1.2.2 Types of Mutants and Mutagens

What are the two types of mutants that are based on conditions in which the mutant phenotype is expressed

1. Non Conditional Mutant - Displays mutant phenotype under all conditions, regardless of the environmental condition

2. Conditional Mutant - - does not always show mutant phenotype, Behavior depends on environmental conditions or presence of other mutants

What are the two types of conditional mutants

1. Temperature-sensitive mutants

2. Suppressor-sensitive mutant

Determine what kind of mutant are a Mutant E.coli that can ONLY grow at 33 degrees celsius, and why is this classified as a mutant

Temperature-sensitive mutants

→ Wild type E. coli grows at 20 to 43 degrees Celsius

these types of mutants have effects that are lethal in a bacterium without such suppressor mechanism

Suppressor-sensitive mutant

What kind of mutation is represented having a Bacteriophage mutant that produces nonsense codons and can replicate only in a host bacterium able to translate the nonsense codon

Suppressor-sensitive mutant

what are the different mutants based on the changes that have occured in the genetic material

1. base substitution or point mutation

2. frame shift mutation

3. Deletion

4. Insertion

This type of Base substitution is the change to the same chemical type of base (purine to purine, and pyrimidine to pyrimidine)

Transition

This type of Base substitution is the change to the other type of base ( purine to pyrimidine, and pyrimidine to purine )

Transversion

What are the different consequences of base pair changes

1. Missense

2. Nonsense

3. Silent

These are Mutation that changes a codon for one amino acid to a codon for another amino acid. Results in an amino acid substitution in the protein product

Missense

Mutation that changes a codon for an amino acid with a codon for chain termination

Nonsense

Change in codon composition that has no effect on the resulting polypeptide due to codon degeneracy ( does not affect the function but affects mRNA stability or translation efficacy)

Silent

how does silent mutations occur

strand slippage and mispairing during DNA replication

What kind of mutation occurs when a mutation is a region of the DNA has been eliminated

Deletion

Effects of Big and Small deletion mutations

Small deletions - frame shift mutation

Big Deletion - loss of genetic material/ chromosome causing severe effects

Deletions or insertions are caused by what events

1. strand slippage for short regions

2. homologous recombination for long sequences

This type of mutation occurs when new bases are added to the DNA

Insertion

his type of mutation results to elimination of a region of the DNA.

Deletion

This type of mutation results to the genetic code being read out of phase

Frameshift mutation

This type of mutation adds or deletes one or two bases (or any non-multiple of three) from a coding sequence in DNA

Frameshift mutation

true or false: If the insertion or deletion is in multiples of three, the reading frame is preserved. This type of mutation is called an in-frame insertion or in-frame deletion. fect: the resulting protein will gain or lose one or more amino acids, but the rest of the sequence downstream stays the same (no frameshift).

true

between in frame mutations and frameshift mutations, which is more detrimental

frameshift mutations are usually more detrimental, Because they change the reading frame, every codon downstream is altered. This can often introduces a premature stop codon, truncating the protein and usually making it nonfunctional.

What type of mutation occurs when thymine becomes cytosine?

Transition

What type of mutation occurs when cytosine becomes adenine?

Transversion

What type of mutation occurs when guanine becomes thymine?

Transversion

What type of mutation occurs when adenine becomes guanine?

Transition

When a new strand is added, forming insertion mutations, how does the frame move? How about when there is looping out?

+1, frame shift forward

looping out of template = deletion, frame shift backward

looping out of the template strand causes, houw about the daughter strand

insertion, deletion

what are the effects of small and big insertions

small - frameshift mutations

big - introduction of new dna segment or duplication of genes or transposons

Their occurrence is most often not related to any adaptive change it may confer on an organism in its environment

Mutation

Also known as nutritional mutants.

Auxotrophs

What is a probable cause of spontaneous mutation that occurs during the normal vital processes that directly implicate the genetic material?

Malfunctioning of natural processes

Aside from malfunctioning of natural processes, what is another probable cause of spontaneous mutation?

