Molecular Biology of the Cell - Volume 1 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1994

Introduction to Cell Biology
How Cells Are Studied
Recombinant DNA Technology

The major challenge of molecular biology today is to understand the detailed mechanisms of cellular processes. We have discussed several sensitive Methods for purifying, analyzing, and tracking Proteins within Cells. This final section focuses on METHODS FOR STUDYING the Structure and function of cellular DNA. The classical approach involves Genetic Methods that allow us to infer Gene function by analyzing the phenotypes of mutant organisms and their offspring. While this approach remains powerful, it has recently been complemented by a suite of techniques collectively known as "Recombinant DNA technology." These methods have vastly expanded The Scope of genetic research, enabling both direct control and detailed chemical analysis of genetic material. Using recombinant DNA methodology, even minor cellular Proteins can be produced in large quantities, thereby facilitating detailed biochemical studies of Cell/13.html">Protein Structure and function.

4.6.1. Recombinant DNA technology has revolutionized cell biology [37]

Until recently, as late as the early 1970s, biochemists regarded DNA as the most difficult cellular component to study. The extremely long, chemically monotonous sequence of NUCLEOTIDES in genetic material could then be investigated only by indirect methods—either by determining The structure of proteins or RNA, or through genetic analysis. Today, the situation has changed dramatically. While DNA analysis once seemed a daunting task to structural biologists, The Development of new methods for sequencing DNA has made such analysis relatively straightforward. It is now possible to isolate specific DNA segments, obtain them in virtually unlimited quantities, and determine their nucleotide sequences at a rate of several hundred nucleotides per day.

Using these same methods, an investigator can alter an isolated gene at will and reintroduce it into The Genome of cultured cells or an animal embryo (which is somewhat more challenging), where the modified gene then becomes functional.

Recombinant DNA technology has had a profound impact on all of cell biology, enabling researchers to tackle problems that once seemed intractable, such as determining the Functions of many newly discovered proteins and their individual domains, and deciphering the complex regulatory mechanisms of Gene Expression IN eukaryotes. Introduction/32.html">Genetic Engineering techniques have made it possible to produce large quantities of many proteins involved in the Regulation of Cell proliferation and development. The application of these methods promises to succeed in the large-scale industrial production of protein Hormones and artificial Vaccines, which previously required enormous effort and resources to obtain.

Recombinant DNA technology encompasses a suite of techniques—both novel and adapted from other disciplines, such as microbial genetics (Table 4-12). The most important of these are:

1) Specific Cleavage of DNA by restriction Nucleases, which greatly facilitates the isolation of and manipulation with individual genes; 2) rapid sequencing of all nucleotides in a purified DNA fragment, allowing the precise boundaries of a gene and the Amino Acid Sequence it encodes to be determined; 3) nucleic acid Hybridization, which enables the detection of specific RNA or DNA sequences with high precision and sensitivity based on their ability to bind complementary nucleic acid sequences; 4) DNA Cloning: the DNA fragment of interest is inserted into a self-replicating genetic element (a plasmid or virus) used to transform Bacteria. After transformation, the bacterial cell replicates this fragment into millions of identical copies; 5) genetic engineering, by which DNA sequences are altered to design modified versions of genes, which are then reintroduced into cells or organisms.

Class="center">Table 4-12. Major Milestones in the development of recombinant DNA technology

To understand recombinant DNA technology, one must have a solid grasp of the natural mechanisms cells use to replicate and decode DNA. We will therefore defer a detailed Discussion of Gene cloning and genetic engineering. In Chapter 5, these topics will be explored after the reader has been introduced to Basic Genetic Mechanisms.

4.6.2. Restriction nucleases cleave DNA at specific nucleotide sequences [38]

To protect themselves against foreign DNA molecules that might enter The Cell and transform it, many bacteria produce restriction nucleases, enzymes that degrade foreign DNA. Each such enzyme recognizes a specific sequence of 4–6 nucleotides in DNA. The corresponding sequences in the bacterium's own genome are protected by the methylation of A and C residues, but any foreign DNA molecule entering the cell is immediately recognized by the nuclease and cleaved in both strands (Fig. 4-60). A large number of restriction nucleases have been isolated from various bacterial species. Today, commercial suppliers produce more than 100 such enzymes, most of which recognize distinct nucleotide sequences.

A given restriction nuclease will cleave a double-stranded DNA molecule of any length into a series of specific pieces, called restriction fragments. By comparing the sizes of the DNA fragments produced after treating a particular region of the genome with a set of restriction nucleases, one can construct a restriction map that shows THE POSITION OF each restriction site relative to other restriction sites (Fig. 4-61). Because a restriction map reflects the arrangement of specific nucleotide sequences in a given region, comparing such maps for two or more related genes allows a rough estimate of their Homology. Consequently, different DNA regions can be compared (by comparing their restriction maps) without determining their nucleotide sequences. For example, a comparison of the restriction maps shown in Fig. 4-62 led to the Conclusion that the chromosomal regions encoding globin chains in humans, orangutans, and chimpanzees have remained virtually unchanged over the last 5–10 million years, i.e., since these species diverged. Restriction maps are also important for DNA cloning and genetic engineering, as they allow a gene to be localized to a specific restriction fragment, as explained below.

