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Exam 2 Flashcards

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1
Q

Alternate name for recessive mutation. How does it act in presence of WT allele?

A

Loss of function. With WT allele, WT phenotype is restored

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2
Q

Alternate name for dominant mutation. How doe sit act in presence of WT allele?

A

Gain of function. Mutant phenotype is expressed even in presence of WT allele.

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3
Q

What can you do to determine whether a mutation is dominant or recessive?

A

Make a partial diploid.

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4
Q

Describe how you make a partial diploid

A

A small region of the chromosome of one strain is introduced into another strain using plasmids or prophages. This is used to test for the ability to complement mutations in the chromosome.

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5
Q

Which type of mutation can be complemented and which cannot, Trans-acting mutations or Cis-acting mutation?

A

Trans-acting –> CAN be complemented

Cis-acting –> can NOT be complemented

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6
Q

What is a genetic library?

A

A collection of DNA fragments from an organism that overlap around the genome. Allows ID of location of mutation through transformation.

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7
Q

How can complementation against a gene library be used to learn more about a mutant?

A

Complementation with a single gene would confirm identity and possibly chromosomal location of the desired gene (see pp. 165-166)

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8
Q

Genetic map

A

Location of genes on chromosome relative to each other.

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9
Q

Isogenic strains

A

A strain harboring a well-defined variation from its progenitor

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10
Q

What are the three mechanisms of gene exchange. Briefly describe each.

A

Transduction (phage mediated), Transformation (naked DNA), Conjugation (direct contact between mating pair)

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11
Q

What is a selectable marker and why is it important in gene exchange experiments?

A

A marker is a way to identify a transformed individual. This is important because relatively few cells participate in gene transfer. Selectable markers allow ID of those few cells that have participated in gene exchange.

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12
Q

What information is used to map genes?

A

Co-transduction (are the genes close enough to be transduced together by the same phage) and Co-conjugation (see figures 3.35, 3.36, 3.37, 3.38, table 3.6)

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13
Q

What is a high frequency recombinant?

A

A cell that has the F plasmid integrated into the chromosome by homologous recombination. It is phenotypically F+ but can transfer chromosomal genes.

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14
Q

What is a constitutively expressed gene?

A

A gene that is always on. Eg. housekeeping genes

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15
Q

Why is regulated gene expression important?

A

It provides organisms with a mechanism to produce gene products appropriate to the needs of the organism without wasting energy producing unnecessary gene products.

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16
Q

What is an operon?

A

Polycistronic genes and their regulatory sequences

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17
Q

Where can transcriptional regulation occur, and which stage is most common? Why?

A

Initiation (most common b/c it saves energy), or after RNA Pol has escaped promoter (transcription attenuation)

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18
Q

Describe positive control

A

An activator regulatory protein enhances association between RNA Pol and promoter.

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19
Q

Describe negative control

A

A repressor regulatory protein interacts with upstream region of a gene, impeding access/progress of RNA pol to promoter and/or coding sequence.

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20
Q

What is an inducer? What are two mechanisms of action of inducers?

A

It is a signaling molecule that increases gene expression. It can do this by activation of an activator or inhibition of a repressor

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21
Q

What is a co-repressor? What are two mechanisms of action of co-repressors?

A

It is a signaling moleucle that inhibits gene expression. It can do this by activation of a repressor or inhibition of an activator.

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22
Q

How doe inducers & co-repressors differ in the types of pathways they work in?

A

Inducers - catabolic pathways (eg. Lac operon)

Co-repressors - biosynthetic pathways (eg. aa synthesis)

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23
Q

What mechanism often controls co-repressor activity?

A

Co-repressors are often activated by the end product of the pathway (if end product is present –> co-repressor is active –> decreased transcription)

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24
Q

What are the components of a negative repressible system and what do they do?

