Genetics and Genomics










Forward genetics
o Phenotype to genotype
Reverse genetics
o Genotype to phenotype
Cell is the basic component of organisms
o Nucleus contains the genes
o Mitochondria have their own genome
o Prokaryotic cells differ
 Genetic material in a nucleoid region
 Cell is organized but has no organelles
Almost everything is encoded in the DNA
o DNA karyotype-lay out chromosomes
Centromere
o Helps the chromosomes migrate from the middle of cell to poles
o Metacentric=middle
o Submetacentric=below the middle
o Telocentic=at the end
Cell division is essential to life
o Mitosis-division (exact copy)
o Meiosis-gametes (not an exact copy due to crossing over)
 Spermato/oogenesis
 2 separations to get haploid cells
o Cell must condense into chromatin
o Spindle attaches to kinetochore via the centromere
DNA replication can induce errors
o Mutations or other changes
o If it was perfect there would be no variation
Source for variation
o DNA replication and repair
o Crossing over and chromosome segregation
Cell cycle is monitored by checkpoints
o G, S, and M
o G0= nondividing cell
o Interphase is G and S
o The checkpoints can let mistakes through
 They check for DNA damage or a failure to replicate
 Something is wrong=apoptosis
Phenotype
o Appearance
o What is expressed












o Could be complex
Genotype
o What do the genes say
o Homo/heterozygous
o Dominant vs recessive
WT vs Mutant
o Wildtype is the normal that is defined
o Mutant is any changes
o Only 1 WT, but many mutants
Mendel
o Found that in the F1 generation only one gene/phenotype dominated
o But if F2 it was a 3:1 phenotypic ratio
o Chose phenotypes coded for by 1 gene
Monohybrid cross
o Only one gene being crossed
o Start with homozygous parental strains
Recessive alleles
o Only expressed when two copies of the gene are present
o In most cases the WT is dominant, but WT can also be recessive
Homozygous
o Two alleles the same
o Can be dominant or recessive
Heterozygous
o Two alleles are different
o Dominant will be expressed in most cases
Hemizygous
o Only one allele present
Dihybrid cross
o Two genes cross to see effect
o 9:3:3:1 outcome in the F2 generation
o Independent assortment
Independent assortment
o Combine the probability of one trait w/ probability of getting another
o Multiply
Test cross
o Can determine genotype if unknown but have a known phenotype
o Difference between homo and heterozygous
o Cross unknown with homo recessive
 If homo- get all dominant expression
 If hetero-get some recessive expression (1/2)
Human crosses







o Multiple different disorders
o Dominant diseases
o Recessive diseases
Pedigrees
o Can follow a disease in a family
o Can determine its genotype
o Recessive-skips generations
o Dominant- in all generations
o X-linked=expressed in more males then females
 Females carry
Expressivity-the overall expression of the disease (how bad it is)
Penetrance-not everyone gets the disease (have the gene but don’t express it)
o Out of the people who have the disease what % express it
Probability and statistics of Mendelian genetics
o P(A,B)=P(A) X P(B)
o P(A or B)= Pa +Pb
o P(a/b)=Pa/Pb
Binomial theorem
o Used to calculate the probability of any specific set of pairs of outcomes among a large #
of potential events
o P=n!/s!t! X asbt
o S=# of a outcomes
o T=# of b outcomes
Chi-square analysis
o Variation between the observed and expected
o See if there is enough variation to reject the null hypothesis which states that nothing is
happening (random chance)
o P must be less than 0.05 to reject the null
o Use a graph of degrees of freedom (# of phenotypes-1) and x squared to determine P
Classes of mutations
o Null mutation
 Destroys the gene
 Removes the allele completely
o Loss of function mutation
 Could be null
 Diminishes expression or function, or destroys a gene
 Usually recessive, need two mutations to alleles
o Gain of function mutation
 Some mutation causes a new function
 Can change the phenotype
 Ex: flies with legs in their head














Dominant mutations
o Why is simple genetic dominance most often observes for geno/phenotype?
 Only need one allele present to function completely
Mutations
o Missense
 Point mutation where the codon and changes the AA and protein
o Neutral
 Changes the codon and AA but not the protein
o Silent
 Changes the codon but not the AA or protein
o Nonsense
 Premature stop codon
Complete dominance
o Homo/heterozygous express the same phenotype
Incomplete dominance
o Heterozygotes have an intermediate phenotype
Codominance
o Express both alleles in the heterozygote
o Ex: blood type
Recessive lethal mutations
o The homozygous recessive is lethal and will not survive
o Do not factor it into the probabilities since they are unable to pass it on
Mixed modes of inheritance modify the 9331 ratio
Epistasis
o The effect of one gene depends on the presence of one or more modifier genes
 Ex: agouti mice-can only get the agouti pattern if colored a certain color
o Recessive or dominant epistasis
Novel phenotypes
o Get something completely unexpected from a cross
Pleiotrophy
o One mutation has a cascade of effects in the body
Complementation
o Helps to determine where in the genome the gene is located
o If in the same place, the cross leads to a mutation
o If in different places the cross leads to a normal phenotype
Sex linked
o Genes located on the x chromosome
o Males have to get their x from the mom and their y from the dad
Pedigrees
o Again help see the expression pattern
Does a genotype always result in the same phenotype


o No, because of penetrance and expressivity
Temperature sensitive phenotypes
o Heat and cold sensitive mutations (conditional)
o See a level of expression changes
Location can also affect expressivity
DNA











Functions
o Replication
o Information storage
o Info expression
Variation through mutation
o Allows new characteristics to evolve
Central Dogma
o DNA
 Transcription
o RNA
 Translation
o Protein
Has to flow in this direction unless a virus goes from RNA to DNA with reverse transcriptase
Ribosome is formed by rRNA
mRNA is loaded into the ribosome
tRNA brings AA to the ribosomes
DNA and genome size
o More genes doesn’t mean more complexity
o Such thing as alternative splicing
DNA as the genetic material
o Griffith’s transformation
 Found that transformation occurred by some molecule
o Avery, Macleod, McCarthy
 Only when using DNAse did transformation not occur
o Hershey-Chase
 Used bacteriophages and labeled molecules
 DNA with phosphate
 Protein with Sulfer
 Found labeled DNA in the cell
RNA can be the genetic material
o Viruses can have ss/ds DNA or RNA
o Reverse transcriptase
o Integration
Discovery of DNA












o X ray crystallography gave the idea of double helix
DNA facts
o DNA is right-handed (right hand rule and thumb up)
o Every strand has a 5’ and 3’ end
o A is always bound to T
o G is always bound to C
o A+T+C+G=1
o G3C
o A2T
o Phosphate connected to sugar, then the sugar is connected to a base
Purines (Double Ring)
o G, A
Pyrimidines (Single Ring)
o C, U, T
Sugar backbone
o RNA has an additional hydroxyl at the 2’ carbon
o DNA lacks the 2’ hydroxyl
When a sugar and base are bonded with phosphate=nucleotide
Without phosphate=nucleoside
o Up to 3 phosphate groups
DNA is made in the 5’ to 3’ direction
o Why cant it be made in the other direction?
 Cant possibly add on to the phosphate group at the 5’ end
Phosphate is negatively charged
o If together-will repel each other
o Need to make up the outsides, with the bases in the middle
o DNA has a negative charge
 Migrates to the Anode
DNA’s density
o G-C bond is more dense due to 3 H-Bonds
o The higher the G-C content the more dense the DNA
o Different DNA melting points as a result
FISH
o Test to detect Nucleic Acids
DNA replication
o Semi-conservative
 Evidence=2 rounds of replication with labeled DNA strands
o Many generations-only trace amounts of the old-mostly new
Bacterial Replication
o Starts at a single origin of replication
o Bidirectional replication










