Molecular Bio Lecs 1-3

What is the definition of a mutation?

An alteration in DNA sequence such as:

• Any base-pair change in sequence

• Single base-pair substitution

• Deletion or intersection of base pairs

• Major alteration in chromosomal structure

Mutations occur in?

Somatic or germ cells, coding or noncoding regions

What are mutations the source and useful for?

• Source for many disease and disorders

• Source of all genetic variation, which further provides the raw material for evolution

• Useful for probing fundamental biological processes

What is a point mutation?

A mutation that involves a single base-pair substitution

In molecular biology, what is a transition mutation?

A base substitution where a purine replaces a purine or a pyrimidine replaces a pyrimidine.

What is a transversion mutation?

A base substitution where a purine is interchanged with a pyrimidine.

According to the provided mnemonic, which three bases are classified as pyrimidines?

Cytosine, Uracil, and Thymine

What is missense mutation?

A point mutation that results in new triplet code for different amino acid and there is a change in amino acid sequence

What is a nonsense mutation?

A mutation that changes a sense codon into a stop codon, causing premature termination of translation.

What is a silent mutation?

A mutation that changes a codon into a synonymous codon without changing the amino acid sequence

What is sickle-cell disease?

A genetic disorder that strikes individuals with two copies of the sickle-cell allele which affects the structure and function of hemoglobin, reducing the oxygen carrying capacity of RBCs

What is the difference between homozygous and heterozygous sickle-cell allele?

Homo: develops sickle-cell disease and often life-threatening

Hetero: develop few harmful effects, but are more likely to survive malaria than homozygous

Why certain populations have a higher prevalence of the sickle cell allele, despite its harmful effects in homozygous individuals?

Because individuals with one copy of the allele have increased resistance to malaria, giving them a survival advantage in regions where malaria is common even though having two copies causes sickle cell disease.

What is frameshift mutations?

Results from the insertion or deletion of nucleotide that shifts the triplet reading frame

Why are frameshift mutations often highly deleterious to protein function?

They alter the entire amino acid sequence following the mutation site.

What is a forward mutation?

A mutation that changes a wild-type phenotype into a mutant phenotype

What is a reverse mutation?

A mutation that changes a mutant phenotype back into the wild-type phenotype.

What is a suppressor mutation?

A second mutation at a different site that hides or suppresses the effect of an original mutation.

How does an intragenic suppressor mutation differ from an intergenic suppressor mutation?

Intragenic A mutation within the same gene as the original mutation that restores or partially restores the wild-type phenotype

intergenic: A mutation in a different gene that reverses or compensates for the effect of a previous mutation, restoring the wild-type phenotype

How do suppressor mutations affect phenotype?

Suppressor mutations lessen or cancel the effects of an original mutation, restoring the phenotype closer to normal.

What defines a neutral mutation?

A mutation that does not affect an organism's fitness or phenotype, which occurs in noncoding region and sometimes in protein-coding region as well

What mutation is this “A codon changes from GAA → GAG”?

Neutral mutation: Both codons code for glutamic acid, so the amino acid sequence does not change and the protein functions normally

What is a lethal mutation?

A mutation that interrupts an essential biological process and results in death

What are lethal conditional mutations?

Mutations that manifest their phenotype only under specific environmental conditions, like temperature

What type of mutation is sickle cell anemia caused by?

Point mutation in the hemoglobin-beta (HBB) gene

What is dominant-negative (DN) mutation?

A mutation where a mutant polypeptide disrupts the activity of the wild-type gene when overexpressed

What makes a mutation dominant?

When only one copy of the gene needs to be mutated to cause a disease phenotype

What makes a mutation negative?

When expression of a mutant protein interferes with the acitivity of a wild-type protein

In which cell type do somatic mutations occur?

Any cell except germ cells (nonreproductive cells) and ARE NOT HERITABLE

What is a germ-line mutation?

A mutation occurring in gametes that can be inherited by offspring.

Human somatic cells are diploid (2n). What is the total number of chromosomes in these cells?

46 because 2(23) = 46

How many sets of chromosomes are found in a human haploid cell (n)?

One set of 23 chromosomes

In humans, how many pairs of chromosomes are autosomes? How many pairs of sex chromosomes?

22 pairs

1 pair of sex chromosomes

Somatic cells vs Sex/Germ cells

Somatic: the body cells that make up tissues and organs

Sex/Germ cells: the reproductive cells

What are homologous chromosomes?

A pair of chromosomes (one maternal, one paternal) with the same length, centromere position, and gene loci

True or False: Different alleles for a particular gene occupy the same locus on homologous chromosomes

True, genes exist in different versions (alleles)

What is the difference between spontaneous and induced mutations?

Spontaneous: A mutation that occurs naturally during DNA replication or cell processes, without any outside influence

Induced: A mutation caused by external factors (mutagens) like chemicals, radiation, or viruses

Autosomal mutations vs X- and Y- linked mutations

Autosomal mutations: Occur within genes located on autosomes

X- and Y- linked mutations: Occur within genes located on X and Y chromosome

What is the function of proofreading by DNA polymerases?

To fix mis-paired bases during the process of DNA synthesis/replication by DNA polymerases

What occurs right after DNA replication?

Repair, which detects and corrects damage throughout the cell cycle

What are tautomers in the context of DNA bases?

