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Unit 6B: Molecular Genetics

Image Credits: Biology (Campbell) 9th edition, copyright Pearson 2011, & The Internet. Provided under the terms of a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
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Christopher Himmelheber

on 6 March 2018

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Transcript of Unit 6B: Molecular Genetics

Universal accross all domains of life.
The Central Dogma
DNA
RNA
Protein
Replication
Transcription
Translation
Once the structure of DNA was determined, it was time to figure out how it worked
Allows for Gene Expression
It is easy to see how DNA is copied by looking at it's structure
"It has not escaped our attention that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material,"
-End of Watson & Crick's 1953 paper
But it still needed to be proven...
"
Semi-Conservative
" Replication
Meselson & Stahl
3 Possible Hypotheses
"The most beautiful experiment in Biology"
Conducted an experiment in 1958 that supported the
semi-conservative model of replication
How Replication Happens:
1. Initiation
Replication can only begin at specific locations ("
origins
") on a chromosome
DNA Polymerase
: Enzyme Responsible for DNA synthesis.

Helicase
: Opens the helix (which causes strand separation)

Single Strand Binding Proteins ("
SSBP's
"): Keep the strand open

Primase
: Puts down a small RNA
primer
which is necessary for DNA polymerase to bind to at the origin.

Topoisomerase
: Rotates the DNA to decrease torque (which would shred the helix.
The addition of new nucleotides into a strand of DNA is
autocatalytic
.

Nucleotides are automatically added to the 3' end of the DNA strand, as determined by the sequence of the nucleotides in the opposite strand.

This means that DNA replication can only occur in the
5' to 3'
direction.

DNA is "
anti-parallel
": Both strands have opposite 5' to 3' orientations (one is "upside-down" compared to the other)
2. Elongation
Nucleotides are added to the new strand of DNA in the 5' to 3' direction.

There is an issue: DNA is anti-parallel

As the replication machinery moves along the chromosome, only one strand of DNA (the "
leading strand
") can be made in a continuous, 5' to 3' piece.
The other strand (the "
lagging strand
") has to be made in smaller, discontinuous 5' to 3' segments ("
Okazaki fragments
") which are then stitched together by the enzyme
ligase
.
Leading Strand
Lagging Strand
The "
Replication Fork
":
The "
Replisome
" Holoenzyme
Once it begins, replication proceeds in two directions from the origin
Prokaryotes vs. Eukaryotes
1 origin vs. Many Origins
The "
Replication Bubble
"
3. Termination
Elongation continues until replication bubbles merge.

The ends of linear eukaryotic chromosomes pose a unique challenge.

Each round of replication shortens the 5' end of the lagging strand (by about 100-200 bp).

If this continued indefinitely, chromosomes would get shorter and shorter after each replication. Information would start to be lost.
"Proof-Reading"
Telomeres:
The ends of eukaryotic chromosomes.
Consist of a short, repeating DNA sequence

Vertebrate Telomere: TTAGGG
Telomerase:
The enzyme responsible for replicating the ends of eukaryotic chromosomes.

Uses an RNA template to add more telomere sequence during replication.

Not active in
senescent
cells.
There are 5 different DNA polymerases described in prokaryotic cells. Eukaryotic cells have ~15.

They serve a variety of functions, well beyond the scope of this course.

We'll focus on one polymerase:

DNA polymerase III
- Is responsible for elongation. Rate of elongation is ~500 bases/second in E. coli

The eukaryotic analog DNA polymerase elongates at a rate of ~50 bases/second.

The initial error rate during elongation is 1 in 10,000 mismatched bases.

This would create 300,000 mutations every time a human cell divided.

There are a series of other DNA polymerases and nucleases responsible for "
proof-reading
" DNA.

Proof-reading reduces the error rate to 1 in 10 billion nucleotides during replication (less than 1 per 3 human cell divisions).

Proof-reading is a continual process.
Allows for
Heritability
DNA
RNA
How transcription happens:
1. Initiation
RNA polymerase attaches to a "
promoter
" region in front ("
upstream
") of a gene
prokaryotes: RNA polymerase binds directly to the promoter.
eukaryotes: RNA polymerase requires an assemblage of "
Transcription factor
" proteins" to be able to bind to the promoter.

