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Genetics 2009
M.T.Yeung
Genetics

Table Of Key Dates
Name
Lamarck (1809)
Observation / discovery
proposed theory of evolution based on inheritance of acquired characteristics
Darwin (1859)
On the origin of Species by Means of Natural Selection published
Mendel (1865)
Experiments on the genetics of peas and formulation of his Two Laws
1868: Friedrich Miescher isolates nucleic acid from pus cells obtained from discarded bandages
Morgan (early 1900s)
pioneered use of Drosophila in genetics experiments and described linkage
Fred Griffith (1920s)
(04AL-I-9)
Found out that a non-lethal strain of bacteria on incubation with a dead lethal strain would become
lethal. This character was inheritable.
Produced evidence suggesting that a chemical ‘ transforming principle’ was responsible for
carrying genetic information
Oswald Avery (1940s)
(04 AL-I-9))

1943: Avery, Macleod and McCarty use bacteria to provide the first evidence that DNA is
the bearer of genetic information
Obtained an extract from the dead lethal bacterial and treated this extract as follows:
http://www.dnaftb.org/dnaftb/17/animation/animation.html
(1) destroyed the protein in it
(2) destroyed the protein and RNA in it
(3) destroyed the protein, RNA and DNA in it
He then incubated a portion of the extract after each treatment with the non-lethal bacteria and
tested the resultant bacteria for their lethal effect.
Chargaff (1950)
Erwin Chargaff discovers regularity in proportions of DNA bases for different species. In all
organisms he studies, the amount of adenine (A) approximately equals that of thymine (T), and
guanine (G) equals cytosine (C). This is the Chargaff’s rule
Hershey and Chase (1952)
1952: Hershey and Chase show that it is the DNA part of the T2 viral particle, not the protein part,
that enters a host cell, furnishing the genetic information for the replication of the virus
Showed DNA to be the hereditary material
James Watson and Francis
Crick (1953) (04 AL-I-9))
1953: Double helix structure of DNA is revealed by Watson, Crick, Franklin, and Wilkins
determined and formulated the detailed molecular structure of DNA
Stahl and Meselson (1959)
described mechanism of semi-conservative replication in DNA
http://www.dnaftb.org/dnaftb/20/concept/index.html
F. Jacob and J. Monod
(1961)
Postulated existence of mRNA in theory on control of protein synthesis
Study of enzyme systems of E. coli
 explain the regulation of enzyme production in term of gene regulation. (Jacob-Monod
Hypothesis)
Others

1961-65: Genetic code is cracked

1972: First successful DNA cloning experiments are carried out in California

1975: Monoclonal antibodies are produced

1975-77: Rapid methods of sequencing DNA are perfected

1977: The first human gene is cloned

1982: Genetically-engineered insulin is approved for use in diabetics in the USA and UK

