扩增子测序法筛选结核分枝杆菌的耐药突变

结核病是严重的公共健康问题之一,而耐药结核病的增加是控制结核病流行的难点之一。快速、准确的诊断是提高结核患者治愈率和降低死亡率的关键因素。本研究建立了基于二代测序技术的扩增子测序方法,对5种一线抗结核药物的17个耐药基因进行检测。在26个临床耐药结核菌株中共鉴定出65个突变,包括33个热点突变,9个稀有突变和23个新突变。对18个新发现的错义突变进行了蛋白质序列保守性和蛋白质局部结构的分析。结果表明,14个新的错义突变在9种分枝杆菌中显示出高度保守性,并且导致了该蛋白质局部结构的改变。根据本研究检测和分析结果,推测这些新发现的突变可能是潜在的耐药突变。在本研究中,构建了扩增子测序的检测方法,可同时检测10株临床结核菌株的17个耐药基因,是一种快速、准确并且全面的检测耐药结核分枝杆菌一线治疗药物耐药突变的方法,该方法不仅能检测热点突变和稀有突变,还能发现一些未报道过的新突变。该检测方法或可用于临床诊断和基础研究。     

酵母与大肠杆菌穿梭定点突变载体系统的构建及应用

利用可以在大肠杆菌中形成单链又可以在大肠杆菌和酵母中穿梭的质粒BFDl9作为寡聚核苷酸介导的定点突变载体,分别在大肠杆菌和酵母中获得了预定的TRP1基因突变体,再利用这个带TRP1突变的质粒BFD25和BFDl9,建立了一个在酵母系统中进行定点突变时可以富集突变体的筛选方法。借助这种方法我们在酵母系统中完成了人表皮生长因子基因表达单元的寡聚核苷酸介导的缺失突变,此突变载体系统在酵母与大肠杆菌中都适用,而且可以直接在酵母细胞中进行突变表型的研究。     

弗氏2a志贺菌htrA基因突变体的构建

目的:构建弗氏2a志贺菌2457T的htrA基因缺失突变株及HtrA酶失活突变株,以便进一步研究HtrA蛋白的功能。方法:用PCR扩增htrA基因上下游同源臂,构建含有kan基因的打靶片段,采用λ-Red重组系统对htrA基因进行缺失,用PCR进行验证;通过定点突变的方法构建HtrA酶失活突变株,并测序验证。结果与结论:构建了2457T htrA缺失突变株和2457T/htrAFSA酶失活突变株。     

乙肝治疗耐药基因突变的焦磷酸测序技术诊断

目的:建立焦磷酸测序技术检测拉米夫定和阿德福韦酯治疗乙肝所致乙肝病毒基因耐药突变的定量检测方法,为临床乙肝耐药诊断和治疗提供依据。方法:针对乙肝病毒DNA聚合酶基因序列上4个常见基因突变位点的6种突变形式,分别克隆构建野生型和突变型质粒作为标准品,应用生物信息学手段设计目标基因通用PCR引物和各突变点的焦磷酸测序引物,建立焦磷酸测序的突变检测方法。对接受拉米夫定、阿德福韦酯治疗的慢性乙型肝炎患者血清标本进行检测。结果:构建了乙肝病毒四种常见耐药性突变的标准株和变异株克隆,建立了分别或同时检测拉米夫定、阿德福韦酯耐药突变的焦磷酸测序方法,对68例临床耐药或疑似耐药的患者血清标本进行检测,双脱氧测序验证,检出拉米夫定耐药突变32例,阿德福韦酯耐药突变5例,其中焦磷酸测序检出20例为混合突变,而双脱氧测序显示为6例。结论:成功建立了焦磷酸测序定量检测拉米夫定、阿德福韦酯耐药基因突变的方法,构建了乙肝病毒耐药基因突变的标准质粒,为临床动态监测乙肝病毒变异病毒株、指导合理用药奠定了基础。     

基于重叠延伸PCR法的定点突变技术

目的:建立一种高效而经济的定点突变方法。方法:采用重叠延伸PCR定点突变技术,引物设计时引入目的突变,以前两次PCR产物为模板,进行第三次PCR,即可获得突变后的目的DNA片段。将此片段连入pMDTM18-T载体后测序验证突变结果。结果:DNA测序表明,待突变位点已由ATTGG突变为ATTTT。结论:成功实现了目的位点的定点突变,重叠延伸PCR法是一种高效且经济的定点突变方法。     

