Deck 17: Applications of Recombinant Dna Technology and Reverse Genetics

Question 1

Suppose you have engineered a plasmid vector in order to produce a functional animal protein in E. coli. But the product you collect, while it has the correct amino acid sequence, is in fact nonfunctional. What might you try that would have a greater chance of success?

A)a eukaryotic expression vector that has sequences for regulating transcription
B)using a host other than E. coli that does not have the same codon bias
C)a eukaryotic expression vector that includes sequences for controlling posttranslational effects
D)treatment of the posttranslation proteins to add signal sequences
E)using yeast artificial chromosomes that can hold longer sequences

Answer 1

D

Question 2

Which of the following is a possible advantage for the environment of having farmers use plants that genetically produce Bt toxin?

A)Plants with genes for the toxin are eaten less often by insects, and therefore less insecticide is needed.
B)Plants that produce Bt toxin must be grown from new seeds each season, and therefore fewer of them are planted.
C)Bt toxin is advantageous to many species of invertebrates that grow in the same fields.
D)Harvesting plants with these genes is less disturbing to the soil layers where they are planted.
E)Plants with these genes must be sprayed with the organism, but need much less than other plants.

Answer 2

A

Question 3

Current sequencing technologies have major advantages over the dideoxy method because they produce more sequence in less time and at less cost. Which of the following would provide further advantage?

A)being able to produce finer analysis of smaller sequences
B)the ability to produce primers for increasingly long sequences
C)the ability to sequence noncoding regions
D)sequencing very rare DNAs from degraded samples
E)sequencing without the need to amplify the DNA

Answer 3

E

Question 4

Snuppy, a male Afghan hound, was created by transferring the nucleus from an ear cell of the male Afghan, Tai, into an enucleated egg from a donor. The embryo was transplanted into a surrogate mother to develop to term. Which of the following animals would be a genetic match for Snuppy if nuclear DNA were tested?

A)Tai
B)the egg donor
C)the surrogate
D)both Tai and the egg donor
E)both Tai and the surrogate

Question 5

Which of the following vectors would be most efficient to use to amplify fragments ~300 kb in length?

A)BACs
B)plasmids
C)YACs
D) $\lambda$ phage
E)cosmids

Question 6

In producing a recombinant plasmid to be used to clone a given donor insert, it is possible to cut both the donor and plasmid with the same restriction enzyme, resulting in complementary sticky ends. Assuming plenty of plasmid DNA is available, why is further selection necessary before the introduction of the plasmid into a cellular system?

A)Contamination will have introduced other donor inserts.
B)Some donor inserts will be single stranded and deteriorate.
C)Some donor inserts will be sensitive to particular antibiotics.
D)Some donor pieces will remain uninserted.
E)Some donor strands will be inserted with an incorrect orientation.

Question 7

In 1978, Arber, Smith, and Nathans won the Nobel Prize in Physiology and Medicine for work with restriction enzymes. Which of the following can still be performed without using these enzymes?

A)PCR of whole genomes
B)genetic mapping
C)sequence cloning
D)vector packaging
E)making recombinant DNA

Question 8

If genes are introduced into a yeast plasmid to serve as a shuttle vector, the plasmid has the ability to __________.

A)move genes from human to yeast
B)replicate in yeast or in bacteria
C)degrade selected yeast genes
D)recombine with yeast nuclear genes
E)degrade other yeast plasmids

Question 9

In using Agrobacterium tumefaciens to transfer genes into plants, what is transferred from bacterium to plant?

A)Ti plasmid
B)T-DNA
C)opines
D)only the recombining sequence
E)the entire bacterium

Question 10

Some restriction enzymes cut at specific and unambiguous sequences (e.g., NotI at GCGGCCGC). As such, the distance between two such sites in a large random DNA sequence is 4⁸, assuming a 50% G+C DNA content. However, the sequence recognized by XhoII is said to be ambiguous (Pu-GATC-Py, where Pu = any purine and Py any pyrimidine). What, therefore, is the expected distance between two XhoII sites in a large random sequence?

A)4 × 4 × 4 × 4
B)1 × 4 × 4 × 4 × 4 × 1
C)2 × 4 × 4 × 4 × 4 × 2
D)4⁶
E)4² × 2²

Question 11

In selecting recombinant bacteria, cells are chosen that are resistant to a specific antibiotic. How are the bacteria made resistant?

