Genetics Exam 3

Explain how Western blotting are used to detect specific proteins

(a) After blotting, a primary antibody is added that binds to the protein of interest. Then a secondary antibody is added that binds to the primary antibody. In this example, the secondary antibody is also attached to an enzyme called alkaline phosphatase. When the colorless compound XP (5-bromo-4-chloro-3-indolyl phosphate) is added, alkaline phosphatase converts it to a dark purple dye. (b) The dark purple band indicates where the primary antibody has recognized the protein of interest.

A mutagen that is a base analog is ethyl methanesulfonate (EMS). 5-bromouracil. UV light. proflavin.

5-bromouracil.

Explain how gene conversion can occur via DNA gap repair synthesis.

A gene is found in two alleles, designated b and B. A double-strand break occurs in the DNA encoding the b allele. Both of these DNA strands are digested away, thereby eliminating the b allele. A complementary DNA strand encoding the B allele migrates to this region and provides the template to synthesize a new double-stranded region. Following resolution, both DNA double helices carry the B allele

Which of the following is not an example of a spontaneous mutation? A mutation caused by an error in DNA replication A mutation caused by a tautomeric shift A mutation caused by UV light All of the above are spontaneous mutations.

A mutation caused by UV light

Define mutagen.

Agents known to alter the structure of DNA and thereby cause mutations

Describe the organization of sequences within different types of transposable elements.

All TEs are flanked by direct repeats (DRs), also called target-site duplications, which are identical base sequences that are oriented in the same direction and repeated. Direct repeats are adjacent to both ends of any TE. The simplest TE is known as an insertion element (IS element). As shown in Figure 20.9a, an IS element has two important characteristics. First, both ends of the element contain inverted repeats (IRs). Inverted repeats are DNA sequences that are identical (or very similar) but run in opposite directions

Which of the following uses of microorganisms is/are important in biotechnology? Production of medicines Food fermentation Biological control All of the above

All of the above

Which of the following was the first living organism to be patented? A strain of E. coli that makes somatostatin A strain of E. coli that makes insulin An oil-eating bacterium A strain of B. thuringiensis that makes an insecticide

An oil-eating bacterium

Which of the following is a key feature of stem cells? They have the ability to divide. They have the ability to differentiate. They are always pluripotent. Both a and b are true of stem cells.

Both a and b are true of stem cells.

Which of the following mechanisms can cause gene conversion? DNA mismatch repair DNA gap repair Resolution of a Holliday junction Both a and b can result in gene conversion.

Both a and b can result in gene conversion.

Common Causes of Induced Mutations - Description

Chemical agents - Chemical substances may cause changes in the structure of DNA. Physical agents - Physical phenomena such as UV light and X-rays can damage DNA.

Distinguish between gene replacement and gene addition.

Gene modification is aimed at altering the sequence of a gene. Different approaches can be followed to modify genes. Also, technologies such as CRISPR-Cas can alter a gene sequence by introducing a specific mutation, such as a missense mutation, into a gene. As discussed later, researchers may want to produce missense mutations in mice that mimic disease-causing mutations in humans. In this way, they can study the effects of the disease in mice. Gene addition involves the insertion of a cloned gene into a genome, such as the genome of a mouse. In some cases, researchers may introduce additional copies of a gene that is already present in the genome. Alternatively, they may introduce a gene that is not already present in the genome.

Distinguish between spontaneous and induced mutations.

Geneticists categorize the causes of mutations in one of two ways: Spontaneous mutations are changes in DNA structure that result from natural biological or chemical processes, whereas induced mutations are caused by environmental agents

Outline how transgenic livestock can produce human medicines in their milk.

Insert the coding sequence of a human hormone gene next to a sheep B-lactoglobulin promoter. This promoter is functional only in mammary cells, so that the protein product is secreted into the melt Inject this DNA into a sheep oocyte the plasmid DNA will integrate into the chromosomal DNA, resulting in the addition of the human hormone gene into the sheeps genome the fertilized oocyte is implanted into a female sheep which then gives birth to a transgenic shee offspring The molk from the transgenic sheep contains a human hormone

Explain how bacteria are genetically engineered to produce human insulin.

Insulin is a hormone composed of two different polypeptide chains, called the A and B chains. To make this hormone using bacteria, the coding sequence of either the A or B chain is placed next to the coding sequence of a native E. coli protein, β-galactosidase This creates a fusion protein comprising β-galactosidase and either the A or B chain. After the fusion proteins are expressed in bacteria, they can be purified and then treated with cyanogen bromide (CNBr), which cleaves the fusion protein after a methionine that is found at the junction between β-galactosidase and the A or B chain. This cleavage step separates β-galactosidase from the Page 541 A or B chain. The A and B chains are then purified and mixed together under conditions in which they refold and form disulfide bonds with each other to make active insulin hormone

Different types of mutation

Neutral mutation does not alter protein function, so it does not affect survival or reproductive success. deleterious mutation, however, decreases the chances of survival and reproduction lethal mutation, which results in the death of a cell or organism beneficial mutation enhances the survival or reproductive success of an organism. conditional mutants, in which the phenotype is affected only under a defined set of conditions.

