Chapter 16

Chapter 16

The Molecular Basis of Inheritance

 Animation: DNA and RNA Structure
Right-click slide / select “Play”
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 The double helix.
5 end
C G
C G Hydrogen bond
3 end
G C
G C T A

3.4 nm
T A
G C G C
C G
A T

1 nm C G
T A
C G
G C
C G A T

A T 3 end
A T
0.34 nm
T A 5 end

(a) Key features of (b) Partial chemical structure

DNA structure
 A model for DNA replication: the basic concept.

A T A T A T A T
C G C G C G C G
T A T A T A T A
A T A T A T A T
G C G C G C G C

(a) Parent molecule (b) Separation of (c) “Daughter” DNA molecules,

strands each consisting of one
parental strand and one
new strand
 Some of the proteins involved in the initiation of DNA replication.

Primase

3
Topoisomerase
5 RNA
3 primer
5
Replication
3
Fork
Helicase

5
Single-strand binding
proteins
 Elongation of a DNA strand during DNA Replication
New strand Template strand
5 3 5 3

Sugar A T A T
Phosphate Base

C G C G

G C G C
DNA
OH
polymerase
3 A T A
T
P P Pi OH
P
P C Pyrophosphate 3 C
OH
Nucleoside 2Pi
triphosphate 5 5
 Figure 16.UN03

Primase synthesizes
DNA pol III synthesizes a short RNA primer
leading strand continuously 3
5

Parental
DNA
Origin of
5 replication
3
5 Lagging strand synthesized
in short Okazaki fragments,
Helicase later joined by DNA ligase

3
5
DNA pol I replaces the RNA
primer with DNA nucleotides
Telomeres.

1 m
 Chromatin Packing in a Eukaryotic Chromosome

Nucleosome
(10 nm in diameter)
DNA double helix
(2 nm in diameter)

H1
Histone
Histones tail
Nucleosomes, or “beads on
DNA, the double helix Histones a string” (10-nm fiber)
Chapter 16

Chapter 17

From Gene to Protein

 Central dogma

DNA RNA Protein

Overview: Eukaryotes: the roles of transcription and translation in the flow of genetic information.

Nuclear
envelope

DNA
TRANSCRIPTION

Pre-mRNA
RNA PROCESSING

mRNA

TRANSLATION Ribosome

Polypeptide

(b) Eukaryotic cell

 Transcription

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The initiation of transcription at a eukaryotic promoter.
1 A eukaryotic promoter
Promoter
Nontemplate strand
DNA
5 T A T A A AA 3
3 A T AT T T T 5
TATA box Template strand
Start point
2 Several transcription
Transcription factors bind to DNA
factors

5 3
3 5

3 Transcription initiation
complex forms

RNA polymerase II
Transcription factors

5 3
3
3 5 5

RNA transcript

Transcription initiation complex

Transcription elongation.
Nontemplate
strand of DNA
RNA nucleotides
RNA
polymerase

A T C C A A
3 T 5
T U
C
3 end
T

G
U
A

G
A C
C A U C C A C
A
5 A 3
T A G G T T

5 Direction of transcription
Template
strand of DNA
Newly made
RNA
The stages of transcription: initiation, elongation, and termination. Promoter Transcription unit

5 3
3 5
DNA
Start point
RNA polymerase
1 Initiation

Nontemplate strand of DNA

5 3
3 5
RNA Template strand of DNA
Unwound
transcript
DNA
2 Elongation

Rewound
DNA
5 3
3 5
3
5
RNA
transcript 3 Termination

5 3
3 5
5 3
Completed RNA transcript

Direction of transcription (“downstream”)

 RNA Processing

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 RNA processing: RNA splicing.

5 Exon Intron Exon Intron Exon 3

Pre-mRNA 5 Cap Poly-A tail
Codon 130 31104 105
numbers
146
Introns cut out and
exons spliced together

mRNA 5 Cap Poly-A tail

1146
5 UTR 3 UTR
Coding
segment
 Translation

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 Translation
• Translation is the RNA-directed synthesis of a polypeptide

• Genetic information flows from mRNA to protein through the

process of translation

• Based on a triplet code:

 A Codon is a triplet of mRNA nucleotides that codes for an

amino acid, the building blocks of proteins

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The Codon Table for mRNA Second mRNA base
U C A G
UUU UCU UAU UGU U
Phe Tyr Cys
UUC UCC UAC UGC C
U Ser
UUA UCA UAA Stop UGA Stop A
Leu

Third mRNA base (3 end of codon)

First mRNA base (5 end of codon) UUG UCG UAG Stop UGG Trp G

CUU CCU CAU CGU U

His
CUC CCC CAC CGC C
C Leu Pro Arg
CUA CCA CAA CGA A
Gln
CUG CCG CAG CGG G

AUU ACU AAU AGU U

Asn Ser
AUC Ile ACC AAC AGC C
A Thr
AUA ACA AAA AGA A
Lys Arg
Met or
AUG start
ACG AAG AGG G

GUU GCU GAU GGU U

Asp
GUC GCC GAC GGC C
G Val Ala Gly
GUA GCA GAA GGA A
Glu
GUG GCG GAG GGG G
 Players for Translation
• Ribosomes
• mRNA
• tRNA with an attached amino acid

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 The initiation of translation.

