Download X. GENE EXPRESSION

UNIT VII – MOLECULAR
GENETICS
Big Campbell – Ch 17, 18, 20
Baby Campbell – Ch 10, 11, 12
UNIT OVERVIEW
• Protein Synthesis
• Regulation of Gene Expression
o Prokaryotes
o Eukaryotes
• Mutations
o Chromosomal
o Gene
o Cancer
• DNA Technology
o
o
o
o
DNA Testing Techniques
PCR
Recombinant DNA
Extensions
I. PROTEIN SYNTHESIS
• Genotype → phenotype
• Central Dogma
I. PROTEIN SYNTHESIS, cont
• History
o Archibald Garrod
 First to suggest genes dictate phenotype through production of
enzymes
 Made in 1909 after studying disease known as alkaptonuria
o George Beadle & Edward Tatum
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Worked with bread mold, Neurospora
Caused mutation of mold’s DNA through repeated X-ray exposure
Mutated Neurospora required enriched medium
Concluded DNA was no longer producing functional enzyme for
metabolic pathways
Their work led to one gene → one enzyme hypothesis
Eventually modified to one gene → one protein
Then, one gene → one polypeptide
Now, one gene → one ??
I. PROTEIN SYNTHESIS, cont
o Working Models of Study of the Central Dogma
C. elegans
I. PROTEIN SYNTHESIS, cont
II. IMPORTANCE OF RNA
• Ribonucleic Acid
o
o
o
II. IMPORTANCE OF RNA, cont
• Types of RNA
o mRNA
(_____________________)
 Disposable copy of gene
 “Coding RNA”
 Exits nucleus via ___________
o tRNA
(______________________)
 Transfers amino acids to
ribosome according to recipe
contained in mRNA
o rRNA
(_______________________)
 Primary component of
ribosomes
 Synthesized in
_____________
II. IMPORTANCE OF RNA, cont
o Non-coding RNAs …
III. TRANSCRIPTION
• Each gene contains a promoter - a specific sequence of nucleotides that
marks the beginning of a gene
• RNA polymerase unzips the DNA and begins moving in nucleotides
o Nucleotides added in a _____________ direction
o No primer is required
o Only one side of the double helix is transcribed; known as the template strand
o
o
• Transcription continues until a termination signal is reached
IV. TRANSLATION
• mRNA is read in groups
of 3 nucleotides known
as a codon
o Sequence of three
nucleotides that
code for an amino
acid
o This is also known
as the reading
frame
o Redundancy
o AUG
o Stop Codons
IV. TRANSLATION, cont
• Transfer RNA
o Function
o Anticodon
• Ribosomes
o Function is to facilitate coupling of
mRNA codon and tRNA anticodon
during protein synthesis.
o Made up of 2 subunits
o Prokaryotic vs Eukaryotic
o rRNA is transcribed from DNA,
then ribosome is constructed
in_______________
IV. TRANSLATION, cont
• tRNA must bind to an amino
acid
o Cytoplasm of every cell stocked
with all 20 amino acids required
for protein synthesis
o Each amino acid is joined to the
correct tRNA through action of
an enzyme known as
aminoacyl-tRNA synthetase
o There are 20 aminoacyl-tRNA
synthetases
 Active site fits a specific
amino acid
 ATP provides the energy
needed to form covalent
bond between tRNA &
corresponding amino acid
IV. TRANSLATION, cont
o Each tRNA anticodon must
match up with the mRNA
codon to insure the correct
amino acid has been
delivered to the ribosome.
o Occurs according to base
pairing rules, however there
are more mRNA codons than
there are tRNAs.
o Certain nitrogen bases in the
third position of the anticodon
will base pair with more than
one corresponding nitrogen
base in a codon. Known as
wobble.
