POB Chapter 15
A researcher is working with a cell line that was derived from a tumor. The cells continuously divide in culture. The researcher transfects the cell line with a gene, and the cells stop dividing. The gene transfected into the cell line is a wild-type tumor _______ gene. Suppressor
A duplication of gene X Which chromosomal mutation would allow protein X to remain functional?
Prenatal screening can be used to identify an embryo or fetus with a disease so that medical intervention can be applied or decisions can be made about whether or not to continue the pregnancy. Newborn babies can be screened so that proper medical intervention can be initiated quickly for those babies who need it. Asymptomatic people who have relatives with a genetic disease can be screened to determine whether they are carriers of the disease-associated allele or are likely to develop the disease themselves. Genetic screening can be done at the level of either the phenotype or the genotype.
How can a gene mutation that causes a disease be mapped and detected before its protein product is known? Mapping a disease-causing mutation can be done by linkage analysis. A polymorphic DNA marker such as an STR can be linked to the occurrence of a disease in many patients. This means that the marker must lie on the chromosome near the mutant disease-causing gene. DNA sequencing can then isolate the gene involved. From the gene sequence or genetic technology, the protein encoded by the gene can then be isolated and its function described. So genotype precedes phenotype.
A nonsense mutation involves a base substitution that causes a stop codon to occur prematurely in the mRNA, resulting in a shortened, and typically nonfunctional, protein. However, if the nonsense mutation is found very near the end of the coding sequence, the protein may still be functional. A nonsense mutation involves a base substitution that causes a stop codon (for translation) to form somewhere in the mRNA (see Figure 15.2D). A nonsense mutation results in a shortened protein, since translation does not proceed beyond the point where the mutation occurred.
In frame-shift mutations, nucleotides may be inserted into, or deleted from, a gene. The reading frame of the mRNA is altered during translation, almost always leading to nonfunctional proteins.
Genetic screening is used to detect human genetic diseases, alleles predisposing people to those diseases, or carriers of those disease alleles. Genetic screening can be done by looking for abnormal protein expression. Review Figure 15.15 DNA testing is the direct identification of mutant alleles. Any cell can be tested at any time in the life cycle.
In gene therapy, a mutant gene is replaced with a normal gene. Both ex vivo and in vivo therapies are being developed.
Many techniques exist for testing whether a sample of DNA carries a mutation. In this animation, we explore the use of two different techniques for identifying whether an individual carries the sickle-cell allele of the β-globin gene. One procedure is called allele-specific oligonucleotide hybridization. In allele-specific oligonucleotide hybridization, the binding of a probe to sample DNA indicates that a particular allele is present in the DNA. The other procedure, DNA testing by allele-specific cleavage, uses restriction enzymes as diagnostic tools. Restriction enzymes are proteins that recognize and cut DNA at specific sequences. Allele-specific cleavage relies on the mutation in the disease allele either adding or eliminating a recognition site for a restriction enzyme.
A DNA test called allele-specific oligonucleotide hybridization. In this procedure, a short DNA fragment—the oligonucleotide (also called the probe)—is created to match the normal version of the gene, and another oligonucleotide is created to match the sickle allele. The normal and sickle probes differ by a single nucleotide. Another way that the sickle-cell allele has been identified is by using enzymes that cleave DNA. In sickle-cell anemia, the sickle-cell allele has a mutation that converts an adenine base to a thymine base. Whereas the normal sequence is digested by the restriction enzyme MstII, this enzyme cannot recognize the sequence in the sickle-cell allele and therefore does not cut it. In this test, you use PCR to amplify the DNA region that includes the mutation site. If you were to digest both samples of DNA with MstII, the normal allele would produce two fragments, and the sickle allele would produce just one. In performing this test, you would digest DNA from the test sample of interest with MstII, as well as digest DNA from a normal β-globin sample and a sickle-cell sample. The digested DNA is then loaded onto an agarose gel for electrophoresis.
A scientist plans to use allele-specific oligonucleotide hybridization to analyze a DNA sample for the presence of a mutation. How many oligonucleotide probes will he have to prepare? C. Two probes: one complementary to the mutation and another complementary to the normal sequence
A mutation in a gene disrupts a restriction enzyme cleavage site. If the DNA containing this mutation is incubated with this restriction enzyme and the resulting fragments separated by agarose gel electrophoresis, what will be observed? A. The mutant DNA will have one fewer fragment than normal DNA.
