Manipulating the Genes of Eukaryotes

I. The Molecular Design of Life
6. Exploring Genes
6.2. Recombinant DNA Technology Has Revolutionized All Aspects of Biology
Figure 6.21. Diagram of a Yeast Artificial Chromosome (YAC). DNA inserts as large as 1000 kb can be propagated
in this vector.
I. The Molecular Design of Life
6. Exploring Genes
6.2. Recombinant DNA Technology Has Revolutionized All Aspects of Biology
Figure 6.22. Chromosome Walking. Long regions of unknown DNA can be explored, starting with a known base
sequence, by subcloning and rescreening. New probes are designed on the basis of the DNA sequences that have been
determined.
I. The Molecular Design of Life
6. Exploring Genes
6.3. Manipulating the Genes of Eukaryotes
Eukaryotic genes, in a simplified form, can be introduced into bacteria, and the bacteria can be used as factories to
produce a desired protein product. It is also possible to introduce DNA into higher organisms. In regard to animals, this
ability provides a valuable tool for examining gene action, and it will be the basis of gene therapy. In regard to plants,
introduced genes may make a plant resistant to pests or capable of growing in harsh conditions or able to carry greater
quantities of essential nutrients. The manipulation of eukaryotic genes holds much promise for medical and agricultural
benefits, but it is also the source of controversy.
6.3.1. Complementary DNA Prepared from mRNA Can Be Expressed in Host Cells
How can mammalian DNA be cloned and expressed by E. coli? Recall that most mammalian genes are mosaics of
introns and exons (Section 5.6). These interrupted genes cannot be expressed by bacteria, which lack the machinery to
splice introns out of the primary transcript. However, this difficulty can be circumvented by causing bacteria to take up
recombinant DNA that is complementary to mRNA. For example, proinsulin, a precursor of insulin, is synthesized by
bacteria harboring plasmids that contain DNA complementary to mRNA for proinsulin (Figure 6.23). Indeed, bacteria
produce much of the insulin used today by millions of diabetics.
The key to forming complementary DNA (cDNA) is the enzyme reverse transcriptase. As discussed in Section 5.3.1, a
retrovirus uses this enzyme to form a DNA-RNA hybrid in replicating its genomic RNA. Reverse transcriptase
synthesizes a DNA strand complementary to an RNA template if it is provided with a DNA primer that is base-paired to
the RNA and contains a free 3 -OH group. We can use a simple sequence of linked thymidine [oligo(T)] residues as the
primer. This oligo(T) sequence pairs with the poly(A) sequence at the 3 end of most eukaryotic mRNA molecules
(Section 5.4.4), as shown in Figure 6.24. The reverse transcriptase then synthesizes the rest of the cDNA strand in the
presence of the four deoxyribonucleoside triphosphates. The RNA strand of this RNA-DNA hybrid is subsequently
hydrolyzed by raising the pH. Unlike RNA, DNA is resistant to alkaline hydrolysis. The single-stranded DNA is
converted into double-stranded DNA by creating another primer site. The enzyme terminal transferase adds
nucleotides for instance, several residues of dG to the 3 end of DNA. Oligo(dC) can bind to dG residues and prime
the synthesis of the second DNA strand. Synthetic linkers can be added to this double-helical DNA for ligation to a
suitable vector. Complementary DNA for all mRNA that a cell contains can be made, inserted into vectors, and then
inserted into bacteria. Such a collection is called a cDNA library.
Complementary DNA molecules can be inserted into vectors that favor their efficient expression in hosts such as E. coli.
Such plasmids or phages are called expression vectors. To maximize transcription, the cDNA is inserted into the vector
in the correct reading frame near a strong bacterial promoter site. In addition, these vectors ensure efficient translation by
encoding a ribosome-binding site on the mRNA near the initiation codon. Clones of cDNA can be screened on the basis
of their capacity to direct the synthesis of a foreign protein in bacteria. A radioactive antibody specific for the protein of
interest can be used to identify colonies of bacteria that harbor the corresponding cDNA vector (Figure 6.25). As
described in Section 6.2.3, spots of bacteria on a replica plate are lysed to release proteins, which bind to an applied
nitrocellulose filter. A 125I-labeled antibody specific for the protein of interest is added, and autoradiography reveals the
location of the desired colonies on the master plate. This immunochemical screening approach can be used whenever a
protein is expressed and corresponding antibody is available.
