Rice (Oryza sativa) is among the world’s most important food crops. With 12 pairs of chromosomes and about 40,000 genes in 400 Mb of DNA, its genome is one of the smallest of the major crop grasses. © Revensis/Dreamstime.com. Genomics, Proteomics, 10 and Genetic Engineering »» CHAPTER ORGANIZATION 10.1 Cloning a DNA molecule takes place in several 10.2 A genomic sequence is like a book without an index, steps. 332 and identifying genes and their functions is a major Restriction enzymes cleave DNA into fragments challenge. 343 with defined ends. 332 The protein-coding potential of an organism is Restriction fragments are joined end to end to contained in its genome sequence. 343 produce recombinant DNA. 334 A genome sequence without annotation is A vector is a carrier for recombinant DNA. 335 meaningless. 343 Specialized vectors can carry very large DNA Comparison among genomes is an aid to fragments. 336 annotation. 344 Vector and target DNA fragments are joined with 10.3 Genomics and proteomics reveal genome-wide DNA ligase. 337 A recombinant cDNA contains the coding sequence patterns of gene expression and networks of protein of a eukaryotic gene. 337 interactions. 347 Loss of b-galactosidase activity is often used to DNA microarrays are used to estimate the relative detect recombinant vectors. 339 level of gene expression of each gene in the Recombinant clones are often identified by genome. 347 hybridization with labeled probe. 342 Microarrays reveal groups of genes that are coordinately expressed during development. 348 Yeast two-hybrid analysis reveals networks of protein interactions. 350 331 773649_CH10_331_364.indd3649_CH10_331_364.indd 333131 99/17/09/17/09 22:22:22 PPMM 10.4 Reverse genetics creates an organism with a The production of useful proteins is a primary designed mutation. 352 impetus for recombinant DNA. 360 Animal viruses may prove useful vectors for gene Recombinant DNA can be introduced into the therapy. 360 germ line of animals. 353 Recombinant DNA can also be introduced into the human connection Pinch of This plant genomes. 356 and a Smidgen of That 355 Transformation rescue is used to determine experimentally the physical limits of a gene. 357 »» Chapter Summary 361 10.5 Genetic engineering is applied in medicine, industry, »» Issues & Ideas 361 agriculture, and research. 358 »» Solutions: Step by Step 362 Animal growth rate can be genetically »» Concepts in Action: Problems for Solution 362 engineered. 358 GENETICS on the web Crop plants with improved nutritional qualities can be created. 358 into a cell or organism to change its genotype in a ere is an incomplete list of mammalian directed, predetermined way. Such genetically engi- genomes that have been sequenced: human, neered organisms are called transgenic organisms. chimpanzee, monkey, lemur, mouse, dog, H Transgenics are often created for experimental stud- cat, bat, squirrel, rabbit, guinea pig, armadillo, ies, but an important application is the development hedgehog, shrew, opposum, horse, elephant, pango- of improved varieties of domesticated animals and lin, sloth, llama, and dolphin. Also sequenced are the crop plants, in which case a transgenic organism genomes of many species of fruit flies, worms, and is often called a genetically modified organism (GMO). fungi, hundreds of bacteria, mitochondria, and chlo- Specific examples of genetically modified organisms roplasts, and thousands of viruses. Together these are considered later in this chapter. genomes represent a colossal amount of sequence data available for analysis and comparison. In addi- tion to the genome sequences, methods are also 10.1 Cloning a DNA molecule takes available for identifying which genes in the genome place in several steps. are transcribed in particular tissue types, at specific times in development, or at different stages of the In genetic engineering, the immediate goal of an cell cycle. These are the raw data of genomics, experiment is usually to insert a particular fragment which deals with the DNA sequence, organization, of chromosomal DNA into a plasmid or a viral DNA function, and evolution of genomes. The counterpart molecule. This is accomplished by techniques for at the level of proteins is proteomics, which aims to breaking DNA molecules at specific sites and for iso- identify all the proteins in a cell or organism (includ- lating particular DNA fragments. ing any post-translationally modified forms), as well as their cellular localization, functions, and interac- Restriction enzymes cleave DNA into tions. Proteomics makes use of methods discussed fragments with defined ends. later in this chapter that identify which proteins in the cell undergo physical contact, thereby revealing DNA fragments are usually obtained by the treatment networks of interacting proteins. of DNA samples with restriction enzymes. Restriction Genomics was made possible by the invention of enzymes are