A

Repressor proteins - turn genes/operons off (inactive in absence of co-repressor)
Co-repressor - a small molecule that activates the repressor

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25
What is a common mechanism in negative repressible systems?
Co-repressor down regulates its own synthesis. Operon encodes enzymes required for synthesis of co-repressor. Thus, genes are expressed in the absence of the co-repressor.
26
Describe how the E. coli trp operon works
It is a negative repressible system. Operon codes genes for tryptophan synthesis. Genes are expressed only in the absence of tryptophan. Trp repressor functions as a dimer and is only active when in association with tryptophan (co-repressor)
27
What are the two types of Positive regulation systems?
Positive inducible systems (ara operon) | Positive repressible systems (fab operon)
28
What are the components of a positive inducible system and how do they work? What type of pathways do they typically regulate?
Activator protein - turns gene(s)/operon on Inducer - small molecule effector that activates the activator Typically regulate Catabolic Pathways
29
Explain how the E. coli ara operon works
The ara operon encodes genes to convert arabinose to a usable sugar form (xylulose-5-phosphate). AraC is the gene that encodes the activator. It functions as a homodimer in two possible ways: w/o arabinose --> anti-activator (loop in pormoter makes it inaccessible to RNA Pol) w/ arabinose --> activator (promoter region is linear and accessible to RNA Pol)
30
What are the components of a positive repressible system and what do they do? What type of pathways do they typically regulate?
Activator protein - turns gene(s)/operon on Co-repressor - a small molecule effector that inhibits the activator Typically regulate biosynthetic pathways
31
Describe the fab operon and how it works
Fatty acid biosynthetic pathway. Fatty acids bind the activator and prevent it from binding to DNA. In absence of fatty acids, activator binds DNA
32
What is transcriptional Attenuation?
A form of regulation that occurs after RNA Pol has already initiated transcription
33
What components are involved in transcriptional attenuation?
Alternative secondary structures in an upstream leader region. The RNA structures are terminators or anti-terminators These are formed in response to a regulatory molecule
34
Describe the transcriptional attenuation of the E. coli trp operon
Txn begins --> Pause --> High trp region If LOW trp --> ribosome slows in this region --> single loop that allows progression forms --> translation If HIGH trp --> ribosome flies through this region --> double loop causing sequestration forms --> termination
35
What are roboswitches
attenuation that is accomplished through interaction between the leader region and a small ligand (often the product of the system - eg. tRNA, vitamins, nucleic acid bases)
36
What type of terminators are roboswitches?
Factor independent terminators (do not use rho)
37
What does catabolite repression describe?
A situation when activation of catabolic genes/operons is repressed due to the presence of a preferred carbon or energy source (eg. glucose)
38
Describe how each of the following work 1. + Lac, - Glc 2. + Lac, + Glc 3. - Lac, + Glc 4. - Lac, - Glc
1. Incr. cAMP and Lac --> lac repressor NOT bound and cAMP/CAP IS bound --> transcription 2. Incr. Lac, Decr. cAMP --> lac repressor NOT bound and cAMP/CAP NOT bound --> NO transcription Blocked by cAMP/CAP not bound 3. Decr. cAMP and Lac --> Lac repressor IS bound. cAMP/CAP NOT bound --> NO transcription Blocked by Lac Repressor 4. Decr. lac, incr. cAMP --> Lac repressor IS bound. cAMP/CAP IS bound --> no transcription Blocked by both lac repressor and cAMP/CAP not bound
39
What are three examples of Extra-chromosomal genetic elements
Plasmids, transposons, bacteriophages
40
What is a replicon?
Any fragment of DNA capable of independent replication (eg. bacterial chromosome or plasmid, but NOT a transposon)
41
How can you define a plasmid?
An independently replicating DNA element that doesn't harbor house-keeping genes. It also has all the cis-acting elements and trans-acting factors of the origin of replication.
42
What are the two mechanisms of plasmid replication?
Theta replication or Rolling Circle Replication
43
Describe Theta replication of plasmids
Uni-or bi-directional replication from the single ori-V. Similar to replication of chromosome from oriC.
44
Describe the basics of Rolling Circle Replication
Stage I produces on DS and on SS molecule | Stage II involves synthesis of complementary strand to the SS molecule to produce a second DS plasmid.
45
How many origins are on plasmids that undergo RC replication?
Two - Double-stranded and Single-stranded origins (DSO and SSO)
46
Describe the details of RC Replication
Plasmid-encoded Rep protein binds DSO and makes a nick in the strand --> free 3' OH and Rep covalently attached to 5' phosphate DNA Pol II extends 3' OH to replicate around the circle and nicked strand is displaced. Host RNA Pol then synthesizes an RNA primer at SSO on the SS Plasmid. Primer is extended by DNA Pol III to complete circle DNA Pol 1 replaces RNA primer with DNA and nick is sealed by DNA Ligase.