Which of the following methods is used to detect a specific RNA within a mixture of many different RNAs? Site-directed mutagenesis Northern blotting Western blotting None of the above

Northern blotting

Which of the following methods use(s) a labeled nucleic acid probe, such as a labeled fragment of DNA? Site-directed mutagenesis Northern blotting Western blotting Both a and b

Northern blotting

Which of the following types of transposable elements rely on an RNA intermediate for transposition? Insertion elements Simple transposons Retrotransposons All of the above

Retrotransposons

Describe how a mutation within the coding sequence of a gene may alter a polypeptide's structure.

Silent mutations are those that do not alter the amino acid sequence of the polypeptide even though the base sequence has changed. Because the genetic code is degenerate, silent mutations can occur in certain bases within a codon, such as the third base, so the specific amino acid is not changed. Missense mutations are base substitutions for which an amino acid change does result. Nonsense mutations involve a change from a normal codon to a stop codon. This change terminates the translation of the polypeptide earlier than expected, producing a truncated polypeptide Frameshift mutations involve the addition or deletion of a number of nucleotides that is not divisible by 3. Because the codons are read in multiples of 3, this shifts the reading frame. The translation of the mRNA then results in a completely different amino acid sequence downstream from the mutation.

Define stem cells, and describe their two key properties.

To accomplish this task, stem cells have two common characteristics. First, they have the capacity to divide, and second, they can differentiate into one or more specialized cell types.

Analyze the results of an Ames test.

To determine if an agent is mutagenic, researchers use testing methods that monitor whether or not the agent increases the mutation rate. TOPIC: What topic in genetics does this question address? The topic is testing for mutagens. More specifically, the question is about the Ames test. INFORMATION: What information do you know based on the question and your understanding of the topic? In the question, you are given data regarding the outcome of four trials using the Ames test. From your understanding of the topic, you may remember that a higher number of colonies on the experimental plates may indicate that a substance is a mutagen. PROBLEM-SOLVING TRATEGY: Make a calculation. Analyze data. To begin to solve this problem, you first need to calculate the mutation rate. To do so, you take the average of the four trials and then divide the average number of mutant colonies by the total number of cells applied to each plate (in this case, 2 million). You also need to conduct a t-test to determine if the control and experimental data are significantly different.

Define point mutation.

a change in a single base pair within the DNA Examples: base substitution, in which one base is substituted for another. A change of a pyrimidine to another pyrimidine, such as C to T, or a purine to another purine, such as A to G, is called a transition. transversion, in which a purine and a pyrimidine are interchanged.

A collection of recombinant vectors that carry fragments of chromosomal DNA is called a genomic library. a cDNA library. a Northern blot. either a or b.

a genomic library.

Double-strand breaks can be repaired by homologous recombination repair (HRR). nonhomologous end joining (NHEJ). nucleotide excision repair (NER). both a and b

both a and b

The function of reverse transcriptase is to copy RNA into DNA. copy DNA into RNA. translate RNA into protein. translate DNA into protein.

copy RNA into DNA.

The restriction enzymes used in gene-cloning experiments _______, which generates sticky ends that can _______. cut the DNA, enter bacterial cells cut the DNA, hydrogen bond with complementary sticky ends methylate DNA, enter bacterial cells methylate DNA, hydrogen bond with complementary sticky ends

cut the DNA, hydrogen bond with complementary sticky ends

Nitrous acid replaces amino groups with keto groups, a process called alkylation. deamination. depurination. crosslinking.

deamination.

During real-time PCR, the synthesis of PCR products is analyzed at the very end of the reaction by gel electrophoresis. at the very end of the reaction by fluorescence that is emitted within the thermocycler. during the PCR cycles by gel electrophoresis. during the PCR cycles by fluorescence that is emitted within the thermocycler.

during the PCR cycles by fluorescence that is emitted within the thermocycler.

When a cloned gene is inserted into a noncritical site in the mouse genome by homologous recombination, the result is gene addition. gene modification. gene knockout. both a and b.

gene addition.

heritable change in the genetic material

mutation

site-specific recombination,

nonhomologous DNA segments are recombined at specific sites. This type of recombination occurs within genes that encode antibody polypeptides.

In the Ames test, a ____________ number of colonies is observed if a substance ________ a mutagen, compared with the number for a control sample that is not exposed to the suspected mutagen. significantly higher, is significantly higher, is not significantly lower, is significantly lower, is not

significantly higher, is

Homologous recombination refers to the exchange of DNA segments that are similar or identical in their DNA sequences. in close proximity to one another. broken due to ionizing radiation. misaligned along a chromosome.

similar or identical in their DNA sequences.

During Western blotting, the primary antibody recognizes the secondary antibody. the protein of interest. an mRNA of interest. a specific fragment of chromosomal DNA.

the protein of interest.

When a dideoxyribonucleotide is incorporated into a growing DNA strand, the strand elongates faster. the strand cannot elongate. the strand becomes more susceptible to DNase I cleavage. none of the above occurs.

the strand cannot elongate.

