Ch 13. Mitochondrial and chloroplast DNA, Extranuclear inheritance

Ch 13. Mitochondrial and chloroplast DNA, Extranuclear inheritance 1 mt genome  Circular (most)  Double stranded  16,500 bp  37 genes  Have o...
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Ch 13. Mitochondrial and chloroplast DNA, Extranuclear inheritance

1

mt genome  Circular (most)

 Double stranded  16,500 bp  37 genes  Have own ribosomes!  Other proteins required for cellular respiration

and mt replication are encoded by nuclear genes and imported into the mt 2

Evolution of mt and chloroplasts = Endosymbiont Theory Box 23.1  primitive bacteria formed symbiotic

relationship with early eukaryotic cells  gradual transfer of mt genes to nucleus  mt genes similar to prokaryotic genes

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mt inheritance  Extranuclear = non-Mendelian = maternal =

uniparental inheritance  

All progeny have phenotype of mother with respect to mt genes Why?  sperm have little cytoplasm

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mt mutations  Neurospora 

[Poky] mutation (1952)



Poky strains grow slowly for days, then growth rate accelerates reaching wild type rate after ~3 days.

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 Human mt mutations 

Leber’s hereditary neuropathy  

Adult optic nerve degeneration  blindness Electron transport chain protein mutations

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DNA sequencing gel – locate the mutation responsible for LHON

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 mt inheritance pedigree

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Normal mitochondria with normal DNA

Mitochondria with mutant DNA

Heteroplasmy – mixed population of mitochondria in a cell 9

Effects of heteroplasmy on egg/offspring

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Bacterial Genomics

Chapter 15

First sequenced Haemophilus influenzae

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Escherichia coli (1997)  Lower intestines of animals

 Pathogenic strains (ex. E. coli 0157)  Genome 

 

4.6 million bp (4.6 megabases) ~ 4000 genes, ~88 % of genome open reading frames Single circular chromosome

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E. coli biology  Prokaryote  nucleoid region contains the chromosome

Neisseria gonorrhoeae.

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E. coli reproduction  Binary fission -> Exponential

growth

~ 3 microns

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Bacterial growth  colony - visible cluster of clones  about 1 million cells /colony  lawn – entire plate covered, no

individual colonies

Growth on agar plate 15

Growth of bacteria (E. coli)  Lag phase - slow or no apparent growth

 Log phase –double every 20’ to 1 X 109/ml  Stationary phase

nutrient and/or oxygen limited  Cell number remains constant  Death phase 



Nutrients gone, toxic products build up, cells die

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Bacterial growth curve

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How to determine the titer of bacteria Titer = number of colonies (cfu) per ml liquid culture 1. plate 100 ul of culture on an agar plate – why 100 ul?

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2. count colonies ~ 400

3. 100 ul plated yielded 400 colonies = 4 X 10 3 cfu /ml

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If there are too many colonies to count, then the original culture must be diluted before plating. Dilute culture 1 :100. Plate 200 ul. Observe 120 colonies. Titer (cfu/ml) ?

1000 ul

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Growth media  minimal media =essentials  

Sugar (carbon source) + salts bacteria synthesize aa, nucleotides, vitamins

 complete media  selective media 

Allows one species to grow while selecting against another

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Solid and liquid culture

Growth in liquid media

Growth on agar plate

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Phenotypes  Prototroph 

synthesize requirements from minimal media

 Auxotroph 



nutritional mutant Requires one or more supplements to grow

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 Resistant to ampicillin = Ampr

 Sensitivity to streptomycin = Strs  auxotroph mutant requires tryptophan = Trp-

trp-leu-thi+tetr Wildtype = +

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Bacterial mutants  Nutritional mutants 

Auxotrophs that require supplement to grow

 Conditional mutants 

The mutation is only expressed in a certain condition

 Resistance mutant 

Antibiotic resistance in bacteria

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quorum sensing  http://www.pbs.org/wgbh/nova/sciencenow/34

01/04.html  What are Vibrio harveyi?  What happens when V. harveyi gather to form a

“quorum”?  What is bioluminescence?  What is meant by bacteria “talking” to each other?

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How do bacteria undergo genetic recombination?