DNA Polym READS from 3’ to 5’ and SYNTHESIZES from 5’ to 3’ a new DNA strand
DNA polym
o I, II, III
o All can proofread and replace their mistakes
Holoenzymes
o Protein machine made up of multiple proteins and TF’s
Bacteria
o Origin of replication is defined by a repeated sequence (9 mer)
o DNAa molecules bind and create an initial bubble of replication
o DNAb/c bind to the bubble and initiate helical unwinding
o Primase adds an RNA primer
o DNA polym starts
Leading vs lagging strand
o All replication proceeds towards the replication fork
o One strand is continuous
o One strand is discontinuous
 Needs multiple primers
 Multiple okazaki fragments
 Ligase sticks together
DNA poly I
o Replaces the RNA primer with DNA
DNA gyrase
o Untangles the DNA helix
Speed
o Euk > Pro because there are multiple origin sites
Euk are not circular
o Have an issue with end of chromosomes
o Some cells have telomerase, which acts as an end primer to avoid losing some of the
telomere
o Most cells don’t have telomerase and lose a small portion with each replication
o Telomerase is only very active during large periods of replications, or when the cell is a
stem cell
Replication and recombination
o Need a single stranded break, then a ligation to a different place
o Crosses with its homologous region and allows for recombination because a piece of
DNA switched from one chromosome to another
Transcription



Transcriptome-all transcripts
Proteome-all proteins
Metabolism-all metabolic compounds










Transcription
o Help get an RNA message
o Need a template strand of DNA to get to RNA
o RNA is identical to the coding strand, but is matched up with the template strand
Prokaryotic cell
o Replication, transcription, translation occur in the nucleus (nucleoid region)
o Need an RNA polymerase
o Scans for an RNA binding site
o Need the sigma subunit to recognize the specific initiation sequence
Nascent RNA
o Transcript
Sigma factor dissociates after a few nucleotides of the RNA strand is built up
o Only necessary for binding and recognizing the promotor
o Recognize TATA box upstream
Operons
o Genes often found in a segment together
o Get a polycistronic mRNA
o Only found in prokaryotes
Ribosomes translate as mRNA is being transcribed
o No posttranslation modification
o Occurs faster than in Euk
o Quickly ramp up protein production
Eukaryotes
o Many more regulation of the mRNA
o Separated into compartments
RNA types
o mRNA
o tRNA
o rRNA
o miRNA
o catalytic RNA
Chromatin in Euk
o Densely packed DNA and organized by histones
o Before transcription may need to modify the chromatin
 Hetero/Euchromatin
o 3 types of RNA polymerase
 I=rRNA
 II= mRNA and snRNA (nucleoplasm)
 III=ssrRNA, tRNA (nucleoplasm)
o RNA Polym II promotors have a core promotor, and enhancer elements
TATA box







o Not a lot have it, but if a gene has it, it is essential to transcription
o Binds the RNA polymerase after binding TATA Binding Protein
o Allows for a transcription regulation
o Brings other RNA poly to site to increase regulation
CAAT box
o Another example of a TATA like binding element
Enhancers
o Specific sequence that can be located in front of, in, or after the gene
o If located in the gene it keeps the gene from being translated
o Can activate or depress depending on location
TF
o Generalized proteins that bind specific sequences to regulate genes and expression level
Transcript
o Eukaryotes need to mature it
o Add a methyl G cap and poly A tail to stabilize
o Alternative splicing
 Exons vs introns
 Introns spliced out
 Immature RNA s always longer than mature RNA (remove introns)
Complexity
o Think about number of proteins, not the number of genes
o Genes also interact with each other in different ways (regulate)
Splicing
o Group 1
 Make rRNA
 Need a guanine to bind to an active site within the intron
 Expressed hydroxyl, this attacks the donor site at the other end of the intron
and splices it out
o Group 2
 mRNA
 needs snRNP’s
 get a complex that forms lariat loops that splice out introns
 exons ligated
modify the transcript
o RNA editing
o Substitution editing
 Get a change of a nucleotide in a transcript
o Two forms of a protein depending on editing
o Insertion/deletion editing
 Can alter the function and shape of protein
 Or bring proteins into the proper reading frame to establish function
Translation



















The codon table
Code for an AA
Start codon AUG begins the open reading frame
Errors:
o Spontaneous mutations lead to base pair changes
o Point mutations: changes a protein
o Frameshift- changes many AA and protein
Length=basepairs/3
Weight=aaX110 daltons
The triplet code is nearly universal and can mostly use the same table
In viruses, they overlap in viruses to save space
Different messages within same transcript
Single mutation can affect multiple genes
Translation of mRNA occurs only when ribosomes and tRNA are present and functional
tRNA=clover shape
o h-bond to the complementary AA
ribosome
o prokaryotes
 70s ribosomes
o Eukaryotes
 80s ribosomes
Changing tRNA’s with AA’s
o Need an empty tRNA
o AA synthetase puts the AA on the tRNA
o Need ATP energy
o Activated enzyme complex (AA, AMP, aminoacyl tRNA) attaches the AA to the tRNA
Factors associated with 3 different phases of translation
o Initiation
o Elongation
o Termination
Ribosome is not formed until the mRNA binds the small subunit
o Then the large subunit binds
3 ribosomal sites
o Aminoacyl site=AA sits in the tRNA
o Peptide=growing peptide chain
o Exit
Stop codon causes the complex to fall apart
o Releases the peptide
Multiple translational complexes form on a single mRNA







Amino Acids
o R Group only thing that changes
o Hydrophilic/hydrophobic
o Polar (charged)
The r group differs in forl/function
o Change the folding by mutations
Protein sequence
o Primary=AA sequence
o Secondary=alpha helix or beta sheet (H-Bond stabilized)
o Tertiary=whole protein folding
o Quaternary=multiple proteins folding
Domain-functional part of protein that has a certain structure
Post protein modifications (post-translational)
o N terminal AA is often modified
o Add carbs to the protein
o Golgi editing
Functions of proteins
o Structural
o Contractile
o Signaling
o Storage
o Transport
o Enzymatic
Roles
o Enzymatic
 Lower activation barrier
o Signal sequence-domain that attracts substrate
o Membrane anchoring
Mutations






