B

____CCTCCGGTAGAG

Figure 9.4 A Portion of the P-hemoglobin Gene with Codes for Normal and Sickle-cell Traits. A. This portion of the normal hemoglobin gene contains a restriction enzyme site, which is underlined. B. The same region shown in A for the hemoglobin of an individual with sickle-cell anemia. The asterisk shows the single nucleotide mutation. This mutation makes the restriction enzymes unable to recognize the site.

So how do we tell the difference between individuals? Remember from chapter 1 that DNA is a double helix held together by complementary base pairing. Recall also that we can now make single-stranded DNA molecules of a specific base sequence in the test tube and use them as primers for polymerase chain reaction. We can use these tools, restriction enzymes and DNA with specific sequences, to distinguish between the DNA from two different individuals. Let's take the case of sickle-cell anemia. A short region of the DNA encoding normal P-hemoglobin is shown in figure 9.4.A. The underlined region indicates a restriction-enzyme site, a place that will be cut by a certain restriction enzyme. The mutation in sickle-cell anemia is a single nucleotide change indicated by an asterisk in figure 9.4.B. Because the base change occurs within the restriction enzyme site, the restriction enzyme no longer recognizes this sequence and so will not cut the mutant DNA at this position. The change in the cutting pattern of DNA is illustrated in figure 9.5.A. This is an example of a single-nucleotide polymorphism (abbreviated SNP) because only a single nucleotide was changed.

Figure 9.5 Restriction Fragment Length Polymorphism of Sickle-cell Anemia. A. Double strands of DNA are depicted with the positions of restriction enzyme cuts marked with scissors. The upper DNA shows a small portion of the normal P-hemoglobin gene from an individual with normal P-hemoglobin; below it is the same section from an individual with sickle-cell anemia. Note that a restriction site is now missing due to the mutation. The strand of complementary DNA is positioned between the two DNA double strands. B. A gel elec-trophoretic analysis of DNA from a sickle-cell anemia patient. The marker lane contains size markers; the sickle lane shows the long single fragment found in the mutant. The normal lane shows the two smaller bands of DNA corresponding to the two DNA fragments created by the restriction enzymes. The carrier lane, as expected, has bands representing both the mutant and the normal DNA.

Figure 9.5 Restriction Fragment Length Polymorphism of Sickle-cell Anemia. A. Double strands of DNA are depicted with the positions of restriction enzyme cuts marked with scissors. The upper DNA shows a small portion of the normal P-hemoglobin gene from an individual with normal P-hemoglobin; below it is the same section from an individual with sickle-cell anemia. Note that a restriction site is now missing due to the mutation. The strand of complementary DNA is positioned between the two DNA double strands. B. A gel elec-trophoretic analysis of DNA from a sickle-cell anemia patient. The marker lane contains size markers; the sickle lane shows the long single fragment found in the mutant. The normal lane shows the two smaller bands of DNA corresponding to the two DNA fragments created by the restriction enzymes. The carrier lane, as expected, has bands representing both the mutant and the normal DNA.

Figure 9.5.B shows the gel electrophoresis pattern of several individuals, two of whom have at least one gene for sickle-cell anemia. The specific bands are differentiated from the background DNA smear because they can bind to the "complementary DNA" probe specific to a region of the P-hemoglobin gene. The complementary DNA can be labeled with a dye for easy detection. No other bands present in the DNA smear binds the complementary DNA, and so they remain invisible. Another example of this technique is shown in box 9.1.

In the sickle-cell anemia example, the defect is a specific change in the DNA sequence of a specific gene , the P-hemoglobin gene. This means that the band of DNA in RFLP is always associated with a sickle-cell defect. However, most often we do not know what gene or what defect in a gene is associated with a disease.

Box 9.1 Identifying Disease Genes Using Restriction Fragment Length Polymorphism

Restriction fragment length polymorphism allows us to identify the piece of DNA associated with a genetic disease. In this procedure, the DNA of affected and unaffected individuals are cut with the same restriction enzyme. If we arranged these pieces by size, we would get a large range, from small to large, and there would not be a distinct group of pieces of a particular size. Recall from chapter 1 that gel electrophoresis is used to separate pieces of DNA by size: if too many pieces of many different sizes are run together, the resulting gel would show the DNA spread out in a smear.

So how can we tell where a particular piece of DNA is in this smear? Recall that DNA has a double-helical structure held together by complementary base pairs. We can make a short piece of DNA with a specific sequence and label it with a dye. After the DNA is cut and put on a gel and separated by size, we can transfer these pieces of cut DNA onto a sheet that prevents them from floating away. By using appropriate chemicals, we can then separate the double strands of the cut DNA pieces. At that point, the specific short piece of labeled DNA is added and allowed to bind to the complementary sequence present somewhere among the cut pieces ofDNA on the sheet. Recall from chapter 1 that short pieces of DNA find the complementary partner faster than large pieces.

Box 9.1 continued

After that, we wash away any labeled DNA that has not bound to its complementary sequence. The band identified by the labeled DNA is a piece of DNA that has within its length a sequence complementary to the labeled DNA. The position of the labeled band determines the size of the DNA piece and how far it moved in the original gel electrophoresis.

Figure B.9.1 RFLP to Identify Congenital Adrenal Hyperplasia. Gel electrophoresis of DNA from normal individuals (norm), those that are heterozygous (het) for congenital adrenal hyperplasia, and congenital adrenal hyperplasia patients (CAH). M provides size markers used to determine the size of the bands shown on the right as numbers of base pairs (bp). Note that both affected individuals (CAH) have a single band at 424 base pairs whereas normal individuals are missing that band and have instead two bands at 298 base pairs and 126 base pairs.

