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©2001 Timothy G. Standish
Isaiah 33:22
22 For the Lord is our judge, the Lord is our lawgiver, the Lord is our king; he will save us.
©2001 Timothy G. Standish
Molecular Basis Molecular Basis Of InheritanceOf Inheritance
Timothy G. Standish, Ph. D.
©2001 Timothy G. Standish
Correlation and InterpretationCorrelation and Interpretation The United States has never lost a war in which the
military used mules Illogical conclusion - If the US is in a war that it wants to
win, it had better use mules Correlation between two variables, in this case victory and
mules, does not mean that a cause-and-effect relationship is at play
The United States has fought wars and won with no mules, i.e., the Persian Gulf war, thus mules are not the magic ingredient for victory
How data are viewed, the questions that are asked and conclusions drawn are frequently the product of researcher’s values and beliefs
©2001 Timothy G. Standish
On Value Free ScienceOn Value Free ScienceThe idea that science should (or can) be value-free is
wrong . . . being steeped in values is part of being human.The success of science comes not from researcher’s attempts
to be objective, but from its adherence to rules (honesty, disclosure of procedures, attempts to disprove hypotheses) and its adversarial nature . . . Scientists’ credibility should rest on openness about uncertainties . . . Scientist should not dictate what society wants, but rather interact vigorously with other scholars and the public to achieve ends that both are feasible and make sense scientifically.Ehrlich, Paul R. 2000. Evolution of an Advocate. Science 287:2159
©2001 Timothy G. Standish
The Scientific MethodThe Scientific Method The Scientific method relies on two types of reasoning: Inductive reasoning - Drawing generalized conclusions
from data. This type of reasoning is used when coming up with a theory
Deductive reasoning - Elimination of possibilities until only one or a very few remain. Hypotheses are testable statements that must be true if a theory is true, thus if the hypothesis is not true, the theory can be deducted from the set of possible theories.
DataHypothesis
Theory
Pass
Beliefs
Indu
ction
Fail
The Scientific MethodThe Scientific Method
Deduction
Test(Experiment)
©2001 Timothy G. Standish
Err
orE
rror
Data
The Scientific MethodThe Scientific MethodDoes Not Always Provide Does Not Always Provide
Definitive AnswersDefinitive Answers
Truth
TimeTime
PresentScience
OldTheory
©2001 Timothy G. Standish
Nucleic AcidsNucleic Acids The double-stranded nucleic acid
2’Deoxyribonucleic Acid (DNA) serves as the genetic material for all living organisms
Other particles that exhibit some, but not all, of the characteristics of life, viruses and viroids, also use nucleic acids as their genetic material but in addition to double-stranded DNA may use single- stranded DNA or Ribonucleic Acid (RNA)
The structure of nucleic acids reveals both why they are excellent molecules for information storage and transmission
©2001 Timothy G. Standish
ContingencyContingency DNA and RNA exhibit no apparent preference for any
sequence of monomers, thus they exhibit a property sometimes called “contingency”
To efficiently code information symbols, sounds or other media used must be made up of elements whose sequence is independent of any physical or chemical constraints
The monomers joined together to make nucleic acids are like letters in the English language, nothing prevents the letters from being arranged in any sequence to create an almost infinite set of words, sentences, paragraphs etc.
