Chapter 10 (Biology 002)

Your biochemist friend has isolated a protein he thinks is responsible for making you feel sleepy. Since he knows you're taking Cell Biology, he wants you to help him isolate the gene encoding this protein. Unfortunately, because your friend could only isolate small amounts of protein, he was only able to obtain three short stretches of amino acid sequence from the protein: (a) H-C-W-K-M (b) R-S-L-L-S (c) D-A-Q-W-Y

(a) H-C-W-K-M. There is the least amount of degeneracy in the nucleotides that could code for this peptide

Which of the following limits the use of PCR to detect and isolate genes? (a) The sequence at the beginning and end of the DNA to be amplified must be known. (b) It also produces large numbers of copies of sequences beyond the 5' or 3' end of the desired sequence. (c) It cannot be used to amplify cDNAs or mRNAs. (d) It will amplify only sequences present in multiple copies in the DNA sample.

(a) The sequence at the beginning and end of the DNA to be amplified must be known.

You want to design a DNA probe used for hybridization to isolate a clone from a cDNA library. Which of the following statements about DNA probes is true? (a) The shorter the DNA probe used to probe the library, the greater the number of colonies to which the probe will hybridize. (b) A DNA probe that contains sequences that span two exons is better suited to the purpose than a DNA probe that only contains sequences from one exon. (c) A DNA probe that contains sequences immediately upstream of the DNA that codes for the first methionine in the open reading frame will usually not hybridize to clones in a cDNA library. (d) Hybridization of a DNA probe to the plasmid of interest will permit the detection of the clone of interest; labeling of the DNA probe is not necessary.

(a) The shorter the DNA probe used to probe the library, the greater the number of colonies to which the probe will hybridize.

You have a piece of circular DNA that can be cut by the restriction nucleases XhoI and SmaI, as indicated in Figure Q10-4. If you were to cut this circular piece of DNA with both XhoI and SmaI, how many fragments of DNA would you end up with? (a) 1 (b) 2 (c) 3 (d) 4

(b) 2

Which of the following statements about PCR is false? (a) PCR uses a DNA polymerase from a thermophilic bacterium. (b) PCR is particularly powerful because after each cycle of replication, there is a linear increase in the amount of DNA available. (c) For PCR, every round of replication is preceded by the denaturation of the doubled-stranded DNA molecules. (d) The PCR reaction will generate a pool of double-stranded DNA molecules, most of which will have DNA from primers at the 5' ends.

(b) PCR is particularly powerful because after each cycle of replication, there is a linear increase in the amount of DNA available.

Which of the following statements about restriction nucleases is false? (a) A reproducible set of DNA fragments will be produced every time a restriction nuclease digests a known piece of DNA. (b) Restriction nucleases recognize specific sequences on single-stranded DNA. (c) Some bacteria use restriction nucleases as protection from foreign DNA. (d) Some restriction nucleases cut in a staggered fashion, leaving short single-stranded regions of DNA at the ends of the cut molecule.

(b) Restriction nucleases recognize specific sequences on single-stranded DNA.

Which of the following statements about gel-transfer hybridization (or Southern blotting) is false? (a) This technique involves the transfer of DNA molecules from gel onto nitrocellulose paper or nylon paper. (b) In this technique, single-stranded DNA is separated by electrophoresis. (c) A labeled DNA probe binds to the DNA by hybridization. (d) The DNA that is separated on a gel is not labeled.

(b). During Southern blotting, double-stranded DNA is loaded onto the agarose gel. The DNA becomes denatured (and thus single stranded) as it gets transferred by the alkali solution from the gel to the nitrocellulose or nylon sheet.

