PANEL 8-1 Review of Classical Genetics
`
`GENES AND PHENOTYPES
`Gene:
`a functional unit of inheritance, usually co rrespond ing
`to the segment of DNA coding for a single protein.
`Genome: an organism's set of genes.
`locus: the site of the gene in the genome
`
`TYPES OF MUTATIONS
`
`~~; .. : .
`
`DELETION: deletes a segment of a chromosome
`
`-----cc. . .. m -
`
`alleles: alternative forms of a gene
`1
`homozygous NA
`
`Wild-type: the normal,
`naturally occurring type
`
`Mutant: differing from the
`wild-type because of a genetic
`change (a mutation)
`
`heterozygous a/A
`
`homozygous a/a
`
`POINT MUTATION: maps to a single site in the genome,
`corresponding to a single nucleotide pair or a very
`small part of a sing le gene
`
`GENOTYPE: the specific set of
`alleles forming the genome of
`an individual
`
`PHENOTYPE: the visi ble
`character of the individual
`
`allele A is dominant (relative to a); allele a is recessive (relative to A)
`In the example above, the phenotype of the heterozygote is the same as that of one of the
`homozygotes; in cases where it is different from both, the two alleles are said to be co-dom inant.
`
`CHROMOSOMES
`
`centromere
`
`a chromosome at the beginning of the cell
`cycle, in G1 phase; the single long bar
`represents one long double helix of DNA
`
`THE HAPLOID-DIPLOID CYCLE OF
`SEXUAL REPRODUCTION
`
`INVERSION: inverts a segment of a chromosome
`
`lethal mutation: causes the developing organism to die
`prematurely.
`conditional mutation: produces its phenotypic effect only
`under certain conditions, ca lled the restrictive conditions.
`Under other conditions-the permissive co nditions-the
`effect is not seen. For a temperature-sensitive mutation, the
`restrictive co ndition typically is high temperature, while the
`permissive condition is low temperature.
`loss-of-function mutation: either reduces or abolishes the
`activity of the gene, These are the commonest class of
`mutations. Loss-of-function mutations are usually
`recessive-the organism can usually f unction normally as
`long as it retains at least one normal copy of the affected
`gene,
`null mutation: a loss-of-function mutation that completely
`abolishes the activity of the gene.
`
`TRANS LOCATION: breaks off a segment from one chromosome
`and attaches it to another
`gain-of-function mutat ion: increases the activity of the gene
`or makes it active in inappropriate circumstances; these
`mutations are usually dominant.
`dominant negat ive mutation: dominant-acting mutation that
`blocks gene activity, causing a loss-of-fu nction phenotype
`even in the presence of a normal copy of the gene. This
`phenomenon occurs when the mutant gene product
`interferes with the function of the normal gene product.
`suppressor mutation: suppresses th e phenotypic effect of
`another mutat ion, so that the double mutant seems norm al.
`An intragenic suppressor mutation lies within the gene
`affected by the first mutation; an extragenic suppressor
`m utation lies in a second gene-often one whose product
`interacts directly with the product of the first.
`
`TWO GENES OR ONE?
`Given two mutations that produce the same phenotype, how can
`we tell whether they are mutations in the same gene? If the
`mutations are recessive (as they most often are), the answer can
`be found by a complementation test
`COMPLEMENTATION:
`MUTATIONS IN TWO DIFFERENT GENES
`
`In the simplest type of complementation test, an individual who is
`homozygous for one mutation is mated with an individual who is
`homozygous for the other. Th e phenotype of the offspring gives the
`answer to the question,
`
`NONCOMPLEMENTATION:
`TWO INDEPENDENT MUTATIONS IN THE SAME GENE
`
`homozygous mutant mother
`
`homozygous mutant father
`
`homozygous mutant mother
`
`homozygous mutant father
`
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`>- :2::
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`hybrid offspring shows normal phenotype:
`one normal copy of each gene is present
`
`hybrid offspring shows mutant phenotype:
`no normal copies of the mutated gene are present
`
`527
`
`short "p" arm
`
`long "q" arm
`
`~ "p" arm
`
`"q " arm
`
`a chromosome at the end of the ce ll cycle, in
`metaphase; it is duplicated and condensed, consisting of
`two ident ical sister chromatids (each contai ning one
`DNA double helix) joined at the centromere.
