Nucleotides and Nucleic
Acids
11/05/2002
Bing Zhou,PhD
Department of Biological Science
and Biotechnology
Tsinghua University
Functions of Nucleotides
? RNA and DNA components
? serving as energy stores for future use in phosphate transfer
reactions,These reactions are predominantly carried out by
ATP,
? forming a portion of several important coenzymes such as
NAD+,NADP+,FAD and coenzyme A.
? second messengers in signal transduction events,The
predominant second messenger is cyclic-AMP (cAMP),a cyclic
derivative of AMP formed from ATP,
? controlling numerous enzymatic reactions through allosteric
effects on enzyme activity,
? serving as activated intermediates in numerous biosynthetic
reactions,These activated intermediates include S-
adenosylmethionine (S-AdoMet) involved in methyl transfer
reactions as well as the many sugar coupled nucleotides
involved in glycogen and glycoprotein synthesis,
DNA as Genetic Material
History of DNA
? 1,The Swiss biologist,Friedrich Miescher,isolated DNA from
salmon sperm in 1868.
? 2,In 1944,Oswald Avery,Colin MacLeod,and Maclyn McCarty
showed that DNA from pathogenic strains of the bacterium
Pneumococcus could be transferred into nonpathogenic
strains,making them (and any succeeding generations)
pathogenic
? 3,Erwin Chargaff reported in 1947 that the quantitities of
adenine and thymine in DNA were very close to the same value,
Similarly,he observed that cytosine and guanine were also
very close to equal in quantity.
? 4,In 1952,Alfred Hershey and Martha Chase showed T2
bacteriophage inject only DNA into cells and this is sufficient to
make more T2 bacteriophage.
? 5,James Watson and Francis Crick proposed the model of the
double helix of DNA in 1953.
? 6,In 1965,Marshall Nirenburg,Philip Leder,and others,
identified the genetic code by which protein is made from
information in DNA.
Levene (1910) - tetranucleotide hypothesis
nucleic acid is a repetitive polymer of four subunits
A:C:G:T in the ratio 1:1:1:1
=> structure seems too simple to carry information
Levene's Tetranucleotide Hypothesis
Early experiments suggested that the four bases occur in DNA in equal
ratios,possibly as a repeating tetramer,The implications was that the
structure of DNA was too simple and too regular to contribute to hereditary
variation,attention thereafter focussed on protein as the heredity substance,
Frederick Griffith
In 1928 a scientist named Frederick Griffith was
working on a project that enabled others to point out
that DNA was the molecule of inheritance,Griffith's
experiment involved mice and two types of pneumonia,
a virulent and a non-virulent kind,He injected the
virulent pneumonia into a mouse and the mouse
died,Next he injected the non-virulent pneumonia into a
mouse and the mouse continued to live,After this,he
heated up the virulent disease to kill it and then injected
it into a mouse,The mouse lived on,Last he injected
non-virulent pneumonia and virulent pneumonia,that
had been heated and killed,into a mouse,This mouse
died,
Why? Griffith thought that the killed virulent bacteria
had passed on a characteristic to the non-virulent one to
make it virulent,He thought that this characteristic was
in the inheritance molecule,This passing on of the
inheritance molecule was what he called transformation,
Griffith's Transformation Experiment
Pneumococcus bacteria include two strains,a virulent IIIS strain with a Smooth
coat that kills mice (left),and a non-virulent IIR Rough strain that does not
(middle),Heating destroys the virulence of IIIS (right),When heat-killed IIIS is
mixed with live IIR and injected into mice,the mouse dies,and its tissue contains
living bacteria with smooth coats like IIIS,and these bacteria are subsequently
virulent to mice,Something in the heat-killed IIIS bacteria has 'transformed' the
biological and hereditary properties of the IIR bacteria,
Oswald Avery
Fourteen years later a scientist named Oswald Avery
continued with Griffith?s experiment to see what the
inheritance molecule was,In this experiment he destroyed
the lipids,ribonucleic acids,carbohydrates,and proteins of
the virulent pneumonia,Transformation still occurred after
this,Next he destroyed the deoxyribonucleic
acid,Transformation did not occur,Avery had found the
inheritance molecule,DNA!
