Development from a caterpillar into a butterfly entail dramatic changes in patterns of gene expression.

The expression levels of thousands of genes can be monitored via DNA arrays.

DNA microarray reveals the expression levels of more than 12,000 human genes

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Recombinant DNA Technology revolutionized biochemistry since 1970 1. Cut, Join, and Replicate DNA and Reverse transcribe RNA. Treat DNA sequences as modules 2. Complementary Hybridization used to construct new combinations of DNA as well as to detect and amplify particular sequences 3. DNA sequencing First small genomes from viruses larger genomes from bacteria eukaryotic genomes including the 3-billion-base-pair human genome.

Scientists are just beginning to exploit genomics ! 4. Deliver foreign DNA into host organisms

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Rise of the Planet of Apes (2011)

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5.1 The Exploration of Genes Relies on Key Tools 1. Restriction-Enzyme analysis. (1978 Nobel Prize) 2. Blotting Techniques. Southern, Northern, Western blotting 3. DNA Sequencing. (1980 Nobel Prize) 4. Solid-phase Synthesis of Nucleic Acids.

Solid-phase synthesis of peptide (1984 Nobel prize) 5. Polymerase Chain Reaction (PCR). (1993 Nobel Prize) to determine the source of a hair left at the scene of a crime, to resurrect genes from fossils. A final set of techniques relies on the computer. (Chapter 6)

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Restriction enzymes, also called restriction endonucleases, recognize specific base sequences in double-helical DNA and cleave, at specific places, both strands of that duplex. To biochemists, these exquisitely precise scalpels are marvelous gifts of nature. Analyzing chromosome structure, sequencing very long DNA molecules, isolating genes, and creating new DNA molecules Werner Arber and Hamilton Smith discovered restriction enzymes, and Daniel Nathans pioneered their use in the late 1960s. (1978 Nobel Prize)

Restriction Enzymes Split DNA into Specific Fragments

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Restriction enzymes are found in a wide variety of prokaryotes. Their biological role is to cleave foreign DNA molecules. The cell’s own DNA is not degraded, because the sites recognized by its own restriction enzymes are methylated. Many restriction enzymes recognize specific sequences of four to eight base pairs and hydrolyze a phosphodiester bond in each strand in this region.

A striking characteristic of these cleavage sites is that they almost always possess twofold rotational symmetry. In other words, the recognized sequence is palindromic, or an inverted repeat, and the cleavage sites are symmetrically positioned. Several hundred restriction enzymes have been purified and characterized. 6

Their names consist of a three-letter abbreviation for the host organism Eco for Escherichia coli, Hin for Haemophilus influenzae, Hae for Haemophilus aegyptius) followed by a strain designation (if needed) a roman numeral (if more than one restriction enzyme from the same strain has been identified). The specificities of several of these enzymes are shown in Figure 5.1.

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Restriction enzymes are used to cleave DNA molecules into specific fragments that are more readily analyzed and manipulated than the entire parent molecule. For example, the 5.l -kb circular duplex DNA of the tumor-producing SV40 virus is cleaved at 1 site by EcoRI, at 4 sites by HpaI, and at 6 sites by HindIII. A piece of DNA produced by the action of one restriction enzyme can be specifically cleaved into smaller fragments by another restriction enzyme. The pattern of such fragments can serve as a fingerprint of a DNA molecule, as will be considered shortly. Indeed, complex chromosomes containing hundreds of millions of base pairs can be mapped by using a series of restriction enzymes.

EcoRI HpaI HindIII Mix

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Electrophoresis of a DNA fragment because the phosphodiester backbone of DNA is negatively charged.

The shorter fragments, the farther the migration. Polyacrylamide gels are used to separate fragments containing up to 1000 base pairs, more-porous agarose gels are used to resolve mixtures of larger fragments (as large as 20 kb). Bands or spots of radioactive DNA in gels can be visualized by autoradiography. Alternatively, a gel can be stained with ethidium bromide, which fluoresces an intense orange when bound to a double-helical DNA molecule (Figure 5.2). A band containing only 50 ng of DNA can be readily seen.

Restriction Fragments Separated by Gel Electrophoresis and Visualized

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Southern Blotting A restriction fragment containing a specific base sequence can be identified by hybridizing it with a labeled complementary DNA strand (Figure 5.3). A mixture of restriction fragments is separated by electrophoresis through an agarose gel, denatured to form single-stranded DNA, and transferred to a nitrocellulose sheet. The positions of the DNA fragments in the gel are preserved on the nitrocellulose sheet, where they are exposed to a 32P-labeled single-stranded DNA probe.

