Concepts Test 2

Which of the following is true regarding your replica plating? (mark all that apply) a. All colonies on the YC-Met-Ura+Glu plates are transformants. b. All colonies on the YC+Met-Ura+Glu plates are showing complementation of a mutation. c. All colonies on the YC-Met-Ura+Gal plates are expressing your protein of interest. d. All transformants containing your pYES2.1-metX plasmid are Schizosaccharomyces pombe

a. All colonies on the YC-Met-Ura+glu plates are transformants. b. All colonies on the YC+Met-Ura+Glu plates are showing complementation of a mutation. c. All colonies on the YC-Met-Ura+Gal plates are expressing your protein of interest.

Scientists have developed conditions to transform: a. Bacteria, Fungi & Mammalian cells b. Fungi c. Bacteria d. Mammalian cells

a. Bacteria, Fungi & Mammalian cells

When an animal is injected with a foreign antigen, the serum collected from the animal: a. Contains a mixture of antibodies that bind to the antigen b. Is identical to serum of animals that have not been injected with the antigen c. Is depleted of antibodies that react with the antigen d. Contains a single antibody that binds the antigen with very high affinity

a. Contains a mixture of antibodies that bind to the antigen

Which of the following statements about antibodies is true? a. Each antibody-producing lymphocyte in an immunized animal secretes a single kind of antibody molecule. b. After multiple rounds of immunization, the serum of an animal contains monoclonal antibodies c. Polyclonal antibodies are produced from hybridoma cells grown in tissue culture d. Polyclonal antibodies are generated by treating hybridoma cells with chemicals that cause them to differentiate along multiple pathways

a. Each antibody-producing lymphocyte in an immunized animal secretes a single kind of antibody molecule.

The plasmid isolation process is based on the physical differences between chromosomal and plasmid DNA. After adding lysis buffer to the denatured cell lysate, it is important to gently invert the tube to mix the buffer with the cell contents. This gentle inversion is necessary because: a. Excessive physical stress can cause the chromosomal DNA to fragment b. Excessive physical stress can cause the cells to lyse c. Gentle inversion is necessary to disrupt the bacterial cell walls d. Gentle inversion allows the plasmid DNA to form aggregates with the chromosomal DNA

a. Excessive physical stress can cause the chromosomal DNA to fragment

Complementation refers to the process in which: a. Foreign DNA supplies a function that is defective in a transformed host b. Cells take up foreign DNA and gain a function that is not normally present in the host species c. Cells take up foreign DNA from the environment d. DNA is transferred from one species to another species

a. Foreign DNA supplies a function that is defective in a transformed host

The GAL1 gene encodes: a. Galactokinase b. Galactose permease c. Enzymes that convert galactose into glucose d. Enzymes that convert glucose into galactose

a. Galactokinase

Both plasmid DNA and linear DNA can be used for transformation. Transformation of yeast with linear pieces of DNA: a. Generates stable strains only when the DNA is incorporated into the chromosome b. Generates stable strains with a similar efficiency as transforming yeast with plasmid DNA c. Depends on enzymes that convert the linear DNA to circular DNA d. Is only successful when the DNA contains a selectable marker

a. Generates stable strains only when the DNA is incorporated into the chromosome

Successful transformation of yeast with a plasmid carrying the yeast MET2 gene will allow which of the following strains to grow in the absence of methionine? a. LEU2 met2 URA3 b. LEU2 met1 ura3 c. leu2 MET1 URA3 d. LEU2 MET2 ura3

a. LEU2 met2 URA3

The sodium hydroxide used in the extraction procedure: a. Leaves the cell intact, but fragile. b. Causes the cell membrane to break and release the cell contents. c. Inhibits proteases in the extracts d. Neutralizes acid released from the yeast vacuole

a. Leaves the cell intact, but fragile.

Proteins account for approximately what percentage of an average cell's dry weight? a. One-half b. One-third c. One-tenth d. One-fourth

a. One-half

Selective media is commonly used to isolate cells that have been transformed with plasmids. Which of the following statements is NOT true about selection schemes used in transformation experiments? a. Selective agents increase the frequency of transformation b. Positive selection schemes use selective media that allow only transformed cells to grow c. Auxotrophic mutants are frequently used as hosts in transformation experiments, because untransformed cells are easily selected against d. When ura3 cells are transformed with a plasmid carrying a URA3 gene, the fraction of transformed cells expected to grow on plates lacking uracil is very high

