1
Q
Evolution
A
GENETIC changes in the composition of a population including:
- emergence of species
- divergence of species
- extinction of species
Involves variation, heredity, and selection:
If variation is not heritable, then it cannot be passed to progeny
-Study diversity that exists in a population and between populations and the factors that can cause diversity.
2
Q
Selection
A
works on entire organisms phenotype so many loci as well as environmental factors are important
3
Q
Genetic change in populations
A
2 step process:
Changes occurs (must be genetic)
- eg. mutation causes new alleles
- eg. recombination causes new combinations of alleles
Then different alleles (or combinations) must increase or decrease in frequency in the gene pool (selection and other factors)
4
Q
What are some factors effecting diversity between populations
A
Genetic Drift Migration Mutations Selection Inbreeding Natural Selection Recombination
5
Q
Genetic drift
A
results in divergence as some populations become fixed for each allelic form
-decreases diversity within populations as alleles are fixed and lost
6
Q
Migration
A
tends to equalize population
7
Q
Selection
A
can cause divergence between populations if different alleles are favored in different populations
8
Q
What does different mutations in different populations allow?
A
populations to diverge
9
Q
Migration and mutation
A
introduce variability within populations by introducing new alleles
10
Q
Inbreeding
A
increases homozygous types with decrease of heterozygous types
11
Q
Natural selection
A
can increase or decrease variability within population depending on type of selection
12
Q
Recombination
A
increases variability
13
Q
Species
A
a group of individuals that actually or potentially interbreed in nature
14
Q
Reproductive isolation
A
species become distinct when they no longer exchange genes
-new species arise
15
Q
Reproductive isolation can occur because:
A
- They don’t choose to mate with each other or cannot mate with each other (PREZYGOTIC)
- Or their progeny are sterile or inviable (POSTZYGOTIC)
16
Q
Biological species concept:
A
members of a species are capable of inter-mating and producing fertile progeny
17
Q
Prezygotic mechanism
A
- mechanism before a zygote has formed
- prevents games from 2 different species from fusing and forming a hybrid zygote
18
Q
Ecological mechanism
A
Prezygotic:
- Differences in habitat; individuals do not meet thus do not reproduce with one another
- different ecological niches
19
Q
Behavioral Mechanism
A
Prezygotic:
differences in mating behavior prevent mating
20
Q
Temporal mechanism
A
Prezygotic:
-reproduction takes place at different times of the year
21
Q
Mechanical Mechanism
A
Prezygotic:
Anatomical differences prevent copulation
22
Q
Gametic Mechanism
A
Prezygotic:
mating between individuals of different species takes place, but the gamete do not form zygotes
-Gametes incompatible or not attracted to each other
23
Q
Postzygotic mechanism
A
mechanism after zygotes have formed
-gametes of two species fuse and form a zygote, but there is not gene flow between the two species, either because the resulting hybrids are inviable or sterile or reproduction breaks down subsequent generations
24
Q
Hybrid Inviability
A
Postzygotic:
- hybrid zygote does not survive to reproduction
- incompatibility between genomes of the two species preventing the hybrid zygote from developing
25
Hybrid Sterility
Postzygotic
- hybrid is sterile
- hybrid embryos compute development but are sterile, so that genes are not passed between species
26
Hybrid Breakdown
Postzygotic
- F1 hybrids are viable and fertile, but F2 are inviable or sterile
- closely related spices are capable of mating and producing viable and fertile F1 progeny. However genes do not flow between the few species because of HYBRID BREAKDOWN, in which further crossing of the hybrids produces inviable or sterile offspring
27
Allopatric speciation
geographic barrier initiates speciation by blocking gene flow
28
Darwins Finches
LOOK IN BOOK
29
Sympatric speciation
arises within a single interbreeding population without geographical barriers to gene flow
30
Races of apple maggot fly
ex of sympatric speciation:
- where resource use is linked to mating preference
- flies feed on fruits of a specific host tree and mating occurs near the fruits so that larvae can grow up on the ripening fruit
31
Another mechanism for sympatric speciaiton?
hybridization that leads to allopolyploidy
32
Anagenesis
evolution within a lineage overtime
33
Cladogenesis
splitting of one lineage into two
- once cladogenesis occurs, the branches evolve separately from each other
- leads to biological diversity since most species exist at the same time
34
Diversity
that different alleles are present at a locus in a population
35
Classical hypothesis
How much heterozygosity is good?
Organisms need low levels of heterozygosity so that they will be well adapted to their environment. Selection favors genotypes that are well-adapted to a specific environment so each organism in a specific environment should had the favorable genotype and there should be little variation in the population.
36
Balance Hypothesis
How much heterozygosity is good?
organisms need high levels of heterozygosity so that they will have the necessary variability to respond to changes in the environment. A successful population would have lots of variability so that it can produce a variety of phenotypes and can allow the population to adapt to a changing environment. Therefore there should be a lot of variability in the population.
37
Neutralist Theory
Why does variation exist?