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Noble Prize for bacterial genetics  Lederberg, Beadle and Tatum 1946> Nobel 1958  Nutritional mutants in E. coli

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1. Conjugation  parasexual mating 

one-way transfer of genetic information from “male” to “female” bacteria

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Plasmid  DNA from donor to recipient bacterium  Plasmid

circular, episomally maintained DNA  F factor plasmid  Encodes F pilus 

1953 Hayes

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F+ cell

F- cell

94,000 bp origin of replication (ori)

Pili cannot attach to other donor cells due to the presence of the proteins coded by the traS and traT genes -- this is called surface exclusion)

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F pilus

Recepient F+

Donor F-

Pilus cannot form between 2 F+ cells

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Conjugation F+

1. 2. 3. 4.

+

F-

=

2F+

Pilus attaches to receipient cell 2. nick DNA -> transfer DNA DNA polymerase makes dsDNA break pilus 33

Note: no chromosomal DNA transferred

Lederberg and Tatum experiment Mix 2 auxotrophs  grow in minimal media Strain A met- bio- thr+ leu+ Strain B met+ bio+ thr- leuOBTAIN --->

a few prototrophs form!

What would the genotype of this prototroph be? 34

rare 1 /10,000,000

Genetic recombination has occurred

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Lederberg and Tatum experiment Davis U-tube conjugation requires cell/cell contact Fig. 15.3

met- bio-

filter

thr- leu-

Filter prevents cell contact

Media can pass  no prototrophs obtained Show that cell-cell contact is required

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Rarely, the plasmid integrates into the bacterial chromosome = Recombination

What happens when an Hfr strain conjugates? 37

• The first DNA to be transferred is the chromosomal DNA ! • Pilus is broken before F factor is transferred • Recipient cell remains F38

Hayes and Cavalli-Sforza

recombination

The transferred DNA MAY undergo homologous recombination

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Why do we care about homologous recombination?  It is a universal biological mechanism

 Bacteria can pick up new traits  Biotechnology  Gene knockouts in mice via homologous

recombination

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 DNA of interest in mouse chromosome

This is the gene targeted for replacement by an engineered construct. Note flanking upstream and downstream DNA sequences. The arrows pointing away from the targeted gene represent the continuous chromosomal DNA

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 1. Prepare construct DNA in lab with

selectable marker

Diagram of engineered construct used to replace the DNA. The upstream and downstream flanking DNA sequences are identical to those which flank the targeted locus.

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 2. Add construct to embryo cells in culture

DNA just prior to homolgous recombination. Amazingly, the DNA construct finds its way into the nucleus and then aligns itself with the targeted locus. The mechanism that performs this alignment is poorly understood but it does work better in some species than others. 43

 3. homologous recombination by cell

The final products of homologous recombination. The chromosome now contains a portion of the flanking DNA as well as all of the engineered construct which has taken the place of the original allele. The original allele has been recombined into the construct and thus is lost over time. From this point on, the cell will replicate the engineered construct as faithfully as any other portion of the chromosome. 44

Summary  homologous sequence of construct flanks existing

gene's DNA sequence upstream and downstream of the gene's location on the chromosome.  cell's nuclear machinery recognizes identical stretches of sequence and swaps out existing gene or portion of gene with constructDNA.  construct DNA is inactive, the swap eliminates, or "knocks out," the function of the existing gene.

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 Hfr strains led to mapping of the E. coli

chromosome  Interrupted mating technique to map genes

on E. coli

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Lederberg’s experiment explained

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Circular chromosome

4.6 million bp (4.6 Mb)

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2. Transformation  Naked DNA enters bacterial cell. Brings new

genes (can change bacteria phenotype)  Bacterium with new DNA is a transformant

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Transformation (rare event)  Natural  Engineered 

CaCl2 treat bacteria  competent cells 

cell membrane permeable to naked DNA

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Plasmids can be cloning vectors Ch 8 pg 175  pUC19

ampr gene ori restriction sites (multiple cloning site)

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Plasmid requirements in biotech 1. Ori for replication

2. Selectable marker ex. ampr 1.

Only cells that take up the plasmid are resistant to amp

3. Restriction enzyme sites to attach foreign DNA 4. High copy number in E. coli (100/cell)

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Transduction –phage mediated transfer of genes into bacteria  Bacteriophage – virus that infects bacteria



Lederberg and Zinder 1952

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phage  DNA or RNA surrounded by protein coat

 genes encode for viral activity, viral parts

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Viral infection lytic cycle 1. Virus adsorbs to cell and injects DNA

