Biochemistry, 4th Edition P41 pps

12.2 What Is a DNA Library? 363 heterologous probes because they are not derived from the homologous (same) organism. Problems arise if a complete eukaryotic gene is the cloning target; eukaryotic genes can be tens or even hundreds of kilobase pairs in size. Genes this size are frag- mented in most cloning procedures. Thus, the DNA identified by the probe may represent a clone that carries only part of the desired gene. However, most cloning strategies are based on a partial digestion of the genomic DNA, a technique that generates an overlapping set of genomic fragments. This being so, DNA segments from the ends of the identified clone can now be used to probe the library for clones carrying DNA sequences that flanked the original isolate in the genome. Re- peating this process ultimately yields the complete gene among a subset of overlap- ping clones. cDNA Libraries Are DNA Libraries Prepared from mRNA cDNAs are DNA molecules copied from mRNA templates. cDNA libraries are con- structed by synthesizing cDNA from purified cellular mRNA. These libraries pre- sent an alternative strategy for gene isolation, especially eukaryotic genes. Because most eukaryotic mRNAs carry 3Ј-poly(A) tails, mRNA can be selectively isolated from preparations of total cellular RNA by oligo(dT)-cellulose chromatography (Figure 12.9). DNA copies of the purified mRNAs are synthesized by first anneal- ing short oligo(dT) chains to the poly(A) tails. These oligo(dT) chains serve as primers for reverse transcriptase–driven synthesis of DNA (Figure 12.10). [Ran- dom oligonucleotides can also be used as primers, with the advantages being less dependency on poly(A) tracts and increased likelihood of creating clones repre- senting the 5Ј-ends of mRNAs.] Reverse transcriptase is an enzyme that synthe- sizes a DNA strand, copying RNA as the template. DNA polymerase is then used to copy the DNA strand and form a double-stranded (duplex DNA) molecule. Linkers are then added to the DNA duplexes rendered from the mRNA Known amino acid sequence: Phe Met Glu Trp His Lys Asn Possible mRNA sequence: UUU UUC AUG GAA GAG UGG CAU CAC AGG AAA AAU AAC 1 2 3 4 5 (a) Cellulose matrix with covalently attached oligo(dT) chains Chromatography column Add solution of total RNA in 0.5 M NaCl Total RNA in 0.5 M NaCl 5 4 3 2 Wash with 0.5 M NaCl to remove residual rRNA, tRNA Eukaryotic mRNA with poly(A) tails hybridizes to oligo(dT) chains on cellulose; rRNA, tRNA pass right through column (b) (c) 0.5 NaCl Elute mRNA from column with H 2 O H 2 O Collect and evaluate mRNA solution ANIMATED FIGURE 12.9 Isolation of eukaryotic mRNA via oligo(dT)-cellulose chromatography. (a) In the presence of 0.5 M NaCl, the poly(A) tails of eukaryotic mRNA anneal with short oligo(dT) chains cova- lently attached to an insoluble chromatographic matrix such as cellulose. Other RNAs, such as rRNA (green),pass right through the chromatography column. (b) The column is washed with more 0.5 M NaCl to remove residual contaminants. (c) Then the poly(A) mRNA (red) is recovered by washing the column with water because the base pairs formed between the poly(A) tails of the mRNA and the oligo(dT) chains are unstable in solutions of low ionic strength. See this figure animated at www.cengage.com/login. Image not available due to copyright restrictions 364 Chapter 12 Recombinant DNA: Cloning and Creation of Chimeric Genes CRITICAL DEVELOPMENTS IN BIOCHEMISTRY Identifying Specific DNA Sequences by Southern Blotting (Southern Hybridization) Any given DNA fragment is unique solely by virtue of its specific nucleotide sequence. The only practical way to find one particu- lar DNA segment among a vast population of different DNA frag- ments (such as you might find in genomic DNA preparations) is to exploit its sequence specificity to identify it. In 1975, E. M. Southern invented a technique capable of doing just that. Electrophoresis Southern first fractionated a population of DNA fragments according to size by gel electrophoresis (see step 2 in figure). The electrophoretic mobility of a nucleic acid is inversely proportional to its molecular mass. Polyacrylamide gels are suitable for separa- tion of nucleic acids of 25 to 2000 bp. Agarose gels are better if the DNA fragments range up to 10 