Many mutations are neutral. This causes polymorphisms to occur in population. Polymorphisms are maintained in the population since neither form has an advantage and the mutant types are not affected by selection.
-eg. two proteins with slightly different amino acid sequences both have proper level of function
38
Selectionist Theory
Why does variation exist?
Many polymorphisms are maintained in the population due to selection. Believe that this will be observed more as we gain more information about the effects of amino acid substitutions.
-Two forms of a protein may allow for optimum performance over a range of cellular conditions.
39
Which theory is true more of the time, neutralist theory or selectionist theory?
Nearly Neutral Model
40
Phylogenetics
study of the relationship among species, individuals, or genes/alleles based on their characteristics
41
Ways to evaluate evolutionary divergence
Morphology
Chromosome Structure
Protein sequences
DNA sequences
42
Dobzhansky
Inversion patterns in D. pseudoobscura correlated with elevation
Different races in Sierra Nevada Mountains:
- morphology similar
- Gene order on chromo #3 differs with altitude
- Gene order differences can be explained by inversion
- REMEMBER INVERSION HETEROZYGOTE CROSSOVER PRODUCTS ARE OFTEN NOT VIABLE SO SELECTION WILL TEND TO SELECT FOR HOMOZYGOTES
43
Minimal Mutation Distance
minimum total of all necessary nucleotide changes for all amino acids in protein
44
How do you evaluate changes in DNA sequence
RFLPs
Microsatellites
DNA sequence
45
DNA sequence analysis
DIFFERENT PARTS OF THE GENOME EVOLVE AT DIFFERENT RATES
- 5' flanking promotor so some sequences will be important for transcription
- leader and trailer sequences are transcribed, but not translated but may contain signals for RNA processing and ribosome attachment
- introns removed
- pseudogenes- don't code for protein
46
Nonsynonymous nucleotide substitution vs synonymous
| dealing with amino acids
NON alter the amino acid
47
Rates of substitution
are lower in amino coding regions of eons but are much higher in nonfunctional DNA such as pseudogenes
48
Molecular Clock
Based on the assumption of constant mutation rate in the change of DNA sequence or amino acid sequence, the difference in sequence between present day organisms can be used to date past evolutionary events
49
Human Globin Genes
- Multi-gene family
| - Evolved by successive gene duplications
50
OTU
Operational Taxonomic Units
| OTU can be a species or a strain of a virus or even different alleles within a species
51
Phylogenetic Trees
are used to show degrees of similarity/relationships between OTUs
52
Unrooted vs Rooted Trees
rooted trees have a common ancestor
53
Terminal Nodes
?
54
Branches
?
55
Internal Nodes
?
56
Constructing Phylogenetic Tress using the parsimony approach
-infers phylogenetic relationships based on the minimum number of evolutionary changes in the sequence that must have taken place since the organisms had a common ancestor
57
Constructing phylogenetic trees using the distance approach
-computing differences to infer relationships on overall similarity of organisms, typically by using multiple phenotypic characteristics or gene sequences
58
Constructing phylogenetic trees using the maximum likelihood/ Bayesian approach
infers relationships based on which gives the maximum probability of obtaining the set of characteristics in the organisms
59
DNA sequence Alignment
Identification of HOMOLOGOUS genes and properly aligning their sequences is critical in determine an accurate tree
-typically performed by computers to minimize the number of evolutionary steps
60
UPGMA
Unweighted Pair Group Method with Arithmetic Mean
| -relatively simple method of constructing a phylogenetic tree based on computing differences in DNA sequences
61
Cancer cells
have uncontrolled cell division-alteration of cell cycle
| Ability to metastasize-spread to other locations
62
Checkpoints
G1 checkpoint
G2 checkpoint
M checkpoint
cdc mutations
63
G1 checkpoint
monitors for proper cell size and undamaged DNA
| 25% of cell cycle
64
G2 checkpoint
holds up cycle until replication and DNA repair are complete
| 25% of cell cycle
65
M checkpoint
proper spindle formation and atachment
66
Protein Kinases and Cyclins
```
Protein kinases (phospohrlate proteins)
Protein cyclins (Structural protein)
```
interact to guide progression through cell cycle
67
Cancer
- mass of tissue cells with unlimited potential to divide/grow and serving no useful function in body
- Error occurs in cell cycle in 1 cell and increases number of affected cells through mitosis
- more than 750,000 new cases per year
- second leading cause of death
CANCER IS GENETIC, BUT CANCER IS RARELY HERITABLE
68
Hyperplasia
uncontrolled cell division
| immortal and invasive (immortal in culture, but also invade deeper into surrounding tissues)
69
Anaplasia
Structure/function of cell is undifferentiated
| -Tumore has lost the differentiated state and are less similar in structure function and cell type
70
Metastasis
ability to move to and establish tumors at other sites in body
71
Tumor
distinct mass of abnormal cells that do not have normal controls on cell division. NOT EVERY TUMOR IS CANCEROUS
72
Benign
abnormal cells remain localized and do not invade surround tissue
73
Malignant
cancer cells invade surrounding tissue
74
Metastatic
cancer cells spread and establish secondary tumors in other sites in the body
75
Genetic causes of cancer
- single gene
- polygenic (more than one gene)
- chromosome aberratoin (Inversion, TRANSLOCATIONS)
- Mutations in somatic cell or in gamete producing cell. (if its in gamete producing cell then it ca be passed to next generation.