IV. TRANSLATION, cont
• Ribosome has 3 binding
“sites” for tRNA
o A Site – Holds the tRNA
carrying the next amino
acid to be added to the
polypeptide chain
o P Site – Holds the tRNA
carrying the growing
polypeptide chain
o E “Site” – Site where
tRNAs exit the ribosome
• Newly added amino
acids form peptide bond
with carboxyl end of
growing polypeptide
Initiation
IV. TRANSLATION, cont
IV. TRANSLATION, cont
Elongation
IV. TRANSLATION, cont
Termination
IV. TRANSLATION, cont
• Polyribosomes
o Multiple ribosomes that translate the same mRNA multiple
times
o Found in both prokaryotic & eukaryotic cells
V. PROKARYOTIC GENE EXPRESSION
• Protein Synthesis
 In transcription, RNA Polymerase recognizes and binds to the promoter
sequence
 Transcription & translation occur virtually simultaneously
VI. REGULATION OF GENE EXPRESSION IN
PROKARYOTES
• Important adaptation
for bacteria
• Two basic mechanisms
for metabolic control
o Regulation of Enzyme
Activity
 Feedback Inhibition
o Regulation of Gene
Expression
 Operons
VI. PRO GENE EXPRESSION REGULATION, cont
• Operon Model
o Operon = Promoter + Operator + all genes required for a given
metabolic pathway
o Operon acts as a single transcription unit
o Promoter → Binding site for RNA polymerase
o Operator → “On-off” switch located either close to or within the promoter
Operator controls whether or not RNA polymerase can bind to the
promoter region
Therefore operator determines whether operon genes are transcribed
& translated
VII. PRO GENE EXPRESSION REGULATION, cont
• Operon Control
o Operon can be turned off by a protein known as a repressor
o Repressor binds to operator and prevents attachment of RNA
polymerase to promoter
o Repressor is a protein controlled by a gene known as a regulatory
gene in a different location on chromosome; not part of operon
 Expressed continuously
 Always a small supply of repressor protein present
VII. PRO GENE EXPRESSION REGULATION, cont
• Types of Operons
o Inducible Operons
 Operons that are usually off;
that is, not usually transcribed
 Can be stimulated when a
specific molecule interacts with
regulatory protein
 Example is the lac Operon
 Regulates transcription of
genes required for
breakdown of lactose
 Typically off; bacterium is
metabolizing glucose,
other carbs; lactose is not
present
VI. PRO GENE EXPRESSION REGULATION, cont
Inducible Operons
 lac Operon, cont
 When lactose is
available, lactose itself
binds with repressor;
inactivates it by
changing its shape
 Repressor cannot bind
to regulator
 Therefore, RNA
polymerase is able to
bind to promoter;
operon is “on”
 3 enzymes required to
metabolize lactose are
synthesized
VII. PRO GENE EXPRESSION REGULATION, cont
o Repressible Operons
 Transcription normally
occurs
 Can be inhibited when a
specific molecule binds
allosterically to regulatory
protein
 Example is the trp Operon
 Operon controls production
of 5 enzymes required to
synthesize amino acid,
tryptophan when it is not
available to bacterium in
surrounding
 Operon normally on;
repressor inactive
VII. PRO GENE EXPRESSION REGULATION, cont
Repressible Operons
 When tryptophan is
present, it binds to the
repressor of the trp
operon, activating the
repressor, and turning
off enzyme
production.
 Tryptophan acts as a
co-repressor, a
molecule that works
with a repressor
protein to switch an
operon off.
VII. PRO GENE EXPRESSION REGULATION, cont
VI. PRO GENE EXPRESSION REGULATION, cont
• Positive Gene Regulation
o In addition to repressors, some operons are also under the control of
proteins known as activators
o Essentially the opposite of repressors
o They “turn up” an operon by making it easier for RNA polymerase to bind to
DNA, therefore facilitating transcription of operon genes
o In the lac operon . . .
