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微生物专题英文文献

Mutation-Screening in Xylanase-Producing Strains by Ion Implantation
班级：生物工程学生：马春玲 2013年12月13日

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试验内容
1. Purpose and meaning 2. Introduction 3. Materials and methods
4.5 正交试验结果
Table 5. Results of ortho.1 Trend curve
Fig.2 Relationship between xylanase and time of fermentation in Aspergillus niger N212

通过对出发菌株注入不同剂量的氮离子，低能氮离子束对菌体细胞均有一定程度的致死和损伤作用，细胞及其损伤DNA又在其修复系统的作用下得到不同程度的修复，从而导致黑曲霉孢子的存活率先下降，后上升，然后又下降，并且菌种的修复出错会使其突变率大大提高，从而提高了菌株的正突变率，从而确定了氮离子最佳注入参数。以上试验可以得出最优培养基的组成（即各组分的最适浓度），而且在以上培养得到了黑曲霉N212（表2），当它发酵60个小时后酶活达到600IU/ml，比之前未优化的菌株减少了12个小时，而且相对于原出发菌株酶活增加了100 ％。试验证明离子注入对微生物进行诱变改良是一种行之有效的诱变技术。
木聚糖酶是植物细胞壁的主要之一，属于非淀粉多糖。可作为生物漂白剂用于造纸工业，也可用于生物转化等等。目前木聚糖酶的生产主要还依靠真菌。
对于产酶微生物的育种，国外多采用基因工程手段构建高产菌，而国内多采用传统的诱变方法，如紫外辐射、化学诱变剂处理等，这些诱变手段获得的突变株一般稳定性差、容易产生回复突变且负突变较多及诱变选育的工作量很大，而20世纪80年代末，人们发现离子束可以引起靶物质原子移位和重排，使细胞表面刻蚀和穿孔，并能影响和改变细胞电性等现象，提出了离子束可以用于细胞加工和基因转移的设想，并陆续得到了研究证实，由此产生了国内外普遍关注的离子束生物技术工程学，而且离子束育种是一项具有我国自主知识产权且被国际所承认的定向遗传改良的集物理诱变和化学诱变于一身的综合诱变方法，具有损伤小、突变谱广、突变率高的特点。

关于生物学的书籍英语作文

关于生物学的书籍英语作文Biology is a vast and fascinating field of study that delves into the intricate workings of living organisms, from the smallest microbes to the most complex multicellular life forms. As an area of scientific inquiry, biology encompasses a wide range of sub-disciplines, each with its own unique focus and set of research methodologies. Whether one's interest lies in the realm of cell biology, evolutionary theory, ecology, or any of the numerous other branches of this discipline, there is a wealth of informative and engaging literature available to explore the myriad aspects of the living world.One of the most valuable resources for those seeking to deepen their understanding of biology is the abundance of high-quality books on the subject. These publications, written by leading experts in the field, offer readers a comprehensive and authoritative exploration of the various concepts, theories, and discoveries that have shaped our knowledge of the biological sciences. From introductory textbooks designed for students to specialized monographs tackling the latest advancements in research, the diversity of biology-focused literature is truly remarkable.For those new to the field, introductory biology books serve as an excellent starting point, providing a solid foundation in the fundamental principles and processes that underpin the study of living organisms. These works often cover a broad range of topics, including the structure and function of cells, the mechanisms of inheritance and genetic expression, the principles of evolution, and the complex interactions between organisms and their environments. By presenting complex scientific concepts in a clear and accessible manner, these introductory texts help to demystify the subject matter and inspire a genuine curiosity in the reader.As one delves deeper into the study of biology, more specialized and advanced books become increasingly valuable. These works delve into the nuances of specific sub-disciplines, offering a more detailed and comprehensive exploration of the research, theories, and methodologies that define these specialized areas of study. For instance, books on cellular and molecular biology might delve into the intricate workings of the cell, examining the various organelles, signaling pathways, and genetic mechanisms that govern the life and function of these fundamental units of life. Similarly, works on evolutionary biology might explore the historical development of life on Earth, the mechanisms of natural selection and adaptation, and the ongoing debates and controversies surrounding the origin and evolution of species.One of the key advantages of biology-focused books is their ability to provide readers with a more in-depth and contextualized understanding of the subject matter, compared to the often-limited scope of scientific articles or online resources. By presenting information in a cohesive and comprehensive manner, these books allow readers to gain a deeper appreciation for the interconnectedness of the various concepts and principles that underlie the biological sciences. Moreover, many biology books also include detailed illustrations, diagrams, and case studies that help to reinforce the key ideas and facilitate a more engaging and interactive learning experience.Beyond the realm of academic and research-oriented literature, there is also a wealth of popular science books that explore the fascinating world of biology in a more accessible and engaging manner. These works, written by talented science communicators, aim to bring the wonders of the living world to a broader audience, often by highlighting the latest discoveries, groundbreaking research, and the profound implications of our understanding of biology. From books that delve into the intricacies of the human body to those that explore the remarkable diversity of life on our planet, these popular science publications serve to inspire a sense of wonder and curiosity in readers of all backgrounds.Regardless of one's level of expertise or specific area of interest within the field of biology, the abundance of high-quality books on the subject provides a valuable resource for learning, exploration, and personal growth. Whether one is seeking to deepen their understanding of the fundamental principles of life or to delve into the latest advancements in a particular sub-discipline, the wealth of biology-focused literature offers something for every curious mind. By engaging with these informative and engaging works, individuals can not only expand their knowledge but also cultivate a deeper appreciation for the incredible complexity and beauty of the living world.。

关于微生物的英文作文

关于微生物的英文作文The World of Microorganisms: Tiny Creatures with Enormous Impact.Microorganisms, often overlooked due to their minute size, play a pivotal role in various aspects of our lives. These tiny creatures, ranging from bacteria and viruses to fungi and protozoa, exist everywhere, from the depths of the ocean to the highest peaks of the mountains. Their influence extends across all ecosystems, influencing everything from the health of our bodies to the fertility of our soils.One of the most remarkable features of microorganisms is their diversity. There are millions of species of bacteria, each with its unique characteristics and functions. Some bacteria are beneficial to humans, aiding in digestion, synthesizing vitamins, and even helping to fight off harmful invaders. Others, however, can cause diseases ranging from minor infections to life-threateningconditions.Viruses, another type of microorganism, are even more enigmatic. They consist of genetic material enclosed within a protein capsid and lack the ability to replicate independently. Instead, they depend on infecting host cells and hijacking their replication machinery to produce more virus particles. This dependence on hosts makes viruses extremely adaptable and able to infect a wide range of organisms, from bacteria to plants and animals.Fungi and protozoa are other significant groups of microorganisms. Fungi, known for their role in bread making and wine fermentation, also play crucial roles in decomposition and nutrient cycling. Protozoa, on the other hand, are single-celled animals that can be eitherparasitic or free-living. Some protozoa, such as amoebas, are known for their unique cell structure and movement patterns.The impact of microorganisms on human health cannot be overstated. The human microbiome, which consists of themicroorganisms that live on and within our bodies, plays a crucial role in maintaining our health. These microorganisms help digest food, synthesize vitamins, and even regulate our immune systems. Dysbiosis, or an imbalance in the microbiome, can lead to various diseases, including inflammatory bowel disease, obesity, and even some types of cancer.Microorganisms also play a vital role in the environment. They are responsible for decomposing organic matter, releasing nutrients back into the soil and water, and supporting the growth of plants and other organisms. Additionally, microorganisms play a key role in the carbon cycle, helping to convert carbon dioxide into organic matter and vice versa.In recent years, the field of microbiology has seen significant advancements, thanks to the development of new technologies such as next-generation sequencing and microscopy. These advances have allowed scientists to explore the microbial world in unprecedented detail, revealing the incredible complexity and diversity of thesetiny creatures.As we delve deeper into the microbial world, it becomes increasingly apparent that these tiny creatures play a crucial role in shaping our planet and our lives. It is essential that we continue to study and understand them,not only to improve our health and environment but also to harness their potential for biotechnological applications and sustainable development.In conclusion, microorganisms, despite their small size, are巨大的影响力on our world. They are essential for maintaining the health of our bodies, the fertility of our soils, and the balance of our ecosystems. As we continue to explore and understand these tiny creatures, we will discover even more remarkable ways that they shape our world.。

关于生物技术的英文文献

关于生物技术的英文文献Biotechnology is such a fascinating field, really bringing the wonders of science to life. You know, I've always been fascinated by how we can manipulate the tiniest building blocks of life to create amazing things.Take genetic engineering, for instance. It's like having a magic wand that can change the DNA of a plant or animal to give it new traits. Imagine growing crops that are resistant to drought or insects, or animals that produce milk with higher nutritional value. It's like science fiction becoming reality.And then there's biopharmaceuticals. Using biotechnology, we can produce drugs and vaccines that are more effective and targeted. It's like having a precision tool to fight diseases, giving us a better chance of curing or preventing them.But it's not just about medicine and agriculture.Biotechnology is also revolutionizing industries like energy and environmental protection. We can now use microbes to produce biofuels, reducing our dependence on fossil fuels and their impact on the environment.Plus, the advances in biotechnology are happening so fast. It's exciting to think about what the future holds. Maybe one day, we'll be able to regenerate organs for transplant using stem cells, or even create synthetic biology systems that can perform complex tasks.The possibilities are endless, and it's truly remarkable to be a part of this era where biotechnology is changing the world for the better. It's not just about scientific discovery; it's about improving lives and making a difference.。

微生物英文文献及翻译—原文

微生物英文文献及翻译—原文本期为微生物学的第二讲，主要讨论炭疽和蛔虫病这两种既往常见而当今社会较为罕见的疾病。

炭疽是由炭疽杆菌所致的一种人畜共患的急性传染病。

人因接触病畜及其产品及食用病畜的肉类而发生感染。

临床上主要表现为皮肤坏死、溃疡、焦痂和周围组织广泛水肿及毒血症症状；似蚓蛔线虫简称蛔虫，是人体内最常见的寄生虫之一。

成虫寄生于小肠，可引起蛔虫病。

其幼虫能在人体内移行，引起内脏幼虫移行症。

案例分析Case 1：A local craftsman who makes garments from the hides of goats visits his physician because over the past few days he has developed several black lesions on his hands and arms. The lesions are not painful, but he is alarmed by their appearance. He is afebrile and his physical examination is unremarkable.案例1：一名使用鹿皮做皮衣的当地木匠来就医，主诉过去几天中手掌和手臂上出现几个黑色皮肤损害。

皮损无痛，但是外观较为骇人。

患者无发热，体检无异常发现。

1. What is the most likely diagnosis?Cutaneous anthrax, caused by Bacillus anthracis. The skin lesions are painless and dark or charred ulcerations known as black eschar. It is classically transmitted by contact with thehide of a goat at the site of a minor open wound.皮肤炭疽：由炭疽杆菌引起，皮损通常无痛、黑色或称为焦痂样溃疡。

生物英文文献.doc

Application of α-amylase and Researchα-amylase to be widely distributed throughout microorganisms to higher plants. The International Enzyme classification number is EC. 3.2.1.1, acting on the starch from the starch molecules within the random cut α a 1,4 glycosidic bond to produce dextrin and reducing sugar, because the end product of carbon residues as Α configuration configuration, it is called α-amylase. Now refers to α-amylase were cut from the starch molecules within the α-1,4 glycosidic bond from the liquefaction of a class of enzymes.α-amylase is an important enzyme, a large number of used food processing, food industry, brewing, fermentation, textile industry and pharmaceutical industries, which account for the enzyme about 25% market share. Currently, both industrial production to large-scale production by fermentation α-amylase. α-amylase in industrial applications1.1 The bread baking industry, as a preservative enzymes used in baking industry, production of high quality products have been hundreds of years old. In recent decades, malt and microbial α-amylase, α-amylase is widely used in baking industry. The enzymes used for making bread, so that these products are much larger, better colors, more soft particles.Even today, baking industry have been α-amylase from barley malt and bacterial, fungal leaf extract. Since 1955 and after 1963 in the UK GRAS level validation, fungal amylase, has served as a bread additive. Now, they are used in different areas. Modern continuous baking process, add in f lour α-amylase can not only increase the fermentation rate and reduce dough viscosity (improving product volume and texture) to increase the sugar content in the dough, improved bread texture, skin color and baking quality, but also to extend the preservation time for baked goods. In the storage process, the bread particles become dry, hard, not crisp skin, resulting in deterioration of the taste of bread. These changes collectively referred to as degenerate. Each year simply because the losses caused by deterioration of bread more than 100 million U.S. dollars. A variety of traditional food additives are used to prevent deterioration and improve the texture and taste of baked goods. Recently, people started to pay attention enzyme as a preservative, preservative agent in improving the role of the dough, as amylopectin, amylase enzyme and a match can be effectively used as a preservative. However, excessive amylase causes a sticky bread too. Therefore, the recent trend is the use of temperature stability (ITS) α a amylase activity are high in starch liquefaction, but the baking process is completed before the inactivation. Despite the large number of microbes have been found to produce α-amylase, but with the temperature stability of the nature of the α-amylase only been found in several microorganisms.1.2 starch liquefaction and saccharification of the main α-amylase starch hydrolysis product market, such as glucose and fructose. Starch is converted into high fructose corn syrup (HFCS). Because of their high sweetness, are used in the soft drink beverage industry sweeteners. The liquefaction process is used in thermal stability at high temperature α-amylase. α-amylase in starch liquefaction ofthe application process is already quite mature, and many relevant reports.1.3 fiber desizing modern fiber manufacturing process in knitting yarn in the process will produce large amounts of bacteria, to prevent these yarn faults, often increase in the surface layer of the yarn can remove the protective layer. The surface layer of the material there are many, starch is a very good choice because it is cheap and easy to obtain, and can be easily removed. Starch desizing α-amylase can be used, it can selectively remove the starch without harming the yarn fibers, but also random degradation of starch dextrin soluble in water, and are easily washed off. 1.4 Paper Industry amylase used in the paper industry mainly to improve the paper coating starch. Paste on the paper is primarily to protect the paper in the process from mechanical damage, it also improved the quality of finished paper. Paste to improve the hardness and strength of paper, enhanced erasable paper, and is a good paper coating. When the paper through two rolls, the starch slurry is added the paper. The process temperature was controlled at 45 ~ 6O ℃, need a stable viscosity of starch. Grinding can also be controlled according to different grades of paper starch viscosity. Nature of the starch concentration is too high for the sizing of paper, you can use part of α-amylase degradation of starch to adjust.1.5 Application of detergents in the enzyme is a component of modern high-efficiency detergents. Enzymes in detergents in the most important function is to make detergents more modest sound. Automatic dishwasher detergents early is very rough, easy to eat when the body hurts, and on ceramics, wood tableware can also cause damage. α-amylase was used from 1975 to washing powder. Now, 90% of the liquid detergents contain an amylase, and automatic dishwasher detergents α-amylase on demand is also growing. α 1 amylase ca2 + is too sensitive to low ca2 + in the stability of poor environment, which limits an amylase in the remover in. And, most of the wild-type strains produced an amylase on raw materials as one of the oxidants detergents are too sensitive. Keep household detergents, this limitation by increasing the number of process steps can be improved. Recently, two major manufacturers of detergents NovozymesandGcncncoreInternational enzyme protein technology has been used to improve the stability of amylase bleaching. They leucine substitution of Bacillus licheniformis α-amylase protein in the first 197 on the methionine, resulting in enzymes of the oxidant component of resistance increased greatly enhanced the oxidation stability of the enzyme stability during storage better. The two companies have been pushing in the market these new products.1.6 Pharmaceutical and clinical chemical analysis with the continuous development of biological engineering, the application of amylase involved in many other areas, such as clinical, pharmaceutical and analytical chemistry. Have been reported, based on the liquid α-amylase stability of reagents have been applied to automatic biochemical analyzer (CibaComingExpress) clinical chemistry system. Amylase has been established by means of a method of detecting a higher content of oligosaccharides, is said this method is more than the effective detection method of silver nitrate.2.1 Research amylase α-amylase enzymes in domestic production and application in 1965, China began to apply for a 7658 BF Bacillus amyloliquefaciens amylase production of one, when only exclusive manufacturing plant in Wuxi Enzyme. 1967 Hangzhou Yi sugar to achieve the application of α-amylase production of caramel new technology can save 7% ~ 10% malt, sugar, increase the rate of 10%.1964, China began a process of enzymatic hydrolysis of starch production of glucose. In September l979 injection of glucose by the enzyme and identification of new technology and worked in North China Pharmaceutical Factory, Hebei Dongfeng Pharmaceutical Factory, Zhengzhou Songshan applied pharmaceutical units and achieved good economic benefits. Compared with the traditional acid to improve the yield of 10% Oh, cost more than 15%. In addition to enzyme for citric acid production in China, glutamic acid fermentation system for beer saccharification, fermentation, rice wine, soy sauce manufacture, vinegar production also has been studied and put into production successfully.2.2 Overseas Researc h α-amylase, present, and in addition a large number of T for conventional mutation breeding, the overseas production has been initially figure out the regulation of α-amylase gene, the transduction of the transformation and gene cloning techniques such as breeding. The Bacillus subtilis recombinant gene into the production strain to increase α-amylase yield of 7 to 10 times and has been used in food and the wine industry, for breeding high-yield strains of α-amylase to create a new way.2.3 domestic and foreign research institutions and major research direction as α-amylase is an important value of industrial enzymes, weekly discussion group and outside it was a lot of research. Representative of the domestic units: Sichuan University, major research produc tion of α-amylase strains and culture conditions; Jiangnan University, the main research structure of α-amylase and application performance, such as heat resistance, acid resistance; Northwest universities, major research denatured α-amylase and the environment on the mechanism of α-amylase; South China University of Technology, the main α-amylase of immobilization and dynamic nature; there Huazhong Agricultural University, Chinese Academy of Sciences Institute of Applied Ecology in Shenyang, Tianjin University, Nankai University, College of Life Sciences, Chinese Academy of Agricultural Sciences, Chinese Academy of Sciences Institute of Microbiology and a number of research institutions on a variety of bacterial α-amylase production of a amylase gene cloning and expression. Representative of foreign research units are: Canada UniversityofBritishColumbia, they were a pancreatic amylase structure and mechanism of in-depth research; Denmark's Carlsberg Research Laboratory of the main structure of barley α-amylase domain and binding sites; U.S. WesternRegionalResearchCenter major study α-amylase in barley and the role of antibiotics and the barley α-amylase active site.3, α-amylase conclusion has become the industrial application of one of the most important enzymes, and a large number of micro-organisms can be used for efficient production of amylase, but large-scale commercial production of the enzyme is still limited to some specific fungi and bacteria. For the effective demandfor α-amylase more and more, this enzyme by chemical modification of existing or improved technology through the white matter are. Benefit from the development of modern biotechnology, α an amylase in the pharmaceutical aspects of growing importance. Of course, the food and starch indust ries is still the main market, α amylase in these areas, a demand is still the largest.Journal of Southeast University(English Edition)2008 24（4）。

