SBI4UAP | Unit 3

DNA Structure, Replication, Protein

Synthesis & Recombinant DNA Technology

MOLECULAR
GENETICS

Learning Objectives: In this unit, you will learn…

1) The experimental history tied to the understanding of DNA structure and function.
2) The structure of DNA and RNA, including “complementary” and “antiparallel” properties.
3) The organization of DNA (genome sets, gene structure and nucleosome packaging).
4) The DNA replication and repair process, and artificial replication techniques (PCR).
5) The mechanism of protein synthesis from DNA to a functional protein.
6) The decoding of DNA and the protein sequence.
7) The types of point and frameshift mutations and their impact on proteins.
8) The regulation of gene expression in bacteria (operon model) and eukaryotes.
9) The applications of genetic engineering and a discussion of their benefits and concerns.
10) The use of restriction enzymes and plasmids in recombinant DNA technology.
11) The application of techniques including PCR, electrophoresis and RFLP.
12) The techniques part of the Human Genome Project and the use of DNA databases.

Name: _____________________ Teacher: ______________

 Lesson ● Eukaryotic Regulation
Unit 3 - Molecular Genetics Outline 8 ● Epigenetics

Lesson
9 “OF” Test – DNA Structure, Replication, Protein Synthesis & Regulation (mid-unit test)
TOPIC 3.1 | DNA DISCOVERY Homework & Assessments
Lesson ● DNA Discovery Scientists
TOPIC 3.6 | BIOTECHNOLOGY Homework & Assessments
1
Lesson ● Biotechnology Examples ***ASSIGNMENT Genetic Engineering
TOPIC 3.2 | DNA STRUCTURE & ORGANIZATION Homework & Assessments 10 ● Genetic Engineering Application Presentation (“of learning”)
Lesson ● DNA Structure & Function DNA Structure Practice Problems
2 ● RNA Structure & Function Lesson ● Genetic Engineering Presentations ***Genetic Engineering Presentation is Due!
● Organization of DNA ***FOR QUIZ: DNA Structure 11

Lab – DNA Extraction

Activity – DNA Modelling TOPIC 3.7 | RECOMBINANT DNA Homework & Assessments
Lesson ● Restriction Enzymes Recombinant Practice Problems
TOPIC 3.3 | DNA REPLICATION Homework & Assessments 12 ● Plasmid Structure
Lesson ● Models of DNA Replication ***FOR QUIZ: DNA Replication ● Gel Electrophoresis
3 ● Meselson-Stahl Experiment
Lesson Activity – Paper Plasmid Paper Plasmid Activity
***ASSIGNMENT DNA Replication Problem 13
Set (“of learning”)
Lesson Lab – Electrophoresis ***LAB Electrophoresis (“of learning”)
Lesson ● DNA Replication & Repair PCR Fill-in-the Blank Review 14
4 ● Polymerase Chain Reaction
***DNA Replication Problem Set is Due!
Demo – PCR TOPIC 3.8 | BACTERIAL VECTOR CLONING Homework & Assessments
Lesson ● Bacterial Vector Cloning ***Electrophoresis Lab is Due!
TOPIC 3.4 | PROTEIN SYNTHESIS Homework & Assessments 15 ● Nucleic Acid Hybridization
Lesson ● Central Dogma Protein Synthesis Practice Proteins Lesson Lab – pGLO Transformation ***LAB pGLO (“of learning”)
4 ● Genetic Code Mutations Practice Proteins 16
Activity – Connecting the Dots to
Disease Genome Database Mining

Lesson ● Transcription Details ***ASSIGNMENT Protein Synthesis Practice

5 ● Translation Details Problems (“of learning”)

Lesson ● Protein Synthesis Review ***Protein Synthesis Problem Set is Due!

6

TOPIC 3.5 | GENE REGULATION Homework & Assessments

Lesson ● Lac Operon EdPuzzle “Operon Modelling”

7 ● Trp Operon

Activity – Playdoh Operon Modelling

 3.1 DNA discovery
Miescher (1869)
Miescher examined pus-soaked bandages
of injured patients and discovered
that the nuclei of white blood cells
contained a substance with large
molecules composed of phosphorus and
nitrogen, which he first called
nuclein because it seemed to come
from cell nuclei. He succeeded in
extracting the protein and acid
components out, and called it nucleic
acid.

Griffith (1928)
Griffith studied the
bacteria responsible for a
pneumonia epidemic in
London. He performed an
experiment using bacteria
and mice that hinted that
DNA was the genetic code
material. He used 2 strains
of pneumonia-inducing
bacteria (virulent and
non-virulent) and injected
mice to discover the
principle of transformation
in bacteria.
Levene (1935)
A Russian-born American chemist who characterized the structure of
DNA and RNA, including their different sugars, bases and how the
three parts of a nucleotide were joined and then strung together in
nucleic acids. He (incorrectly) proposed the tetranucleotide model.

Avery, MacLeod and McCarty (1944)

Set up treatments, each selectively degrading protein, RNA and DNA;
injected mice like Griffiths experiment. Suggested that DNA was the
transforming agent in bacteria, thereby presenting the first
experimental evidence that DNA was hereditary material.
Chargaff (1949)
Purines pair with pyrimidine nitrogen bases (A & T pair and G & C
pair)…called “Chargaff’s Rule”. Evidence based on comparative
studies of organisms and their nitrogen base ratio patterns.

Hershey and Chase (1952)

Used radioactively-labelled viruses (bacteriophages) to infect
bacteria. Observed that the infected bacterial cells contained
radioactively labelled P originating from the viral DNA. Suggested
that DNA is hereditary material in viruses that reprogram the
infected cell.
Pauling (1953)
Pauling was the titan of 20th-century chemistry who was the world
expert on chemical bonding and led the way in working out the
structure of big biological molecules. Working without X-ray
pictures, he published a paper suggesting that DNA was a “triple
helix” that had nitrogen bases to the exterior and sugar-phosphate
groups on the interior based on molecular modelling. He failed to
take into account that DNA is acidic so phosphate groups could not
be neutralized and that phosphate groups would repel in close
proximity.

Watson and Crick (1953)

Francis Crick trained and worked as a physicist, but switched to

biology after WWII. After co-discovering the structure of DNA, he
went on to crack the genetic code that translates DNA into protein.
James Watson went to Chicago University at age 15, and teamed up
with Crick in Cambridge in late 1951. After solving the double
helix, he went on to work on viruses and RNA, another genetic
information carrier. He also
helped launch the human genome
project.

They deciphered the DNA double

helix structure using a molecular
modelling approach, with the
final model aided by Franklin’s
“photo 51” to determine that DNA
was a double helix structure.
Wilkins and Franklin (1953)

Franklin trained as a chemist and was an

expert in deducing the structure of
molecules by firing X-rays through them.
Her images of DNA - disclosed without
her knowledge - put Watson and Crick on
the track toward the right structure.
She went on to do pioneering work on the
structures of viruses. The X-ray
diffraction technique involved purifying
DNA and reconstructing a 3D model of DNA
through painstaking
measurements.