If both glucose and lactose are available, bacterium utilizes glucose
until its supplies are depleted
As glucose ↓, concentration of cyclic AMP (cAMP) ↑
Increase in cAMP triggers release of activator protein known as CAP;
CAP binds to promoter, facilitates binding of RNA polymerase to
promoter of operon to enhance synthesis of enzymes of lac operon
When glucose concentration is high, decrease in cAMP results in
decrease in CAP → RNA polymerase has very low affinity for lac
operon promoter so lactose metabolism does not occur
VI. PRO GENE EXPRESSION REGULATION, cont
VII. EUKARYOTIC GENE EXPRESSION
 Transcription
o Within the promoter is a
DNA sequence known as
the TATA box – repeated
Ts and As that identify the
transcription site
o Proteins known as
transcription factors
recognize the TATA box,
bind, and allow for
attachment of RNA
polymerase
VII. EUKARYOTIC GENE EXPRESSION, cont
• Transcription, cont
o Transcription continues
until polyadenylation
signal (AAUAAA). mRNA
is released 10-35
nucleotides downstream
from polyadenylation
signal although
transcription continues
o At this point, RNA strand is
known as the RNA
transcript or pre-mRNA
VII. EUKARYOTIC GENE EXPRESSION, cont
• Transcription, cont
o Editing the mRNA
 Each gene has long
segments of noncoding DNA known
as introns
 Introns must be cut
out of mRNA,
remaining regions
known as exons are
spliced together,
exit the nucleus,
and are expressed
in the translated
proteins
VII. EUKARYOTIC GENE EXPRESSION, cont
• Transcription, cont
o Modifying the mRNA
 5’ end of mRNA is “capped” with a guanine nucleotide
Known as 5’ cap
 3’ end has an additional 50-250 adenine nucleotides added
after polyadenylation signal
Known as poly A tail
 Both modifications appear to help mRNA leave the nucleus,
protect the mRNA, and facilitate the attachment of ribosomes to
the 5’ end of the mRNA
VII. EUKARYOTIC GENE EXPRESSION, cont
VII. EUKARYOTIC GENE EXPRESSION, cont
VIII. REGULATION OF GENE EXPRESSION IN EUKARYOTES
• Early in development, eukaryotic cells are totipotent
o Mammalian embryos remain totipotent until 16-cell stage
• Cells are described as pluripotent once extra-embryonic membranes
(placenta, etc) are formed
o AKA embryonic stem cells
VIII. REGULATION OF EUK GENE EXPRESSION, cont
• As development continues, cells
of multicellular organisms
differentiate
o Differentiation due to differential
gene expression in each cell, not
different genes
o Some organisms can dedifferentiate
 Regeneration in animals
 In plants, root cells can grow
into mature plant
 IPS – Induced Pluripotent
Stem Cells
VIII. EUKARYOTIC GENE EXPRESSION REGULATION, cont
• Gene expression
is regulated by
three mechansims
o Regulation of
chromatin
structure
o Regulation of
initiation of
transcription
o Posttranscriptional
regulation
VIII. EUKARYOTIC GENE EXPRESSION REGULATION, cont
Regulation of Chromatin Structure
 2-3 m of DNA per cell is elaborately folded
 DNA wraps around proteins called histones. Charge attraction holds
DNA to histones.
 Cluster of histones forms nucleosome.
 Stretches of DNA between nucleosomes are known as linkers
VIII. EUKARYOTIC GENE EXPRESSION REGULATION, cont
Regulation of Chromatin Structure
• Folding of DNA is highly specific
• Generally, the more condensed the
DNA is, the less likely it is to be
transcribed.
o
• During interphase, DNA is visible as
irregular clumps of chromatin. Two
types:
o Heterochromatin
o Euchromatin
VIII. EUKARYOTIC GENE EXPRESSION REGULATION, cont
Regulation of Chromatin Structure
• Modification of Histones
o Acetyl (-COCH3) group added to N-end of histone “tail”
o Neutralizes + charge
o Histone less attracted to nucleosome, coil loosens, DNA
becomes more transcribable.
• DNA Methylation
o Addition of methyl groups to certain bases in DNA
 Most often involves cytosine
o Deactivates DNA
o For example, in females, inactivated X chromosome is highlymethylated
VIII. EUKARYOTIC GENE EXPRESSION REGULATION, cont
Transciptional/Translational Regulation
• Regulation of Initiation of Transcription
o Transcription Factors
 Bind to TATA box
 Form Transcription Complex that allows RNA Polymerase to bind to DNA
o Enhancer Sequences
 DNA sequences
 May be located up to 20,000 bp “upstream” from the promoter
 Bind activator proteins
o Silencers
 Bind repressor proteins
o Work together to determine rate of transcription
VIII. EUKARYOTIC GENE EXPRESSION REGULATION, cont
Transciptional/Translational Regulation
• Post-Transcriptional Regulation
 Alternative RNA Splicing
VIII. EUKARYOTIC GENE EXPRESSION REGULATION, cont
Transcriptional/Translational Regulation
• Post-Transcriptional Regulation, cont
 Degradation of mRNA
 Translation
 Protein Processing & Degradation
VIII. EUKARYOTIC GENE EXPRESSION REGULATION, cont
• Post-Transcriptional Regulation, cont
“Other” RNAs
MicroRNAs (miRNAs)
 Formed from longer RNA strand that folds onto itself to create a hairpin
loop
 Enzyme called Dicer trims it into a short double-stranded fragment
 One strand is degraded; the remaining strand can bind to any
complementary mRNA
 Blocks translation
Small interferring RNAs (siRNAs)
 Similar in mechanism to miRNAs
 Original RNA strand longer, more “hairpins”; generates many more siRNAs
VIII. EUKARYOTIC GENE EXPRESSION REGULATION, cont
• miRNA
IX. MUTATIONS
o Change in the nucleotide
sequence
o May be spontaneous mistakes
that occur during replication,
repair, or recombination
o May be caused by mutagens;
for example, x-rays, UV light,
carcinogens
o Two categories
 Gene Mutations
 Chromosomal Mutations
IX. MUTATIONS, cont
• Gene Mutations
o Point mutations – change in a gene involving a single nucleotide
pair; 2 types
 Substitution – Further subdivided into . . .
