that reports on the thermodynamic effects of amino acid mutations in protein structures. This hybrid biophysical and evolutionary model accounts for nucleotide transition/transversion rate bias, multi‐nucleotide codon changes, the number of codons per amino acid, and thermodynamic protein stability. We find that our theoretical model accurately recapitulates the complex yet universal pattern observed in common global amino acid substitution matrices used in phylogenetics. These results suggest that selection for thermodynamically stable proteins, coupled with nucleotide mutation bias filtered by the structure of the genetic code, is the primary driver behind the global amino acid substitution patterns observed in proteins throughout the tree of life.