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
3
mt inheritance Extranuclear = non-Mendelian = maternal =
uniparental inheritance
All progeny have phenotype of mother with respect to mt genes Why? sperm have little cytoplasm
4
mt mutations Neurospora
[Poky] mutation (1952)
Poky strains grow slowly for days, then growth rate accelerates reaching wild type rate after ~3 days.
5
Human mt mutations
Leber’s hereditary neuropathy
Adult optic nerve degeneration blindness Electron transport chain protein mutations
6
DNA sequencing gel – locate the mutation responsible for LHON
7
mt inheritance pedigree
8
Normal mitochondria with normal DNA
Mitochondria with mutant DNA
Heteroplasmy – mixed population of mitochondria in a cell 9
Effects of heteroplasmy on egg/offspring
10
Bacterial Genomics
Chapter 15
First sequenced Haemophilus influenzae
11
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
12
E. coli biology Prokaryote nucleoid region contains the chromosome
Neisseria gonorrhoeae.
13
E. coli reproduction Binary fission -> Exponential
growth
~ 3 microns
14
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
16
Bacterial growth curve
17
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?
18
2. count colonies ~ 400
3. 100 ul plated yielded 400 colonies = 4 X 10 3 cfu /ml
19
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
20
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
21
Solid and liquid culture
Growth in liquid media
Growth on agar plate
22
Phenotypes Prototroph
synthesize requirements from minimal media
Auxotroph
nutritional mutant Requires one or more supplements to grow
23
Resistant to ampicillin = Ampr
Sensitivity to streptomycin = Strs auxotroph mutant requires tryptophan = Trp-
trp-leu-thi+tetr Wildtype = +
24
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
25
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?
26
How do bacteria undergo genetic recombination?
27
Noble Prize for bacterial genetics Lederberg, Beadle and Tatum 1946> Nobel 1958 Nutritional mutants in E. coli
28
1. Conjugation parasexual mating
one-way transfer of genetic information from “male” to “female” bacteria
29
Plasmid DNA from donor to recipient bacterium Plasmid
circular, episomally maintained DNA F factor plasmid Encodes F pilus
1953 Hayes
30
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)
31
F pilus
Recepient F+
Donor F-
Pilus cannot form between 2 F+ cells
32
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
35
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
36
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
39
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
40
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
41
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.
42
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.
45
Hfr strains led to mapping of the E. coli
chromosome Interrupted mating technique to map genes
on E. coli
46
Lederberg’s experiment explained
47
Circular chromosome
4.6 million bp (4.6 Mb)
48
2. Transformation Naked DNA enters bacterial cell. Brings new
genes (can change bacteria phenotype) Bacterium with new DNA is a transformant
49
Transformation (rare event) Natural Engineered
CaCl2 treat bacteria competent cells
cell membrane permeable to naked DNA
50
Plasmids can be cloning vectors Ch 8 pg 175 pUC19
ampr gene ori restriction sites (multiple cloning site)
51
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)
52
Transduction –phage mediated transfer of genes into bacteria Bacteriophage – virus that infects bacteria
Lederberg and Zinder 1952
53
phage DNA or RNA surrounded by protein coat
genes encode for viral activity, viral parts
54
Viral infection lytic cycle 1. Virus adsorbs to cell and injects DNA
55
2. normal bacterial activity is shut down and bacterium becomes a “phage factory”
56
3. host DNA broken into pieces, new viruses released to infect new cells
57
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
58
59
Bacteriophage phenotypes virulent phage - always lytic, cannot
become a prophage temperate phage - lysogenic
60
Temperate phage and lysogenic pathway Phage DNA integrated
into specific location in chromosome Prophage is lysogenic Phage gene represses
lytic cycle
61
Gene therapy with virus (Ch 10) Objective : insert normal gene into human DNA
Candidates: people with single gene disorders Use virus as vector
62
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
65
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
66
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
67
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
69
LacZ gene is only transcribed when lactose sugar is present
b- gal is an inducible enzyme
70
DNA ->
Proteins ->
promoter = regulates transcription of ZYA operator = must be unbound for P to be “open”
71
REPRESSOR PROTEIN (I) Encoded by
Lac I gene
Binds to operator Prevents RNA pol from binding to promoter
72
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
74
• Lactose (the inducer) enters the cell Binds repressor protein causing a conformational change
75
No lactose: repressor binds to operator polymerase cannot bind promoter no transcription of ZYA genes
76
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 … ?
78
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
79
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
80
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