生物科学中英文对照外文翻译文献

中英文资料外文翻译文献译文标题：传统意大利榛子的体外繁殖用于当地遗传资源库的稳定和保存译文：关键词：欧洲榛，榛属，传统种质，体外繁殖摘要：在地中海盆地，榛子（欧洲榛）是非常重要的一种作物。

体外繁殖能够有效的稳定当地遗传资源库。

为了提高榛子微组织繁殖实验记录的精确性，各种不同的研究已经在进行。

这些研究通常以重要的品种为材料，然而，微组织繁殖实验记录应用在这些幼小品种上比起传统方法通常会产生相反的结果，这种技术在幼小品种上很少取得成功。

本实验的目的是为重要品种微组织繁殖的操作积累相关的知识和信息。

实验过程中需要设计不同成分的培养基，灭菌时间和培养时间都要进行详细的讨论。

传统意大利品种植株茎芽中的N6-异戊烯腺嘌呤的作用是改善这种状态。

生根阶段是榛属微组织繁殖应用于大型商业生产的关键步骤。

欧洲榛在欧洲特别是生物地理分布区地中海盆地代表一种重要的经济类林木。

榛子主要产于土耳其，意大利，美国和西班牙（分别是每年55,000, 110,000, 25,000, 18,000+吨），其次是法国，希腊，葡萄牙。

大约90%的产品被去皮并且以树芯的形式卖出，然而剩余的10%则作为树苗消费。

极好的营养成分和营养制品的特性也使该物种产生很高的利润。

此外，在一些特有的栽培地区，传统和文化身份严重受榛子产量的影响，文化身份常常会促进贫瘠土地的回收和利用。

即使这样，在一些地区，这种林业作物仍然不是重要的农业资源，然而，就当地足够维持的生产式系统和作为宝贵的食物的传统而言，它却是一种有趣的收入来源。

世界第二大生产商意大利说一些传统的品种主要种植在Campania ,Latium, Piedmont,在西西里岛有大量的属典型种。

近几年，一些主要品种由于质量和传统特性获得了欧洲质量印模。

此外，这些品种还被引进其他国家特定的果园中以增大他们的生长范围。

没有经过检验的物质可能会传播疾病，也可能会导致原因不明的物质的出现。

微组织繁殖法等生物技术的应用会促进健康的合乎本性的物质的产生(Nas et al.,2004)，并且提高这种林木的经济价值。

分子生物学英文文献1

Chapter 5An Efficient Protocol for VZV BAC-Based MutagenesisZhen Zhang, Ying Huang, and Hua ZhuAbstractVaricella-zoster virus (VZV) causes both varicella (chicken pox) and herpes zoster (shingles). As a member of the human herpesvirus family, VZV contains a large 125-kb DNA genome, encoding 70 unique open reading frames (ORFs). The genetic study of VZV has been hindered by the large size of viral genome, and thus the functions of the majority of these ORFs remain unclear. Recently, an efficient protocol has been developed based on a luciferase-containing VZV bacteria artificial chromosome (BAC) system to rapidly isolate and study VZV ORF deletion mutants.Key words:Varicella-zoster virus, Bacterial artificial chromosome, Deletion mutagenesis, Bioluminescence1. I ntroductionVaricella-zoster virus (VZV) is a common human herpesvirus thatis a significant pathogen in the United States, with more than 90%of the US population harboring the virus (1). Primary infectionof VZV leads to varicella (chicken pox). VZV establishes lifelonglatency in the host, specifically in trigeminal ganglia and dorsalroot ganglia (2). The VZV reactivation results in herpes zoster(shingles), which often leads to chronic postherpetic neuralgia(2, 3). As a member of human alpha-herpesvirus subfamily, VZVhas a 125-kb long double-stranded DNA genome, which encodesat least 70 unique open reading frames (ORFs). The genomes ofseveral different VZV strains were sequenced and a few of theVZV genes genetically analyzed (4).It has been extremely difficult to generate VZV site-specificmutations using conventional homology recombination meth-ods. This was mainly due to the high cell-associated nature ofVZV infection in vitro, which leads to the difficulties in isolatingJeff Braman (ed.), In Vitro Mutagenesis Protocols: Third Edition,Methods in Molecular Biology, vol. 634,DOI 10.1007/978-1-60761-652-8_5, © Springer Science+Business Media, LLC 20107576Zhang, Huang, and Zhuviral DNA and purifying recombinant virus away from wild-type virus. In the last few years, a popular method for VZV in vitro mutagenesis involves a four-cosmid system covering the entire viral genome (5–7). Using the cosmid system to generate recom-binant VZV variants involves technically challenging steps such as co-transfection of four large cosmids into permissive mammalian cells and multiple homologous recombination events within a single cell to reconstruct a full-length viral genome. The highly cell-associated nature of VZV also makes the downstream appli-cations of traditional virology methods such as plaque assay-based titering and plaque purification difficult. To date, the functions of the majority of VZV ORFs remain uncharacterized (8).In order to create recombinants of VZV more efficiently, the full-length VZV (P-Oka strain, a cloned clinical isolate of VZV) genome has been successfully cloned as a VZV bacteria artificial chromosome (BAC) (9, 10). This VZV BAC combined with a highly efficiently E. coli homologous recombination system allows quick and easy generation of recombinant VZV. To further ease the downstream virus quantification assays, a firefly luciferase reporter gene, was inserted into the VZV BAC to generate a novel luciferase-expressing VZV (10). In this protocol, we show the generation and analyses of VZV full-length ORF deletion mutants and genetic revertants as examples to demonstrate the utility and efficiency of this versatile system for VZV mutagenesis in vitro. Furthermore, this protocol can be easily modified to broaden its applications to a variety of genetic maneuvers including making double ORF deletions, partial ORF deletions, insertions, and point mutations.1. Human melanoma (MeWo) cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 U penicillin–streptomycin/ml, and2.5 m g amphotericin B/ml at 37°C in a humidified incubator with 5% CO 2. All tissue culture reagents were obtained from Sigma (St. Louis, MO).2. VZV luc was recently developed in the laboratory (10). It con-tains a full-length VZV P-Oka genome with a firefly luciferase cassette (see Note 1). The BAC vector was inserted between VZV ORF60 and ORF61, which includes a green fluorescent protein (GFP) expression cassette and a chloramphenicol resistance cassette (Cm R ).3. pGEM-oriV/kan was previously constructed (11) in the lab-oratory and used as a PCR template to generate the expres-sion cassettes for the kanamycin or ampicillin resistance genes (Kan R and Amp R ).2. M aterials2.1. Cells, VZV luc , Plasmids, and E. coliStrain77An Efficient Protocol for VZV BAC-Based Mutagenesis 4. pGEM-lox-zeo was derived from pGEM-T (Promega, Madison, WI) (12) and was used to generate the rescue clones of VZV ORF deletion mutants. 5. E. coli strain DY380 was obtained from Neal Copeland and Craig Stranthdee and used for mutagenesis (13). 6. A cre recombinase expression plasmid pGS403 was a gift from L. Enquist (Princeton University, NJ). 1. All primers were synthesized by Sigma-Genosys (Woodlands, TX) and stored in TE buffer (100 m M). 2. HotStar Taq DNA polymerase (Qiagen, Valencia, CA) was used for PCR reactions and Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA) could be used for optional hi-fidelity PCR reactions (see Note 2). 3. PCR purification was carried out using a PCR purification kit (Qiagen, Valencia, CA). 4. The amplified linear DNAs were suspended in sterile ddH 2O and w ere q uantified b y s pectroscopy (NanoDrop T echnologies, Wilmington, DE). 5. DpnI (New England Biolabs, Ipswich, MA) restriction treat-ment following PCR was carried out in order to eliminate circular template DNA. 6. Electroporation was carried out with a Gene Pulser II Electroporator (Bio-Rad, Hercules, CA). 1. All antibiotics were obtained from Sigma (St. Louis, MO). LB plates containing specific antibiotics were used for appro-priate selections (Table 1).2. A 37°C air shaker and a 37°C water bath shaker were used for bacterial culturing.2.2. Primers, PCR, PCRpurification, DpnITreatment, andElectroporation2.3. AntibioticsSelection and BACDNA Purification Table 1Antibiotics concentrations for selectionFor BACs (single or low copy numbers)For plasmids (high copynumbers)78Zhang, Huang, and Zhu3. NucleoBond Xtra Maxi Plasmid DNA purification kits (Clontech Laboratories, Inc., Palo Alto, CA) were used to purify VZV BAC DNA from E. coli .4. Kimwipes (Kimberly-Clark Global Sales, Inc., Roswell, GA) were used as small filters in BAC DNA Mini-preparations.5. Phenol/chloroform, isopropanol, and ethanol were obtained from Sigma (St. Louis, MO) and were used as additional reagents in BAC DNA preparations.6. Hin dIII (New England Biolabs, Ipswich, MA) digestions were performed to check the integrity of BAC DNA.1. FuGene6 transfection kit (Roche, Indianapolis, IN) was used for transfecting viral BAC DNA into MeWo cells (ATCC).2. An inverted fluorescent microscope was used to observe and count plaque numbers.3. Tissue culture media containing 150 m g/ml d -luciferin (Xenogen, Alameda, CA) was used as substrate for in vitro bioluminescence detection.4. An IVIS Imaging 50 System (Xenogen, Alameda, CA) was used to record bioluminescence signal from virally infected cells.5. Bioluminescence data were quantified by using Living Image analysis software (Xenogen, Alameda, CA).In order to generate VZV ORF deletion mutants using this new VZV luc system, we took advantage of an efficient recombination system for chromosome engineering in E. coli DY380 strain (13). A defective lambda prophage supplies the function that protects and recombines linear DNA. This system is highly efficient and allows recombination between homologies as short as 40 bp. The experimental design is summarized in Fig. 1.1. The first step in making any specific VZV ORF deletion was to amplify a Kan R cassette containing 40-bp flanking sequences of the targeted ORF.2. Primers were stored in TE buffer (100 m M). The Kan R expres-sion cassette was amplified from pGEM-oriV/Kan using a HotStar DNA polymerase kit following a standard protocol.3. PCR product was purified using a PCR purification kit fol-lowing the manufacturer’s protocol.2.4. Transfectionand SubsequentVirological Assays(Tittering and Growth Curve Analysis)3. M ethods3.1. Generation of VZVORF Deletion BACClones 3.1.1. Making a Kan R Cassette Targeting a Specific VZV Open Reading Frame79An Efficient Protocol for VZV BAC-Based Mutagenesis 4. The purified PCR product was treated with DpnI in order to eliminate the template DNA. This step greatly reduces the background in later selections.5. PCR product was purified again as above (step 3) and the amplified linear DNA was suspended in sterile ddH 2O and was quantified by spectroscopy (see Note 3).1. DY380 cells were grown at 32°C until the OD 600nm measure-ment reached 0.5 (see Note 5).2. The culture was shifted to 42°C by placing the flask into a 42°C water bath with vigorous shaking for 10–15 min (see Note 4).3.1.2. Induction of theLambda RecombinationSystem and Preparationof Electroporation-Competent DY380Fig. 1. Generating ORF deletion mutants (ORFD). (a ) The E. coli DY380 strain provides a highly efficient homologous recombination system, which allows recombination of homologous sequences as short as 40 bp. The homologous recombination system is strictly regulated by a temperature-sensitive repressor, which permits transient switching on by incubation at 42°C for 15 min. VZV luc BAC DNA is introduced into DY380 by electroporation. Electro-competent cells are prepared with homologous recombination system activation. (b ) Amplification of the Kan R expression cassette by PCR using a primer pair adding 40-bp homologies flanking ORFX. (c ) About 200 ng of above PCR product are transformed into DY380 carrying the VZV luc BAC via electroporation. (d ) Homologous recombination between upstream and downstream homologies of ORFX replaces ORFX with the Kan R cassette, creating the ORFX deletion VZV clone. The recombinants are selected on LB agar plates containing kanamycin at 32°C. (e ) The deletion of ORFX is confirmed by testing antibiotic sensitivity and PCR analysis. The integrity of viral genome after homologous recombination is examined by restriction enzyme Hin dIII digestion. (f ) VZV luc BAC DNA with ORFX deletion is propagated and isolated from DY380. (g ) Purified BAC DNA is transfected into MeWo cells. (h ) 3–5 days after transfection, the ORFX deletion virus is visualized under a fluores-cent microscope due to EGFP expression given nonessentiality of ORFX.Select for kan Rat 32°Cseqs. (40 bp)ORFX kan R ORFE. coli 32°C ts λ cI repressorVZV-BAC Defective l prophage D BkanR E BAC DNATransfect MeWo cells ProducerecombinantVZV (givenORFX is notessential)x G Hx MR WTORFXDORFXR Confirm recombinant VZV by antibiotic sensitivity, PCR and HindIII digestion80Zhang, Huang, and Zhu3. The culture was immediately transferred to an ice–water slurry for 30 min. (see Note 6).4. After incubation on ice, the culture was then pelleted at 6,000 × g for 10 min 4°C, washed with ice-cold sterile ddH 2O, and repelleted.5. Prechilled 10% glycerol (use about 1% of original volume of culture) was used to resuspend cells, and a 40-m l aliquot (>1 × 1010 cells) was used for each electroporation reaction. 1. Two microliters of Kan R cassette DNA (greater than 200 ng) were electroporated into competent DY380 cells harboring the VZV luc BAC. Homologous recombination took place between the 40-bp ORF flanking sequences and the targeted BAC ORF was replaced by the linear Kan R cassette creating the expected VZV ORF deletion clones. 2. Electroporation was carried out at 1.6 kV, 200 W , and 25 m F in a Gene Pulser II electroporator. Two microliters of con-centrated linear DNA cassette (greater than 200 ng) were used in each reaction. 3. The bacteria were immediately transferred to 1 ml LB medium after electroporation and incubated at 32°C for 1 h before plating. The resultant recombinants were selected on LB agar plates containing kanamycin at 32°C for 16–24 h (see Note 7). 4. Antibiotic sensitivity: it is important to further test that kanamycin-resistant colonies are resistant to kanamycin but not to ampicillin because the circular pGEM-oriV/Kan R (containing Amp R ) was used as the PCR template. This can be tested by re-streaking single colonies on mul-tiple LB agar plates containing different antibiotics. VZV ORFX deletion clones should be resistant to chloram-phenicol (from BAC vector), hygromycin (from luciferase cassette), and kanamycin (VZV ORF replacement cassette), but sensitive to ampicillin (potentially from pGEM-oriV/Kan R ; see Note 8). 1. Mini-BAC DNA preparations.(a) A single DY380 clone containing the recombinant VZV BAC was inoculated in 5 ml LB supplemented with the appropriate antibiotics and cultured at 32°C overnight.(b) BAC DNA was isolated by pelleting the bacteria, resus-pending in 1 ml resuspension buffer supplemented with RNase A (Buffer RES), lysing in 1 ml NaOH/SDS lysis buffer (Buffer LYS), and neutralizing in 1 ml potassium acetate neutralization buffer (Buffer NEU) for 5 min for each step (NucleoBond Xtra Maxi Plasmid DNA purifi-cation kit).3.1.3. Electroporation andRecombinant Screening3.1.4. BAC DNAPurification and BACClone Verification81An Efficient Protocol for VZV BAC-Based Mutagenesis (c) The cloudy solution was centrifuged at 4,500 × g for 15 min at 4°C. The supernatant was filtered through a small piece (cut to 4 × 4 cm) of Kimwipe tissue (Kimberly-Clark Global Sales, Inc., Roswell, GA).