You cut a vector using the PclI restriction nuclease. Which of the following restriction nucleases will generate a fragment that can be ligated into this cut vector with the addition of only ligase and ATP? (a) HindIII (b) NcoI (c) MmeI (d) NspV

(b). However, you will not be able to excise the fragment after ligation, because you will destroy both the PclI site and the NcoI site, creating a new site with the sequence 5'-ACATGG-3' 3'-TGTACC-5'

Recombinant DNA technologies involve techniques that permit the creation of custom-made DNA molecules that can be introduced back into living organisms. These technologies were first developed in the ______. (a) 1930s (b) 1950s (c) 1970s (d) 1990s

(c) 1970s

Which of the following statements about RNA interference (or RNAi) is false? (a) RNAi is a natural mechanism used to regulate genes. (b) During the process of RNAi, hybridization of a small RNA molecule with the mRNA degrades the mRNA. (c) Because RNAi depends on the introduction of a double-stranded RNA into a cell or an organism, it is not a process that can cause heritable changes in gene expression. (d) In C. elegans, RNAi can be introduced into the animals by feeding them with bacteria that produce the inhibitory RNA molecules.

(c) Because RNAi depends on the introduction of a double-stranded RNA into a cell or an organism, it is not a process that can cause heritable changes in gene expression.

Why are dideoxyribonucleoside triphosphates used during DNA sequencing? (a) They cannot be incorporated into DNA by DNA polymerase. (b) They are incorporated into DNA particularly well by DNA polymerases from thermophilic bacteria. (c) Incorporation of a didoxyribonucleoside triphosphate leads to the termination of replication for that strand. (d) Dideoxyribonucleotide triphosphates are more stable than deoxyribonucleoside triphosphates.

(c) Incorporation of a didoxyribonucleoside triphosphate leads to the termination of replication for that strand.

Which of the following statements about genomic DNA libraries is false? (a) The larger the size of the fragments used to make the library, the fewer colonies you will have to examine to find a clone that hybridizes to your probe. (b) The larger the size of the fragments used to make the library, the more difficult it will be to find your gene of interest once you have identified a clone that hybridizes to your probe. (c) The larger the genome of the organism from which a library is derived, the larger the fragments inserted into the vector will tend to be. (d) The smaller the gene you are seeking, the more likely it is that the gene will be found on a single clone.

(c) The larger the genome of the organism from which a library is derived, the larger the fragments inserted into the vector will tend to be.

Which of the following describes a feature found in bacterial expression vectors but not in cloning vectors? (a) origin of replication (b) cleavage sites for restriction nucleases (c) promoter DNA sequences (d) a polyadenylation signal

(c) promoter DNA sequences

You have purified DNA from your recently deceased goldfish. Which of the following restriction nucleases would you use if you wanted to end up with DNA fragments with an average size of 70 kilobase pairs (kb) after complete digestion of the DNA? The recognition sequence for each enzyme is indicated in the righthand column. (a) Sau3AI GATC (b) BamHI GGATCC (c) NotI GCGGCCGC (d) XzaI GAAGGATCCTTC

(c). A restriction nuclease that has a 4-base-pair recognition sequence cuts on average once every 44 or 256 base pairs; one that has a 6-base-pair recognition sequence cuts once every 46 or 4096 base pairs; one that has an 8-base-pair recognition sequence cuts once every 48 or 65,536 base pairs; one that has a 12-base-pair recognition sequence cuts once every 412 or 16 million base pairs. Thus, to obtain fragments of about 70 kb in size, you would cut with a nuclease that recognizes an 8-base-pair site.

Your friend works at the Centers for Disease Control and has discovered a brandnew virus that has recently been introduced into the human population. She has just developed a new assay that allows her to detect the virus by using PCR products made from the blood of infected patients. The assay uses primers in the PCR reaction that hybridize to sequences in the viral genome. Your friend is distraught because of the result she obtained (see Figure Q10-35) when she looked at PCR products made using the blood of three patients suffering from the viral disease, using her own blood, and using a leaf from her petunia plant. You advise your friend not to panic, as you believe she is missing an important control. Which one of the choices listed below is the best control for clarifying the results of her assay? Explain your answer. (a) a PCR reaction using blood from a patient who is newly infected but does not yet show symptoms (b) a PCR reaction using blood from a dog (c) a PCR reaction using blood from an uninfected person (d) repeating the experiments she has already done with a new tube of polymerase