Promoters have characteristic DNA sequences (ex "
TATA Box
" in eukaryotes)
2. Elongation
Similar to DNA replication, RNA production occurs in a 5' to 3' direction.

The "
template strand
" of DNA is the one that the RNA transcript is being produced off of (its sequence is opposite to the transcript)

The "
nontemplate strand
" or "
coding strand
" of the DNA will have the same sequence as the RNA transcript (with thymines replaced by uracils in the transcript)
3. Termination
Transcript production continues until the end of the transcription unit is reached.

There are multiple mechanisms of termination, two examples from prokaryotes:
Rho-independent
: the transcript bases hydrogen bond with themselves, fold back and pull the transcript out of RNA polymerase.

Rho-dependent
: The Rho protein destabilizes the RNA-DNA hydrogen bonding at RNA polymerase and ceases transcription.
What Happens Next?
Many Kinds of RNA
Unlike DNA, RNA plays many roles in the cell
There are ~10 described types of RNA, each with different functions, but there are three major types:

Messenger RNA (
mRNA
): Carries DNA sequence information to the ribosome
Transfer RNA (
tRNA
): Carries specific amino acids to the ribosome
Ribosomal RNA (
rRNA
): Major structural building block of ribosomes


We'll just focus mRNA for now, since it has the information that will become protein. In prokaryotes, the mRNA transcript is immediately translated.

In eukaryotes, the mRNA transcript is extensively processed in the nucleus before it leaves to be translated.
Post-Transcriptional mRNA Processing
(Eukaryotes ONLY!)
5'Cap and poly-A tail
Exon Splicing
A modified nucleotide is added to the 5' end of the transcript.

A tail of several hundred adenine residues is put on the 3' end of the transcript.

These modifications function in nuclear export and maintenance of the mRNA
Eukaryotic genes contain large stretches of non-coding DNA ("
introns
") interspersed between coding DNA ("
exons
")
To produce a functional protein, the introns must be removed and the exons must be spliced together prior to the movement of the mRNA transcript to the nucleus.

This process is accomplished by a
spliceosome
(a type of enzymatic RNA molecule)
Why Introns?
It's a great question. Not really answered.

Evolutionary baggage? Selfish genes?

We do know that having multiple exons in a gene allows eukaryotes to make multiple functional proteins from one gene ("
alternative splicing
")
RNA
polypeptide
The Genetic Code:
The Ribosome
tRNA
The site of protein synthesis.

The only "non-membrane" bound organelle.

All cells have ribosomes.

Composed of two subunits.

Has three "sites":
A site
: "Aminoacyl"- where amino acids enter the ribosome
P site
: "peptidyl"- where the growing polypeptide is kept.
E site
: "exit"- where empty tRNA molecules leave.
Transfer RNA molecules.

Responsible for bringing amino acids to the ribosome
Amino acids are added to tRNA molecules through the action of "
amino-acyl tRNA synthetase
" enzymes

A tRNA with the an amino acid attached is said to be "
charged
"
Triplet code: mRNA is read in units of three bases ("
codons
")

There are 64 possible codons (for 20 possible amino acids).

The code is redundant and unambiguous.

The code has "
start
" and "
stop
" punctuation.
pig with GFP from a jellyfish
tobacco plant with luciferase from a firefly
How translation happens:
1. Initiation
The mRNA attaches to the small ribosomal subunit.

Methionine
is brought to the start codon (AUG) by the methionine tRNA.

The ribosome assembles so that the start codon (
AUG
) is in the P-site.

This is called the "
translation initiation complex
".
2. Elongation
The next codon determines the next amino acid to be brought to the ribosome.
The incoming charged tRNA enters at the A-site.
The growing polypeptide is transfered to the new tRNA molecule. A
peptide bond
is formed.
The ribosome shifts ("
Translocates
"). The tRNA with the polypeptide is now in the P-site.

The uncharged amino acid is now in the E-site.
The next codon is now available in the A-site for the next incoming charged tRNA
3. Termination
When a stop codon (
UAG
,
UAA
, or
UGA
) is encountered, a release factor binds to the A-site.