(d) The filtered solution was extracted with an equal volume of phenol/chloroform and the BAC DNA precipitated with two volumes of ethanol.(e) After the final spin at 4,500 × g for 30 min at 4°C, the DNA pellet was air-dried and resuspended in 20 m l sterile ddH 2O. 2. PCR verification: multiple colonies with the correct antibiotic sensitivities were picked for the mini-BAC DNA preparations. The ORF deletions with Kan R replacements were confirmed by PCR using a HotStar DNA polymerase kit following a standard protocol. The target ORF should be absent in ORF deletion clones while the adjacent ORFs should remain intact as positive controls. 3. Maxi-BAC DNA preparations: the large-scale BAC DNA preparations using the NucleoBond Xtra Maxi Plasmid DNA purification kit (Clontech Laboratories, Inc., Palo Alto, CA) started with 500 ml of overnight cultures. The standard man-ufacturer’s protocol for BAC DNA purification was followed. The final DNA products were resuspended in 250 m l sterile ddH 2O and quantified by spectroscopy (see Note 9). 4. Hin dIII digestion profiling: PCR verified clones were selected for maxi-BAC DNA preparations. To confirm that no large VZV genomic DNA segment is deleted, Hin dIII digestion profiling was routinely carried out (see Note 10). Three micrograms of BAC DNA from maxi-preparations were digested with 20 U of Hin dIII in a 20-m l reaction at 37°C overnight. Hin dIII digestion patterns were compared by electrophoresis on ethidium bromide stained 0.5% agarose gels. As shown in Fig. 1, Hin dIII digestion patterns of each VZV ORF deletion clone were highly comparable with the parental wild-type VZV luc clone (see Note 11).The generation of VZV ORF deletion revertants is necessary to prove that the deleted ORF is responsible for any phenotype (usu-ally a growth defect) observed in analyses of the deletion mutants. The viral revertants should be able to fully restore the wild-type phenotype. As an example, generating the VZV ORFX deletion rescue virus is described to demonstrate the approach (see Fig. 2).1. VZV ORFX was amplified from wild-type VZV luc BAC DNAby PCR. Two unique restriction enzyme sites and two addi-tional 6-bp random sequences were added to the ends of the PCR product. A hi-fidelity PCR kit could be used in order to minimize unwanted mutations during PCRs (see Note 2).3.2. Generation of VZVORF DeletionRevertant BAC Clones82Zhang, Huang, and Zhu2. The ORFX gene was directionally cloned into pGEM-zeo to form pGEM-ORFX-zeo. The cloned ORFX was verified by sequencing analysis.3. ORFX-zeoR cassette was made by PCR using pGEM-ORFX-zeo as template (Fig. 2). The PCR product contained 40-bp homologies of flanking sequences of Kan R cassette, which was also used to generate the ORFX deletion mutant.4. The subsequent procedures are similar to producing the ORFX deletion mutant. Briefly, the linear ORFX-zeoR cas-sette was treated with DpnI and electroporated into compe-tent DY380 cells harboring VZV luc ORFX deletion BAC. Similarly, homologous recombination functions were tran-siently induced by switching the culture temperature from 32 to 42°C for 10–15 min when electroporation-competent cells were prepared. The recombinants were selected on LB agar plates containing zeocin. After verification, the ORFX dele-tion rescue BAC DNA was isolated from E. coli .Because of VZV’s highly cell-associated nature in cell culture,conventional virology techniques, including plaque purification and plaque assay, become troublesome. By developing andexploiting the new luciferase VZV BAC system, the resulting virus has a removable EGFP expression cassette and a built-in 3.3. Transfectionand Subsequent Virological Assayszeo R lox mcs mcskan lox zeo R ORFX lox lox E. ORFXR D. ORFXR-zeo B. C. ORFXD zeo R ORFX zeo ORFX ORFX Fig. 2. Generating an ORFX deletion rescue clone (ORFXR). (a ) To generate the ORFXR clone, ORFX was amplified by PCR from the wild-type VZV BAC DNA. The ORFX was directionally cloned into plasmid pGEM-lox-zeo to form pGEM-zeo-ORFX. (b ) Amplification of the ORFX-Zeo R cassette by PCR using a primer pair adding 40 bp homologies flanking ORFX. (c ) Such PCR product was transformed into DY380 carrying the VZV luc ORFXD BAC via electroporation. (d ) Homologous recombination between upstream and downstream homologies of ORFX replaced Kan R with the ORFX-Zeo R cassette, creating the ORFXR clone. (e ) Zeo R was removed while generating virus from BAC DNA by co-transfecting a Cre recombinase expressing plasmid.83An Efficient Protocol for VZV BAC-Based Mutagenesis luciferase reporter. In this protocol, an alternative biolumines-cence quantification approach has been provided to significantly increase the reproducibility of results. This approach has also been successfully used in monitoring VZV growth in vivo (10). 1. VZV BAC DNA from maxi-preparations was transfected into MeWo cells using the FuGene6 transfection kit according to the manufacturer’s standard protocol. 2. One and a half micrograms of BAC DNA and 6 m l of transfec-tion reagent were used for a single reaction in one well of 6-well tissue culture plates (see Note 12). 3. As an option, 0.5 m g of Cre expression plasmid was co-transfected with the VZV BAC DNA to remove the BAC sequence flanked by two loxP site from the viral genome (see Note 13). 4. In order to prevent the precipitation of BAC in solution, 1.5 m g BAC DNA were diluted in serum-free tissue culture medium, and the volume of DNA solution was adjusted to 50 m l (see Note 14). 5. The DNA solution was slowly added to the transfection reagent with gentle stirring using pipet tips. 6. Because of GFP expression from the BAC vector, VZV plaques were usually visually discernable using a fluorescent microscope within 3–5 days after transfection given deleted ORF is dispensable (see Note 15). If a VZV ORF is essential for viral replication, no plaque will be observed. 7. Since VZV is highly cell-associated in tissue culture, mutant VZV-infected MeWo cells were harvested and stored in liquid nitrogen for future studies.Recombinant viruses were titered by infectious focus assay. MeWo cells were seeded in 6-well tissue culture plates and inoculated with serial dilutions of VZV-infected MeWo cell suspensions. Plaques were counted by fluorescent microscopy 3 days after inoculation and viral titer was determined. 1. MeWo cells were infected with 100 PFU of infected MeWo cell suspensions in 6-well tissue culture plates. 2. After every 24-h interval, cell culture media was replaced with media containing 150 m g/ml d -luciferin.3. After incubation at 37°C for 10 min, the bioluminescent sig-nal was quantified and recorded using an IVIS ImagingSystem following the manufacturer’s instructions. 4. Fresh tissue culture medium was added to replace the luciferin-containing medium for further incubation at later time points.3.3.1. Transfection of BACDNA into MeWo Cells3.3.2. Titering by InfectiousFocus Assay3.3.3. Growth CurveAnalyses Based onBioluminescence Imaging(See Fig. 3 and Note 16)84Zhang, Huang, and Zhu5. Measurements from the same plate were repeated every day for 7 days.6. Bioluminescence signal data from each sample was quantified by manual designation of regions of interest and analyzed using Living Image analysis software (see Note17).1. The luciferase expression cassette, driven by an SV40 early pro-moter, was inserted between VZV ORF65 and ORF66. The cassette also contains a hygromycin B resistance gene (Hyg R ).2. Platinum Taq DNA polymerase can be used alternatively if a hi-fidelity PCR product is preferred.3. In order to achieve optimum results, the final concentration of the linear DNA cassette for the subsequent electroporation was adjusted to at least 100 ng/m l.4. The 42°C temperature shift is critical for the success of the homologous recombination. The temperature needs to be adjusted accurately to 42°C and remain constant. Too much recombination system activity is detrimental to E. coli and harm the integrity of BAC DNA. On the other hand, inade-quate induction of the recombination system in DY 380 leads to inefficient recombination. Ten to fifteen minutes might need to be adjusted carefully in order to achieve optimized efficiency of homologous recombination.5. E. coli DY380 strain needs to be cultured at 32°C all the time except when the recombination system is transiently activated and expressed by shifting the culture to 42°C.6. Beyond this point, every step needs to be carried out at a low temperature (0–4°C). All reagents, centrifuge rotor and glass-ware need to be prechilled.4. N otes Growth curve analysisVZVluc infectedMeWo cells / animal. a b c d Bioluminescenceimaging Image acquisition Fig. 3. Growth curve analyses based on bioluminescence imaging. (a ) Small animals/tissue culture can be infected with VZV luc . (b ) After administration of an enzyme substrate, luciferin, bioluminescence emitting from living animals/cultured cells can be detected and monitored by using a bioluminescence imaging system (a CCD camera mounted on top of a light-tight imaging dark chamber). (c ) Data can be stored in a connected PC and quantified by using region-of-interest analysis. (d ) Viral growth kinetics can be analyzed based on quantification of bioluminescence signals.85 An Efficient Protocol for VZV BAC-Based Mutagenesis7. Recombinants often have multiple antibiotic resistances. For instance, VZV ORFX/Kan clone will have Kan R, Cm R (from BAC vector), and Hyg R (from luciferase cassette). Screening for recombinants with more than one antibiotic is optional. However, the growth rate under such conditions could be much slower than selection under one antibiotic.8. If a clone also has Amp R, it should count as a false positive result.9. Due to the large size, handling BAC DNAs needs to avoid any harsh physical sheering force including vortexing or quickly passing through fine pipette tips. Freeze and thaw should also be avoided. BAC DNA solutions should always be stored at 4°C.10. Although it has been shown that VZVluc DNA is highly stablein E. coli (10) under the conditions described in this protocol, large undesirable deletions in the BAC clones were observed if homo l ogous recombination system in DY380 was over-induced.11. Since many large DNA fragments are generated by a Hin dIIIdigestion of the VZV genome, smaller genetic alterations, including replacement of an ORF by a Kan R cassette, would be difficult to recognize by this assay.12. The ratio of BAC DNA and FuGene6 reagent might need tobe adjusted to maximize transfection efficiency.13. The ORFX rescue clone was generated by introducing the wild-type ORFX back into the deletion viral genome along with a Zeo R cassette flanked by two loxP sites. By following this optional step in transfection, Zeo R will be removed from the genome by Cre-mediated recombination. The resulting virus will have a wild-type copy of ORFX restored in the same direction and loca-tion as the parental wild-type strain except a remaining loxP site(34 bp) in the 3¢ noncoding region of ORFX.14. Highly concentrated (greater than 250 m g/m l) BAC DNAsolutions are viscous and BAC DNA molecules easily precipi-tate out of solution when added to transfection reagent solu-tions. When such precipitation becomes visible, it is irreversible and the result of the transfection assays is often poor.Therefore, we predilute each BAC DNA in media before gen-tly mixing with the transfection reagent.15. Transfection efficiency was easy to monitor because of theresulting GFP expression from the BACs.16. Growth curve analyses were traditionally carried out by aplaque assay-based method.17. See ref. 14 for more detailed methods and more applicationof in vivo bioluminescence assay.86Zhang, Huang, and Zhu References1. Abendroth A, Arvin AM (1999) Varicella-zoster virus immune evasion. Immunol Rev 168:143–1562. Gilden DH, Kleinschmidt-DeMasters BK,LaGuardia JJ, Mahalingam R, Cohrs RJ (2000) Neurologic complications of the reactivation of varicella-zoster virus. N Engl J Med 342:635–6453. Arvin AM (2001) Varicella-zoster virus. In:Knipe DM, Howley PM (eds) Fields virology, vol 2. Lippincott Williams & Wilkins, Philadelphia, PA, pp 2731–27674. Davison AJ, Scott J (1986) The completeDNA sequence of varicella zoster virus. J Gen Virol 67:1759–18165. Cohen JI, Seidel KE (1993) Generation ofvaricella-zoster virus (VZV) and viral mutants from cosmid DNAs: VZV thymidylate syn-thetase is not essential for replication in vitro.Proc Natl Acad Sci USA 90:7376–73806. Mallory S, Sommer M, Arvin AM (1997)Mutational analysis of the role of glycoproteinI in varicella-zoster virus replication and itseffects on glycoprotein E conformation and trafficking. J Virol 71:8279–82887. Niizuma T, Zerboni L, Sommer MH, Ito H,Hinchliffe S, Arvin AM (2003) Construction of varicella-zoster virus recombinants from P-Oka cosmids and demonstration that ORF65 protein is dispensable for infection of human skin and T cells in the SCID-hu mouse model. J Virol 77:6062–60658. Cohen JI, Straus SE, Arvin AM (2007)Varicella-zoster virus replication, pathogene-sis, and management. In: Knipe DM, HowleyPM (eds) Fields virology, vol 2. Lippincott Williams & Wilkins, Philadelphia, PA, pp 2773–28189. Nagaike K, Mori Y, Gomi Y, Yoshii H,Takahashi M, Wagner M, Koszinowski U, Yamanishi K (2004) Cloning of the varicella-zoster virus genome as an infectious bacterial artificial chromosome in Escherichia coli.Vaccine 22:4069–407410. Zhang Z, Rowe J, Wang W, Sommer M, ArvinA, Moffat J, Zhu H (2007) Genetic analysis of varicella zoster virus ORF0 to 4 using a novel luciferase bacterial artificial chromosome sys-tem. J Virol 81:9024–903311. Wang W, Patterson CE, Yang S, Zhu H(2004) Coupling generation of cytomegalovi-rus deletion mutants and amplification of viral BAC clones. J Virol Methods 121:137–143 12. Netterwald J, Yang S, Wang W, Ghanny S,Cody M, Soteropoulos P, Tian B, Dunn W, Liu F, Zhu H (2005) Two gamma interferon-activated site-like elements in the human cyto-megalovirus major immediate-early promoter/enhancer are important for viral replication.J Virol 79:5035–504613. Yu D, Ellis HM, Lee EC, Jenkins NA,Copeland NG, Court DL (2000) An efficient recombination system for chromosome engi-neering in Escherichia coli. Proc Natl Acad Sci USA 97:5978–598314. Tang QY, Zhang Z, Zhu H (2010) Bioluminesc-ence imaging for herpesvirus studies in vivo.In: Gluckman TR (ed) Herpesviridae: viral structure, life cycle and infections. Nova Science, Huntington, in press。