Define mapping.

the term mapping refers to the experimental process of determining the relative locations of genes or other segments of DNA along individual chromosomes.

Compare and contrast a genomic library with a cDNA library.

(a) To make a genomic library, chromosomal DNA is first digested to produce many fragments. The fragment containing the gene of interest is highlighted in red. Following ligation, each vector carries a different piece of chromosomal DNA. The library shown here is composed of four colonies. An actual genomic library would contain thousands of different bacterial colonies, each one carrying a different piece of chromosomal DNA. (b) To make a cDNA library, oligonucleotide linkers that contain a restriction site are attached to the cDNAs, so they can be inserted into vectors.

Explain how gene conversion can occur via mismatch repair.

A branch migrates past a homologous region that contains slightly different DNA sequences. This produces two heteroduplexes: DNA double helices with mismatches. The mismatches can be repaired in four possible ways by the mismatch repair system described in Chapter 19. Two of these ways result in gene conversion. The repaired base is shown in red.

Explain how changes in chromosome structure may affect gene expression.

A change in chromosome structure can also be associated with an alteration in the expression of single genes. In some cases, a chromosomal rearrangement may affect a gene because a chromosomal breakpoint—a region where two chromosome pieces break and rejoin with other chromosome pieces—occurs within a gene. A breakpoint in the middle of a gene is very likely to inhibit gene function because it separates the gene into two pieces. In other cases, a gene may be left intact, but its expression may be altered when it is moved to a new location. When this occurs, the change in gene location is said to have a position effect. (a) A chromosomal inversion has repositioned the core promoter of gene A next to the regulatory sequences for gene B. Because regulatory sequences are often bidirectional, the regulatory sequences for gene B may regulate the transcription of gene A. (b) A translocation has moved a gene from a euchromatic to a heterochromatic chromosome. This type of position effect inhibits the expression of the relocated gene.

Distinguish between germ-line and somatic mutations.

A germ-line mutation can occur directly in a sperm or egg cell, or it can occur in a precursor cell that produces the gametes. If a mutant gamete participates in fertilization, all cells of the resulting offspring will contain the mutation. Likewise, when an individual with a germ-line mutation produces gametes, the mutation may be passed along to future generations of offspring. The somatic cells are all cells of the body excluding the germ-line cells. Therefore, in the adult, a portion of the body contains the mutation. The size of the affected region depends on the timing of the mutation. In general, the earlier the mutation occurs during development, the larger the affected region. An individual that has somatic regions that differ genotypically from each other is called a genetic mosaic.

Common Causes of Spontaneous Mutations - Description

Aberrant recombination - Abnormal crossing over may cause deletions, duplications, translocations, and inversions (see Chapter 8). Aberrant segregation - Abnormal chromosomal segregation may cause aneuploidy or polyploidy (see Chapter 8). Errors in DNA replication - A mistake by DNA polymerase may cause a base pair mismatch (see Chapter 11). Toxic metabolic products - The products of normal metabolic processes, such as reactive oxygen species, may be chemically reactive agents that can alter the structure of DNA. Transposable elements - Transposable elements can insert themselves into the sequence of a gene (see Chapter 20). Depurination - On rare occasions, the linkage between a purine (i.e., adenine or guanine) and deoxyribose can spontaneously break. If not repaired, this can lead to mutation. Deamination - Cytosine or 5-methylcytosine can spontaneously deaminate to create uracil or thymine. Tautomeric shifts - Spontaneous changes in base structure can cause mutations if they occur immediately prior to DNA replication.

Which of the following DNA repair systems may involve the removal of a segment of a DNA strand? Base excision repair Nucleotide excision repair Mismatch repair All of the above

All of the above

Which of the following is an example of a somatic mutation? A mutation in an embryonic muscle cell A mutation in a sperm cell A mutation in an adult nerve cell Both a and c are examples of somatic mutations.

Both a and c are examples of somatic mutations.

Which of the following may be used as a vector in a gene-cloning experiment? mRNA Plasmid Virus Both b and c

Both b and c

Distinguish among cytogenetic, linkage, and physical mapping.

Cytogenetic mapping (also called cytological mapping) is aimed at determining the locations of specific sequences, such as gene sequences, within chromosomes that are viewed microscopically. When stained, each chromosome of a given species has a characteristic banding pattern, and genes are mapped cytogenetically relative to a band location. Such studies, known as linkage mapping, use the frequency of genetic recombination between different genes to determine their relative spacing and order along a chromosome. In eukaryotes, linkage mapping involves crosses among organisms that are heterozygous for two or more genes. The number of recombinant offspring provides a relative measure of the distance between genes, which is computed in map units (mu). A third approach is physical mapping in which DNA-cloning techniques are used to determine the location of and distance between genes and other DNA regions. In a physical map, the distances are computed as the number of base pairs between genes.

Describe the three steps of a PCR cycle.