Missense mutations are *base substitution changes that alter the genetic code such that one amino acid substitutes for another in a protein (see Figure 15.2C). A specific example is the mutation that causes sickle-cell disease, a serious heritable blood disorder. The disease occurs in people who carry two copies of the sickle allele of the gene for β-globin—a subunit of hemoglobin, the protein in human blood that carries oxygen. The sickle allele differs from the wild-type allele by one base pair, resulting in a polypeptide that differs by one amino acid from the wild-type protein. Individuals who are homozygous for this recessive allele have defective, sickle-shaped red blood cells.
A point mutation is the addition or subtraction of a single nucleotide, or the substitution of one nucleotide base for another. There are two kinds of base substitution: 1. A transition is the substitution of one purine for the other purine, or one pyrimidine for the other 2. A transversion is the substitution of a purine for a pyrimidine, or vice versa:
This phenomenon of expanding triplet repeats has been found in more than a dozen other diseases, such as myotonic dystrophy (involving repeated CTG triplets) and Huntington's disease (in which CAG is repeated). Such repeats, which may be found within a protein-coding region or outside it, appear to be present in many other genes without causing harm. How the repeats expand is not known; one hypothesis is that DNA polymerase may slip after copying a repeat and then fall back to copy it again. expanding triplet repeat: A three-base-pair sequence in a human gene that is unstable and can be repeated a few to hundreds of times. Often, the more the repeats, the less the activity of the gene involved. Expanding triplet repeats occur in some human diseases such as Huntington's disease and fragile-X syndrome.
About one-fifth of all males who have the fragile-X chromosomal abnormality are phenotypically normal, as are most of their daughters. But many of those daughters' sons are intellectually disabled. In a family in which fragile-X syndrome appears, later generations tend to show earlier onset and more severe symptoms of the disease. It is almost as if the abnormal allele itself is changing—and getting worse. And that's exactly what is happening. The gene associated with fragile-X syndrome (FMR1) contains a repeated triplet, CGG, at a certain point in the promoter region (Figure 15.9). In normal people, this triplet is repeated 6 to 54 times (the average is 29). In intellectually disabled people with fragile-X syndrome, the CGG sequence is repeated 200 to 2,000 times.
How are chromosomal mutations detected? Hint: See Figure 11.20. Chromosomal mutations can be detected by staining dividing cells with dyes specific for each chromosome. Stained chromosomes can then be identified, and missing pieces or translocated pieces can be observed. Inversions can be detected by a special method called banding, whereby dyes on chromosomes produce banding patterns instead of colors. In this case, a reversal of bands can be seen.
Changes in single nucleotides are not the most dramatic changes that can occur in the genetic material. Whole DNA molecules (that is, whole chromosomes) can break and rejoin, grossly disrupting the sequence of genetic information. There are four types of such chromosomal mutations: deletions, duplications, inversions, and translocations. These mutations can be caused by severe damage to chromosomes resulting from mutagens or by drastic errors in chromosome replication. A deletion occurs by the removal of part of the genetic material and can happen if a chromosome breaks at two points and then rejoins, leaving out the DNA between the breaks (Figure 15.3A). A duplication can be produced at the same time as a deletion and can occur if homologous chromosomes break at different positions and then reconnect to the wrong partners (Figure 15.3B). One of the two chromosomes ends up with a deleted segment, and the other has two copies (a duplication) of the same segment. An inversion can also result from the breaking and rejoining of a chromosome, and can occur if a segment of DNA becomes "flipped," so that it runs in the opposite direction from its original orientation (Figure 15.3C). A translocation results when a segment of a chromosome breaks off and becomes attached to a different chromosome. As we mentioned in Key Concept 11.5, a translocation of a large segment of chromosome 21 is one cause of Down syndrome. Translocations may involve reciprocal exchanges of chromosome segments, as in Figure 15.3D.
Allele-specific oligonucleotide hybridization is a widely used test that allows researchers to distinguish between two alleles that differ by a single nucleotide. The oligonucleotide probes are created so that each type hybridizes to DNA from just one of the alleles. Another probe hybridizes to a second allele. The probes need to be perfectly matched to the alleles, because a single mismatch will prevent hybridization of the probe to the DNA.
DNA testing by allele-specific cleavage uses restriction enzymes as diagnostic tools. In the example of sickle-cell anemia, the mutation that causes the disease also eliminates an MstII recognition site from the mutated β-globin gene. For this reason, the allele-specific cleavage test can be used to determine a person's genotype. However, many mutations that cause disease do not affect the recognition sites of restriction enzymes, thereby limiting the use of this particular technique in diagnosing disease.
DNA testing: In human genetics, the determination of genotype by analysis of DNA sequence.
DNA testing is the direct analysis of DNA for a mutation, and it offers the most direct and accurate way of detecting an abnormal allele. Now that the mutations responsible for many human diseases have been identified, any cell in the body can be examined at any time of life for mutations. The amplification power of PCR means that only one or a few cells are needed for testing. These methods work best for diseases caused by only one or a few different mutations.