分子生物学英文文献6

Chapter19Detection and Quantitative Analysis of Small RNAs by PCR Seungil Ro and Wei YanAbstractIncreasing lines of evidence indicate that small non-coding RNAs including miRNAs,piRNAs,rasiRNAs, 21U endo-siRNAs,and snoRNAs are involved in many critical biological processes.Functional studies of these small RNAs require a simple,sensitive,and reliable method for detecting and quantifying levels of small RNAs.Here,we describe such a method that has been widely used for the validation of cloned small RNAs and also for quantitative analyses of small RNAs in both tissues and cells.Key words:Small RNAs,miRNAs,piRNAs,expression,PCR.1.IntroductionThe past several years have witnessed the surprising discovery ofnumerous non-coding small RNAs species encoded by genomesof virtually all species(1–6),which include microRNAs(miR-NAs)(7–10),piwi-interacting RNAs(piRNAs)(11–14),repeat-associated siRNAs(rasiRNAs)(15–18),21U endo-siRNAs(19),and small nucleolar RNAs(snoRNAs)(20).These small RNAsare involved in all aspects of cellular functions through direct orindirect interactions with genomic DNAs,RNAs,and proteins.Functional studies on these small RNAs are just beginning,andsome preliminaryﬁndings have suggested that they are involvedin regulating genome stability,epigenetic marking,transcription,translation,and protein functions(5,21–23).An easy and sensi-tive method to detect and quantify levels of these small RNAs inorgans or cells during developmental courses,or under different M.Sioud(ed.),RNA Therapeutics,Methods in Molecular Biology629,DOI10.1007/978-1-60761-657-3_19,©Springer Science+Business Media,LLC2010295296Ro and Yanphysiological and pathophysiological conditions,is essential forfunctional studies.Quantitative analyses of small RNAs appear tobe challenging because of their small sizes[∼20nucleotides(nt)for miRNAs,∼30nt for piRNAs,and60–200nt for snoRNAs].Northern blot analysis has been the standard method for detec-tion and quantitative analyses of RNAs.But it requires a relativelylarge amount of starting material(10–20μg of total RNA or>5μg of small RNA fraction).It is also a labor-intensive pro-cedure involving the use of polyacrylamide gel electrophoresis,electrotransfer,radioisotope-labeled probes,and autoradiogra-phy.We have developed a simple and reliable PCR-based methodfor detection and quantiﬁcation of all types of small non-codingRNAs.In this method,small RNA fractions are isolated and polyAtails are added to the3 ends by polyadenylation(Fig.19.1).Small RNA cDNAs(srcDNAs)are then generated by reverseFig.19.1.Overview of small RNA complementary DNA(srcDNA)library construction forPCR or qPCR analysis.Small RNAs are polyadenylated using a polyA polymerase.ThepolyA-tailed RNAs are reverse-transcribed using a primer miRTQ containing oligo dTsﬂanked by an adaptor sequence.RNAs are removed by RNase H from the srcDNA.ThesrcDNA is ready for PCR or qPCR to be carried out using a small RNA-speciﬁc primer(srSP)and a universal reverse primer,RTQ-UNIr.Quantitative Analysis of Small RNAs297transcription using a primer consisting of adaptor sequences atthe5 end and polyT at the3 end(miRTQ).Using the srcD-NAs,non-quantitative or quantitative PCR can then be per-formed using a small RNA-speciﬁc primer and the RTQ-UNIrprimer.This method has been utilized by investigators in numer-ous studies(18,24–38).Two recent technologies,454sequenc-ing and microarray(39,40)for high-throughput analyses of miR-NAs and other small RNAs,also need an independent method forvalidation.454sequencing,the next-generation sequencing tech-nology,allows virtually exhaustive sequencing of all small RNAspecies within a small RNA library.However,each of the clonednovel small RNAs needs to be validated by examining its expres-sion in organs or in cells.Microarray assays of miRNAs have beenavailable but only known or bioinformatically predicted miR-NAs are covered.Similar to mRNA microarray analyses,the up-or down-regulation of miRNA levels under different conditionsneeds to be further validated using conventional Northern blotanalyses or PCR-based methods like the one that we are describ-ing here.2.Materials2.1.Isolation of Small RNAs, Polyadenylation,and Puriﬁcation 1.mirVana miRNA Isolation Kit(Ambion).2.Phosphate-buffered saline(PBS)buffer.3.Poly(A)polymerase.4.mirVana Probe and Marker Kit(Ambion).2.2.Reverse Transcription,PCR, and Quantitative PCR 1.Superscript III First-Strand Synthesis System for RT-PCR(Invitrogen).2.miRTQ primers(Table19.1).3.AmpliTaq Gold PCR Master Mix for PCR.4.SYBR Green PCR Master Mix for qPCR.5.A miRNA-speciﬁc primer(e.g.,let-7a)and RTQ-UNIr(Table19.1).6.Agarose and100bp DNA ladder.3.Methods3.1.Isolation of Small RNAs 1.Harvest tissue(≤250mg)or cells in a1.7-mL tube with500μL of cold PBS.T a b l e 19.1O l i g o n u c l e o t i d e s u s e dN a m eS e q u e n c e (5 –3 )N o t eU s a g em i R T QC G A A T T C T A G A G C T C G A G G C A G G C G A C A T G G C T G G C T A G T T A A G C T T G G T A C C G A G C T A G T C C T T T T T T T T T T T T T T T T T T T T T T T T T V N ∗R N a s e f r e e ,H P L CR e v e r s e t r a n s c r i p t i o nR T Q -U N I r C G A A T T C T A G A G C T C G A G G C A G GR e g u l a r d e s a l t i n gP C R /q P C Rl e t -7a T G A G G T A G T A G G T T G T A T A G R e g u l a r d e s a l t i n gP C R /q P C R∗V =A ,C ,o r G ;N =A ,C ,G ,o r TQuantitative Analysis of Small RNAs299 2.Centrifuge at∼5,000rpm for2min at room temperature(RT).3.Remove PBS as much as possible.For cells,remove PBScarefully without breaking the pellet,leave∼100μL of PBS,and resuspend cells by tapping gently.4.Add300–600μL of lysis/binding buffer(10volumes pertissue mass)on ice.When you start with frozen tissue or cells,immediately add lysis/binding buffer(10volumes per tissue mass)on ice.5.Cut tissue into small pieces using scissors and grind it usinga homogenizer.For cells,skip this step.6.Vortex for40s to mix.7.Add one-tenth volume of miRNA homogenate additive onice and mix well by vortexing.8.Leave the mixture on ice for10min.For tissue,mix it every2min.9.Add an equal volume(330–660μL)of acid-phenol:chloroform.Be sure to withdraw from the bottom phase(the upper phase is an aqueous buffer).10.Mix thoroughly by inverting the tubes several times.11.Centrifuge at10,000rpm for5min at RT.12.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.13.Measure the volume using a scale(1g=∼1mL)andnote it.14.Add one-third volume of100%ethanol at RT to the recov-ered aqueous phase.15.Mix thoroughly by inverting the tubes several times.16.Transfer up to700μL of the mixture into aﬁlter cartridgewithin a collection bel theﬁlter as total RNA.When you have>700μL of the mixture,apply it in suc-cessive application to the sameﬁlter.17.Centrifuge at10,000rpm for15s at RT.18.Collect theﬁltrate(theﬂow-through).Save the cartridgefor total RNA isolation(go to Step24).19.Add two-third volume of100%ethanol at RT to theﬂow-through.20.Mix thoroughly by inverting the tubes several times.21.Transfer up to700μL of the mixture into a newﬁlterbel theﬁlter as small RNA.When you have >700μL of theﬁltrate mixture,apply it in successive appli-cation to the sameﬁlter.300Ro and Yan22.Centrifuge at10,000rpm for15s at RT.23.Discard theﬂow-through and repeat until all of theﬁltratemixture is passed through theﬁlter.Reuse the collectiontube for the following washing steps.24.Apply700μL of miRNA wash solution1(working solu-tion mixed with ethanol)to theﬁlter.25.Centrifuge at10,000rpm for15s at RT.26.Discard theﬂow-through.27.Apply500μL of miRNA wash solution2/3(working solu-tion mixed with ethanol)to theﬁlter.28.Centrifuge at10,000rpm for15s at RT.29.Discard theﬂow-through and repeat Step27.30.Centrifuge at12,000rpm for1min at RT.31.Transfer theﬁlter cartridge to a new collection tube.32.Apply100μL of pre-heated(95◦C)elution solution orRNase-free water to the center of theﬁlter and close thecap.Aliquot a desired amount of elution solution intoa1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap carefully because it might splashdue to pressure buildup.33.Leave theﬁlter tube alone for1min at RT.34.Centrifuge at12,000rpm for1min at RT.35.Measure total RNA and small RNA concentrations usingNanoDrop or another spectrophotometer.36.Store it at–80◦C until used.3.2.Polyadenylation1.Set up a reaction mixture with a total volume of50μL in a0.5-mL tube containing0.1–2μg of small RNAs,10μL of5×E-PAP buffer,5μL of25mM MnCl2,5μL of10mMATP,1μL(2U)of Escherichia coli poly(A)polymerase I,and RNase-free water(up to50μL).When you have a lowconcentration of small RNAs,increase the total volume;5×E-PAP buffer,25mM MnCl2,and10mM ATP should beincreased accordingly.2.Mix well and spin the tube brieﬂy.3.Incubate for1h at37◦C.3.3.Puriﬁcation 1.Add an equal volume(50μL)of acid-phenol:chloroformto the polyadenylation reaction mixture.When you have>50μL of the mixture,increase acid-phenol:chloroformaccordingly.2.Mix thoroughly by tapping the tube.Quantitative Analysis of Small RNAs3013.Centrifuge at10,000rpm for5min at RT.4.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.5.Add12volumes(600μL)of binding/washing buffer tothe aqueous phase.When you have>50μL of the aqueous phase,increase binding/washing buffer accordingly.6.Transfer up to460μL of the mixture into a puriﬁcationcartridge within a collection tube.7.Centrifuge at10,000rpm for15s at RT.8.Discard theﬁltrate(theﬂow-through)and repeat until allof the mixture is passed through the cartridge.Reuse the collection tube.9.Apply300μL of binding/washing buffer to the cartridge.10.Centrifuge at12,000rpm for1min at RT.11.Transfer the cartridge to a new collection tube.12.Apply25μL of pre-heated(95◦C)elution solution to thecenter of theﬁlter and close the cap.Aliquot a desired amount of elution solution into a1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap care-fully because it might be splash due to pressure buildup.13.Let theﬁlter tube stand for1min at RT.14.Centrifuge at12,000rpm for1min at RT.15.Repeat Steps12–14with a second aliquot of25μL ofpre-heated(95◦C)elution solution.16.Measure polyadenylated(tailed)RNA concentration usingNanoDrop or another spectrophotometer.17.Store it at–80◦C until used.After polyadenylation,RNAconcentration should increase up to5–10times of the start-ing concentration.3.4.Reverse Transcription 1.Mix2μg of tailed RNAs,1μL(1μg)of miRTQ,andRNase-free water(up to21μL)in a PCR tube.2.Incubate for10min at65◦C and for5min at4◦C.3.Add1μL of10mM dNTP mix,1μL of RNaseOUT,4μLof10×RT buffer,4μL of0.1M DTT,8μL of25mM MgCl2,and1μL of SuperScript III reverse transcriptase to the mixture.When you have a low concentration of lig-ated RNAs,increase the total volume;10×RT buffer,0.1M DTT,and25mM MgCl2should be increased accordingly.4.Mix well and spin the tube brieﬂy.5.Incubate for60min at50◦C and for5min at85◦C toinactivate the reaction.302Ro and Yan6.Add1μL of RNase H to the mixture.7.Incubate for20min at37◦C.8.Add60μL of nuclease-free water.3.5.PCR and qPCR 1.Set up a reaction mixture with a total volume of25μL ina PCR tube containing1μL of small RNA cDNAs(srcD-NAs),1μL(5pmol of a miRNA-speciﬁc primer(srSP),1μL(5pmol)of RTQ-UNIr,12.5μL of AmpliTaq GoldPCR Master Mix,and9.5μL of nuclease-free water.ForqPCR,use SYBR Green PCR Master Mix instead of Ampli-Taq Gold PCR Master Mix.2.Mix well and spin the tube brieﬂy.3.Start PCR or qPCR with the conditions:95◦C for10minand then40cycles at95◦C for15s,at48◦C for30s and at60◦C for1min.4.Adjust annealing Tm according to the Tm of your primer5.Run2μL of the PCR or qPCR products along with a100bpDNA ladder on a2%agarose gel.∼PCR products should be∼120–200bp depending on the small RNA species(e.g.,∼120–130bp for miRNAs and piRNAs).4.Notes1.This PCR method can be used for quantitative PCR(qPCR)or semi-quantitative PCR(semi-qPCR)on small RNAs suchas miRNAs,piRNAs,snoRNAs,small interfering RNAs(siRNAs),transfer RNAs(tRNAs),and ribosomal RNAs(rRNAs)(18,24–38).2.Design miRNA-speciﬁc primers to contain only the“coresequence”since our cloning method uses two degeneratenucleotides(VN)at the3 end to make small RNA cDNAs(srcDNAs)(see let-7a,Table19.1).3.For qPCR analysis,two miRNAs and a piRNA were quan-titated using the SYBR Green PCR Master Mix(41).Cyclethreshold(Ct)is the cycle number at which theﬂuorescencesignal reaches the threshold level above the background.ACt value for each miRNA tested was automatically calculatedby setting the threshold level to be0.1–0.3with auto base-line.All Ct values depend on the abundance of target miR-NAs.For example,average Ct values for let-7isoforms rangefrom17to20when25ng of each srcDNA sample from themultiple tissues was used(see(41).Quantitative Analysis of Small RNAs3034.This method ampliﬁes over a broad dynamic range up to10orders of magnitude and has excellent sensitivity capable ofdetecting as little as0.001ng of the srcDNA in qPCR assays.5.For qPCR,each small RNA-speciﬁc primer should be testedalong with a known control primer(e.g.,let-7a)for PCRefﬁciency.Good efﬁciencies range from90%to110%calcu-lated from slopes between–3.1and–3.6.6.On an agarose gel,mature miRNAs and precursor miRNAs(pre-miRNAs)can be differentiated by their size.PCR prod-ucts containing miRNAs will be∼120bp long in size whileproducts containing pre-miRNAs will be∼170bp long.However,our PCR method preferentially ampliﬁes maturemiRNAs(see Results and Discussion in(41)).We testedour PCR method to quantify over100miRNAs,but neverdetected pre-miRNAs(18,29–31,38). 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生物英文文献.pdf

