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104 terms

Micro 10 Transcription and Translation

STUDY
PLAY
Transcription DNA -> RNA the 1st step to making
protein.
The single strand of RNA that results from reading the DNA is called
messenger RNA (mRNA)
1st step RNA polymerase binds to a promoter
is just a codon that says "start here"
T T C G A G C A
DNA
A A G C U C G U
RNA
Transcription RNA polymerase binds to the
promoter
RNA Transcription, Moves down the strand of
mRNA reading each base and building RNA's complimentary base.
RNA Transcription Arrives at a STOP signal and
detaches.
RNA Transcription
Result is a single strand of mRNA
Translation mRNA ->
protein
Translation, Ribosomal RNA (rRNA) binds to the
mRNA
Ribosomal RNA
rRNA
Translation, The rRNA "translates" the mRNA into an
amino acid sequence
The entire process of transcription and translation is are referred to as
gene expression.
Gene expression, Every gene ultimately results in the production of
protein.
Phenotype
The physical appearance or functional expression of a trait. (blonde hair, green eyes, estrogen receptor)
Genotype
The genetic code underlying a single trait or set of traits. (specific DNA sequence for blonde hair, green eyes, estrogen receptor)
Regulating Gene Expression, How are transcription and translation controlled
60-80% of genes are constitutive
60-80% of genes are constitutive and
Produced at a fixed rate
How are transcription and translation controlled
Two mechanisms control gene expression Repression & Induction
Two mechanisms control gene expression
Repression & Induction
Repression Inhibits
Transcription
Repression is Usually due to a
build up of end-product
Repression , As the end-product builds up, repressors bind to the
promoter region of DNA and prevent RNA polymerase from binding
Transcription is turned on by an
inducer.
Enzymes that require an inducer to be produced are called
inducible enzymes
Lactose acts as an inducer for
b-galactosidease (ONPG)
b-galactosidease breaks lactose into
glucose and galactose.
Mutations are changes in the
hereditary message of an organism.
Two categories of mutations
Sequence changes & Changes in Gene position
Sequence changes
Base substitution, Chemical modification, DNA breaks, Slipped mispairing , Triplet expansion
Changes in Gene position
Chromosomal rearrangement & Insertional inactivation
Sequence Changes
Base substitution, Normal C G T C A, Base sub C G G C A, Occurs during replication, Also called a point mutation
Sequence Changes
Chemical modification, A base becomes chemically modified by a mutagenic chemical, Another class of point mutation
Sequence changes DNA
breaks
Sequence changes Caused by
Ionizing radiation
Sequence changes Causes
double strand breaks
Sequence changes Results in the
loss (deletion) of short segments
Sequence changes Slipped
Mispairing
Sequence changes Results in
a deletion
Sequence changes Also called a
frameshift mutation
Sequence changes, A sequence is present in
more than one place on a strand and pairs out of order
Sequence changes Like a shirt buttoned on the
wrong button
Triplet Expansion is When a
3-base sequence is repeated several times in a gene.
Triplet Expansion Results in
mutant proteins
More than 15 human diseases are attributed to
triplet expansion
Mutations - Changes in Gene Position
Chromosomal Rearrangement
Translocation
Part of one chromosome becomes a part of another chromosome
Inversion
Orientation of a portion of a chromosome is reversed
Duplication (gene abnormalities)
Entire regions of chromosome become duplicated
Deletion(gene abnormalities)
Similar to the deletion on a nucleotide level but much larger amounts of DNA are deleted
Aneuploidy (gene abnormalities)
Whole chromosomes are lost or gained
Polyploidy(gene abnormalities)
An entire SET of chromosomes are added.
Genetic Recombination Technology, Modern scientists can insert or delete gene segments to
form new combinations of genes
Genetic Recombination Technology
Genes are usually inserted
Genetic Recombination Technology, Vaccine development is trying to delete
pathogenic genes.
What do the letters PCR stand for
Polymerase, Chain, Reaction
PCR is the amplification of a
DNA sequence
PCR is the name of the assay that scientists use to look at what genes are being expressed in a
cell, tissue, or organism.
Why amplification? We have not yet developed the technology to
detect 1 copy of DNA although it is fast approaching.
Orientation to DNA, DNA is made up of
4 nucleotides
DNA , 4 nucleotides 1
A - Adenine
DNA , 4 nucleotides 2
T - Thymine
DNA , 4 nucleotides 3
C - Cytosine
DNA , 4 nucleotides 4
G - Guanine
A =
T ALWAYS
C=
G ALWAYS
DNA Replications Major Players
DNA, DNA polymerase, Nucleotide Bases (A's, T's, C's G's)
DNA Polymerase...what is it? It is the enzyme in your body responsible for
naturally replicating your DNA
DNA Polymerase...what is it? It is the most accurate enzyme making less than
one mistake for every billion bases
DNA Polymerase...what is it? It has a built in
proofreading capability
DNA Replication, Where to start and stop? Mother nature uses
"codons"
DNA Replication, Where to start and stop? Scientists use
primers
Primers let scientists designate where they would like
amplification to begin and end.
What is PCR?
The amplification of a DNA sequence
What is in a PCR tube?
DNA, Primers, A's T's C's and G's, DNA Polymerase
PCR reaction, PCR is broken into
cycles
PCR reaction 94°C for 1 min
denaturation
PCR reaction 55°C for 20sec
annealing
PCR reaction 72°C for 2min
elongation
Taq Polymerase The reason that
PCR is possible
Taq Polymerase was Found in the bacteria that exist in
hot springs
PCR Applications, Why would you ever need to amplify the whole genome?
Forensics, Archeology, Disease Diagnosis
Who Pioneered PCR?
Dr Kary B. Mullis
Dr Kary B. Mullis Won the
Nobel Prize for Chemistry in 1993.
PCR in Research Scientists rarely have a need to look at
the whole genome
PCR in Research Scientists want to look at expression of
specific genes.
How are specific genes expressed? Does every cell carry the entire genetic code?
YES
How are specific genes expressed? Does every cell express the entire genetic code?
NO
(RT-PCR) What does the RT stand for?
Reverse Transcription
Reverse Transcription (RT-PCR)How?
Reverse Transcriptase
Reverse Transcriptase
Retro-viruses have it
Reverse Transcriptase VERY
sloppy enzyme, 1 mistake in every 2,000 bases
Inside an RT-PCR tube Ingredients
mRNA, Primers, Taq, Reverse Transcriptase, dNTP's
Reverse Transcriptase, Primers, and dNTP's make the first strand of
cDNA.
After the first strand of cDNA is made the other primer binds and Taq helps to make the first
dsDNA
After the creation of dsDNA, PCR continues on as before
Denaturing, Annealing, elongation
(Primers) It is generally accepted that if you find "part" of the message you have found
the entire message
(Analyzing RT-PCR)What is Gel Electrophoresis?
Forcing molecules through a gel with electric charge.
Analyzing RT-PCR Big/Heavy molecules vs
Small/Light molecules.
What charge does DNA carry?
Negative
How do we see DNA in a gel?
Ethidium Bromide
Ethidium Bromide, How does it work?
Intercalating agent.......Fluoresces under UV light
Analyzing a gel
DNA bp ladder, Positive Control, Negative Control, Samples