Molecules with the same formula but different connectivity that allow noncomplementary base pairing and increases the chances of mispairing during DNA replication

Tautomeric shifts typically result in?

Transition mutation in the complementary strand

What is depurination? What is deamination?

Depurination: The spontaneous loss of a purine base (Adenine or Guanine) from DNA, leading to an apurinic site (without purine)

Deamination: The loss of an amino group from a DNA base, most frequently affecting Cytosine.

True or False: The spontaneous depurination and deamination reactions breaks the phosphodiester backbone of DNA

False, it does not! It just creates serious DNA damage in cells

What does the machinery do after depurination occurs?

• During replication, the machinery may skip the missing purine, producing a daughter strand missing one nucleotide pair.

• Alternatively, it may insert an incorrect base, usually adenine (A), across from the missing purine, causing a mutation

What does the machinery do after deamination of cytosine occurs?

Inserts an adenine when it encounters a uracil on the template strand, resulting in a C → T transition after two replication cycles and if uncorrected causes a base substitution mutation

Explain the steps of deamination and what occurs after?

• Converts cytosine → uracil, which pairs with adenine instead of guanine.

• During replication, the machinery inserts an A opposite uracil.

• After two replication cycles, this results in a C → T transition.

• If uncorrected, it causes a base substitution mutation.

What is a null mutation?

A mutation that results in the complete loss of function of a gene product.

What are intercalating agents?

Chemical compounds with flat (planar) structures that insert, or “wedge”, between adjacent DNA base pairs and disrupt the structure of the double helix

How do free radicals directly/indirectly affect DNA?

• Alter purines and pyrimidines

• Break phosphodiester bonds

• Produce deletions, translocations, and fragmentation

What are free radicals?

Highly reactive, unstable molecules that steal electrons from other molecules and cause damage to other molecules, such as DNA, lipids, and proteins

What is a nutritional (biochemical) mutation?

A mutation that converts a prototroph into an auxotroph

In a pedigree analysis, if a trait appears equally in males and females and affected people have at least one affected parent, it is likely _____.

Autosomal dominant

What is the primary characteristic of a gain-of-function mutation?

The appearance of a new trait or function, or expression in an inappropriate tissue or time.

Alkylating agents induce mutations by donating a(n) _____ group to DNA bases.

Alkyl

Which chemical mutagen is specifically used to add a hydroxyl group to DNA bases?

Hydroxylamine

Intercalating agents cause DNA damage by inducing base-pair distortions and DNA _____.

Unwinding

In the electromagnetic spectrum, as the wavelength of radiation decreases, its energy _____.

Increases

What creates pyrimidine dimers?

UV radiation

A thymine dimer is made up of?

Two adjacent thymine bases (on the same strands) have become covalently attached to each other

Why are thymine dimers particularly hazardous during DNA replication?

They distort DNA conformation and cause the replication machinery to stall.

How does ionizing radiation typically interact with tissues compared to UV light?

It penetrates deeply into tissues.

What is the Ames Test?

• A lab test used to see if a chemical can cause mutations in DNA.

• It uses bacteria like Salmonella or E. coli

What is a revertant (reverse mutation) in the Ames test?

A revertant is a bacterium whose original mutation is reversed, restoring its ability to make the needed amino acid and grow.

What is an auxotroph?

A mutant microorganism or cell line that through mutation, has lost the ability to synthesize one or more substances required for growth

What is a minimal medium?

A medium containing only the essential nutrients needed to support the growth and reproduction of wild-type strains of an organism

What are the characteristics of essential nutrients?

• Required for normal physiological function

• Cannot be synthesized by the body

• Must be obtained from the diet

What is a complete medium?

A minimal medium plus a full range of amino acids, vitamins, etc

When can an auxotrophic bacterium grow on minimal medium?

An auxotrophic bacterium cannot grow on minimal medium unless it is reverted back to the wild type or it is given something that restores the function of the unsynthesizable element

Why is rat liver extract included in the Ames Test procedure?

It helps determine whether a substance is metabolized into a more or less mutagenic form and stimulates in vivo metabolism

In an Ames Test, what does a significantly higher colony count on the treatment plate compared to the control plate indicate?

A positive result, which indicates that the chemical is mutagenic and therefore may act as a carcinogen

If a chemical is only mutagenic in the Ames Test after being treated with liver enzymes, it is considered a(n) _____ mutagen.

Indirect (or metabolically activated)

Which specific oxidative species is a by-product of normal cellular processes and can damage DNA, lipids, and proteins?

Free radicals (or Reactive Oxygen Species).

What is the purpose of the control plate in an Ames Test experiment?

To determine the baseline rate of spontaneous reverse mutations.

Base Analogs

Chemical mutagens with structures similar to normal nitrogenous bases that can be incorporated into DNA during replication.

In the context of the Ames Test, what does the term 'revertant' refer to?

A bacteria that has mutated back to a wild-type state (His+) and can grow on minimal medium.

What is the primary site of damage when skin cells are exposed to sunlight?

DNA (specifically the formation of thymine dimers).

What is the term for a wild-type strain that can grow on minimal medium without additional nutrient supplements?

Prototroph (implied as opposite of auxotroph).

What is the functional result of liver enzymes 'detoxifying' a compound in the context of the Ames Test?

The compound becomes less mutagenic.