CHROMOSOMAL ABNORMALITIES Chromosomal abnormalities also cause human diseases. Such abnormalities result from the gain or loss of complete chromosomes (aneuploidy) (see Figure 11.19), or from the gain or loss of chromosomal segments (see Figure 15.3). About 1 newborn in 200 has a chromosomal abnormality. This may be inherited from a parent who also has the abnormality, or it may result from an error in meiosis during the formation of gametes in one of the parents. One example is fragile-X syndrome, which is a constriction in the tip of the X chromosome that can result in intellectual disability (Figure 15.8). About 1 male in 3,000 and 1 female in 7,000 are affected. Although the basic pattern of inheritance is that of an X-linked recessive trait, there are departures from this pattern. Not all people with the fragile-X chromosomal abnormality are intellectually disabled, as we will see.
Disease-causing mutations may involve a single base pair, a long stretch of DNA, multiple segments of DNA, or even entire chromosomes (as we saw for Down syndrome in Key Concept 11.5). POINT MUTATIONS Sickle-cell anemia is just one of many diseases caused by a point mutation. In some cases (sickle-cell anemia, for example), everyone with the disease has the same genetic mutation. In other cases, different loss-of-function point mutations in one gene can lead to the same disease (as we saw above for PKU). Think about it: the three-dimensional structure of an enzyme protein depends on its secondary structure, so any change in the amino acid sequence of a protein has the potential to affect its structure, and consequently its function. LARGE DELETIONS Larger mutations may involve many base pairs of DNA. For example, deletions in the X chromosome that include the gene for the protein dystrophin result in Duchenne muscular dystrophy. Dystrophin is important in organizing the structure of muscles, and people who have only the abnormal form have severe muscle weakness. Sometimes only part of the dystrophin gene is missing, leading to an incomplete but partly functional protein and a mild form of the disease. In other cases, deletions span the entire sequence of the gene, so that the protein is missing entirely, resulting in a severe form of the disease. In yet other cases, deletions involve millions of base pairs and cover not only the dystrophin gene but adjacent genes as well; the result may be several diseases in the same person.
ABNORMAL HEMOGLOBIN As mentioned in Key Concept 15.1, sickle-cell disease is caused by a recessive, missense mutation. This blood disorder most often afflicts people whose ancestors came from the tropics or from the Mediterranean region. Recall that human hemoglobin is composed of four globin subunits—two α-chains and two β-chains—as well as the pigment heme (see Figure 3.11). In sickle-cell disease, one of the 146 amino acids in the β-globin polypeptide chain is abnormal: at position 6, glutamic acid is replaced by valine. This replacement changes the charge of the protein (glutamic acid is hydrophilic and valine is hydrophobic), causing it to form long, needlelike aggregates in the red blood cells. The phenotypic result is sickle-shaped red blood cells and an impaired ability of the blood to carry oxygen. The sickled cells tend to block narrow blood capillaries, resulting in tissue damage and eventually death by organ failure.
Genetic mutations are often expressed phenotypically as proteins that differ from normal (wild-type) proteins. Abnormalities in enzymes, receptor proteins, transport proteins, structural proteins, and most of the other functional classes of proteins have all been implicated in genetic diseases. The disease results from an abnormality in a single enzyme, phenylalanine hydroxylase (PAH),
There are two approaches to somatic cell gene therapy: 1. Ex vivo gene therapy: Target cells are removed from the patient, given the new gene, and then reinserted into the patient. This approach is being used, for example, for diseases caused by defects in genes that are expressed in white blood cells. 2. In vivo gene therapy: The gene is actually inserted directly into a patient, targeted to the appropriate cells. An example is a treatment for lung cancer in which a solution with a therapeutic gene is squirted onto a tumor.
If a cell lacks an allele that encodes a functional product, an optimal treatment would be to provide a functional allele. The objective of gene therapy is to add a new gene that will be expressed in appropriate cells in a patient. What cells should be targeted? There are two approaches: 1. Germ line gene therapy: The new gene is inserted into a gamete (usually an egg) or the fertilized egg. In this case, all cells of the adult will carry the new gene. Ethical considerations preclude the use of this method in humans. 2. Somatic cell gene therapy: The new gene is inserted into somatic cells involved in the disease. This method is being tried for numerous diseases, ranging from inherited genetic disorders to cancer.
What are the differences between point mutations that cause phenotypic changes and those that don't? Point mutations that cause phenotypic changes could have resulted in a different amino acid in the encoded protein that consequently changes a protein's function; changed a promoter so a gene's expression is significantly altered; or created a stop codon that terminates expression prematurely, resulting in a shorter nonfunctional protein. Point mutations that are phenotypically silent may arise in codons where redundancy ensures no amino acid change; cause codon changes that result in amino acid changes that are not significant to protein function; or occur in noncoding regions of DNA, such as introns.
