Cell Biology Lab Concept Test II

analyze pattern of bands on stained SDS-PAGE gels (5)

1) SimplyBlue: analyze the proteins in complex extracts 2) Western Blots: detect Met and LacZ fusion proteins 3) smaller proteins migrate quicker than larger proteins 4) migration rate inversely proportional to the log MW of a proteins 5) standards allow size to be determined - higher MW standards for lower concentrations (y-shaped that bind to epitopes)

Selective media is commonly used to isolate cells that have been transformed with plasmids. Which of the following statements is true about selection schemes used in transformation experiments? (3) 1) 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. 2) Positive selective schemes use selective media that allow only transformed cells to grow. 3) Auxotrophic mutants are frequently used as hosts in transformation experiments, because untransformed cells are easily selected against. 4) Selective agents increase the frequency of transformation.

1) 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. 2) Positive selective schemes use selective media that allow only transformed cells to grow. 3) Auxotrophic mutants are frequently used as hosts in transformation experiments, because untransformed cells are easily selected against.

antibodies

1) antibodies: proteins produced by vertebrates w/adaptive immune systems capable of responding to foreign antigens

monoclonal antibodies (3)

1) bind to the same epitope on an antigen 2) produced via cultured hybridoma cells 3) we used mouse

functional elements that have been engineered in lab plasmids (6)

1) can transfer foreign DNA into a cell 2) carry up to 10kb of foreign DNA 3) easily isolated from microorganisms for manipulation 4) have selectable marker, allow transformed cells to grow under non-permissive conditions - ours contain the ampR gene (allow to grow in ampicillin) and URA3 gene (allow to grow in absence of uracil) 5) MET mutations inserted downstream of GAL1, allow for manipulation 6) plasmids encode tags that can be used for other procedures (such as purification)

process of transformation at molecular level (6)

1) causes a change in phenotype due to uptake of DNA 2) use variety of chemicals to alter cell to be able to take up DNA (destabilize the membrane + physical stress - such as heat or voltage) 3) structure of DNA changes the transformation efficiency (how many transformants per microgram of DNA) - supercoiled are much more efficient than linear (cells enter more readily or are less susceptible to endonuclease digestion 4) lithium ions neutralize negative charges + general small holes of phospholipid bilayer 5) carrier DNA, single stranded, protects plasmid DNA from endonucleases 6) PEG brings DNA closer to membrane + promotes membrane fusion

explain prep of yeast cell extracts (3)

1) disruption of plasma membrane by mechanical/chemical treatments 2) mechanical treatments: sonication, high pressure, beating with glass beads and must be vigorous because cell walls are tough but run the risk of damaging the proteins due to heat + foam 3) chemical: detergents solubilize membranes , SDS detergent used here, unfolds proteins by breaking thousands of weak bonds that stabalize structures

analyze protein expression through western blots

1) electrophoretic transferase of proteins from SDS-PAGE gel to membrane (PVDF) using transfer sandwich 2) blocking of nonspecific protein binding sites on transfer membranes - using 5% lowfat milk 3) incubation of membrane with primary (monoclonal) antibody that recognizes epitope tags (V5 epitope) 4) incubation of secondary antibody (polyclonal) that recognizes primary antibodies 5) visualization of bound antibodies - TMB used to show bands, DI water used to stop

physical properties of plasmids + use in purification (3)

1) encodes tags that allow for protein purification 2) small, super-coiled pieces of DNA 3) resistant to denaturation

effects of different carbon sources on transcription genes controlled by yeast GAL1 promoter (7)

1) glucose is the preferred carbon source 2) genes involved in metabolism of other carbon sources are repressed when glucose is present 3) when glucose isn't present, other available energy sources are metabolized - galactose 4) galactose increases transcription of enzymes that bring galactose into the cell and convert it into G6P (glucose-6-phosphate) - GAL1 promoter is the 1st of these 5) in presence of glucose, repressor proteins bind to negative regulatory sites 6) Gal4p inactivates since it is a transcriptional activator at a positive regulatory site by binding to Gal80p 7) raffinose is composed of galactose, fructose, and glucose and can induce GAL1 expression

how to isolate plasmids from transformed strains of E coli (5)