Wilkins was trained as a

physicist and was involved
with the Manhattan Project
to build the nuclear bomb.
Wilkins worked with
Franklin in the same lab
at King's College London,
although their
relationship was strained.
He helped to verify Watson
and Crick's model, and
shared the 1962 Nobel with
them.
 3.2 DNA STRUCTURE & ORGANIZATION
NUCLEIC ACID STRUCTURE
▪ Two kinds of nucleic acids:

1) Deoxyribose nucleic acid (DNA) contains hereditary information.

2) Ribose nucleic acid (RNA) functions in protein synthesis.

▪ A nucleic acid subunit is a NUCLEOTIDE, consisting of 3 parts:

1) phosphate group Label the following bonds:

2) pentose sugar
❑ Phosphodiester bond
3) nitrogen base
❑ Glycosyl bond
Types of nucleotide sugars:

DEOXYRIBOSE (in DNA) RIBOSE (in RNA)

Types of nitrogen bases:

PYRIMIDINES

PURINES
Structural Differences Between DNA & RNA:
i) ______________________________________________________________

ii) ______________________________________________________________

iii) ______________________________________________________________

DNA PROPERTIES
● DNA is ______________________ meaning it twists in a counter-clockwise direction.
 CHARGAFF’S
● DNA is ____________________
BASE-PAIR RULES
meaning single strands of DNA are held
together by hydrogen bonds between A pairs with ____ via ____ H-bonds.

matching base pairs. C pairs with ____ via ____ H-bonds.

● DNA is ______________ meaning single strands of DNA run opposite to one another.
If the percentage of A in a DNA strand is 22%, what is the percentage of G?

Which of the two DNA strands requires more energy to denature?

Strand A = GGCTAGGCCCAGTAAGGCCG
Strand B = ATATCGTTTAAAGGATCGAT

Identify which bases these are. WITHOUT NOTES!

 DNA Structure Practice Problems

1) In the diagram below:

a) Label the 3 and 5 position carbons on the deoxyribose.

b) Indicate the overall direction of the 3’ and 5’ strands.
c) Circle and label a pyrimidine and a purine base.
d) Label a phosphodiester and glycosyl bond.
e) Complete the following chart.

Found between…
Phosphodiester bond
Glycosyl bond
Hydrogen bond
2) State two things that are wrong in the DNA strand shown. Identify the bases by number.

3) Use the following chart to compare the structure and function of mRNA, tRNA and DNA.

mRNA tRNA DNA

General
Sketch

Structure

Function

4) In order to undergo processes like replication and protein synthesis, an array of proteins
interact with DNA. State why DNA-binding proteins have positively-charged regions.
 ORGANIZATION OF DNA IN EUKARYOTES
Eukaryotes can possess 2 or 3 genome sets:

NUCLEAR GENOME

Linear, double-stranded chromosomes.

MITOCHONDRIAL & CHLOROPLAST GENOMES

Mitochondria and chloroplasts have circular plasmids that capable of self-replicating and
include genes that are used in cell respiration (mitochondria) or photosynthesis (chloroplast).
 In prokaryotes, there is a single CIRCULAR bacterial chromosome. Most of the DNA in a genome
codes for protein (or tRNA and rRNA), with a small amount of noncoding DNA, primarily regulators.

In eukaryotes, chromosomes consist of LINEAR DNA. Most of the DNA (about 98.5% in humans)
does not code for protein or RNA and includes: regulatory sequences, introns and repetitive DNA.

EUKARYOTIC GENE STRUCTURE

__________________ = regulatory region found at start of gene

__________________ = regulatory region found at end of gene

__________________ = coding regions

__________________ = non-coding regions

 PACKAGING OF DNA
The appearance of DNA during:

METAPHASE INTERPHASE
Looped domains coil and fold to produce the Chromatin is much less condensed than
condensed form of the chromosome. the chromatin of mitosis.

▪ Eukaryotic chromosomes contain a great amount of DNA relative to their condensed

length…if each DNA molecule were extended, it would be 6 cm long! Now multiply
this by 45 more chromatids for a human cell!

▪ It all fits into the nucleus through an elaborate, multilevel system of DNA packing.

▪ Chromosomes contain a fibre of chromatin wrapped around packaging proteins

called ___________________ that form the “beads on a string” structure with the
basic packing unit called a ____________________.

NUCLEOSOME STRUCTURE

DNA wrapped around a central core of

eight histone proteins fastened by a
second type of histone (H1).
 3.3 DNA REPLICATION
The complementary nature of the DNA molecule gave scientists an idea of how DNA might
self-replicate and three hypotheses emerged:

SEMI-CONSERVATIVE REPLICATION
Watson and Crick hypothesized that DNA would be replicated semi-conservatively, but the
experimental evidence came from experiments conducted by Meselson and Stahl (1958):

They grew E. coli in a medium using ammonium ions (NH4+) as the

source of nitrogen for DNA, but this nitrogen was the “heavy”
isotope (15N). DNA was extracted from the first generation bacteria
and spun it in a centrifuge where the 15N “band” settled at a lower
position on a cesium chloride density gradient.

The bacteria were transferred to a medium containing the normal

15
N isotope and allowed them to divide just once to form the “first
generation”. The DNA in this new generation of cells was exactly
intermediate in density between that of the previous generation.

However, when the bacteria were allowed to divide again in normal

ammonium ions (14NH4+) in the second generation, the two distinct
densities of DNA were formed: half the DNA was normal and half
was intermediate.
The Meselson & Stahl Experiment Confirming
Semi-Conservative Replication (1958)
 STEPS OF DNA REPLICATION

STEP 1 - STRAND SEPARATION

The enzyme helicase is responsible for unzipping the DNA helix by breaking hydrogen
bonds between complementary bases so that the strands are available to copy. Special
proteins – single-stranded binding proteins (SSBPs) - bind to help stabilize the unwound
DNA strands to facilitate replication.

The helicase enzymes begin to work at areas called the origin of replication. There are
various origins of replication on DNA molecules. The helicase enzymes move away from
the origins of replications in both directions, exposing areas called replication bubbles,
where replication is progressing.
 STEP 2 - PRIMING

In order to initiate DNA replication,

the enzyme primase attaches a short
fragment of RNA called a primer to
the complementary strand. RNA
primers are needed in order for
further chain growth to happen.

STEP 3 - POLYMER SYNTHESIS

New DNA strands are formed in the 5’ to 3’ direction using the enzyme DNA polymerase
III which adds DNA nucleotides complementary to the template at the free 3’ end of the
growing chain, working first from the primer nucleotides.
 DNA Replication ALWAYS follows the 5’→ 3’ direction rule!

