10 Генетическая инженерия (1160079)
Текст из файла
Лекция 10.Генетическая инженерияC H A P T E RRecombinant DNA TechnologyPaul BergStanley N. CohenHerbert Boyer984In our final chapter we describe a technology that in less than twodecades has become fundamental to the advance of biochemistry. Ithelps to define present and future biochemical frontiers and illustratesmany important principles of biochemistry. As the laws governing enzymatic catalysis, macromolecular structure, cellular metabolism, andinformation pathways continue to be elucidated, new research is directed at ever more complex biochemical processes.
Cell division, theimmune response, developmental processes in eukaryotes, vision,taste, oncogenesis, the cognitive processes in your brain as you readthese words—all are orchestrated in an elaborate symphony of molecular and macromolecular interactions. As increasingly greater effortsare focused on understanding the biochemistry that underlies theseprocesses, the real promise and implications of the biochemical journeybegun in the nineteenth century become clear. Human beings not onlycan understand life, they can alter it.The biochemical approach to understanding a complex biologicalprocess is to isolate and study the individual components in vitro withthe goal of understanding the overall process in the whole organism.Perhaps the most fertile source for molecular insights into these processes lies in the cell's own information storehouse, its DNA. The sheersize of cellular chromosomes, however, presents us with an enormousbarrier.
How does one find and study a particular gene encoding aprotein or RNA molecule with a molecular function we can only guessat, when that gene is only one of perhaps 100,000 genes scatteredamong the billions of base pairs that make up a mammalian genome?The answers began to appear in the mid-1970s.Decades of advances in genetics, biochemistry, cell biology, andphysical chemistry came together in the laboratories of Paul Berg,Herbert Boyer, and Stanley Cohen to yield techniques for locating,isolating, preparing, and studying small segments of DNA derivedfrom much larger chromosomes.
Taken together, these techniques areknown as DNA cloning. DNA cloning has opened opportunities unimaginable just a few decades ago, including the identification and studyof genes involved in almost every known biological process. These newmethods are transforming basic research, agriculture, forensics, medicine, ecology, and many otherfields,while at the same time presentingsociety with bewildering choices and serious ethical dilemmas.Revolutionary as it is, this technology is grounded in the most fundamental biological and biochemical principles. The first two parts ofthis chapter outline these fundamentals, drawing on our understand-Chapter 28 Recombinant DNA Technologying of the chemistry and enzymes of nucleic acid metabolism describedin the previous five chapters.
We then turn to topics that help illustratethe range of applications and the potential of this technology.DNA Cloning: The BasicsTo clone means to make identical copies; it is a term that was oncerestricted to the procedure of isolating one cell from a larger populationof cells, then allowing it to reproduce itself to generate many identicalcells.
In such a way, sufficient quantities of a single cell type weremade available for study. By analogy, DNA cloning involves separatinga specific gene or segment of DNA from its larger chromosome andattaching it to a small molecule of carrier DNA, then replicating thismodified DNA thousands or even millions of times. The result is aselective amplification of that particular gene or DNA segment. Cloning a segment of DNA, either prokaryotic or eukaryotic, entails fivegeneral procedures:1. A method for cutting DNA at precise locations. The discovery ofsequence-specific endonucleases (restriction endonucleases) provided the necessary molecular scissors.2.
A method for joining two DNA fragments covalently. DNA ligasecan do this.3. Selection of a small molecule of DNA capable of self-replication.Segments of DNA to be cloned can be joined to plasmids or viralDNAs (cloning vectors). These composite DNA molecules containing covalently linked segments derived from two or more sourcesare called recombinant DNAs.4. A method for moving recombinant DNA from the test tube into ahost cell that can provide the enzymatic machinery for DNA replication.5.
Methods to select or identify those host cells that contain recombinant DNA.The methods used to accomplish these and related tasks are collectively referred to as recombinant DNA technology, or more informally as genetic engineering. We now turn to these methods, withemphasis on their biochemical origins.In this initial discussion we will focus on DNA cloning in the bacterium E. coli, which was the first organism used for recombinant DNAwork and is still the most common host cell. E. coli has many advantages: its DNA metabolism (and many other biochemical processes) arewell understood; many naturally occurring cloning vectors such as bacteriophages and plasmids associated with E.
coli are well characterized; and effective techniques are available for moving DNA from onebacterial cell to another. DNA cloning in other organisms will be addressed later in the chapter.Restriction Endonucleases and DNA LigaseYield Recombinant DNAParticularly important to recombinant DNA technology is a set of enzymes made available by decades of research on nucleic acid metabo-985Part IV Information Pathways986EukaryoticchromosomeCloningvector(plasmid)cleavage with restrictionendonuclease, separationof fragmentscleavage withrestrictionendonucleaselism (Table 28-1). Two enzymes in particular lie at the heart of thegeneral approach to generating and propagating a recombinant DNAmolecule as outlined in Figure 28-1. First, restriction endonucleases cleave DNA at specific sequences to generate a set of smallerfragments.