a. Selective agents increase the frequency of transformation

Restriction endonucleases (REs) are often used to fingerprint DNA. Which of the following statements are true? a. The lengths of the DNA fragments produced by a particular RE vary widely b. Restriction endonucleases are robust enzymes that resist denaturation under conditions that denature DNA c. Cleavage sites for a particular RE are regularly spaced along a DNA molecule, separated by an interval characteristic of the particular RE d. Restriction endonucleases nick one strand of the DNA helix, generating nicked fragments that can be separated by electrophoresis

a. The lengths of the DNA fragments produced by a particular RE vary widely

Yeast contains a variety of proteases capable of degrading proteins in the cell extracts. How does our extraction procedure minimize proteolytic destruction of yeast proteins? a. The procedure relies on rapid denaturation of proteases by boiling with SDS, a denaturing detergent. b. The extraction solution includes protease inhibitors c. The procedure leaves the yeast vacuole intact, which is where most of the proteases are located. d. During the procedure, cells are boiled in sodium hydroxide to inactivate proteases.

a. The procedure relies on rapid denaturation of proteases by boiling with SDS, a denaturing detergent.

Which of the following is NOT important when planning your RE digest? a. The sequence of DNA upstream of your MET gene in the yeast genome b. The sequence of the lacZ gene c. The sequence of your MET gene d. The sequence of the pYES2.1 plasmid, including its URA3 gene and origins of replication

a. The sequence of DNA upstream of your MET gene in the yeast genome

What genetic element of the pYES2.1 plasmid construct allows for positive selection in S.cerevisiae met mutant strains? a. URA3 gene b. Gene encoding beta-lactamase c. GAL1 promoter sequence d. Yeast ORF sequence

a. URA3 gene

The recognition site for EcoRI is GAATTC. What would be the average size of the restriction fragments generated in a restriction digest of human genomic DNA? a. 24,000 bp b. 4,096 bp c. 240 bp d. 1,024 bp

b. 4,096 bp

Which of the following statements best describes the transfer process used for western blots? a. A stained SDS-PAGE gel is placed against a membrane and included in a cassette, which is oriented so that proteins migrate toward the negative (black) pole b. An unstained SDS-PAGE gel is placed against a membrane and included in a cassette, which is oriented so that proteins migrate toward the positive (red) pole c. An unstained SDS-PAGE gel is placed against a membrane and included in a cassette, which is oriented so that proteins migrate toward the negative (black) pole d. A stained SDS-PAGE gel is placed against a membrane and included in a cassette, which is oriented so that proteins migrate toward the positive (red) pole

b. An unstained SDS-PAGE gel is placed against a membrane and included in a cassette, which is oriented so that proteins migrate toward the positive (red) pole

Yeast transformation reactions usually include all of the following EXCEPT: a. Polyethylene glycol (PEG) b. Bovine serum albumin (BSA) c. Single stranded (denatured) DNA d. Lithium acetate

b. Bovine serum albumin (BSA)

The denaturation step of plasmid isolation achieves all of the following objectives EXCEPT: a. DNA is denatured b. Contaminants are removed from the preparation c. Native proteins are denatured d. The bacterial cell wall is lysed

b. Contaminants are removed from the preparation

The rate at which a protein moves through in an SDS-PAGE gel could be increased by: a. Reversing the polarity of the electric field b. Decreasing the concentration of bis-acrylamide used in the gel mixture c. Boiling the samples for twice the normal amount of time before loading them on the gel d. Using a larger running gel

b. Decreasing the concentration of bis-acrylamide used in the gel mixture

A variety of mechanical methods can be used to prepare extracts from yeast. Possible methods include all of the following EXCEPT: a. Sonification (high frequency sound waves) b. Denaturation with detergents c. High pressure d. Bead beating

b. Denaturation with detergents

Which of the following do you hypothesize to be true? a. Galactose grown yeast will yield more total protein than glucose grown cultures. b. Glucose grown yeast will yield more total proteins than galactose grown cultures. c. Transformants containing pYES-LacZ plasmid will not grow in YC+Raffinose. d. A met16 mutant will have more total protein than other mutants.

b. Glucose grown yeast will yield more total proteins than galactose grown cultures.

Which of the following is NOT true about the yeast cell wall? a. It protects yeast from mechanical and osmotic stresses. b. It is approximately half lipid and half protein. c. It surrounds the cell membrane. d. It is rich in polysaccharides.

b. It is approximately half lipid and half protein.