- Viruses
76
Enviromental agents of cancer
Carcinogens:
- can cause mutations
- can alter gene expression
EX: nicotine, radiation, certain types of plastics
77
Multi hit model of cancer
Most cancers are sporatic and influenced by environment (usually)
- siblings are rarely affected by the same cancer
- Populations that migrate to new regions tend to get cancer rates typical of that region indicating factors in the environment are very important, especially diet.
Cancers develop over time (usually)
- changes in cancer rates due to new environment (ex. smoking) tend to take decades
- incidense of cancer rises with age
This is consistent with a multi-hit model where cancer arises over time with multiple genetic changes
78
Pancreatic cancer
most deaths per new cases per year. More significant mortality rate.
79
What are the two types of normal genes that can "go bad" and cause tumor or cancer development
Tumor supressor genes prevent bad cells from dividing. Work cell cycle checkpoints and stall the cell to make sure everything is ok, if ok then allow the cell to continue in the cell cycle
Proto-oncogenes allow good cells to divide.
- Oncogene-when it isn't work
- Protogene- good form
80
Tumor Suppressor Genes
Recessive action, have to nock out both to lose function of this gene.
- normal gene prevents uncontrolled growth
- abnormal gene- no inhibition- results in tumor if no normal allele present
- must disrupt both copies of the gene to lose cell cyle regulation (recessive action)
81
Retinoblastma- RB Gene
Inherited Retinoblastoma:
- An individual inherits an inactivating mutation in one of its RB genes.
- The other RB gene is inactivated by a somatic mutation during eye development
Sporadic Retinoblastoma
- An individual inherits two active RB genes
- Both of the RB genes are inactivated by Somatic mutations during eye development
---40% of cases are inherited (1 bad gene present in zygote)
--Normal protein responsible for regulation at G1 checkpoint
82
Knudson's Two hit hypothesis
both copies have to be defective in same cell to allow tumor to develop
RB gene
83
BRCA1 and BRCA2
These 2 genes thought to account for about 10% of breast cancers
- BRCA1-associated with half of hereditary breast cancers
- 90% of women with mutation in BRCA1 get breast cancer
Strong family history of ovarian cancer may indicate a mutation in one of these genes
Men with these mutations:
- Have an increased risk of breast cancer (esp. BRCA2)
- Have increase risk of prostate cancer
Testing by Myriad Genetics:
- Cost 300-3000$
- Often covered by insurance
Carriers of a BRCA1 or BRCA2 mutation are much more likely to get breast cancer and ovarian cancer than those who do not have one defective allele.
84
p53
- chromosome 17
- Tumor suppressor gene
- Functions at G1 checkpoint
- Mutated form seen in diverse cancer types: colon, lung, breast, brain, and is found in altered form in 50% of human tumors
- The fork in the road: if DNA is damaged, p53 delays cell division until damage is repaired or programs cell to die
- If p53 not working properly, cell division occur even though DNA is damaged and occurs in unregulated manner
85
p53 mechanism
?
86
Haploinsufficiency
When a diploid organism has the loss of function of one copy of a gene and is left with only one functional copy of a gene and this functional copy does not produce enough of the gene product to exhibit the wild-type phenotype.
Although, typically, both copies of a tumor suppressor gene have to be altered to result in cancer, there are some cases where a higher susceptibility to cancer occurs when only one copy of the normal allele is present.
Example: Bloom Syndrome
87
Bloom Syndrome
Due to a defective DNA helicase enzyme that is important in repairing double stranded breaks of DNA
Individuals homozygous for mutated BLM gene have a very high rate of cancer
Heterozygous individuals have an elevated risk for colorectal cancer
Similar pattern in mice: Those heterozygous for the BLM were more than twice as likely to develop intestinal tumors than those with two normal alleles
88
Oncogenes
PROTO-ONCOGENES: normally promote cell division, but must be activated to regulated properly
Mutation in proto-oncogene results in ONCOGENE that allows uncontrolled cell division
Only need mutation in 1 copy of a proto-oncogene to get tumor whereas in tumor-suppressor gene, both copies must be mutated to get tumor
89
Ras oncogene
Cellular Location of Product: Cell membrane
| Function of Proto-oncogene:GTP binding and GTPase
90
MYC oncogene
Cellular location of product: Nucleus
| Function of Proto-oncogene: Transcripton Factor
91
Burkitts Lymphoma
Abnormal function of B cells
tumor in the lymph nodes that affect the B cells, therefore can't produce antibodies
reciprocal transloction between chromosome 8 and 14 places c-myc (oncogene) next to enhancer. Thought that enhancer lead to abnormally high function of c-myc gene.