`
`A normal diploid chromosome set, as
`seen in a metaphase spread,
`prepared by bursting open a cell at
`metaphase and staining the scattered
`chromosomes. In the example shown
`schematically here, there are three
`pairs of autosomes (chromosomes
`inherited symmetrically from
`both parents, regardless of sex) and
`two sex chromosomes-an X from
`the mother and a Y from the father.
`Th e numbers and types of sex
`chromosomes and their role in sex
`determination are variable from one
`class of organisms to another, as is
`the number of pairs of autosomes.
`
`ma<omal' ~
`~ paternal3
`
`maternal 3
`
`paternal 2 /f maternal 2
`
`sex chromosomes
`
`MEIOSIS AND GENETIC RECOMBINATION
`
`maternal chromosome
`
`A
`paternal chromosome
`I O rm!
`a
`
`diploid germ cell
`
`genotype AB
`ab
`
`S26
`
`8
`
`MEIOSIS AND
`RECOMBINATION
`
`site of crossing-over
`
`genotype aB o w
`
`a
`
`haploid gametes (eggs or sperm)
`
`c§, ~ cSliej)
`
`:
`
`DIPLOID
`MEI
`
`:
`
`@
`
`HAPLO~
`
`SEXUAL FUSION (FERTILIZATION )
`
`DIPLOID
`
`maternal
`chromosome
`
`paternal
`chromosome
`
`For simplicity, the cycle is shown for only one
`chromosome/chromosome pair.
`
`The greater the distance
`between two loci on a single
`chromosome, the greater is th e
`chance that they wil l be
`separated by crossi ng-over
`occurring at a site between
`them. If two genes are thus
`reasserted in x% of gametes,
`they are said to be separated on
`a chromosome by a genetic map
`distance of x map units (or
`x centimorgans).
`
`00054
`
`EX1010 = pt. 3
`
`

`

`Figure 8-55 lnsertional mutant of
`the snapdragon, Antirrhinum.
`A mutation in a single gene coding for a
`regulatory protein causes leafy shoots to
`develop in place of flowers. The mutation
`allows cells to adopt a character that
`would be appropriate to a different part
`of the normal plant. The mutant plant is
`on the left, the normal plant on the r ight.
`(Courtesy of Enrico Coen and Rosemary
`Carpenter.)
`
`The Classical Approach Begins with Random Mutagenesis
`
`Before the advent of gene cloning technology, most genes were identified by the
`processes disrupted when the gene was mutated. This classical genetic
`approach-identifying the genes responsible for mutant phenotypes-is most
`easily performed in organisms that reproduce rapidly and are amenable to
`genetic manipulation, such as bacteria, yeasts, nematode worms, and fruit flies.
`Although spontaneous mutants can sometimes be found by examining extremely
`large populations-thousands or tens of thousands of individual organisms(cid:173)
`the process of isolating mutants can be made much more efficient by generating
`mutations with agents that damage DNA. By treating organisms with mutagens,
`very large numbers of mutants can be created quickly and then screened for a
`particular defect of interest, as we will see shortly.
`An alternative approach to chemical or radiation mutagenesis is called
`insertional mutagenesis. This method relies on the fact that exogenous DNA
`inserted randomly into the genome can produce mutations if the inserted frag(cid:173)
`ment interrupts a gene or its regulatory sequences. The inserted DNA, whose
`sequence is known, then serves as a molecular tag that aids in the subsequent
`identification and cloning of the disrupted gene (Figure 8-55). In Drosophila, the
`use of the transposable P element to inactivate genes has revolutionized the
`study of gene function in the fruit fly. Transposable elements (see Table 5-3, p. 287)
`have also been used to generate mutants in bacteria, yeast, and in the flowering
`plant Arabidopsis. Retroviruses, which copy themselves into the host genome
`(see Figure 5-73), have been used to disrupt genes in zebrafish and in mice.
