Todays Plan: 2/16/11 Bellwork: Talk about yesterday and the
test (30 mins) Casting gels for tomorrow (30 mins) DNA Tech Notes
(the rest of class)
Slide 2
Todays Plan: 2/18/2010 Bellwork: Test discussion (15 mins)
Transformation Notes, if time
Slide 3
Todays Plan: 2/19/2010 Bellwork: Flies and Test Corrections (15
mins) AP Lab 6 Lab Bench Intro to the Molecular Biology Lab (30
mins) Continue notes (the rest of class) Pack/Wrap-up (last few
mins of class)
Slide 4
Todays Plan: 2/22/10 Bellwork: Cast Gels (30 mins) Count flies
and look at bacteria (while gel is dissolving Practice and run gels
(45 mins) Continue with notes (the rest of the period)
Slide 5
Todays Plan: 2/23/10 Bellwork: Read Gels/answer questions/fly
counts (30 mins) Set up for Lunch demos (30 mins) Continue notes
(the rest of class)
Slide 6
Todays Plan: 2/24/10 Bellwork: Flies/Test Q&A (20 mins) DNA
Tech test (the rest of class)
Slide 7
Todays Plan: 9/15/09 Bellwork: Intstructions (5 mins) Research
for Discussion (30 mins) Bioethics discussion (the rest of
class)
Slide 8
Todays Plan: 9/16/09 Bellwork: Last Presentation (10 mins)
Senses Stations (50 mins) Continue with DNA Tech notes (the rest of
class)
Slide 9
Todays Plan: 9/17/09 Bellwork: Class Optional Taste Demo (10
mins) Finish Senses Stations (50 mins) Finish DNA Tech notes (the
rest of class)
Slide 10
Todays Plan: 9/18/09 Bellwork: Test Q&A (15 mins) DNA Tech
Test (as needed) If you finish your test early, work on the senses
questions.
Slide 11
Regulating Gene Expression Prokaryotes (Bacteria) Often live in
erratic environments Need to turn on and off genes in response to
the environment Use Operons to regulate genes. These are DNA
sections that are regulated by repressors which can turn off the
promoter site, and keep transcription from happening. Operons
contain several related genes, each with its own start and stop
codon The convenience of the operon is that there only has to be 1
on/off switch for all of the genes
Slide 12
Regulating Operons Operons contain a promoter region
(consisting of an attachment point for RNA polymerase and an
operator for the repressor to attach to when not needed), and the
genes for a specific job Repressors come from a regulatory gene at
a point away from the operon Repressible operons are always on
unless a repressor is bound (ex: trp operon-repressor is inactive
until bound to a tryptophan molecule) Inducible operons are off
unless an inducer is present to inhibit the repressors hold on the
DNA (ex: lac operon-repressor is active unless bound to
allolactose)
Figure 17-8 lacl promoter DNA Promoter of lac operon Operator
lacZlacYlacA lac operon
Slide 15
Figure 17-7 Repressor present, lactose absent: Repressor
present, lactose present: No repressor present, lactose present or
absent: Transcription occurs. Repressor synthesized DNA lacl + RNA
polymerase bound to promoter (blue DNA) lacZ lacY TRANSCRIPTION
BEGINS -Galactosidase Permease mRNA lacZ lacY RNA polymerase bound
to promoter (blue DNA) Lactose-repressor complex Repressor
synthesized No functional repressor synthesized mRNA TRANSCRIPTION
BEGINS -Galactosidase Permease lacZ lacY RNA polymerase bound to
promoter (blue DNA) Lacl Repressor binds to DNA. No transcription
occurs. Lactose binds to repressor, causing it to release from DNA.
Transcription occurs (lactose acts as inducer). Normal lacl gene
Normal lacl gene lacl + Mutant lacl gene The repressor blocks
transcription
Slide 16
Figure 17-9 When tryptophan is present, transcription is
blocked. When tryptophan is absent, transcription occurs. Repressor
Tryptophan No transcription Operator RNA polymerase bound to
promoter RNA polymerase bound to promoter TRANSCRIPTION 5 genes
coding for enzymes involved in tryptophan synthesis
Slide 17
Figure 17-10 lac operontrp operon Catabolism Anabolism
(breakdown of lactose) (synthesis of tryptophan) Repressor Lactose
Tryptophan Lactose binds to repressor Tryptophan binds to repressor
Lactose- repressor complex releases from operator Operator
Tryptophan- repressor complex binds to operator No more
transcription of trp operon Transcription of lac operon
TRANSCRIPTION
Slide 18
Positive Gene Regulation Bacteria needs to sense whether or not
glucose as well as lactose are present in its environment. Bacteria
prefer to use glucose for glycolysis, and therefore only use
lactose when there isnt glucose available Cyclic AMP (cAMP)
accumulates when glucose is scarce. cAMP binds to a regulatory
protein, catabolite activator protein (CAP) and the complex becomes
an activator that binds to the DNA just upstream of the promoter.
