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

Molecular Organization of Cells
Basic Genetic Mechanisms
DNA Cloning and Genetic Engineering

The discoveries discussed in this chapter arose from the desire of scientists to understand Cell life and the basic mechanisms of heredity. In recent years, however, this fundamental knowledge has found Structure/179.html">Practical Applications. DNA Cloning and Introduction/32.html">Genetic Engineering techniques make it possible to isolate specific genes in sufficient quantities, "tailor" them as desired, and then reintroduce them into various Cells and organisms. These Methods represent only a portion of the broader toolkit known as Recombinant DNA technology (which we discussed in Chapter 4). The advent of recombinant DNA technology has truly revolutionized the Study of Living cells. Furthermore, it has opened new avenues for medicine and industry to obtain sufficient quantities of Proteins that were previously either completely unavailable or obtainable only in very small amounts.

5-48

5-49

5-50

5.6.1. Restriction Nucleases facilitate Gene cloning [55]

In the 1960s, the possibility of isolating a single gene seemed infinitely remote. Unlike a protein, a gene in a cell is not a discrete unit; it is merely a small segment of a much larger DNA molecule. Although DNA molecules can be broken into many segments by mechanical shearing, this Cleavage occurs at random, and the fragment containing the gene of interest can easily be lost among millions of other fragments. How can a specific gene be obtained in purified form? Because all DNA molecules are composed of the same four NUCLEOTIDES in roughly equal proportions, they cannot be easily separated like proteins based on differences in charge or their ability to bind to specific Reagents (see Section 4.4.3). Furthermore, even if a purification scheme could be devised, a vast amount of DNA would be required to obtain the desired gene in quantities sufficient for subsequent experiments. The solution to all these problems became possible with the discovery of Enzymes called restriction nucleases (restriction enzymes). These enzymes, which can be purified from bacterial cells, cleave the DNA double helix at specific sequences of 4–8 nucleotides, cutting it into fragments of strictly defined size (known as restriction fragments). The sequence Specificity of restriction nucleases varies among different bacterial species, making it relatively easy to select a restriction enzyme that will excise a small DNA fragment containing a specific gene. The size of this restriction fragment then serves as a guide for the partial purification of the gene (isolating it from the mixture).

Class="center">

Figure 5-78. Cohesive ends produced by many restriction nucleases (see Fig. 4-63) allow two DNA fragments to join through complementary base-pairing. DNA fragments joined in this way are then covalently linked in a highly efficient reaction catalyzed by DNA ligase. Shown here is The formation of a recombinant DNA molecule consisting of a plasmid and an inserted chromosomal DNA fragment.

Another property of restriction nucleases makes them convenient tools for gene cloning. Many of them make staggered cuts, producing short, single-stranded "tails" at both ends of the DNA fragment. These are called cohesive ends because they can pair with any other end produced by the same enzyme. Thanks to the cohesive ends generated by a restriction nuclease, two fragments of a DNA double helix from different genomes can be joined together by base-pairing (Fig. 5-78). For example, a DNA fragment containing a human gene can be joined in vitro to the chromosome of a bacterial virus, and the resulting recombinant DNA molecule can then be introduced into a bacterial cell. Starting with just this single recombinant DNA molecule infecting a single bacterial cell, the viral Replication machinery can produce more than 10¹² identical Viral DNA molecules in less than a day, thereby amplifying the attached human DNA fragment to the same extent. A virus used for this purpose is called a cloning vector.

5-51

5.6.2. Construction of a DNA library using viral or Plasmid Vectors [56]

Cloning a particular gene begins with the construction of a DNA library in a viral or plasmid vector. The principles underlying gene cloning are the same for both types of vectors, although the methods may differ in detail. For simplicity, we will ignore these differences in this chapter and illustrate the methods using Plasmids as an example.

Figure 5-79. Purification and Amplification of a specific DNA sequence by DNA cloning in bacterial cells.

Plasmid vectors used in gene cloning are small, circular, double-stranded DNA molecules derived from larger plasmids naturally occurring in bacterial, Yeast, and mammalian cells (see Section 5.5.11). Typically, they constitute only a small fraction of the host cell's total DNA, but because of their small size, they can be easily separated from chromosomal DNA molecules, which, being much larger, sediment to the bottom of the tube during centrifugation. Cloning begins by treating purified circular plasmid DNA molecules with a restriction enzyme to produce linear DNA molecules. Next, the genomic DNA from which the library is to be constructed is cleaved with the same restriction enzyme, and the resulting restriction fragments (including those containing the gene to be cloned) are added to the cut plasmids and allowed to anneal so that recombinant circular DNA molecules can form. Covalent linkage by DNA ligase completes the formation of these recombinant molecules containing foreign DNA inserts (see Fig. 5-78).

The next step in constructing a DNA library is to introduce the recombinant circular DNA molecules into cells (usually bacterial or yeast) that have been rendered temporarily permeable to DNA; the cells are said to be transfected with the plasmids. As these cells grow and divide, the recombinant plasmids also replicate, eventually producing a vast number of copies of the circular molecules containing the foreign DNA (Fig. 5-79). Many bacterial plasmids carry Antibiotic Resistance genes. This property can be used to select transfected cells from untransfected ones: if Bacteria are grown in the presence of the antibiotic, only those cells containing the plasmids will survive and form colonies. These surviving cells constitute the DNA library. However, only a few of them carry the recombinant plasmids containing the gene to be isolated. We must be able to identify them to obtain the DNA of interest in purified form and in sufficient quantity. Before discussing how this can be achieved, we should describe another pathway for constructing a DNA library that is also used in cloning.

5.6.3. Two Types of DNA libraries are used for different purposes [57]

The fragmentation of a genome with a restriction nuclease, described above as one of the steps in cloning, is sometimes called "shotgun cloning". This produces a vast number of DNA fragments—up to a million for a mammalian genome—which means that the number of different colonies of transfected cells must also reach millions. Each such colony represents a clone, i.e., a population of descendants from a single progenitor cell, and the recombinant plasmid in every cell of the clone carries the exact same inserted genomic DNA sequence. The plasmids in a single colony contain a genomic DNA clone, and the entire collection of plasmids constitutes a genomic DNA library. However, because the fragmentation of genomic DNA is random, only some of the resulting fragments contain complete genes; many contain only a portion of a gene, and in most genomic DNA clones derived from the DNA of higher Eukaryotic cells...

Figure 5-80. Synthesis of cDNA. Reverse Transcriptase synthesizes a DNA copy (cDNA) of an mRNA molecule, thereby forming a hybrid DNA/RNA helix. Under alkaline conditions, the RNA strand of this hybrid helix is degraded into individual nucleotides, after which the remaining single-stranded cDNA is copied by DNA polymerase to form double-stranded cDNA. As shown in the diagram, both enzymes require a primer to initiate synthesis. For reverse transcriptase, the primer is a short oligonucleotide; in this case, oligo(dT) is annealed to the long polyA sequence present at the 3' end of almost all mRNAs.

Note that the double-stranded cDNA molecule formed here lacks cohesive ends; such "blunt-ended" DNA molecules can be cloned using several Procedures similar to the one shown in Fig. 5-78, though less efficiently. For example, one can ligate synthetic oligonucleotides containing restriction sites to the ends of the cDNA, or enzymatically add single-stranded "tails" to the ends of the cDNA molecule to facilitate its insertion into a cloning vector, only non-coding DNA is present, which, as is well known, constitutes the major part of such a genome (see Section 9.1.3).

