(英汉对照)分子生物学-- 实验技术

mao44

Appendix D:

Experimental Techniques               
附录D:

实 验 技 术       


Elucidating the molecular basis of life is no easy task.  Our current understanding of complex processes like replication, transcription or translation has required thousands of scientists laboring for decades.  Yet, considering the tiny scale on which molecular biologists work, the amount known is admirable.  This understanding is due to a powerful set of techniques, tools that allow our immense hands to cut and past molecules, and our blurry eyes to peer inside cells.   In this chapter we review the most important of these techniques used today.                要弄清楚生命的分子基础并不是一件容易的事。我们现在了解的复杂过程，如复制、转录或转译，是几千位科学家通过几十年的辛勤研究得来的。不过，考虑到分子生物学家工作的对象是如此微小，已获得的知识还是值得称道的。这样的理解得益于一套有效的技术，这是一些使我们的大手能够去切割并粘贴分子和使我们模糊的眼睛能够窥探细胞内部的工具。本章我们回顾现今使用的一些最重要的技术。       

D.1  DNA Manipulation
        Techniques                D.1  DNA操作技术       

D.1.1  Gel Electrophoresis and
        Southern Blotting                D.1.1  凝胶电泳与
        Southern印迹法       

Gel electrophoresis is a technique used to separate a mixture of DNA molecules according to length (Figure D.1). DNA molecules are pulled through a gel by a voltage that attracts the negative charges on phosphodiester bonds.  The gel is a tangle of polymers that is quite dense, but contains holes through which DNA molecules can pass.  Shorter molecules fit more easily through these holes than larger molecules; as a result, the speed at which a DNA molecule moves down the gel is directly related to its size.                凝胶电泳是用来将DNA分子混合物根据它们的长度分开的技术（图D.1）。DNA分子的磷酸二酯键上带有负电荷，在电场作用下会在凝胶中发生移动。凝胶是一团密度较大但有孔的聚合物，DNA分子可以从孔中穿过。较小的分子比大分子更容易进入这些孔；结果，一个DNA分子在凝胶中的移动速度直接与它的大小有关。       

After the gel has been ‘run’ for some time, DNA molecules of different lengths will each occupy different places on the gel, appearing as individual bands.  Very fine gels can be made that can allow DNA molecules to be resolved even if they differ in length by only one base pair.  Note that gel electrophoresis does not differentiate molecules according to sequence.  Two molecules with different sequences but with the same number of base pairs will appear as one band on a gel.                在凝胶被‘跑’了一段时间以后，每种不同长度的DNA分子将在凝胶上占据不同的位置，呈现出不同的条带。甚至可以制备出很精细的凝胶用于分开长度只差一个碱基对的DNA分子。注意，凝胶电泳不能根据序列区分分子。具有相同长度但序列不同的两个分子将出现在凝胶的同一个位置。       


Figure D.1  DNA gel electrophoresis. (a) DNA molecules migrate towards anode. The smaller molecules move faster than the larger ones. (b) DNA bands on the gel after electrophoresis and staining.                图D.1  DNA凝胶电泳。（a）DNA分子向正极迁移。较小的分子比大分子移动更快。（b）电泳后凝胶经过染色显示的DNA条带。       


Gel electrophoresis is often used to determine whether a specific DNA molecule is present in a mixture.  Size is often enough to identify the specific molecule on a gel.  The gel is stained with a chemical that colors DNA, so that all bands become apparent.  Meanwhile, in a parallel lane of the gel, a set of DNA molecules of known size is run.  By comparing the bands of experimental DNA to the DNA bands of known size, a good estimate of the size of the experimental DNA molecules can be assessed.                凝胶电泳也常用于确定某种特殊的DNA分子是否出现在某个混合物中。通常根据分子的大小就足以鉴定在凝胶上是否有这一特殊分子。凝胶用一种化学物质将DNA染上颜色，使所有条带都清楚易见。同时，在凝胶的平行泳道上跑一套已知大小的DNA分子。通过比较实验DNA的条带与已知大小DNA的条带，就可以很好地估计实验DNA分子的大小。       


Figure D.2  Southern blotting. The probe binds to a band of complementary sequence and makes it visible.                图D.2  Southern印迹法。探针与互补序列条带结合而让它能被看到。       


Sometimes it is necessary to check for the presence of a specific DNA molecule on a gel even though its size is not known.  In this case, a technique called Southern blotting is used (Figure D.2).  Short pieces of DNA called ‘probes’ are made that are complementary to the DNA molecule of interest, and therefore bind only to that molecule.  The probes are made to be radioactive or fluorescent.  They are then applied to the gel (or an imprint of the gel).  If they bind to a DNA molecule in the gel, the band where that DNA is present will become visible by the radioactivity or the fluorescence of the probe.  Southern blotting is also used when there are so many genes on the gel that size alone cannot be used to determine the presence of a specific gene, as other genes may have the same size.                  有时甚至在不知道实验DNA分子大小的情况下也需要检查某种特殊的DNA分子是否在凝胶上。这时，需要采用称为Southern印迹法的技术（图D.2）。先制备与感兴趣的DNA分子互补的称为‘探针’的DNA小片段，探针只能与那个分子结合。之后使探针带上放射性或荧光标记。然后将它们与凝胶（或凝胶的印迹）混合。如果它们能与凝胶上的某种DNA分子结合，那么这一DNA出现的地方会由于探针上的放射性或荧光而被看到。Southern印迹法也用在许多基因出现在凝胶中仅靠分子大小不能确定某种特殊基因是否存在的情况下，因为其它基因可能具有相同的大小。       

D.1.2  Polymerase Chain Reaction
        (PCR)                D.1.2  聚合酶链式反应
        （PCR）       


Figure D.3  The polymerase chain reaction                图D.3  聚合酶链式反应       


Polymerase chain reaction is a technique used to copy specific regions of a DNA molecule (Figure D.3).  As with normal replication in cells, PCR depends on DNA polymerase and deoxyribonucleotides in synthesis.  However, most other enzymes used in normal replication are replaced during PCR by machine functions and artificial molecules.                聚合酶链式反应是用来拷贝DNA分子特殊区域的技术（图D.3）。与细胞中的正常复制一样，PCR在合成中需要依靠DNA聚合酶和脱氧核糖核苷酸。但是，在正常复制中用到的大多数其它酶被PCR仪器的功能和其它人造分子取代。       

A mixture of components, including DNA molecules, is placed into a machine that controls temperature.  The machine initially raises the temperature of the mixture, denaturing the double-stranded DNA into single-strands.  This essentially mimics the function of helicase.  DNA polymerase can use these single strands as template for synthesis.  However, before it can do so, primers must present on the DNA.  In PCR, primers are artificially-made DNA oligomers that are added to the reaction mixture.  They are not RNA, and they are not made by primase.  The machine lowers the reaction temperature to allow the DNA primers to hydridize or ‘anneal’ to the DNA template.  Following this, DNA polymerase is able to copy each strand, beginning at the primer.                将各种成分的混合物（包括DNA分子）放进能控制温度的仪器里。PCR仪先升高混合物的温度，将双链DNA变性成为单链。这实际上模仿了解旋酶的功能。DNA聚合酶能够使用这些单链为模板进行合成。但在它开始合成前，必须有引物出现在DNA上。在PCR中，引物是人工制备的DNA寡聚物，它们也被加到了反应混合物中。它们不是RNA，也不是由引发酶产生的。PCR仪将温度降下来以便DNA引物与DNA模板杂交或‘退火’。接着，DNA聚合酶便能够从引物开始拷贝每一条链了。       

After one round of synthesis, two DNA molecules have been made for each initial template molecule present.  Now another cycle begins.  The machine heats the reaction once again, denaturing the DNA molecules – including the newly made ones – then cools to allow annealing of primers and synthesis.  From the two molecules made in the previous round of PCR, four new molecules can be made.  In the next round, these four molecules will be used to make 8 new molecules.  Thus, with every round of PCR, the number of DNA molecules increases exponentially.  After several dozen rounds, billions of copies can be made.                在完成一轮合成后，从最初的一个模板分子得到了两个DNA分子。现在另一个循环开始了。PCR仪又一次加热使DNA分子变性──包括新合成的分子──然后降温使引物退火并进行DNA合成。从前一轮PCR得到的两个分子出发可以产生四个新的分子。下一轮合成中，这四个分子将被用来产生8个新的分子。这样，随着每一轮PCR的进行，DNA分子的数量呈指数式增长。经过几十轮后，可以产生几十亿个拷贝。       

A powerful feature of PCR is that specific regions of the template molecule can be copied, rather than the whole template molecule (Figure D.4).  The region that will be replicated is determined by which sites on the template the artificial primers are designed to anneal to.  One primer determines one end of the region to be copied, and a second primer determines the other end.  Each primer always binds to one site and only binds to one strand of the DNA.   Using PCR, many copies can be made of any gene in a cell’s genome.                PCR的一个有用之处是它可以用来拷贝模板分子的特殊区域，而不是整个模板分子（图D.4）。将要被复制的区域由根据模板上的退火位点设计的人工引物决定。一个引物决定了需要拷贝区域的一个末端，另一个引物决定了另一个末端。每个引物总是与一个位点结合并且只与模板DNA的一条链结合。应用PCR可以产生细胞基因组中任何基因的许多拷贝。       