The polypeptide chain is released.

The ribosome disassembles.
tRNA binding at the ribosome is mediated by an "
anti-codon
" loop in the tRNA molecule
tRNA "anti-codon"
Protein Synthesis in Total
Prokaryote & Eukaryote
Translation Specifics
Prokaryotes
Since prokaryotes do not have a nucleus, transcription and translation can be coupled.

Polyribosomes
: simultaneous translation of a transcript (even while that transcript is still being made.
Eukaryotes
Eukaryotes can not couple transcription and translation.

Eukaryotes also need to target different polypeptides to different areas of the cell.
Mutations
It becomes clear how changes in DNA can affect changes in protein structure, and in physiology. There are 2 major types of DNA-level mutations:

Point mutations
: One DNA base is replaced by another DNA base.
Frame-shift mutations
: DNA bases are inserted or deleted.

Each type of mutation can have different effects, depending on the situation.
Point Mutations
Silent
The substitution changes a codon to another codon for the same amino acid

Can sometimes lead to defects
Codon usage bias
Missense:
The substitution changes a codon to a codon for a different amino acid.
Nonsense:
The substitution changes a codon to a stop codon
Frameshift Mutations
Extensive missense
The reading frame of the ribosome is altered so that all amino acids downstream from the mutation are altered
Immediate nonsense
The reading frame of the ribosome is altered so that a stop codon is introduced prematurely
Limited Effect
The reading frame is restored when mutations occur in multiples of three.
PS:
The code was cracked largely by Marshall Nirenberg

Put synthetic RNA into "cell free" E. coli extract and analyzed the polypeptides that were made.

Nobel Prize: 1968
Big Questions
Make Sure You Can


Signal Peptide
: a small signal peptide sequence on polypeptides that need to be made at the endoplasmic reticulum. Recruits an SRP protein, which modulates "docking" of the ribosome to the rough ER.
An RNA "
hairpin
" loop, similar to what happens in Rho-independent termination
Sickle Cell Anemia is due to a point mutation.
How is the structure of DNA related to its function?

How does DNA allow for heritability?

How does DNA allow for traits in an organism?

How do mutations affect DNA structure and function?
Explain all steps of replication, transcription and translation, the enzymes required for each and the flow of information from DNA to RNA to protein.

Compare repliacation, transcription and translation in prokaryotic and eukaryotic biological systems.

Interpret the genetic code and use it to determine the amino acid sequence of a polypeptide if given the DNA sequence.

Explain the relationship between DNA sequence, protein sequence, and phenotype of an organism.

Describe the possible effects of DNA-level mutations on protein structure and organismal phenotype.
DNA Replication
Transcription
Translation
Mutation
Sickle Cell
The Genetic Code
Splicing
Regulation of Gene Expression
Bacteria
Eukaryotes
In bacteria, transcription and translation happen simultaneously (they are "coupled")

Bacteria regulate gene expression (and therefore their metabolism) almost entirely by regulating transcription.

The lack of a nucleus makes this very efficient.
Eukaryotes regulate gene expression (and therefore their metabolism) at every step of protein synthesis from pre-transcription to post-translation
Refer to organized clusters of genes that all contribute to a particular metabolic task.

Bacteria only!

2 Major Flavors:
Inducible
and
Repressible
Operons:
Inducible:
Repressible:
"Up Regulation":
Precursor
Enzyme 1
Enzyme 2
Enzyme 3
-
-
Gene 1
Gene 2
Gene 3
Gene 4
Gene 5
Product
Inhibition of
enzyme activity
Inhibition of
transcription
Francois Jacob & Jacques Monod
Nobel Prize: 1965
For metabolic (catabolic) pathways that are usually "off"

Ex: The
Lac Operon
(digests lactose)
For metabolic (anabolic) pathways that are usually "on"