(c). Primers can sometimes hybridize to unintended sequences and produce unintended products. The appropriate control for your friend's experiment would be DNA from an uninfected person; in that way she would be able to determine whether the bands present in the PCR from her blood truly correspond to product generated from viral DNA rather than cross hybridization to DNA sequences in the human genome, because the bands would be absent from a person uninfected by the virus in the former case only. Doing PCR from an infected but asymptomatic person would not be useful (choice (a)), because it would not allow your friend to distinguish whether she is infected. Although doing PCR from dog blood (choice (b)) should not give any viral bands, any nonspecific products from a dog would probably be different from those in your friend. The absence of PCR fragments in the petunia lane suggests that there is no viral contaminant in any of your friend's reagents, so using a new tube of polymerase is not the solution (choice (d)).

A DNA library has been constructed by purifying chromosomal DNA from mice, cutting the DNA with the restriction enzyme NotI, and inserting the fragments into the NotI site of a plasmid vector. What information cannot be retrieved from this library? (a) gene regulatory sequences (b) intron sequences (c) sequences of the telomeres (the ends of the chromosomes) (d) amino acid sequences of proteins

(c). The very ends of all of the chromosomes are unlikely to be NotI sites, meaning that the fragments containing the ends of the chromosomes will not be able to insert into the bacterial vector (because they have not been cut by NotI at both ends) and will be lost from the library. All sequences present in genomic DNA (which includes regulatory sequences and introns) should be present in a genomic library. The coding sequence of the gene (and hence the amino acid sequence of the encoded protein) is also present in a genomic clone, although it is interrupted by intron sequences and is therefore somewhat difficult (but not impossible) to determine.

You are interested in a single-stranded DNA molecule that contains the following sequence: 5'- ..... GATTGCAT .... -3' Which molecule can be used as a probe that will hybridize to your sequence of interest? (a) 5'-GATTGCAT-3' (b) 5'-TACGTTAG-3' (c) 5'-CTAACGTA-3' (d) 5'-ATGCAATC-3'

(d) 5'-ATGCAATC-3'

You have been hired to create a cat that will not cause allergic reactions in catlovers. Your co-workers have cloned the gene encoding a protein found in cat saliva, expressed the protein in bacteria, and shown that it causes violent allergic reactions in people. But you soon realize that even if you succeed in making a knockout cat lacking this gene, anyone who buys one will easily be able to make more hypoallergenic cats just by breeding them. Which of the following will ensure that people will always have to buy their hypoallergenic cats from you? (a) Inject the modified ES cells into embryos that have a genetic defect to prevent the mature adult from reproducing. (b) Implant the injected embryos into a female cat that is sterile as a result of a genetic defect. (c) Sell only the offspring from the first litter of the female cat implanted with the injected embryos. (d) Surgically remove the sexual organs of all the knockouts before you sell them.

(d) Surgically remove the sexual organs of all the knockouts before you sell them.

You have a piece of circular DNA that can be cut by the restriction nucleases EcoRI, HindIII, and NotI, as indicated in Figure Q10-5. Which of the following statements is false? (a) One piece of DNA will be obtained when this DNA is cut by NotI. (b) A piece of DNA that cannot be cut by EcoRI will be obtained by cutting this DNA with both NotI and HindIII. (c) Two DNA fragments that cannot be cut by HindIII will be obtained when this DNA is cut by EcoRI and NotI. (d) Two DNA fragments of unequal size will be created when this DNA is cut by both HindIII and EcoRI.

(d) Two DNA fragments of unequal size will be created when this DNA is cut by both HindIII and EcoRI.

In situ hybridization can be used to determine the _________________. (a) sequence of a cloned gene (b) distribution of proteins in tissues (c) size of a gene (d) distribution of a given type of mRNA in different tissues.

