Mutations and Mutagens in Molecular Biology: Types, Effects, and Mechanisms

What induces mutations?

mutagens

List examples of mutagens

chemicals (ethylene bromide), UV (sunlight), viruses (human papilloma), transposons, and infidelity of DNA replication and repair (DNA polymerase makes mistakes)

Types of mutations

simple mutations: base substitution, base deletion, and base insertions.

sequence changes: allelic and non-allelic

(No DNA polymerase is involved)

Mutations can lead to disadvantages, neutral, or _______ to the effected

advantages.

-all of these lead to variable frequencies of different mutations seen in the human population.

The concept of the molecular clock

The mutations that bring neutral effects occur at a constant rate. The number of such mutations in the human population is therefore determined solely by time

Heritable mutations in a population must be

-in germ-line cell

-not lethal to a haploid cell

-Not imparing the reproductive capability of the affected

( ex: cancer is not really heritable/ genetic disease)

Simple mutations are mainly due to

DNA polymerase/ infidelity.

-Inherent error rate of DNA polymerase: 1/10^8 nucleotides leads to base substitution, 30 errors/ haploid genome.

-replication slippage-base deletions/insertions

replication slippage leads to

base deletion or base insertion

Effects of mutations in coding DNA

Silent (synonymous) mutations

nonsense

missense

Silent (synonymous) mutations

ACC to ACU is still threonine

-No change in peptides

-most frequent

Nonsense mutations

Codon becomes a stop codon which shortens the protein

-rare generates unstable mRNA or truncated peptide (small readable protein)

missense mutations

change in first or 3rd position

The original codon becomes a new codon

Frequencies of mutations as seen in humans are NOT random. Give examples

(1) The frequency of transitions is unexpectedly higher than that of transversions.

(2) The frequency of mutations in non-coding DNAs is higher than that in coding DNAs.

(3) The frequencies of mutations in different genes are different.

Nondegenerate site vs 2 and 4 fold degenerate sites

2 fold can be swapped out for 2 nucleotides and sequences won't change vs 4 fold can be swapped out with 4. Degenerate becasuet eh DNA is redundant.

More ________ than transversions exist

transitions

Transition vs Transversion

transition - substituting purine for purine, e.g. (transItion = Identical type)

transversion - substituting purine for pyrimidine, e.g. (transVersion = conVersion between types)

More mutations are seen in this kind of DNA

Non coding DNA

Variable Number Tandem Repeat examples

Microsatellite DNA

Minisatellite DNA

Alu sequences (large repeat sequences, not short)

Backward slippage causes

insertion

forward slippage causes

deletion

allelic sequence exchange

consists of equal and unequal crossing over

Basis for DNA fingerprint

mini-satellite because it's the most polymorphic and more variation

Unequal crossing over and unequal sister chromatid exchange lead to

insertions and deletions

gene conversion

The donor gene remains unchanged while the acceptor is changed by having some or all of its sequence replaced by a sequence copied from the donor.

Allelic vs nonallelic gene conversion

Allelic gene conversion happens between two different alleles of the same gene on homologous chromosomes, while non‑allelic (ectopic) gene conversion happens between similar sequences located at different genomic loci, often paralogs.

Mismatch repair

he DNA repair pathway that fixes base–base mismatches and insertion/deletion loops that escape DNA polymerase proofreading. It is one of the most important systems for maintaining genomic stability, and defects in MMR cause microsatellite instability

Constitutive gene expression

The expression of house-keeping genes is not affected by the tissue type, the embryo development, or enviornemental factors

Examples of housekeeping genes

ribosomal RNA, actin, b-tubulin, NA/K ATPase

Types of restrictive gene expression

-Spatial restriction

-temporal restriction

-environmental restriction

Spatial restriction example

organ tissue, and cell type dependent

Environmental vs temporal restriction

ER-The insulin gene is induced by blood glucose

TR-no expression at earlier embryo, orderly expression, cell cycle-dependent

Transcriptional regulation of gene expression

modulated by the binding of proteins to the regulatory region in nearby flanking or intronic sequences

Additional changes that are heritable but that do not depend on a change in the genome sequence are

epigenetic

overview of gene expression

transcription regulation, post-transcriptional regulation, and epigenetic mechanisms and long-range control of gene expression

RNA polymerase 1

rRNA gene, and uses UCE and CPE

Post-transcriptional regulation at the RNA level

Alternative splicing ( different isoforms and untranslated sequences) and RNA editing (changing codon in RNA level

RNA editing

involves enzyme-mediated insertion or deletion of nucleotides or substitution of single nucleotides at the RNA level. A to I RNA editing

-dADAR and RNA editing enzyme does C to U editing

-APOBEC1 a cytosine deaminase, is involved.

epigenetic inheritance not determined by DNA sequences

DNA methylation

Genomic imprinting

X chromosome inactivation

consequences of DNA methylation

leads to gene silencing either through interference with transcription factor binding or through the recruitment of repressors that bind sites containing methylated CpG islands.