An investigator digests bacteriophage DNA with three different restriction endonucleases in separate reactions. One reaction contains Pst I, which recognizes the sequence TTATAA. The second reaction contains Hae III, which recognizes the sequence GGCC, and the third reaction contains Not I, which recognizes the sequence GCGGCCGC. The products of the three reactions are separated on an agarose gel. Which of the reactions should you expect to generate the smallest number of fragments. a. Hae III b. Not I c. They should all be expected to produce the same number of fragments d. Pst I

b. Not I

Which of the following best describes how polyacrylamide gels differ from agarose gels? a. Polyacrylamide gels are used to determine the sizes of molecules. b. Polyacrylamide gels are held together by covalent, rather than non covalent, bonds c. Changing the pore size of Polyacrylamide gels changes the rate at which molecules move through the gel d. Polyacrylamide gels contain a network of pores

b. Polyacrylamide gels are held together by covalent, rather than non covalent, bonds

Which of the following is true about polymerization of SDS-PAGE gels? a. Polymerization occurs when a solution of acrylamide cools. b. Polymerization is catalyzed by ammonium persulfate and TEMED c. Polymerization is catalyzed by hydrogen peroxide and UV light d. Polymerization is catalyzed by bis-acrylamide molecules

b. Polymerization is catalyzed by ammonium persulfate and TEMED

Like all enzymes, restriction endonucleases (REs) need to be handled very carefully to ensure that they do not denature. Which of the following is NOT recommended to protect these fragile structures from denaturation? a. Investigators should minimize the number of air bubbles introduced into RE solutions during pipetting b. REs activity is best preserved by freezing the RE and thawing it out for brief periods of time as needed c. REs should be stored at low temperatures, since the rate of denaturation increases with temperature d. REs should be stored in glycerol solutions, which do not freeze at temperatures below the freezing point of water

b. REs activity is best preserved by freezing the RE and thawing it out for brief periods of time as needed

Which of the following is true? a. Running gels have a lower pH and lower ionic strength than the stacking gels. b. Running gels have a higher pH and higher ionic strength than the stacking gels. c. Running gels have a lower pH and higher ionic strength than the stacking gels. d. Running gels have a higher pH and lower ionic strength than the stacking gels.

b. Running gels have a higher pH and higher ionic strength than the stacking gels.

Which of the following is NOT true about transformation? a. Chemicals are used to increase the permeability of cell walls to foreign DNA b. Salmon sperm DNA should never be boiled before use in a transformation reaction c. Depending on conditions, transformed cells may not have a selective advantage over non-transformed cells d. Transformation is a very inefficient process

b. Salmon sperm DNA should never be boiled before use in a transformation reaction

Western blots include a series of steps with blocking reagents. Blocking reagents: a. Are used in similar concentration to the antibodies in western blots b. Saturate non-specific sites that would otherwise bind antibody c. React with proteins that are not able to bind antibody d. Are proteins that are unable to bind the protein being detected in the western blot

b. Saturate non-specific sites that would otherwise bind antibody

Our plasmid isolation procedure involves an alkaline lysis step, followed by a neutralization step. What happens to the plasmid during the neutralization step? a. The plasmid shears into small fragments because of its small molecular weight b. The plasmid renatures due to its supercoiled structure c. The plasmid renatures because plasmid DNA is completely unaffected by strong denaturants d. The plasmid precipitates out with the denatured proteins because its mass is small to those of bacterial proteins

b. The plasmid renatures due to its supercoiled structure

Which of the following best describes the procedures used in western blots? a. Western blots use antibodies to detect DNA molecules b. Western blots use antibodies to detect protein molecules c. Western blots use molecular hybridization to detect RNA molecules d. Western blots use molecular hybridization to detect DNA molecules

b. Western blots use antibodies to detect protein molecules

Some restriction endonucleases are referred to as four-cutters. What makes an enzyme a four-cutter and how frequently would you expect a four-cutter to cut DNA? a. Four-cutters recognize four different nucleotide sequences and cuts about once every 256 base pairs on average b. Four-cutters have four subunits that function together and cut about once every ~ 4,000 bp c. Four-cutters recognize specific nucleotide sites that are four nucleotides long and cut about once every 256 bp on average d. Four-cutters cleave all sequences four times and cut about once ever ~ 4,000 bp

c. Four-cutters recognize specific nucleotide sites that are four nucleotides long and cut about once every 256 bp on average