1987: First genetically-engineered microorganisms are used in field experiments
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Genetics 2009
M.T.Yeung
I. Structure of Nucleic Acids
- all living organisms contain nuclei acids
- in form of deoxyribonucleic acid (DNA) & ribonucleic acid (RNA)
- Some viruses may contain only RNA (e.g. tobacco mosaic virus) while others have DNA (phages)
1. Nucleotides
- the monomeric units of nucleic acid
- structure of nucleotides:
a. a phosphoric acid
- they link the pentoses in a linear pattern.
b. a 5-carbon sugar ( pentose )
- there are two types:
i. ribose
ii. deoxyribose [with one oxygen atom less than ribose at carbon 2, a deoxyl group]
c. an organic nitrogenous base ( purine or pyrimidine )
i. purines (two rings):
- adenine (A)
- guanine (G)
ii. pyrimidines (one ring only)
- cytosine (C)
- thymine (T, in DNA)
or uracil (U, in RNA)
- types of nucleotides
a. Polymer ( polynucleotide ) :
- includes RNA and DNA
---> heredity
---> protein synthesis
b. Non-polymer : - e.g.
i. ATP (Adenosine triphosphate, a mononucleotide)
---> energy rich compound, release energy by bond breaking
ii. NAD (Nicotinamide adenine dinucleotide, a dinucleotide)
---> as a coenzyme
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Genetics 2009
M.T.Yeung
2. Polynucleotide
- nucleotides joined together via Condensation
[removing a water molecule from the pentose sugar ( at carbon 3 ) of one nucleotide & the
phosphate group of another nucleotide ( at carbon 5 )]
--->
long polynucleotide chain (single strand):
- a backbone composed of alternating sugar
3. Structure of DNA ( Watson and Crick Model )
In 1953, two English scientists James Watson and Francis Crick, based on x-ray diffraction
patterns of the 3-D structure of DNA, they concluded that a DNA molecule consisted of :
- two parallel polynucleotide chains (double stranded)
- running in opposite direction (anti-parallel) and twisted (double helix :with 10 base pairs of 3.4
nm per turn)
- backbone of each strand with alternating sugar (deoxyribose) and phosphate group
- bases projecting inwards
- crossed linked between the complementary bases on the two strands at regular intervals with
hydrogen bonds:
A & T pairs (2 hydrogen bonds)
G & C pairs (3 hydrogen bonds)
 A:T=1:1,
G:C=1:1,
 purines : pyrimidines = 1:1
5’End
3’End
C3 position
attaché to another
nucleotide
3’End
5’End
Why is DNA so stable ?
- i.
The two strands are held tightly by a large number of H-bond between the base pairs.
ii.
the base pairings are highly specific. This maximizes the number of effective hydrogen
bonds formed.
iii.
the two strands coil tightly round each other.
 DNA has a very stable molecular structure!
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Genetics 2009
M.T.Yeung
DNA as genetic materials
1. stable complementary nature of the two strands of DNA
 i. makes it suitable for storing genetic information and transmitting the information from
one generation to another
ii. the H bonds between the base pairs tend to drive the double helix to reform
spontaneously after uncoiling in replication and transcription
2. discrete segment of DNA molecule composed of a particular sequence of nucleotides (gene)
can act as a unit of inheritance of a single characteristic or polypeptide synthesis. (see “Gene
concept” )
3. genetic information between DNA in different chromosomes and be exchanged (see “ Meiosis” )
or be changed (see “Mutation”)
 allow variation among different individuals, evolution or forming new species.
4. Structure of RNA
- single-stranded
- ribose
- with uracil instead of thymine
- hydrogen bonds:
A & U pairs (2 hydrogen bonds)
C & G pairs (3 hydrogen bonds)
- four major types:
a. Ribosomal RNA ( rRNA )
- associates with ribosomal proteins
---> major structural component of ribosomes.
b. Transfer RNA ( tRNA )
- free in cytosol
---> tRNA carry specific amino acids form the
cytosol to the surface of ribosomes during
protein synthesis.
- at least 20 types of tRNA
- at an intermediate point along the chain the 'anticodon' (a sequence of 3 bases which
complementary to codon on mRNA) is formed.
- with an end for amino acid attachment
c. Messenger RNA ( mRNA )
- the transcripts (like a mirror copy) of some regions of chromosomal DNA
---> as a template which carry the genetic information form genes to ribosomes where
translation occurs.
- easily and quickly broken down
d. Nuclear RNA ( nRNA )
- RNA in nucleus
- e.g. precursors of mRNA, tRNA & rRNA.
[HKALE 86 I]
1. Distinguish between
(b) the function of messenger RNA and transfer RNA.
Ans:
(2 marks)
The function of mRNA is to copy the message from DNA and carries it from the nucleus
into the cytoplasm where it direct the assembly of a protein.
The function of tRNA is to deliver its corresponding amino acid to the mRNA where amino
acids are assemble to form the protein.
(2)
Genetics 2009
5. Comparison between DNA & RNA
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Genetics 2009
M.T.Yeung
Summary:
Adenine
Guanine
Purines
(double-ringed)
Organic bases
Cytosine
Thymine (only in DNA)
Pyrimidines
(single-ringed)
Uracil (only in RNA)
Deoxyribose
Pentose ring
Condensation
Ribose
Inorganic Phosphate group
Mononucleotides
e.g. ATP (Adenosine triphosphate)
as an energy carrier
Nucleotides
Condensation
Dinucleotides
