Mutations

Question 1

mutant

Answer 1

genetic state of an organism, different from the wild type

Question 2

mutation

Answer 2

structural alteration of DNA in a mutant, always change in base sequence of DNA

Question 3

mutagen

Answer 3

physical or chemical agent that causes mutations

Question 4

mutagenesis

Answer 4

process of producing a mutation

spontaneous vs. induced mutagenesis (background level of mutation, about 1 per 107-109 divisions)

Question 5

TYPES OF MUTATIONS 5

Answer 5

1. wildtype - normal state of the MO
2. base substitution
3. base addition or insertion
4. base rearrangement (recombination)
5. base deletion

Question 6

Point mutation

Answer 6

single base pair changed

Question 7

Multiple mutation

Answer 7

2 or more bp change

Question 8

_____ sequence> __ ___ sequence >___ ___ ____

Answer 8

base sequence, AA sequence, 3D protein structure

Question 9

Silent mutation

Answer 9

does not alter AA sequence

Question 10

Neutral mutation

Answer 10

AA substitution - no effect- does not change structure of protein

Question 11

Leaky mutation

Answer 11

reduction but not loss of activity

not in active site

Question 12

Conditional Mutations

Answer 12

affected by temperature - exhibit mutant phenotype under certain conditions.

Question 13

Missense mutation

Answer 13

change in AA - substitution

Question 14

Nonsense mutation (chain termination)

Answer 14

(amber, ochre, opal - stop codons) - codon stops protein synthesis

Question 15

Detection of Mutants - Screening

Answer 15

1. plate a large number of bacteria on agar with antibiotic or any compound in agar
2. sugar-utilization mutants
   color indicator plates
   EMB agar (pH colonies) incorporate dyes or not (Lac+ black or Lac- white)
   MacConkey, tetrazolium agars
3. leucine requiring - mutant (Leu-)

Question 16

Replica plating for Ts mutants

Answer 16

• grow at different temperatures.

Question 17

ENRICHMENT FOR MUTANTS

Selection

Answer 17

• only mutant cells will grow
  1. antibiotic resistance mutants
  2. grown on substrates that normally cannot be used

Question 18

ENRICHMENT FOR MUTANTS

Answer 18

1. spontaneous mutation - Leu- 1 per 107 cells, 5x104 plates
2. induced mutation rate - 1x105, 500 plates
3. need to favor growth of mutant over wildtype
   Example: Penicillin Selection Techinque
   cells in Leu- media, synthesize Leu+ grow & explode
   99% die Leu-, now 1/104, need only 50 plates
   2nd cycle repeat pen enrichment 1 leu-/102

Question 19

Origin of Spontaneous Mutations

Sources

Answer 19

1. errors during replication
   corrected by Pol III proofreading
   corrected by recombination repair
2. spontaneous alteration of a nucleotide - deamination
   spontaneously deaminated MeC->T
3. depurination - N glycosylic bond for purine breaks

Question 20

errors during replication
corrected by Pol III proofreading
corrected by recombination repair
can happen if:

Answer 20

-tautomerision of nucleotides pg. 197

boat & chair forms

Question 21

Types of Mutagens

Answer 21

1. Base-analogue mutagens
2. Chemical mutagens
3. UV light
4. INtercalating agents
5. Transposable Elements

Question 22

1. Base-analogue mutagens

Answer 22

tautomerize, two modes of H bonding
a. 5-bromouracil (BU) analogue of thymine
usually pairs A, keto form pairs with A ,*rare enol form pairs with G AT -> GC

1. pair with G –> G:C pair
2. occassionally BU analogue for C can pair the other way rarely to cause a mutation GC -> AT

b. 2-aminopurine (AP) substitute for A base pair w/T or C

Question 23

Chemical Mutagens

Answer 23

substance alters a base already in DNA helix, change H-bond properties
oxidative
a. nitrous acid - amino groups ——————-> keto groups
deamination
cytosine & adenine are deaminated
cytosine -> uracil -> UA - next round of replication
adenine -> hypoxanthine -> HC (in vitro reaction)

b. hydroxylamine - reacts with C –> converts to a base that pairs with A
c. EMS (ethyl methane sulfonate) (used w/eukaryotes)

Question 24

alkylating agent

Answer 24

react with G, sometimes T

• add alkyl group (alkane minus one hydrogen CH3CH2) to N-7 position of the purine ring
  1. G pairs with T in ionized form
  2. effects SOS repair system
  2. depurination occurs, another base inserted before repair mechanism can fix original loss

Question 25

3. UV light

Answer 25

thymine dimers - SOS repair system

Question 26

4. Intercalating agents

Answer 26

x. acridine orange, proflavine, acriflavine, EB (dyes) -planar, 3 rings, same size as base pair - intercalation causes frameshift mutations class=Section3> used in very low concentrations also prevent plasmids from replicating/partitioning (plasmid curing)

