Download Flip Folder 6 KEY - Madison County Schools

Flip Folder #6 - Unit 6: Molecular Biology KEY
1. DNA Discovery
a. Griffith – experiment summary
Conclusion: Transformation (The transfer of genetic material from one cell to another cell or from one
organism to another organism) can occur
b. Hershey and Chase – phages - experiment summary
DNA, and not proteins, is the hereditary material.
c. Watson, Crick, and Franklin - contributions to discovery of DNA structure
Double Helix
d. Wilkins and Franklin
X-ray crystallography (picture of DNA)
2. Structure of DNA
a. Double helix
b. Nucleotide Structure
c. Chargaff’s Rule
In all living organisms, % of A=% of T and % of C=% of G
d. Pyrimidines, purines
1. What are pyrimidines and what are the examples?
Single ringed bases; C, T, and U
2. What are purines and what are the examples?
Double ringed bases; A and G (Purines Are Good)
e. Antiparallel
One strand runs 5’ to 3’ while the other runs 3’ to 5’
f. Semiconservative model
Each of the original sides of the DNA double helix serve as a template to build an entirely new double helix. The
new double helix has one of the original strands and one new strand (1/2 old and ½ new).
g. Telomeres, why do we need them?
Non-coding, repeating sequences at the end of a chromosome. The lagging strand loses a small portion of DNA at
the end of each chromosome during each replication; therefore, the tip of the DNA of the chromosome is lost with
each division. The length of our telomeres determines how many times the cell can divide (longer telomeres = more
possible divisions).
This occurs when the last RNA primer is removed. DNA polymerase has to have a primer to begin
replication. Without one on the tip, DNA polymerase cannot add the complementary nucleotides.
i. Telomerase**
Makes more of the telomere. It is on during embryonic development (because our cells have to divide so many
times). They turn off once we are born. Cancer cells turn these back on (so they can continue dividing).
3. Prokaryote v Eukaryote DNA
a. Similarities and differences
Similarities
 Use same nucleotides (A, T, C, G)
 Same codon chart (AUG would code for the amino acid Methionine in both)
Differences
Prokaryotes
Eukaryotes
single, circular chromosome
multiple linear chromosomes
haploid (1 copy of their DNA)
eukaryotes diploid (pairs of homologous
chromosomes)
DNA in nucleoid region (transcription and translation
Nucleus (transcription in nucleus and translation in
happen in the cytoplasm)
cytoplasm)
No histone proteins
Histones
Control gene expression by operons
Control gene expression by DNA methylation, histone
acetylation, heterochromatin, RNA alternative splicing,
etc.
No introns
introns
b. Different levels of Eukaryotic DNA packing
i. Nucleosomes
The nucleosome is the fundamental subunit of chromatin. Each nucleosome is composed of a little less than two
turns of DNA wrapped around a set of eight proteins called histones.
ii. Histones and chromatin
DNA is loosely associated with histones.
iii. Histones and Chromosomes
Chromosomes are tightly wrapped around histone proteins. This form of DNA cannot be used.
iv. Euchromatin
DNA that is not wrapped around histones are called euchromatin (true chromatin) because it can be used.
v. Heterochromatin
DNA wrapped around histones is called heterochromatin (different chromatin) because it can’t be used.
4. DNA synthesis
a. Origins of Replication – DNA sequences where DNA polymerase will bind and start replicating.
i. replication bubbles
Replication is occurring at many points along the DNA strand (to speed up the process). The bubbles eventually
connect.
ii. antiparallel Elongation
If the leading strand is elongating to the left on the top strand then the lagging strand is elongating to the right on the
bottom strand.
b. leading strand, lagging strand
Both are involved in DNA replication. Leading strand adds continuously to the 3’ end toward the
replication fork. The lagging strand must add to the 3’ end away from the replication fork; therefore,
it is built in segments that are then joined together by DNA ligase.
c. Okazaki fragments
Fragment on lagging strand that occur because the lagging strand has to wait for helicase to open the replication fork
and then work backward. These gaps occur when the RNA primer is removed.
d. Ligase
“Links” Okazaki fragments on lagging strand together by adding complementary nucleotides to gaps.
e. 5’ – 3’ elongation
Polymerase can only add nucleotides to the 3’ end of a DNA strand. This is because Phosphate is in the way on the
5’ end. There is a hydroxyl group (-OH) on the 3’ end. Remember that covalent bonds are made by dehydration
(remove OH from one molecule and H from another to remove water/create covalent bond).