Ex: The
Trp Operon
(synthesizes tryptophan)
When Lactose is absent:
The repressor protein (made by the
LacI
gene) is able to attach to the
operator
(a region of the promoter)
RNA polymerase cannot transcribe the
structural genes
(
LacZ
,
LacY
and
LacA
) that the cell needs to be able to digest lactose, since it can not attach to the promoter.
This is how things remain in the cell as long as there is no lactose present.
When Lactose is present:
The
inducer
molecule (
allolactose
, a form of lactose) binds to the repressor protein.
This changes the shape of the repressor protein so that it can not attach to the promoter.
RNA polymerase is then able to transcribe the structural genes that the cell needs to be able to digest lactose.
This is how things remain in the cell until lactose is digested
When tryptophan is absent:
The repressor protein (made by the
TrpR
gene) is unable to attach to the operator
RNA polymerase can transcribe the structural genes (
TrpE
,
TrpD
,
TrpC
,
TrpB
, &
TrpA
) that the cell needs to be able to synthesize tryptophan.
This is how things remain in the cell as long as there is no tryptophan present.
When Tryptohan is present:
The
corepressor
molecule (
tryptophan
) binds to the repressor protein.
This changes the shape of the repressor protein so that it can attach to the promoter.
RNA polymerase can not transcribe the structural genes that the cell uses to synthesize tryptophan.
This is how things remain in the cell until tryptophan is no longer present.
A way to increase the rate of transcription of an operon

Ex: The
CAP/cAMP
System
When Lactose is present and glucose is low:
Low glucose means the amount of
cyclic AMP
(
cAMP
) is high
cAMP binds to the
Catabolite Activator Protein
(
CAP
), activating it
The Activated
cAMP/CAP complex
increases the rate of transcription of the lac operon (and ~100 other catabolic operons), boosting the rate of transcription several fold.
When Glucose and Lactose are both present:
Normal glucose level means the amount of cyclic AMP (cAMP) is low
cAMP is not bound to CAP
CAP is inactive.
Very little lac structural gene transcription occurs.
Levels of Control
1.DNA Access
2.Pre-Transcription
3.Post-Transcription
Recent research has uncovered many forms of "
non-coding" RNA
(
ncRNA
) molecules in cells.










We'll look at one example, active in regulation of gene expression post-transcription and pre-tranlstion
4.Pre-Translation
5.Post-Translation
The
decoupling of

Transcription
&
Translation

allows for
more control
points
Only keep necessary genes accessible.
In eukaryotes, DNA is "wound" around
histone
proteins.

The addition of
acetyl

groups
(
-CH2CH3
) to histones causes them to become less tightly packed, allowing for access to the DNA.

Heterochromatin
: more tightly packed DNA, unavailable for transcription.

Euchromatin
: less tightly packed DNA, available for transcription.

Cool fact: Histone acetylation patterns seems to be heritable. Epigenetics strikes again!
Recent research shows that even in interphase, the "loose" chromosomes occupy distinct nuclear regions.

"
Transcription factories
": Areas of the nucleus where active regions of different chromosomes interface. May be associated with common functions

Thousands of transcription factories in any nucleus
All the actors need to be present for the play to begin.
Eukaryotic genes interact with many "upstream" regulatory elements. These are DNA sequences that preceede a transcription unit that need to have specific proteins present for RNA polymerase to begin transcription.
The proteins that mediate RNA Polymerase are known as "
Transcription Factors
":
Control of transcription factor availability is one of the major ways that cells of a multicellular organism accomplish "
differential gene expression
", which in turn allows cells to "
differentiate
" to serve different functions in the organism.

This is crucial!!!
So many options lead to so many outcomes.
Following transcription,
5' capping
and
3' poly-adenylation
are necessary for eukaryotic mRNA to remain functional and be transported to the cytoplasm for translation.
"
Alternative splicing
" of exons allows for multiple functional (or non-functional) gene products to be made from a single primary transcript.

Anywhere from 75 - 100 percent of human genes with multiple exons probably undergo alternative splicing.

Shown here: the Troponin T gene produces 2 different mRNA sequences to produce 2 different gene products.

Not shown: A Drosophila gene that has enough exons to produce 19,000 different transcripts.
Lots still left to learn
RNA Interference (
RNAi
):
Mediated by a group of tiny RNA molecules ("
Micro RNA
" or
miRNA
).