e.g. NAD (Nicotinamide
Adenine Dinucleotide) as a
coenzyme
Ribosomal RNA (rRNA)
Transfer RNA
(tRNA)
Messenger RNA (mRNA)
Polynucleotide single chains
(Nucleic Acid strands:- DNA / RNA)
- linear
- backbone with alternating sugar and phosphate
Watson-Crick Model of DNA
-
- 2 // single-stranded DNA running in opposite
direction
- twisted and form a double helix
- backbone of each strand with alternating sugar
(deoxyribose) and phosphate group
- crossed linked between the complementary
bases on the two strands at regular intervals with
hydrogen bonds (A=T ; G≡C)
- bases projecting inwards
Genetics 2009
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M.T.Yeung
DNA Spooling (rough DNA extraction from fresh tissues) -----3 Basic Steps
1.
2.
3.
Break the cell to release DNA and denature cell proteins
The cell and nucleus must be lysed (broken open) to release the DNA. Remove chromosomal proteins.
denature or inactivate DNAase
Protect DNA from degradation by enzymes that will cause shearing.
Precipitate DNA in alcohol
Result:
milk layer
Procedures:
containing DNA at
1. homogenize tissue in a blender
the middle layer
 break cell to release DNA
2. adding detergents
 break cell membranes and alter protein structure
Confirmation:
3. adding salt solution
adding Universal indicator
 denature the cell proteins and sink to the tube bottom
cause the extract turns
4. heating (60oC water bath)
orange yellow in colour
to denature the DNAase enzymes that cause shearing in DNA
(i.e. acidic)
5. ice bath
 to slow the enzyme action that degrade DNA
6. stirring and filtering
7. adding protease
(meat tenderizer has papain, an enzyme that helps clean the protein from the
DNA that can contaminate it. Papaya juice and pineapple juice also contains this
enzyme.)
8. adding iced alcohol to uncoils and precipitate the DNA
(In order for the cell to be lysed, the lipid walls must be broken down. The detergent and salt solutions accomplish this. Cell walls,
cell membranes, and nuclear membranes are also broken down by the action of the blender. Some references state that a temperature
of 60oC is necessary to denature the DNAase enzymes that cause shearing in DNA while DNA is denatured about 80 oC. But other
references state that DNA can denature at 60oC.)
Someone suggested that the use of heat can be eliminated in most cases. As the DNA had been sheared when
heat was used. Heat may destroy the enzymes as well as the DNA.
Genetics 2009
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M.T.Yeung
II. Replication of DNA (Semiconservative Replication)
The mode of DNA replication is described as semi-conservative. It means that only half of the replicated
product is derived from the original DNA molecules, while the other half is newly formed.
A. Mechanisms
- replication start at one or a few fixed points on a chromosome (replication origins)
- (by helicase) unwinding and separate of duplex by breaking the hydrogen bonds between them (use ATP)
- each separated strand serving as template for synthesis of a new complementary strand by attracting the
surrounding free nucleotides.
-
initiate synthesis of new DNA chains by primase (Priming)
- the bases of each strand then pair with the bases of the free nucleotides in a complementary pattern:
AT; GC
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M.T.Yeung
- newly lined up nucleotides are joined together by condensation with sugar-to-phosphate bonds
---> a new chain formed and join ( by hydrogen bond between bases) complementary to the old one
- grow of the new strands by DNA polymerase (from 5' to 3' end):
leading strand undergoes continuous replication
lagging strand undergoes discontinuous replication (ligase for ligation between fragments)
fragments joined together
by ligase
Genetics 2009
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M.T.Yeung
- new and old strands wound together
---> yield 2 'daughter' double-helix:
each consisting of one-half parental DNA and one-half new material
B. Evidence (The Meselson-Stahl Experiment, 1958)
The semi-conservative replication of DNA was demonstrated by Matthew Meselson and Franklin Stahl in
1958 with the use of Escherichia coli ( E. coli, a kind of colon bacterium)
- E. coli :
grow in medium with 15N (heavy nitrogen) for many generation
----> with heavy (higher density) DNA molecules with 15N in bases of DNA molecules
grow in 14N (light nitrogen) medium for one generation
----> hybrid DNA of 15N and 14N
grow in 14N medium for one more generation
----> half the offspring with hybrid DNA
half the offspring with 14N DNA only
- CsCl density gradient
centrifugation at - pH7
---> idea of :
1. each replicated chromosome contains one parental strand and one
new strand (i.e. semi-conservative replication); or
2. DNA strand breakage (fragments of different mass---> different
bands)
- pH12 (DAN denatured into separated single strands)
-- since no molecule of intermediate mass
---> ruled out the possibility of DNA strand breakage
Genetics 2009
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Genetics 2009
M.T.Yeung
III. Gene Concept
Gene
- unit of inheritance
---> i. determine an organism's characteristics
ii. through them characteristics passed on to the offspring
hence, must:
i. contain information for development
ii. able to replicate without losing its information