The activator makes it more likely that RNA polymerase will attach
to the operon and transcribe
Slide 19
Figure 17-14 Glucose inhibits the activity of the enzyme
adenylyl cyclase, which catalyzes production of cAMP from ATP. The
amount of cAMP and the rate of transcription of the lac operon are
inversely related to the concentration of glucose. ATP Adenylyl
cyclase Glucose inhibits this enzyme cAM P Two phosphate groups
Infrequent transcription of lac operon (Cell continues to use
glucose as energy source.) CAP does not bind to DNA CAP LOW cAMP
INACTIVE adenylyl cyclase HIGH glucose concentration LOW glucose
concentration ACTIVE adenylyl cyclase HIGH cAMP CAP CAP-cAMP
complex binds to DNA Frequent transcription of lac operon (Cell
uses lactose if lactose is present.)
Slide 20
Prokaryote vs. Eukaryote Genomes Smaller Genome Fewer Genes
Higher gene density (more genes in a smaller segment of DNA
Relatively few noncoding regions and protein genes are continuous
Large Genome Many more genes Lower gene density Many noncoding
regions (introns) and protein genes are not continuous
Slide 21
Eukaryotic Gene Regulation Recall that in complex organisms,
the complete genome is in all cells, but only the genes necessary
for the function of the individual cell are turned on in that
individual cell In stead of regulating just transcription, as
bacteria do, eukaryotic cells can regulate gene expression at any
step from DNA to protein
Slide 22
Figure 18-1 Nucleus Chromatin (DNA-protein complex) 1.
Chromatin remodeling 2. Transcription Open DNA (Some DNA not
closely bound to proteins) 3. RNA processing Primary transcript
(pre-mRNA) Cap Tail Mature mRNA Cytoplasm 4. mRNA stability 5.
Translation Degraded mRNA (mRNA lifespan varies) mRNA Polypeptide
Active protein 6. Post-translational modification (folding,
transport, activation, degradation of protein)
Slide 23
DNA Regulation Chromatin is DNA that is packaged with proteins,
called histones. The basic unit of chromatin is called the
nucleosome Under normal conditions, the lysine tails of histones
extend out from the nucleosome and are attracted to other
nucleosomes Histone acetylation attaches acetyl groups to these
tails, making them no longer attracted to other histones, which
loosens up the chromatin to make transcription easier Its also been
shown that methyl groups are also added to the histone tails, which
can promote condenstion of the chromatin DNA Methylation Methyl
groups can be attached to cytosine, again causing condensation of
the chromatin Some research shows that heavily methylated areas
recruit deacetylation enzymes, which dually promotes condensation
This appears to be an important regulatory step from embryo to
mature organism. In cases where a template strand is methylated,
the cell matches the methylation in the daughter strand after
replication so that the cell stays specialized Epigenetic
Inheritance Inheritance of traits not directly involved with the
DNA sequence, such as alteration of methylation patterns
Slide 24
Figure 18-2 Nucleosomes in chromatin Nucleosomes DNA Nucleosome
structure Linker DNA H1 protein attached to linker DNA and
nucleosome DNA Group of 8 histone proteins Nucleosome In some
cases, nucleosomes may be grouped into 30-nanometer fibers. 30
nm
Slide 25
Figure 18-4 Condensed chromatin Decondensed chromatin Acetyl
group on histone
Slide 26
The Eukaryotic Gene Recall that even Eukaryotic genes contain a
promoter site, on which the transcription initiation complex
assembles There are introns and exons within the gene and control
elements that dont code but bind proteins
Slide 27
Figure 18-7 Start site Exon Intron EnhancerPromoterEnhancer
Intron Exon Promoter- proximal element Enhancer
Slide 28
Regulating Transcription Transcription factors bind to the DNA
and make it easier for RNA polymerase to bind These can be general
transcription factors, if they are necessary for all protein-coding
genes, and if they result in a low rate of transcription Specific
transcription factors are proteins that attach to only certain
genes and generally produced a high rate of transcription in cells
needing particular genes Enhancers are generally found thousands of
nucleotides upstream from the gene and are called Distal control
elements Activators and repressors can bind to these elemets to
regulate the initiation of transcription by interacting with
mediator proteins The DNA can also be bent so that these form a
transcription initiatinon complex The actual number of activators
is small, but its the combination of control elements (proteins,
activators, etc) that is unique to each gene
Slide 29
Figure 18-10 THE ELEMENTS OF TRANSCRIPTIONAL CONTROL: A MODEL
Regulatory transcription factor Chromatin remodeling complex (or
HATs) 1. Regulatory transcription factors recruit chromatin-
remodeling complex, or HATs. Chromatin decondenses. Exposed DNA
Promoter-proximal element Promoter Exon Intron Exon Promoter
Transcribed portion of gene for muscle-specific protein
Co-activators Regulatory transcription factors Promoter-proximal
element Basal transcription complex TRANSCRIPTION RNA polymerase II
Basal transcription complex 2. When chromatin decondenses, a region
of DNA is exposed, including the promoter. 3. Regulatory
transcription factors recruit proteins of the basal transcription
complex to promoter. Note looping DNA. 4. RNA polymerase II
completes the basal transcription complex; transcription begins.