PCR reactions often produce a variety of undesirable side-products.  Gel electrophoresis is quite useful for separating the desired product from the side-products and purifying it.                PCR反应经常会产生一些不希望出现的副产物。凝胶电泳在将需要的产物从副产物中分离出来方面相当有用。       


Figure D.4  PCR is used to copy specific region of DNA.                图D.4  PCR用来拷贝DNA的特殊区域。       


D.1.3  Recombining DNA                D.1.3  重组DNA       

In addition to being copied and separated, DNA molecules can also be rearranged in the laboratory.  Whole regions of DNA can be moved around, deleted, and reattached, for example, using common techniques.                除了能被拷贝和分离外，在实验室里也能对DNA分子进行重排。例如，可以使用常规技术对DNA的整个区域进行移动、删除和重新连接。       

Recombinant DNA technology is made possible by restriction endonucleases, which are proteins that cut DNA molecules at precise sequences (Figure D.5). Usually, the target sequences are palindromes, meaning they read the same backward and forward.  The enzyme makes two single-stranded cuts several bases apart on separate strands.  This produces two ends, each with one single-strand slightly longer than the other.  There are hundreds of different restriction enzymes, each recognizing different sites.  However, every time a particular restriction enzyme cuts a DNA molecule, it produces the same exact ends.                限制性内切核酸酶使重组DNA技术成为可能，这是一些能在准确的位置切割DNA分子的蛋白质（图D.5）。通常，它们的目标序列具有回文结构，即从反向读和从正向读它们都是一样的。这种酶在两条链上相距几个碱基各产生一个单链切口。这样就得到了两个末端，每个末端各有一条单链比另一条略长。共有几百种不同的限制酶，每一种识别不同的位点。不过，每次一种特殊的限制酶切割DNA分子时，它产生的都是完全相同的末端。       


Figure D.5  EcoR I, a restriction endo- nuclease, recognizes a palindromic sequence and cleaves it to yield sticky ends.                图D.5  限制性核酸内切酶EcoR I识别回文序列并将其切割产生粘性末端。       


The staggered ends produced by restriction enzymes are often called sticky ends.  This is because one end can bind to a complementary end, sticking two molecules together.  In fact, after a DNA molecule is cut by a restriction enzyme, the two complementary ends produced often continue to stick together.  But they don’t have to.  If another DNA molecule is cut with the same enzyme, the same sticky ends will be produced.  These ends are also complementary to those in the first molecule, and can bind to them (Figure D.6).                限制酶产生的交错切口常被称为粘性末端。这是因为一个末端可以与另一个互补的末端结合，把两个分子粘合在一起。事实上，在DNA分子被限制酶切开后，两个互补的末端常常继续粘合在一起。但它们并不是非这样不可。如果另外一个DNA分子也用相同的酶切割，就会得到相同的粘性末端。这些末端与从第一个分子得到的粘性末端是一样的，也能结合在一起（图D.6）。       


Figure D.6  Recombinant DNA molecule created with restriction enzyme and ligase.                图D.6  由限制酶和连接酶产生的重组DNA分子。       


This is a very useful property, because it allows two different molecules to be glued to each other in a specific way.  If different molecules are cut with the same restriction enzymes, the ends produced at each one have the capacity to bind to each other, joining together the two different molecules.  To create covalent bonds where the sticky ends have hybridized, an enzyme called ligase is required.                这是一种很有用的性质，因为它可以让两个不同的分子以特殊的方式结合在一起。如果不同的分子用相同的限制酶切割，从每个分子产生的末端都能互相结合，从而将两种不同的分子连接在一起。要在粘性末端已经发生杂交的地方形成共价键，还需要一种称为连接酶的酶。       

D.1.4  DNA Sequencing                D.1.4  DNA序列测定       

Understanding, manipulating, and identifying a DNA molecule often requires knowing the exact sequence of bases it contains.  A base sequence would be important, for example, in making probes for Southern blotting, or in determining which restriction enzymes can cut a gene.  One of the most convenient aspects about working with DNA in the laboratory is the ease with which its sequence can be determined.  There are various ways of determining a DNA sequence, and new techniques are being rapidly developed.  The first very successful method, which is still quite popular, is called the dideoxy method.                了解、操作和鉴定DNA分子常常需要知道它含有哪些确切的碱基序列。例如，在为Southern印迹法制备探针或确定哪种限制酶能够切割某一基因的时候，清楚地知道它的碱基序列就很重要。在实验室中对DNA开展工作最方便的一个方面就是可以很容易地测得它的序列。有几种测定DNA序列的方法，新技术也在快速发展。第一个最成功的、目前仍然相当通行的方法称为双脱氧法。       

In this technique a DNA molecule is copied in a reaction similar to PCR.  However, small amounts of nucleotides called dideoxyribonucleotides are present in the reaction mixture (Figure D.7).  These special nucleotides do not contain the 3’-OH group present on normal nucleotides.  As a result, once they are incorporated into a growing chain, no further nucleotides can be attached to the DNA molecule, and synthesis is terminated.                在这一技术中，DNA分子在一个 PCR类似的反应中被拷贝。但是，反应混合物中含有少量的双脱氧核糖核苷酸（图D.7）。这些特殊的核苷酸没有正常核苷酸中所具有的3’-OH。结果，一旦它们被整合到生长链中，将不再有核苷酸能够连接到DNA分子上，合成就终止了。       



Figure D.7  Structure of a dideoxycytocine- 5’-triphosphate                图D.7  双脱氧胞嘧啶核苷-5’-三磷酸的结构       


To sequence DNA, four separate reactions are prepared (Figure D.8).  Each one receives a small amount of a different dideoxy - G, A, T, or C, as well as larger quantities of all four normal nucleotides.  Let us see what happens in the mixture that receives dideoxy-C (Figure D.8a).  Many DNA synthesis reactions are occurring in the mixture at the same time.  Each time that DNA polymerase must add a C to the growing DNA molecule, there is a chance that the dideoxy-C will be incorporated.  Sometimes this happens early, so synthesis stops early and a small DNA molecule is produced.  Sometimes it happens late, and long molecule is produced before synthesis stops.  In short, adding dideoxy-C causes a variety of DNA molecules with different lengths to be synthesized, each one ending in C.                为了DNA进行测序，需要分开准备四组反应（图D.8）。每组含有少量不同的双脱氧-G、A、T或C以及大量的所有四种正常的核苷酸。让我们来看一看含有双脱氧-C的混合物中会发生什么情况（图D.8a）。在这一混合物中同一时间发生着许多DNA合成反应。每次DNA聚合酶向生长中的DNA分子添加C的时候，双脱氧-C都会有机会被整合进去。有时这样的事发生得较早，这样合成终止得也早，得到的是一个较小的DNA分子。有时它发生得较晚，则在合成终止前得到了较长的分子。简言之，添加上双脱氧-C导致合成一系列不同长度的DNA分子，每个分子的结尾处都是C。       

Next, the length of each of these molecules is determined by gel electrophoresis.  The length of a DNA molecule is directly related to the number of bases.  We also know that all DNA molecules from the dideoxy-C reaction end in C.  By determining the lengths of these various molecules, each position of the DNA that contains C can be determined.  For example, if the molecules are 24, 27, and 32 base pairs in length, this means that C is present at positions 4, 7, and 12 in the normal molecule (after subtraction of 20, length of the primer).  The same process is repeated using the other three dideoxy nucleotides, and this way, the entire sequence of the DNA molecule can be determined (Figure D.8 b ~ d).                之后，用凝胶电泳来测定这些分子的长度。DNA分子的长度与它具有的碱基数直接相关联。我们还知道，来自于双脱氧-C反应的所有DNA分子都以C结尾。通过测定这些不同分子的长度，DNA中每个含有C的位置能够被确定下来。例如，如果得到的分子长度是24、27和32个碱基对长，这就意味着在正常的分子中，C出现在第4、第7和第12的位置（在减去引物长度20后得出）。再用其它三种双脱氧核苷酸重复同样的过程，这样，DNA分子的全部序列就能被测出（图D.8 b ~ d）。       



Figure D.8  (a)~(d): Products of sequencing reactions with ddCTP, ddTTP, ddATP and ddGTP, respectively. (e) Electrophoresis of the products.                图D.8  （a）~（d）：分别使用ddCTP、ddTTP、ddATP和ddGTP进行的测序反应产物。（e）反应产物的电泳图。       


D.1.5  Molecular Cloning                D.1.5  分子克隆       

The first step in studying a gene is to isolate it, and to have access to many identical copies of it.  This is called ‘cloning’ the gene.  The process by which a gene is cloned is called molecular cloning (Figure D.9). This technique draws on the more basic techniques discussed above.                研究基因的第一步是分离它并获得许多与它完全相同的拷贝。这称为‘克隆’基因。一个基因被克隆的过程称为分子克隆（图D.9）。这一技术牵涉到上面讨论过的更基本的技术。       

Cloning begins with a PCR reaction in which the many copies are made of the gene of interest.  It might seem as though the process of molecular cloning could end with PCR.  The gene has been isolated, and many copies are available.  However, the PCR reaction often makes mistakes, and the product contains a mixture of accurate copies and inaccurate copies.  Whether the gene will be sequenced, recombined, or expressed, such a hodgepodge is not desirable.                克隆开始于PCR反应，先从中获得许多目的基因拷贝。看起来好像分子克隆的过程可以在PCR这儿结束了，因为基因已经被分离了，也获得了很多拷贝。然而，PCR反应常常会出错，得到的产物是准确拷贝和不准确拷贝的混合物。不管是要对它进行测序、重组或表达，这样一堆乱七八糟的东西总是不行的。       