Figure B.9.1 RFLP to Identify Congenital Adrenal Hyperplasia. Gel electrophoresis of DNA from normal individuals (norm), those that are heterozygous (het) for congenital adrenal hyperplasia, and congenital adrenal hyperplasia patients (CAH). M provides size markers used to determine the size of the bands shown on the right as numbers of base pairs (bp). Note that both affected individuals (CAH) have a single band at 424 base pairs whereas normal individuals are missing that band and have instead two bands at 298 base pairs and 126 base pairs.

Now, we can treat this band of DNA on the gel as a genetic trait in the same way we view a person's blood type. Figure B.9.1 shows an example of identifying the genetic defect for congenital adrenal hyperplasia. Congenital adrenal hyperplasia is a recessive disease caused by a defect in an enzyme important for salt balance. Infants with this disease can develop life-threatening dehydration or shock. This disease is treatable with hormones.

Even in cases where the disease mutation itself does not produce different banding patterns in RFLP, this technique can help track down the gene responsible for a disease. This is because a special banding pattern may be associated with a disease. Why might this be? This would be the case if a particular DNA sequence is closely linked to the disease gene, that is, located close to the disease gene on the chromosome. Recall that recombination between genes is caused by the breaking and rejoining of chromosomes. The recombination frequency depends upon the distance between the two genes. So we can consider a band on a gel as a gene. If the rate of recombination between a particular DNA sequence and the actual defective gene that causes the disease is low, the distance between them must be very short, that is, that piece of DNA must be close to the disease gene. By this logic, one can find a fragment of DNA that is close to the defective gene. The closer the piece of DNA represented by the restriction fragment, the more likely that piece will be inherited together with the defective gene. Since the restriction fragment can be detected by gel electrophoresis, we can locate the defective gene closely associated with it. Researchers analyze the DNA of large families in which the disease exists; affected family members' DNA should show a higher chance of being associated with a specific DNA sequence close to the disease gene, whereas those of unaffected members should not. It is by this technique that scientists have closed in on genes responsible for many human genetic diseases.

Box 9.2 Identifying a Disease-Resistance Gene in Barley Through Map-Based Cloning

The most interesting and important genes, such as disease-causing genes in humans or disease-resistance genes in plants, are often the most difficult to identify because we do not know what proteins they code for or what their function might be. Cloning a gene is the first step toward learning how it functions and using it to suit our needs.

Stem rust is a fungal disease in barley that routinely reduced crop yields until the early 1940s. In a particularly bad epidemic of the disease in 1935, Sam Lykken, a farmer from Kindred, North Dakota, identified a single healthy plant in his field, which was otherwise totally decimated by stem rust. This single healthy plant was saved and became the source of resistance genes in barley strains. It turns out that this strain of barley contained the dominant stem

Box 9.2 continued rust-resistance gene that has since been given the name "Rpgl." The presence of this gene has prevented any significant losses due to this disease since then.

To learn more about the stem rust-resistance gene in barley, a team of scientists led by Andris Kleinhofs at Washington State University in Pullman, Washington, set out to clone this gene. How can one identify this gene or other genes that are known only by their phenotype? We can look for DNA markers or bands on gels linked to the trait. The barley genome is huge; thus a gene can be hundreds of thousands of base pairs away from the DNA marker to which it is linked! So the scientists put large pieces of the barley genome into bacteria in such a way that each bacterium contained a portion of the barley genome. Recombinant DNA molecules like these are called bacterial artificial chromosomes, or BAC clones. One can identify the BAC clone of interest with the DNA markers linked to Rpgl. Because the BAC clones contain random pieces of the genome, if one has enough of them overlapping pieces can be found. Chromosome walking is a process used to follow the sequence of DNA in overlapping pieces of DNA like BAC clones. Once a number of overlapping DNA clones including the gene of interest are found, they are sequenced to identify potential genes and candidates for the gene of interest (figure B.9.2.A)

We say candidate genes at this point in the process because we still do not know which gene is the one responsible for disease resistance. Searching among the candidate genes to identify the resistance gene is difficult. The DNA sequences must be compared between resistant and susceptible strains to identify which gene is consistently different between them. If a particular gene always shows a difference, this is strong evidence, but not proof, that the correct gene has been identified. Kleinhofs's team used this method to identify the Rpgl gene, but they also transformed barley with the Rpgl gene. If a gene is indeed the one that confers resistance to stem rust, the transformed barley should be resistant. Indeed the transformation of a susceptible barley strain with the Rpgl gene conferred resistance to the stem-rust pathogen! Thus Rpgl is truly the stem rust-resistance gene! Interestingly, the transgenic plants continued on next page

Figure B.9.2 Identifying the Disease-Resistance Gene in Barley. A. A diagram illustrating map-based cloning. Over half a million base pairs of the barley genome containing Rpgl, the stem rust-resistance gene, are shown at the top. The two DNA markers linked to Rpgl are shown below the bacterial artificial chromosomes (BAC clones) that contain this DNA sequence. Additional contiguous BAC clones used to "walk" to the disease resistance gene are shown in dark mottled lines. B. Photographs of barley leaves treated with spores of the fungus that causes stem-rust disease. On the left is the susceptible strain, in the middle is the resistant strain, and on the right, a susceptible strain transformed with the resistance gene Rpgl. Note that the transformed strain shows no evidence of rust, and is thus more resistant than the original resistant strain. Photo courtesy David Hansen.

were more resistant to stem rust than the original resistant strain from which the gene was isolated (figure B.9.2.B). Though this was an arduous process that took years, the identification of the resistance gene by the Kleinhofs's team allows researchers to understand disease resistance better and to use this knowledge and this gene for agricultural purposes.

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