1) laboratories use commercial kits - using Zyppy kits 2) lysis and denaturation (weaken bacterial cell wall, using a strong base and detergent (to solubilize the cell wall) to strongly denature, irreversible, breaks h-bonds of two DNA helices) 3) neutralization (allows complimentary DNA strands to reanneal and proteins precipitate, DNA cannot renature tho - must shake gently to prevent fragmentation of DNA) 4) centrifugation (plasmid DNA separates from chromosomal DNA) 5) additional purification (adding resin or organic extraction)

polyclonal antibodies

1) mixtures of antibodies that bind to different epitopes on an antigen 2) an animal's response to an antigen - those in the lab come from animals that were inoculated with an antigen 3) we used rabbit

different functional regions of antibodies + how they are used in western blots

1) provide molecular probes that can be used to detect the expression of proteins on western blots 2) y-shaped that bind to epitopes + produced by lymphocytes 3) secrete by hybridoma cells 4) structured as immunoglobins - 2 Fab regions (antigen-binding) - 1 Fc region process antigen-antibody complexes 5) antibodies bind to antigens, change their structure + some produce antibodies + reproduce in high affinity 6) each plasma cell secretes a single antibody with high affinity for antigens 7) each plasmid encodes a epitope tag(s) - ours are V5 virus + His6-tag to purify

principles that govern protein separation on discontinuous SDS-PAGE gels (9)

1) resolving gel: proteins are resolved on the basis of their molecular weight - large amounts of glycine (ionization rate is critical) - pH 8.8 (akaline) 2) stacking gel: proteins are concentrated prior to entering the resolving gel 3)electric field is used to move proteins through a matrix depending on the properties of the gel and molecules 4) properties of the gels: size + geometry matters, can be changed by changing the gel monomer within a certain range 5) (in this case changing the concentration of acrylamide or the ratio of acrylamide to bisacrylamide - raising the concentration of either will make pores smaller) 6) properties of the molecules: smaller, highly charged molecules travel faster 7) proteins are smaller so polyacrylamide gels are used for separation - must be denatured prior to being run to impart a uniform mass to charge ratio- boiled in SDS, denaturation detergent and 2-mercarp + tracking dye (bromophenol blue) 8) chloride ions move towards the front (negative charge) and go forwards, leading everything else to follow while glycine molecules move to the back - set up a voltage gradient in stacking gel - glycine moves back to the front in the running gels w/a neg charge 9) once stained, proteins become fixed to the gel

experimental considerations of transformation/complementation (3)

1) use URA3 to determine if mutated strains are successfully transformed, well0tested and reliable 2) issues with the GAL1 promoter 3) regulatory balances of GA1 promoter is off in plasmid DNA

Proteins account for approximately what percentage of an average cell's dry weight?

1/2

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.)

4

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?

4096 bp

cleavage probability of DNA

6-cutter (which recognize a 6-nucletoide sequence), you take 4^6 = 4096 bp

Brilliant Blue is used to stain proteins after their separation on SDS-PAGE. Which of the following statements about staining is NOT true? 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. 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. Brilliant Blue will not stain your protein of interest. 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.

Brilliant Blue will not stain your protein of interest.

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

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

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

Epitopes are recognized by the constant regions of antibodies.

Many restriction endonucleases recognize palindromes in 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.)

GATATC

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

Glucose growth yeast will yield more total protein than galactose grown cultures.

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

It is approximately half lipid and half protein.

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?

LEU2 met2 URA3

what online tools are used to ID recognitions sites for REs in a DNA molecule

NEBCutter program to ID REs sites in plasmid seq (make sure circular is checked off)

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 HaeIII, 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 would you expect to generate the smallest number of fragments?