5. Other proteins involved in DNA synthesis
a. Helicase
separates strands of DNA by breaking hydrogen bonds between bases
b. Topoisomerase
Enzyme that clips only a couple base pairs in front of helicase to prevent the DNA double helix from “kinking” or
“twisting” too much in front of the replication fork.
c. Single-strand binding proteins
The 2 strands of a double helix bind together by hydrogen bonds between the bases. These bonds are essentially like
magnets (weak bonds between opposing charges). Single strand binding proteins hold the single strands away from
each other so they don’t reanneal behind helicase.
d. Primase, RNA primer
DNA polymerase does not actually create bonds BETWEEN STRANDS (i.e. the hydrogen bond between A and T).
Those occur naturally. It actually the phosphodiester (covalent) bonds between the 3’ carbon and the phosphate of
nucleotides in a single strand; therefore, it must have a primer down to begin building (primase puts down this
primer). RNA is used for the primer because it is eventually removed (remember RNA is a cheap copy).
Polymerase reads the other strand to determine what complementary base that it should add.
e. Ligase
“Links” Okazaki fragments on lagging strand together by adding complementary nucleotides to gaps.
6. Central dogma (flow of genetic information): DNA  RNA  proteins
a. Transcription
a. What is it? DNA to mRNA (just a cheap copy of DNA) by RNA Polymerase
b. Where does it happen? nucleus (because DNA is involved)
c. Why does it happen? DNA is too important to take out of the nucleus and risk it getting messed
up. Also, DNA is too big to get out of the nucleus.
b. Translation
a. What is it? mRNA to proteins
b. Where does it happen? ribosomes (in the cytoplasm or rough ER)
c. Why does it happen? to make proteins that do all of the work in the cell
i. universal code**
If the DNA of one organism is placed into another, the new organism will create the same proteins (Which is
why recombinant organisms is possible). The “DNA code” is the same for all organisms.
ii. Start and stop Codons**
a. AUG = start (or codes for Methionine if it is in the middle of the RNA strand)
b. UAA, UAG, UGA = stop. These all code for a Release factor (enzyme) enters the A site causing a
hydrolysis reaction to occur that releases the protein from the last tRNA molecule (which is sitting in
the P site).
a. After the hydrolysis reaction occurs, the ribosome detaches and the sub units separate to be
reused.
iii. redundancy in code: Give example**
UCA and UCG both code for the amino acid serine.
7. RNA
a. Structure
Built from RNA nucleotides. Same as DNA nucleotide but ribose sugar and uracil instead of thymine nitrogenous
base.
b. Base Pairing
A to U and C to G
c. mRNA, tRNA, rRNA
 Messenger RNA = takes code for making proteins from DNA in nucleus to ribosomes in cytoplasm. This is
the blueprint at the construction site (DNA is the blueprint at the office)
 Transfer RNA = transfers amino acids from the cytoplasm to the ribosomes to be added to the growing
protein. These are the delivery truckers.
 Ribosomal RNA = Ribosomes are made of rRNA and proteins. These read mRNA and build proteins.
These are the construction site.
d. RNAi
RNA interference: These are essentially responsible for degrading mRNA so that the protein/enzyme it codes for
isn’t made after the body no longer needs it.
8. The Genetic Code
a. Codons and reading frame
1. Answer the following questions concerning codons
a. Where are they located? mRNA
b. How many bases are they made of? 3
c. What do they “code” for? amino acid (monomer of protein)
d. Using the answer given in part c, how many of they does each codon “code” for? 1
b. Template strand vs. Non-template
Template strand is the DNA strand used to make the mRNA (so it is the template for building the mRNA).
Non-template is the side not used to make the mRNA.
c. Coding vs. Non-coding
Coding = non-template. This may seem backwards, but this is called the coding strand because it has the same
sequence as the mRNA (instead of T’s in place of U’s). This is because they are both complementary to the template
strand.
Non-coding = template
9. Transcription
a. RNA polymerase
Reads DNA and build RNA
b. Transcription Initiation
i. TATA box
ii. Transcription factors
I.
A protein called a Transcription Factor attaches to the TATA box to determine the direction the
“factory” will proceed. The TATA box is part of the promoter sequence. (Look at the TATA sequence,
can you see it running in different directions. This orients the “factory” to the direction it will transcribe.)
II.