Many restriction endonucleases recognize palindromes in a DNA. Which of the following DNA sequences is a palindrome? (Remember that DNA is a double helix with two antiparallel strands. Only one strand of the helix is shown.) a. GATCGA b. GATTAG c. GATATC d. GATGAT

c. GATATC

Which of the following growth conditions should yield a protein that can be recognized by the anti-V5 antibody that we are using in this lab? a. Growth of a transformant containing pBG1805-METX in YC+Galactose b. Growth of a transformant containing pBG1805-METX in YC+Glucose c. Growth of a transformant containing pYES-METX in YC+Galactose d. Growth of a transformant containing pYES-METX in YC+Glucose

c. Growth of a transformant containing pYES-METX in YC+Galactose

Hybridoma cells are widely used in industry for antibody production. Which of the following statements about hybridoma cells is true? a. Hybridoma cells are derived by fusing a myeloma cell with a liver cell b. Hybridoma cell lines are used to produce primary antibodies c. Hybridoma cell lines secrete antibodies that recognize a single epitope on an antigen d. Hybridoma cells divide twenty-eight times in culture, after which they enter senescence and die

c. Hybridoma cell lines secrete antibodies that recognize a single epitope on an antigen

The SDS-PAGE procedure that we are using depends on the differential ionization of glycine molecules in the running and stacking gels. Glycine: a. Molecules are positively charged in the running gel b. Molecules move more rapidly than proteins in the stacking gel because of their smaller size c. Is an amino acid with no net charge at neutral pH d. Molecules are larger than most proteins, so they move more slowly through the stacking gel than proteins

c. Is an amino acid with no net charge at neutral pH

SDS-PAGE is commonly used to estimate the molecular weight of proteins in samples. Molecular weight estimates are based on all of the following assumptions, EXCEPT: a. Proteins in the sample are unfolded and surrounded by negative charges b. Proteins in the sample have a similar geometry c. Proteins in the sample have a similar quaternary structure d. Proteins in the sample have a similar charge-to-mass ratio

c. Proteins in the sample have a similar quaternary structure

A yeast cell extract was prepared by boiling cells in the presence of SDS and mercaptoethanol. Which statement best describes the migration of the proteins on SDS-PAGE gels? a. Proteins with a net positive charge in their native state migrate more rapidly than proteins that have a net negative charge in their native state. b. Larger proteins migrate more rapidly than smaller proteins. c. Smaller proteins migrate more rapidly than larger proteins. d. Proteins with a net negative charge in their native state migrate more rapidly than proteins that have a net positive charge in their native state.

c. Smaller proteins migrate more rapidly than larger proteins.

A yeast strain with the genotype MATa ura3 leu2 his3 met8 is transfomred with a plasmid carrying yeast URA3 and MET1 genes. Which of the following plates would the transformed strain be expected to grow upon? Check all that apply. a. YC minus methionine b. YC minus leucine c. YC minus uracil d. YC minus histidine

c. YC minus uracil

When you get your protein samples back to load into your SDS-PAGE gel, which of the following are true? a. If a pellet exists in your sample tube, you should not vortex it, the pellet contains no protein. b. Your samples contain only your protein of interest (METX) c. Your samples will be in loading dye already d. Your samples are already denatured

c. Your samples will be in loading dye already d. Your samples are already denatured

An investigator is using a plasmid carrying a LEU2 gene for transformation. The genotypes of several potential host strains are shown below. Which of the following strains would make the best host for the transformation reactions? a. met1 LEU1 ura3 b. met 3 LEU2 URA3 c. met3 leu2 ura3 d. met3 leu1 URA3

c. met3 leu2 ura3

Which of the following is true about restriction endonucleases (REs)? a. A hypothetical RE that is a 3 cutter would cut ever ~ 32 base pairs in a sequence b. Most commercially available REs are purified from their species of origin c. Bacterial species are named for the REs that are present in their cytoplasm d. Bacterial cells can contain multiple REs that recognize different DNA sequences

d. Bacterial cells can contain multiple REs that recognize different DNA sequences

Brilliant Blue is used to stain proteins after their separation on SDS-PAGE. Which of the following statements about staining is NOT true? a. If Protein A is twice as long as Protein B; 1000 molecules of Protein A will generate a band that is just as intense as the band corresponding to 1000 molecules of Protein B b. If Extract 1 has twice the amount of Protein A as Extract 2, the intensity of the Protein A band in Extract 1 will be twice as great as that of the Protein A band in Extract 2. c. Brilliant Blue dyes stains are not specific for particular proteins, so they can be used to obtain a general overview of proteins in a sample. d. Brilliant Blue will not stain your protein of interest

d. Brilliant Blue will not stain your protein of interest

Transformation refers to the process in which: a. Plasmids provide functions that are missing in a host cell b. Cells acquire mutations that produce a new phenotype c. Microorganisms assume a new phenotype d. Cells take up DNA from the environment

d. Cells take up DNA from the environment

To increase the pore size of an SDS-PAGE gel, a researcher could: a. Increase the concentration of bis-acrylamide, while maintaining the same concentration of acrylamide b. Increase the pH of the gel buffer c. Increase the concentration of acrylamide d. Decrease the concentration of bis-acrylamide, while maintaining the same concentration of acrylamide

d. Decrease the concentration of bis-acrylamide, while maintaining the same concentration of acrylamide