Mobility of transposon elements

When there is a spontaneous structural alteration of nucleotide bases, a/an _______ form of thymine can pair with guanine.

Enol

When there is a spontaneous structural alteration of nucleotide bases, a rare imino form of _______ can pair with cytosine.

When there is a spontaneous structural alteration of nucleotide bases, a rare imino form of _______ can pair with adenine.

When there is a spontaneous structural alteration of nucleotide bases, a/an _______ form of guanine can pair with thymine.

adenine

cytosine

enol

one of the mechanisms to how an organism maintain its stability despite changes in their DNA via having mutations to regain its characteristics

Reversion or Reverse mutation

The organism that has reverted is called a

Revertant

what are the two types of reversion and their differences

a. True reversion - restores the wild type sequences, a back mutation

b. pseudoreversion - restores the wild type phenotype by compensating gene sequences change through a second mutationn

what kind of mutations falls under true reversion, and how does this back mutation occur

only insertion mutations

back mutation occurs via the Precise deletion (“excision”) of the added DNA sequence

true or false: deletion mutation cannot be restored by true reversion, and why

true, because the DNA is completely lost. At best, a suppressor mutation can compensate its functionality. But the original sequences are forever gone

this kind of mutation Restores the wild-type phenotype by a compensating gene sequence change (a second mutation)

pseudoreversion

what are the possible locations where pseudoreversion can occur

1. same gene as the original forward mutation ( Intragenic Suppressor )

2. different gene or sequence ( Intergenic suppressor )

this is described to be a Second mutation within the same gene that restores function of the mutant gene product ( same gene as the original mutation )

Intragenic suppressor

what are the possible mutational phenomena that can occur under intragenic suppressors

1. Base substitution mutations

   a. same codon = same nucleotide ( same chemical properties )

   b. same codon = different nucleotide ( entirely different amino acid )

2. Frameshift mutations

true or false: the flexibility of the genetic code→ protein can sometimes rescue the function

true

what is the phenomena describing as another site in the same gene would compensate for the mutation such as frameshift mutations. allow the protein to fold or function again/ The sequence made is not the same as the wild type, but the protein structure activity is restored, restoring downstream processes

intragenic suppressor

this kind of psuedomutation does not fix the genes but reorganizes cellular processes to compensate for the mutation

Intergenic suppressor

A second, compensating mutation in a different gene

Intergenic suppressor

what are the different types or phenomena associated with Intergenic suppressor

1. informational suppressor

2. interaction suppressor

3. overproduction suppressor

4. bypass suppressor

5. physiological suppressor

this is a type of intergenic suppressor where A second mutation that restores function of a primary mutation by altering the way the gene is expressed occurs

Informational suppressor

what kind of intergenic suppressor is described in the situation : Mutations that alter a tRNA allowing it to misread a nonsense (stop) codon

Informational suppressor, specifically nonsense suppressor tRNA - the tRNA changes its anti-codon that it would insert and amino acid to restore its partial function

what kind of intergenic suppressor is described in the situation : Mutations that alter a tRNA allowing it to misread a codon through frameshift mutation

Informational suppressor, specifically frameshift suppressor tRNA - allows reading of 4 bases instead of three, compensating for the frameshift mutation

what kind of intergenic suppressor is described in the situation : Mutations in ribosomal genes that affect the fidelity of translation

Informational suppressor, specifically ribosome fidelity mutations → allows the ribosome to tolerate mismatches caused by mutations

what kind of intergenic suppressor is described in the situation : Affects translation of mutant mRNA

Informational suppressor

what kind of intergenic suppressor is described to Restores the functional association of two residues in different gene products. Either two different polypeptides, two different nucleic acids molecules, or a polypeptide and a nucleic acid molecule

Interaction suppressor

what kind of intergenic suppressor is described to have mutations in one subunit of a protein complex, causing destabilization, but a mutation in another subunit restores its stability