A)They are pre-selected for the experiment on this basis.
B)The antibiotic resistance gene is encoded in the vector.
C)The antibiotic resistance gene is encoded on the donor insert.
D)Resistance is activated by the recombination event.
E)Resistance is activated when the cells are provided with the antibiotic

Question 12

In the construction of a YAC library, each chromosome has the advantage of being able to hold very long DNA sequences. However, besides selectable marker(s)the YAC vector must minimally include which of the following?

A)a bacterial origin (ori)
B)cos site(s)and origin
C)centromere, telomeres, and yeast origin
D)centromere and origin
E)centromere and telomeres

Question 13

In nature, what is the order of events in processing the product of the gene for insulin?

A)cleavage of 35 amino acids from the middle of the polypeptide to make two chains that are then joined by disulfide bonds
B)cleavage of 24 amino acids from the N terminus to form proinsulin that is subsequently cleaved to make A and B chains
C)removal of 5′ leader sequence from the mRNA, and splicing out of a large central intron to produce the mRNA that codes for the final form of polypeptide
D)removal of large mRNA sequences by RNA editing, followed by splicing of the products of the two insulin genes
E)transcription of the two insulin genes A and B, translation, and posttranslational joining of the two polypeptides

Question 14

One use of PCR has been to monitor the trafficking of endangered organisms. Suppose that one suspect was accused of illegally importing a specific animal, but the suspect asserts that the young animal had been bred in this country from two resident zoo animals. How would you best proceed?

A)Use blood cells to obtain DNA from the animals and look for sequences found only in illegally traded ones.
B)Use DNA obtained from biopsy of the suspect organism and use a probe for infectious agents found more frequently in its native country.
C)Use DNA obtained from the possibly imported animal and treat with restriction enzyme; use a probe for disease genes common in its native country.
D)Use DNA from hair follicles of supposed zoo parents and of the supposed offspring to compare sequences amplified with primers for a highly polymorphic region.
E)Amplify highly polymorphic regions from zoo and native populations to compare with the suspect organism.

Question 15

When a transgene is used in mice to knock out a specific function, two problems that might occur are the unintended introduction of multiple copies or abnormal gene expression. The second of these problems is related to __________.

A)position effect
B)homologous recombination
C)vector insufficiency
D)mitotic recombination
E)unequal crossing over

Question 16

You have a DNA sequence of 1600 bp. To characterize it, you decide to make a restriction map. Cutting with the first enzyme, you show by electrophoresis that the fragments produced are 400 and 1200 bp, respectively. After using the second enzyme, you recover 700-bp and 900-bp fragments. After treatment with both enzymes, fragments were 300, 400, and 900 bp. Which is the correct map?

A)-900 ------/400--/3---
B)-400/--900-----/300 ---
C)-300-/--400--/--900----
D)-400-/300-/---900-----
E)-400-/-100/--300--/900

Question 17

Snuppy, a male Afghan hound, was created by transferring the nucleus from an ear cell of the male Afghan, Tai, into an enucleated egg from a donor. The embryo was transplanted into a surrogate mother to develop to term. Which of the following animals would be a genetic match for Snuppy if nuclear DNA were tested and if Snuppy were not a clone, but the product of in vitro fertilization?

A)Tai
B)the egg donor
C)the surrogate
D)both Tai and the egg donor
E)both Tai and the surrogate

Question 18

Snuppy, a male Afghan hound, was created by transferring the nucleus from an ear cell of the male Afghan, Tai, into an enucleated egg from a donor. The embryo was transplanted into a surrogate mother to develop to term. Which of the following animals would be a genetic match for Snuppy if mitochondrial DNA were tested?

A)Tai
B)the egg donor
C)the surrogate
D)both Tai and the egg donor
E)both Tai and the surrogate

Question 19

Whether libraries are cDNA or genomic, whether amplification is based on PCR or cloning, and whether screening is done with northern or Southern blotting or probes, are all decisions based on which of the following?

A)complementarity
B)complementation
C)reverse transcription
D)reverse translation
E)gene expression

Question 20

In forensics, the laws in the United States and elsewhere allow the establishment of databases of PCR amplification of short tandem repeats (STRs)of the Y chromosome. Which of these statements would provide an appropriate basis for making such databases divided by ethnicity?

A)Different heritage groups have different levels of such polymorphisms.
B)It would be easier to evaluate suspects of different ethnicities.
C)It would show detectives which ethnic category they should be looking for.
D)Most crimes of certain types are found among certain groups.
E)Y polymorphisms increase with the probability of criminal behavior.

Question 21

A hypothetical restriction sequence is 5′-TCTAGA-3′. The palindromic sequence is 3′-________-5′.

Question 22

What technique of genomic library construction allows the genome to be cut by a given restriction enzyme at some but not all recognition sequences?