Fig. 4-60. The nucleotide sequences recognized by three widely used restriction nucleases (recognition sites). Such sequences often contain six nucleotides and are "palindromic," meaning that The nucleotide sequence reads the same in both directions. The two DNA strands are cleaved within or near the recognition site; in many cases, the cleavage is staggered rather than straight across, resulting in single-stranded cohesive ends (sticky ends), such as those produced by Eco RI or Hind III. Restriction nucleases are obtained from various bacteria: Hpa I from Haemophilus parainfluenzae, Eco RI from Escherichia coli, and Hind III from Haemophilus influenzae.

Fig. 4-61. A simple example illustrating the relative positions of recognition sites for different restriction nucleases (also called "restriction sites") on a DNA double helix, which together form a restriction map. Conclusion: Enzyme A cleaves near one end of the molecule. Enzyme B must cleave either near the same end or near the other end. The sizes of the fragments produced by Digestion with both enzymes rule out the first possibility and establish the order of restriction sites shown below.

Fig. 4-62. Restriction map of the gene cluster encoding Hemoglobin in humans and various primates. The two colored boxes in each map indicate the position of the region corresponding to the globin gene. Each letter indicates a site cleaved by a different restriction nuclease. The positions of the cleavage sites were determined by comparing the sizes of the DNA fragments produced by digesting the DNA with various restriction nucleases individually and in different combinations. (Courtesy of Elisabeth Zimmer and Alan Wilson.)

4.6.3. DNA cloning allows any DNA sequence to be produced in large quantities [39]

Many restriction nucleases make staggered cuts in the two DNA strands, leaving short single-stranded tails at the ends of the fragments. These single-stranded ends can form complementary Base Pairs with any other single-stranded end produced by the same enzyme, and are therefore called cohesive ends (or "sticky ends") (Fig. 4-63). Cohesive ends produced by restriction enzymes make it easy to join any two DNA fragments together, provided they were generated by the same restriction nuclease (or another nuclease that generates identical cohesive ends). Thus, a DNA fragment of any origin can be inserted into the purified DNA of a self-replicating genetic element, typically a plasmid or a bacterial virus. A bacterial clone containing such a plasmid or virus can be thought of as a factory for producing this DNA fragment. The starting fragment can be derived directly from genomic DNA or from cDNA (complementary DNA), which is DNA synthesized by copying Messenger RNA. These methods are discussed in detail in Chapter 5.

The first step in obtaining genomic DNA clones typically involves isolating the DNA and cleaving it with a restriction enzyme. This generates a vast number of different DNA fragments; for instance, a mammalian genome yields between 10⁵ and 10⁷ fragments. During the cloning process, the investigator obtains millions of cell colonies (clones), most of which contain different DNA fragments. The most challenging step in cloning is identifying the specific clone that contains the desired DNA fragment.

Fig. 4-63. Many types of restriction nucleases produce DNA fragments with cohesive (single-stranded) ends. DNA fragments with such ends join through complementary base-pairing in the cohesive end region, as shown in the diagram. The two DNA fragments being joined here were produced by the restriction nuclease Eco RI (see Fig. 4-60).

The preparation of cDNA clones begins with the isolation of mRNA from cells. This mRNA is then used as a template for Reverse Transcriptase, an enzyme synthesized by certain Viruses whose DNA is produced by copying an RNA sequence (a process reverse to the usual Transcription process, in which RNA is synthesized from a DNA template). The enzyme makes a DNA copy (cDNA) of each of the represented RNA molecules. These single-stranded DNA molecules are then converted into double-stranded DNA (see Section 5.6.3). They are cloned using methods similar to those used to clone genomic DNA fragments.

It should be kept in mind that There is a fundamental difference between genomic and cDNA clones, which is explained, in particular, by the splicing process characteristic of higher eukaryotes (see Section 9.4.8). Most likely, it is cDNA clones that contain continuous nucleotide sequences encoding proteins.

4.6.4. Gel Electrophoresis allows rapid fractionation of DNA molecules of different sizes [40]

In the early 1970s, it was shown that gel electrophoresis techniques, which had proved so useful for analyzing protein chains, could accurately determine the length and purity of DNA molecules. This method is much simpler than the modification used for proteins; each nucleotide in a nucleic acid molecule already carries a negative charge, so there is no need to add the negatively charged detergent SDS, which "forces" protein molecules to move toward the positive electrode. Special polyacrylamide gels were developed to separate DNA fragments up to 500 nucleotides long, differing by as little as a single nucleotide (Fig. 4-64, A). Unfortunately, the pores in polyacrylamide gels are too small for large DNA molecules; to separate them by size, special agarose-based gels (a polysaccharide isolated from seaweed) were developed (Fig. 4-64, B). Both of these DNA Separation Methods are widely used for analytical and preparative purposes.

Fig. 4-64. Gel electrophoresis is a powerful method for separating DNA molecules by size. In the three Examples shown, electrophoresis was run from top to bottom, so larger DNA molecules are at the top of the gel. A. A fine-pore polyacrylamide gel was used to fractionate single-stranded DNA. In the size range of 10 to 500 nucleotides, DNA molecules differing by only a single nucleotide can be resolved. In this case, lanes 1-4 are loaded with the products of four independent DNA sequencing reactions, where the chain ends contain dideoxyribonucleotides G, A, T, and C (see Fig. 4-68 in parentheses); since these reactions use DNA molecules previously labeled with radioisotopes, the Location of these radioisotopes can be detected autoradiographically, as shown in the figure. B. A medium-pore agarose gel is used to separate double-stranded DNA molecules. This method is most convenient for separating molecules ranging from 300 to 5000 nucleotide pairs in length. These are the restriction fragments of bacteriophage, detected by fluorescence after staining with ethidium bromide. C. Pulsed-field gel electrophoresis in agarose gel was used to separate 16 different Yeast Chromosomes (Saccharomyces cerevisiae), whose sizes range from 220,000 to 2,500,000 nucleotide pairs. In these gels, molecules up to 10⁷ nucleotide pairs in length can be resolved. (A - courtesy of Linder Laufer, Peter Walter; B - courtesy of Ken Kreutzer, C - courtesy of D. Wolrath, A.W. Davies - Nucl. Acid Res., 15, 7876, 1988.)