An alternative method begins the cloning process by selecting those DNA sequences that are transcribed into RNA and, therefore, presumably correspond to genes. This is accomplished by extracting mRNA (or a purified subfraction of mRNA) from cells and then preparing a complementary DNA (cDNA) copy of each mRNA molecule present; this reaction is catalyzed by reverse transcriptase, a retroviral enzyme that synthesizes a DNA strand on an RNA template (see Section 5.5.8). The single-stranded DNA molecules synthesized by reverse transcriptase are converted into double-stranded DNA molecules by DNA polymerase, and these double-stranded molecules are inserted into plasmids to form clones (Fig. 5-80). Each clone obtained in this way is called a cDNA clone, and the entire collection of clones derived from a single mRNA preparation constitutes a cDNA library.

As shown in Fig. 5-81, genomic DNA clones and cDNA clones differ significantly. Genomic clones represent a random sample of all the DNA sequences of a given Organism. The composition of a genomic library is independent of The Cell type chosen for its preparation. In contrast, cDNA clones contain only those Regions of the genome that are transcribed into mRNA, and because different Tissues synthesize different sets of mRNA molecules, cDNA libraries vary depending on the cell types used to construct them.

Figure 5-81. Diagram illustrating the differences between cDNA clones and genomic DNA clones. In this example, gene A is transcribed rarely, whereas gene B is transcribed frequently. Both types of RNA transcripts undergo splicing, which removes the single intron shown here during mRNA formation. Most genes contain many introns (see Table 9-1).

Gene cloning from cDNA libraries offers several advantages. The first of these is that many proteins are produced by specialized cells in very large quantities, meaning that the mRNA encoding such a protein is also synthesized in Abundance. A cDNA library from these cells is naturally enriched with cDNA molecules encoding this specific protein (Fig. 5-81). This abundance of the desired cDNA molecules greatly simplifies the task of identifying the target clone in the library. Hemoglobin, for example, is synthesized in large amounts in developing red Blood Cells, which is precisely why globin genes were among the first to be cloned.

Another advantage of cDNA clones is that their coding nucleotide sequence is uninterrupted. Eukaryotic genes, as is well known, consist of coding DNA sequences separated by non-coding ones, so mRNA synthesis from these genes must be accompanied by the removal of non-coding (intronic) regions from the initial RNA transcript and splicing of the remaining fragments, i.e., the coding sequences. Neither bacterial nor yeast cells are capable of carrying out such Processing of RNA transcribed from a gene of a higher Eukaryotic Cell. Therefore, if the goal of cloning is to determine the Amino Acid Sequence of a protein from a given DNA or to obtain large quantities of the protein by expressing the cloned gene in a bacterial or yeast cell, it is preferable to start with cDNA.

Genomic DNA and cDNA libraries are an inexhaustible resource for such work, and researchers often share them; the number of such libraries supplied by specialized companies is also steadily growing.

Fig. 5-82. The Use of subtractive Hybridization to purify those rare cDNA clones that correspond to mRNA molecules present in T lymphocytes but absent in B lymphocytes. Since these two cell types are very similar, most of the mRNA should be common to both. This Procedure, therefore, represents a highly efficient way to enrich for the specialized molecules by which these two cell types differ from each other.

10-5

5.6.4. cDNA libraries can be prepared from selected populations of mRNA molecules [58]

If cDNA is prepared from cells in which the expression level of the gene of interest is very high, most cDNA clones will contain The nucleotide sequence of this gene, making its Selection straightforward. For genes that are transcribed less intensely, some methods are required to enrich the existing mixture of mRNA molecules with the desired species before constructing the cDNA library. If, for example, Antibodies to a given protein are available, they can be used to selectively precipitate those isolated polyribosomes that contain its growing polypeptide chains. These Ribosomes will also contain the mRNA encoding this protein, which can enrich the precipitate for the mRNA of interest by up to 1000-fold.

Subtractive hybridization is another highly efficient method for enriching a mixture of molecules with specific nucleotide sequences prior to cDNA cloning. This selection procedure can be used, for example, when two closely related cell types from the same organism differ only in that one produces the protein (or proteins) of interest. This method was first used to identify receptor proteins present On the surface of T lymphocytes but absent on B lymphocytes. It can also be used if a cell producing a certain protein has a mutant counterpart lacking this ability. The first step involves synthesizing cDNA using mRNA from the cell type that produces the desired protein. These cDNAs are then hybridized with a large excess of mRNA molecules from the second cell type. Consequently, a small fraction of the cDNA nucleotide sequences will not find a partner, i.e., a complementary mRNA sequence. Obviously, these few cDNA sequences correspond to mRNA sequences present only in the first cell type. They can be purified using a simple biochemical procedure that separates single-stranded Nucleic Acids from double-stranded ones (Fig. 5-82). cDNA libraries obtained by subtractive hybridization are useful not only for cloning genes whose products are restricted to a specific differentiated cell type; they also allow for the determination of differences in Gene Expression between any two closely related cell types.

5-52

5.6.5. Hybridization with a radioactive DNA probe can be used to identify the desired clones in a gene library [59]

In gene cloning, the most difficult task is identifying those rare colonies in a library that contain the DNA fragment of interest. This is especially true for Genomic Libraries, where finding a specific mammalian gene requires searching for a single cell among a million. Most often, a special form of hybridization in situ is used for this purpose, based on the high specificity of

Fig. 5-83. An efficient method widely used to identify a bacterial colony containing a specific DNA clone (see also Fig. 5-79). Each bacterial cell carrying a recombinant plasmid gives rise to a colony of identical cells, which appears as a white spot on nutrient Agar. By pressing a filter paper disc against The surface of the plate, a replica of the bacterial culture is obtained. This replica is treated with alkali to lyse the cells adhering to the paper and denature the plasmid DNA, followed by hybridization with a highly radioactive DNA probe. Bacterial colonies that have bound the DNA probe are detected by autoradiography.

complementary interactions between two complementary nucleic acid molecules. Bacterial colonies growing on plates are blotted with a piece of filter paper. In this process, some cells from each colony adhere to the paper. These adhering colonies—referred to as replicas—are treated with alkali and then incubated with a radioactive DNA probe containing a portion of the nucleotide sequence of the gene of interest (Fig. 5-83). If necessary, millions of bacterial clones can be screened in this manner to identify the clone capable of hybridizing with the probe.

Fig. 5-84. Selection of regions with a known amino acid sequence for the preparation of synthetic oligonucleotide probes. In reality, only one specific nucleotide sequence encodes the given protein. However, due to the degeneracy of METABOLISM/28.html">The Genetic Code, several different nucleotide sequences can yield the same amino acid sequence, so it is impossible to know in advance which one is correct. It is desirable for the correct nucleotide sequence to constitute the largest fraction in the mixture of oligonucleotides used as a probe; therefore, regions with the minimum number of possibilities are selected, as shown in the figure. Once the mixture of oligonucleotides is chemically synthesized, the 5' end of each oligonucleotide is radioactively labeled (see Fig. 4-65, B).