(d) distribution of a given type of mRNA in different tissues.

DNA can be introduced into bacteria by a mechanism called ____________. (a) transcription (b) ligation (c) replication (d) transformation

(d) transformation

After which of the five treatments listed below can the plasmid shown in Figure Q10-19 re-form into a circle simply by treating it with DNA ligase? Assume that after treatment any small pieces of DNA are removed, and it is the larger portion of plasmid that will reassemble into a circle. After digestion with __________. 1. SalI alone 2. SalI and XhoI 3. SalI and PstI 4. SalI and SmaI 5. SmaI and PstI

1 and 2. When SalI and XhoI cut DNA, the staggered ends left behind will match up by base pairing and can therefore be joined by ligase alone.

In which of the treatments can the plasmid re-form into a circle by the addition of DNA ligase after treating the cut DNA with DNA polymerase in a mixture containing the four deoxyribonucleotides? Again assume that you are trying to get the larger portion of plasmid to reassemble into a circle.

1, 2, and 4. SmaI cuts and leaves a blunt end. Addition of DNA polymerase and the four deoxynucleotides will fill in the 5ƍ overhangs generated by digestion with SalI and XhoI, leaving blunt ends. DNA ligase joins the blunt ends. However, 3' overhangs (that is, those generated by PstI) will not be filled in, because DNA polymerase moves in a 5' to 3' direction. DNA ligase will not join 3' overhangs to blunt-ended DNA, which are the situations presented in treatments 3 and 5.

Name three features that a cloning vector for use in bacteria must contain. Explain your answers.

A cloning vector for use in bacteria must contain the following: 1. a bacterial replication origin (to allow the plasmid to be replicated) 2. at least one unique restriction site (to allow easy insertion of foreign DNA) 3. an antibiotic-resistance gene or some other selectable marker gene (to allow selection for bacteria that have taken up the recombinant plasmids)

A nuclease hydrolyzes the __________________ bonds in a nucleic acid. Nucleases that cut DNA only at specific short sequences are known as __________________. DNA composed of sequences from different sources is known as __________________. __________________ can be used to separate DNA fragments of different sizes. Millions of copies of a DNA sequence can be made entirely in vitro by the __________________ technique. DNA sequencing phosphodiester endonucleases polymerase chain reaction exonucleases recombinant DNA gel electrophoresis restriction nucleases hydrogen ribonucleases

A nuclease hydrolyzes the *phosphodiester* bonds in a nucleic acid. Nucleases that cut DNA only at specific short sequences are known as *restriction nucleases*. DNA composed of sequences from different sources is known as *recombinant DNA*. *Gel electrophoresis* can be used to separate DNA fragments of different sizes. Millions of copies of a DNA sequence can be made entirely in vitro by the *polymerase chain reaction* technique.

Your friend has isolated a protein present in the cheek cells of all straight-A seniors at your school. She says that this protein helps you remember everything you read and therefore will help cut down on the number of hours needed to study for exams. She sequences the protein, which she calls "geniuszyme", and designs a probe to isolate the gene that encodes it. To make sure she designed the probe correctly, she consults with the company that cloned Factor VIII. They have 100% confidence that her probe will work. She also obtains a high-quality liver cDNA library from the company and uses her probe to try to isolate the gene for geniuszyme. Unfortunately, she is unable to isolate any clones. A. What is the likely explanation for her failure? B. Not to be discouraged, your friend has obtained some genomic DNA isolated from the nuclei of liver cells and has made a genomic library from that DNA. Do you expect she will succeed in isolating the geniuszyme gene from this library? Why or why not?

A. Geniuszyme is not expressed in the liver. Because cDNA is made from mRNA, a cDNA library reflects the genes expressed in a particular tissue. B. Yes, she should be able to isolate the gene, because genomic DNA is essentially the same in all tissues.

