SBI4U - Unit 2: Molecular Genetics & Biotechnology
EXPECTATIONS
D2.1 Use appropriate terminology related to molecular genetics
D2.2 Analyze a strand of DNA to determine the genetic code and base pairing of DNA
D3.1 Explain the model of DNA replication and describe the different repair
mechanisms that can correct mistakes in DNA sequencing
D3.2 Compare the structures and functions of RNA and DNA, and explain their roles in
the process of protein synthesis
D3.3 Explain the steps involved in the process of protein synthesis and how genetic
expression is controlled in prokaryotes and eukaryotes by regulatory proteins
D3.4 Explain how mutagens (radiation, chemicals, etc.) can cause mutations by
changing the genetic material in the cells
D3.5 Describe some examples of genetic modification and how it is applied in industry
and agriculture
D3.6 Describe the functions of some of the cell components used in biotechnology
D3.7 Describe on the basis of research some of the historical scientific contributions
that have advanced our understanding of molecular genetics
IMPORTANT RESEARCHERS & THEIR DISCOVERIES
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Gregor Mendel discovered the fundamental laws of inheritance (Mendel’s law)
Frederick Griffith discovered the “transformation principle”; lead to discovery
that DNA acts as a carrier for genetic information
Joachin Hammerling discovered that the nucleus is the storehouse of hereditary
material
Oswald Avery, Maclyn McCarthy, and Colin MacLeod discovered that DNA is the
material of which chromosomes and genes are made of
Alfred D. Hershey and Martha Chase discovered that only the DNA of a virus
needs to enter a bacterium to infect it
Rosalind Franklin (and Maurice Wilkins) discovered the structure of DNA (photo
51)
June Lindsey discovered the double-helix structure of DNA
James Watson and Francis Crick discovered the double-stranded, twistedladder structure of DNA
Friedrich Miescher identified DNA as a distinct molecule in 1869
DNA SYNTHESIS AND REPLICATION SUMMARY
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DNA replicates semi-conservatively; one coding strand and one newly
synthesized strand
○ discovered by Meselson and Stahl (1958)
step-by-step process
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proteins bind at origin
of replication (ori site)
enzyme DNA helicase
unzips DNA by breaking
H-bonds between base
pairs
SSBs bind to exposed
DNA strands to prevent
from rebonding
enzyme DNA gyrase
relieves tension in DNA during unwinding - prevents supercoiling
DNA polymerase III builds the complementary strands
○ continuously synthesizes the leading strand as it moves in the same
direction as helicase - adding free nucleotides to their complementary
base pair
○ synthesizes the lagging strand in short segments (Okazaki fragments) as
DNA pol III and helicase move in opposite directions
○ can only build in the 5’ to 3’ direction and cannot start building a new
strand by itselfm
○ additionally acts as proofreader for mutations in DNA (with DNA pol I) by
cutting out mismatched nucleotides and replacing them with the correct
ones, and then polymerase continues synthesizing
an RNA primer is required for DNA pol III to bind to and start building the
complementary strands; it is synthesized by enzyme primase
○ 1 required for leading strand
○ continuously added to lagging strand as replication fork (junction where
strands are still joined) moves
DNA polymerase I removes RNA primers to replace with correct
deoxyribonucleotides
○ acts as proofreader for mistakes/mutations in DNA (with DNA pol III)
enzyme DNA ligase joins the Okazaki fragments together into one strand with
phosphodiester bonds
errors missed by DNA pol I & III are corrected by other mechanisms after
replication
PROTEIN SYNTHESIS OVERVIEW/SUMMARY
● DNA is first transcribed into mRNA which is then translated; mainly occurs in
nucleus (protective nuclear envelope)
● major classes of RNA; messenger (mRNA), transfer (tRNA), ribosomal (rRNA)
○ mRNA gets translated into proteins by ribosomes
○ tRNA carries amino acids to ribosome for protein synthesis
rRNA is a structural component (along w proteins) of ribosomes &
provides site for polypeptide assembly
genetic code; 4 nitrogenous bases (3 RNA bases) code for 20 amino acids, each
triplet called a codon
○ each codon codes for one amino acid (except stop codons); more than
one codon can code for a single amino acid
○ start codon: AUG (codes for methionine) - signals initiation
○ stop codons: UAA, UAG, UGA (do not code for any amino acids) - signals
termination
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TRANSCRIPTION SUMMARY
● initiation begins when RNA polymerase binds to DNA (opens the double helix) at
the promoter (segment of DNA upstream from a gene)
○ rich in As + Ts (double bonds easier to break than Cs + Gs triple bond)
● initiation, transcription factors must first bind to promoter before RNApol binds
(in eukaryotes)
● elongation occurs when RNApol builds RNA in the same direction (3’ to 5’
direction) and does not require primer or helicase