枯草杆菌蛋白酶嗜热性改造单突变效果评判方法研究

对蛋白质进行嗜热性改造是蛋白质工程的主要问题之一,残基突变方法被广泛运用于其中。本文以枯草杆菌蛋白酶（SUBTILISIN BPN＇）为研究对象,旨在建立评判嗜热性改造效果的方法,选取了有可靠实验资料的9个突变点,运用分子动力学模拟方法,在四种不同模拟条件下,对其中的6个突变体和1个野生型蛋白进行了多种参量的对比分析,提取4个特征有效参量,建立了蛋白酶嗜热性改造单突变效果评判方法;利用该方法对其它3个突变效果进行评判,评判结果与实验资料完全吻合,证明该方法可用于枯草杆菌蛋白酶嗜热性改造单突变效果的评判。     

乙肝治疗耐药基因突变的焦磷酸测序技术诊断

目的：建立焦磷酸测序技术检测拉米夫定和阿德福韦酯治疗乙肝所致乙肝病毒基因耐药突变的定量检测方法,为临床乙肝耐药诊断和治疗提供依据。方法：针对乙肝病毒DNA聚合酶基因序列上4个常见基因突变位点的6种突变形式,分别克隆构建野生型和突变型质粒作为标准品,应用生物信息学手段设计目标基因通用PCR引物和各突变点的焦磷酸测序引物,建立焦磷酸测序的突变检测方法。对接受拉米夫定、阿德福韦酯治疗的慢性乙型肝炎患者血清标本进行检测。结果：构建了乙肝病毒四种常见耐药性突变的标准株和变异株克隆,建立了分别或同时检测拉米夫定、阿德福韦酯耐药突变的焦磷酸测序方法,对68例临床耐药或疑似耐药的患者血清标本进行检测,双脱氧测序验证,检出拉米夫定耐药突变32例,阿德福韦酯耐药突变5例,其中焦磷酸测序检出20例为混合突变,而双脱氧测序显示为6例。结论：成功建立了焦磷酸测序定量检测拉米夫定、阿德福韦酯耐药基因突变的方法,构建了乙肝病毒耐药基因突变的标准质粒,为临床动态监测乙肝病毒变异病毒株、指导合理用药奠定了基础。     

一种简便快速的单引物PCR定点突变方法

为了解决在一些特殊位点上利用Quick Change方法进行定点突变时会在突变位点处额外插入引物序列导致突变失败的问题,对Quick Change法进行了改良。改良方法为:合成在突变位点处点突变的一对反向互补引物,分别进行单引物PCR扩增,将两种扩增产物混合,变性复性后加入Dpn I进行酶切,酶切产物转化大肠杆菌DH5α,抗性筛选阳性克隆进行测序验证。利用此法成功突变紫穗槐二烯合酶(amorpha-4,11-diene synthase,ADS)基因中多个利用常规方法突变均因引入额外引物而无法成功的特殊位点,证明此方法实践上可行,而且也可以避免插入额外引物序列,这也从侧面证明额外引物插入的原因是双引物同时反应。     

福氏2a志贺氏菌2457T株argT基因突变体的构建

目的：构建福氏2a志贺氏菌2457T株argT基因缺失突变体和ArgT蛋白非降解突变体,以进行后续ArgT功能研究。方法：根据福氏2a志贺氏菌2457T株基因组全序列,采用λ-Red重组系统对argT基因进行缺失,并经PCR验证;采用定点突变的方法构建ArgT非降解株,并经SDS-PAGE验证;对野生株、argT缺失突变株和ArgT非降解突变株37℃时的生长曲线及生化反应进行比较研究。结果：构建了2457T的argT缺失突变株和ArgT非降解突变株;2种突变株初始生长均较慢,但最终和野生株状态一致;2种突变株利用甘露醇的能力都比野生株强,而利用葡萄糖的能力降低。结论：获得了福氏2a志贺氏菌2457T株argT基因缺失突变体和ArgT蛋白非降解突变体。     

基于高通量测序数据构建斑马鱼隐性遗传突变筛选系统

目的:针对斑马鱼高通量测序数据,通过一系列质量控制和突变过滤,构建一套有效隐性遗传突变筛选系统。方法:A)经过与斑马鱼参考基因组比对,利用GATK获得初始的VCF格式突变信息。B)使用perl语言编写本地脚本,对突变信息进行过滤,注释等。C)通过绘制纯合突变点分布图,找出突变富集区域。整合以上步骤,构建出一套针对斑马鱼高通量测序数据的遗传性突变筛选系统。结果:对获得的突变位点进行了一系列的质量控制,定位到具体染色体上的特定区域,并且对获得的突变进行了功能上的注释。结论:本过滤筛选系统能有效控制检测的假阳性率,对斑马鱼致病性的隐性突变位点筛查提供有力参考。     