92
Chronic Myelogenous Leukemia
Fatal uncontrolled replication of myeloid stem cells
- 90% of patients have the philadelphia chromosome
- Reciprocal Translocation involving chromosomes 9 and 22 places 2 oncogenes near each other.
93
Bcr/c-abl fusion protein
Transcription and translation occurs forming the fusion protein. Thought that the protein affects cell cycle control and leads to cancer associated with white blood cells
94
Oncogenes vs Tumor-suppressor genes
Oncogenes: Dominant-acting mutation
| Tumor-suppressor genes: Recessive-acting
95
Palladin Gene
Pancreatic cancer is the 4th leading cause of cancer death even though only about 37,000 new cases occur each year.
- The paladin gene codes for a CYTOSKELETON PROTEIN that is important in maintaining cell shape. Cells that metastasize generally have poor cytoskeleton structure causing them to detach from the tumor mass easily
96
Most cancers are not due to one gene, BUT TO ACCUMUALATIONOS OF MUTations in several genes
!!
97
Colon cancer
- Tumor suppressor and oncogenes defective
- Progression: Benign adenomas to malignant tumors to metastasis
Results in very small polyps and larger polyps
98
Retroviruses
can cause cancer by :
- Mutating and Rearranging proto-oncogenes
- Inserting Strong promoter near proto-oncogenes
99
Clonal Evolution
Over time, tumor cells acquire more mutations that allow them to be progressively more aggressive in proliferation.
These mutations can affect:
- Cell cycle regulation
- Signal Transduction
- DNA Repair
- Telomere Length
- Chromosome segregation (many tumor cells are aneuploid)
- Vascularization
100
Signal Transduction:
external signal triggers a cascade of intracellular reactions to produce a specific response
101
Ras Signal-Transuction pathways
Stimulates the cell cyle
102
Defective Nucleotide Excision Repair
Xeroderma Pigmentosum
103
Defective Mismatch Repair
Colorectal, endometrial, stomach cancers
104
Role of Telomere Length in Cancer
- Typically Telomeres shorten as a cell ages and this ultimately contributes to the death of the cell
- Normally, telomerase works in germ line cells, but not in somatic cells: allows somatic cells to die, be replaced, etc.
- Tumor cells often have telomerase expression, which is thought to contribute to the "immortality" of cancer cells.
- Mutations in genes that regulate telomerase activity may be important in cancer, but role of telomerase is not clear at this time.
- Possible cancer therapy could be to block telomerase activity
105
Cancer cell kayotypes
abnormal
106
Angiogenesis
(growth of new blood vessels) is important to tumor progression
- Growth factors and other proteins involved in angiogenesis are often over expressed in tumor cells
- Angiogenesis inhibitors may be inactivated or under expressed
107
Is it possible to fight to cancer by preventing angiogenesis?
Metastasis is the cause of death in90% of human cancer cancers
108
Classic Cancer Model
- All tumor cells can from new tumors and are therefore equally tumorigenic
- Unregulated growth is due to a serial acquisition of genetic events leading to the expression of genes that promote cell proliferation while silencing the growth of inhibitory genes and blunting cell death
- Cancer is a proliferative disease
109
Cancer Stem Cell Theory
- Tumors arise from "cancer stem cells" (CSCs) that are SELF RENEWING and MULTIPOTENT
- CSCs persist in tumors and may be the cause of relapses and metastasis
- This might explain why many cancers are difficult to treat
110
Chemotherapy
may kill the bulk of tumor cells, but may not affect the CSCs, leading to a recurrence of the cancer
-Development of specific therapies targeting CSCs may provide hope for increased survival and quality of life especially for metastatic disease.
111
Restriction Enzyme
?
112
Palindrome
?
113
Joining TWO DNA molecules
1) digestion with HindIII (or other restriction enzyme) causing 5' and 3' sticky ends
2) Nick in sugar phosphate backbone joining the two different strands
4) Ligase to repair gaps
114
Cloning Vector
- Vector is a carrier DNA
- Molecule that is capable of independent replication into which a DNA fragment can be cloned. Its purpose is to carry foreign DNA into the cell
115
What does the ideal cloning vector contain?
1) origin of replication
2) Selectable/screenable markers
3) A single cleavage site for each restriction enzyme
116
Plasmid Vectors
can carry 100 bp- 10kb insert (usually 6-7 kb)
117
What does plasmid vectors contain?
need origin of replication
| need 2 marker genes
118
Lambda virus as a vector
- Lambda can be lytic or lysogenic: We want LYTIC since want to replicate inserted sequence
- DNA is made as a CONCATAMER (many copies joined together)
- It is cut to proper size at locations called "cos" sites. Cos sites are important in proper packaging of DNA into viral head
- DNA is packaged as a piece 45 kb long and size is critical in packaging.