times this size. Most preparations of genomic DNA show a broad spectrum of sizes, from less than 1 kbp to more than 20 kbp. Typically, no discrete-size fragments are evident following electrophoresis, just a “smear” of DNA through- out the gel. Blotting Once the fragments have been separated by electrophoresis (step 3), the gel is soaked in a solution of NaOH. Alkali dena- tures duplex DNA, converting it to single-stranded DNA. After the pH of the gel is adjusted to neutrality with buffer, a sheet of absorbent material soaked in a concentrated salt solution is then placed over the gel, and salt solution is drawn through the gel in a direction perpendicular to the direction of electrophoresis (step 4). The salt solution is pulled through the gel in one of three ways: capillary action (blotting), suction (vacuum blotting), or electrophoresis (electroblotting). The movement of salt solution through the gel carries the DNA to the absorbent sheet, which binds the sing le-stranded DNA molecules very tightly, effectively immobilizing them in place on the sheet. Note that the distribu- tion pattern of the electrophoretically separated DNA is main- tained when the single-stranded DNA molecules bind to the ab- sorbent sheet (step 5 in figure). The sheet is then dried. Next, in the prehybridization step, the sheet is incubated with a solution containing protein (serum albumin, for example) and/or a detergent such as sodium dodecylsulfate. The protein and detergent molecules saturate any remaining binding sites for DNA on the absorbent sheet, so no more DNA can bind nonspecifically. Hybridization To detect a particular DNA within the electrophoretic smear of countless DNA fragments, the prehybridized sheet is incubated in a sealed plastic bag with a solution of specific probe molecules (step 6 in figure). A probe is usually a single-stranded DNA of de- fined sequence that is distinctively labeled, either with a radioac- tive isotope (such as 32 P) or some other easily detectable tag. The nucleotide sequence of the probe is designed to be complemen- tary to the sought-for or target DNA fragment. The single-stranded probe DNA anneals with the single-stranded target DNA bound to the sheet through specific base pairing to form a DNA duplex. This annealing, or hybridization as it is usually called, labels the target DNA, revealing its position on the sheet. For example, if the probe is 32 P-labeled, its location can be detected by autoradi- ographic exposure of a piece of X-ray film laid over the sheet (step 7 in figure). Southern’s procedure has been extended to the identification of specific RNA and protein molecules. In a play on Southern’s name, the identification of particular RNAs following separation by gel electrophoresis, blotting, and probe hybridization is called North- ern blotting. The analogous technique for identifying protein mol- ecules is termed Western blotting. In Western blotting, the probe of choice is usually an antibody specific for the target protein. ᮣ The Southern blotting technique involves the transfer of electrophoretically separated DNA fragments to an absorbent sheet and subsequent detection of specific DNA sequences. A preparation of DNA fragments [typically a restriction digest, (1)] is separated according to size by gel electrophoresis (2). The separa- tion pattern can be visualized by soaking the gel in ethidium bromide to stain the DNA and then illuminating the gel with UV light (3). Ethidium bromide mol- ecules intercalated between the hydrophobic bases of DNA are fluorescent under UV light.The gel is soaked in strong alkali to denature the DNA and then neutralized in buffer.Next, the gel is placed on a sheet of DNA-binding material and concentrated salt solution is passed through the gel (4) to carry the DNA fragments out of the gel where they are bound tightly to the sheet (5). Incubation of the sheet with a solution of labeled, single-stranded probe DNA (6) allows the probe to hybridize with target DNA sequences complementary to it.The location of these target sequences is then revealed by an appropriate means of detection, such as autoradiography (7). 12.2 What Is a DNA Library? 