6.3.2. Gene-Expression Levels Can Be Comprehensively Examined
Most genes are present in the same quantity in every cell namely, one copy per haploid cell or two copies per diploid
cell. However, the level at which a gene is expressed, as indicated by mRNA quantities, can vary widely, ranging from
no expression to hundreds of mRNA copies per cell. Gene-expression patterns vary from cell type to cell type,
distinguishing, for example, a muscle cell from a nerve cell. Even within the same cell, gene-expression levels may vary
as the cell responds to changes in physiological circumstances.
Using our knowledge of complete genome sequences, it is now possible to analyze the pattern and level of expression of
all genes in a particular cell or tissue. One of the most powerful methods developed to date for this purpose is based on
hybridization. High-density arrays of oligonucleotides, called DNA microarrays or gene chips, can be constructed either
through lightdirected chemical synthesis carried out with photolithographic microfabrication techniques used in the
semiconductor industry or by placing very small dots of oligonucleotides or cDNAs on a solid support such as a
microscope slide. Fluorescently labeled cDNA is hybridized to the chip to reveal the expression level for each gene,
identifiable by its known location on the chip. (Figure 6.26).
The intensity of the fluorescent spot on the chip reveals the extent of transcription of a particular gene. DNA chips have
been prepared that contain oligonucleotides complementary to all known open reading frames, 6200 in number, within
the yeast genome (Figure 6.27). An analysis of mRNA pools with the use of these chips revealed, for example, that
approximately 50% of all yeast genes are expressed at steady-state levels of between 0.1 and 1.0 mRNA copy per cell.
This method readily detected variations in expression levels displayed by specific genes under different growth
conditions. These tools will continue to grow in power as genome sequencing efforts continue.
6.3.3. New Genes Inserted into Eukaryotic Cells Can Be Efficiently Expressed
Bacteria are ideal hosts for the amplification of DNA molecules. They can also serve as factories for the production of a
wide range of prokaryotic and eukaryotic proteins. However, bacteria lack the necessary enzymes to carry out
posttranslational modifications such as the specific cleavage of polypeptides and the attachment of carbohydrate units.
Thus, many eukaryotic genes can be correctly expressed only in eukaryotic host cells. The introduction of recombinant
DNA molecules into cells of higher organisms can also be a source of insight into how their genes are organized and
expressed. How are genes turned on and off in embryological development? How does a fertilized egg give rise to an
organism with highly differentiated cells that are organized in space and time? These central questions of biology can
now be fruitfully approached by expressing foreign genes in mammalian cells.
Recombinant DNA molecules can be introduced into animal cells in several ways. In one method, foreign DNA
molecules precipitated by calcium phosphate are taken up by animal cells. A small fraction of the imported DNA
becomes stably integrated into the chromosomal DNA. The efficiency of incorporation is low, but the method is useful
because it is easy to apply. In another method, DNA is microinjected into cells. A fine-tipped (0.1- µ m-diameter) glass
micropipet containing a solution of foreign DNA is inserted into a nucleus (Figure 6.28). A skilled investigator can inject
hundreds of cells per hour. About 2% of injected mouse cells are viable and contain the new gene. In a third method,
viruses are used to bring new genes into animal cells. The most effective vectors are retroviruses. As discussed in
Section 5.3.1, retroviruses replicate through DNA intermediates, the reverse of the normal flow of information. A
striking feature of the life cycle of a retrovirus is that the double-helical DNA form of its genome, produced by the action
of reverse transcriptase, becomes randomly incorporated into host chromosomal DNA. This DNA version of the viral
genome, called proviral DNA, can be efficiently expressed by the host cell and replicated along with normal cellular
DNA. Retroviruses do not usually kill their hosts. Foreign genes have been efficiently introduced into mammalian cells
by infecting them with vectors derived from Moloney murine leukemia virus, which can accept inserts as long as 6 kb.