`Such studies are well suited for dissecting biological processes in worms and
`flies, but how can we study gene function in humans? Unlike the organisms we
`have been discussing, humans do not reproduce rapidly, and they are not inten(cid:173)
`tionally treated with mutagens. Moreover, any human with a serious defect in an
`essential process, such as DNA replication, would die long before birth.
`There are two answers to the question of how we study human genes. First,
`because genes and gene functions have been so highly conserved throughout
`evolution, the study of less complex model organisms reveals critical informa(cid:173)
`tion about similar genes and processes in humans. The corresponding human
`genes can then be studied further in cultured human cells. Second, many muta(cid:173)
`tions that are not lethal-tissue-specific defects in Iysosomes or in cell-surface
`receptors, for example-have arisen spontaneously in the human population.
`Analyses of the phenotypes of the affected individuals, together with studies of
`their cultured cells, have provided many unique insights into important human
`cell functions. Although such mutations are rare, they are very efficiently dis(cid:173)
`covered because of a unique human property: the mutant individuals call atten(cid:173)
`tion to themselves by seeking special medical care.
`
`Genetic Screens Identify Mutants Deficient in
`Cellular Processes
`
`Once a collection of mutants in a model organism such as yeast or flies has been
`produced, one generally must examine thousands of individuals to find the
`altered phenotype of interest. Such a search is called a genetic screen. Because
`obtaining a mutation in a gene of interest depends on the likelihood that the
`gene will be inactivated or otherwise mutated during random mutagenesis, the
`larger the genome, the less likely it is that any particular gene will be mutated.
`Therefore, the more complex the organism, the more mutants must be exam(cid:173)
`ined to avoid missin g genes. The phenotype being screened for can be simple or
`complex. Simple phenotypes are easiest to detect: a metabolic deficiency, for
`example, in which an organism is no longer able to grow in the absence of a
`particular amino acid or nutrient.
`Phenotypes that are more complex, for example mutations that cause
`defects in learning or memory, may require more elaborate screens (Figure
`8-56). But even genetic screens that are used to dissect complex physiological
`systems should be as simple as possible in design, and, if possible, should permit
`
`Figure 8-56 Screens can detect
`mutations that affect an animal's
`behavior. (A) Wild-type C. elegans engage
`in social feeding. The worms swim around
`until they encounter their neighbors and
`commence feeding. (B) Mutant animals
`feed by themselves. (Courtesy of Cornelia
`Bargmann, Ce// 94:cover. 1998. © Elsevier.)
`
`1 mm
`
`the examination of large numbers of mutants simultaneously. As an example,
`one particularly elegant screen was designed to search for genes involved in
`visual processing in the zebrafish. The basis of this screen, which monitors the
`fishes' response to motion , is a change in behavior. Wild- type fish tend to swim
`in the direction of a perceived motion, while mutants with defects in their visual
`systems swim in random directions-a behavior that is easily detected. One
`mutant discovered in this screen is called lakritz, which is missing 80% of the
`retinal ganglion cells that help to relay visual signals from the eye to the brain.
`As the cellular organization of the zebrafish retina mirrors that of all vertebrates,
`the study of such mu tants should also provide insights into visual processing in
`humans.
`Because defects in genes that are required for fundamental cell processes(cid:173)
`RNA synthesis and processing or cell cycle control, for example-are usually
`lethal , the functions of these genes are often studied in temperature-sensitive
`mutants. In these mutants the protein product of the mutant gene functions
`normally at a medium temperature, but can be inactivated by a small increase
`or decrease in temperature. Thus the abnormality can be switched on and off
`experimentally simply by changing the temperature. A cell containing a temper(cid:173)
`ature-sensitive mutation in a gene essential for survival at a non-permissive
`temperatu re can nevertheless grow at the normal or permissive temperature
`(Figure 8-57). The temperature-sensitive gene in such a mutant usually contains
`a point mutation that causes a subtle change in its protein product.
