Phenotype Switching and Mutations in Random Environments

Cell populations can benefit from changing phenotype when the environment changes. One mechanism for generating these changes is stochastic phenotype switching, whereby cells switch stochastically from one phenotype to another according to genetically determined rates, irrespective of the current environment, with the matching of phenotype to environment then determined by selective pressure. This mechanism has been observed in numerous contexts, but identifying the precise connection between switching rates and environmental changes remains an open problem. Here, we introduce a simple model to study the evolution of phenotype switching in a finite population subject to random environmental shocks. We compare the successes of competing genotypes with different switching rates, and analyze how the optimal switching rates depend on the frequency of environmental changes. If environmental changes are as rare as mutations, then the optimal switching rates mimic the rates of environmental changes. If the environment changes more frequently, then the optimal genotype either maximally favors fitness in the more common environment or has the maximal switching rate to each phenotype. Our results also explain why the optimum is relatively insensitive to fitness in each environment.     

哺乳动物中RNA干扰沉默基因表达的研究进展

RNA干扰及相关基因沉默导致的代谢通路变化已经彻底改变了人们对基因调控的理解。基因沉默技术已经被用来作为一种研究工具来控制某些细胞基因的表达,特异的si RNA导入不同的细胞需要不同的转染载体才能达到最大的转染效率,并且不会产生较大的细胞毒性。近年来尤其是碳纳米管等新型纳米材料载体的应用拓展了人们对传统脂质体和病毒载体的认识。首先介绍RNA干扰技术及其发展历程,随后对利用几种不同的载体来设计和进行RNA干扰试验进行了比较,最后对最初的脂质体到生物病毒再到高分子纳米材料介导的RNA干扰在疾病的治疗和临床诊断进行了展望。     

Suppression of TGA Mutations in the Bacillus subtilis spoIIR Gene by prfB Mutations

An unexpectedly high proportion of TGA nonsense mutations was obtained in a collection of chemically induced mutations in the spoIIR locus of Bacillus subtilis. Of 11 different mutations obtained, TGA mutations were found in four codons, whereas only three codons yielded missense mutations. Six suppressors of the TGA mutations were isolated, and five of the suppressing mutations were mapped to the prfB gene encoding protein release factor 2. These are the first mutations shown to map to the B. subtilis prfB locus. The sequence of the prfB gene was completed, and two revisions of the published sequence were made. The five prfB mutations also resulted in suppression of the catA86-TGA mutation to between 19 and 54% of the expression of catA86⁺, compared to the readthrough level of 6% in the prfB⁺ strain. N-terminal sequencing of suppressed catA86-TGA-specified protein demonstrated that the amino acid inserted at UGA because of the prfB1 mutations was tryptophan.     

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

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

Evolution by Gene Duplication and Compensatory Advantageous Mutations

Relaxation of selective constraint is thought to play an important role for evolution by gene duplication, in connection with compensatory advantageous mutant substitutions. Models were investigated by incorporating gene duplication by unequal crossing over, selection, mutation and random genetic drift into Monte Carlo simulations. Compensatory advantageous mutations were introduced, and simulations were carried out with and without relaxation, when genes are redundant on chromosomes. Relaxation was introduced by assuming that deleterious mutants have no effect on fitness, so long as one or more genes free of such mutations remain in the array. Compensatory mutations are characterized by the intermediate deleterious step of their substitutions, and therefore relaxation by gene redundancy is important. Through extensive Monte Carlo simulations, it was found that compensatory mutant substitutions require relaxation in addition to gene duplication, when mutant effects are large. However when mutant effects are small, such that the product of selection coefficient and population size is around unity, evolution by compensatory mutation is enhanced by gene duplication even without relaxation.     