 Silent
 Nonsense
 Missense
 Frameshift – due to addition or deletion of nucleotide pairs
Normal mRNA
X
IX. MUTATIONS, cont
• Gene Mutations & Phenotype
o Traits may be described as dominant, recessive, etc . based on
the effect of the abnormal allele on the organism’s phenotype
o Vast majority of proteins encoded in genes are enzymes
o Abnormal allele → Defective enzyme
 If the enzyme produced by the normal allele is present in
sufficient quantities to catalyze necessary reactions,
No noticeable effect on phenotype
Defective allele is classified as recessive
 If the lack of normal enzyme production by defective allele
cannot be overcome by normal allele,
Organism’s phenotype is affected
Defective allele is classified as dominant
IX. MUTATIONS, cont
• Chromosomal Mutations
o Chromosome Number Mutations/Disorders
o Alterations in Chromosome Structure
 Often due to mistakes made during __________________
X. A CLOSER LOOK AT CANCER
• In early 1900s, scientists realized there are viruses that can cause
cancer, including Human Papilloma virus, Epstein-Barr virus, and
HTLV.
• Research led to discovery of cancer-causing genes called
oncogenes
• We now know there are two important categories of genes in which
mutations may lead to cancer
o Oncogenes/Proto-oncogenes
o Tumor Suppressor Genes
X. A CLOSER LOOK AT CANCER, cont
• Oncogenes
 Amplification – Increases number of copies of proto-oncogene; will increase protein production
 Point mutation in the promoter for an proto-oncogene, or in the gene itself
 Movement of DNA - May change the rate at which gene at which gene is transcribed,
therefore, translated
 Translocation
 Transposons
 “Jumping Genes”
 Genes that are moved due to folding of DNA, cut (or copy) & paste mechanism
X. A CLOSER LOOK AT CANCER, cont
Oncogenes & Transposons
X. A CLOSER LOOK AT CANCER, cont
X. A CLOSER LOOK AT CANCER, cont
• Tumor-Suppressor Genes
o Encode for proteins that inhibit cell division therefore any mutation that
inhibits activity of tumor-suppressor gene may lead to abnormal cell
growth and formation of tumors.
o Act by producing proteins that repair damaged DNA, control densitydependent inhibition & anchorage dependence, or act as CDKs
o Gene that is most often defective in human cancers codes for
transcription factor known as p53
 Known as the “guardian angel of the genome”
 Serves as the master brake on the cell cycle when DNA damage has
occurred
X. A CLOSER LOOK AT CANCER, cont
• Tumor Suppressor
Genes, p53 cont.
 When stimulated by DNA
damage, p53 activates
several genes with multiple
effects
 Genes activated to halt
cell cycle
 DNA repair genes
turned on
 If DNA damage cannot
be repaired, “suicide
genes” are activated;
results in apoptosis
X. A CLOSER LOOK AT CANCER, cont
X. A CLOSER LOOK AT CANCER, cont
• Tumor-Suppressor Genes,
cont
o BRCA 1, BRCA 2 genes
o BRCA 1
• Women who inherit one
mutant allele have ~
60% chance of having
breast cancer by 50
• Individuals with two
normal alleles have ~
2% chance
X. A CLOSER LOOK AT CANCER, cont
DNA TECHNOLOGY & GENOMICS
I. TECHNIQUES IN DNA TECHNOLOGY
• Restriction Enzymes
o Used by bacteria to “chop up” viral
DNA
o Bacterial DNA protected by _________
o Very specific
 Each enzyme recognizes a particular
nucleotide sequence
 Called a restriction sequence or
restriction site
 Palindromic
 Cuts made at specific points
 May create “sticky ends”
o Used in gel electrophoresis
o Also used to form recombinant DNA
 Fragments may be pasted together
with DNA ligase to form recombinant
DNA
I. TECHNIQUES, cont
• Polymerase Chain
Reaction (PCR)
o In vitro method of
amplifying small amounts
of DNA
 DNA is heated to
separate the double
helix.