Denaturation - DNA is first denatured by heat treatment, causing the strands to separate Primer Annealing - s the temperature is lowered, oligonucleotide primers bind Page 520 to the DNA Primer Extension - Once the primers have annealed, the temperature is raised slightly, and Taq polymerase catalyzes the synthesis of complementary DNA strands in the 5′ to 3′ direction, starting at the primers

Describe how reactive oxygen species alter DNA structure and cause mutations.

If ROS accumulate, they can damage cellular molecules, including DNA, proteins, and lipids. Oxidative stress refers to an imbalance between the production of ROS and an organism's ability to break them down. If ROS overaccumulate, one particularly harmful consequence is oxidative DNA damage, which refers to changes in DNA structure that are caused by ROS. DNA bases are very susceptible to oxidation. Guanine bases are particularly vulnerable to oxidation, which can lead to several different oxidized products.

Nucleotide Excision Repair

In NER, several nucleotides in the damaged strand are removed from the DNA, and the intact strand is used as a template for resynthesis of a normal complementary strand. NER is found in all eukaryotes Page 482 and prokaryotes, although its molecular mechanism is better understood in prokaryotic species. A protein complex consisting of two UvrA molecules and one UvrB molecule tracks along the DNA in search of damaged DNA. Such DNA has a distorted double helix, which is sensed by the UvrA/UvrB complex. When a damaged segment is identified, the two UvrA proteins are released, and UvrC binds to the site. The UvrC protein makes cuts in the damaged strand on both sides of the damaged site. Typically, the damaged strand is cut 8 nucleotides from the 5′ end of the damaged site and 4-5 nucleotides away from the 3′ end. After this process, UvrD, which is a helicase, recognizes the region and separates the two strands of DNA. This releases a short DNA segment that contains the damaged region, and UvrB and UvrC are also released. Following the excision of the damaged DNA, DNA polymerase fills in the gap, using the undamaged strand as a template. Finally, DNA ligase makes the covalent connection between the newly made DNA and the original DNA strand.

Examples of Mutagens

Mutagen - Effect(s) on DNA Structure Chemical Nitrous acid - Deaminates bases Nitrogen mustard - Alkylates bases Ethyl methanesulfonate - Alkylates bases Proflavin - Intercalates within DNA helix 5-Bromouracil - Functions as a base analog 2-Aminopurine - Functions as a base analog Physical X-rays - Cause base deletions, single-stranded nicks in DNA, crosslinking, chromosomal breaks, and oxidized bases UV light - Promotes pyrimidine dimer formation, such as thymine dimers

Explain how Northern blotting are used to detect specific RNAs

RNA is extracted and purified from living cells. This RNA can be isolated from a particular cell type under a given set of conditions or during a particular stage of development. Any particular cell produces thousands of different types of RNA molecules, because cells express many genes at any given time. After the RNA is extracted from cells and purified, it is loaded onto an agarose gel that separates the RNA transcripts according to their size. The RNAs within the gel are then blotted onto a nylon membrane and probed with a labeled fragment of DNA from a cloned gene. RNAs that are complementary to the DNA probe are detected as labeled bands

Outline the method of real-time PCR, and discuss why it is used.

Real-Time PCR Is Used to Quantitate the Amount of a Specific Gene or mRNA in a Sample real-time PCR allows a researcher to follow the amount of a specific PCR product in real time as PCR is taking place in a thermocycler. The procedure is carried out in a thermocycler that has the capacity to measure changes in the level of fluorescence that is emitted from probes that are added to the PCR mixture. The fluorescence given off by the probes depends on the amount of the PCR product. The reporter molecule emits fluorescence at a certain wavelength, but that fluorescence is largely absorbed by the nearby quencher. Therefore, the close proximity of the reporter molecule to the quencher molecule prevents the detection of fluorescence from the reporter molecule. . During the primer extension step, the 5′ to 3′ exonuclease activity of Taq polymerase cleaves the oligonucleotide in the TaqMan probe into individual nucleotides, thereby separating the reporter from the quencher. This allows the reporter to emit (unquenched) fluorescence that can be measured within the thermocycler. As PCR products accumulate, more and more of the TaqMan probes are digested, and therefore the level of fluorescence increases.

Examples of Intergenic Mutations

Redundant Function - a first mutation inhibits the function of a protein, and a second mutation alters a different protein to carry out that function. Common Pathway - Two or more different proteins may function as enzymes in a common pathway. A mutation that causes a defect in one enzyme may be compensated for by a mutation that increases the function of a different enzyme in the same pathway Multimeric Protein - a mutation in a gene encoding one protein subunit that inhibits function may be suppressed by a mutation in a gene that encodes a different subunit. The double mutant as restored function Transcription factor - a first mutation causes a loss of function of a particular protein. A second mutation may alter a transcription factor and cause it to activate the expression of another gene, This other gene encodes a protein that can compensate for the loss of function by the first mutation

Outline the steps of reproductive cloning in mammals.

Reproductive cloning refers to methods that produce two or more genetically identical individuals. This happens occasionally in nature; identical twins are genetic clones that began from the same fertilized egg. Similarly, researchers can take mammalian embryos at an early stage of development (e.g., the two-cell to eight-cell stage), separate the cells, implant them into the uterus, and obtain multiple births of genetically identical individuals. Donor sheep's mammary cell is extracted and grown in a tissue culture flask.