365 1 2 345 6 7 +– DNA Digest DNA with restriction endonucleases DNA restriction fragments Perform agarose gel electrophoresis on the DNA fragments from different digests Buffer solution Agarose gel DNA fragments fractionated by size (visible under UV light if gel is soaked in ethidium bromide) Longer DNA fragments Shorter DNA fragments Transfer (blot) gel to absorbent sheet using Southern blot technique Soak gel in NaOH, neutralize Sheet of DNA- absorbing material Gel Wick Buffer Weight Absorbent paper DNA fragments are bound to the sheet in positions identical to those on the gel Hybridize sheet with radioactively labeled probe Radioactive probe solution Expose sheet to X-ray film; resulting autoradiograph shows hybridized DNA fragments 366 Chapter 12 Recombinant DNA: Cloning and Creation of Chimeric Genes templates, and the cDNA is cloned into a suitable vector. Once a cDNA derived from a particular gene has been identified, the cDNA becomes an effective probe for screening genomic libraries for isolation of the gene itself. Because different cell types in eukaryotic organisms express selected subsets of genes, RNA preparations from cells or tissues in which genes of interest are selec- tively transcribed are enriched for the desired mRNAs. cDNA libraries prepared from such mRNA are representative of the pattern and extent of gene expression that uniquely define particular kinds of differentiated cells. cDNA libraries of many normal and diseased human cell types are commercially available, including cDNA libraries of many tumor cells. Comparison of normal and abnormal cDNA libraries, in conjunction with two-dimensional gel electrophoretic analysis (see Appendix to Chapter 5) of the proteins produced in normal and abnormal cells, is a promising new strategy in clinical medicine to understand disease mechanisms. Expressed Sequence Tags When a cDNA library is prepared from the mRNAs syn- thesized in a particular cell type under certain conditions, these cDNAs represent the nucleotide sequences (genes) that have been expressed in this cell type under these conditions. Expressed sequence tags (ESTs) are relatively short (ϳ200 nucleo- tides or so) sequences obtained by determining a portion of the nucleotide se- quence for each insert in randomly selected cDNAs. An EST represents part of a gene that is being expressed. Probes derived from ESTs can be labeled, radioactively or otherwise, and used in hybridization experiments to identify which genes in a ge- nomic library are being expressed in the cell. For example, labeled ESTs can be hy- bridized to a gene chip (see following discussion). mRNA 5' 3' Anneal oligo(dT) 12-18 primers mRNA 5' 3' Add reverse transcriptase and substrates dATP, dTTP, dGTP, dCTP (a) First-strand cDNA synthesis mRNA cDNA 5' 3' 3' 5' Heteroduplex Add RNase H, DNA polymerase, and dATP, dTTP, dGTP, dCTP; mRNA degraded by RNase H (b) 5' 3' 3' 5' DNA polymerase copies first-strand cDNA using RNA segments as primer 5' 3' 3' 5' DNA fragments joined by DNA ligase DNA polymerase 5' 3' 3' 5' cDNA duplex (c) (d) EcoRI linkers, T4 DNA ligase EcoRI-ended cDNA duplexes for cloning (e) cDNA cDNA P ACTIVE FIGURE 12.10 Reverse transcriptase–driven synthesis of cDNA from oligo(dT) primers annealed to the poly(A) tails of purified eukary- otic mRNA. (a) Oligo(dT) chains serve as primers for synthesis of a DNA copy of the mRNA by reverse tran- scriptase. Following completion of first-strand cDNA synthesis by reverse transcriptase, RNase H and DNA polymerase are added (b). RNase H specifically digests RNA strands in DNAϺRNA hybrid duplexes. DNA poly- merase copies the first-strand cDNA, using as primers the residual RNA segments after RNase H has created nicks and gaps (c). DNA polymerase has a 5Ј→3Ј exonuclease activity that removes the residual RNA as it fills in with DNA.The nicks remaining in the second-strand DNA are sealed by DNA ligase (d), yielding duplex cDNA. EcoRI adapters with 5Ј-overhangs are then ligated onto the cDNA duplexes (e) using phage T4 DNA ligase to create EcoRI-ended cDNA for insertion into a cloning vector. Test yourself on the concepts in this figure at www.cengage.com/login. 12.2 What Is a DNA Library? 