- bound by start codon and stop codon
- a segment of DNA in chromosome that determine
i. the synthesis (amino acid sequence) of a polypeptide chain; or
ii. a protein of single polypeptide
DNA molecule:
gene A
--> synthesis
of polypeptide A
gene B
--> synthesis
of polypeptide B
- Cistron
when a gene defined as the unit of the genetic material that control the synthesis of a polypeptide chain
(a chemical function gene), it is called a cistron
- Muton
when a gene defined as a unit of genetic material that has undergone mutation to become another form
(a different allele), which may produce and inheritable change in the phenotype of the organism, it is
called a muton.
Relationship of gene, DNA and chromosome:
DNA molecule
(with many genes along different region of the molecule)
coiled
Chromatin
(individual threads are invisible under microscope before cell division)
coiled (become most condensed when a cell is preparing to divide)
Chromosome
(individual threads condense and become visible under microscope)
Proteins called histones
play a key role in
packaging DNA within
chromosomes.
to form units called
nucleosomes.
Centromere
During cell division, the spindle
fibers that connect and move
the centromeres around. This
process ensures that each
chromosome moves to its
proper place.
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Genetics 2009
M.T.Yeung
IV. Protein Synthesis
A. Genetic codes (Triplet Codon / Triplet code )
- each 3 nucleotides on a DNA molecules forms a coding unit (DNA codon)
- different sequence of nucleotides in a codon may be specific for one amino acid
(some codons, nonsense codon, do not specify any amino acids. Their function is to ensure that the
synthesis of a polypeptide chain terminates at a fixed point. So they are also called stop codons)
Features of genetic code:
1. Triplet Codon / Triplet code
- a group of three nucleotide is necessary to encode for a specific amino acid.
2. non-overlapping
- a nucleotide will only be used once for each translation process / will not be used twice in
adjacent codes in one translation.
[i.e codons within a gene never share / overlap their nucleotides]
3. Universal
:- same triplet of nucleotides is responsible for coding the same amino acid in all organism.
[i.e. all codons are universal. They are called universal codes.]
(hence, genetic exchange between individuals of different species become possible!!).
4. degenerate
- 4 different nucleotides to form the triplet 64 available codes (i.e. 43=64, but there are only
20+ amino acids), some amino acids have more than one code.
- the codons are transcribed into the mRNA codons
- tRNAs have anticodons that complementary to the mRNA codons
DNA contains codes
The Genetic Code (mRNA codon):
Three bases in DNA code for one amino acid. The DNA
code is copied to produce mRNA. The order of amino
acids in the polypeptide is determined by the
sequence of 3-letter codes in mRNA.
mRNAs are synthesis in nucleus and than leaves the nucleus through the nuclear pore to the ribosomes in
the cytoplasm to direct the synthesis of protein there
[i.e. mRNA codons are translated into the amino acid sequence in a polypeptide]
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Genetics 2009
M.T.Yeung
B. How the genes determine body characteristics
We have known that :
1. Most chemicals within the cells are similar.
2. The protein and DNA of different species are different.
Role of DNA and RNA in protein synthesis
transcription
translation
Gene in DNA
RNA
(provide genetic codes)
(mRNA codons)
metabolism
amino acid sequence
(polypeptide / protein)
phenotype
•
Sequence of bases in DNA provides genetic codes to govern the sequence of amino acids arranged
in a polypeptide chain
•
Enzymes and many functional molecules are made of protein
i.e. metabolic activities are indirectly controlled by DNA
mRNA
Phenotype
Necessary conditions for protein synthesis in a cell
1. Sufficient supply of amino acids
2. Sufficient supply of energy in form of ATP
3. Presence of suitable enzymes for transcription and translation process
Mechanism
1. Synthesis of non-essential amino acids (Transamination) or
obtain of essential amino acids from diet
2. Transcription
= a process by which a complementary mRNA copy is made from a specific region (a cistron) of a
template DNA molecule.
- broken of hydrogen bond between base pairs in the cistron of DNA
- by RNA polymerase, free complementary RNA nucleotide pair with the bases in anti-sense strand of
the DNA (the template) and jointed together to form a single strand of mRNA (in 5' to 3' direction)
- a number of mRNA will be transcribed before the DNA strands wind up into a double helix again.
- mRNA strand passes through nuclear pores to ribosomes in cytoplasm
Genetics 2009
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M.T.Yeung
3. Activation of Amino acid
- each kind of amino acid combine with the amino acid binding site of a transfer RNA with specific
anticodon(s) (catalyst by enzyme, energy from ATP)
- tRNAs with anticodons which are complementary to the mRNA codons
 help to recognize correct positioning of amino acids on a growing polypeptide , according to the
specific sequence of mRNA codons.
- may have more than one tRNA (each with a specific triplet anticodon) code for each amino acid