Because original strands of DNA are antiparallel, replication of each new strand cannot
occur in the same way. One strand is copied continuously, from the origin of replication, and
following the path of the helicase enzyme and the moving replication fork; this is called the
leading strand of replicated DNA.

The other strand is copied discontinuously, in many fragments (called “Okazaki fragments”)
that begin at the location of the helicase enzyme and replication fork and move toward the
origin of replication; this is called the lagging strand.

Nucleoside triphosphates (also called

deoxynucleotides) serve as building blocks
and energy molecules for DNA synthesis.
Two phosphate groups are released to
create a phosphodiester bond.
Successive fragments
form as the helicase
enzyme and replication
fork continue moving.
The fragments are
called Okazaki
fragments, and are
forming the lagging
strand of replicated
DNA.

STEP 4 – PRIMER REMOVAL

A summary of the enzymes involved in the completion of the lagging strand of replicated
DNA, including primer removal, is described in the diagram.
 KEY EVENTS IN DNA REPLICATION
1) STRAND SEPARATION
▪​ REPLICATION BUBBLE: Form when the double helix opens at a replication origin; replication
forks are Y-shaped regions forming at the initiation point where new strands grow.

▪​ GYRASES (TOPOISOMERASES): Enzymes that “relieve stress” by untwisting the tightly coiled
DNA.

▪​ HELICASES: Enzymes that unzip the double helix by breaking hydrogen bonds.

▪​ SINGLE-STRANDED BINDING PROTEINS (SSBPs): Proteins that bind to the

sugar-phosphate backbone. that keep the separated strands apart (keeps replication bubble open).

2) PRIMING
▪​ PRIMASE: Enzyme that adds short fragments of RNA PRIMERS are added to complementary
DNA; these RNA primers are needed to be in place in order for further chain growth to happen.

3) SYNTHESIS OF NEW DNA STRANDS

▪​ DNA POLYMERASE III: Enzyme that adds complementary nucleotides, beginning at the
primer, in the 5’ to 3’ direction; nucleotides are added only to the 3’ end of the growing strand.

▪​ NUCLEOSIDE TRIPHOSPHATES: Nucleotides that serve as building blocks and energy

molecules for DNA synthesis; energy from breaking two phosphate groups is used to create a
phosphodiester bond in the sugar-phosphate backbone.

▪​ LEADING STRAND: DNA strand synthesized continuously in 5’→ 3’ direction TOWARD the
replication fork.

▪​ LAGGING STRAND: DNA strand is discontinuously synthesized AGAINST overall direction of

replication, but still in the 5’→ 3’ direction; in the lagging strand, short fragments called
OKAZAKI FRAGMENTS are added between RNA primers.

4) REMOVAL OF PRIMERS
▪​ DNA POLYMERASE I: Removes RNA primers and replaces these regions with DNA bases.

▪​ DNA LIGASE: When DNA replaces RNA primer regions, “gaps” in the sugar-phosphate
backbone remain; DNA ligase scans the backbone and when it finds gaps, it recreates the
missing phosphodiester bond.

5) PROOFREADING
▪​ POLYMERASES I, II, III: Act as “proof-reading” enzymes called EXONUCLEASES which cut
out nucleotides when pairing errors occur and replacing with correct bases.
 STEP 5 - PROOF-READING & REPAIR OF DNA

Mutations (incorrect base pairing) occur at a

rate of one error per 10,000 base pairs, but
proofreading and repair lower the final error
rate to one per billion nucleotides!

MISMATCH REPAIR

A repair mechanism that initiates removes

bases from a nick created distant to the
mismatched base then refilled with correct
bases using DNA polymerases I, II & III
(EXONUCLEASES).

But, DNA Replication Is Not Per fect!

THE PROBLEM:

Limitations in DNA polymerase

create provides no way to
complete the 5’ ends of daughter
DNA strands, producing shorter
and shorter DNA molecules.
THE SOLUTIONS:

The ends of linear DNA molecules are

#1 known as “telomere regions” which
contain special repeating sequences that
are non-coding. In human telomeres, this
sequence is typically TTAGGG, repeated
between 100 and 1,000 times. Telomeres
protect genes from being eroded through
multiple rounds of DNA replication.

Eukaryotic cells have evolved

#2 a mechanism to restore
shortened telomeres by using
enzymes called telomerases.
These use a short molecule of
RNA as a template to extend
the 3’ end of the telomere, now
creating room for primase and
DNA polymerase to extend the
5’ end. Thus, telomerases
don’t repair the 3’-end
“overhang,” instead, they
lengthen the telomere.

Telomerase __________ present in most cells. Thus, telomere length

may be a limiting factor in the life span of certain tissues/organism.
Some cells

have Telomerase ________ present in germ-line cells

telomera se,
(sperm/egg) to ensure long telomeres.

some don’t … Telomerase ________ present in cancer cells. This overcomes the
progressive shortening that would eventually lead to self-destruction
of the cancer.
When DNA Repair Doesn’t Work …
UV Light Damage
UV can make two T bases join to each
other (“thymine dimer”) which buckles
DNA, interfering with DNA replication.
Humans have an enzyme called
photolyase that uses visible light
energy to break the covalent bonds
between adjacent thymines and reform
the normal A-T base pairs. Individuals
with mutant photolyase enzyme suffer
from a “sun-allergy” condition called
xeroderma pigmentosum.

Cancer
p53 has been described as "the guardian of the genome" because of its role in conserving
stability by preventing genome mutation. Hence p53 is classified as a tumor suppressor
gene. So, mutations affecting p53 lead to cancerous cells.
General Purpose: To amplify small quantities of DNA artificially.

Developed By: Kary Mullis, 1983

Apparatus Used: Thermocycler

Applications of the Technique:

•​ Diagnosis of genetic & infectious diseases
•​ Genetic fingerprinting (forensics, paternity testing)
•​ DNA sequencing (as in human genome project)
•​ Genetic diversity studies, species identification
•​ Molecular phylogeny and evolution studies

Reaction Ingredients:

There is a
doubling of
DNA with every
cycle.
Illustrated Diagram of PCR:
 Polymera se Chain Reaction (PCR) Review!

PCR was developed in the late 1980s, and is useful for making many copies Bacteria
of DNA through repeated cycles of strand _______________ and replication. Billion
Prior to the development of this technique, numerous copies of DNA could Complementary
only be made through the use of a ___________. Constant
Degraded
This method however, is not always desirable as it requires the tasks of Diagnosis
extracting the plasmid and extracting the desired DNA fragment. PCR is a Direct
much more ____________ method, and thus much more efficient. DNA
Gyrase
The method of PCR resembles the process DNA _______________ that all Heat
cells undergo prior to mitosis and ______________. In DNA replication, the Hydrogen
enzymes _____________ and helicase separate the DNA, whereas in PCR, Investigations
_________ in the range of 94-96 degrees Celsius is used to separate the Meiosis
strands through breaking the ___________ bonds. One
Plasmid
Unlike DNA replication which uses ________ primers, PCR uses _______ Repeated
primers. The primers used must be complementary to the ends of the target Replication
DNA. Research
RNA
As well, it is DNA polymerase III that helps build the _______________ Separation
strands during DNA replication at about body temperature (37°C); but, for Small
PCR, the polymerase enzyme ________ polymerase (after its origin, which is Speed
from ____________ named Thermus aquaticus, that live in thermal springs) Taq
at much higher temperatures of 50-65 degrees Celsius. These higher Variable
temperatures likely help ________ up the reaction rate.

