Diverse pathogenicity of Burkholderia cepacia complex strains in the Caenorhabditis elegans host model

A fast screening method was developed to assess the pathogenicity of a diverse collection of environmental and clinical Burkholderia cepacia complex isolates in the nematode Caenorhabditis elegans. The method was validated by comparison with the standard slow-killing assay. We observed that the pathogenicity of B. cepacia complex isolates in C. elegans was strain-dependent but species-independent. The wide range of observed pathogenic phenotypes agrees with the high degree of phenotypic variation among species of the B. cepacia complex and suggests that the taxonomic classification of a given strain within the complex cannot predict pathogenicity.     

食细菌线虫Caenorhabditis elegans的取食偏好性

通过设置平板培养试验,以模式种线虫 Caenorhabditis elegans 为材料,观察了食细菌线虫的取食行为。结果表明：C. elegans 在取食细菌时对原位土壤中分离的一种Pseudomonas sp细菌存在最大的取食偏好性。这种取食偏好性表现在大部分C. elegans 在24 h内都直接朝Pseudomonas sp迁移,说明C. elegans能通过某种机制识别Pseudomonas sp。距离迁移试验及C. elegans迁移率表明它可能是通过辨别细菌所发出的气味识别它喜欢的食物。C. elegans的繁殖率跟其取食的偏好性是相关的,在迁移率较高的细菌培养基中线虫表现出更高的繁殖率。结果还表明：相对于G+细菌而言, C. elegans 偏好取食G-细菌。为进一步了解土壤生态系统中食细菌线虫与细菌群落结构间相互关系提供了帮助。     

Trehalose extends longevity in the nematode Caenorhabditis elegans

Trehalose is a disaccharide of glucose found in diverse organisms and is suggested to act as a stress protectant against heat, cold, desiccation, anoxia, and oxidation. Here, we demonstrate that treatment of Caenorhabditis elegans with trehalose starting from the young‐adult stage extended the mean life span by over 30% without any side effects. Surprisingly, trehalose treatment starting even from the old‐adult stage shortly thereafter retarded the age‐associated decline in survivorship and extended the remaining life span by 60%. Demographic analyses of age‐specific mortality rates revealed that trehalose extended the life span by lowering age‐independent vulnerability. Moreover, trehalose increased the reproductive span and retarded the age‐associated decrease in pharyngeal‐pumping rate and the accumulation of lipofuscin autofluorescence. Trehalose also enhanced thermotolerance and reduced polyglutamine aggregation. These results suggest that trehalose suppressed aging by counteracting internal or external stresses that disrupt protein homeostasis. On the other hand, the life span‐extending effect of trehalose was abolished in long‐lived insulin/IGF‐1‐like receptor (daf‐2) mutants. RNA interference‐mediated inactivation of the trehalose‐biosynthesis genes trehalose‐6‐phosphate synthase‐1 (tps‐1) and tps‐2, which are known to be up‐regulated in daf‐2 mutants, decreased the daf‐2 life span. These findings indicate that a reduction in insulin/IGF‐1‐like signaling extends life span, at least in part, through the aging‐suppressor function of trehalose. Trehalose may be a lead compound for potential nutraceutical intervention of the aging process.     

Adaptation of the nematode Caenorhabditis elegans to medium high temperature

Action of high temperature (36°C) on the nematode Caenorhabditis elegans organism was manifested in errors of the motor program of swimming induced by a mechanical stimulus (37 ± 2 min), the complete, but reversible cessation of locomotion (57 ± 3 min), while damage—in thermal death (215 ± 5 min). Addition into medium of atropine (10^?8–10^?9 M) and chemical stimuli (10^?8–10^?6 cAMP or lysine) causes considerable changes of thermal stability of the worm locomotion. Analysis of these data has shown that the cause of the reversible thermal disturbance of the C. elegans locomotion is disintegration of neurons in the nervous centers regulating behavior. The obtained data indicate the presence in the simple organism of C. elegans of adaptations increasing stability of processes of integration of neurons to a high temperature, which were found earlier in arthropods and vertebrates.     