Avery's isolation of the "Transforming Principle"
Avery repeated Griffith's experiment of combining heat-killed virulent IIIS
bacteria with non-virulent IIR bacteria,In order to isolate the transforming
substance,he fractionated the heat-killed IIIS cells and selectively
removed carbohydrates and lipids,leaving behind proteins and nucleic
acids,
Biochemical assays for transformation
Avery combined living IIR bacteria with various enzynmatic treatments of killed
IIIS material that selectively destroy protein,RNA,and DNA, Only the last
treatment destroys the transformative capacity of IIIS,which suggests that the
transforming substance is DNA,
Hershey & Chase (1952) - 'blender experiment'
bacteriophages are grown in radioactive medium
proteins labeled with 35S
DNA labeled with 32P
during transformation of E,coli by bacteriophages,
32P goes in,35S stays out
=> DNA is the transforming principle
T1 Bacteriophages ("Phage")
Bacteriophages are viruses that attack bacteria,They
attach to the outside membrane of bacteria by means
of the thin "tail",the round "head" contains DNA,The
name means "bacteria eaters" and is commonly
shortened to just "phage",Early biochemical and
genetic studies of phage are the foundations of
modern molecular biology,
The Hershey & Chase Experiment
Growing bacteriophage (a virus that grows on bacteria) was differentially
labelled with radioactive phosphorus (which labels nucleic acid) or
radioative sulfur (which labels protein)
The Hershey & Chase Experiment
During the process of infection,a quick spin in a blendor shears phage away from the
outside of the bacteria,Analysis of the bacterial and supernatant fractions afterward
shows that phosphorus-labelled DNA inside the phage shows up in bacteria (left),
whereas the sulfur-labelled protein in the external coat remains in the supernatant
(right),Further,bacteria with labelled DNA go on to produce progeny phage that are
also labelled,This biological experiment confirms Avery's conclusion that DNA is the
transforming substance,and shows that transformation is heritable,
Bases,Nucleosides,and
Nucleotides
Bases
T
U
C G
A
Structure of T and C
Structure of A and G
Deoxyribose and Ribose
Structure of Deoxyribose
syn-Adenosine and anti-Adenosine
Structure of dA
Nucleosides and Nucleotides
dNTP
Bases,Nucleosides,and Nucleotides
? Cytosine,Cytidine,Cytidine monophosphate
CMP,CDP,CTP,and dCTP etc,
? Uracil,Uridine,Uridine monophosphate
UMP
? Thymine,Thymidine,Thymidine monophosphate
TMP
? Adenine,Adenosine,Adenosine monophosphate
AMP
? Guanine,Guanosine,Guanosine monophosphate
GMP
Unusual Nucleotides
? The nucleotide uridine is never found in DNA and
thymine is almost exclusively found in DNA,
Thymine is found in tRNAs but not rRNAs nor
mRNAs,
? Less common bases found in DNA and RNA
include 5-methylcytosine,
? A variety of modified bases appear in the tRNAs,
Many modified nucleotides are encountered
outside of the context of DNA and RNA that serve
important biological functions.
Synthetic Nucleotide Analogs
?A large family of analogues are used as anti-tumor
agents,because they interfere with the synthesis of
DNA and thereby preferentially kill rapidly dividing cells
such as tumor cells,Often used in chemotherapy,6-
mercaptopurine,5-fluorouracil,5-iodo-2'-deoxyuridine
and 6-thioguanine,Each of these compounds disrupts
the normal replication process by interfering with the
formation of correct Watson-Crick base-pairing,
?Nucleotide analogs are used as antiviral agents,Several
analogs are used to interfere with the replication of HIV,
such as AZT (azidothymidine) and ddI (dideoxyinosine).
?Dideoxy nucleotides are used in sequencing reactions,
Polynucleotides
?Polynucleotides are formed by the condensation of
nucleotides between the alcohol of a 5'-phosphate
of one nucleotide and the 3'-hydroxyl of a second,
with the elimination of H2O,forming a
phosphodiester bond,The formation of
phosphodiester bonds in DNA and RNA exhibits
directionality,The primary structure of DNA and
RNA (the linear arrangement of the nucleotides)
proceeds in the 5' ----> 3' direction,
?The common representation of the primary
structure of DNA or RNA molecules is to write the
nucleotide sequences from left to right
synonymous with the 5' ----> 3' direction as shown:
5'-pGpApTpC-3?,or GATC
Chemical Structures of RNA/DNA
Example of DNA Backbone,
5'-d(CGAAT):
Phosphodiester bonds in
nucleic acids are very stable to
hydrolysis in the absence of a
catalyst (such as an acid or a
nuclease).
However,DNA hydrolysis is
thermodynamically favorable.
Synthesis of a phosphodiester bond in
nucleic acids requires energy input,As a
result,the nucleoside monophosphates in
nucleic acids are built up from hydrolysis
of nucleoside triphosphates,Cleaving a
pyrophosphate from a nucleoside
triphosphate yields a nucleoside
monophosphate and enough free energy
to make the formation of polynucleoside
monophosphates (i.e.,polynucleotides)
thermodynamically favorable.
RNA Backbone is
Sensitive to Base
Hydrolysis
Solving DNA Structure
Although deoxyribonucleic acid,or DNA,was discovered in the late 1860s,the
substance was largely ignored for nearly a century because it seemed too simple
to serve any significant purpose,This view changed dramatically in the 1940s,At
this time scientists discovered that chromosomes,which were known to carry
hereditary information,consisted of DNA and proteins,Experiments conducted
throughout the 1940s showed that,contrary to the prevailing opinion that proteins
carry the genetic information,DNA actually seemed to be the genetic material,
However,it was still not known what the structure of DNA was,and how such a
simple molecule could contain all the information needed to produce a human
being or other living organisms.
James Watson and Francis Crick,a postdoctoral fellow and a graduate student,
started to solve the mystery of DNA structure,The partnership began when
Watson,an American,took a research position at Cambridge University in
England in 1951,Crick was also at Cambridge,studying protein structure with a
technique called Xray crystallography,By their own admission,both were more
interested in the prevailing scientific problems of that day than in their own work,
and the structure of DNA was definitely an interesting problem,Over the next few
years,Watson and Crick would collect,by coincidence,hard work,and a little luck,
key pieces of information that they would use to solve the DNA puzzle.