● only one strand of DNA is transcribed (other strand is called coding strand)
● termination begins when RNA polymerase transcribes a termination sequence at
the end of a gene - stops polymerizing (usually TTATTT in eukaryotes) - RNApol
and mRNA disassociate
● 3 post-transcriptional modifications are made to primary mRNA transcripts in
the nucleus before released into cytoplasm for translation (eukaryotic cells)
○ 5’ cap
■ modified guanine nucleotide is added to the 5’ end (beginning) of
the transcript
■ required for initiation of translation & protects mRNA from
digestion by nucleases and phosphates
○ poly-A tail
■ enzyme poly-A polymerase adds 50-250 adenine nucleotides to the
3’ end of the transcript
■ helps transport transcript out of nucleus and protects mRNA from
digestion
○ removal of introns
■ non-coding sequences (introns) are interspersed among coding
sequences (exons)
■ called RNA splicing performed by spliceosomes (made of RNA and
proteins)
■ introns are cut out, exons are joined forming final mRNA transcript
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alternative splicing - different polypeptides encoded by the same
gene
TRANSLATION SUMMARY
● mRNA enters cytoplasm after post-transcriptional modifications - ribosome
recognizes 5’ cap and binds to mRNA
● ribosomes are made of 2 subunits (60S large subunit, 40S small subunit combine to form intact ribosome of 80S)
○ clamp mRNA between subunits
○ ribosome moves in a 5’ to 3’ direction
● transfer (t)RNA - single stranded ribonucleic acid that delivers amino acids to
ribosome
● exposed nucleotides at 3’ end of tRNA bind to a specific amino acid to form
aminoacyl-tRNA
○ aminoacyl-tRNA synthetases add amino acids to tRNA
○ anticodon of tRNA recognizes codon of mRNA (e.g. AGU - UCA)
○ each tRNA carries a specific amino acid - can recognize more than one
■ only first two bases are important - third base referred to as
“wobble position” due to its “flexibility”
● initiation begins when aminoacyl-tRNA carrying methionine binds to small
ribosomal subunit, then binds to 5’ cap of mRNA and slides to start codon
● anticodon of tRNA forms H-bonds with AUG on mRNA - large ribosomal subunit
joins complex
● elongation occurs in 3 sites in the ribosome - P (peptide) site, A (acceptor) site,
and E (exit) site
○ tRNA carrying methionine is in the P site - anitcodon of tRNA (UAC) is
bound to the start codon
○ next tRNA (that matches the following mRNA codon) binds to the A site
○ met is released from its tRNA and shifts over to the amino acid at the A
site
○ ribosome moves down the mRNA (translocates) one codon
■ i.e second tRNA is now in the P site, the first tRNA (lacking an
amino acid) enters the E site and is released from the ribosome
○ ribosome translocates and process repeats
● termination occurs when stop codon is reached (stop codons do not have
corresponding tRNA) - protein release factor binds to A site, releasing
polypeptide and both ribosomal subunits fall off
● proteins require post-translational modifications (they are rarely in active form
after synthesis)
○ glycosylation (addition of oligosaccharide to amino acids)
○ phosphorylation (addition of phosphate groups to amino acids)
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chain may also be cut in specific places by enzymes or modifications are
made to individual amino acids
CONTROL MECHANISMS SUMMARY
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methods of controlling protein synthesis based on a cell’s needs
housekeeping genes are those that are constantly being transcribed and
translated and are required in order to carry our vital life functions
gene regulation (turning on and off of specific genes) increases cellular
efficiency and is used for cellular differentiation
gene regulation can occur at 4 levels
○ transcriptional (supercoiled & methylated DNA is not transcribed,
transcription factors are used)
○ post-transcriptional (capping, splicing, and addition of a poly-A tail is
controlled)
○ translational (controls how often and how rapidly mRNA is translated into
a polypeptide)
○ post-translational (most proteins must be altered before they become
active)
operons (groups of several genes under the control of one promoter and one
operator) are used for prokaryotic transcriptional regulation and gene
expression
operators are the location on DNA where DNA repressor proteins can bind (in
order to prevent transcription)
lac operon
MUTATIONS SUMMARY
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errors in DNA can have a deleterious, positive, or no effect
point mutations: specific base pairs are altered
○ can be silent (no change in amino acid sequence) or non-silent (change in
amino acid sequence
substitution mutations: one base pair is changed to another
○ can be missense (codon for different amino acid is created) or nonsense
(stop codon is produced)
frameshift mutations: reading frame (set of 3 bases of a codon) is altered
○ more drastic change to polypeptide structure as it affects the folding of
the protein structure (can possibly render it dysfunctional)
deletion mutations: removal of one or more nucleotides from reading frame
