Thursday, November 8, 2012

质粒载体 病毒 dna rna 病毒是一种不能进行独立代谢只能在活的细菌或细胞内生长和增殖的微生物。 ... 肿瘤病毒按照其所含的核酸不同分为两大类,即DNA肿瘤病毒和RNA


质粒载体

求助编辑百科名片

质粒载体 即,在由限制性核酸内切酶修饰过的质粒DNA序列中插入外源的目的基因,以质粒为载体,将目的基因通过转化或转导的方法导进宿主细胞,进行重组、筛选、扩增的过程。

编辑本段质粒载体简介

质粒是小型环状DNA分子,在基因工程中作为最常用,最简单的载体,必须包括三部分:遗传标记基因,复制区,目的基因。 质粒在所有的细菌类群中都可发现,它们是独立于细菌染色体外自我复制的DNA分子。自然界中,质粒是在营养充足时出现的,它在结构大小、复制方式,每个细菌的拷贝数,在不同的细菌体内的繁殖力,在菌种之间的转移力等方面都会变化,可能最重要的是质粒所携带的特征的改变。大多数原核生物的质粒是双链环状的DNA分子;但是无论是在革兰式阳性还是阴性菌体内都可以发现线状质粒。质粒大小变化很大,可从几个到数百个kb。质粒依靠宿主细胞提供的蛋白质进行复制,但也可以使宿主细胞获得质粒编码的功能。质粒复制可以与细菌的细胞周期同步,导致菌体内质粒的拷贝数较低,质粒复制也可独立于细胞周期,使每个菌体内扩增了成百上千个质粒拷贝。一些质粒在菌种间可自由地转移它们的DNA分子,另一些只转移质粒给同种细菌,而有些却根本不转移它们的DNA。质粒带有具有许多功能的基因,这些功能包括对抗生素和重金属道德抗性、对诱变原的敏感性、对噬菌体的易感或抗性、产生限制酶、产生稀有的氨基酸和毒素、决定毒力、降解复杂有机分子,以及形成共生关系的能力和在生物界内转移DNA的能力。

编辑本段质粒的基本特征

质粒(plasmid)是细菌或细胞染色质以外的,能自主复制的,与细菌细胞共生的遗传成分。其特点如下:

编辑本段是染色质外的双链共价闭合环形DNA

covalently closed circuar DNA,cccDNA),可自然形成超螺旋结构,不同质粒大小在2-300kb之间,<15kb的小质粒比较容易分离纯化,>15kb的大质粒则不易提取。

能自主复制,是能独立复制的复制子

autonomous replicon)。一般质粒DNA复制的质粒可随宿主细胞分裂而传给后代。按质粒复制的调控及其拷贝数可分两类:严紧控制(stringent control)型质粒的复制常与宿主的繁殖偶联,拷贝数较少,每个细胞中只有1个到十几个拷贝;另一类是松弛控制(relaxed control)型质粒,其复制宿主不偶联,每个细胞中有几十到几百个拷贝。每个质粒DNA上都有复制的起点,只有ori能被宿主细胞复制蛋白质识别的质粒才能在该种细胞中复制,不同质粒复制控制状况主要与复制起点的序列结构相关。有的质粒的可以整合到宿主细胞染色质DNA中,随宿主DNA复制,称为附加体,例如细菌的性质粒就是一种附加体,它可以质粒形式存在,也能整合入细菌的DNA,又能从细菌染色质DNA上切下来。F因子携带基因编码的蛋白质能使两个细菌间形成纤毛状细管连接的接合(conjugation),通过这细管遗传物质可在两个细菌间传递。