Erwin Chargaff
To understand the DNA molecule better scientists were trying
to make a model to understand how it works and what it
does,In the 1940?s another scientist named Erwin Chargaff
noticed a pattern in the amounts of the four bases,adenine,
guanine,cytosine,and thymine,He took samples of DNA of
different cells and found that the amount of adenine was almost
equal to the amount of thymine,and that the amount of guanine
was almost equal to the amount of cytosine,Thus you could
say,A=T,and G=C,This discovery later became Chargaff?s
Rule.
Franklin & Wilkins' X-ray Crystallography
Two scientists named,Rosalind Franklin and Maurice Wilkins,decided
to try to make a crystal of the DNA molecule,If they could get DNA to
crystallize,then they could make an x-ray pattern,thus resulting in
understanding how DNA works,These two scientists were successful
and obtained an x-ray pattern,The pattern appeared to contain rungs,
like those on a ladder between two strands that are side by side,It also
showed by an,X” shape that DNA had a helix shape,
2 or 3 Strands?
The structure of B-
DNA
Structure of DNA
Watson-Crick model
?The bases are in the interior of the helix aligned at
a nearly 90 degree angle relative to the axis of the
helix,
?Purine bases form hydrogen bonds with
pyrimidines,in the crucial phenomenon of base
pairing,
?Base-pairs composed of G and C contain three H-
bonds,whereas those of A and T contain two H-
bonds,This makes G-C base-pairs more stable
than A-T base-pairs.
DNA Replication
Photo by A.C,Barrington Brown,courtesy of Cold Spring Harbor Laboratory Archives.
1962 Francis
Crick,James
Watson,and
Maurice
Wilkins
receive the
Nobel Prize
for
determining
the
molecular
structure of
DNA.
Base Pairs
A-T Base Pair G-C Base Pair
dA-dT Base Pair
dG-dC Base Pair
Structure of DNA Double Helix
Space-filling
model of DNA.
View Down the Helix Axis
Summary of B-DNA Structure
? 1,The two chains in the double helix are antiparallel (one goes 5'
to 3' whereas the complementary strand goes 3' to 5').
? 2,Phosphate groups link together the sugar backbone via
phosphodiester bonds.
? 3,The bases on the two chains pair in a complementary fashion,
Adenine (A) pairs with thymine (T) and guanine (G) pairs with
cytosine (C).
? 4,Hydrogen bonds between bases (3 for G-C,2 for A-T) provides
the pairing specificity.
? 5,There are ten bases per turn of the helix,so the helix rotates 36
per base.
? 6,The rise of the double helix is the distance parallel to the axis
of the helix from the level of one base to the level of the adjacent
base,The pitch in B-DNA is 3.4 nm because the rise is 0.34 nm
and there are ten base pairs per turn of the helix.
? 7,B-DNA possesses a major and a minor groove
The double helix of DNA has been shown to exist in
several different forms,depending upon sequence
content and ionic conditions of crystal preparation,The
B-form of DNA prevails under physiological conditions
of low ionic strength and a high degree of hydration,
Regions of the helix that are rich in pCpG dinucleotides
can exist in a novel left-handed helical conformation
termed Z-DNA,This conformation results from a 180
degree change in the orientation of the bases relative to
that of the more common A- and B-DNA.
Structure of B-DNA and Z-DNA
Z-form DNA
?Helix has left-handed sense
?Can be formed in vivo,given proper sequence
and superhelical tension,but function remains
obscure,
?Narrower,more elongated helix than A or B,
?Major "groove" not really groove
?Narrow minor groove
?Conformation favored by high salt
concentrations,some base substitutions,but
requires alternating purine-pyrimidine sequence,
?Zigzag backbone due to C sugar conformation
compensating for G glycosidic bond
conformation
?Conformations,
?G,syn,C2'-endo; C,anti,C3'-endo
B and Z-DNA comparison
? Z-Form
? Left
? 12 base pairs/turn
? Major groove Flat,Minor
groove narrow and deep
? Orientation of N-
glycosidic Bond,Anti for
Py,Syn for Pu
? Occurs in stretches of
alternating purine-
pyrimidine base pairs
? B Form
? Right
? 10 base pairs/turn
? Major groove wide and
deep,Minor groove narrow
and deep
? Orientation of N-glycosidic
Bond,All Anti
? most prevalent within cells
A-form DNA
?Observed occasionally in vitro when loss of water,
Shorter,wider helix than B,
?Deep,narrow major groove not easily accessible to
proteins
?Wide,shallow minor groove accessible to proteins,
but lower information content than major groove,
?Favored conformation at low water concentrations
Base pairs tilted to helix axis and displaced from
axis
?Sugar pucker C3'-endo (in RNA 2'-OH inhibits C2'-
endo conformation)
Nucleic Acid Properties
Forces in dsDNA
Forces Affecting dsDNA Formation
? Three types of forces contribute to maintaining the
stability of the DNA double helix one against it,
? 1 Hydrophobic interactions,The base pairs in the interior
of the DNA molecule create a hydrophobic environment,
with the negatively charged phosphates along the
backbone being exposed to the solvent,
? 2 Stacking interactions,relatively weak but additive van
der waals forces
? 3 Hydrogen bonding,relatively weak but additive.
? 4 Electrostatic replusion by negatively charged
phosphates along the DNA backbone destabilize the
double helix.
Thermal Denaturation of DNA
? Tm,In the process of thermal denaturation,Tm is a point at
which 50% of the DNA molecule exists as single strands.