The method for obtaining a specific DNA probe depends on the information available regarding the gene to be cloned. In many cases, the protein of interest can be identified by chemical methods and isolated, at least in small amounts, in a purified form. A few micrograms of pure protein are sufficient to determine The sequence of the first thirty Amino Acids in its molecule. From this amino acid sequence, the corresponding nucleotide sequence can be deduced based on the genetic code. Once these data are obtained, two sets of DNA oligonucleotides, each approximately 20 nucleotides in length, are chemically synthesized and radioactively labeled. The two sets are used to ensure matching to two different regions of the predicted nucleotide sequence of the cloned gene (Fig. 5-84). Cell colonies that show The ability to hybridize with both sets of DNA probes are highly likely to contain the desired gene; therefore, these are selected and saved for further study (see below).

5.6.6. Isolation of overlapping DNA clones ('chromosome walking') allows for the identification of a gene adjacent to one that has already been cloned [60]

Many genes of greatest interest (such as those regulating development) are known to us only from genetic analysis of mutants in organisms like the fruit fly Drosophila or the nematode C. elegans. The protein products of these genes have not been isolated; they may be present only at a specific developmental stage. Nevertheless, by studying the genetic linkage of various Mutations, chromosome maps can be constructed to show the relative positions of these genes. If one of the genes mapped in this way has been cloned, clones in a genomic DNA library corresponding to neighboring genes can be identified using a technique known as 'chromosome walking'. This involves using two different genomic DNA libraries prepared from the same DNA by digesting it with two different restriction nucleases. Fig. 5-85 shows how a clone from one library can serve as a DNA probe to identify an overlapping clone from the other library. A DNA probe is then prepared from this new clone and used to find another overlapping clone in the first library, and so on. In this way, by finding clone after clone, one can walk along the chromosome, moving a distance of about 30,000 nucleotide pairs or more each time. How do we know, however, when we have finally reached the gene of interest (originally identified by some deleterious mutation)? A common approach is to continuously compare the sizes of restriction DNA fragments from mutant and normal Chromosomes using Southern blot analysis, using each new clone as a probe during the 'walk'. Some mutants arise from small deletions or, conversely, insertions of DNA sequences in the corresponding gene, and such disruptions are usually easy to detect. For example, it is known that among deleterious mutations affecting a typical human gene, about one in ten is a deletion easily detected by Southern blot analysis. The number of molecular defects responsible for human Hereditary diseases identified in this way has been steadily increasing.

Fig. 5-85. The use of overlapping fragments to map a gene of interest by 'chromosome walking'. To reduce the 'walking' time, genomic libraries containing very large cloned DNA molecules are most suitable. The probe for each subsequent clone is a short ³²P-labeled DNA fragment from one end of the previously identified clone. If, for example, the 'right' end is used, the movement is to the 'right', as in the case shown in this figure. A short terminal fragment is also convenient as a probe because it reduces the likelihood of a repetitive DNA sequence being present in the probe, which could hybridize with many clones from different PARTS OF THE genome and thus disrupt the 'walk'.

Using Similar Methods, almost the entire set of large genomic clones can be ordered (mapped) along the chromosomes of C. elegans. These large clones, each about 30,000 bp, are inserted into special vectors derived from bacteriophage λ, known as cosmids, which are suitable for carrying large DNA inserts. A few thousand cosmid clones are sufficient to cover the entire genome of an organism like C. elegans or Drosophila, whereas mapping The Human Genome in this way would require more than 100,000 cosmid clones. This procedure would, of course, be very time-consuming, but it is technically feasible. Furthermore, human DNA fragments ten times larger than cosmid clones (300,000 bp) can be cloned as yeast artificial chromosomes; in principle, the human genome could be mapped using 10,000 clones of this type (see Fig. 9-5).

Fig. 5-86. Hybrid-selection technique. Purified mRNA molecules are eluted from the filter under conditions that cause the RNA-DNA double helix to dissociate into single strands.

In the near future, researchers will undoubtedly be able to purchase ordered sets of genomic clones from specialized centers housing DNA libraries. There, a complete library for any common research organism will be available, with catalogs specifying for each DNA insert both its chromosome of origin and its order relative to all other DNA fragments from the same chromosome. Beginning a "chromosome walk" will then be as simple as obtaining from the library all clones spanning the region containing the mutant gene of interest. These clones will then serve to prepare DNA probes that will allow precise localization of the altered gene. Ultimately, this approach will make it possible to isolate many of the mutant genes responsible for human hereditary diseases.

5.6.7. In vitro Translation facilitates the identification of the appropriate DNA clone [61]

Although highly effective, the isolation of genomic DNA and cDNA clones by in situ hybridization is somewhat inconvenient because such experiments typically select many "false-positive" clones. Additional techniques are required to distinguish them from true positives. The task is simplified if the desired clone encodes a protein that has already been characterized by other methods. In this case, each cloned DNA fragment to be tested is used to select its complementary mRNA molecules from a mixture of cellular mRNAs by a process called hybrid selection; an excess of the DNA fragment is denatured into single strands, immobilized on a filter, and incubated with the mRNA mixture to select complementary mRNA molecules by RNA-DNA hybridization (Fig. 5-86). The mRNA purified in this way is then used for Protein Synthesis in a cell-free system containing radioactive amino acids. The resulting radioactive protein is analyzed and compared with the protein product expected to be encoded by the clone. A match in their characteristics in such an assay usually justifies the Conclusion that the cloned DNA fragment encodes the protein.

5.6.8. Expression vectors can be used for protein overexpression [62]

Very often, no biochemical data are available for the protein encoded by a cloned DNA fragment. This is the case, for example, when the clone in question has been identified by subtractive hybridization or by "chromosome walking" to the corresponding mutant gene. Moreover, in such cases, the mRNA encoding this protein is often present in such small amounts or in so few cells that hybrid selection of the complementary mRNA is unfeasible. Other methods must then be used to characterize the protein product of the cloned gene. One method is to synthesize a short protein fragment (an oligopeptide) corresponding to the deduced amino acid sequence of the protein product of the sequenced cDNA molecule, and then raise antibodies against this oligopeptide. In many cases, these antibodies will recognize the same amino acid sequence within the native protein, allowing the detection, localization, and purification of the protein encoded by the original cDNA. This immunological approach, combined with cloning by subtractive hybridization, is effective both as a way to identify cell-type-specific proteins and as a path to studying the pattern of differentiation, as well as the Properties and Functions of each cell type in a multicellular organism.

However, the simplest way to characterize a protein encoded by a cloned cDNA is to have the cDNA itself direct protein synthesis in a host cell. Plasmids or Viruses serve as expression vectors in such cases; they are designed to link the cloned cDNA to a sequence that acts as a strong promoter for Transcription. Researchers have a fairly wide range of expression vectors at their disposal, each adapted to function in the specific cell type in which the protein is to be synthesized. Using this genetic engineering approach, bacterial, yeast, and mammalian cells can be made to produce large quantities of various valuable proteins, such as human Growth Hormone, interferon, or viral antibodies used for vaccine preparation. Bacterial cells with plasmid or viral vectors designed in this way are particularly convenient for protein production; the inserted foreign gene often accounts for over 10% of the total protein synthesized by the cell. Since synthesizing such a large amount of a single protein is often an overwhelming metabolic burden for the cell, special "inducible" promoters have been developed, allowing transcription to be turned on only a few hours before harvesting the cells for Protein Purification. Some plasmid expression vectors, for example, contain a promoter derived from bacteriophage λ. Its function is regulated by a Temperature-sensitive protein product of a repressor gene; by raising the temperature of the bacterial cells to 42°C, the promoter can be "switched on" at any time to rapidly obtain a large amount of the required protein.