In certain regions of DNA, many of the cytosine residues have methyl groups added at their 5 positions, forming 5−methylcytosine. Methylation plays an important role in gene regulation (discussed in Key Concept 16.4). DNA sequencing has revealed that mutation "hot spots" are often located where cytosines have been methylated. Figure 15.4 shows unmethylated cytosine, which can lose its amino group, either spontaneously (see Figure 15.4A) or because of a chemical mutagen, to form uracil (see Figure 15.4B). This type of error is usually detected by the cell and repaired, because uracil is recognized as inappropriate for DNA. However, when 5−methylcytosine (methylated cytosine) loses its amino group, the product is thymine, a natural base for DNA (Figure 15.5). The DNA repair mechanism ignores this thymine. During replication, however, the mismatch repair mechanism recognizes that G-T is a mismatched pair, although it cannot tell which base is incorrect. Half of the time it matches a new C to the G, but the other half of the time it matches a new A to the T, resulting in a mutation.
The DNA bands will migrate more slowly across the gel and will appear to be larger than they are. Challenge this Question Suppose you run gel electrophoresis using the standard voltage, but the spaces between the polymers of the gel are smaller than usual because of an error in making the gel. The samples are commonly run, so you neglect to include a lane with standards that would indicate the sizes of the bands. What is the most likely result? Please choose the correct answer from the following choices, and then select the submit answer button.
Incidences of most forms of cancer increase with age. Which of the following explanations is most plausible? Cancer usually requires several somatic mutations.
Induced mutations occur when some agent from outside the cell—a mutagen—causes a permanent change in DNA. As we mentioned above, retroviruses can function as mutagens. In addition, certain chemicals and radiation can cause mutations: 1. Some chemicals can alter nucleotide bases. For example, nitrous acid (HNO2) and similar molecules can react with cytosine and convert it to uracil by deamination. More specifically, they convert an amino group on the cytosine (—NH2) into a keto group (—C=O) (Figure 15.4B). This alteration has the same result as spontaneous deamination: instead of a G, DNA polymerase inserts an A (see Figure 15.4C). 2. Some chemicals add groups to the bases. For instance, benzopyrene, a component of cigarette smoke, adds a large chemical group to guanine, making it unavailable for base pairing. When DNA polymerase reaches such a modified guanine, it inserts any one of the four bases at random. Three-fourths of the time the inserted base is not cytosine, and a mutation results. 3. Radiation damages the genetic material. Radiation can damage DNA in two ways. First, ionizing radiation (including X rays, gamma rays, and radiation from unstable isotopes) produces highly reactive chemicals called free radicals. Free radicals can change bases in DNA to forms that are not recognized by DNA polymerase. Ionizing radiation can also break the sugar-phosphate backbone of DNA, causing chromosomal abnormalities. Second, ultraviolet radiation (from the sun or a tanning lamp) is absorbed by thymine, causing it to form covalent bonds with adjacent bases. This, too, plays havoc with DNA replication by distorting the double helix.
It is useful to distinguish between mutations that are spontaneous or induced, based on their causes. Spontaneous mutations are permanent changes in the genetic material that occur without any outside influence. The movement of transposons is an example of spontaneous mutation. Spontaneous mutations can also occur because cellular processes are imperfect, and may occur by several mechanisms: 1. A transient rearrangement in the structure of a nucleotide base can result in mistakes during replication. Each base can exist in two different forms (called tautomers), one of which is common and one rare. When a base temporarily forms its rare tautomer, it can pair with the wrong base. For example, C normally pairs with G, but if C takes on the form of its rare tautomer at the time of DNA replication, it pairs with (and DNA polymerase will insert) an A. If this is passed on to a daughter cell after cell division, the result is a point mutation: G → A (Figure 15.4A and C). 2. A chemical reaction may alter the structure of a DNA base. For example, a deamination reaction can result in loss of the amino group (NH2) attached to carbon 4 in cytosine. If this occurs in a DNA molecule, the error will usually be repaired. However, since the repair mechanism is not perfect, the altered nucleotide will sometimes remain during replication. In these cases, DNA polymerase will add an A (which base-pairs with U on the template DNA) instead of G (which normally pairs with C). 3. DNA polymerase can make errors in replication (see Key Concept 13.4)—for example, by inserting a T opposite a G. Most of these errors are repaired by the proofreading function of the replication complex, but some errors escape detection and become permanent. 4. Meiosis is not perfect. Nondisjunction—the failure of homologous chromosomes to separate during meiosis—can occur, leading to one too many chromosomes or one too few (see Figure 11.19). Random chromosome breakage and rejoining can produce deletions, duplications, inversions, or translocations.