质粒对宿主生存并不是必需的

这点不同于线粒体,线粒体DNA也是环状双链分子,也有独立复制的调控,但线粒体的功能是细胞生存所必需的。线粒体是细胞的一部分,质粒也往往有其表型,其表现不是宿主生存所必需的,但也不妨碍宿主的生存。某些质粒携带的基因功能有利于宿主细胞的特定条件下生存,例如,细菌中许多天然的质粒带有抗药性基因,如编码合成能分解破坏四环素、氯霉素、氨芐表霉素等的酶基因,这种质粒称为抗药性质粒,又称R质粒,带有R质粒的细菌就能在相应的抗生素存在生存繁殖。所以质粒对宿主不是寄生的,而是共生的。医学上遇到许多细菌的抗药性,常与R质粒在细菌间的传播有关,F质粒就能促使这种传递。
现在分子生物学使用的质粒载体都已不是原来细菌或细胞中天然存在的质粒,而是经过了许多的人工的改造。从不同的实验目的出发,人们设计了各种不同的类型的质粒载体,近年来发展很快,新的有特定用途的质粒不断被创建。图20-2给出最常用的大肠杆菌克隆用质粒pUC19的图谱,此质粒的复制起点处序列经过改造,能高频率起动质粒复制,使一个细菌pUC19的拷贝数可达500-700个;质粒携带一个抗氨芐青霉素基因,编码能水解β-内酰胺环,从而破坏氨芐青霉素的酶,当用pUC19转化细菌后放入含氨芐青霉素的培养基中,凡不含pUC19者都不能生长,结果长出的细菌就是都含有pUC19的;pUC19还携带细菌lac操纵元中的lacI和lacZ基因编码,β-半乳糖苷酶N端状146个氨基酸的段落,当培养基中含有诱导物IPTG和Xgal时,lacz ' 基因被诱导表达产生的β-半乳糖苷酶N端肽与宿主菌表达的C端肽互补而具有β-半乳糖苷酶活性(质粒和宿主编码的肽段各自都没有酶活性,两都融为一体而具酶活性,称为α-互补,α-complementation),半乳糖苷酶水解Xgal而使菌落呈现蓝色;在lacz '中间又插入了一段人工设计合成的DNA序列,其中密集多个常用的限制性核酸内切酶的位点,使外来的基因和序列能很方便地被插入此位置,当外来序列插入后则破坏了lacz '编码的半乳糖苷酶活性,生长的菌落就呈白色,这种颜色标志的变化就很容易区分和挑选含有和不含有插入序列或基因的转化菌落,称为蓝白筛选法。
除常用的大肠杆菌质粒载体外,近年来发展了许多人工构建的其它能用于微生物、酵母、植物等的质粒载体。含有不止一个ori、能携带插入序列在不同种类宿主细胞中繁殖的载体称为穿梭载体(shuttle vectors)。

编辑本段质粒的分类

按复制形式

分为严紧型和松弛型复制。

按基因转移性

分两类:(1)传递性质粒:常为大型、严紧型质粒;
(2)非传递性质粒:多为小型、松弛型质粒。

按遗传性状、产物分类

(1)抗生素抗性:如氨苄西林、氯霉素抗性;
(2)限制酶-修饰酶(R-M)系统:如hsdR-菌株可使DNA甲基化,但不限制外源DNA;hsd-菌株为限制和甲基化双缺陷型。

编辑本段人工构建的质粒载体分类

高拷贝数的质粒载体

ColE1、pMB1派生质粒具有高拷贝数的特点。适合大量增殖克隆基因,或需要大量表达的基因产物。

低拷贝数的质粒载体

由 pSC101派生来的载体特点是分子量小的拷贝数。它有特殊的用途:当有些被克隆的基因的表达产物过多时会严重影响寄主菌的正常代谢活动,导致寄主菌的死亡时,就需要低拷贝的载体。

失控的质粒载体

这是一类温度敏感型复制控制质粒。如pBEU1、 pBEU2。
插入失活型克隆载体。载体的克隆位点位于其某一个选择性标记基因内部。如pDF41、pDF42。

正选择的质粒载体

直接选择转化后的细胞。只有带有选择标记基因的转化菌细胞才能在选择培养基上生长。

编辑本段质粒载体的筛选特征

选择质粒载体的要素是要了解可用到的载体的特征和预测重组克隆所用于的实验。
所有的质粒载体都有三个共同的特征:一个复制子、一个选择性标志和一个克隆位点。复制子是含有DNA复制起始位点的一段DNA(ori),也包括表达由质粒编码的复制必需的RNA和蛋白质的基因。选择性标志对于质粒在细胞内持续存在时必不可少的。克隆位点是限制性内切酶切割位点,外源性DNA可由此插入质粒内,而且并不影响质粒的复制能力,或为宿主提供选择性表型。
选择性标志:编码抗生素抗性的基因对质粒载体来说是最普遍的细菌选择性标志(如pBR322);主要的抗生素选择基因有:氨苄青霉素,氯霉素,四环素和卡那霉素四种。转化的宿主菌一般都是抗生素敏感型的,获得质粒后,它能在有抗生素的平板上生长,从而达到筛选的要求。另一个显性的选择标志就是对λ噬菌体感染的免疫(也就是λ阻抑物)。有时也会用的一些隐性标志,如leuB-(亮氨酸存在时就不能生长)。