Germline vs soma
o Much more dangerous in germline, passed onto future generations
Classes= LOF, GOF, null
Transition
o Purine changed into a different purine
Transversion
o Purine changed for pyrimidine
Repeat expansion
o Continue to get repeated sequences
Genetic analysis
o Use mutations to ID mutations and their resultant phenotyoes



o Induce many mutations to get a specific mutation
Origin
o Proofreading errors
 DNA replication
 But you do get a lot of repair of these mutations
o Tautomeric shift
 One H switches position within nucleotide
 Leads to mispairing and replication errors
 T to G and C to A
 When replicated back to their normal binding partner, causes mutation
o Deamination
 Amino group in C or A converted to a keto group, which changes the basepairing
o Depurination
 Lose a nucleotide within the DNA
o Oxidative damage
 Oxygen damages the DNA
o Transposons
 Pieces of DNA that can insert or move within the genome
o Replication slippage
 Multiple repeats
 Get an increased # of copy number variants
o Base Analogs
 Incorporates a different nucleic acid
 5 bromouracil (binds to A)
o Alkylation
 Donate methyl or ethyl groups to amino or keto groups
 Guanine to 6-ethylguanine
o UV radiation
 Thymine dimers
 Repaired by nucleotide excision repair
Accessing genotoxicity
o Before anything is released used the Ames Test
o Have a control side and get the number of random background mutations
o Add the mutagen, see if any difference than the background rate
Repair
o DNA polymerase can proofread
o Mismatch repair
 Mut S/L/and H scan the DNA for the incorrect base pairs
 Then stick on the DNA and recruit DNA polymerase
o Excision repair (during DNA replication)
 DNA polymerase finds a lesion, it skips over
o
o
o
o
 REC-A comes back and fills in the gap
 DNA ligase ligates it together
SOS repair
 Last resort
 Induces more mutations
 Only occurs when there is massive mutation
Photoreactivation repair
 Dimer forms
 Dimer repaired
 Normal pairing restored
Base excision repair
 Recognizes a single wrong nucleotide
 Base removed by DNA glycosolase
 AP endonuclease recognizes lesion and nicks DNA
 DNA polymerase fills gap
Double stranded break repair
 Multiple lesions makes DNA unstable
 Activated during late S/early G2 stage
 When sister chromatids are available to serve as templates
Evolutionary Genetics







Darwinian evolution
o Species have a common ancestor
Neodarwinism
o Discovery of genetics
Evolution requires:
o Variation between organisms
o Competition between individuals
o Selection
Descent from common ancestors
o Can use genetics to find these relationships
Two forms
o Micro/macroevolution
o Large and small scale
Phylogenetic tree-shows relationship between species
o Stasic-doesn’t change
o Anagenesis-one species evolved into a different one
o Cladogenesis-species diverged into 2 separate ones
Morphology
o Species based on the way they look?
o Not a great model due to different looking organisms being of the same species














Biological species concept
o Define a species based on the ability to reproduce and have offspring
Selection and fitness
o Advantage for one characteristic
o Get some fitness affect
o Fitness-measure in the success of breeding
Mutations usually aren’t good
o However may be advantageous
o Selected for
Stabilizing selection
o Less genetic variation
o When the environment is stable
Directional selection
o Shift towards one side
o When the environment changes
Disruptive selection
o Environment heterogeneous
o Can harbor two different organisms
Maintain genetic variation
o Variation is not limited
o Sequence the genome to see the differences
o Change environment, some mutations become advantageous and are selected for
Cost of variation
o Protective effects of sickle cell anemia against malaria
o Fitness to genotype changes with the environment
Speciation
o Pre/postzygotic barriers
o Ex: geographical separation
Population genetics
o Hardy Weinberg
 Describes an ideal population’s allele and genotype frequencies
 P2+2pg+q2=1
 P+q=1
 Can predict what will happen in the next generation if no natural selection
occurs
Stronger selection against the recessive allele if homo recessive is fatal
Can be small or weak selection against an allele (or large)
Just mutations
o Takes many years for mutations to become a part of the species unless the environment
changes
Genetic drift



o The changes in allele frequencies due to chance
o More of an effect in a smaller population
Founder effect
o When a new population is started due to migration
o Will not have the same allele frequencies as before
Inbreeding
o Inbreeding depression (lose heterozygotes)
o No new influx of genetic material
o F value
 F=1 all homozygous
 F=0 no inbreeding
Distance apart in years=# of mutations X mutation rate
DNA organization











Simple chromosomes
o Viral and bacterial chromosomes often consist of single DNA molecules
o Bacteriophage=lambda (lollipop head)
Circular replication
o Cut bu a nuclease
o Copied discontinuously and continuously
Bacterial DNA packaging
o Ecoli supercoils the DNA
o DNA has no tension due to turns
Eukaryotes
o Organize using histone proteins
o Condensed state get G-bands (dark and light)
o Can alter the packaging to get to genes
 DNA loops out of chromosomes when needed
Nucleosome
o Histone octamer
Solenoid
o Group of 6 nucleosomes
Looped domains
Chromatin fiber
Chromatid
Net packing ratio of 500:1
Repetitive DNA
o 98% is repetitive DNA
o Centromeres
 Sister chromatid cohesion
 Assembly site for kinetochore
o









CEN
 The minimal DNA required for centromere function
o Satellite DNA
 Repetitive pieces of 2 or 3 nucleotides that are constantly repeated
o DNA isolation
 Satellite DNA has a lower density
 Less dense with more A-T bonds
o VNTR
 Variable number of tandem repeats
o STR
 Short tandem repeats
 Very short 5 or less bases
o LINE
 Long interspersed nuclear elements (transposon)
o SINE
 Short “ “
o Ribosomal genes
 Repeated in the DNA
Epigenetics
o Histone modification
o Can be passed on
o Reversible
Epigenators
o Environmental signals (internal or external)
o Signal is transduced to the cell
Histone modification
o Histones have a tail that can be modified
Acetylation
o Opens up
o Deacetylation closes
Methylation
o Opens or closes depending on location
HDAC
o Histone deacetylation complex
o Closes the DNA up
HAT
o Histone acetylation complex
o Opens dna up
CPG islands
o Sites where the DNA is methylated
Imprinting
o
o
o
IGF2 not turned off
Hypo/hyper methylation
Epigenetic inheritance can lead to cancer
Variation in chromosome number and arrangement