- To use lambda as a vector, remove lysogenic genes (15kb) in center and ad 15 kb insert- size is critical!
- Virus containing recombinant molecule injects DNA into cell and can replicate
119
Cosmids
- Contains cos genes from lambda ask that it can be packaged into a viral coat and get into the bacterial cell like a virus
- Contains origin of replication for bacteria so that it replicates like a plasmid in bacterial cell
- polycloning site
- selectable marker (amp^r)
- Holds about 44kb insert (big piece)
120
Expression vectors
- allow the inserted gene product to be produced
- Must contain sequences required for transcription and translation of the gene in addition to other vector characteristics
121
Expression vectors contain:
-operon sequeces that allow inserted DNA to be transcribed and translated.
-Include sequences that regulate- turn on or turn off- the desired gene
-
122
Bacterial Artificial chromosome
- holds about 300 kb insert
| - uses F factor genes for replication
123
Yeast Artificial Chromosome
- Yeast telomere and add centromere regions
- selective markers
- origin of replication
- restriction sites for cloning
- can hold BIG pieces of DNA (several hundred kb)
124
Shuttle Vectors
- can replicate in 2 or more host organisms
- eg. Yep24- replicates in yeast and e. coli
- contains selective markers for E. coli and yeast and sequences which allow replication in both hosts
125
Insertional Inactivation
The inserted DNA inactivates a gene in the vector by inserting into that gene. This allows cells which contain the recombinant DNA molecule to be identified.
126
Identification of different cell types
cells with no uptake, cells that took up the original vector and cells that took up the recombinant plasmid. In this example, the cells that took up the recombinant plasmid will be ampr, tets. There was insertional inactivation of the tetracycline resistance gene
127
Ligation experiment
conducted to join foreign DNA to vector. The foreign DNA and the vector are both cut with the same restriction enzyme. The DNA's are mixed and DNA ligase is added. Some recombinant molecules should form.
128
Polylinker
DNA sequence within marker gene that contains unique recognition sites for many restriction enzymes into which foreign DNA can be inserted
129
Transformation experiment
Conducted to allow cells to take up products from ligation experiment
Products from ligation are put into bacteria
Then cells containing recombinant plasmid are identified: use of marker genes
130
Selectable/Screenable Marker
gene (on vector) that can be used to differentiate between cells with and without the vector or that can be inactivated by insertion to differentiate between cells that do or do not have the insert
131
Cells that take up nothing or take up only foreign DNA are?
amp^s and tet^s
132
Cells that take up original vector are?
amp^r and tet^r
133
Cell that take up recombinant plasmid are?
amp^r and tet^s
134
Blue-white Screening
?
135
Library
-set of clones derived from 1 approach
136
Type of libraries
Genomic
Chromosome Specific:: use micro dissection or flow cytometry to isolate a specific chromosome
cDNA libraries:
- cDNA=DNA that is a copy of mRNA
- only biologically active genes
- cDNA is already properly processed for expression
137
Reverse Transcriptase
RNA dependent DNA polymerase
| -used to make cDNA
138
isolating mRNA
1) a specific column contains short olgio (dt) chains linked to cellulose
2) Total cellular RNA is isolated from cells and passed through the column
3) The poly(A) tails of mRNA pair with oligo(dT) chains and the mRNA is retained in the column where as the rest of the RNA passes through
4) The mRNA is washed front he column by adding a buffer that breaks down the hydrogen bonds between the poly(A) tails and the oligo(T) chains leaving only mRNA with poly (A) tails
139
making cDNA after isolating mRNA with poly(A) tails
1) oligo (dT) primers anneal to the poly(A) tails of the mRNA and provide 3' -OH groups for DNA synthesis
2) Reverse Transcriptase synthesizes a DNA strand by using the mRNA as a template.
3) The RNA-DNA hybrid molecule is briefly treated with RNase, which partly digest the RNA strand
4) DNA polymerase synthesizes the second DNA strand by using the short undigested RNA pieces as primers and the nicks in the sugar-phosphate backbone are sealed by DNA ligase
140
Screening a library
1) a disc of nitrocellulose or other membrane is laid on top of the bacterial colonies
2) A few cells from each colony adhere to the nitrocellulose filter
3) The cells are disrupted, and their DNA is denatured and fixed to the filter
4) A labeled probe hybridizes with any complementary DNA
5) Excess probe is washed off and the membrane is overlaid with X-ray film which detects the presence of the probe
6) Comparison of the membrane with the master plate reveals which bacterial colonies have the DNA of interest
141
In situ Hybridization to locate Specific sequence
?