If a cDNA library is constructed in an expression vector, each clone will produce a specific protein, and the clones of interest can then be identified by their protein products rather than their nucleotide sequences. In this case, radioactively labeled antibodies are typically used as probes for the clones. Alternatively, a direct assay detecting the biological activity of the gene product can sometimes be performed. This method is particularly effective when searching for eukaryotic genes responsible for secreted growth factors. For example, cDNA clones from cells producing a specific growth factor can be inserted into an expression vector that replicates in mammalian cells. A mixture of transfected cells containing many such different clones is grown in a small dish, where each cell expressing the gene secretes the growth factor into the medium. Samples of the medium are then assayed for the presence of the growth factor by adding them to cultures of other cells capable of responding to it. The assay is so sensitive that a positive response is obtained even if only one cell in a thousand in the starting culture contains the gene encoding the growth factor. Repeated testing allows the single clone producing the factor to be identified from the mixture. In this way, previously unknown growth factors have been discovered and isolated in a matter of months; if the procedure had been carried out using conventional Biochemical Methods, a million-fold purification would have required extremely painstaking work lasting for years.

5-53

5.6.9. Genes can be "engineered" to produce proteins with desired Amino acid sequences [63]

By engineering the coding sequence and regulatory regions of a gene, one can alter the Functional Properties of its protein product, The amount of protein synthesized, and, finally, the cell type capable of producing the protein.

Highly significant changes can be made to the coding sequence; for example, a portion of it can be joined to another gene, resulting in a new hybrid gene that encodes a fusion protein. Such proteins are frequently used to identify the functions of different domains of a protein molecule. For instance, most Nuclear Proteins are known to contain specific short amino acid sequences that are recognized as signals for the immediate import of these proteins into the Cell Nucleus. By artificially attaching—via Gene Fusion—various parts of nuclear protein molecules to a cytoplasmic protein, one can identify these "signal Peptides" responsible for nuclear import.

More subtle structural reorganization of a gene (resulting in the replacement of one or more amino acids in the encoded protein) requires Special Methods. First, a small DNA molecule containing the altered portion of the gene's nucleotide sequence is chemically synthesized. This synthetic oligonucleotide is then hybridized to a single-stranded plasmid DNA containing the sequence to be altered; hybridization is carried out under conditions that allow mismatch pairing (Fig. 5-87). The synthetic oligonucleotide serves as a primer for DNA polymerase, which synthesizes DNA to produce a molecule containing the gene with the altered sequence incorporated. The modified gene is then inserted into an expression vector, allowing the altered protein to be produced in sufficient quantities for detailed study. By replacing specific amino acids in the protein of interest in this way, one can determine which parts of the polypeptide chain are responsible for fundamental processes such as protein folding, protein-Ligand interaction, or Enzymatic Catalysis.

To determine which part of a eukaryotic gene is responsible for regulating its expression, the putative regulatory DNA sequences of that gene can be joined to the coding sequence of another gene chosen because it encodes an easily detectable marker enzyme normally absent in eukaryotic cells. The bacterial protein chloramphenicol acetyltransferase (CAT) is most commonly used as such a marker enzyme. The resulting recombinant DNA molecule is introduced into a eukaryotic cell to test whether the putative regulatory sequences indeed stimulate CAT gene expression. This is most easily done by transfecting cells in culture and then, after overnight incubation, assaying them for chloramphenicol acetyltransferase activity. Among the transfected cells, many will transiently express the foreign gene, but only a very small number of cells retain this gene stably. To achieve stable transformation, one must select those rare cell clones in which the recombinant DNA molecules have integrated into their chromosomes. By transfecting different cell types with such recombinant DNA molecules, it is possible to identify the regulatory DNA sequences that allow a given gene to be expressed in some cells but not in others (Section 10.2.8).

Fig. 5-87. Use of synthetic oligonucleotides to modify specific coding regions of a gene. Only two Examples of many possible types of such modifications are shown here. Using an appropriate oligonucleotide, one can, for example, replace several amino acids simultaneously, or cause the deletion of one or more amino acids. The diagram shows that in this procedure, only one of the two DNA strands in the parental recombinant plasmid is altered, so that after replication, only half of the transfected cells will contain the plasmid with the desired mutant gene. Only a small portion of the entire plasmid sequence is shown here.

5.6.10. Engineered genes can be introduced into the Germ Cells of mice or fruit flies to produce transgenic organisms [64]

To determine the function of an altered protein, it is necessary to introduce its gene into an organism and observe the effect. Ideally, one would want to replace the normal gene with the altered one, so that The Effect of the mutant protein can be observed in the absence of the normal protein. The only eukaryotic organism in which this procedure can be performed with relative ease is yeast. DNA fragments introduced into growing yeast cells integrate quite efficiently as single copies into homologous chromosomal sites by Homologous Recombination, replacing the endogenous copy of the gene with the engineered gene. This

opens up broad opportunities for studying the functions of various genes in yeast (see Section 4.6.15).

The situation is different for mammalian cells. Here, we cannot yet control how or where the introduced DNA integrates into the chromosome. On average, only about one integration event in a thousand results in gene replacement. Instead, cellular enzymes rapidly ligate linear DNA fragments end-to-end, and such a long tandem array usually integrates into the chromosome at seemingly random sites. Fertilized mammalian eggs behave similarly to other mammalian cells in this respect. If 200 copies of a linear DNA molecule are injected into a mouse egg, it often develops into a mouse that has these tandemly repeated copies of the introduced gene integrated into a random site in one of its chromosomes (Fig. 5-88). If the modified chromosome is also present in the germ cells (eggs or sperm), the mouse will transmit these newly acquired genes to its offspring; animals with such a stable genetic alteration are called transgenic. Because their normal gene is usually retained, Transgenic Animals are mostly unsuitable for demonstrating the effects of gene alteration as convincingly as in yeast; however, they have already provided important insights into how mammalian genes are regulated (see p. 10.3.11) and how certain genes (called oncogenes) cause Cancer (see Section 21.2.5).

Similar experiments can be performed with the fruit fly Drosophila, using a technique that allows a single copy of any gene of interest to be integrated into a random site in the fly genome. The main task here is to insert the DNA fragment of interest between the two terminal sequences of a specific Drosophila mobile element, the so-called P element. These terminal sequences allow the P element to integrate into Drosophila chromosomes, provided that a specific enzyme catalyzing this integration (P-element transposase; see Section 5.5.10) is also present in the cell. Therefore, to obtain transgenic fruit flies, one must inject the appropriately modified DNA fragment into a fly embryo at a very early stage of development, while simultaneously introducing a separate plasmid containing the transposase gene. In this case, the injected gene often integrates as a single copy (via transposition) into the germline cells (Fig. 5-88).