Some genes introduced by this retroviral vector into the genome of a transformed host cell are efficiently expressed.
Two other viral vectors are extensively used. Vaccinia virus, a large DNA-containing virus, replicates in the cytoplasm
of mammalian cells, where it shuts down host-cell protein synthesis. Baculovirus infects insect cells, which can be
conveniently cultured. Insect larvae infected with this virus can serve as efficient protein factories. Vectors based on
these large-genome viruses have been engineered to express DNA inserts efficiently.
6.3.4. Transgenic Animals Harbor and Express Genes That Were Introduced into
Their Germ Lines
Genetically engineered giant mice illustrate the expression of foreign genes in mammalian cells (Figure 6.29). Giant
mice were produced by introducing the gene for rat growth hormone into a fertilized mouse egg. Growth hormone
(somatotropin), a 21-kd protein, is normally synthesized by the pituitary gland. A deficiency of this hormone produces
dwarfism, and an excess leads to gigantism. The gene for rat growth hormone was placed on a plasmid next to the mouse
metallothionein promoter (Figure 6.30). This promoter site is normally located on a chromosome, where it controls the
transcription of metallothionein, a cysteine-rich protein that has high affinity for heavy metals. Metallothionein binds to
and sequesters heavy metals, many of which are toxic for metabolic processes (Section 17.3.2). The synthesis of this
protective protein by the liver is induced by heavy-metal ions such as cadmium. Hence, if mice contain the new gene, its
expression can be initiated by the addition of cadmium to the drinking water.
Several hundred copies of the plasmid containing the promoter and growth-hormone gene were microinjected into the
male pronucleus of a fertilized mouse egg, which was then inserted into the uterus of a foster mother mouse. A number
of mice that developed from such microinjected eggs contained the gene for rat growth hormone, as shown by Southern
blots of their DNA. These transgenic mice, containing multiple copies ( ~ 30 per cell) of the rat growth-hormone gene,
grew much more rapidly than did control mice. In the presence of cadmium, the level of growth hormone in these mice
was 500 times as high as in normal mice, and their body weight at maturity was twice normal. The foreign DNA had
been transcribed and its five introns correctly spliced out to form functional mRNA. These experiments strikingly
demonstrate that a foreign gene under the control of a new promoter site can be integrated and efficiently expressed in
mammalian cells.
6.3.5. Gene Disruption Provides Clues to Gene Function
A gene's function can also be probed by inactivating the gene and looking for resulting abnormalities. Powerful methods
have been developed for accomplishing gene disruption (also called gene knockout) in organisms such as yeast and mice.
These methods rely on the process of homologous recombination. Through this process, regions of strong sequence
similarity exchange segments of DNA. Foreign DNA inserted into a cell thus can disrupt any gene that is at least in part
homologous by exchanging segments (Figure 6.31). Specific genes can be targeted if their nucleotide sequences are
known.
For example, the gene knockout approach has been applied to the genes encoding gene regulatory proteins (also called
transcription factors) that control the differentiation of muscle cells. When both copies of the gene for the regulatory
protein myogenin are disrupted, an animal dies at birth because it lacks functional skeletal muscle. Microscopic
inspection reveals that the tissues from which muscle normally forms contain precursor cells that have failed to
differentiate fully (Figure 6.32). Heterozygous mice containing one normal myogenin gene and one disrupted gene
appear normal, indicating that the level of gene expression is not essential for its function. Analogous studies have
probed the function of many other genes to generate animal models for known human genetic diseases.