Interaction suppressor

what kind of intergenic suppressor is described to Increases amount of defective gene product to provide enough residual activity to restore function

Overproduction suppressor

what kind of intergenic suppressor is described to activate a new pathway to replace a blocked metabolic pathway

Bypass suppressor

what kind of intergenic suppressor is described to modify the environment (ph, temp etc), to allows mutant protein to function

Physiological suppressor

this is the process of generating genetic mutants that are heritable - the source of variation

mutagenesis

the physical or chemical agents that change the genetic material

mutagens

what are the two types of mutagens and their examples

1. Physical mutagens

   a. Electromagnetic radiation

   b. particulate radiations

2. chemical mutagens

   a. deaminating agents

   b. base analogs

   c. alkylating agents

   d. intercalating agents

3. PCR site directed mutagenesis

Physical mutagens include high energy radiations that penetrate living cells that damages cells directly or indirectly through the formation of ____

free radicals

true or false: The effect of radiation exposure is directly proportional to the degree of penetration of the radiation

true

What are the two type of electromagnetic radiations that can cause mutations to the DNA

1. Ionizing

   a. X-rays

   b. Gamma Rays

2. Non-ionizing radiations

   a. UV rays

what are the differences in effect of Ionizing and non ionizing radiations in the dna

Ionizing - causes breakage in the phosphodiester bond in the DNA ( leads to chromosomal rearrangements, deletions or translocations )

Non-ionizing - causes pyrimidine dimers, causing replication errors

indirect of effect of both

Formation of free radicals or Reactive Oxygen Species

energy in electromagnetic spectrum is carried through what particle

Photons

true or false: the shorter the wavelength, the higher the frequency, the higher energy, and more mutagenic like x-rays and gamma rays

true

true or false: longer wavelengths has little mutagenetic effects to no mutagenic effects

true

what are the effects of the following wavelengths to the body

radiowaves, microwave, infrared, visible light, UV, x-rays, and gamma rays

radiowaves - no mutagenic effects

microwave - tissue heating

infrared - heat effect

visibile light - non - mutagenic

UV- causes pyrimidine dimers and can cause caner

x-rays - dna strands break

gamma rays - high penetration, high severity dna and tissue damage

what is the correlation or mechanism of high energy with the ability to disrupt the dna

high energy radiation can remove tightly bonded electrons from the atoms, creationg ions and free radicals

why is it that ionizing radiations have detrimental effects to the organisms

because the result is a single or double strand breaks in the DNA molecules. These breaks are hard to repair because they don’t often leave a phosphate tacked 9attached) onto the 3’OH where the break occurs

true or false: Penetration power is inversely proportional to their wavelength

true

these are high energy particles or subatomic particles released during radioactive decay the can cause damage to the dna

particulate radiations

what are the different types of particulate radiations

1. alpha particles

2. beta particles

3. neutrons

In terms of penetrating power, which is more penetrant, beta or alpha

In terms of penetrating power, beta particles > alpha

particles due to smaller size

this sub atomic particles are heavy, less penetrating and cause dense ionization along short tracks

a-particles

this sub atomic particle is lighter, more penetrating the alpha particles

b-particles

this sub-atomic particles are highly penetrating, and colludes with nuclei inside the cells, causing breaks and severe ionization. This results to DNA fragmentation, base loss or chromosomal breakage

neutrons

what is the indirect effect of ionizinf radiation and it mechanism of action

formation of free radicals

MOA: Radiolysis of water - radiation interacts with water molecules inside the cells, generating reactive oxygen species. The unpaired electrons here combine with oxygen

this is the most destructive reactive oxygen species , which are also the primary culprits of oxidative DNA damage

HYDROXIDE group

the most biologically significant non-ionizing mutagen

Ultraviolet light

what are the different UV lights and their effects to the organisms

UV-A - longest wavelength and has the lowest energy, can cause ROS

UV-B - most mutagenic, can cause pyrimidine dimers, main cause of sunburn and skin cancers