Just like DNA replication, PCR builds the new strands in the 5’ to 3’ direction.
Once the complementary strands are built, the process is ______________.
After about 30 cycles, more than 1 __________ copies of the target DNA
have been produced!

Note that initially, DNA strands of _____________ lengths are produced, but
as the process is repeated more and more, more ____________ length
strands consisting only of the desired DNA are produced.

The process of PCR is useful for criminal _____________________, medical

_________________, and genetic ______________. It is an extremely
beneficial technique as it only requires a ___________ amount of DNA to
work; a sample from just __________ cell or in a hair follicle will suffice. As
well, the DNA does not have to be fresh, and can even be __________!
3.4 PROTEIN SYNTHESIS
“Central Dogma” represents the flow of information in a cell:
 Protein Synthesis: A Two-Step Process
1.​ Transcription = Formation of mRNA in the nucleus by copying (“transcribing”) the
DNA code and carrying the copied message out of the nucleus to the ribosome.

2.​ Translation = Decoding (“translating”) the mRNA message by the ribosomes in the
cytoplasm in order to form a polypeptide chain (sequence of amino acids).

There are 3 types of RNA: Each has a special function in protein synthesis.
(but many more types of RNA are known that play a role in protein synthesis or gene regulation)

1)​ mRNA stands for “__________________ RNA”, and its function is to carry a copy
of the coded message from DNA to
ribosomes.

2)​ rRNA stands for “__________________ RNA”, and its function is to combine with
proteins in order to form a complete ribosome.

3)​ tRNA stands for “__________________ RNA”, and its

function is to bind to a specific amino acid and transfer it to
the ribosome for protein synthesis.
 HOW THE CODE IS READ

Uses a “Triplet Code” = bases read in 3’s (codon), which specifies an amino acid.

●​ The mRNA ________________ must match with the tRNA _________________ in

order to have the correct amino acid attached.

●​ One codon can code for the same amino acid, but each codon specifies only 1 amino acid.

Codon = 3 nucleotides on the mRNA that codes for a specific amino acid

Anticodon = 3 nucleotides on the tRNA complementary to the codon

The mRNA code is read using the CODON TABLE:

Start Codon =

Stop Codons =

A mRNA codon table shows the following patterns:

1)​ REDUNDANCY – each amino acid may be coded from by more than one codon
(the 3rd amino acid in the codon is referred to as the “wobble position”)

2)​ SPECIFICITY – no ambiguity (each codon codes for 1 amino acid only)

3)​ UNIVERSALITY – same codons code for the same amino acids across all organisms
(some exceptions in select organisms, as well mitochondria + chloroplasts codes)

What if the genetic code consisted of a single nucleotide or even

pairs of nucleotides per amino acid?
 TRANSCRIPTION
Three key steps in transcription:

1 Occurs at the promoter sequence on the DNA in order to

start the transcription of a specific gene.

2 Is performed by RNA polymerase in order to transcribe

the mRNA strand.

3 Occurs when the mRNA transcript is released and the

RNA polymerase detaches from the DNA.
A)​ INITIATION

PROMOTER REGION
Regulatory (non-coding)
region ahead of the gene
that includes the TATA box.

TRANSCRIPTION
FACTORS
Proteins that bind onto the
promoter region that signal
the binding of RNA
polymerase II to start
transcription.

RNA POLYMERASE II
Once all transcription
factors have bound the
promoter, RNA polymerase
II binds in order to start
transcription.
B)​ ELONGATION

SENSE STRAND
DNA strand
(non-template) that is the
same as the mRNA
sequence.

ANTISENSE STRAND
DNA strand (template)
complementary to mRNA
sequence.
C)​ TERMINATION

TERMINATOR SEQUENCE
Regulatory sequence at the
end of a gene that triggers
the unbinding of RNA
polymerase II to stop
transcription.

HAIRPIN LOOP
Pairing of complementary G-C bases at the terminal end of mRNA to form a folded
shape that triggers the release of the RNA polymerase to end transcription.
 POST-TRANSCRIPTIONAL MODIFICATION
▪​ In eukaryotes, an extra step happens before translation called RNA processing in order
to remove intron regions.

▪​ Primary Transcript = Unedited mRNA produced in transcription that contains introns.

RNA processing in eukaryotes involves the following steps:

1)​ ADDITION OF CAP & TAIL:

5’ GUANINE CAP 3’ POLY-A-TAIL

Functions in orienting the mRNA String of adenines added that
at the ribosome. protects mRNA from being degraded
by cytoplasmic RNAses.

2)​ SPLICING:

SPLICEOSOME
A “cutting pasting machine” that
includes numerous proteins and
catalytic snRNA functioning in intron
removal and exon joining.

RIBOZYME
snRNA (small nuclear RNA) acts as
catalysts (just like protein enzymes)
to mediate the removal of introns
from mRNA.
 ALTERNATIVE RNA SPLICING
Gives rise to two or more different polypeptides, depending on
which segments are treated as exons.

This counters the “one gene, one protein” view!

Alternative splicing is more widespread than originally thought, estimated to occur in more
than 60% of human genes. It is believed to provide a “selective” advantage because exon
shuffling may lead to new proteins through novel combinations of functions.

An example of alternative splicing…

Alternative splicing of alpha-tropomyosin pre-mRNA produces components of
muscle cells in different locations in body (introns = black lines, exons = blue bars).
 TRANSLATION

TRANSFER RNA (tRNA)

Carries a specific amino acid
determined by the complement
sequence of the anticodon.

AMINOACYL-tRNA SYNTHETASE
Enzyme that joins correct tRNA to
its amino acid using energy; there
are 20 different synthetases to
match the 20 different amino acids.
 RIBOSOME STRUCTURE
Ribosomes form by uniting
its large and subunit
together. Each subunit is
made up of dozens of
proteins plus rRNA.
Prokaryotic ribosomes are
smaller than eukaryotic
ribosomes.

Three key steps in translation:

▪​ Translation can be divided into 3 stages: initiation, elongation and termination.

o​ All three phase require protein “factors” that aid in the translation process.
o​ Both initiation and chain elongation require energy provided by GTP hydrolysis.
A)​INITIATION
▪​ The small ribosomal subunit attaches near 5’ end of mRNA, utilizing translation factors.
▪​ tRNA carrying methionine attaches to the mRNA at the start codon AUG via H-bonds.