`Many temperature-sensitive mutants were isolated in the genes that encode
`the bacterial proteins required for DNA replication by screening populations of
`mutagen -treated bacteria for cells that stop making DNA when they are warmed
`from 30°C to 42°C. These mutants were later used to identify and characterize
`the corresponding DNA replication proteins (discussed in Chapter 5). Tempera(cid:173)
`ture-sensitive mutants also led to the identification of many proteins involved in
`regulating the cell cycle and in moving proteins through the secretory pathway
`in yeast (see Panel 13- 1). Related screening approaches have demonstrated the
`function of enzymes involved in the principal metabolic pathways of bacteria and
`yeast (discussed in Chapter 2), as well as discovering many of the gene products
`
`colonies replicated
`onto two identical
`plates and incubated
`at two different
`tempratures
`
`mutagenized cells plated
`out in Petri dish grow into
`colonies at 23°C
`
`mutant cell that divides
`at the permissive
`temperature but fa ils to
`divide at the restrictive
`temperature
`
`Figure 8-57 Screening for
`temperature-sensitive bacterial or
`yeast mutants. Mutagenized cells are
`plated out at the permissive temperature.
`The resulting colonies are transferred to
`two identical Petri dishes by replica
`plating; one of these plates is incubated at
`the permissive temperature, the other at
`the non-permissive temperature. Cells
`containing a temperature-sensitive
`mutation in a gene essential for
`proliferation can divide at the normal.
`permissive temperature but fai l to divide
`at the elevated, non-permissive
`temperature.
`
`528
`
`Chapter 8: MANIPULATING PROTEINS. DNA.AND RNA
`
`STUDYING GENE EXPRESSION AND FUNCTIO N
`
`529
`
`>-o--
`c2
`
`(:(.)
`- '
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`(_)
`Cl
`LU
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`... -----!:
`(./) er.
`LY
`0 z
`
`00055
`
`

`

`Figure 8-58 Using genetics to
`determine the order of function o f
`genes. In normal cells. proteins are
`loaded into vesicles. which fuse with the
`plasma membrane and secrete their
`contents into the extracellular medium. In
`secretory mutant A. proteins accumulate
`in the ER. In a different secretory mutant
`B, proteins accumulate in the Golgi. In the
`double mutant AB, proteins accumulate in
`the ER; this indicates that the gene
`defective in mutant A acts before the gene
`defective in mutant B in the secretory
`pathway.
`
`responsible for the orderly development of the Drosophila embryo (discussed in
`Chapter 21).
`
`Golgi
`secretory
`apparatus vesicles
`
`ER
`
`A Complementation Test Reveals Whether Two Mutations
`Are in the Same or in Different Genes
`
`A large-scale genetic screen can turn up many different mutants that show the
`same phenotype. These defects might lie in different genes th at function in the
`same process, or they might represent different mutations in the same gene.
`How tan we tell, then, whether two mutations that produce the same phenotype
`occur in the same gene or in different genes? If the mutations are recessive-if,
`for example, they represent a loss of function of a particular gene-a comple(cid:173)
`mentation test can be used to ascertain whether the mutations fall in the same
`or in different genes. In the simplest type of complementation test, an individual
`tha t is homozygous for one mutation-that is, it possesses two identical alleles
`of the mutant gene in question-is mated with an individual that is homozygous
`for the other mutation. If the two mutations are in the same gene, the offspring
`show the mutant phenotype, because they still will have no normal copies of the
`gene in question (see Panel 8-1 , pp. 526-527). If, in contrast, the mutations fall
`in different genes, the resulting offspring show a normal phenotype. Th ey retain
`one normal copy (and one mutant copy) of each gene. The mutations thereby
`complement one another and restore a normal phenotype. Complementation
`testing of mutants identified during genetic screens has revealed, for example,
`that 5 genes are required for yeast to digest the sugar galactose; that 20 genes
`are needed fo r E. coli to build a functional flagellum; that 48 genes are involved
`in assembling bacteriophage T4 viral particles; and that hundreds of genes are
`involved in the development of an adult nematode worm from a fertilized egg.