Enhancer
Slide 30
Do Eukaryotes have Operons? While there are co-expressed genes
in Eukaryotes, each gene has its own promoters Some co-expressed
genes are clustered, while others are on different chromosomes
Coordinate control of co-expressed genes seems to be regulated by
the genes having the same combination of control elements at the
same time, usually in response to a signal outside of the cell
Slide 31
Figure 18-14 Cytoplasm Signaling molecule Cell-surface receptor
Inactive STAT protein (two single polypeptide chains) Activated
STAT protein (dimer of two polypeptide chains) Nuclear envelope
Enhancer TRANSCRIPTION Transcription activated Nucleus
Slide 32
Post-transcriptional Gene Regulation RNA Processing-Alternative
RNA splicing The same transcript may result in different mature RNA
depending on which segments are treated as introns mRNA Degradation
In Prokaryotes, mRNA is degraded within a few minutes, but
EukaryotesmRNA can last for days or weeks Initiation of Translation
Proteins can block the 5 end of an mRNA, preventing attachment by
the ribosome In other cases, the poly-A tail is not synthesized
long enough until the organism is ready for the protein Protein
processing and Degradation Many protiens require post-translation
modifications, such as reversible phorphorylation Some proteins are
tagged with ubiquitin, which alerts the proteasomes to their
presence and degrades them
Slide 33
Figure 18-12 Tropomyosin gene Intron Exon Processed mRNAs
Skeletal muscle Smooth muscle Some exons are specific to
tropomyosin in skeletal or smooth muscle; some exons are common to
both muscle types
Slide 34
Noncoding RNAs These are other molecules, like tRNA and rRNA
Many more RNAs are discovered frequently and have a variety of
functions within the cell Apparently, not all DNA is supposed to
code for proteins, and in fact, doesnt
Slide 35
MicroRNAs These are small pieces of RNA that are complimentary
to mRNA Called miRNA Formed from a large primary transcript that
bends into one or more hairpin turns An enzyme, called a dicer cuts
these away, forming double-stranded mRNA One strand degrades, while
the other forms a complex with a protein These complexes can bond
with and interfere with mRNA (if theyre complimentary at some
part), and can degrade it (if theyre complimentary along the
length) Another type of RNA, small interfering RNA (siRNA) can also
interfere with mRNAs function. These are formed from larger,
double-stranded precursor RNA molecules Collectively, this is
called RNAi (RNA interference)
Slide 36
Figure 18-13 miRNAs TARGET CERTAIN mRNAS FOR DESTRUCTION RNA
hairpin 1. Transcription of a microRNA gene. DNA RNA polymerase
Precursor miRNA Cytoplasm Enzyme Mature miRNA Single-stranded miRNA
RISC protein complex Target mRNA 2. Initial transcript is processed
into a precursor micro RNA (miRNA). 3. Enzyme in cytoplasm cuts out
hairpin loop, forming a mature miRNA. 4. miRNA becomes
single-stranded and binds to RISC protein complex. 5. miRNA, held
by RISC, binds to complementary sequence on target mRNA. 6. Enzyme
inside RISC cuts mRNA.
Slide 37
Gene Expression and Embryonic Development The genome (including
the cytoplasmic genome) contains a program for cell differentiation
in the embryo Cytoplasmic determinants (RNA and DNA in the
cytoplasm-matrolineal) get divided unevenly, which may contribute
to cellular differentiation The embryos own cells may also induce
changes in the other embryonic cells
Slide 38
Sequential Regulation of Gene Expression during Differentiation
Once a cell begins the process of differentiation, it is
irreversible-even if the cell is moved to another part of the
embryo Each cell type produces its own tissue-specific proteins
from transcribed mRNA in genes that are turned on
Slide 39
Figure 21-7 VISUALIZING mRNAs BY IN SITU HYBRIDIZATION 1. Start
with a single- stranded DNA or RNA probe, complementary in sequence
to target mRNA. 4. Treat preserved cells or tissues to make them
permeable to probe. Add many copies of probe. 3. Preserve the
specimen (in this case, a Drosophila embryo). 2. Add label to probe
(a radioactive atom or an enzyme that catalyzes a color- producing