编辑本段多克隆位点

如今的载体都含有一多克隆位点(Multiple cloning site, MCS)或多位点接头[包含约20个串联排列的限制性内切核酸酶位点(如pUC19)]这些位点在载体内通常是独一无二的,这样就可以防止插入片段插入不恰当的位置,如载体其他的特征导致破裂的位点。多克隆位点的存在可以确保载体合适大部分的DNA片段,可以针对插入片段提供特定的酶切位点图谱,在质粒重组操作方面具有更大的灵活性。在选择质粒载体时既要考虑在多接头处出现的是何位点,又要考虑到这个位点的顺序,因为必须确保特定的位点存在于多接头里。

编辑本段质粒载体大小

大的质粒(大于15kb)不会很好转化而且DNA产量通常很低。在设计实验时要考虑到加入插入片段的最终载体大小,尽量用更小的载体。
兼容性
当多于一个质粒载体必须同时存在于同一个细菌细胞中,这两个质粒的复制子必须是兼容的。当他们不能稳定地共存时,则认为这两个质粒是不兼容的。
选择/检测插入片段:
为了鉴别质粒中是否含有插入片段,对一些载体进行了工程改造,这样产生的多位点接头的分离会引起可见的表型。例如:lacZ基因的应用(多位点接头的分离会引用白细胞的生长而抑制蓝细胞的生长,如pU19)和cccdB基因(调控细胞死亡)的应用(分离会促进细胞存活)。
参考资料:
质粒载体的定向克隆
质粒载体的定向克隆
[1]
质粒裁体
质粒裁体(1张)
词条图册更多图册
参考资料
  • 1. 质粒载体
扩展阅读:
  • 1
    精编分子生物学实验指南,分子克隆指南,细胞实验指南
开放分类:
分子生物学技术 基因工程 DNA克隆
百度百科中的词条正文与判断内容均由用户提供,不代表百度百科立场。如果您需要解决具体问题(如法律、医学等领域),建议您咨询相关领域专业人士。
本词条对我有帮助
合作编辑者
如果您认为本词条还需进一步完善,百科欢迎您也来参与 编辑词条 在开始编辑前,您还可以先学习如何编辑词条
如想投诉,请到百度百科投诉中心;如想提出意见、建议,请到百度百科吧



病毒- 维基百科,自由的百科全书

zh.wikipedia.org/zh-hk/病毒頁庫存檔 - 類似內容 - 轉為繁體網頁
这些简单的生物体可以利用宿主的细胞系统进行自我复制, 但无法独立生长和复制。 ... 病毒由两到三个成份组成:病毒都含有遺傳物質(RNA或DNA,只由蛋白质组成 ...

粒子物理与原子核物理专业优秀论文)反馈控制作用双头布朗马达 .生物大分子在力作用下的随机行走、分子马达..

粒子物理与原子核物理专业优秀论文)反馈控制作用双头布朗马达 ...
www.docin.com/p-300576733.html頁庫存檔 - 轉為繁體網頁
2011年12月4日 – 人们对于爱因斯坦论布朗运动的意义、一维随机行走与布朗运动、生物大分子在力作用下的随机行走、分子马达(蛋白质高分子在相7 郑州人学硕I+ ...

Trading With VWAP And MVWAP

http://www.investopedia.com/articles/trading/11/trading-with-vwap-mvwap.asp#axzz2Bg0Tcygh

VWAP is often used in algorithmic trading.

VWAP is often used in algorithmic trading.

In finance, volume-weighted average price (VWAP) is the ratio of the value traded to total volume traded over a particular time horizon (usually one day). It is a measure of the average price a stock traded at over the trading horizon.[1]
VWAP is often used as a trading benchmark by investors who aim to be as passive as possible in their execution. Many pension funds, and some mutual funds, fall into this category. The aim of using a VWAP trading target is to ensure that the trader executing the order does so in-line with volume on the market. It is sometimes argued[by whom?] that such execution reduces transaction costs by minimizing market impact costs (the additional cost due to the market impact, i.e. the adverse effect of a trader's activities on the price of a security).
VWAP can be measured between any two points in time but is displayed as the one corresponding to elapsed time during the trading day by information provider.
VWAP is often used in algorithmic trading. Indeed, a broker may guarantee execution of an order at the VWAP and have a computer program enter the orders into the market in order to earn the trader's commission and create P&L. This is called a guaranteed VWAP execution. The broker can also trade in a best effort way and answer to the client the realized price. This is called a VWAP target execution; it incurs more dispersion in the answered price compared to the VWAP price for the client but a lower received/paid commission. Trading algorithms that use VWAP as a target belong to a class of algorithms known as volume participation algorithms.

