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Viruses, Bacteria, Eukaryotic and DNA Tech
Viruses, Bacteria, Eukaryotic and DNA Tech
Capsomere
of capsid
RNA
A capsid is the
protein shell that
encloses the viral
genome and can have
various structures
18 250 mm
20 nm
Tobacco mosaic virus
Capsomere
DNA
Glycoprotein
7090 nm (diameter)
50 nm
Adenoviruses
Membranous
envelope
Capsid
RNA
Glycoprotein
80200 nm (diameter)
50 nm
Influenza viruses
Head
Tail
sheath
Tail
fiber
DNA
80 225 nm
50 nm
Bacteriophage T4
VIRUS
Entry into cell and
uncoating of DNA DNA
Capsid
Transcription
Replication
HOST CELL
Viral DNA
mRNA
Viral DNA
Capsid
proteins
Self-assembly of
new virus particles
and their exit from cell
Attachment
Phage assembly
Release
Synthesis of viral
genomes and proteins
Phage
DNA
Daughter cell
with prophage
Many cell divisions
produce a large
population of
bacteria infected with
the prophage.
Phage DNA
circularizes
Phage
Bacterial
chromosome
Lytic cycle
The cell lyses, releasing phages.
Occasionally, a prophage
exits the bacterial chromosome,
initiating a lytic cycle.
Lysogenic cycle
Certain factors
determine whether
Lytic cycleorLysogenic cycle
is induced
is entered
Reproductive Cycles of
Animal
Viruses
Two key variables in classifying viruses
that infect animals:
DNA or RNA?
Single-stranded or double-stranded?
Viral Envelopes
Many viruses that infect animals have a
membranous envelope
Viral glycoproteins on the envelope
bind to specific receptor molecules
on the surface of a host cell
Capsid
RNA
HOST CELL
Envelope (with
glycoproteins)
Viral genome (RNA)
Template
mRNA
ER
Glycoproteins
Capsid
proteins Copy of
genome (RNA)
New virus
Glycoprotein
Viral envelope
Capsid
Reverse
transcriptase
RNA
(two identical
strands)
HIV
Membrane of
white blood cell
HOST CELL
Reverse
transcription
Viral RNA
0.25 m
RNA-DNA
hybrid
DNA
NUCLEUS
Chromosomal
DNA
RNA genome
for the
next viral
generation
mRNA
Provirus
Prion
Original
prion
Many prions
Normal
protein
New
prion
Transformation and
Transduction
Transformation is the alteration of a
Phage DNA
A+ B+
A+ B+
Donor
cell
A+
Crossing
over
A+
A B
Recipient
cell
A+ B
Recombinant cell
Conjugation and
Plasmids
Sex pilus
5 m
Mixture
Mutant
strain
arg+ trp
Mutant
strain
arg trp+
Mixture
Mutant
strain
arg+ trp
Mutant
strain
arg trp+
No
colonies
(control)
Colonies
grew
No
colonies
(control)
F plasmid
Bacterial chromosome
F+ cell
Mating
bridge
F cell
F+ cell
F+ cell
Bacterial
chromosome
Conjunction and transfer of an F plasmid from and F+ donor to an F recip
Hfr cell
F+ cell
F factor
Hfr cell
F cell
Temporary
Recombinant F
partial
bacterium
diploid
Conjugation and transfer of part of the bacterial chromosome from an
Hfr donor to an F recipient, resulting in recombination
antibiotics
When a bacterial population is exposed to an
antibiotic, individuals with the R plasmid will
survive and increase in the overall population
Transposition of Genetic
The DNA of a Elements
cell can also undergo
Insertion Sequences
The simplest transposable elements, called
insertion sequences, exist only in
bacteria
An insertion sequence has a single gene for
transposase, an enzyme catalyzing
movement of the insertion sequence from
one site to another within the genome
Insertion sequence
5
3
3
5
Inverted
repeat
Transposase gene
Inverted
repeat
Transposons
Transposable elements called transposons are
longer and more complex than insertion
sequences
In addition to DNA required for
transposition, transposons have extra
genes that go along for the ride, such as
genes for antibiotic resistance
Transposon
Insertion
sequence
Antibiotic
resistance gene
Insertion
sequence
5
3
3
5
Inverted repeat
Transposase gene
trp operon
Promoter
Promoter
DNA
mRNA
trpE
trpR
Regulatory
gene
trpD
trpC
trpB
trpA
Operator
Stop codon
RNA
Start codon
polymerase
mRNA 5
Protein
Genes of operon
E
Inactive
repressor
DNA
mRNA
Active
repressor
Protein
Tryptophan
(corepressor)
Tryptophan present, repressor active, operon off
DNA
No RNA made
mRNA
Active
repressor
Protein
Tryptophan
(corepressor)
Tryptophan present, repressor active, operon off