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2. normal bacterial activity is shut down and bacterium becomes a “phage factory”

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3. host DNA broken into pieces, new viruses released to infect new cells

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Generalized transduction  A piece of chromosomal DNA gets packaged

into a virus = faulty head stuffing  This transducing phage infects a new cell

and transfers genes from the first bacterium  Homologous recombination occurs

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Bacteriophage phenotypes  virulent phage - always lytic, cannot

become a prophage  temperate phage - lysogenic

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Temperate phage and lysogenic pathway  Phage DNA integrated

into specific location in chromosome  Prophage is lysogenic  Phage gene represses

lytic cycle

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Gene therapy with virus (Ch 10)  Objective : insert normal gene into human DNA

 Candidates: people with single gene disorders  Use virus as vector

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Gene Therapy ADA 1990  Gene for adenosine deaminase  

ADA normally eliminates deoxyadenosine from degraded DNA Deoxyadenosine toxic to lymphocytes

  Severe immune deficiency

20q12-q13.11 63

Ashanti Disilva was 4 and dying 1. remove viral replication genes from virus 2. insert normal ADA gene in virus 3. remove wbc from patient 4. infect wbc in lab with engineered virus 5. infuse into patient 6. repeat every few months 64

The Lac Operon 1961, Jacob and Monod E. coli and other bacteria  Bacterial Genes 

Many genes constitutively expressed 



“housekeeping” genes

Other genes regulated 

Can be turned on, or off depending on cell needs

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Operon  group of coordinately regulated genes  One promoter for a number of genes  Polycistronic mRNA  1 mRNA molecule has info from multiple genes  Inducer molecule – turns operon on

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E. Coli

Lac Operon

 E. coli cells can convert lactose to glucose and

galactose  Betagalactosidase enzyme  

Lac Z gene turned off when cell grown in glucose 1000X increase in enzyme when cell grown in lactose

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The Lac Operon allows for coordinate gene expression

Note: 1 mRNA, promoter 68

3 STUCTURAL GENES = Z, Y, A Lac Z

gene encodes b-galactosidase enzyme

b-gal lactose ------------- glucose + galactose substrate products

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LacZ gene is only transcribed when lactose sugar is present 

b- gal is an inducible enzyme

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DNA ->

Proteins ->

promoter = regulates transcription of ZYA operator = must be unbound for P to be “open”

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REPRESSOR PROTEIN (I)  Encoded by

Lac I gene

 Binds to operator  Prevents RNA pol from binding to promoter

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Is this operon ON or OFF? Is lactose PRESENT or ABSENT?

Lac I, P, O, ZYA genes are CIS elements 73

INDUCER (LACTOSE SUGAR)  LACTOSE PRESENT • •

• • •

Lactose enters Binds repressor protein causing conformational change This pulls repressor off operator RNA polymerase transcribes genes Cell metabolizes lactose

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• Lactose (the inducer) enters the cell Binds repressor protein causing a conformational change

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No lactose: repressor binds to operator polymerase cannot bind promoter no transcription of ZYA genes

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 NO LACTOSE

Lac operon animation 77

Operon mutants Mutant

Mutant Phenotype

lac I-

constitutive expression because…

Oc

constitutive expression because …

Plac Z-

no expression of operon because … ?

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Partial diploid cells contain a plasmid (F factor) F’ I+ I-P+O+Z+Y+A+

F’

I- P- O+ Z+Y-AI+P+O+Z- Y+A+

Inducible? (yes)

no

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 Remember, repressor and polymerase are

proteins which are diffusible  

proteins bind DNA act in TRANS

 promoter, operator, and ZYA and I are genes

and cannot move 

act in CIS

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DNA technology Ch. 10 Human Growth Hormone

(hGH) cloned into bacteria (1980s)  Pituitary dwarfism mutation in hGH

gene  hGH protein is a 191 aa peptide produced by pituitary gland  Pre-1985 hGH from cadaver brains Drawbacks?  Today 

hGH cloned

26 inches tall

Cloning steps:  1. isolate hGH mRNA

from normal human  2. reverse transcribe to cDNA (no introns)  3. ligate hGH cDNA into plasmid vector

 4. transform bacteria  grow bacteria and they

express hGH  5. purify protein

Other cloned drugs made by bacteria Human insulin for diabetics Factor VIII for hemophiliacs Hepatitis B vaccine

bGH to increase milk yield in cows

Advantages of rbacteria/drugs  Clean

 Worlds supply in one lab  Cheap ?

