Lab 1 Flashcards
1
Q
Lab
approaches used to identify genes in bacteria
A
- mutagenesis
- relies on loss of phenotype
- causes changes in DNA structure that in turn results in phenotype changes
- Allows rapid identification of genetics associated with a phenotype change
- assesses role for a given gene in a particular function
- Two common methods
- use of transposons (mobile genetic elements)
- use of chemicals
- disadvantages
- organism may have several related structural genes expressing the same phenotype
- loss of a particular phenotype may be the result of mutations in a gene that is important in the expression of a phenotype, but is not the actual structural gene for that phenotype
- i.e. regulatory genes involved in the expression of the target gene
- cloning for function
- relies on gain of phenotype (gene function)
- offers a direct method to isolate and identify a gene by expression of its phenotype in another organism
- Reverse genetics
- opposite of forward genetics (mutations and direct cloning)
- working with genome sequences to identify gene function through mutagenesis or other forms of altering gene expression
- becoming the new standard
- easier to sequence genome and decipher function
- Biochemical approach
- isolate protein
- determine amino acid sequence
- design degenerate primers for PCR
- identify gene
2
Q
Lab
Transposon mutagenesis
A
- highly preferred
- uses transposons (mobile genetic elements)
- advantage
- transposon insertions automatically result in a physical “tagging” of the gene location
- the mutated gene can be easily identified by physically locating the presence of the transposon in genome
- for example
- cloning transposon or portion of it, w/DNA region where transposon inserted
- DNA sequences flanking transposons can be used as probes to identify homologous sequences from wildtype DNA
- for example
- Successful use contingent on contingent upon the tractability (susceptibility) of the host organism
- disadvantages
- Lack of host susceptibility
- potential for less random insertions throughout the genome
- may insert in regions that are “hot spots” (specific nucleotide sequences preferred for insertion), as opposed to “cold spots” (specific nucleotide sequences where they don’t insert).
3
Q
Lab
Chemical mutagenesis
A
- DNA changes are not readily detected at the physical level by molecular methods that are less sensitive than nucleotide sequencing
- recovery of the corresponding wildtype gene is based on restoration (complementation) of the mutated phenotype
- requires transferring an entire library of clones that collectively make up the genome of the wildtype organism into the mutant strain
- advantage
- most organisms are susceptible to mutagens
- several chemicals that differ in mutation modes of action are available for use
- less likely to induce mutations in “hot spots”
- Manipulation of conditions influencing mutation rate can generate single- or multiple mutations per cell
4
Q
Lab
Cloning for function (direct cloning)
A
- relies on gain of phenotype (gene function)
- expression of a gene/group of genes originating from one bacterium in a different bacterium that lacks the phenotype (heterologous expression)
- conducted by constructing a genetic “library” of clones consisting of a number of DNA fragments each contained in a plasmid vector
- can consist of fragments of an organism’s entire genome, or a subset
- library is transferred to a second organism
- individual clones are maintained separately in individual cells
- library then screened for in a second, heterologous organism for phenotype acquisition
5
Q
Lecture
Genotype
A
genetic information encoded within the genome of an organism
6
Q
Lecture
Phenotype
A
- observable characteristic
- based on genotype and environment
7
Q
Lecture
mutation
A
- A heritable change in an organism’s genome (genotype)
- caused by
- alteration of single base units in DNA
- deletion
- insertion
- rearrangement of larger sections of DNA in genes or chromosomes
- can occur
- Naturally. Spontaneous mutations
- during DNA replication
- rate of 10-4 to 10-10 per generation: 1 in 10,000 to 1 in 10 billion
- between 10-1 and 10-2 per individual
- cellular factors maintain replication fidelity such as proofreading and mismatch repair
- mutagens
- agents that speed up the natural rate of mutation
- Directed mutagenesis/site specific mutations
- through genetic manipulation
- Naturally. Spontaneous mutations
8
Q
Lecture
Effects of Mutagens
A
- Physical manifestation
- structural change of DNA molecule
- Results in genetic change during replication
- Internally coded
- inheritable information
- change in genetic information – change in base sequence
9
Q
Lecture
Structural changes
What chemical alterations result in mutations?
A
- Tautomeric shifts
- transient rearrangement of bonding to form a structural isomer
- results in altered base pairing during replication
- deamination
- loss of exocyclic amino group
- changes base, and thus base pairing capabilities
- i.e. Cytosine to Uracil
- loss of bases by depurination and depyrimidination
- physical changes to DNA (eg. Cross linking or thymine/pyrimidine dimers)
10
Q
Lecture
Structural changes
Tautomeric shifts
A
- transient rearrangement of bonding to form a structural isomer
- results in altered base pairing during replication
- i.e.