Explain how LTR retrotransposons move to new locations in a genome.

Retrotransposons Use Reverse Transcriptase for Retrotransposition the movement of LTR retrotransposons requires two key enzymes: reverse transcriptase and integrase In a series of steps, reverse transcriptase uses this RNA as a template to synthesize a double-stranded DNA molecule. The long terminal repeats (LTRs) at the ends of the double-stranded DNA are then recognized by integrase, which catalyzes the insertion of the DNA into the target chromosomal DNA The integration of a retrotransposon can occur at many locations within the genome Furthermore, because a single retrotransposon can be copied into many RNA transcripts, retrotransposons may accumulate rapidly within a genome.

Explain how reverse-transcriptase PCR is carried out.

Reverse Transcriptase PCR Is Used to Amplify RNA RNA is isolated from a sample and mixed with reverse transcriptase, a primer that binds near the 3′ end of the RNA of interest, and deoxyribonucleotides This generates a single-stranded cDNA, which then can be used as template DNA in a conventional PCR reaction. The end result is that the RNA has been amplified to produce many copies of DNA.

Possible Consequences of Gene Mutations Outside of a Coding Sequence

Sequence - Promoter mutations that increase transcription are termed up promoter mutations. Mutations that make a sequence more like the consensus sequence are likely to be up promoter mutations. In contrast, a down promoter mutation causes the promoter to become less like the consensus sequence, decreasing its affinity for transcription factors and decreasing the transcription rate. Regulatory element/operator site - May disrupt the ability of the gene to be properly regulated 5′-UTR/3′-UTR - May alter the ability of mRNA to be translated; may alter mRNA stability Splice recognition sequence - May alter the ability of pre-mRNA to be properly spliced

Compare and contrast the different types of DNA repair mechanisms.

System - Description Direct repair - An enzyme recognizes an incorrect alteration in DNA structure and directly converts the structure back to the correct form. Base excision repair and nucleotide excision repair - An abnormal base or nucleotide is first recognized and removed from the DNA, and a segment of DNA in this region is excised. Then the complementary DNA strand is used as a template to synthesize a normal DNA strand. Mismatch repair - Similar to excision repair except that the DNA defect is a base pair mismatch in the DNA, not an abnormal nucleotide. The mismatch is recognized, and a segment of DNA in this region is removed. The parental strand is used to synthesize a normal daughter strand of DNA. Homologous recombination - Occurs at double-strand breaks or when DNA repair damage causes a gap in synthesis during DNA replication. The strands of a normal sister chromatid are used to repair a damaged sister chromatid. Nonhomologous end joining Occurs at double-strand breaks. The broken ends are recognized by proteins that keep the ends together; the broken ends are eventually rejoined.

Summarize the studies of McClintock, and explain how they revealed the existence of transposable elements.

The colorless background of a kernel was due to the transposition of Ds into the C allele, which would inactivate that allele. In a few cells, Ds occasionally transposed out of the C allele during kernel growth (see drawing below). During transposition, Ds moved out of the C allele to a new location, and the two parts of the C allele were rejoined, thereby restoring its function. As the kernel grew, such a cell would continue to divide, resulting in a red secto

Define biological control and describe how microorganisms may play a role in these two processes.

The term biological control refers to the use of living organisms or their products to alleviate plant diseases or damage from environmental conditions. Biological control may involve the use of microorganisms living in the field. Another biological control agent is Bacillus thuringiensis, usually referred to as Bt (pronounced "bee-tee"). This naturally occurring bacterium produces toxins that are lethal to many caterpillars and beetles that feed on a wide variety of food crops and ornamental plants

Define bioremediation, and describe how microorganisms may play a role in these two processes.

The term bioremediation refers to the use of living organisms or their products to decrease pollutants in the environment. During bioremediation via microorganisms, enzymes produced by a microorganism modify a toxic pollutant by altering or transforming its structure. This event is called biotransformation. In many cases, biotransformation results in biodegradation, in which the toxic pollutant is degraded, yielding less complex, nontoxic metabolites.

Describe the double-strand break model for homologous recombination.

The top chromatid exeriences a double-strand break strand degratdation occurs at the double-strand break site to yield single-stranded ends the near strand of the bottom chromatid invades the opening in the to chromatid to from a D-loop Gap repair synthesis fills in the vacant regions producing two Holliday junctions *Depending on the way these are resolved, the end result is nonrecombinant or recombinant chromosomes, each containing a short heteroduplex.

Explain how gene knockins are made in mice, and list some of their important uses.

To accomplish gene addition in mice, researchers can produce a gene knockin, in which a gene of interest has been inserted into a particular site in the mouse genome The gene of interest is cloned and flanked with pieces of DNA that are homologous to a noncritical site in the mouse genome. The cloned DNA is introduced into a fertilized oocyte. The gene of interest inserts into the noncritical site by homologous recombination

What is the key reason why the A and B chains of insulin are made as fusion proteins with β-galactosidase? To make purification easier To prevent their degradation To be secreted from the cell All of the above are reasons for making the chains as fusion proteins.