? Tm is primarily dependent upon 1) Base composition of
that DNA molecule,2) Chemical nature of the solvent and
3) the identities and concentrations of ions in the solution
? When thermally melted DNA is cooled,the
complementary strands will again re-form the correct
base pairs,in a process termed annealing or
hybridization.
Hyperchromicity
DNA solution characteristically shows an absorbance
maximum at 260nm (in the UV region of the
spectrum),If the same DNA solution is melted,the
absorbance at 260nm increases,This property is
termed "hyperchromicity." The hyperchromic shift is
due to the fact that unstacked bases absorb more light
than stacked bases.
Tm vs GC Content
Relaxed and Supercoiled DNA
Conformations of Single-strand
Nucleic Acids
DNA Separation by Size
Agarose Electrophoresis,agarose,a carbohydrate
polymer purified from a salt water algae,It is a
copolymer of mannose and galactose that when
melted and re-cooled forms a gel with pores sizes
dependent upon the concentration of agarose,The
phosphate backbone of DNA is highly negatively
charged,therefore DNA will migrate in an electric
field,The size of DNA fragments can then be
determined by comparing their migration in the gel
to known size standards.
Pulsed-Field Gel Electrophoresis
Extremely large molecules of DNA are effectively
separated in agarose gels using pulsed-field gel
electrophoresis (PFGE),This technique employs two
or more electrodes,placed orthogonally with respect
to the gel,that receive short alternating pulses of
current,PFGE allows whole chromosomes and large
portions of chromosomes to be analyzed.
Genome of Various Organisms
Central Dogma
DNA <-----?RNA -----?Protein
Transcription,RNA is Made on DNA
Templates
?mRNA,messenger RNA,coding for
proteins,In eukaryotes,it is often poly-
adenylated,In higher eukaryotes,RNA
precursor is often much larger than matured
mRNA
?tRNA,transfer RNA,the mediator between
mRNA and protein formation
?rRNA,ribosomal RNA,in ribosome
Introns and Exons
? Some parts of RNA are spliced out during RNA
maturation,the corresponding regions of the
spliced-out parts in DNA are called introns,
? In higher eukaryotes,one gene normally has
multiple introns,The exon number = intron
number + 1,
? Coding region is continuous in exons,
? cDNA (complementary DNA) is reverse
transcribed from mRNA and carries no introns.
“Junk” DNA
Most DNA regions in higher eukaryotes do
not code for proteins and have no known
functions,This majority part of genome is
sometimes called,junk DNA”,Junk DNA,
however,may have functions,For example,
genomic DNA often gives higher expression
level in transgenic animals than cDNA.
mRNAs Code for Polypeptides
?Polycistronic,a single mRNA code for
several polypeptide chains
?Monocistronic,code for one peptide chains,
In eucaryotes,most mRNAs are
monocistronic.
DNA Mutation
? DNA change that causes genetic alteration is called
mutation,
? Most carcinogens cause DNA mutations,Most mutagens
are carcinogens,Many food preservatives such as sodium
nitrate and sodium nitrite also cause DNA mutations,
? DNA also undergoes spontaneous mutations,for instance,
deamination (loss of amino group)
? Many aging mutants identified so far are DNA repairing
mutants,
? Radiation also causes DNA damage,UV causes
pyrimidine dimers,X-ray causes DNA breakages.
? Oxidative radicals might be the major mutation stress
under normal physiological conditions.
Fruit fly has many kinds
of mutant strains,all
caused by DNA
mutations
? Human individual difference is mainly because of the
existence of multiple SNPs,
SNP=Single Nucleotide Polymorphism,It occurs about
once every kilobases,
? Human genetic diseases can be caused by various DNA
mutations.
Point Mutations
Small deletions and Insertions
Big DNA or chromosomal alterations
Genetic Difference is Due to DNA
Change
Shows 23 pairs of chromosomes,Twenty two pairs are called autosomes,1 pair
is called the sex chromosomes ; XY in a male,Males are associated with
secondary sexual characteristics – abundant facial hair,voice,others,
Differences are also evident in the genitalia.
Normal Male
HUMAN GENETICS
Normal Female
Shows 23 pairs as in the male,but sex chromosomes are represented by
XX,Females also have different physical features from males,A Barr body
is also present in cells of the female,representing an inactivated X
chromosome.
Klinefelter's Syndrome
Three sex chromosomes are associated with Klinefelter rather than the
expected 2 - XXY,These individuals are males with some development of
breast tissue normally seen in females,Little body hair is present,and such
person are typically tall,with or without evidence of mental retardation,
Males with XXXY,XXXXY,and XXXXXY karyotypes have a more severe
presentation,and mental retardation is expected.
Down's Syndrome
Normally associated with 3 copies of chromosome number 21(trisomy of
chromosome 21),rather than the 2 found normally,Down syndrome is
characterized by differing degrees of mental retardation,a skin fold over the
eye,typically short stature,and short hands with a deep crease in the palm,
Down is also known as mongolism (mongoloid).
Turner's Syndrome
Only 1 sex chromosome is present -X0,or X_,The expected Y chromosome
is missing,Turner syndrome is associated with underdeveloped ovaries,
short stature,webbed/.bull neck,and broad chest,Individuals are sterile,
and lack expected secondary sexual characteristics,Mental retardation
typically not evident.