Then additional transcription factors (proteins and enzymes) are added to the “factory”.
III.
Finally, RNA Polymerase II joins to complete the factory. The whole “factory” is called a Transcription
Initiation Complex. (Can you see the definition in the term? Transcription is the process being done.
Initiation refers to the beginning process. Complex indicates we have many parts involved in making the
structure.)
IV.
This is a step by step controlled process. (The cell “controls” each step to help make sure nothing goes
wrong.)
c. Transcription Elongation
i. 5’ – 3’
RNA is built 5’ to 3’ just like DNA.
10. Modification of mRNA
a. Poly-A tail
Back end (3’) modification of the mRNA molecule.
a. A Poly A Tail is added. (“poly” means “many”; 50-250 Adenines will be added onto the tail.)
b. This acts as protection against digestive enzymes in the cytoplasm. (Remember, it is a construction site
and things are being broken down as well as being built.)
b. 5’ Cap
Front end (5’) modification of the mRNA molecule.
a. A 5’ protective cap is added. (This would be like you putting on a hard hat to protect your head when you
go outside into a “construction site”.)
b. This cap acts as a signal to the ribosome particles, telling it where to attach.
c. Internal modification of internal mRNA
i. Splicesomes
During this step, remove the non-coding introns (These act as spacers) using Spliceosomes. (A spliceosome is a
collection of snRNPs.)
i. snRNP’s (small nuclear ribonucleic proteins act as scissors.) Remember, Ribozymes are RNA
molecules that act as enzymes.
ii. snRNP
snRNP’s (small nuclear ribonucleic proteins act as scissors.) Remember, Ribozymes are RNA molecules that act as
enzymes.
iii. Introns, exons
Introns are the non-coding sections (not translated into proteins) section of DNA. These acts as spacers. They stay
inside the nucleus.
The coding sections that exit the nucleus are called exons. They are rearrange the separated coding exons
(important blueprint pieces.) to the needed configuration. This is why it is called alternative; pieces (exons) can be
rearranged in different orders. (This is REALLY important in the making of antibodies by our immune system.
Remember the variable portion of the antibody structure.)
iv. Alternative mRNA splicing
Modification of the Primary Transcript for EUKARYOTIC Cells (This also occurs in the nucleus.)
1. Middle modification of the mRNA molecule. (This modification is referred to as RNA
Alternative Splicing.)
a. “Stitch” the pieces together to make the finalized secondary mRNA transcript
that is know ready for transport to the ribosomes for translation into proteins.
2. One mRNA’s exons can be put together in different orders to code for different proteins.
(123, 132, 231, etc.)
11. Translation (Molecular Components)
a. tRNA
The amino acid is connected to the 3’ end of the tRNA molecule.
A. Remember, the tRNA molecule is a nucleotide sequence; so there is a phosphate on the 5’ end and an
open bond on the 3’ end… so this is where the amino acid gets attached so that it can be transported to
the ribosome (construction site).
b. aminoacyl-tRNA synthetase
This connection between the tRNA molecule and the amino acid is constructed using the Aminoacyl – tRNA
synthetase enzyme.
c. wobble and third base of codons
45 different tRNAs are used for 61 possible codons. Inosine (acts as a “wild card”) makes it possible for
a cell to conserve materials and energy.
II.
The use of Inosine creates the “Wobble effect” - It does not fit perfectly, but gets the job done.
III.
Inosine is found in the third slot of the anticodon only.
A. Remember, that the ANTICODON is found on the tRNA molecule, NOT the mRNA.
I.
d. Codons, Anti-Codons
The Anticodon “matches” the codon on the mRNA molecule ensuring the correct amino acid is brought to the
construction site of the Ribosome. If they DO NOT match … it is the wrong Amino Acid!
12. Translation Stages
a. Initiation - This is building the factory needed to make the protein.
i. AUG
I.
The small sub-unit attaches to the 5’ cap. This interaction signals the large sub unit.
II.
AUG (the start codon on the mRNA molecule) brings in the tRNA (using the anticodon) molecule with
Methionine attached. This starts production of our protein.
ii. GTP for energy**
Then the large sub-unit is brought in using initiation factors (these are enzymes) and uses GTP for energy in the
process. (Remember, GTP is “like” ATP…both are energy molecules.)
iii. 5’-3’ polypeptide elongation**
The large sub-unit is aligned so that Methionine is in the P site. The A site is open for the addition of the next
tRNA molecule.