Fitness Is Strongly Influenced by Rare Mutations of Large Effect in a Microbial Mutation Accumulation Experiment

Our understanding of the evolutionary consequences of mutation relies heavily on estimates of the rate and fitness effect of spontaneous mutations generated by mutation accumulation (MA) experiments. We performed a classic MA experiment in which frequent sampling of MA lines was combined with whole genome resequencing to develop a high-resolution picture of the effect of spontaneous mutations in a hypermutator (ΔmutS) strain of the bacterium Pseudomonas aeruginosa. After ∼644 generations of mutation accumulation, MA lines had accumulated an average of 118 mutations, and we found that average fitness across all lines decayed linearly over time. Detailed analyses of the dynamics of fitness change in individual lines revealed that a large fraction of the total decay in fitness (42.3%) was attributable to the fixation of rare, highly deleterious mutations (comprising only 0.5% of fixed mutations). Furthermore, we found that at least 0.64% of mutations were beneficial and probably fixed due to positive selection. The majority of mutations that fixed (82.4%) were base substitutions and we failed to find any signatures of selection on nonsynonymous or intergenic mutations. Short indels made up a much smaller fraction of the mutations that were fixed (17.4%), but we found evidence of strong selection against indels that caused frameshift mutations in coding regions. These results help to quantify the amount of natural selection present in microbial MA experiments and demonstrate that changes in fitness are strongly influenced by rare mutations of large effect.     

siRNA抑制丙型肝炎病毒IRES介导的基因表达

以HCVIRES为靶位 ,应用T7RNA多聚酶体外转录合成了 5条小干扰RNA(siRNA) .脂质体转染法将其导入HCVIRES介导萤光素酶表达的转基因细胞 (HepG2 .970 6 )中 ,通过测定萤光素酶的量 ,评价T7siRNA对HCV介导基因表达的抑制作用 .结果表明 ,所合成的 5条T7siRNAs ,均能特异性地抑制萤光素酶基因的表达 ,抑制率分别为 94 31%、80 0 1%、78 0 1%、80 33%、85 6 4% ,其中以靶向HCVIRES第二茎环结构的T7siRNA1抑制率最高 ,且对HCV基因的抑制作用有剂量依赖性 ,随T7siRNA1量的增加 ,抑制率逐渐增强 .siRNA抑制HCV基因的作用具有良好的特异性 ,改变其中 1个核苷酸即无显著抑制作用 .     

siRNA干扰HSV-1 UL30 DNA聚合酶基因的研究

目的：筛选高效沉默HSV-1UL30基因的siRNA,研究siRNA沉默UL30基因后对HSV-1繁殖的影响。方法：设计并化学合成12对靶向UL30基因的siRNA,与pEGFP-N1-Fi融合表达质粒共转染VERO细胞,流式细胞术筛选高效抑制Fi-EGFP融合蛋白的siRNA,实时荧光定量PCR检测siRNA对感染细胞内UL30mRNA表达的抑制效果,CPE法和空斑减数实验评价siRNA对HSV-1繁殖的抑制效果。结果：共转染实验筛选出高效抑制Fi-EGFP融合蛋白的siRNA4、siRNA10及siRNA8,这3对siRNA均能显著降低感染细胞内UL30mRNA的表达水平及病变细胞释放到培养上清的子代病毒滴度,siRNA4和siRNA10在感染后36h对HSV-1的繁殖有明显的抑制效果,其病斑分别比对照组减少61.17%、51.46%（P〈0.05）,siRNA4、siRNA10及siRNA8组最终形成的空斑直径分别比对照组减小29.94%、23.49%、21.69%（P〈0.01）。结论：筛选到高效抑制UL30的3对siRNAs,siRNA4及siRNA10在病斑形成早期对HSV-1的繁殖有明显的抑制效果,说明siRNA4、siRNA10及siRNA8均能延缓病斑的扩大和病斑数目的增长,对病毒的繁殖有一定的抑制效果。     

心肌肌钙蛋白T基因突变在心肌病发病机制中的研究进展

肥厚型和扩张型心肌病中,基因缺陷分别占发病的50％和35％,其病理生理机制,主要包括肌小节蛋白基因突变引起的收缩力产生缺陷,细胞骨架蛋白基因突变引起的收缩力传递缺陷等。心肌肌钙蛋白T将肌钙蛋白C和肌钙蛋白I连接到肌动蛋白和原肌球蛋白上,在心肌细胞收缩和舒张过程中发挥重要作用。在肥厚型和扩张型心肌病中发现了多种心肌肌钙蛋白T的基因突变,围绕心肌肌钙蛋白T的研究有助于阐明心肌病的发病机制。本文总结了心肌肌钙蛋白T基因突变在心肌病发病机制中的研究情况。     