Promoter
Regulatory
gene
Operator
lacl
DNA
lacZ
No
RNA
made
3
mRNA
5
Protein
RNA
polymerase
Active
repressor
lac operon
DNA
lacl
mRNA
5
lacY
-Galactosidase
Permease
lacA
RNA
3 polymerase
mRNA 5
Protein
Allolactose
(inducer)
lacZ
Inactive
repressor
Transacetylase
Promoter
DNA
lacl
lacZ
CAP-binding site
cAMP
Inactive
CAP
RNA
Operator
polymerase
Active can bind
and transcribe
CAP
Inactive lac
repressor
Promoter
DNA
lacl
lacZ
CAP-binding site
Operator
Inactive
CAP
RNA
polymerase
cant bind
Inactive lac
repressor
Nucleosomes, or Beads on
a String
2 nm
DNA double helix
Histones
Histone
tails
Histone H1 10 nm
Linker DNA
(string)
Nucleosome
(bead)
30 nm
Nucleosome
30-nm
fiber
Protein scaffold
Loops
300 nm
Looped domains (300-nm
fiber)
Scaffold
700 nm
1,400 nm
Metaphase
chromosome
Differential Gene
Expression
Signal
NUCLEUS
Chromatin
DNA
Gene available
for transcription
Gene
Transcription
RNA
Exon
Primary transcript
Intro
RNA processing
Tail
Cap
mRNA in nucleus
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
Degradation
of mRNA
Translation
Polypeptide
Cleavage
Chemical modification
Transport to cellular
destination
Active protein
Degradation of protein
Degraded protein
Regulation of Chromatin
Structure
Histone Modification
In histone acetylation, acetyl groups are attached
to positively charged lysines in histone tails
This process seems to loosen chromatin structure,
thereby promoting the initiation of
transcription
Histone
tails
DNA
double helix
Amino acids
available
for chemical
modification
Unacetylated histones
Acetylated histones
DNA Methylation
DNA methylation, the addition of methyl groups
to certain bases in DNA, is associated with reduced
transcription in some species
In some species, DNA methylation causes longterm inactivation of genes in cellular
differentiation
In genomic imprinting, methylation turns of either
the maternal or paternal alleles of certain genes at
the start of development lions, tigers, ligers?
The Roles of
Transcription Factors
Enhancer
Proximal
(distal control elements) control elements
Exon
Intron
Exon
Poly-A signal
Termination
sequence
region
Intron Exon
DNA
Upstream
Promoter
Primary RNA
transcript 5
(pre-mRNA)
Exon
Intron
Intron RNA
Downstream
Transcription
Poly-A signal
Exon
IntronExon
Cleaved 3 end
of primary
transcript
RNA processing:
Cap and tail added;
introns excised and
exons spliced together
Coding segment
mRNA
3
5 Cap 5 UTR
(untranslated
region)
Start Stop
codon codon
3 UTR Poly-A
(untranslatedtail
region)
Distal control
element
Activators
Promoter
Gene
DNA
Enhancer
TATA
box
General
transcription
factors
DNA-bending
protein
Group of
mediator proteins
RNA
polymerase II
RNA
polymerase II
Transcription
Initiation complex
RNA synthesis
Liver cell
nucleus
Available
activators
Lens cell
nucleus
Available
activators
Enhancer Promoter
Control
elements
Albumin
gene
Crystallin
gene
Albumin
gene not
expressed
Albumin
gene
expressed
Crystallin gene
not expressed
Liver cell
Crystallin gene
expressed
Lens cell
DNA
Primary
RNA
transcript
RNA splicing
mRNA
or
mRNA Degradation
The life span of mRNA molecules in the
cytoplasm is a key to determining the protein
synthesis
The mRNA life span is determined in part by
sequences in the leader and trailer regions
Protein
complex
Degradation of mRNA
Dicer
OR
miRNA
Target mRNA
Hydrogen
bond
Blockage of translation
Ubiquitin
Proteasome
Protein to
be degraded
Proteasome
and ubiquitin
to be recycled
Ubiquitinated
protein
Protein entering a
proteasome
Protein
fragments
(peptides)
Proto-oncogene
DNA
Translocation or transposition:
gene moved to new locus,
under new controls
Gene amplification:
multiple copies of the gene
New
promoter
Normal growth-stimulating
protein in excess
Point mutation
within a control
element
Oncogene
Normal growth-stimulating
protein in excess
Point mutation
within the gene
Oncogene
Normal growth-stimulating
Hyperactive or
protein in excess
degradationresistant protein
Tumor-Suppressor Genes
Tumor-suppressor genes encode proteins
that inhibit abnormal cell division
Any decrease in the normal activity of a