生物科学论文中英文资料外文翻译文献

中英文对照翻译Carotenoid Biosynthetic Pathway in the Citrus Genus: Number of Copies and Phylogenetic Diversity of SevenGeneThe first objective of this paper was to analyze the potential role of allelic variability of carotenoid biosynthetic genes in the interspecifi diversity in carotenoid composition of Citrus juices. The second objective was to determine the number of copies for each of these genes. Seven carotenoid biosynthetic genes were analyzed using restriction fragment length polymorphism (RFLP) and simple sequence repeats (SSR) markers. RFLP analyses were performed with the genomic DNA obtained from 25 Citrus genotypes using several restriction enzymes. cDNA fragments of Psy, Pds, Zds, Lcyb, Lcy-e, Hy-b, and Zep genes labeled with [R-32P]dCTP were used as probes. For SSR analyses, two primer pairs amplifying two SSR sequences identified from expressed sequence tags (ESTs) of Lcy-b and Hy-b genes were designed. The number of copies of the seven genes ranged from one for Lcy-b to three for Zds. The genetic diversity revealed by RFLP and SSR profiles was in agreement with the genetic diversity obtained from neutral molecμLar markers. Genetic interpretation of RFLP and SSR profiles of four genes (Psy1, Pds1, Lcy-b, and Lcy-e1) enabled us to make inferences on the phylogenetic origin of alleles for the major commercial citrus species. Moreover, the resμLts of our analyses suggest that the allelic diversity observed at the locus of both of lycopene cyclase genes, Lcy-b and Lcy-e1, is associated with interspecific diversity in carotenoid accumμLation in Citrus. The interspecific differences in carotenoid contents previously reported to be associated with other key steps catalyzed by PSY, HY-b, and ZEP were not linked to specific alleles at the corresponding loci.KEYWORDS: Citrus; carotenoids; biosynthetic genes; allelic variability; phylogeny INTRODUCTIONCarotenoids are pigments common to all photosynthetic organisms. In pigment-protein complexes, they act as light sensors for photosynthesis but also prevent photo-oxidation induced by too strong light intensities. In horticμLtural crops, they play a major role in fruit, root, or tuber coloration and in nutritional quality. Indeed some of these micronutrients are precursors of vitamin A, an essential component of human and animal diets. Carotenoids may also play a role in chronic disease prevention (such as certain cancers), probably due to their antioxidant properties. The carotenoid biosynthetic pathway is now well established. Carotenoids are synthesized in plastids by nuclear-encoded enzymes. The immediate precursor of carotenoids (and also of gibberellins, plastoquinone, chlorophylls,phylloquinones, and tocopherols) is geranylgeranyl diphosphate (GGPP). In light-grown plants, GGPP is mainly derivedcarotenoid, 15-cis-phytoene. Phytoene undergoes four desaturation reactions catalyzed by two enzymes, phytoene desaturase (PDS) and β-carotene desaturase (ZDS), which convert phytoene into the red-colored poly-cis-lycopene. Recently, Isaacson et al. and Park et al. isolated from tomato and Arabidopsis thaliana, respectively, the genes that encode the carotenoid isomerase (CRTISO) which, in turn, catalyzes the isomerization of poly-cis-carotenoids into all-trans-carotenoids. CRTISO acts on prolycopene to form all-trans lycopene, which undergoes cyclization reactions. Cyclization of lycopene is a branching point: one branch leads to β-carotene (β, β-carotene) and the other toα-carotene (β, ε-carotene). Lycopene β-cyclase (LCY-b) then converts lycopene intoβ-carotene in two steps, whereas the formation of α-carotene requires the action of two enzymes, lycopene ε- cyclase (LCY-e) and lycopene β-cyclase (LCY-b). α- carotene is converted into lutein by hydroxylations catalyzed by ε-carotene hydroxylase (HY-e) andβ-carotene hydroxylase (HY-b). Other xanthophylls are produced fromβ-carotene with hydroxylation reactions catalyzed by HY-b and epoxydation catalyzed by zeaxanthin epoxidase (ZEP). Most of the carotenoid biosynthetic genes have been cloned and sequenced in Citrus varieties . However, our knowledge of the complex regμLation of carotenoid biosynthesis in Citrus fruit is still limited. We need further information on the number of copies of these genes and on their allelic diversity in Citrus because these can influence carotenoid composition within the Citrus genus.Citrus fruit are among the richest sources of carotenoids. The fruit generally display a complex carotenoid structure, and 115 different carotenoids have been identified in Citrus fruit. The carotenoid richness of Citrus flesh depends on environmental conditions, particμLarly on growing conditions and on geographical origin . However the main factor influencing variability of caro tenoid quality in juice has been shown to be genetic diversity. Kato et al. showed that mandarin and orange juices accumμLated high levels of β-cryptoxanthin and violaxanthin, respectively, whereas mature lemon accumμLated extremely low levels of carotenoids. Goodner et al. demonstrated that mandarins, oranges, and their hybrids coμLd be clearly distinguished by their β-cryptoxanthin contents. Juices of red grapefruit contained two major carotenoids: lycopene and β-carotene. More recently, we conducted a broad study on the organization of the variability of carotenoid contents in different cμLtivated Citrus species in relation with the biosynthetic pathway . Qualitative analysis of presence or absence of the different compounds revealed three main clusters: (1) mandarins, sweet oranges, and sour oranges; (2) citrons, lemons, and limes; (3) pummelos and grapefruit. Our study also enabled identification of key steps in the diversification of the carotenoid profile. Synthesis of phytoene appeared as a limiting step for acid Citrus, while formation of β-carotene and R-carotene from lycopene were dramatically limited in cluster 3 (pummelos and grapefruit). Only varieties in cluster 1 were able to produce violaxanthin. In the same study , we concluded that there was a very strong correlation between the classification of Citrus species based on the presence or absence of carotenoids (below,this classification is also referred to as the organization of carotenoid diversity) and genetic diversity evaluated with biochemical or molecμLar markers such as isozymes or randomLy amplified polymorphic DNA (RAPD). We also concluded that, at the interspecific level, the organization of the diversity of carotenoid composition was linked to the global evolution process of cμLtivated Citrus rather than to more recent mutation events or human selection processes. Indeed, at interspecific level, a correlation between phenotypic variability and genetic diversity is common and is generally associated with generalized gametic is common and is generally associated with generalized gametic disequilibrium resμLting from the history of cμLtivated Citrus. Thus from numerical taxonomy based on morphological traits or from analysis of molecμLar markers , all authors agreed on the existence of three basic taxa (C. reticμLata, mandarins; C. medica, citrons; and C. maxima, pummelos) whose differentiation was the resμLt of allopatric evolution. All other cμLtivated Citr us species (C. sinensis, sweet oranges; C. aurantium, sour oranges; C. paradisi, grapefruit; and C. limon, lemons) resμLted from hybridization events within this basic pool except for C. aurantifolia, which may be a hybrid between C. medica and C. micrantha .Our previous resμLts and data on Citrus evolution lead us to propose the hypothesis that the allelic variability supporting the organization of carotenoid diversity at interspecific level preceded events that resμLted in the creation of secondary speci es. Such molecμLar variability may have two different effects: on the one hand, non-silent substitutions in coding region affect the specific activity of corresponding enzymes of the biosynthetic pathway, and on the other hand, variations in untranslated regions affect transcriptional or post-transcriptional mechanisms.There is no available data on the allelic diversity of Citrus genes of the carotenoid biosynthetic pathway. The objective of this paper was to test the hypothesis that allelic variability of these genes partially determines phenotypic variability at the interspecific level. For this purpose, we analyzed the RFLPs around seven genes of the biosynthetic pathway of carotenoids (Psy, Pds, Zds, Lcy-b, Lcy-e, Hy-b, Zep) and the polymorphism of two SSR sequences found in Lcy-b and Hy-b genes in a representative set of varieties of the Citrus genus already analyzed for carotenoid constitution. Our study aimed to answer the following questions: (a) are those genes mono- or mμLtilocus, (b) is the polymorphism revealed by RFLP and SSR markers in agreement with the general history of cμLtivated Citrus thus permitting inferences about the phylogenetic origin of genes of the secondary species, and (c) is this polymorphism associated with phenotypic (carotenoid compound) variations.RESΜLTS AND DISCUSSIONGlobal Diversity of the Genotype Sample Observed by RFLP Analysis. RFLP analyses were performed using probes defined from expressed sequences of seven major genes of the carotenoid biosynthetic pathway . One or two restriction enzymes were used for each gene. None of these enzymes cut the cDNA probe sequence except HindIII for the Lcy-e gene. Intronic sequences and restriction sites on genomic sequences werescreened with PCR amplification using genomic DNA as template and with digestion of PCR products. The resμLts indicated the absence of an intronic sequence for Psy and Lcy-b fragments. The absence of intron in these two fragments was checked by cloning and sequencing corresponding genomic sequences (data not shown). Conversely, we found introns in Pds, Zds, Hy-b, Zep, and Lcy-e genomic sequences corresponding to RFLP probes. EcoRV did not cut the genomic sequences of Pds, Zds, Hy-b, Zep, and Lcy-e. In the same way, no BamHI restriction site was found in the genomic sequences of Pds, Zds, and Hy-b. Data relative to the diversity observed for the different genes are presented in Table 4. A total of 58 fragments were identified, six of them being monomorphic (present in all individuals). In the limited sample of the three basic taxa, only eight bands out of 58 coμLd not be observed. In the basic taxa, the mean number of bands per genotype observed was 24.7, 24.7, and 17 for C. reticμLata, C. maxima, and C. medica, respectively. It varies from 28 (C. limettioides) to 36 (C. aurantium) for the secondary species. The mean number of RFLP bands per individual was lower for basic taxa than for the group of secondary species. This resμLt indicates that secondary species are much more heterozygous than the basic ones for these genes, which is logical if we assume that the secondary species arise from hybridizations between the three basic taxa. Moreover C. medica appears to be the least heterozygous taxon for RFLP around the genes of the carotenoid biosynthetic pathway, as already shown with isozymes, RAPD, and SSR markers.The two lemons were close to the acid Citrus cluster and the three sour oranges close to the mandarins/sweet oranges cluster. This organization of genetic diversity based on the RFLP profiles obtained with seven genes of the carotenoid pathway is very similar to that previously obtained with neutral molecμLar markers such as genomic SSR as well as the organization obtained with qualitative carotenoid compositions. All these resμLts suggest that the observed RFLP and SSR fragments are good phylogenetic markers. It seems consistent with our basic hypothesis that major differentiation in the genes involved in the carotenoid biosynthetic pathway preceded the creation of the secondary hybrid species and thus that the allelic structure of these hybrid species can be reconstructed from alleles observed in the three basic taxa.Gene by Gene Analysis: The Psy Gene. For the Psy probe combined with EcoRV or BamHI restriction enzymes, five bands were identified for the two enzymes, and two to three bands were observed for each genotype. One of these bands was present in all individuals. There was no restriction site in the probe sequence. These resμLts lead us to believe that Psy is present at two loci, one where no polymorphism was found with the restriction enzymes used, and one that displayed polymorphism. The number of different profiles observed was six and four with EcoRV and BamHI, respectively, for a total of 10 different profiles among the 25 individuals .Two Psy genes have also been found in tomato, tobacco, maize, and rice . Conversely, only one Psy gene has been found in Arabidopsis thaliana and in pepper (Capsicum annuum), which also accumμLates carotenoids in fruit. According to Bartley and Scolnik, Psy1 was expressed in tomato fruit chromoplasts, while Psy2 was specific to leaf tissue. In the same way, in Poaceae (maize, rice), Gallagher et al. found that Psy gene was duplicated and that Psy1 and notPsy2 transcripts in endosperm correlated with endosperm ca rotenoid accumμLation. These resμLts underline the role of gene duplication and the importance of tissue-specific phytoene synthase in the regμLation of carotenoid accumμLation.All the polymorphic bands were present in the sample of the basic taxon genomes. Assuming the hypothesis that all these bands describe the polymorphism at the same locus for the Psy gene, we can conclude that we found allelic differentiation between the three basic taxa with three alleles for C. reticμLata, four for C. maxima, and o ne for C. medica.The alleles observed for the basic taxa then enabled us to determine the genotypes of all the other species. The presumed genotypes for the Psy polymorphic locus are given in Table 7. Sweet oranges and grapefruit were heterozygous with one mandarin and one pummelo allele. Sour oranges were heterozygous; they shared the same mandarin allele with sweet oranges but had a different pummelo allele. Clementine was heterozygous with two mandarin alleles; one shared with sweet oranges and one with “Willow leaf” mandarin. “Meyer” lemon was heterozygous, with the mandarin allele also found in sweet oranges, and the citron allele. “Eureka”lemon was also heterozygous with the same pummelo allele as sour oranges and the citron allele. The other acid Citrus were homozygous for the citron allele.The Pds Gen. For the Pds probe combined with EcoRV, six different fragments were observed. One was common to all individuals. The number of fragments per individual was two or three. ResμLts for Pds led us to bel ieve that this gene is present at two loci, one where no polymorphism was found with EcoRV restriction, and one displaying polymorphism. Conversely, studies on Arabidopsis, tomato, maize, and rice showed that Pds was a single copy gene. However, a previous study on Citrus suggests that Pds is present as a low-copy gene family in the Citrus genome, which is in agreement with our findings.The Zds Gene. The Zds profiles were complex. Nine and five fragments were observed with EcoRV and BamHI restriction, respectively. For both enzymes, one fragment was common to all individuals. The number of fragments per individual ranged from two to six for EcoRV and three to five for BamHI. There was no restriction site in the probe sequence. It can be assumed that several copies (at least three) of the Zds gene are present in the Citrus genome with polymorphism for at least two of them. In Arabidopsis, maize, and rice, like Pds, Zds was a single-copy gene .In these conditions and in the absence of analysis of controlled progenies, we are unable to conduct genetic analysis of profiles. However it appears that some bands differentiated the basic taxa: one for mandarins, one for pummelos, and one for citrons with EcoRV restriction and one for pummelos and one for citrons with BamHI restriction. Two bands out of the nine obtained with EcoRV were not observed in the samples of basic taxa. One was rare and only observed in “Rangpur” lime. The other was found in sour oranges, “V olkamer” lemon,and “Palestine sweet” lime suggesting a common ancestor for these three genotypes.This is in agreement with the assumption of Nicolosi et al. that “V olkamer” lemon resμLts from a complex hybrid combination with C. aurantium as one parent. It will benecessary to extend the analysis of the basic taxa to conclude whether these specific bands are present in the diversity of these taxa or resμLt from mutations after the formation of the secondary species.The Lcy-b Gene with RFLP Analysis.After restriction with EcoRV and hybridization with the Lcy-b probe, we obtained simple profiles with a total of four fragments. One to two fragments were observed for each individual, and seven profiles were differentiated among the 25 genotypes. These resμLts provide evidence that Lcy-b is present at a single locus in the haploid Citrus genome. Two lycopene β-cyclases encoded by two genes have been identified in tomato. The B gene encoded a novel type of lycopene β-cyclase whose sequence was similar to capsanthin-capsorubin synthase. The B gene expressed at a high level in βmutants was responsible for strong accumμLation ofβ-carotene in fruit, while in wild-type tomatoes, B was expressed at a low level.The Lcy-b Gene with SSR Analysis. Four bands were detected at locus 1210 (Lcy-b gene). One or two bands were detected per variety confirming that this gene is mono locus. Six different profiles were observed among the 25 genotypes. As with RFLP analysis, no intrataxon molecμLar polymorphism was found within C. Paradisi, C. Sinensis, and C. Aurantium.Taken together, the information obtained from RFLP and SSR analyses enabled us to identify a complete differentiation among the three basic taxon samples. Each of these taxons displayed two alleles for the analyzed sample. An additional allele was identified for “Mexican” lime. The profiles for all secondary species can be reconstructed from these alleles. Deduced genetic structure is given in. Sweet oranges and clementine were heterozygous with one mandarin and one pummelo allele. Sour oranges were also heterozygous sharing the same mandarin allele as sweet oranges but with another pummelo allele. Grapefruit were heterozygous with two pummelo alleles. All the acid secondary species were heterozygous, having one allele from citrons and the other one from mandarins ex cept for “Mexican” lime, which had a specific allele.柑桔属类胡萝卜素生物合成途径中七个基因拷贝数目及遗传多样性的分析摘要：本文的首要目标是分析类胡萝卜素生物合成相关等位基因在发生变异柑橘属类胡萝卜素组分种间差异的潜在作用；第二个目标是确定这些基因的拷贝数。