Novel Mutations in the Human HPRT Gene

Inherited mutation of a purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT), gives rise to Lesch-Nyhan Syndrome (LNS) or HPRT-related gout. Here, we report five novel independent mutations in the coding region of the HPRT gene from five unrelated male patients manifesting different clinical phenotypes associated with LNS: exon 2: c.133A > G, p.45R > G; c.35A > C, p.12D > A; c.88delG; exon 7: c.530A > T, p.177D > V; and c.318 + 1G > C: IVS3 + 1G > C splice site mutation.     

Rational Design of ssODN to Correct Mutations by Gene Editing

Biochemistry (Moscow) - Gene editing allows to make a variety of targeted changes in genome, which can potentially be used to treat hereditary human diseases. Despite numerous studies in this area,...     

角毛壳菌几丁质酶基因chi58多点突变及在毕赤酵母中的高效表达

应用重叠延伸PCR技术(gene splicing by overlap extension PCR,gene SOEing),简称SOE-PCR对角毛壳菌(Chaetomium cupreum)的几丁质酶基因chi58进行多点突变.依据毕赤酵母密码子偏爱性,将毕赤酵母中编码Arg使用频率几乎为0的密码子CGC突变为使用频率高的AGA,构建了含有正确突变的酵母表达载体pPIC9K-chi58A,电转化毕赤酵母GS115,获得的重组酵母株在诱导120 h酶活力最高,平均可达101.71 U/mL±3.33 U/mL;其活力比未优化重组酵母株(31.83 U/mL±4.85 U/mL)提高了约3倍,且经10代传代培养后遗传稳定性良好.表达产物的SDS-PAGE分析表明,酶蛋白分子大小为58 kD.     

沉默Gpr3基因表达对猪卵泡颗粒细胞凋亡的影响

G蛋白偶联受体3（G protein-coupled receptor 3,Gpr3）属于G蛋白偶联受体超家族成员,能够维持卵泡卵母细胞减数分裂的前期阻滞,但在卵泡颗粒细胞中的作用不清。该研究利用RNAi技术,以化学合成的siRNA转染体外培养的猪卵泡颗粒细胞,并利用Real-time PCR和Western blot技术检验Gpr3基因的沉默效果;利用MTT（四甲基偶氮唑盐）、流式细胞术和Real-time PCR技术检测沉默Gpr3基因表达对猪卵泡颗粒细胞凋亡以及凋亡相关基因表达的影响。结果显示,Gpr3-siRNA能够有效地抑制猪卵泡颗粒细胞中Gpr3基因mRNA和蛋白的表达（P〈0.01）;在沉默Gpr3基因表达后,猪卵泡颗粒细胞的细胞活性由0.419升高至0.586,同时细胞凋亡率由2.67%下降至0.42%,并在显著上调Bcl-2表达的同时,下调了Bax的表达（P〈0.05）。结果表明,沉默Gpr3基因的表达抑制了猪卵泡颗粒细胞的凋亡,其机制可能与调控Bcl-2和Bax表达有关。     

Matrilineal Fertility Inheritance Detected in Hunter–Gatherer Populations Using the Imbalance of Gene Genealogies

Fertility inheritance, a phenomenon in which an individual's number of offspring is positively correlated with his or her number of siblings, is a cultural process that can have a strong impact on genetic diversity. Until now, fertility inheritance has been detected primarily using genealogical databases. In this study, we develop a new method to infer fertility inheritance from genetic data in human populations. The method is based on the reconstruction of the gene genealogy of a sample of sequences from a given population and on the computation of the degree of imbalance in this genealogy. We show indeed that this level of imbalance increases with the level of fertility inheritance, and that other phenomena such as hidden population structure are unlikely to generate a signal of imbalance in the genealogy that would be confounded with fertility inheritance. By applying our method to mtDNA samples from 37 human populations, we show that matrilineal fertility inheritance is more frequent in hunter–gatherer populations than in food-producer populations. One possible explanation for this result is that in hunter–gatherer populations, individuals belonging to large kin networks may benefit from stronger social support and may be more likely to have a large number of offspring.