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.

Accepted Answer

P element-mediated transformation involves the insertion of P elements into the genome, but this process does not target specific positions in the genome. Instead, the insertion sites are relatively random, although there may be some preferences for certain genomic regions.

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

Accepted Answer

Eukaryotic protein production in E. coli typically involves optimizing codon usage, using cDNAs to avoid intron-related issues, and employing vectors with appropriate promoter and translation initiation sequences. A negative selectable marker for nonhomologous recombination is not relevant to this process, as it pertains more to genome editing or cloning strategies where specific genomic integration or maintenance is critical.

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.

Accepted Answer

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.

Accepted Answer

The Cre-lox recombination system, derived from bacteriophage P1, allows for site-specific recombination events to occur within an organism's DNA. Cre recombinase recognizes loxP sites, specific DNA sequences, to catalyze the excision, insertion, or inversion of DNA segments between these sites. This system is widely used in genetic engineering, including the removal of selectable marker genes from transgenic organisms, making option D correct. Options A, B, and C are incorrect as they describe characteristics of other systems or organisms. Option E is also incorrect because the Cre-lox system is extensively used for manipulating DNA both in vitro and in vivo, not limited to in vitro applications.

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.

Accepted Answer

The restriction map can be determined from the given data. When cut with the first enzyme, the DNA yields fragments of 400 and 1200 bp, indicating a single cut. The second enzyme produces 700 and 900 bp fragments, suggesting a different single cut. When both enzymes are used, the fragments are 300, 400, and 900 bp. The presence of a 400 bp fragment in both single enzyme digests and the double digest confirms its position. The 900 bp fragment from the second enzyme digest remains unchanged in the double digest, indicating it's at one end. The 300 bp fragment appears only after the double digest, suggesting it's a part of the 700 bp fragment when cut further by the first enzyme. Therefore, the correct order is 400 bp, followed by 300 bp, ending with the 900 bp fragment.

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

Accepted Answer

The position effect refers to the phenomenon where the expression of a gene is influenced by its location within the genome. In vertebrates with larger genes and more heterochromatin, a transgene might be inserted into a region that is tightly packed or not usually expressed, leading to abnormal expression patterns. This is because heterochromatin is generally transcriptionally inactive, and the large size of genes could mean that regulatory elements are far from the transgene's insertion point, affecting its expression.

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.

Accepted Answer

The antibiotic resistance gene is typically encoded in the vector used for cloning. This allows for the selection of bacteria that have successfully taken up the vector by growing them on media containing the antibiotic; only those bacteria with the vector, and thus the resistance gene, will survive.

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'

Accepted Answer

PstI recognizes and cleaves the palindromic sequence 5'-CTGCAG-3'. The complementary sequence to 5'-CTG-3' on the opposite strand must be 3'-GAC-5', which when read in the 5' to 3' direction is 5'-CAG-3', making the complete sequence 5'-CTGCAG-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

Accepted Answer

The answer of Suppose you have engineered a plasmid vector...

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

Accepted Answer

The answer of The human genome is 3 × 10⁹...

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.

Accepted Answer

Homologous recombination occurs less frequently than illegitimate (or non-homologous) recombination in the process of creating transgenic animals, making option C false.

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.

Accepted Answer

cDNA libraries are made from mRNA and represent the genes that are being expressed in the tissue from which the mRNA was isolated. Since a genomic library contains all the sequences present in the genome, including those not being expressed in a particular tissue, sequences in a tissue-specific cDNA library (like muscle) should also be present in any tissue's genomic library (like brain), because the genomic library represents the entire set of genetic information in an organism, regardless of tissue-specific expression.

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

Accepted Answer

The correct sequence for genetic therapy involves first reprogramming cells into a pluripotent state (II), then removing the reprogramming factors (III), followed by correcting the genetic defect via CRISPR-Cas9 (V), differentiating the corrected cells into the desired cell type (I), and finally transplanting these cells back into the patient (IV).

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.

Accepted Answer

Bacteria producing white colonies on plates containing X-gal and ampicillin indicates that the lacZ gene has been disrupted by the insertion of a DNA fragment, preventing the production of functional β-galactosidase. This enzyme is responsible for cleaving X-gal and producing a blue product. Therefore, the presence of white colonies suggests that β-galactosidase is not functional in these bacteria.

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.

Accepted Answer

Plants genetically modified to produce Bt toxin are less susceptible to insect damage, reducing the need for chemical insecticides, which can have harmful environmental impacts.

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

Accepted Answer

The T-DNA (Transfer DNA) region of the Ti plasmid is transferred from Agrobacterium tumefaciens into the plant genome during genetic transformation.

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

Accepted Answer

The answer of Assume that a plasmid is 4000 base...

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

Accepted Answer

The answer of What size fragments you would expect from...

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

Accepted Answer

The correct order for producing human insulin in E.coli during the 1970s starts with reverse translating the human insulin genes to create synthetic genes (III), followed by adding restriction sites for cloning (IV), then cloning each insulin chain into an expression vector (I), and finally inducing the formation of disulfide bonds (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

Accepted Answer

The correct order for cDNA library preparation is: II. Isolate mRNA, V. Create a single-stranded DNA molecule using reverse transcriptase, I. Partially degrade the mRNA with RNase H, III. Synthesize a strand of cDNA using DNA polymerase, and IV. Ligate cDNAs into a cloning vector.

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

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

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

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

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?

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

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

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?

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') .

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

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

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?

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

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

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.

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