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What is a mutation?

A change in the DNA sequence that can be transmitted

Germline Somatic
Inherited Acquired
mutations mutations
Mutations and biotechnology
- Generating mutants - traditionally
selective breeding
inducing mutations (x-rays, chemicals)

- Generating mutants biotechnology


Adding genes with positive traits (transgenic technology;
gene therapy)
Gene editing techniques (CRISPR)
Deleting portions of genes (+ and effects)
Modifying (changing 1 to several bases)
Creating new functions fusing two genes
Tagging genes (e.g. with GFP); biosensors; track proteins
or cells.
Biotechnology and mutation
- Use of mutants to understand life (medicine, cell bio, dev bio,
evolution, biochemistry....)
- How scientists use mutants and genetics -
Break things and figure out how stuff works by looking at what
happens next
- Examples of biotech discoveries involving mutants:
- Medical biotech (Mapping disease, antibiotic resistance, drug
discovery and bio-pharma)
- Microbial biotech (cloning and editing genes, antibiotic
resistance, DNA as hereditary material)
- Plant biotech (GMO plants with new/desirable traits)
- Bioremediation (mutant plants and microbes)
DNA Mutations
Induced or Spontaneous

. PHYSICAL
Radiation: extra energy changes stability
UV light
, , rays (think Chernobyl)
X-rays
Example: UV light causes Cs and Ts to dimerize
. CHEMICAL
Free radicals, tar, asbestos, charred steaks
. CHANCE
This chance is increased over multiple cycles of replication.
Cycles of replication are triggered by several causes
growth, repair, replenish (and some tissues are constantly
regenerating, like the G.I. epithelium)
Mutations: Causes and Consequences
Mutation change in the nucleotide sequence of DNA
Major cause of genetic diversity
Can also be detrimental
Types of Mutations
Point/Substitution mutations
Silent mutations
Missense mutations
Nonsense mutations
Frameshift mutations
Insertion or deletion mutations
viruses,
Transposable elements (jumping genes),
chromosomal translocations
Mutations: Types
Proofreading
Point mutation
Chromosomal mutations
Repair of DNA errors
Error rate of 1 for every 105 bases
replicated.
If uncorrected, ~60,000 mutations each
division
At least three mechanisms of repair
Proofreading
Mismatch repair
Excision repair
What Do Mutations Do?
DNA codes for proteins via transcription and
translation
A mutated mRNA transcript provides faulty
directions for protein formation
Different order of bases = different codons
As a result, the order or length of the amino
acid sequence may be changed
This affects protein SHAPE, and hence, its
function (remember?)
Mutations in non-translated RNA are also very
dangerous. Think, telomers, tRNA, rRNA
Mutations: Consequences

Mutations are a major cause of genetic diversity


Human genomes are approximately 99.9% identical
0.1% differences in DNA between individuals, or one base
out of every thousand
Roughly 3 million differences between different individuals
Majority of these occur as SNPs and the others are in the form
of deletions and repetitions
Most have no obvious effects; other mutations strongly
influence cell functions, behavior, and susceptibility to
genetic diseases
Control of Gene Expression
Gene expression refers to the production of a
functional gene product (RNA or protein)
All cells of an organism contain the same genome,
so how and why are skin cells different from brain
cells or liver cells?
Cells regulate the genes they express
Gene regulation is how genes are turned on or off in
response to different signals
Cells maintain their identity following cell division
Regulation of Gene Expression
Points of Control
Control of gene expression can occur at any step in the
pathway from a gene to the functional protein.
Unpacking DNA
Transcription (promoters, activating elements,
transcription factors)
Post-transcriptional modifications (transport out of the
nucleus, access to ribosomes, protection against
degradation)
Translation
Post-translational modifications
Protein degradation
DNA

Chromatin Structure
double
helix
(2-nm
diameter)

Histones
DNA is packed, coiled, around Beads on
a string
histones, a kind of protein.

Epigenetic Regulation Nucleosome


(10-nm diameter)
- Based on winding/unwinding of DNA
- Changes access to RNA polymerase
No change in DNA sequence
Methylation of DNA and histones
Acetylation of histones
Tight helical fiber
(30-nm diameter) Supercoil
(200-nm diameter)

700
nm

Metaphase chromosome
Copyright 2003 Pearson Education, Inc. publishing as
Benjamin Cummings
Epigenetic Modifications
Methylation:
-CH3

Heterochromatin
Tight association
between histones
and DNA

Acetylation:
- CH2CO
Euchromatin
Loose association
between histones
and DNA
Transcriptional regulation
Controlling the amount of mRNA transcribed from
a particular gene
Conserved sequences in the promoter region affect the
affinity of RNA polymerase
TATA box at -35 (TATAAT - pribnow box in procaryotes)
CAAT box at -80
RNA polymerase cannot bind to promoter region without
presence of transcription factors
Enhancer sequences bind to regulatory regions called
activators
Upstream Activating Sequences are 50 bp or more
upstream or even downstream
Repressors feed back inhibition
Transcriptional Regulation
Transcription factors are themselves
proteins
So what regulates their expression?

1. Feedback loops
the presence or absence of the product the protein acts
upon may be a switch

2. Signals from other cells


hormones or other signals may switch on gene expression,
including that of transcription factors
RNA and Protein Synthesis
Micro RNA (miRNA) - 20-25 bases regulate gene expression by
silencing gene expression through blocking translation of mRNA
or by causing degradation of mRNA
Small interfering RNA (siRNA) dsRNA molecules, typically
exogenous, 20-26 nucleotide long, originally discovered in plants
undergoing transgene silencing
Antisense RNA ssRNA complimentary to mRNA
RNAi RNA interference and prevention of mRNA to be
translated
RISC RNA induced Silencing Complex, which is a close
association of RNA and Dicer enzyme (endoribonuclease)
that degrades mRNA, preventing it from being translated
and hence effectively silenced
RNA interference

Rich Jorgenesen (1986) Thought adding additional genes for


purple coloration will make the purple petunias more intense
Surprised by the appearance of regions of hypopigmentation
Explained the phenomenon as co-suppresion

Later the phenomenon was explained in C. elegans by Fire and


Mello (1998)
Showed that exogenous addition of dsRNA interfered with the
expression of the complimentary mRNA
won the Nobel Prize in 2006
Gene Expression and Biotechnology
Flavr Savr tomato (1994) Non-browning Arctic Apples (2015)
- Antisense Technology - RNAi with dsRNA
Regulatory RNAs
DNA Replication
Priming RNAs
Modulating RNAs
Transcription
snRNA small nuclear RNA (mRNA splicing - snRPs)
snoRNA small nucleolar RNA (ribosomal RNA maturation)
gRNA guiding RNA
Post-transcriptional
RNA dependent DNA polymerase
RNA dependent RNA polymerase
ncRNAs non-coding RNAs
lncRNA long non-coding RNA
PiRNA Piwi (P element Induced Wimpy testes) interacting
RNA, known to control the expression of retrotransposons
An updated view of the Molecular Central Dogma
Bacterial Gene Expression Control
Bacteria use operons to regulate gene expression
Operons are modular genetic units
Clusters of several related genes located together and
controlled by a single operator
Operator region lies within the promotor region
Can use operons to regulate gene expression in response
to their nutrient requirements
Lac Operon

Either all three genes are turned on, in the presence of lactose
Or all three genes are turned off in the absence of lactose
When lactose binds the repressor protein, its conformation changes and it can no
longer bind to the promoter thus inhibiting RNA polymerase to interact with DNA