B)​ELONGATION
▪​ tRNA molecules deliver the correct amino acid to the A-site where a peptide bond
form between the growing chain and the new amino acid (uses GTP).
C)​TERMINATION
▪​ When a stop codon enters the A site, a protein called release factor binds to the
mRNA and causes the release of the polypeptide.

A ribosome requires less than a minute to translate an average-sized mRNA into a protein.

POLYRIBOSOMES
Multiple ribosomes may
trail along the same
mRNA, allowing a single
mRNA to make many
copies of a polypeptide
simultaneously.
 Prokaryotes can transcribe and translate the same gene simultaneously.

There are two populations of ribosomes in eukaryotes.

FREE RIBOSOMES BOUND RIBOSOMES

Suspended in the cytosol Attached to the cytosolic side of the RER and
and synthesize proteins that synthesizes proteins of the endomembrane
reside in the cytosol. system as well as proteins secreted from the cell.
POST-TRANSLATIONAL MODIFICATION (changes made to the polypeptide)

1)​ Protein Folding: A polypeptide folds to its 3D shape spontaneously in the RER
lumen, and chaperone proteins may aid correct folding.

2)​ Polypeptide Chain Modification: Additions like sugars, lipids, or phosphate group
to amino acids can be added to polypeptide chains, or two polypeptides may join to
make a quaternary protein, or a portion of the polypeptide may even be cleaved.
Protein modification occurs along the endomembrane pathway, notably in the Golgi.
 An example of
INSULIN PROTEIN
post-translational modifications.

POST-TRANSLATIONAL SORTING (determining where the protein needs to go)

Each polypeptide has a “postal” code that ensures delivery to correct cellular location.

SIGNAL PEPTIDE SIGNAL RECOGNITION PARTICLE (SRP)

Lead region of polypeptide about Consists of a protein-RNA complex that binds
20 amino acids long that signals to the signal peptide, then attaches it and its
its destination for the ribosome to a receptor in the ER membrane.
endomembrane system. After binding, the SRP leaves and protein
synthesis resumes with the growing
polypeptide feeding into the ER lumen. The
signal peptide is then cleaved.
Point & Frameshift Mutations
A quick review…

Base-Pair Substitution Base-Pair Insertion or Deletion

SILENT: no effect on amino acid FRAMESHIFT CAUSING EXCESSIVE

sequence (ie, substitution occurs in MISSENSE: leads to string of incorrect amino
“wobble” position) acids

MISSENSE: results in different amino FRAMESHIFT CAUSING IMMEDIATE

acid in the polypeptide chain, leading NONSENSE: leads to truncation of the amino
to non-functional protein acid sequence

NONSENSE: amino acid substituted INSERTION/DELETION OF 3 NUCLEOTIDES

with a stop codon, creating shorter CAUSING NO EXTENSIVE FRAMESHIFT:
polypeptide chain leads to omission or addition of an amino acid;
may/may not affect protein function
 Protein Synthesis Practice Problems
Shown below is a complete DNA sequence that codes for a protein that is separated into three
fragments in a mixed-up order. One of the fragments includes an 11 base promoter region.

FRAGMENT A: T C T T C C C T C C T A A A C G T T C T T A A T C C G C C G C C A G G G A C T

FRAGMENT B: A A A T T A A T T A A T A C A A A A C C G T A A A C A C A G A C C A A C T C C G C A A C

FRAGMENT C: T C A G A C G T T T T T G C C C T T G T T A C A A C A T G G T C A T A A A C G T C A

1. Circle/highlight and label the fragment where the 11 base promoter region is located. State is the
function of the promoter (ie, what molecules interact with/bind onto the promoter)?

2. Identify the beginning, middle & end of the DNA sequence. Beginning? ____ Middle? ____ End? ____

Explain how you determined this order.

3. Omitting the 11-base promoter sequence, divide the sequences into triplets by putting a slash between
each. Number the codons beginning in order of the first, middle and last fragments.

Three introns are found in this DNA sequence:

Intron #1 - codons 4-11 (NOTE: Try highlighting these sequences and label them as introns!)
Intron #2 – codons 15-24
Intron #3 – codons 28-36

Determine the mRNA sequence after splicing. Be sure to indicate the 5’ and 3’ ends.
4. RNA processing involves modifications to the mRNA before shipping out of the nucleus.

a) To the above mRNA sequence, label the “G cap” and a “polyA tail” to the appropriate locations.

b) State the function of each of these modifications.

G cap function:

Poly A tail function:

c) State where RNA processing occurs in a cell (state location)?

d) State the structure that accomplishes this processing?

e) Determine the resulting amino acid sequence following translation?

f) Describe the impact of the following mutations on the polypeptide sequence:

Codon 25: TCA

changes to TCG

Codon 3: ACC
changes to ACT

Codon 13: G is
inserted after GAC

Codon 34: C is
deleted

g) Discuss three differences between protein synthesis in a prokaryote and a eukaryote.

i)

ii)

iii)
PRACTICE: Identifying Mutations & Predicting Their Consequences

Nucleotide sequences of mRNA transcribed from different forms of an imaginary gene are
given below in the table. For each of the mutants, choose the type of mutation from the list
below and predict the possible impact (if any) on the protein.
a.​ no effect substitution
b.​ missense substitution
c.​ nonsense substitution
d.​ insertion or deletion frameshift with (extensive) missense
e.​ insertion or deletion frameshift with (immediate) nonsense
f.​ insertion or deletion with no extensive frameshift

Gene Nucleotide Sequence Mutation Impact (if any)

Type (from template DNA strand) Type of Mutation on
(choose letter) Protein
Wild AUGACACAUCGAGGGGUGGUAAACCCUAAG ____ ____
(normal)

Mutant AUGACACAUCCAGGGGUGGUAAACCCUAAG
1

Mutant AUGACACAUCGAGGGUGGUAAACCCUAAG
2

Mutant AUGACGCAUCGAGGGGUGGUAAACCCUAAG
3

Which of the above mutations is the most severe in terms of impact on the protein?

Explain using an example of a hypothetical protein that would be found in the body.
3.5 CONTROL OF GENE EXPRESSION
▪​ Cells express a small fraction of their genes.

▪​ The gene expression profile of each cell type is different; gene expression is controlled on
a long-term basis during CELL DIFFERENTIATION.

▪​ Thousands of genes in the cell are continually turned on and off in response to signals
from their internal and external environments.

▪​ There are two main gene expression patterns:

INDUCIBLE EXPRESSION CONSTITUTIVE EXPRESSION

Some genes are “turned on Some genes are

and off” in response to “housekeeping genes” needed
external signals. for basic cellular function.