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Northern blotting Similarly, RNA molecules 1. Separated by gel electrophoresis 2. Transfer to nitrocellulose 3. Hybridization 4. Identify specific sequences A further play on words accounts for the term Western blotting, which refers to a technique for detecting a particular protein by staining with specific antibody. Southern : DNA Northern: RNA Western: protein

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The analysis of DNA structure and its role in gene expression also have been markedly facilitated by the development of powerful techniques for the sequencing of DNA molecules. The key to DNA sequencing is the generation of DNA fragments whose length depends on the last base in the sequence. Collections of such fragments can be generated through the controlled termination of replication (Sanger dideoxy method), a method developed by Frederick Sanger and coworkers. This technique has superseded alternative methods

DNA Sequencing by Controlled Termination of Replication

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The synthesis is primed by a chemically synthesized fragment that is complementary to a part of the sequence known from other studies. In addition to the four deoxyribonucleoside triphosphates (radioactively labeled), each reaction mixture contains a small amount of the 2', 3'-dideoxy analog of one of the nucleotides, a different nucleotide for each reaction mixture The incorporation of this analog blocks further growth of the new chain because it lacks the 3' -hydroxyl terminus needed to form the next phosphodiester bond.

The concentration of the dideoxy analog is low enough that chain termination will take place only occasionally. The polymerase will insert the correct nucleotide sometimes and the dideoxy analog other times, stopping the reaction.

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For instance, if the dideoxy analog of dATP is present, fragments of various lengths are produced, but all will be terminated by the dideoxy analog (Figure 5.4). Importantly, this dideoxy analog of dATP will be inserted only where a T was located in the DNA being sequenced. Thus, the fragments of different length will correspond to the positions of T.

Four sets of chain-terminated fragments (one for each dideoxy analog) then undergo electrophoresis, and the base sequence of the new DNA is read from the autoradiogram of the four lanes.

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Fluorescence detection is a highly effective alternative to autoradiography. A fluorescent tag is incorporated into each dideoxy analog a differently colored one for each of the four chain terminators (e.g., A, C, G, T). With the use of a mixture of terminators, a single reaction can be performed and the resulting fragments are then subjected to electrophoresis. The separated bands of DNA are detected by their fluorescence as they emerge following electrophoresis; the sequence of their colors directly gives the base sequence.

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DNA strands, like polypeptides (Section 3.4), can be synthesized by the sequential addition of activated monomers to a growing chain that is linked to an insoluble support.

DNA probes and genes synthesized by automated Solid-Phase Methods

Each cycle takes 10 minutes and 99% yield 16

This solid-phase approach is ideal for the synthesis of DNA, because the desired product stays on the insoluble support. At the end of each step, soluble reagents and by-products are washed away from the resin that bears the growing chains. At the end of the synthesis, NH3 is added to remove all protecting groups and release the oligonucleotide from the solid support. the desired chain is the longest length with various lengths of by-products Purified by high-pressure liquid chromatography or by electrophoresis DNA chains of as many as 100 nucleotides can be readily synthesized

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Yield: (0.99)100 = 0.36 Rxn Time: 100 × 10 min / 60 min = 16.7 hr

The ability to rapidly synthesize DNA chains of any selected sequence opens many experimental avenues. For example, a synthesized oligonucleotide labeled at one end with 32P or a fluorescent tag can be used to search for a complementary sequence in a very long DNA molecule or even in a genome consisting of many chromosomes. The use of labeled oligonucleotides as DNA probes is powerful and general. serve as the starting point of an exploration of adjacent uncharted DNA. Such a probe can be used as a primer to initiate the replication of neighboring DNA by DNA polymerase. An exciting application of the solid-phase approach is the synthesis of new tailor-made genes. New proteins with novel properties can now be produced in abundance by the expression of synthetic genes.

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In 1984, Kary Mullis devised an ingenious method called the polymerase chain reaction (PCR) for amplifying specific DNA sequences. Consider a DNA duplex consisting of a target sequence surrounded by nontarget DNA. Millions of the target sequences can be readily obtained by PCR if the flanking sequences of the target are known. PCR is carried out by adding the following components to a solution containing the target sequence: (1) a pair of primers that hybridize with the flanking sequences of the target (2) all four deoxyribonucleoside triphosphates (dNTPs) (3) a heat-stable DNA polymerase. A PCR cycle consists of three steps.

Selected DNA Sequences Can Be Greatly Amplified by the Polymerase Chain Reaction (PCR)

1993 Chem

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1. Strand separation. The two strands of the parent DNA molecule are separated by heating the solution to 95°C 2. Hybridization of primers. The solution is then abruptly cooled to 54°C to allow each primer to hybridize to a DNA strand. Parent DNA duplexes do not form, ? because the primers are present in large excess. Primers are from 20 to 30 nucleotides long. ?

