Lecture 7

Question 1

Why might an organism regulate expression of genes and synthesis of proteins?

Answer 1

Control mechanisms ensure that genes are active only when their products are required.
Enzymes are only produced as they are needed and prevent the waste of energy and materials.

Question 2

What is an operon?

Answer 2

a DNA sequence where a regulatory protein binds

Question 3

What roles do each of the following components of an operon play: structural genes?

Answer 3

often encoding one or more enzymes

Question 4

What roles do each of the following components of an operon play: promoter signal?

Answer 4

the DNA sequence where RNA polymerase binds

Question 5

A regulatory protein

Answer 5

that binds to the operator

Question 6

What is the difference between repressor and activator proteins

Answer 6

Repressor proteins prevent transcription when bound to the operator

Activator proteins promote transcription when bound to the operator

Question 7

What is the difference between inducer vs corepressor signals?

Answer 7

Inducers stimulate binding of activator proteins, inhibit repressors

Corepressors stimulate binding of repressor proteins to the operator

Question 8

A signal

Answer 8

usually a substrate or product of enzymes encoded by the operon

Question 9

What is the function of the genes encoded in the lac operon in E. coli, and why would this be regulated by lactose?

Answer 9

The lac operon in E. coli has 3 genes encoding enzymes involved in utilization of lactose (sugar found in milk) as a carbon and energy source.

The operon consists of 3 structural genes, a promoter, and an operator that contains sequences recognized by the repressor protein
Regulatory protein: Transcriptional repressor
Signal: Lactose acts as an inducer

Question 10

lac operon in E. coli, why would this be regulated by lactose

Answer 10

In the presence of lactose, lactose acts as in inducer and binds to the repressor.

This prevents the repressor from binding to the operator, resulting in transcription.

Question 11

Be able to describe the basic mechanism of regulation of the lac operon in E. coli in response to the presence and absence of the sugar lactose…what is the signal, what type of regulatory protein is in action, and what happens to expression of the genes in the lac operon in the presence and absence of lactose?

Answer 11

The operon consists of 3 structural genes, a promoter, and an operator that contains sequences recognized by the repressor protein
Regulatory protein: Transcriptional repressor
Signal: Lactose acts as an inducer
In the absence of lactose, the repressor binds to the operator and prevents transcription

Question 12

lac operon

Answer 12

slide 7-9

ON: presence of lactose inactivates the repressor, releasing it from the DNA and allowing transcription to occur

OFF: repressor prevents transcription in the absence of lactose

In the presence of lactose, lactose acts as in inducer and binds to the repressor.
This prevents the repressor from binding to the operator, resulting in transcription.
Additional regulation not shown prevents transcription in the presence of glucose (preferred substrate)

Question 13

What is the difference between a repressible operon and an inducible operon?

Answer 13

REPRESSIBLE:often encode anabolic enzymes, Typically expressed (“on”) under normal conditions.
Expression is repressed by excessive amounts of product.
Example: Amino acid synthesis pathway; synthesis only occurs when amino acid is needed.

INDUCIBLE:often encode for catabolic enzymes.
Typically not expressed (“off”) in the absence of substrate.
Expression is induced in the presence of the specific catabolic substrate.
Example: Lactose utilization (lac) operon; genes not expressed if their substrate is not present.

Question 14

Which of these two does the lac operon fall under?

Answer 14

Inducible operons, Lactose utilization (lac) operon; genes not expressed if their substrate is not present.

***Lac operon is inducible (turned “on” in the presence of lactose), but it is controlled by a transcriptional repressor (that prevents transcription unless lactose is present).

Question 15

What is phase variation?

Answer 15

when bacteria turn on or off a set of genes in a reversible, heritable way that leads to obvious phenotypic changes

Question 16

What types of traits does phase variation typically affect, and why might this affect whether or not a pathogen that undergoes phase variation can be recognized by the human immune system?

Answer 16

This term is most often applied to traits affecting the bacterial cell surface

Describes the ability of bacteria to change components of their surface marked for targeting the host’s immune system
Can help evade recognition by the host immune system
Can also influence the bacterium’s ability to attach to surfaces
Allows microbes to adapt to, and stick in, different environments

Question 17

• What is a mutation?