生物论文英文总结范文

Introduction:Climate change has emerged as one of the most pressing challenges of our time, with significant implications for biodiversity. This paper aims to provide a comprehensive review of the existing literature on the impact of climate change on biodiversity, highlighting the major findings and discussing the potential consequences for ecosystems and species.1. Temperature and Precipitation Changes:One of the primary impacts of climate change is the alteration of temperature and precipitation patterns. The Intergovernmental Panel on Climate Change (IPCC) has projected that global temperatures will rise by 1.5 to 6 degrees Celsius by the end of the 21st century. This rise in temperature can lead to various consequences for biodiversity.1.1 Species Distribution Shifts:Many species are expected to shift their distribution ranges in response to changing temperatures. Warmer temperatures may allow species to colonize new areas, while others may face extinction if they cannot adapt or migrate to suitable habitats. This phenomenon has been observed in various ecosystems, including forests, grasslands, and marine environments.1.2 Altered Life Cycles:Climate change can disrupt the timing of species' life cycles, leading to mismatches between species and their resources. For instance, earlier flowering times in plants may result in earlier insect emergence, causing a mismatch in timing for pollination and seed dispersal.2. Ocean Acidification:The increased absorption of carbon dioxide by the ocean has led to a decrease in pH levels, a process known as ocean acidification. This change in ocean chemistry has significant implications for marine biodiversity, particularly for calcifying organisms such as corals, mollusks, and certain plankton species.2.1 Coral Bleaching:Ocean acidification has been linked to the occurrence of coral bleaching events, where corals expel the algae living in their tissues, resulting in a loss of color. This phenomenon can lead to reduced coral cover, affecting the diversity and functioning of coral reef ecosystems.2.2 Shell Formation and Growth:Ocean acidification can also hinder the shell formation and growth of calcifying organisms, potentially leading to reduced population sizes and altered community structures.3. Habitat Loss and Fragmentation:Climate change-induced changes in temperature, precipitation, and sea-level rise can lead to habitat loss and fragmentation, further impacting biodiversity.3.1 Forests:Many forest ecosystems are at risk of habitat loss due to changing climate conditions. Deforestation, combined with climate change, can exacerbate the loss of biodiversity, particularly in tropical regions.3.2 Mountainous Areas:Mountainous areas are particularly vulnerable to climate change, as warming temperatures can lead to the melting of glaciers and the loss of alpine habitats. This can result in the extinction of species adapted to specific mountainous environments.Conclusion:In conclusion, climate change has significant implications for biodiversity, with various direct and indirect effects on species and ecosystems. The alterations in temperature, precipitation, ocean chemistry, and habitat availability can lead to species distribution shifts, altered life cycles, habitat loss, and fragmentation. Understanding the complex interactions between climate change and biodiversity is crucial for developing effective conservation strategiesand mitigating the potential consequences of climate change on our planet's ecosystems.。

PNAS生物英文文献分享

Methods
Patient Behavior and Voxel-Based Morphometry Analyses.
Electrophysiological Recordings Behavior Tests In Vivo Imaging Quantifying Microglial Chemotaxis. Blood and Spleen Cell Harvest. Isolation of Microglia from Adult Mouse Brain. Quantitative Real-Time PCR. Primary Mouse Microglia ProteinExtractionandELISAs Immunohistochemistry. NF-κB Reporter Assay. Statistical Analysis.
Microglial NFκB-TNFα hyperactivation induces obsessive–compulsive behavior in mouse models of progranulin-deficient frontotemporal dementia
Reporter：
Background
理毛行为(grooming behavior) 是指动物个体对其本身或对同种其他个体身体表面(毛发、皮肤或羽毛) 各
种形式的照看和关注,包括对身体表面有条理的梳理,有时也用嘴唇和舌头舔毛发和皮肤。在此基础上,Pérez et al . 提出理毛行为是通过观察或接触对身体表面的一个或多个位点近距离探查,同时分开毛发捡出盐粒和皮肤寄生物,这一定义被多数学者广泛接受。理毛行为可分为:自我理毛(autogrooming) 和相互理毛(allogrooming)，自我理毛(autogrooming) 多用于研究抑郁、焦虑、恐惧或躯体不适对个体行为的影响；相互理毛除对上述问题研究外，还可观察动物的社会行为。

英文生物学范文

英文生物学范文The wonders of biology are as vast as they are intricate, encompassing the study of life in all its forms. From the microscopic world of cells to the complex ecosystems of our planet, biology reveals the interconnectedness of all living things.In the realm of genetics, we discover the blueprint of life itself. DNA, the double helix structure, carries the genetic information that defines each species and individual. It's a testament to the precision and complexity ofbiological systems.Evolution, another cornerstone of biology, explains the diversity of life on Earth. Through the process of natural selection, species adapt and evolve over time, shaping the rich tapestry of life we see today.The study of ecology delves into the relationships between organisms and their environments. It's a dance of interdependence, where every creature plays a role in the balance of ecosystems, from the smallest insect to thelargest mammal.Biotechnology, a modern application of biological knowledge, has revolutionized medicine, agriculture, and environmental management. It's a field where science meets creativity, offering solutions to some of the world's mostpressing challenges.Conservation biology is a critical discipline that focuses on the preservation of species and habitats. It's a reminder of our responsibility to protect the natural world and ensure its survival for future generations.In the study of human biology, we explore the intricacies of our own bodies. From the cellular level to the systemsthat keep us alive and healthy, this knowledge is vital for understanding and improving human health.Lastly, the field of microbiology opens up a world thatis invisible to the naked eye. Microbes, both beneficial and harmful, play a crucial role in our health, the environment, and even the production of food and medicine.Biology is not just a science; it's a lens through which we can better understand the world around us and our place within it. It's a field that is constantly evolving, with new discoveries waiting to be made and mysteries to be solved.。

(英文)生物外文文献

• 染色质免疫共沉淀技术的原理是在保持组蛋白和DNA联合的同时，通过运用对应于一个特定组蛋白标记的生物抗体，染色质被切成很小的片断，并沉淀下来。是目前唯一研究体内DNA与蛋白质相互作用的方法。这项技术主要用来分析目标基因有没有活性、或者分析一种已知蛋白（转录因子）的靶基因有哪些。 • ChIP-Seq的原理是：首先通过染色质免疫共沉淀技术（ChIP）特异性地富集目通量测序。研究人员通过将获得的数百万条序列标签精确定位到基因组上，从而获得全基因组范围内与组蛋白、转录因子等互作的DNA区段信息。
58%
FIG. 3
SK-BR-3 cell，HER2过表达的乳腺癌细胞，STAT3可以通过特定的细胞因子激活。HER2是重要的乳腺癌预后判断因子，HER2阳性（过表达或扩增）的乳腺癌，其临床特点和生物学行为有特殊表现，治疗模式也与其他类型的乳腺癌有很大的区别。
36%
FIG. 4 LIF刺激
These findings are consistent with the observed reductions in STAT3 DNA binding and gene expression.
These findings indicate that GRN enhances cytokine-induced STAT3 activation and nuclear accumulation.
• 三阴性乳腺癌特指雌激素受体、孕激素受体及人表皮生长因子受体 2均阴性的乳腺癌患者。三阴乳腺癌的预后与肿瘤大小和淋巴结状况关系不大，复发相对迅速，1-3年是复发高峰，脑转移发生率高。 • 目前还没有特有的针对三阴性乳腺癌的治疗指南。因此其治疗一般按乳腺癌常规标准治疗进行。
STAT3

生物学英文文献通用引用参考文献格式

生物学英文文献通用引用参考文献格式全文共6篇示例，供读者参考篇1How to Properly Credit the Smart ScientistsHave you ever wondered how all those big books and fancy papers about plants, animals, and tiny creatures get written? Well, the super smart scientists who study biology don't just make everything up themselves. They read what other scientists have already learned and discovered, and then they build upon that knowledge with their own experiments and observations.But here's the tricky part – they have to give proper credit to all the scientists whose work they used and referenced. It's like if you copied your friend's math homework without giving them credit – that would be considered cheating, right? Well, in the science world, it's super important to always give credit where it's due. That's why there are special rules and formats for how scientists cite (that's a fancy word for "credit") the work of other scientists in their own writing.These citation rules make sure that every scientist gets properly recognized for their hard work and brilliant discoveries.It also helps other scientists easily find and double-check the original sources that a new piece of writing is based on. Pretty neat, huh?So, what do these special citation formats look like? Well, there are a few different styles that scientists use, but one of the most common for biological papers is called the "Author-Date" style. Let me break it down for you.Let's say a scientist named Jane Doe wrote an awesome book in 2018 all about the mating habits of gorillas. If another scientist, like John Smith, wanted to mention some of Jane's findings in his own paper, he would cite her work like this in the body of his writing:"Gorillas have been observed engaging in some pretty wacky mating rituals (Doe, 2018)."See how John gave credit to Jane Doe right there, along with the year her book was published? That's an in-text citation. Pretty simple, right?But that's not all! At the end of John's paper, there would also be a "References" section where he lists out all the full citations for every source he mentioned, like this:Doe, J. (2018). The Wild Mating Rituals of Gorillas. Primate Press.This full citation includes the author's full name, the publication date, the title of the work, and even which publishing company put it out. With all those details, any other scientist could easily look up Jane's original book if they wanted to learn more.Sometimes, scientists might cite a chapter from an edited book, a scientific journal article, a website, or some other type of source instead of a whole book. But the same basic principles apply – always give credit to the original authors and include key details like titles, dates, and publishers. That way, the proper scientists get the kudos they deserve!These citation rules might seem a bit fussy at first, butthey're actually super important for maintaining integrity and giving props in the scientific community. So the next time you read an incredible book about the mysteries of biology, remember – behind all those cool facts and discoveries are tons of scientists meticulously crediting each other's work. How's that for teamwork?篇2How to Cite Biology Papers ProperlyHi there! My name is Jamie and I'm going to teach you all about how to cite biology papers and books in the right way. Citing means giving credit to the authors who wrote the stuff you used for your report or project. It's really important to do this so you don't accidentally steal someone else's hard work!There are a few different styles for citing biology sources, but the main ones are APA and CSE. APA stands for American Psychological Association, and CSE means Council of Science Editors. Both of these styles have very specific rules on how to list out the citations.Let's start with APA style, since that's probably the one you'll use the most in school. When you cite something from a biology journal in APA, it looks like this:Author's Last Name, First Initial. Second Initial. (Year Published). Article title capitalized like a book title. Journal Name in Italics, Volume#(Issue#), Page range.So for example, if I wanted to cite a paper called "Frog Feeding Behaviors" by scientists John Smith and Jane Doe that was published in 2018 in volume 25, issue 2 of the Journal of Amphibian Research on pages 125-138, it would look like this:Smith, J. D., & Doe, J. (2018). Frog feeding behaviors. Journal of Amphibian Research, 25(2), 125-138.For books in APA style, it's a little different:Author's Last Name, First Initial. Second Initial. (Year Published). Book title capitalized like a sentence. Publisher Location: Publisher Name.Like if I cited a book called "The Lives of Reptiles" written by James Lee and published in 2015 by Oxford University Press in New York City, it would be:Lee, J. (2015). The lives of reptiles. New York City: Oxford University Press.Now let's talk about CSE style. This one is used a lot in super professional science journals and papers. For journal articles in CSE, the citation looks like this:Author(s) Surname(s) Followed by Initials. Article Title With Only Proper Nouns Capitalized. Journal Name in Normal Typeface. Year;Volume(Issue):Full Page Range.So for that same frog paper I cited earlier by Smith and Doe in APA style, it would be:Smith JD, Doe J. Frog feeding behaviors. J Amphib Res. 2018;25(2):125-138.For books in CSE style, it's:Author(s) Last Name Initials. Book Title Capitalized Like a Sentence. Place of Publication: Publisher Name; Year.So that reptile book by James Lee would be cited as:Lee J. The lives of reptiles. New York City: Oxford University Press; 2015.Those are the basics of citing in APA and CSE styles! There are some little extra rules too, like if there are more than 7 authors you write out the first 6 and then "et al." And for website sources you include the URL and access date. But overall those examples cover the most common types of citations you'll need for biology papers and reports when you're in elementary and middle school.The most important thing is to just make sure you carefully write down ALL the details about the sources you used - the authors' names, the article or book title, the journal or publisher, the year, volume/issue, and page numbers. That way you can put together a citations section at the end and give proper credit to all the scientists and scholars whose work you learned from.Citing is just a way to be responsible and play by the rules of good science - so get into that habit early! Let me know if you need any other citation formatting tips.篇3How to Cite Biology Books and PapersHi there! Today I'm going to teach you all about how scientists and students cite or reference the books and papers they use when writing about biology. It's really important to give credit to the authors and researchers whose work you are using. That way, other people can find and read the original sources too!There are a few different styles that are commonly used for citations in biology. The main ones are APA, MLA, CSE, and Chicago. Each one has a specific way of formatting the citations so they look neat and consistent. It's kind of like having different uniform styles for different sports teams!APA StyleLet's start with APA style, which stands for the American Psychological Association. This one is used a lot in fields like biology, psychology, and medicine.the year the book was published in parentheses, the book title in italics, and then the publisher information. For example:Dawkins, R. (1976).The selfish gene. Oxford University Press.For a paper or journal article in APA, it goes: Author(s) last name, initials. (Year). Article title.Journal Name, Volume(Issue), page numbers.Like this:Watson, J. D., & Crick, F. H. (1953). A structure for deoxyribose nucleic acid.Nature, 171(4356), 737-738.MLA StyleNext up is MLA, the Modern Language Association style. This one is more common for English and literature classes, but some biology courses use it too.first name, a period, thebook titlein italics, the publisher, a comma, and the publication year. Like:Dawkins, Richard.The Selfish Gene. Oxford UP, 1976.For an article, it's the author's last name, a comma, first name, a period, "Article Title."Journal NameVolume.Issue (Year): page range. For example:Watson, James D., and Francis H. Crick. "A Structure for Deoxyribose Nucleic Acid."Nature171.4356 (1953): 737-38.CSE & Chicago StylesTwo other common styles in biology are CSE (Council of Science Editors) and Chicago. I'll show you examples of how those look.CSE book citation:Dawkins R. 1976.The selfish gene. Oxford (United Kingdom): Oxford University Press.CSE journal article:Watson JD, Crick FH. 1953. A structure for deoxyribose nucleic acid.Nature171(4356):737-738.Chicago book style:Dawkins, Richard.The Selfish Gene. Oxford: Oxford University Press, 1976.Chicago journal article:Watson, James D., and Francis H. Crick. "A Structure for Deoxyribose Nucleic Acid."Nature171, no. 4356 (1953): 737-38.Those all look a little different, but they include the same key information - author(s), title, publication details, and page numbers for articles. The styles just format and order things in a particular way.Why Cite Sources?You might be wondering, why is it so important to cite sources properly? Well, there are a few big reasons:It gives credit to the authors and researchers whose ideas and discoveries you are using in your own work. That's only fair!It allows your readers to find the original sources if they want to read more about a particular topic. The citations act like thorough reference lists.It shows that you have done solid research from reliable, published sources rather than just making things up.Citing correctly prevents you from accidentally plagiarizing someone else's writing, which is unethical.Following the appropriate style shows you understand the rules and expectations for academic writing in your field.So in summary, always be sure to keep track of the books, journal articles, and other sources you use when writing about biology topics. Then cite them properly using the style required by your teacher, publication, or discipline. It's a crucial skill for any student or scientist!Let me know if you need any clarification or have additional questions about citation styles. Proper sourcing is super important, so I want to make sure you've got it down!篇4How to Cite Sources in Biology PapersHi kids! Today we're going to learn about how to properly cite the sources you use when writing biology papers and reports. Citing sources is really important because it gives credit to the people who did the original research and wrote the books or articles you are using. It also allows people reading your paper to find and check those original sources themselves if they want to learn more.When you cite a source, you include some key pieces of information about it, such as the name(s) of the author(s), the title of the book/article, the year it was published, and some other details depending on whether it's a book, journal article, website, etc. This information is formatted in a specific way according to established citation styles used in biology and other sciences.The most common citation style used in biology is called "Author-Date" style. It was developed by the Council of Science Editors (CSE) and has two main components:In-text citations within the body of your paperA reference list at the end listing all the sources you citedLet's start with in-text citations first. Whenever you mention a fact, idea, or finding that came from one of your sources, you need to include a brief citation inside parentheses. For a book or article by one or two authors, you list the author's last name(s) and the year of publication like this:(Miller 2012)or(Jackson and Smith 2018)If there are three or more authors, you write the last name of the first author listed followed by "et al." which means "and others":(Williams et al. 2020)If you're quoting something word-for-word from the source, you also include the page number(s) where the quote appears:(Miller 2012, p. 297)The other key part is the reference list at the very end of your paper. This is where you list out all of the complete citation details for every source you cited within your paper. The references are listed in alphabetical order by the last name of the first author. The format looks like this:Book:Author(s) Last Name, First Initial(s). Year Published. Book Title (italicized). Place of Publication: Publisher Name.For example:Hickman, C.P., L.S. Roberts, and F.M. Hickman. 1988. Integrated Principles of Zoology. St. Louis: Times Mirror/Mosby College Publishing.Journal Article:Author(s) Last Name, First Initial(s). Year Published. Article Title. Journal Name (italicized) Volume(Issue):Page Numbers.For example:Weber, J.N. and B.K. Baksi. 1976. Geological implications of a redetermination of the decay constant for 187Re. Earth and Planetary Science Letters 31(1):129-138.For websites, you include the author(s) if available, the date the website was published or last updated, the title of the page or document, the URL, and the date you accessed the website.There are a few other small variations for things like books with editors instead of authors, translated books or articles, articles with DOI numbers, and other specific cases. But those cover the most common citation formats you'll need.Citing sources properly shows that you've done your research, gives credit to other scholars, and allows your readers to trace your information back to the original publications. Getting used to this format now will make it much easier when you get to higher levels of education. Let me know if you have any other questions!篇5How to Cite Science Books and Papers Like a ProHave you ever read a really cool book or paper about animals, plants, or other living things? Maybe it taught you fascinating facts about dinosaurs, or how bees make honey, or the different kinds of frogs in the rainforest. After learning all that amazing stuff, you probably wanted to share what you discovered with your friends and family!But wait, there's something important you need to do first. Whenever you talk about facts and information you learned from a book, website, or scientific paper, you have to give credit to the author or authors who did all the hard work of researching and writing about that topic. That's called a citation.Citations are like thank-you notes for writers and scientists. They let everyone know where you got your information from, and that you didn't just make it all up yourself. Plus, if someone wants to learn more about that subject, the citation tells them exactly which book or paper to look for.There's a special way to write citations for science books and papers, called a "reference format." It's kind of like a secret code that researchers around the world use to share sources with each other. Here's how it works:For a book, you list:The author's last name and first initialThe year the book was publishedThe title of the bookThe city where the book was publishedThe name of the publisherSo if you learned about elephants from a book called "Amazing Elephants" written by Jane P. Doe and published in 2020 by Creature Books in New York City, the citation would look like this:Doe, J.P. (2020). Amazing Elephants. New York City: Creature Books.For a paper published in a scientific journal, you list:The author's last name and first initialThe year the paper was publishedThe title of the paperThe name of the journalThe volume and issue number of the journalThe page numbers where the paper appearsLet's say you read a fascinating paper called "How Butterflies Get Their Colors" by scientists Sam M. Itton and Alex Z. Ander, published in 2022 in Volume 12, Issue 3 of the journal "Incredible Insects" on pages 57-68. The citation would be:Itton, S.M. & Ander, A.Z. (2022). How Butterflies Get Their Colors. Incredible Insects, 12(3), 57-68.Writing citations might seem like a bit of work, but it's an important skill to learn. Following the proper reference format shows that you respect other people's hard work and ideas. It also makes it much easier for people to find and read the same books and papers you enjoyed so they can learn new things too!So next time you discover mind-blowing facts about the natural world, make sure to jot down all the details you need to cite your sources. Your teachers, parents, and all the other science lovers out there will be really impressed by your responsible research skills!篇6A Curious Kid's Guide to Citing Bio SourcesHey there, young scientists! Have you ever wondered how all those big brainy biologists keep track of all the books, journals, and websites they use for their research? Well, let me tell you, it's not just a messy pile of papers and scribbled notes. They follow some pretty cool systems to give proper credit to the smart folks whose work they're building on.These systems are called citation styles, and they're like special codes or formats that tell you exactly how to write down the details about a source you used. That way, if someone else wants to check out that same source, they know right where to look!One of the most common citation styles in biology is called APA, which stands for American Psychological Association. These APA rebels have their own unique way of formatting things. For example, if you cited a book, it would look something like this:Author's Last Name, First Initial. (Year Published). Book Title: Subtitle if Any. Publisher Name.See? It's got the author, the year it was published, the title, and who put it out there for the world. Neat and tidy!But what if you need to cite a science journal article instead? No problem, the APA has you covered:Author(s) Last Name(s), Initial(s). (Year Published). Article title. Journal Name, Volume(Issue), Page range.This format includes the author(s), the year, the article name, the journal it was published in, the volume and issue numbers, and the pages it was on. Phew, that's a mouthful!Now, some of you young bio brainiacs might be using a different style guide called CSE, which is preferred by the Council of Science Editors. Their formats look a little different:Book:Author(s) Initial(s). Title of Book. Edition if not first. Place of Publication: Publisher Name; Year.Journal:Author(s) Initial(s). Article title. Abbreviated Journal Name. Year;Volume(Issue):Inclusive page range.See how the CSE style separates things a bit differently and includes some extra details like the edition and place of publication? Scientists sure are sticklers for the nitty-gritty!The key thing to remember is that whenever you use someone else's words or ideas in your own bio writing, you need to give them props by citing exactly where you got thatinformation from. It's only fair, right? You wouldn't want someone swiping YOUR brilliant science finds without giving you credit!So there you have it, a whistle-stop tour of how thosebig-kid biologists cite their sources. Pretty snazzy, huh? Who knows, maybe one day you'll be the ones writing upcutting-edge research that other scientists have to cite! But for now, just focus on learning all the amazing things biology has to teach us about this wildly wonderful world.Keep exploring and asking questions, young naturalists! The adventure of science awaits!。