Because nucleic acids are made up of subunits whose sequence is uninfluenced by chemical interactions between the subunits they excel as information storage molecules
DNA
mRNA
Transcription
IntroductionIntroduction
The Central Dogma The Central Dogma of Molecular Biologyof Molecular Biology
Cell
Polypeptide(protein)
TranslationRibosome
©1998 Timothy G. Standish
OH
OCH2
Sugar
H
HH
A NucleotideA NucleotideAdenosine Mono Phosphate (AMP)Adenosine Mono Phosphate (AMP)
OH
NH2
N
N N
N
BaseP
O
OH
HO O
Phosphate
2’3’
4’
5’
1’Nucleotide
Nucleoside
H+
-
Pyrimidines
NH2
O
N
N NH
N
Guanine
N
N
Adenine
N
N
NH2
N O
NH2
N O
NH2
NCytosine
Purines
Uracil(RNA)CH3
N ON
O
NH
N ON
O
NH
Thymine(DNA)
NO
H
NO
N
NH C
ytosine
H
O
NN
N
N
N
H
H
Guanine -+
+
+
-
-
Base PairingBase PairingGuanine And CytosineGuanine And Cytosine
CH 3
N
O
N
ON
H+
- ThymineN
NN
N
HN H
-
+Adenine
Base PairingBase PairingAdenine And ThymineAdenine And Thymine
Base PairingBase PairingAdenine And CytosineAdenine And Cytosine
NO
H
NO
N
NH C
ytosine-
+
-
N
NN
N
HN
H
-
+
Adenine
Base PairingBase PairingGuanine And ThymineGuanine And Thymine
CH
3
NO
N
O
NH+
- Thymine
H
O
NN
N
N
N
H
H
Guanine
+
+
-
©2001 Timothy G. Standish
SU
GA
R-P
HO
SP
HA
TE
BA
CK
BO
NE
H
P
O
HO
O
O
CH2
HOH
P
O
O
HO
O
O
CH2
H
P
O
OH
HO
O
O
CH2
NH2
N
N
N
N
O
O
NH2N
NH
N
N
N O
NH2
N
B A
S E
S
DDNNAA
OH
P
O
HO
O
O
CH2
HO
O
H 2N
NHN N
N H
H
P HO
O
O
CH2
OO
N
O
H 2N
NH
H2O
H OH
P
O
HO
O
O
CH2
CH 3
O
O
HNN
H2O
5’Phosphate group
3’Hydroxyl group
5’Phosphategroup
3’Hydroxyl group
Because of specific base paring, any single- stranded sequence of DNA or RNA can be used as a template for production of the complimentary strand
©2001 Timothy G. Standish
The Watson - Crick The Watson - Crick Model Of DNAModel Of DNA
3.4 nm1 nm
0.34 nm
Majorgroove
Minorgroove
A T
T AG C
C G
C GG C
T A
A T
G CT A
A TC G
--
-
-
---
--
--
--
-
--
--
-
---
--
--
--
-
-
©2001 Timothy G. Standish
Forms of the Double HelixForms of the Double Helix
0.26 nm
2.8 nmMinorgroove
Majorgroove
C GA T
T AG C
C G
G CT A
A T
G CT A
A TC G
A T
G C
1.2 nm
A DNA
1 nm
Majorgroove
Minorgroove
A T
T AG C
C G
C G
G CT A
A T
G CT A
A TC G
0.34 nm
3.9 nm
B DNA
+32.7o Rotation/Bp11 Bp/turn
-30.0o Rotation/Bp12 Bp/turn
+34.6o Rotation/Bp10.4 Bp/turn
C GG C
G CC G
C G
G CG C
G CC G
G CC G
0.57 nm
6.8 nm
0.9 nm
Z DNA
©2001 Timothy G. Standish
C-DNA:– Exists only under high dehydration conditions– 9.3 bp/turn, 0.19 nm diameter and tilted bases
D-DNA:– Occurs in helices lacking guanine– 8 bp/turn
E-DNA:– Like D-DNA lack guanine– 7.5 bp/turn
P-DNA:– Artificially stretched DNA with phosphate groups found inside
the long thin molecule and bases closer to the outside surface of the helix
– 2.62 bp/turn
Even More Forms Of DNAEven More Forms Of DNA
B-DNA appears to be the B-DNA appears to be the most common form most common form in in vivovivo. However, under . However, under some circumstances, some circumstances, alternative forms of DNA alternative forms of DNA may play a biologically may play a biologically significant role.significant role.