Answer 17

A mutation is a heritable change to the nucleotide sequence in the genome, i.e. it is passed down from generation to generation

Question 18

wild type-mutation mean?

Answer 18

a microorganism that exhibits a natural, nonmutated characteristic (“normal” for the organism or population)

Question 19

What is the difference between spontaneous and induced mutations?

Answer 19

Spontaneous mutations occur due to errors in DNA replication
Induced mutations can be caused by exposure to radiation (UV, x-rays) or chemicals that cause damage to DNA that is not repaired

Question 20

What are some examples of things that can induce mutations?

Answer 20

? Can involve the loss of base pairs (deletion), the addition of base pairs (insertion), a change in individual bases (point mutations) or a rearrangement in the order of base pairs (inversion).
Mutations in genes will affect the resulting RNA sequence and potentially the resulting protein sequence

Question 21

• What are three categories of mutations based on their effects (i.e. their phenotypes)?

Answer 21

Deleterious mutations:can have harmful effects on the cell, and can be lethal
Neutral mutations: have neither harmful or beneficial effects
Beneficial mutations: are more rare, and can result in helpful changes that can improve cell growth

Question 22

Be able to identify the difference between point mutations, insertion and deletion mutations, and back mutations.

Answer 22

Point mutations are changes in DNA sequences at individual positions
Point mutations in genes are categorized by their effects on products (RNA, protein)

Insertions and deletions: gain or loss of DNA at a specific site of the genome.
Can be small (1 base pairs) or large (many 1000’s of bp)

Back mutations are mutations that return the genotype to the wild-type sequence

Question 23

• What effects do missense, nonsense, silent, and frameshift mutations in a protein-coding gene have on the resulting protein (Table 8.8)? SLIDE 15

Answer 23

MISSENSE:missense mutation leading to amino acid switch

NONSENSE:base substitution: nonsense,premature stop codon–>truncated protein
SILENT: base substitution: silent (no change in function)
FRAMESHIFT:

Question 24

• Give two examples of mechanisms of DNA repair, and explain how excision repair works.

Answer 24

DNA repair: Repair of ultraviolet (UV)-induced damage by photolyase
Repair of point mutations by excision repair
Repair of Mutations

Excision:1. The first enzyme complex
recognizes one or several
incorrect bases. These bases are then removed from the newly synthesized DNA strand.
2. The second enzyme complex (DNA polymerase I and ligase)
places correct bases by base pairing and seals the gaps.
3. Repaired DNA

Question 25

What are two enzymes that are involved in both DNA replication and excision repair?

Answer 25

DNA Ligase, DNA polymerase

Question 26

• How could mutation in a bacterium to an antibiotic resistant form, followed by exposure of the population to that antibiotic, result in an increase in abundance of the mutant form over the wild-type?

Answer 26

When the environment changes, some mutants may be better equipped to survive in the new environment than the wild type.
Natural selection will then favor growth of the mutant
Acquired drug resistance is a clear model for this type of selection and adaptation: in the presence of an antibiotic, if only a few cells have resistance, they will eventually grow to replace non-resistant members.This can result in prevalent resistance drugs (e.g. HIV) or vaccines (influenza virus) in some viruses.

Question 27

• Do viruses typically have lower or higher mutation rates than cellular organisms?

Answer 27

Viruses often have very high mutation rates in comparison to cellular organisms

Question 28

• What is recombination, and what is a recombinant?

Answer 28

Recombination involves exchange of DNA.
Recombination: In this context, it is an event in which one bacterium (the donor) donates DNA to another bacterium (the recipient)
The recipient is called a recombinant

Question 29

What sorts of DNA can be transferred by recombination…plasmid or chromosomal DNA, or both?

Answer 29

Can occur with DNA other than plasmids, e.g. exchange of portions of chromosomal DNA

Question 30

• What is horizontal gene transfer, and how does it differ from normal vertical descent of genomic DNA?

Answer 30

This transfer from one bacterium to another is called.
It differs from vertical gene transfer from a mother cell to daughter cells during reproduction

Any transfer of DNA that results in organisms acquiring new genes that did not come directly come from parent organisms
Typically involves small pieces of DNA in the form of plasmids or chromosomal fragments

Question 31

• What factors are involved in conjugation?