`Once a set of genes involved in a particular biological process has been iden(cid:173)
`tified, the next step is to determine in which order the genes function. Deter(cid:173)
`mining when a gene acts can facilitate the reconstruction of entire genetic or
`biochemical pathways, and such studies have been central to our understand(cid:173)
`ing of metabolism, signal transduction , and many other developmental and
`physiological processes. In essence, untangling the order in which genes func(cid:173)
`tion requires careful characterization of the phenotype caused by mutations in
`each different gene. Imagine, for example, that mutations in a handful of genes
`all cause an arrest in cell division during early embryo development. Close
`examination of each mutant may reveal that some act extremely early, prevent(cid:173)
`ing the fertilized egg from dividing into two cells. Other mutations may allow
`early cell divisions but prevent the embryo from reaching the blastula stage.
`To test predictions made about the order in which genes function, organ(cid:173)
`isms can be made that are mutant in two different genes. If these mutations
`affect two different steps in the same process, such double mutants should have
`a phenotype identical to that of the mutation that acts earliest in the pathway. As
`an example, the pathway of protein secretion in yeast has been deciphered in
`this manner. Different mutations in this pathway cause proteins to accumulate
`aberrantly in the endoplasmic reticulum (ER) or in the Golgi apparatus. When a
`cell is engineered to harbor both a mutation that blocks protein processing in
`the ER and a mutation that blocks processing in the Golgi compartment, pro(cid:173)
`teins accumulate in the ER. This indicates that proteins must pass through the
`ER before being sent to the Golgi before secretion (Figure 8- 58).
`
`Genes Can Be Located by Linkage Analysis
`
`With mutants in hand , the next step is to identify the gene or genes that seem to
`be responsible for the altered phenotype. If insertional mutagenesis was used
`for the original mutagenesis, locating the disrupted gene is fairly simple. DNA
`fragments containing the insertion (a transposon or a retrovirus, for example)
`are collected and amplified, and the nucleotide sequence of the flanking DNA is
`determined. This sequence is then used to search a DNA database i-o identify the
`gene that was interrupted by insertion of the transposable element.
`
`(1
`
`normal cell
`
`secretory mutant A
`
`secretory mutant B
`
`double mutant AB
`
`protein secreted
`
`protein accumulates
`in ER
`
`protein accumulates
`in Golgi apparatus
`
`protein accumulates
`in ER
`
`If a DNA-damaging chemical was used to generate the mutants, identifying
`the inactivated gene is often more laborious and can be accomplished by several
`different approaches. In one, the first step is to determine where on the genome
`the gene is located. To map a newly discovered gene, its rough chromosomal
`location is first determined by assessing how far the gene lies from oth er known
`genes in the genome. Estimating the distance between genetic loci is usually
`done by linkage analysis, a technique that relies on the fact that genes that lie
`near one another on a chromosome tend to be inherited together. The closer the
`genes are, the greater the likelihood they will be passed to offspring as a pair.
`Even closely linked genes, however, can be separated by recombination during
`meiosis. The larger the distance between t\-vo genetic loci, the greater the chance
`that they will be separated by a crossover (see Panel 8- 1, pp. 526-527). By calcu(cid:173)
`lating the recombination frequency between two genes, the approximate dis(cid:173)
`tance between them can be determined.
`Because genes are not always located close enough to one another to allow
`a precise pinpointing of their position, linkage analyses often rely on physical
`markers along the genome for estimating the location of an unknown gene.