- in guanine or thymine
- C=O (keto) converted to C-OH (enol)
- double bond moves from exocyclic to endocyclic
- in adenine and cytosine
- NH2 (amino) to NH (imino)
- double bond moves from endocyclic to exocyclic
- in guanine or thymine
11
Q
Lecture
Structural changes
Tautomeric shifts: Altered Pairing
A
- in guanine or thymine
- C=O (keto) converted to C-OH (enol)
- in adenine and cytosine
- NH2 (amino) to NH (imino)
- T’ (enol) pairs with G
- C’ (imino) pairs with A
- G’ (enol) pairs with T (keto)
- A’ (imino) pairs with C (amino)
12
Q
Lecture
Structural changes
Deamination
A
- loss of exocyclic amino group
- changes base, and thus base pairing capabilities
- i.e. Cytosine to Uracil
- Loss of an amine (NH2) group and is replaced by an Oxygen
- changes C to U
- Uracil is equivalent to T
- Cytosine should base pair with G, but now base pairs with A
- 5-Methylcytosine deamination occurs naturally but less than Cytosine deamination
13
Q
Lecture
Structural changes
A
- depurination and depyrimidination
- loss of a base from the phosphate sugar backbone of a DNA polymer
- results in random insertion of base during replication, subsequently changing the G to T
- in image
- 1 = shows GC pairing
- 2 = depurination of G (missing)
- 3 = G is randomly replaced with A by DNA polymerase
- 1 in 4 chances it will pick the right one
- better than terminating replication early because of missing base
- 4 = A is then base paired with T
14
Q
Lecture
Structural changes
physical changes to DNA
A
- Cross linking or thymine/pyrimidine dimers
- Pyrimidine dimers (typically TT) form when DNA is exposed to Ultraviolet light
- results in covalent bond
- on the same DNA strand
- physical linkage does not allow dna polymerase to read the strand correctly
15
Q
Lecture
Types of Mutagens
Environmental
A
- UV light
- Ionizing Radiation: structural damage or breakage
- damage is done by oxidative damage
- i.e. conversion of Guanine to 8-oxo-7-hydro-guanine (GO)
- See Scenario 1 in image
- unrepaired GO causes a transversion, a base change from pyrimidine to purine or vice versa
16
Q
Lecture
Types of Mutagens
Chemical - Alkylating agents
A
- broad group of compounds that function in base modification by adding an alkyl (methyl or ethyl) group to DNA base
- Prevents correct base reading during replication
- Can lead to
- depurination/depyrimidination
- dimers/covalent linkages that lead to DNA strand breakage
- examples
- Aflatoxins
- naturally occurring toxins produced by fungi
- involved in rot fruits, vegetable and grains
- Nitrosoguanidine
- aka N-methyl-N-nitro-N-nitrosoguanidine, NTG or MNNG
- used in experiments conducted in this class
- can perform alkylation or depurination
- functions primarily at replication
- need replicating bacteria, in exponential phase
- can cause multiple mutations/clustering
- Aflatoxins
- Most susceptible site for alkylation w/in DNA
- the 6’-O position on guanine bases
- Oxygen is attacked by an electrophilic mutagen
- An alkyl is added converting G → O-6-Ethylguanine
- This leaves only two N sites for H-bonds in O-6-Ethylguanine
- O-6-eG pairs with T instead of C
- transition conversion: purine to purine change (or pyrimidine to pyrimidine)
17
Q
Lecture
Types of Mutagens
Chemical - Cross-linking agents
A
- between strands
- e.g. eg mustard or mitomycin
- Prevents strand separation, blocking replication
- categorized as alkylating agents
- covalent bond formation results in strand breakage
- If DNA repair occurs, it typically results in regions of DNA deletion
18
Q
Lecture
Types of Mutagens
Chemical - Base analogs
A
- molecules which have a very similar structure to one of the four nitrogenous bases used in DNA: adenine, guanine, cytosine or thymine
- They form a structure similar to one of the DNA nucleotides
- can be used to form the new strand in semi conservative replication
- causes mispairings
- e.g.
- 5 bromouracil
- has two forms, a keto and enol
- keto pairs with A, and enol paris with G
- 5-methylcytosine
- similar in structure to cytosine and thymine
- continues to pair with G
- 5 bromouracil