insertion mutations: addition of one or more nucleotides
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translocation mutations: caused by segments of DNA that move from one
location to another (called transposons or “jumping genes”)
○ typically lead to loss of function of a particular allele or mutagenesis by:
○ disrupting the coding of a gene
○ alternating the transcriptional start site
○ potentially overriding the transcriptional program of a gene’s promoter
mutagenic agents (chemical or physical, capable of inducing mutations in DNA)
often cause mutations in DNA from spontaneous errors in DNA polymerase
○ chemicals, carcinogens (cause DNA adducts; piece of DNA covalently
bond to a chemical), uv light (bp pair with each other instead of
complementary bp), radiation, etc.
EPIGENETICS SUMMARY
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study of how behaviours and environment(s) can cause changes that impact the
function of certain genes
○ changes when exposed to environmental factors such as metals (arsenic,
nickel, methyl mercury), benzyne, air pollution, organic pollutants
(industrial chemicals), radiation, etc.
○ changes from lifestyle factors such as stress, diet, irregular sleep
schedules (working night shifts), physical activity, smoking, high alcohol
intake, etc.
reversible and do not alter DNA sequences; can alter how an organism’s body
reads/processes a DNA sequence
3 classes: DNA methylation, histone modifications, non-coding RNA action
changes allow for another layer on top of DNA (called epigenomes) that signals
for the start or silencing of different genes
GENE ORGANIZATION & CHROMOSOMAL STRUCTURE SUMMARY
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chromosomes; the structures that organize the genome (composed of proteins
and one unbroken double-stranded DNA helix molecule per chromosome)
genome; the sum of all genetic material in an organism
○ about 3 billion bp in human haploid genome - about 21,000 genes code
for proteins (1.5% of genome) - rest consists of regulatory sequences,
RNA genes, pseudogenes, other non-coding sections (transposons, viral
DNA)
packaging DNA; in order to fit within nucleus of a cell (1.8m long of DNA of a
single human cell) must be packaged tightly - supercoiled
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1. DNA coils around histones; group of 8 proteins, positively charged, allowing
negatively charged DNA to bind tightly
a. each coil is made up of about 200 nucleotides; called nucleosome
2. series of nucleosomes coil into chromatin fibres (DNA is in a semi-relaxed state,
i.e. chromatin fibres, most of the time)
3. chromatin fibres supercoil into a higher level of coiling; occurs during mitosis
when chromosome condenses
● DNA →nucleosome →chromatin →supercoil →chromosome
● human genetics; 46 chromosomes divided into 23 pairs ordered by size and
function
● 44 chromosomes are somatic (numbered 1-22 largest to smallest), 2 are sex
chromosomes (X & Y)
● individual’s set of chromosomes called a karyotype
● pseudogenes are a nucleotide sequence similar to a functioning gene - may be
crippled copies of other genes
● some non-coding regions of chromosomes are filled with variable number
tandem repeats (VNTRs - also known as microsatellites)
○ repeated sequences of DNA (e.g. TGATGATGA) - differs between
individuals in length and location
○ used in forensics - all are unique except identical twins
● VNTRs can be found within genes, the telomere, or centromere
○ within genes: e.g Huntington’s disease - usually disrupts genes
○ telomere: region at the end of chromosomes to protect from fraying; in
humans, there is a repeat of TTAGGG - protects cells from losing valuable
genomic material during DNA replication & used to set limit on the
lifespan of a cell and prevent chromosomes from fusing to one another
○ centromere: centre part of chromosomes - holds two replicated strans
(sister chromatids) together and binds to spindle fibres during mitosis
BIOTECHNOLOGY SUMMARY:
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can be used to investigate & treat genetic disorders, alter organisms to produce
chemicals (e.g insulin), and solve crimes (forensics)
transformation involves the insertion of a gene into an organism in order to
change its traits
○ industry: produce large volumes of material (like insulin)
○ agriculture: giving plants resistance to frost, pests, or spoilage
○ biomediation: giving bacteria the genes needed to digest toxins (like oils)
○ medicine: beginning to be used to correct defective genes that cause
disease
bacteria are primarily used for transformation studies as they reproduce
quickly, have a simply & easy to manipulate genome, can be frozen (stored),
tend to be cheaper, and animal sources have been shown to cause allergic
reactions
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prokaryotic chromosomes are circular whereas eukaryotic are more linear bacterial cells contain plasmids, which are independent from chromosomes, in
order to express genes (e.g digestive enzymes, protective proteins, etc.)