…often dependent on cell …such as enzymes of

position and developmental glycolysis and regulators
stage. of cell cycle.
 Cells cope with environmental
fluctuations by using metabolic
control by NEGATIVE FEEDBACK
which regulates gene expression in
either of 2 ways:

1) via ENZYME ACTIVITY

(allosteric inhibition)

2) via GENE EXPRESSION

(enhancing/repressing genes)

▪​ Control of gene expression occurs at any point in the pathway, from gene to protein:

o​ Prokaryotes - The expression of specific genes is commonly regulated solely at

the level of transcription by DNA-binding proteins that interact with other proteins
and with external signals.

o​ Eukaryotes – Also control gene expression at the transcriptional level primarily.

However, with their greater complexity, there are greater opportunities for
controlling gene expression at additional stages.
 PROKARYOTIC GENE EXPRESSION
▪​ Bacteria use what is called an OPERON to control gene expression at transcriptional level.

▪​ Allows bacteria to TURN ON a gene (eg. transcribed) when that protein is NEEDED or
TURN OFF a gene so that the protein is no longer transcribed when NOT NEEDED.

▪​ An operon consists of:

A promoter = A sequence of DNA bases where RNA polymerase binds.

A set of structural genes = A cluster of genes that work together in a

metabolic pathway (i.e., synthesis of tryptophan) or to achieve a specific function in
the cell (breakdown of lactose).

An operator = A sequence of DNA nucleotides where a repressor protein binds,

blocking binding by RNA polymerase to the promoter.

The basics behind the workings of an operon:

1.​ When a protein product such as an enzyme is needed, the operon is turned ‘on’ and an mRNA
transcript is produced which travels to a ribosome to yield the protein.

2.​ When a protein product is not needed, the operon is turned ‘off’
due to the binding of a repressor protein to the operator, which
blocks the binding of RNA polymerase, and thus will not produce
an mRNA transcript to be used for protein production.
Two well-studied prokaryotic operons are the lac operon and the trp operon:

1) LAC OPERON - Codes for the enzyme beta-galactosidase which digests the
disaccharide lactose, the sugar in milk products, into glucose and galactose, so that
energy of lactose can be harvested. The lac operon consists of three genes:
HOW THE LAC OPERON WORKS…
‘ON’ - Lactose is present → it binds to the repressor protein → repressor protein changes shape →
r.p + lactose complex cannot bind to the operator site of the operon → RNA polymerase is thus able
to bind to the promoter → cluster of genes coding for beta-galactosidase is transcribed into mRNA →
mRNA travels to ribosome where the enzyme is made → enzyme digests lactose so cell can harvest
energy.

‘OFF’ – Lactose is absent → repressor protein maintains its original shape → r.p. is able to bind to
the operator site of the operon → RNA polymerase is blocked from binding to the promoter → no
transcription can occur → no mRNA produced → thus no enzyme beta galactosidase produced.

Allolactose, an isomer of lactose, is called an inducer because its binding to the repressor stops the
repressor from doing its job of repressing (stopping) the genes of the operator from being
transcribed. Thus, induces (helps) gene transcription.

Repressor protein is produced (via a gene upstream from the operon) in its active form – i.e. – a
form which is the proper shape such that it can bind to the operator.
 Negative Gene Control

Gene is either turned off or on.

Positive Gene Control

Transcription is either sped up or slowed down.

HOW DOES LAC SPEED UP?

RNA polymerase has high affinity for the lac promoter only when the activator protein (CAP)
is bound to a DNA site at the upstream end of the promoter. CAP attaches to its DNA site
only when associated with cyclic AMP (cAMP), whose concentration in the cell rises when the
glucose concentration falls. Thus, when glucose is present, even if lactose is also available,
the cell preferentially catabolizes glucose and makes very little lactose-digesting enzymes.
2) TRP OPERON – Codes for tryptophan, an amino acid that aids in protein
formation, needed when the diet does not provide tryptophan through consumed foods.

HOW THE TRP OPERON WORKS…

Repressor protein details – produced (via a gene upstream from the operon) in an inactive form – i.e –
a form in which the shape is such that it cannot bind to the operator

‘ON’ – Tryptophan is absent → repressor protein maintains its original shape → r.p. cannot bind to the
operator → RNA is thus able to bind to the promoter → cluster of genes coding for tryptophan is
transcribed into mRNA → mRNA is translated at a ribosome into tryptophan → thus larger protein
products can be made by the cell

‘OFF’ – Tryptophan is present → it binds to the repressor protein → r.p. + tryptophan complex
changes shape → complex is able to bind to the operator → RNA polymerase is blocked from binding
to the promoter → no transcription occurs → no mRNA produced → thus no more additional
tryptophan produced

Tryptophan is called a corepressor because its binding to the repressor helps the repressor do its
job, and thus, in effect represses (stops) gene transcription. *
 EUKARYOTIC GENE EXPRESSION
▪​ Each stage in the entire process of gene expression provides a potential control point
where gene expression can be turned on or off, speeded up or slowed down.

▪​ The following are ways to control gene expression in eukaryotes:

1.​ BEFORE TRANSCRIPTION

Densely condensed regions of chromatin are
usually not expressed because transcription
proteins cannot reach the DNA.

2.​ DURING TRANSCRIPTION

Control over which DNA genes are transcribe
or the rate of transcription of those DNA genes.

3.​ POST TRANSCRIPTION

Adding of the 5’ cap and poly A tail can be
prevented or introns not spliced, which
ultimately leads to mRNA being degraded in the
cytoplasm by RNAses.

4.​ DURING TRANSLATION

mRNA can fail to latch onto the ribosome
machinery to prevent translation.

5.​ POST TRANSLATIONAL

The protein can be prevented from being
functional by not adding necessary side groups
like phosphates, carbohydrates, etc. or the
protein fails to reach its destination where it
needs to function.

TRANSCRIPTION
is the most important and universally
used control point in gene expression.
1) CHROMOSOME LEVEL CONTROL

CHROMATIN ORGANIZATION

Level of packing is one way that gene expression is regulated:

HETEROCHROMATIN = Densely packed areas where genes are inactivated (not being transcribed).

EUCHROMATIN = Loosely packed areas where genes are being actively transcribed.

Genes of densely condensed

heterochromatin are usually not
expressed, presumably because
transcription proteins cannot
reach the DNA.

CHEMICAL MODIFICATION OF CHROMATIN

HISTONE ACETYLATION DNA METHYLATION

Acetylated histones grip DNA less tightly, Addition of a methyl group to cytosine bases
providing easier access for transcription inactivates DNA by preventing transcription
proteins in this region. Some of the enzymes machinery from binding. It allows for long-term
responsible for acetylation or deacetylation inactivation of genes during cell
are associated with or are components of differentiation. DNA methylation does not
transcription factors that bind to promoters. alter DNA sequence, so it is an epigenetic
mechanism of controlling gene expression.
2) TRANSCRIPTION LEVEL CONTROL
POLYCISTRONIC mRNA (Prokaryotes)
Genes are often clustered into an operon with a single promoter and
the genes are transcribed into a single mRNA and translated together.