分子生物学英文文献

Mobile Genetic Elements 2:6, 267-271; November/December, 2012; © 2012 Landes BioscienceLETTER TO THE EDITORLETTER TO THE EDITORGAA repeats were shown to be the most unstable trinucleotide repeats in the pri-mates genome evolution by comparison of orthologous human and chimp loci.2 The instability of the GAA repeat in the first intron of the frataxin gene X25 is particu-larly well studied since it causes an inher-ited disorder, Friedreich ataxia (FRDA).3-6 I n Friedreich ataxia, once the length of the GAA repeat inside the frataxin gene (FXN GAA) reaches a certain threshold, the combined probability of its expan-sions and deletions in progeny of affected parents is about 85%.7 Deletions and con-tractions of the repeat in intergenerational transmissions can reach hundreds of base pairs.7 However, the FXN GAA repeat is much more stable in somatic cells.8 I t is relatively stable in blood, but shows some instability in dorsal root ganglia,9 which is responsible for some of the neurodegen-erative symptoms of Friedreich ataxia.5 GAA repeats were shown to be stable in FRDA fibroblasts cell lines and neuronal stem cells.10The question why the FXN GAA repeat is so much more stable in somatic cells than in intergenerational transmis-sions remains open. Recent studies in FRDA iPSCs that are closer to embryonic cells than somatic cells models, showed expansions of the GAA repeat with 100% probability.10,11 It is intriguing that all cells Complexes between two GAA repeats within DNA introduced into Cos-1 cellsMaria M. KrasilnikovaPennsylvania State University; University Park, PA USAKeywords: replication, GAA repeat, Friedreich ataxia, genome instability, chromatinCorrespondence to: Maria M. Krasilnikova; Email: muk19@ Submitted: 10/08/12; Revised: 12/09/12; Accepted: 12/10/12/10.4161/mge.23194in the iPSC cell lines that were analyzed were synchronously adding about two GAA repeats in each replication.The studies focused on the FXN GAA repeat provided many valuable insights; however, human genome contains many other GAA repeats: the human X chro-mosome, for instance, contains 44 GAA stretches with more than 100 repeats in each. About 30 GAA repeats were detected on the chromosome 4.12 GAA repeats mostly originated from the 3' end of the poly A associated with Alu elements.13I t is not known what makes repeats with the GAA motif most unstable com-pared with other trinucleotide repeats. It is possible that GAA repeats instability is caused by their ability to form non-B DNA structures. In vitro, GAA repeats can form triplexes,14,15 and sticky DNA structures.16 At the same time, hairpins 17 and paral-lel duplexes 18 have also been observed. When transcription is going through a GAA repeat, it can also form an R-loop, a DNA-RNA complex that leaves one of the complementary strands single-stranded.19 However, it is unclear whether these struc-tures indeed form in mammalian cells. If we assume that the instability of the GAA repeat is indeed associated with the struc-ture formation, it is still unclear why the structures would form in early embryo-genesis when the GAA expansion event in Friedriech ataxia is believed to occur,7 and do not form in somatic cells where the GAA repeat was shown to be more stable. I n our recent study, we hypothesize that the differences in chromatin structure are at least partially responsible for the differ-ences in the GAA repeats stability.1The propensity of GAA repeat to form a triplex structure may strongly depend on the structure of chromatin at the repeat and surrounding area.1 Consistent with other studies, we observed that formation of chromatin at an SV40-based plasmid introduced into mammalian cells occurs gradually: 8 h after transfection there are only occasional nucleosomes at the plas-mid, while by 72 h the nucleosome struc-ture is already regular.20 Our analysis of replication stalling at the repeat revealed that the repeat affects replication only in the first replication cycle, when chromatin is still at the formation stage. We believe that replication stalling at GAA is caused by a triplex structure that the GAA repeat adopts during transfection or inside the cell. In the subsequent replication cycles, replication was completely unaffected by the presence of the repeat, which is likely to be due to the inhibition of triplex for-mation by tight chromatin packaging.1Here we show the data that strengthen our previous observations and extend it to one more structure: a complex betweenWe have recently shown that GAA repeats severely impede replication elongation during the first replication cycle of transfected DNA wherein the chromatin is still at the formation stage.1 Here we extend this study by showing that two GAA repeats located within the same plasmid in the direct orientation can form complexes upon transient transfection of mammalian Cos-1 cells. However, these complexes do not form in DNA that went through several replication rounds in mammalian cells. We suggest that formation of such complexes in mammalian genomes can contribute to genomic instability.We studied replication of several SV40-based plasmids that contained two (GAA)57 repeats located at different posi-tions in Cos-1 cells (Figs. 1–3). I n each case, the cells were transiently transfected with 1 μg of each plasmid, and replication intermediates were isolated after about 30 h. This allowed us to observe replica-tion arcs that resulted from the plasmids that replicated for more than two rounds. However, some residual amount of the first replication cycle arcs can also be registered. Replication stalling at GAA repeats only occurs during the first repli-cation cycle of an SV40-based plasmid,1 hence we did not observe it in our system.For each of the plasmids, and for all patterns of restriction digests that we studied, we observed complexes between the two (GAA)57 repeats (indicated by red arrows in each of the figures). The migration of those complexes was differ-ent depending on the digest pattern, so the complexes were at different positions in 2D gel patterns, in agreement with our expectations based on their shapes.We did not observe replication stalling associated with these complexes. When this complex is formed, it migrates signifi-cantly slower on 2D gels; the replication of plasmids that contain such complexes should result in an extra replication arc originating from the complex position. However, the number of molecules that form this complex is significantly lower than the overall number of plasmids, and there may be not enough material to observe their replication.For the situation when the two GAA repeats were located in two different frag-ments upon PvuI digest (Fig. 1), we showed that the spot 3 (Fig. 1D ) (also indicated by an arrow in Fig. 1A ) con-tains both fragments: the spot hybridized to the probes corresponding to either of them (Fig. 1A and B ). However, the com-plex appears at the position that migrates slower than unreplicated plasmid in the second dimension upon AflI I I and ScaI digest when both repeats belong to the same fragment (Fig. 2). We suggest that this fragment contains a loop generated by the interaction between the two GAA repeats (Fig. 2C ) that slows it down in the second dimension, and has little effect on the mobility in the first dimension sinceThe method of two-dimensional elec-trophoresis 22,23 allowed us to analyze the replication progression through a DNA fragment containing two GAA repeats. In this method, the replication intermediates are isolated under non-denaturing condi-tions, digested by a restriction enzyme, and separated on two consecutive gel runs in perpendicular directions. The first direction runs in 0.4% agarose (that separates mostly by mass), and the second direction runs in 1% agarose (that sepa-rates by both mass and shape of a DNA molecule).two GAA stretches. Two GAA repeats has been shown to readily form complexes, such as “sticky DNA,” in vitro,16 but it is not obvious whether it can also form inside the mammalian cells. The sticky DNA requires more than one GAA stretch to form.21 We studied the interaction of two GAA repeats located within the same plas-mid, but since the human genome contains at least several hundreds of long GAA motif repeats,12,13 this structure can theoretically form in the genomic environment as well. However, more experiments are needed to detect its formation in genome.Figure 1. A complex between two (GAA)57 repeats within the same plasmid. Plasmid replication intermediates were isolated from Cos-1 cells 30 h after transient transfection. Intermediates were digested by restriction enzymes indicated in the plasmid maps, and separated by two-dimension-al neutral-neutral agarose gel electrophoresis as described previously.1 The gel was transferred to a nylon membrane and hybridized to one of the probes indicated in the plasmid maps as green lines. A position of the complex at the 2D gel pattern depends on the restriction digest of the replication intermediates (indicated by a red arrow). (A ) Two-dimensional electrophoresis of replication intermediates digested by PvuII that places each repeat within a separate fragment. The membrane was hybridized with probe 1 indicated in (C ). The names of the plasmids GAAGAA, GAACTT, etc., reflect the orientation of the GAA repeats within the plasmid (GAAGAA means that the two GAA stretches are in the direct orientation). (B ) The same membrane was stripped of probe 1, and re-hybridized with probe 2 (C ). (C ) The scheme of the plasmid that was used in the experiment in (A and B ). Two different fragments that resulted from the PvuII digest are shown in red and blue. The positions of GAA repeats are shown in black. They can be in the direct or the reverse orientations in this plasmid. (D ) The scheme of the 2D gel in (A ). Spot 1: unreplicated blue fragment, spot 2: unreplicated red fragment that appears because of the cross-contamination of probe 1 with probe 2 due to their preparation from the same plasmid with a restriction digest. Spot 3: a complex between the red and the blue fragments. Spikes 3-2 and 3-4 may result from double-stranded breaks in the plasmid during transfection that make one of the arms of the complex in spot 3 shorter.it has the same mass as the unreplicated fragment.The complexes formed only when the two GAA stretches were positioned on a plasmid as direct repeats (GAAGAA and CTTCTT in the plasmid names). The inverted repeats GAACTT and CTTGAA did not form complexes as shown in Figures 1 and 2. This is in agreement with the sticky DNA formation in supercoiled plasmids containing two GAA repeats that has been previously shown in vitro.16 In sticky DNA, the two GAA strands that are in the antiparallel orientation, and the CTT strand, form a stable complex stabilized by Mg2+-dependent reverse-Hoogsteen triads. However, the sticky DNA complex fell apart upon heating in the presence of EDTA, which removes the Mg2+ ions necessary for its stability,16 while we did not detect any changes in spot 3 upon heating the intermediates with EDTA (Fig. 3B). We suggest that in our case the complex may be different from the canonical sticky DNA. It may be based on Hoogsteen base pairing where the Mg2+ is not needed and a slightly acidic pH has a stabilizing effect.24 It has been shown that this type of structure forms within long GAA stretches in vitro even at a pH that is close to neutral.15 We also cannot exclude that the complexes are hemicatenated molecules connected at GAA repeats with Watson-Crick pairing.25The complexes between the two GAA repeats persisted only until the plasmids went through one replication round. DpnI restriction enzyme is a frequent-cutter that digests all DNA that contains strands syn-thesized in bacteria: it cleaves DNA that is methylated at GATC by dam methyl-ase, which is only present in bacteria, but not in mammalian cells. Extensive DpnI digest that we performed, cleaved the ini-tial DNA used in transfection, as well as the products of the first replication cycleA complex between the two repeats within the same fragment slow down its progres-sion in the second dimension of the 2D gel. The same plasmid as in the Figure 1 was used in this experiment, however, they were digested with different enzymes. (A) Replication intermediates were digested with AflIII and ScaI, placing both repeats within the same fragment. The complex of two GAA repeats results in a slowly migrating structure that is shown by a red arrow. (B) A map of the digest of the same plasmid as in Figure 1 with restriction enzymes ScaI and AflIII. Here both of the repeats are located within the same fragment shown in blue. (C) The scheme of the 2D gel in . Spots 1 and 2 are the same as in Figure 1D. Spot 3: a looped intermediate that resulted from the interaction of the two GAA repeats.A complex between the two GAA repeats does not form in plasmids that went through more than two replication rounds in mammaliancells. Two-dimensional gels of replication intermediates of a plasmid containing two (GAA)57 repeats were obtained as described in the Figure 1) Two-dimensional gel of replication intermediates digested by AflIII (placing the two repeats at two different fragments). A red arrow indi-cates the position of the complex between the two GAA-containing fragments. (B) The same replication intermediates were incubated at 80°C in the presence of 10 mM EDTA for 10 min; the pattern of the 2D gel did not change. (C) The same intermediates were additionally digested with10 units of DpnI for 2 h prior to loading. The spot at the position indicated by an arrow in Figure 1 A is not present in this picture. An additional spot that appeared in this pattern is likely not a part of the pattern, and is probably due to some contamination. (D) A map of the plasmid that was used inFigure 1A and B. Spot 1, unreplicated blue fragment; spot 2, unreplicated red fragment (which appeared due to contamination of probe 1 with other plasmid sequences). Spot 3, a complex between the blue and the red fragments. A very faint duplicate Y arc from replication of the second fragment originates from spot 2. The spikes originating from spot 3 can be interpreted the same way as11. Ku S, Soragni E, Campau E, Thomas EA, Altun G, Laurent LC, et al. Friedreich’s ataxia induced plu-ripotent stem cells model intergenerational GAA·TTC triplet repeat instability. Cell Stem Cell 2010; 7:631-7; PM I D:21040903; /10.1016/j.stem.2010.09.014.12. Siedlaczck I , Epplen C, Riess O, Epplen JT. Simple repetitive (GAA)n loci in the human genome. Electrophoresis 1993; 14:973-7; PM I D:7907288; /10.1002/elps.11501401155.13. Chauhan C, Dash D, Grover D, Rajamani J, MukerjiM. Origin and instability of GAA repeats: insights fromAlu elements. J Biomol Struct Dyn 2002; 20:253-63;PMID:12354077; /10.1080/07391102.2002.10506841.14. Gacy AM, Goellner GM, Spiro C, Chen X, Gupta G, Bradbury EM, et al. GAA instability in Friedreich’sAtaxia shares a common, DNA-directed and intraal-lelic mechanism with other trinucleotide diseases. MolCell 1998; 1:583-93; PMI D:9660942; http://dx.doi.org/10.1016/S1097-2765(00)80058-1.15. Potaman VN, Oussatcheva EA, Lyubchenko YL, Shlyakhtenko LS, Bidichandani SI, Ashizawa T , et al.Length-dependent structure formation in Friedreich ataxia (GAA)n*(TTC)n repeats at neutral pH. NucleicAcids Res 2004; 32:1224-31; PMID:14978261; http:///10.1093/nar/gkh274.16. Sakamoto N, Chastain PD, Parniewski P , OhshimaK, Pandolfo M, Griffith JD, et al. Sticky DNA: self-association properties of long GAA.TTC repeats inR.R.Y triplex structures from Friedreich’s ataxia. MolCell 1999; 3:465-75; PMID:10230399; http://dx.doi.org/10.1016/S1097-2765(00)80474-8.17. Heidenfelder BL, Makhov AM, Topal MD. Hairpinformation in Friedreich’s ataxia triplet repeat expansion.J Biol Chem 2003; 278:2425-31; PMI D:12441336;/10.1074/jbc.M210643200.18. LeProust EM, Pearson CE, Sinden RR, Gao X. Unexpected formation of parallel duplex in GAA and TTC trinucleotide repeats of Friedreich’s ataxia. J MolBiol 2000; 302:1063-80; PM D:11183775; http:///10.1006/jmbi.2000.4073.19. McIvor EI, Polak U, Napierala M. New insights into repeat instability: role of RNA•DNA hybrids. RNABiol 2010; 7:551-8; PMI D:20729633; http://dx.doi.org/10.4161/rna.7.5.12745.20. Chandok GS, Kapoor KK, Brick RM, Sidorova JM, Krasilnikova MM. A distinct first replication cycleof DNA introduced in mammalian cells. NucleicAcids Res 2011; 39:2103-15; PMID:21062817; http:///10.1093/nar/gkq903.21. Sakamoto N, Ohshima K, Montermini L, Pandolfo M, Wells RD. Sticky DNA, a self-associated complexformed at long GAA*TTC repeats in intron 1 of thefrataxin gene, inhibits transcription. J Biol Chem2001; 276:27171-7; PMI D:11340071; http://dx.doi.org/10.1074/jbc.M101879200.22. Krasilnikova MM, Mirkin SM. Analysis of tripletrepeat replication by two-dimensional gel electro-phoresis. Methods Mol Biol 2004; 277:19-28;PMID:15201446.23. Friedman KL, Brewer BJ. Analysis of replicationintermediates by two-dimensional agarose gel elec-trophoresis. Methods Enzymol 1995; 262:613-27; PM I D:8594382; /10.1016/0076-6879(95)62048-6.24. Frank-Kamenetskii MD, Mirkin SM. T riplex DNA structures. Annu Rev Biochem 1995; 64:65-95; PM D:7574496; /10.1146/annurev.bi.64.070195.000433.25. Lucas I, Hyrien O. Hemicatenanes form upon inhibition of DNA replication. Nucleic Acids Res 2000; 28:2187-93; PM D:10773090; /10.1093/nar/28.10.2187.26. McLay DW, Clarke HJ. Remodelling the paternal chromatin at fertilization in mammals. Reproduction 2003; 125:625-33; PM D:12713425; /10.1530/rep.0.1250625.because they contain one strand synthe-sized in bacteria.The replication intermediates digested with DpnI did not contain the spot 3, cor-responding to the complex between two GAA stretches (Fig. 3C ). We suggest that the absence of the complex is due to the chromatin coverage of the plasmid that accompanies replication. This is similar to our observation that GAA repeats only block replication during the first replica-tion round, until the chromatin is formed. The replication blockage that we have pre-viously observed is consistent with forma-tion of a triplex that occurs in transfected DNA only prior to nucleosome cover-age.1 Here, the complex between the two (GAA)57 repeats also occurred only with-out the chromatin structure. The absence of the complex in replicated DNA also shows that the complexes that we observe are not an artifact of the isolation and sub-sequent treatment of our intermediates, since then they would exist in at least some fraction of the replicated DNA as well.The question remains whether the non-B DNA structures can form within GAA repeats in mammalian cells since their formation requires DNA stretches that are not folded in chromatin. A win-dow when these complexes can form during development is the spermatogen-esis when the maturing sperm chromatin changes from nucleosome- to protamine-bound assembly.26 Another opportunity to form complexes comes when the chroma-tin of a sperm and an egg restructure after the fusion of the gamets.26,27 This is asso-ciated with degradation of protamines and nucleosome deposition, as the zygote DNA may lack a compact chromatin structure.28 It should be noted that the expansions in Friedreich ataxia were traced to the early divisions of the zygote.7An opportunity for the complexes to form may also exist in cancer cells. It is known that some regions of their genome are overmethylated and convert in hetero-chromatin, while other regions are under-methylated, which may promote a loose chromatin packaging.29,30It is not clear whether the two-repeats complexes would compete with triplex structure formation within each individ-ual (GAA)57 repeat. It is possible that both of them exist and contribute to overallgenomic instability. However, a separate study is necessary to determine whether these structures indeed have a biological role.Disclosure of Potential Conflicts of InterestNo potential conflicts of interest weredisclosed.Acknowledgments This study was supported by NI H grant GM087472, and research grant from Friedreich’s Ataxia Research Alliance toMMK.References 1. Chandok GS, Patel MP , Mirkin SM, Krasilnikova MM.Effects of Friedreich’s ataxia GAA repeats on DNAreplication in mammalian cells. Nucleic Acids Res2012; 40:3964-74; PM D:22262734; http://dx.doi.org/10.1093/nar/gks021.2. Kelkar YD, Tyekucheva S, Chiaromonte F , MakovaKD. The genome-wide determinants of humanand chimpanzee microsatellite evolution. GenomeRes 2008; 18:30-8; PMI D:18032720; http://dx.doi.org/10.1101/gr.7113408.3. Sharma R, Bhatti S, Gomez M, Clark RM, MurrayC, Ashizawa T, et al. The GAA triplet-repeat sequencein Friedreich ataxia shows a high level of somaticinstability in vivo, with a significant predilection forlarge contractions. Hum Mol Genet 2002; 11:2175-87; PM I D:12189170; /10.1093/hmg/11.18.2175.4. Pandolfo M. The molecular basis of Friedreichataxia. Adv Exp Med Biol 2002; 516:99-118;PM D:12611437; /10.1007/978-1-4615-0117-6_5.5. Pandolfo M. Friedreich ataxia. Arch Neurol 2008;65:1296-303; PM I D:18852343; http://dx.doi.org/10.1001/archneur.65.10.1296.6. Campuzano V, Montermini L, Moltò MD, PianeseL, Cossée M, Cavalcanti F , et al. Friedreich’s ataxia:autosomal recessive disease caused by an intronic GAAtriplet repeat expansion. Science 1996; 271:1423-7; PM ID:8596916; /10.1126/sci-ence.271.5254.1423.7. De Michele G, Cavalcanti F , Criscuolo C, Pianese L,Monticelli A, Filla A, et al. Parental gender, age at birthand expansion length influence GAA repeat intergener-ational instability in the X25 gene: pedigree studies andanalysis of sperm from patients with Friedreich’s ataxia.Hum Mol Genet 1998; 7:1901-6; PMI D:9811933; /10.1093/hmg/7.12.1901.8. De Biase I , Rasmussen A, Monticelli A, Al-MahdawiS, Pook M, Cocozza S, et al. Somatic instability of theexpanded GAA triplet-repeat sequence in Friedreich ataxia progresses throughout life. Genomics 2007;90:1-5; PMID:17498922; /10.1016/j.ygeno.2007.04.001.9. De Biase I , Rasmussen A, Endres D, Al-Mahdawi S,Monticelli A, Cocozza S, et al. Progressive GAA expan-sions in dorsal root ganglia of Friedreich’s ataxia patients.Ann Neurol 2007; 61:55-60; PMID:17262846; http:///10.1002/ana.21052.10. Du J, Campau E, Soragni E, Ku S, Puckett JW,Dervan PB, et al. Role of mismatch repair enzymesin GAA·TTC triplet-repeat expansion in Friedreichataxia induced pluripotent stem cells. J Biol Chem2012; 287:29861-72; PMID:22798143; http://dx.doi.org/10.1074/jbc.M112.391961.29. Watanabe Y, Maekawa M. Methylation of DNAin cancer. Adv Clin Chem 2010; 52:145-67; PM D:21275343; /10.1016/S0065-2423(10)52006-7.30. Kulis M, Esteller M. DNA methylation and cancer.Adv Genet 2010; 70:27-56; PMID:20920744; /10.1016/B978-0-12-380866-0.60002-2.27. Spinaci M, Seren E, Mattioli M. Maternal chromatinremodeling during maturation and after fertilization in mouse oocytes. Mol Reprod Dev 2004; 69:215-21; PM ID:15293223; /10.1002/mrd.20117.28. I mschenetzky M, Puchi M, Gutierrez S, MontecinoM. Sea urchin zygote chromatin exhibit an unfolded nucleosomal array during the first S phase. J Cell Biochem 1995; 59:161-7; PM D:8904310; /10.1002/jcb.240590205.。