To prevent their degradation

autonomous vs nonautonomous TEs

Transposable elements are considered to be complete, or autonomous elements, when they contain all of the information necessary for transposition or retrotransposition to take place. However, TEs are often incomplete, or nonautonomous. A nonautonomous element typically lacks a gene such as one that encodes transposase or reverse transcriptase, which is necessary for transposition to occur.

Discuss the effects of transposable elements on gene function.

Transposable elements have a variety of effects on chromosome structure and gene expression (Table 20.4). Many of these outcomes are likely to be harmful. Consequence - Cause Chromosome Structure Chromosome breakage - Excision of a TE. Chromosomal rearrangements - Homologous recombination between TEs located at different positions in the genome. Gene Expression Mutation - Incorrect excision of TEs. Gene inactivation - Insertion of a TE into a gene. Alteration in gene regulation - Transposition of a gene next to regulatory sequences or the transposition of regulatory sequences next to a gene. Alteration in the exon content of a gene - Insertion of exons into the coding sequence of a gene via TEs. This phenomenon is called exon shuffling.

Explain how transposons move to new locations in a genome.

Transposase Catalyzes the Excision and Insertion of Transposons Transposase monomers first bind to the inverted repeat sequences at the ends of the TE. The monomers then dimerize, which brings the inverted repeats close together The DNA is cleaved between the inverted and direct repeats, excising the TE from its original site within the chromosome Transposase carries the TE to a new site and cleaves the target DNA sequence at staggered recognition sites The TE is then inserted and ligated into the target DNA. the ligation of the transposable element into its new site initially leaves short gaps in the target DNA. Notice that the DNA sequences in these gaps are complementary to each other (in this case, ATGCT and TACGA). Therefore, when they are filled in by DNA gap repair synthesis, the repair produces direct repeats that flank both ends of the TE. These direct repeats are common features found adjacent to all TEs

One way that TNRE may occur involves the formation of _________ that disrupts ____________. a double-strand break, chromosome segregation an apurinic site, DNA replication a hairpin, DNA replication a free radical, DNA structure

a hairpin, DNA replication

Outline the steps in automated DNA sequencing via the dideoxy method

a sample containing many copies of the single-stranded recombinant vector is mixed with many primers that will bind to the primer-annealing site. All four types of deoxyribonucleotides are added at a high concentration and all four dideoxyribonucleotides (ddA, ddT, ddG, and ddC), which are fluorescently labeled, are added at a low concentration. DNA polymerase is then added, which causes the synthesis of strands that are complementary to the target DNA sequence After the samples have been incubated for several minutes, mixtures of DNA strands of different lengths are made, depending on the number of nucleotides attached to the primer. These DNA strands are separated according to their lengths by running them on a slab gel or more commonly by running them through a gel-filled capillary tube. Because we know the color with which each dideoxyribonucleotide is labeled, we also know which base is at the very end of each DNA strand separated on the gel. Therefore, we can deduce the DNA sequence that is complementary to the target DNA by reading which base is at the end of every DNA strand and matching this sequence with the length of the strand.

During mammalian reproductive cloning, _________ is fused with __________. a somatic cell, a stem cell a somatic cell, an egg cell a somatic cell, an enucleated egg cell an enucleated somatic cell, an egg cell

a somatic cell, an enucleated egg cell

During the molecular process of homologous recombination between homologous chromosomes, a Holliday junction forms. branch migration occurs. a heteroduplex region forms. all of the above occur.

all of the above occur.

The function of transposase is to recognize inverted repeats. to remove a TE from its original site. to insert a TE into a new site. all of the above.

all of the above.

A mutation in one gene that reverses the phenotypic effects of a mutation in a different gene is an intergenic suppressor. an intragenic suppressor. an up promoter mutation. a position effect.

an intergenic suppressor.

A point mutation could be caused by depurination. deamination. tautomeric shift. any of the above.

any of the above.

Which is the proper order of the following steps in a gene-cloning experiment involving vectors? Add DNA ligase. Incubate the chromosomal DNA and the vector DNA with a restriction enzyme. Introduce the DNA into living cells. Mix the chromosomal DNA and vector DNA together. a. 1, 2, 3, 4 c. 2, 4, 1, 3 b. 2, 3, 1, 4 d. 1, 2, 4, 3

c. 2, 4, 1, 3

Describe how cDNA is made.

cDNA Can Be Made from mRNA via Reverse Transcriptase mRNAs, which naturally contain a polyA tail at their 3′ ends, are purified from a sample of cells mRNAs are mixed with primers composed of a string of thymine-containing nucleotides This short strand of DNA is called a poly-dT primer. The poly-dT primer is complementary to the 3′ end of mRNAs. Reverse transcriptase and deoxyribonucleotides (dNTPs) are then added to make a DNA strand that is complementary to the mRNA. One way to make the other DNA strand is to use RNaseH, which partially digests the RNA, generating short RNAs that are used as primers by DNA polymerase to make a second DNA strand that is complementary to the strand made by reverse transcriptase. Finally, DNA ligase seals any nicks in this second DNA strand. When DNA is made using an RNA template, the DNA is called complementary DNA (cDNA). The term originally referred to the single strand of DNA that is complementary to the RNA template. However, cDNA now refers to any DNA, whether it is single- or double-stranded, that is made using RNA as the starting material.