47
What are three additional functions of oriV?
Host range determination, Regulation of copy number, and incompatibility
48
What are some features of a broad host range plasmid?
They encode most (or all) proteins and RNAs required for INITIATION of replication. Also promoters must be recognizable by RNA Pols from a variety of bacteria
49
What is the difference between stringent and relaxed control of plasmid replication?
Stringent --> Low copy number: REplicate once per cell cycle | Relaxed --> high copy number: Replication is inhibited once number of plasmids reaches a threshold level.
50
Describe the role of ColE1 in controlling copy number of plasmids
RNA II is required for replication. RNA I is an antisense RNA that paire with RNA II. Both are produced at the same rate, but stability determines ratio in cell.
51
What is incompatibility
If two plasmids can stably co-exist in the same cell they are COMPATIBLE and belong to DIFFERENT Inc groups. If they cannot coexist, they are INCOMPATIBLE and belong to the SAME Inc group
52
What are two mechanisms resulting in incompatibility of plasmids?
Regulation of each other's replication | Regulation of each other's partitioning (par) during distribution of replicated plasmids to daughter cells.
53
Why are dimers/multimers a problem in plasmids, and how are they resolved?
They will impact distribution to daughter cells (particularly in low copy number plasmids) THey use site-specific recombination system of the host. Utilizing plasmid-specific cis-acting sequences (cer) and host XerCD recombinase (no FtsK)
54
How do plasmids partition into daughter cells during cell replication?
Par system: cis acting (parC) and trans-acting (parM and parR) ParR attaches to parC --> parM polymerization making a filament that pushes parC-parR plasmid complexes apart. ParM filament is dynamically unstable --P hydrolysis at oldest region (center) --> plasmids at opposite poles.
55
How can partitioning impact plasmid compatibility
Plasmids can be incompatible if they share the same par system even if they are in different Inc groups.
56
What is Toxin-Antitoxin or Plasmid Addiction Systems?
1. Plasmid encodes stable toxin and less stable antitoxin. 2. When cell divides without a plasmid, the stable toxin lingers, while the antitoxin already present diminishes. Without a plasmid to produce more antitoxin, the cell witll succumb to the toxin.
57
What is the goal of plasmid cloning vectors?
To move a gene from organism A to organism B by being a vehicle to harbor a cloned fragment of DNA
58
What are three important parts of a cloning vector plasmid?
Origin of replication Selectable markers/genes Multiple cloning sites
59
What is a major characteristic that distinguishes pUC-type vectors?
Blue-White Screen allows screening for insertion into the multiple cloning site
60
What is alpha-complementation and what is its role in cloning vectors?
This is how the Blue/White screen works. Beta galactosidase is a tetramer with each monomer consisting of two domains, alpha and omega. If the alpha fragment is deleted, the gene is non-functional. In cloning vectors, a LacZ alpha deleted mutant is used in combination with plasmid that has an antibiotic resistance gene plus the LacZ alpha gene with a multiple cloning site within it. When a population of transformed cells are plated on antibiotic + X-gal plates: If no plasmid is present, it will not grow due to the antibiotic If plasmid is present, but gene is not inserted, it will grow and will be blue (due to functional beta-gal) If plasmid is present w/ gene inserted at MCS in LacZ alpha, it will grow, but will be white due to non-functional beta-gal.
61
What are three types of cloning vectors?
pUC-type vectors, Bacterial artificial chromosomes, shuttle vectors
62
Describe bacterial artificial chromosomes
Low copy number plasmids that are based on the F replicon. They can harbor large fragments of DNA --> useful in moving big chunks of DNA.
63
Describe shuttle vectors
Plasmids that can replicate in two or more organisms. Allows genetic manipulation to be done in an easy to work with organism (eg. E. coli), then allows transfer to a more complicated organism.
64
What are self-transmissible plasmids
Plasmids that encode all functions required for their own transmission - Encode the sex pilus, as well as other factors for conjugation
65
What are mobilizable plasmids?
Plasmids that can be transferred but require many functions provided in trans by self-transmissible plasmids
66
What is transmitted during bacterial conjugation?
Single stranded DNA, which is then used as a template for replication --> dsDNA.
67
What are three genetic features of bacteria that can undergo conjugation?
They have tra (transfer) genes. There are three origins (ori). Only one is functional (oriV). THey also harbor a par system to ensure post-replication segregation to daughter cells.
68
What are the two components of tra genes?