3. DNA synthesis. The solution is then heated to 72°C, the optimal temperature for Taq DNA polymerase. Taq derived from Thermus Aquaticus, a thermophilic bacterium that lives in hot springs.

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Cycle of the PCR amplification can be carried out repetitively just by changing the temperature. The thermostability of the polymerase makes it feasible to carry out PCR in a closed container; no reagents are added after the first cycle. At the completion of second cycle, four duplexes have been generated(Figure 5.8). Of the eight DNA strands, two short strands constitute only the target sequence –the sequence including and bounded by the primers. After n cycles, the desired sequence is amplified 2n-fold. The amplification is a million fold after 20 cycles and a billion fold after 30 cycles, which can be carried out in less than an hour. 106 molecules × 100 bases/molecule= 108 bases = 10-14 mol 109 molecules × 100 bases/molecule= 1011 bases = 10-11 mol 21

Several features of this remarkable method for amplifying DNA are noteworthy. First, the sequence of the target need not be known. All that is required is knowledge of the flanking sequences. Second, the target can be much larger than the primers. Targets larger than 10 kb have been amplified by PCR. Third, primers do not have to be perfectly matched to flanking sequences to amplify targets. With the use of primers derived from a gene of known sequence, it is possible to search for variations on the theme. In this way, families of genes are being discovered by PCR. Fourth, PCR is highly specific at relatively high temperature. Stringency is the required closeness of the match between primer and target, which can be controlled by temperature and salt. At high temperatures, the only DNA that is amplified is that situated between primers that have hybridized Fifth, PCR is sensitive. A single DNA molecule can be amplified and detected.

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PCR can provide valuable diagnostic information in medicine. Bacteria and viruses can be readily detected with the use of specific primers. PCR can reveal the presence of human immunodeficiency virus (HIV) in persons who have not mounted an immune response to this pathogen. In these patients, assays designed to detect antibodies against the virus would yield a false negative test result. Finding Mycobacterium tuberculosis bacilli in tissue specimens is slow and laborious. With PCR, as few as 10 tubercle bacilli PCR million human cells can be readily detected.

PCR Is a Powerful Technique in Medical Diagnostics, Forensics, and Studies of Molecular Evolution.

Tuberculosis: 결핵 23

PCR is a promising method for the early detection of certain cancers. This technique can identify mutations of certain growth-control genes, such as the ras genes (Chapter 14). The capacity to greatly amplify selected regions of DNA can also be highly informative in monitoring cancer chemotherapy. Tests using PCR can detect when cancerous cells have been eliminated and treatment can be stopped; they can also detect a relapse and the need to immediately resume treatment. PCR is ideal for detecting leukemias caused by chromosomal rearrangements.

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PCR is also having an effect in forensics and legal medicine. An individual DNA profile is highly distinctive because many genetic loci are highly variable within a population. For example, variations at a specific one of these locations determines a person's HLA type (human leukocyte antigen type; Section 34.5); organ transplants are rejected when the HLA types of the donor and recipient are not sufficiently matched. PCR amplification of multiple genes is being used to establish biological parentage in disputed paternity and immigration cases. Analyses of blood stains and semen samples by PCR have implicated guilt or innocence in numerous assault and rape cases. The root of a single shed hair found at a crime scene contains enough DNA for typing by PCR (Figure 5.9).

D: defendant Blood stain on the pants and shirt of a defendant V: victim TS: control

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DNA is a remarkably stable molecule, particularly when relatively shielded from air, light, and water. Under such circumstances, large fragments of DNA can remain intact for thousands of years or longer. PCR provides an ideal method for amplifying such ancient DNA molecules so that they can be detected and characterized. As will be discussed in Chapter 6, sequences from these PCR products can be sources of considerable insight into evolutionary relationships between organisms.

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The pioneering work of Paul Berg, Herbert Boyer, and Stanley Cohen in the early 1970s led to the development of recombinant DNA technology, which has taken biology from an exclusively analytical science to a synthetic one. New combinations of unrelated genes can be constructed in the laboratory by applying recombinant DNA techniques. These novel combinations can be cloned–amplified many-fold–by introducing them into suitable cells, where they are replicated by the DNA-synthesizing machinery of the host. The inserted genes are often transcribed and translated in their new setting. What is most striking is that the genetic endowment of the host can be permanently altered in a designed way.