©2001 Timothy G. Standish
Negatively (twisting to
the left) supercoiled
DNA
SupercoilingSupercoiling
Opening negatively supercoiled DNA may contribute to strand separation
Opened negatively supercoiled
DNA
Open circle DNA with no supercoiling
©2001 Timothy G. Standish
DNA Structure Influences DNA Structure Influences Migration Through GelsMigration Through Gels
H
P
O
-O
O
O
CH2
HOH
P
O
O
-O
O
O
CH2
H
P
O
O-
-O
O
O
CH2
NH2
N
N
N
N
O
O
NH2N
NH
N
N
N O
NH2
N
OH
P
O
O
O
CH2
O
H 2N
NHN N
N
H
P O-
O
O
CH2
O
O
N
O
H 2N
N
H OH
P
O
O-
O
O
CH2
CH 3
O
O
HNN
O-
O-
Distribution Of Negative Charge Distribution Of Negative Charge Prevents DNA AnnealingPrevents DNA Annealing
NaCl
H
P
O
-O
O
O
CH2
HOH
P
O
O
-O
O
O
CH2
H
P
O
O-
-O
O
O
CH2
NH2
N
N
N
N
O
O
NH2N
NH
N
N
N O
NH2
N
OH
P
O
O
O
CH2
O
H 2N
NHN N
N
H
P O-
O
O
CH2
O
O
N
O
H 2N
N
H OH
P
O
O-
O
O
CH2
CH 3
O
O
HNN
O-
O-
NaCl
Cl-
Na+
Salts Allow DNA AnnealingSalts Allow DNA Annealing
Cat ions can cancel out the negative charge carried on the sugar phosphate backbone.
Na+
Na+
Na+
Na+
Na+
Na+
Na+
H
P
O
-O
O
O
CH2
HOH
P
O
O
-O
O
O
CH2
H
P
O
O-
-O
O
O
CH2
NH2
N
N
N
N
O
O
NH2N
NH
N
N
N O
NH2
N
OH
P
O
O
O
CH2
O
H 2N
NHN N
N
H
P O-
O
O
CH2
O
O
N
O
H 2N
N
H OH
P
O
O-
O
O
CH2
CH 3
O
O
HNN
O-
O-
Salts Allow DNA AnnealingSalts Allow DNA Annealing
Na+
Na+
Na+
Na+
Na+
Na+
OH
P
O
O
O
CH2
O
H 2N
NHN N
N
H
P O-
O
O
CH2
O
O
N
O
H 2N
N
H OH
P
O
O-
O
O
CH2
CH 3
O
O
HNN
O-
O-
Salts Allow DNA AnnealingSalts Allow DNA Annealing
H
P
O
-O
O
O
CH2
HOH
P
O
O
-O
O
O
CH2
H
P
O
O-
-O
O
O
CH2
NH2
N
N
N
N
O
O
NH2N
NH
N
N
N O
NH2
N
Na+
Na+
Na+
Na+
©2001 Timothy G. Standish
HybridizationHybridization The bases in DNA will only pair in very specific ways, G
with C and A with T In short DNA sequences, imprecise base pairing will not be
tolerated Long sequences can tolerate some mispairing only if -G
of the majority of bases in a sequence exceeds the energy required to keep mispaired bases together
Because the source of any single strand of DNA is irrelevant, merely the sequence is important, DNA from different sources can form a double helix as long as their sequences are compatible
Thus, this phenomenon of base pairing of single-stranded DNA strands to form a double helix is called hybridization as it may be used to make hybrid DNA composed of strands which came from different sources
©2001 Timothy G. Standish
HybridizationHybridization
DNA from source “Y”
TACTCGACAGGCTAG
CTGATGGTCATGAGCTGTCCGATCGATCAT
DNA from source “X”
TACTCGACAGGCTAG
HybridizationHybridization
©2001 Timothy G. Standish
HybridizationHybridization Because DNA sequences will seek out and hybridize with
other sequences with which they base pair in a specific way much information can be gained about unknown DNA using single-stranded DNA of known sequence
Short sequences of single-stranded DNA can be used as “probes” to detect the presence of their complimentary sequence in any number of applications including:– Southern blots– Northern blots (in which RNA is probed)– In situ hybridization– Dot blots . . .