Question 23

In a cDNA library, not all expressed genes will be represented. On what does this depend?

Question 24

In the formation of transgenic animals, DNA is usually inserted into eggs or embryos. If some cells of the embryo do not receive the introduced DNA, the result is an organism with cells of different genotypes; the organism is known as what?

Question 25

In the case of Jesse Gelsinger, a young man who died during gene therapy, the therapy involved delivery via a viral vector. Was this germ-line or somatic therapy?

Question 26

By what method is T-DNA integrated into the plant genome?

Question 27

One way to genetically engineer S. cerevisiae is by homologous recombination. If a given sequence is thus introduced and is integrated via two regions of homology, is it more probably introduced as a linear or as a circular entity?

Question 28

In creating a recombinant DNA molecule, what are two methods that prevent re-ligation of the cloning vector that contains no insert DNA?

Question 29

Specific sequences in the $\lambda$ genome that interact with coat proteins to package genomes into phage particles are known as ________ sites.

Question 30

In the pUC plasmids, a polylinker region that includes multiple restriction sites is embedded in the lacZ component. If a sequence of interest is inserted into this region, what is the effect on lacZ?

Question 31

If a gene is replaced with a heterologous sequence such that the gene is then nonfunctional, what is the result usually called?

Question 32

If a researcher is cloning a sequence by using pUC and uses the plasmid to transform bacteria, which are then plated on X-gal medium, would the recombinant plasmids be found in blue or white colonies or both?

Question 33

Snuppy, a male Afghan hound, was created by transferring the nucleus from an ear cell of the male Afghan, Tai, into an enucleated egg from a donor. The embryo was transplanted into a surrogate mother to develop to term. Which of the following animals would be a genetic match for Snuppy if mitochondrial DNA were tested and if Snuppy were not a clone, but the product of in vitro fertilization?

A)Tai
B)the egg donor
C)the surrogate
D)both Tai and the egg donor
E)both Tai and the surrogate

Question 34

In grape cultivation, growers often find that heterozygous forms are more desirable. To propagate the heterozygotes without having to interbreed in each generation, what method can they use?

Question 35

Which type of DNA library (genomic or cDNA)would include introns and promoters?

Question 36

How can you use the known human cDNA gene sequence for cytC that you already have in an experiment(s)to identify and compare the sequences from other vertebrates?

Question 37

Agrobacterium tumefaciens is often used in the production of transgenic plants because of its Ti plasmid. This plasmid is usually "disarmed" by deleting its tumor genes along with the genes for producing what products that would have been advantageous to the bacterium?

Question 38

For a transgenic animal line to be produced from a chimera, the transgene must be carried by what?

Question 39

In producing an E. coli plasmid to be an expression vector, an experimenter includes genes for selectable markers and a promoter for the expression of the introduced genes. She uses a cDNA sequence to ensure the insert contains no introns. The gene inserted is transcribed, but not translated. What sequence did she omit from the plasmid?

Question 40

If a plant species cannot readily have a new gene integrated into it via a Ti plasmid, it must then be subjected to different methods. These often include removing the cell wall and inserting DNA, after treating the membrane with heat or electricity to do what to the membrane?

Question 41

In species X, the UUU codon for phenylalanine is used significantly more frequently than other phenylalanine codons, and knowing this allows the scientist to manipulate translation efficiency. This unequal frequency is known as ________.

Question 42

Suppose a gene from a cow is introduced into a frog. This results in a ________ transgene.

Question 43

Discuss the ethical and societal reasons for and against germ-line gene therapy.

Question 44

If recombination results in random integration of a DNA sequence so that it produces a non-homologous region, the recombination is ________.

Question 45

Your student is trying to create a "Gene X" knockout mouse by introducing a recombinant construct into embryonic stem (ES)cells, selecting transformants, and introducing the transformed cells into mouse blastocysts. He designed his construct as follows:
Neo^R----region of homology to Gene X---HSV tk--- region of homology to Gene X
where Neo^R is the neomycin-resistance gene and HSV tk is the Herpes Simplex virus thymidine kinase gene. He transforms the ES cells and exposes them to G418 and ganciclovir. Explain to your student he failed to recover transformed ES cells.

Question 46

What characteristics of plants allow them to be cloned relatively easily, whereas cloning in animals is rare and difficult to achieve?

Question 47

In the 1990s, gene therapy was seen as a probable cure for many genetic diseases. List three or four reasons that it has so far been of limited success.

Question 48

Describe two or more ethical issues arising from sequencing the genomes of individuals.