The miRNAs are produced after the transcript for them is cleaved into multiple fragments by a "
dicer
" protein.

The miRNAs complex with proteins.

Any mRNA with a sequence complementary to an miRNA is "tagged" with the miRNA/protein complex.

Tagged miRNA molecules are not translated.

Nobel Prize: 2006
Don't let unnecessary proteins hang around.
Ubiquitin
(a protein so plentiful in all eukaryotic cells it is "ubiquitous"), will tag unnecessary proteins for transport to a proteasome. Inside the
proteasome
, the protein is broken down.
Nobel Prize: 2004
Proteasomes are abundant in eukaryotic cells (why?)
Polyribosomes demonstrating the coupling of transcription and translation in prokaryotes:
Prokaryotic control of metabolism through control of transcription:
Wikipedias "list of RNAs"
(Feb. 2017):
Coding RNAs
RNAi
"
Central Dogma
": Term coined by Francis Crick to explain how information flows in cells.

Unit 6B:Molecular Genetics
DNA:A History
Back to the 20th Century
Frederick Griffith: Transformation
Avery, McCarty & MacLeod:
Griffith Refined
Hershey & Chase:
The "Blender" Experiment
Erwin Chargaff's "Rules"
Watson & Crick,
Franklin & Wilkins
The First Puzzle Solved!
1928
1944
1952
1950 - 1952
1953
By the middle of the 20th century, genetics was well established as a field of study.

It was known that traits were inherited, but it was not known how that process happened.

Mathematical analysis can only go so far. What were genes made of? How did they work?

Nobody knew.

The discovery of DNA's role in inheritance is arguably the most significant contribution to understanding how life works.

It was not the result of any one person, but the final result of decades of investigation by many different researchers.
Chromosomes
Observation of chromosomes during cell division demonstrates that they act in a way consistent with molecules of heredity.

Chromosomes are made of 2 ingredients: DNA and protein.

This suggests that heritability is controlled by one of these two molecules.

Before 1940 (or so), most biologists thought that protein was probably the molecule responsible for inheritance (why?).

No one had any idea about DNA's structure or function.
A Scottish Microbiologist

Discovered that bacteria could give other bacteria heritable traits, even after they were dead.

That's where he left it.
The two forms of Streptococcus pneumoniae
R (rough, on left) is harmless
S (smooth, on right) is pathogenic.
How do you explain this?!?
Oswald
Avery
Maclyn
McCarty
Colin
MacLeod
Refined Griffith's Experiment

Exposed R-strain Streptococcus to purified S-strain protein, and purified S-strain DNA

Only the bacteria exposed to the S-strain DNA were transformed

Not enough evidence for the haters
Alfred
Hershey
Martha
Chase
Worked with
bacteriophages


Conclusively demonstrated that DNA was the molecule of heredity by tagging phage DNA and protein with radioactive atoms and tracking the transmission of that radioactivity to infected bacteria

Nobel Prize: Hershey (1969)
A colorized electron micrograph showing bacteriophages infecting an E. coli cell by injecting DNA
AHA!
An Austrian Biochemist

Demonstrated two major rules of DNA composition
1. All species have different amounts of adenine, thymine, cytosine and guanine in their DNA.

2. In every species:
the amount of adenine = the amount of thymine
and
the amount of cytosine = the amount of guanine
Why does this matter?
James
Watson
Francis
Crick
Rosalind
Franklin
Maurice
Wilkins
Two competing teams to determine the structure of DNA

Watson and Crick used X-ray diffraction data developed by Rosalind Franklin to develop their "double helix" model of DNA

Nobel Prize: Watson, Crick & Wilkins (1962)
Photo 51: The crucial data used by Watson & Crick
The Double-Helix Model of DNA
Understanding DNA structure helps explain its role in heredity
Bases on one strand are covalently bonded to each other ("
phosphodiester bonds
")

Bases on opposite strands are hydrogen bonded to each other ("
base pairs
").