Multiple Molecular Forms of Acetylcholinesterase in the Nematode Caenorhabditis elegans

Abstract: Extracts of the nematode Caenorhabditis elegans contain five molecular forms of acetylcholinesterase (AChE) activity that can be separated by a combination of selective solubilization, velocity sedimentation, and ion-exchange chromatography. These are called form IA (5.2s), form IB (4.9.s), form II (6.7s), form III (11.3s), and form IV (13.0s). All except form III are present in significant amounts in rapidly prepared extracts and are probably native; form III is probably derived autolytically from form IV. Most of forms IA and IB can be solubilized by repeated extractions without detergent, whereas forms II, III, and IV require detergent for effective solubilization and may therefore be membrane-bound. High salt concentrations are not required for, and do not aid in, the solubilization of these forms. For all forms, molecular weights and frictional ratios have been estimated by a combination of gel permeation chromatography and velocity sedimentations in both H₂O and D₂O. The molecular weight estimates range from 83,000 to 357,000 and only form II shows extensive asymmetry. The separated forms have been characterized with respect to substrate affinity, substrate specificity, inhibitor sensitivity, thermal inactivation, and detergent sensitivity. Judging by these properties, C. elegans is like other invertebrates in that none of its cholinesterase forms resembles either the “true” or the “pseudo” cholinesterase of vertebrates. However, internal comparison of the C. elegans forms clearly distinguishes forms IA, III, and IV as a group from forms IB and II; the former are therefore designated “class A” forms, the latter “class B” forms. Genetic evidence indicates that separate genes control class A and class B forms, and that these two classes overlap functionally. Several factors, including kinetic properties, molecular asymmetry, molecular size, and solubility, all suggest that a molecular model of the multiple cholinesterase forms observed in vertebrate electric organs probably does not apply in C. elegans. Potential functional roles and subunit structures of the multiple AChE forms within each C. elegans class are discussed.     

线虫PCR:直接针对秀丽线虫进行PCR扩增基因组DNA的新方法

目的:直接针对秀丽线虫进行PCR反应,以便快速扩增基因组DNA,从而提高钓取目的基因和鉴定基因组是否发生突变的效率.方法:根据生物信息学分析,针对不同基因设计单重或多重PCR引物;在不含砌DNA聚合酶的PCR反应体系中加入蛋白酶K消化秀丽线虫染色体中的组蛋白,然后加入Taq酶,直接针对野生型或突变型秀丽线虫个体进行PC...     

The burrowing behavior of the nematode Caenorhabditis elegans: a new assay for the study of neuromuscular disorders

The nematode Caenorhabditis elegans has been a powerful model system for the study of key muscle genes relevant to human neuromuscular function and disorders. The behavioral robustness of C. elegans, however, has hindered its use in the study of certain neuromuscular disorders because many worm models of human disease show only subtle phenotypes while crawling. By contrast, in their natural habitat, C. elegans likely spends much of the time burrowing through the soil matrix. We developed a burrowing assay to challenge motor output by placing worms in agar‐filled pipettes of increasing densities. We find that burrowing involves distinct kinematics and turning strategies from crawling that vary with the properties of the substrate. We show that mutants mimicking Duchenne muscular dystrophy by lacking a functional ortholog of the dystrophin protein, DYS‐1, crawl normally but are severely impaired in burrowing. Muscular degeneration in the dys‐1 mutant is hastened and exacerbated by burrowing, while wild type shows no such damage. To test whether neuromuscular integrity might be compensated genetically in the dys‐1 mutant, we performed a genetic screen and isolated several suppressor mutants with proficient burrowing in a dys‐1 mutant background. Further study of burrowing in C. elegans will enhance the study of diseases affecting neuromuscular integrity, and will provide insights into the natural behavior of this and other nematodes.     

Predicting aging/longevity-related genes in the nematode Caenorhabditis elegans

We present a novel mathematical/computational strategy for predicting genes/proteins associated with aging/longevity. The novelty of our method arises from the topological analysis of an organismal longevity gene/protein network (LGPN), which extends the existing cellular networks. The LGPN nodes represent both genes and corresponding proteins. Links stand for all known interactions between the nodes. The LGPN of C. elegans incorporated 362 genes/proteins, 160 connecting and 202 age-related ones, from a list of 321 with known impact on aging/longevity. A 'longevity core' of 129 directly interacting genes or proteins was identified. This core may shed light on the large-scale mechanisms of aging. Predictions were made, based upon the finding that LGPN hubs and centrally located nodes have higher likelihoods of being associated with aging/longevity than do randomly selected nodes. Analysis singled-out 15 potential aging/longevity-related genes for further examination: mpk-1, gei-4, csp-1, pal-1, mkk-4, 4O210, sem-5, gei-16, 1O814, 5M722, ife-3, ced-10, cdc-42, 1O776Co, and 1O690.     