Recently, a modification of agarose gel electrophoresis, called pulsed-field gel electrophoresis (PFGE), was introduced. It allows the separation of very large, indeed giant, DNA molecules. Ordinary gel electrophoresis cannot resolve such molecules because the constant electric field causes them to adopt a snake-like configuration. Molecules in this configuration move through the gel at a constant rate regardless of their length. However, if the direction of the electric field is changed frequently, the migration rate of the molecules will depend on their ability to reorient in response to this change. This process takes much longer for larger molecules, causing them to lag behind. On gels after pulsed-field electrophoresis, intact bacterial or yeast chromosomes are resolved as distinct bands (Fig. 4-64, C), making it easy to detect chromosomal rearrangements. Furthermore, by using hybridization of cloned DNA molecules to search for complementary sequences in the gel, it has been possible to map many genes in yeast (see Section 4.6.8).

Unless DNA is labeled or stained, the bands in agarose or polyacrylamide gels remain invisible. One of the most effective Methods for Staining DNA is to soak the gel after electrophoresis in a solution of the dye ethidium bromide, which fluoresces under ultraviolet light when bound to DNA (Fig. 4-64, B and C). An even more sensitive detection method is based on incorporating radioisotopes into DNA molecules prior to electrophoresis; for this, ³²P is commonly used, which is incorporated into DNA phosphates and emits a β-particle of sufficiently high energy to be detected by autoradiography (Fig. 4-64, A).

4.6.5. Purified DNA molecules can be radioactively labeled in vitro [41]

Two methods are widely used to label purified DNA molecules with radioisotopes. In the first method, radioactively labeled nucleotides are incorporated into the DNA molecule using E. coli DNA polymerase I (Fig. 4-65, A). This yields radioactive "DNA probes" used in nucleic acid hybridization reactions (see below). In the second method, the enzyme polynucleotide kinase from a bacteriophage is used to transfer individual ³²P-labeled phosphates from ATP to the 5' end of each DNA strand (Fig. 4-65, B). Because each DNA strand is labeled with only a single ³²P atom by the kinase, the DNA molecules are usually not radioactive enough to be used as DNA probes; however, the fact that the DNA strands are labeled at only one end makes them highly suitable for sequencing and "footprinting", which will be discussed below.

4.6.6. Isolated DNA fragments can be easily sequenced [42]

In the late 1970s, methods were developed for the simple and rapid Determination of the nucleotide sequence (sequencing) of any purified DNA fragments. Following this, the complete nucleotide sequences of many mammalian genes were determined, including those encoding hemoglobin, Insulin, and cytochrome c. The volume of DNA sequence information is so vast (many millions of nucleotides) that computers must be used to store and analyze the available data. Several long DNA sequences containing more than 10⁵ nucleotide pairs have been sequenced, including the complete genome of the Epstein-Barr virus (which causes infectious mononucleosis in humans) and the complete genome of plant Chloroplasts. Currently, two different DNA Sequencing Methods are widely used; the principles underlying the chemical method are illustrated in Figs. 4-66 and 4-67, while the enzymatic method is explained in Fig. 4-68.

These methods are so rapid and reliable that when a researcher is faced with the task of determining The amino acid sequence of a protein, it is practical to sequence the corresponding gene and reconstruct the amino acid sequence based on the genetic code. Although any DNA can in principle be read in six different reading frames (three in each strand), the correct reading frame is determined by the following property: it is usually the only reading frame in which stop codons are rare (see Section 5.1.6). To ensure that we have not made an error in deducing the amino acid sequence of a protein from the nucleotide sequence of the corresponding gene,

Fig. 4-65. Two enzymatic methods commonly used to prepare radioactive DNA molecules. A. DNA polymerase I labels all nucleotides in a DNA molecule, yielding highly radioactive DNA probes. B. Polynucleotide kinase labels only the 5' ends of DNA. If DNA labeling is combined with restriction nuclease Treatment, as shown in the figure, the fraction of DNA molecules containing single strands labeled at the 5' end can be detected quite easily.

Fig. 4-66. A family of 5'-end-labeled DNA fragments generated by cleavage at a specific nucleotide (in this case, base A). The strand being analyzed is the product of denaturing a double-stranded molecule isolated by the method described in Fig. 4-65, B. This strand was subjected to mild chemical treatment that removes a small fraction of one of the four nucleotides from the chain; most of these nucleotides remain in the chain. Since only the fragments shown on the left contain the 5' end and its ³²P-phosphate group, only these fragments are detected after gel autoradiography. This Procedure forms The basis of the chemical method of DNA sequencing described in Fig. 4-67. One should directly sequence a small region of the amino acid sequence in the purified protein.