Why The Market Ignored GOOG's Plunge (If Only Briefly)



Tyler Durden's picture




Via Michael Faso of FBN Securities,
GOOG’s ill timed oops in the early afternoon dumped the S&P 500 approximately 12 handles from what been shaping up previously as a fourth straight “checkmark” session. The technology behemoth provided another example of a non-financial firm’s missing earnings expectations by a country mile such that companies in aggregate have fallen short of bottom line and revenue estimates for the quarter when factoring out the figures from big banks. With large broker-dealer announcements mostly in the rearview mirror, the prospects for the rest of the reporting season appear dim.
Despite the shocking nature of the disappointment, the TICK never registered a print worse than –925 in the immediate wake of the surprise headline, a highly unusual phenomenon given the aggressiveness of the downward move.
Here is a chart showing an adjusted TRIN vs the S&P futures - no broad-based selling pressure as the index fell and recovered...


This suggests large institutions stayed with their VWAP buy programs (as in the chart below) out of confusion or necessity.

I can envision only two scenarios for such adherence to purchasing in the face of clear extremely negative news on, what was at the time, the third biggest stock in America.
First, in anticipation of this morning’s expiry, the baskets could have reflected a desire to add to share positions to the balance sheet and/or to protect the very important 1450 strike price for the S&P 500. Furthermore, the buying could have arisen simply from the need to put money to work especially in light of the standard October fiscal year end window dressing for many mutual funds. Regardless of the reason, both of these impetuses are transient as the momentum may tail off as early as today with the arrival of the “double witch.” This puts equities in a very tenuous state especially considering the huge increase in open interest for the futures after Wednesday’s third consecutive session of solid gains hinting that managers are still trying to expand their portfolio beta.
The economic data continues to improve aggressively with the Philly Fed easily outdistancing consensus despite its two most important components, Employment and New Orders, ticking down sequentially. Some bulls were encouraged by the 1% increase in Leading Indicators versus the August numbers; however, the data series historically has offered nothing of predictive value, save that of a contrarian indicator. For example, the previous instance of such a leap occurred arrived three days ahead of the April, 2010 top that spawned a 17% selloff for the blue chip index. Although the survey did increase with similar magnitude in April, 2009, it produced several encouraging readings near the apex of both the Dot Com bubble and financial crisis.
Regardless of how I spin these numbers, I am forced to concede that the recovery has appeared to accelerate in September. This optimism conflicts with what corporations are saying via their earnings releases, preannouncements, and conference calls. I had argued through the fall of 2011 that in the case of such a divergence, I always will defer to what companies have projected as opposed to the typically stale figures the government has released. Thus, I subsequently remained steadfastly bullish at the time. This dynamic currently has flipped such that I do not expect a trough in earnings growth, but rather a peak in economic strength. Overseas weakness, fiscal uncertainty, and a central bank that has fired its last bullet by signaling the launch of the QE3 only gives me comfort for this thesis.
Other warning signs remain intact as the small caps continue to lag as the Russell 2000 underperformed the S&P 500 again yesterday despite the heavy weighting of GOOG and AAPL for the latter. The average intraday range for the blue chip index continues to calculate to approximately 12 handles, a dangerously skittish level that consistently has led to significant pullbacks*** as many managers step aside during selloffs for fear of taking heavy losses soon after entering or adding to a position. Finally, as we inch closer to the Election in the face of a tight race, the probability of an exogenous negative shock on November 6 and in the intermediate aftermath rises daily. Moreover, with Congress scheduled to go on vacation on December 14, the window available to solve the fiscal cliff will shut quickly in the wake of a bitter campaign season.

http://www.forexabode.com/technical-analysis/moving-averages

http://www.forexabode.com/technical-analysis/moving-averages

tradermust Some Traders Caught Flat-Footed

LAURICELLA And JONATHAN CHENG
Wednesday turned out to be a bad day for investors betting on a Mitt Romney win.

With Mr. Romney and President Barack Obama virtually neck and neck going into Tuesday's election, traders said that many investors had bets aimed at capturing any rise in stocks should Mr. Romney pull out a win. Their wager: Many largely expected Mr. Obama to be victorious, therefore any selloff would be minimal, with the reward seen as bigger than the risk.

[image]

But things didn't go as planned. Even before U.S. trading started, unexpected bad economic news out of Europe sent financial markets into "risk-off" mode. The euro, and subsequently prices of stocks and other investments such as commodities perceived as riskier, fell following the announcement that the European Union had cut euro-zone growth forecasts.

When U.S. trading got under way, traders found themselves in a mad dash to sell at the opening bell. The result was a postelection drop in U.S. stock prices. And those who had bet on gains were caught flat-footed.