Another form of DNA, called a transposon or transposable element, can also insert itself into genes and cause mutations. As you will see in Chapter 17, transposons are widespread in both prokaryotic and eukaryotic genomes. A transposon is a DNA sequence of a few hundred to a few thousand base pairs that can move from one position in the genome to another. It usually carries genes that encode the enzymes needed for this movement. Some transposons remove themselves from their positions in the genome and then insert themselves into other sites (the "cut and paste" mode of transposition). These transposons do not always excise cleanly, but leave behind short sequences of a few base pairs that become permanent mutations in the affected genes. Other transposons first replicate themselves, and then the new copies are inserted into new sites in the genome (the "copy and paste" mode). A sequence of genomic DNA is sometimes carried along with the transposon DNA when it moves, and gene duplication occurs. As you'll see, gene duplication plays an important role in evolution.
Key Concept 14.2 described how certain viruses called retroviruses can insert their genetic material into the host cell's genome. Such insertions happen at random, and if one occurs within a gene, it can cause a loss-of-function mutation in that gene. In many cases, the viral DNA remains in the host genome and is passed on from one generation to the next. When this happens the virus is called an endogenous retrovirus. Endogenous retroviruses are common—in fact, they make up 5 to 8 percent of the human genome.
Large DNA molecules can be cut into smaller pieces by restriction digestion and then sorted by gel electrophoresis. PCR is used to amplify sequences of interest from complex samples. These techniques are used in DNA fingerprinting to analyze DNA polymorphisms for the purpose of identifying individuals. Genes involved in disease can be identified by first detecting the abnormal DNA sequence and then the protein that the wild-type allele encodes. Scientists hope to be able to identify all species using DNA analysis.
Large DNA molecules can be cut into smaller pieces by restriction digestion and then sorted by gel electrophoresis. PCR is used to amplify sequences of interest from complex samples. These techniques are used in DNA fingerprinting to analyze DNA polymorphisms for the purpose of identifying individuals. Genes involved in disease can be identified by first detecting the abnormal DNA sequence and then the protein that the wild-type allele encodes. Scientists hope to be able to identify all species using DNA analysis.
SUPPLYING THE MISSING PROTEIN An obvious way to treat a disease caused by the lack of a functional protein is to supply that protein. This approach is used to treat hemophilia A, a disease in which blood factor VIII is missing and blood clotting is impaired (see Table 15.2). In the past, the missing protein was obtained from blood. Sometimes, however, blood carries contaminants, such as viruses (e.g., HIV) or other pathogens that could harm the recipient
METABOLIC INHIBITORS We described how drugs that are inhibitors of various cell cycle processes are used to treat cancer. Drugs are also used to treat the symptoms of many genetic diseases. As biologists have gained insight into the molecular characteristics of these diseases and the specific proteins involved, a more specific approach to treatment is taking shape. Targeted therapies are being developed, especially for cancer, and some have resulted in life-saving interventions. This is a major area of applied research.
The example of cancer illustrates how many common phenotypes, including ones that cause disease, are multifactorial; that is, they are caused by the interactions of many genes and proteins with one or more factors in the environment. When studying genetics, we tend to call individuals either normal (wild type) or abnormal (mutant); however, in reality every individual contains thousands or millions of genetic variations that arose through mutations. Our susceptibility to disease is often determined by complex interactions between these genotypes and factors in the environment, such as the foods we eat or the pathogens we encounter.
Many chromosomal and point mutations have been described in cancer cells. Such mutations affect *oncogenes, whose products stimulate cell division, or tumor suppressor genes, whose products inhibit cell division. More than two gene mutations are usually needed for full-blown cancer.
A silent mutation does not usually affect protein function, because the nucleotide substitution results in a codon that calls for the same amino acid as found in the normal protein.
Missense mutations cause one amino acid to substitute for another in a protein. The resulting protein may be defective, but if the substituted amino acid is similar enough to the original, the change in protein function may be minor. A missense mutation may result in a defective protein, but often has no effect on the protein's function.
reversion mutation: A second- or third-round mutation that reverts the DNA to its original sequence or to a new sequence that results in a non-mutant phenotype.
Most point mutations can be reversed; reversion mutations result when a gene is mutated a second time so that the DNA reverts to its original sequence or to a coding sequence that results in the non-mutant phenotype. When reversion mutations occur, the phenotype reverts to wild type.