The Modification of the DNA sequencing method shown in Figs. 4-66 and 4-67 can be used to identify DNA sequences recognized by DNA-binding proteins. The binding of these proteins to regulatory regions of DNA (which are usually located outside the coding regions of genes) apparently plays an important role in determining which genes are active in a given cell type. Understanding the function of these proteins is extremely important for identifying the specific sequences to which they bind. To identify such sequences, a method called DNA footprinting is commonly used. First, a purified DNA fragment is labeled at one end with ³²P and then cleaved with a nuclease or a chemical compound that makes random cuts in the DNA double helix. The fragments generated from the labeled strand are separated on a gel and detected by autoradiography; the pattern of DNA bands produced in the presence and absence of DNA-binding proteins is then compared. If binding has occurred, nucleotides at the cleavage site are protected from the action of the nuclease. As a result, labeled fragments containing the binding site are absent, creating a gap on the gel that contains no DNA fragments, known as a "footprint" (Fig. 4-69, A). Fig. 4-69, B shows a footprint of proteins that activate Eukaryotic Transcription.

Fig. 4-67. Chemical method of DNA sequencing. The procedure described in Fig. 4-66 is performed simultaneously on four identical DNA samples. Chemical agents are used that cleave DNA at T in the first case, at C in the second, at G in the third, and at A in the fourth. The resulting samples are subjected to electrophoresis in parallel lanes of a single gel, as shown in Fig. 4-64, A. By analyzing the electrophoresis results, the DNA sequence can be determined. Thus, the bottom-most band corresponds to the nucleotide located at the 5' end. The lane in which the band is located is identified—in this case, it is T. To determine the complete sequence, the same procedure is performed for the second band, then the third, and so on. In this diagram, the method is idealized; in reality, the chemical treatment is less specific than shown here.

Molecules terminating in each of the dideoxyribonucleotides are detected as colored bands based on the corresponding colored primers. In this way, The sequence of the complementary DNA strand can be read directly as the bands pass the detector

Fig. 4-68. Nucleic acid sequencing method based on the enzymatic incorporation of a chain-terminating nucleotide. Key to this method is The Use of dideoxyribonucleoside triphosphates, which lack the 3'-OH group of deoxyribose present in normal nucleotides; when incorporated into a DNA strand, such a modified nucleoside blocks The addition of the next nucleoside. A. In vitro synthesis of a DNA molecule in the presence of a primer and a small amount of one of these modified nucleosides leads to The formation of a "ladder" of DNA fragments, as shown in Fig. 4-66. If radioactive DNA is used to generate these fragments, four separate synthesis reactions are performed, each using a different chain-terminating nucleoside, and electrophoretic analysis is carried out on four parallel gel lanes, the nucleotide sequence of the DNA can be determined (see Fig. 4-67 and Fig. 4-64, A). B. A more modern modification of the method, in which four sets of differently labeled fragments are automatically analyzed by fluorescence as they migrate down a single gel lane.

Fig. 4-69. DNA footprinting method. A. A protein binds tightly to a specific eight-nucleotide region of DNA and protects it from a cleaving agent. If the reaction is performed without the DNA-binding protein, a complete set of bands will appear on the gel (not shown). B. An actual footprint used to determine the binding site for a human protein that stimulates transcription of certain eukaryotic genes. The results indicate that this site is located 60 nucleotides upstream of the RNA Synthesis initiation site. A low-molecular-weight iron-containing organic compound was used as the cleaving agent. This substance normally cleaves each phosphodiester bond with nearly equal frequency. (B - courtesy of Michelle Savadogo, Robert Roeder.)

4.6.7. Nucleic acid hybridization is a sensitive method for detecting specific nucleotide sequences [43]

If an aqueous solution of DNA is heated to 100 °C and made highly alkaline (pH 13), the complementary base pairs holding the two strands of The Double Helix together will disrupt, and the DNA will rapidly dissociate into two single strands. This process, known as DNA Denaturation, was previously thought to be irreversible. However, in 1961, it was discovered that if complementary DNA strands are incubated at 65 °C, they readily reassociate, restoring the double-helix structure (a process termed renaturation or hybridization). Similar hybridization processes can occur between any two single strands of Nucleic Acids (DNA–DNA, RNA–RNA, DNA–RNA), provided they contain complementary nucleotide sequences.

The rate of double-helix formation is limited by the probability of collision between two complementary nucleic acid sequences, which in turn is determined by their concentration in solution. The rate of hybridization can be used to determine the concentration of any RNA or DNA sequence in a mixture containing other nucleic acid sequences. This assay requires a pure single-stranded DNA fragment complementary to the sequence to be detected. This DNA fragment can be obtained by cloning, or, if the sequence is short, it can be synthesized chemically. In either case, the DNA fragment is heavily labeled with ³²P (see Fig. 4-65) to monitor the incorporation of this molecule into duplexes during the hybridization reaction. The single-stranded DNA molecule used here as an indicator is called a DNA probe; it can contain from 15 to 1000 nucleotides.

Hybridization assays using DNA probes are so sensitive and selective that they can identify sequences present at a concentration of 1 molecule per cell (Fig. 4-70). This allows researchers to determine how many copies of a DNA sequence complementary to the DNA probe are present in a cell's genome. The same method is highly effective for finding non-identical but related genes; for example, once mouse or chicken genes of interest have been cloned, their sequences can be used to search for corresponding genes in The Human Genome.