Cloning into plants (GM) pg 282  Transgenic plants 

Plants acquire a new genetic trait by direct introduction of gene



We have been modifying plant genes by breeding for 1000’s of years

Getting a gene into a plant nucleus A natural system:

Agrobacterium tumefaciens bacteria infects plants  crown gall disease (tumor) at wound sites

Agrobacterium tumefaciens cells attached to a plant cell. From Genome News Network and Martha Hawes.

Agrobacterium with plasmid Ti (tumor inducing) Note T DNA region

Infection stimulates excision of 30 kb region of Ti called T-DNA  insertion into chromosome Ti plasmid is 200kb

ssT-DNA ~20kb excised

tumor

 Why is this referred to as horizontal gene

transfer?  How could this be used to introduce engineered genes into a plant cell?

1. Engineer Ti plasmid  

Remove tumor inducing genes Include  Excision, transfer, and insertion sequences  Gene of interest

 2. transform agrobacterium with Ti plasmid

 3. wound plant and infect with agrobacterium

 4. gene of interest transferred to plant cell

 5. grow explant into a plant  = transgenic plant

Benefits

Drawbacks

 Increased crop yield  Resistance to drought, freezing

 Decreased use of pesticides  Decreased use of herbicides  Increased nutrition  Increased shelf life

 Can remove allergens

insects?

increased seed costs pesticide resistant bugs resistant weeds new allergens may spread to other plants harmful to

GM foods: Bt corn Ch 10 pg 284  Corn plant engineered with gene that

codes for a Bt protein lethal to the corn borer  Bt protein normally made by bacteria

Golden Rice

hits market 2011 not in textbook

 Vitamin A deficiency 

Leading cause of childhood blindness (500,000 new cases /year)

 Engineer rice to produce genes needed for

carotene in endosperm 

(phytoene synthase and phytoene desaturase)



Tissue specific expression vector

 Do we need legislation for labeling of GM foods?  Should GM genes, plants, animals, be

patented?

Cloning genes into animals  A transgenic animal carries a foreign gene

deliberately inserted into its genome.

Transgenic goats Ch. 9 Produce human protein (drug) in milk

Pharming

Transgenic animals to produce human protein in milk Isolate human EPO gene 2. Ligate to tissue-specific promoter 1.



Promoter ONLY active in mammary gland  protein only made in milk

microinjection 1. Inject gene construct into animal fertilized egg, it integrates into chromosome

2. Implant embryo into surrogate mother -> kid is born  How do we know if kid

is transgenic (has human EPO gene in its DNA/every cell) ?

Probed gel of goat kid DNA

3. How can we get the transgenic kid to produce human drug? Only active in mammary tissue

4. purify drug (protein) from milk • One herd can supply the world’s need • Clean, disease free

Pail of milk with EPO

Bottled EPO drug

Other proteins made in transgenic sheep and goat milk • Spider silk (BioSteel) – The dragline form of spider silk is regarded as the strongest material known; 5 times stronger than steel and twice as strong as Kevlar.

genus Araneus

Mouse model organism  These mice are

models for human disease (Alzheimer)

 This mouse is

genetically modified to be diabetic

Knockout mice  Normal gene (in embryo) has been replaced

with non-functional gene

Agriculture  This pig is genetically engineered to be able

to digest more and produce less manure

 Other pigs produce meat high in omega 3

fatty acids

Xenotransplantation  Pigs have similar sized organs to humans

 Knock out pig cell surface antigens

to prevent hyperacute rejections

100,000 in US await organ transplantation - ~ 20,000 will get organs

Fish farming  genetically engineered salmon grow faster

More than 99% of the salmon are triploid (sterile), fish farmed inland, in tanks fitted with filters to imprison eggs and fish

AquaAdvantage salmon

Patenting  Raw products of nature are not patentable.

 DNA products become patentable when they

have been isolated, purified, or modified to produce a unique form not found in nature.  Millions of patents  Can patent a gene, a method, an animal etc..