I.
II.
III.
IV.
V.
VI.
b. Translation Elongation
i. APE sites
The Small sub-unit - This part acts as a platform for work; much like your desk.
The Large sub-unit - This part is the factory for making the protein.
The A site - This is where the next tRNA molecule is ADDED in the “factory”.
The P site - This is the part of the “factory” where the PROTEIN is attached.
The E site - This is where the “used tRNA molecule” EXITS the “factory” to be reused.
Remember, these are NOT organelles. All cells possess these structures.
I.
ii. Peptide bond formation
The ribosome “walks” down the mRNA one codon at a time until it gets to the stop codon at the end of the
mRNA molecule. Thus having completed the “message” on how to make that particular protein. This
“walking” is called Translocation. (Can you see function in the name?)
c. Translation Termination
i. stop codons
I.
This occurs when a termination codon reaches the A site.
ii. release factor
I.
A Release factor (enzyme) enters the A site causing a hydrolysis reaction to occur that releases the
protein from the last tRNA molecule (which is sitting in the P site).
II.
After the hydrolysis reaction occurs, the ribosome detaches and the sub units separate to be reused.
13. Mutations
a. Prevention of mutations - Proofreading and Repairing DNA
i. nuclease
Enzyme that cuts out mismatched nucleotides.
ii. nucleotide excision repair
b. Substitutions: missense, nonsense, silent
POINT mutations - A single nucleotide mutates thus affecting a single codon.
 Silent Point Mutation– The mutation causes no change in the amino acid coded for.
(We would never know because it has no effect. This can happen because the codon coding is redundant,
remember?)
 Missense Point Mutation – The mutation changes the amino acid coded for. (MIStake)
(This is best seen in the mutation that causes Sickle cell.)
 Nonsense Point Mutation – The mutation changes from coding for an amino acid to coding
for a STOP codon. NO protein will be made. (NO sense)
c. Frameshift -Insertions and deletions
These mutations alter the codon sequence.
 Insertion – adding nucleotides to the sequence.
For Example: THE BIG TAN DOG RAN
with Inserted Letter: THE BOI GTA NDO GRA N
 Deletion – taking out nucleotides from the sequence.
For Example: THE BIG TAN DOG RAN
with Deleted Letter: THE BGT AND OGR AN
14. DNA Mutation Diseases – cause and effect
a. PKU
PKU, is a rare inherited disorder that causes an amino acid called phenylalanine to build up in your body because
your body cannot metabolize it.
b. Sickle Cell – Heterozygous Advantage
a. The 6th Amino Acid in the hemoglobin molecule is changed (Glutein  Valine) in the primary sequence
needed to make red blood cells. (The easy way to remember this is: 666 is the number of the beast. 6 is
the amino acid that changed to create this horrible disease. It went from good [glutein] to very bad
[valine].)
b. Sickle- cell trait (“trait” is used to refer to individuals that are carriers.)
i.
These individuals have resistance to Malaria because of the one recessive allele they
possess but mainly have normal red blood cells for carrying oxygen.
ii.
This is referred to as the Heterozygote Advantage. They have an advantage over
individuals that are homozygous dominant or homozygous recessive. Homozygous
dominant are not resistant to Malaria. Homozygous recessive are also resistant to Malaria;
BUT they have the disease to contend with.
c. These sickle shaped cells have reduced oxygen carrying ability. They also are painful when the points of
the cell jab into the walls of the blood vessels.
c. Cystic Fibrosis
The disorder creates a faulty Chloride ion (Cl-) protein carrier on cell membranes in the lungs. This causes fluid
(water) to build up in the lung tissues.
i.
People drown in their own fluid.
ii.
They are also prone to get multiple infections in the lungs.
15. Structure of Viruses
a. Capsids and envelopes
Viruses are made from 2 components – protein capsid (coat) for movement/attachment AND an interior nucleic
acid (DNA or RNA).
Some have a membrane envelope which they get from their host as they exit the cell. It is essentially a cloak
covering the virus so they have the glycoproteins of the host (and the immune system cannot detect that there is
a virus inside).
b. Genetic information
DNA or RNA (retroviruses) – This is what they inject into you to infect your cell (and make you make more
viruses).
c. phages
Virus that infects bacteria. Used in Hershey-Chase experiments.