UV-C - has the highest energy, strongly absorbed by the DNA, extremely mutagenic, but is absorbed by the ozone layer

what is the MOA of UV rays

these has lower energy than ionizing radiations thus cannot eject electrons from the atoms BY can excite electrons, leading to chemical changes in the molecules. Its direct absorption in the DNA, specifically by C and T forms covalent bonds, forming dimers, blocking replication and transcription

these are chemical substances that alters the DNA structure leading to mutations, it modified the bases by inserting between them or substitution them

chemical mutagens

this type of chemical mutagen causes the loss of an amino group leading to different bases ( causing transition mutations )

deamination agents

what are some examples of deaminating agents

nitric oxide, nitrous acid, N-nitrosoindoles

memorize the effects of deaminating agents in the n-bases

okay

these chemical mutagens Structurally resemble purines and pyrimidines and maybe incorporated into DNA in place of normal bases during DNA Replication, causing mispairing or base pair substitution

base analogs

true or false: base analogs are said to be More prone to tautomeric shifts than the natural bases, thus Results in higher frequency of mutation

true

Base analogs may exist in two alternative forms:

keto or enol form

these chemical mutagens are the ones that donate alkyl groups (methyl, ethyl) to other

molecule. These are highly mutagenic, carcinogenic, and cytotoxic, and causes damage to the DNA backbone

alkylating agents

MOA for alkylating agents

Alkylated base may then degrade to yield a baseless site ( DEPURINATION )

different types of alkylating agents and their mechanisms

methylmethane sulphonate (MMS) - similar to EMS

ethyl methane sulfonate (EMS)

N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) - potent alkylating agent that methylates guanine at the O6 position causing mispairing with thymine to cause mutations

mustard gas - chemical warfare agent that crosslinks dna

Nitrosammone - from tobaccos

cyclophosphamide busulfan - anti cancer drugs that can kill rapidly dividing cells by DNA cross linkage

these are flat aromatic planar molecules that may insert between bases in DNA, increasing the distance between the base pairs, disrupts the dna structure, interfering with replication and transcription by inserting or deleting a base, opposite to the added intercalating molecule, leading to frameshift mutations

intercalating agents

examples of intercalating agents

acridine derivatives (acridine orange, proflavine,

acriflavine), nitrogen mustards, ethidium bromide
ICR-191

what are the disadvantages of creating mutations through the traditional method (physical and chemical mutagens)

1. mutations are random and uncontrolled

2. requires screening for large population of mutants to find the desired one

3. often induces multiple mutations

4. time consuming

process of pcr site directed mutagenesis

General Steps in Site-Directed Mutagenesis

1. Design primers with the desired mutation

   • Primers are synthesized so that they carry the intended base change(s).

2. Amplify the plasmid (or DNA template) with these mutagenic primers

   • PCR is performed, and the primers introduce the mutation into the newly synthesized DNA strands.

3. Mutation incorporated into amplified DNA

   • The PCR products now contain the desired mutation within the plasmid sequence.

4. Digest the parental (template) DNA with DpnI

   • DpnI specifically cleaves methylated or hemimethylated DNA (the original parental plasmid), leaving only the newly synthesized, unmethylated mutant DNA intact.

5. Transform the mutated DNA into host cells (e.g., E. coli)

   • The host cells propagate the mutant plasmid, allowing colonies carrying the mutation to be recovered and analyzed.

specifically cleaves methylated or hemimethylated DNA (the original parental plasmid), leaving only the newly synthesized, unmethylated mutant DNA intact.

DpnI

inverse pcr steps

• Restriction digestion of vector – The plasmid is cleaved by a restriction enzyme at a single site near the mutation site, generating a linear vector.

• Primer design – Two primers anneal to the ends of the linearized plasmid; one primer carries the desired mutation.

• Amplification – PCR amplifies across the linearized plasmid, introducing the mutation.

• Ligation – The amplified linear DNA must be ligated to regenerate a circular plasmid.

• Transformation – The circularized mutant plasmid is transformed into E. coli for propagation.