3 types of cloning  1.

gene cloning Recombinant bacteria (as in lab)  Transgenic plants  Transgenic animals 

 2. reproductive cloning  Yields an organism  Embryo twinning or nuclear transfer  3. therapeutic cloning  nuclear transfer for stem cells to treat disease

Reproductive cloning Embryo twinning 

1 sperm + 1 egg - 2 embryos (genetically identical)

 http://learn.genetics.utah.edu/units/cloning/wh

atiscloning/

Nuclear transfer method - The clone’s DNA is a genetic copy of the donor

SCNT = somatic cell nuclear transfer

1997 Ian Wilmut

http://learn.genetics.utah.edu/units/cloning/



Obtain somatic cell from donor ewe



Place nucleus into enucleate egg

 

Grow embryo for 6 days in lab Implant into surrogate mother

277 embryos -> 1 lamb (Dolly)

Our somatic nuclei (DNA from a differentiated cell) can be reprogrammed to embryonic state

Why clone animals?  Models for disease

 Pharming  Endangered species – ex. Mouflon sheep,

the surrogate mother was a domestic sheep  Reproduce deceased pet  Help infertile couples? Cloning game http://learn.genetics.utah.edu/units/cloning/cloni ngornot/

Problems with reproductive cloning

 High failure rate < 3% success rate  Enucleate egg may not function  Embryo may not divide  Embryo may not implant  Miscarriage

 Large offspring syndrome (LOS)  With abnormally large organs that don’t function correctly  Abnormal gene expression  We don’t understand how the nucleus is reprogrammed (its old DNA in a new egg!)  Telomere problems  Older DNA has shortened telomeres, but some clones show lengthened telomeres

All countries have banned human reproductive cloning.

Therapeutic cloning  Obtain embryonic stem

(ES) cells 

1. Isolate nucleus from a

somatic cell – which? 2. Remove egg nucleus from donated egg

How many chromosomes in nucleus of somatic cell?

 Somatic cell nuclear transfer

3. inject somatic cell nucleus into enucleate egg 4. Grow to blastocyst stage

3 day embryo (morula)

5 day blastocyst

Cells at this stage are undiffferentiated

Blastocyst ~ 100 cells, day 4 Hollow ball of cells with inner cell mass

ICM -> embryo

5. Take inner cell mass, transfer to flask, and ES cells reproduce.

~100 cells

How do we get the cells to differentiate into what we want?

Stem cells

Questions  Sperm?

 Fertilization?  Embryo?

Types of stem cells  Totipotent stem cells (ES) can differentiate into

any cell type including placenta 

Example: early embryo

 Pluripotent stem cells (ES) - 5 day embryo  blastocyst can differentiate into any body cell type

 Multipotent stem cells give rise to a number

of cell types 

example: stem cells in bone marrow

Sources of stem cells  Therapeutic cloning (SCNT)  Advantage = no immune rejection  Not dependent on transplant from another person  Left over in vitro fertilization embryos  Donated sperm and eggs  Umbilical cord blood, placental blood, bone marrow

Therapeutic cloning is not reproductive cloning ES cells/embryo

Therapeutic cloning Reproductive cloning -> Implant into female (uterus)->- birth ILLEGAL, rarely successful in animals

Cells divide to produce more ES cells

Use to treat /cure disease

Uses of ES cells 1. tissue transplants – new liver cells, pancreas cells

2. Replace lost cells: Alzheimer disease, spinal cord injury, Parkinson’s disease, multiple sclerosis, diabetes, burned tissue, stroke, lung disease, heart disease, arthritis NOTE – ES cells cannot develop into a fetus – why?

Libraries Ch 10 

How to find a gene to clone  

If sequence is known  PCR If sequence is not known  library

 Genomic library = Collection of clones that contain entire genome  Need > 50,000 bacterial clones to hold the entire human genome

Each colony contains different fragment of DNA fragments unordered

Need many plates

Caveats Restriction enzymes may cut within genes 2. Need a lot of rbacteria to represent entire genome 1.

 cDNA library  



Isolate mRNA cDNA Coding regions only Tissue specific

Tissue specific expression

Alcohol dehydrogenase

Lane 1 RNA marker Lane 2 total RNA (Liver) Lane 3 Brain Lane 4 Cerebellum Lane 5 Cerebrum Lane 6 Kidney Lane 7 Liver Lane 8 Lung Lane 9 Spleen Lane 10 Thymus Lane 11 Testis

Northern blot to assay mRNA levels in various tissues144

Chromosome specific library

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