Recently, gene replacement in mice has been achieved by an indirect, rather laborious route. The procedure is as follows. First, a DNA fragment containing the desired mutant gene is transfected into a specific line of pluripotent Embryonic Stem Cells grown in culture. The cells are allowed to proliferate for some time, after which Southern blot analysis is used to identify rare colonies in which homologous recombination has resulted in gene replacement, and individual cells from these colonies are injected into an early mouse embryo. In this new environment, the embryonic stem cells often proliferate, contributing to large areas of the normal mouse body. Mice in which the gene replacement has occurred in the germline cells are bred to obtain males and females that are heterozygous for this trait (i.e., carrying one normal and one mutant copy of the gene). If these animals are then mated, one-fourth of their offspring will be homozygous for the altered gene. Studying such homozygotes allows the function of the altered gene to be observed in the absence of its normal allele.

Fig. 5-88. Comparison of standard procedures used to produce a transgenic mouse and a transgenic fruit fly. In these examples, the gene introduced into the mouse egg causes A change in coat color, while the gene introduced into the fruit fly embryo causes a change in eye color. In both cases, in some of the transgenic organisms, DNA inserts are found in multiple chromosomal sites rather than just one.

The ability to generate Transgenic Mice and Drosophila provides researchers with a powerful new tool for studying gene function in the intact organism.

5.6.11. Selected DNA segments can be cloned in vitro by the Polymerase Chain Reaction [65]

The availability of purified DNA polymerases and chemically synthesized DNA oligonucleotides has made it possible to rapidly clone specific DNA sequences outside of a living cell. This technique, known as the polymerase chain reaction (PCR), allows a selected region of The Genome to be amplified more than a million-fold, provided that at least part of its nucleotide sequence is known. The flanking regions of this sequence, surrounding the area selected for amplification, are used to design two synthetic DNA oligonucleotides, each complementary to one of the two strands of the DNA molecule. These oligonucleotides serve as primers for in vitro DNA Synthesis catalyzed by DNA polymerase; they define the ends of the resulting DNA fragment (Fig. 5-89, A).

THE PRINCIPLE OF the PCR technique is illustrated in Fig. 5-89, B. Each reaction cycle begins with a brief heating of the DNA to separate the two strands of The Double Helix (step 1). Subsequent cooling of the DNA in the presence of a large excess of the two DNA oligonucleotides leads to specific hybridization of these oligonucleotides to their complementary DNA sequences (step 2). After annealing, the mixture is incubated with DNA polymerase and the four deoxyribonucleoside triphosphates, resulting in the selective synthesis of DNA regions downstream of the primers (step 3). Efficient DNA amplification requires 20 to 30 reaction cycles. In each successive cycle, the amount of DNA doubles compared to the previous cycle. A single cycle takes about 5 minutes; thus, automating the entire procedure for 'cell-free molecular cloning' of a DNA fragment takes only a few hours, whereas conventional cloning procedures take several days.

The PCR method is extremely sensitive, capable of detecting a single DNA molecule in a sample. The same approach is suitable for analyzing trace amounts of RNA; for this purpose, RNA is converted into DNA sequences using reverse transcriptase (see Section 5.6.3). PCR-based cloning is rapidly replacing Southern blot analysis in fields such as Prenatal Diagnosis of Hereditary diseases and detection of viral infections. This method also holds great promise in forensics, as it allows the unambiguous identification of a single cell as belonging to a specific individual.

Figure 5-89. Polymerase chain reaction (PCR) used to amplify specific nucleotide sequences in vitro. A. DNA isolated from cells is heated to separate its complementary strands. These strands are then annealed by adding an excess of two chemically synthesized DNA oligonucleotides (15-20 nucleotides each), designed to be complementary to sequences separated by a distance of X nucleotides (the value of X varies from 50 to 2000). These two oligonucleotides serve as specific primers for in vitro DNA synthesis catalyzed by DNA polymerase, which copies the DNA between the corresponding sequences. B. Repeated cycles of the reaction yield A large number of copies of a single DNA fragment (X nucleotides in length).

Figure 5-90. Detection of restriction fragment length polymorphism (RFLP) by Southern blot analysis. For simplicity, only a few restriction sites are shown on the chromosomes, although in reality there are thousands. If the corresponding region is amplified by PCR prior to restriction nuclease Digestion, the same test can be performed without radioisotopes, eliminating The Need for Southern blotting.

10-31

5.6.12. The use of recombinant DNA greatly facilitates the mapping and analysis of large genomes [66]

Recently developed methods allow the construction of detailed maps of very large genomes. There are two categories of maps: 1. Physical maps, based on The structure of the DNA molecules that make up each chromosome. These include restriction maps and ordered genomic DNA clone libraries. 2. Genetic linkage maps, constructed based on the frequency of co-transmission to offspring of two or more traits—genetic markers—that differ between the parents and are assigned to a specific chromosomal region. Traditionally, genes whose expression is detected by their phenotypic effect (such as genes causing genetic diseases, e.g., muscular dystrophy) have been used as markers. Relatively recently developed recombinant DNA techniques have made it possible to use short DNA sequences containing a restriction site and varying among individuals as genetic markers. Such sequences are particularly useful for genetic mapping because restriction nuclease digestion produces fragments of varying lengths, and this restriction fragment length polymorphism (RFLP) can be easily detected by Southern blot analysis using an appropriate DNA probe (Fig. 5-90).

If two genetic markers are on different chromosomes, they are unlinked, meaning the chances of their co-transmission to offspring are 50:50. The same is true for markers located at opposite ends of the same chromosome, due to the high probability of their Separation by Crossing-over, which occurs at a very high frequency during Meiosis in the formation of eggs and sperm (see Section 15.2.3). The closer two markers are to each other on the same chromosome, the more likely they are to escape separation by crossing-over and thus be inherited together. By screening large families for the co-inheritance of a gene of interest (such as a disease gene) and a large number of individual RFLP markers, one can identify several RFLP markers flanking the gene. In this way, DNA sequences near the gene, and ultimately the DNA corresponding to the gene itself, can be localized (Fig. 5-91). This method is used to map many genes responsible for human diseases. Once such a gene is isolated, its protein product can be analyzed in detail (see Section 4.6.12).

To facilitate these and other studies, intensive work is currently underway to construct a detailed RFLP map of the human genome, which will feature thousands of markers spaced, on average, 10⁶ bp apart (two markers separated by this distance are co-inherited in 99 out of 100 cases). With such a map in hand, genes previously identified only by their phenotypic effects in humans can be localized, based on genetic linkage data, within one or more large clones in an ordered genomic DNA library. Following this, isolating the gene will, in most cases, take no more than a few months.

Figure 5-91. Genetic linkage analysis reveals the co-transmission to offspring of a gene causing a specific human phenotype (in this case, a particular disease) and a neighboring RFLP marker. Such analysis makes it possible to clone many human genes and study their products.

Rapid advances in recombinant DNA technology will soon make it possible to sequence long stretches of DNA at relatively low cost. This means that sequencing entire genomes of bacteria, yeast, nematodes, and Drosophila, as well as vast regions of the genomes of various Higher Plants and vertebrates, including humans, will become feasible.