Coding SNPs (mutations)

SNPs cause amino acid changes

Non-coding SNPs (silent mutations)

SNPs don't cause amino acid change

How to genotype SNP

-DNA sequencing

-allele specific PCR

-SNaP shot (single base sequencing)

-Restriction fragment length polymorphism

the 2 SNaP shot methods

homozygotes and heterozygotes

Pseudogenes

-similar to real genes in sequence and location

-multiple copies of pseudo genes

-notranscribable or nontranslatable

Minisatellite DNA

-up to 1000 repeats

-highly polymorphic

-great for DNA fingerprinting

Microsatellite DNA

1-4 bp repeats (sucah as a run of A)

Cell differentiation begins with stem cells.

Is cell differentiation reversible?

yes

What does beta-catenin. bicoid gene, and Hox genes do? give examples

(1) In Frog, Beta-catenin induces dorsal development.

(2) In fruit fly, the bicoid gene encodes an anterior morphogen

(3) Pattern formation is carried out by Hox genes which specify positions.

(4) Hedgehog signaling.

Development contains

(1) Cell divisions:

(2) Pattern formation:

-Body Plan, axes

-Germ layers: ectoderm, mesoderm, endoderm

(3) Morphogenesis:

Gastrulation is to form the gut.

(4) Cell Differentiation:

(5) Cell growth:

Tandem trinucleotide repeats can cause a variety of human diseases. List 2

1) Huntington's disease

2) Fragile X syndrome

Molecular pathology

seeks to explain why a given genetic change should result in

a particular clinical phenotype.

Mutational effects:

Amorph (null allele): no product or activity

Hypomorph: less product or activity

Hypermorph: more product or activity

Neomorph: novel product or activity

Antimorph: antagonizes the activity of the normal product

Epigenetic modification can abolish gene function without a DNA sequence change.

DNA methylation

-chromatin structure

-imprinting

-protein conformation (prions)

3. Most of genes are haplosufficient and only a few genes are not.

Gain of function mutations

1. Overexpression

2. Constitutive expression of a receptor protein

3. Chimeric gene.

4. Structural change of the protein

Gene mutations and clinical phenotypes: from gene to disease.

1. The phenotype depends on the level of gene product.

2. Mutations in the same gene will cause different diseases.

3. The same disease can be caused by mutations in several different genes.

4. Mutations in different members of a gene family can underlie a series of related

syndromes.

5. Microdeletion syndromes bridge the gap between single gene and chromosomal

syndromes.

Proteomics

the large-scale study of proteins, particularly their structures and functions.

The human mitochondrial genome structure

(1) It is small in size (16,569 bp), but numerous in copy number per cell.

(2) It is a circular double-stranded DNA. The heavy strand vs. the light strand.

Short VNTR vs long VNTR polymorphism (variable number of tandem repeat)

-Short VNTR polymorphism is caused mainly through the replication slippage.

- Long VNTR polymorphism is caused mainly through the unequal crossover which gives rise

to frequent insertions and deletions.

epigenetic modification leads to gene function loss. List examples

DNA methylation, chromatin structures, imprinting, and protein folding

a-Globin vs B-globin shows how some genes are mor susceptivle to mutations than others explain how

a-globin has a decrased change of a nonsynomyous mutation than b-globin. On the other hand, a-globin has a higher rate of synonmyous mutaion than b-globin.

Explain process of gene conversion

you have donor and acceptor DNA, followed by strand invasion and heteroduplex formation, then mismathc repair and synthesis of complimentary sequence.

(gene conversiono assumes donor is correct when that's not always the case and it happnes with 2 very similiar DNA.

Tri-nucleotide repeats in the promoter sequence leads to what syndrome?

fragile X syndrome because of mulitple CGG repeats

Mutations based on structural changes:

Mutation, Deletion, Insertion, and point mutations

Mutations based on phenotypic changes:

Amorph, Hypomorph, Hypomorph, Neomorph, and Antimorph

Many different changes to a gene can cause loss of function, explain them

-small dletions and insertions may cause a frameshift

-nonsense mutations trigger nonsense-mediated mRNA decay

-splicing mutations alter teh conserved GT....AG sequences at the ends of introns, splicng enhancers or silencers, and teha ctivatino of cryptic splice sites

the PAX3 gene is an example of _______ of function

loss

Differences between Duchenne or Bechker muscular dystrophy gene deletion

-Duchenne-severe deletion leads to frameshift

-Becker-mild in frame deletion, X-linked recessive and most patients are male

Most genes are haplosufficient true or false

true

Loss of function mutations are usually recessive because heterozygotes function normall with a few exceptions:

-Haploinsufficiency-you need both alleles for a normal product

-Domiant negative effects-mutant polypeptide not only loses its own function but also interferes with the product of the normal allele in a heterozygote

mutations in protein mulitmers lead to dominant negative effect which means:

the mutant interferes with normal protein to make normal product

Mutations in the same gene may cause different diseases, True or False?