生物英文文献

2002 by International and Japanese Gastric Case reportAn oral anticancer drug, TS-1, enabled a patient with advanced gastric cancer with Virchow’s metastasis to receive curative resectionT akashi I wazawa 1, M asakatsu K inuta 1, H iroshi Y ano 1, S higeo M atsui 1, S hinji T amagaki 1, A tsushi Y asue 1,K azuyuki O kada 1, T oshiyuki K anoh 1, T akeshi T ono 1, Y oshiaki N akano 1, S higeru O kamoto 2,and T akushi M onden 11Department of Surgery, NTT West Osaka Hospital, 2-6-40 Karasugatsuji, Tennouji-ku, Osaka 543-8922, Japan 2Department of Pathology, NTT West Osaka Hospital, Osaka, Japanconsidered to be stage IV, with distant metastasis (M1),according to the Japanese classiﬁcation of gastric cancer [1], and is usually not an indication for surgery. The prognosis of unresectable stage IV gastric cancer is ex-tremely poor, and several chemotherapy regimens have been introduced to attempt to prolong survival [2,3] or to achieve downstaging, followed by curative resection [4,5]. However, to the best of our knowledge, few pre-vious reports have documented chemotherapy that enables the curative resection of gastric cancer with metastasis to Virchow’s lymph node, even though the response rate of recent combined chemotherapeutic modalities is 30% to 50%. We encountered a patient with gastric cancer with Virchow’s lymph node meta-stasis, who subsequently received curative resection following treatment with the newly developed oral anti-cancer drug, TS-1. There were no signiﬁcant adverse reactions to the chemotherapy.Case reportA 67-year old woman, who had complained of upper abdominal discomfort for 3 months, presented on June 6, 2000, with advanced gastric cancer, with swelling of Virchow’s lymph nodes. Gastrointestinal ﬁberscopy (GIF) and upper gastrointestinal series (UGI) showed a type 2 tumor, i.e., ulcerated carcinomas with sharply demarcated and raised margins, on the greater curva-ture in the middle third of the stomach (Fig. 1A,C). A biopsy specimen showed poorly-to-moderately differ-entiated adenocarcinoma. Four swollen lymph nodes,up to 1.5cm diameter, in the left supraclavicular area were considered to be metastasis to Virchow’s lymph node, based on ﬁne-needle aspiration cytology (Fig. 2).Abdominal computed tomography (CT) and ultra-sound sonography (USG) showed swelling of several paraaortic lymph nodes (Fig. 3A; USG is not shown).Abdominal magnetic resonance imaging (MRI) showedGastric Cancer (2002) 5: 96–101AbstractWe encountered a patient with advanced gastric cancer, with V irchow’s lymph node metastasis, who subsequently under-went curative resection after neoadjuvant chemotherapy with the newly developed oral anticancer drug, TS-1. The patient was a 67-year-old woman who had a type 2 tumor in the middle third of the stomach, and Virchow’s lymph node me-tastasis, which was diagnosed by ﬁne-needle aspiration cytol-ogy; she also had swollen paraaortic lymph nodes. Curative resection was considered impossible, and TS-1 (100mg/day)was administered for 28 days in one course, mainly in the outpatient clinic. Although grade 2 stomatitis interrupted the therapy on day 21 of the second course and on day 7 of the third course, the type 2 tumor showed marked remission (par-tial response; PR) and the metastasis in the V irchow’s and paraaortic lymph nodes had completely disappeared after the third course (complete response; CR). Eleven weeks after the completion of the TS-1 treatment, total gastric resection with D3 lymph node dissection was performed. Histopatho-logical examination revealed tumor involvement only in the mucosal and submucosal layers of the stomach and the no. 4d lymph node. Most of the tumor was replaced with ﬁbrosis with granulomatous change in the muscularis propria of the stom-ach and in the no. 3, no. 6, and no. 7 lymph nodes. This may be the ﬁrst report of a patient with advanced gastric cancer with V irchow’s lymph node metastasis who successfully received curative resection following neoadjuvant chemotherapy with a single oral anticancer drug.Key words TS-1 · Virchow’s lymph node metastasis · Gastric cancer · Gastrectomy · Neoadjuvant chemotherapyIntroductionGastric cancer with involvement of lymph nodes in the left supraclavicular fossa (Virchow’s lymph node) isOffprint requests to: T. IwazawaReceived: August 7, 2001 / Accepted: January 28, 2002thickening of the gastric wall and tumor invasion to the gastric serosa (Fig. 1E). The patient was given a diagno-sis of stage IV (cT3, cN3, cP0, cH0, cM1) advanced gastric cancer with extensive lymph node metastasis,even though no distant metastasis to the liver, lung,bone, or peritoneum was diagnosed by CT, USG, or bone scintigraphy. The performance status of this pa-tient was grade 0, and laboratory examination results were within the normal ranges, except for a high level of carbohydrate antigen (CA)19-9 (283U/ml). Because curative resection was impossible for this patient, che-motherapy, using TS-1, was started, on 22 June, 2000.TS-1, 100mg, was administered orally every day for 28 days, followed by a 14-day cessation as one course;the drug was administered mainly in the outpatient clinic. One course resulted in the complete disappear-ance of the swollen Virchow ’s lymph nodes, but slightly swollen paraaortic lymph nodes still remained on USG.GIF showed marked reduction of the gastric tumor, but the presence of malignant cells was demonstrated by biopsy. During the second course of TS-1 administra-tion, the patient had grade 2 stomatitis, so that the administration of TS-1 was interrupted on day 21. Al-though the third course of the treatment started after 35days of rest, grade 2 stomatitis interrupted the adminis-tration again, on day 7. After this treatment, effective-ness was evaluated with UGI, GIF, CT, USG, and MRI.UGI and GIF revealed that the type 2 tumor had changed to an ulcer scar with fold convergence and a small elevated lesion (Figs. 1B,D; 4A), in which adeno-carcinoma was proven by histological examination of the biopsy specimen. Dynamic abdominal MRI showed marked reduction of wall thickness after TS-1 adminis-tration and, ﬁnally, no serosal tumor invasion was dem-onstrated (Fig. 1F). Abdominal CT did not show anyregional or paraaortic lymph node swelling (Fig. 3B),Fig. 1.A,B Upper gastrointestinal series shows a protruding lesion with a crater on the greater cur-vature of the middle third of the stomach before treatment (A ); the crater and margin of the tumor were ﬂattened after treatment (B ). Evaluation of the response was partial response (PR). C,D Endo-scopic ﬁndings show an irregularly shaped tumor with a crater before treatment (C ); the lesion had markedly regressed and ﬂattened after treatment (D ). E,F Abdominal dynamic magnetic resonance imaging (MRI) shows the thickness of gastric wall with high intensity (arrow ) before treatment (E );the gastric wall became thinner (arrow ) and the serosal surface became smooth after treatment (F )abdominal USG showed a marked reduction of para-aortic lymph nodes (data not shown), and Virchow ’s lymph node was not palpable. The serum level of CA19-9 decreased to within the normal range, at 18U/ml.Therefore, we concluded that curative resection could be achieved.Total gastrectomy with splenectomy and D3 lymph node dissection was performed on November 8, 2000,and no invasion to neighboring organs, no peritoneal dissemination, and no hepatic metastasis was recog-nized, and peritoneal cytology was negative. Histo-pathological examination showed submucosal invasion of a predominantly mucinous adenocarcinoma in a small elevated gastric lesion. Only xanthoma cells in ﬁl-trated the ulcer scar (Fig. 4A,B,C,D,E). Metastasis was found in only one lymph node, along the right gastroepi-ploic vessels (no. 4d), and no cancer cells were found in any other lymph nodes, including the paraaortic lymph nodes (no. 16a2 and 16b1). Necrosis and disappearance of the tumor, with granulomatous change, were ob-served in lymph nodes along the lesser curvature (no.3), infrapyloric lymph nodes (no. 6), and lymph nodes along the left gastric artery (no. 7) (Fig. 4F). Eventually,the ﬁnal stage was determined to be T1, N1, P0, CY0,H0, Mx, and the curability of the surgical procedure was B (no residual tumors, but not evaluable as “curability A ”). No signs of recurrence have been revealed by any examination 1 year after the surgery.DiscussionUnresected gastric cancer has been treated by several regimens of combined chemotherapy. Some random-ized control reports have documented that chemo-therapy for gastric cancer patients with grade 0–2performance status improved survival compared with best supportive care [6–8]. FAMTX (5-ﬂuorouracil [5-FU] combined with Adriamycin [ADM] and Meth-otrexate [MTX]) is considered to be standard chemo-therapy in Western countries [9,10]; however, it has not been accepted in Japan because of its severe toxicity.Fig. 2.Fine-needle aspiration cytology of Virchow ’s lymph node revealed a large number of adenocarcinoma cells with clear nuclei, a high nuclear/cytoplasmic (N/C) ratio, and mitosis. Papanicolaou stain, ϫ1000Fig. 3A,B.Enhanced abdominal com-puted tomography (CT) showed that lymph nodes around the abdominal aorta (no. 16a2) were swollen (arrow ) before treatment (A ); the swollen lymph nodes disappeared after treatment (B )According to a J apanese Clinical Oncology Group (J COG) study, FP (5-FU combined with cisplatin [CDDP]) therapy has better response rates than UFTM (UFT [tegafur, uracil] combined with mitomycin C [MMC]) and 5-FU alone [11], and continuous low-dose FP is used widely in Japan because of its high response rate, which is equal to that of the original FP protocol, with less toxicity [12]. The response rates of these che-motherapy regimens have improved to 30%–50%, but no regimen yields better survival than continuous injec-tion of 5-FU alone. Most patients suffer side effects, and often need long hospitalization for systemic chemo-therapy, and thus, more effective anticancer drugs with milder toxicity are needed.TS-1 is a newly developed oral anticancer drug, which consists of tegafur, gimeracil, and oteracil potassium at a molecular ratio of 1:0.4:1, based on the biochemical modulation of 5-FU [13]. Gimeracil competitively in-hibits dihydropyrimidine dehydrogenase, is produced in various organs, including tumor tissues, and rapidly degrades 5-FU [14]. Oteracil potassium is an inhibitor of orotate phosphoribosyltransferase that catalyzes theFig. 4.A Macroscopic ﬁndings show anulcer scar with fold convergence andneighboring small elevated lesion on thegreater curvature in the middle third ofthe stomach B–F Histological ﬁndingsshowed that the gastric tumor was re-placed by ﬁbrosis with granulomatouschanges and xanthoma cell inﬁltration inthe ulcer scar (B, C, D) and regionallymph nodes (F). Dominantly mucinousadenocarcinoma with tubular adenocarci-noma invaded the submucosal layer of theelevated lesion (B, F). B H&E, ϫ4; CH&E, ϫ40; D H&E, ϫ100; E H&E, ϫ40;F H&E, ϫ100phosphorylation of 5-FU, a process that is considered to be responsible for the toxic effects of 5-FU. Oteracil potassium is mainly distributed in the gastrointestinal tract after oral administration to rats, and induces ame-lioration of the gastrointestinal toxicity induced by 5-FU [15]. TS-1 induced a 53.6% response rate for gastric cancer in an early phase II study [16] and a response rate of 49% in a late phase II study [17], with a 35.7% adverse reaction rate in the early phase II study and a 20% adverse reaction rate in the late phase II study. Not only is the response rate the highest for a single agent but also this oral anticancer drug does not require patient hospitalization, because of its mild toxicity. TS-1 was more effective for lymph node metastasis (re-sponse rate of cervical lymph nodes, 68.4%; abdominal lymph nodes, 49.2%) than for the primary lesion (32.6%), lung metastasis (22.2%), or liver metastasis (35.1%) in a phase II study. The present patient had lymph node metastasis at a distant site (Virchow’s and paraaortic lymph nodes), but no metastasis to other organs, and no invasion to surrounding organs. Also,the performance status of the patient was grade 0, andshe had no problems ingesting food. Therefore, this patient was a good candidate for neoadjuvant chemo-therapy with the oral anticancer drug, TS-1. In fact, the metastasis in Virchow’s lymph node completely disap-peared (CR) and the gastric tumor was reduced by more than 50% by TS-1; consequently, curative surgical resection could be performed. Histopathological exami-nations showed viable cancer cells to exist only in the mucosal and submucosal layer of stomach and in a group 1 lymph node, but not in any group 2 and group 3 lymph nodes, including paraaortic lymph nodes. More than two-thirds of the gastric tumor was replaced by ﬁbrosis with granulomatous changes, in particular in the submucosal lesion and some regional lymph nodes. The response was classiﬁed as category grade 2, moderate change, according to Japanese classiﬁcation of gastric carcinoma. The ﬁnal stage was T1 N1 P0 CY0 H0 Mx, and the level of curability of the surgery was B.A few reports in Japan have documented the success-ful treatment of gastric cancer with Virchow’s lymph node metastasis by chemotherapy. Ohyama et al. [18] reported a patient with advanced gastric cancer with Virchow’s and paraaortic lymph node metastasis who completely responded to a four-drug combination che-motherapy. Pathological examination revealed no tu-mor cells in the primary lesion or in any dissected nodes, including Virchow’s nodes, although recurrence devel-oped 18 months after surgery [18]. Three reports have documented that Virchow’s lymph node metastasis disappeared after low-dose FP chemotherapy, and that, consequently, gastrectomy could be performed, al-though paraaortic lymph node metastasis was histologi-cally proven in all three patients [19–21]. Nakaguchi et al. [22] reported that Virchow’s lymph node metastasis disappeared after treatment with an oral anticancer drug, 5Ј-deoxy-5-ﬂuorouridine (5Ј-DFUR), and, con-sequently, distal gastrectomy was performed, but it was not curative surgery because of paraaortic lymph node metastasis. Therefore, the present report appears to be the ﬁrst report of curative resection of advanced gastric cancer after the disappearance of Virchow’s lymph node metastasis induced by neoadjuvant chemotherapy with a single oral anticancer drug. Complete response should be conﬁrmed by longer observation. Although it is not yet known whether the overall survival of this patient has been improved, this case suggests that TS-1 is an effective anticancer drug for advanced gastric cancer with extended lymph node metastasis. More-over, this oral agent has the advantage of not requiring hospitalization for patients with good performance sta-tus, because of its mild toxicity.Acknowledgment The authors gratefully acknowledge the assistance of Dr. Ogura, who provided UGI ﬁlms before the treatment.References1.J apanese Gastric Cancer Association. J apanese classiﬁcation ofgastric carcinoma. 2nd English ed. Gastric Cancer 1998;1:10–24.2.Preusser P, Wilke H, Achterrath W, Fink U, Lenaz L, HeinickeA, et al. Phase II study with the combination etoposide, doxoru-bicin, and cisplatin in advanced measurable gastric cancer. J Clin Oncol 1989;7:1310–7.3.Boku N, Ohtsu A, Shimada Y, Shirao K, Seki S, Saito H, et al.Phase II study of a combination of irinotecan and cisplatin against metastatic gastric cancer. J Clin Oncol 1999;17:319–23.4.Nakajima T, Ota K, Ishihara S, Oyama S, Nishi M, Ohashi Y, et al.Combined intensive chemotherapy and radical surgery for incur-able gastric cancer. Ann Surg Oncol 1997;4:203–8.5.Yonemura Y, Sawa T, Kinoshita K, Matsuki N, Fushida S,Tanaka S, et al. Neoadjuvant chemotherapy for high-grade advanced gastric cancer. World J Surg 1993;17:256–62.6.Murad AM, Santiago FF, Petroianu A, Rocha PRS, RodriguesMAG, Rausch M. Modiﬁed therapy with 5-ﬂuorouracil, doxoru-bicin, and methotrexate in advanced gastric cancer. Cancer 1993;72:37–41.7.Glimelius B, Hoffman K, Haglund U, Nyren O, Sjoden PO. Initialor delayed chemotherapy with best supportive care in advanced gastric cancer. Ann Oncol 1994;5:189–90.8.Pyrhonen S, Kuitunen T, Nyandoto P, Kouri M. Randomizedcomparison of ﬂuorouracil, epidoxorubicin and methotrexate (FEMTX) plus supportive care with supportive care alone in patients with non-resectable gastric cancer. Br J Cancer 1995;71: 587–91.9.Wils JA, Klein HO, Wagener DJT, Bleiberg H, Reis H, KorstenF, et al. Sequential high-dose methotrexate and ﬂuorouracil com-bined with doxorubicin – a step ahead in the treatment of advanced gastric cancer: a trial of the European Organization for Research and Treatment of Cancer Gastrointestinal Tract Cooperative Group. J Clin Oncol 1991;9:827–31.10.Kelsen D, Atiq OT, Saltz L, Niedzwiecki D, Ginn D, Chapman D,et al. FAMTX versus etoposide, doxorubicin, and cisplatin: a random assignment trial in gastric cancer. J Clin Oncol 1992;10: 541–8.11.Shimada Y, Shirao K, Ohtsu A, Hyodo I, Saito H, Yamamichi N,et al. Phase III study of UFT ϩ MMC versus 5-FU ϩ CDDP versus 5-FU alone in patients with advanced gastric cancer: JCOG study 9205. Proc Am Soc Clin Oncol 1999;18:1043.12.Chung YS, Yamashita Y, Nakata B, Nitta A, Inoue T, HirayamaK, et al. Combination therapy of 5-FU and low dose CDDP for advanced and recurrent gastric cancer. Jpn J Cancer Chemother 1995;22:149–51.13.Shirasaka T, Nakano K, Takechi T, Satake H, Uchida J, FujiokaA, et al. Antitumor activity of 1M tegafur-0.4M 5-chloro-2,4-dihydroxypyridine-1M potassium oxonate (S-1) against human colon carcinoma orthotopically implanted into nude rats. Cancer Res 1996;56:2602–6.14.Shirasaka T, Shimamoto Y, Ohshimo H, Yamaguchi M, Kato T,Yonekura K, et al. Development of a novel form of an oral 5-ﬂuorouracil derivative (S-1) directed to the potentiation of the tumor selective cytotoxicity of 5-ﬂuorouracil by two biochemical modulators. Anticancer Drugs 1996;7:548–57.15.Takechi T, Nakano K, Uchida J, Mita A, Toko K, Takeda S, et al.Antitumor activity and low intestinal toxicity of S-1, a new formu-lation of oral tegafur, in experimental tumor models in rats.Cancer Chemother Pharmacol 1997;39:205–11.16.Sugimachi K, Maehara Y, Horikoshi N, Shimada Y, Sakata Y,Mitachi Y, et al. An early phase II study of oral S-1, a newly developed 5-ﬂuorouracil derivative for treating patients with advanced and recurrent gastrointestinal cancers. Oncology 1999;57:202–10.17.Sakata Y, Ohtsu A, Horikoshi N, Sugimachi K, Mitachi Y,Taguchi T. Late phase II study of novel oral ﬂuoropyrimidineanticancer drug S-1 (1M Tegafur-0.4M gimestat-1M otastat potassium) in advanced gastric cancer patients. Eur J Cancer 1998;34:1715–20.18.Ohyama S, Komatsu O, Nakajima T, Ohta K, Takahashi T,Yanagisawa A. Case report of pathological complete remission with FLEP therapy. In: Nakajima T, Yamaguchi T, editors.Multimodality therapy for gastric cancer. Berlin Heidelberg New York Tokyo: Springer-Verlag; 1999. pp. 104–7.19.Umehara Y, Okubo T, Sano Y, Sakamoto R, Nakamura T,Tsuchiya Y, et al. A case of advanced gastric remnant carcinoma with Virchow’s metastasis treated with neoadjuvant chemo-therapy (low dose CDDP ϩ 5-FU) followed by surgical resection.Jpn J Cancer Chemother 1995;22:277–9.20.Kajihara K, Ishikawa H, Akama, F, Ninomiya H, Shigeta K, SanoI, et al. A case of advanced gastric cancer with Virchow’s metasta-sis responding remarkably to combination chemotherapy of low-dose CDDP and 5-FU. Jpn J Cancer Chemother 1998;25:585–8.21.Wada Y, Kamiya N, Asano S, Shinya F. A case of advancedgastric cancer with Virchow’s and paraaortic lymph node me-tastases successfully resected after combined chemotherapy of low-dose CDDP and 5-FU. Jpn J Cancer Chemother 2001;28:79–82.22.Nakaguchi K, Nakano Y, Kitahara T, Onoe K, Nagamine H,Fukuda H. A resected case of gastric carcinoma with complete remission of Virchow’s node metastasis by 5Ј-DFUR administra-tion. Jpn J Cancer Chemother 1990;17:2101–4.。

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High Mobility Group Box-1 Protein
Tethered particle motion
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背景知识
V(D)J Recombination
V(D)J重组
V（D）J代表可变区V，多样区D，连接区J。重组是在T和B细胞成熟的早期阶段仅发生在淋巴细胞中的基因重组的独特机制。它是B和T细胞上发现的免疫球蛋白（Ig）和T细胞受体（TCR）的高度多样性的组成部分。 V（D）J重组负责组装在B和T淋巴细胞发育期间抗原受体的可变区。在V（D）J 重组期间，同时位于特定染色体上的V，D和J片段被重排到功能性V（D）J或者有特异性决定簇的B细胞受体或免疫球蛋白（Ig）和 T细胞受体（TCR）VJ等位基因中。与V，D和J基因区段相邻的是由高度保守的七聚体（共有序列5‘CACAGTG-3’）和九聚体（共有序列5‘-ACAAAAACC-3’）组成的重组信号序列（RSS），被相对不保守的间隔序列12bp 或23bp（分别称为12RSS和23RSS）分开。
— 3—
背景知识
Recombination activating gene
RAG
重组激活基因（RAG）是在编码免疫球蛋白和T细胞受体分子的基因的重排和重组中起重要作用的酶。它有两种称为RAG-1和RAG-2的重组激活基因产物，其细胞表达在其发育阶段限于淋巴细胞。RAG-1和RAG-2对于产生成熟B和T淋巴细胞是两种细胞类型是必需的。 RAG蛋白启动V（D）J重组，这是pre-B和pre-T细胞的成熟所必需的。研究表明RAG-1和RAG-2必须以协同方式工作以激活VDJ重组。当分离和转染成纤维细胞样品时，显示RAG-1低效诱导VDJ基因的重组活性。当 RAG-1与RAG-2共转染时，重组频率增加了1000倍，这一发现促进了新修订的理论，RAG基因不仅可以帮助VDJ重组，而是直接诱导VDJ基因的重组。
In the presence of 5 nM RAG1/2c and 80 nM HMGB1 for 1 h。
结果
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Results
C
(i) paired complex formation and excision of the looped DNA faster than we can resolve with our method (ii) a pathway for cutting that does not require the two RSS sites to synapse
讨论
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Discussion
我们发现HMGB1增加RAG-RSS复合物中DNA长度的减少量。这种观察结果与在12RSS-RAG-HMGB1或23RSS-RAG-HMGB1复合物中检测到的大弯曲的HMGB1依赖性一致，尽管那些研究使用短的寡核苷酸底物。我们还使用这种DNA链系长度的减少量来确定在RAG1 / 2c和 HMGB1存在下12RSS和23RSS的Kd，并且发现HMGB1与单独的RAG1 / 2c相比降低了两个Kd值，并且它们是相似的，与前面的观察一致。我们研究HMGB1依赖过程的单分子实验可以扩展到研究除RAG1 / 2c以外的其它可以与DNA结合并且可以被HMGB1促进的蛋白质（例如，肿瘤抑制蛋白p53）。
讨论
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Thank You
其中B box是发挥炎症的功能区域，A box是B box的拮抗位点。A box和B box都能够与DNA结合，并参与DNA双链的折叠与扭曲。
HMGB1
— 5—
背景知识
Tethered particle motion
TPM
Tethered particle motion (TPM)是用生物物理方法研究各种聚合物如DNA及其与其他物质（例如蛋白质）的相互作用。
讨论
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Discussion
在我们实验中使用的RAG1 / 2c和HMGB1的浓度下，在HMGB1存在（或不存在）下与RAG1 / 2c相关的结合事件相对频繁，但是该结合（配对复合物形成）的下游产物相对稀少 ; 在我们的实验中，珠子结合到DNA上在配对的复合物状态中仅花费总观察时间的4％。我们直接观察到在DNA底物上存在12RSS和23RSS时配对复合物的形成。当仅存在 12RSS（或不存在RSS）时，我们没有看到复合物形成的证据，并且我们确认当使用1,200和1,800-bp间距时配对复合物时的表观长度与已经被缩短了12/23的信号间距的系链一致。我们无法确定是否一个或两个 RSSs在配对的复杂形成之前被占领，我们需要进行更多的研究来解决这个重要机制问题。
实时检测单个RAG-RSS复合物。
结合动力学显示RAG-RSS复合物的平均停留时间。
HMGB1改变RAG-RSS复合物的结合性质。
直接观察平行复合物形成。
12/23规则调节珠释放作为RAG1 / 2c和HMGB1的函数的动力学。
讨论
— 18 —
Discussion
我们观察到在没有HMGB1的情况下通过RAG的RSS结合的DNA长度的明显减少，这与先前的工作一致，显示单独的RAG蛋白在RSS处弯曲 DNA。这种计算提供了RAG-RSS交互的Kd。我们期望，我们的单分子方法可用于测量非共有RSS的Kd值，从而提供对RSS和RAG活性之间的关系的更完整的理解。该方法还允许首先测定RAG1 / 2c复合物结合到12RSS或23RSS的平均停留时间。我们发现RAG1 / 2c-RSS复合物是相当稳定的，停留时间在几分钟的量级。特别地是，我们发现，与23RSS（〜230s）相比，复合物在12RSS（〜430s）处保持更长的结合时间，与两个位点之间的Kd的差异一致;
5 nM RAG1/2c-----the red line is the rms trajectory of the bead and the black dashed line is the resulting segmentation between long and short states of the DNA tether.
结果
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fig.7
Dynamics of 12/23 Rule-Regulated Bead Release as a Function of RAG1/2c and HMGB1.
Results
结果
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Results
结果
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fig.8
Results
结果
— 17 —
Discussion
结果
— 12 —
fig.6
2900bp
Results
Direct Observation of Paired Complex Formation.
Display distinctly different DNA lengths of 1,360 bp for the 1,200bp intersignal substrate and 880 bp for the 1,800-bp intersignal substrate
结果
— 8—
fig.3
Detecting Single RAG–RSS Complexes in Real Time.
As such, we interpret these periods of reduced rms motion to be binding events that change the effective tether length by 64 ± 5 bp for a single 12RSS binding site and 57 ± 5 bp for a single 23RSS binding site
475 ± 5 bp
结果
482 ± 5 bp
— 10 —
fig.5
Results
HMGB1 Alters the Binding Properties of RAG–RSS Complexes.
结果
— 11 —
Results
We observe that HMGB1 alters the reduction in DNA tether length for RAG1/2c alone from 64 ± 5 bp and 57 ± 5 bp for a single 12RSS and 23RSS to 101 ± 8 bp and 132 ± 4 bp, respectively.
讨论
— 21 —
Discussion
最后，我们通过观察珠子释放推断发夹结构的形成作为研究RAGHMGB1-RSS介导的DNA上的切割。在这些实验中，我们证实了珠释放和RAG1 / 2c和HMGB1的浓度之间的关系。我们发现珠粒释放在含有 12RSS / 23RSS对的底物上被显着增强，需要催化活性RAG，并且当信号间距离缩短超过以前显示抑制RAG介导的切割时V（D） J重组。利用这些工具，现在可以对V（D）J反应过程进行大量的系统研究是有可能的。我们的结果已经显示，大量的RSS和它们的序列可能改变重组动力学，并且对确定在淋巴发育期间产生Ig和TCR基因的各种12/23规则调节的V，D和J反应的突触和发夹形成的定量规则是有很大意义的。
Single-molecule analysis of RAG-mediated V(D)J DNA cleavage
PNAS 2015 112 (14) 4193-4194; doi:10.1073/pnas.ss11214
• reporter：
背景知识
V(D)J Recombination
Recombination Activating Gene
— 9—
fig.4
Results