Deck 15: Analysis of Gene Function by Forward Genetics and Reverse Genetics

Question 1

Which of the following is FALSE regarding P element- mediated transformation?

A) The gene of interest is typically inserted in the vector plasmid between the P element inverted repeat end sequences.
B) The selectable marker is typically inserted in the vector plasmid between the P element inverted repeat end sequences.
C) The transposase activity inserts P elements into the genome at specific positions in the genome.
D) P elements transpose only in the germ- line cells of Drosophila.
E) A second plasmid can be used to supply the transposase activity in trans.

Answer 1

C

Question 2

Efficient production of eukaryotic proteins in E. coli may require all of the following except?

A) altered codon usage within the heterologous sequences to approximate the codon bias in E. coli
B) the use of cDNAs, that are free of introns, as eukaryotic transgenes
C) an E. coli expression vector with a promoter sequence that binds RNA polymerase
D) an E. coli expression vector with a Shine- Dalgarno sequence for efficient translation
E) a negative selectable marker to select against nonhomologous recombination events

Answer 2

E

Question 3

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

Answer 3

D

Question 4

Which of the following is true regarding the Cre- lox recombination system?

A) Cre recombinase can recognize the P element inverted repeat end sequences.
B) This site- specific recombination system is derived from Drosophila.
C) Cre recombinase initiates conjugative transfer of the T- DNA in Agrobacterium tumefaciens.
D) It can be used to remove selectable markers in transgenic organisms.
E) It is limited to manipulating DNA sequences in vitro.

Question 5

You have a linear DNA fragment of 1600 bp. To characterize it, you decide to make a restriction map. Cutting with one enzyme, you show by electrophoresis that the fragments produced are 400 and 1200 bp. Cutting the same DNA with another enzyme you recover 700 and 900 bp fragments. After treatment with both enzymes, fragments were 300, 400, and 900 bp. Which of the following order of fragments, if any, best represents the restriction map?

A) 400/ 300/ 900
B) 900/ 400/ 300
C) 400/ 900/ 300
D) 300/ 400/ 900
E) It is not possible to determine the order.

Question 6

Why might the expression of a transgene be especially abnormal in vertebrates with larger average size of genes and amount of heterochromatin in their genomes?

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

Question 7

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

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

Question 8

Half of the palindromic restriction site that the endonuclease PstI recognizes, binds, and cleaves is 5'- CTG - 3'. What is the complete sequence?

A) 5'- CTGCAG- 3'
B) 5'- CTGGTC- 3'
C) 5'- CTGCTG- 3'
D) 5'- CTGGAG- 3'
E) 5'- CTGGAC- 3'

Question 9

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

Question 10

The human genome is 3 × 109 bp in length. Calculate how many fragments would result from the complete digestion of the human genome with EcoRI (5'- G˙AATTC- 3').

A) 1.17 × 107
B) 4.32 × 105
C) 7.32 × 105
D) 9.72 × 105
E) 4.58 × 104

Question 11

Which of the following is FALSE regarding the formation of transgenic animals?

A) 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 a chimera.
B) DNA is usually inserted into eggs or embryos since totipotency is not characteristic of most animal cells.
C) Homologous recombination occurs much more frequency than illegitimate recombination.
D) Either integration of multiple copies of the transgene or position effect can lead to variability in the expression of the transgene.
E) For a transgenic animal line to be produced from a chimera, the transgene must be carried by germ line or germ cells.

Question 12

Which of the following is true of the sequences found in DNA libraries from human tissues?

A) Those in a muscle cDNA library should all be present in a brain genomic library.
B) Those in a brain cDNA library should not be found within a muscle genomic library.
C) Those in a brain cDNA library should all be present a muscle cDNA library.
D) Those in a muscle genomic library should not be found within a brain genomic library.
E) Those in a muscle cDNA library should all be present in a brain cDNA library.

Question 13

Which lists the potential steps for genetic therapy for mice with sickle cell disease (fiS/fiS) in the correct order? I. Differentiation of iPS cells into hematopoietic progenitor (HP) cells by infecting with retrovirus expressing HoxB4.
II) Reprogramming tail fibroblasts into induced pluripotent stem (iPS) cells by infecting them with vectors containing transcription factors.
III) Infection with a viral vector expressing Cre recombinase to remove transcription factor(s) from f iPS cells.
IV) Transplantation of corrected (þA/þA) hematopoietic cells back into irradiated mice.
V) Homologous recombination with fiS and wild- type fiA allele via CRISPR- Cas9 genome editing

A) V, III, IV, I, II
B) V, III, I, IV, II
C) I, II, III, V, IV
D) II, III, V, I, IV
E) II, III, IV, V, I

Question 14

If bacteria transformed with a recombinant pUC18 plasmid produce white colonies when grown on plates containing ampicillin and Xgal, which of the following is least likely?

A) They produce functional fi- galactosidase that cleaves X- gal.
B) They carry a vector with the bla gene used as the selectable marker.
C) They carry a vector with a lacZ gene that has been disrupted and rendered nonfunctional.
D) They carry a vector that contains a DNA fragment inserted into the multiple cloning site.
E) They produce fi- lactamase that provides resistance to ampicillin.

Question 15

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

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

Question 16

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

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

Question 17

Assume that a plasmid is 4000 base pairs in length and has restriction sites at the following locations: 600, 1000, 2200, and 3800. Give the expected sizes of the restriction fragments following complete digestion.