5.2 Recombinant DNA Technology Has Revolutionized All Aspects of Biology

Paul Berg 1980 Chem

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Plasmids (naturally occurring circles of DNA that act as accessory chromosomes in bacteria) and bacteriophage lambda (λ phage), a virus, are choice vectors for cloning in E. coli. The vector can be prepared for accepting a new DNA fragment by cleaving it at a single specific site by a restriction enzyme.

Restriction Enzymes and DNA Ligase Are Key Tools in Forming Recombinant DNA Molecules

The staggered cuts made by this enzyme produce complementary single-stranded ends, which have specific affinity for each other and hence are known as cohesive or sticky ends. Any DNA fragment can be joined if it has the same cohesive ends. The DNA fragment and the cut plasmid can be annealed and then joined by DNA ligase

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What if the target DNA is not naturally flanked by the appropriate restriction sites? This cohesive-end DNA molecules can be made by adding a short, chemically synthesized DNA linker that can be cleaved by restriction enzymes. First, the linker is covalently joined to the ends of a DNA fragment or vector. For example, a decameric linker and a DNA molecule are phosphorylated by polynucleotide kinase and then joined by the T4 DNA ligase

This ligase can form a covalent bond between blunt-ended double-helical DNA. Cohesive ends are produced when these terminal extensions are cut by an appropriate restriction enzyme. Thus, cohesive ends corresponding to a particular restriction enzyme can be added to virtually any DNA molecule. 29

Plasmids are circular double-stranded DNA molecules that occur naturally in some bacteria. Plasmids carry genes for the inactivation of antibiotics, the production of toxins, and the breakdown of natural products. These accessory chromosomes can replicate independently. A bacterial cell may have no plasmids at all or it may house as many as 20 copies

pBR322 Plasmid. Insertion of DNA at the SalI restriction site inactivate the gene for tetracycline resistance, an effect called insertional inactivation. Cells containing pBR322 with a DNA insert at one of these restriction sites are resistant to ampicillin but sensitive to tetracycline, and so they can be readily selected.

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Plasmids & λ Phage Are Choice Vectors for DNA Cloning in Bacteria

•  Transformation: Insert plasmid into E. coli –  Not efficient à Why markers are important

How to Find Transformed E. coli

Normal E.coli cell are sensitive to ampicillin and cannot grow with

Blue means non-recombinant colonies Failed in inserting gene

Survival means Amp res White means gene insertion

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Another class of plasmids have been optimized for use as expression vectors for the production of large amount of protein. They contain promoter sequences designed to drive the transcription of large amounts of a protein-coding DNA sequence. Plasmid vector often features a polylinker region that includes many unique restriction sites within its sequence. This polylinker can be cleaved with many different restriction enzymes or combinations of enzymes, providing great versatility in the DNA fragments that can be inserted.

Lambda Phage. Another widely used vector, λ phage, enjoys a choice of life styles: this bacteriophage can destroy its host or it can become part of its host (Figure 5.14).

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In the lytic pathway, viral DNA and proteins are quickly produced and packaged into virus particles, leading to the lysis (destruction) of the host cell In the lysogenic pathway, the phage DNA becomes inserted into the host-cell genome and can be replicated together with host-cell DNA for many generations, remaining inactive. Certain environmental changes can trigger lytic pathway. Large segments of the 48-kb DNA of λ phage are not essential for productive infection and can be replaced by foreign DNA, thus making λ phage an ideal vector.

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However, a suitably long DNA insert (such as 10 kb) between the two ends of λ DNA enables such are combinant DNA molecule to be packaged. Another advantage of using these modified viruses as vectors is that they enter bacteria much more easily than do plasmids. Cosmid essentially a hybrid of λ phage and a plasmid that can serve as a vector for large DNA inserts (as large as 45 kb).

Mutant λ phages designed for cloning have been constructed. An especially useful one called λgt-λβ The middle segment of this λ DNA molecule can be removed. The two remaining pieces have a combined length equal to 72%. This amount of DNA is too little to be packaged

Much larger pieces of DNA can now be propagated in Bacterial Artificial Chromosomes (BACs) or Yeast Artificial Chromosomes (YACs). BACs are highly engineered versions of the E. coli fertility (F factor) that can include inserts as large as 300 kb. YACs contain a centromere. an autonomously replicating sequence (ARS, where replication begins), a pair of telomeres (normal ends of eukaryotic chromosomes), selectable marker genes, and a cloning site. Inserts are as large as 1000 kb can be cloned into YAC vectors.