MONOCISTRONIC mRNA
(Eukaryotes)
Genes coding for the enzymes of a
metabolic pathway may be scattered
over different chromosomes. Even if
genes are on the same chromosome;
each gene has its own promoter and is
individually transcribed.

A eukaryotic gene includes control elements located upstream of the promoter

region that regulate transcription by binding transcription factors.
Transcriptional control in eukaryotes involves:

Promoters = Bind RNA polymerase and initiate transcription.

The transcription initiation complex assembles on the promotor sequence
and involves transcription factors and RNA polymerase.

Enhancers = Increase transcription of associated genes.

Distant control elements may be found thousands of nucleotides away from
the promoter or even downstream of the gene or within an intron. Bending
of DNA enables transcription factors called activators bind to enhancers to
contact the protein initiation complex at the promoter.

Silencers = Suppress transcription of associated genes.

Repression may operate mostly at the level of chromatin modification.

Distant control elements may be found thousands of nucleotides away

from the promoter or even downstream of the gene or within an intron.
Bending of DNA enables transcription factors called activators bind
to enhancers to contact the protein initiation complex at the promoter.
3) POST-TRANSCRIPTIONAL LEVEL CONTROL

ALTERNATIVE SPLICING

Different mRNA molecules are produced from the same primary transcript,
depending on which RNA segments are included as exons.

mRNA DEGRADATION

Typically, prokaryotic mRNA degrades after a few minutes, while eukaryotic mRNA
degrades after hours, days, or weeks using 2 different mechanisms:

DECAPPING

Involves shortening of the

poly(A) tail, followed by removal
of the 5’ cap. Degradation of the
mRNA by nucleases happens in
the 5’ to 3’ and 3’ to 5’ direction.
4) PRE-TRANSLATIONAL LEVEL CONTROL

Translation of specific mRNAs can

be blocked by regulatory proteins
that bind to specific sequences or
structures within the 5’ leader
region of mRNA - this prevents
attachment to ribosomes.

5) POST-TRANSLATIONAL LEVEL CONTROL

Eukaryotic polypeptides must often be processed to yield functional proteins, and these
modifications include cleavage, chemical modifications, and transport to the appropriate
destination…thus, regulation may occur at any of these steps.

PROTEOSOME
Proteins intended for degradation are
marked by attachment of ubiquitin proteins
which are recognized by giant proteasome
complexes to degrade the tagged protein.
3.6 BIOTECHNOLOGY INTRODUCTION
BIOTECHNOLOGY: Manipulation of biological organisms to create useful products for human use.

Examples:

RECOMBINANT DNA TECHNOLOGY: Gene(s) from different sources (i.e.,

between individuals of same or different species) put into a host for expression.

Basic Technique Involved… (for example to create human insulin)

EXAMPLES OF GENETIC ENGINEERING

GM MEDICATION
Transgenic bacteria have been used to produce various
drugs such as insulin to treat diabetes, clotting factors to
treat hemophilia and human growth hormone to treat various
forms of dwarfism. These recombinant proteins are safer
than the products they replaced. Transgenic bacteria are
also used to produce hepatitis vaccines.

SPIDER SILK
Artificial spider silk can be made by
inserting the silk gene from an
orb-weaving spider into a fertilized goat
egg. These “spider-goats” produce milk
that can be manufactured into strong,
light-weight fibers that are reportedly five
times stronger than steel. These can be
used to make rope, sutures, artificial
tendons and bandages for burn victims.

GM SALMON
GM Atlantic salmon has a growth
hormone-regulating gene from
Pacific Chinook salmon that allows
it to grow year-round instead of
only during spring and summer.
The fish grows to market size in 16
to 18 months rather than three
years.

OIL-DEGRADING BACTERIA
While there are a few natural oil-digesting
bacteria found in nature, these GM bacteria
were engineered to consume oil faster and
more efficiently, which is important when
trying to clean up oil spills to minimize
ecological impacts of the oil.
 ENVIROPIGS
Cereal grains including corn, soybean and
barley contain 50 to 75% of their
phosphorus in the form of phytic acid.
Enviropigs produce the enzyme phytase in
their salivary glands that allows the pigs to
use the phosphorus in the grains rather
than needing to add a mineral supplement.
This reduces cost and gives the pigs a
more balanced diet. As a result of better
digestion, significantly less phosphorus is
found in the manure, thus less phosphorus
leaches into aquatic systems to cause algal
growth (eutrophication cascade).

CONCERNS:

GOLDEN RICE
Genes for making beta-carotene
are taken from daffodils and
inserted into the genome of a strain
of rice. When consumed,
beta-carotene (a vitamin A
precursor) protects undernourished
people from blindness caused by a
lack of vitamin A, which kills 6000
children per day.

CONCERNS:
BT CORN Bacillus thuringiensis (Bt) is a bacterium that produces a protein toxin
(Cyr toxin) that is harmful to some insects. This toxic gene was isolated and put into
crops such as corn, as well as cotton and potatoes. Bt crops produce this toxic protein to
prevent insect damage to crops that lower yield. The use of Bt crops also has farmers
requiring less chemical pesticides applied to crops.

CONCERNS:

ROUNDUP-READY SOYA Monsanto created a GM soya (as well as corn,

cotton, alfalfa, canola) that is resistant to the herbicide Roundup so farmers require less
chemical herbicides to kill weeds that could lower their crop yields. Roundup ready
seeds sold by Monsanto are known as “terminator seeds” because they are sterile which
requires farmers to purchase new seeds from the company every year.

CONCERNS:
3.7 RECOMBINANT DNA TECHNOLOGY
Tools of the Trade

I. Restriction Endonucleases (aka “____________________”)

Where are they found?

Found in bacteria (not humans!).

What is their function?

Bacteria use restriction enzymes to cut viral DNA

(their form of defence against viruses that infect
bacteria called “bacteriophages”).

How do they work?

Restriction enzymes scan DNA looking for a “target sequence” which they will cut.
Key Features:

SPECIFICITY: Locate and cut recognition sequences typically 4-8 base pairs long.

PALINDROMIC: Recognition sequences can be read both front and backward.

Types of Cuts:

STICKY Create “staggered” (zig-zag) cuts by breaking phosphodiester and H-bonds.

ENDS More USEFUL in genetic engineering

BLUNT Create straight cuts by breaking phosphodiester bonds only.

ENDS NOT useful in genetic engineering

How does a bacterium protect itself from its own restriction enzymes?

The addition of methyl groups to the bases of the recognition site by a methylase
enzyme…acts like a “cloaking device” to hide the target sequence.

This is called “__________________________________”.

How can sticky ends be rejoined?