Which of the following statements is NOT true? a. Molecular cloning is often used to attach epitopes to a protein being studied b. Epitopes are associated with antigens that provoke immune responses c. A single antigen may have multiple epitopes d. Epitopes are recognized by the constant regions of antibodies

d. Epitopes are recognized by the constant regions of antibodies

Bacteriophage lambda has a 48 kb linear genome. If you digest lambda DNA with an enzyme known to recognize three sites in its sequence, how many bands would you expect to see on an agarose gel? (Our MW standards are derived from restriction digests of lambda DNA.) a. Three b. Two c. One d. Four

d. Four

What genetic component of the pYES2.1 plasmid allows for positive selection in bacteria? a. Yeast URA3 gene b. GAL1 promoter sequence c. The size of the plasmid d. Gene encoding beta-lactamase

d. Gene encoding beta-lactamase

The preferred carbon source for yeast is: a. Galactose b. Fructose c. Raffinose d. Glucose

d. Glucose

The GAL1 promoter: a. Is repressed by galactose b. Is induced by glucose c. Is constitutively active in yeast cells d. Is induced by galactose

d. Is induced by galactose

An investigator carefully isolates plasmid DNA with an alkaline lysis method, which is designed to minimize damage to the plasmid structure. The plasmid contains 4,300 bp of DNA. What should the investigator expect to see on an agarose gel? a. A single band with the size expected for a 4.3 kb piece of linear DNA. b. No discrete bands. c. A smear of DNA fragments along the gel lane. d. Multiple bands, none of which migrates with a 4.3 kb piece of linear DNA

d. Multiple bands, none of which migrates with a 4.3 kb piece of linear DNA

Which of the following statements about the membranes used in western blots is NOT true? a. Membranes used in western blotting have high protein binding capacity b. During the transfer procedure, proteins bind to the membrane, generating a replica of the SDS-PAGE gel c. A variety of different membranes are used in western blots d. PVDF membranes are most frequently used for western blots because they are hydrophilic

d. PVDF membranes are most frequently used for western blots because they are hydrophilic

Selectable markers on a plasmids are important because: a. Selectable markers cause the yeast ORF to be translated b. The plasmid maintains its circular structure c. Transcription factors can bind to the selectable marker on the plasmid d. Researchers can distinguish cells that have been transformed with the plasmid

d. Researchers can distinguish cells that have been transformed with the plasmid

Which of the following is not true about plasmids? a. Naturally occurring plasmids are found in both prokaryotic and eukaryotic cells b. Shuttle vectors have more than one origin of replication c. Transcription factor binding sites are sometimes engineered into plasmids to drive overexpression of a particular protein d. Selectable markers give cells transformed with plasmids a competitive advantage over nontransformed cells under non-selective growth conditions

d. Selectable markers give cells transformed with plasmids a competitive advantage over nontransformed cells under non-selective growth conditions

What physical feature about plasmid DNA explains the answer in question #4? a. Plasmids contain two origins of replication b. Plasmids autonomously replicate from the yeast genome c. Plasmids can be cleaved with restriction enzymes d. Supercoiling allows plasmids to assume a variety of different conformations

d. Supercoiling allows plasmids to assume a variety of different conformations

Methods to isolate plasmids depend on: a. The fact that plasmids replicate independently of the chromosomal DNA b. The circular structure of plasmid DNA, in contrast to the linear structure of bacterial chromosomal DNA c. The presence of multiple copies of a plasmid in a bacterial cell d. The ability of plasmids to renature after cells are treated with denaturants

d. The ability of plasmids to renature after cells are treated with denaturants

If you discovered a bacterial cell that contained no restriction endonucleases, which of the following would you expect to happen? a. The cell would be unable to replicate its DNA b. The cell would become an obligate parasite c. The cell would create incomplete plasmids d. The cell would be easily killed by bacteriophages

d. The cell would be easily killed by bacteriophages

Western blots provide very sensitive detection of a protein of interest against a background of unrelated proteins. Western blots include multiple steps, each of which amplifies the signal of the preceding step. Which of the following statements does not describe a step in a western blot? a. The primary antibody recognizes an epitope in protein extracts b. The secondary antibody binds to the constant region of primary antibody heavy chains c. Horseradish peroxidase conjugates to a secondary antibody catalyses the formation of a colored reaction product d. The secondary antibody binds to the variable region of primary antibody light chains

d. The secondary antibody binds to the variable region of primary antibody light chains