Acids
11/05/2002
Bing Zhou,PhD
Department of Biological Science
and Biotechnology
Tsinghua University
Functions of Nucleotides
? RNA and DNA components
? serving as energy stores for future use in phosphate transfer
reactions,These reactions are predominantly carried out by
ATP,
? forming a portion of several important coenzymes such as
NAD+,NADP+,FAD and coenzyme A.
? second messengers in signal transduction events,The
predominant second messenger is cyclic-AMP (cAMP),a cyclic
derivative of AMP formed from ATP,
? controlling numerous enzymatic reactions through allosteric
effects on enzyme activity,
? serving as activated intermediates in numerous biosynthetic
reactions,These activated intermediates include S-
adenosylmethionine (S-AdoMet) involved in methyl transfer
reactions as well as the many sugar coupled nucleotides
involved in glycogen and glycoprotein synthesis,
DNA as Genetic Material
History of DNA
? 1,The Swiss biologist,Friedrich Miescher,isolated DNA from
salmon sperm in 1868.
? 2,In 1944,Oswald Avery,Colin MacLeod,and Maclyn McCarty
showed that DNA from pathogenic strains of the bacterium
Pneumococcus could be transferred into nonpathogenic
strains,making them (and any succeeding generations)
pathogenic
? 3,Erwin Chargaff reported in 1947 that the quantitities of
adenine and thymine in DNA were very close to the same value,
Similarly,he observed that cytosine and guanine were also
very close to equal in quantity.
? 4,In 1952,Alfred Hershey and Martha Chase showed T2
bacteriophage inject only DNA into cells and this is sufficient to
make more T2 bacteriophage.
? 5,James Watson and Francis Crick proposed the model of the
double helix of DNA in 1953.
? 6,In 1965,Marshall Nirenburg,Philip Leder,and others,
identified the genetic code by which protein is made from
information in DNA.
Levene (1910) - tetranucleotide hypothesis
nucleic acid is a repetitive polymer of four subunits
A:C:G:T in the ratio 1:1:1:1
=> structure seems too simple to carry information
Levene's Tetranucleotide Hypothesis
Early experiments suggested that the four bases occur in DNA in equal
ratios,possibly as a repeating tetramer,The implications was that the
structure of DNA was too simple and too regular to contribute to hereditary
variation,attention thereafter focussed on protein as the heredity substance,
Frederick Griffith
In 1928 a scientist named Frederick Griffith was
working on a project that enabled others to point out
that DNA was the molecule of inheritance,Griffith's
experiment involved mice and two types of pneumonia,
a virulent and a non-virulent kind,He injected the
virulent pneumonia into a mouse and the mouse
died,Next he injected the non-virulent pneumonia into a
mouse and the mouse continued to live,After this,he
heated up the virulent disease to kill it and then injected
it into a mouse,The mouse lived on,Last he injected
non-virulent pneumonia and virulent pneumonia,that
had been heated and killed,into a mouse,This mouse
died,
Why? Griffith thought that the killed virulent bacteria
had passed on a characteristic to the non-virulent one to
make it virulent,He thought that this characteristic was
in the inheritance molecule,This passing on of the
inheritance molecule was what he called transformation,
Griffith's Transformation Experiment
Pneumococcus bacteria include two strains,a virulent IIIS strain with a Smooth
coat that kills mice (left),and a non-virulent IIR Rough strain that does not
(middle),Heating destroys the virulence of IIIS (right),When heat-killed IIIS is
mixed with live IIR and injected into mice,the mouse dies,and its tissue contains
living bacteria with smooth coats like IIIS,and these bacteria are subsequently
virulent to mice,Something in the heat-killed IIIS bacteria has 'transformed' the
biological and hereditary properties of the IIR bacteria,
Oswald Avery
Fourteen years later a scientist named Oswald Avery
continued with Griffith?s experiment to see what the
inheritance molecule was,In this experiment he destroyed
the lipids,ribonucleic acids,carbohydrates,and proteins of
the virulent pneumonia,Transformation still occurred after
this,Next he destroyed the deoxyribonucleic
acid,Transformation did not occur,Avery had found the
inheritance molecule,DNA!