Figure D.9  A general process of molecular cloning (plasmid size not drawn to scale)                图D.9  分子克隆的一般过程（质粒大小未按比例画）       


In order to have accurate copies of the original gene, it needs to be placed into a living cell, usually E. coli, which has the ability to copy genes with very few errors.  DNA copied by PCR is linear, and will be degraded if placed in an E. coli cell.  Therefore, the PCR product must first be inserted into plasmids, circular pieces of DNA which act as artificial chromosomes.                为了获得原始基因的准确拷贝，需要将它放入活细胞（通常是大肠杆菌）中，活细胞具有准确地拷贝基因的能力（很少出错）。PCR拷贝得到的DNA是线状的，如果放入大肠杆菌细胞的话会被降解掉。因此，PCR产物必须先插入到质粒中，质粒是环状DN**段，能起到人工染色体的作用。       

Placing a linear piece of DNA into a circular plasmid requires recombinant DNA techniques discussed above.  Both the PCR product and the plasmid are cut with the same restriction enzymes, in two places.  This produces sticky ends on either side of the gene fragment, and also creates an opening in the plasmid where the fragment can enter.  Because the gene and the plasmid have complementary sticky ends after cutting, they can be joined together. Afterwards they are covalently linked by the protein ligase.                将线状DN**段放入环状质粒需要采用上面讨论过的DNA重组技术。PCR产物和质粒均用相同的限制酶在两个位置进行切割，在基因片段的两端产生粘性末端，同时在质粒上打开一个缺口以便基因片段进入。由于酶切后基因和质粒具有相同的粘性末端，所以它们能结合在一起。之后由连接酶将它们共价连接起来。       

Once the gene fragments made by PCR have been inserted in plasmids, the plasmids must be placed inside E. coli cells.  In this case, the insertion of DNA into a cell is called transformation.  Usually, the plasmid is mixed with a population of E. coli cells, and then a shock is applied, such as heat or electricity, that temporarily makes the cells permeable to large molecules like DNA.  In order to distinguish cells that have taken the plasmid from those that have not, plasmids are usually designed to contain a gene for resistance to an antibiotic.  After transformation, the cells are placed on medium containing antibiotic.  Only those cells that have taken up the plasmid are able to grow on the antibiotic medium.                一旦从PCR来的基因片段被插入到质粒上，质粒必须被送入大肠杆菌细胞中去。这种将DNA送入细胞的过程称为转化。通常，将质粒与一个大肠杆菌细胞群体混合，之后给一个强刺激（如热刺激或电刺激）使细胞产生短暂的通透性，让DNA这样的大分子能够进入。为了区别已经摄入了质粒和没有摄入质粒的细胞，通常将质粒设计成含有抗生素抗性的基因。在转化之后，将细胞涂到含有抗生素的培养基上。只有那些摄入了质粒的细胞才能在含有抗生素的培养基上生长。       

The end result is a plate containing spots of E. coli called colonies.  All of the E. coli cells in a colony are exactly the same because they are descended from one cell.  Whenever a copy of the gene is needed, these colonies can be placed in a highly nutritious environment and grown.  Copies of the plasmid with the desired gene are then easily extracted from the cells.  Before choosing a particular colony to grow and extract the gene, it is wise to verify the quality of the gene received by that colony.  Recall that PCR often makes mistakes in replication, and the copy of the gene ligated into the plasmid may have a mutation.  The integrity of the gene can be checked by extracting a small quantity and having its sequence determined.  The sequence should be identical to the known sequence of the gene.                最后得到的是一块含有大肠杆菌斑点（称为菌落）的平板。一个菌落中的所有大肠杆菌细胞是完全一样的，因为它们都来自于一个细胞。在需要这一基因的任何时候都可以将这些菌落取出放到营养丰富的环境中让它们生长。之后可以很容易地从细胞中提取含有目的基因的质粒拷贝。由于是选择一个特别的菌落去生长和提取基因，检查一下那一菌落里基因的质量是明智的。回想一下，PCR在复制时常常会出错，连接到某一质粒上的基因拷贝或许带有一个突变。基因的完整性可以通过提取少量基因并对其测序而进行鉴定。测出的序列应该与基因的已知序列相同。       

D.1.6  Manipulating RNA                D.1.6  操作RNA       

RNA is more difficult to work with in the laboratory than DNA because it is much more easily degraded.  RNA molecules can be separated by gel electrophoresis, very similar to DNA.  Individual molecules are also identified using oligonucleotide probes, except that the technique is called a Northern blotting, instead of a Southern blotting, when RNA is involved.  Many experimental procedures involving RNA depend on first converting the RNA to DNA.  This process is called reverse transcription.                在实验室中操作RNA要比操作DNA困难得多，因为它很容易被降解。RNA分子可以通过凝胶电泳分开，这一点与DNA很相似。单一RNA分子也可以使用寡核苷酸探针进行鉴定，但当研究对象是RNA时这一技术称为Northern印迹法，而不是Southern印迹法。许多涉及RNA的实验过程依赖于先将RNA转变成DNA，这一过程称为反转录。       

D.2  Manipulating Proteins                D.2  操作蛋白质       

D.2.1  Protein Purification and
        Separation                D.2.1  蛋白质纯化与分离       


In order to study an individual protein present in a mixture it is often necessary to separate and purify it.  Proteins can be separated according to properties such as size, charge, and ability to bind a specific molecule.                为了研究出现在混合物中的某种蛋白质，常常需要对它进行分离、纯化。可以根据蛋白质的大小、电荷以及与特殊分子结合的能力等性质而分离蛋白质。       

1. Column Chromatography                1. 柱层析       

Column chromatography is the most common method to purify proteins, and can be applied for all three criteria just mentioned (Figure D.10a).  In column chromatography, a mixture of proteins is passed through a column.  The speed with which a protein passes through the column, and thus the time at which it exits, depends on the unique properties of the protein and on the contents of the column.  Proteins are separated by collecting the solution that exits the column in separate tubes every couple of minutes.                柱层析是最常见的蛋白质纯化方法，能够在上面提到的所有三种情况下应用（图D.10a）。在柱层析中，让蛋白质混合物通过一根层析柱。某种蛋白质通过层析柱的速度以及它流出层析柱的时间取决于蛋白质的独特性质和层析柱的填充物。通过每隔几分钟用不同的管子收集从柱中流出的液体可以将蛋白质分离开。       

Ion-exchange chromatography is a kind of column chromatography that separates proteins by charge (Figure D.10b).  The column contains beads that are either positively or negatively charged.  As a protein mixture is forced through the column, the speed with which the protein passes through depends on its charge relative to the charge of the beads.  If the column has positively charged beads, positively charged proteins will pass most quickly and negatively charged proteins, which are attracted to the beads, will pass most slowly.                离子交换层析是通过电荷特性对蛋白质进行分离的一种柱层析（图D.10b）。层析柱中含有带正电荷或负电荷的珠子。当蛋白质混合物被迫通过层析柱的时候，某种蛋白质通过的速度取决于它所带电荷与珠子上电荷之间的关系。如果层析柱中的珠子带正电荷，那么带正电荷的蛋白质通过的速度最快，而带负电荷的蛋白质通过的速度最慢，因为它们会被带正电荷的珠子吸引。       

In gel-filtration chromatography, proteins are separated based on size (Figure D.10c).  Beads in the column contain small tunnels in which proteins of various sizes can get stuck.  Smaller proteins pass more slowly through the column because they become easily stuck, whereas larger proteins pass more quickly.  The name ‘gel-filtration’ comes from the fact that the beads are made of a kind of gel.                凝胶过滤层析根据蛋白质的大小将它们分开（图D.10c）。层析柱中的珠子含有小的通道，不同大小的蛋白质会被卡住。较小的蛋白质通过的速度很慢，因为它们很容易被卡住，而较大的蛋白质通过得很快。‘凝胶过滤’这一名称来自于这样一个事实，即层析柱中的珠子是用一种凝胶制备而来。       


Figure D.10  Column chromatography                图D.10  柱层析       


Affinity chromatography, another kind of column chromatography, can be used to separate proteins based on their ability to bind a particular molecule (Figure D.10d).  Beads in the column are coated with the molecule.  Most proteins pass quickly through the column; however proteins that have an affinity for the molecule on the bead exit late.                亲和层析是另一种柱层析方法，它根据蛋白质与某种特殊分子的结合能力来分离蛋白质（图D.10d）。层析柱中的珠子用这种特殊分子包裹。大多数蛋白质很快地通过层析柱；而与珠子上的分子有亲和性的蛋白质流出的时间更晚。       