142
PCR
Polymerase Chain Reactoin
- Denature DNA by heating to 95 Celsius (breaks hydrogen bonds and allow strands to separate)
- Each strand serves as a template for replication
- Primers anneal (50-65 C) to identify target that will be amplified
- Taq polymerase adds nucleotides to 3' end of primer (72 C). Repeat many times
143
Kary Mullis
Developed PCR protocol
144
Thermal Cycler
machine that is programmed to alter temperatures so that PCR can be conducted
145
Limitations of PCR
- Must know something about sequence surround gene of interest in order to use PCR to clone a gene
- PCR reactions are easily contaminated from other DNA in the lab
- Taq polymerase does not proofread and correct errors (error rate about 1 in 20000BP)
- Fragments amplified by PCR are relatively small
146
Electrophoresis
separating out pieces of DNA based on size
- Distance migrates is inversely proportional to the log of the fragment size
- DNA loaded at negative pole and migrates to positive pole "RUN to RED"
- Small fragments migrate faster than large fragments
- Sizes can be determined based on comparing fragment migration to that of a control of known size
147
Blotting
process of transferring molecules that were previously separated (on a gel, etc.) to a membrane that is better able to support additional testing
148
Southern Blot
DNA fragments separated based on length
149
Northern Blot
RNA fragments separated based on length
150
Western Blot
Proteins that are separated on molecular weight, isoelectric point, electric charge, etc.
151
Southern Blot Process
1) Single Stranded DNA fragments from a gel are transferred to nylon membrane using capillary action in the Southern Blot
2) The nylon membrane is incubated with labeled, single stranded probe DNA
3) The probe binds to complimentary DNA fragments on the nylon
4) The position of the probe is identified
152
Probe
Single Stranded DNA that is a sequence we are interested in
153
Dideoxy Sequencing Reaction
DNA Sequencing!
Developed by Sanger
- DNA replication reaction proceeds until a dideoxy nucleotide is incorporated
-No further extension occurs
-Detect at which position the dideoxy nucleotide was incorporated.
154
Reporter
Foreign Gene is used as a reporter- on or off but can detect when it is on
155
GFP
Reporter GENE
- GFP linked to a protein allows us to visualize when protein is expressed in living cells over time
- GFP=Green Fluorescent Protein (from a jellyfish)
- GFP gene is joined to protein gene
- Gene is put into organism
- Organism produces a fusion protein
- Shine UV light on organism
- GFP fluoresces
- Allows expression of protein to be seen over time in living organism
156
Therapeutic proteins
- Production of Eukaryotic Proteins
| - Human growth hormone, insulin, blood clotting factors
157
Industrial Applications
- Production of Eukaryotic Proteins
- Rennin In cheese manufacturing
- Enzymes such as bacterial proteases in detergents and meat tenderizers, amylases to degrade complex sugars
158
Transgenic Plants and animals
Transgenic plant or animal is one in which a foreign or exogenous gene has been introduced into its genome, thereby altering its genetic constitution
159
Ti Plasmid
Ti plasmid is a tumor inducing plasmid from Agrobacterium tumefaciens. It causes crown gall disease in the normal infection process
-The Ti plasmid inserts into the plant chromosome to cause the disease
Goals:
- Remove tumor causing genes
- Leave transfer functions intact
- Harness the transfer functions so that the plant will be "infected" with a gene of interest
- Allow inserted gene to function in plant
160
Transposon
mobile piece of DNA flanked by terminal repeat sequences and typically containing genes coding for transposition (jumping genes)
161
Luciferase gene from firefly
emits a glowing color
- attempted to put into tobacco Plant
- T-DNA + insert inserted into 1 plant chromosome-heterozygous plant that glows
**Mendelian Inheritance pattern of 3 glowing: 1 non-glowing
162
Why do we use luciferase gene from the firefly?
Its used as a reporter gene so that if place regulatory sequences upstream form laciferase gene, regulation of luciferase can be monitored during developement
163
Forward genetics
start with a mutant phenotype and seek out the gene that causes that phenotype
-characterize that mutant type- is it dominant, recessive, etc.
164
Reverse Genetics
Start with a DNA sequence ( a genotype), alter its function or prevents its expression and observe the effects on the phenotype.
165
Transgenic mice
useful for forward vs reverse genetics
- Gene Function Studies
- Inject gene of interest into fertilized egg
- Implant in female
- Test progeny for presence of gene
- Mate to obtain mice homozygous for gene
- Study gene function
166
Knock-out mice to study Gene function
- insertion of neo gene into another gene will inactivate the other gene and provide resistance to the antibiotic G418
- injected altered gene into embryonic stem cells and select stem cells that have taken up gene
- Inject those cells into early mouse embryos. Isolate mice that show sectors with injected cells (black coast stem cells are often used as a visible marker
- Mate to get homozygous knock-out mice. Phenotype of mouse identifies gene function
167
RFLP
Restriction Fragment Length Polymorphism
- coexistence of two or more restriction fragment mapping patterns revealed by hybridization to a single probe
- Location of probe relative to the variable cut site is important in determining fragment sizes that will be seen.
-RFLP pattern within gene correlates with genotype in pedigree
168
Gene Therapy
- Introducing function copies of a gene into individuals who have only defective copies of that gene.