Answer 31

Conjugation: Requires the attachment of two related species and the formation of a bridge from a donor to a recipient cell that can transport DNA

Donor cell with pilus
Fertility plasmid in donor
Both donor and recipient alive
Bridge forms between cells to transfer DNA

Question 32

? Which of these processes requires cell-to-cell contact? Be able to compare and contrast these three major mechanisms of horizontal gene transfer (Table 8.7 in the textbook).

Answer 32

Conjuncation requires cell-to-cell contact

Three mechanisms of horizontal gene transfer
Conjugation: Requires the attachment of two related species and the formation of a bridge from a donor to a recipient cell that can transport DNA

Transformation: Entails the transfer of naked DNA and requires no special vehicle

Transduction: DNA carried from one bacterium to another via a bacteriophage

Question 33

• Explain the roles of the following in conjugation involving the F factor (F plasmid): F+ cell, F- cell, F pilus (or “sex” pilus), conjugation bridge, DNA replication. What happens to the recipient cell after conjugation is complete?

Answer 33

Donor cells (F+) contain the Fertility factor (F factor) plasmid
This plasmid allows the synthesis of a conjugative pilus (F pilus)
Recipient cell (F-) do not contain the F plasmid, but they do have a recognition site for the F pilus on its surface.
Contact is made when a pilus grows out from the F+ cell, attaches to the surface of the F- cell, contracts, and draws the two cells together.
A conjugation bridge is then formed between donor and recipient
The plasmid is replicated and a single strand copy is transferred to the recipient F- cell,
The single strand is then replicated in the recipient, which now contains the F plasmid and becomes F+

F+: cell that has the plasmid (donor)
F-: cell that lacks the plasmid (recipient)
Contact is made when a pilus grows out from the F+ cell, attaches to the surface of the F- cell, contracts, and draws the two cells together

**Reciepient is now F+ and can act as a donor

Question 34

• How does conjugation between an Hfr strain and a recipient differ from conjugation involving transfer of the F plasmid only? SLIDE 26/27

Answer 34

Fertility factor (F factor)F+: cell that has the plasmid (donor)
F-: cell that lacks the plasmid (recipient)
Contact is made when a pilus grows out from the F+ cell, attaches to the surface of the F- cell, contracts, and draws the two cells together
The plasmid is replicated and a single strand copy is transferred to the F- cell,
The single strand is then replicated in the recipient which becomes F

Hfr:High-frequency recombination (Hfr) donors transfer chromosomal DNA to the recipient cell.
The F plasmid becomes integrated into the F+ donor chromosome
Initial steps of conjugation occur as normal
F pilus on F+ donor cell attaches to F- recipient cell
Conjugation bridge is formed
During replication and transfer, some chromosomal genes get transferred to the recipient along with a portion of the plasmid.
The donated DNA is then incorporated into the recipient chromosome.
Usually not all plasmid genes are transferred, so recipient may not be F+ after transfer is complete

Question 35

• What are R plasmids (or R factors) and how do they play a role in spread of antibiotic resistance via conjugation?

Answer 35

Resistance (R) plasmids or factors: Plasmids capable of conjugation that contain genes for resisting antibiotics and other drugs

Conjugation is efficient: because the recipient becomes a donor, conjugal plasmids such as R plasmids can spread rapidly through a population of bacteria
Can result in rapid spread of resistance to antimicrobial drugs

Question 36

• What does competency mean, in terms of uptake of DNA by transformation?

Answer 36

competent: cells that are capable of accepting genetic material

Requires no special appendages; donor and recipient cells do not have to be in direct contact

Question 37

• What acts as the vector during transduction?

Answer 37

Bacteriophage, or phage: another term for a virus that infects bacteria
In transduction, bacteriophage acts as a vector (something that facilitates the transfer of DNA)

Question 38

What are some differences between generalized and specialized transduction (which regions of the chromosome can be transferred? Which types of bacteriophage are capable of each)?