367 DNA Microarrays (Gene Chips) Are Arrays of Different Oligonucleotides Immobilized on a Chip Robotic methods can be used to synthesize combinatorial libraries of DNA oligonucleotides directly on a solid support, such that the completed library is a two-dimensional array of different oligonucleotides (see the Critical Developments in Biochemistry box on combinatorial libraries, page 361). Synthesis is performed by phosphoramidite chemistry (Figure 11.29) adapted into a photochemical HUMAN BIOCHEMISTRY The Human Genome Project Completed in 2003, the Human Genome Project was a 13-year col- laborative international, government- and private-sponsored effort to map and sequence the entire human genome, some 3 billion base pairs distributed among the two sex chromosomes (X and Y) and 22 autosomes (chromosomes that are not sex chromosomes). A primary goal was to identify and map at least 3000 genetic mark- ers (genes or other recognizable loci on the DNA), which were evenly distributed throughout the chromosomes at roughly 100-kb intervals. At the same time, determination of the entire nucleotide sequence of the human genome was undertaken. J. Craig Venter and colleagues working at Celera, a private corporation, took an alternative approach based on computer alignment of sequenced human DNA fragments. A working draft of the human genome was completed in June 2000 and published in February 2001. An ancillary part of the project has focused on sequencing the genomes of other species (such as yeast, Drosophila melanogaster [the fruit fly], mice, and Arabidopsis thaliana [a plant]) to reveal comparative aspects of genetic and sequence organization (Table 12.1). Information about whole genome sequences of organisms has created a new branch of science called bioinformatics: the study of the nature and organization of biological information. Bioinformatics includes such approaches as functional genomics and proteomics. Functional genomics addresses global issues of gene expression, such as looking at all the genes that are activated dur- ing major metabolic shifts (as from growth under aerobic to growth under anaerobic conditions) or during embryogenesis and development of organisms. Transcriptome is the word used in functional genomics to define the entire set of genes expressed (as mRNAs transcribed from DNA) in a particular cell or tissue under defined conditions. Functional genomics also provides new in- sights into evolutionary relationships between organisms. Pro- teomics is the study of all the proteins expressed by a certain cell or tissue under specified conditions. Typically, this set of proteins is revealed by running two-dimensional polyacrylamide gel elec- trophoresis on a cellular extract or by coupling protein separation techniques to mass spectrometric analysis. The Human Genome Project has proven to be very beneficial to medicine. Many human diseases have been traced to genetic de- fects whose position within the human genome has been identi- fied. As of 2007, the Human Gene Mutation Database (HGMD) listed more than 56,000 mutations in more than 2100 nuclear genes associated with human disease. Among these are cystic fibrosis gene the breast cancer genes, BRCA1 and BRCA2 Duchenne muscular dystrophy gene* (at 2.4 megabases, one of the largest known genes in any organism) Huntington’s disease gene neurofibromatosis gene neuroblastoma gene (a form of brain cancer) amyotrophic lateral sclerosis gene (Lou Gehrig’s disease) melanocortin-4 receptor gene (obesity and binge eating) fragile X-linked mental retardation gene* as well as genes associated with the development of diabetes, a variety of other cancers, and affective disorders such as schizophre- nia and bipolar affective disorder (manic depression). *X-chromosome–linked gene. As of 2007, more than 295 disease-related genes have been mapped to the X chromosome (source: the GeneCards website at the Weizmann Institute of Science, Israel.) Genome Year Genome Size 2 Completed Bacteriophage ␾X174 0.0054 1977 Bacteriophage ␭ 0.048 1982 Marchantia 3 chloroplast genome 0.187 1986 Vaccinia virus 0.192 1990 Cytomegalovirus (CMV) 0.229 1991 Marchantia 3 mitochondrial genome 0.187 1992 Variola (smallpox) virus 0.186 1993 Haemophilus influenzae 4 1.830 1995 (Gram-negative bacterium) Mycobacterium genitalium 0.58 1995 (mycobacterium) Escherichia coli (Gram-negative 4.64 1996 bacterium) Saccharomyces cerevisiae (yeast) 12.1 1996 Methanococcus jannaschii 1.66 1998 (archaeon) Arabidopsis thaliana (green plant) 115 2000 Caenorhabditis elegans (simple 88 1998 animal: nematode worm) Drosophila melanogaster (fruit fly) 117 2000 Homo sapiens (human) 3038 2001 Pan troglodytes (chimpanzee) 3109 2005 1 Data available from the National Center for Biotechnology Information at the National Library of Medicine. Website: http://www.ncbi.nlm.nih.gov/ 2 Genome size is given as millions of base pairs (mb). 