○ plasmids are small circular double stranded pieces of DNA (range in size
from 1,000-200,000 bp) and are used as vectors in transformation studies
gene segments are inserted into the plasmids, plasmids are then placed in
bacteria, and finally, bacterial cellular machinery produce the desired protein
○ artificial plasmids are engineered to contain one restriction site for
multiple restriction enzymes (also contain genes for antibiotic resistance the host body kills cell that do not contain antibiotic resistance)
DNA ligase is required to join DNA fragments together by forming
phosphodiester bonds in DNA backbone using a condensation reaction
in order to get the foreign gene into bacteria, a vector and a competent cell is
required (most cells are not naturally competent, but can be altered to be so)
○ heat shock: chilling cells in a solution of CaCl2 causes the cell membranes
to become permeable to plasmids - cells are then placed in a water bath
(42oC for 30-120 seconds) which allows for DNA to enter the cell
○ electroporation: cells are electrically shocked - disrupts the cell
membrane allowing plasmids to enter the cell
○ particle bombardment: small gold or tungsten particles are coated with
DNA and shot into cells - some genetic material will stay in the cells mostly used in plant cells as bacterial cells are too small & fragile
restriction enzymes function as molecular scissors that can cut double-stranded
DNA at specific base pair sequences (called restriction sites - often palindromic)
○ each type of RE will recognize its own restriction site & will only cut that
position (cuts phosphodiester bonds in the backbone of DNA)
○ H-bonds are also disrupted, however any thermal energy causes them to
fully break between base pairs
cuts produce two fragments of DNA, which will have either sticky or blunt ends
○ sticky ends (short, single-stranded overhangs) can join to other sticky end
fragments cut with the same RE
○ blunt ends (fully base paired) require (special) T4 DNA ligase to cut
enzymes that modify an organism’s own restriction site to prevent digestion are
called methylases (a methyl group is added to a base in the restriction site)
○ can be used by molecular biologists to protect gene fragments from
being cleaved in the wrong location
gel electrophoresis is used to separate charged particles by size (treating DNA
with REs produces a variety of DNA fragment sizes) - like a molecular sieve useful for isolating genes excised from its source DNA
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jell-O like substance with wells placed at one end - then placed in a
container filled with buffer (contains electrolytes)
DNA solution is mixed with a dye (to see progression) and loaded into
wells
negative & positive charge are placed at either end of gel (creates
electromagnetic current) - DNA will migrate towards positive charge
(short fragments of DNA migrate further)
DNA fragment sizes are determined by comparing with a size marker
(contains fragments of known size by # of base pairs)
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producing recombinant DNA (DNA combination of two or more sources):
1.
cut desired gene with a RE & isolate gene using gel electrophoresis
2.
cut plasmid with same RE
3.
put gene fragment & plasmid into the same buffer solution (sticky ends
will aneal with plasmid)
4.
add DNA ligase to rebuild phosphodiester bonds between fragments
(circular plasmid has now been reformed & carries foreign gene) gene has been cloned
5.
place plasmid into bacterial cell - when the cell replicates, plasmid (and
gene) will be replicated as well
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transgenic cells must be isolated in order to determine whether transformation
was successful - can be done by selective plating and checking for foreign gene
○ selective plating: plasmids used to make recombinant DNA contain
antibiotic resistance genes - if transformation was successful, bacteria
will be able to grow on media that contain the antibiotic
○ checking for the foreign gene: a single bacterial colony is removed from
the plate and allowed to proliferate in a liquid media - DNA is extracted
from bacteria, cut with a RE and run through an electrophoresis gel - if
transformation was successful, band produced by gene will be present