`These markers are generally nucleotide fragments, with a known sequence a nd
`genome location, that can exist in at least two allelic forms. Single-nucleotide
`polymorphisms (S Ps), for example, are short sequences that differ by one or
`more nucleotides among individuals in a population. SNPs can be detected by
`hybridization techniques. Many such physical markers, distributed all along the
`length of chromosomes, have been collected for a variety of organisms, includ(cid:173)
`ing more than 106 for humans. If the distribution of these markers is sufficiently
`dense, one can, through a linkage analysis that tests for the tight coinheritance
`of one or more SNPs with the mutant phenotype, narrow the potential location
`of a gene to a chromosomal region that may contain only a few gene sequences.
`These are then considered candidate genes, and their structure and function
`can be tested directly to determine which gene is responsible for the original
`mutant phenotype.
`Linkage analysis can be used in the same way to identify the genes respon(cid:173)
`sible for heritable human disorders. Such studies require that D A samples b e
`collected from a large number of families affected by the disease. These samples
`are examined for the presence of physical markers such as SNPs that seem to be
`closely linked to the disease gene-these sequences would always be inherited
`by individuals who have the disease, and not by their unaffected relatives. The
`disease gene is then located as described above (Figure 8-59). The genes for cystic
`fibrosis and Huntington's disease, for example, were discovered in this manner.
`
`Searching for Homology Can Help Predict a Gene's Function
`
`Once a gene has been identified, its function can often be predicted by identi(cid:173)
`fying homologous genes whose functions are already known. As we discussed
`earlier, databases containing nucleotide sequences from a variety of organ(cid:173)
`including the complete genome sequences of many dozens of microbes,
`isms-
`C. elegans, A. thaliana, D. melanogaster, and human-can be searched for
`sequences that are similar to those of the uncharacterized target gene.
`
`530
`
`Chapter 8 : MA NIPULATING PROTEIN S, DNA. AN D RNA
`
`STUDYI N G GENE EXPRESSION AND FUNCTION
`
`531
`
`00056
`
`

`

`chromosome pair in
`mother with disease
`
`same chromosome pair
`in disease-free father
`
`defective gene
`causing diseas~
`
`SNP marker on - (cid:173)
`this copy of
`chromosome only
`
`egg - - - - . . . - - - - - sperm
`
`+
`+
`
`+
`+
`
`+
`+
`TESTS PERFORMED ON 7 CHILDREN
`
`+
`
`disease
`SNP marker
`
`CONCLUSION· gene causing disease is coinherited with SNP marker from diseased mother in
`75% of the diseased progeny. If this same correlation is observed in other families that have been
`examined, the gene causing disease is mapped to this chromosome close to the SNP. Note that a SNP
`that is either far away from the gene on the same chromosome. or located on a different chromosome
`than the gene of interest, will be coinherited only 50% of the time.
`
`When analyzing a newly sequenced genome, such a search serves as a first(cid:173)
`pass attempt to assign functions to as many genes as possible, a process called
`annotation. Further genetic and biochemical studies are then performed to
`confirm whether the gene encodes a product with the predicted function, as we
`discuss shortly. Homology analysis does not always reveal information about
`function: in the case of the yeast genome, 30% of the previously uncharacterized
`genes could be assigned a putative function by homology analysis; 10% had
`homologues whose function was also unknown; and another 30% had no homo(cid:173)
`logues in any existing databases. (The remaining 30% of the genes had been
`identified before sequencing the yeast genome.)
`In some cases, a homology search turns up a gene in organism A which pro(cid:173)
`duces a protein that, in a different organism, is fused lo a second protein that is
`produced by an independent gene in organism A. In yeast, for example, two sep(cid:173)
`arate genes encode two proteins that are involved in the synthesis of trypto(cid:173)
`phan; in E. coli, however, these two genes are fused into one (Figure 8-60).