Avery's isolation of the "Transforming Principle"
Avery repeated Griffith's experiment of combining heat-killed virulent IIIS
bacteria with non-virulent IIR bacteria,In order to isolate the transforming
substance,he fractionated the heat-killed IIIS cells and selectively
removed carbohydrates and lipids,leaving behind proteins and nucleic
acids,
Biochemical assays for transformation
Avery combined living IIR bacteria with various enzynmatic treatments of killed
IIIS material that selectively destroy protein,RNA,and DNA, Only the last
treatment destroys the transformative capacity of IIIS,which suggests that the
transforming substance is DNA,
Hershey & Chase (1952) - 'blender experiment'
bacteriophages are grown in radioactive medium
proteins labeled with 35S
DNA labeled with 32P
during transformation of E,coli by bacteriophages,
32P goes in,35S stays out
=> DNA is the transforming principle
T1 Bacteriophages ("Phage")
Bacteriophages are viruses that attack bacteria,They
attach to the outside membrane of bacteria by means
of the thin "tail",the round "head" contains DNA,The
name means "bacteria eaters" and is commonly
shortened to just "phage",Early biochemical and
genetic studies of phage are the foundations of
modern molecular biology,
The Hershey & Chase Experiment
Growing bacteriophage (a virus that grows on bacteria) was differentially
labelled with radioactive phosphorus (which labels nucleic acid) or
radioative sulfur (which labels protein)
The Hershey & Chase Experiment
During the process of infection,a quick spin in a blendor shears phage away from the
outside of the bacteria,Analysis of the bacterial and supernatant fractions afterward
shows that phosphorus-labelled DNA inside the phage shows up in bacteria (left),
whereas the sulfur-labelled protein in the external coat remains in the supernatant
(right),Further,bacteria with labelled DNA go on to produce progeny phage that are
also labelled,This biological experiment confirms Avery's conclusion that DNA is the
transforming substance,and shows that transformation is heritable,
Bases,Nucleosides,and
Nucleotides
Bases
T
U
C G
A
Structure of T and C
Structure of A and G
Deoxyribose and Ribose
Structure of Deoxyribose
syn-Adenosine and anti-Adenosine
Structure of dA
Nucleosides and Nucleotides
dNTP
Bases,Nucleosides,and Nucleotides
? Cytosine,Cytidine,Cytidine monophosphate
CMP,CDP,CTP,and dCTP etc,
? Uracil,Uridine,Uridine monophosphate
UMP
? Thymine,Thymidine,Thymidine monophosphate
TMP
? Adenine,Adenosine,Adenosine monophosphate
AMP
? Guanine,Guanosine,Guanosine monophosphate
GMP
Unusual Nucleotides
? The nucleotide uridine is never found in DNA and
thymine is almost exclusively found in DNA,
Thymine is found in tRNAs but not rRNAs nor
mRNAs,
? Less common bases found in DNA and RNA
include 5-methylcytosine,
? A variety of modified bases appear in the tRNAs,
Many modified nucleotides are encountered
outside of the context of DNA and RNA that serve
important biological functions.
Synthetic Nucleotide Analogs
?A large family of analogues are used as anti-tumor
agents,because they interfere with the synthesis of
DNA and thereby preferentially kill rapidly dividing cells
such as tumor cells,Often used in chemotherapy,6-
mercaptopurine,5-fluorouracil,5-iodo-2'-deoxyuridine
and 6-thioguanine,Each of these compounds disrupts
the normal replication process by interfering with the
formation of correct Watson-Crick base-pairing,
?Nucleotide analogs are used as antiviral agents,Several
analogs are used to interfere with the replication of HIV,
such as AZT (azidothymidine) and ddI (dideoxyinosine).
?Dideoxy nucleotides are used in sequencing reactions,
Polynucleotides
?Polynucleotides are formed by the condensation of
nucleotides between the alcohol of a 5'-phosphate
of one nucleotide and the 3'-hydroxyl of a second,
with the elimination of H2O,forming a
phosphodiester bond,The formation of
phosphodiester bonds in DNA and RNA exhibits
directionality,The primary structure of DNA and
RNA (the linear arrangement of the nucleotides)
proceeds in the 5' ----> 3' direction,
?The common representation of the primary
structure of DNA or RNA molecules is to write the
nucleotide sequences from left to right
synonymous with the 5' ----> 3' direction as shown:
5'-pGpApTpC-3?,or GATC
Chemical Structures of RNA/DNA
Example of DNA Backbone,
5'-d(CGAAT):
Phosphodiester bonds in
nucleic acids are very stable to
hydrolysis in the absence of a
catalyst (such as an acid or a
nuclease).
However,DNA hydrolysis is
thermodynamically favorable.
Synthesis of a phosphodiester bond in
nucleic acids requires energy input,As a
result,the nucleoside monophosphates in
nucleic acids are built up from hydrolysis
of nucleoside triphosphates,Cleaving a
pyrophosphate from a nucleoside
triphosphate yields a nucleoside
monophosphate and enough free energy
to make the formation of polynucleoside
monophosphates (i.e.,polynucleotides)
thermodynamically favorable.
RNA Backbone is
Sensitive to Base
Hydrolysis
Solving DNA Structure
Although deoxyribonucleic acid,or DNA,was discovered in the late 1860s,the
substance was largely ignored for nearly a century because it seemed too simple
to serve any significant purpose,This view changed dramatically in the 1940s,At
this time scientists discovered that chromosomes,which were known to carry
hereditary information,consisted of DNA and proteins,Experiments conducted
throughout the 1940s showed that,contrary to the prevailing opinion that proteins
carry the genetic information,DNA actually seemed to be the genetic material,
However,it was still not known what the structure of DNA was,and how such a
simple molecule could contain all the information needed to produce a human
being or other living organisms.
James Watson and Francis Crick,a postdoctoral fellow and a graduate student,
started to solve the mystery of DNA structure,The partnership began when
Watson,an American,took a research position at Cambridge University in
England in 1951,Crick was also at Cambridge,studying protein structure with a
technique called Xray crystallography,By their own admission,both were more
interested in the prevailing scientific problems of that day than in their own work,
and the structure of DNA was definitely an interesting problem,Over the next few
years,Watson and Crick would collect,by coincidence,hard work,and a little luck,
key pieces of information that they would use to solve the DNA puzzle.