reaction). 5. Probe binds to target mRNA. Labeled probe that does
not bind to target mRNA is excess, and is washed away. 6. In this
case, target mRNAs are concen- trated in the anterior end of the
embryo. The label shows up as black in this image. Posterior
Anterior Target mRNA DNA probe DNA probe Embryo DNA probe
Label
Slide 40
Setting up the body plan Pattern formation is the organization
of the body and is regulated by cytoplasmic determinants and
inductive signals from neighboring cells Early on, positional
information, such as where the head and tail are to be, is
established In Drosophila, a series of homeotic (hox) genes control
the segmentation of the body and position of body parts Body Axis
is determined by maternal effect genes- when there is a mutant in
the mother, there are mutations in the offspring, regardless of the
offsprings genotype Bicoids are two-tailed mutants come from
mutations in these maternal effect genes These genes produce
Morphogens, that concentrate in certain segments and determine what
that segment will become
Slide 41
Figure 21-6 A normal fruit-fly embryoA bicoid mutant Abdominal
segments Abdominal segments Abdominal segments Thoracic segments
Head segments
Slide 42
Figure 21-13 Head The location of Hox genes on the mouse
chromosome correlates with their pattern of expression in mouse
embryos. Hox genes The location of Hox genes on the fly chromosome
correlates with their pattern of expression in fly embryos. Fly
embryo Mouse embryo Hox genes Thorax Abdomen
Slide 43
Figure 21-12 Homeotic mutant Normal fruit fly Homeotic mutant
Legs in place of antennae Wings in place of halteres Haltere
Antennae
Slide 44
Wrapping up Eukaryotic Genomes Eukaryotic Genomes consist of
many non- coding and repetitive sequences that scientists now
suspect actually serve important purposes within the cells
Transposable Elements-Jumping Genes Transposons are genes that move
via a DNA intermediate Retrotransposons are what most transposable
elements are and they move via an RNA intermediate
Slide 45
Figure 20-5 HOW LINE TRANSPOSABLE ELEMENTS SPREAD DNA 7. New
copy of LINE is integrated into genome. New copy Cytoplasm LINE
protein Nuclear envelope Ribosome Original copy cDNA mRNA Reverse
transcriptase Integrase Reverse transcriptase Original location of
LINE (15 kb) RNA polymerase LINE mRNA LINE mRNA and LINE proteins
Gene for reverse transcriptase Gene for integrase 6. Integrase cuts
chromosomal DNA and inserts LINE cDNA. 5. Reverse transcriptase
makes LINE cDNA from mRNA, then makes cDNA double stranded. 4. LINE
mRNA and proteins enter nucleus. 3. LINE mRNA exits nucleus and is
translated. 2. RNA polymerase transcribes LINE, producing LINE
mRNA. 1. A long interspersed nuclear element (LINE) exists in
DNA.
Slide 46
Figure 20-8 GENE DUPLICATION BY UNEQUAL CROSSOVER Homologous
chromosomes 1 1 1 1 1 2 2 2 2 2 3 3 3 3 4 4 4 4 4 5 5 5 5 5 6 6 6 6
6 Gene deletion Gene duplication 1. Start with two homologous
chromosomes containing the same genes (numbered 16). 2. The genes
misalign during meiosis I. Crossing over and recombination occur.
3. Gene 3 has been deleted from one chromosome and duplicated in
the other chromosome. 12334 56
Slide 47
The Molecular Biology of Cancer As we learned before, cancers
are unregulated cells. Scientists think that that oncogenes-are the
cancer- causing genes as well as random mutation from DNA damage
Proto-oncogenes are the normal genes that code for proteins that
regulate the cell cycle Tumor supressor genes inhibit cell division
There are generally 3 ways that protooncogenes become oncogenes DNA
movement w/in a genome Point mutations Amplification of a
proto-oncogene
Slide 48
The development of Cancer Cancer requires multiple mutations
and at least 1 oncogene Cancers can begin as benign polyps, tumors,
etc, but the longer that these exist, the longer there is for the
necessary mutations to accumulate Viruses also play a role in the
development of some cancers Retroviruses have oncogenes that can be
donated to the host cell The viral DNA may also be inserted in such
a way that it disrupts a tumor-supressing gene.