Answer 38

Generalized transduction
Occurs during the process of assembly of phage particles in an infected cell (can happen either for lytic or lysogenic phages…see below)
Results from accidental packaging of a fragment of the host cell’s DNA, instead of the viral genome, into the phage protein capsid
This capsid is able to inject the packaged (non-infectious DNA) into a new host; this DNA can then be integrated into the new host’s genome

Specialized transduction
Also occurs during the assembly of phage particles in an infected cell
ONLY occurs when the phage is capable of lysogeny, or integration into the host cell’s genome at a specific site
When the phage genome is excised from the host genome, a portion of the neighboring genome is included.
This is then packaged and transferred to a new host cell.
Transfer of DNA is specific (only near the phage integration site)

Question 39

• What is a restriction enzyme, and what is meant by “sticky ends” produced by restriction enzyme cleavage of DNA?

Answer 39

Enzymes capable of recognizing foreign DNA and breaking the phosphodiester bonds between adjacent nucleotides on both strands of DNA

“Sticky ends” produced by a given restriction enzyme are complementary to other ends produced by the same enzyme acting on different DNA sequences.
This allows portions of DNA sequences from different organisms to be combined into a single molecule, e.g. on a plasmid

Question 40

How can restriction enzymes be useful in genetic engineering and molecular cloning?

Answer 40

“Restriction” comes from the fact that they are endonucleases (cut DNA internally, rather than at the ends) that only cut DNA at specific sequences
Allows biotechnologists to cleave DNA at desired sites
Necessary for recombinant DNA technology

Question 41

• Explain briefly how gel electrophoresis can be used to separate DNA fragments based on their size.

Answer 41

Gel electrophoresis can be used to separate and visualize restriction fragments

Analysis of DNA fragments by gel electrophoresis
Produces a readable pattern of DNA fragments
Samples are placed in compartments in a soft agar gel and subjected to an electrical current
Phosphate groups have a negative charge, which causes DNA to move toward the positive pole in the gel
Larger fragments migrate more slowly; smaller fragments faster
Position of fragments are determined by staining the gel
Creates a genetic fingerprint

Question 42

• What are five important components in molecular cloning?

Answer 42

DNA SEQUENCE TO BE CLONED (often called an “insert”)
A PLASMID VECTOR r to put the DNA insert into
A RESTRICTION ENDONUCLEASE to cut both the plasmid and insert
DNA LIGASE ENZYME to connect the insert to the plasmid
A HOST ORGANISM that will take up the recombinant plasmid (insert + vector) by transformation and will allow its replication

Question 43

• What are three very basic steps in molecular cloning (slide 41)? What enzymes are used, and what method of horizontal gene transfer are used in this process?

Answer 43

1) Treat both insert and plasmid with the same restriction enzyme
2) Mix the fragments and treat with ligase
3) Transform the recombinant plasmid into a host cell, e.g. E. coli

Question 44

Factors involved in Transformation

Answer 44

Entails the transfer of naked DNA and requires no special vehicle

Free donor DNA (fragment)
live, competent recipient cell

Question 45

Factors involved in Transduction?

Answer 45

DNA carried from one bacterium to another via a bacteriophage

Donor is lysed bacterial cell.
Defective bacteriophage is carrier of donor DNA. Live reciepient cell of same species as donor

Question 46

Generalized transduction

Answer 46

Occurs during the process of assembly of phage particles in an infected cell (can happen either for lytic or lysogenic phages…see below)
Results from accidental packaging of a fragment of the host cell’s DNA, instead of the viral genome, into the phage protein capsid
This capsid is able to inject the packaged (non-infectious DNA) into a new host; this DNA can then be integrated into the new host’s genome

Question 47

Specialized transduction

Answer 47

Also occurs during the assembly of phage particles in an infected cell
ONLY occurs when the phage is capable of lysogeny, or integration into the host cell’s genome at a specific site
When the phage genome is excised from the host genome, a portion of the neighboring genome is included.
This is then packaged and transferred to a new host cell.
Transfer of DNA is specific (only near the phage integration site)

Question 48

What are three important components of a vector used in molecular cloning?

Answer 48

Vector: Used to “transport” the insert into a host cell
Often a plasmid is used. Important components of a plasmid vector are…
Origin of replication
Restriction endonuclease recognition sequences
Antibiotic resistance gene