DNA probes are also used in hybridization reactions with RNA to detect the expression of a given gene in cells. In this case, a DNA probe containing a portion of the gene sequence is hybridized with RNA isolated from the analyzed cell. If hybridization occurs, expression is quantified. More advanced techniques involve treating the DNA probe with specific nucleases to detect regions that hybridize with cellular RNA. In this way, the start and end regions of RNA transcripts can be determined (Fig. 4-71); this same method can be useful for determining the precise boundaries of regions excised from RNA transcripts during RNA splicing.

During embryonic development, large groups of genes are switched on and off in a coordinated manner. Hybridization of a DNA probe with cellular RNAs allows one to determine whether a specific gene is active or silent; moreover, when the level of gene expression changes, one can find out whether this change depends on control,

Fig. 4-70. Measuring the copy number of a specific gene in a DNA sample using DNA hybridization. The single-stranded radioactive DNA fragment used in such experiments is called a DNA probe. In this case, the chromosomal DNA does not contain radioactive atoms.

Fig. 4-71. Use of nucleic acid hybridization to determine the region of a cloned DNA fragment that is transcribed into mRNA. This method involves treatment with a nuclease that cleaves DNA strands not paired with a complementary RNA strand. This method allows precise identification of the start and end of the RNA molecule. Similar Procedures are also effective for mapping introns (non-coding sequences of eukaryotic genes).

operating at the level of DNA Transcription, RNA splicing, or the Translation of mature mRNA molecules into protein. Hybridization methods are so widely used in modern cell biology that it is difficult to imagine how Gene Structure and expression could be studied without them.

4.6.8. Northern and Southern blotting methods allow hybridization of nucleic acid molecules previously fractionated by electrophoresis [44]

To detect nucleic acid molecules with sequences complementary to the entire probe or a portion of it, DNA probes are often used in combination with gel electrophoresis. A mixture containing many different RNA and DNA molecules is fractionated by electrophoresis according to size, followed by a hybridization reaction; if the probe labels molecules of one or more specific sizes, one can be confident that the hybridization is sufficiently specific. In some cases, even information about the size of the hybridized DNA molecules is highly valuable. This is illustrated by the following example.

Suppose a researcher aims to determine The Nature of a defect in a mutant mouse that synthesizes abnormally low amounts of albumin (a protein normally secreted into the Blood by Liver cells in large quantities). To do this, liver tissue samples must first be taken from mutant and normal mice (the latter serving as controls) and the cells treated with a strong detergent to inactivate cellular nucleases, which would otherwise degrade the nucleic acids. RNA and DNA are then separated from all other cellular components: the proteins present are completely denatured and removed by sequential extraction with phenol, a powerful organic solvent. The nucleic acids remain in the aqueous phase. To separate them from low-molecular-weight cellular compounds, alcohol precipitation is performed. After this, DNA is separated from RNA based on their differing solubilities in alcohols and treated with highly specific enzymes (RNase or DNase, respectively) to eliminate unwanted nucleic acid contaminants.

To analyze albumin-encoding RNAs using a DNA probe, Northern blotting is employed. In the first step, intact RNA molecules from mutant and control liver cells are fractionated by gel electrophoresis, yielding a pattern of bands. To make the RNA molecules in the gel more accessible to the DNA probe, the fractionated RNA molecules are transferred (blotted) from the gel onto a sheet of nitrocellulose. In the next step, the nitrocellulose sheet is incubated with a solution containing the labeled DNA probe. The RNA bands that hybridize with the probe are detected by autoradiography (Fig. 4-72). It is well known that the migration rate of nucleic acid molecules in a gel depends on their size: during electrophoresis, smaller molecules move faster than larger ones. By comparing the migration rate of the molecules in our sample of interest with RNA molecules of known size (RNA standards), the size of each probe-binding molecule can be determined. It may turn out that the liver cells of mutant mice synthesize albumin RNA in normal amounts, and the size of these molecules is normal. If this were not the case, a reduced amount of normal albumin RNA molecules would be detected. Alternatively, the RNA molecules from mutant liver cells might be truncated and, as a result, migrate faster through the gel than normal. In this latter case, the blot containing the defective RNA molecules can be re-hybridized with more sensitive DNA probes to map the missing regions.

Fig. 4-72. Northern and Southern blotting methods. After electrophoretic fractionation of a mixture of DNA or RNA molecules in an agarose gel, the various nucleic acid fragments are transferred to a sheet of nitrocellulose or nylon ("blotting"). This sheet is then incubated with a radioactive DNA probe for an extended period under conditions that favor hybridization. The sheet is then thoroughly washed, so that only those fragments that hybridize with the DNA probe remain radioactively labeled. On the autoradiograph obtained from the nitrocellulose sheet, these fragments appear as bands.

To analyze the STRUCTURE OF THE albumin gene in mutant mice, Southern blotting was used. In this case, DNA is analyzed instead of RNA. Isolated DNA is first digested with restriction nucleases, then the resulting fragments are separated by size using gel electrophoresis, and those complementary to the albumin DNA probe are detected by transfer and hybridization, as described for RNA (see Fig. 4-72). By repeating this procedure with different restriction nucleases, a detailed restriction map of the genome in the region of the albumin gene can be constructed (see Section 4.6.2). Analyzing this map can reveal whether the albumin gene in mutant animals carries rearrangements, such as deletions or insertions of short DNA fragments.