Slide 49
What about Genetic Predisposition? It makes sense, that if
oncogenes are partially responsible for cancer, that certain
cancers should run in families Examples of cancers with a
strongly-heritable component are colorectal cancer and breast
cancer In Breast cancer, mutations in the BRCA1 or BRCA2 genes
appear to be responsible for many breast cancers These genes play a
role in the cells DNA damage repair proteins It makes sense, then,
that avoiding mutagens would lower the risk of cancer, even if one
has the mutations in his/her genome
Slide 50
Viruses At their simplest, these are a piece of genetic
material with a protein coat (called the capsid) These are
considered non-living b/c they have no metabolism, homeostasis,
growth, and require a host cell to carry out their functions Are
extraordinarily small, since they are active inside of cells. They
can contain traditional, double-stranded DNA, single-stranded DNA,
or even RNA Recall that theyre specific to their hosts-the capsid
must fit into a receptor on the host cell in order to infect the
cell, and theres a lot of variety in the capsid A few, like
influenza, have a viral envelope, derived from the membranes of
their host cell
Slide 51
Figure 35-7 Nonenveloped virus Genome (in this case, DNA)
Capsid (protein) Enveloped virus Genome (in this case, RNA) Capsid
(protein) Envelope (phospholipid bilayer) Host protein Viral
protein
Figure 35-21 Leaf infected with virus Healthy leaf
Slide 55
Figure 35-1-1 Heart coxsackie Trachea and lungs parainfluenza
RSV influenza adenovirus Lymphatic and immune systems Epstein-Barr
HIV paramyxovirus (e.g., measles) Brain and CNS encephalitis rabies
polio herpes zoster yellow fever Ebola dengue West Nile
Slide 56
Figure 35-1-2 Digestive track and liver hepatitis A, B, C, D, E
rotavirus Blood vessels and blood cells erythrovirus Ebola
hantavirus Skeletal muscles coxsackie Skin rubella variola
papillomavirus herpes 1 molluscum contagiosum Reproductive organs
herpes 2 papillomavirus Peripheral nerves rabies
Slide 57
Figure 35-15-Table 35-2
Slide 58
Viral Cycles All cycles begin with the virus binding to the
host cell Some are taken in by endocytosis Others inject their
genome Lytic Viruses Also called virulent phages, because these
infect, degrade the hosts DNA, reproduce, and kill the host cell
right away (rhinovirus, influenza, T4 bacteriophage) Lysogenic
Viruses These are called temperate phages because these inject
their genome (prophage), and integrate it within the hosts DNA, so
they can hide inside of the host until theyre triggered (ex: HSV,
HPV, lambda phage) Retroviruses These are special lysogenic viruses
whose prophage is made of RNA, so they must inject reverse
transcriptase (rt) as well as the prophage in order to integrate
with the hosts chromosomes (ex: HIV)
Slide 59
Figure 35-8a LYTIC REPLICATION RESULTS IN A NEW GENERATION OF
VIRUS PARTICLES AND THE DEATH OF THE HOST CELL. Virus particle
ProteinmRNA DNA Host-cell genome Protein DNA 1. Viral genome enters
host cell. 2. Viral genome is transcribed; viral proteins are
produced. 3. Viral genome is replicated. 5. Particles exit to
exterior. 4. Particles assemble inside host. 6. Free particles in
tissue or environment are transmitted to new host.
Slide 60
Figure 35-8b LYSOGENIC REPLICATION RESULTS IN VIRUS GENES BEING
TRANSMITTED TO DAUGHTER CELLS OF THE HOST. 1. Viral genome enters
host cell. 2. Viral genome integrates into host- cell genome. 3.
Host-cell DNA polymerase copies chromosome. 4. Cell divides. Virus
is transmitted to daughter cells.
Slide 61
Figure 35-14 Double-stranded DNA First, reverse transcriptase
synthesizes cDNA from RNA Then, reverse transcriptase synthesizes
double-stranded DNA from cDNA cDNA template cDNA RNA template
Slide 62
Preventing Viruses Some cells have evolved defenses against
these viruses in the form of restriction enzymes that can destroy
the viral DNA Vaccination helps animals avoid viral infection Being
infected allows the immune system to learn to detect and fight
existing strains of viruses
Slide 63
Figure 35-9 HOW VACCINATION WORKS 1. Viral antigens (in red)
are intro- duced into the body. 2. Antigens bind to receptors on
certain immune system cells. 3. These cells stimulate other immune
system cells to produce antibodies (in green) to the virus. 4.
Later, if the host organism is exposed to actual virus particles,
the antibody-producing cells are activated. The virus particles
become coated with antibodies. 5. Viruses that are coated with
antibodies are destroyed by immune system cells. The antigens are
usually protein components of a virus capsid or envelope The cells
that produce specific antibodies remain active for a long timeyears
or decades Virus
Slide 64
Emerging Viruses New Viruses occur because of 3 main causes:
Mutation of existing viruses-especially RNA viruses, which mutate
faster Viruses coming from a small, isolated human population
Viruses jumping from one species to another- especially in
closely-related species Epidemic=emergence of a new strain of an
existing virus Pandemic=global epidemic
Slide 65
Plants and viruses Yes, plants get viruses too Transmission
occurs in 1 of 2 ways: Horizontal transmission-plant is infected by
an external source of virus, especially if the epidermis of the
plant is damaged (herbivore damage is especially bad b/c herbivores
can act as horizontal transmitters) Vertical transmission-plant
inherits the virus from the parent. The virus can spread through
the plasmodesmata
Slide 66
Viroids and Prions Viroids=circular pieces of RNA that infect
plants These reproduce inside of the plants cells and cause errors
in the regulation of growth Infected plants typically exhibit
stunted growth Prions=infectious proteins (ex: BSE=mad cow disease)
These develop slowly (up to 10 year incubation period) These are
indestructible Scientists believe that these are abnormally- folded
proteins, that, when they enter a cell that has the normal
proteins, corrupt these
Slide 67
DNA Technology Involves a number of techniques for identifying,
copying, cutting, and modifying DNA These are all part of the field
of biotechnology Genetic engineering-directly manipulating the DNA
of an organism, is also part of biotechnology
Slide 68
DNA Cloning Involves copying DNA-useful for studying specific
genes, since you can keep a library of cloned genes, rather than
search an entire genome for them Most cloning is done with
bacterial plasmids-circular pieces of DNA in a bacteria that
contain only a few genes and are separate from the bacterias main
chromosome (these are called cloning vectors) In recombinant DNA, a
plasmid is removed from the bacteria and spliced with a new piece
of DNA. This can be re-inserted into the bacteria, which will both
express the gene and copy it every time the cell divides The gene
we inserted is called the donor gene The process of putting the
gene back into the bacteria is called transformation
Slide 69
Figure 19-2 GENES CAN BE CLONED BY INSERTING THEM INTO PLASMIDS
Recognition site 5 5 3 3 Plasmid 5 5 3 3 Recognition site
Restriction endonuclease (EcoR1) Plasmid Recombinant plasmid 1.