`Knowledge that these two proteins in yeast correspond to two domains in a sin(cid:173)
`gle bacterial protein means that they are likely to be functionally associated, and
`probably work together in a protein complex. More generally, this approach is
`used to establish functional links between genes that, for mosl organisms, are
`widely separated in the genome.
`
`Reporter Genes Reveal When and Where a Gene Is Expressed
`Clues to gene function can often be obtained by examining when and where a
`gene is expressed in the cell or in the whole organism. Determining the pattern
`and timing of gene expression can be accomplished by replacing the coding
`
`gene 1
`
`gene 2
`
`organism B
`
`gene 3
`
`Figure 8-59 Genetic linkage analysis
`using physical markers on the DNA
`to find a human gene. In this example
`one studies the coinheritance of a specific
`human phenotype (here a genetic disease)
`with a SNP marker. If individuals who
`inherit the disease nearly always inherit a
`particular SNP marker, then the gene
`causing the disease and the SNP are likely
`to be close together on the chromosome.
`as shown here. To prove that an observed
`linkage is statistically significant, hundreds
`of individuals may need to be examined.
`Note that the linkage will not be absolute
`unless the SNP mar ker is located in the
`gene itself.Thus, occasionally the SNP will
`be separated from the disease gene by
`meiotic crossing-over during the
`formation of the egg or sperm: this has
`happened in the case of the chromosome
`pair on the far right. When working with a
`sequenced genome, this procedure wou ld
`be repeated with SN Ps located on either
`side of the initial SNP, until a I 00%
`coinheritance is found.
`
`portion of the gene under study with a reporter gene. In most cases, the expres(cid:173)
`sion of the reporter gene is then monitored by tracking the fluorescence or enzy(cid:173)
`matic activity of its protein product (pp. 518-519).
`As discussed in detail in Chapter 7, gene expression is controlled by regula(cid:173)
`tory DNA sequences, located upstream or downstream of the coding region,
`which are nol generally transcribed. These regulatory sequences, which control
`which cells will express a gene and under what conditions, can also be made to
`drive the expression of a reporter gene. One simply replaces the target gene's
`coding sequence with that of the reporter gene, and introduces these recombi(cid:173)
`nant DNA molecules into cells. The level, timing, and cell specificity of reporter
`protein production reflect the action of the regulatory sequences that belong to
`the original gene (Figure 8-61).
`Several other techniques, discussed previously, can also be used to deter(cid:173)
`mine the expression pattern of a gene. Hybridization techniques such as Northern
`analysis (see Figure 8-27) and in situ hybridization for RNA detection (see Fig(cid:173)
`ure 8-29) can reveal when genes are transcribed and in which tissue, and how
`much mRNA they produce.
`
`Microarrays Mon itor the Exp ression of Thousands
`of Genes at Once
`So far we have discussed techniques that can be used to monitor the expression
`of only a single gene at a time. Many of these methods are fairly labor-intensive:
`generating reporter gene constructs or GFP fusions requires manipulating DNA
`and transfecting cells with the resulting recombinant molecules. Even Northern
`analyses are limited in scope by the number of samples that can be run on an
`agarose gel. Developed in the 1990s, DNA microarrays have revolutionized the
`way in which gene expression is now analyzed by allowing the RNA products of
`thousands of genes to be monitored at once. By examining the expression of so
`many genes simultaneously, we can now begin to identify and study the gene
`expression patterns that underlie cellular physiology: we can see which genes are
`switched on (or off) as cells grow, divide, or respond to hormones or to toxins.
`
`for protein X -- •.
`
`coding sequence
`
`start site for RNA
`synthesis
`
`.