- In successful gene therapy, the transgene will make the missing gene product and restore normal phenotype
- Somatic Cell (non-heritable): treats, but does not cure the disease. All current gene therapies are somatic cell therapies
- Germ-line (heritable): major moral and ethical considerations
169
When introducing normal genes into cells this requires?
the use of viruses:
- Retroviral vectors integrate into the DNA of the host cell
- Transgene is transmitted to all progeny cells in the cell lineage
- Transgene may integrate so that it disrupts function of another gene
170
Transgene
introduced copy of the gene
171
NIH requirements for Gene Therapy
- Gene must be cloned, well characterized and available in pure form
- An effective method must be available for delivering gene to desired cells and/or tissues
- The risks of gene therapy to the patient must have been carefully evaluated and shown to be minimal
- The disease cannot be treatable by other methods
- Data must be available from preliminary experiments with animal models or human cells and must indicate that the proposed therapy should be effective
172
PCR as diagnostic Tool's Goal
determine if a particular DNA sequence is present in a sample of DNA
EX: is the blood sample infected by HIV
173
PCR as diagnostic tools method
- conduct PCR of a blood sample using primers for known HIV sequences
- Separate fragments on gel and determine if they are the proper length of the known HIV fragments
174
Real-Time PCR
- Quantifies the amount of nucleic acid present after each cycle of PCR
- Uses a fluorescent probe to identify the DNA of interest
* *Can be combined with reverse transcription to quantify mRNA (or other RNAs) and evaluate gene expression and the amount of mRNA produced under variable conditions
EX: does a drug affect production of a particular mRNA?
-Compare the amount of mRNA produced by cells that are treated with a drug and control cells
175
STR and VNTR
Short tandem Repeats
Variable Number of Tandem Repeats Micro satellite regions
-The allele is based on the length of the DNA segment with different alleles having different lengths because they have different number of copies of a short repeated DNA sequence
-Useful loci for paternity/forensics purposes must be polymorphic (many forms)
176
CODIS
- Combined DNA index System
- DNA databases funded by FBI
- Uses 13 polymorphic regions used for forensic identification
-3 tiered system: separate federal, state, and local databases
177
CODIS:
| Where do the DNA profiles come from?
- more than 9.4 million come from offenders
- about 360,000 from crime scenes
- DNA from missing person, relative of missing persons and unidentified human remains
178
STR analysis
Power of STR analysis:
-Ability to use small amounts of forensic material
-Statistical power of discrimination
13 core STR loci that are currently used for discrimination
These assort independently so product rule applies regarding probability of a particular combination of alleles
- However, accuracy of probabilities does depend on accurate probability estimates for particular alleles in the population
- Typically estimates are about 1 in 1 billion
- Data for one population is not necessarily correct for another population
179
Capillary Electrophoresis
- Same principle of separating DNA fragments based on their size
- Easy to test multiple genes in one automated run
180
FBI database
- if you were detained for some federal offenses, your DNA sample can be taken and put in the DNA database- if four not guilty can petition to get it removed
- If you are detained as immigrant (illegal or not), your DNA sample can be taken and put in the DNA database
- They do no need a warrant (in some states) to collect DNA from suspects
181
Ethical Issues with GN testing
Use of PARTIAL MATCH DATA: search databases for partial matches to help identify suspects
- Familial DNA testing
- Conflict betweens solving crimes and protecting privacy
There have been 311 people who were falsely convicted released from prison
182
Genetics
study of one or a few genes at a time
183
Genomics
Study of whole genomes- all of the genes, the interaction between genes and interactions with the enviroment
184
Genome Science
Goal is to study the structure, function and evolution of whole genomes. This complements the study of quantitative molecular and developmental genetics
185
Genome-Wide Association Studies
GWAS
- studies that look for non-random association between the presence of a trait (phenotype) and alleles at many different loci scattered across the genome
- Especially useful for identifying QTLs-those loci that are important in quantitative traits
- Find association between molecular marker and look for candidate gene can affect a that that located near the marker
186
DGRP
Drosophila Genetic Reference Panel
- IS AT NCSU
- 205 fully sequenced inbred lines from a natural population
- Used to identify QTLs
187
Mouse Collaborative Cross
Started with 8 inbred mouse lines:
-3 wild-derived + 5 lab lines
Mated to randomize genes resulting in about 1000 inbred lines that are mixtures of the 8 original lines
188
What could genomics mean for you?