---> more than 20 tRNA of different anticodons is found
(since there are 20 types of amino acids commonly found in proteins)
= Anticodon is a three-base sequence on a tRNA molecule while
codon is a three-base sequence on a mRNA molecule.
= Anticodon is complementary to codon.
4. Translation
= the process that translate the mRNA codon into amino acid sequence in polypeptide chains
- combination of mRNA with small subunit of a ribosome
( combination of mRNA with small ribosomal subunit, formylated methionine tRNA and initiation factors to be a complex, then binding of large
ribosomal subunit to the complex)
- formylated methionine tRNA bound to the1st vacant site in large subunit of the completed ribosome,
with its anticodon pair with the initiation mRNA codon (start codon, AUG)
- a second tRNA with a specific amino acid recognizes the adjacent mRNA codon and binds to the2nd
vacant site in the ribosomal
- a peptide bond is formed between the amino acids that are attached to the tRNAs at the 1st vacant site
and the 2nd vacant site
- ribosome move along the mRNA (from 5’ end to the 3’ end, which corresponds to the N-terminal in the completed protein),
tRNA at 1st vacant site is ejected and tRNA at 2nd vacant site is shifted to the 1st site
- empty 2nd vacant site accept the next tRNA-amino acid complex with its anticodon complementary to
the next mRNA codon
- repeat the steps of peptide bond formation and shift of tRNA in the 1st and 2nd vacant site,
polypeptide is elongated until a stop codon in the mRNA is read
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- one mRNA molecule in cytoplasm may be translated by more than one ribosome at the same time
(polyribosome / polysome ------a chain of ribosomes attaching to an mRNA molecule)
Each t-RNA has a specific anticodon consists of three
ribonucleotide bases (1), these pair up in a
complementary manner with the codon bases on the m-RNA
(1) bringing the amino acid with it (½). Adjacent
amino acids join by peptide bond (½)to form polypetide.
Ribosome moves along m-RNA (½), amino acids add on one
at a time (½).
* the ejected tRNA is free to bind with another free amino-acid of the same type again.
* released polypeptide may assemble to form proteins by folding into the secondary, tertiary or quaternary
structures.
* mRNA molecules will be broken down to regenerate nucleotides later.
Summary : http://www.dnaftb.org/dnaftb/22/concept/index.html
Summary of Central Dogma
Genetics 2009
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Genetics 2009
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A DNA molecule consists many genes, how is the protein synthesis occurring in it ?
Ans.: See the following polysomes:
Ref: http://biotechnology.usask.ca/Posters/Transcription/Transcription.html
(genotype)
Enzyme and other
proteins produced
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Genetics 2009
C.
M.T.Yeung
Regulation of transcription
If need for a protein decreases, it is usually more energy efficient to simply shut off the transcription of the
gene and not make an unnecessary mRNA.
 Gene expression must be under precise control so that genes function only in specific tissues at the
right developmental stage or time.
Jacob-Monod Hypothesis
Study of enzyme systems of E.coli by F. Jacob and J. Monod in 1961
 explain the regulation of enzyme production in term of gene regulation.
- Operon
= a group of adjacent genes which act together (hence located on a particular segment of a same
chromosome)
- mainly consists of two parts:
1. Structural genes
- closely linked genes
(same linkage group)
- responsible for synthesis
of functionally related
polypeptides
2. Operator gene
- in front of the structural gene in the operon
- controls the turning on or off of the following structural genes
- Repressor
-- protein produced by regulator gene outside an operon
-- binds to operator of the operon to repress the transcription of the following structural genes
-- may be activated (turn on of repressible operon) / inactivated (turn off of inducible operon) by
presence of certain substances (e.g. substrate of a metabolic reaction)
- Inducible operon
-- usually in the 'off ' state by repressor
-- requires inducer to turn it 'on'
-- e.g. turning on the genes of lactose utilizing enzymes (including lactase) in E. coli by the
presence of lactose (inducer) which inactivate the repressor protein
( E. coli normally uses glucose as the source of energy for growth and metabolic activities. But if it is grown
on a medium containing lactose, it will produce enzymes that allow itself to utilize lactose. This results
shows that some genes can be turned on when the enzymes they specify have to be produced.)
- Repressible operon
-- usually in the 'on' state
-- turned 'off ' only in special conditions by repressor (usually the end product of a metabolic
pathway)
-- e.g. turning off the genes of the enzyme tryptophan synthetase in E. coli by activation of
repressor protein when the later combine with excess trytophan (corepressor)
 example of feedback inhibition working at the gene level.
( E. coli normally manufactures its own tryptophan (an a.a. essential for growth) with tryptophan synthetase.
If tryptophan is added to culture medium, production of the enzyme will be stopped.)