Karyotype
o Group chromosomes and banding patterns
Aneuploidy
o 2n±x chromosomes
Euploidy
o Multiples of n chromosomes
Polyploidy
o Multiples of the same gene
o Auto/allopolyploidy
 Auto=duplication of whole genome
 Allo=duplication of 2 diff species
Nondisjunction
o Doesn’t separate
o Leads to trisomy
o Trisomy 21=downs
o Trisomy 13=patau
o Trisomy 18=Edwards
Chromosomal rearrangements
o Need breakage of a chromosome
o Terminal deletion (lose piece at origin)
o Intecalary deletion
 Form a deletion look and it ejects a gene out of a chromosome
o Deletion loop
Duplication
o Unequal crossover between 2 sets of homologous chromosomes
o rRNA present in many copies
CNV (copy # variants)
o Chunks of repeated DNA in chromosomes due to duplication
o Can be present within promotor regions
o Can cause an increase in the replication of the gene
Inversion
o May express new genes
o Can happen due to loop
o Forms a 4 part breakage
Paracentric
o Doesn’t change arm length







Pericentric
o Changes the length of the arms
Consequences during chromosomal inversion
o Inversion heterozygote=one inverted and ore normal chromosome
Crossing over leads to nonfunctional chromosomes
Nonreciprocal translocation
o One chromosomes steals from another
Reciprocal
o They share-just changes chromosomes
o Forms a cruciform tetrad during meiosis
Robertsonian translocation
o Exchange of small arm of one chromosome for the large arm of another
o Can get familial downs
Fragile X
o Pieces of the X can break off at the end
o Its so thin because the DNA isn’t as condensed
Microbial genetics







Lag, log, then stationary phases
Auxotrophs-cant produce certain compounds and need it to be added
Grow bacteria on selective media
o Only grow with additions
2 life cycles
o Lytic
 Phage DNA is injected into the cell
 Cell begins to produce phage components
 Cell lyses and releases phages
o Lysogenic
 DNA integrated into the host
 Dormant
 All subsequent cells have viral DNA
 Eventually when stressed, the cell produces viruses
U tube experiments
o Just the medium allowed to pass
Conjugation
o Needs a sex pilus and attachment between cells to pass the DNA
o Also need a plasmid F factor
o Will not happen in U tube
HFR cells
o Have the F gene in the DNA itself
o Will conjugate but will not pass on the F gene to the other cell
















R factor encodes for antibiotic resistance
Horizontal gene transfer
o Within one generation
Vertical gene transfer
o Inherit from generation to generation
F factor can integrate into the genome
o Then it’s the HFR cell
Transformation
o Take up free genomic DNA from the environment and incorporate it
o Will occur in U tube experiments
Transduction
o The DNA is inserted via bacteriophage into another cell
o Will occur in U tube experiments
Genetic mapping
o Use recombination between the regions to map for mutations
o Deletion mapping-map consequences
o Recombination mapping-based on genetic exchange
Linkage
o Two genes on a single pair of homologs
o No exchange occurs
Distance matters in recombination
o Count the recombinants and parental
o Map distance=REC/(total) X 100
o 1cm= 1% recombination observed
Two and three point mapping
o Consider single and double crossovers
Double cross overs
o Frequency is the product of the two SCO’s
Table
o The highest #’s are the parental strains
o The lowest numbers are the double crossovers
Order of the genes is based off of which one is in the middle
o For the double crossover, the one that appears to change is the middle
C=coefficient of coincidence
o DCO observed/expected
o Interference=1-C
 Never the expected due to one crossover inhibiting a second
Somatic cell hybridization
o Linkage mapping
Sister chromatid exchanges
o Don’t see phenotype exchanges

o Exactly the same genes but cant see changes without mutations
GWAS (Genome wide association study)
o The goal is to map phenotypes and where they appear on chromosomes based on maps
Extranuclear inheritance









Inheritance of genes that are not contained in the nucleus
Chloroplasts and mitochondria
Both only inherited from mother
o Chloroplasts inherited from the MT+ parent
Mitochondrial inheritance can be tested with colonies
o Petite colonies indicate something is wrong with mitochondria
 Segregational=nuclear (1/2 petite)
 Neutral=cytoplasmic (all normal)
 Supressive=cytoplasmic (1/2 petite)
Chloroplasts
o Larger DNA than mitochondria (more introns)
MtDNA
o Smaller
o Goes missing
o No introns
The origin of mitochondria is via the endosymbiosis theory
Nuclear contributions to the mitochondria and chloroplasts
o Via nuclear genes
o Passed on via regular genetics
Mitochondrial diseases
o MERRF, LHON, KSS
Genetic elements + viruses



IS elements (bacteria)
o Insertion sequence
o Defined by inverted terminal repeats
o Flanked on both sides of the gene
o Transposons
 Recognizes inverted terminal sequences specific for an IS
 Inserts the sequence somewhere else in the genome
o DNA bases transposon elements (tn)
 Can be larger
Heteroduplex
o The complementary sequence that helps it bud off
In the presence of Ac, Ds is not transposable
o But Ac alone can transpose
o
o








Ac must still have its transposase gene
Ds lost its transposase function
 But still have the inverted sequences
Nonreplicative/replicative transposons
Rearrangements are mediated by pairs of tns
o Deletion between two transposons
o Get crossovers between repeats
o Get circular deletion
o Separate from chromosome
RNA based TE’s
o Retrovirus
 LTR=attracts RNA polymerase to make its products
o LINE
o SINE
Replication (copy elements)
o Transcribed into RNA and protein
o Can silence the transposon DNA
o Target for destruction
Retrovirus
o Integrase
 Mediates integration of DNA into genome
o Retroviral integration
 ssRNA to dsDNA
 reverse transcriptase cant proofread
o retroviral budding
 products packaged and moved to the PM
DNA viruses
o Have a lytic/lysogenic life cycle
RNA virus
o Remain RNA always
o Can be + or – stranded
 +=no rdrp (translated directly)
 -=rdrp to transcribe to + strand
o RDRP=RNA dependent RNA polymerase
Zoonoses=movement of virus from animal to human
Recombinant DNA



Cut a plasmid vector with restriction enzyme (vector)
Cloned DNA is cut with same RE
Then the two pieces of DNA get linked together
o Then introduced to host cells via transformation











Select cells with recombinant DNA by antibiotic resistance selection
Libraries
o Collections of clones
o CDNA library (complementary DNA)
o Higher complexity means the more coverage
o Need 5 times the number of clones to cover a whole genome
 Make sure all overlaps
Vectors
o Plasmid vectors have a small amount of DNA
o Phage/cosmid vectors are larger
o Artificial chromosomes
o Expression vectors
o Shuttle vectors
Restriction Enzymes
o Endonucleases with a specific recognition restriction site where it cuts DNA
o Leaves sticky ends
o Cut every 4N base pairs
 N is number of bases in RE recognition site
cDNA
o get ds cDNA with reverse transcriptase and mRNA
need a selective marker in the vector
o screen for the vectors
PCR
o 95=denature
o 50=annealing
o 75=polymerization
o Amplify the DNA experimentally
Real time PCR
o Probe on the template
o See the florescence level
Restriction mapping
o Cut the DNA with different enzymes and see how the DNA is put together
Southern Blot=DNA
Northern Blot= RNA
Genomics