True

Mutations in different genes may not cause the same disease

False, mutations in deifferent genes may cause the saem disease

Microdeltion syndromes brdige the gap between

single gene and chromosoamle syndromes

Mutations in members of a gene family cause related ________. For example, mutations in Fibroblast growth factor receptor 1,2,and 3 lead to bone or cartilage disorders/

syndromes

The 2 types of transcriptome sequencing

Open system: sequencing-based and involves the sampling cDNA population

close system: hybridization-based and involves the use of DNA microarray.

define Open system (for analysis of gene expression)

-random sampling of cDNA libraries

-Analysis of EST databases

-differntial display PCR

-Serial analysis of gene expression (SAGE)

Examples of serial analysis of gene expression (SAGE)

1. Isolate mRNA

2. Convert to cDNA

3. Cut with restriction enzyme

4. Add adapters + cut again

5. Link tags together (ditags → concatamers)

6. Clone and sequence

7. Count tags

Spotted DNA microarray

A microarray made by physically spotting (printing) known DNA fragments onto a glass slide. cDNA from samples is fluorescently labeled and hybridized to the spots. The intensity of fluorescence at each spot reflects gene expression levels. Typically compares two samples (e.g., healthy vs diseased) using different colors.

High-density oligonucleotide array

A microarray made using in situ synthesis to create thousands-millions of short, known DNA sequences (oligonucleotides) directly on a chip. Labeled sample RNA/cDNA hybridizes to these probes, and signal intensity is measured to determine gene expression. Offers higher precision, sensitivity, and genome-wide coverage compared to spotted arrays.

Reasons Mendel chose the pea plant

-easy to grow

-easy to observe

-grow to maturity in a single season

Genotype

the specific genetic genome of an individual in the form of DNA. It codes for the phenotype of that individual.

phenotype

is iether its total physcial appearance and consititution or a specific manifestation fo a trai suchs as eye color, size, or behaviour taht varies between individuals.

locus

fixed postion on a chromosome, such as the position of a gene

Mendel's postulates

unit factors exist in pairs that determine trait

-when these pairs are not same, one is doiminant the other is recessive

when germ cells are formed paired unit factors segregate randomly

unit factors assort independently when germ cells fuse

Mendelian Character

determined by a gentoype at a single locus (about 10,000)

-dominance and recessiveness in mendelian inheritance are propertines of phenotypes not the ones of genotypes.

Huntingtons disease is categorized as this kind of genetic disease

autosomal dominant

Basic Mendelian Pedigree Inheritance Patterns

Autosomal dominant and recessive

Y linked

X linked recessive and dominant

Autosomal dominant

each generation has effected individual

-either sex may be affected

transmitted by either sex

affected parent

wooly hair and achondroplasia are passed by what kind of inheritance

autosomal dominant

Autosomal recessive

either sex may be affected

-transmitted by either sex

unaffected parents

typical to see 1 generation with no individual effected definitley recessive

albinism is what kind of genetic inheritance

autosomal recessive

X linked dominant

more affected females than affected males

transmitted by either sex

Faulty enamel is an exmaple of

X linked domiannt

X linked recessive

affectws mainly males

-trasmitted by unaffected mothers who are carriers

if maternal grandfather effected so will maternal grandson

Hemophilia and Testicular feminazation syndrome is an example of

X linked recessive

Y linked

only males are affected

-transmitted by father to all sons

Defintion of Possible distortions

common recessive traits may appear dominant----pseduodominance

-Age dependence

Possible distortions examples

non-penetrance-hapsburg jaws

sex-dependence-pattern baldness

in males both homo and hetero will leads to baldness vs in females only homozygous

-autosomal dominance for males vs autosomal recessivesness for females

-genetic imporinting-only genes form mother of father expressed from gene methylation

-novel mutation-cystic fibrosis and sickle cell anemia

-fragil x syndrome and genetic anticipation symptoms become sever over generations and age

the genome in biology is equivalent to

the periodic table in chemistry

all life forms may be reduced to genes on chromosomes

critics said

big biology is bad biology

why sequence junk DNA sequences

impossible to do