Not I

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

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

Which of the following best describes how polyacrylamide gels differ from agarose gels?

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

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: Proteins in the sample are unfolded and surrounded by negative charges. Proteins in the sample have a similar geometry. Proteins in the sample have a similar quaternary structure. Proteins in the sample have a similar charge-to-mass ratio.

Proteins in the sample have a similar quaternary structure.

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? 1) REs should be stored in glycerol solutions, which do not freeze at temperatures below the freezing point of water. 2) REs should be stored at low temperatures, since the rate of denaturation increases with temperature. 3) Investigators should minimize the number of air bubbles introduced into RE solutions during pipetting. 4) RE activity is best preserved by freezing the RE and thawing it out for brief periods of time as needed

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

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? The primary antibody recognizes an epitope in protein extracts. The secondary antibody binds to the constant region of primary antibody heavy chains. The secondary antibody binds to the variable region of primary antibody light chains. Horseradish peroxidase conjugated to a secondary antibody catalyses the formation of a colored reaction product.

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

what genetics element of pYES2.1 plasmid construct allows for positive selection in S. cerevisiae met mutant strains

URA3 gene

A yeast strain with the genotype MATa ura3 leu2 his3 met8 is transformed with a plasmid carrying yeast URA3 and MET1 genes. Which of the following plates would the transformed strain be expected to grow upon?

YC-uracil

Which of the following is true regarding your replica plating?

all colonies on the YC-Met-Ura+Glu plates are transformants + showing complementation of a mutation

process of complementation at molecular level

all plasmids carry normal URA3 gene - complementation will occur because the mutant strains are defective in ura2

Which of the following statements best describes the transfer process used for western blots?

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

Scientists have developed conditions to transform

bacteria, fungi & mammalian cells

Which is true about restriction endonucleases (REs)?

bacterial cells can contain multiple REs that recongize different DNA sequences

Transformation refers to the process in which

cells take up DNA from the environment

When an animal is injected with a foreign antigen, the serum collected from the animal

contains a mix of antibodies that bind to the antigen

The denaturation step of plasmid isolation achieves all of the following objectives EXCEPT: 1) DNA is denatured. 2) contaminants are removed from the preparation. 3) the bacterial cell wall is lysed. 4) native proteins are denatured.

contaminants are removed from the preparation

To increase the pore size of an SDS-PAGE gel, a researcher could

decrease the concentration of bis-acrylamide, while maintaining the same concentration of acrylamide

The rate at which a protein moves through in an SDS-PAGE gel could be increased by

decreasing the concentration of bis-acrylamide used in the gel mix

A variety of mechanical methods can be used to prepare extracts from yeast. Possible methods include all of the following, except: bead beating denaturation with detergents high pressure sonication (high frequency sound waves)

denaturation with detergents

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:

excessive physical stress can cause chromosomal DNA to fragment

Complementation refers to the process in which

foreign DNA supplies a function that is defective in a transformed host

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?

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

interpret pattens of REs on agarose gel (2)

fragments of 4000 bp are useful because they contain coding seq for proteins and are well-resolved on agarose gels the sizes of the fragments produced allow researchers to determine the original DNA molecules

The GAL1 gene encodes

galactokinase

What genetic component of the pYES2.1 plasmid allows for positive selection in bacteria?

gene encoding beta-lactamase

Both plasmid DNA and linear DNA can be used for transformation. Transformation of yeast with linear pieces of DNA

generates stable strains only when the DNA is incorporated into the chromosome

The preferred carbon source for yeast is

glucose

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?

growth of a transformant containing pYES-METX in YC+Galactose

strategies to distinguish DNA molecules using REs (5)

how many restriction fragments will be generated? what are the predicted sizes? will the be clearly resolved on the gel? will the fragments be distinctive? how expensive?