4.6.9. Synthetic DNA probes allow Prenatal Diagnosis of Hereditary diseases [45]

While microbiologists were developing DNA Cloning METHODS, organic chemists perfected techniques for synthesizing short DNA fragments. Today, this is done using instruments capable of automatically synthesizing any sequence of up to 80 nucleotides overnight. The ability to obtain DNA molecules of any specified sequence makes it possible to engineer genes, which is a key aspect of genetic engineering (Chapter 5).

An important application of DNA oligonucleotides is the prenatal diagnosis of hereditary diseases. More than 500 human Genetic Disorders are associated with defects in a single gene. In most cases, these Mutations are recessive, meaning that the disease develops only if an individual inherits defective copies of the gene from both parents. One goal of modern medicine is to detect such abnormal embryos before birth, inform the mother, and offer her the option to terminate the Pregnancy. For example, in Sickle-Cell Anemia, the exact nucleotide substitution in the mutant gene is known (the sequence GAG is replaced by GTG in the DNA strand encoding the ß-chain of hemoglobin). In this case, two oligonucleotides are synthesized. One corresponds to the sequence of the normal gene in the region of the suspected mutation, while the other carries the mutation that causes the disease. When these sequences are short enough (about 20 nucleotides) and at a hybridization Temperature where only perfectly matched strands remain stable, radioactive probes can be used. The test involves isolating DNA from embryonic cells in the Amniotic Fluid (obtained via amniocentesis) and using it for Southern blotting with radioactive DNA probes. A defective embryo is easily identified because its DNA will hybridize only with the oligonucleotide complementary to the mutant DNA sequence. Unfortunately, for most hereditary diseases, the defect at the DNA level has not yet been deciphered; however, the range of diseases for which prenatal diagnosis is available is constantly expanding. This has been made possible by utilizing The phenomenon of restriction fragment length polymorphism (RFLP). In this approach, hybridization is used to detect the presence or absence of specific restriction sites tightly linked to the defective genes.

4.6.10. Hybridization also allows the detection of distantly related genes [46]

The Emergence of new genes during evolution is linked to the divergence and duplication of ancestral genes, as well as the recombination of gene segments in new combinations. For this reason, most genes belong to families of related sequences within the genome, some of which likely share similar functions. Isolating a DNA clone corresponding to the first member of such a gene family is a highly laborious procedure (Section 5.6.5). However, isolating the remaining genes in the family is simplified because the first gene can be used as a probe. Since related genes are unlikely to share identical sequences, hybridization with DNA probes is typically performed under less stringent conditions. Consequently, even an incomplete match to the probe sequence can form a stable double helix (Fig. 4-73).

Although using non-stringent hybridization increases the likelihood of false signals from random, short regions of homology in unrelated parts of DNA, such hybridization represents one of the most successful Applications of recombinant DNA technology. For example, this approach led to the Isolation of the entire family of DNA-binding proteins that function as key regulators of gene expression during early embryonic development in Drosophila (see Section 16.5.19). The same approach was used to identify genes related to this family in other organisms, including humans.

Fig. 4-73. Comparison of several modifications of the hybridization method differing in stringency. In the reaction on the left (high stringency), the temperature of the solution is maintained only a few degrees below the denaturation temperature of the fully complementary DNA helix (its melting temperature). Under these conditions, mismatched helices that form at lower stringency (see right) are unstable. On the right, the hybridization conditions used to search for related genes that are not completely identical to gene A are shown.

4.6.11. In situ hybridization is used to localize specific nucleic acid sequences in chromosomes and cells [47]

All cellular macromolecules, including nucleic acids, occupy strictly defined positions within Tissues and cells. Extracting these molecules from tissues or cells by homogenization results in the loss of information regarding their spatial distribution. Therefore, methods have been developed to localize specific nucleic acid sequences in situ—in isolated chromosomes or specific cell types—where DNA and RNA probes are used in much the same way as labeled Antibodies. This method is called in situ hybridization. It is used to analyze DNA in chromosomes and RNA in cells. Highly radioactive nucleic acid probes are hybridized with chromosomes after a brief exposure to high pH to separate the DNA base pairs. The chromosomal regions that bind the radioactive probes during hybridization are detected by autoradiography. The spatial resolution of this method can be increased by using chemically labeled rather than radioactively labeled DNA probes. Typically, probes are synthesized using nucleotides containing a biotin side chain, and the hybridized probes are detected by staining with streptavidin (whose molecules form a network) or other marker molecules (Fig. 4-74). In situ hybridization methods have also been developed to determine the distribution of specific RNA molecules in cells within tissues. In this case, the tissues are not exposed to high pH, so the chromosomal DNA remains double-stranded and cannot bind the probe. However, if the tissue is gently fixed,

the RNA it contains can hybridize when the tissue is incubated with a complementary DNA probe. In this way, it was possible to observe the differential gene activity in Drosophila (Fig. 4-75). This approach has led to significant progress in studying the MOLECULAR MECHANISMS OF differentiation in various embryonic cells.

4.6.12. Recombinant DNA technology makes it possible to study even minor cellular proteins [48]

Until recently, the Study of Cellular proteins was limited to major fractions, i.e., proteins present in cells in relatively large amounts. Using conventional chromatography and electrophoresis methods, about 0.1 g (100 mg) of a major protein, constituting 1% or more of the total cellular protein, can be obtained from several hundred grams of cell mass. This amount of protein is quite sufficient for sequencing its Amino Acids, detailed analysis of its biological or enzymatic activity (if any), and raising antibodies that can be used to localize the protein in cells. Furthermore, if suitable crystals can be grown, x-ray crystallography can be used to determine the three-dimensional structure of the molecule. It was in this manner that the structure and function of many abundant proteins, including hemoglobin, Trypsin, immunoglobulin, and Lysozyme, were determined.