 Mixture is allowed to
cool, DNA primers
attach to target
 Heat-stable
polymerase is used to
extend the primers in
the 5’–3’ direction.
I. TECHNIQUES, cont
• Gel Electrophoresis
o Separates DNA
fragments based on size
o Restriction fragment
analysis
 DNA treated with
restriction enzymes
 Resulting fragments
migrate based on size
 Produce a pattern
characteristic of
original DNA and
restriction enzyme
used
I. TECHNIQUES, cont
• Southern Blotting
 Designed by Dr.
Southern
 Detects particular
DNA sequences
• Northern Blotting
 Detects particular
mRNA sequences
• Western Blotting
 Used to detect
proteins
II. EXTENSIONS IN DNA TECHNOLOGY
• Recombinant DNA
 DNA containing
nucleotides from
other sources
 Process utilizes
restriction enzymes
that make jagged cuts
in DNA; creates
sticky ends
 When DNA from
different sources
treated with same
restriction enzyme,
sticky ends “mix &
match”
 Often use reporter
genes to determine
success; for example,
ampicillin resistance
II. EXTENSIONS, cont
• cDNA - complementary DNA
o Procedure for “cloning DNA” that uses mRNA, reverse transcriptase
o
• STRs – short tandem repeats
o Short segments of DNA that are highly repetitive, polymorphic
o Repeat patterns are inherited
o Useful for identifying individuals
• SNPs – single nucleotide polymorphisms
o Single base-pair that shows variation in a significant % of population
o SNPs that alter the fragment length following exposure to restriction
enzymes called RFLPs (restriction fragment length polymorphisms)
o Genetic markers
II. EXTENSIONS, cont
• DNA Microarray
Assays
o AKA DNA
Chips
o Test used to
determine gene
function, gene
interactions
o May be used to
determine
agressiveness
of cancers,
method of
treatment, etc
II. EXTENSIONS, cont
• Gene Cloning
o Process of preparing multiple copies of a particular segment of DNA
o Requires host and vector
o Hosts
 Initially done using bacterial cells
 Now eukaryotic hosts are used
 Yeast
 Plants
o Vector
 Should have 4 characteristics
 Ability to replicate independently of host cell DNA
 Recognition sequence
 Reporter gene
 Small size
 Possible vectors include
 Plasmids
 Viruses
 YAC = Yeast Artificial Chromosome
II. EXTENSIONS, cont
• Gene Cloning
 Use of plasmid as
vector
 Plasmid isolated from
bacterial cell
 Foreign DNA
inserted into plasmid
 Plasmid returned to
bacterial cell;
described as
recombinant
bacterium
 Foreign gene is
cloned as bacteria
reproduce
 Common bacterium
used for plants is
Agrobacterium
tumefactiens
II. EXTENSIONS, cont
A CLOSER
LOOK AT
GENE
CLONING
II. EXTENSIONS, cont
• Reproductive Cloning
 Nuclear Transplantation
 Process of using unfertilized
egg cell & replacing nucleus
with DNA
 In 1997, scientists were able to
produce first reproductive
clone, “Dolly”, by culturing
somatic cells in a nutrient-poor
medium to de-differentiate
them and force them back to
totipotency.
 Reproductive cloning in
animals has enjoyed limited
success.
II. EXTENSIONS, cont
• Gene Silencing
o Knockout Genes
Use of genetic recombination
to create an inactive ,
“knocked out” gene
Mutated allele introduced into
embryonic stem cells
Forms chimeras
Often used in mice to study
gene expression
II. EXTENSIONS, cont
o RNAi
Based on principal of
microRNA
Small-interfering RNA
(siRNA) synthesized
complementary to mRNA
Base-pairing occurs
Translation is blocked
Has been used to block
production of growth
factors in certain cancers
III. GENOMICS
• Human Genome Project
 International government effort begun in 1990
 Goals
o identify all the approximately 20,000-25,000 genes in human DNA,
o determine the sequences of the 3 billion chemical base pairs that
make up human DNA,
o store this information in databases,
o improve tools for data analysis,
o transfer related technologies to the private sector, and
o address the ethical, legal, and social issues (ELSI) that may arise
from the project.
 Celera Genomics
o Shotgun sequencing
 Completed early and under-budget in 2003
 Genomics has given rise to proteonomics