Personalized medicine that takes into account the molecular events underlying a disease and your "genotype" to determine the best treatment option for you
- we know that not everyone responds the same to a particular medicine or therapy
- Tailoring medicine to individuals should provide better medical care and prevent harmful side effects
EX: tamoflaxin, some anesthetics
189
Warfarin
Blood thinner
- widely prescribed anti-coagulant (coumadin- prescribed in the from called)
- Greater than 10 fold inter-individual variability in the dose required to attain a therapeutic response
- Required dose also varies depending on individuals diet and is not the same for each person
2 genes influence effect dose
- CYP2C9 (warfarin metabolic enzyme)
- VKORC1 ( vitamin K epoxide reductase complex 1)
190
SNP
Single Nucleotide Polymorphism
- A SNP is a specific site in the genome where the DNA base varies in at least 1% of the population
- There are about 10 million SNPs in the human genome
- SNPs located near each other in the genome are often inherited together and can often be grouped as a HAPLOTYPE (a sequence of SNP patterns along the length of a chromosome)
191
Linkage Disequilibrium
The non-random association of certain variants with each other so that these combinations occur more frequently in the population than expected based on independent assortment
192
TagSNPs
Linkage Disequilibrium
tagSNPs= the few SNPs used to identify a haplotype
-A haplotype of 1000s of SNPs can be identified by only a few SNPs
-Thought that about 100,000 SNPs can be used to identify most haplotypes in humans
193
Using SNPs in association studies
Correlate Presence/absence of specific SNP or SNP haplotype with presence/absence of genetic disorder
Advantage:
-SNP association studies identify DNA difference between individuals more quickly/more easily that sequencing entire genomes
Problem:
- Correlation is not equal to CAUSATION ( but is a starting point for research)
- It takes a lot of research to identify if a particular SNP is meaningful for a specific disorder
194
Ethical Questions regarding genomics
- How do you control/regulate the spread of use of your genetic information
- Is the information going to make life simpler or more complex?
- Who has access to your genetic information?
195
GINA
Genetic Information Nondiscrimination Act
- Health insurance companies cannot decrease coverage or increase rates based on rates of genetic tests
- Employers cannot discriminate (hire/fire, etc.) based on results of genetic tests
- Neither Health insurance Companies or employers can require genetic testing
196
Mapping, Sequencing and analyzing the structure and function of whole genomes involve:
- collecting sequence data
- Correlating genetic, cytological and physical chromosome maps
- identifying DNA sequences that contain genes of interest in the genome and analyzing the function of those gene products
197
What companies are doing Direct to consumer testing?
23 and me
deCODE me
Navigenics
198
Genetic map of a chromosome
constructed from recombination frequencies in units of CENTIMORGANS. Includes RFLP sites
199
Cytological maps of a chromosome
Banding patterns based on chromosome staining
200
Physical maps of a chromosome
molecular distance in bp,kb, or mb
-physical maps often show restriction enzyme recognition sites, locations of particular clones or sequence-tagged sites (STS)
201
Anchor Markers
used to correlate different maps
| -are mapped both genetically and physically so can be used to correlate the genetic and physical maps
202
STS
Sequence tagged sites
-short, unique DNA sequences (200-500 bp) used to link physical and genetic maps. These sequences are hybridized to overlapping clones on physical maps and are hybrized to cytological maps using in situ hybridization to anchor map types
203
EST
expressed-sequence tags
-short cDNA sequences that are used to link genetic and physical maps. These cDNA sequences are used as hybridization probes to anchor maps
204
Contig
overlapping sets of clones that give a physical map of part of a chromosome
205
Positional Cloning
isolating a clone of a gene by searching its general position in the genome
206
Human Genome Project
1988-congress funds NIH and DOE
1991- project begins
2001- first draft published (90% complete)
2003- second draft compete (99% of euchromatin)
Project still continues with focus on variation
Craig Venter and FRANCIS COLLINS
207
What are the two approached to the human genome project
1) Map based sequencing:
- Make genomic library
- Fingerprint library and orient clones relative to each other
- Screen the library with markers that allow you to relate it to the recombinant map
- Map based sequencing relies on detailed genetic and physical maps to align sequence fragments
2) Whole genome Shotgun approach
- Small-insert clones (plasmids) prepared directly from genomic data and sequenced
- computer programs assembly by examining overlap
- Advantage of being highly automated
208
Orthologs
homologous sequences found in different species
209
Paralogs
homologous genes int eh same species and arrive through gene duplication
210
Microarray Analysis for Gene expression
allows study of gene expression including ability to quantify expression level between issues or strains of organism
211
Microarray analysis of RNA from cancer and Noncancerous cells
Red-higher expression in cancer cells
Green: higher expression in non cancer cells
Yellow: equal gene expression in both cell types
212
Bioinformtics
combines computer science and biology
Goals include:
- Maintaining and analyzing data bases of sequence data
- Comparing structural and functional features of DNA sequences and resulting proteins EX: BLAST
- determining evolutionary relationships among genomes
GN 311
flashcards
Decks in class (17)
# Cards
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Test 1127
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Extra20
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Test 2173
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Test 2 problem session19
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Lec 1732
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Lec 1851
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Lec1937
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Lec 2052
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Lec 2132
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Lec 2219
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EXTRA TEST 50
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Test2 problem session31
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Lecture 23: mutations, biochemical pathways, and repair mechs44
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Lec 26: Prokaryote regulation47
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Lec 27: Regulation of Eukaryotes37
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Lec 2931
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Test 5212