`
`EXPRESSION PATTERN
`OF GENE X
`
`cells
`A B C D E F
`
`ca=r=-
`
`pattern of normal gene X
`expression
`
`(A) STARTING DNA MOLECULES
`
`normal
`
`1
`'-
`
`3
`
`/
`
`2
`reg u l~tory
`DNA sequences
`that determine the
`expression of gene X
`
`2
`
`3
`
`(B) TEST DNA MOLECULES
`
`3
`
`2
`
`2
`
`coding sequence for
`reporter protein Y
`
`EXPRESSION PATTERN OF
`REPORTER GENE Y
`
`EXPRESSION PATTERN OF
`REPORTER GENEY
`
`Figure 8-60 D omain fusions reveal
`relationships betwee n functionally
`linked ge nes. In this example, the
`functional interaction of genes I and 2 in
`organism A is inferred by the fusion of
`homologous domains into a single gene
`(gene 3) in organism B.
`
`(C) CONCLUSIONS
`
`-regulatory sequence 3 turns on gene X in cell B
`-regulatory sequence 2 turns on gene X in cells D, E, and F
`-regulatory sequence 1 turns off gene X in cell D
`
`-:(
`:2:
`C:t:
`(
`I
`_J
`
`>-o--
`c2 co
`
`_J
`-..J
`<-.(
`C>
`Cl
`
`LU ---
`
`Figure 8-61 Using a reporter protein
`to determine the pattern of a gene's
`expression. (A) In this example the
`coding sequence for protein X is replaced
`by the coding sequence for protein Y.
`(B) Various fragments of DNA containing
`candidate regulatory sequences are added
`in combinations. The recombinant DNA
`molecules are then tested for expression
`after their transfection into a variety of
`different types of mammalian cells, and the
`results are summarized in (C). For
`experiments in eucaryotic cells, two
`commonly used reporter proteins are the
`enzymes P-galactosidase ( [J.gal) and green
`fluorescent protein or GFP (see Figure
`9-44). Because these are bacterial
`enzymes, their presence can be monitored
`by simple and sensitive assays of enzyme
`activity, without any interference from
`host cell enzymes. Figure 7-39 shows an
`example in which the P-gal receptor gene
`is used to monitor the activity of the eve
`gene regulatory sequence in a Drosophila
`embryo.
`
`532
`
`Chapter 8 : MANIPULATING PROTEINS, DNA.AND RNA
`
`STUDYIN G GENE EXPRESSION AND FUNCTION
`
`533
`
`00057
`
`

`

`•
`
`collection of gene-specific DNA molecules
`+
`PCR amplification
`+
`robotic 'printing' onto glass slide
`+
`
`mRNA from
`sample 1 labeled
`with red
`fluorochrome
`
`mRNA from
`sample 2 labeled
`with green
`fluorochrome
`
`HYBRIDIZE
`+
`WASH
`+
`SCAN RED AND GREEN SIGNALS
`AND COMBINE IMAGES
`
`' . . ,,
`. '
`
`0
`
`small region of microarray representing
`expression of 11 O genes from yeast
`
`•
`
`'
`• •
`•
`
`4
`
`'
`
`Figure 8-62 Using DNA microarrays to monitor the expression of
`thousands of genes simultaneously. To prepare the microarray, DNA
`fragments--each corresponding to a gene-are spotted onto a slide by a
`robot. Prepared arrays are also available commercially. In this example,
`mRNA is collected from two different cell samples for a direct comparison
`of their relative levels of gene expression. These samples are converted to
`cDNA and labeled, one with a red fluorochrome, the other with a green
`fluorochrome. The labeled samples are mixed and then allowed to hybridize
`to the microarray. After incubation, the array is washed and the fluorescence
`scanned. In the portion of a microarray shown, which represents the
`expression of I IO yeast genes, red spots indicate that the gene in sample I is
`expressed at a higher level than the corresponding gene in sample 2; green
`spots indicate that expression of the gene is higher in sample 2 than in
`sample I. Yellow spots reveal genes that are expressed at equal levels in both
`cell samples. Dark spots indicate little or no expression in either sample of
`the gene whose fragment is located at that position in the array. For details
`see Figure 1-45. (Microarray c