16. Virus life cycles
a. Lytic Life Cycles
i. virulency
Virus infects host cell by injecting DNA/RNA. Makes host cell copy their nucleic acid (DNA or RNA) using their
DNA/RNA polymerase. Makes host cell make more of their protein capsid using the host cells ribosomes. The cell
eventually overfills with viruses, lyses, and the virus is released.
ii. restriction enzymes
Virus infects bacteria by injecting their DNA. Bacteria have restriction enzymes to cut up the viral DNA before it
can take over the cell.
iii. Herpes/Chicken Pox/Shingles**
All are viruses that work by using a lytic life cycle.
b. Lysogenic Life Cycles
Lysogenic cycle – does not destroy host cell immediately (can remain dormant)
• Virus then reproduced each time cell replicates
i. temperate phages
Temperate phages are basically bacteriophages which can choose between a lytic and lysogenic pathway of
development. The lytic pathway is similar to this of virulent phages. In the lysogenic pathway virus remains dormant
until induction.
ii. prophage
• Virus DNA inserted into host cells DNA, phase DNA called prophage
17. HIV
a. HIV**
• RNA virus that infects Helper T Cells. A retrovirus uses mRNA as its genetic material as well. The newly made
“viral DNA” is then incorporated into the host cells genome.
• The host cell then transcribes and translates the viral DNA every time it does the same processes for itself.
b. AIDS**
Outwardly the AIDS virus looks like the flu or mumps (membranous envelope and glycoprotein spikes)
Inwardly, it has 2 identical copies of its RNA and reverse transcriptase enzymes.
People who have AIDS don’t die from AIDS. They die from their specific immune system (lymphocytes – B,
Helper T, Cytotoxic T cells) not working; therefore, they die from the common cold or some other virus.
•
c. Reverse transcriptase
The retrovirus uses its own reverse transcriptase enzyme to make DNA from its mRNA (the reverse of the usual
transcription process, hence the “retro”virus)
18. Viroids and Prions
a. Viroids
These are naked, infectious RNA molecules. They attack plants only. (“oid” means “like”… they are “like” viruses
as they are infectious.)
b. Prions
These are infectious proteins. Mad Cow is one example. It destroys brain cells thus driving the cow “mad” until it
dies. The human version is Kruetzfeldt-Jacob Disease. (KJD)
19. Generating genetic variation in bacteria
a. transformation
Transformation – uptake of DNA from surrounding other cells or other organisms (Griffith’s expt)
• Bacterial DNA
• They possess one circular strand of DNA that is located in the nucleoid region.
• In addition to this strand, they also have Plasmids.
• These are small, circular, exchangeable pieces of DNA.
• These are in addition the main large circular DNA strand.
• These help to increase variation and survival.
b. transduction
Transduction – bacteriophage (virus) DNA taken up by bacteria (Hershey and Chase)
c. Conjugation and Plasmids
i. Conjugation and plasmids
Conjugation – bacterial cells mating; Male has sex pili which attaches to female, outside layers of cells fuse and
create cytoplasmic bridge, DNA from male “donor” cell passes to female and male copies his DNA as it transfers so
he doesn’t lose any genes. They transfer plasmids.
ii. F plasmids
F factor – has genes to make sex pili and other things necessary for conjugation as well as having an origin of
replication
iii. R plasmids
R plasmids – These transfer antibiotic resistance between bacteria.
20. Transposition of Genetic Elements
a. Prokaryotes
i. transposition
DNA moving from one position to another (either in one chromosome or from one chromosome to another)
ii. transposons
A transposable element (TE or transposon) is a DNA sequence that can change its position within a genome,
sometimes creating or reversing mutations and altering the cell's genome size. For prokaryotes, these typically
function to block transcription.
a. Basic Insertion
i. This is the simplest form.
ii. Transposase – enzyme that allows the DNA to “jump” from location to location.
b. Composite (means “complex”)
i. Transposase on both sides of a resistance gene are “jumping” as a unit.
b. Eukaryotes
i. transposons
Same basic thing as in prokaryotes
ii. retrotransposons
First, they are transcribed from DNA to RNA, and the RNA produced is then reverse transcribed to DNA. This
copied DNA is then inserted back into the genome at a new position. The reverse transcription step is catalyzed by a
reverse transcriptase, which is often encoded by the TE itself. The characteristics of retrotransposons are similar to
retroviruses, such as HIV.
iii. multigene families
Used to include groups of genes from the same organism that encode proteins with similar sequences either over
their full lengths or limited to a specific domain. DNA duplications can generate gene pairs.
iv. pseudogenes
Pseudo = false. So these are false genes.
