Ch. 11: Gene regulationsHow is cloning possible?

Every cell has the same chromosomes

Then….. 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?

Differentiation

Differentiation is controlled by turning specific sets of genes on or off

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

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

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

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 X

Active X

Eukaryotic gene expression

– Each gene has its own promoter and terminator

– Are controlled by interactions between numerous regulatory proteins and control sequences

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

Enhancers

Otherproteins

DNA

Transcriptionfactors

Activatorproteins

RNA polymerase

Promoter

Gene

Bendingof DNA

Transcription

Alternative RNA splicing

– Can involve removal of an exon with the introns on either side

Animation: RNA Processing

1

or

Exons

DNA

RNA splicing

RNAtranscript

mRNA

2 3 4 5

1 2 3 4 5

1 2 4 51 2 3 5

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

miRNA 1

Translation blockedORmRNA degraded

Target mRNA

Protein

miRNA-proteincomplex

2

3 4

Control of gene expression also occurs with

– Breakdown of mRNA

– Initiation of translation

– Protein activation

– Protein breakdown

Folding ofpolypeptide andformation ofS—S linkages

Initial polypeptide(inactive)

Folded polypeptide(inactive)

Active formof insulin

Cleavage

Ex. Insulin formation

Epigenetic Inheritance

This can be accomplished by acetylation or methylation of histones

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

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

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

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

Signaling cell

DNA

Nucleus

Transcriptionfactor(activated)

Signaling molecule Plasma

membraneReceptorprotein

Relayproteins

TranscriptionmRNA

Newprotein

Translation

Target cell

2

1

3

4

5

6

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.

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

To clone or not to clone….

Benefits of reproductive cloning?

Disadvantages of cloning?

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

Ch 12: DNA Technology

1. DNA profiling

2. Genetically modified organisms/recombinant DNA technology

3. Gene therapy

4. Genomics

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

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

STR site 1

Crime scene DNA

STR site 2

Suspect’s DNA

Number of short tandemrepeats match

Number of short tandemrepeats do not match

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

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

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

Mixture of DNAfragments ofdifferent sizes

Completed gel

Longer(slower)molecules

Gel

Powersource

Shorter(faster)molecules

Crime sceneDNA isolated1

Suspect 1 Suspect 2

DNA of selectedmarkers amplified

2

Amplified DNA compared

3

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

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

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

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

Advantages of recombinant DNA products

Genetically modified (GM)

Transgenic organisms contain at least one gene from another species

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

Pros?

GM plants

GM animals

Cons?

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

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)

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

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

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

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%)