Question 27

Transposable Elements (mobile DNA segments)

Answer 27

100's-1000's bp in length -copies of Tn can be inserted at other locations on the genome one copy at original site with a new copy in another site. -some contain sequences that terminate transcription big problem in polycistronic MRNA -can find Tn when you induce mutation

Question 28

SITE SPECFIC MUTAGENESIS

Answer 28

1. Production of deletions with endonucleases (restriction) 2 cuts, remove piece of DNA and then reanneal 2. Point mutations at random sites in a particular region remove fragment as in #1 treat fragment with mutagenic agents reinsert into DNA 3. Point mutations at a particular base sequence a. synthetic piece of DNA is made - M13 method pg. 365, Fig. 15.9 b. dut ung method

Question 29

MUTATOR GENES

Answer 29

genes that in a mutant state cause mutations to occur in other genes actually gene function is to keep mutation rate low; defective product of mutator gene, then accompanying mutations occur

Question 30

MUTATOR GENES- mechanism

Answer 30

1. polymerase is defective - (altered), stops the editing function 2. dam gene for methylation of DNA, 3'-->5" editing function for mismatch repair if no methylation doesn't know which base to repair in which strand, daughter or parent. 3. enzyme altered that would carry out excision step (mismatch repair) 4. proteins changed that keep SOS turned off.

Question 31

Mutational Hot Spots

Answer 31

map location of mutants on the genome - certain sites altered with high frequency explanations (20-500 times more often)

Question 32

Reasons for Hot Spots

Answer 32

1. area that breathes more often 2. area accept base analogue or SOS repair 3. average in DNA - 1% prok., 5% euk. cytosines are methylated (MeC), when these are deaminated (usually spontaneously) --> 5-methyluracil or thymine

Question 33

____ doesn't occur w/ high frequency in areas where it affects a ___ _____

Answer 33

MeC, gene product

Question 34

REVERSION

Answer 34

(back mutation, reverse mutation) | result of spontaneous mutagenesis - random

Question 35

some mutants that don't revert at a detectable frequency | reasons:

Answer 35

1. double mutant (2 base sequences changed) | 2. deletion of many consecutive bases - replacement is very rare

Question 36

Intragenic Reversion

Answer 36

-inside the gene 1. Same site reversion - rare - true reversion 2. Second site or suppressor mutation reversion not at the 1st mutation site but by a mutation at a second site (reversion doesn't return to genotype, but does exhibit the same phenotype)

Question 37

Types of Reversion

Answer 37

a. Charge of AA | b. Size of AA

Question 38

Types of Intragenic Revertants:

Answer 38

1. wildtype - return to wt genotype 2. same site revertant 3. second site revertant

Question 39

Second site - frameshift reversion (shift back to right reading pattern)

Answer 39

Two requirements for this reversion: 1. reverting event - near mutation site 2. polypeptide chain area can be withstand the alterations (outside active site or regulatory sites)

Question 40

Suppression or Intergenic Reversion

Answer 40

change in second gene that eliminates or suppress first mutation...

Question 41

Information suppressors

Answer 41

- change cell's translational machinery - read mutation as "normal" a. mutations in tRNA genes b. mutations in ribosomal genes - usually these are unhealthy mutations

Question 42

Interaction suppressors

Answer 42

- two proteins that interact - mutation in second gene restores overall protein function - used to study protein interactions

Question 43

Overproduction suppressors

Answer 43

- mutant protein has lower activity (leaky mutation) | - mutation in regulatory gene or regulatory site on protein -> overproduction of mutant protein

Question 44

Bypass suppressors

Answer 44

-turn on a new pathway that eliminates need for mutant gene

Question 45

Physiological suppressors

Answer 45

-suppression by general changes in cell metabolism, mutations that increase conc of "chaperone" proteins (shock proteins)

Question 46

tRNA Suppressors

Answer 46

suppress original mutation & others as well. | -"suppressor-sensitive mutations"

Question 47

Nonsense or chain termination mutations

Answer 47

presence of a stop codon is not enough to stop protein synthesis a. altered tRNA - reads a stop codon as an AA codon

Question 48

Reasons tRNA terminator suppressors cells are viable

Answer 48

a. Protein factors are also involved in chain termination and will terminate even if tRNA is bound to stop codon - much of the time - get less of the protein. b. Often chain termination codons are in pairs - second terminator works.

Question 49

Missense Suppressors

Answer 49

(exchange one AA or another) a. mutant tRNA that can recognize 2 codons b. mutant tRNA incorrectly recognized by an aminoacyl synthetase c. mutant animoacyl synthetase that puts wrong AA on the tRNA

Question 50

Ames Test

Answer 50

test for mutagenesis/carcinogenic 1. His- reverting to His+ 2. microsomal fraction (isolated from a rat liver) converts some chemical to carcinogenic form