Restriction enzymes, which are made by microorganisms as a defense against viruses, bind to and cut DNA at specific recognition sequences (also called restriction sites), producing smaller fragments of DNA. This cutting process is known as restriction digestion. Restriction enzymes can be used in the laboratory to produce small fragments of DNA for study.
Multifactorial diseases are caused by the interactions of many genes and proteins with the environment. They are much more common than diseases caused by mutations in a single gene.
Does every mutation have a phenotypic effect? Not necessarily. Some mutations have effects on proteins and their function, and some do not (Focus: Key Figure 15.1): 1. A silent mutation does not usually affect protein function (see Figure 15.1B). It can be a mutation in a region of DNA that does not encode a protein, or it can be in the coding region of a gene but not affect the amino acid sequence. Because of the redundancy of the genetic code, a base change in a coding region will not always cause a change in the amino acid sequence when the altered mRNA is translated (see Figure 15.2). Silent mutations are common, and they usually result in genetic diversity that is not expressed as phenotypic differences. 2. A loss-of-function mutation affects protein function (see Figure 15.1C). Such a mutation may cause a gene to not be expressed at all, or the gene may be expressed but produces a dysfunctional protein that can no longer play its cellular role, such as its catalytic function if it is an enzyme. Loss-of-function mutations almost always show recessive inheritance in diploid organisms, because the presence of one wild-type allele will usually result in sufficient functional protein for the cell. For example, recall from Key Concept 12.1 that the familiar wrinkled seed allele in pea plants, originally studied by Mendel, is due to a recessive loss-of-function mutation in the SBE1 (starch branching enzyme) gene. Normally the protein made by this gene catalyzes the branching of starch as seeds develop. In the mutant, the SBE1 protein is not functional, and that leads to osmotic changes, causing the wrinkled appearance. 3. A gain-of-function mutation leads to a protein with an altered function (see Figure 15.1D). A gain-of-function mutation usually shows dominant inheritance, because the presence of the wild-type allele does not prevent the mutant allele from functioning. This type of mutation is common in cancer. For example, there are mutations in oncogenes that result in proteins that constantly stimulate cell division.
Mutations are classified generally by the type of cell in which they occur: 1. Somatic mutations occur in somatic (nongamete) cells. These mutations are passed on to the daughter cells during mitosis, and to the daughters of those cells in turn, but are not passed on to sexually produced offspring. For example, a mutation in a single human skin cell could result in a patch of skin cells that all have the same mutation, but it would not be passed on to the person's children. 2. Germ line mutations occur in the cells of the germ line—the specialized cells that give rise to gametes. A gamete with the mutation passes it on to a new organism at fertilization. The new organism will have the mutation in every cell of its body and will be able to pass the mutation on to its offspring. The BRCA1 mutation that Angelina Jolie, her aunt, and her mother inherited from Angelina's grandmother is a germ line mutation.
How do metabolic inhibitors function in treating genetic diseases such as cancer? Metabolic inhibitors block important chemical transformations in cancer cells. An inhibitor may either block the accumulation of a harmful substance or block cancer-specific transformations to harmful substances.
Mutations can occur in "hot spots" where cytosine has been methylated to 5-methylcytosine.
Genetic screening can be done by examining the phenotype Genetic screening can involve examining a protein or other chemical that is relevant to a phenotype associated with a particular disease.
Nucleic acid hybridization (see Figure 14.6) can be used to detect the presence of a specific DNA sequence, such as a sequence containing a particular mutation. Samples of DNA are collected from people who may or may not carry the mutation, and PCR is used to amplify the region of DNA where the mutation may occur. Short synthetic DNA strands called oligonucleotide probes are hybridized with the denatured PCR products. The probe is labeled in some way (e.g., with radioactivity or a fluorescent dye) so that hybridization can be readily detected
phenylalanine; tyrosine People with PKU often have too much of the amino acid _______ and too little of the amino acid _______.
Prenatal screening is much more feasible today, in part because very few cells are needed. What technology has been most responsible for the reduction in the number of cells required? PCR
Altering the phenotype of a genetic disease so that it no longer harms an individual is commonly done in one of three ways: by restricting the substrate of a deficient enzyme, by inhibiting a harmful metabolic reaction, or by supplying a missing protein product
RESTRICTING THE SUBSTRATE Restricting the substrate of a deficient enzyme is the approach taken when a newborn is diagnosed with PKU. In this case, the deficient enzyme is phenylalanine hydroxylase, and the substrate is phenylalanine
Gel electrophoresis is a common and convenient technique for separating or purifying DNA fragments. Because of its phosphate groups, DNA is negatively charged at neutral pH; therefore, because opposite charges attract, the DNA fragments move through the gel toward the positive end of the field. Because the spaces between the polymers of the gel are small, small DNA molecules can move through the gel faster than larger ones.