Describe how specialized DNA polymerases are able to synthesize DNA over a damaged region.

cells are equipped with specialized DNA polymerases that assist the replicative DNA polymerases during the process of translesion synthesis (TLS)—the synthesis of DNA over a template strand that harbors some type of DNA damage. These translesion-replicating polymerases, which are also described in Chapter 11 (see Table 11.4), contain an active site with a loose, flexible pocket that can accommodate aberrant structures in the template strand. When a replicative DNA polymerase encounters a damaged region, it is swapped with a lesion-replicating polymerase. A negative consequence of translesion synthesis is low fidelity. Due to their flexible active site, translesion-replicating polymerases are much more likely to incorporate the wrong nucleotide into a newly made daughter strand. The mutation rate is typically in the range 10-2-10-3. This phenomenon is called error-prone replication. By comparison, replicative DNA polymerases are highly intolerant of the geometric distortions imposed on DNA

In one PCR cycle, the correct order of steps is primer annealing, primer extension, denaturation. primer annealing, denaturation, primer extension. denaturation, primer annealing, primer extension. denaturation, primer extension, primer annealing.

denaturation, primer annealing, primer extension.

Outline how mutations arise by depurination, deamination, and tautomeric shifts.

depurination - the removal of a purine (adenine or guanine) from the DNA. The covalent bond between deoxyribose and a purine base is somewhat unstable and occasionally undergoes a spontaneous reaction with water that releases the base from the sugar, thereby creating an apurinic site Deamination - removal of an amino group from the cytosine base. This produces uracil. As discussed later, DNA repair enzymes can recognize uracil as an inappropriate base within DNA and subsequently remove it. However, if such a repair does not take place, a mutation may result because uracil hydrogen bonds with adenine during DNA replication. Therefore, if a DNA template strand has uracil instead of cytosine, a newly made strand will incorporate adenine into the daughter strand instead of guanine. tautomeric shift - the tautomers are bases, which exist in keto and enol or amino and imino forms. These forms can interconvert by a chemical reaction that involves the migration of a hydrogen atom and a switch of a single bond and an adjacent double bond. The common, stable form of guanine and thymine is the keto form; the common form of adenine and cytosine is the amino form (Figure 19.9a). At a low rate, G and T can interconvert to an enol form, and A and C can change to an imino form. Though the amounts of the enol and imino forms of these bases are relatively small, their presence can cause a mutation because these rare forms do not conform to the AT/GC rule of base pairing. Instead, if one of the bases is in the enol or imino form, hydrogen bonding will promote TG and CA base pairs, as shown in

In nucleotide excision repair in E. coli, the function of the UvrA/UvrB complex is to detect DNA damage. make cuts on both sides of the damage. remove the damaged piece of DNA. replace the damaged DNA with undamaged DNA.

detect DNA damage.

A down promoter mutation causes the promoter of a gene to be _______ like the consensus sequence and _________ transcription. less, stimulates more, stimulates less, inhibits more, inhibits

less, inhibits

In reverse transcriptase PCR, the starting biological material is chromosomal DNA. mRNA. proteins. all of the above.

mRNA.

A mutation changes a codon that specifies tyrosine into a stop codon. This type of mutation is a missense mutation. nonsense mutation. frameshift mutation. neutral mutation.

nonsense mutation.

An advantage of translesion-replicating polymerases is that they can replicate _________________, but a disadvantage is that they _________________. very quickly, have low fidelity over damaged DNA, have low fidelity when resources are limited, are very slow over damaged DNA, are very slow

over damaged DNA, have low fidelity

transposition.

small segments of DNA, called transposable elements, can move to multiple locations within chromosomal DNA.

Compare and contrast intragenic and intergenic suppressors.

suppressor mutations - this name is meant to indicate that a suppressor mutation acts to suppress the phenotypic effects of another mutation. A suppressor mutation differs from a reversion, because it occurs at a different site in the DNA from the first mutation. When the second mutation site is within the same gene as the first, the mutation is termed an intragenic suppressor. a suppressor mutation can occur in a different gene from the first mutation—an intergenic suppressor. Examples:

Explain how no LTR retrotransposons move to new locations in a genome.

target-site primed reverse transcription (TPRT) the retrotransposon is first transcribed into RNA with a polyA tail at the 3′ end . The target DNA site is recognized by an endonuclease, which may be encoded by the retrotransposon. This endonuclease recognizes a consensus sequence of 5′-TTTTA-3′, and initially cuts just one of the DNA strands. The polyA tail of the retrotransposon RNA binds to this nicked site due to AT base pairing. Reverse transcriptase then uses the target DNA as a primer and makes a DNA copy of the RNA, which is why it is named target-site primed reverse transcription. To be fully integrated into the target DNA, the endonuclease makes a second cut in the other DNA strand usually about 7-20 nucleotides away from the first cut. The retrotransposon DNA is then ligated into the target site within a chromosome, perhaps by nonhomologous end joining. The mechanism for synthesis of the other DNA strand of the retrotransposon is not completely understood.