6.3.6. Tumor-Inducing Plasmids Can Be Used to Introduce New Genes into Plant Cells
The common soil bacterium Agrobacterium tumefaciens infects plants and introduces foreign genes into plants cells
(Figure 6.33). A lump of tumor tissue called a crown gall grows at the site of infection. Crown galls synthesize opines, a
group of amino acid derivatives that are metabolized by the infecting bacteria. In essence, the metabolism of the plant
cell is diverted to satisfy the highly distinctive appetite of the intruder. Tumor-inducing plasmids (Ti plasmids) that are
carried by Agrobacterium carry instructions for the switch to the tumor state and the synthesis of opines. A small part of
the Ti plasmid becomes integrated into the genome of infected plant cells; this 20-kb segment is called T-DNA
(transferred DNA; Figure 6.34).
Ti plasmid derivatives can be used as vectors to deliver foreign genes into plant cells. First, a segment of foreign DNA is
inserted into the T-DNA region of a small plasmid through the use of restriction enzymes and ligases. This synthetic
plasmid is added to Agrobacterium colonies harboring naturally occurring Ti plasmids. By recombination, Ti plasmids
containing the foreign gene are formed. These Ti vectors hold great promise as tools for exploring the genomes of plant
cells and modifying plants to improve their agricultural value and crop yield. However, they are not suitable for
transforming all types of plants. Ti-plasmid transfer is effective with dicots (broad-leaved plants such as grapes) and a
few kinds of monocots but not with economically important cereal monocots.
Foreign DNA can be introduced into cereal monocots as well as dicots by applying intense electric fields, a technique
called electroporation (Figure 6.35). First, the cellulose wall surrounding plant cells is removed by adding cellulase; this
treatment produces protoplasts, plant cells with exposed plasma membranes. Electric pulses are then applied to a
suspension of protoplasts and plasmid DNA. Because high electric fields make membranes transiently permeable to
large molecules, plasmid DNA molecules enter the cells. The cell wall is then allowed to reform, and the plant cells are
again viable. Maize cells and carrot cells have been stably transformed in this way with the use of plasmid DNA that
includes genes for resistance to antibiotics. Moreover, the transformed cells efficiently express the plasmid DNA.
Electroporation is also an effective means of delivering foreign DNA into animal cells.
The most effective means of transforming plant cells is through the use of "gene guns," or bombardment-mediated
transformation. DNA is coated onto 1- µ m-diameter tungsten pellets, and these microprojectiles are fired at the target
cells with a velocity greater than 400 m s-1. Despite its apparent crudeness, this technique is proving to be the most
effective way of transforming plants, especially important crop species such as soybean, corn, wheat, and rice. The genegun technique affords an opportunity to develop genetically modified organisms (GMOs) with beneficial characteristics.
Such characteristics could include the ability to grow in poor soils, resistance to natural climatic variation, resistance to
pests, and nutritional fortification. These crops might be most useful in developing countries. The use of genetically
modified organisms is highly controversial at this point because of fears of unexpected side effects.
The first GMO to come to market was a tomato characterized by delayed ripening, rendering it ideal for shipment. Pectin
is a polysaccharide that gives tomatoes their firmness and is naturally destroyed by the enzyme polygalacturonase. As
pectin is destroyed, the tomatoes soften, making shipment difficult. DNA was introduced that disrupts the
polygalacturonase gene. Less of the enzyme was produced, and the tomatoes stayed fresh longer. However, the tomato's
poor taste hindered its commercial success.
I. The Molecular Design of Life
6. Exploring Genes
6.3. Manipulating the Genes of Eukaryotes
Figure 6.23. Synthesis of Proinsulin by Bacteria. Proinsulin, a precursor of insulin, can be synthesized by transformed
(genetically altered) clones of E. coli. The clones contain the mammalian proinsulin gene.
I. The Molecular Design of Life
6. Exploring Genes
6.3. Manipulating the Genes of Eukaryotes
Figure 6.24. Formation of a cDNA Duplex. A cDNA duplex is created from mRNA by using reverse transcriptase to
synthesize a cDNA strand, first along the mRNA template and then, after digestion of the mRNA, along that same newly
synthesized cDNA strand.