Erwin Chargaff
To understand the DNA molecule better scientists were trying
to make a model to understand how it works and what it
does,In the 1940?s another scientist named Erwin Chargaff
noticed a pattern in the amounts of the four bases,adenine,
guanine,cytosine,and thymine,He took samples of DNA of
different cells and found that the amount of adenine was almost
equal to the amount of thymine,and that the amount of guanine
was almost equal to the amount of cytosine,Thus you could
say,A=T,and G=C,This discovery later became Chargaff?s
Rule.
Franklin & Wilkins' X-ray Crystallography
Two scientists named,Rosalind Franklin and Maurice Wilkins,decided
to try to make a crystal of the DNA molecule,If they could get DNA to
crystallize,then they could make an x-ray pattern,thus resulting in
understanding how DNA works,These two scientists were successful
and obtained an x-ray pattern,The pattern appeared to contain rungs,
like those on a ladder between two strands that are side by side,It also
showed by an,X” shape that DNA had a helix shape,
2 or 3 Strands?
The structure of B-
DNA
Structure of DNA
Watson-Crick model
?The bases are in the interior of the helix aligned at
a nearly 90 degree angle relative to the axis of the
helix,
?Purine bases form hydrogen bonds with
pyrimidines,in the crucial phenomenon of base
pairing,
?Base-pairs composed of G and C contain three H-
bonds,whereas those of A and T contain two H-
bonds,This makes G-C base-pairs more stable
than A-T base-pairs.
DNA Replication
Photo by A.C,Barrington Brown,courtesy of Cold Spring Harbor Laboratory Archives.
1962 Francis
Crick,James
Watson,and
Maurice
Wilkins
receive the
Nobel Prize
for
determining
the
molecular
structure of
DNA.
Base Pairs
A-T Base Pair G-C Base Pair
dA-dT Base Pair
dG-dC Base Pair
Structure of DNA Double Helix
Space-filling
model of DNA.
View Down the Helix Axis
Summary of B-DNA Structure
? 1,The two chains in the double helix are antiparallel (one goes 5'
to 3' whereas the complementary strand goes 3' to 5').
? 2,Phosphate groups link together the sugar backbone via
phosphodiester bonds.
? 3,The bases on the two chains pair in a complementary fashion,
Adenine (A) pairs with thymine (T) and guanine (G) pairs with
cytosine (C).
? 4,Hydrogen bonds between bases (3 for G-C,2 for A-T) provides
the pairing specificity.
? 5,There are ten bases per turn of the helix,so the helix rotates 36
per base.
? 6,The rise of the double helix is the distance parallel to the axis
of the helix from the level of one base to the level of the adjacent
base,The pitch in B-DNA is 3.4 nm because the rise is 0.34 nm
and there are ten base pairs per turn of the helix.
? 7,B-DNA possesses a major and a minor groove
The double helix of DNA has been shown to exist in
several different forms,depending upon sequence
content and ionic conditions of crystal preparation,The
B-form of DNA prevails under physiological conditions
of low ionic strength and a high degree of hydration,
Regions of the helix that are rich in pCpG dinucleotides
can exist in a novel left-handed helical conformation
termed Z-DNA,This conformation results from a 180
degree change in the orientation of the bases relative to
that of the more common A- and B-DNA.
Structure of B-DNA and Z-DNA
Z-form DNA
?Helix has left-handed sense
?Can be formed in vivo,given proper sequence
and superhelical tension,but function remains
obscure,
?Narrower,more elongated helix than A or B,
?Major "groove" not really groove
?Narrow minor groove
?Conformation favored by high salt
concentrations,some base substitutions,but
requires alternating purine-pyrimidine sequence,
?Zigzag backbone due to C sugar conformation
compensating for G glycosidic bond
conformation
?Conformations,
?G,syn,C2'-endo; C,anti,C3'-endo
B and Z-DNA comparison
? Z-Form
? Left
? 12 base pairs/turn
? Major groove Flat,Minor
groove narrow and deep
? Orientation of N-
glycosidic Bond,Anti for
Py,Syn for Pu
? Occurs in stretches of
alternating purine-
pyrimidine base pairs
? B Form
? Right
? 10 base pairs/turn
? Major groove wide and
deep,Minor groove narrow
and deep
? Orientation of N-glycosidic
Bond,All Anti
? most prevalent within cells
A-form DNA
?Observed occasionally in vitro when loss of water,
Shorter,wider helix than B,
?Deep,narrow major groove not easily accessible to
proteins
?Wide,shallow minor groove accessible to proteins,
but lower information content than major groove,
?Favored conformation at low water concentrations
Base pairs tilted to helix axis and displaced from
axis
?Sugar pucker C3'-endo (in RNA 2'-OH inhibits C2'-
endo conformation)
Nucleic Acid Properties
Forces in dsDNA
Forces Affecting dsDNA Formation
? Three types of forces contribute to maintaining the
stability of the DNA double helix one against it,
? 1 Hydrophobic interactions,The base pairs in the interior
of the DNA molecule create a hydrophobic environment,
with the negatively charged phosphates along the
backbone being exposed to the solvent,
? 2 Stacking interactions,relatively weak but additive van
der waals forces
? 3 Hydrogen bonding,relatively weak but additive.
? 4 Electrostatic replusion by negatively charged
phosphates along the DNA backbone destabilize the
double helix.
Thermal Denaturation of DNA
? Tm,In the process of thermal denaturation,Tm is a point at
which 50% of the DNA molecule exists as single strands.