Adenine = Thymine
Cytosine = Guanine
Chromosomes are densely packed double-stranded DNA molecules (with hundreds of millions of base pairs). Chromosomal proteins help mediate this packing.
Friedrich Miescher

Discovered nucleic acids (1869)
To put it another way...
Chargaff's Ratios:
The Helix Unwound
A way to produce many copies of a DNA molecule without the use of cells.

Nobel Prize: 1993
Cloning produces specific cells to be used for medical purposes.

Requires collection of "
pleuripotent stem cells
" from an early embryo, or from an adult.
Copying a genetic sequence. A single gene can be cloned. That's not really what people mean when they talk about "cloning".

They mean creating new organisms without sexual reproductive events.

Normal in most domains of life.

Controversial when used for organisms that do not naturally reproduce asexually.

Has been achieved in all domains of life.
Biotechnology
Applications
Tools & Techniques
Restriction Enzymes
Electrophoresis & Labeling
PCR
DNA Sequencing
Genetic Engineering
Genetic Testing
Vectors
Microarrays
Gene Libraries
Cloning
What they Are:
What they Are:
What it is:
What they are:
What it is:
What they are:
What it is:
What it is:
How they work:
How they work:
How it works:
How it works:
How it works:
How they work:
How it works:
How it works:
Officially: "
Restriction Endonucleases
"
Only cut DNA at specific sequences (hence "restriction")
Cutting locations: "
Restriction sites
" Usually 4-8 bp
Hundreds have been isolated

Probably serve as a bacterial immune system against phages

Nobel Prize: 1972
EcoRI: Cuts at GAATTC

Restriction Enzymes are named for the bacteria they are isolated from
There are many different types of restriction enzymes.

Generally speaking:
They recognize "palindromic DNA sequences"
They either cut in the middle of the sequence (
blunt ends
), or produce a overhang of a few bases (
sticky ends
).
Different restriction enzymes recognize different restriction sites.
DNA from different sources can be recombined by treatment with the same, sticky end making, restriction enzyme.

Ligase can be used to seal the break between strands.
DNA molecules that can be modified to store and replicate other DNA sequences.

Examples: Bacterial plasmids, phages, viruses, artificial chromosomes.
We'll focus on
plasmids
To be useful, plasmids must minimally have:
Origin of replication
Multiple cloning site
Many unique restriction sites
Selectable marker gene
Enable identification of cells that have successfully taken up the plasmid
Usually
antibiotic resistance
There is a size limit on plasmids.

To get more genes in to cells,
artificial chromosomes
can be used.

In 2010, the J. Craig Venter Institute created the first cell with a completely synthetic chromosome.
A way to separate fragments of DNA based on their size.
The DNA is placed into a matrix gel made of
agarose
.

The gel is exposed to an electric field.

DNA migrates to the positive electrode.

Different sized fragments move through the gel at different rates (smaller = faster)

"
DNA Fingerprint
": The unique banding pattern of a particular restriction enzyme digest of a particular DNA sequence.
A way to identify sequences of interest.
DNA fragments are isolated and denatured (strands separated).

The fragments are exposed to a complementary
oligonucleotide
probe, tagged with radioactivity (old) or flourescence (new).

The oligonucleotide binds to the complementary sequence among the fragments.
The "Polymerase Chain Reaction"
A "target" DNA sequence is analyzed.

Oligonucleotide
primers
that bracket the sequence are created.

The DNA, primers, free nucleotides, coenzymes, and special
Taq polymerase
are put in a thermocycler.

The
thermocycler
is programmed and it runs.

One sequence can be copied 2 times overnight.
1.
Denaturation
:
94-98 degrees Celsius
DNA strands separate
2.
Annealing
:
50-65 degrees Celsius
primers bond to strands
3.
Extension
:
75-80 degrees Celsius
Taq polymerase attaches to primers and replicates target sequence
Taq Polymerase
For PCR to work, a polymerase that is not destroyed by high temperatures is required.

Taq polymerase: isolated from Thermus aquaticus, a bacterium first found in hot springs in Yellowstone National Park
30
collections of DNA sequences, stored in vectors.
large sequences are cut into smaller pieces.

the pieces are placed in to vectors.

the vectors are introduced into cells.
A way to determine the sequence of bases in a piece of DNA
Nobel Prize: 1980
Kary Mullis
Fred Sanger
(his second Nobel Prize)
DNA is replicated in the presence of
dideoxynucleotides
(
ddNT's
).