2. Protein Gels                2. 蛋白质凝胶       

Proteins can also be separated by gel electrophoresis (Figure D.11).  The principle of a protein gel is similar to a DNA gel.  When a voltage is applied to a mixture of proteins in the gel, they are separated according to their unique characteristics.  Unlike DNA, normal protein migration depends on charge and folded shape, as well as length.  To separate proteins solely according to length, a molecule called sodium dodecyl sulfate (SDS) is added to the protein mixture.  SDS denatures proteins and coats them with a uniform charge.  The result is that the protein is stripped of most of its unique characteristics except for its length.                蛋白质也可以通过凝胶电泳的方法进行分离（图D.11）。蛋白质凝胶的原理与DNA凝胶的相似。当向凝胶中的蛋白质混合物施加电压时，能根据它们的特性而将它们分开。与DNA不同，正常蛋白质在凝胶中的移动取决于它所带的电荷、折叠形状以及它的长度。为了只根据长度来分离蛋白质，需要在蛋白质混合物中加入一种称为十二烷基硫酸钠（SDS）的分子。SDS使蛋白质发生变性并使它们带上统一的电荷。结果是，蛋白质被剥夺了除长度以外的大多数特性。       


Figure D.11  SDS denatures the protein and masks it with negative charges.                图D.11  SDS使蛋白质变性并用负电荷屏蔽蛋白质。       


3. Antibodies and Western Blotting                3. 抗体与Western印迹法       

When working with DNA, oligomer probes are used to bind to specific DNA molecules and identify them.  The equivalent for proteins is called an antibody.  Antibodies are proteins made by the immune system, where they are used to bind invasive molecules.  Because the immune system must be prepared for a huge variety of invaders, a huge variety of antibodies exist to bind to essentially any molecule imaginable.  This is very valuable in the laboratory, as antibodies can be made to specifically bind to any protein desired.  Usually, this involves injecting the protein in question into a live animal, and purifying the antibodies that the animal subsequently makes against the protein (Figure D.12).                当研究对象是DNA时，使用寡聚物探针与特异DNA分子结合并鉴定它们。对蛋白质而言，其对等物为抗体。抗体是由免疫系统产生的蛋白质，它们用来结合入侵的分子。由于免疫系统必须准备好对付数量众多的入侵者，因此存在数量众多的抗体，它们能够对付几乎任何可能出现的分子。在实验室里这是非常有价值的，因为可以利用这一特性制备出能特异性地与任何所希望的蛋白质发生结合的抗体。通常，这需要将所研究的蛋白质注射到动物活体内，之后将该动物随后产生的针对此蛋白质的抗体纯化出来（图D.12）。       


Figure D.12  Preparation of specific antibodies (a) and their specific binding to different proteins (b).                图D.12  特异抗体的制备（a）及其它们与不同蛋白质的特异性结合（b）。       


Antibodies can be used to detect the presence of a specific protein on a gel.  A radioactive or fluorescent antibody against the experimental protein is added to the filter with protein blots.  If the protein in question is present, the antibody will bind to it and make its location visible.  This is called a Western blotting (Figure D.13).                抗体可以用来探测在一块凝胶上是否存在某种特殊的蛋白质。向带有蛋白质印迹的滤膜加入一种针对实验蛋白的具有放射性或能发出荧光的抗体。如果有那种蛋白存在的话，抗体将与其结合，使它所在的位置能够被看见。这叫做Western印迹法（图D.13）。       


Figure D.13  Western blotting. The labeled antibody binds to a specific band of protein and makes it visible.                图D.13  Western印迹法。标记的抗体与蛋白质特异条带结合而让它显示出来。       


Antibodies have many other uses as well.  They can be used in purification of a protein by affinity chromatography, for example, or for visualization of a protein within a living cell.                抗体也具有其它多种用途。例如，它们可以用在亲和层析中纯化某种蛋白，或者用来观察活细胞中的某种蛋白。       

D.2.2  Protein Identification                D.2.2  蛋白质鉴定       

Antibodies are usually used to search for known proteins that are expected, or suspected, to be present.  To find the identity of a completely unknown protein, say a random band on a gel, a technique called mass spectrometry is used.  The sample of unknown protein is placed in a machine which breaks it into smaller pieces. The pieces are then placed in an electric field, which accelerates them until they collide with a detector.  The time for the pieces to reach the detector depends on their mass.  By analyzing the masses of various small pieces of a larger protein, short sequences of the protein can be determined.  These sequences are then compared to a collection of DNA sequences to find which gene the protein corresponds to.  Protein identification by this technique usually requires that the protein’s gene has been already sequenced.                抗体通常用来搜寻认为存在的或怀疑有可能存在的已知蛋白。为了鉴定一种完全未知的蛋白质，如凝胶上的一条带，需要采用一种称为质谱的技术。未知蛋白样品被放进一仪器内，由仪器将它打断成小片段。之后这些片段被放到电场中，电场将它们加速直到它们撞上探测器。这些片段到达探测器的时间取决于它们的质量。通过分析较大蛋白的不同小片段的质量，能够确定下来该蛋白的短序列。之后将这些序列与某个DNA序列的集合体进行比较，就有可能找出与该蛋白相应的基因。用此技术鉴定蛋白质一般要求该蛋白的基因已经被测序。       

One of the great conveniences of mass spectrometry is that it only requires tiny samples of proteins.  Before mass spectrometry, protein sequences were determined by a chemical reaction that removes amino acids from the end of the protein one by one.  This is called the Edman degradation reaction.  It requires much larger quantities of the protein, and is much more laborious.                质谱的最大方便之一是它只需要很少一点蛋白质。在质谱技术发展出来以前，蛋白质序列是用化学反应方法进行测定的，化学反应从蛋白质的末端一次移去一个氨基酸。这一方法称为爱德曼降解反应，它需要用很多蛋白质，并且也很费时费力。       

D.2.3  Other Protein Manipulations                D.2.3  其它蛋白质操作       

1. Copying a Protein                1. 复制蛋白质       

There is no method for directly copying a protein, as is possible for DNA with PCR.  However, it is possible to obtain many copies indirectly, by first isolating the gene for the protein and then transcribing/translating the gene.  The gene is placed into an expression vector, which contains promoter sequences that strongly drive transcription.  Proteins can be experimentally expressed in vitro or in vivo.  (These are commonly used terms, derived from Latin, which mean ‘in an artificial setting’ and ‘in a live organism’, respectively.)  In vivo protein expression usually involves inserting the expression vector into E. coli, yeast, or cultured insect cells.  After some incubation time, cells are lysed and the protein is purified from the extract.                 还没有方法可以用来直接拷贝一种蛋白质，DNA则可以用PCR来直接拷贝。然而，可以采用间接的方法获得蛋白质的许多拷贝，就是先分离出该蛋白的基因然后转录/转译这一基因。把该基因放入表达载体，表达载体含有强启动子序列来启动转录。蛋白质可以在离体（in vitro）或活体（in vivo）中进行试验性的表达。（in vitro和in vivo是两个被普遍使用的术语，来源于拉丁语，意思分别是‘在人工背景中’和‘在活生物体内’。）活体中表达蛋白质通常需要将表达载体送入大肠杆菌、酵母或昆虫细胞中。在经过一段时间的培养后，将细胞裂解，蛋白质可从裂解液中纯化出来。       

2. Recombining Proteins                2. 重组蛋白质       

Proteins can be cut by enzymes called proteases, but these are not as numerous or as useful as restriction enzymes are when working with DNA.  Furthermore, proteins that have been cleaved cannot be easily rejoined to other proteins to create the rearrangements possible with recombinant DNA technology.  Recombinant proteins are created indirectly, by first recombining their DNA, and then expressing the protein from the recombinant DNA.                蛋白质可以被称为蛋白酶的酶切割，但它们并不像作用于DNA的限制酶那样数量很多也很有用。再者，被切断的蛋白质不容易重新连接在一起，不像重组DNA技术那样可以容易地得到重排的序列。重组蛋白是用间接的方法得到的，首先将它们的DNA重组，然后从重组DNA表达出蛋白质。       

D.3  The Function of Genes
        and their Proteins                D.3  基因及其蛋白质
        的功能       

We have now introduced basic methods for dealing with proteins, DNA, and RNA.  These techniques form the basis of more complex methods that help us to answer a range of questions about gene and protein function.  We list below the most important of these questions, and the experimental techniques commonly used to answer them.                到现在为止，我们已经介绍了研究蛋白质、DNA和RNA的基本方法。这些技术形成了一些更复杂方法的基础，要回答关于基因和蛋白质功能方面的一系列问题需要这些更复杂方法的帮助。下面我们列出这些问题中最重要的部分，以及回答这些问题需要的常用实验技术。       

D.3.1  What is the protein’s
        structure?                D.3.1  蛋白质具有什
        么样的结构？       

There is a strong relationship between the structure of a protein and its function.  Knowing the structure of a protein often allows significant understanding about what it does, as well as how it works.  As we know, the DNA sequence of a gene fully determines the amino acid sequence, and the amino acid sequence is generally sufficient to determine the three-dimensional structure of a functional protein.  Thus, it is theoretically possible to look at the sequence of a gene and know the shape of the protein it codes for.  Actually, not enough is known yet about the physics of protein folding for scientists to make good structural predictions from sequence.  Still, DNA sequences can be used to some extent to predict structures within the protein.                蛋白质的结构与它的功能之间有着非常密切的联系。知道了一种蛋白质的结构常常能让我们获得有关它的作用以及如何发挥作用等方面的重要知识。如我们所知，基因的DNA序列完全决定了氨基酸的序列，而氨基酸的序列一般也足以确定功能蛋白的三维结构。这样，理论上就有可能从基因的序列上看出它编码的蛋白质的形状。实际上，对于蛋白质折叠的物理学还知道得不够多，科学家还不能从序列出发来准确地预测结构。尽管如此，在一定程度上DNA序列还是可以用来预测蛋白质结构中的某些部分。       