Genetic variation for outcrossing among Caenorhabditis elegans isolates

The evolution of breeding systems results from the existence of genetic variation and selective forces favoring different outcrossing rates. In this study we determine the extent of genetic variation for characters directly related to outcrossing, such as male frequency, male mating ability, and male reproductive success, in several wild isolates of the nematode Caenorhabditis elegans. This species is characterized by an androdioecious breeding system in which males occur with hermaphrodites that can either self-fertilize or outcross with males. We find genetic variation for all characters measured, but also find that environmental variation is a large fraction of the total phenotypic variance. We further determine the existence of substantial genetic variation for population competitive performance in several laboratory environments. However, these measures are uncorrelated with outcrossing characters. The data presented here contribute to an understanding of male maintenance in natural populations through their role in outcrossing.     

利用模式生物线虫评价精对苯二甲酸废水的毒性

应用模式生物秀丽隐杆线虫,通过生命周期、半数致死天数、生殖速度、产卵数、头部摆动频率和身体弯曲次数等指标对精对苯二甲酸(PTA)废水毒性进行了研究.结果表明,与对照组相比,660 mg·L^-1 PTA废水暴露下的线虫生命周期有一定的延长,产卵时间延迟,头部摆动频率降低,身体弯曲次数减少(P<0.05),且PTA废水对线虫生殖能力的影响极显著(P<0.01),暴露于废水中的线虫产卵数大约只有正常产卵数的1/4.最敏感效应指标——产卵数,有望成为该类废水毒性预警预报的潜在生物标志物.     

Tests for parental imprinting in the nematode Caenorhabditis elegans

Summary The mutation him-6(e1423) leads to generalized chromosomal nondisjunction during meiosis in oogenesis and spermatogenesis of C. elegans. As a result, gametes nullisomic or disomic for each of the six chromosomes occur at appreciable frequency. Crosses utilizing marked him-6 strains were used to generate and identify exceptional euploid progeny which had received both homologues of a marked autosome either from the male parent or from the female parent. Examples of all ten possible exceptions were identified and found to be viable and fertile. These results (together with previous data for the X chromosome) indicate that major chromosomal imprinting effects do not occur during gametogenesis in this organism.     

细菌与秀丽隐杆线虫之间互作关系的研究进展

秀丽隐杆线虫(Caenorhabditis elegans)以其个体小、易培养、生活周期短等优势成为生物发育、衰老、神经及免疫相关机制研究的模式生物.它在实验室培养时主要靠饲喂大肠杆菌OP50,有报道,细菌及其代谢物对线虫的代谢、行为和寿命有至关重要的影响.因此,作为一个遗传模型,秀丽隐杆线虫可以帮助研究微生物与宿主相...     

以秀丽线虫为模式生物评价蓖麻碱毒性的研究

以秀丽线虫作为评价蓖麻碱毒性的模式生物,通过测定不同浓度的蓖麻碱提取物对线虫的半致死浓度、生殖能力和体内酶活性的影响,对蓖麻碱的毒性进行初步评价。结果表明,蓖麻碱提取物的48h的LD50为0.977mg/mL,72h的LD50为0.821mg/mL;随着蓖麻碱提取物浓度从0.5mg/mL增加到2.0mg/mL,虫体的SOD活性由(80.669±3.2)U/mg降低至(1.532±0.2)U/mg;CAT活性由(70.947±2.7)U/mg降低至(0.234±2.1)U/mg。说明蓖麻碱提取物浓度越大,毒性越强,线虫体内酶活越低,蓖麻碱提取物可使秀丽线虫生殖能力降低或丧失。     

Investigating the molecular mechanisms of learning and memory using Caenorhabditis elegans

Learning is an essential biological process for survival since it facilitates behavioural plasticity in response to environmental changes. This process is mediated by a wide variety of genes, mostly expressed in the nervous system. Many studies have extensively explored the molecular and cellular mechanisms underlying learning and memory. This review will focus on the advances gained through the study of the nematode Caenorhabditis elegans. C. elegans provides an excellent system to study learning because of its genetic tractability, in addition to its invariant, compact nervous system (~300 neurons) that is well-characterised at the structural level. Importantly, despite its compact nature, the nematode nervous system possesses a high level of conservation with mammalian systems. These features allow the study of genes within specific sensory-, inter- and motor neurons, facilitating the interrogation of signalling pathways that mediate learning via defined neural circuits. This review will detail how learning and memory can be studied in C. elegans through behavioural paradigms that target distinct sensory modalities. We will also summarise recent studies describing mechanisms through which key molecular and cellular pathways are proposed to affect associative and non-associative forms of learning.