Sanger sequencing
o Able to do short segments of the genome (about 1000)
Next gen sequencing
o Sequences the entire genome
Clone by clone sequencing
o
o
o











Omics
Cut up the genome into pieces using RE’s
Smaller and smaller pieces
Then insert into a large plamid
 YAC/BAC
o Fit together with overlapping clones
Shotgun based sequencing
o Use different RE’s to cut
o Sequence contigs (next gen sequencing)
o Overlap contigs using a computer system
 Repetitive DNA is hard to overlap
Gene models
o ID the UtR, initiation site, promoter, regulator elements, introns, and exons
Sequence the cDNA or RNA so that you know what is expressed in a mature cell
Can also get different mRNA based on alternative splicing
Determine the expressed pieces of a genome
o Computer reads all three frames
o Best when there are no introns
Databases
o BLAST
 Uses an algorithm to see how close a protein overlaps with the alignment of
known proteins
 Determine % overlap
 Also can determine functional domains
Human Genome Project (HGP)
o 20,000 genes
o 3 billion base pairs
o 98% noncoding
o The noncoding DNA may have a regulatory function
o Made partial chromosomal maps
Genes cluster
o Deserts in between genes
Disease maps
o Map the genes that cause diseases and where it is located on the chromosome
ENCODE
o Look at hetero/euchromatin and changes from cell/cell
o Shows where the genes are going to be expressed
CHIP
o Chromatin immunoprecipitate
o tag the protein with antibodies to find the protein of interest












variation in genome size and gene number in bacterial genomes
o not sure what the minimum # of genes is to make an organism
Organization is circular or linear
Eukaryotes
o Less gene dense (have introns)
o More DNA
o More noncoding and repetitive portions
Dogs used to compare to humans
o Have similar genes
o Chromosome 15 responsible for the size of dogs
Linkage disequilibrium
o Tendency of two alleles to remain linked through meiosis
o Synthany=tells how similar genes are in terms of the order of genes
Comparative genomics
o Genes can evolve based on exon duplication and exon shuffling
o Can trace the domains back
Alpha and beta globulin
o Similar on the molecular level
o Make up a subfamily of proteins
Multigene family
o Evolve by deplication and divergence
o Phylogenetic tree
o Conservation of gene structure
Superfamily
o All the related proteins
Global Ocean Sampling (GOS)
o Metagenomics
 Looked at the spectrum
 Look at everything as a whole and draw conclusions
 Too large to look individually
Human Microbiome Project (HMP)
o Transcriptomics
 Look at all the transcripts to see what is expressed
o Analyze the transcripts
 Microarrays-specific
 Checks for a small sequence
 Checks a lot of different small sequences
 Very good and cheaper to check for cancer
 RNA sequencing= everything
 Sequence all the RNA
Typify diseases based on the RNA expression





Checking for cancer question
o Check for the DNA having the code
 DNA microarray specific polymorphism (change)
 Full sequence if looking for cnv, or other repeats
o Transcribed to RNA
 RNA seq
o Translated to protein
 Western blot
Microarray
o Looks at a small subset of the genes or RNA and not the whole cell
Proteomics
o Look at the proteins present in the cell
o Western blotting
Cut proteins into pieces using trypsn
o Use mass spec to separate based on mass to charge ratio
Systems biology
o Make sense of all the data
o Connect concepts and networks
o How the pathways interact with one another
Human Genetics







Females XX (homogametic)
Males XY (heterogametic)
Theory-the embryo can develop into male/female
o At one point important changes due to the presence of the Y chromosome set the male
into action
Y chromosome
o PAR region (pseudo autosomal region)-allows the Y chromosome to pair with the X for
mitosis and meiosis
o MSY region (male specific)-genes that make a male a male
o SRY region (sex determining region)-induces the development of testes
 Expressed 6-8 weeks into development
o TDF=testes determining factor
X inactivation
o Due to dosage compensation
o Creates a barr body
 Utilizes the Xic region and the T-six gene
Single Nucleotide polymorphisms (SNPs)
o Differences in genomes between organisms, or genetic variation
Genomic variation (types of SNPs)
o RFLP




 Restriction fragment length polymorphisms
 One of the first ways to distinguish between genomes
 Appearance/disappearance of specific restriction sites
o VNTR
 Variable # of tandem repeats
 The more repeats, the earlier disease onsets
o SNP’s
 Single mutations at a specific location
o CNV
 Copy number variants
 Large piece of DNA repeated
GWAS database
o Links phenotypes to genotypes
o Associates SNP’s with genomes
Need to adjust the p value when you have such a large sample size
o Bonferroni-corrected significance cutoff
 Original p / N (sample size)
Pharmacogenomics
o Try to associate peoples genomes to the way a drug functions
o Responsiveness
 The % of effectiveness
 Determined by the genome
o Drug ex: Herception
 Need to sequence first, using microarrays to determine if the expression
correlates to the disease
 Can only use if specific HER-2 mutation
o Personalized medicine
 Based on a person’s genome
Adverse drug reactions
o Cost billions of dollars
o People process drugs in different ways
 Ultrarapid metabolizer > extensive metabolizer (normal) > Poor metabolizers
Prokaryotic gene regulation


Operons
o The idea of an operon is that in prokaryotes, many genes that are expressed together
are under the control of the same promoter elements
Inducible operons (also known as adaptive, facultative)
o Only expressed when necessary
o System can be turned on/off depending on environmental stimuli
o Positive control






 Inducer in the system that turns on gene expression
o Negative control
 Genes that are normally on get shut off by the presence of the molecule
Constitutively active
o Always on
Lac operon (inducible)
o Cis acting regulatory sites are present upstream of gene clusters
o 3 genes
 LacZ=B-galactosidase
 Lactose to glucose and galactose
 LacY=lactose permease
 Facilitates entry of lactose into the cell
 LacA=lactose transacetylase
 Detoxifying enzyme
o Repression
 Lac I
 Expressed and binds to the operator site to stop transcription
o Polycistronic RNA is created after transcription
o Repression of the Lac operon
 LacI repressed when present
 Binds the operator regon
 Only leaves when lac is present and binds to the repressor (and glucose is
absent)
Mutations
o LacI mutants
 Cant bind to the promotor
 Stays on constantly
o LacI mutants
 Can bind to the promoter but not lac, so always off
o Operator region
 Wont bind the repressor-always on
 Known as the Oc mutation because it is constitutively active
Make diploids to see mutation effects (Merodiploids)
o The operator needs to be in front of the genes
 So if a mutated operator is in the plasmid, will not have an effect
o Repressor can be made anywhere and travel to bind the promoter
o IPTG can induce the lac operon expreeion
Glucose is the preferred carbon source
o Less energy cost to the cell
o Glucose levels high, cAMP levels low
cAMP levels are high when no glucose
o