Some protein domains have characteristic amino acid sequences that can be identified in the protein’s gene sequence.  For example, proteins that pass through the lipid plasma membrane have regions dominated by hydrophobic amino acids.  In such cases, patterns of codons for hydrophobic amino acid in a DNA sequence are usually enough to reveal whether a protein passes through a lipid membrane or not, even if the full structure of the protein cannot be determined.                有些蛋白质功能域具有特征性的氨基酸序列，这些序列可以在该蛋白的基因序列中被鉴别出来。例如，穿过脂质膜的蛋白质具有以疏水氨基酸为主的区域。在这样的情形中， DNA序列中疏水氨基酸密码子出现的样式时常足以揭示该蛋白是否穿过脂膜，即使在整个蛋白质的结构没能被测定的情况下也能做出这样的判断。       

Obtaining the complete three dimensional structure of a protein is currently a very difficult task.  The best method is X-ray crystallography (Figure D.14).  Crystallogra- phy requires very pure and highly concentrated solutions of the protein of interest.  Previously, these were obtained by grinding up large numbers of cells and organs, and purifying proteins from them.  Now, a cloned version of the gene is used to express the protein purely and in large concentrations.                获得蛋白质完整的三维结构目前还是一件很难完成的任务。最好的方法是X射线晶体衍射（图D.14）。晶体衍射需要很纯、浓度很高的目的蛋白溶液。以前，它们是从研磨很大数量的细胞和器官纯化而来。现在，可以用克隆到的基因表达出纯的高浓度蛋白。       


Figure D.14  X-ray crystallography                图D.14  X射线晶体衍射       


The composition of the pure protein solution is then manipulated such that the protein molecules are induced to bind to each other and arrange themselves in a regular pattern, called a crystal.  The protein crystal is then bombarded with X-rays.  Because the wavelength of X-rays is comparable to the size of atoms, a phenomenon called diffraction occurs, in which the X-ray waves are split.  The pattern of diffraction can be analyzed by computer programs to give a very clear image of the 3-D arrangement of proteins at the atomic level.  Crystal structures are quite difficult to obtain, mainly because the conditions for crystallization differ for each protein and are difficult to determine.  Crystallization is especially difficult for membrane proteins and proteins with floppy, unstructured domains.                然后对纯的蛋白质溶液成分进行操作诱导蛋白质分子互相结合并以规则的方式排列，即形成晶体。之后，用X射线轰击得到的蛋白质晶体。由于X射线的波长与原子的大小差不多，因而会发生一种称为衍射的现象，衍射现象使X射线波分开。衍射的样式可以用计算机程序进行分析，从而在原子水平上给出非常清晰的蛋白质3-D排列图象。晶体结构是很难获得的，主要原因是每种蛋白质的结晶条件不一样并且很难确定。对那些膜蛋白和具有松弛、非结构化区域的蛋白质进行结晶尤其困难。       

D.3.2  What other proteins is it
        related to?                D.3.2  它与其它哪些
        蛋白质有关？       

Functional insight about a gene often comes from comparing it to other genes.  Two genes, or regions of a gene, with very similar sequence often have related functions.  Computers can be used to make alignments between thousands of sequences to determine which ones are similar to each other.  Programs that perform these alignments can be quite sophisticated.  Instead of simply determining how many bases gene A shares with gene B, they can take into consideration more subtle factors – such as the fact that some difference in bases would not cause as serious differences in structure as others.                对一个基因功能的深入了解常常来自于将它与其它基因进行比较。具有非常相似的序列的两个基因或基因区域常常具有相关的功能。可以用计算机来对几千条序列进行比对，从而确定哪些序列互相之间是相似的。能执行这种比对功能的一般都是一些比较高级的程序。它们并不是简单地确定基因A中有多少碱基与基因B中相同，它们能够考虑到更多细微的因素──比如有些碱基的不同不会像其它碱基的不同那样会导致结构上的严重差异。        

Structures of proteins can also be used to make guesses about functional similarities between genes.  As with DNA sequences, protein domain structures can be compared by computer programs.  This is increasingly common as more and more protein structures become available.                蛋白质结构也能用来猜测基因之间功能的相似性。与DNA序列一样，蛋白质结构域可以用计算机程序来进行比较。在越来越多的蛋白质结构已经弄清楚的情况下，这样的比较正在变得越来越普遍。       

D.3.3  When is the protein
        expressed?                D.3.3  这种蛋白质在
        什么时候表达？       

We have already seen that mixtures of proteins can be separated, by columns or gels, and then individual proteins can be identified using antibodies or mass spectrometry.  These same methods are used to detect the presence of proteins in the cell as a whole.  A mixture of all of the cell’s proteins, called a cell extract, is made.  The set of techniques chosen to detect an individual protein within this extract will depend on the properties of the protein and how much is known about. The most efficient method to detect an individual protein is usually to use affinity chromatography with a molecule, possibly an antibody that binds to the protein in question.                我们已经看到，蛋白质混合物可以用层析柱或凝胶分离，然后单一的蛋白质可以用抗体或质谱进行鉴定。同样是这些方法也被用来探测细胞作为一个整体含有哪些蛋白质。先制备一种含有细胞所有蛋白质的混合物（叫做细胞提取物）。被选择用来从这一提取物中检测某种蛋白的整套技术取决于该蛋白的性质以及对它了解的程度。检测某种蛋白最有效的方法通常是采用一种能与该蛋白结合的分子（比如抗体）进行亲和层析。       

A somewhat similar technique for detecting a protein is called immunoprecipitation (Figure D.15).  A cell extract is mixed directly with an antibody against the protein in question.  An antibody-binding protein is also present.  It causes the antibodies to clump together, as well as the cell extract proteins to which the antibody binds.  This aggregation of antibodies and target proteins is called a precipitate.  This precipitate can be run on a gel to identify the target protein.                一种有些相似的蛋白质检测技术称为免疫沉淀法（图D.15）。细胞提取物直接与针对该蛋白的抗体混合。同时加入另一种与抗体结合的蛋白，它能使抗体发生聚集，那些与抗体结合的细胞提取物中的蛋白也会因此而聚集在一起。这种抗体与目标蛋白之间的聚集会产生沉淀。该沉淀可以用凝胶对目标蛋白进行鉴定。       


Figure D.15  An immunoprecipitation
        procedure                图D.15  免疫沉淀过程       


The expression of proteins can also be tested in a less direct way by examining the mRNAs produced by a cell.  It is reasonable to assume that expressed proteins will have mRNA present in the cell, whereas the mRNA of proteins that are not expressed will generally be absent.  The presence of mRNA can be tested by a Northern blot.  The RNA in a cell is extracted and run on a gel.  The presence or absence of specific RNAs in the cell is then detected using radioactive or fluorescent oligonucleotide probes complementary to RNA in question.  Although Northern blots give reasonable evidence as to whether a protein is expressed or not, they are not 100% reliable.  mRNAs in a eukaryotic cell have different life spans and different rates of translation, so the quantity of mRNA in a cell does not always reflect the quantity of corresponding proteins.                蛋白质的表达还可以通过检查细胞产生的mRNA这种较为间接的方式进行试验。有理由假设，得到表达的蛋白质在细胞中应该有相应的mRNA，而没有表达的蛋白质一般来说也不会有mRNA。是否存在相应的mRNA可以用Northern印迹进行试验。先将细胞中的RNA提取出来并在凝胶上跑电泳。之后用含放射性标记或荧光标记的寡核苷酸探针（与要研究的RNA互补）检测特异RNA是否存在。虽然Northern印迹给出了符合常理的证据来说明某种蛋白表达与否，但它们不是100%可靠的。真核细胞中的mRNA具有不同的存活期和不同的转译速率，因此细胞中mRNA的量并不总是能反映出相应蛋白质的量。       


Figure D.16  Principle of DNA microarray technology                图D.16  DNA芯片技术的原理       


A tool called a DNA microarray is used to assess how the concentration of thousands of mRNAs changes between various conditions.  This gives important clues about which proteins are expressed under different conditions (Figure D.16).  Thousands of genes from an organism are uniquely represented on a small chip, each gene receiving one spot.  Each spot contains a high concentration of an oligomer that is complementary only to its gene.  mRNA is then extracted from a cell under one condition.  The mRNA is turned into DNA by reverse transcription, and dyed red.  Then, mRNA is extracted from a cell under a different condition.  The mRNA is turned into DNA and dyed green.                一种称为DNA芯片的工具被用来估计几千种mRNA的浓度是如何随不同条件而变化的。这给出了关于在不同条件下有哪些蛋白质得到表达的重要线索（图D.16）。一种生物的几千个基因以独特的方式被体现在一块小的芯片上，每个基因占据一个点。每个点含有一种高浓度的寡聚物，它只与对应的基因互补。之后从处于一种条件下的细胞中提取mRNA。通过反转录将mRNA转变成DNA并带上红色标记。然后，从处于另一条件下的细胞中提取mRNA。将此mRNA转变成DNA并带上绿色标记。       