Functionless relatives of genes that have lost their gene expression in the cell or their ability to code protein.
Pseudogenes often result from the accumulation of multiple mutations within a gene whose product is not required
for the survival of the organism.
21. Prokaryotic control of gene expression - Operons
a. general structures: promoter, operator, repressor protein, regulatory gene
Operon - A group of prokaryotic genes with a related function that are often grouped and transcribed together. In
addition, the operon has only one promoter region for the entire operon.
Promoter-The nucleotide sequence that can bind with RNA polymerase to start transcription. This sequence also
contains the operator region.
Operator-The nucleotide sequence that can bind with repressor protein to inhibit transcription.
Regulatory protein – Protein made by the regulatory gene that turns the operator on or off.
Regulatory gene- Gene that lies outside the operon region and produces a protein called a repressor that can inhibit
the transcription of an operon by attaching to the operator.
Structural genes- genes that are related and code for related enzymes in a biochemical pathway.
b. trp operon
Bacteria producing the amino acid tryptophan.
c. lac operon
Bacteria making lactase to break down lactose when glucose is not present (so they need to break lactose down to
get glucose to do cellular respiration to make ATP).
d. repressible vs. inducible operons**
Repressible = normally on, has to be turned off. Ex. Trp operon. This is because the bacteria generally needs this
amino acid.
Inducible = normally off, has to be turned on. Ex. Lac operon. This is because the bacteria only needs to make
lactase when GLUCOSE IS ABSENT AND LACTOSE IS PRESENT.
22. Eukaryotic control of gene expression
a. Pre-transcriptional regulation
i. Enhancers
I.
Enhancers and Activators - These help control the rate of transcription. They are segments of DNA
that basically “grab” the factory, using a bending protein, and move it down the DNA faster thus
enhancing the process of transcription. They are “Pushers”.
A. They are always in front of gene to be transcribed.
ii. Histone acetylation
This is the attaching of acetyl (COCH3 ) groups to the histones lysine amino acids.
This attaching breaks the bond between the DNA and the histones by covering up the positive charges
thus creating NO attraction for each other.
III.
This allows for RNA Polymerase and transcription factors to attach to the “freed” DNA so that
transcription may occur.
I.
II.
1.
2.
iii. Methylation
This refers to putting a heavy “coat” of methyl (CH3 ) groups of the DNA, thus preventing transcription from
occurring. The Methyl groups attach to Cytosine or Adenine nucleotides.
This is the source of Genomic Imprinting that occurs in gamete production. It essentially “erases” information”.
iv. Epigenetics
The term epigenetics refers to heritable changes in gene expression (active versus inactive genes) that does not
involve changes to the underlying DNA sequence; a change in phenotype without a change in genotype.
Epi = outside. So basically this is DNA methylation and histone acetylation.
v. Activators and repressors
Activators – bind to enhancer to turn it on
Repressor or Silencer - These control proteins sit on the TATA box – they prevent transcription from occurring.
This silences or represses the gene from being expressed. Remember the TATA box is where RNA Polymerase
binds. Repressor block the access.
b. Post-transcriptional regulation
i. RNA processing (5’cap, poly-AAA tail, Alternative splicing)
ii. miRNA
Micro RNA (miRNA) and small interfering RNA (siRNA) – together these are RNAi.
1. These are little pieces of RNA that attach to mRNA and thus control transcription of the mRNA.
2. They degrade mRNA
iii. proteosomes
Proteosomes (special protein digesting Lysosomes) control HOW LONG the protein lasts
c. Post-translational modification of polypeptides
i. protein folding and locations of folding
I.
POST (means “after”) Translation Modification (This is the protein folding that must occur for the
protein to be functional.)
A. If the 1’ sequence enters a Chaperonin, the protein will stay inside the cell.
1. Entry is “guarded” by a Signal Recognition Particle (SRP) inside the bottom piece.
B. If the 1’ sequence enters the RER, the protein will be exported out of the cell.
1. Signal Peptide on the 1’ sequence. (This acts as a siren. It is “like” yelling “Take me to
the RER!”)
2. Signal Recognition Particle (SRP) - This particle acts as a guide leading the 1’
sequence to the RER. It attaches to the Signal.
23. Genetic Engineering
a. Plasmids
This process involves the bacterial plasmids and another DNA source.