In addition, the renaturation or hybridization of DNA in solution can tell much about the nature of organism’s genomes
©2001 Timothy G. Standish
Inverted RepeatsInverted Repeats
Inverted repeat
Single-stranded DNA or RNA
©2001 Timothy G. Standish
Inverted RepeatsInverted Repeats
©2001 Timothy G. Standish
Inverted RepeatsInverted Repeats
©2001 Timothy G. Standish
5’TGCTAATACGCGATCAGCGCGTACTGGTAT3’
Inverted RepeatsInverted Repeats
5’TGCTAATACGCGATCAGCGCGTACTGGTAT3’
Inverted repeat
©2001 Timothy G. Standish
Inverted RepeatsInverted Repeats
5’AUGCUAAUACGCG-CGCGUACUGGUAUC3’A
C
GAU
∆G = Energy released in base pair formation (∆Gx)+ Energy required to bend loop (∆Gu)
= >-2.0 kcal/mol + 4.4 kcal/mol = >+2.4 kcal/mol not spontaneous/unlikely
©2001 Timothy G. Standish
Inverted RepeatsInverted Repeats
5’AUGCUAAUACGC-GCGUACUGGUAUC3’G-CA
C
GAU
©2001 Timothy G. Standish
Inverted RepeatsInverted Repeats
5’AUGCUAAUACG-CGUACUGGUAUC3’C-GG-CA
C
GAU
©2001 Timothy G. Standish
Inverted RepeatsInverted Repeats
5’AUGCUAAUAC-GUACUGGUAUC3’
C-GG-C
G-CA
C
GAU
©2001 Timothy G. Standish
Inverted RepeatsInverted Repeats
5’AUGCUAAUA-UACUGGUAUC3’C-GG-C
G-CC-G
A
C
GAU
©2001 Timothy G. Standish
Inverted RepeatsInverted RepeatsMuch of the -∆G from base pairing comes from base stacking excluding water and thus hydrophobic interrelations between bases. ∆G is calculated in terms of base stacking.
+4.4 Hairpin loop (∆Gu)
-2.0 CG/GC-3.4 GC/CG-2.0 GC/CG-2.1 AC/UG-1.1 UA/AUA-U
G-C
G-CC-G
C-G
A
C
GAU
5’AUGCUAAU-ACUGGUAUC3’
∆Gx=-10.6
∆Gx + ∆Gu =
With a series of stacked base pairs due to an inverted repeat, the formation of a duplex becomes thermodynamically likely:∆Gu = Energy required to hold bases in an unpaired state (positive)∆Gx = Energy released by base pair formation (negative)
-10.6 + 4.4 = -6.2 kcal/mol Stable∆G =
©2001 Timothy G. Standish
Base StackingBase Stacking
N
N
NN
H
H
NAdenineUracilN
H
N
ON
O
N
NN
N
HHN
Adenine
Uracil
N
H
N
O
N
O
5’3’
5’
3’
5’AA3’
3’UU5’
©2001 Timothy G. Standish
N
N
NN
H
H
NAdenineUracilN
H
N
ON
O
N
NN
N
H HN
AdenineUracil
N
H
N
O
N
O
5’3’
5’
3’
5’AU3’
3’UA5’
Base StackingBase Stacking
©2001 Timothy G. Standish
Double-StrandedDouble-StrandedInverted RepeatsInverted Repeats
©2001 Timothy G. Standish
Double-StrandedDouble-StrandedInverted RepeatsInverted Repeats
©2001 Timothy G. Standish
Double-StrandedDouble-StrandedInverted RepeatsInverted Repeats
©2001 Timothy G. Standish
AZTAZT3’-Azido-3’-deoxythymidine3’-Azido-3’-deoxythymidine
N3
OCH2
Deoxyribose
H
OH
CH3
N ON
O
NHThymine
©2001 Timothy G. Standish
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