Mpf (mating pair formation) and dtr (DNA transfer and conjugal replication) components.
69
What do mpf genes encode?
Sex plius and coupling protein
70
What do dtr genes encode?
Components that process and prepare the plasmid for transfer. Includes a relaxase and a site-specific endonuclease and a primase
71
How does the site-specific endonuclease of dtr genes function?
1. Indroduces nick in oriT (origin of transfer) 2. Guides the ssDNA plasmid to the recipient 3. Recircularizes ssDNA by sealing the nick once in the recipient.
72
How are tra genes regulated?
TraJ is primary regulatory protein. It is a transcriptional activator. TraJ activates tra functions soon after F enters cell. TraJ is inhibited by finP (antisense RNA) and finO (stabilizes traJ/finP interaction) --> inhibition of translation and targeting of traJ for destruction
73
Can DNA be transferred in both directions during conjugation?
No, DNA transfer is polar from donor (male) to recipient (female)
74
What are the three states the F plasmid can exist in in the cell?
Independent plasmid Episome (integrated into chromosome) F' (Independent plasmid w/ host chromosomal genes)
75
What is the outcome of conjugation of an independent plasmid?
One strand of F transferred to F- cell Single strands --> Double strand in host F+ stays F+, F- converted to F+ NO chromosomal genes transferred
76
What is the outcome of conjugation of an episome?
F is integrated into the chromosome Cell is phenotpyically and genotypically F+ The donor is known as an Hfr strain Recipient usually remains F- because only part of F is transferred Chromosomal genes ARE transferred
77
What is the outcome of conjugation of F'?
F' mating F- results in recipient with the same chromosomal genes as in the original F' parent These transconjugants are merodiploids (partial diploids)
78
What is a mobilizable plasmid and what are the key requirements?
A plasmid that is not capable of self-transmission but requires help from a conjugative plasmid. They require oriT but all other requirements can be provided in trans (from conjugative plasmid)
79
What is an integrating conjugative element? What is it lacking?
It is a self-transmissible island of DNA found on chromosomes. It has no origin of replication.
80
What do Integrating conjugative elements encode?
They encode tra function for their transmission and do NOT transfer chromosomal genes because ICE is excised prior to transfer.
81
What is Transformation?
Uptake of naked DNA from the environment
82
What type of bacteria is most commonly naturally competent?
Gram Positive
83
What is natural competence
The natural ability to undergo transformation. Few bacteria exhibit this feature and those that do only do so under certain conditions (quorum sensing).
84
What is transferred during natural transformation?
Linear DNA
85
Where do competence (com) genes reside?
In multiple operons
86
What are the steps of transformation in G+ organisms?
Binding of DS DNA at cell surface Cleavage of DS DNA into ~10kb fragments Degradation of one strand of DNA Translocation of SS DNA into cell
87
How does transformation differ from G+ in G- organisms?
They need a protein pore in teh outer membrane. One strand of DS DNA is degraded as it is threaded through the inner membrane.
88
What happens to SS DNA when it enters the cell by Transformation?
It is coated with SSB to protect from degradation. If homology exists, recombination may occur If no homology, DNA will eventually degrade.
89
What has to occur for transformation of plasmids?
They must be converted to linear SS DNA (from circular DS DNA). Two or more copies are linearized at different points. They are co-transformed. The SS linear plasmids hybridized and are repaired --> regeneration of the plasmid.
90
What are the two methods of artificially induced competence? How do each work?
Chemical Induction - cells treated w/ Ca2+ or Rb2+ then heat shock --> membrane permeable to DNA Electroporation - Strong electric field applied --> opening of rapidly self-sealing holes in the membrane, allowing DNA entry.
91
What is recombination
The interaction and exchange between two DNA molecules by crossing over.
92
What are the two general types of recombination?
General --> any site | Site-specific --> at a certain site
93
What is the result of linear donor DNA and a single crossover event
Chromosome break
94
What is the result of linear donor DNA and two crossover event
Reciprocal exchange of DNA
95
What is the result of circular donor DNA and one crossover event
Integration of donor and duplication of homologous sequences
96
What is the result of circular donor DNA and two crossover events
Reciprocal exchange of DNA
97
What is the difference between intragenic and extragenic complementation?
Intragenic - occurs in different regions of the same gene. | Extragenic - occurs in a different gene.
98
What is a suicide vector?
A cloning vector that cannot replicate in the cell, so it is degraded/diluted if it does not insert.

Micro 450 (3 decks)

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