Ch. 11: Gene regulations How is cloning possible?
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Ch. 11: Gene regulationsHow is cloning possible?
Every cell has the same chromosomesThen….. Why does a heart muscle cell look different from a skin cell?
Organisms respond to their environment by altering gene expression
Central question: what regulates gene expression?
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Differentiation
Differentiation is controlled by turning specific sets of genes on or off
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DNA Packing eukaryotic chromosomes condense during
prophase of Mitosis helps regulate gene expression by preventing
transcription– Nucleosomes
– Tight helical fiber =
– Supercoil = coiling of the tight helical fiber
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DNA double helix(2-nm diameter)
“Beads ona string”
Linker
Histones
Metaphasechromosome
Tight helical fiber(30-nm diameter)
Nucleosome(10-nm diameter)
Supercoil(300-nm diameter) 700 nm
Animation: DNA Packing
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X-chromosome inactivation
– female mammals– one of the two X chromosomes is highly
compacted and transcriptionally inactive (Barr body)
– Occurs early in embryonic development, thus all cellular descendants have the same inactivated chromosome
– Tortoiseshell fur coloration is due to inactivation of X chromosomes in heterozygous female cats
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Two cell populationsin adult
X chromosomes
Early embryo
Allele forblack fur
Inactive X
Black furAllele fororange fur
Orange fur
Cell divisionand random
X chromosomeinactivation Active X
Inactive XActive X
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Eukaryotic gene expression
– Each gene has its own promoter and terminator
– Are controlled by interactions between numerous regulatory proteins and control sequences
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Regulatory proteins • Transcription factors - help RNA
polymerase bind to the promoter• Activators –
• Silencers - Control sequences
– Promoter– Enhancer
– Related genes located on different chromosomes can be controlled by similar enhancer sequences
Animation: Initiation of Transcription
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Enhancers
Otherproteins
DNA
Transcriptionfactors
Activatorproteins
RNA polymerase
PromoterGene
Bendingof DNA
Transcription
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Alternative RNA splicing
– Can involve removal of an exon with the introns on either side
Animation: RNA Processing
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1
or
Exons
DNA
RNA splicing
RNAtranscript
mRNA
2 3 4 5
1 2 3 4 5
1 2 4 51 2 3 5
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Small RNAs control gene expression RNA interference (RNAi)
– small, complementary RNAs bind to mRNA transcripts, blocking translation
MicroRNA (miRNA)
– MicroRNA + protein complex binds to complementary mRNA transcripts, blocking translation
Animation: Blocking Translation Animation: mRNA Degradation
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miRNA 1
Translation blockedORmRNA degraded
Target mRNA
Protein
miRNA-proteincomplex
2
3 4
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Control of gene expression also occurs with – Breakdown of mRNA– Initiation of translation– Protein activation– Protein breakdown
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Folding ofpolypeptide andformation ofS—S linkages
Initial polypeptide(inactive)
Folded polypeptide(inactive)
Active formof insulin
Cleavage
Ex. Insulin formation
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Epigenetic Inheritance
This can be accomplished by acetylation or methylation of histones
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Regulation of Chromatin Structure
Chemical modification of histone tails can affect the configuration of chromatin and thus gene expression
(a) Histone tails protrude outward from a nucleosome
DNAdouble helix
Histonetails
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Addition of methyl groups to certain bases in DNA is associated with reduced transcription in some species
(b) Acetylation of histone tails promotes loose chromatin structure that permits transcription
Unacetylated histones Acetylated histones
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NUCLEUS
DNA unpackingOther changes to DNA
Addition of cap and tail
Chromosome
Gene
RNA transcript
GeneTranscription
Intron
Exon
Splicing
CapmRNA in nucleus
Tail
Flow throughnuclear envelope
Broken-downmRNA
CYTOPLASM
Breakdown of mRNA
Translation
mRNA in cytoplasm
Broken-downprotein
Cleavage / modification /activation
Breakdown of protein
Polypeptide
Active protein
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Why so much control over gene expression?
It allows cells to respond appropriately to their environment
Signal transduction pathways convert messages received at the cell surface to responses within the cell via gene expression
Three steps:1.Reception – 2.Amplification/transduction –
3.Response - transcription factor is activated, enters nucleus, transcribes specific genes
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Signaling cell
DNA
Nucleus
Transcriptionfactor(activated)
Signaling molecule Plasma
membraneReceptorprotein
Relayproteins
TranscriptionmRNANewprotein
Translation
Target cell
2
1
3
4
5
6
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Cloning: How? Nuclear transplantation– Replacing the nucleus of an egg cell with a
nucleus from an adult somatic cell. Allow embryo to form. Embryo can be used in:
– Reproductive cloning
– Therapeutic cloning– Grow embryonic stem cells in culture – Induce stem cells to differentiate and grow
into organs, tissues, etc.
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Removenucleusfrom eggcell
Implant blastocyst insurrogate mother
Add somatic cellfrom adult donor
Donorcell
Remove embryonicstem cells fromblastocyst andgrow in culture
Reproductivecloning
Nucleus fromdonor cell
Grow in cultureto produce anearly embryo(blastocyst)
Therapeuticcloning
Clone ofdonor is born
Induce stemcells to formspecialized cells
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To clone or not to clone….
Benefits of reproductive cloning?