When a ddNT is incorporated into a strand of DNA, it terminates replication.

The results of the process are read, either manually (old school) or automatically (modern way).
A way to visualize simultaneous gene expression for multiple genes
The microarray contains a complementary sequence for different mRNAs on thousands of microscopic spots.
mRNA is isolated from a cell and used to make fluorescent cDNA.
The flourescence pattern is analyzed.
"
Expression analysis
"
What it is:
Combining genetic information from two or more organisms to create a "
transgenic
" organism that expresses new traits.

Involves restriction enzymes and vectors.

Has been accomplished in all domains of life.

Used in medicine, agriculture, industry, research
Details:
cDNA:
Recombinant
Screening
The frequency of successful engineering events is quite low.

How do you know if an organism is expressing the engineered trait?

Reporter genes
: Genes in vectors that enable detection of successful engineering.

Examples: Ampicillin resistance, GFP, luciferase
"
Complementary DNA
"

DNA copies of RNA molecules (produced using reverse transcriptase).

Increases stability, removes introns for prokaryotic expression of eukaryotic genes
Eukaryotic
Engineering
More difficult than prokaryotic engineering due to nuclear membrane.

Requires different strategies.
Gene Therapy
Engineering cells in a multi-cellular organism.

Tricky and mixed results to this point in time.
Complex
Engineering
Engineering multi-gene traits is becoming more common.
What it is:
Using biotechnological tools to look for the presence/absence of particular genetic sequences.

Restriction Enzymes for DNA digestion. PCR for amplification, Gel Electrophoresis for analysis.
Health:
Identity
Research
Look for genetic markers associated with various traits.
Typically analyze "
Restriction Fragment Length Polymorphisms
" (aka "
RFLP's
") , though modern approaches also compare expression patterns.
In this example, the RFLP is due to a
single nucleotide polymorphism
(
SNP
) leading to loss of a DdeI restriction site, which can be visualized on a gel.
Look at areas of the genome that have many repeating sequences ("
Short Tandem Repeats
":
STR's
).

Soon, whole-genome sequencing will be cheap enough to be covered by most insurance plans.
The only limitation for genetic testing in research is experimental creativity,
What it is:
Reproductive Cloning
Cloning produces a new organism.
Somatic Cell Nuclear Transfer
Therapeutic Cloning
The Thermocycler
Essentially, a very fancy oven that can move between specific temperatures very quickly.
GFP Expression in these bacteria identifies successful transformants.
Ampicillin resistance identifies all bacteria who took in the plasmid.
The development of GFP as a reporter gene has spawned a variety of applications outside of genetic engineering.

Shown here: various flourescent proteins showing gene expression during Drosophila development.

Nobel Prize in 2008.
Agrobacterium is a bacterium that naturally inserts a plasmid (the "
Ti plasmid
") into plant cells during it's life cycle.

The Agrobacterium plasmid induces the formation of a tumor in the plant, where the bacterium lives.

By engineering the Ti plasmid, scientists can introduce novel genes into plants.
This is a "
gene gun
", which can be used to shoot micro-pellets coated with DNA into a eukaryotic cell's nucleus.
This goat produces a human blood clotting protein in its milk, an example of what is called a "
pharm animal
".
Golden Rice
is an engineered variety of rice that expresses beta carotene (a precursor of vitamin A) in its grain.

1-2 million people die of vitamin A defficiency every year
The gene "
cassette
" that was constructed to create Golden Rice.
xkcd.com
CC, the first cloned cat
The Banding Pattern is visualized with DNA stains (ethidium bromide shown)
Steps of PCR:
Old School:
Making Recombinant DNA Molecules
PCR
PCR!
PCR!?!
Sanger Sequencing
DNA Microarrays
https://www.neb.com/products/restriction-endonucleases
By comparing enough of these areas, identity can be confirmed or ruled out.
Two Major Questions:
What is the genetic material (DNA or Protein)?
What is the structure of the genetic material?
Full transcript