Plasmid DNA contains a recognition site for a restriction
endonuclease. 2. Attach the same recognition site to the gene that
will be inserted into the plasmid. 3. A restriction endonuclease
makes staggered cuts at each of the recognition sites, creating
sticky ends. 4. Sticky ends on plasmid and on gene to be inserted
bind by complementary base pairing. 5. Use DNA ligase to catalyze a
phosphodiester bond at points marked by green arrows, sealing the
inserted gene. Sticky end
Slide 70
Restriction Enzymes These are enzymes that cut DNA at specific
recognition sequences (usually palindromic) Useful for many
biotechnology applications because we know their recognition
sequences Each resulting restriction fragment (DNA cut with a
restriction enzyme), has sticky- ends so that it is easy to
splice
Slide 71
Storing Cloned Genes Genomic Library=cell clones containing the
recombinant plasmid Sometimes, phages are used as genomic libraries
b/c they can carry bigger inserts Scientists have also found mRNA
extracts useful in producing libraries b/c of the poly-A tail The
tail is a useful primer for reverse transcriptase, and can be used
to make cDNA (complimentary DNA) The cDNA can then be inserted into
the cloning vector Bacterial Artificial Chromosomes (BAC) can also
act as libraries This is simply a large plasmid which contains the
inserts and genes necessary for replication
Slide 72
Figure 19-3-1 CREATING A cDNA LIBRARY THAT CONTAINS THE HUMAN
GROWTH HORMONE GENE mRNA Single- stranded cDNA Reverse
transcriptase Double- stranded cDNA 3. Make the cDNA double-
stranded. 2. Use reverse transcriptase to synthesize a cDNA from
each mRNA. 1. Isolate mRNAs from cells in pituitary gland.
Slide 73
Screening a Library for a Gene This involves creating a nucleic
acid probe that has a complimentary sequence to the DNA were
looking for We can then see where this probe hybridizes to find the
gene
Slide 74
Figure 19-4 Labeled probe USING A DNA PROBE TO FIND A TARGET
SEQUENCE IN A COLLECTION OF MANY DNA SEQUENCES 1. Single-stranded
DNA probe has a label that can be visualized. 2. Expose probe to
collection of single-stranded DNA sequences. 3. Probe binds to
complementary sequences in target DNAand only to that DNA. Target
DNA is now labeled and can be isolated.
Slide 75
Expressing Eukaryotic cloned DNA Eukaryotic expression in
bacteria is sometimes difficult b/c the promoters and control
sequences are often different Scientists use an expression vector,
a vector that has a very active promoter region upstream from the
donor gene Scientists also occasionally need to use cDNA donor
genes b/c of the presence of introns in the eukaryotic genes,
making them unwieldy Yeasts can be used as cloning vectors to
completely bypass this problem Yeast Artificial Chromosomes (YACs)
combine the necessary origin for DNA replication, centromeres, and
telomeres, with the donor genes Sometimes, you need to use a
eukaryotic vector b/c only it is capable of the post-translational
protein modifications necessary for the protein to function
Slide 76
PCR Polymerase Chain Reaction allows the scientist to amplify a
sample of DNA Produces results within hours, rather than days
Involves thermal cycling to denature (unzip) the DNA molecule with
heat, then cooling to promote annealing (hydrogen bonding), and
uses a heat- stable DNA polymerase molecule
Slide 77
Figure 19-6 PCR primers must be located on either side of the
target sequence, on opposite strands. 3 When target DNA is single
stranded, primers bind and allow DNA polymerase to work. 5 3 3 3 3
3 5 5 5 5 5 Primer Region of DNA to be amplified by PCR
Slide 78
Figure 19-7 THE POLYMERASE CHAIN REACTION IS A WAY TO PRODUCE
MANY IDENTICAL COPIES OF A SPECIFIC GENE 1. Start with a solution
containing template DNA, synthesized primers, and an abundant
supply of the four dNTPs. dNTPs Primers 5 5 3 3 3 3 5 5 3 3 5 5 5
One cycle 5 5 5 5 5 3 3 3 3 2. Denaturation Heating leads to
denaturation of the double-stranded DNA. 3. Primer annealing At
cooler temperatures, the primers bind to the template DNA by
complementary base pairing. 4. Extension During incubation, Taq
polymerase uses dNTPs to synthesize complementary DNA strand,
starting at the primer. 5. Repeat cycle of three steps (24) again,
doubling the copies of DNA. 6. Repeat cycle again, up to 2030
times, to produce millions of copies of template DNA.