Large
ribosomal
subunit
3 U A C 5 P site
Met 5 A U G 3 Met

Pi
Initiator 
tRNA GTP GDP
E A
mRNA
5 5
3 3
Start codon
Small
mRNA binding site ribosomal Translation initiation complex
subunit
 Amino end of
polypeptide

E
mRNA 3
Ribosome ready for P A
site site
next aminoacyl tRNA 5 GTP

GDP  P i

E E

P A P A

GDP  P i

GTP

P A
 The termination of translation.

Release
factor Free
polypeptide

5
3 3
2 GTP 3
5 5
2 GDP  2 P i
Stop codon
(UAG, UAA, or UGA)
 The molecular basis of sickle-cell disease: a point mutation.

Wild-type hemoglobin Sickle-cell hemoglobin

Wild-type hemoglobin DNA Mutant hemoglobin DNA
3 C T T 5 3 C A T 5
5 G A A 3 5 G T A 3

mRNA mRNA
5 G A A 3 5 G U A 3

Normal hemoglobin Sickle-cell hemoglobin

Glu Val
 Types of Small-Scale Mutations

• Point mutations within a gene can be divided into two general

categories

1. Nucleotide-pair substitution replaces one nucleotide and its

partner with another pair of nucleotides

2. Nucleotide-pair insertions
3. Nucleotide-pair deletions

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 1. Nucleotide-pair Substitutions
• A nucleotide-pair substitution replaces one nucleotide and
its partner with another pair of nucleotides
• A Silent mutation results in a change in the third base of a
codon that has no effect on the amino acid
• A Missense mutation results in a change in the codon that
will change the amino acid
• Nonsense mutations results in a change in the codon that
changes an amino acid codon into a stop codon, nearly
always leading to a nonfunctional protein

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Wild type
DNA template strand 3 T A C T T C A A A C C G A T T 5
5 A T G A A G T T T G G C T A A 3

mRNA5 A U G A A G U U U G G C U A A 3
Protein Met Lys Phe Gly Stop
Amino end Carboxyl end

(a) Nucleotide-pair substitution: silent

A instead of G
3 T A C T T C A A A C C A A T T 5
5 A T G A A G T T T G G T T A A 3
U instead of C
5 A U G A A G U U U G G U U A A 3
Met Lys Phe Gly Stop
 What Is a Gene? Revisiting the Question

• The idea of the gene has evolved through the history of genetics

• A gene as

– A discrete unit of inheritance

– A region of specific nucleotide sequence in a chromosome

– A DNA sequence that can be expressed to produce a final

functional product, either a polypeptide or an RNA
molecule

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Chapter 16

Chapter 18

Regulation of Gene
Regulation of GeneExpression
Expression
 Cells differ from each other by regulating Gene
Expression: Selectively turning genes on or off in the cell.

Pancreas cell White blood cell Nerve cell

Glycolysis
Enzyme
Antibody
Insulin gene
Hemoglobin
gene

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 Bacteria
• A bacterial cell can exert metabolic control by two main
mechanisms.

1. Adjustment of activity of enzymes already present in the cell

(feedback inhibition)

2. Regulation of production of enzymes (gene expression)

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 Inducible operons

Genes not expressed Genes expressed

Promoter
Operator
Genes

Active repressor: Inactive repressor:

no inducer present inducer bound
 Repressible Operon

Genes expressed Genes not expressed

Promoter
Genes

Operator Active repressor:

corepressor bound
Inactive repressor:
no corepressor present Corepressor
Eukaryotic gene expression is regulated at many stages

• Almost all the cells in an organism are genetically identical-same

genome

• Differences between cell types result from differential gene

expression, the expression of different genes by cells with the
same genome

 Gene expression is regulated at many stages though regulation

of gene transcription is one of the most important.