A Eukaryotic Cell contains thousands of different proteins, but the vast majority of these, including the most interesting ones, are present in small amounts. Some of them are sometimes extremely difficult, if not impossible, to obtain in pure form in quantities exceeding a few micrograms. The development of recombinant DNA technology has made any cellular protein (including minor proteins) available in large quantities. To achieve this, the gene for the desired protein is cloned and then inserted into a special plasmid called a cloning vector. This vector is designed so that when introduced into bacteria, yeast, or mammalian Cells of the appropriate type, it directs the large-scale Synthesis of the protein. Thus, whereas previously only a few proteins were available for detailed structural or functional studies, virtually any cellular protein can now be subjected to such investigation.

Fig. 4-74. Localization of a gene on a Drosophila polytene chromosome by in situ hybridization with a cloned biotin-labeled DNA probe. The DNA of this giant chromosome is partially denatured to allow hybridization with the probe. After hybridization and washing of the probe, the chromosomes are treated with an enzyme complex in which horseradish peroxidase is conjugated to streptavidin (see Fig. 4-58, B). To detect the binding site of the probe, the preparation is treated with hydrogen peroxide and stained; the peroxidase is detected as a dark band on the preparation (arrow). (Courtesy of Tod Leverty, Gerald Rubin.)

Fig. 4-75. Autoradiograph of a section of a very early Drosophila embryo subjected to in situ hybridization using a radioactive DNA probe complementary to a gene involved in segment formation, ftz. The probe hybridizes with the embryonic RNA, and the distribution of silver grains after development indicates that the RNA synthesized by the ftz gene is localized in regular bands extending across the entire embryo. The width of the bands corresponds to three to four cells. At these Selection/3.html">Stages of development (cellular blastoderm), the embryo consists of approximately 6000 cells. (From E. Hafen, A. Kuroiwa, W. J. Gehring. Cell, 37; 833-841, 1984.)

4.6.13. Gene function is most clearly revealed in mutant organisms [49]

Suppose someone has succeeded in cloning a gene encoding a newly discovered protein. How can the intracellular function of this protein be determined? This is a very difficult task, as neither the three-dimensional structure of the protein nor the complete nucleotide sequence of its gene allows one to deduce its function. Furthermore, many proteins, such as structural proteins and components of complex multi-enzyme complexes, do not exhibit their normal activity when separated from the Other components of the complex functional unit to which they belong.

One approach we have already discussed (see Section 4.5.6) is to inactivate a specific protein with specific antibodies and observe which cellular processes are affected as a result. In some cases, this method allows for a fairly reliable Determination of protein function, but it is not very effective for intracellular proteins because microinjected antibodies are diluted during cell proliferation or destroyed by intracellular degradation. A more successful solution to this problem is possible through genetic approaches. Mutants that lack a particular protein or, more conveniently, synthesize a temperature-sensitive form of it (inactivated by a slight increase or decrease in temperature) are extremely useful when investigating protein function. They have been used to identify the functions of enzymes involved in major bacterial metabolic pathways. This approach also led to the discovery of many gene products responsible for the orderly development of Drosophila embryos. Typically, this method is used to study organisms with short generation times, such as bacteria, yeast, Roundworms, and fruit flies. By treating them with substances that cause Changes in DNA (mutagens), one can quickly obtain a large number of mutants and select those carrying a specific defect of interest. For example, from a mutagenized bacterial population, cells were selected that stop DNA Synthesis when the ambient temperature is raised from 30° to 42°C. Among these, a significant number of temperature-sensitive mutants were identified, with mutations affecting bacterial proteins involved in DNA Replication. These mutants were later used to identify and characterize the proteins required for DNA replication.

The human reproductive cycle is very long. Furthermore, no one would intentionally expose humans to mutagens. Moreover, a human fetus with severe defects in vital processes, such as DNA replication, would die long before birth. However, many mutations may have virtually no effect on viability—for example, tissue-specific defects in Lysosomes or cell-surface receptors that arise spontaneously in the human population. Analyzing the phenotype of these patients, as well as studying their cells in culture, provides a unique opportunity to investigate important cellular functions. Although such mutations are extremely rare, they are effectively identified because those who carry them seek medical attention.

4.6.14. Cells and organisms containing altered genes can be engineered [50]

Obtaining Mutants with Impaired DNA replication or, for example, eye development is in principle quite simple. However, linking this defect to an alteration in a specific protein can take years. Recombinant DNA technology has provided researchers with a completely different approach: the analysis begins with the protein and ends with the creation of a mutant cell or a whole Organism. Because this approach is the reverse of classical genetic analysis, which proceeds from gene to protein, it is commonly called reverse genetics.

Reverse genetics begins with isolating the desired protein from the cell. Using the methods described in Chapter 5, the gene for this protein is cloned and sequenced; this sequence is then altered by Biochemical Methods to create a mutant gene encoding an altered form of the protein. This gene is then introduced into a cell, where it can integrate into a chromosome via Homologous Recombination, thereby becoming a permanent part of the genome. If the integrated gene is expressed, the host cell and all its progeny will synthesize the altered protein. When an in vitro altered gene is introduced into a fertilized egg, a multicellular mutant organism is produced. Some of these transgenic organisms will pass this gene on to their offspring as a permanent element of germline cells (Fig. 4-76). Such genetic transformation is now becoming a routine procedure for fruit flies and mammals. In principle, human transformation is also entirely feasible today, but such experiments are not performed out of concern for potential genetic disorders that cannot be ruled out in individuals undergoing such procedures.