Hybridoma cells are widely used in industry for antibody production. Which of the following statements about hybridoma cells is true?

hybridoma cell lines secrete antibodies that recognize a single epitope on an antigen

mechanism of plasmid replication (3)

independently of the chromosomes in the microorganisms that harbor them depends on host cell polymerases - rely on host enzymes to carry out their replication plasmids in yeast have distinct ori sequences that bind to yeast DNA polymerase ours contain: pBR322 - replicayed in E coli

The SDS-PAGE procedure that we are using depends on the differential ionization of glycine molecules in the running and stacking gels. Glycine

is an amino acid with no net charge at neutral pH

The GAL1 promoter

is induced by galactose

The sodium hydroxide used in the extraction procedure

leaves the cells intact but fragile

Yeast transformation reactions usually include all of the following

lithium acetate single stranded (denatured) DNA polyethylene glycol (PEG)

An investigator is using a plasmid carrying a LEU2 gene for transformations. The genotypes of several potential host strains are shown below. Which of the following strains would make the best host for the transformation reactions?

met3 leu2 ura3

An investigator carefully isolates plasmid DNA with an alkaline lysis method, which is designed to minimize damage to the plasmid structure. The plasmid contains 4300 bp of DNA. What should the investigator expect to see on an agarose gel? What physical feature about plasmid DNA explains the answer in question #4?

multiple bands, none of which migrates with a 4.3 kb piece of linear DNA --- supercoiling allows plasmids to assume a variety of different conformations

design a selection strategy to isolate transformed strains

must have a selectable marker

describe the biological origins of REs

naturally occur in bacteria

Which of the following is true about polymerization of SDS-PAGE gels?

polymerization is catalyzed by ammonium persulfate and TEMED

analyze ability of plasmid-encoded MET genes to complement met deficiencies

replica plating process

Selectable markers on a plasmid are important because:

researchers can distinguish cells that have been transformed with the plasmid

sticky ends (2)

restriction fragments generate in a reaction that have short single-stranded tails on the 5' ends because the can form h-bonds with complimentary seq important for recombinant DNA tech

Which of the following statements describes the differences between running and stacking gels used in SDS-PAGE?

running gels have a higher pH and higher ionic strength than the stacking gels

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

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

saturate non-specific sites that otherwise would bind antibody

what is not true about plasmids? 1) shuttle vectors have more than one origin of replication 2) selectable markers give cells transformed with plasmids a competitive advantage over nontransformed cells under non-selective growth conditions 3) transcription factor binding sites are sometimes engineered into plasmids to drive overexpression of a particular protein 4) naturally occurring plasmids are found in both prokaryotic and eukaryotic cells

selectable markers give cells transformed with plasmids a competitive advantage over non-transformed cells under non-selective growth conditions

structure of plasmids (2)

small, circular DNA molecules most plasmids have a restricted host range

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?

smaller proteins migrate more rapidly than larger proteins

fusion proteins (2)

testing to see if they function normally additonal sequences at C-termini

Methods to isolate plasmids depend on

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?

the cell would be easily killed by bacteriophages

Restriction endonucleases (REs) are often used to fingerprint DNA. What is true?

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

Our plasmid isolation procedure involves an alkaline lysis step, followed by a neutralization step. What happens to the plasmid during the neutralization step?

the plasmid renatures due to its supercoiled structure

Yeast contain a variety of proteases capable of degrading proteins in the cell extracts. How does our extraction procedure minimize proteolytic destruction of yeast proteins?

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

describe the functions of REs (3)

they recognize short stretches of nucleotides in DNA and catalyze double-strand breaks at/or near the recognition site part of the bacterial defense system against foreign DNA protected from cleavage because they have been modified using a methyltransferase

Which of following best describes the procedures used in western blots?

western blots use antibodies to detect protein molecules

When you get your protein samples back to load into your SDS-PAGE gel, which of the following are true?

your samples are already denatured + will be in loading dye already