高通量测序技术结合正向遗传学手段在基因定位研究中的应用

传统的利用正向遗传学方法的基因定位一般是通过构建遗传连锁图谱进行的,该过程步骤繁琐、耗时耗力,很多情形下定位精确度低、区间大。随着高通量测序技术的快速发展以及测序成本的不断降低,多种简单快捷的利用测序手段定位基因的方法被开发出来,包括对突变体基因组直接测序定位、突变体材料构建混池测序定位和遗传分离群体测序构建图谱定位等,还可以对转录组和部分基因组进行测序定位。这些方法可以在核苷酸水平鉴定突变位点,并已推广到复杂的遗传背景中。近期报道的一些测序定位甚至是在不依赖于参考基因组序列、遗传杂交和连锁信息的情况下完成的,这使得很多非模式物种也能开展正向遗传学研究。本文就这些新技术及其在基因定位中的应用进行了综述。     

Polishing the craft of genetic diversity creation in directed evolution

Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success. Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating “smart” libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field.     

Investigation of morphogenesis of inflorescence and determination of the nature of inheritance of “supernumerary spikelets” trait of bread wheat (Triticum aestivum L.) mutant line

Using C-banding and FISH methods, the karyotype of MC1611 induced mutant of bread wheat, which develop additional spikelets at a rachis node (trait “supernumerary spikelets”) was characterized. It was determined that the mutant phenotype is not associated with aneuploidy and major chromosomal rearrangements. The results of genetic analysis showed that supernumerary spikelets of the line are caused by a mutation of the single Bh-D.1 gene, influenced by the genetic background. The mutation causes abnormalities of inflorescence morphogenesis associated with the development of ectopic spikelet meristems in place of floral meristems in the basal part of the spikelets, causing the appearance of additional spikes at a rachis node. The mutant phenotype suggests that the Bh-D gene determines the fate of the lateral meristems in ear, which develops as floral meristem and gives rise to floral organs in wild-type inflorescences. In the bh-D.1 mutant, the floral meristem identity is impaired. The characterized mutant can be used in further studies on molecular genetic basis of development of wheat inflorescence.     

Negative effect of genetic bottlenecks on the adaptability of vesicular stomatitis virus

Muller's ratchet is a principle of evolutionary genetics describing mutant accumulation in populations that are repeatedly subjected to genetic bottleneck. The immediate effect of Muller's ratchet, overall loss of fitness, has been confirmed in several viral systems belonging to different groups. This report shows that in addition to fitness loss, genetic bottlenecks also have longer-term effects, namely changes in the capacity of viral populations to adapt. Thus, vesicular stomatitis virus strains with a history of genetic bottleneck have lower adaptability than strains maintained at relatively large population sizes. This lower adaptability is illustrated by their reduced ability to regain fitness and by their inability to outcompete wild-type populations in situations where the initial fitness of the bottlenecked mutant is the same or even higher than the initial fitness of the wild-type.     

The pathogenesis of familial hypertrophic cardiomyopathy: early and evolving effects from an alpha-cardiac myosin heavy chain missense mutation

Familial hypertrophic cardiomyopathy (FHC) is a genetic disorder resulting from mutations in genes encoding sarcomeric proteins. This typically induces hyperdynamic ejection, impaired relaxation, delayed early filling, myocyte disarray and fibrosis, and increased chamber end-systolic stiffness. To better understand the disease pathogenesis, early (primary) abnormalities must be distinguished from evolving responses to the genetic defect. We did in vivo analysis using a mouse model of FHC with an Arg403Gln alpha-cardiac myosin heavy chain missense mutation, and used newly developed methods for assessing in situ pressure-volume relations. Hearts of young mutant mice (6 weeks old), which show no chamber morphologic or gross histologic abnormalities, had altered contraction kinetics, with considerably delayed pressure relaxation and chamber filling, yet accelerated systolic pressure rise. Older mutant mice (20 weeks old), which develop fiber disarray and fibrosis, had diastolic and systolic kinetic changes similar to if not slightly less than those of younger mice. However, the hearts of older mutant mice also showed hyperdynamic contraction, with increased end-systolic chamber stiffness, outflow tract pressure gradients and a lower cardiac index due to reduced chamber filling; all 'hallmarks' of human disease. These data provide new insights into the temporal evolution of FHC. Such data may help direct new therapeutic strategies to diminish disease progression.     