You have accidentally torn the labels off two tubes, each containing a different plasmid, and now do not know which plasmid is in which tube. Fortunately, you have restriction maps for both plasmids, shown in Figure Q10-6. You have the opportunity to test just one sample from one of your tubes. You have equipment for agarose-gel electrophoresis, a standard set of DNA size markers, and the necessary restriction enzymes. A. Outline briefly the experiment you would do to determine which plasmid is in which tube. B. Which restriction enzyme or combination of restriction enzymes would you use in this experiment?

A. You would first digest your sample with a combination of restriction enzymes selected so that they give a set of fragment sizes that could have come from only one of the plasmids. Then you would run the resulting mixture of DNA fragments on a gel alongside a set of size markers and determine the size of each fragment. By looking at the restriction maps, you should then be able to match your results to one of the plasmids. B. Digestion with any of the following combinations will enable you to distinguish which plasmid you have: HindIII + BglII; EcoRI + BglII; EcoRI + BglII + HindIII. The plasmids are the same size, so you cannot distinguish between them simply by making a single cut (with HindIII) and determining the size of the complete DNA by gel electrophoresis. Nor can you distinguish them by cutting with all four restriction nucleases, Because the set of fragment sizes produced from both plasmids will be the same. Cutting with BamHI or EcoRI on their own is not sufficient because you will get bands of the same size from both plasmid A and plasmid B. The only difference between the two plasmids is in the location of the BglII site relative to the two BamHI sites, so if you cut with an enzyme that cuts outside the BamHI fragment and with BglII, you will get different-sized fragments from the two plasmids.

You have an oligonucleotide probe that hybridizes to part of gene A from a eukaryotic cell. Indicate whether a cDNA library or a genomic DNA library will be more appropriate for use in the following applications. A. You want to study the promoter of a gene A. B. Gene A encodes a tRNA and you wish to isolate a piece of DNA containing the full-length sequence of the tRNA. C. You discover that gene A is alternatively spliced and you want to see which predicted alternative splice products the cell actually produces. D. You want to find both gene A and the genes located near gene A on the chromosome. E. You want to express gene A in bacteria to produce lots of protein A.

A.) Outcome 1 would occur. If the mRNA is degraded from the 5' end, it will still be reverse transcribed and will end up in the library as a clone lacking its 5' end. B.) Outcome 4 would occur. If the mRNA is degraded from the 3' end, it will lack its 3' poly A tail. In the construction of a cDNA library, only molecules that still have their poly A tail will be reverse transcribed, so mRNAs lacking their 3' end will not be represented in the library. C.) Outcome 1 would occur. If the 5' end hybridizes to sequences in the middle of the gene, the "hairpin" formed when the single-stranded DNA loops back on itself to form the primer for DNA polymerase will be very large. After this loop has been digested, the remaining double-stranded DNA fragment will lack the 5' end of the gene. D.) Outcome 2 would occur. If the gene has a long stretch of internal A's, the poly T primer used in the reverse transcription step can hybridize to the internal poly A stretch rather than to the poly A tail, and the resulting cDNA will have lost its 3' end.

Insulin is a small protein that regulates blood sugar level, and it is given by injection to people who suffer from the disease diabetes. Diabetics once used insulin purified from pig pancreas to control their diabetes. Give two reasons why the drug companies that produce insulin wanted to clone the human insulin gene to provide an alternative source of the hormone.

Any two of the following would be acceptable. 1. Cloning the gene allows human insulin to be produced in large quantities from bacteria or other cells carrying the cloned DNA sequence. 2. It is easier and less costly to extract the same amount of insulin from a bacterial culture than from pig pancreas. 3. Insulin made in a bacterial culture and then purified will be free of any possible contaminating viruses that pigs (and any other whole animal) tend to harbor. 4. The pig protein has slight differences from the human protein, which can lead to side effects on prolonged use. Whenever possible, a human protein would be preferred for clinical treatment of this sort.