cAMP binds to CAP (catobolite activating protein)
 CAP induces expression of the lac operon (assuming lac is present)
Lac repressor
o Homotetramer
o Inserts 4 O sequences that then are pulled together to form a repression loop and stop
transcription
Trp operon
o Repressible system
o Opposite of lac
o The presence of trp shuts off the operon
o The lack of trp turns it on
o The repressor is bound to the operon when it is bound to trp
Trp mutants
o trpR mutants
 always on
o trpO mutants
 always on because it cant be blocked
o trpP mutants
 always off
attenuation
o an interaction between transcription and translation that regulates expression
o leader region is in front of the trp operon
 transcribed onto the mRNA and has a regulatory function
 trp present=terminator hairpin and no transcription
 trp absent=anti-terminator hairpin and transcription
 charged tRNA’s determine if trp is present or not
 if charged tRNA present-there is trp present
o mediated by trp RNA binding attenuating protein (TRAP)
 TRAP enables formation of the transcription terminator hairpin if it binds to
enough trp
 ANTI-TRAP
 No binding of trp, forms the antiterminator hairpin loop
Arabinose operon
o Under both inducible and repressible control
o 3 genes and a CAP binding site in the E.coli
o Both types of control are mediated by Ara C
Don’t invest in the synthesis of any other sugar if glucose is present
Eukaryotic gene expression

Domains separate chromosomes
o Chromosome territories






o Interchromosomal domains between chromosomes
Compactness of DNA
o Acetylation and methylation of histones and DNA regulate the compactness of
chromatin
o Chromatin remodeling complex (swi/snf)
 Opens up the DNA
 Needs ATP to remodel
 Moves the nucleosomes apart
o Less tightly wound makes the DNA more accessible to transcription
o Insulator elements prevent the spread of chromatin remodeling
Cis acting sites in chromosomal DNA bind to transcriptional regulatory proteins
o Promoters
o Enhancers
o Silencers
Promoters
o Focused
 Always initiates transcription from the same site
o Dispersed
 Initiates transcription from multiple sites
 Get multiple transcripts
o Focused promoter elements
 BRE
 B recognition elements-affect complex binding
 TATA
 INR
 MTE
 Motive 10 elements-help RNA polym bind
 DPE
 Downstream promoter elements-help RNA polym bind
 CAAT box
 Required for initiation
 GC box
 Binds TF’s
o Effect of mutations
 Mutate promoter elements-reduce the transcription level
Cis acting elements bind TF’s
o TF’s often expressed in time and tissue specific patterns and can recruit or interact with
RNA polymerase, and other Tf’s, and respressor proteins
Basal transcription level vs induced transcription level
Functional domains of TF’s
o Can screen the genome and ID the TF’s based on their properties
o




DNA binding domains
 Helix turn helix
 Trans-activated domains (repressors)
 Zinc finger DNA binding domain
 Basic leucine zipper
Assembly of TF’s
o Ex: RNA polymerase
o TBP (Tata Binding Protein)
 Binds to the sequence and brings in TAF (TATA associated factors)
o Polymerase comes in and forms the complex
o TBP and TAF stays in place to recruit additional transcription complexes
Enhancers
o More upstream
o Help attract TF’s
o Can affect how fast a complex is made
o Increase the rate of DNA unwinding and RNA polymerase release from the promoter to
initiate transcription
o Ex: UASg
 Constitutively active
post translational regulation
o alternative splicing
o ex: sex determination in Drosophila
 SLX gene is only active in females
 Get female only splicing that leads to the production of the DSX-F protein
 DSX-M protein present in males
o mRNA stability control
 control the half life of the mRNA
 depends on the transcription rate, processing, and degredation
Protein level
o Autoregulation
 Ex: tubulin subunits bind to the growing polypeptide chain
 Can stall the translation
 Get RNAse to degrade the mRNA
o Iron regulation
 Regulates the ferrin gene
 No translation if an Iron regulatory protein is bound (which means no iron is in
the cell since iron binds to release it)
 Too much iron?
 Binds to IRP, which down-regulates the mRNA (which is only stable
when IRP is bound to it)
o miRNA and siRNA
o
o


RISC


RITS

both bind to the RICS and RITS complexes
created from dsRNA via the dicer protein
Degradation of mRNA complementary to the sequence of the small RNA
Downregulates the mRNA that is not exactly complementary but close
Goes directly into the cell nucleus and downregulates the production of the
gene directly
Gene Function






Forward genetics
o Genome wide genetic screens for mutants with specific phenotypes
o Id the genotype that creates the phenotype
Reverse genetics
o Define every gene in the genome based on sequence analyses
o Reduce/eliminate functions of specific genes and assess the phenotypic impacts
Model organisms
o Easy to grow
o Short generation
o Abundant progeny
o Can cross in large numbers
Yeast
o Simplest eukaryote
o Haploid and diploid alternating generations
o Phenotypes are evident in haploid
o Diploid allows for recessive lethal mutations to be studied
Drosophila
o No meiotic crossing over in males
o Diploid
o Recessive lethal mutations are maintained in strains heterozygous for balancer
chromosomes
P-Elements
o DNA transposons that insert into the genome
o Can enable transformation
 Wild type or altered copy of the gene to assess transgene function
 Reporter gene in which enhancer/promoter drives expression of beta-gal or
other detectible genes
o Need a positive selective marker
o Can use this to destroy genes
 Randomly inserts itself into the open reading frame
o P elements either insert or destroy gene






Mice
o Genomic synteny with humans
o Large scale genomic screens difficult
o Creating transgenics and gene knockouts/replacements is more feasible
Mutagenization
o Mutagenize parental strain, then perform crosses to generate progeny that can be
assessed for phenotypes of interest
Types of mutations
o Chemical
 EMS, ENU
o Radiation
 X-Rays, gamma radiation
Screen mutations
o Genetic screen helps select out the ones which were mutated
o Can look at yeast and determine the stages of cell cycle
 See any arrested development
 Will not grow if mutated
o Replica plating
 Get the same colony and grow under different stressors to see if mutations are
sensitive or if new mutations appear under stress
o Screening mutants (Balancer Chromosomes)
 Can screen for recessive mutations in diploids by creating a collection of
mutagenized chromosomes in balanced heterozygotes
 Assess the phenotypic impact of homo/hemizygocity
 Retain mutations of interest
Untangling paths
o The order of generation is based on epistasis
o The effect of mutation in one gene masks or modifies the mutation in another gene
o Pathways affected by this-because every gene must be present to make a product
o Use epistasis analysis to determine which gene is at the top and which is at the bottom
 Can determine pathway order with mutation to genes
o Screens for suppressor mutations can ID additional genes in a pathway not Id’ed in an
initial screen (second round of mutagenesis)
 Second mutation by chance mutates the other one t try and bypass the initial
mutation
 Modify the original phenotype
o Suppressor mutants
 Diminish/eliminate the phenotype caused by the initial mutation
Gene product sequence
o May reveal gene function
o Presence of domains known to have specific functions