Slide 79
DNA Sequences Gel Electrophoresis Uses charge and size to pull
fragments of DNA across a Gel Useful for generating characteristic
banding patterns, but also for looking at differences in sequences,
as the DNA fragments are cut with restriction enzymes Southern
Blotting Is a combination of gel electrophoresis and DNA
hybridization Probe is radioactive
Slide 80
Figure 20-7b Lane sources: X: An unrelated individual M: A
mother B: A boy the mother claims is her own U: Undisputed children
of the mother A gel showing minisatellite seqences from unrelated
and related individuals X M B U U U
Slide 81
Figure 19-8l Location of restriction endonuclease cuts Sample 1
Samples from four individuals Double- stranded DNA Double-stranded
DNA SOUTHERN BLOTTING: ISOLATING AND FINDING A TARGET DNA IN A
LARGE COLLECTION OF DNA FRAGMENTS 1. Restriction endonucleases cut
DNA sample into fragments of various lengths. Each type of
restriction endonuclease cuts a specific sequence of DNA. 2. A
sample consists of all the DNA fragments of various lengths. The
sample is loaded into a gel for electrophoresis. 3. During
electrophoresis, a voltage gel separates DNA fragments by size.
Small fragments run faster. Power supply 1 2 3 4
Slide 82
Figure 19-8r Single- stranded DNA Stack of blotting paper
Labeled probe DNA Filter Gel Sponge in alkaline solution SOUTHERN
BLOTTING: ISOLATING AND FINDING A TARGET DNA IN A LARGE COLLECTION
OF DNA FRAGMENTS 4. The DNA fragments are treated to make them
single stranded. 5. Blotting. An alkaline solution wicks up through
the gel into blotting paper. DNA fragments from the gel are carried
to the filter, where they are permanently bound. 6. Hybridization
with labeled probe. The filter is put into a solution containing
labeled probe DNA. The probe binds to DNA fragments containing
complementary sequences. 7. Visualize fragments bound by probe.
Fluorescence or autoradiography (see BioSkills 7) is used to find
label. 1 2 3 4
Slide 83
DNA Sequencing This is when the sequence of bases on the
molecule is determined Mostly, this is automated now. Dideoxy Chain
Method of Sequencing is one of these methods, using fluorescent
dyes and can sequence a segment up to about 800 bps
Slide 84
Figure 19-9 DIDEOXY SEQUENCING 3 3 5 5 Normal dNTP (extends DNA
strand) ddNTP (terminates synthesis) ddGTPs Template DNA 3 No OH 3
5 Labeled primer Non-template DNA ddCTPs ddATPs ddTTPs 5 end3 end
Smaller fragments Larger fragments Non-template DNA 5 5 3 3
Template DNA 1. Incubate a large number of normal dNTPs with a
small number of ddNTPs (in this case starting with ddGTPs),
template DNA, a primer for the target sequence, and DNA polymerase.
2. Collect DNA strands that are produced. Each strand will end with
a ddGTP (corresponding to a C on the template strand). 3. Repeat
process three more times using ddCTPs, ddATPs, and ddTTPs, which
will terminate synthesis where Gs, Ts, and As occur on the template
strand, respectively. 5 4. Line up different-length strands by size
using gel electrophoresis to determine DNA sequence. DNA
sequence
Slide 85
Figure 19-10 FLUORESCENT MARKERS IMPROVE SEQUENCING EFFICIENCY.
DNA polymerase Template DNA Long fragments Short fragments
Capillary tube Output 1. Do one sequencing reaction instead of
four. Reaction mix contains ddATP, ddTTP, ddGTP, ddCTP with
distinct fluorescent markers. (With radioactive labels, four
reactions are neededone labeled ddNTP at a time.) 2. Fragments of
newly synthesized DNA that result have distinctive labels. 3.
Separate fragments via electrophoresis in mass- produced,
gel-filled capillary tubes. Automated sequencing machine reads
output.
Slide 86
Sequencing Whole Genomes HGP was set up to create chromosome
maps of the human genome This was done with a 3-step approach The
first step was to create a linkage map (like we did with Sordaria
Next, a physical map was constructed, using linkage mapping data
Finally, the genes were sequenced (dideoxy sequencing) Shotgun
sequencing Uses cut-ups of human DNA, inserted into bacteria for
cloning, then analysis of the small sequences and
reconstruction
Slide 87
Figure 20-2 SHOTGUN SEQUENCING A GENOME 160 kb fragments
Genomic DNA BAC library 1-kb fragments Shotgun clones Shotgun
sequences BAC Main bacterial chromosome 1. Cut DNA into fragments
of 160 kb, using sonication. 2. Insert fragments into bacterial
artificial chromosomes; grow in E. coli cells to obtain large
numbers of each fragment. 3. Purify each 160-kb fragment, then cut
each into a set of 1-kb fragments, using sonication, so that 1-kb
fragments overlap. 4. Insert 1-kb fragments into plasmids; grow in
E. coli cells. Obtain many copies of each fragment. 5. Sequence
each fragment. Find regions where different fragments overlap.