3 Marchantia is a bryophyte (a nonvascular green plant). 4 The first complete sequence for the genome of a free-living organism. TABLE 12.1 Completed Genome Nucleotide Sequences 1 368 Chapter 12 Recombinant DNA: Cloning and Creation of Chimeric Genes process that can be controlled by light. Computer-controlled masking of the light allows chemistry to take place at some spots in the two-dimensional array of grow- ing oligonucleotides and not at others, so each spot on the array is a population of identical oligonucleotides of unique sequence. The final products of such proce- dures are referred to as “gene chips” because the oligonucleotide sequences syn- thesized upon the chip represent the sequences of chosen genes. Typically, the oligonucleotides are up to 25 nucleotides long (there are more than 10 15 possible sequence arrangements for 25-mers made from four bases), and as many as 100,000 different oligonucleotides can be arrayed on a chip 1 cm square. The oligonucleotides on such gene chips are used as the probes in a hybridization ex- periment to reveal gene expression patterns. Figure 12.11 shows one design for gene chip analysis of gene expression. 1 2 OR PCR amplification purification Laser 1 Laser 2 Excitation Robotic synthesis of oligonucleotide arrays ESTs or other DNA clones Hybridize target to microarray Reverse transcription Label with fluor dyes Test Reference Gene chip Emission Computer analysis (a) (b) FIGURE 12.11 Gene chips (DNA microarrays) in the analysis of gene expression. Here is one of many analytical possibilities based on DNA microarray technology: (1) Gene segments (for example, ESTs) are isolated and amplified by PCR (see Figure 12.18), and the PCR products are robotically printed onto coated glass microscope slides to create a gene chip.The gene chip usually is considered the “probe”in a “targetϺprobe” screening experiment. (2) Target preparation:Total RNA from two sets of cell treat- ments (control and test treatment) are isolated, and cDNA is produced from the two batches of RNA via reverse transcriptase. During cDNA production, the control is la- beled with a specific fluorescent marker (green, for example) and the test treatment is labeled with a different fluorescent marker (red, for example), so the wavelength of fluorescence allows discrimination between the two different sets of cDNAs.The two batches of labeled cDNA are pooled and hybridized to the gene chip. Laser excitation of the hybridized gene chip with light of appropriate wavelength allows collection of data indicating the intensities of fluorescence, and hence the degree of hybridization of the two different probes with the gene chips.Because the location of genes on the gene chip is known, which genes are expressed (or not) and the degree to which they are expressed is revealed by the fluorescent patterns. (Adapted from Figure 1 in Duggan, D.J., et al.,1999. Expression profiling using cDNA microarrays. Nature Genetics 21 supple- ment:10–14.) 12.3 Can the Cloned Genes in Libraries Be Expressed? 369 12.3 Can the Cloned Genes in Libraries Be Expressed? Expression Vectors Are Engineered So That the RNA or Protein Products of Cloned Genes Can Be Expressed Expression vectors are engineered so that any cloned insert can be transcribed into RNA, and, in many instances, even translated into protein. cDNA expression li- braries can be constructed in specially designed vectors. Proteins encoded by the various cDNA clones within such expression libraries can be synthesized in the host cells, and if suitable assays are available to identify a particular protein, its corre- sponding cDNA clone can be identified and isolated. Expression vectors designed for RNA expression or protein expression, or both, are available. RNA Expression A vector for in vitro expression of DNA inserts as RNA transcripts can be constructed by putting a highly efficient promoter adjacent to a versatile cloning