I. The Molecular Design of Life
6. Exploring Genes
6.3. Manipulating the Genes of Eukaryotes
Figure 6.25. Screening of cDNA Clones. A method of screening for cDNA clones is to identify expressed products by
staining with specific antibody.
I. The Molecular Design of Life
6. Exploring Genes
6.3. Manipulating the Genes of Eukaryotes
Figure 6.26. Gene Expression Analysis Using Microarrays. The expression levels of thousands of genes can be
simultaneously analyzed using DNA microarrays (gene chips). Here, analysis of 1733 genes in 84 breast tumor samples
reveals that the tumors can be divided into distinct classes based on their gene expression patterns. Red corresponds to
gene induction and green corresponds to gene repression. [Adapted from C. M. Perou et al., Nature 406(2000):747.]
I. The Molecular Design of Life
6. Exploring Genes
6.3. Manipulating the Genes of Eukaryotes
Figure 6.27. Monitoring Changes in Yeast Gene Expression. This microarray analysis shows levels of gene
expression for yeast genes under different conditions. [Adapted from Iyer et al., Nature 409(2000):533.]
I. The Molecular Design of Life
6. Exploring Genes
6.3. Manipulating the Genes of Eukaryotes
Figure 6.28. Microinjection of DNA. Cloned plasmid DNA is being microinjected into the male pronucleus of a
fertilized mouse egg.
I. The Molecular Design of Life
6. Exploring Genes
6.3. Manipulating the Genes of Eukaryotes
Figure 6.29. Transgenic Mice. Injection of the gene for growth hormone into a fertilized mouse egg gave rise to a giant
mouse (left), about twice the weight of his silbling (right). [Courtesy of Dr. Ralph Brinster.]
I. The Molecular Design of Life
6. Exploring Genes
6.3. Manipulating the Genes of Eukaryotes
Figure 6.30. Rat Growth Hormone-Metallothionein Gene Construct. The gene for rat growth hormone (shown in
yellow) was inserted into a plasmid next to the metallothionein promoter, which is activated by the addition of heavy
metals, such as cadmium ion.
I. The Molecular Design of Life
6. Exploring Genes
6.3. Manipulating the Genes of Eukaryotes
Figure 6.31. Gene Disruption by Homologous Recombination. (A) A mutated version of the gene to be disrupted is
constructed, maintaining some regions of homology with the normal gene (red). When the foreign mutated gene is
introduced into an embryonic stem cell, (B) recombination takes place at regions of homology and (C) the normal
(targeted) gene is replaced, or "knocked out," by the foreign gene The cell is inserted into embryos, and mice lacking the
gene (knockout mice) are produced.
I. The Molecular Design of Life
6. Exploring Genes
6.3. Manipulating the Genes of Eukaryotes
Figure 6.32. Consequences of Gene Disruption. Sections of muscle from normal (A) and gene-disrupted (B) mice, as
viewed under the light microscope. Muscles do not develop properly in mice having both myogenin genes disrupted.
[From P. Hasty, A. Bradley, J. H. Morris, D. G. Edmondson, J. M. Venuti, E. N. Olson, and W. H. Klein, Nature 364
(1993):501.]
I. The Molecular Design of Life
6. Exploring Genes
6.3. Manipulating the Genes of Eukaryotes
Figure 6.33. Tumors in Plants. Crown gall, a plant tumor, is caused by a bacterium (Agrobacterium tumefaciens) that
carries a tumor-inducing plasmid (Ti plasmid).
I. The Molecular Design of Life
6. Exploring Genes
6.3. Manipulating the Genes of Eukaryotes
Figure 6.34. Ti Plasmids. Agrobacteria containing Ti plasmids can deliver foreign genes into some plant cells. [After
M. Chilton. A vector for introducing new genes into plants. Copyright ©1983 by Scientific American, Inc. All rights
reserved.]
I. The Molecular Design of Life
6. Exploring Genes
6.3. Manipulating the Genes of Eukaryotes