4.6.15. Specific dominant mutations can be created using artificial genes encoding antisense RNAs [51]

When mutant genes are introduced into bacterial or yeast cells, which are typically haploid, they will recombine with their normal homologs quite frequently. As a result, cells can be selected in which the mutant gene has replaced the single copy of the normal gene (Fig. 4-77, A), such as cells synthesizing a mutant form of a specific protein. The function of the normal protein can usually be determined from the phenotype of the mutant cells. In higher eukaryotes, such as mammals or fruit flies, methods to easily replace a normal gene with a cloned mutant gene have not yet been developed. Genetic Transformation of these organisms usually results in the insertion of the cloned gene into random sites in the genome, so that the cell or organism contains the mutant gene alongside its normal copy (Fig. 4-77, B).

Fig. 4-76. Comparison of a normal Drosophila larva and two mutant larvae containing defective ftz genes. One of the defective (ftz') larvae was transformed after injection of cloned DNA containing the normal ftz gene sequence into the egg from which it developed. This additional DNA sequence integrated into one of the fly's chromosomes and was subsequently inherited and expressed normally. The ftz gene is required for normal development, and its addition to the defective genome, as shown by the experiment, restores the larval segments missing in ftz' organisms. METHODS FOR PRODUCING Transgenic Animals are discussed later (see Fig. 5-88). (Courtesy of Walter Gehring.)

Fig. 4-77. A gene with an altered nucleotide sequence can be introduced into the chromosome of a host organism. In bacteria and yeast, mutants can be selected in which (A) the altered gene has replaced the normal one as a result of genetic recombination. In this case, only mutant genes are retained in the cells. In higher eukaryotes, gene addition occurs instead of replacement (B). Their transformed cells or organisms contain mutant genes In addition to normal ones. It is believed that in organisms characterized by an excess of DNA, gene replacements occur quite rarely because it is necessary for the mutant gene to "find" its normal homolog among many other sequences and pair with it.

Fig. 4-78. Use of the antisense RNA strategy to obtain dominant mutations. Mutant genes have been engineered to synthesize RNA with a sequence complementary to the RNA synthesized by normal genes. These Two Types of RNA are capable of pairing to form double-stranded molecules. If a significant excess of antisense RNA is synthesized, it can hybridize with and thereby inactivate most of the normal RNA synthesized by gene X. It is believed that this approach could eventually be used to inactivate any gene. Currently, this technique is applicable only to certain genes.

It would be extremely useful to create specific dominant mutations in higher Eukaryotic cells by introducing mutant genes that would eliminate The activity of their normal counterparts in the cell. To achieve this, a highly ingenious and promising approach is used, based on the specificity of hybridization reactions between two complementary DNA strands. It is known that, under normal conditions, only one of the two DNA strands in a given region is transcribed into RNA, and this is always the same strand for a given gene. If, however, a cloned gene has been engineered so that only the oppositely oriented DNA strand is transcribed, an antisense RNA is produced with a sequence complementary to normal RNA transcripts. When such antisense RNA is synthesized in sufficiently large amounts, it will hybridize with high frequency to the "sense" RNA synthesized by normal genes, thereby inhibiting the synthesis of the corresponding protein (Fig. 4-78). If this protein is vital for the cell or organism, the dominant mutants described here will die, making it impossible to study the protein's function. To prevent this, genes can be engineered to synthesize antisense RNA on demand—for example, in response to a temperature change or in the presence of a specific signaling molecule. Cells or organisms containing such inducible antisense genes will be deprived of the specific protein at a particular time, allowing the resulting effect to be monitored. Of course, this method is not yet fully developed technically, but it is already clear that it is highly promising for determining protein function in higher organisms.

Conclusion

Recombinant DNA technology has revolutionized cell biology. Today, restriction nucleases can be used to excise any segment of cellular DNA and insert it into a self-replicating genetic element (a plasmid or virus) to obtain a "genomic DNA clone." Alternatively, a DNA copy of any RNA molecule can be used to obtain a "cDNA clone." In this way, unlimited quantities of highly purified DNA can be produced, and its nucleotide sequence can be determined (at a rate of hundreds of nucleotides per day), as well as the amino acid sequence of the protein it encodes. Genetic engineering techniques make it possible to synthesize mutant genes and introduce them into cell chromosomes, where they subsequently become a permanent part of the genome. If a fertilized egg is used as the recipient for gene transfer, transgenic organisms can be obtained that express the mutant gene and pass it on to their offspring. For cell biology, it is exceptionally important that these methods allow cells to be modified in a highly targeted manner, which in turn makes it possible to assess The impact of altering a specific protein's structure on the cell.

The applications of recombinant DNA technology open up broad Prospects. Using these methods, bacterial, yeast, and mammalian cells can be converted into "factories" for the large-scale production of virtually any protein. This enables detailed Analysis of Protein structure and function, or their use as therapeutic agents. In addition, recombinant DNA technology is the basis for producing highly specific DNA probes, which are used to study gene expression in tissues, localize genes on chromosomes, and identify genes with related functions.

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