Restriction enzyme digestion is used to manipulate DNA in the laboratory so that mutations can be identified and analyzed. After a laboratory sample of DNA has been cut with one or more restriction enzymes, the DNA is in fragments, which must be separated to identify (map) where the cuts were made. Because the recognition sequence does not occur at regular intervals, the fragments are not all the same size, and these size differences can be used to separate the fragments from one another. Separating the fragments is necessary to determine the number and molecular sizes (in base pairs) of the fragments produced, or to identify and purify an individual fragment for further analysis or for use in an experiment.
Identifying a mutant gene requires finding a marker that is closely linked to the gene of interest, a process called linkage analysis. The reference points for gene isolation are genetic markers. Genetic markers such as STRs and SNPs can be used as landmarks to find a gene of interest, if the gene also has multiple alleles (for example, normal and disease-causing alleles). The key to this method is the well-established observation that if two genes are located near each other on the same chromosome, they are usually passed on together from parent to offspring (see Key Concept 12.4). The same holds true for any pair of DNA genetic markers. In the case of linkage analysis, the idea is to find markers that are progressively closer to the gene of interest.
Short tandem repeats (STRs) are short, repetitive DNA sequences that occur side by side on the chromosomes, usually in the noncoding regions. These repeat patterns, which contain one to five base pairs, are also inherited. For example, at a particular locus on chromosome 15 there may be an STR of "AGG." An individual may inherit an allele with six copies of the repeat (AGGAGGAGGAGGAGGAGG) from her mother and an allele with two copies (AGGAGG) from her father. Again, PCR is used to amplify DNA fragments containing these repeated sequences, and then the amplified fragments, which have different sizes because of the different lengths of the repeats, are distinguished by gel electrophoresis (Figure 15.13A).
restriction enzyme: Any of a type of enzyme that cleaves double-stranded DNA at specific sites; extensively used in recombinant DNA technology. Also called a restriction endonuclease. restriction digestion: An enzymatic reaction in which a molecule of DNA is fully cleaved by a restriction enzyme. There are many such restriction enzymes, each of which cleaves DNA at a specific sequence of bases called a recognition sequence or a restriction site.
Some bacteria defend themselves against such invasions by producing restriction enzymes (also known as restriction endonucleases), which cut double-stranded DNA molecules—such as those injected by bacteriophages—into smaller, noninfectious fragments (Figure 15.11). These enzymes break the bonds of the DNA backbone between the 3′ hydroxyl group of one nucleotide and the 5′ phosphate group of the next nucleotide. This cutting process is called restriction digestion.
conditional mutation: A mutation that results in a characteristic phenotype only under certain environmental conditions.
Some mutations have effects on phenotype only under certain conditions. For example, a conditional mutation affects phenotype only under restrictive conditions and is not detectable under what are called permissive conditions. Many conditional mutations are temperature-sensitive, resulting in proteins with reduced stability at high temperatures.
Once a linked DNA region is identified, many methods are available to identify the actual gene responsible for a genetic disease. The complete sequence of the region can be searched for candidate genes, using information available from databases of genome sequences. With luck a scientist can make an educated guess, based on biochemical or physiological information about the disease, along with information about the functions of candidate genes, as to which gene is responsible for the disease. The identification of DNA polymorphisms within candidate genes that correlate with the presence or absence of disease can also help narrow down the search. A variety of techniques, such as analyzing mRNA levels of candidate genes in diseased and healthy individuals, are used to confirm that the correct gene has been identified. The isolation of the BRCA1 gene that is involved in breast cancer (described in the chapter opening) offers a good illustration of molecular techniques used to identify genes associated with disease
To narrow down the location of a gene, a scientist must find a genetic marker that is always inherited with the gene. To do this, family medical histories are taken and pedigrees are constructed. If a genetic marker and a genetic disease are inherited together in many families, then they must be near each other on the same chromosome (Figure 15.14). This situation recalls the conclusion reached in the classic studies of inheritance undertaken by Thomas Hunt Morgan and discussed in Key Concept 12.4: two genes do not always assort independently. Genes that are "linked" on the same chromosome will sometimes be inherited together—especially when the two loci are close to one another on the chromosome.
A major challenge has been getting the therapeutic gene into cells. Uptake of DNA into eukaryotic cells is a rare event, and once the DNA is inside a cell, its entry into the nucleus and expression are rarer still. One solution to these problems is to insert the gene into a carrier virus (a viral vector) that can infect human cells but has been altered genetically to prevent viral replication. An example is the DNA virus called adeno-associated virus, which has been widely used in human gene therapy clinical trials. This virus has a small genome that can be spliced into a human gene; infects most human cells, including nondividing cells such as neurons; is harmless to humans; does not provoke rejection by the immune system; and enters the cell nucleus, where its DNA with the new gene can be expressed.