A) 200 bp, 400 bp, 600 bp, 1200 bp, 1800 bp
B) 400 bp, 800 bp, 1000 bp, 1800 bp
C) 400 bp, 800 bp, 1200 bp, 1600 bp
D) 200 bp, 600 bp, 1200 bp, 1600 bp
E) 200 bp, 800 bp, 1200 bp, 1800 bp

Question 18

What size fragments you would expect from an EcoRI/ XhoI double digest of the plasmid below?

A) 2 kb, 3.75 kb, 4.25 kb
B) 2 kb, 8 kb
C) 1.25 kb, 2 kb, 2.5 kb, 3 kb
D) 1.25 kb, 2.5 kb, 3 kb, 3.25 kb
E) 2.5 kb, 3 kb, 3.25 kb

Question 19

Which of the following best lists the correct order of the steps taken to produce human insulin in E.coli in the 1970s?
I) Each insulin chain was cloned into an expression vector as a continuation of the lacZ reading fra resulting in a fusion protein for each chain.
II) Disulfide bonds were induced to form between the cysteine residues of the purified insulin A a chains.
III) Both human insulin genes were reverse translated to create synthetic genes.
IV) EcoRI and BamHI restriction sites were added to the ends of the DNA to facilitate directional cloning of each into a separate plasmid vector.

A) III, I, II, IV
B) I, II, III, IV
C) I, III, IV, II
D) III, IV, I, II
E) IV, III, I, II

Question 20

Which of the following lists the steps for cDNA library preparation in the correct order? I. Partially degrade the mRNA with RNase H
II) Isolate mRNA from a specific cell/ tissue with an oligo- dT primer that hybridizes to poly- A tail
III) Synthesize a strand of cDNA using DNA polymerase with mRNA fragments as primers
IV) Ligate cDNAs into a cloning vector
V) Create a single- stranded DNA molecule complementary to the mRNA using reverse transcriptase

A) II, V, IV, III, I
B) II, V, I, III, IV
C) I, II, III, IV, V
D) II, III, V, I, IV
E) V, III, I, IV, II

Question 21

If bacteria containing non- recombinant pUC18 are plated on X- gal medium, would colonies appear blue or white colonies or both?

Question 22

Which of the following statements is FALSE regarding gene therapy?

A) Approaches with iPS cells avoid problems of immune system incompatibility, but are limited to diseases, like blood disorders, in which cells can be isolated, corrected, and reintroduced.
B) The integration of a transgene into the genome of the target cell by a viral vector is not permanent, and therefore it will require repeated treatments.
C) iPS cells that continue to express the Yamanaka factors are predisposed to become cancerous.
D) Both somatic and germinal gene therapy have been successful in animal systems; but for ethical reasons, only somatic gene therapy has been attempted in humans.
E) The insertion of a vector may cause a detrimental mutation.

Question 23

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 24

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

Question 25

Snuppy, a male Afghan hound, was created by transferring the nucleus from an ear cell of the male Afghan hound, 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) the egg donor
B) Tai
C) the surrogate
D) both Tai and the egg donor
E) both Tai and the surrogate

Question 26

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

Question 27

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

Question 28

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 29

Which of the following is FALSE regarding grape cultivation?

A) the shoots are often genetically identical and chosen on the basis of their fruit phenotype
B) the vines are usually chimeric
C) clonal propagation helps to maintain highly heterozygous cultivars
D) phenotypes are usually maintained through self- fertilization or crossing with another cultivar
E) the roots are often genetically identical and chosen for being well adapted to soil conditions

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

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

Question 32

Cloning animals by nuclear implantation involves all of the following except?

A) induction of cell division and implantation of blastocyst in the surrogate mother
B) removal of an egg cell, extraction of the nucleus, and injection of the diploid donor nucleus
C) fertilization of the cell egg with a sperm cell followed by implantation in a surrogate mother
D) a low frequency of success likely due to variations in the completeness of epigenetic programming of the somatic cell nucleus
E) removal of somatic cells and extraction of a diploid donor nucleus from the animal to be cloned

Question 33

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 34

Genetic therapy for mice with Duchenne muscular dystrophy demonstrated that which method could be used to delete a mutant exon of the dystrophin gene in muscle cells?

Question 35

The ends left by some restriction enzymes that do not have 5' or 3' overhangs are called what?

Question 36

Snuppy, a male Afghan hound, was created by transferring the nucleus from an ear cell of the male Afghan hound, 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) the surrogate
B) Tai
C) the egg donor
D) both Tai and the egg donor
E) both Tai and the surrogate

Question 37

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 38

To ensure that a human gene is translated properly in a bacterial cell, what should you clone instead of the genomic DNA fragment that contains the gene?

Question 39

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

Question 40

What are the short single- stranded overhangs created by cleavage of DNA by specific restriction enzymes?

Question 41

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

Question 42

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:
NeoR---- region of homology to Gene X--- HSV tk- -- region of homology to Gene X where NeoR 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 43

The binary approach uses both a transformation vector that contains the T- region flanked
by border sequences and a disarmed conjugative transfer.

Question 44

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 45

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 46

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

Question 47

You have isolated a plasmid from E. coli. You get the following results, using three restriction enzymes, 1 XhoI, 2 BamHI, and 3 EcoRI to perform six different digestions: single digests using each enzyme alone and double diges using each combination of two enzymes. Draw a restriction map of the plasmid.

Question 48

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

Question 49

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

Question 50

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