site. Figure 12.12 depicts such an expression vector. Linearized recombi- nant vector DNA is transcribed in vitro using SP6 RNA polymerase. Large amounts of RNA product can be obtained in this manner; if radioactive or fluorescent- labeled ribonucleotides are used as substrates, labeled RNA molecules useful as probes are made. Protein Expression Because cDNAs are DNA copies of mRNAs, cDNAs are unin- terrupted copies of the exons of expressed genes. Because cDNAs lack introns, it is feasible to express these cDNA versions of eukaryotic genes in prokaryotic hosts that cannot process the complex primary transcripts of eukaryotic genes. To ex- press a eukaryotic protein in E. coli, the eukaryotic cDNA must be cloned in an ex- pression vector that contains regulatory signals for both transcription and transla- tion. Accordingly, a promoter where RNA polymerase initiates transcription as well as a ribosome-binding site to facilitate translation are engineered into the vector just upstream from the restriction site for inserting foreign DNA. The AUG initiation codon that specifies the first amino acid in the protein (the translation start site) is contributed by the insert (Figure 12.13). Strong promoters have been constructed that drive the synthesis of foreign pro- teins to levels equal to 30% or more of total E. coli cellular protein. An example is the hybrid promoter, p tac , which was created by fusing part of the promoter for the E. coli genes encoding the enzymes of lactose metabolism (the lac promoter) with part of the promoter for the genes encoding the enzymes of tryptophan biosynthesis (the trp promoter) (Figure 12.14). In cells carrying p tac expression vectors, the p tac pro- moter is not induced to drive transcription of the foreign insert until the cells are ex- posed to inducers that lead to its activation. Analogs of lactose (a ␤-galactoside) such as isopropyl-␤-thiogalactoside, or IPTG, are excellent inducers of p tac . Thus, expression of the foreign protein is easily controlled. (See Chapter 29 for detailed discussions of inducible gene expression.) Perhaps the most widely used protein expression system is based on the pET plas- mid. Transcription of the cloned gene insert is under the control of the bacterio- phage T7 RNA polymerase promoter in pET. This promoter is not recognized by the E. coli RNA polymerase, so transcription can only occur if the T7 RNA po- lymerase is present in host cells. Host E. coli cells are engineered so that the T7 RNA polymerase gene is inserted in the host chromosome under the control of the lac promoter. IPTG induction triggers T7 RNA polymerase production and subsequent transcription and translation of the pET insert. The bacteriophage T7 RNA po- lymerase is so active that most of the host cell’s resources are directed into protein expression and levels of expressed protein approach 50% of total cellular protein. The bacterial production of valuable eukaryotic proteins represents one of the most important uses of recombinant DNA technology. For example, human insulin for the clinical treatment of diabetes is now produced in bacteria. 2 1 3 Polylinker cloning site Foreign DNA RNA transcription by SP6 RNA polymerase Runoff SP6 RNA transcri p t SP6 RNA p olymerase Insert foreign DNA at polylinker cloning site SP6 promoter Linearize ANIMATED FIGURE 12.12 Expression vectors carrying the promoter recognized by the RNA polymerase of bacteriophage SP6 are useful for the pro- duction of multiple RNA copies of any DNA inserted at the polylinker. Before transcription is initiated, the circu- lar expression vector is linearized by a single cleavage at or near the end of the insert so that transcription termi- nates at a fixed point. See this figure animated at www.cengage.com/login. 