The DNA from both conditions is then applied to the chip.  DNA from a particular gene will hybridize to that gene’s spot on the chip.  If a gene is expressed more strongly under the ‘red’ condition, then mostly red DNA will bind to the gene’s spot, and the spot will appear red.  If a gene is expressed more strongly under the ‘green’ condition, then mostly green DNA will bind to the gene’s spot, and the spot will appear green.  If expression is equal between the two conditions, the spot will have an intermediate color like yellow or orange.  There are thousands of spots on a chip, and therefore thousands of genes can be analyzed simultaneously to determine under which condition each one is more highly expressed.  By better understanding when a gene is expressed, we can sometimes derive clues about its function.  For example, a gene that is only expressed in the presence of a certain nutrient may be responsible for metabolism of that nutrient.                然后将从两种条件下得到的DNA加到芯片上。来自于一个特殊基因的DNA将与芯片上代表那一基因的点发生杂交。如果一个基因在‘红色’条件下表达更强烈，那么大多数红色DNA将结合到该基因的点上，这一点显示红色。如果一个基因在‘绿色’条件下表达更强烈，那么大多数绿色DNA将结合到该基因的点上，这一点显示绿色。如果在两种条件下一个基因的表达情况相同，则该点具有一种中间色如黄色或橙色。在一块芯片上有几千个点，因此可以同时测定几千个基因中的每个基因在哪种条件下表达量更高。通过更好地理解基因在什么时候表达，我们有时能够获得关于它的功能的线索。例如，某个基因只在存在某种营养成分的时候表达，说明该基因可能负责这种营养成分的代谢。       

D.3.4  When is the protein active?                D.3.4  这种蛋白质什么
        时候有活性？       


Figure D.17  In vitro assay of protein activity                图D.17  蛋白质活性的离体分析       


In many cases the expression of a protein does not reflect its activity.  A protein may be always present, but only active under certain conditions.  One way to determine whether or not a protein is active at a given time is to remove it from a cell under that condition and test its activity in vitro (Figure D.17).  The outline of this test will vary widely depending on the normal activity of the protein.  Usually, the activity of a protein with respect to a target protein or molecule is examined.                在很多时候，一种蛋白质的表达并不反映出它的活性。一种蛋白质可能一直出现，但只在一定的条件下才有活性。检测某种蛋白质在某一时间点有无活性的一种方法是在那一条件下将蛋白质取出并在离体条件下检测它的活性（图D.17）。这种检测的总体框架将随该蛋白质正常活性的不同而有很大差异。通常，需要检查该蛋白相对于一种目标蛋白或目标分子的活性。       

Take the example of protein A that can degrade protein B.  We want to know if protein A degrades protein B during mitosis.  A cell extract can be made by grinding up cells that are going through mitosis.  Then this extract can be mixed with protein B, and the presence of protein B detected with time.  If protein B is degraded, then protein A is probably active during mitosis; if not, then it may well be inactive.  In vitro tests are always imperfect because the artificial conditions can cause unusual protein behaviors.                举例说，蛋白A能够降解蛋白B。我们想要知道在有丝分裂过程中蛋白A是否降解了B。可以将正在进行有丝分裂的细胞磨碎并制备出细胞提取物。之后将此提取物与蛋白B混合并随时间的推移检测B的量。如果蛋白B被降解，那么蛋白A在有丝分裂过程中或许是有活性的；如果蛋白B没有被降解，则说明蛋白A是无活性的。离体试验总是有一定缺陷的，因为人工的条件会导致蛋白质产生不寻常的行为。       


Figure D.18  Detection of protein modifications. (a) Ubiquitination. (b) Phosphorylation.                图D.18  蛋白质修饰的检测。（a）泛素化。（b）磷酸化。       


Another way to understand a protein’s activity is to examine its state of modification, as the function of many proteins is determined by their state of modification.  Different tests exist for different modifications.  Proteins that have been ubiquitinated (attachment of a small protein tag), for example, are usually destined for destruction by the cell.  Ubiquintinated proteins often appear as several close but distinct bands on the gel (Figure D.18a).  Proteins that have been phosphorylated can also be detected.  In order to do so, cells can be grown on radioactive phosphorous.  If a phosphate group is attached to a protein, the protein will be detectable by radiation when it is run on a gel (Figure D.18b).  Mass spectrometry can also be a useful technique for observing covalent modifications to proteins.  Mass measurements can be sensitive enough to detect the increase in mass caused by attachment of even the smallest chemical groups.                  另一种了解蛋白质活性的方法是检查它们的修饰状态，因为许多蛋白质的功能都由它们的修饰状态决定。针对不同的修饰作用有不同的试验方法。例如，已经被泛素化（加上一个小的蛋白质标签）了的蛋白质通常注定要被细胞摧毁。泛素化蛋白在凝胶上常常以几条靠近但可以分清的条带出现（图D.18a）。被磷酸化的蛋白质也能被检测出来。为了达成此目的，可以将细胞培养在含有放射性磷的培养基中。如果磷酸基团被加到了某种蛋白质上，则在凝胶上可以检测到该蛋白的放射性（图D.18b）。质谱也是对蛋白质共价修饰进行观察的有效技术。对质量的检测非常灵敏，即使只有极小的化学基团被加到了蛋白质上也能被探测到。       

D.3.5  Where is it expressed?                D.3.5  它在哪里表达？       

The function of a protein is often tied to its location in the cell.  Therefore, knowing the location of a protein can give you clues about its function.  Proteins localized to the inside of the lysosome, for example, often have roles in protein degradation.  Knowing where a protein is not present can also rule out possible functions.  Fore example, a protein that is never found in the nucleus is unlikely to be directly involved in splicing.                蛋白质的功能常常与它在细胞中的位置相关联。因此，知道了某种蛋白质的位置能够给你关于它的功能的线索。例如，位于溶酶体中的蛋白质通常具有降解蛋白质的功能。知道某种蛋白质不出现在哪里同样也能排除它具有某种功能的可能性。例如，一种从来没有在细胞核中发现过的蛋白质不太可能直接与剪接有关。       

The most powerful way to determine the localization of a protein within the cell is t**in fluorescent or radioactive.  Fluorescent proteins emit characteristic wavelengths of light when exposed to a certain wavelength of light, and can be detected at high magnifications by special microscopes.  The most popular way to make a protein fluorescent is to fuse it to a small fluorescent protein (Figure D.19).  Green fluorescent protein (GFP) is commonly used.  Genetic engineering can be used to combine any gene with the gene for GFP.  When this recombinant gene is expressed in the cell, the gene’s protein will be fused to GFP.  Generally, GFP is attached at one end of the protein and does not significantly alter the attached protein’s function.                确定某种蛋白在细胞中位置的最有效方法是让该蛋白带上荧光标记或放射性标记。荧光蛋白暴露在一定波长的光下时能发射出不同波长的光，这种光能用特殊的显微镜在很高的放大倍数下进行检测。让蛋白质带上荧光标记最通行的方法是将它与一个小的荧光蛋白融合（图D.19）。常用的是绿色荧光蛋白（GFP）。可以应用基因工程的方法将任何基因与GFP基因进行组合。当这样的重组基因在细胞中表达的时候，该基因的蛋白质将与GFP融合在一起。一般情况下，GFP是挂在该蛋白的一个末尾上，不会明显地改变该蛋白的功能。       


Figure D.19  Determination of protein localization.  (a) Preparation of fusion protein. (b) Subcelullar localization of proteins made visible by tagging with GFP.
(Source: www.wikipedia.org)                图D.19  蛋白质位置的测定。（a）制备融合蛋白。（b）用GFP做标签使蛋白质的亚细胞位置可见。
        （来源：www.wikipedia.org）       

D.3.6  What other proteins does
        it bind to?                D.3.6  它与其它哪些
        蛋白质结合？       

Most proteins function in conjunction with other proteins.  Knowing how a protein associates with others can reveal a lot about its function.  There are several techniques to search for binding.                许多蛋白质与其它蛋白质一起发挥作用。知道了某种蛋白质怎样与其它蛋白质联合能够揭示许多关于其功能方面的信息。有几种技术可以用来研究蛋白质结合方面的信息。       



Figure D.20  Co-immunoprecipitation                图D.20  免疫共沉淀法       


Recall that in immunoprecipitation an antibody is used to isolate a target protein from a mixture, usually an extract from a cell.  If the target protein binds to other proteins, these other proteins might be indirectly precipitated by the antibody.  Thus an immunoprecipi- tation can actually be used to identify groups of proteins that bind to each other; in this case, the technique is called a co-immunoprecipitation (CoIP) (Figure D.20).  To identify the proteins present after a CoIP, the precipitate is run by gel electrophoresis.  The protein that was attached to the antibody, as well as all the proteins attached to that protein, should produce separate bands.  The identity of proteins in each band can then be easily assessed by mass spectrometry.                回忆一下，在免疫沉淀法中抗体被用来从混合物（通常是细胞提取物）中分离目标蛋白。如果目标蛋白与其它蛋白结合，这些其它蛋白可能也会被抗体间接地沉淀出来。因此，免疫沉淀法实际上可以用来鉴定互相结合在一起的成组蛋白；在这种情况下，这一技术称为免疫共沉淀法（CoIP）（图D.20）。为了用CoIP鉴定这些蛋白质，对该沉淀进行凝胶电泳。与抗体结合的蛋白将与所有其它结合蛋白一起产生多条分开的带。之后，每条带中的蛋白质可以用质谱仪容易地进行估计。
       