nucleases that cleave DNA wherever it techniques originally devised for the manipulation contains a particular short sequence of nucleotides of genes and the creation of genetically engineered that matches the restriction site of the enzyme (see organisms with novel genotypes and phenotypes. Section 6.6). Most restriction sites consist of four or We refer to this approach as recombinant DNA, six nucleotides, within which the restriction enzyme but it also goes by the names gene cloning or genetic makes two single-strand breaks, one in each strand, engineering. The basic technique is quite simple: DNA generating 3Ј-OH and 5Ј-P groups at each position. is isolated and cut into fragments by one or more About a thousand restriction enzymes, nearly all restriction enzymes; then the fragments are joined with different restriction site specificities, have been together in a new combination and introduced back isolated from microorganisms. 332 CHAPTER 10 Genomics, Proteomics, and Genetic Engineering 773649_CH10_331_364.indd3649_CH10_331_364.indd 333232 99/17/09/17/09 22:22:22 PPMM Restriction sites for EcoRI GAATTC GAATTC GAATTC CTTAAG CTTAAG CTTAAG G AATTC G CTTAA G CTTAA ATTCA G AATTC G C TTAA G Any two Circularization “sticky” ends produced by Circularization the same A T A T A C restriction G T T A A T T G enzyme can G A C C T A come together C T A G and form base pairs. Circularization of DNA fragments produced by a restriction enzyme. The red arrowheads indicate the EcoRI cleavage sites. Most restriction sites are symmetrical in the sense • The breaks need not be directly opposite one that the sequence is identical in both strands of the another in the two DNA strands. DNA duplex. For example, the restriction enzyme • Enzymes that cleave the DNA strands asymmet- EcoRI, isolated from E. coli, has the restriction site rically generate DNA fragments with comple- 5Ј-GAATTC-3Ј; the sequence of the other strand is mentary ends. 3Ј-CTTAAG-5Ј, which is identical but written with the 3Ј end at the left. EcoRI cuts each strand between These properties are illustrated for EcoRI in . the G and the A. The term palindrome is used to Most restriction enzymes are like EcoRI in that denote this type of symmetrical sequence. they make staggered cuts in the DNA strands, pro- Soon after restriction enzymes were discovered, ducing single-stranded ends called sticky ends observations with the electron microscope indicated that can adhere to each other because they contain that the fragments produced by many restriction complementary nucleotide sequences. Some restric- enzymes could spontaneously form circles. The circles tion enzymes (such as EcoRI) leave a single-stranded Ј could be made linear again by heating. On the other overhang at the 5 end ( , part A); oth- Ј hand, if the circles that formed spontaneously were ers leave a 3 overhang. A number of restriction treated with DNA ligase, which joins 3Ј-OH and 5Ј-P enzymes cleave both DNA strands at the center of groups, then they could no longer be made linear with symmetry, forming blunt ends. Part B of Figure heat because the ends were covalently linked by the 10.2 shows the blunt ends produced by the enzyme DNA ligase. This observation was the first evidence for BalI. Blunt ends also can be ligated by DNA ligase. three important features of restriction enzymes: However, whereas ligation of sticky ends re-creates the original restriction site, any blunt end can join • Restriction enzymes cleave DNA molecules in with any other blunt end and not necessarily create palindromic sequences. a restriction site. 10.1 Cloning a DNA Molecule Takes Place in Several Steps 333 773649_CH10_331_364.indd3649_CH10_331_364.indd 333333 99/17/09/17/09 22:22:22 PPMM EcoRI restriction site Bal I restriction site (A) Cuts asymmetrically (B) Cuts on line of symmetry GAATTC TGGCCA CTTAAG ACCGGT When the cleavage is When the cleavage asymmetrical, the Separation of Separation of is symmetrical, the over-hanging ends are fragments fragments resulting ends are “sticky” (complementary). blunt. G ATTCA T G G C C A CTTAA G ACC GGT Sticky-end fragments Blunt-end fragments Two types of cuts made by restriction enzymes. The red arrowheads indicate the cleavage sites. (A) Cuts made in each strand at an equal distance from the center of symmetry of the restriction site. (B) Cuts made in each strand at the center of symmetry of the restriction site. Most restriction enzymes recognize their restric- tion sequence without regard to the source of the DNA. Thus, This principle will be seen to be one of the founda- tions of recombinant DNA technology. Because most restriction enzymes recognize a unique sequence, the number of cuts made in the DNA of an organism by a particular enzyme is limited. For example, an E. coli DNA molecule contains 4.6 106 base pairs, and any enzyme that cleaves a six-base restriction site will cut the molecule into about a thou- sand fragments.