Draft sequence 6. Assemble all the 1-kb fragments from each
original 160-kb fragment by matching overlapping ends. 7. Assemble
sequences from different BACs (160-kb fragments) by matching
overlapping ends.
Slide 88
Analyzing Gene Expression Northern Blotting Same basic
procedure as Southern Blotting, but were looking for mRNA in cells
at different stages of development to see if the protein were
studying is needed at these steps Reverse-transcriptase PCR Will
accomplish the same thing as Northern Blotting, but uses rt to make
cDNA from the mRNA, which is then put through PCR and run on a gel
The gene were observing only occurs in samples that contained the
mRNA with that gene DNA Microarray Assays Hybridization of cDNA
with a pre-fixed slide of mRNA This helps scientists to see which
genes may be turned on at the same time and thus working
together
Slide 89
Figure 20-11 Exon 286 Exon 287 Exon 288 Microarray slide Each
spot on the slide contains many single- stranded copies of a
different exon
Slide 90
Figure 20-12 PROTOCOL FOR A MICROARRAY EXPERIMENT Normal
temperature High temperature 1. Use reverse transcriptase to
prepare single-stranded cDNA from mRNA of control cells and
treatment cells. 2. React with labeled nucleotides to add
fluorescent green label to control cDNA and fluorescent red label
to treatment cDNA. 3. Probe a microarray with the labeled cDNAs.
Probe cDNA will bind and label spots containing complementary
sequences. 4. Shine laser light to induce fluorescence. Analyze the
pattern of hybridization between the two cDNAs and the DNA on the
microarray. cDNA mRNA cDNA probes Microarray Microarray computer
output: Green = genes transcribed in control cells Yellow = genes
transcribed equally in both cells Dark = low gene expression Red =
genes transcribed in treatment cells
Slide 91
Determining Gene Function Usually, scientists disable a gene
which has been identified by DNA tech, then observe the
consequences in the cell This is called in vitro mutagenesis
Slide 92
Cloning Organisms Plants can be cloned using single-cell
cultures Differentiated cells from the root can be grown in culture
and become entire organisms, genetically identical to the parent
When mature cells are capable of dedifferentiating and
redifferentiating, theyre called totipotent Recall that through
propogation, plants are cloned as well! Animals can be cloned via
nuclear transfer Originally, an unfertilized egg was used, which
worked, except that the ability of the new nucleus to control the
resultant clone decreased with donor nucleus age Dolly was
different because she was made from an already- differentiated
mammary cell. Dedifferentiation was accomplished by culturing the
cell in a nutrient poor medium Dolly died at age 6, when she was
euthanized after suffering from a lung disease that usually effects
much older sheep, leading scientists to speculate that clones
werent as vigorous as the original organism.
Slide 93
Figure 21-3 Surrogate mother Cloned sheep Dolly Early embryo
Fused cell Mammary cellsEgg cell Mammary-cell donor sheep Egg-cell
donor sheep CLONING A SHEEP 1. Start with two female sheep. Each
will donate one cell. 5. Grow in culture. Embryo begins
development. 6. Implant early embryo in uterus of third sheep. 7.
Embryo develops normally, resulting in lamb that is genetically
identical to mammary-cell donor. This result supports the
hypothesis that mature cells contain all the genes in the genome.
2. Culture mammary- gland cells. Remove nucleus from egg cell. 3.
Fuse the mammary-gland cell to enucleated egg cell. 4. Egg cell now
contains nucleus from mammary- gland cell.
Slide 94
Problems with Organismal Cloning Cloning is inefficient-only a
small percent of cloned embryos develop normally, and there are
often defects (like pneumonia, obesity, liver failure, and
premature death) Scientists are working to improve the efficiency
of cloning by studying systematic changes to the chromatin as the
nucleus matures
Slide 95
Stem Cells These are unspecialized cells Ultimately, this is
what scientists would like to achieve through cloning for the
treatment of disease The most common place to find these is in
embryos (these are pluripotent- can develop into a wide variety of
cell types), although, there are some less flexible stem cells in
adults
Slide 96
Applications of Biotechnology Medical Applications Diagnosis of
disease Gene Therapy Pharmaceuticals Forensic Evidence
Environmental Clean-up Ag Apps old school=selective breeding
Slide 97
Ethics Issues with Biotechnology Safety questions about GMOs
Problems with the technologies leading to super bugs and
maldeveloped mutants Creating organisms with medical issues since
clones arent as vigorous Obtaining Stem Cells Where to draw the
line with research?