Treatment of a human genetic disease may involve an attempt to modify the abnormal phenotype by restricting the substrate of a deficient enzyme, inhibiting a harmful metabolic reaction, or supplying a missing protein. By contrast, gene therapy aims to address a genetic defect by inserting a normal allele into a patient's cells.
GEL ELECTROPHORESIS PROVIDES... 1. The number of fragments. The number of fragments produced by digestion of a DNA sample with a given restriction enzyme depends on how many times that enzyme's recognition sequence occurs in the sample. Thus gel electrophoresis can provide some information about the presence of specific DNA sequences (the restriction sites) in the DNA sample. 2. The sizes of the fragments. DNA fragments of known size (size markers) are often placed in one well of the gel to provide a standard for comparison. The size markers are used to determine the sizes of the DNA fragments in samples in the other wells. By comparing the fragment sizes obtained with two or more restriction enzymes, the locations of their recognition sites relative to one another can be worked out (mapped). 3. The relative abundance of a fragment. In many experiments, the investigator is interested in how much DNA is present. The relative intensity of a band produced by a specific fragment can indicate the amount of that fragment.
Two types of polymorphisms are especially informative: Single nucleotide polymorphisms (SNPs; pronounced "snips") are inherited variations involving a single nucleotide base—they are point mutations. These polymorphisms have been mapped for many organisms. If one parent is homozygous for the base A at a certain point in the genome, and the other parent is homozygous for a G at that point, the offspring will be heterozygous: one chromosome will have A at that point and the other will have G. If a SNP occurs in a restriction enzyme recognition site, such that one variant is recognized by the enzyme and the other isn't, then individuals can be distinguished from one another very easily using the *polymerase chain reaction (PCR). A fragment containing the polymorphic sequence is amplified by PCR from samples of total DNA isolated from each individual. The fragments are then cut with the restriction enzyme and analyzed by gel electrophoresis.
Why do many mutations involve G-C base pairs? C can be methylated to 5-methylcytosine. When deaminated spontaneously or by a mutagen, this base forms T. This is a normal base and is not removed by DNA repair. Other base changes are repaired.
What distinguishes the various kinds of chromosomal mutations: deletions, duplications, inversions, and translocations? - Deletions are missing part of a chromosome; - Duplications have an extra copy (or copies) of a chromosomal region - Inversions, a chromosome region is out of sequence - Translocations, a piece of one chromosome breaks off and attaches to another chromosome.
There are two main approaches to treating genetic diseases: 1. Modifying the disease phenotype 2. Replacing the defective gene (modifying the genotype)
What is the advantage of screening for genetic mutations by allele-specific oligonucleotide hybridization relative to screening phenotype differences in enzyme activity? DNA analysis can be done on any tissue at any time in the life cycle of an individual. In addition, heterozygotes can be detected. Phenotype analysis by enzyme activity requires gene expression in an accessible tissue at a certain time and place. In many cases, heterozygotes cannot be detected.
with all insertions and deletions whose sizes are not multiples of three nucleotides. Frame-shift mutations occur in?
What is the common treatment for hemophilia A? Correct: Supplying a missing protein
A bacterial cell has been exposed to a powerful mutagen. The first line below is the sequence of the ancestral cell, and the second line is for the descendant cell. Intervening sequences where the ancestral and descendant cells have the identical sequences are represented by dots. Ancestral: ...ACT...GCA...CAA...CTG... Descendant: ...ATT...GTA...CAG...CAG... There have been _______ transitions and _______ transversions. 3 ; 1
Which mutation requires that two homologous chromosomes break? Duplications only.
Painkillers are provided to an individual with sickle-cell disease. Which therapeutic scenario does not illustrate the concept of treating a genetic disease by modifying its full phenotype?
exhibit the disease; not exhibit the disease If a disease is due to a completely dominant mutation, the DNA of individuals that hybridize both normal and disease probes in allele-specific oligonucleotide hybridization should _______. If a disease is due to a completely recessive mutation, the DNA samples of individuals that hybridize both normal and disease probes in this test should _______.
short synthetic DNA strands. In allele-specific oligonucleotide hybridization for genetic screening, the probes are?
loss-of-function; recessive The wrinkled seed allele in pea plants (one of Mendel's traits) is due to a _______ mutation and is _______
During DNA replication, DNA polymerase inserts a T opposite a G, thereby introducing a(n) _______ mutation. Correct: spontaneous
low in phenylalanine. Lofenelac, the milk-based product given to children with PKU, is?