370 Chapter 12 Recombinant DNA: Cloning and Creation of Chimeric Genes Analogous systems for expression of foreign genes in eukaryotic cells include vec- tors carrying promoter elements derived from mammalian viruses, such as simian virus 40 (SV40), the Epstein–Barr virus, and the human cytomegalovirus (CMV). A sys- tem for high-level expression of foreign genes uses insect cells infected with the bac- ulovirus expression vector. Baculoviruses infect lepidopteran insects (butterflies and moths). In engineered baculovirus vectors, the foreign gene is cloned downstream of the promoter for polyhedrin, a major viral-encoded structural protein, and the recombinant vector is incorporated into insect cells grown in culture. Expression from the polyhedrin promoter can lead to accumulation of the foreign gene prod- uct to levels as high as 500 mg/L. Technologies for the expression of recombinant proteins in mammalian cell cul- tures are commercially available. These technologies have the advantage that the unique post-translational modifications of proteins (such as glycosylation; see Chap- ter 31) seen in mammalian cells take place in vivo so that the expressed protein is produced in its naturally occurring form. Screening cDNA Expression Libraries with Antibodies Antibodies that specifically cross-react with a particular protein of interest are often available. If so, these anti- bodies can be used to screen a cDNA expression library to identify and isolate cDNA clones encoding the protein. The cDNA library is introduced into host bacteria, which are plated out and grown overnight, as in the colony hybridization scheme pre- viously described. DNA-binding nylon membranes are placed on the plates to obtain a replica of the bacterial colonies. The nylon membrane is then incubated under con- ditions that induce protein synthesis from the cloned cDNA inserts, and the cells are treated to release the synthesized protein. The synthesized protein binds tightly to the nylon membrane, which can then be incubated with the specific antibody. Binding of the antibody to its target protein product reveals the position of any cDNA clones ex- pressing the protein, and these clones can be recovered from the original plate. Like other libraries, expression libraries can be screened with oligonucleotide probes, too. Fusion Protein Expression Some expression vectors carry cDNA inserts cloned directly into the coding sequence of a vector-borne protein-coding gene (Figure 12.15). Translation of the recombinant sequence leads to synthesis of a hybrid pro- tein or fusion protein. The N-terminal region of the fused protein represents amino acid sequences encoded in the vector, whereas the remainder of the protein is en- coded by the foreign insert. Keep in mind that the triplet codon sequence within the cloned insert must be in phase with codons contributed by the vector se- quences to make the right protein. The N-terminal protein sequence contributed by the vector can be chosen to suit purposes. Furthermore, adding an N-terminal p t a c o r i a m p r HindIII EcoRI EcoRI EcoRI Pst I Bgl I Polylinker cloning site pUR278 5.2 kbp ANIMATED FIGURE 12.14 A p tac protein expression vector contains the hybrid promoter p tac derived from fusion of the lac and trp promoters. Isopropyl-␤-D-thiogalactoside, or IPTG, induces expres- sion from p tac . See this figure animated at www .cengage.com/login. Image not available due to copyright restrictions 12.3 Can the Cloned Genes in Libraries Be Expressed? 371 signal sequence that targets the hybrid protein for secretion from the cell simpli- fies recovery of the fusion protein. A variety of gene fusion systems have been de- veloped to facilitate isolation of a specific protein encoded by a cloned insert. The isolation procedures are based on affinity chromatography purification of the fu- sion protein through exploitation of the unique ligand-binding properties of the vector-encoded protein (Table 12.2). Reporter Gene Constructs Are Chimeric DNA Molecules Composed of Gene Regulatory Sequences Positioned Next to an Easily Expressible Gene Product Potential regulatory regions of genes (such as promoters) can be investigated by placing these regulatory sequences into plasmids upstream of a gene, called a reporter gene, whose expression is easy to measure. Such chimeric plasmids are Ec o RI ClaI HindIII XbaI Sa l I BamHI Ec o RI P st I a m p r l a c Z o r i Cloning site pUR278 5.2 kbp p tac Codon: Cys Gln Lys Gly Asp Pro Ser Thr Leu Glu Ser Leu Ser Met Cloning site: TGT CAA AAA GGG GAT CCG TCG ACT CTA GAA AGC TTA TCG ATG BamHI SalI XbaI HindIII ClaI ANIMATED FIGURE 12.15 A typical expression vector for the synthesis of a hybrid protein. The cloning site is located at the end of the coding region for the protein ␤-galactosidase. Insertion of foreign DNAs at this site fuses the foreign sequence to the ␤-galactosidase coding region (the lacZ gene). IPTG induces the transcription of the lacZ gene from its promoter p lac , causing expression of the fusion protein. (Adapted from Figure 2, Rüther, U., and Müller-Hill, B., 1983. EMBO Journal 2:1791–1794. See this figure animated at www.cengage.com/login. Fusion Protein Secreted?