©2001 timothy g. standish isaiah 33:22 22for the lord is our judge, the lord is our lawgiver, the...

©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.


Molecular Basis Molecular Basis Of InheritanceOf Inheritance

Timothy G. Standish, Ph. D.


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


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


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)


Err

orE

rror

Data

The Scientific MethodThe Scientific MethodDoes Not Always Provide Does Not Always Provide

Definitive AnswersDefinitive Answers

Truth

TimeTime

PresentScience

OldTheory


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


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


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

+

+

-


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


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

--

-

-

---

--

--

--

-

--

--

-

---

--

--

--

-

-


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


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.


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


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-


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-


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+


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


HybridizationHybridization

DNA from source “Y”

TACTCGACAGGCTAG

CTGATGGTCATGAGCTGTCCGATCGATCAT

DNA from source “X”

TACTCGACAGGCTAG

HybridizationHybridization


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


Inverted RepeatsInverted Repeats

Inverted repeat

Single-stranded DNA or RNA


5’TGCTAATACGCGATCAGCGCGTACTGGTAT3’


5’TGCTAATACGCGATCAGCGCGTACTGGTAT3’

Inverted repeat



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



5’AUGCUAAUACGC-GCGUACUGGUAUC3’G-CA

C

GAU



5’AUGCUAAUACG-CGUACUGGUAUC3’C-GG-CA

C

GAU



5’AUGCUAAUAC-GUACUGGUAUC3’

C-GG-C

G-CA

C

GAU



5’AUGCUAAUA-UACUGGUAUC3’C-GG-C

G-CC-G

A

C

GAU


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 =


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’


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


Double-StrandedDouble-StrandedInverted RepeatsInverted Repeats


AZTAZT3’-Azido-3’-deoxythymidine3’-Azido-3’-deoxythymidine

N3

OCH2

Deoxyribose

H

OH

CH3

N ON

O

NHThymine

©2001 timothy g. standish isaiah 33:22 22for the lord is our judge, the lord is our lawgiver, the...

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