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Bacterial and Yeast artificial chromosomes (BAC & YAC)

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Ingenious cloning and selection methods have made it possible to isolate small stretches of DNA in a genome containing more than 3 × 106 kb. The approach is to prepare a large collection (library) of DNA fragments and then to identify those members of the collection that have the gene of interest. To clone a gene in an entire genome, two critical components must be available: A specific oligonucleotide probe for the gene of interest DNA library that can be screened rapidly How is a specific probe obtained?

Specific Genes Can Be Cloned from Digests of Genomic DNA

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A probe of a gene can be prepared if a part of the amino acid sequence of the protein encoded by the gene is known. However, a problem arises because a single peptide sequence can be encoded by a number of different oligonucleotides. Thus, peptide sequence containing tryptophan and methionine are preferred because these amino acids are specified by a single codon. All the DNA sequences are synthesized by the solid-phase method and made radioactive by phosphorylating their 5’ ends with 32P. Alternatively, probes can be obtained from the corresponding mRNA from cells in which it is abundant.

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To prepare DNA library, a sample containing many copies of total genomic DNA is first mechanically sheared or partly digested by restriction enzymes into large fragments

This process yields a random population of overlapping DNA fragments, then separated by gel electrophoresis to the set of fragments about 15 kb long. 1. Add synthetic linkers for cohesive ends 2. Inserted into a vector, such as λ phage DNA 3. Infect E. coli with these recombinant phages. The resulting lysate contains fragments of human DNA housed in a sufficiently large number of virus particles to ensure that nearly the entire genome is represented. These phages constitute a genomic library. Phages can be propagated indefinitely, and so the library can be used repeatedly over long periods.

This genomic library is then screened to find the very small number of phages harboring the gene of interest. For the human genome, a calculation shows that a 99% probability of success requires screening about 500,000 clones; hence, a very rapid and efficient screening process is essential. Rapid screening can be accomplished by DNA hybridization.

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Recombinant phages is first plated on a lawn of bacteria. Plaque containing identical phages develops on the plate. Intact bacteria on this sheet are lysed with NaOH, which also serves to denature the DNA so that it becomes accessible for hybridization with a 32P-labeled probe. The presence of a specific DNA sequence in a single spot on the replica can be detected by using a radioactive complementary DNA or RNA molecule as a probe. A single investigator can readily screen a million clones in a day.

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Complementary DNA prepared from mRNA can be expressed in host cells

The preparation of eukaryotic DNA libraries presents unique challenges, especially if the researcher is interested in the protein-coding region. Mammalian gene are mosaics of introns and exons. These interrupted genes cannot be expressed by bacteria, which lack the machinery to splice introns out of the primary transcript. This difficulty can be circumvented by causing bacteria to take up recombinant DNA that is complementary to mRNA, where the intronic sequences have been removed. The key to forming complementary DNA is the enzyme reverse transcriptase. A retrovirus uses this enzyme to form a DNA-RNA hybrid (Sec. 4.3)

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Oligo T as primer Terminal transferase Oligo C as primer Synthetic linkers for ligation to a suitable vector Complementary DNA for all mRNA that a cell contains can be made, inserted into vectors, and then inserted into bacteria. Such a collection is called a cDNA library.

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Complementary DNA molecules can be inserted into expression vectors to enables the production of the corresponding protein of interest. Clones of cDNA can be screened on the basis of their capacity to direct the synthesis of a foreign protein in a bacteria, a technique referred to as expression cloning. A radioactive antibody specific for the protein of interest can be used to identify colonies of bacteria that express corresponding protein product. This immunochemical screening approach can be used whenever a protein is expressed and corresponding antibody is available.

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Complementary DNA has many applications beyond the generation of genetic libraries. The overproduction and purification of most eukaryotic proteins in prokaryotic cells necessitates the insertion of cDNA into plasmid vectors. For example, proinsulin, a precursor of insulin, is synthesized by bacteria harboring plasmids that contain DNA complementary to mRNA for proinsulin. Indeed, bacteria produce much of the insulin used today by millions of diabetics

Much has been learned about genes and proteins by analyzing the effects that mutations have on their structure and function. In the classic genetic approach, mutations are generated randomly throughout the genome of a host organism, an those individuals exhibiting a phenotype of interest are selected. Analysis of these mutants then reveals which genes are altered, and DNA sequencing identifies the precise nature of the changes. Recombinant DNA technology now makes the creation of specific mutations feasible in vitro. We can construct new genes with designed properties by making three kinds of directed changes: deletions, insertions, and substitutions.