tumor-suppressor protein may contribute to
cancer
MUTATION
Growth
factor
Hyperactive
Ras protein
(product of
oncogene)
issues signals
on its own
G protein
Cell cycle-stimulating
pathway
Receptor
Protein kinases
(phosphorylation
cascade)
NUCLEUS
Transcription
factor (activator)
DNA
Gene expression
Protein that
stimulates
the cell cycle
Cell cycle-inhibiting
pathway
Protein kinases
MUTATION
Defective or
missing
transcription
factor, such as
p53, cannot
activate
transcription
Active
form
of p53
UV
light
DNA damage
in genome DNA
Protein that
inhibits
the cell cycle
Effects of
mutations
EFFECTS OF MUTATIONS
Protein overexpressed
Protein absent
Activation of
ras oncogene
Loss of
tumorsuppressor
Colon wallgene APC (or
other)
Normal colon
epithelial cells
Small benign
growth (polyp)
Loss of
tumorsuppressor
gene DCC
Loss of
tumorsuppressor
gene p53
Additional
mutations
Larger benign
growth (adenoma)
Malignant tumor
(carcinoma)
Transposable Elements
and Related Sequences
The first evidence for wandering DNA segments
came from geneticist Barbara McClintocks
breeding experiments with Indian corn
McClintock identified changes in the color of corn
kernels that made sense only by postulating that
some genetic elements move from other
genome locations into the genes for kernel color
Movement of Transposons
and Retrotransposons
Eukaryotic transposable elements are
of two types:
Transposons, which move within a
genome by means of a DNA intermediate
Retrotransposons, which move by
means of an RNA intermediate
Transposon
DNA of genome
Transposon
is copied
New copy of
transposon
Insertion
Mobile transposon
Transposon movement (copy-and-paste mechanism)
Retrotransposon
New copy of
retrotransposon
DNA of genome
RNA
Reverse
transcriptase
Retrotransposon movement
Insertion
DNA
RNA transcripts
Non-transcribed
spacer
Transcription unit
DNA
18S
5.8S
28S
rRNA
28S
5.8S
18S
Part of the ribosomal RNA gene family
-Globin
Heme
Hemoglobin
-Globin
-Globin gene family
Chromosome 16
Embryo
1 2 1
Fetus
and adult
Embryo Fetus
Adult
Bacterium
Bacterial
Plasmid
chromosome
Recombinant
DNA (plasmid)
Gene of
interest
Plasmid put into
bacterial cell
DNA of
chromosome
Recombinant
bacterium
Host cell grown in culture
to form a clone of cells
containing the cloned
gene of interest
Gene of
interest
Protein expressed
by gene of interest
Copies of gene
Basic
research
on gene
Protein harvested
Basic research and
various applications
Basic
research
on protein
Restriction site
DNA 5
3
3
5
Sticky end
Bacterial cell
Isolate plasmid DNA
and human DNA.
lacZ gene
Human
(lactose
cell
breakdown)
Restriction
site
Gene of
interest
Sticky
ends
Human DNA
fragments
Colony carrying
recombinant
plasmid with
disrupted lacZ gene
Bacterial
clone
3
Target
sequence
Genomic DNA
Cycle 1
yields
2
molecules
5
Denaturation:
Heat briefly
to separate DNA
strands
Annealing:
Cool to allow
primers to form
hydrogen bonds
with ends of
target sequence
Primers
Extension:
DNA polymerase
adds nucleotides to
the 3 end of each
New
primer
nucleotides
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
Gel Electrophoresis
One indirect method of rapidly analyzing and
comparing genomes is gel electrophoresis
This technique uses a gel as a molecular sieve
to separate nuclei acids or proteins by size
Cathode
Power
source
Mixture
of DNA
molecules
of different sizes
Shorter
molecules
Gel
Glass
plates
Anode
Longer
molecules
175 bp
Ddel
201 bp
Ddel
Large fragment
Ddel
Ddel
376 bp
Ddel
Large fragment
Ddel
Ddel
Large
fragment
201 bp
175 bp
376 bp
Medical Applications
One benefit of DNA technology is
identification of human genes in which
mutation plays a role in genetic diseases
Scientists can diagnose many human
genetic disorders by using PCR and
primers corresponding to cloned disease
genes, then sequencing the amplified
product to look for the disease-causing
mutation
Even when a disease gene has not been
cloned, presence of an abnormal allele
can be diagnosed if a closely linked RFLP
RFLP marker
DNA
Restriction
sites
Disease-causing
allele
Normal allele
Cloned gene
Insert RNA version of normal allele
into retrovirus.