You have the amino acid sequence of a protein and wish to search for the gene encoding this protein in a DNA library. Using this protein sequence, you deduce a particular DNA sequence that can encode this protein. Why is it unwise to use only this DNA sequence you have deduced as the probe for isolating the gene encoding your protein of interest from the DNA library?

Because most amino acids can be encoded by more than one codon, a given sequence of amino acids could be encoded by several different nucleotide sequences. Probes corresponding to all these possible sequences have to be synthesized, to be sure of including the one that corresponds to the actual nucleotide sequence of the gene and thus will hybridize with it.

Why is a heat-stable DNA polymerase from a thermophilic bacterium (the Taq polymerase) used in the polymerase chain reaction rather than a DNA polymerase from E. coli or humans?

The PCR technique involves heating the reaction at the beginning of each cycle to separate the newly synthesized DNA into single strands so that they can act as templates for the next round of DNA synthesis. Using a heat-stable polymerase avoids having to add it afresh for each round of DNA replication.

What is the main reason for using a cDNA library rather than a genomic library to isolate a human gene from which you wish to make large quantities of the human protein in bacteria?

The gene isolated from a genomic library would still contain introns, and bacteria do not contain the biochemical machinery for removing introns by RNA splicing. The same gene isolated from the cDNA library will already have had its introns removed.

The technique of __________________ hybridization can be used to detect a specific RNA expression in a particular region of the brain. Northern blotting detects a specific sequence in __________________. Southern blotting detects a specific sequence in __________________. A short, single-stranded DNA is a(n) __________________. A piece of DNA used to detect a specific sequence in a nucleic acid by hybridization is known as a(n) __________________. DNA polymerase chain reaction in situ probe in vitro RNA oligonucleotide vector

The technique of in situ hybridization can be used to detect a specific RNA expression in a particular region of the brain. Northern blotting detects a specific sequence in RNA. Southern blotting detects a specific sequence in DNA. A short, single-stranded DNA is called a(n) oligonucleotide. A piece of DNA used to detect a specific sequence in a nucleic acid by hybridization is known as a(n) probe.

Two fragments of DNA can be joined together by __________________. Restriction enzymes that cut DNA straight across the double helix produce fragments of DNA with __________________. A fragment of DNA is inserted into a __________________ in order to be cloned in bacteria. A __________________ library contains a collection of DNA clones derived from mRNAs. A __________________ library contains a collection of DNA clones derived from chromosomal DNA. blunt ends DNA polymerase RNA cDNA genomic staggered ends DNA ligase probe vector

Two fragments of DNA can be joined together by *DNA ligase*. Restriction enzymes that cut DNA straight across the double helix produce fragments of DNA with *blunt ends*. A fragment of DNA is inserted into a *vector* in order to be cloned in bacteria. A *cDNA* library contains a collection of DNA clones derived from mRNAs. A *genomic* library contains a collection of DNA clones derived from chromosomal DNA.

Note that cutting DNA with EcoRI produces a staggered end, whereas cutting DNA with HaeIII produces a blunt end. Why must polymerase be added in this reaction? (a) Polymerase will fill in the staggered end to create a blunt end. (b) Polymerase is needed to seal nicks in the DNA backbone. (c) Polymerase will add nucleotides to the end produced by the HaeIII restriction nuclease. (d) Without polymerase, there will not be enough energy for the reaction to proceed.

a) Polymerase will fill in the staggered end to create a blunt end.

During gel electrophoresis, DNA fragments _______________________. (a) travel through a matrix containing a microscopic network of pores (b) migrate toward a negatively charged electrode (c) can be visualized without stains or labels (d) are separated on the basis of their sequence

a) travel through a matrix containing a microscopic network of pores

A double-stranded DNA molecule can be separated into single strands by heating it to 90°C because _______________________. (a) heat disrupts the hydrogen bonds holding the sugar-phosphate backbone together (b) DNA is negatively charged (c) heat disrupts hydrogen bonding between complementary nucleotides (d) DNA is positively charged

c) heat disrupts hydrogen bonding between complementary nucleotides