Summary

In DNA cloning, the fragment containing the gene of interest is typically identified using a radioactive DNA probe or, following GENE EXPRESSION IN a host cell, using antibodies that detect the protein encoded by this gene. Cells carrying the DNA fragment are then allowed to multiply, producing large numbers of copies of both the gene itself and its product molecules. For genetic engineering purposes, the nucleotide sequence of such a cloned DNA fragment can be altered, joined to another DNA sequence, and then reintroduced into cells. The combination of DNA cloning and genetic engineering provides cell biologists with an extremely powerful research tool. In principle, it is possible to design a gene encoding a protein with any desired amino acid sequence and link it to a promoter DNA sequence that allows control over the timing and tissue specificity of gene expression. This new gene can be introduced either into cells grown in culture or into the germ line of mice or fruit flies. In transgenic animals, the effects of the introduced gene's expression can be observed in many different Cells and Tissues.

References

General

Judson H. F. The Eighth Day of Creation: Makers of the Revolution in Biology, New York, Simon and Schuster, 1979. (History derived from personal interviews.)

Lewin B. Genes, 3rd ed. New York, Wiley, 1987.

Stent G.S. Molecular Genetics: An Introductory Narrative, San Francisco, Freeman, 1971. (Clear descriptions of the classical experiments.) Watson J. D., Hopkins N. H.,

Roberts J. W., Weiner A. M. Molecular Biology of the Gene, 4th ed.Menlo Park, CA, Benjamin-Cummings 1987.

Cited

1. Chamberlin M. Bacterial DNA-dependent RNA polymerases. In: The Enzymes, 3rd ed., Vol. 15B (P. Boyer ed.), pp. 61-108. New York, Academic Press, 1982.

McClure W. Mechanism and control of Transcription initiation in prokaryotes.

Annu. Rev. Biochem., 54, 171-204, 1985.

Yager T.D., Hippel P.H. Transcript elongation and termination in E. coli. In: Escherichia coli and Salmonella typhimurium: CELLULAR AND MOLECULAR Biology (F.C. Niedhardt ed.), pp. 1241-1275. Washington DC, American Society for Microbiology, 1987.

2. Hawley D. K., McClure W.R. Compilation and Analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res., 11, 2237-2255, 1983.

Siebenlist U., Simpson R., Gilbert W. E. coli RNA polymerase interacts homologously with two different promoters. Cell, 20, 269-281, 1980.

3. Rich A., Kim S. H. The three-dimensional Structure of Transfer RNA. Sci. Am., 238(1), 52-62, 1978.

Schimmel P. R., Soll D., Abelson J. N., eds. Transfer RNA: Structure, Properties and Recognition, and Biological Aspects (2 volumes). Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1980.

4. Schimmel P. Aminoacyl tRNA synthetase: general scheme of structure-function relationships in the Polypeptides and recognition of Transfer RNAs. Annu. Rev. Biochem., 56, 125-158, 1987.

5. Crick F.H.C. The genetic code. III. Sci. Am., 21(4), 55-62, 1966. The Genetic Code. Cold Spring Harbor Symp. Quant. Biol., Vol. 31, 1966. (The original experiments that defined the code.)

6. Moore P. B. The ribosome returns. Nature, 331, 223-227, 1988.

Noller H.F. Structure of ribosomal RNA. Annu. Rev. Biochem., 53, 119-162, 1984.

Spirin A. S. Ribosome Structure and Protein Synthesis. Menlo Park CA, Benjamin-Cummings, 1986.

7. Clark B. The elongation step of Protein Biosynthesis. Trends Biochem. Sci., 5, 207-210, 1980.

Watson J. D. The involvement of RNA in the synthesis of proteins. Science, 140, 17-26, 1963. (A description of how ribosome function was initially deciphered.)

8. Caskey C. T. Peptide chain termination. Trends Biochem. Sci., 5, 234-237, 1980. Craigen W.J., Caskey C. T. The function, structure and

regulation of E. coli peptide chain release factors. Biochemie, 69, 1031-1041, 1987.

9. Gold L. Posttranscriptional regulatory mechanisms in Escherichia coli. Annu. Rev. Biochem., 57, 199-233, 1988.

Hunt T. The initiation of protein synthesis. Trends Biochem. Sci., 5, 178-181, 1980. Pain V.M. Initiation of protein synthesis in mammalian cells. Biochem. J., 235, 625-637, 1986.

10. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and Organelles. Microbiol. Rev., 47, 1-45, 1983.

Kozak M. An analysis of 5' noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res., 15, 8125-8147, 1987.

Steitz J. A. Genetic signals and nucleotide sequences in Messenger RNA. In: Biological Regulation and Development (R. Goldberg ed.), Vol. 1, pp. 349-399. New York, Plenum, 1979.

11. Rich A. Polyribosomes. Sci. Am., 209(6), 44-53, 1963.

12. Hunt T. Phosphorylation and the control of protein synthesis. Philos. Trans. R. Soc. Lond. (Biol.), 302, 127-134, 1983.

Jagus R., Anderson W. F., Safer B. The regulation of initiation of mammalian protein synthesis. Prog. Nucleic Acid Res. Mol. Biol., 25, 127-185, 1981.

13. Kirkwood T.B., Rosenberger R.F., Galas D. J., eds. Accuracy in Molecular Processes: Its Control and Relevance to Living Systems. London, Chapman and Hall, 1986. (Chapters 4, 5, 6 and 11.)

Thompson R. C. EFTu provides an internal kinetic standard for translational accuracy. Trends Biochem. Sci., 13, 91-93, 1988.

14. Jimenez A. Inhibitors of translation. Trends Biochem. Sci., 1, 28-30, 1976.

15. Alberts B.M. The function of the hereditary Materials: biological catalyses reflect the cell's evolutionary history. Am. Zool., 26, 781-796, 1986.

Orgel L.E. RNA catalysis and THE ORIGIN OF life. J. Theor. Biol. 123, 127-149, 1983.

Weiner A. M., Maizels N. tRNA-like structures tag the 3' ends of genomic RNA molecules for replication: implications for the origin of protein synthesis. Proc. Natl. Acad. Sci. USA, 84, 7383-7387, 1987.

16. Friedberg E. C. DNA Repair. San Francisco, Freeman, 1985.

Lindahl T. DNA repair enzymes. Annu. Rev. Biochem., 51, 61-88, 1982.

17. Dickerson R., Geis I. The Structure and Action of Proteins, pp. 59-66. New York, Harper and Row, 1969.

Wilson A. C., Carlson S. S., White T. J. Biochemical evolution. Annu. Rev. Biochem., 46, 573-639, 1977.

Wilson A. C., Ochman H., Prager E. M. Molecular time scale for evolution. Trends Genet., 3, 241-247, 1987.

18. Drake J. W. Comparative rates of spontaneous mutation. Nature, 221, 1132, 1969.

19. Cairns J. Cancer: Science and Society. San Francisco. Freeman, 1978. (Chapter 7 discusses The Role of mutation in The Development of cancer.)

Ohta T., Kimura M. Functional Organization of genetic material as a product of molecular evolution. Nature, 233, 118-119, 1971. (The mutation rate limits the maximum number of genes in an organism.)

20. Schrodinger E. What is Life? Cambridge UK, Cambridge University Press, 1945.

21. Kornberg A. DNA replication. San Francisco, Freeman, 1980. (Chapter 4 describes DNA polymerase. Chapter 8 describes DNA ligase, and Chapter 16 covers the general enzymology of DNA repair.)

22. Cleaver J. E., Karentz D. DNA repair in man: regulation by a multigene family and association with human disease. Bioessays, 6, 122-127, 1987.