Viral RNA
Retrovirus
capsid
Inject engineered
cells into patient.
Bone
marrow
Pharmaceutical Products
Some pharmaceutical applications of
DNA technology:
Large-scale production of human hormones
and other proteins with therapeutic uses
Production of safer vaccines
Forensic Evidence
DNA fingerprints obtained by analysis
of tissue or body fluids can provide
evidence in criminal and paternity cases
A DNA fingerprint is a specific pattern of
bands of RFLP markers on a gel
The probability that two people who are
not identical twins have the same DNA
fingerprint is very small
Exact probability depends on the number
of markers and their frequency in the
population
Victims
blood (V)
PCR 1
PCR 2
Gel Electrophoresis 1
Gel Electrophoresis 2
DNA Fingerprinting
Construction of a DNA Library
DNA Restriction Enzymes
Restriction Enzyme Digestion of DNA
Restriction Endonucleases
Principles of Biotechnology
Applications of Biotechnology
Constructing Vaccines
DNA Probe (DNA Hybridization)
Restriction Length Ploymorphisms
cDNA
Southern Blot
Entry of Virus into Host Cell
Replication Cycle of a Retrovirus
Microarray
Sanger Sequencing
DNA Fingerprinting
RNA Interface
miRNA
Dicer
DNA Microarrays
Bozeman - DNA Fingerprinting
Bozeman - Viral Replication
repressor gene
promoter
inducer
repressor protein
all of the above
repressor gene
promoter
inducer
repressor protein
all of the above
46%
32%
13%
1.5%
46%
32%
13%
1.5%
protein is
not made.
The RNA fragments enhance protein synthesis by
the mRNA.
The RNA fragments bind the ribosome to enhance
use of the mRNA and protein synthesis.
The target mRNA is blocked from being used
in translation.
The RNA fragments act on the ribosome to shut
down translation of all mRNAs.
mRNA
transcription factor
ribosome
myoblast
homeotic
embryonic lethal
Wild type
myoD
Ras
Eye
wild-type
Mutant
homeotic
embryonic lethal
Wild type
myoD
Ras
Eye
wild-type
Mutant
gene suppression
translocation
amplification
point mutation
retroviral activation
gene suppression
translocation
amplification
point mutation
retroviral activation
to be control elements.
Only control element 3 appears to be a
control element.
All three appear to be control
elements.
None of the possible control elements
appear to be actual control elements.
RNA
phospholipids
proteins
glycoproteins
DNA
RNA
phospholipids
proteins
glycoproteins
DNA
grazed on by herbivores.
Sap from one plant is rubbed on the leaves of a
second plant; both plants eventually show disease
symptoms.
Seeds are planted and reared under protected
conditions, but mature plants show disease
symptoms.
After a gardener prunes several plants with the same
shears, they all show disease symptoms.
grazed on by herbivores.
Sap from one plant is rubbed on the leaves of a
second plant; both plants eventually show disease
symptoms.
Seeds are planted and reared under protected
conditions, but mature plants show disease
symptoms.
After a gardener prunes several plants with the same
shears, they all show disease symptoms.
Which is an incorrect
statement about STRs (Short
Tandem
They are repeats)?
tandemly repeated units of 5
to 10 nucleotide sequences
The number of repeats is polymorphic
from person to person
Two alleles of an STR may differ in an
individual
They occur in specific regions of the
genome
PCR is used to amplify particular STRs.
Which is an incorrect
statement about STRs (Short
Tandem repeats)?
Resistance to drought
Resistance to herbicides
Resistance to salinity
Superweeds
Resistance to drought
Resistance to herbicides
Resistance to salinity
Superweeds
3-1-2
3-2-1
1-2-3
2-3-1
1-3-2
2-1-3
target sequence
ExtensionDNA polymerase adds nucleotides to the 3 end of each
primer
DenaturationHeat briefly to separate DNA strands
3-1-2
3-2-1
1-2-3
2-3-1
1-3-2
2-1-3