Gene product function
o Investigated further using molecular genetic tools and techniques
o Many different methods
 Take gene see protein
 Take protein see function
 Gene knockout/replacement
 Where protein is expressed and what its function is
o Can ID the cDNA responsible for protein via library
o Use antibodies against the protein of interest to screen expression vectors/libraries
 Expressed clone contains sequences for the gene of interest
o Cloning genes by complementation of genetic defects in heterologous or homologous
cell/cell lines
 Can recover the function with human gene inserted into the yeast
 Associate with the function
Gene expression
o Want to ID where and what the gene is doing in the cells
o Place and time of gene expression
o Tagged immunochemistry/florescence to see where and when the protein is expressed
Mice
o Selection for insertion of positive selectable marker disrupting the target gene
o Get recombinant and negative selection against nonhomologous insertions
o Introduce knockout isolated cells into blastocyst
 Get a chimera mouse
 Need the chimera to get the homozygous line after getting heterozygous
knockouts
Chip-Chip Sequencing
o Assess epigenetic state
RNAi
o Ran interference
o Homologous RNA
 Get the RISC complex to degrade the target RNA leading to gene knockout
Bioengineering




transgenic pigs engineered to express green florescence protein (GFP)
genetically engineered biopharm. Products
o cell lines genetically engineered to produce a medicine/drug
biologics produced using bacteria, fungi, and cell lines as bioreactors
o Can create insulin for example
o Extract the A and B proteins from different cells and combine them to form fully
functional insulin
Biopharming












o Use of GMP for production of biologics
Biologics
o Genetically engineered biopharm products
Expression in bacteria, yeast, and mammalian cells
Vaccines
o Either inactive or attenuated sample of a virus
o Can be edible or injected
Subunit vaccine
o One or more surface proteins from a pathogen
o Get an immune response
Inject proteins into a person-still get the immune response
Plant genetic engineering
o Higher yields
o Drought prevention
o Started from artificial selection
 Select the best ones and breed them
EPSP
o Important to produce aeromatic DNA
o We don’t make some of these AA’s
 Tyrosine, threonine
o Bacteria and plants make them
o Destroy operation of aeromatic AA’s so that the plant dies
o Put a strong promoter in to get a high EPSP synthase
Locating animals for production of biologics and protection against mastitis (staph)
o GM lysostaphin production cleaves the cell wall of the protein
Use florescence to detect things
o Constitutively on promoter turns on when the object is present
Synthetic bio
o What is the minimum genome
o Can then begin to incorporate other things
Fetal karyotyping and genotyping
o Amniocentesis
 Stick a needle in and take amniotic cells
o Chorionic villus sampling
 Take a sample from the placenta
o Fetal cell sorting
 Blood sample from mother (some fetal cells)
o Helps get a karyotype and genotype on fetal cells
o Preimplantation diagnosis
 PCR amplifying DNA
RFLP









o Detect 5-10% of genome wide sequence variation
Aso testing (allele specific oligonucleotide)
o Short oligonucleotide of defined sequence based on SNP’s hybridization PCR
amplification of genomic DNA from sample
Array based genotyping
o Microarray
o Look for gene expression and level
o check for SNP’s/CNV variation
p53 genechip
o any of the 500 mutations that could lead to cancer
can see the genes required for infection, propagation, and pathogenesis
can see which genes are involved in fighting viruses
gene therapy for people with SCIDs
o only a one gene fix
o never officially proven
MMLV virus used to insert the correct gene
o Virus that effects once and shuts down
Majority of delivery vesicles are viruses
o Randomly integrate-so need better control
Concerns
o Capacity only 8kb
o Could provoke an immune response
Body Plan







Developmental genetics
o Genetic and molecular mechanisms underlying cellular and organismal development,
homeostasis, aging and senescence
Development
o Develop tissues
o Death of specific tissues
o Balance between growth and death
Specification
o When genetic and positional cues confer a spatially discrete ID on cells
Determination
o Cells time when a specific developmental state becomes fixed
Differentiation
o Process by which a cell achieves its final form and function
Hypothesis
o Development-attainment of a different state by all somatic cells in an organism
Variable gene activity hypothesis
o Differential expression and action of genes














Controls development
o When and where are genes expressed and active
o How is gene expression regulated
Preformation
o Sperm had little human inside that became bigger
Fertilization occurs when an egg and sperm fuse
o Maternal cytoplasmic components
o mRNA and proteins
 first components to trigger development
 without these nothing would happen
body plan
o very similar in organisms within the same species
o Pattern of organization-characteristics and recognizable traits
Pattern formation
o Aspects of development of the body plan
o Leads to genesis of patterns or structures that make up the body plan
Number of axes (primary)
o Anterior
o Posterior
o Dorsal
o Ventral
Animal body plans are segmented
o The body plan has 11 segments
o Often has appendages
Drosophila
o Homologies among embryonic, larval, and adult body plans
o Governed by a set of genes
o Different segments develop into different parts
Segmental organization of embryonic and adult tissues is homologous
Segmental disks develop into extremities
o Imaginal discs rise to external structures
Mutations that alter the body plan affect the pattern formation
o 3rd segment develops into a second segment-fly has 2 sets of wings
Embryogenesis over 24 hours get the body plan and imaginal discs
Syncytial blastoderm (multiple nuclei)
o Followed by nuclear migration and cellularization
o The pole cells form at the posterior and are the precursors to a germ cell line
o Maternal functions direct the AP and DV axes
Zygotic genes
o Part of the genome but regulated by maternal effect genes
o Gap genes, pair rule genes, and segmental polarity genes form the body plan
o









Then homeotic genes (HOX genes) determine the fate of cells and specify the type of
cell they will become
Nuslein-Volhard and Wieschaus
o Determined which genes are important to body plan
Maternal effect genes
o Form the anterior posterior gradiants
o Gap genes are triggered (they are TF’s)
 Trigger certain genes in gap genes to form the band regions
o Formation of discrete bands triggers pair rule genes
 Divide gap gene bands into smaller regions
o Activation of pair rule genes activate segment polarity genes
 Even more divided
o Then the hox genes are activated and specify the ID of each segment
Gap genes are zinc finger TF’s
o Activate the next set of genes
Pair rule genes
o Often encode helix turn helix TF’s
o Overlap of TF’s or non overlap specifies specific segments
Mutations
o Runt (mutated RunX2 protein) encodes a TF
o Mouse doesn’t have proper muscle/bone development
o Humans get cleidocranial dysplasia
o Autosomal dominant diseases
Two Hox genes clusters in drosophila
o Antennapedia complex and Bithorax complex
Hox genes and TF’s with Homeobox
o DNA binding homeodomain
o Different complexes influence the further specification of segments into specific cell
types
Gene organization with Hox genes
o Hox genes have a logical order in the DNA
o Not intermixed
o Collinear with expression patterns in the embryo
Humans
o 4 human hox gene clusters
 39 total
o Control A-P patterning in humans (and other vertebrates)
o 5’ end genes
o Limb development
o Humans don’t get mutated very often (need a double mutation since diploid)
 get smaller changes (ex: polydactyly)