     

细菌介导的发夹结构双链RNA对秀丽小杆线虫中par-1基因的RNA干扰

秀丽小杆线虫(Caenorhabditis elegans)是一种重要的模式生物,目前在C.elegans中发现的许多基因在序列和功能上与哺乳动物的基因有很高的同源性,对其基因功能的研究有助于阐明哺乳动物的基因功能。为检测细菌介导的通过发夹结构表达双链RNA(dsRNA)的RNA干扰(RNAi)在C.elegans中的作用效果,我们设计并构建了可以转录表达母源极性基因par-1的发夹dsRNA的RNAi载体,并转入E.coli HT115中,经过IPTG诱导后浓缩。浓缩菌液在25℃下喂食C.elegans48h后,观察C.elegans胚胎发育情况,同时以含有空载体的菌液作为对照,并与par-1突变体及野生型比较。结果显示:发夹结构表达的dsRNA对par-1基因进行RNAi后,虫体内par-1 mRNA几乎消失,C.elegans早期胚胎分裂不对称性丧失,par-1(RNAi)干扰率在95%以上。     

必需氨基酸延缓秀丽隐杆线虫衰老的研究

利用模式生物秀丽隐杆线虫,考察8种人体必需氨基酸对衰老的影响。首先建立秀丽隐杆线虫寿命模型,以雷帕霉素为阳性对照药,分别考察8种必需氨基酸对线虫生存时间的影响。再用筛选出的氨基酸处理线虫21d,通过秀丽隐杆线虫-绿脓杆菌感染模型,考察氨基酸对线虫的抗感染能力的影响,利用实时荧光定量Real-Time RT-PCR方法检测氨基酸处理线虫后DAF-16/FOXO下游基因和免疫相关基因的表达水平。结果表明8种必需氨基酸中苏氨酸和异亮氨酸既能延长野生型线虫的寿命又能延长daf-16突变型线虫的寿命,同时还能增强秀丽隐杆线虫抗绿脓杆菌感染的能力,并提高免疫相关基因lys-7、clec-67的表达水平,而DAF-16/FOXO下游基因表达没有明显变化。因此苏氨酸和异亮氨酸能延长线虫寿命、提高抗感染能力,且对线虫寿命的延长作用不完全依赖于DAF-16/FOXO转录因子。     

秀丽隐杆线虫在高校遗传学实验中的应用

秀丽隐杆线虫(Caenorhabditiselegans)是模式生物中的重要成员之一,因其实验成本低,实验周期短,非常适宜用于高校的遗传学实验教学中。线虫在实验教学中的使用,一方面可以有效地丰富高校实验教学的内容,另一方面也可以很好地激发学生的学习兴趣。本文介绍了线虫在遗传学实验教学中的应用实例,如生活周期观察、单因子杂交、单核苷酸多态性研究、RNA干扰(RNAi)实验等;对实验设置、操作要求、实验相关准备工作等进行了较为细致的描述,为线虫在高校遗传学实验教学中的应用提供了详实案例,可为线虫在高校遗传学实验或其他相关实验课程如细胞生物学实验、模式生物与发育生物学实验中的应用提供参考。     

Genome characterization, analysis of virulence and transformation of Microbacterium nematophilum, a coryneform pathogen of the nematode Caenorhabditis elegans

A coryneform bacterium designated Microbacterium nematophilum has previously been reported to act as a pathogen for Caenorhabditis elegans. This bacterium is able to colonize the rectum of infected worms and cause localized swelling, constipation and slowed growth. Additional isolates and analysis of this bacterium are described here. Tests of pathogenicity on other Caenorhabditis nematodes show that M. nematophilum infection is lethal to most species in the genus, in contrast to its relatively mild effects on C. elegans. The size and geometry of the pathogen genome have been determined as a closed circular molecule of 2.85 Mb with high G+C content. Bacteria also harbor a 55 kb plasmid, pMN1, which is largely composed of a lysogenic bacteriophage genome. Mutagenesis experiments have yielded stable avirulent mutants of M. nematophilum. As a first step towards molecular genetic analysis, methods for low-efficiency transformation of M. nematophilum have been developed.