Complementary sticky ends can be rejoined using DNA LIGASE to

recreate the phosphodiester linkages.
II. Plasmids (aka “__________________________”)

Sticky ends are useful for creating recombinant DNA

because they can be inserted into plasmid vectors.

Why are plasmids good cloning vectors?

● small size
● stable & easy to manipulate
● have restriction cutting sites
● self-replicating
● carry antibiotic resistance
III. Electrophoresis (aka “_________________________”)

What is the purpose of electrophoresis?

Technique used to separate different-sized

pieces of DNA using a polysaccharide
matrix under an electrical current.

What are the general steps of the technique?

(1) An agarose gel is cast that has wells located at one end of the gel.
(2) DNA fragments are inserted into the well, and a buffer containing salts is poured over gel.
(3) An electrical current is applied and the ions in the buffer conduct an electrical current.
(4) The DNA fragments migrate away from the negative electrode towards the positive
(5) electrode because DNA carries a net negative charge.
(6) DNA fragments separate into a pattern of bands, in what is known as DNA Fingerprint.
(7) The DNA fragments are visualized after staining.
What are uses of DNA fingerprinting?

1. Research (medical, evolutionary studies).

2. Genetic testing for disease/infection.

3. Paternity testing or ancestry tracing.

4. Crime or disaster forensics.

 Recombinant DNA Practice Problems

QUESTION 1
The segment of DNA shown in the figure below has restriction
sites I and II, which create restriction fragments A, B, and C.
Which of the gels produced by electrophoresis shown below
would represent the separation and identity of these fragments?

QUESTION 2
Examine the restriction map of the circular plasmid for E.coli. This plasmid contains 7,896 base
pairs. There is an EcoRI site at base pair 1. The locations of other restriction sites are shown on
the map. The numbers tell the base pair number that the enzyme cleaves the DNA.
a) How many pieces result and what are their sizes when you cut with these enzymes?

EcoRI: _____ pieces of the following sizes: ________________________________

BamHI: _____ pieces of the following sizes: ________________________________

HindIII: _____ pieces of the following sizes: ________________________________

b) Sketch what the gel would look like after cutting with these pieces. Label the wells
and indicate the electrodes found at each end of the gel.

c) If the uncut plasmid was introduced into a bacterium, would it survive in a medium
with the antibiotic tetracycline? Would it survive in the presence of ampicillin or
penicillin? Explain.
QUESTION 3

Examine the figure that is the restriction map of the

circular plasmid YIP5. This plasmid contains 5,541
base pairs. There is an EcoRI site at base pair 1.
The locations of other restriction sites are shown
on the map. The numbers after the enzymes tell at
what base pair that enzyme cleaves the DNA.

Determine the digest products in the following mixes then sketch each mix on a gel.

d) Mix #1 – The plasmid was cut with both the enzymes EcoRI and EagI?

e) Mix #2 - The plasmid was cut with both the enzymes HindIII and ApaI?

f) Mix #3 - The plasmid was cut with both three enzymes HindIII, ApaI and PvuI?

g) Mix #4 - The digestion products from Mix #1 are digested with PvuI.
 3.8 BACTERIAL VECTOR CLONING
▪ What is the purpose of bacteria vector cloning?

▪ What are the general steps of the technique?

1) Isolate bacterial plasmid

and human DNA that
includes the gene of
interest.

2) Cut both DNA with the

same restriction enzyme
to insert gene of interest
into the plasmid; seal with
ligase.

3) Insert recombinant
plasmid into bacteria by
transformation.

4) Select recombinant
plasmid using antibiotic
and X-gal screening.

5) Identify correct
recombinant plasmid by
nucleic acid hybridization.
 Synthesizing Insulin By
BACTERIAL VECTOR CLONING
STEP-BY-STEP

PURPOSE: To have bacteria express a foreign gene and produce its protein.

What you need to start:

Bacterial Plasmid Human DNA

STEP 1: CUT PLASMID & HUMAN DNA WITH THE SAME RESTRICTION ENZYME

Plasmid and human DNA digested with the same enzyme to create the same sticky ends.
 STEP 2: MAKE RECOMBINANT PLASMIDS USING LIGASE (3 possibilities)

Complementary sticky ends are joined by DNA ligase, thus creating a mixture of
recombinant DNA molecules (some plasmids are unsuccessfully recombined
while some plasmids have either the gene of interest or another piece of cut DNA).

STEP 3: TRANSFORMATION OF BACTERIA (4 possibilities)

Bacteria are transformed (plasmid is taken up) in order to make a diverse pool of bacteria
(some bacteria have taken up the desired recombinant plasmid DNA, but other have not).
 BACTERIAL TRANSFORMATION TECHNIQUES

During cloning, some bacteria take up a plasmid readily (“competent”), but most
bacteria require chemical or physical induction to be transformed:

Heat-Shock Transformation: Bacterial cells bathed in CaCl2 salt solution

at the freezing point to stabilize cell membrane chemically and physically.
Plasmid introduced into the solution, followed by a quick heat shock treatment.
Allows plasmids to sweep in.

Electroporation: Brief electrical pulses create a temporary hole in the

plasma membrane through which plasmids (especially larger ones) can enter.
 STEP 4: ANTIBIOTIC SCREENING (eliminates one possibility)

Only bacteria having plasmids with the antibiotic resistance gene will grow.
This step determines transformation success.

STEP 5: X-GAL SCREENING (eliminates one possibility)

Bacteria with plasmids lacking foreign DNA stain blue when beta-galactosidase hydrolyzes X-gal.
Bacteria with plasmids containing foreign DNA are white because they lack beta-galactosidase.
This step determines recombination success.
 STEP 6: NUCLEIC ACID HYBRIDIZATION (selects correct clone)

A radioactive single-stranded DNA complement of the gene of interest is used as a

probe in order to locate bacterial colonies that include the gene of interest.
 GENE SOURCES FOR CLONING

1. GENOMIC LIBRARIES = DNA isolated directly from an organism

DNA is isolated and cut into thousands of pieces using restriction enzymes, then these
pieces are inserted into plasmids, which are then introduced into bacteria via
transformation.

2. cDNA LIBRARIES = Complementary DNA synthesized from mRNA templates

Complementary DNA (cDNA) is made in the laboratory using mRNA as the template
using the enzyme reverse transcriptase. The mRNA gene product has the introns
removed, therefore the cDNA gene is more manageable in size and has the potential
of being translated into protein by bacterial cells that lack the ability of RNA
processing. To be expressed, the cDNA attaches to other DNA sequences containing
a promoter.
Creating a cDNA Library using Reverse Transcriptase:

1. Processed mRNA (lacking introns) is isolated.

2. Viral reverse transcriptase enzyme is added in order to create a cDNA strand.

Advantage is that the cDNA has the potential to be translated

into protein by bacterial cells that lack the ability for mRNA processing.