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 Signal
Stages in gene expression that can be regulated in eukaryotic cells

NUCLEUS
Chromatin
Chromatin modification:
DNA unpacking involving
histone acetylation and
DNA demethylation
DNA
Gene available
for transcription
Gene
Transcription
RNA Exon
Primary transcript
Intron
RNA processing
Tail
Cap mRNA in nucleus

Transport to cytoplasm

CYTOPLASM
mRNA in cytoplasm

Degradation Translation
of mRNA

Polypeptide
Protein processing, such
as cleavage and
chemical modification

Active protein
Degradation
of protein
Transport to cellular
destination

Cellular function (such

as enzymatic activity,
structural support)
 Enhancers Promoter
Gene

DNA
Activator
proteins
Transcription
factors Other
proteins

RNA polymerase

Bending
of DNA

Transcription
 18.5: Cancer results from genetic changes that affect cell
cycle control
Cancer is a variety of diseases in which cells

 Experience changes in gene expression and

 Escape from the control mechanisms that normally limit

their growth and division.

Cancer can be caused by mutations to genes that regulate cell

growth and division

 Conversion of a proto-oncogene to an oncogene (bad guy,

cancer causing gene)

 Mutation that disables the function of a tumor suppressor gene

like p53 (the good guys)
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 Genetic changes that can turn proto-oncogenes into oncogenes.

Proto-oncogene
DNA

Translocation or
transposition: gene Gene amplification: Point mutation:
moved to new locus, multiple copies of within a control within
under new controls the gene element the gene

New Oncogene Oncogene

promoter

Normal growth- Normal growth-stimulating Normal growth- Hyperactive or

stimulating protein in excess stimulating degradation-
protein in excess protein in resistant
excess protein
 Tumor-Suppressor Genes
Tumor-suppressor genes help prevent uncontrolled cell growth

Tumor-suppressor proteins

– Repair damaged DNA

– Control cell adhesion

– inhibit cell division and prevent uncontrolled cell growth

Mutations that result in decreased or absent tumor-suppressor

proteins in the cell may contribute to cancer onset

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 Signaling pathways that regulate cell division.

Proto-oncogene converted to Mutation/Loss of function of

ONCOGENES TUMOR SUPPRESSOR GENE
EFFECTS OF MUTATIONS
Protein Protein absent
overexpressed

Cell cycle Increased cell Cell cycle not

overstimulated division inhibited

(c) Effects of mutations

 A multistep model for the development of colorectal cancer.

Colon

1 Loss
of tumor- 4 Loss
suppressor 2 Activation of tumor-
gene APC of ras suppressor
(or other) oncogene gene p53

3 Loss 5 Additional
Colon wall of tumor- mutations
Normal colon Small benign suppressor Larger Malignant
epithelial cells growth gene DCC benign growth tumor
(polyp) (adenoma) (carcinoma)
Chapter 16

Chapter 19

Viruses
 Structure of Viruses

• Viruses are not cells

• A virus is a very small infectious particle consisting of

 Nucleic acid –DNA or RNA

 Protein coat-capsid/capsomeres

 in some cases, surrounded by a membranous envelope

• Viruses replicate only in host cells

• Viruses are obligate intracellular parasites, which means they

can replicate only within a host cell

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 The lytic versus lysogenic cycles of phage
Phage The phage attaches to a
DNA host cell and injects its DNA.

Bacterial
chromosome Prophage

Lytic cycle Lysogenic cycle

 Retroviruses

• Retroviruses carry an enzyme called reverse

transcriptase that transcribes DNA using RNA as a
template.

• This provides RNA  DNA information flow.

• Human immunodeficiency virus (HIV), the virus that

causes AIDS (acquired immunodeficiency syndrome),
is a retrovirus.

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 Glycoprotein Viral envelope

Capsid
RNA (two
Reverse identical HOST
transcriptase strands) CELL
HIV
Reverse
Viral RNA transcriptase
RNA-DNA
hybrid

DNA

NUCLEUS
Chromosomal Provirus
DNA
RNA genome
for the
next viral mRNA
generation

New virus
 Examples of emerging viruses
– HIV
– Ebola virus
– West Nile virus
– RNA coronavirus causing severe acute respiratory syndrome (SARS)
– Avian flu virus
– Swine flu virus H1N1- 2009 Mexico and the United States- 2009:
– H7N9 Bird Flu 2013- 2013
 Cases In China Rise To 108; Shandong Province Has First Illness (4/23/13
http://www.forbes.com)

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Examples of Emerging viruses

1 m
(a) 2009 pandemic H1N1 (b) 2009 pandemic
influenza A virus screening

(c) 1918 flu pandemic

 Viroids & Prions
Two classes of pathogens are even smaller than
viruses.
1. Viroids are small, circular RNA molecules that infect plants.

2. Prions are misfolded proteins that somehow convert normal

proteins to the misfolded prion version, leading to disease.