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Proteins with New Functions Can Be Created Through Directed Changes in DNA

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Deletions. A specific deletion can be produced by cleaving a plasmid at two sites with a restriction enzyme and ligating to form a smaller circle. This simple approach usually removes a large block of DNA. A smaller deletion can be made by cutting a plasmid at a single site. The ends of the linear DNA are then digested by an exonuclease that removes nucleotides from both strands. The shortened piece of DNA is then ligated to form a circle that is missing a short length of DNA about the restriction site.

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Suppose that we want to replace a particular serine residue with cysteine. This mutation can be made if (1) we have a plasmid containing the gene or cDNA for the protein and (2) we know the base sequence around the site to be altered. If the serine of interest is encoded by TCT , we need to change the C to a G to get cysteine because cysteine is encoded by TGT. This type of mutation is called a point mutation because only one base is altered. The mismatch of 1 of 15 base pairs is tolerable at an appropriate temperature.

Substitutions: Oligonucleotide-Directed Mutagenesis. Mutant proteins with single amino acid substitutions can be readily produced by oligonucleotide-directed mutagenesis.

Insertions: Cassette Mutagenesis. In cassette mutagenesis, a variety of mutations, including insertions, deletions, and multiple point mutations, can be introduced into the gene of interest. A plasmid harboring the original gene is cut with a pair of restriction enzymes to remove a short segment A synthetic double-stranded oligonucleotide (the cassette) carrying the genetic alterations of interest is prepared with cohesive ends that complementary to the ends of the cut plasmid. Ligation of the cassette into plasmid yields the desired mutated gene product

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Designer Genes. Novel proteins can also be created by splicing together gene segments that encode domains that are not associated in nature. For example, a gene for an antibody can be joined to a gene for a toxin to produce a chimeric protein that kills cells that are recognized by the antibody. These immunotoxins are being evaluated as anticancer agents. Furthermore, noninfectious coat proteins of viruses can be produced in large amounts by recombinant DNA methods. They can serve as synthetic vaccines that are safer than conventional vaccines prepared by inactivating pathogenic viruses. A subunit of the hepatitis B virus produced in yeast is proving to be an effective vaccine against this debilitating viral disease. Finally, entirely new genes can be synthesized de novo by the solid-phase method. These genes can encode proteins with no known counterparts in nature.

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5.3 Complete Genomes Have Been Sequenced and Analyzed

The methods just described are for fragments of DNA. However, the genomes from viruses to human comprise longer sequences Is it possible to sequence complete genomes and analyze them? Sanger and coworkers determined the complete sequence of the 5,386 bases in the DNA of the Φ174 DNA virus in 1977. This tour was followed by the sequence of human mitochondrial DNA, a double stranded circular DNA molecule containing 16,569 base pairs. Many other viral genomes were sequenced in subsequent years. However, the genomes of free-living organisms presented a great challenge because even the simplest comprises more than 1 million base pairs. Thus, sequencing projects require both rapid sequencing techniques and efficient methods for assembling many short stretches of 300 to 500 base pairs into a complete sequence.

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The Genomes of Organisms Ranging from Bacteria to Multicellular Eukaryotes Have Been Sequenced

With the development of automatic DNA sequencers based on fluorescent dideoxynucleotide chain terminators, The genome sequence of the bacterium Haemophilus influenzae was determined in 1995 by using a "shotgun" approach.

The genomic DNA was sheared randomly into fragments that were then sequenced. Computer programs assembled the complete sequence by matching up overlapping regions between fragments. The H. influenzae genome comprises 1,830,137 base pairs and encodes approximately 1740 proteins determined the sequences of more than 100 bacterial and archaeal species

The first eukaryotic genome to be completely sequenced was that of baker's yeast, Saccharomyces cerevisiae, in 1996. comprises 12 million base pairs, distributed on 16 chromosomes, and encodes more than 6,000 proteins. In 1998 by the first complete sequencing of the genome of a multicellular organism, the nematode C. elegans, which contains 97 million bps. includes more than 19,000 genes. The genomes of many organisms have now been sequenced, including those of the fruit fly Drosophila melanogaster, the model plant Arabidopsis thaliana, the mouse, the rat and the dog. various stages from "draft” through "completed" to "finished." Even after a sequence has been declared “finished,” some sections may be missing because these DNA sequences are very difficult

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The ultimate goal of much of genomics research has been the sequencing and analysis of the human genome. Given that the human genome comprises approximately 3 billion base pairs of DNA distributed among 24 chromosomes, the challenge of producing a complete sequence was daunting. the human genome has now progressed from a draft sequence first reported in 2001 to a finished sequence reported in late 2004

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The Sequencing of the Human Genome Has Been Finished

At the beginning, the number of genes was estimated to be approximately 100,000. This estimate was reduced to 30,000 to 35,000. With the finished sequence, the estimate fell to 20,000 to 25,000. We will use the estimate of 23,000 throughout this book.