A key difference between the original Holliday model and the double-strand break model is the way that the DNA strands are initially broken. branch migration occurs. a heteroduplex is formed. resolution occurs.

the DNA strands are initially broken.

Outline the procedure for cloning a gene into a vector.

the chromosomal DNA is isolated and digested with a restriction enzyme. This enzyme cuts the chromosomes into many small fragments. he plasmid DNA is also cut with the same restriction enzyme However, the plasmid has only one unique site for the restriction enzyme. The digested chromosomal DNA and plasmid DNA are mixed together and incubated under conditions that promote the binding of these complementary sticky ends. DNA ligase is then added to catalyze the covalent linkage between adjacent DNA backbones. In some cases, the two ends of the vector simply ligate back together, restoring the vector to its original structure. This is called a recircularized vector. The vector containing a piece of chromosomal DNA is a recombinant vector. Only a very small percentage of bacterial cells actually take up a plasmid.

Define mutation rate.

the likelihood that a gene Page 478 will be altered by a new mutation. This rate is commonly expressed as the number of new mutations in a given gene per cell generation. The spontaneous mutation rate for a particular gene is typically in the range from 1 in 100,000 to 1 in 1 billion, or 10-5-10-9 per cell generation. The rate of new mutation is different from the concept of mutation frequency. The mutation frequency for a gene is the number of mutant genes divided by the total number of copies of that gene within a population. If 1 million bacteria were plated and 100 were found to carry a mutation in a particular gene, the mutation frequency for that gene would be 1 in 10,000, or 10-4.

gene conversion

the phenomenon in which one allele is converted to the allele on the homologous chromosome

Homologous recombination

the process whereby identical or similar DNA segments are exchanged between homologous chromosomes. As described in Chapter 3, homologous recombination occurs when chromosomes cross over during meiosis. Not only does homologous recombination enhance genetic diversity, it also helps to repair DNA and ensures the proper segregation of chromosomes. (a) Homologous recombination between sister chromatids. (b) Homologous recombination between homologous chromatids. This second form of homologous recombination may lead to a new combination of alleles, which is called a recombinant (or nonparental) genotype.

According to the selfish DNA hypothesis, TEs exist because they offer the host a selective advantage. they have characteristics that allow them to multiply within the chromosomal DNA of living cells. they promote the expression of certain beneficial genes. all of the above.

they have characteristics that allow them to multiply within the chromosomal DNA of living cells.

The function of photolyase is to repair double-strand breaks. apurinic sites. thymine dimers. all of the above.

thymine dimers.

Distinguish between totipotent, pluripotent, multipotent, and unipotent stem cells.

totipotent describes a stem cell that has the genetic potential to produce an entire individual. A somatic plant cell or a fertilized egg is totipotent. pluripotent describes a stem cell that can differentiate into all or nearly all the types of cells of the adult organism. multipotent a type of stem cell that can differentiate into several different types of cells. unipotent decribes a stem cell that can differentiate into only a single type of cell.

Explain the mechanism of trinucleotide repeat expansion.

trinucleotide repeat expansion (TNRE) - the phenomenon in which a repeated sequence of three nucleotides can readily increase in number from one generation to the next. Though it may occur in more than one way, researchers have determined that a key aspect of TNRE is that the triplet repeat can form a hairpin, also called a stem-loop. A consistent feature of the triplet sequences associated with TNRE is they contain at least one C and one G (see Table 19.5). As shown in Figure 19.11a, such a sequence can form a hairpin due to the formation of CG base pairs. The formation of a hairpin during DNA replication can lead to an increase in the length of a DNA region if it occurs in the newly made daughter strand (Figure 19.11b). After the hairpin forms, DNA polymerase may temporarily slip off the template strand. Next, DNA polymerase essentially backs up and hops back onto the template strand and resumes DNA replication from the end of the hairpin. When this occurs, DNA polymerase is synthesizing most of the hairpin region twice.

Describe the Holliday model

two homologous chromatids are aligned with each other. a break or nick occurs at identical sites in one strand of each of the two homologous chromatids The strands then invade the opposite helices and base-pair with the complementary strands and covalently link to the strands to the right of the nicks to form a Holliday junction the swapping of the DNA strands during branch migration may produce a heteroduplex, a region in the double-stranded DNA that contains base mismatches. The strands that were not originally nicked are broken the strands are connected to create recombinant chromosomes with a short heteroduplex region

One strategy for producing a protein in the milk of a cow is to place the coding sequence of the gene of interest next to a __________ and then inject the gene into a _________. lac operon promoter, cow oocyte β-lactoglobulin promoter, cow oocyte lac operon promoter, cow mammary cell β-lactoglobulin promoter, cow mammary cell

β-lactoglobulin promoter, cow oocyte