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 Biotechnology

• Recombinant DNA: A DNA molecule made in vitro with DNA from

different sources

• Often different species

• Gene cloning or DNA cloning: Generation of multiple identical

copies of a gene or DNA segment

• Genetic Engineering: Using recombinant DNA technology to

manipulate genes for practical purposes including genetic analysis
& developing therapeutic products

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 How do you make Recombinant DNA molecules?

• Key factors involved in generation of recombinant DNA molecule

include:
1. Cloning vector, often bacterial plasmids
2. Restriction enzymes
3. DNA Ligase

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 Animation: Restriction Enzymes
Right-click slide / select “Play”
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Using a restriction enzyme and DNA ligase to make recombinant DNA
Restriction site

DNA 5 3
3 5

1 Restriction enzyme
cuts sugar-phosphate
backbones.

Sticky end

2 DNA fragment added

from another molecule
cut by same enzyme.
Base pairing occurs.

One possible combination

3 DNA ligase
seals strands.

Recombinant DNA molecule

 Examples of
gene use

Genes may be inserted

Recombinant into other organisms
DNA Gene
plasmid of interest
9 Genes or proteins
7 Put plasmid are isolated from the
into bacterium cloned bacterium
by transformation
Recombinant
bacterium
Harvested
proteins
8 Allow bacterium may be
to reproduce used directly

Clone
of cells
Examples of
protein use
Making complementary DNA (cDNA) from eukaryotic genes.
DNA in
nucleus
mRNAs in
cytoplasm

Reverse
transcriptase Poly-A tail
mRNA
5 A A A A A A 3
3 T T T T T 5
DNA Primer
strand

5 A A A A A A 3
3 T T T T T 5

5 3
3 5
DNA
polymerase

5 3
3 5
cDNA
Gel electrophoresis TECHNIQUE

Mixture of Power
DNA mol- source
ecules of – Cathode Anode +
different
sizes

Gel
1

Power
source
– +
Longer
molecules

2 Shorter
molecules

RESULTS
 Southern Blotting Heavy
Restriction I II III weight
DNA + restriction enzyme fragments Nitrocellulose
membrane (blot)

Gel

Sponge

I Normal II Sickle-cell III Heterozygote Paper

-globin allele Alkaline
solution towels
allele
1 Preparation of restriction fragments 2 Gel electrophoresis 3 DNA transfer (blotting)

Radioactively labeled
probe for -globin gene

Probe base-pairs
I II III with fragments I II III

Fragment from
sickle-cell
-globin allele Film
over
Fragment from blot
normal -globin
Nitrocellulose blot allele
4 Hybridization with radioactive probe 5 Probe detection
Figure 20.8 TECHNIQUE 5 3

Target
sequence
Genomic DNA 3 5

1 Denaturation 5 3

3 5
2 Annealing
Cycle 1
yields Primers
2
molecules

3 Extension

New
nucleotides

Cycle 2
yields
4
molecules

Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
Figure 20.19
TECHNIQUE
Mammary Egg cell
cell donor donor
1 Egg 2
cell from
ovary Nucleus
3 Cells fused removed
Cultured
mammary
cells

Nucleus from
4 Grown in culture mammary cell

Early embryo
5 Implanted in uterus
of a third sheep

Surrogate
mother
6 Embryonic
development
RESULTS Lamb (“Dolly”) genetically
identical to mammary cell donor
Working with stem cells Embryonic stem cells Adult stem cells
Early human embryo From bone marrow
at blastocyst stage in this example
(mammalian equiva-
lent of blastula)

Cells generating Cells generating

all embryonic some cell types
cell types

Cultured
stem cells

Different
culture
conditions

Different Liver cells Nerve cells Blood cells

types of
differentiated
cells
Cloned Human Gene Ther
gene
1 Insert RNA version of normal allele
into retrovirus.

Viral RNA

2 Let retrovirus infect bone marrow cells

Retrovirus that have been removed from the
capsid patient and cultured.

3 Viral DNA carrying the normal

allele inserts into chromosome.

Bone
marrow
cell from
patient

4 Inject engineered Bone

cells into patient. marrow
STR analysis used to release an innocent man
from prison.

This photo shows

Washington just before
his release in 2001,
after 17 years in prison.

Source of STR STR STR

sample marker 1 marker 2 marker 3

Semen on victim 17,19 13,16 12,12

Earl Washington 16,18 14,15 11,12

Kenneth Tinsley 17,19 13,16 12,12

(b) These and other STR data exonerated Washington

and led Tinsley to plead guilty to the murder.
 Genetic Engineering in Plants

Genetic engineering in plants has been used to transfer many

useful genes including genes for
- herbicide resistance
- increased resistance to pests
- increased resistance to salinity
-improved nutritional value of crops