Coulondre C., Miller J. H., Farabaugh P. J., Gilbert W. Molecular Basis of base substitution hotspots in E. coli. Nature, 274, 775-780, 1978. Lindahl T. New class of enzymes acting on damaged DNA. Nature, 259, 64-66, 1976.

Sancar A.Z., Sancar G.B. DNA repair enzymes. Annu. Rev. Biochem. 57, 29-67, 1988.

23. Lindahl T., Sedgwick B., Sekiguchi M., Nakabeppu Y. Regulation and expression of the adaptive response to alkylating agents. Annu. Rev. Biochem., 57, 133-157, 1988.

Ossanna N., Peterson K. R., Mount D. W. Genetics of DNA repair in bacteria. Trends Genet., 2, 55-58, 1986.

Walker G. C. Inducible DNA repair systems. Annu. Rev. Biochem., 54, 425-457, 1985.

24. Alberts B.M. Prokaryotic DNA Replication Mechanisms. Philos. Trans. R. Soc. Lond. (Biol.), 317, 395-420, 1987.

Kornberg A. DNA Replication. San Francisco, Freeman, 1980.

McMacken R., Silver L., Georgopoulos C. DNA replication. In: Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (F. C. Neidhardt ed.), pp. 564-612. Washington DC. American Society for Microbiology, 1987.

25. Joyce C.M., Steitz T.A. DNA polymerase I: from crystal structure to function via genetics. Trends Biochem. Sci., 12, 288-292, 1987.

Kornberg A. Biologic synthesis of deoxyribonuclear acid. Science, 131, 1503-1508. (Nobel prize address.)

Meselson M., Stahl F. W. The replication of DNA in E. coli. Proc. Natl. Acad. Sci. USA, 44, 671-682, 1958.

26. Cairns J. The bacterial chromosome and its manner of replication as seen by autoradiography. J. Mol. Biol., 6, 208-213, 1963.

Inman R. B., Schnos M. Structure of branch points in replicating DNA: presence of single-stranded connections in lambda DNA branch parts. J. Mol. Biol., 56, 319-325, 1971. (Electron Microscopy demonstrates that the Replication fork is asymmetric.)

Ogawa T., Okazaki T. Discontinuous DNA replication. Annu. Rev. Biochem., 49, 421-457, 1980.

27. Brutlag D., Kornberg A. Enzymatic synthesis of deoxyribonucleic acid: a proofreading function for the 3' to 5' exonuclease activity in DNA polymerases. J. Biol. Chem., 247, 241-248, 1972.

Fersht A. R. Enzymatic editing mechanisms in PROTEIN SYNTHESIS AND DNA replication. Trends Biochem. Sci., 5, 262-265, 1980.

Topal M., Fresco J. R. Complementary base pairing and the origin substitution mutations. Nature, 263, 285-289, 1976.

28. Rowen L., Kornberg A. Primase, the dnaG protein of Escherichia coli: an enzyme which starts DNA chains. J. Biol. Chem., 253, 758-764, 1978.

29. Abdel-Monem M., Hoffmann-Berling H. DNA unwinding enzymes. Trends Biochem. Sci., 5, 128-130, 1980.

Alberts B. M., Frey L. T4 bacteriophage gene 32: a structural protein in the replication and recombination of DNA, Nature, 227, 1313-1318, 1970.

Lohman T. M., Bujalowski W., Overman L. B. E. coli single strand binding protein: a new look at helix-destabilizing proteins. Trends Biochem. Sci., 13, 250-254, 1988.

30. Alberts B. M. Protein machines mediate the basic genetic processes. Trends Genet., 1, 26-30, 1985.

Kornberg A. DNA replication. J. Biol. Chem., 263, 1-4, 1988.

LeBowitz J., McMacken R. The E. coli dnaB replication protein is a DNA helicase. J. Biol. Chem., 261, 4738-4748, 1986.

31. Modrich P. DNA mismatch correction. Annu. Rev. Biochem., 56, 435-466, 1987.

Radman M., Wagner R. The high fidelity of DNA duplication. Sci. Am., 259(2), 40-47, 1988.

32. Bramhill D., Kornberg A. Duplex opening by dnaA protein at novel sequences in initiation of replication at the Water/144.html">Origin of the E. coli chromosome. Cell, 52, 743-755, 1988.

Dodson M. et al. Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: localized unwinding of duplex DNA by a six-protein reaction. Proc. Natl. Acad. Sci. USA, 83, 7638-7642, 1986.

Zyskind J. W., Smith D. W. The bacterial origin of replication, ori C. Cell, 46, 489-490, 1986.

33. DiNardo S., Voelkel K., Sternglanz R. RNA topoisomerase II mutant of Saccharomyces cerevisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication. Proc. Natl. Acad. Sci. USA, 81, 2616-2620, 1984.

Wang J. C. DNA topoisomerases. Annu. Rev. Biochem., 54, 665-698, 1985.

34. Campbell J. L. Eucaryotic DNA replication. Annu. Rev. Biochem., 55, 733-771, 1986.

35. Low K. B. ed. The Recombination of Genetic Material. San Diego, Academic Press, 1988.

Stahl F. W. Genetic recombination. Sci. Am., 256(2), 90-101, 1987.

Whitehouse H.L.K. Genetic Recombination: Understanding the Mechanisms. New York, Wiley, 1982.

36. Dressler D., Potter H. MOLECULAR MECHANISMS OF genetic recombination. Annu. Rev. Biochem., 51, 727-762, 1982.

Smith G. R. Mechanism and control of homologous recombination in Escherichia coli. Annu. Rev. Genet., 21, 179-201, 1987.

37. Wetmur J. G., Davidson N. Kinetics of renaturation of DNA. J. Mol. Biol., 31, 349-370, 1968.

38. Cox M. M., Lehman I. R. Enzymes of general recombination. Annu. Rev. Biochem., 56, 229-262, 1987.

Kowalczykowski S. C. Mechanistic aspects of the DNA strand exchange activity of E. coli recA protein. Trends Biochem. Sci., 12, 141-145, 1987.

Radding C. M. Recombination activities of E. coli recA protein. Cell, 25, 3-4, 1981.

39. Holliday R. A mechanism for Gene Conversion in Fungi. Genet. Res., 5, 282-304, 1964.

Meselson M. S., Radding C. M. A general model for genetic recombination. Proc. Natl. Acad. Sci. USA, 72, 358-361, 1975.

Sigal N., Alberts B. Genetic recombination: The Nature of a cross-strand exchange between two homologous DNA molecules. J. Mol. Biol., 71, 789-793, 1972.

40. Fincham J. R. S., Day P. R., Radford A. Fungal Genetics, 4th ed. Berkeley, University of California Press, 1979.

Kourilsky P. Molecular mechanisms of gene conversion in higher cells. Trends Genet., 2, 60-62, 1986.

41. Campbell A. Some general questions about movable elements and their implications. Cold Spring Harbor Symp. Quant. Biol., 45, 1-9, 1980.

Craig N. The Mechanism of conservative Site-Specific Recombination. Annu. Rev. Genet., 22, 77-105, 1988.

Grindley N. D., Reed R. R. Transpositional recombination in prokaryotes. Annu. Rev. Biochem., 54, 863-896, 1985.