Second, the DNA fragment to be cloned can be isolated andjoined to a suitable cloning vector using DNA ligase to seal the DNAmolecules together. The recombinant vector is then introduced into ahost cell, which "clones" it as the cell undergoes many generations ofcell divisions.Table 28-1 Some of the enzymes used in recombinant DNAtechnologyRecombinantvectortransformation(or viral infection)of bacterialhost cellcell propagationFigure 28-1 Schematic illustration of DNA cloning. A fragment of DNA of interest to the researcher is obtained by cleaving a eukaryotic chromosome with a restriction endonuclease. Afterisolating the fragment and ligating it to a cloningVector that has also been cleaved with a restrictionendonuclease, the resulting recombinant DNA isintroduced into a host cell where it can be propagated (cloned). Note that the size of the E.
colichromosome relative to that of a typical cloningvector such as a plasmid is much greater than depicted here.Enzyme(s)FunctionType II restrictionendonucleasesDNA ligaseDNA polymerase I(E. coli)Reverse transcriptasePolynucleotide kinaseCleaving DNAs at specific base sequencesJoining two DNA molecules or fragmentsFilling in gaps in duplexes by stepwiseaddition of nucleotides to 3' endsMaking a DNA copy of an RNA moleculeAdding a phosphate to the 5'-OH end of apolynucleotide to label it or permit ligationAdding homopolymer tails to the 3'-OH endsTerminal transferaseof a linear duplexRemoving nucleotide residues from the 3' endsExonuclease IIIof a DNA strandBacteriophage A exonuclease Removing nucleotides from the 5' endsof a duplex to expose single-stranded 3' endsRemoving terminal phosphates from eitherAlkaline phosphatasethe 5' or 3' end or bothRestriction endonucleases are found in a wide range of bacterialspecies.
Werner Arber discovered that their biological function is torecognize and cleave foreign DNA (e.g., the DNA of an infecting virus);such DNA is said to be restricted. The cell's own DNA is not cleavedbecause the sequence recognized by the restriction endonuclease ismethylated (and thereby protected) by a specific DNA methylase. Therestriction endonuclease and the corresponding methylase in a bacterium are sometimes referred to as a restriction-modification system. There are three types of restriction endonucleases, designated I,II, and III. Types I and III are generally large, multisubunit complexescontaining both the endonuclease and methylase activities.
Type I restriction endonucleases cleave DNA at random sites that can be 1,000base pairs or more from the recognition sequence. Type III enzymescleave the DNA about 25 base pairs from the recognition sequence.Both types of enzyme move along the DNA in a reaction that requiresthe energy of ATP. The type II restriction enzymes, first isolated byHamilton Smith, are simpler, require no ATP, and cleave the DNAwithin the recognition sequence itself. The extraordinary utility of thetype II enzymes was first demonstrated by Daniel Nathans, and theseare the enzymes used most widely for recombinant DNA work.Chapter 28 Recombinant DNA TechnologyMore than 800 restriction endonucleases have been discovered indifferent bacterial species.
Характеристики
Тип файла PDF
PDF-формат наиболее широко используется для просмотра любого типа файлов на любом устройстве. В него можно сохранить документ, таблицы, презентацию, текст, чертежи, вычисления, графики и всё остальное, что можно показать на экране любого устройства. Именно его лучше всего использовать для печати.
Например, если Вам нужно распечатать чертёж из автокада, Вы сохраните чертёж на флешку, но будет ли автокад в пункте печати? А если будет, то нужная версия с нужными библиотеками? Именно для этого и нужен формат PDF - в нём точно будет показано верно вне зависимости от того, в какой программе создали PDF-файл и есть ли нужная программа для его просмотра.
![](https://s.studizba.com/z.php?f=/templates/si/i/noavatar.png&w=100&h=100&t=1"){kind=avatar}