* Affinity Ligand ␤-Galactosidase No p-Aminophenyl-␤-D-thiogalactoside (APTG) Protein A Yes Immunoglobulin G (IgG) Chloramphenicol acetyltransferase Yes Chloramphenicol (CAT) Streptavidin Yes Biotin Glutathione-S-transferase (GST) No Glutathione Maltose-binding protein (MBP) Yes Starch Hexahistidine tag No Nickel or cobalt Hemagglutinin (HA) peptide No HA-peptide antibody *This indicates whether combined secretion–fusion gene systems have led to secretion of the protein product from the cells, which simplifies its isolation and purification. TABLE 12.2 Gene Fusion Systems for Isolation of Cloned Fusion Proteins 372 Chapter 12 Recombinant DNA: Cloning and Creation of Chimeric Genes then introduced into cells of choice (including eukaryotic cells) to assess the po- tential function of the nucleotide sequence in regulation because expression of the reporter gene serves as a report on the effectiveness of the regulatory ele- ment. A number of different genes have been used as reporter genes. A reporter gene with many inherent advantages is that encoding the green fluorescent pro- tein (or GFP), described in Chapter 4. Unlike the protein expressed by other re- porter gene systems, GFP does not require any substrate to measure its activity, nor is it dependent on any cofactor or prosthetic group. Detection of GFP re- quires only irradiation with near-UV or blue light (400-nm light is optimal), and the green fluorescence (light of 500 nm) that results is easily observed with the naked eye, although it can also be measured precisely with a fluorometer. Figure 12.16 demonstrates the use of GFP as a reporter gene. EGFP is an engineered ver- sion of GFP that shows enhanced fluorescent properties. Specific Protein–Protein Interactions Can Be Identified Using the Yeast Two-Hybrid System Specific interactions between proteins (so-called protein–protein interactions) lie at the heart of many essential biological processes. One method to identify specific protein–protein interactions in vivo is through expression of a reporter gene whose transcription is dependent on a functional transcriptional activator, the GAL4 pro- tein. The GAL4 protein consists of two domains: a DNA-binding (or DB) domain and a transcriptional activation (or TA) domain. Even if expressed as separate proteins, these two domains will still work, provided they can be brought together. The method depends on two separate plasmids encoding two hybrid proteins, one consisting of the GAL4 DB domain fused to protein X and the other consisting of the GAL4 TA do- main fused to protein Y (Figure 12.17a). If proteins X and Y interact in a specific protein–protein interaction, the GAL4 DB and TA domains are brought together so Nerve cord Oviducts Ovaries FIGURE 12.16 Green fluorescent protein (GFP) as a re- porter gene. In the experiment here, GFP expression depends on the promoter for the Drosophilia melano- gaster Tdc2 gene. Tdc2 encodes a neuronal tyrosine de- carboxylase (TDC) whose expression is necessary for egg laying in fruit flies. (Bottom) Green fluorescence highlights neuronal projections expressing the Tdc2 gene. (Top) Diagram of a fly, its nervous system, and ovaries. Note that Tdc2 neurons innervate the ovaries and oviducts of flies. (See Cole, S. H., et al., 2005.Two func- tional but noncomplementing Drosophila tyrosine decarboxy- lase genes. Journal of Biological Chemistry 280:14948–14955. GFP image courtesy of Shannon H. Cole and Jay Hirsh, the University of Virginia. Fly image derived from the Atlas of Drosophila Devel- opment by Volker Hartenstein, http://flybase.bio.indiana.edu/ allied-data/lk/interactive-fly/atlas/00contents.htm.) TA DB lacZ Reporter Gene (b) (a) lacZ Reporter Gene X X Y Y TA DB FIGURE 12.17 The yeast two-hybrid system for identify- ing protein–protein interactions. If proteins X and Y interact, the lacZ reporter gene is expressed. Cells expressing lacZ exhibit ␤-galactosidase activity.