Disadvantages of cloning?
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Human stem cell research Ethical concerns with reproductive cloning Ethical concerns with therapeutic cloning?
Benefits:
Human embryos – have the greatest potential to give rise to all cell types
– Adult stem cells (bone marrow) or cord blood cells
– can give rise to many but not all types of cells
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Ch 12: DNA Technology
1. DNA profiling2. Genetically modified organisms/recombinant
DNA technology3. Gene therapy4. Genomics
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1. DNA profiling = analysis of DNA fragments to determine whether they come from a particular individual
3 steps:1..
2.Amplify (copy) markers for analysis –
3.Compare sizes of amplified fragments by gel electrophoresis
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1. Select genetic marker to analyze
Short tandem repeats (STRs) are genetic markers used in DNA profiling
– STRs =
– STR analysis compares the lengths of STR sequences at specific regions of the genome
– Current standard for DNA profiling is to analyze 13 different STR sites
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STR site 1
Crime scene DNA
STR site 2
Suspect’s DNA
Number of short tandemrepeats match
Number of short tandemrepeats do not match
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Polymerase chain reaction (PCR) = method of amplifying a specific segment of a DNA molecule
Relies upon a pair of primers =
Repeated cycle of steps for PCR:1.Sample is heated to separate DNA strands2.Sample is cooled and primer binds to
specific target sequence3.Target sequence is copied with DNA
polymerase
2. Amplify the DNA sample
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Cycle 1yields 2 molecules
21 3
GenomicDNA
Cycle 3yields 8 molecules
Cycle 2yields 4 molecules
3 5 3 5 3 5
Targetsequence
Heat toseparateDNA strands
Cool to allowprimers to formhydrogen bondswith ends oftarget sequences
35
3 5
35
35 35
Primer New DNA
5
DNApolymerase addsnucleotidesto the 3 endof each primer
5
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3. Gel electrophoresis separates DNA molecules based on size
– DNA samples placed at one end of a porous gel– Current is applied and DNA molecules move
from the negative electrode toward the positive electrode
– DNA fragments appear as bands, visualized through staining or radioactivity or fluorescence
Video: Biotechnology Lab
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Mixture of DNAfragments ofdifferent sizes
Completed gel
Longer(slower)molecules
GelPowersource
Shorter(faster)molecules
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Crime sceneDNA isolated1
Suspect 1 Suspect 2
DNA of selectedmarkers amplified
2
Amplified DNA compared
3
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Longer fragmentsmove slower
Powersource
Shorter fragmentsmove faster
Mixture of DNA fragments
A “band” is acollection of DNAfragments of oneparticular length
DNA attracted to +pole due to PO4
– groups
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Applications of DNA profiling
Forensics - to show guilt or innocence Establishing paternity Identification of human remains Species identification
– Evidence for sale of products from endangered species
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2. Recombinant DNA technology/ Genetically Modified organisms
– Recombinant DNA is formed by joining DNA sequences from two different sources:
1. .2. .
– Bacterial Plasmids (small, circular DNA molecules independent of the bacterial chromosome) are often used as vectors
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Recombinant cells and organisms can mass-produce gene products
– Common prokaryotic host: E. coli bacterium
– Has many advantages in gene transfer, cell growth, and quantity of protein production
– Common eukaryotic hosts:– Yeast: S. cerevisiae– “Pharm” animals
– Will secrete gene product in milk
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Advantages of recombinant DNA products
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Genetically modified (GM)
Transgenic organisms contain at least one gene from another species
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Agrobacterium tumefaciens
DNA containinggene for desired trait
Tiplasmid Insertion of gene
into plasmid
RecombinantTi plasmid
1
Restriction site
Plant cell
Introductioninto plantcells
2
DNA carrying new gene
Regenerationof plant
3
Plant with new trait
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Pros?
GM plants
GM animals
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Cons?
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3. Gene therapy
One possible procedure: – insert functional gene into a virus– virus delivers the gene to an affected cell– Viral DNA & gene insert into the patient’s
chromosome – Return the cells to the patient for growth
and division
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Insert normal geneinto virus
1
Viral nucleic acid
Retrovirus
Infect bone marrowcell with virus
2
Viral DNA insertsinto chromosome
3
Inject cellsinto patient
4
Bone marrowcell from patient
Bonemarrow
Cloned gene(normal allele)
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4. Genomics
Genomics =
Applications:– Evolutionary relationships: Genomic studies
showed a 96% similarity in DNA sequences between chimpanzees and humans
– Medical advancement: Functions of human disease-causing genes have been determined by comparisons to similar genes in yeast
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Human Genome Project
Goals:– To determine the nucleotide sequence all
DNA in the human genome – To identify the location and sequence of
every human gene
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Results of the Human Genome Project
– 21,000 genes in 3.2 billion nucleotide pairs– Only 1.5% of the DNA codes for proteins– The remaining 88.5% of the DNA contains
– Control regions (promoters, enhancers)– Unique noncoding DNA– Repetitive DNA
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RepetitiveDNA thatincludestransposableelementsand relatedsequences(44%)
RepetitiveDNA unrelated totransposableelements(15%)
UniquenoncodingDNA (15%)
Introns andregulatorysequences(24%)
Exons (regions of genes coding for proteinor giving rise to rRNA or tRNA) (1.5%)