A method called a pull-down assay detects the binding of cellular proteins to a protein in a column (Figure D.21).  This is essentially the same as affinity chromatography.  In a usual pull-down, a test protein is engineered to be fused to a special domain called GST (glutathione S-transferase), which binds very tightly to a molecule called glutathione.  A column is prepared with an inner surface that is coated with glutathione. The column allows cell extracts to enter at the top, and exit at the bottom.  First, the test protein, which has been fused to GST, is passed through the column.  Because of the GST domain, it becomes stuck to the glutathione coated surface.                一种称为下拉分析的方法可以用来检测细胞蛋白与层析柱中某种蛋白的结合（图D.21）。这在本质上与亲和层析是一样的。在普通的下拉分析中，一种供试蛋白用基因工程的方法融合到称为GST（谷胱甘肽S-转移酶）的特殊结构域上，GST能与谷胱甘肽分子非常紧密地结合在一起。制备一根内表面用谷胱甘肽包裹的层析柱。让细胞提取物从顶端进入层析柱并从底端流出。首先，让已经与GST融合的供试蛋白通过层析柱。由于带有GST结构域，它会被吸附到谷胱甘肽包裹的表面上。       


Figure D.21  A GST-pull down assay                图D.21  GST下拉分析       


Next, a cell extract is passed through the column.  Proteins that do not bind to the test protein will simply pass through the column.  However, proteins that do bind will become stuck to the surface of the column.  Finally, the GST domain is released from glutathione using a special solution.  The complex of bound proteins that then exits the column can be recovered, and the identity of the proteins identified by mass spectrometry.                其次，让细胞提取物通过层析柱。不能与供试蛋白结合的蛋白将直接通过层析柱。然而，那些确实能结合的蛋白将被吸附到柱的表面。最后，用一种特殊的溶液将GST结构域从谷胱甘肽上释放。收集此时才流出柱子的结合蛋白复合体并用质谱仪对它们进行鉴定。       


Figure D.22  The yeast two-hybrid                图D.22  酵母双杂交       


Fusion proteins are also the basis for a completely different protein-binding assay, called yeast two-hybrid (Figure D.22).  This technique is often used to check for binding between two proteins.  Recall that transcriptional activators are modular proteins, meaning they have one domain that binds to DNA, and a distinct domain that activates transcription.  Both domains are required for activating transcription of a specific gene.  In a two-hybrid assay, one test protein is fused to the DNA binding domain of an activator.  Another test protein is fused to the activation domain of the activator.  Both proteins are then expressed in yeast cells that have a reporter gene.  The reporter gene produces a signal, usually a color, when stimulated by the activator.  Neither of the fusion test proteins alone is able to activate transcription.  However, if the two test proteins bind to each other, then the DNA binding domain will become attached to the activation domain.  This reconstituted activator can then fuel transcription of the reporter gene.                融合蛋白也是另一种完全不同的蛋白质结合分析技术的基础，它就是酵母双杂交（图D.22）。这一技术常常被用来检查两种蛋白之间的结合。回忆一下，转录激活蛋白是模块化的蛋白，就是说它们具有一个与DNA结合的结构域，又有一个分开的激活转录的功能域。在激活一个基因的转录时，这两个域都是需要的。在双杂交分析中，将一种供试蛋白与某种激活蛋白的DNA结合域融合。而将另一种供试蛋白与该激活蛋白的激活域融合。之后让两种蛋白都在具有一种报告基因的酵母细胞中表达。如果该报告基因受到激活蛋白的刺激，它能产生一种信号（一般是某种颜色）。两种融合蛋白中的任何一种都不能够激活转录。但是，如果这两种供试蛋白能够互相结合在一起，那么DNA结合域就能与转录激活域连接在一起。这样，这种重新组织起来的激活蛋白就能够启动报告基因的转录。       

D.3.7  What DNA sequences does
        it bind to?                D.3.7  它与什么样的
        DNA序列结合？       

Assays also exist to test for binding of proteins to other molecules, such as DNA.  A technique called chromatin immunoprecipi- tation (ChIP) can be used to identify numerous sites on DNA to which a test protein binds (Figure D.23).  First, cells are exposed to a chemical that creates covalent bonds between DNA and proteins bound to DNA.  This essentially freezes DNA-binding proteins in position.  Next, the DNA is extracted and chopped into smaller pieces, several hundred nucleotides each.  Although the DNA is no longer fully intact, the proteins remain attached to their original sites.  Then an antibody against the test protein is added to the extract, causing the test protein to precipitate along with the pieces of DNA to which it is still attached.  The idea is very similar to a co-immunopreci- pitation.  Finally, the precipitate is isolated and the links between the test protein and DNA are reversed.  The various sequences of DNA that were attached to the protein can then be detected.                也存在用来分析蛋白质与其它分子（如DNA）结合的方法。一种称为染色质免疫沉淀法（ChIP）的技术能被用来鉴定DNA上能与供试蛋白结合的无数个位点（图D.23）。首先，细胞暴露在一种能在DNA与其结合蛋白质之间形成共价键的化学物质中。这基本上将DNA结合蛋白固定在了它们的位置上。其次，将DNA提取出来并切成小片段，每个片段几百个核苷酸。虽然DNA不再完整，但是蛋白质还保留在它们原来的位置上。之后，将一种针对供试蛋白的抗体加入提取物中，使供试蛋白与它结合的DN**段一起发生沉淀。这种构思与免疫共沉淀法很相似。最后，分离出此沉淀并将供试蛋白与DNA之间的连接逆转。之后就能对与该蛋白结合的不同DNA序列进行检测了。       


Figure D.23  Chromatin immunoprecipi- tation (ChIP)                图D.23  染色质免疫沉淀法（ChIP）       


Instead of finding all of the DNA sequences to which a protein binds, other techniques can find all of the proteins to which a DNA sequence binds.  One method is a gel-shift assay (Figure D.24).  Protein extract from a cell is mixed with a sequence of DNA, and the solution is run on a gel by electrophoresis.  If a protein binds to the DNA sequence, its migration down the gel will be slowed, because the DNA will pass with more difficulty through the gaps in the gel.  Every different protein that binds to the DNA sequence slows the migration by a different amount, depending on its size and charge.  Thus, a series of bands will appear on the gel, each one corresponding to the DNA sequence bound to a different protein.  The proteins in each band can then be identified.                除了发现能与一种蛋白结合的所有DNA序列外，还有技术可以用来发现能与一种DNA序列结合的所有蛋白质。其中之一就是凝胶移位分析（图D.24）。一个细胞的蛋白质提取物用来与一条DNA序列混合，得到的溶液在凝胶上跑电泳。如果有某种蛋白结合在DNA序列上，那么它在凝胶上的迁移速率会变慢，因为它要通过凝胶内的空隙更困难。每种与该DNA序列结合的蛋白质都会不同程度地降低它的迁移速率，这取决于蛋白质的大小和它们所带的电荷。这样，在凝胶上可以得到一系列条带，每条对应着与该DNA结合的一种不同的蛋白质。之后便可以对每条带上的蛋白质进行鉴定。       


Figure D.24  The gel-shift assay                图D.24  凝胶移位分析       


Affinity chromatography can also be performed using DNA.  The inside of a column in coated with a specific DNA sequence.  Proteins extracted from a cell are then passed through the column.  Those that do not bind to the sequence pass quickly through the column.  Those with strong binding to the sequence are identified because they exit last and only under solution conditions that disrupt the binding.                亲和层析也可应用在DNA上。层析柱的内部用一种特殊的DNA序列包裹。然后使从细胞中提取的蛋白质通过该柱。那些不能与DNA序列结合的蛋白质很快地通过层析柱。而那些与DNA序列紧密结合的蛋白质能被鉴定出来，因为它们只有在能打断这种结合的溶液条件下才最后流出层析柱。       

D.3.8  What happens if it is
        removed or altered?                D.3.8  如果它被移去或
        改变会怎样？       

The methods described so far aim to understand proteins in their natural condition.  What do they normally bind to?  Where are they normally located?  Sometimes, however, a lot can be learned about the function of a protein by disrupting its normal state.  For example, by mutating a protein and analyzing the effect of this mutation in the cell, we can learn a lot about the normal function of the protein.  The reasoning holds for domains of proteins as well as whole proteins.                到现在为止所介绍的方法目的都是要弄清自然条件下蛋白质的性质。比如，正常情况下它们与什么分子结合？正常情况下它们的位置在哪儿？然而，有时候通过扰乱蛋白质的正常状态可以让我们对蛋白质的功能有更多的了解。例如，通过使一种蛋白质发生突变并分析该突变在细胞中造成的影响，我们可以获得许多关于该蛋白正常功能的知识。这一推理对蛋白质的功能域以及整个蛋白质来说都是成立的。       

1.  Eliminating Proteins: Knock-
        downs, Knockouts, and More                1.  排除蛋白质：
        基因敲低、基因敲除
        及更多方法       


