碱性蛋白酶基因在Bacillus licheniformis 2709中的整合和扩增

将含有去掉启动子的枯草杆菌碱性蛋白酶基因和Ｃｍ抗性基因的整合质粒ｐＭＰ以原生质体ＰＥＧ法转化Ｂ．ｌｉｃｈｅｎｉｆｏｒｍｉｓ２７０９在１０ｕ／ｍｌＣｍ平板上筛选整合子,并通过不断提高整合子的抗氯霉素能力来扩增染色体碱性蛋白酶基因,最终选到一株产酶比生产菌２７０９提高６０％的高表达整合菌ＢＬ－２０３,该菌在无选择压力下遗传性状相当稳定。     

Gated ion fluxes involved in photophobic responses of the blue-green alga,Phormidium uncinatum

The sensory transduction chain of photophobic responses in the blue-green alga, Phormidium uncinatum seems to involve a gating cation transport through membrane bound ion channels which provides an effective amplification.The calcium conducting ionophore A23187 inhibits the photophobic response totally and induces frequent reversals which resemble phobic responses but occur without any light stimulation. This indicates that the electrogenic ion conductance may depend on a gradient of divalent cations, esp. calcium. The calcium conductance during a photophobic response is further confirmed by the inhibitory effect of ruthenium red and lanthanum, blockers of the electrogenic calcium transport. In the case of lanthanum this inhibition is found at a concentration at which neither the number of motile filaments nor the average speed of movement is impaired.Incorporation of ionophores for monovalent cations (gramicidin and valinomycin) only partially impairs the response. Similarly, inhibition of the Na⁺/K⁺ pump by ouabain is less effective. Thus, the existence of a countercurrent of monovalent cations during the response, which has been described for e.g. ciliates, is yet obscure in blue-green algae.     

Pre-PCR processing

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.     

核酸体外扩增技术

核酸体外扩增是分子生物学研究的基础。随着生物技术的发展 ,出现了越来越多的核酸体外扩增技术。根据其特点可分为两类 :一类是靶核酸的直接扩增 ,如聚合酶链式反应、链替代扩增、连接酶链式反应、核酸依赖的扩增、Qβ复制、转录介导的扩增和滚环扩增等 ;另一类是信号放大扩增 ,如支链DNA、侵染探针等。就现有的核酸扩增技术的原理、特点及应用进行介绍。     

Selective Amplification of Classical and Atypical Prions Using Modified Protein Misfolding Cyclic Amplification

With the development of protein misfolding cyclic amplification (PMCA), the topic of faithful propagation of prion strain-specific structures has been constantly debated. Here we show that by subjecting brain material of a synthetic strain consisting of a mixture of self-replicating states to PMCAb, selective amplification of PrP^Sc could be achieved, and that PMCAb mimicked the evolutionary trend observed during serial transmission in animals. On the other hand, using modified PMCAb conditions that employ partially deglycosylated PrP^C (dgPMCAb), an alternative transmissible state referred to as atypical protease-resistant form of the prion protein (atypical PrPres) was selectively amplified from a mixture. Surprisingly, when hamster-adapted strains (263K and Hyper) were subjected to dgPMCAb, their proteinase K digestion profile underwent a dramatic transformation, suggesting that a mixture of atypical PrPres and PrP^Sc might be present in brain-derived materials. However, detailed analysis revealed that the proteinase K-resistant profile of PrP^Sc changed in response to dgPMCAb. Despite these changes, the 263K strain-specific disease phenotype was preserved after passage through dgPMCAb. This study revealed that the change in PrP^Sc biochemical phenotype does not always represent an irreversible transformation of a strain, but rather demonstrated the existence of a wide range of variation for strain-specific physical features in response to a change in prion replication environment. The current work introduced a new PMCA technique for amplification of atypical PrPres and raised a number of questions about the need for a clever distinction between actual strain mutation and variation of strain-specific features in response to a change in the replication environment.     

Evidence of Cotton leaf curl Burewala virus Variant and its Associate Betasatellite Causing Yellow Mosaic of Eggplant (Solanum melongena) in Pakistan

Eggplant (Solanum melongena L.) plants with severe leaf mosaic and mottling were found in a kitchen garden near cotton fields in Pakistan. Rolling Circle Amplification products from six of the naturally infected eggplant plants, subjected to PCR, successfully amplified expected products of 2.8 and 1.4 kb using begomovirus and betasatellite‐specific primers, respectively. Based on 99% nucleotide sequence identity, the virus was identified as a variant of Cotton leaf curl Burewala virus (CLCuBuV) (GenBank Accession No. HG428709). Likewise, the sequenced betasatellite with a maximum of 97% nucleotide sequence identity was recognized as a new variant of Cotton leaf curl Multan betasatellite (CLCuMuB^Mul) (GenBank Accession No. HG428708). The symptomatic induction of Cotton leaf curl disease in CLCuBuV susceptible cotton genotype CIM‐496 by back‐indexing further confirmed the presence of CLCuBuV in eggplant. This is the first report of CLCuBuV and its associate betasatellite in naturally infected plants of eggplant.     

Does habitat disturbance increase infectious disease risk for primates?

Many studies have suggested that ecosystem conservation protects human and wildlife populations against infectious disease. We tested this hypothesis using data on primates and their parasites. First, we tested for relationships between species' resilience to human disturbance and their parasite richness, prevalence and immune defences, but found no associations. We then conducted a meta‐analysis of the effects of disturbance on parasite prevalence, which revealed no overall effect, but a positive effect for one of four types of parasites (indirectly transmitted parasites). Finally, we conducted intraspecific analyses of malaria prevalence as a function of mammalian species richness in chimpanzees and gorillas, and an interspecific analysis of geographic overlap and parasite species richness, finding that higher levels of host richness favoured greater parasite risk. These results suggest that anthropogenic effects on disease transmission are complex, and highlight the need to define the conditions under which environmental change will increase or decrease disease transmission.     

A framework for studying transient dynamics of population projection matrix models

Empirical models are central to effective conservation and population management, and should be predictive of real-world dynamics. Available modelling methods are diverse, but analysis usually focuses on long-term dynamics that are unable to describe the complicated short-term time series that can arise even from simple models following ecological disturbances or perturbations. Recent interest in such transient dynamics has led to diverse methodologies for their quantification in density-independent, time-invariant population projection matrix (PPM) models, but the fragmented nature of this literature has stifled the widespread analysis of transients. We review the literature on transient analyses of linear PPM models and synthesise a coherent framework. We promote the use of standardised indices, and categorise indices according to their focus on either convergence times or transient population density, and on either transient bounds or case-specific transient dynamics. We use a large database of empirical PPM models to explore relationships between indices of transient dynamics. This analysis promotes the use of population inertia as a simple, versatile and informative predictor of transient population density, but criticises the utility of established indices of convergence times. Our findings should guide further development of analyses of transient population dynamics using PPMs or other empirical modelling techniques.     

从ICR小鼠脂肪组织对resistin基因的扩增

目的 PCR扩增ICR小鼠脂肪组织的resistin基因,为进一步在ICR小鼠中开展resistin的相关研究提供必要的基础.方法 在脂肪组织中抽提总RNA,用RT-PCR进行resistin基因的体外扩增,经电泳检测和测序后用DNAMAN软件进行碱基序列分析.结果 抽提的总RNA完整性较好,RT-PCR产物电泳检测获...