The Dow Jones Industrial Average dropped 312.95 points, or 2.4%, to 12932.73. That handed the blue-chip average its biggest one-day fall since Nov. 9, 2011, and sent the Dow to its lowest level since August.


Wednesday's decline marked the fifth-largest selloff after Election Day. The largest was four years ago, when, in the midst of the financial crisis, the Dow fell 5.1% following President Obama's election to a first term.

Jerry Harris, chief investment officer at Sterne Agee in Birmingham, Ala., which manages $17 billion in assets, was among those who had placed bets on Mr. Romney winning the election. Mr. Harris had put money into some health-care stocks on Monday and Tuesday, predicting they would get a bump. While some health-care stocks rose, others sold off.

Stocks skidded to their biggest one-day loss in a year amid post-election concerns and worries about the coming fiscal cliff battle in Congress. Paul Vigna has details on The News Hub. (Photo: AP)

Last August, President Obama and Congress put the U.S. economy on course to go over a "fiscal cliff." With the 2012 presidential election decided, WSJ's David Wessel tells you everything you need to know about the "cliff" but were afraid to ask.

Uncertainty over the fiscal cliff added to trading volatility following election day. Tom Lauricella reports on Markets Hub. Photo: Reuters.

President Obama will face a divided Congress, making the threat of that the U.S. will go over the fiscal cliff likely. University of Chicago economics professor Randall Kroszner discusses on Markets Hub. Plus, the re-election of Obama means he gets to nominate the next Federal Reserve Chief. Who might it be? Photo: AFP/GettyImages.

Mr. Harris now regrets his move, though he said the damage to his portfolio was minimal. "It wasn't a big risk, but I did the two [trades] with the deliberate intention of thinking we'd get a boost out of stocks if Romney won," he said.

Lawrence McDonald, former Lehman trader and author of "A Colossal Failure of Common Sense," joins Markets Hub to discuss how Wall Street is likely to view a second Obama term. Photo: AFP/GettyImages.

On Wednesday, Mr. Harris said he told himself: "You've been caught leaning. You know better than to have strong opinions before an event like that."

The selloff surprised Thomas Lee, stock-market strategist at J.P. Morgan, who before Tuesday had called for a postelection rally through year-end no matter which candidate won. "There were probably more people than we realized hoping for a Romney win," he said.

Among the more vocal predictions on Wall Street that Mr. Romney would win came from veteran investment newsletter writer Dennis Gartman. On Monday, Mr. Gartman wrote that Mr. Romney would win, "perhaps quite handily." On Wednesday, the first words of his newsletter were, "We were wrong."

"I've been very good at this for 30 years and I missed this one," said Mr. Gartman, who said he didn't have any investments explicitly tied to the election outcome. "It's a shame that I missed this one, but it's probably not going to be my last mistake."

Associated Press
A trader on the floor of the New York Stock Exchange looks at the front page of a newspaper on Wednesday morning, the day after President Barack Obama was re-elected.

Especially hard hit Wednesday were financial and energy stocks, which experienced a double whammy. A win by Mr. Romney likely would have provided an easier regulatory climate for both. In addition, both are sectors that often see aggressive selling on "risk-off" days.

Exxon Mobil XOM -1.26%fell 3.1% and Chevron CVX -1.53%lost 2.6%. A number of coal stocks, seen as big winners under a Romney presidency, saw declines of 10% or more. Among financial stocks, Bank of America BAC +1.73%tumbled 7.1% and J.P. Morgan Chase JPM -0.20%gave up 5.6%.


Kent Engelke, chief economic strategist at brokerage firm Capitol Securities in Richmond, Va., on Monday reiterated what even he described as a "brazen" call that Mr. Romney would win by seven percentage points. That followed a late summer call that no matter who won the presidency, the "fiscal cliff"—tax increases and spending cuts that take effect on Jan. 1—would send stocks lower.


On Tuesday, before the polls closed, Mr. Engelke, wrote a short client update anticipating a triumph by Mr. Romney. Instead, he got up Wednesday morning and penned an acknowledgment that he was wrong. "I've never been this wrong before," he said Wednesday afternoon.The response from clients was supportive, he said. Then, on his way back from a meeting, he stopped to help some folks whose car had run out of gas. It turned out all three had worked for the Obama campaign. "All three shook my hand," he said. "Maybe this is an example of bipartisanship we need."

LPL Financial Chief Market Strategist Jeff Kleintop on what the election results mean for the economy and the markets, especially with the impending fiscal cliff on the horizon. Photo: Reuters.

Write to Jonathan Cheng at jonathan.cheng@wsj.com

"Volume Weighted Moving Average software "

Volume Weighted Moving Average software (without quotes):