Mizuuchi K., Craigie R. Mechanism of bacteriophage mu transposition. Annu. Rev. Genet., 20, 385-429, 1986.

42. Joklik W. K. ed. Virology, 2nd ed. Norwalk CT, Appleton and Lange, 1985. Shapiro J. A. ed. Mobile Genetic Elements. New York, Academic Press, 1983.

Watson J. D., Hopkins N. H., Roberts J. W., Steitz J. A., Weiner A. M. Molecular Biology of the Gene, 4th ed. Menlo Park CA, Benjamin-Cummings, 1987. (Chapter 24 provides a modern Overview of Eukaryotic Viruses.)

43. Hershey A. D., Chase M. Independent functions of viral PROTEIN AND NUCLEIC acid in growth of bacteriophage. J. Gen. Physiol., 36, 39-56, 1952.

Luria S. E., Darnell J. E., Baltimore D., Campbell A. General Virology, 3rd ed.

New York, Wiley, 1978. (The introductory chapter provides a Historical Overview of the development of virology.)

44. Simons K., Garoff H., Helenius A. How an animal virus gets in and out of its host cell. Sci. Am., 246(2), 58-66, 1982.

45. Gierer A., Schramm G. Infectivity of ribonucleic acid from tobacco mosaic virus. Nature, 177, 702-703, 1956.

46. Cohen S. S. Virus-Induced Enzymes. New York, Columbia University Press, 1968.

47. Kornberg A. DNA Replication. San Francisco, Freeman, 1980. (Chapters 14 and 15 provide up-to-date, beautifully illustrated Description of the varieties of ways phage and animal viral DNA's replicate.)

48. Campbell A.M. Bacteriophage lambda as a model system. Bioessays, 5, 277-280, 1986.

Daniels D., Sanger F., Coulson A. R. Features of bacteriophage lambda: Analysis of the complete nucleotide sequence. Cold Spring Harbor Symp. Quant. Biol., 47, 1009-1024, 1982.

Lwoff A. Lysogeny. Bacteriol. Rev., 17, 269-337, 1952.

49. Watson J.D., Hopkins N.H., Roberts J. W., Steitz J.A., Weiner A.M. Molecular Biology of the Gene, 4th ed. Menlo Park CA, Benjamin-Cummings, 1987. (Chapter 26 describes the viruses that can cause tumors.)

50. Baltimore D. Viral DNA-dependent DNA polymerase. Nature, 226, 1209-1211, 1970.

Gallo R.C. The AIDS virus. Sci. Am., 256(1), 46-56, 1987.

Temin H. M., Mizutani A. RNA-dependent DNA polymerase in virions of Rous Sarcoma virus. Nature, 226, 1211-1213, 1970.

Varmus H. Retroviruses. Science, 240, 1427-1435, 1988.

51. Boeke J.D., Garfinkel D.J., Styles C.A., Fink G.R. Ty elements transpose through an RNA intermediate. Cell, 40, 491-500, 1985.

Fink G. R., Boeke J. D., Garfinkel D. J. The mechanisms and consequences of retrotransposition. Trends Genet., 2, 118-123, 1986.

52. Cohen S.N., Shapiro J.A. Transposable genetic elements. Sci. Am., 242(2), 40-49, 1980.

Mizuuchi K. Mechanism of transposition of bacteriophage Mu: polarity of the strand transfer reaction at the initiation of transposition. Cell, 39, 395-404, 1984.

53. Novick R.P. Plasmids. Sci. Am., 243(6), 102-127, 1980.

Reisner D., Gross H.J. Viroids. Annu. Rev. Biochem., 54, 531-564, 1985.

Rossmann M.G. The evolution of RNA viruses. Bioessays, 7, 99-103, 1987.

54. Drlica K. Understanding DNA and Gene Cloning. New York, Wiley, 1984.

Sambrook J., Fritsch E. F., Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989.

Watson J.D., Tooze J., Kurtz D. T. Recombinant DNA: A Short Course. New York, W.H. Freeman, 1983.

55. Smith H. O. Nucleotide sequence specificity of Restriction Endonucleases. Science, 205, 455-462, 1979.

56. Cohen S., Chang A., Boyer H., Helling R. Construction of biologically functional bacterial plasmids in vitro. Proc. Natl. Acad. Sci. USA, 70, 3240-3244, 1973.

57. Maniatis T. et al. The isolation of structural genes from libraries of eukaryotic DNA. Cell, 15, 687-701, 1978.

Maniatis T., Kee S. G., Efstratiadis A., Kafatos F. C. Amplification and characterization of a β-globin gene synthesized in vitro. Cell, 8, 163-182, 1976.

58. Hedrick S. M., Cohen D. I., Nielsen E. A., Davis M. M. Isolation of cDNA clones encoding T cell-specific membrane-associated proteins. Nature, 308, 149-153, 1984.

59. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. USA, 72, 3961-3965, 1975.

Suggs S. V., Wallace R.B., Hirose T., Kawashima E.H., Itakura K. Use of synthetic oligonucleotides as hybridization probes: isolation of cloned sequences for human β2-microglobulin. Proc. Natl. Acad. Sci. USA, 78, 6613-6617, 1981.

60. Coulson A., Sulston J., Brenner S.. Karn J. Toward a physical map of the genome of the nematode Caenorhabaitis elegans. Proc. Natl. Acad. Sci. USA, 83, 7821-7825, 1986.

Proustka A., Lehrach H. Jumping libraries and linking libraries: the next generation of molecular tools in mammalian genetics. Trends Genet., 2, 174-179, 1986.

61. Pelham H.R.B., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem., 67, 247-256, 1976.

62. Geysen H. M. et al. Chemistry of Antibody Binding to a protein. Science, 235, 1184-1190, 1987.

Gilbert W., Villa-Komaroff L. Usefull proteins from recombinant bacteria. Sci. Am., 242(4), 74-94, 1980. Yokata T. et al. Use of a cDNA expression vector for isolation of mouse interleukin 2 cDNA clones: expression of T-cell growth-factor activity after transfection of monkey cells. Proc. Natl. Acad. Sci. USA, 82, 6872, 1985.

Young R. A., Davis R. W. Efficient Isolation of Genes by using antibody probes. Proc. Natl. Acad. Sci. USA, 80, 1194-1198, 1980.

63. Smith M. In vitro mutagenesis. Annu. Rev. Genet., 19, 423-462, 1985.

64. Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc. Natl. Acad. Sci. USA, 75, 1929-1933, 1978.

Palmiter R. D., Brinster R. L. Germ line transformation of mice. Annu. Rev. Genet, 20, 465-499, 1986.

Spradling A.C., Rubin G. M. Transposition of cloned P elements into Drosophila germ line chromosomes. Science, 218, 341-347, 1982.

Thomas K. R., Capecchi M. R. Site-Directed Mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell, 51, 503-512, 1987.

65. Marx J.L. Multiplying genes leaps and bounds. Science, 240, 1408-1410, 1988.

Saiki R. K. et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239, 487-491, 1988.

66. Botstein D., White R. L., Skolnick M., Davis R. W. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet., 32, 314-331, 1980. Mapping and Sequencing the Human Genome. Washington DC, National Academy Press, 1988.

White R., LaLouel J.-M. Chromosome mapping with DNA markers. Sci. Am. 258(2), 40-49, 1988.