? Tm is primarily dependent upon 1) Base composition of
that DNA molecule,2) Chemical nature of the solvent and
3) the identities and concentrations of ions in the solution
? When thermally melted DNA is cooled,the
complementary strands will again re-form the correct
base pairs,in a process termed annealing or
hybridization.
Hyperchromicity
DNA solution characteristically shows an absorbance
maximum at 260nm (in the UV region of the
spectrum),If the same DNA solution is melted,the
absorbance at 260nm increases,This property is
termed "hyperchromicity." The hyperchromic shift is
due to the fact that unstacked bases absorb more light
than stacked bases.
Tm vs GC Content
Relaxed and Supercoiled DNA
Conformations of Single-strand
Nucleic Acids
DNA Separation by Size
Agarose Electrophoresis,agarose,a carbohydrate
polymer purified from a salt water algae,It is a
copolymer of mannose and galactose that when
melted and re-cooled forms a gel with pores sizes
dependent upon the concentration of agarose,The
phosphate backbone of DNA is highly negatively
charged,therefore DNA will migrate in an electric
field,The size of DNA fragments can then be
determined by comparing their migration in the gel
to known size standards.
Pulsed-Field Gel Electrophoresis
Extremely large molecules of DNA are effectively
separated in agarose gels using pulsed-field gel
electrophoresis (PFGE),This technique employs two
or more electrodes,placed orthogonally with respect
to the gel,that receive short alternating pulses of
current,PFGE allows whole chromosomes and large
portions of chromosomes to be analyzed.
Genome of Various Organisms
Central Dogma
DNA <-----?RNA -----?Protein
Transcription,RNA is Made on DNA
Templates
?mRNA,messenger RNA,coding for
proteins,In eukaryotes,it is often poly-
adenylated,In higher eukaryotes,RNA
precursor is often much larger than matured
mRNA
?tRNA,transfer RNA,the mediator between
mRNA and protein formation
?rRNA,ribosomal RNA,in ribosome
Introns and Exons
? Some parts of RNA are spliced out during RNA
maturation,the corresponding regions of the
spliced-out parts in DNA are called introns,
? In higher eukaryotes,one gene normally has
multiple introns,The exon number = intron
number + 1,
? Coding region is continuous in exons,
? cDNA (complementary DNA) is reverse
transcribed from mRNA and carries no introns.
“Junk” DNA
Most DNA regions in higher eukaryotes do
not code for proteins and have no known
functions,This majority part of genome is
sometimes called,junk DNA”,Junk DNA,
however,may have functions,For example,
genomic DNA often gives higher expression
level in transgenic animals than cDNA.
mRNAs Code for Polypeptides
?Polycistronic,a single mRNA code for
several polypeptide chains
?Monocistronic,code for one peptide chains,
In eucaryotes,most mRNAs are
monocistronic.
DNA Mutation
? DNA change that causes genetic alteration is called
mutation,
? Most carcinogens cause DNA mutations,Most mutagens
are carcinogens,Many food preservatives such as sodium
nitrate and sodium nitrite also cause DNA mutations,
? DNA also undergoes spontaneous mutations,for instance,
deamination (loss of amino group)
? Many aging mutants identified so far are DNA repairing
mutants,
? Radiation also causes DNA damage,UV causes
pyrimidine dimers,X-ray causes DNA breakages.
? Oxidative radicals might be the major mutation stress
under normal physiological conditions.
Fruit fly has many kinds
of mutant strains,all
caused by DNA
mutations
? Human individual difference is mainly because of the
existence of multiple SNPs,
SNP=Single Nucleotide Polymorphism,It occurs about
once every kilobases,
? Human genetic diseases can be caused by various DNA
mutations.
Point Mutations
Small deletions and Insertions
Big DNA or chromosomal alterations
Genetic Difference is Due to DNA
Change
Shows 23 pairs of chromosomes,Twenty two pairs are called autosomes,1 pair
is called the sex chromosomes ; XY in a male,Males are associated with
secondary sexual characteristics – abundant facial hair,voice,others,
Differences are also evident in the genitalia.
Normal Male
HUMAN GENETICS
Normal Female
Shows 23 pairs as in the male,but sex chromosomes are represented by
XX,Females also have different physical features from males,A Barr body
is also present in cells of the female,representing an inactivated X
chromosome.
Klinefelter's Syndrome
Three sex chromosomes are associated with Klinefelter rather than the
expected 2 - XXY,These individuals are males with some development of
breast tissue normally seen in females,Little body hair is present,and such
person are typically tall,with or without evidence of mental retardation,
Males with XXXY,XXXXY,and XXXXXY karyotypes have a more severe
presentation,and mental retardation is expected.
Down's Syndrome
Normally associated with 3 copies of chromosome number 21(trisomy of
chromosome 21),rather than the 2 found normally,Down syndrome is
characterized by differing degrees of mental retardation,a skin fold over the
eye,typically short stature,and short hands with a deep crease in the palm,
Down is also known as mongolism (mongoloid).
Turner's Syndrome
Only 1 sex chromosome is present -X0,or X_,The expected Y chromosome
is missing,Turner syndrome is associated with underdeveloped ovaries,
short stature,webbed/.bull neck,and broad chest,Individuals are sterile,
and lack expected secondary sexual characteristics,Mental retardation
typically not evident.