o more complex development
signaling pathways
o cell signaling/signal transduction
o central to development
o wnt path, TGF-B path, hedgehog path, RTK path, notch signal path
o cell signaling paths determine cell fate
o depends on cell/cell interactions-need to talk to each other
o mediated by ligands and receptors
notch signal path example
o Membrane bound ligand stimulates membrane spanning receptor, a portion of the
receptor is transported to the nucleus and affects target gene expression
o Human NOTCH 1-4 mutations
 Alagille syndrome
 Lymphoblastic leukemia
 Spondylocostal dysnstosis
o Developmental program initiated by NCID going into the nucleolus (after the delta
protein binds notch receptor)
o Activates TF’s
Notch in C elegans
o 959 cells exactly
o Know exactly how all cells form
o Starts with the zygote then splits into adult over series of steps
o All divisions occur by specific developmental stages
Uterine
o Development is random between 2 cells
o Lag 2=signals Lin12 (receptor)
 Both cells expressing proteins
o By chance one will express more signal than the other (more Lag 2)
 This inhibits Lag 2 in the other cell and get more receptor in that cell
o More signal= anchor cell
o More receptor= ventral uterine precurson cell
o Once the anchor cell is determined it increased production of Lin3
o Cells closest to the anchor get the most lin3
 Those cells are primary development cells (into the vulva)
 Cells around that, secondary development cells
 no signal from the anchor-develop into skin cells
depends not only on maternal factors, but other factors as well
o help from gradients that trigger specific developmental programs
cells removed during development
o don’t stop dividing-cancer
o produced cells need to be removed
o
o
o
o
o
o
occurs in c elegans to get to the 959 cells
 hermaphrodites 131/1090 cells die
 males 147/1178 cells die
15 cell death (ced) genes
Apoptosis=cell death (programmed)
Ced9 expressed, shuts down ced3-4 and the cell survives
 Vice versa, no ced 9, get cell death
Gain of function mutation in ced9- leads to no cell death
BCL-2 is the human version of ced9
 Overexpression prevents cell death
Cancer genomics







Disease of somatic cells
o 25-33% of the human population is affected
o Kept in check y autoimmune surveillance and cell death
Somatic cell dysfunction is due to dysregulation of cell growth and movement
Uncontrolled cell division and the avoidance of cell death
o No apoptosis
Dysfunctions
o Proliferation-excess cell growth
o Metastasis-movement of cancer cells
o Benign tumor- local mass of cells
o Malignant tumor-cells metastasize and the tumor has access to a blood supply
o Primary tumor-the initial site
o Secondary tumor-the site where the tumor spreads to
Genetic theory of cancer
o Cancer is the result of multiple gene mutations
o Accumulation of mutations in different genes due to genetic or epigenetic variation
o Up to 1010 mutations over a human lifetime
Genomic instability
o Mutator phenotype
o Aneuploidy
o Rearrangement
 Translocation
 Inversion
 Deletion
o SNP’s
o Amplification
Clonality
o Tumors are comprised of clonal cell populations that all originate from a single founder
cell
o
o










Disregulated growth and then disregulated movement
Are all cells dividing?
 Think that cancer stem cells are the only ones dividing
Proto-onco genes
o Genes that promote/ stimulate normal cell division and growth
o Gain of function: by overexcitation or loss of regulation, proto-onco genes become onco
genes, which stimulate hyperproliferation
Tumor suppressor genes
o Genes required for negative control
o Shut down cell division if activated
o So if you lose control via a Loss of function mutation, it leads to cancer
1-2% of cancer is hereditary
Ex: FAP (Familial adenomatous polyposis)
o Heritable cancer based on mutated copy (single) of APC gene on chromosome 5
o Keep growing-don’t stop division
o APC=tumor suppressor gene
o Role in contact mediated growth inhibition
o Get polyps in the SI
Driver mutations
o Confer growth advantage to cancer cells
o Cancer becomes worse with these mutations
Passenger mutations
o Other mutations that happen in the course of cell division that do not confer growth
Epigenetic variation
o Demethylation or acetylation of chromatin encompassing genes that stimulate cell
division/migration
o Hypermethylation or histone deacetylation accompany genes that arrest cell division or
mediate cell death
Cell cycle control
o Altered function of genes regulating the cell cycle can lead to dysregulation of cell
division and excessive cell proliferation
o Abundance of different cyclins during the cell cycle that regulate transitions from one
part to the next
o Mutate cyclins
 Get cell division when cell shouldn’t be dividing
Apoptosis
o Programmed cell death
o BCL 2 level important (low for cell death)
o Cell death triggered by caspases
o Apoptotic bodies are engulfed by phagocytosis
Bax homodimer promotes apoptosis
o







P53 induces BAX transcription
 Inhibits BCL2 transcription
 This stimulated cell death
o P53 low function in cancer
 BAX transcription low
 BCL2 high
 Cell doesn’t die
RAS (protoonco gene)
o GF stimulates the cell proliferations
o Activated when bound to GTP
o Tells the cell to proliferate
o Mutated and constantly active-cell always proliferates
P53 (tumor suppressor)
o DNA damage repair
o DNA damage promotes cell cycle arrest and fixing of the DNA
o Mutation rate increases if p53 not working
RB1 (tumor suppressor)
o Inhibits TF’s when not phosphorylated
o Can be inherited- one copy damaged
o Triggers a cascade of genes that pushes the cell through the cell cycle
o No longer binding E2F?
 Constantly pushes the cell through the cell cycle
o Only one good allele needed
Migration of metastatic cells away from the primary tumor site
o Establishes itself at secondary tumor site
o Get blood vessels to oxygenate (angiogenesis)
Metastatic cells
o Reduced expression of E-cadherin glycoprotein (reduced cell-cell adhesion)
o Increased expression of tissue metalloproteinases (TMP’s) (increase cell migration)
o Reduced interaction with tissue inhibitors of TMP’s (increase cell migration)
o LOF mutation in metastatic genes-leads to metastasis
o Or GOF mutation
Viral contributions to cancer
o Many people believe that cancer is caused by a virus
o Onco gene retroviruses
 Acute transformation retroviruses
 First IDed in chickens by Rous (RSV gene)
 RSV translated portions of a cellular gene that stimulates cell division (C-SRC)
 May pick up the gene from the genome while spreading
 Inserted into another genome and leads to overexpression (now 2 copies)
Environmental contributions

o Radiation
o Smoking
o Other factors
Drug design
o Use the exact path to develop drugs
o Many times specific to a certain mutation
o Gleevec
 Acts as ATP and binds the site that allows BCR-ABL to stimulate cell division
when bound to ATP
o Trastuzumab
 Binds to HER-2 and induces its removal (down regulation)
 HER signals less intense and the cell therefore divides less often