The reduction in this estimate is due, in part, to the realization that there are a relatively large number of pseudogenes, many of which are formerly functional genes that have picked up mutations and are no longer expressed. For example, more than half of the genomic regions that correspond to olfactory receptors key molecules responsible for our sense of smell are pseudogenes. The corresponding regions in the genomes of other primates and rodents encode functional olfactory receptors. Nonetheless, the surprisingly small number of genes belies the complexity of the human proteome. Many genes encode more than one protein through mechanisms such as alternative splicing of mRNA and posttranslational modifications of proteins. The different proteins encoded by a single gene often display important variations in functional properties.

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The human genome contains a large amount of DNA that does not code for proteins. A great challenge in modern biochemistry and genetics is to elucidate the roles of this noncoding DNA. Much of this DNA is present because of the existence of mobile genetic elements. These elements, related to retroviruses, have inserted themselves throughout the genome over time. More than 1 million Alu sequences, 300 bases in length, in the human genome. Alu sequences are examples of SINES, short interspersed elements. The human genome also includes nearly 1 million LINES, long interspersed elements The roles of these elements as neutral genetic parasites or instruments of genome evolution are under current investigation.

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Apr. 2008

Aug. 2009

Sep. 2009

2001Draft Sequence (Science) 2004 Finished Sequence (Nature)

Nov. 2008

Apr. 2010

Oct. 28 2010

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“Next-Generation” Sequencing methods enable the rapid determination of a whole genome sequence

Recent development of “next-generation” sequencing has extended this capability to formerly unforeseen levels. By combining technological breakthroughs in the handling of very small amount of liquid, high-resolution optics, and computer power, these methods enable the parallel sequencing of more than 400,000 individual DNA fragments at several hundred bases per fragment. A single 10-hour sequencing experiment can generate more than 100,000,000 bases (100 megabases). Although significant hurdles remain, this sequencing capacity suggests that the rapid sequencing of anyone’s genome at low cost is a very real possibility. Individual genome sequences may usher in an era of personalized medicine.

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Next Generation Sequencing

454 Roche, Nature, Sep. 2005

Illumina Solexa, Nature, Nov 2008

Helicos, Science, Apr. 2008

Pacific Bioscience, Science, Jan. 2009

Oxford Nanopore, Nature Nanotech, Feb. 2009

ion torrent, Nature, Jul 2011

Sep 2010

2011/6/15

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Comparative genomics has become a powerful research tool

Comparisons with genomes from other organisms are a source of insight into the human genome. Comparisons reveal that an astonishing 99% of human genes have counterparts in these rodent genomes. However, these genes have been substantially reassorted among chromosomes in the estimated 75 million years of evolution since humans and rodents had a common ancestor.

The genomes of other organisms also have been determined specifically for use in comparative genomics. For example, the genomes of two species of puffer fish have been determined. These genomes were selected because they are very small and lack much of the intergenic DNA present in such abundance in the human genome.

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The puffer fish genomes include fewer than 400 megabase pairs (Mbp), one eighth of the number in the human genome, yet it contains essentially the same number of genes. Furthermore, comparison of the two species of puffer fish, which had a common ancestor approximately 25 million years ago, is a source of insight into more recent events in evolution. Comparative genomics is a powerful tool, both for interpreting the human genome and for understanding major events in the origin of genera and species.

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5.4 Eukaryotic Genes Can Be Quantitated and Manipulated with Considerable Precision

How gene and protein function in the context of a whole cell or organism ? How the expression of a particular gene is regulated ? How mutations in the gene affect the function of the corresponding protein ? How the behavior of an entire cell or model organism is altered by the introduction of mutations within specific genes?

Quantitative Analysis

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Gene-Expression Levels Can Be Comprehensively Examined Most genes are present in the same quantity in every cell namely, one copy per haploid cell or two copies per diploid cell. However, the level at which a gene is expressed, as indicated by mRNA quantities, can vary widely, ranging from no expression to hundreds of mRNA copies per cell. Gene-expression patterns vary from cell type to cell type, distinguishing, for example, a muscle cell from a nerve cell. Even within the same cell, gene-expression levels may vary as the cell responds to changes in physiological circumstances. Note that mRNA levels sometimes correlate with the levels of proteins expressed, but this correlation does not always hold. Thus, care must be exercised when interpreting the results of mRNA levels alone.

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The quantity of mRNA determined by quantitative PCR (qPCR), or real time PCR RNA is isolated and cDNA is prepared with the use of reverse transcriptase.