Figure D.25  Gene knockdown                图D.25  基因敲低       


Various methods exist to completely eliminate a protein’s expression.  The most common method used for eukaryotic cells is RNA interference (RNAi), introduced in chapter 7.  The technique is also called gene knockdown (FigureD.25).  RNAi is a natural pathway used by the cell to destroy specific mRNAs.  Introduction of a short double-stranded RNA (called siRNA) into the cell will stimulate destruction of any mRNAs with regions complementary to one of the siRNA strands.  Thus, any mRNA can be specifically targeted for destruction by injecting cells with siRNAs designed to be complementary to that mRNA.                存在着多种彻底排除蛋白质表达的方法。应用于真核生物的最常见方法是第7章中介绍过的RNA干涉（RNAi）。这一技术也被称为基因敲低（图D.25）。RNAi是细胞用来摧毁特殊mRNA分子的自然途径。将一小段双链RNA（叫做siRNA）引入细胞将会刺激对那些能与siRNA中的一条链互补的mRNA的降解作用。因此，通过向细胞中注射siRNA（设计成与目标mRNA互补），任何mRNA都能被特异性地锁定为摧毁的目标。       

For small organisms, such as C. elegans, siRNAs can be used to prevent expression of a protein in all cells.  However, for larger model organisms like mice, it is so far impossible to introduce the siRNAs into all of the body’s cells.  Instead, a technique called gene knockout is used.  Knockout causes the removal of a gene from the genome of an organism.                对小的生物如线虫而言，siRNA可以用来防止某种蛋白在所有细胞中的表达。然而，对更大的模式动物如小鼠而言，目前还不可能通过将siRNA引入所有的体细胞。此时可以采用另一种技术，即基因敲除。敲除使某个基因从一种生物的基因组中去除。       

A fragment of DNA is made that has sequences homologous to both sides of the gene to be removed.  However, the fragment does not contain a functional copy of the gene.  The fragment is introduced into a plate containing millions of embryonic cells (Figure D.26).  Some cells take up the fragment.  In a very small percentage of the cells, recombination occurs between the gene in the chromosome and the artificial fragment.  This replaces the normal gene with the nonfunctional version present in the fragment.  The rare cells in which this occurs are identified by various selective properties present on the artificial fragment, such as resistance to drugs that normally kill cells.  These recombinant cells are now heterozygous for the targeted gene.  The copy on one chromosome is normal, but the other copy on the other chromosome is disrupted.                制备一个DN**段，它具有与需要去除的基因的两边同源的序列。然而，该片段并不含有目标基因的功能性拷贝。将此片段引入到含有几百万个胚细胞的平板中（图D.26）。有些细胞能吸收这一片段。在很小比例的细胞中染色体和该人工构建的片段之间会发生重组。这样的重组会导致正常的基因被片段中的无功能版本取代。发生了这种情况的少数细胞可以通过人工构建片段上的不同选择特性而进行鉴定，比如对某些药剂的抗性，这些药剂一般来说会杀死细胞。对目标基因来说这些重组细胞现在是杂合的。该基因在一条染色体上的拷贝是正常的，但在另一条染色体上的拷贝是被破坏了的。       



Figure D.26  Preparation of embryonic stem cells with an interrupted gene                图D.26  制备某一基因被打断了的胚胎干细胞       


The cells are multiplied, and some are introduced into the early embryo of an animal, usually a mouse (Figure D.27).  The recombinant cells form normal body tissues like the other cells in the organism.  This animal is called a chimera.  Some of its tissues descended from the recombinant cells and are heterozygous for the targeted gene, other tissues are normal.                将这些细胞增殖，并将其中一些引入到一种动物（通常是小鼠）的早期胚胎中（图D.27）。这些重组细胞将与其它细胞一样在该动物中形成正常的体细胞组织。这种动物叫做嵌合动物。它的一些组织来自于重组细胞，对目标基因来说是杂合的，而其他组织则是正常的。       


Figure D.27  Creating a knockout mouse                图D.27  创造基因敲除小鼠       


Ideally an animal can be produced that is homozygous for the disrupted gene in all tissues.  This can be accomplished when an animal is produced in which the germ cells, which produce sperm or egg, are derived from the heterozygous recombinant cells.  In this case, half of the sperm or eggs produced will carry the disrupted gene.  If two animals of this kind are bred with each other, one fourth of the offspring will have no functional copies of the gene.  This produces a knockout animal.                理想情况下可以产生一只所有组织中对于目标基因来说是纯合的动物。当产生这只动物的性细胞（产生精子或卵子的细胞）是来自于杂合的重组细胞时，就有可能得到这样的动物。在这种情形下，产生的一半精子或卵子将携带被破坏了的基因。如果两只这样的动物互相交配，它们的子代个体中就会有四分之一具有该基因的无功能拷贝。这就产生了一只基因敲除动物。       

Knockout and knockdown animals and/or cells can be closely examined for changes in phenotype.  These changes give clues about the function of the eliminated protein.  For example, if knockout of a gene in mice causes them to turn white, then this gene may normally be involved in producing hair color.                之后可以对基因敲除和基因敲低动物和/或细胞的表型变化进行仔细检查。这些变化给出了被排除掉的蛋白质功能的线索。例如，某个基因被敲除的小鼠变成了白色，那就说明该基因正常情况下可能在产生毛发的颜色方面起作用。       

2.  Altering a Protein                2.  改变蛋白质       

Methods also exist to modify proteins within the cell without actually eliminating them.  This may be useful, for example, when studying the importance of domains or amino acids to the function of the greater protein.  It may also be used to investigate one function of a protein with several functions.  Mutations to proteins can be made in various ways.  Large insertion, deletions, and fusions to other proteins can be made by the recombinant DNA technology described above.  Mutations to individual bases, called site-specific mutagenesis, can be made using a variation of PCR.                也存在可以对细胞中的蛋白质进行改变而不必将它们去除的方法。在研究某些结构域或氨基酸对更大的蛋白质的重要性时，这些方法很有用。它也可以用来研究多功能蛋白质功能中的一种。可以用不同的方法让蛋白质产生突变。可以用上述重组DNA技术产生大的插入、缺失以及与其它蛋白融合。还可以用PCR的一种改进方法来产生单个碱基的突变，称为位点定向诱变。       

Mutated proteins can be tested in vitro or in vivo.  To introduce the mutated protein into an entire animal, a very similar procedure as that which creates knockout mice is used.  To introduce the mutated protein into cells, the recombinant gene is inserted into the cell, often using viruses.  The normal gene’s expression is then repressed, often by RNAi.  This method can also be used to express unusually large quantities of a normal gene.  The phenotype from such overexpression can sometimes give clues as to the gene’s function.                突变了的蛋白可以在离体或活体条件下进行检验。将突变蛋白引入整个动物体的方法非常类似于产生基因敲除小鼠的过程。将突变蛋白引入细胞，通常使用病毒将重组基因带进细胞里。这样，正常基因的表达被阻遏（一般通过RNAi）。这一方法也能被用来大量地表达一个正常基因。这种过量表达表型有时能够提供该基因功能的线索。       

Most of the approaches described above are designed to elucidate the functions of a specific gene or protein.  In many cases, however, researchers investigating the cause of a phenotype do not yet know which gene they should study!  Instead, they start with a phenotype and work backwards to find out which gene or genes are responsible.  Because this was the approach commonly used before gene cloning methods were developed, it is called ‘classical genetics’ or forward genetics.  Beginning with a gene and investigating its function is called reverse genetics.  We leave discussion of classical genetics for other books.                上面描述的大多数方法都是被设计成去弄清一个特殊的基因或蛋白质的功能的。然而，在很多时候，调查一种表型起因的研究人员并不知道他们应该研究哪个基因！相反，他们从表型开始，回溯其起因，找出哪个基因或哪些基因是造成这一表型的原因。由于这是一种在基因克隆方法诞生以前的常用方法，所以被称为‘经典遗传学’或正向遗传学。从基因开始研究它的功能称为反向遗传学。我们就把对经典遗传学的讨论留给其它书本吧。
       

Vocabulary                词  汇       

DNA manipulation                DNA操作       
gel electrophoresis                凝胶电泳       
Southern blotting                Southern印迹法       
Polymerase Chain Reaction (PCR)                聚合酶链式反应       
restriction endonuclease                限制性内切核酸酶       
sticky end                粘性末端       
ligase                连接酶       
DNA Sequencing                DNA序列测定       
dideoxy method                双脱氧法       
dideoxyribonucleotide                双脱氧核糖核苷酸       
molecular cloning                分子克隆       
plasmid                质粒       
transformation                转化       
colony                菌落       
Northern blotting                Northern印迹法       
reverse transcription                反转录       
column chromatography                柱层析       
ion-exchange chromatography                离子交换层析       
gel-filtration chromatography                凝胶过滤层析       
affinity chromatography                亲和层析       
sodium dodecyl sulfate (SDS)                十二烷基硫酸钠       
Western blot                Western印迹       
mass spectrometry                质谱       
in vitro                离体       
in vivo                活体       
protease                蛋白酶       
X-ray crystallography                X射线晶体衍射       
immunoprecipitation                免疫沉淀法       
DNA microarray                DNA芯片       
co-immunoprecipitation (CoIP)                免疫共沉淀法       
pull-down assay                下拉分析       
glutathione                谷胱甘肽       
yeast two-hybrid                酵母双杂交       
chromatin immunoprecipitation (ChIP)                染色质免疫沉淀法       
gel-shift assay                凝胶移位分析       
gene knockdown                基因敲低       
gene knockout                基因敲除       
overexpression                过量表达       
forward genetics                正向遗传学       
reverse genetics                反向遗传学

[ 本帖最后由 mao44 于 2009-6-6 13:21 编辑 ]

mao44

实验图片传不上去,sorry...>_<

anjingdeqingtia

谢了