Genetic approaches to the study of protein-protein interactions.

This article describes genetic approaches to the study of heterologous protein-protein interactions, focusing on the yeast Saccharomyces cerevisiae as a useful eukaryotic model system. Several methods are described that can be used to search for new interactions, including extragenic suppression, multicopy suppression, synthetic lethality, and transdominant inhibition. Strategies for screening, genetic characterization, and clone identification are described, along with recent examples from the literature. In addition, genetic methods are discussed that can be used to further characterize a newly discovered protein-protein interaction. These include the creation of mutant libraries of a given protein by chemical mutagenesis or polymerase chain reaction, the production of dominant-negative mutants, and strategies for introducing these mutant alleles back into yeast for analysis. Although these genetic methods are quite powerful, they are often just a starting point for further biochemical or cell biological experiments.     

The induction of pigmentation change in a non-acid-fast strain ofMycobacterium phlei by ultraviolet radiation

Five scotochromogenic mutants and 11 achromogenic mutants were induced by UV irradiation of the non-acid-fast photochromogenic PN strain ofMycobacterium phlei. Spontaneous scotochromogenic and achromogenic mutants were not obtained. Colonies of the scotochromogenic mutants are orange, except for one mutant which is ochre. Three mutants are resistant to STM. Out of 11 achromogenic mutants 3 were induced by UV treatment of the original photochromogenic strain, 8 were prepared from the scotochromogenic mutant. No significant differences in the sensitivity to UV rays were found among the scotochromogenic mutant, achromogenic mutant and the photochromogenic PN strain ofMycobacterium phlei under the given experimental conditions. Scotochromogenic mutants and most achromogenic mutants are stable and suitable for further genetic investigation. Pigmentation changes can be used as genetic marker in mutation studies.     

COMPOSITION OF CEREBRAL LIPIDS IN MURINE LEUCODYSTROPHY: THE QUAKING MUTANT

The composition of sphingolipids and phospholipids of mouse brain during myelination was determined in the Quaking mutant, which manifests a genetic disorder of myelin formation, and in littermate controls. The biochemical changes during myelination in the brains of the controls corresponded quantitatively with previous findings in a different strain of mice. The Quaking mutant exhibited concentrations of sphingolipids and phospholipids in brain which were comparable to those of controls in the early stage of myelination but the tissue content failed to increase with maturation. The greatest differences occurred in the cerebrosides which at 65 days of postnatal age were only 10 per cent of control levels. During development the pattern of cerebral levels of sphingomyelin, plasmalogen and total phospholipid in the mutants tended to resemble that of the cerebrosides. The defect in the Quaking mutant is compatible with a failure in maturation of myelin. These findings have been compared with those in the Jimpy mutant, a different genetic disorder of myelin in the mouse previously studied in a similar fashion. The Jimpy mutant is characterized by a quantitatively more pronounced deficiency of myelin lipids and a decline in cerebrosides during brain development.     

Dopamine modulates metabolic rate and temperature sensitivity in Drosophila melanogaster

Homeothermal animals, such as mammals, maintain their body temperature by heat generation and heat dissipation, while poikilothermal animals, such as insects, accomplish it by relocating to an environment of their favored temperature. Catecholamines are known to regulate thermogenesis and metabolic rate in mammals, but their roles in other animals are poorly understood. The fruit fly, Drosophila melanogaster, has been used as a model system for the genetic studies of temperature preference behavior. Here, we demonstrate that metabolic rate and temperature sensitivity of some temperature sensitive behaviors are regulated by dopamine in Drosophila. Temperature-sensitive molecules like dTrpA1 and shi(ts) induce temperature-dependent behavioral changes, and the temperature at which the changes are induced were lowered in the dopamine transporter-defective mutant, fumin. The mutant also displays a preference for lower temperatures. This thermophobic phenotype was rescued by the genetic recovery of the dopamine transporter in dopamine neurons. Flies fed with a dopamine biosynthesis inhibitor (3-iodo-L-tyrosine), which diminishes dopamine signaling, exhibited preference for a higher temperature. Furthermore, we found that the metabolic rate is up-regulated in the fumin mutant. Taken together, dopamine has functions in the temperature sensitivity of behavioral changes and metabolic rate regulation in Drosophila, as well as its previously reported functions in arousal/sleep regulation.