A plasmid is a small ring of DNA found in bacteria in addition to the main large circular DNA strand found in the
nucleoid region.
I.
b. Restrictions Enzymes
Restriction Enzymes are used to cut a DNA plasmid and DNA from other donor source.
1. Restriction enzymes cut DNA at specific nucleotide sequences.
The specific DNA sequence is referred to as the Restriction Site.
2.
3.
4.
c. Vector
Genes of interest (and antibiotic resistance genes) are cut into Restriction Fragments.
When the DNA is cut, “Sticky Ends”, or single-stranded DNA ends are created.
The same restriction enzyme must be used on both the plasmid and the DNA donor source.
a. Therefore, the “sticky ends” of the plasmid and the genes of interest will match and
can be joined.
Vector is a carrier of a desired trait. Bacteria that contain a plasmid with recombinant DNA would be a vector
because they carry a desired trait.
d. GMO/GMF
Genetically modified organisms or food. Contain recombinant DNA from procedure described above.
e. Cloned Animals
In reproductive cloning, researchers remove a mature somatic cell, such as a skin cell, from an animal that they wish
to copy. They then transfer the DNA of the donor animal's somatic cell into an egg cell, or oocyte, that has had
its own DNA-containing nucleus removed.
24. Techniques
a. PCR
Polymerase Chain Reaction (PCR) – This process requires no organism in the production of new
DNA
molecules.
1.
The process is used to turn a single molecule of DNA into a large, workable sample of
100% identical DNA molecules. This is widely used in criminal forensics (Murder cases).
2. Nobel Prize is the top award a scientist can receive for their research. It would be like an
MVP award in sports or an Oscar for actors or a Grammy for singers.
B. Process
1. The DNA sample is placed in a PCR Thermal Cycler machine.
a. The machine uses heat, DNA Primers, enzymes and a constant supply of
nucleosides to build new DNA molecules that are identical in nucleotide
sequence to the original molecule.
b.
c.
d.
e.
f.
g.
First step: Heat is used to separate the DNA double helix so that replication can
occur.
Second step: The attachment of a DNA Primer to the template DNA strand will
start replication.
Third step: The DNA polymerase enzyme works 5’3’ attaching nucleosides to
the growing “new” side of the replicated DNA molecule.
Fourth step: Cool the mixture to recombine and stabilize the DNA back into a
double strand.
Repeat the cycle many more times to get large, workable sample of the DNA.
Analyze the amplified DNA fragments.
b. Electrophoresis
This process is used to create a “DNA fingerprint”.
A. Different individual’s DNA samples, but from the same region of a chromosome are exposed to the same
restriction enzyme.
1. This creates Restriction Fragment Length Polymorphisms (RFLP’s)
a. These are fragments of DNA having different lengths that were created using
restriction enzymes. (Can you see that in the term?)
B. The DNA RFLP’s are loaded into an agarose gel.
C. Turn on the electricity. (Remember, DNA is negatively charged because of the phosphate backbone, so it will
be repelled on the negative end [Black] and pulled by the positive end [Red].) (Electricity should flow from the
Black  Red strips when performing this process.)
D. The RFLP’s will separate according to length/size of the fragments.
1. Big pieces move slowly through the gel.
Small pieces move quickly through the gel.
E. The DNA fragments are stained for ease of viewing.
F. The DNA bands create a unique “fingerprint” of the individual’s DNA.
c. DNA sequencing
DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes
any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and
thymine—in a strand of DNA.
I.
DNA Nucleotide Sequencing processes were involved in the above project.
A. DNA Microarray Assay (This process looks like a Light-Brite toy.)
1. Uses radioactively labeled and colored nucleotides to create a visible sequence on a
monitor.
2. Follow the colored sequence to determine the DNA sequence. (For example, red =
adenine)
B. Dideoxy Chain Termination Method
1. Run like a PCR, but has special Dideoxyribonucleotides added to the mix. (This stops
replication.)
2. The procedure produces chains of different length due to termination by dideoxy
nucleotide.
3. Then the fragments are run through a gel and scanned using a laser to identify the
dideoxy.
4. Pieces are then combined together using the Dideoxy hits to “create” the nucleotide
sequence.
d. RFLP (Restriction Length Polymorphism)
This creates Restriction Fragment Length Polymorphisms (RFLP’s)
These are fragments of DNA having different lengths that were created using restriction enzymes. (Can you
see that in the term?)