DNA with dye SYBR Green I In initial PCR, not enough After repeated PCR cycles, fluorescence intensity exceed the detection threshold (CT). Proportional to the number of copies of the original template. After the relation between the original copy numbers and the CT has been established with the use of a known standard, subsequent qPCR experiments can be used to determine the number of copies in original sample

Transcriptome pattern and level of expression of all genes in a particular cell or tissue. One of the most powerful methods developed to date for this purpose is based on hybridization. Oligonucleotides or cDNAs are fixed to a solid support such as a microscope slide, creating a DNA microarray. Fluorescently labeled cDNA is hybridized to the chip to reveal the expression level for each gene, identifiable by its known location on the chip The intensity of the fluorescent spot on the chip reveals the extent of the transcription of a particular gene.

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DNA chips have been prepared that contain oligonucleotides complementary to all known open reading frames, 6200 in number, within the yeast genome An analysis of mRNA pools with the use of these chips revealed, for example, that approximately 50% of all yeast genes are expressed at steady-state levels of between 0.1 and 1.0 mRNA copy PCR cell. This method readily detected variations in expression levels displayed by specific genes under different growth conditions.

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New genes inserted into eukaryotic cells can be efficiently expressed

Bacteria are ideal hosts for the amplification of DNA molecules. However, bacteria lack the necessary enzymes to carry out posttranslational modifications. Thus, many eukaryotic genes can be correctly expressed only in eukaryotic host cells. The introduction of recombinant DNA molecules into cells of higher organisms can also be a source of insight into how their genes are organized and expressed. How are genes turned on and off in embryological development? How does a fertilized egg give rise to an organism with highly differentiated cells that are organized in space and time? These central questions of biology can now be fruitfully approached by expressing foreign genes in mammalian cells.

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Recombinant DNA molecules can be introduced into animal cells in several ways. In one method, foreign DNA molecules precipitated by calcium phosphate are taken up by animal cells. A small fraction of the imported DNA becomes stably integrated into the chromosomal DNA. The efficiency of incorporation is low, but the method is useful because it is easy to apply. In another method, DNA is microinjected into cells. A fine tipped glass micropipet containing a solution of foreign DNA is inserted into a nucleus. About 2% of injected mouse cells are viable.

In a third method, viruses are used to introduce new genes into animal cells. The most effective vectors are retroviruses. Retroviruses do not usually kill their hosts. Foreign genes have been efficiently introduced into mammalian cells by infecting them with vectors derived from Moloney murine leukemia virus, which can accept insert as long as 6 kb. Two other viral vectors are extensively used. Vaccinia virus, a large DNA-containing virus, replicates in the cytoplasm of mammalian cells, where it shuts down host-cell protein synthesis. Baculovirus infects insect cells, which can be conveniently cultured. Insect larvae infected with this virus can serve as efficient protein factories.

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Transgenic mice are a powerful means of exploring the role of a specific gene in the development, growth, and behaviors of an entire organism. Useful models for a particular disease process Amyotrophic lateral sclerosis (ALS): Lou Gehrigh Disease

Transgenic Animals Harbor and Express Genes That Were Introduced into Their Germ Lines

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A gene's function can also be probed by inactivating the gene and looking for resulting abnormalities. Powerful methods have been developed for accomplishing gene disruption (also called gene knockout) in organisms such as yeast and mice. These methods rely on the process of homologous recombination.

Gene Disruption Provides Clues to Gene Function

Through this process, regions of strong sequence similarity exchange segments Foreign DNA inserted into a cell can thus disrupt any gene that is at least in part homologous by exchanging segments. Specific genes can be targeted if their nucleotide sequences are known.

When a double-stranded RNA molecule is introduced into an appropriate cell, the RNA is cleaved by an enzyme referred to as Dicer into fragments approximately 21 nucleotides in length. small interfering RNAs (siRNAs) are each incorporated into a different enzyme referred to as RNA-induced silencing complex (RISC). RISC to cleave mRNA molecules Thus, levels of such mRNA molecules are dramatically reduced. Moreover, initial clinical trials of therapies based on RNA interference are underway.

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RNA Interference Provides an Additional Tool for Disrupting Gene Expression

The field of gene therapy attempts to express specific genes within the human body in such a way that beneficial results are obtained. The gene targeted for expression may be already present or specially introduced. Alternatively, gene therapy may attempt to modify genes containing sequence variations that have harmful consequences. A tremendous amount of research remains to be done before gene therapy becomes practical. Nonetheless, considerable progress has been made. Future research promises to transform gene therapy into an important tool for clinical medicine.

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Human Gene Therapy Holds Great Promise for Medicine